A Dosage Range-finding Embryo-fetal Development Study of ...

FINAL REPORT

Testing Facility Study No. 20024505

A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a

Preliminary Evaluation in Non-Pregnant Rabbits

SPONSOR: RegeneRx Biopharmaceuticals, Inc. 15245 Shady Grove Road, Suite 470

Rockville, MD 20850 United States

TESTING FACILITY: Charles River Laboratories

Preclinical Services, Pennsylvania (PCS-PA) 905 Sheehy Drive, Building A

Horsham, PA 19044 United States

22 March 2013

of 333

Final Report Testing Facility Study No. 20024505

TABLE OF CONTENTS

1. LIST OF FIGURES .................................................................................................................4

2. LIST OF TABLES ..................................................................................................................5

3. LIST OF APPENDICES .........................................................................................................6

4. COMPLIANCE STATEMENT ..............................................................................................7

5. QUALITY ASSURANCE STATEMENT ..............................................................................8

6. RESPONSIBLE PERSONNEL ...............................................................................................9 6.1. Testing Facility ..............................................................................................................9 6.2. Principal Investigator (PI) at Testing Facility ................................................................9 6.3. Principal Investigator (PI) at Testing Facility-designated Test Sites .............................9

7. SUMMARY ..........................................................................................................................10

8. INTRODUCTION .................................................................................................................12

9. MATERIALS AND METHODS ..........................................................................................12 9.1. Test and Control Articles .............................................................................................12 9.1.1. Test Article...................................................................................................................12 9.1.2. Control Article .............................................................................................................12 9.2. Test and Control Article Characterization ...................................................................13 9.3. Reserve Samples ..........................................................................................................13 9.4. Test and Control Article Inventory and Disposition ....................................................13 9.5. Safety ...........................................................................................................................13 9.6. Dose Formulation and Analysis ...................................................................................13 9.6.1. Preparation of Control Article .....................................................................................13 9.6.2. Preparation of Test Article ...........................................................................................13 9.6.3. Sample Collection and Analysis ..................................................................................14 9.7. Test System ..................................................................................................................15 9.7.1. Receipt .........................................................................................................................15 9.7.2. Justification for Test System and Number of Animals ................................................15 9.7.3. Animal Identification ...................................................................................................15 9.7.4. Environmental Acclimation .........................................................................................15 9.7.5. Mating - Part B.............................................................................................................15 9.7.6. Selection, Assignment, and Replacement of Animals .................................................15 9.7.7. Disposition ...................................................................................................................16 9.7.8. Husbandry ....................................................................................................................16 9.8. Experimental Design ....................................................................................................18 9.8.1. Part A (Nonmated Rabbits) ..........................................................................................18 9.8.2. Part B (Time-Mated Rabbits) .......................................................................................18 9.8.3. Administration of Test Materials .................................................................................18 9.8.4. Justification of Route and Dose Levels .......................................................................18 9.9. In-life Procedures, Observations, and Measurements ..................................................19 9.9.1. Viability Checks (Part A and B) ..................................................................................19 9.9.2. Clinical Observations (Part A and B) ..........................................................................19 9.9.3. Body Weights ...............................................................................................................19

Final Report Page 2Testing Facility Study No. 20024505


9.9.4. Food Consumption .......................................................................................................19 9.10. Laboratory Evaluations ................................................................................................19 9.10.1. Bioanalysis and Toxicokinetic Evaluation - Part B .....................................................19 9.10.2. Anti-therapeutic Antibody Evaluation - Part B ...........................................................20 9.11. Terminal Procedures ....................................................................................................21 9.11.1. Method of Euthanasia - Part A and B ..........................................................................21 9.11.2. Part A ...........................................................................................................................21 9.11.3. Part B ...........................................................................................................................22 9.11.4. Tissue Collection and Preservation - Part B ................................................................23 9.11.5. Fetal Examinations - Part B .........................................................................................23

10. COMPUTERIZED SYSTEMS .............................................................................................23

11. STATISTICAL ANALYSIS .................................................................................................24

12. RETENTION OF RECORDS, SAMPLES, AND SPECIMENS .........................................24

13. RESULTS ..............................................................................................................................25 13.1. Dose Formulation Analyses .........................................................................................25 13.2. Part A ...........................................................................................................................25 13.3. Part B ...........................................................................................................................25 13.4. Mortality, Clinical and Necropsy Observations...........................................................25 13.5. Maternal Body Weights and Body Weight Changes ...................................................25 13.6. Maternal Food Consumption .......................................................................................26 13.7. Caesarean-Sectioning and Litter Observations ............................................................26 13.8. Fetal Gross Examinations ............................................................................................26 13.9. Toxicokinetic Evaluations ...........................................................................................26

14. RECOMMENDATION .........................................................................................................28

15. REPORT APPROVAL ..........................................................................................................29

16. SCIENTIFIC REPORT REVIEW .........................................................................................30

17. REFERENCES ......................................................................................................................31



1. LIST OF FIGURES

Figure 1 Body Weights - Part A ...........................................................................................32

Figure 2 Maternal Body Weights - Part B ............................................................................34



2. LIST OF TABLES

Table 1 Clinical and Necropsy Observations - Summary - Part A ....................................36

Table 2 Body Weights and Body Weight Changes - Summary - Part A ...........................38

Table 3 Clinical and Necropsy Observations - Summary - Part B ....................................40

Table 4 Maternal Body Weights - Summary - Part B ........................................................42

Table 5 Maternal Body Weight Changes - Summary - Part B ...........................................45

Table 6 Maternal Absolute Food Consumption Values (g/day) - Summary - Part B ........47

Table 7 Maternal Relative Food Consumption Values (g/kg/day) - Summary - Part B ....................................................................................................................49

Table 8 Caesarean-Sectioning Observations - Summary - Part B .....................................51

Table 9 Litter Observations (Caesarean-Delivered Fetuses) - Summary - Part B .............53

Table 10 Fetal Gross External Alterations - Caesarean-Delivered Live Fetuses (Day 29 of Gestation) - Summary - Part B ..............................................55



3. LIST OF APPENDICES

Appendix 1 Protocol, Amendments, and Deviations ................................................................57

Appendix 2 Test and Control Article Characterization ............................................................97

Appendix 3 Dose Formulation Analysis Report .....................................................................102

Appendix 4 Clinical Observations - Individual Data - Part A ................................................144

Appendix 5 Body Weights - Individual Data - Part A ............................................................146

Appendix 6 Necropsy Observations - Individual Data - Part A .............................................148

Appendix 7 Clinical Observations - Individual Data - Part B ................................................150

Appendix 8 Maternal Body Weights - Individual Data - Part B ............................................153

Appendix 9 Maternal Food Consumption Values - Individual Data - Part B ........................159

Appendix 10 Necropsy Observations - Individual Data - Part B .............................................165

Appendix 11 Caesarean-Sectioning Observations - Individual Data - Part B ..........................167

Appendix 12 Litter Observations (Caesarean-Delivered Fetuses) - Individual Data - Part B ..................................................................................................................169

Appendix 13 Fetal Sex, Vital Status, Body Weight and Gross External Alterations - Individual Data - Part B ......................................................................................171

Appendix 14 Individual Data for Toxicokinetic Rabbits - Part B ............................................174

Appendix 15 Bioanalysis Report ..............................................................................................180

Appendix 16 Toxicokinetic Report ..........................................................................................292

Appendix 17 Historical Control Data .......................................................................................310

Appendix 18 Tabulated Summary of the Common Technical Document ...............................330



4. COMPLIANCE STATEMENT

The study was performed in accordance with the U.S. Department of Health and Human Services, Food and Drug Administration (FDA), United States Code of Federal Regulations, Title 21, Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies and as accepted by Regulatory Authorities throughout the European Community (OECD Principles of Good Laboratory Practice) and Japan (MHLW).

Any portion of this study conducted by Charles River Laboratories in Canada was performed in accordance with the OECD Principles of Good Laboratory Practice and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA) and Japan (MHLW).

One exception from the FDA Good Laboratory Practice (GLP) for Nonclinical Laboratory Studies regulation occurred. Analyses conducted to support the information cited in the Certificate of Analysis for the test and control articles were conducted in accordance with current FDA Good Manufacturing Practice regulations. The outcome or interpretation of the study was not adversely affected by this exception, because the FDA GLP requires that “The integrity, strength, purity, and composition or other characteristics which will appropriately define the test or control article shall be determined for each batch and shall be documented [58.105(a)].” This requirement is met, therefore there is no effect on the study.

This study was conducted in accordance with the procedures described herein. All deviations authorized/acknowledged by the Study Director are documented in the Study Records. The report represents an accurate and complete record of the results obtained.

There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.



6. RESPONSIBLE PERSONNEL

6.1. Testing Facility

Study Director Valerie A. Sharper, MS Principal Research Scientist Address as cited for Testing Facility

Testing Facility Management Alan M. Hoberman, PhD, DABT, Fellow ATS Executive Director, Site Operations and Toxicology Address as cited for Testing Facility

Director of Operations Matthew J. Vaneman, BS

Senior Manager Technical Operations Joseph W. Lech, BS, LAT

Senior Manager Technical Resources James Yeager, BS, LAT

Senior Manager Laboratory Sciences Julian Gulbinski, III, BS, MBA

6.2. Principal Investigator (PI) at Testing Facility

Dose Formulation Analysis Jason Sarsoza, BSc Scientist II Charles River Laboratories Preclinical Services 905 Sheehy Drive, Building A Horsham, PA 19044

6.3. Principal Investigator (PI) at Testing Facility-designated Test Sites

Bioanalysis [Effective 12 September 2012] Robert Jenkins Senior Research Scientist Charles River Laboratories Preclinical Services Montreal 22022 Transcanadienne Senneville, Quebec, Canada H9X 3R3 Nathalie Proulx Senior Research Scientist Charles River Laboratories Preclinical Services Montreal 22022 Transcanadienne Senneville, Quebec, Canada H9X 3R3

Toxicokinetics Andrew M. Vick, PhD Seventh Wave Laboratories LLC 743 Spirit 40 Park Drive, Suite 209 Chesterfield, MO 63005-1121



7. SUMMARY

The objectives of this study were to provide information for selection of dosages to be used in a subsequent embryo-fetal development study in rabbits and to provide a preliminary evaluation of the effects of Thymosin beta 4 (Tβ4), formulated as Tβ4 Injectable Solution (RGN-352) on nonmated rabbits and on pregnancy and embryo-fetal development. This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C to D of the reproductive process. In addition, the toxicokinetic (TK) characteristics of RGN-352 were determined.

Part A

Twelve non-mated New Zealand White [Hra:(NZW)SPF] female rabbits were randomly assigned to four dose groups, 3 rabbits per group. Rabbits were administered the control article or test article formulation via intravenous injection at doses of 0, 10, 30 and 90 mg/kg/day on Days 1 through 5 of study (DSs 1 through 5). The study design for Part A was as follows:

Text Table 1 Experimental Design

Group No. Test Material

Dose Level (mg/kg)

Concentration (mg/mL)

Dose Volume (mL/kg) No. of Study Rabbits

1 Control Article 0 0 0.9 3 2

RGN-352 10 100 0.1 3

3 30 100 0.3 3 4 90 100 0.9 3

The following parameters and end points were evaluated in Part A of this study: viability, clinical signs, body weights, body weight changes, food consumption and gross necropsy findings.

All rabbits survived to scheduled sacrifice. The only adverse clinical observation was purple discoloration of both ears (the injection site) of one rabbit in the 90 mg/kg/day dose group. Body weights and body weight gains were generally comparable among the four dose groups throughout the dosing period and no dose-dependent trends were observed. No gross lesions were identified at necropsy.

Based on these data, dosages of 0, 3, 10, 30 and 90 mg/kg/day of Tβ4 Injectable Solution were considered to be appropriate for the dose-range developmental toxicity portion of the study in mated rabbits. None of these dosages were expected to cause notable maternal toxicity.

Part B

Twenty-five time-mated New Zealand White [Hra:(NZW)SPF] female rabbits were randomly assigned to five dose groups, 5 rabbits in each of Groups 5 through 9. An additional twelve rabbits, 3 rabbits in each of Groups 6 through 9 were assigned for use in TK sample collection. Rabbits were administered the control article or test article formulation via intravenous injection at doses of 0, 3, 10, 30 and 90 mg/kg/day on Days 7 through 19 of presumed gestation (DGs 7 through 19). The study design for Part B was as follows:





Dose Level (mg/kg)


Dose Volume (mL/kg)

No. of Main Study

Rabbits

No. of TK Study

Rabbits 5 Control Article 0 0 0.9 5 N/A 6

RGN-352

3 100 0.03 5 3 7 10 100 0.1 5 3 8 30 100 0.3 5 3 9 90 100 0.9 5 3

The following parameters and end points were evaluated in Part B of this study: viability, clinical signs, body weights, body weight changes, food consumption, bioanalytical, TK parameters, ovarian and uterine parameters, fetal examinations (sex and external alterations) and gross necropsy observations.

All rabbits survived to scheduled sacrifice. All clinical observations were considered unrelated to the test article because the incidences were not dose dependent and/or the observations occurred in only a single rabbit. No gross lesions were identified at necropsy. Reductions in body weight gain occurred in the 90 mg/kg/day dose group on DGs 7 to 10, 13 to 16 and 20 to 24; however, on DG 29, there was no reduction in average body weight as compared to control. Body weight gains were variable in the five dose groups throughout the study and overall, dose-dependent changes were not observed. Absolute and relative food consumption values were generally comparable among the five dose groups throughout the study.

Fetal body weights were reduced in the 90 mg/kg/day dose group; however, all values were within the ranges observed historically at the Testing Facility. No other Caesarean-sectioning or litter parameters were affected by dosages of Tβ4 Injectable Solution as high as 90 mg/kg/day. There were no test article-related fetal alterations.

Evidence of systemic plasma exposure to Tβ4 was observed in all RGN-352-treated TK rabbits following both single and repeat intravenous administration. Tβ4 exposure increased with increasing dose and was reasonably proportional to dose on both TK collection days (DGs 7 and 19). Mean AUCall values on DG 19 for Tβ4 were decreased 26%, 14%, 30% and 20% compared to AUCall values on DG 7 for the 3, 10, 30, and 90 mg/kg/day dose groups, respectively. Based on the data; there was no clear or consistent evidence of accumulation of Tβ4 in plasma with repeated intravenous dosing of RGN-352 over the study duration evaluated.

Based on these data, dosages of 0 (Control Article), 10, 30 and 90 mg/kg/day of Tβ4 Injectable Solution are recommended for the developmental toxicity study in rabbits. The 10 mg/kg/day dosage is expected to be a no-observable-adverse-effect level (NOAEL) for both maternal and embryo-fetal toxicity, and the 90 mg/kg/day dosage is expected to produce minimal or no maternal and developmental toxicity.



8. INTRODUCTION

The objectives of this study were to provide information for selection of dosages to be used in a subsequent embryo-fetal development study in rabbits and to provide a preliminary evaluation of the effects of Thymosin beta 4, formulated as Tβ4 Injectable Solution (RGN-352) on nonmated rabbits and on pregnancy and embryo-fetal development. This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C to D of the reproductive process. In addition, the TK characteristics of RGN-352 were determined.

The Study Director signed the protocol on 16 May 2012, and dosing was initiated on 21 May 2012 (Part A) and 20 Jun 2012 (Part B). The in-life phase of the study was completed on 26 May 2012 (Part A) and 12 Jul 2012 (Part B). The study protocol, protocol amendments, and deviations are presented in Appendix 1.

9. MATERIALS AND METHODS

9.1. Test and Control Articles

9.1.1. Test Article

Identification: Thymosin beta 4 (Tβ4) formulated as RGN-352 (Tβ4 Injectable Solution)

Batch (Lot) No.: 330-05-001

Receipt Date: 22 Aug 2011

Expiration Date: 01 Dec 2012

Physical Description: Clear, colorless liquid, 2.25 mL/vial

Concentration: 100 mg Tβ4/mL

Storage Conditions: Refrigerated (2°C to 8°C)

Supplier: Sponsor

9.1.2. Control Article

Identification: RGN-352 Placebo (Vehicle for RGN-352)

Batch (Lot) No.: 330-06-001

Receipt Date: 22 Aug 2011

Expiration Date: 27 Aug 2012

Physical Description: Clear, colorless liquid, 5.5 mL/vial


Supplier: Sponsor



9.2. Test and Control Article Characterization

The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition and stability for the test and control articles. Certificates of Analysis were provided to the Testing Facility and are presented in Appendix 2.

The Sponsor has appropriate documentation on file concerning the method of synthesis, fabrication or derivation of the test and control articles, and this information is available to the appropriate regulatory agencies should it be requested.

9.3. Reserve Samples

For each batch (lot) of test and control article, a reserve sample (1 vial) was collected and maintained under the appropriate storage conditions by the Testing Facility.

9.4. Test and Control Article Inventory and Disposition

Records of the receipt, distribution, and storage of test and control articles (including empty containers) were maintained. With the exception of reserve samples, all unused test and control articles were retained for use in future studies. All empty containers were maintained for the duration of the study (See Appendix 1, Protocol, Amendments and Deviations). Any residual volumes were discarded before issuance of the Final Report.

9.5. Safety

The following safety instructions applied to this study:

Gloves, dust-mist/HEPA-filtered mask, appropriate eye protection and uniform/lab coat were worn during formulation preparation and dosing. Tyvek® sleeves were worn during dose administration.

The Material Safety Data Sheet (MSDS) is maintained in the raw data.

9.6. Dose Formulation and Analysis

9.6.1. Preparation of Control Article

The control article, RGN-352 Placebo, was administered as received. The control article was dispensed daily for administration to Group 1 and 5 control animals. The control article was removed from the refrigerator at least 30 minutes before dosing to allow it to equilibrate to room temperature.

9.6.2. Preparation of Test Article

The test article, Tβ4 Injectable Solution (RGN-352), was administered as received. The test article was dispensed daily for administration to Groups 2 through 4 and Groups 6 through 9. The test article was removed from the refrigerator for at least 30 minutes before dosing to allow it to equilibrate to room temperature.



9.6.3. Sample Collection and Analysis

Dose formulation samples were collected for analysis as indicated in Text Table 3.

Text Table 3 Dose Formulation Sample Collection Schedule

Interval Concentration Homogeneity Stability Before First Administration (Part A) 0 and 100 mg/mL N/A N/A Last Day of Administration (Part B) 0 and 100 mg/mL N/A N/A

N/A = Not applicable.

All samples analyzed were transferred (ambient conditions) to the analytical laboratory at the Testing Facility. Upon receipt at the analytical laboratory, the samples were stored refrigerated (2°C to 8°C) until analysis. Any residual analytical samples (and test article used in analysis) were discarded before issue of the Final Report.

9.6.3.1. Analytical Method

Analyses described below were performed by HPLC-UV using validated analytical procedure TYMN03 - Analytical Procedure for the Analysis of Thymosin beta 4 (RGN-352) in RGN-352 Placebo Dose Formulation by HPLC-UV; validated under Charles River Preclinical Services, Pennsylvania (PCS-PA) Protocol No. 20019481.

9.6.3.2. Concentration Analysis

Duplicate samples (two 2.25 mL samples of placebo and two 2.25 mL vials of test article) were taken before the first day of dose administration (Part A) and the last day of dose administration (Part B). A single sample (2.25 mL) was transferred to the analytical laboratory at the Testing Facility for analysis; the remaining samples were retained at the Testing Facility as backup samples and stored refrigerated (2°C to 8°C). (See Appendix 1, Protocol, Amendments and Deviations). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it were within or equal to ± 15%. After acceptance of the analytical results, backup samples of placebo were discarded and backup samples of test article (unopened vials) were retained for use on future studies.

9.6.3.3. Homogeneity Analysis

The test article Tβ4, formulated and vialed as RGN-352 (100 mg/mL), yields a sterile aqueous, homogeneous solution. Therefore, homogeneity analysis of the test article was not conducted. The test article was not mixed with the placebo (RGN-352 excipients minus Tβ4) in this study.

9.6.3.4. Stability Analysis

Stability of the formulated test article for the duration of the study was confirmed by comparison of the results of the concentration analysis conducted for samples collected before the first dose administration and samples collected on the last day of administration.



9.7. Test System

9.7.1. Receipt

9.7.1.1. Part A

Eight nonmated New Zealand White [Hra:(NZW)SPF] female rabbits were received from Covance Research Products, Inc., Denver, PA. Rabbits were approximately 5 ½ months of age at arrival at the Testing Facility. The body weight range was 2.7 kg to 3.5 kg on the day of arrival. Twelve rabbits, 3 rabbits in each of Groups 1 through 4, were assigned to Part A of the study.

9.7.1.2. Part B

Thirty-eight time-mated New Zealand White [Hra:(NZW)SPF] female rabbits were received from Covance Research Products, Inc., Denver, PA. Rabbits were approximately 6 ½ months of age at arrival at the Testing Facility. The body weight range was 3.2 kg to 4.1 kg on the day of arrival and 3.1 kg to 4.0 kg at randomization and assignment to study (gestation day 0, DG 0). Twenty-five female rabbits (5 per dose group) were assigned to the main study and 12 female rabbits (3 rabbits in each of Groups 6 through 9) were assigned to the TK study.

9.7.2. Justification for Test System and Number of Animals

The test system was selected because: 1) it is a standard species accepted for use in embryo-fetal development studies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the Testing Facility.

The number of animals chosen for this study was the smallest number considered necessary to select dosages for subsequent studies.

9.7.3. Animal Identification

Female rabbits were given unique permanent identification numbers when assigned to the study and permanently identified using Monel® self-piercing ear tags.

9.7.4. Environmental Acclimation

After receipt at the Testing Facility, the rabbits were acclimated for 4 or 5 days prior to initiation of dose administration.

9.7.5. Mating - Part B

The female rabbits were naturally bred at the Supplier by breeder male rabbits of the same source and strain before shipment to the Testing Facility. The day mating occurred was designated DG 0. The rabbits were shipped to the Testing Facility after mating, to arrive on DG 2.

9.7.6. Selection, Assignment, and Replacement of Animals

9.7.6.1. Part A

After an acclimation period, healthy female rabbits were assigned to groups using a computer-based randomization procedure.



9.7.6.2. Part B

Before shipment of the rabbits, the Supplier forwarded breeding records and DG 0 body weights. A computer-generated (weight-ordered) randomization procedure was used to assign healthy mated female rabbits to dose groups based on this information.

9.7.7. Disposition

The disposition of all rabbits was documented in the study records. Any remaining rabbits not selected for study were euthanized.

9.7.8. Husbandry

9.7.8.1. Housing

All cage sizes and housing conditions are in compliance with the Guide for the Care and Use of Laboratory Animals1.

9.7.8.1.1. Part A

The rabbits were individually housed in a plastic rabbit caging system with stainless steel racks (See Appendix 1, Protocol, Amendments and Deviations).

9.7.8.1.2. Part B

The rabbits were individually housed in units of six to eight stainless steel cages. No nesting materials were supplied because the female rabbits were euthanized before parturition was expected.

9.7.8.2. Environmental Conditions

The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 61°F to 72°F (16°C to 22°C); relative humidity was targeted at 30% to 70% (See Appendix 1, Protocol, Amendments and Deviations). An automatically controlled 12-hour light:12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (± 30 minutes). On DG 7, 20 June 2012 and on DG 19, 2 July 2012, the room lights were turned on during the dark period for 61 and 46 minutes, respectively, to facilitate blood collection.



9.7.8.3. Food

Analyses were routinely performed by the food supplier. No contaminants at levels exceeding the maximum concentration limits for certified food or deviations from expected nutritional requirements were detected by these analyses.

The Study Director was not aware of any potential contaminants likely to be present in the certified food at levels that would have interfered with the results of this study. Therefore, no analyses other than those routinely performed by the food supplier or those mentioned in this protocol were conducted.

9.7.8.3.1. Part A

Approximately 150 g of Certified Rabbit Chow® #5322 (PMI® Nutrition International) was available to each rabbit each day. The certified feed was available from individual stainless steel "J-type" feeders attached to each cage.

9.7.8.3.2. Part B

Approximately 150 g of Certified Rabbit Chow®#5322 (PMI® Nutrition International) was available to each rabbit each day until the first day of dosing, at which time approximately 180 g to 185 g of the same certified food was offered to each rabbit each day. The certified food was available from individual stainless steel "J-type" feeders attached to each cage.

9.7.8.4. Water

Local water (Horsham, PA), that had been processed by passage through a reverse osmosis membrane (R.O. water), was available to the rabbits ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.

The processed water is analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, PA) and monthly for possible bacterial contamination (QC Laboratories, Southampton, PA).

The Study Director was not aware of any potential contaminants likely to be present in the drinking water at levels that would interfere with the results of this study. Therefore, no analyses other than those mentioned in this protocol were conducted.

9.7.8.5. Animal Enrichment

For psychological enrichment, rabbits were provided with items such as an enrichment toy.

9.7.8.6. Veterinary Care

Veterinary care was available throughout the course of the study and rabbits were examined by the veterinary staff as warranted by clinical signs or other changes. During the course of the study, rabbits with reduced food consumption and scant fecal output were provided dry timothy cubes daily, a secondary water source and/or bunny blocks as needed (replaced if consumed or soiled). All veterinary examinations were documented in the study records.



9.8. Experimental Design

The experimental design is shown in Text Table 4 and Text Table 5, below.

9.8.1. Part A (Nonmated Rabbits)



Dose Level (mg/kg/day)


Dose Volume (mL/kg) Rabbit Numbers

1 Control Article 0 0 0.9 1450 - 1452 2

RGN-352 10 100 0.1 1453 - 1455

3 30 100 0.3 1456 - 1458 4 90 100 0.9 1459 - 1461

9.8.2. Part B (Time-Mated Rabbits)





Dose Volume (mL/kg)

Main Study Rabbit

Numbers TK Rabbits

Numbers 5 Control Article 0 0 0.9 2176 - 2180 N/A 6

RGN-352

3 100 0.03 2181 - 2185 2130 - 2132 7 10 100 0.1 2186 - 2190 2133 - 2135 8 30 100 0.3 2191 - 2195 2136 - 2138 9 90 100 0.9 2196 - 2200 2139 - 2141

N/A = Not Applicable

9.8.3. Administration of Test Materials

9.8.3.1. Part A

Female rabbits (nonmated) were administered the test article or the control article by once daily intravenous bolus injection via the marginal ear vein on DSs 1 through 5 (the first day of dose administration was designated as DS 1). Doses were adjusted based on body weights recorded immediately before dosing and given at approximately the same time each day.

9.8.3.2. Part B

Female rabbits (presumed pregnant) were administered the test article or the control article by once daily intravenous bolus injection via the marginal ear vein on DGs 7 through 19. Doses were adjusted based on the most recently recorded body weight and administered at approximately the same time each day.

9.8.4. Justification of Route and Dose Levels

The intravenous route was selected for use because it is one of the proposed systemic routes for clinical use.



Dose level selections for the test article in this study bracket the dose level selections planned for RGN-352 in future clinical trials.

9.9. In-life Procedures, Observations, and Measurements

The in-life procedures, observations, and measurements listed below were performed for all main study rabbits and TK rabbits; with the exception that food consumption was not recorded for the rabbits assigned to Part A and Part B TK study.

9.9.1. Viability Checks (Part A and B)

The rabbits were assessed for viability at least twice daily during the study.

9.9.2. Clinical Observations (Part A and B)

9.9.2.1. General Appearance

The rabbits were observed for general appearance once during the predose period, daily before administration during the dose period, and once daily during the postdose period.

9.9.2.2. Postdose Observations

On the first day of dosing (Part A), postdose observations were recorded at approximately hourly intervals for the first four hours and at the end of the normal working day. Beginning on the second day and on all subsequent days of dose administration (Part A and Part B), postdose observations were recorded between one and two hours after dose administration (See Appendix 1, Protocol, Amendments and Deviations).

9.9.3. Body Weights

Body weights (Part A) were recorded on the day of arrival at the Testing Facility, daily during the dosing period and on the day of scheduled euthanasia.

Body weights (Part B) were recorded on DG 0 by the Supplier, the day of arrival at the Testing Facility and daily during the dose and postdose periods.

9.9.4. Food Consumption

An estimated percentage of food consumed was recorded daily after arrival at the Testing Facility for rabbits assigned to Part A and rabbits assigned to the Part B TK study.

Food consumption values (Part B) were recorded daily after arrival at the Testing Facility (values not tabulated), and daily during the dose and postdose periods.

9.10. Laboratory Evaluations

9.10.1. Bioanalysis and Toxicokinetic Evaluation - Part B

9.10.1.1. Bioanalytical Sample Collection

On DGs 7 and 19, blood samples (1.0 mL) were collected from the medial auricular artery from each rabbit assigned to the TK study according to Text Table 6. The samples were processed as described in Section 9.10.1.2 (Bioanalytical Sample Processing).



Text Table 6 TK Sample Collection Schedule

Group No.

Sample Collection Time Points (Time Post Dose) on DGs 7 and 19

Predose 5 m 10 m 20 m 1 h 2 h 6 h 12 h 6-9 X X X X X X X X

m = minute; h = hour; X = blood collected

9.10.1.2. Bioanalytical Sample Processing

Samples were mixed gently and kept on crushed wet ice immediately following collection. The samples were centrifuged in a refrigerated centrifuge at 2700 rpm for approximately 10 minutes. The resultant plasma was separated, transferred to uniquely labeled clear polypropylene cryovials, and frozen immediately over dry ice or in a freezer set to maintain -80°C.

The plasma samples were shipped frozen on dry ice to Charles River Laboratories, Preclinical Services Montreal for bioanalysis.

9.10.1.3. Bioanalytical Sample Analysis

Plasma samples were analyzed for concentration of Tβ4, formulated as RGN-352 (Tβ4 Injectable Solution). Analysis was performed by LC-MS/MS under Analytical Procedure 142476.PL.02, using a validated analytical procedure (Study No. 142474).

Incurred sample reanalysis (ISR) was performed for this study as per the appropriate SOP(s) of the bioanalytical laboratory.

The bioanalytical results are available in Appendix 15.

9.10.1.4. Toxicokinetic Evaluation

Results of plasma Tβ4 analysis were provided to the Principal Investigator for calculation of noncompartmental exposure parameters.

The TK results are available in Appendix 16.

9.10.2. Anti-therapeutic Antibody Evaluation - Part B

9.10.2.1. Anti-therapeutic Antibody Sample Collection

Blood samples (1.0 mL) were collected from the medial auricular artery of rabbits in the main study (5/group) before the first dose and on DG 29. The samples were processed as described in Section 9.10.2.2 (Anti-therapeuptic Antibody Sample Processing).

9.10.2.2. Anti-therapeutic Antibody Sample Processing

Blood samples were transferred into labeled serum separator tubes allowed to clot for at least 30 minutes and centrifuged at room temperature for approximately 10 minutes. The resulting sera samples were frozen on dry ice as soon as possible and maintained in a freezer set to maintain -80°C at the Testing Facility. These samples were not analyzed and are retained at the Testing Facility.



9.11. Terminal Procedures

9.11.1. Method of Euthanasia - Part A and B

A euthanasia solution (390 mg pentobarbital sodium and 50 mg phenytoin sodium) was used to euthanize nonmated and time-mated rabbits (via intravenous injection) and live fetuses (via intraperitoneal injection).

9.11.2. Part A

Terminal procedures are summarized in Text Table 7.

Text Table 7 Terminal Procedures - Part A

Group No.

No. of Animals

Scheduled Euthanasia

Day

Necropsy Procedures

Histology Histopathology

Ovarian/ Uterine

Examination NecropsyTissue

CollectionOrgan

Weights

1 3

DS 6 Pregnancy Status X - -

- -

2 3 - -

3 3 - - 4 3 - -

- = Not applicable.

On DS 6, all rabbits were euthanized and a gross necropsy of the thoracic, abdominal and pelvic viscera was conducted.



9.11.3. Part B

Terminal procedures are summarized in Text Table 8.

Text Table 8 Terminal Procedures for Main and Toxicokinetic Rabbits - Part B

Group No.

No. of Animals


Day

Necropsy Procedures


Ovarian/ Uterine

Examination NecropsyTissue

CollectionOrgan

Weights Toxicokinetic Animals

6 3

20 Pregnancy Status - - -

- -

7 3 - -

8 3 - - 9 3 - -

Main Study Animals

5 5

29 Full Exam X X -

- - 6 5 - -

7 5 - -

8 5 - -

9 5 - - X = Procedure conducted; - = Not applicable.

9.11.3.1. Scheduled Euthanasia

On DG 20, female rabbits assigned to the TK portion of the study were euthanized after the last blood sample collection and examined for pregnancy status. Conceptuses and carcasses were discarded without further evaluation.

On DG 29, female rabbits assigned to the main portion of the study were Caesarean-sectioned, and examined for gross lesions as further described in Section 9.11.3.2 (Ovarian and Uterine Examination) and Section 9.11.3.3 (Necropsy).

9.11.3.2. Ovarian and Uterine Examinations

For rabbits assigned to the main study, the reproductive tract was dissected from the abdominal cavity. The uterus was opened, and the contents were examined. The fetuses were removed from the uterus and placed in individual containers.

The ovaries and uterus of each rabbit were examined for number and distribution of corpora lutea, implantation sites, placentae (size, color or shape), live and dead fetuses, and early and late resorptions. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption.



Uteri of apparently nonpregnant rabbits were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rabbits were retained in 10% neutral buffered formalin and were discarded when authorized by the Study Director.

9.11.3.3. Necropsy

All main study rabbits were subjected to a gross necropsy examination, which included an evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Unless specifically cited in Section (Tissue Collection and Preservation), all tissues were discarded.

9.11.4. Tissue Collection and Preservation - Part B

Representative samples of the tissues identified in Text Table 9 were collected from each rabbit assigned to Part B and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Text Table 9 Tissue Collection and Preservation

Tissue Collected Comment Cervix X Collected with uterus. All nonpregnant rabbits. Ovaries X All nonpregnant rabbits. Uterus X All nonpregnant rabbits.

X = Procedure conducted.

9.11.5. Fetal Examinations - Part B

Fetuses were examined for external abnormalities. Late resorptions were examined for external abnormalities and sex to the extent possible. The body weight of each fetus was recorded. All fetuses were examined internally to determine sex. Fetuses with external abnormalities were fixed in Bouin's solution; all other fetuses were discarded.

Representative photographs of fetal external abnormalities were taken and are retained as electronic images and archived with the raw data.

10. COMPUTERIZED SYSTEMS

Critical computerized systems used in the study are listed below or presented in the appropriate Phase Report. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.



Text Table 10 Critical Computerized Systems

System Name Version No. Description of Data Collected and/or Analyzed Dispense 7.0.3 Test article/control article receipt

Argus Automated Data Collection and Management

System

13.11.18.9, 13.11.18.10

Clinical observations, body weights, food consumption, Caesarean-sectioning observations, statistical analyses

Vivarium Temperature and Relative Humidity Monitoring System

2.0 Study room temperature and humidity

Quattro Pro 8 Body weight graph

11. STATISTICAL ANALYSIS

Statistical analyses were descriptive, rather than inferential. Averages and percentages were calculated. Litter values were used where appropriate. Only body weights of live fetuses were used to determine litter mean fetal body weight.

Data collected for rabbits assigned to TK sample collection were not summarized or analyzed statistically.

12. RETENTION OF RECORDS, SAMPLES, AND SPECIMENS

All study-specific raw data, documentation, protocol, samples, specimens, and final reports from this study were available at the Testing Facility during the study and will be transferred to the Testing Facility archive by no later than the date of Final Report issue. One year after issue of the audited draft report, the Sponsor will be contacted to determine the disposition of materials associated with the study.

All records, samples, specimens, and reports generated from phases or segments performed by Testing Facility-designated subcontractors will be sent to the Testing Facility for archiving.



13. RESULTS

13.1. Dose Formulation Analyses

(Appendix 3)

Mean measured Tβ4 concentrations sampled at the start and end of the study were within the acceptable limits of ± 10% of nominal concentrations. Individual Tβ4 concentrations for all samples were within the acceptable limits of ± 15% of nominal concentrations. There was no test article contamination of the control article.

13.2. Part A

(Figure 1, Table 1, Table 2, Appendix 4, Appendix 5 and Appendix 6)

All rabbits survived to scheduled sacrifice. The only adverse clinical observation was purple discoloration of both ears (the injection site) of one rabbit in the 90 mg/kg/day dose group on Days 2 through 6 of study (DSs 2 through 6). Body weights and body weight gains were generally comparable among the four dose groups throughout the dosing period and no dose-dependent trends were observed. On DS 1 average body weights were 103%, 103% and 109% of the control group value in the 10, 30 and 90 mg/kg/day dose groups, respectively. On DS 6, average body weights were 102%, 100% and 107% of the control group value in these same respective groups. No gross lesions were identified at necropsy.

Based on these data, dosages of 0, 3, 10, 30 and 90 mg/kg/day of Tβ4 Injectable Solution were considered to be appropriate for the dose-range developmental toxicity portion of the study in time-mated rabbits, Part B. None of these dosages were expected to cause notable maternal toxicity.

13.3. Part B

13.4. Mortality, Clinical and Necropsy Observations

(Table 3, Appendix 7 and Appendix 10)

All rabbits survived to scheduled sacrifice. All clinical observations were considered unrelated to the test article because the incidences were not dose dependent and/or the observations occurred in only a single rabbit. One rabbit in the 90 mg/kg/day dose group had purple discoloration of one ear on gestation Days (DGs) 12 to 16; this discoloration was likely due to trauma at the injection site rather than a reaction to the test article. One rabbit in the 3 mg/kg/day dose group was found out of its cage (which was located at the top of the rack) at the morning viability check on DG 10 and had limited use of the left hindlimb and/or an irregular gait from DGs 10 through 29. No gross lesions were identified at necropsy.

13.5. Maternal Body Weights and Body Weight Changes

(Figure 2, Table 4, Table 5 and Appendix 8)

Reductions in body weight gain occurred in the 90 mg/kg/day dose group on DGs 7 to 10, 13 to 16 and 20 to 24; however, on DG 29, there was no reduction in average body weight as compared to control. Body weight gains were variable in the five dose groups throughout the study and overall, dose-dependent changes were not observed. Body weight gain for the entire



study period after the initiation of dosing (DGs 7 to 29) were 52%, 121%, 88% and 74% of the control group value in the 3, 10, 30 and 90 mg/kg/day dose groups, respectively, while average body weights on DG 29 were 95%, 102%, 97% and 98% of the control group value in these same respective dose groups.

13.6. Maternal Food Consumption

(Table 6, Table 7 and Appendix 9)

Absolute and relative food consumption values were generally comparable among the five dose groups throughout the study. Absolute food consumption values for the entire study period after the initiation of dosing (DGs 7 to 29) were 94%, 117%, 99% and 92% of the control group value in the 3, 10, 30 and 90 mg/kg/day dose groups, respectively.

13.7. Caesarean-Sectioning and Litter Observations

(Table 8, Table 9, Appendix 10, Appendix 11, Appendix 12 and Appendix 13)

Caesarean-sectioning observations were based on 5 (100%), 4 (80.0%), 4 (80.0%), 4 (80.0%) and 5 (100%) pregnant rabbits in Groups 5 through 9, respectively.

Fetal body weights were reduced in the 90 mg/kg/day dose group; total, male and female average fetal weights were 90%, 87% and 92% of the control group values, respectively. However, all values were within the ranges observed historically at the Testing Facility (Appendix 17).

No other Caesarean-sectioning or litter parameters were affected by dosages of Tβ4 Injectable Solution as high as 90 mg/kg/day. The litter averages for corpora lutea, implantations, percent preimplantation loss, litter sizes, live fetuses, early and late resorptions, percent postimplantation loss, percent resorbed conceptuses, and percent live male fetuses were comparable among the five dose groups. No doe had a litter consisting of only resorbed conceptuses, and there were no dead fetuses.

13.8. Fetal Gross Examinations

(Table 10 and Appendix 13)

There were no test article-related fetal alterations. One fetus in the 30 mg/kg/day dose group had gastroschisis and a cleft snout. The litter and fetal incidences of these malformations were within the ranges observed historically at the Testing Facility (Appendix 17). No other fetal gross alterations occurred.

13.9. Toxicokinetic Evaluations

(Appendix 14)

All rabbits assigned to TK sample collection survived to scheduled sacrifice. Clinical observations and body weights in these rabbits were comparable to those in the main study groups. All rabbits assigned for use in TK sample collection were pregnant. Data for these rabbits are presented in the individual data tables only.

Evidence of systemic plasma exposure to Tβ4 was observed in all RGN-352-treated TK rabbits (Groups 6 through 9) following both single and repeat intravenous administration. Peak plasma



concentrations of Tβ4 following intravenous administration were observed at the first time point (0.083 hour) for both DG 7 and DG 19 TK collections.

Following intravenous bolus injection, Tβ4 plasma concentrations exhibited a polyphasic pattern of decay, characterized by a rapid distribution phase followed by a log-linear elimination phase. Although occasional differences were noted, mean CL, Vd, and HLλz values appeared to be dose-independent between the 3 and 90 mg/kg dose levels for DGs 7 and 19. Group mean CL and Vd values on DG 19 ranged from 128 to 185 mL/h/kg and 133 to 167 mL/kg, respectively, and these values were 14 to 41% and 16 to 59% higher, respectively, than those obtained on DG 7. No clear change in HLλz values was noted following repeat-dosing of Tβ4 (relative to DG 7), with group mean values ranging from 0.531 to 0.751 hours across DGs 7 and 19.

Tβ4 exposure (as assessed by mean Cmax and AUCall) following intravenous administration increased in a dose-related fashion over the dose range evaluated on both DG 7 and DG 19. Over the dose range evaluated, the observed increase in exposure was reasonably proportional to dose on both TK collection days. On DG 7, mean Tβ4 Cmax and AUCall values increased 28.3- and 30.7-fold over a 30-fold dose range. On DG 19, mean Tβ4 Cmax and AUCall values increased 31.1- and 33.0-fold over a 30-fold dose range.

Mean AUCall values on DG 19 for Tβ4 were decreased 26%, 14%, 30%, and 20% compared to AUCall values on DG 7 for the 3, 10, 30, and 90 mg/kg dose groups, respectively. Based on the data; there was no clear or consistent evidence of accumulation of Tβ4 in plasma with repeated intravenous dosing of RGN-352 over the study duration evaluated.

Selected toxicokinetic parameters are presented in Text Table 11.

Text Table 11 Selected Tβ4 Toxicokinetic Parameters

DG Dose (mg/kg)

Cmax (µg/mL)

AUCall (µg*h/mL)

AUCINFobs (µg*h/mL)

CL (mL/h/kg)

Vd (mL/kg)

HLλz

(h)

7

3 37.4 24.5 22.3 147 121 0.587 10 137 95.8 89.6 112 101 0.631 30 396 280 279 109 105 0.671 90 1060 753 751 120 116 0.671

19

3 35.0 18.2 16.8 185 140 0.531 10 137 82.7 79.9 128 133 0.706 30 333 195 195 154 167 0.751 90 1090 601 599 154 159 0.719



14. RECOMMENDATION

Based on these data, dosages of 0 (Control Article), 10, 30 and 90 mg/kg/day of Tβ4 Injectable Solution are recommended for the developmental toxicity study in rabbits. The 10 mg/kg/day dosage is expected to be a no-observable-adverse-effect level (NOAEL) for both maternal and embryo-fetal toxicity, and the 90 mg/kg/day dosage is expected to produce minimal or no maternal and developmental toxicity.



17. REFERENCES 1. National Research Council. Guide for the Care and Use of Laboratory Animals.

Washington, D.C.: National Academy Press. 1996.



Figure 1 Body Weights - Part A


3.00

3.02

3.04

3.06

3.08

3.10

3.12

3.14

3.16

3.18

3.20

3.22

3.24

3.26

3.28

3.30

3.32

3.34

3.36

WE

IGH

T (K

G)

1 2 3 4 5 6

0 (CONTROL ARTICLE) MG/KG/DAY

10 (RGN-352) MG/KG/DAY



BODY WEIGHTS - PART AFigure 1

PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS

DAY OF STUDY



Figure 2 Maternal Body Weights - Part B


3.40

3.50

3.60

3.70

3.80

3.90

4.00

4.10

4.20

4.30

WE

IGH

T (K

G)

0 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

0 (CONTROL ARTICLE) MG/KG/DAY





MATERNAL BODY WEIGHTS - PART BFigure 2

PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS

DAY OF GESTATION



Table 1 Clinical and Necropsy Observations - Summary - Part A


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 1 (PAGE 1): CLINICAL AND NECROPSY OBSERVATIONS - SUMMARY - PART A ------------------------------------------------------------------------------------------------------------------------------------ GROUP 1 2 3 4 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ MAXIMUM POSSIBLE INCIDENCE 18/ 3 18/ 3 18/ 3 18/ 3 MORTALITY 0 0 0 0 INJECTION SITE(S): PURPLE 0/ 0 0/ 0 0/ 0 5/ 1 NO GROSS LESIONS WERE IDENTIFIED AT NECROPSY ------------------------------------------------------------------------------------------------------------------------------------ MAXIMUM POSSIBLE INCIDENCE = (DAYS x RABBITS)/NUMBER OF RABBITS EXAMINED PER GROUP ON DAYS 1 THROUGH 6 OF STUDY N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RABBITS WITH OBSERVATION a. Dosage occurred on Days 1 through 5 of study.



Table 2 Body Weights and Body Weight Changes - Summary - Part A


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 2 (PAGE 1): BODY WEIGHTS AND BODY WEIGHT CHANGES - SUMMARY - PART A ------------------------------------------------------------------------------------------------------------------------------------ GROUP 1 2 3 4 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 3 3 3 3 BODY WEIGHT (KG) DAY 1 MEAN±S.D. 3.03 ± 0.12 3.11 ± 0.08 3.12 ± 0.02 3.30 ± 0.38 DAY 2 MEAN±S.D. 3.01 ± 0.12 3.12 ± 0.09 3.07 ± 0.01 3.29 ± 0.37 DAY 3 MEAN±S.D. 3.02 ± 0.09 3.14 ± 0.08 3.07 ± 0.01 3.29 ± 0.39 DAY 4 MEAN±S.D. 3.01 ± 0.08 3.12 ± 0.07 3.06 ± 0.00 3.29 ± 0.38 DAY 5 MEAN±S.D. 3.05 ± 0.08 3.12 ± 0.06 3.04 ± 0.02 3.34 ± 0.45 DAY 6 MEAN±S.D. 3.09 ± 0.10 3.14 ± 0.09 3.08 ± 0.01 3.32 ± 0.45 BODY WEIGHT CHANGE (KG) DAYS 1 - 2 MEAN±S.D. -0.02 ± 0.00 +0.00 ± 0.04 -0.05 ± 0.03 -0.01 ± 0.02 DAYS 2 - 3 MEAN±S.D. +0.01 ± 0.03 +0.02 ± 0.01 +0.00 ± 0.02 +0.00 ± 0.03 DAYS 3 - 4 MEAN±S.D. -0.01 ± 0.01 -0.02 ± 0.03 -0.01 ± 0.00 +0.00 ± 0.01 DAYS 4 - 5 MEAN±S.D. +0.04 ± 0.02 +0.00 ± 0.01 -0.02 ± 0.02 +0.06 ± 0.06 DAYS 5 - 6 MEAN±S.D. +0.03 ± 0.03 +0.02 ± 0.04 +0.04 ± 0.03 -0.02 ± 0.02 DAYS 1 - 6 MEAN±S.D. +0.05 ± 0.02 +0.02 ± 0.06 -0.04 ± 0.02 +0.03 ± 0.06 ------------------------------------------------------------------------------------------------------------------------------------ DAY(S) = DAY(S) OF STUDY a. Dosage occurred on Days 1 through 5 of study.



Table 3 Clinical and Necropsy Observations - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 3 (PAGE 1): CLINICAL AND NECROPSY OBSERVATIONS - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ MAXIMUM POSSIBLE INCIDENCE 115/ 5 115/ 5 115/ 5 115/ 5 115/ 5 MORTALITY 0 0 0 0 0 UNGROOMED COAT 0/ 0 4/ 1 0/ 0 7/ 2 1/ 1 SPARSE HAIR COAT: LIMB(S) 0/ 0 0/ 0 0/ 0 0/ 0 6/ 1 INJECTION SITE(S): PURPLE 0/ 0 0/ 0 0/ 0 0/ 0 5/ 1 RED SUBSTANCE ON LINER 0/ 0 0/ 0 1/ 1 0/ 0 0/ 0 SCANT FECES 7/ 2 11/ 2 0/ 0 0/ 0 0/ 0 SOFT OR LIQUID FECES 1/ 1 1/ 1 0/ 0 0/ 0 0/ 0 LIMITED USE OF LEFT HINDLIMB 0/ 0 20/ 1 0/ 0 0/ 0 0/ 0 IRREGULAR GAIT 0/ 0 8/ 1 0/ 0 0/ 0 0/ 0 NO GROSS LESIONS WERE IDENTIFIED AT NECROPSY ------------------------------------------------------------------------------------------------------------------------------------ MAXIMUM POSSIBLE INCIDENCE = (DAYS x RABBITS)/NUMBER OF RABBITS EXAMINED PER GROUP ON DAYS 7 THROUGH 29 OF PRESUMED GESTATION N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RABBITS WITH OBSERVATION a. Dosage occurred on Days 7 through 19 of presumed gestation.



Table 4 Maternal Body Weights - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 4 (PAGE 1): MATERNAL BODY WEIGHTS - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N 5 4 4 4 5 MATERNAL BODY WEIGHT (KG) DAY 0 MEAN±S.D. 3.47 ± 0.13 3.48 ± 0.14 3.52 ± 0.14 3.42 ± 0.08 3.49 ± 0.14 DAY 7 MEAN±S.D. 3.73 ± 0.03 3.71 ± 0.14 3.74 ± 0.14 3.67 ± 0.09 3.75 ± 0.14 DAY 8 MEAN±S.D. 3.79 ± 0.04 3.76 ± 0.16 3.78 ± 0.13 3.74 ± 0.14 3.77 ± 0.14 DAY 9 MEAN±S.D. 3.79 ± 0.05 3.77 ± 0.18 3.78 ± 0.13 3.70 ± 0.11 3.77 ± 0.17 DAY 10 MEAN±S.D. 3.80 ± 0.05 3.74 ± 0.24 3.79 ± 0.13 3.72 ± 0.11 3.78 ± 0.16 DAY 11 MEAN±S.D. 3.82 ± 0.06 3.76 ± 0.23 3.81 ± 0.13 3.74 ± 0.14 3.79 ± 0.16 DAY 12 MEAN±S.D. 3.83 ± 0.08 3.76 ± 0.24 3.84 ± 0.15 3.76 ± 0.13 3.82 ± 0.18 DAY 13 MEAN±S.D. 3.87 ± 0.10 3.81 ± 0.25 3.88 ± 0.16 3.77 ± 0.14 3.83 ± 0.20 DAY 14 MEAN±S.D. 3.91 ± 0.11 3.83 ± 0.23 3.90 ± 0.17 3.78 ± 0.14 3.84 ± 0.20 DAY 15 MEAN±S.D. 3.98 ± 0.12 3.81 ± 0.21 3.96 ± 0.20 3.81 ± 0.12 3.86 ± 0.21 DAY 16 MEAN±S.D. 3.99 ± 0.14 3.77 ± 0.18 4.01 ± 0.16 3.86 ± 0.17 3.88 ± 0.23 DAY 17 MEAN±S.D. 3.99 ± 0.16 3.78 ± 0.18 4.00 ± 0.16 3.84 ± 0.17 3.89 ± 0.22 DAY 18 MEAN±S.D. 3.97 ± 0.14 3.78 ± 0.23 4.02 ± 0.18 3.86 ± 0.18 3.92 ± 0.23 DAY 19 MEAN±S.D. 3.97 ± 0.16 3.83 ± 0.25 4.04 ± 0.17 3.88 ± 0.18 3.94 ± 0.22 DAY 20 MEAN±S.D. 3.97 ± 0.19 3.83 ± 0.24 4.08 ± 0.16 3.91 ± 0.21 3.96 ± 0.24 ------------------------------------------------------------------------------------------------------------------------------------ DAY = DAY OF GESTATION a. Dosage occurred on Days 7 through 19 of gestation.


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 4 (PAGE 2): MATERNAL BODY WEIGHTS - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N 5 4 4 4 5 MATERNAL BODY WEIGHT (KG) DAY 21 MEAN±S.D. 3.98 ± 0.19 3.84 ± 0.24 4.08 ± 0.19 3.92 ± 0.20 3.97 ± 0.25 DAY 22 MEAN±S.D. 4.04 ± 0.15 3.87 ± 0.26 4.11 ± 0.21 3.94 ± 0.18 3.99 ± 0.26 DAY 23 MEAN±S.D. 4.07 ± 0.16 3.89 ± 0.26 4.14 ± 0.19 3.98 ± 0.19 4.00 ± 0.28 DAY 24 MEAN±S.D. 4.09 ± 0.16 3.89 ± 0.25 4.15 ± 0.19 4.00 ± 0.20 4.00 ± 0.30 DAY 25 MEAN±S.D. 4.11 ± 0.16 3.90 ± 0.26 4.18 ± 0.19 4.03 ± 0.19 4.03 ± 0.31 DAY 26 MEAN±S.D. 4.11 ± 0.16 3.91 ± 0.24 4.18 ± 0.16 4.00 ± 0.22 4.06 ± 0.31 DAY 27 MEAN±S.D. 4.12 ± 0.13 3.91 ± 0.24 4.18 ± 0.18 4.03 ± 0.19 4.06 ± 0.29 DAY 28 MEAN±S.D. 4.11 ± 0.13 3.91 ± 0.24 4.21 ± 0.16 4.05 ± 0.18 4.05 ± 0.25 DAY 29 MEAN±S.D. 4.15 ± 0.12 3.93 ± 0.24 4.24 ± 0.18 4.04 ± 0.19 4.06 ± 0.22 ------------------------------------------------------------------------------------------------------------------------------------ DAY = DAY OF GESTATION a. Dosage occurred on Days 7 through 19 of gestation.



Table 5 Maternal Body Weight Changes - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 5 (PAGE 1): MATERNAL BODY WEIGHT CHANGES - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N 5 4 4 4 5 MATERNAL BODY WEIGHT CHANGE (KG) DAYS 0 - 7 MEAN±S.D. +0.26 ± 0.10 +0.22 ± 0.12 +0.22 ± 0.04 +0.24 ± 0.08 +0.26 ± 0.04 DAYS 7 - 10 MEAN±S.D. +0.07 ± 0.07 +0.04 ± 0.12 +0.06 ± 0.05 +0.05 ± 0.03 +0.02 ± 0.05 DAYS 10 - 13 MEAN±S.D. +0.07 ± 0.05 +0.06 ± 0.04 +0.09 ± 0.04 +0.05 ± 0.04 +0.05 ± 0.04 DAYS 13 - 16 MEAN±S.D. +0.12 ± 0.07 -0.04 ± 0.09 +0.14 ± 0.05 +0.09 ± 0.05 +0.05 ± 0.05 DAYS 16 - 20 MEAN±S.D. -0.02 ± 0.19 +0.06 ± 0.12 +0.06 ± 0.02 +0.06 ± 0.05 +0.07 ± 0.04 DAYS 7 - 20 MEAN±S.D. +0.23 ± 0.19 +0.12 ± 0.21 +0.34 ± 0.06 +0.24 ± 0.13 +0.21 ± 0.14 DAYS 20 - 24 MEAN±S.D. +0.12 ± 0.06 +0.06 ± 0.04 +0.08 ± 0.04 +0.09 ± 0.03 +0.04 ± 0.10 DAYS 24 - 29 MEAN±S.D. +0.06 ± 0.07 +0.04 ± 0.08 +0.09 ± 0.09 +0.04 ± 0.05 +0.06 ± 0.11 DAYS 20 - 29 MEAN±S.D. +0.18 ± 0.12 +0.10 ± 0.09 +0.16 ± 0.07 +0.13 ± 0.03 +0.10 ± 0.13 DAYS 7 - 29 MEAN±S.D. +0.42 ± 0.14 +0.22 ± 0.15 +0.51 ± 0.10 +0.37 ± 0.12 +0.31 ± 0.09 DAYS 0 - 29 MEAN±S.D. +0.68 ± 0.22 +0.45 ± 0.26 +0.72 ± 0.14 +0.62 ± 0.18 +0.57 ± 0.11 ------------------------------------------------------------------------------------------------------------------------------------ DAYS = DAYS OF GESTATION a. Dosage occurred on Days 7 through 19 of gestation.



Table 6 Maternal Absolute Food Consumption Values (g/day) - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 6 (PAGE 1): MATERNAL ABSOLUTE FOOD CONSUMPTION VALUES (G/DAY) - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N 5 4 4 4 5 MATERNAL FOOD CONSUMPTION (G/DAY) DAYS 7 - 10 MEAN±S.D. 164.4 ± 22.3 176.8 ± 9.4 175.8 ± 13.0 158.8 ± 23.6 151.1 ± 26.1 DAYS 10 - 13 MEAN±S.D. 155.4 ± 36.9 167.7 ± 17.6 175.0 ± 13.8 156.4 ± 31.2 152.9 ± 24.6 DAYS 13 - 16 MEAN±S.D. 137.5 ± 50.5 125.3 ± 55.4 167.2 ± 18.6 142.4 ± 34.8 132.2 ± 31.4 DAYS 16 - 20 MEAN±S.D. 127.6 ± 79.1 125.0 ± 80.4 179.8 ± 5.2 151.8 ± 31.4 141.2 ± 33.2 DAYS 7 - 20 MEAN±S.D. 144.8 ± 39.0 146.9 ± 35.0 174.9 ± 11.6 152.3 ± 29.2 144.1 ± 28.1 DAYS 20 - 24 MEAN±S.D. 151.9 ± 25.9 125.3 ± 65.4 169.0 ± 9.5 142.6 ± 44.0 122.6 ± 45.7 DAYS 24 - 29 MEAN±S.D. 117.6 ± 35.4 98.2 ± 26.0 131.8 ± 55.0 97.6 ± 25.4 95.6 ± 29.1 DAYS 20 - 29 MEAN±S.D. 132.8 ± 14.6 110.3 ± 34.6 148.4 ± 33.3 117.6 ± 32.2 107.6 ± 32.6 DAYS 7 - 29 MEAN±S.D. 139.9 ± 24.8 131.9 ± 30.0 164.0 ± 16.5 138.1 ± 29.4 129.2 ± 25.4 ------------------------------------------------------------------------------------------------------------------------------------ DAYS = DAYS OF GESTATION a. Dosage occurred on Days 7 through 19 of gestation.



Table 7 Maternal Relative Food Consumption Values (g/kg/day) - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 7 (PAGE 1): MATERNAL RELATIVE FOOD CONSUMPTION VALUES (G/KG/DAY) - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N 5 4 4 4 5 MATERNAL FOOD CONSUMPTION (G/KG/DAY) DAYS 7 - 10 MEAN±S.D. 43.5 ± 5.8 47.2 ± 2.3 46.7 ± 4.3 42.8 ± 5.7 40.0 ± 5.6 DAYS 10 - 13 MEAN±S.D. 40.5 ± 9.2 44.4 ± 3.4 45.8 ± 4.2 41.6 ± 7.7 40.0 ± 4.8 DAYS 13 - 16 MEAN±S.D. 34.7 ± 12.2 33.4 ± 16.0 42.5 ± 4.3 37.4 ± 8.8 34.0 ± 6.1 DAYS 16 - 20 MEAN±S.D. 31.8 ± 19.3 33.1 ± 21.8 44.7 ± 2.5 39.0 ± 6.9 35.8 ± 6.7 DAYS 7 - 20 MEAN±S.D. 37.1 ± 9.5 39.1 ± 10.4 44.9 ± 3.5 40.1 ± 6.9 37.3 ± 5.5 DAYS 20 - 24 MEAN±S.D. 37.6 ± 5.1 32.4 ± 17.1 41.2 ± 4.2 35.8 ± 10.3 30.4 ± 9.7 DAYS 24 - 29 MEAN±S.D. 28.6 ± 8.9 25.2 ± 6.7 31.7 ± 13.9 24.2 ± 6.0 23.6 ± 7.0 DAYS 20 - 29 MEAN±S.D. 32.6 ± 3.6 28.4 ± 9.1 36.0 ± 9.3 29.4 ± 7.5 26.6 ± 7.2 DAYS 7 - 29 MEAN±S.D. 35.2 ± 5.5 34.6 ± 8.6 41.1 ± 5.4 35.6 ± 6.9 32.8 ± 4.7 ------------------------------------------------------------------------------------------------------------------------------------ DAYS = DAYS OF GESTATION a. Dosage occurred on Days 7 through 19 of gestation.



Table 8 Caesarean-Sectioning Observations - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 8 (PAGE 1): CAESAREAN-SECTIONING OBSERVATIONS - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ RABBITS TESTED N 5 5 5 5 5 PREGNANT N(%) 5(100.0) 4( 80.0) 4( 80.0) 4( 80.0) 5(100.0) RABBITS PREGNANT AND CAESAREAN-SECTIONED ON DAY 29 OF GESTATION N 5 4 4 4 5 CORPORA LUTEA MEAN±S.D. 9.8 ± 2.4 10.8 ± 2.4 8.2 ± 2.5 8.8 ± 2.8 11.4 ± 3.4 IMPLANTATIONS MEAN±S.D. 9.4 ± 2.3 10.0 ± 2.2 6.8 ± 3.8 8.8 ± 2.8 10.4 ± 2.8 % PREIMPLANTATION LOSS MEAN±S.D. 4.0 ± 5.8 6.8 ± 4.8 24.0 ± 26.1 0.0 ± 0.0 8.0 ± 4.6 LITTER SIZES MEAN±S.D. 9.2 ± 2.3 9.5 ± 1.3 6.8 ± 3.8 8.5 ± 2.4 9.8 ± 2.2 LIVE FETUSES N 46 38 27 34 49 MEAN±S.D. 9.2 ± 2.3 9.5 ± 1.3 6.8 ± 3.8 8.5 ± 2.4 9.8 ± 2.2 DEAD FETUSES N 0 0 0 0 0 RESORPTIONS MEAN±S.D. 0.2 ± 0.4 0.5 ± 1.0 0.0 ± 0.0 0.2 ± 0.5 0.6 ± 0.9 EARLY RESORPTIONS N 0 0 0 1 2 MEAN±S.D. 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.5 0.4 ± 0.5 LATE RESORPTIONS N 1 2 0 0 1 MEAN±S.D. 0.2 ± 0.4 0.5 ± 1.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.4 % POSTIMPLANTATION LOSS MEAN±S.D. 2.0 ± 4.5 3.8 ± 7.7 0.0 ± 0.0 2.1 ± 4.2 4.9 ± 6.7 DOES WITH ANY RESORPTIONS N(%) 1( 20.0) 1( 25.0) 0( 0.0) 1( 25.0) 2( 40.0) DOES WITH ALL CONCEPTUSES RESORBED N(%) 0( 0.0) 0( 0.0) 0( 0.0) 0( 0.0) 0( 0.0) DOES WITH VIABLE FETUSES N(%) 5(100.0) 4(100.0) 4(100.0) 4(100.0) 5(100.0) PLACENTAE APPEARED NORMAL N(%) 5(100.0) 4(100.0) 4(100.0) 4(100.0) 5(100.0) ------------------------------------------------------------------------------------------------------------------------------------ % PREIMPLANTATION LOSS = [(NUMBER OF CORPORA LUTEA - NUMBER OF IMPLANTATIONS) / NUMBER OF CORPORA LUTEA] x 100 % POSTIMPLANTATION LOSS = [(NUMBER OF IMPLANTATIONS - NUMBER OF LIVE FETUSES) / NUMBER OF IMPLANTATIONS] x 100 a. Dosage occurred on Days 7 through 19 of gestation.



Table 9 Litter Observations (Caesarean-Delivered Fetuses) - Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 9 (PAGE 1): LITTER OBSERVATIONS (CAESAREAN-DELIVERED FETUSES) - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ LITTERS WITH ONE OR MORE LIVE FETUSES N 5 4 4 4 5 IMPLANTATIONS MEAN±S.D. 9.4 ± 2.3 10.0 ± 2.2 6.8 ± 3.8 8.8 ± 2.8 10.4 ± 2.8 LIVE FETUSES N 46 38 27 34 49 MEAN±S.D. 9.2 ± 2.3 9.5 ± 1.3 6.8 ± 3.8 8.5 ± 2.4 9.8 ± 2.2 % LIVE MALE FETUSES/LITTER MEAN±S.D. 47.9 ± 20.0 44.7 ± 7.9 69.0 ± 21.6 44.9 ± 22.1 33.6 ± 20.4 LIVE FETAL BODY WEIGHTS (GRAMS)/LITTER MEAN±S.D. 42.00 ± 4.57 40.54 ± 2.69 46.59 ± 4.46 41.36 ± 5.63 37.81 ± 3.77 MALE FETUSES MEAN±S.D. 43.94 ± 5.65 42.62 ± 3.04 45.88 ± 4.78 42.43 ± 6.44 38.38 ± 2.35 [ 4]b FEMALE FETUSES MEAN±S.D. 39.91 ± 4.43 38.98 ± 2.79 45.92 ± 3.06 40.71 ± 4.79 36.57 ± 4.49 [ 3]c % RESORBED CONCEPTUSES/LITTER MEAN±S.D. 2.0 ± 4.5 3.8 ± 7.7 0.0 ± 0.0 2.1 ± 4.2 4.9 ± 6.7 ------------------------------------------------------------------------------------------------------------------------------------ [ ] = NUMBER OF VALUES AVERAGED a. Dosage occurred on Days 7 through 19 of gestation. b. Litter 2196 had no male fetuses. c. Litter 2190 had no female fetuses.



Table 10 Fetal Gross External Alterations - Caesarean-Delivered Live Fetues (Day 29 of Gestation) -

Summary - Part B


Final Report Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS TABLE 10 (PAGE 1): FETAL GROSS EXTERNAL ALTERATIONS - CAESAREAN-DELIVERED LIVE FETUSES (DAY 29 OF GESTATION) - SUMMARY - PART B ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 6 7 8 9 TEST MATERIAL CONTROL ARTICLE RGN-352 RGN-352 RGN-352 RGN-352 DOSE LEVEL (MG/KG/DAY)a 0 3 10 30 90 ------------------------------------------------------------------------------------------------------------------------------------ LITTERS EVALUATED N 5 4 4 4 5 LITTERS WITH LIVE FETUS(ES) N 5 4 4 4 5 FETUSES EVALUATED N 46 38 27 34 49 LIVE N 46 38 27 34 49 ------------------------------------------------------------------------------------------------------------------------------------ BODY: GASTROSCHISIS LITTER INCIDENCE N(%) 0( 0.0) 0( 0.0) 0( 0.0) 1( 25.0) 0( 0.0) FETAL INCIDENCE N(%) 0( 0.0) 0( 0.0) 0( 0.0) 1( 2.9)b 0( 0.0) SNOUT: CLEFT LITTER INCIDENCE N(%) 0( 0.0) 0( 0.0) 0( 0.0) 1( 25.0) 0( 0.0) FETAL INCIDENCE N(%) 0( 0.0) 0( 0.0) 0( 0.0) 1( 2.9)b 0( 0.0) ------------------------------------------------------------------------------------------------------------------------------------ a. Dosage occurred on Days 7 through 19 of gestation. b. Fetus 2192-10 had other gross external alterations.



Appendix 1 Protocol, Amendments, and Deviations


FINAL PROTOCOL


A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a

Preliminary Evaluation in Non-Pregnant Rabbits






16 May 2012

Page 1 of 29



TABLE OF CONTENTS

1. OBJECTIVES ..........................................................................................................................3

2. PROPOSED STUDY SCHEDULE ........................................................................................3

3. GUIDELINES FOR STUDY DESIGN ...................................................................................4

4. REGULATORY COMPLIANCE ...........................................................................................4

5. QUALITY ASSURANCE .......................................................................................................4

6. SPONSOR ...............................................................................................................................5

7. RESPONSIBLE PERSONNEL ...............................................................................................6

8. TEST AND CONTROL ARTICLES ......................................................................................7

9. SAFETY ..................................................................................................................................8

10. DOSE FORMULATION AND ANALYSIS ..........................................................................9

11. TEST SYSTEM .....................................................................................................................10

12. HUSBANDRY ......................................................................................................................12

13. EXPERIMENTAL DESIGN .................................................................................................14

14. IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS .........................15

15. LABORATORY EVALUATIONS .......................................................................................16

16. TERMINAL PROCEDURES ...............................................................................................18

17. FETAL EXAMINATIONS ...................................................................................................22



20. AMENDMENTS AND DEVIATIONS ................................................................................22

21. RETENTION OF RECORDS, SAMPLES, AND SPECIMENS .........................................23

22. REPORTING .........................................................................................................................24

23. ANIMAL WELFARE ...........................................................................................................24

24. REFERENCES ......................................................................................................................26

25. TESTING FACILITY APPROVAL .....................................................................................27

26. SPONSOR APPROVAL .......................................................................................................29



1. OBJECTIVES The objectives of this study are to provide information for selection of dosages to be used in a subsequent embryo-fetal development study in rabbits and to provide a preliminary evaluation of the effects of thymosin beta 4 (formulated as RGN-352) on pregnancy and embryo-fetal development. This study evaluates ICH Harmonised Tripartite Guideline stages C to D of the reproductive process. In addition, the toxicokinetic characteristics of RGN-352 will be determined.

2. PROPOSED STUDY SCHEDULE Proposed study dates are listed below. Actual applicable dates will be included in the Final Report.

2.1. Part A Animal Arrival - Acclimation Begins: 16 May 2012

Initiation of Dosing: 21 May 2012

Completion of In-life (Day 6 of Study): 26 May 2012 (Date of necropsy)

Unaudited Letter Report: 31 May 2012

2.2. Part B Animal Arrival: 15 Jun 2012

Initiation of Dosing: 20 Jun 2012

Toxicokinetic Sample Collection: 20 Jun 2012 - 21 Jun 2012 (Day 7 and 8 [12-hour] of presumed gestation)

Toxicokinetic Sample Collection: 02 Jul 2012 - 03 Jul 2012 (Day 19 and 20 [12-hour] of presumed gestation)

Completion of In-life Toxicokinetic Study: 03 Jul 2012

(Day 20 of presumed gestation)

Completion of In-life Main Study: 12 Jul 2012

(Day 29 of presumed gestation)

Shipment of Toxicokinetic Samples: 24 Jul 2012

Unaudited Letter Report: 19 Jul 2012

Dose Formulation Draft Report: 06 Sep 2012 (Expected ship date from PI to Study Director)



Bioanalytical Draft Report: 06 Sep 2012 (Expected ship date from PI to Study Director)

Toxicokinetic Draft Report: 06 Sep 2012 (Expected ship date from PI to Study Director)

Audited Draft Report: 21 Sep 2012

3. GUIDELINES FOR STUDY DESIGN The design of this study was based on the study objectives, the overall product development strategy for the test article, and the following study design guidelines:

• ICH Harmonised Tripartite Guideline M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals

• ICH Harmonised Tripartite Guideline S3a. Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies.

• ICH Harmonised Tripartite Guideline S5 (R2). Guideline for Industry: detection of toxicity to reproduction for medicinal products & toxicity to male fertility.

4. REGULATORY COMPLIANCE The study will be performed in accordance with the United States Department of Health and Human Services, Food and Drug Administration (FDA), Code of Federal Regulations, Title 21, Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies (GLPs) and as accepted by Regulatory Authorities throughout the European Community (OECD Principles of Good Laboratory Practice) and Japan (MHLW).

Any portion of this study conducted by Charles River Laboratories in Canada will be performed in accordance with the OECD Principles of Good Laboratory Practice and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA) and Japan (MHLW).

Exceptions to GLPs include the following study elements:

• Characterization of the test and control articles was performed by a Sponsor subcontractor at a facility that follows FDA current Good Manufacturing Practice (cGMP) regulations.

5. QUALITY ASSURANCE

5.1. Testing Facility The Testing Facility Quality Assurance Unit (QAU) will monitor the study to assure the facilities, equipment, personnel, methods, practices, records, and controls are in conformance with Good Laboratory Practice regulations. The QAU will review the protocol, conduct inspections at intervals adequate to assure the integrity of the study, and audit the Final Report to assure that it accurately describes the methods and standard operating procedures and that the reported results accurately reflect the raw data of the study.



The Testing Facility QAU contact for this study is indicated below:

Karen J. Waetjen, M.S., RQAP-GLP Address as cited for Testing Facility Tel: 215.443.8710 Fax: 215.443.8587 E-mail: [email protected]

5.2. Testing Facility-designated Subcontractors The following study phases performed by Testing Facility-designated subcontractors will be audited by the Testing Facility QAU:

• Dose formulation analysis

The following study phases performed by Testing Facility-designated subcontractors will be audited by the respective subcontractor QAU(s):

• Bioanalysis

• Toxicokinetics

For all study phase(s) inspected by subcontractor QAU(s), copies of each periodic inspection report will be made available to the Study Director, Testing Facility Management, and the Testing Facility QAU.

6. SPONSOR Sponsor Representative and Study Monitor David Crockford, Vice President Clinical and Regulatory Affairs RegeneRx Biopharmaceuticals, Inc. Address as cited for Sponsor Tel: 301.208.9191 Cell: 617.285.5588 Fax: 301.208.9194 E-mail: [email protected]



7. RESPONSIBLE PERSONNEL Study Director Valerie A. Sharper, MS Address as cited for Testing Facility Tel: 267.532.3838 Fax: 215.443.8587 E-mail: [email protected]

Management Contact Alan M. Hoberman, PhD, DABT, Fellow ATS Address as cited for Testing Facility Tel: 215.443.8710 Fax: 215.443.8587 E-mail: [email protected]

Principal Investigators (PI) at Testing Facility-designated Test Site(s) Dose Formulation Analysis Jason Sarsoza, BSc

Scientist I Charles River Laboratories Preclinical Services 905 Sheehy Drive, Building A Horsham, PA 19044 Tel: 267.532.3771 Fax: 215.443.8587 E-mail: [email protected]

Bioanalysis Nathalie Proulx Senior Research Scientist Charles River Laboratories Preclinical Services Montreal 22022 Transcanadienne Senneville, Quebec, Canada H9X 3R3 Tel: +1.514.630.8200, ext. 8184 Fax: +1.514.630.8230 E-mail: [email protected]

Toxicokinetics Andrew M. Vick, PhD Seventh Wave Laboratories LLC 743 Spirit 40 Park Drive, Suite 209 Chesterfield, MO 63005-1121 Tel: 636.519.4885 Fax: 636.519.4886 E-mail: [email protected]



Each PI is required to report any deviations or other circumstances that could affect the quality or integrity of the study to the Study Director in a timely manner. Each PI will provide a report addressing their assigned phase of the study, which will be included as an appendix to the Final Report. The phase report will include the following:

• A Statement of Compliance

• A QA Statement (if audited by a QAU other than that of the Testing Facility)

• The archive site and storage duration for all records, samples, specimens and reports generated from the phase or segment

• A listing of critical computerized systems used in the conduct and/or interpretation of the assigned study phase

8. TEST AND CONTROL ARTICLES

8.1. Test Article(s) Identification: Thymosin beta 4 (Tβ4) formulated as RGN-352 (Tβ4 Injectable

Solution)

Supplier: Sponsor

Batch (Lot) Number: 330-05-001

Expiration Date: To be documented in the raw data

Physical Description: colorless, clear liquid, 2.25 mL/vial



8.2. Control Article Identification: RGN-352 Placebo (Vehicle for RGN-352)

Supplier: Sponsor

Batch (Lot) No.: 330-06-001

Expiration Date: To be documented in the raw data

Physical Description: colorless, clear liquid, 5.5 mL/vial


8.3. Test Material Shipment All test material shipments should be addressed to the attention of Julian Gulbinski ([email protected]), Senior Manager Laboratory Sciences, at the previously cited Testing Facility address.



Shipments should include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment.

8.4. Test and Control Article Characterization The Sponsor will provide to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test and control articles. A Certificate of Analysis or equivalent documentation will be provided for inclusion in the Final Report. The Sponsor will also provide information concerning the regulatory standard that was followed for these evaluations.

The Sponsor has appropriate documentation on file concerning the method of synthesis of the active ingredient, Tβ4, and fabrication or derivation of the test and control articles, and this information is available to the appropriate regulatory agencies should it be requested.

8.5. Analysis of Test Article Stability of the formulated test article for the duration of the study will be confirmed by comparison of the results of the concentration analysis conducted for samples collected before the first dose administration and samples collected on the last day of administration.

8.6. Reserve Samples For each batch (lot) of test and control articles, a reserve sample (1 vial) will be collected and maintained under the appropriate storage conditions by the Testing Facility, pursuant to 21 CFR 58.190.

8.7. Test and Control Article Inventory and Disposition Records of the receipt, distribution, storage, and disposition of test and control articles (including empty containers) will be maintained. With the exception of reserve samples, all unused Sponsor-supplied bulk test and control articles will be retained for use in future studies with this test article or returned to the Sponsor before issue of the final report. All empty containers will be maintained for the duration of the study.

9. SAFETY The following safety instructions apply to this study:

Gloves, dust-mist/HEPA-filtered mask, appropriate eye protection and uniform/lab coat.

The Material Safety Data Sheet (MSDS) will be maintained in the raw data.



10. DOSE FORMULATION AND ANALYSIS

10.1. Preparation of Control Article The control article, RGN-352 Placebo, will be administered as received. Upon receipt, the control article will be dispensed daily for administration to Group 1 control animals. The control article will be removed from the refrigerator at least 30 minutes before dosing to allow it to come to room temperature.

Any residual volumes will be discarded before issuance of the Final Report.

10.2. Preparation of Test Article The test article, formulated as RGN-352, will be administered as received. Upon receipt, the test article will be dispensed daily for administration to Groups 2 and 3. The prepared test article will be removed from the refrigerator at least 30 minutes before dosing to allow it to come to room temperature.

Any residual volumes will be discarded before issuance of the Final Report.

10.3. Sample Collection and Analysis Dose formulation samples will be collected for analysis as indicated in the following table. Additional samples may be collected and analyzed at the discretion of the Study Director.

Dose Formulation Sample Collection Schedule

Interval Concentration Homogeneity Stability Before First Administration (Part A)a All groupsa N/A N/A Last Day of Administration (Part B) All groups N/A N/A

N/A = Not applicable. a. If the results of the concentration analysis of the last day of administration samples from the most recently

previously performed study (Testing Facility Study No. 20024504) are within the acceptance criteria and are within 30 days from the first administration on this study (Testing Facility Study No. 20024505), analysis of the predose samples will not be required. If analyses are not required, a protocol amendment will be issued.

Samples to be analyzed will be submitted on the date prepared. All samples to be analyzed will be transferred (ambient conditions) to the analytical laboratory at the Testing Facility. Upon receipt at the analytical laboratory, the samples will be stored refrigerated (2°C to 8°C) until analysis. Any residual/retained analytical samples (and test article used in analysis) will be discarded before issue of the Final Report.

10.3.1. Analytical Method Analyses described below will be performed by HPLC using a validated analytical procedure [TYMN03 - Analytical Procedure for the Analysis of Thymosin beta 4 (RGN-352) in RGN-352 Placebo by HPLC-UV; validated under PCS-PA Protocol No. 20019481].



10.3.1.1. Concentration Analysis Samples for Analysis: Single sample from one vial; transferred to Charles River’s

Analytical Chemistry Laboratory.

Backup Samples: Single sample from one vial; maintained at the Testing Facility. Backup samples may be analyzed at the discretion of the Study Director.

Sample Volume: 2.25 mL


Acceptance Criteria: The criteria for acceptability will be mean sample concentration results within or equal to ± 10% of theoretical concentration. Each individual sample concentration result within or equal to ± 15%.

10.3.1.2. Homogeneity Analysis The test article Tβ4, formulated and vialed as RGN-352 (100 mg/mL), yields a sterile aqueous, homogeneous solution. Therefore, homogeneity analyses of the test article will not be conducted. The test article will not be mixed with the placebo (RGN-352 excipients minus Tβ4) in this study.

10.3.1.3. Stability Analysis Stability of the formulated test article for the duration of the study will be confirmed by comparison of the results of the concentration analysis conducted for samples collected before the first dose administration and samples collected on the last day of administration, as described in Section 10.3, Sample Collection and Analysis.

11. TEST SYSTEM Species: Rabbit

Strain: New Zealand White [Hra:(NZW)SPF]

Gender: Female

Source: Covance Research Products, Inc., Denver, PA

Condition:

Part A: Non-mated

Part B: Timed-mated (day mating is observed = Day 0 of presumed gestation)

Number Ordered:

Part A: 13

Part B: 38



Number Assigned To Study:

Part A: 12

Part B: 37

Target Age at Arrival: 5 to 7 months

Target Weight at Arrival: 2.5 kg to 5.5 kg

The actual body weight will be recorded the day of receipt. The actual age, weight, and number of animals received will be listed in the Final Report.

11.1. Justification of Test System and Number of Animals The test system was selected because: 1) it is a standard species accepted for use in embryo-fetal development studies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the Testing Facility.

The number of animals chosen for this study is the smallest number considered necessary to select dosages for subsequent studies.

11.2. Animal Identification Rabbits are permanently identified using Monel® self-piercing ear tags. Female rabbits are given unique permanent identification numbers when assigned to the study.

11.3. Environmental Acclimation After receipt at the Testing Facility, the rabbits will be acclimated for at least 4 days prior to initiation of dose administration.

11.4. Mating, Part B The female rabbits will be naturally bred at the Supplier, by breeder male rabbits of the same source and strain, before shipment to the Testing Facility. The day mating occurs will be designated Day 0 of presumed gestation. The rabbits will be shipped to the Testing Facility after mating, to arrive on Day 2 of presumed gestation.

11.5. Selection, Assignment, and Replacement of Animals Assigned animals that die or are euthanized for reasons considered unrelated to toxicity will be replaced if possible, at the discretion of the Study Director, with spare animals. Any animal replacement following randomization will be documented in the study file and presented in the final report.

11.5.1. Part A After an acclimation period, healthy female rabbits will be assigned to groups using a computer-based randomization procedure.



11.5.2. Part B Before shipment of the rabbits, the Supplier will forward breeding records and Day 0 of presumed gestation body weights. A computer-generated (weight-ordered) randomization procedure will be used to assign healthy mated female rabbits to dose groups based on this information.

11.6. Animal Disposition The disposition of all animals will be documented in the study records. Any remaining animals not selected for study will be assigned to the Testing Facility’s general population or humanely euthanized.

12. HUSBANDRY

12.1. Housing All cage sizes and housing conditions are in compliance with the Guide for the Care and Use of Laboratory Animals1

12.1.1. Part A

.

The rabbits will be individually housed in units of six to eight stainless steel cages.

12.1.2. Part B The rabbits will be individually housed in units of six to eight stainless steel cages. No nesting materials will be supplied because the female rabbits will be euthanized before parturition is expected.

12.2. Environmental Conditions The targeted conditions for animal room environment will be as follows:

Temperature: 61°F to 72°F (16°C to 22°C)

Humidity: 30% to 70%

Ventilation: At least 10 changes per hour of 100% fresh air that has been passed through 99.97% HEPA filters.

Light Cycle: An automatically controlled 12-hour light:12-hour dark fluorescent light cycle will be maintained. Each dark period will begin at 1900 hours (± 30 minutes). The light cycle may be adjusted by the Study Director or designee if deemed necessary to accommodate scheduled laboratory activities. Any such adjustment will be documented in the raw data.



12.3. Food

12.3.1. Part A Approximately 150 g of Certified Rabbit Chow® #5322 (PMI® Nutrition International) will be available to each rabbit each day. The certified feed will be available from individual stainless steel "J-type" feeders attached to each cage.

12.3.2. Part B Approximately 150 g of Certified Rabbit Chow®#5322 (PMI® Nutrition International) will be available to each rabbit each day until the first day of dosing, at which time approximately 180 g to 185 g of the same certified feed will be offered to each rabbit each day. The certified feed will be available from individual stainless steel "J-type" feeders attached to each cage.

12.4. Water Water will be available ad libitum from individual bottles attached to the cages and/or from an automatic watering access system (Horsham, PA). All water will be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processed water as a bacteriostat; processed water is expected to contain no more than 1.2 ppm chlorine at the time of analysis. Water is analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination.

12.5. Animal Enrichment Suspended chain link devices hanging from the stainless steel cages will be supplied to all rabbits during the course of the study. Other enrichment devices or food supplements (e.g., Timothy cubes) may be used as directed by the veterinary staff, if necessary

12.6. Contaminants The Study Director is not aware of any potential contaminants likely to be present in the certified food, enrichment devices or in the drinking water at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the food supplier or those mentioned in this protocol will be conducted.

12.7. Veterinary Care Veterinary care will be available throughout the course of the study and animals will be examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, will be documented in the study records.



13. EXPERIMENTAL DESIGN

13.1. Part A

Experimental Design




Dose Volume (mL/kg) No. of Study Animals

1 Control Article 0 0 0.9 3 2

RGN-352 10 100 0.1 3

3 30 100 0.3 3 4 90 100 0.9 3

13.2. Part B

Experimental Design




Dose Volume (mL/kg)

No. of Main Study Animals

No. of Toxicokinetic

Animals 5 Control Article 0 0 0.9 5 N/A 6

RGN-352

3 100 0.03 5 3 7 10 100 0.1 5 3 8 30 100 0.3 5 3 9 90 100 0.9 5 3

NA = Not Applicable

13.3. Administration of Test and Control Articles

13.3.1. Part A Female rabbits (nonmated) will be administered the test article and/or the control article once daily on Days 1 through 5 of study (the first day of dose administration will be designated as Day 1 of study). Doses will be adjusted based on body weights recorded immediately before dosing and given at approximately the same time each day.

13.3.2. Part B Female rabbits (presumed pregnant) will be administered the test article and/or the control article once daily on Days 7 through 19 of presumed gestation. Doses will be adjusted based on the most recently recorded body weight and administered at approximately the same time each day.

13.4. Justification of Route and Dose Levels The intravenous route was selected for use because it is one of the proposed systemic routes for clinical use.



Dose level selections for the test article in this study bracket the dose level selections planned for RGN-352 in future clinical trials.

14. IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS The in-life procedures, observations, and measurements listed below will be performed for all main study rabbits and toxicokinetic rabbits (Part B); food consumption will not be recorded for the rabbits assigned to the toxicokinetic study.

14.1. Viability Checks (Part A and B) Frequency: At least twice daily.

Procedure: Viability will be recorded.

14.2. Clinical Observations (Part A and B) Clinical observations may be recorded more frequently than cited in the following sections.

14.2.1. General Appearance Frequency: At least once during the predose period, daily before

administration during the dosing period, and once daily during the postdose period.

Procedure: Clinical observations will be recorded.

14.2.2. Postdose Observations Frequency:

Part A: On the first day of dosing, postdose observations will be recorded at approximately hourly intervals for the first four hours and at the end of the normal working day. Subsequent postdose observations will be recorded at intervals deemed appropriate by the Study Director or designee after determination of peak pharmacologic/toxicologic effects.

Part B: Postdose observations will be recorded at intervals determined appropriate during the conduct of Part A. Time intervals for postdose observations may be adjusted if deemed appropriate by the Study Director or designee during the course of the study. Such adjustments will be documented in the raw data.

Procedure: Clinical observations will be recorded.



14.3. Body Weights Frequency:

Part A: Day of arrival at the Testing Facility, daily during the dosing period and on the day of scheduled euthanasia.

Part B: Day 0 of presumed gestation and on the day of arrival at the Testing Facility and daily during the dosing and postdose periods.

Procedure: Body weights will be recorded.

14.4. Food Consumption Frequency:

Part A: Daily after arrival at the Testing Facility (values not tabulated).

Procedure: Estimated percentage of food consumed will be recorded.

Part B (Main Study): Daily after arrival at the Testing Facility (values not tabulated), and daily during the dosing and postdose periods.

Procedure: Weights of food left and food fed will be recorded.

15. LABORATORY EVALUATIONS

15.1. Bioanalysis and Toxicokinetic Evaluation - Part B

15.1.1. Bioanalytical Sample Collection Blood will be collected from medial auricular artery from each rabbit assigned to the toxicokinetic study. If necessary, blood may be collected from an alternate site (marginal auricular vein or saphenous vein); if so, the alternate site will be documented in the raw data. Samples will be collected according to the following table.

TK Sample Collection Schedule

Group No.

Sample Collection Time Points (Approximate Time Postdose) on Days 7 and 19 of Presumed Gestation

Predose 5 m 10 m 20 m 1 h 2 h 6 h 12 h 2 X X X X X X X X 3 X X X X X X X X 4 X X X X X X X X 5 X X X X X X X X

m = minute; h = hour; X = blood collected

Target Volume: 1 mL

Anticoagulant: K2EDTA



15.1.2. Bioanalytical Sample Processing Samples will be mixed gently and kept on crushed wet ice immediately following collection. The samples will be centrifuged in a refrigerated centrifuge at 2700 rpm for approximately 10 minutes. The resultant plasma will be separated, transferred to uniquely labeled clear polypropylene cryovials, and frozen immediately over dry ice or in a freezer set to maintain -80°C.

Samples to be analyzed will be submitted at the completion of the in-life phase of the study.

The plasma samples will be shipped on dry ice to:

Frank Alleruzzo Coordinator II Sample Management Attn: Nathalie Proulx Senior Research Scientist Charles River Laboratories Preclinical Services Montreal 22022 Transcanadienne Senneville, Quebec, Canada H9X 3R3 Tel: +1.514.630.8200, ext. 8974 Fax: +1.514.630.8230 E-mail: [email protected]

The bioanalytical laboratory will be notified before shipment of the samples. Upon receipt at the bioanalytical laboratory, the samples will be stored at or below -70°C.

15.1.3. Bioanalytical Sample Analysis Plasma samples will be analyzed for concentration of Tβ4, formulated as RGN-352 (Tβ4 Injectable Solution). Analysis will be performed by LC-MS/MS under Analytical Procedure 142475.PL.xx (where ‘xx’ denotes the version number), using a validated analytical procedure (Study No. 142473). Data collection will be performed using Analyst from AB Sciex. Statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy and precision will be performed using Watson Laboratory Information Management System (LIMS) and Microsoft Excel.

Incurred sample reanalysis (ISR) will be performed for this study as per the appropriate SOP(s) of the bioanalytical laboratory.

Any residual/retained bioanalytical samples will be discarded before issue of the Final Report.



15.1.4. Toxicokinetic Evaluation

Results of plasma Tβ4 analysis will be provided to the Principal Investigator for calculation of noncompartmental exposure parameters (using WinNonlin, Version 5.2) as appropriate, such as peak concentration (Cmax), time to peak concentration (Tmax), and area under the concentration-time curve (AUC). When data permit, the slope of the terminal elimination phase of each arithmetic mean concentration versus time curve will be determined by log-linear regression, and disposition parameters (e.g., AUC0-inf, clearance, volume of distribution, and elimination half-life) will also be estimated. No statistical analyses other than calculation of descriptive statistics (e.g., mean, SD, %CV, etc.) will be performed on the resulting parameters. The Principal Investigator for toxicokinetic analysis will be responsible for all toxicokinetic-delegated phase activities and will issue a formal toxicokinetic report from the data generated. The toxicokinetic report will be included as an appendix to the Final Report.

16. TERMINAL PROCEDURES

16.1. Part A Terminal procedures are summarized in the following table:

Terminal Procedures for All Rabbits

Group No.

No. of Animals


Day

Necropsy Procedures

Histology Histopathology Gross Necropsy Tissue

Collection Organ

Weights 1 3

6 X - -

- -

2 3 - -

3 3 - - 4 3 - -

Unscheduled Deaths X - - - -



16.2. Part B Terminal procedures are summarized in the following table:

Terminal Procedures for Main Study and Toxicokinetic Animals

Group No.

No. of Animals


Day

Necropsy Procedures


Ovarian/ Uterine

Examination Necropsy Tissue

Collection Organ

Weights Toxicokinetic Animals

6 3

20 Pregnancy Status - - -

- -

7 3 - -

8 3 - - 9 3 - -

Unscheduled Deaths Pregnancy Status X - - - -

Main Study Animals 5 5

29 Full Exam X X -

- - 6 5 - -

7 5 - -

8 5 - -

9 5 - -

Unscheduled Deaths Full Exam X X - - - X = Procedure to be conducted; - = Not applicable.

16.3. Method of Euthanasia A euthanasia solution (390 mg pentobarbital sodium and 50 mg phenytoin sodium) will be used to euthanize rabbits (via intravenous injection) and live fetuses (via intraperitoneal injection).

16.4. Unscheduled Deaths

16.4.1. Part A Rabbits that die or are euthanized before scheduled termination will be examined for the cause of death or condition as soon as possible after the observation is made. The rabbits will be examined for gross lesions. When not precluded by autolysis, the heart, lungs, liver, kidneys, stomach, and spleen will be retained in neutral buffered 10% formalin for possible histological evaluation. Additional tissues may be retained at the discretion of the Study Director.



16.4.2. Part B Rabbits that die or are euthanized before scheduled termination will be examined for the cause of death or condition as soon as possible after the observation is made. The rabbits will be examined for gross lesions. When not precluded by autolysis, the heart, lungs, liver, kidneys, stomach, and spleen will be retained in neutral buffered 10% formalin for possible histological evaluation. Additional tissues may be retained at the discretion of the Study Director.

Pregnancy status will be recorded, and uteri of apparently nonpregnant rabbits will be examined while being pressed between glass plates to confirm the absence of implantation sites. Uterine contents will be recorded, and aborted fetuses, conceptuses in utero and/or delivered kits will be examined to the extent possible, using the same methods described for term fetuses. Uteri (with cervix) and ovaries of apparently nonpregnant rabbits will be retained in neutral buffered 10% formalin and may be discarded when authorized by the Study Director.

16.5. Scheduled Euthanasia

16.6. Part A On Day 6 of study, all rabbits will be euthanized and a gross necropsy of the thoracic, abdominal and pelvic viscera will be conducted.

16.7. Part B On Day 20 of presumed gestation, female rabbits assigned to the toxicokinetic portion of the study will be euthanized after the last blood sample collection and examined for pregnancy status. Uteri of apparently nonpregnant does will be examined while being pressed between glass plates to confirm the absence of implantation sites. Conceptuses and carcasses will be discarded without further evaluation.

On Day 29 of presumed gestation, female rabbits assigned to the main portion of the study will be Caesarean-sectioned, and examined for gross lesions as further described in Sections 16.7.1 (Ovarian and Uterine Examinations) and 16.8 (Necropsy).

16.7.1. Ovarian and Uterine Examinations The reproductive tract will be dissected from the abdominal cavity. The uterus will be opened and the contents will be examined. The fetuses will be removed from the uterus and placed in individual containers (or a tray).

The ovaries and uterus will be examined for number and distribution of:

• Corpora Lutea

• Implantation Sites

• Placentae (size, color or shape) – any abnormalities will be recorded

• Live and Dead Fetuses

• Early and Late Resorptions



Uteri of apparently nonpregnant animals will be examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant animals will be retained in 10% neutral buffered formalin and may be discarded when authorized by the Study Director.

16.8. Necropsy A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed at scheduled euthanasia for each rabbit assigned to Part A, each rabbit assigned to Part B, and all rabbits that are found dead or euthanized prior to scheduled euthanasia. Unless specifically cited in Section 16.9 (Tissue Collection and Preservation), all tissues will be discarded.

Images may be generated for illustration of or consultation on gross observations. Generation of such images will be documented. Images and associated documentation will be retained and archived.

16.9. Tissue Collection and Preservation Representative samples of the tissues identified in the Tissue Collection and Preservation table will be collected from each rabbit assigned to Part A, each rabbit assigned to Part B, and all rabbits that are found dead or euthanized prior to scheduled euthanasia and preserved in 10% neutral buffered formalin, unless otherwise indicated. Additional tissues may be collected to elucidate abnormal findings. Unless specifically cited below, all other tissues will be discarded.

Corresponding tissues will be retained from one rabbit in the control group in Part B of the study, in order to provide comparative tissues for any possible histopathological evaluations of gross lesions.

Tissue Collection and Preservation

Tissue Collect Comment Cervix X Collect with uterus. All nonpregnant rabbits.

Gross lesions/masses X All rabbits. Heart X Rabbits found dead or euthanized before scheduled termination.

Salivary Gland X Rabbits with expressed salivation (ptyalism) at any time during the study Kidney X Rabbits found dead or euthanized before scheduled termination. Liver X Rabbits found dead or euthanized before scheduled termination Lung X Rabbits found dead or euthanized before scheduled termination.

Ovaries X All nonpregnant rabbits. Spleen X Rabbits found dead or euthanized before scheduled termination.

Stomach X Rabbits found dead or euthanized before scheduled termination. Uterus X Retain for all nonpregnant rabbits.

X = Procedure to be conducted.



17. FETAL EXAMINATIONS Fetuses will be examined for external abnormalities. Late resorptions and dead fetuses will be examined for external abnormalities and sex to the extent possible. The body weight of each fetus will be recorded. All fetuses will be examined internally to determine sex. Fetuses with external abnormalities will be fixed in Bouin's solution; all other fetuses will be discarded. Representative photographs of fetal external abnormalities will be taken at the discretion of the Study Director. Photographs will not be included in the report, but will be retained as electronic images and archived with the raw data.

18. COMPUTERIZED SYSTEMS The following proposed critical computerized systems may be used in the study. The actual computerized systems used will be specified in the Final Report.

Data for parameters not required by protocol, which are automatically generated by analytical devices used will be retained on file but not reported. Statistical analysis results that are generated by the program but are not required by protocol and/or are not scientifically relevant will be retained on file but will not be included in the tabulations.

Proposed Critical Computerized Systems

System Name Description of Data Collected and/or Analyzed Dispense Test article/vehicle/control article/component receipt

Argus Automated Data Collection and Management System

Clinical observations, body weights, food consumption, organ weights, Caesarean-sectioning observations, statistical analyses

Vivarium Temperature and Relative Humidity Monitoring System Study room temperature and humidity

Quattro Pro Body weight graph

19. STATISTICAL ANALYSIS Statistical analysis will be descriptive, rather than inferential. Averages and percentages will be calculated. Litter values will be used where appropriate. Only body weights of live fetuses will be used to determine litter mean fetal body weight. Additional procedures and/or analyses may be performed if deemed appropriate.

20. AMENDMENTS AND DEVIATIONS Changes to the approved protocol shall be made in the form of an amendment, which will be signed and dated by the Study Director and the Sponsor.

All protocol and SOP deviations will be documented in the study records. Deviations from the protocol and/or SOP related to the phase(s) of the study conducted at a Test Site shall be documented, acknowledged by the PI, and reported to the Study Director for authorization/acknowledgement. The Study Director will notify the Sponsor of deviations that may result in a significant impact on the study as soon as possible.



21. RETENTION OF RECORDS, SAMPLES, AND SPECIMENS All study-specific raw data, documentation, protocol, samples, specimens, and interim (if applicable) and final reports from this study are the property of the Sponsor. These materials will be available at the Testing Facility during the study and will be transferred to the Testing Facility archive by no later than the date of final report issue. One year after issue of the audited draft report, the Sponsor will be contacted to determine the disposition of materials associated with the study.

Electronic data generated by the Testing Facility will be archived, and the software and hardware required to produce it in a readable form will be maintained and available.

All records, samples, specimens and reports generated from phases or segments performed by Testing Facility-designated subcontractors will be returned to the Testing Facility for archiving.

Records to be maintained will include, but will not be limited to, documentation and data for the following:

• Protocol, protocol amendments, and deviations

• Study schedule

• Study-related correspondence

• Test and control article receipt, identification, preparation, and analysis

• Test system receipt, health, and husbandry

• Mating history

• Selection, assignment and replacement of animals

• Animal disposition

• Veterinary care, if required

• Administration of test and control articles

• In-life measurements and observations

• Clinical pathology sample collection and evaluation, if required

• Bioanalytical sample collection and evaluation, if required

• Gross and/or microscopic observations and related data

• Ovarian/Uterine and Fetal Observations

• Photographs, if required

• Packing and/or Shipment Lists



22. REPORTING A comprehensive Draft Report will be prepared following completion of the study and will be finalized following consultation with the Sponsor. The report will include all information necessary to provide a complete and accurate description of the experimental methods and results and any circumstances that may have affected the quality or integrity of the study.

An unaudited preliminary data report for the purpose of dosage selection for Part B of this study or the full (definitive) study will be prepared following completion of Part A and Part B of this study.

The Sponsor will receive an electronic version of the Draft and Final Report provided in Adobe Acrobat PDF format (hyperlinked and searchable at final) along with a Microsoft Word version of the text. The PDF document will be created from native electronic files to the extent possible, including text and tables generated by the Testing Facility. Report components not available in native electronic files and/or original signature pages will be scanned and converted to PDF image files for incorporation. An original copy of the report with the Testing Facility’s handwritten signatures will be retained.

A tabulated data summary following the appropriate format as outlined in the ICH Harmonized Tripartite Guideline, The Common Technical Document for the Registration of Pharmaceuticals for Human Use: Safety – M4S (R2), Nonclinical Overview and Nonclinical Summaries of Module 2, Organisation of Module, will be provided as an appendix to the Draft and Final Reports.

Reports should be finalized within 6 months of issue of the audited Draft Report. If the Sponsor has not provided comments to the report within 6 months of draft issue, the report will be finalized by the Testing Facility unless other arrangements are made by the Sponsor.

23. ANIMAL WELFARE This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (Code of Federal Regulations, Title 9), the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare, and the Guide for the Care and Use of Laboratory Animals from the National Research Council.1,2 The protocol and any amendments or procedures involving the care or use of animals in this study will be reviewed and approved by the Testing Facility Institutional Animal Care and Use Committee before the initiation of such procedures.



The Testing Facility’s attending veterinarian is responsible for implementation of programs for the evaluation of the health status of study animals, the recommendation of treatment for health conditions, the evaluation of response to treatment, as well as the diagnosis of pain or distress. The veterinary staff will communicate to the Study Director signs of animal illness, injury, pain and distress, and recommendations on treatment of the animal(s) and/or alteration of study procedures. In nonemergency situations, decisions regarding the study animals, including treatments, alterations in study design, and/or justification of action(s) will be documented, and will be approved in advance by the Study Director, and in consultation with the Sponsor as appropriate. If euthanasia is deemed necessary, it will be in accordance with the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia and with the procedures outlined in the protocol.3 The Sponsor will be fully informed of any such events.

If an animal is determined by the veterinary staff to be in overt pain/distress, or appears moribund and is beyond the point where recovery appears reasonable, the animal will be euthanized for humane reasons in accordance with the AVMA Guidelines on Euthanasia.

By approving this protocol, the Sponsor affirms that there are no acceptable non-animal alternatives for this study, that this study is required by a relevant government regulatory agency(ies) and that it does not unnecessarily duplicate any previous experiments.



24. REFERENCES 1. National Research Council. Guide for the Care and Use of Laboratory Animals.

Washington, D.C.: National Academy Press. 1996.

2. Office of Laboratory Animal Welfare. Public Health Services Policy on Humane Care and Use of Laboratory Animals. Bethesda, MD: National Institutes of Health. August 2002.

3. American Veterinary Medical Association. AVMA Guidelines on Euthanasia. June 2007.


Final Report Page 58 Testing Facility Study No. 20024505

DEVIATIONS All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. All protocol deviations and those SOP deviations that could have impacted the quality or integrity of the study are listed below. Minor SOP deviations that did not impact the quality or integrity of the study have been included at the discretion of the Study Director.

Unless otherwise indicated below, none of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Dose Formulation and Analysis

• On the last day of dose administration (02 Jul 2012), one vial of the placebo was retained as a backup sample, in addition to a 2.25 mL backup sample. This deviation did not adversely affect the outcome or interpretation of the study because the additional backup sample is of no consequence.

• On 23 May 2012, vial #884 of Thymosin beta 4 (Tβ4) formulated as RGN-352 was discarded at the Testing Facility; all vials were to be retained. This deviation did not adversely affect the outcome or interpretation of the study because it was a single vial; there will be no additional analysis of these vials.

Husbandry

• On study days 1 through 6 (DSs 1 through 6), 16 through 26 May 2012, all rabbits assigned to Part A of the study were housed in a plastic rabbit caging system with stainless steel racks, rather than in stainless steel caging. This deviation did not adversely affect the outcome or interpretation of the study because the caging used was appropriate for this species (Allentown plastic rabbit caging system with stainless steel rack, stainless steel panels for upper cage and back and sides, stainless steel doors, and injection molded cages with circular floor perforations).

Environmental Conditions

• The temperature was outside of the 61°F to 72°F range at one timepoint recorded throughout the course of the study. The online vivarium environmental monitoring system records temperature and humidity values no less frequently than four times hourly. The out-of-range temperature was 77.1°F on 26 June 2012. This deviation did not adversely affect the outcome or interpretation of the study because this elevation occurred at a single timepoint during the study.

• The sensitivity of the relative humidity recording equipment is ±3%. Documentation of all excursions is available in the raw data. Excursions out of the 30% to 70% (±3%) relative humidity range are reported below.



Date Study Room

Total Duration (hours)

Peak Humidity (%)

Approximate Duration of Peak Humidity

17 May 2012 55 9 26 1 hour 20 May 2012 55 1 26 1 hour 28 Jun 2012 55 1 74 1 hour 28 Jun 2012 55 5 74 1 hour 30 Jun 2012 55 4 73 1 hour 02 Jul 2012 55 1 74 1 hour 03 Jul 2012 55 3 74 1 hour

These deviations did not adversely affect the outcome or interpretation of the study because the magnitude of the humidity deviations was small.

In-Life Procedures, Observations and Measurements

• On day 9 of presumed gestation (DG 9), 29 June 2012, post dose observations were performed 1 minute early for rabbit 2197 in the 90 mg/kg/day dose group (Part B). This deviation did not adversely affect the outcome or interpretation of the study because the time deviated was minor.



Appendix 2 Test and Control Article Characterization



Appendix 3 Dose Formulation Analysis Report


FINAL REPORT

Study Phase: Analytical Chemistry


A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits,

Including a Preliminary Evaluation in Non-Pregnant Rabbits




Preclinical Services, Pennsylvania (PCS-PA) 905 Sheehy Dr., Building A


Page 1 of 41


Final Analytical Report Testing Facility Study No. 20024505

TABLE OF CONTENTS

1. LIST OF TABLES ......................................................................................................3

2. LIST OF APPENDICES .............................................................................................3

3. COMPLIANCE STATEMENT ..................................................................................4

4. RESPONSIBLE PERSONNEL...................................................................................5

5. INTRODUCTION .......................................................................................................6

6. MATERIALS AND METHODS ................................................................................6 6.1. Test Article.........................................................................................................6 6.2. Control Article ...................................................................................................6 6.3. Sample Receipt and Storage ..............................................................................7 6.4. Sample Analysis.................................................................................................7 6.5. Test and Control Article Inventory and Disposition ..........................................7 6.6. Computerized Systems.......................................................................................7

7. STATISTICAL ANALYSIS .......................................................................................7

8. RETENTION OF RECORDS .....................................................................................7

9. RESULTS ....................................................................................................................8

10. CONCLUSIONS .........................................................................................................9

11. REPORT APPROVAL ..............................................................................................10



1. LIST OF TABLES

Table 1 Study Samples – Thymosin beta 4 Concentration .....................................11


Appendix 1 Deviations .................................................................................................12

Appendix 2 Certificates of Analysis .............................................................................14

Appendix 3 Analytical Procedure .................................................................................19

Appendix 4 Dose Formulation Analysis Reports .........................................................35



3. COMPLIANCE STATEMENT This phase of this study was conducted in accordance with the U.S. Department of Health and Human Services, Food and Drug Administration, United States Code of Federal Regulations, Title 21, Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies and as accepted by Regulatory Authorities throughout the European Community (OECD Principles of Good Laboratory Practice) and Japan (MHLW)

Exceptions to GLPs include the following study elements:

• Characterization of the test and control articles was performed by a Sponsor subcontractor at a facility that follows FDA current Good Manufacturing Practice (cGMP) regulations. This exception to the GLP regulation had no effect on the overall integrity of the study or the interpretation of the study results and conclusions because the material was appropriately characterized.

This phase of this study was conducted in accordance with the procedures described herein. All deviations authorized/acknowledged by the Principal Investigator and Study Director are documented in the Study Records. The report represents an accurate and complete record of the results obtained.



4. RESPONSIBLE PERSONNEL Principal Investigator Jason Sarsoza, BSc

Charles River Laboratories, PCS-PA

Research Assistant IV Phinh xu Ngo Charles River Laboratories, PCS-PA

Research Assistant II Shichei Andega, MS Charles River Laboratories, PCS-PA

Senior Manager, Laboratory Sciences Julian Gulbinski, III, BS, MBA Charles River Laboratories, PCS-PA

Director of Operations Matthew J. Vaneman, BS

Executive Director, Laboratory Sciences North America

Alan Bartlett, CChem, FRSC



5. INTRODUCTION The purpose of this project was to determine the concentration of Thymosin beta 4 in the test and control articles from Study No. 20024505 titled “A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a Preliminary Evaluation in Non-Pregnant Rabbits.” The study was sponsored by RegeneRx Biopharmaceuticals, Inc., Rockville, MD where David Crockford, Vice President, served as the Sponsor Representative. Valerie A. Sharper, MS, Charles River Laboratories Preclinical Services, Horsham, PA, was the Study Director. Jason Sarsoza, BSc, Charles River Laboratories Preclinical Services, Horsham, PA, was the Principal Investigator for this study phase.

This study phase was started on 18 May 2012 and completed on 06 Jul 2012.

6. MATERIALS AND METHODS

6.1. Test Article Identity: RGN-352 (also known as Thymosin beta 4 (Tβ4) Injectable

Solution)

Batch/Lot No.: 330-05-001

Expiration Date: 01 December 2012

Purity/Potency/Assay: 99.54% (assumed 100%)



Manufacturer/Supplier: Sponsor

The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test article, RGN-352. A Certificate of Analysis was provided to the Testing Facility and is presented in Appendix 2.

6.2. Control Article Identity: RGN-352 Placebo (also known as RGN-352 Vehicle)

Batch/Lot No.: 330-06-001

Expiration Date: 27 August 2012


Manufacturer/Supplier: Sponsor



The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the control article, RGN-352 Placebo. A Certificate of Analysis was provided to the Testing Facility and is presented in Appendix 2.

6.3. Sample Receipt and Storage Two sets of samples, RGN-352 and RGN-352 Placebo, were received from the Testing Facility on 18 May 2012 and 02 Jul 2012. The samples were received in satisfactory condition. Samples were stored at 2°C to 8°C prior to analysis or were analyzed upon receipt. The samples were analyzed within the established stability period.

6.4. Sample Analysis Samples of RGN-352 and RGN-352 Placebo were analyzed for Thymosin beta 4 according to the validated method described in PCS-PA Analytical Procedure TYMN03 “Analytical Procedure for the Analysis of Thymosin Beta 4 in RGN-352 Placebo Dose Formulations by HPLC-UV.” A copy of the most recent version of the Analytical Procedure is contained in Appendix 3.

6.5. Test and Control Article Inventory and Disposition Records of the receipt, distribution, and storage of the test article (RGN-352) and control article (RGN-352 Placebo) were maintained. All unopened vials of test and control article were maintained at the Testing Facility.

6.6. Computerized Systems Critical computerized systems used in this study phase are listed below (see Text Table 1).

Text Table 1 Computerized Systems

System Name Version No. Description of Data Collected and/or Analyzed

TotalChrom® (PerkinElmer®) 6.2.1. Acquisition of HPLC data, assessment of system suitability, and integration of the peak area of the

analyte

Excel (Microsoft Office) 1997 or later Regression analysis and calculation of concentrations and descriptive statistics

7. STATISTICAL ANALYSIS Regression analysis and descriptive statistics (such as means and relative standard deviations) were used to determine the dose formulation concentrations.

8. RETENTION OF RECORDS All study-specific raw data, documentation and the final report from this study phase are the property of the Sponsor. These materials will be available at the Testing Facility during the progress of the study and will be transferred to the Testing Facility archive by no



later than the date of final report issue. One year after issue of the audited draft report, the Sponsor will be contacted to determine the disposition of materials associated with the study. Archival material will be indexed by Study No. 20024505.

9. RESULTS Concentration results are summarized in Table 1. Results and conclusions for each analytical run are provided in the Dose Formulation Analysis Reports, which are contained in Appendix 4.

Thymosin beta 4 concentrations for all doses of RGN-352 were within the acceptable limits (± 10% of nominal concentration for the mean result and ± 15% of nominal concentration for the individual results).



10. CONCLUSIONS Samples of RGN-352 and RGN-352 Placebo were analyzed for Thymosin beta 4 by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The method was validated for the analysis of Thymosin beta 4 in RGN-352 Placebo at concentrations ranging from 50 mg/mL to 100 mg/mL.

The dose formulations were within specification.



Table 1 Study Samples – Thymosin beta 4 Concentration

Occasion (Sampling Date) Group

Theoretical Concentration

(mg/mL) Sampling Location

Measured Concentration

(mg/mL) Bias

Start of Study (18 May 2012)

1 0 Middle ND NA ND NA

Mean - NA

2 to 4 100 Middle 100 0.0 100 0.0

Mean 100 0.0

End of Study (02 July 2012)

5 0 Middle <LOD NA ND NA

Mean - NA

6 to 9 100 Middle 98.0 -2.0 99.5 -0.5

Mean 98.8 -1.2 Group 1 and 5: Control article, RGN-352 Placebo Group 2 to 4 and Group 6 to 9: Test Article, RGN-352 LOD = Limit of detection (0.0038 mg/mL). NA = Not applicable. ND = None detected.



Appendix 1 Deviations



DEVIATIONS

All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. Only minor SOP deviations that did not impact the quality or integrity of the study occurred during the course of the study.

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.



Appendix 2 Certificates of Analysis



Appendix 3 Analytical Procedure



Appendix 4 Dose Formulation Analysis Reports



DOSE FORMULATION ANALYSIS REPORT

Sponsor: RegeneRx Biopharmaceuticals, Inc. Study Facility: Charles River Laboratories Preclinical Services, Pennsylvania Protocol Number: 20024505 Analyte: Thymosin beta 4 (formulated as RGN-352) Analytical Facility: Charles River Laboratories Preclinical Services, Pennsylvania Batch ID: 20024505-1-001-1 Sampling Criteria: Start of Study Concentration Analysis Vehicle: RGN-352 Placebo Storage Conditions: 2°C to 8°C Analytical Procedure: TYMN03 Revision 00 Analysis Date: May 18, 2012 RESULTS: (Concentrations in mg/mL, ND = none detected)

CALIBRATION STANDARDS

Standard Nominal Response Calculated % "X" = Criteria Standard Description Conc. Area Conc. Bias Exclude Limit Pass/Fail Cal Std A1 0.100 137207 0.0983 -1.7 5% PASS Cal Std B1 0.200 279280 0.199 -0.5 5% PASS Cal Std A2 0.400 565596 0.403 +0.8 5% PASS Cal Std B2 0.600 849985 0.605 +0.8 5% PASS Cal Std A3 0.800 1114811 0.793 -0.9 5% PASS Cal Std B3 1.00 1409064 1.00 0.0 5% PASS

CHECK STANDARDS

Standard Nominal Response Dilution Conc. % Criteria Standard Description Conc. Area Factor Found Bias Limit Pass/Fail

Check Std A3 0.800 1121892 1 0.798 -0.2 5% PASS Check Std A3 0.800 1113733 1 0.792 -1.0 5% PASS



Project Number: 20024505Analysis of Thymosin beta 4 in RGN-352

Batch ID: 20024505-1-001-1

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 0.2 0.4 0.6 0.8 1 1.2


Res

pons

e (A

rea)

Slope: 1407400Y-Int: -1149.2Corr: 0.99993

n: 6



SAMPLES

Nominal Total

Sample Prep Sample Response Dilution mg/mL % Description Date Conc. Replicate Area Factor Found Bias

Group 1 08/22/11 0 A 0 50 ND Group 1 08/22/11 0 B 0 50 ND

Groups 2 - 4 08/22/11 100 A 1127929 125 100 0.0 Groups 2 - 4 08/22/11 100 B 1126173 125 100 0.0

Nominal Sample Sample Mean %

Description Conc. Conc. Error Group 2 - 4 100 100 0.0

CONCLUSIONS: Results indicate that the formulations are within the acceptable limits of ±10% of nominal concentration for the mean result and ±15% of the nominal concentration for the individual results. ACTIONS TAKEN: None.



DOSE FORMULATION ANALYSIS REPORT

Sponsor: RegeneRx Biopharmaceuticals, Inc. Study Facility: Charles River Laboratories Preclinical Services, Pennsylvania Protocol Number: 20024505 Analyte: Thymosin beta 4 (formulated as RGN-352) Analytical Facility: Charles River Laboratories Preclinical Services, Pennsylvania Batch ID: 20024505-1-002-1 Sampling Criteria: End of Study Concentration Analysis Vehicle: RGN-352 Placebo Storage Conditions: 2°C to 8°C Analytical Procedure: TYMN03 Revision 01 Analysis Date: July 05, 2012 RESULTS: (Concentrations in mg/mL, ND = none detected, LOD = Limit of Detection)

CALIBRATION STANDARDS

Standard Nominal Response Calculated % "X" = Criteria Standard Description Conc. Area Conc. Bias Exclude Limit Pass/Fail Cal Std A1 0.100 141859 0.100 0.0 5% PASS Cal Std B1 0.200 278932 0.200 0.0 5% PASS Cal Std A2 0.400 554571 0.399 -0.2 5% PASS Cal Std B2 0.600 839405 0.605 +0.8 5% PASS Cal Std A3 0.800 1099500 0.793 -0.9 5% PASS Cal Std B3 1.00 1389446 1.00 0.0 5% PASS

CHECK STANDARDS

Standard Nominal Response Dilution Conc. % Criteria Standard Description Conc. Area Factor Found Bias Limit Pass/Fail

Check Std A3 0.800 1095153 1 0.790 -1.3 5% PASS Check Std A3 0.800 1095098 1 0.790 -1.3 5% PASS



Project Number: 20024505Analysis of Thymosin beta 4 in RGN-352

Batch ID: 20024505-1-002-1

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 0.2 0.4 0.6 0.8 1 1.2


Res

pons

e (A

rea)

Slope: 1382300Y-Int: 3092.5

Corr: 0.99993n: 6



SAMPLES

Nominal Total

Sample Prep Sample Response Dilution mg/mL % Description Date Conc. Replicate Area Factor Found Bias

Group 5 08/22/11 0 A 1479 50 <LOD Group 5 08/22/11 0 B 0 50 ND

Groups 6 to 9 08/22/11 100 A 1086960 125 98.0 -2.0 Groups 6 to 9 08/22/11 100 B 1103171 125 99.5 -0.5

Nominal Sample Sample Mean %

Description Conc. Conc. Error Group 6 to 9 100 98.8 -1.2

CONCLUSIONS: Results indicate that the formulations are within the acceptable limits of ±10% of nominal concentration for the mean result and ±15% of the nominal concentration for the individual results. ACTIONS TAKEN: None.



Appendix 4 Clinical Observations - Individual Data - Part A


Final Report Page 133 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 4 (PAGE 1): CLINICAL OBSERVATIONS - INDIVIDUAL DATA - PART A ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # DESCRIPTION ------------------------------------------------------------------------------------------------------------------------------------ GROUP 1 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1450 NO ADVERSE FINDINGS 1451 NO ADVERSE FINDINGS 1452 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 2 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1453 NO ADVERSE FINDINGS 1454 NO ADVERSE FINDINGS 1455 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 3 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1456 NO ADVERSE FINDINGS 1457 NO ADVERSE FINDINGS 1458 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 4 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1459 NO ADVERSE FINDINGS 1460 NO ADVERSE FINDINGS 1461 DS( 2- 6) INJECTION SITE(S): PURPLE a ------------------------------------------------------------------------------------------------------------------------------------ CLINICAL OBSERVATIONS APPEARED NORMAL UNLESS NOTED OTHERWISE DS = DAY OF STUDY a. Observation confirmed at necropsy.



Appendix 5 Body Weights - Individual Data - Part A


Final Report Page 135 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 5 (PAGE 1): BODY WEIGHTS - INDIVIDUAL DATA - PART A ------------------------------------------------------------------------------------------------------------------------------------ DAY 1 2 3 4 5 6 ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 1 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1450 2.94 2.92 2.95 2.95 2.98 3.00 1451 2.99 2.97 2.98 2.98 3.04 3.06 1452 3.17 3.14 3.12 3.10 3.13 3.20 ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 2 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1453 3.03 3.06 3.09 3.10 3.10 3.10 1454 3.20 3.22 3.23 3.19 3.18 3.24 1455 3.11 3.07 3.10 3.06 3.07 3.07 ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 3 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1456 3.12 3.06 3.07 3.06 3.02 3.09 1457 3.15 3.08 3.06 3.06 3.04 3.07 1458 3.10 3.08 3.08 3.07 3.05 3.07 ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 4 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1459 3.09 3.08 3.07 3.08 3.10 3.09 1460 3.74 3.72 3.74 3.73 3.86 3.84 1461 3.06 3.08 3.06 3.06 3.07 3.03 ------------------------------------------------------------------------------------------------------------------------------------ DAY = DAY OF STUDY ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.



Appendix 6 Necropsy Observations - Individual Data - Part A


Final Report Page 137 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 6 (PAGE 1): NECROPSY OBSERVATIONS - INDIVIDUAL DATA - PART A ------------------------------------------------------------------------------------------------------------------------------------ RABBIT DAY OF DOSES NUMBER NECROPSY ADMINISTERED OBSERVATIONS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 1 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1450 DS 6 5 ALL TISSUES APPEARED NORMAL. 1451 DS 6 5 ALL TISSUES APPEARED NORMAL. 1452 DS 6 5 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 2 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1453 DS 6 5 ALL TISSUES APPEARED NORMAL. 1454 DS 6 5 ALL TISSUES APPEARED NORMAL. 1455 DS 6 5 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 3 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1456 DS 6 5 ALL TISSUES APPEARED NORMAL. 1457 DS 6 5 ALL TISSUES APPEARED NORMAL. 1458 DS 6 5 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 4 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 1459 DS 6 5 ALL TISSUES APPEARED NORMAL. 1460 DS 6 5 ALL TISSUES APPEARED NORMAL. 1461 DS 6 5 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ DS = DAY OF STUDY



Appendix 7 Clinical Observations - Individual Data - Part B


Final Report Page 139 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 7 (PAGE 1): CLINICAL OBSERVATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # DESCRIPTION ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2176 DG( 18- 19) SCANT FECES 2177 NO ADVERSE FINDINGS 2178 NO ADVERSE FINDINGS 2179 NO ADVERSE FINDINGS 2180 DG( 17- 21) SCANT FECES DG( 18 ) SOFT OR LIQUID FECES ------------------------------------------------------------------------------------------------------------------------------------ GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2181 DG( 15- 22) SCANT FECES DG( 25 ) SCANT FECES 2182a DG( 10- 29) LIMITED USE OF LEFT HINDLIMB DG( 11 ) IRREGULAR GAIT DG( 13 ) IRREGULAR GAIT DG( 15 ) IRREGULAR GAIT DG( 17 ) IRREGULAR GAIT DG( 19- 22) IRREGULAR GAIT DG( 21 ) SOFT OR LIQUID FECES 2183 DG( 17- 18) SCANT FECES 2184 DG( 21- 24) UNGROOMED COAT 2185 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2186 NO ADVERSE FINDINGS 2187 DG( 26 ) RED SUBSTANCE ON LINER 2188 NO ADVERSE FINDINGS 2189 NO ADVERSE FINDINGS 2190 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ CLINICAL OBSERVATIONS APPEARED NORMAL UNLESS NOTED OTHERWISE DG = DAY OF PRESUMED GESTATION a. On morning of Day 10 of gestation, rabbit 2182 was found outside of cage.


Final Report Page 140 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 7 (PAGE 2): CLINICAL OBSERVATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # DESCRIPTION ------------------------------------------------------------------------------------------------------------------------------------ GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2191 NO ADVERSE FINDINGS 2192 DG( 9 ) UNGROOMED COAT DG( 17- 18) UNGROOMED COAT DG( 21- 23) UNGROOMED COAT 2193 DG( 24 ) UNGROOMED COAT 2194 NO ADVERSE FINDINGS 2195 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2196 NO ADVERSE FINDINGS 2197 NO ADVERSE FINDINGS 2198 DG( 24- 29) SPARSE HAIR COAT: LIMB(S)a 2199 DG( 12- 16) INJECTION SITE(S): PURPLE 2200 DG( 24 ) UNGROOMED COAT ------------------------------------------------------------------------------------------------------------------------------------ CLINICAL OBSERVATIONS APPEARED NORMAL UNLESS NOTED OTHERWISE DG = DAY OF PRESUMED GESTATION a. Observation confirmed at necropsy.



Appendix 8 Maternal Body Weights - Individual Data - Part B


Final Report Page 142 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 8 (PAGE 1): MATERNAL BODY WEIGHTS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2176 P 3.64 3.77 3.77 3.74 3.74 3.73 3.71 3.71 3.80 3.82 3.78 3.71 3.76 2177 P 3.42 3.74 3.84 3.85 3.87 3.89 3.91 3.97 4.00 4.11 4.10 4.14 4.13 2178 P 3.58 3.76 3.80 3.75 3.79 3.82 3.82 3.88 3.79 3.88 3.91 3.98 3.96 2179 P 3.40 3.70 3.74 3.76 3.79 3.81 3.84 3.89 3.95 4.02 4.04 4.08 4.05 2180 P 3.33 3.70 3.80 3.84 3.82 3.85 3.87 3.90 4.02 4.06 4.11 4.02 3.94 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 21 22 23 24 25 26 27 28 29 ------------------------------------------------------------------------------------------------------------------------------------ 2176 P 3.77 3.80 3.80 3.85 3.92 3.91 3.93 3.92 3.96 3.96 4.02 2177 P 4.17 4.18 4.19 4.22 4.27 4.29 4.32 4.32 4.31 4.32 4.35 2178 P 3.97 4.03 4.05 4.05 4.06 4.06 4.05 4.07 4.07 4.06 4.09 2179 P 4.07 4.10 4.11 4.14 4.19 4.21 4.24 4.22 4.18 4.13 4.18 2180 P 3.88 3.75 3.75 3.94 3.93 3.96 4.02 4.02 4.07 4.06 4.11 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.


Final Report Page 143 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 8 (PAGE 2): MATERNAL BODY WEIGHTS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2181 P 3.67 3.80 3.88 3.90 3.90 3.92 3.92 3.98 3.95 3.93 3.85 3.81 3.74 2182 P 3.45 3.59 3.64 3.62 3.46 3.48 3.44 3.48 3.54 3.53 3.50 3.53 3.49 2183 P 3.47 3.86 3.91 3.94 3.98 3.98 3.98 4.02 4.06 4.00 3.91 3.94 4.04 2184 NP 3.59 3.75 3.83 3.84 3.85 3.90 3.92 3.88 3.89 3.89 3.90 3.82 3.79 2185 P 3.33 3.58 3.60 3.61 3.64 3.67 3.69 3.75 3.76 3.79 3.81 3.86 3.87 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 21 22 23 24 25 26 27 28 29 ------------------------------------------------------------------------------------------------------------------------------------ 2181 P 3.77 3.76 3.77 3.75 3.77 3.78 3.78 3.86 3.91 3.91 3.93 2182 P 3.52 3.53 3.54 3.57 3.60 3.62 3.62 3.63 3.62 3.62 3.65 2183 P 4.11 4.10 4.10 4.16 4.19 4.20 4.21 4.20 4.20 4.20 4.24 2184 NP 3.71 3.69 3.79 3.79 3.82 3.83 3.82 3.80 3.77 3.86 3.92 2185 P 3.91 3.94 3.96 3.99 3.99 3.97 3.98 3.95 3.92 3.90 3.91 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.


Final Report Page 144 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 8 (PAGE 3): MATERNAL BODY WEIGHTS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2186 P 3.66 3.82 3.87 3.84 3.80 3.87 3.88 3.93 3.94 3.99 4.00 4.03 4.06 2187 NP 3.42 3.67 3.72 3.71 3.75 3.76 3.77 3.81 3.79 3.77 3.81 3.71 3.66 2188 P 3.51 3.76 3.80 3.82 3.85 3.84 3.88 3.91 3.92 3.98 4.08 4.05 4.06 2189 P 3.58 3.83 3.87 3.89 3.91 3.92 3.97 4.01 4.08 4.16 4.18 4.16 4.18 2190 P 3.32 3.53 3.60 3.59 3.60 3.62 3.63 3.65 3.68 3.69 3.79 3.77 3.76 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 21 22 23 24 25 26 27 28 29 ------------------------------------------------------------------------------------------------------------------------------------ 2186 P 4.06 4.10 4.14 4.14 4.15 4.18 4.18 4.16 4.15 4.18 4.21 2187 NP 3.75 3.89 3.80 3.83 3.87 3.89 3.92 3.96 4.00 4.02 4.05 2188 P 4.10 4.14 4.13 4.19 4.17 4.21 4.29 4.28 4.32 4.35 4.40 2189 P 4.19 4.22 4.24 4.29 4.34 4.34 4.32 4.31 4.32 4.32 4.35 2190 P 3.80 3.85 3.80 3.81 3.88 3.88 3.91 3.95 3.95 3.99 4.00 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.


Final Report Page 145 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 8 (PAGE 4): MATERNAL BODY WEIGHTS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2191 NP 3.74 3.89 3.86 3.84 3.83 3.87 3.92 3.87 3.75 3.86 3.84 3.90 3.90 2192 P 3.45 3.77 3.90 3.78 3.82 3.89 3.88 3.88 3.89 3.92 4.04 3.97 4.03 2193 P 3.53 3.71 3.77 3.78 3.79 3.80 3.82 3.83 3.86 3.88 3.92 3.96 3.96 2194 P 3.39 3.57 3.57 3.54 3.57 3.57 3.57 3.57 3.58 3.64 3.63 3.61 3.61 2195 P 3.33 3.62 3.70 3.71 3.68 3.71 3.76 3.79 3.77 3.80 3.83 3.82 3.83 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 21 22 23 24 25 26 27 28 29 ------------------------------------------------------------------------------------------------------------------------------------ 2191 NP 3.93 3.90 3.85 3.93 3.96 3.96 4.01 4.02 4.03 4.03 4.06 2192 P 4.02 4.09 4.07 4.07 4.10 4.14 4.14 4.15 4.20 4.21 4.22 2193 P 4.00 4.02 4.04 4.07 4.12 4.14 4.14 4.13 4.09 4.11 4.10 2194 P 3.63 3.62 3.64 3.68 3.70 3.72 3.75 3.68 3.76 3.80 3.78 2195 P 3.89 3.92 3.93 3.94 3.98 4.02 4.08 4.04 4.08 4.09 4.08 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.


Final Report Page 146 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 8 (PAGE 5): MATERNAL BODY WEIGHTS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2196 P 3.65 3.96 3.98 4.03 4.03 4.04 4.10 4.14 4.15 4.19 4.25 4.23 4.28 2197 P 3.46 3.70 3.72 3.68 3.65 3.68 3.70 3.68 3.72 3.73 3.72 3.73 3.73 2198 P 3.47 3.70 3.72 3.75 3.78 3.78 3.82 3.84 3.88 3.91 3.92 3.93 3.97 2199 P 3.59 3.81 3.82 3.82 3.82 3.84 3.84 3.88 3.83 3.85 3.86 3.90 3.92 2200 P 3.30 3.59 3.59 3.58 3.61 3.62 3.63 3.62 3.62 3.64 3.66 3.68 3.72 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 21 22 23 24 25 26 27 28 29 ------------------------------------------------------------------------------------------------------------------------------------ 2196 P 4.29 4.33 4.36 4.40 4.46 4.49 4.55 4.59 4.55 4.46 4.41 2197 P 3.75 3.74 3.76 3.81 3.83 3.86 3.89 3.93 3.98 3.97 4.03 2198 P 3.97 4.02 4.04 4.00 3.97 3.96 3.95 3.94 3.94 3.96 3.95 2199 P 3.96 3.97 3.96 4.00 4.04 4.03 4.05 4.06 4.05 4.07 4.08 2200 P 3.74 3.73 3.74 3.73 3.72 3.67 3.73 3.77 3.78 3.78 3.81 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.



Appendix 9 Maternal Food Consumption Values - Individual Data - Part B


Final Report Page 148 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 9 (PAGE 1): MATERNAL FOOD CONSUMPTION VALUES - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAYS 7 - 8 8 - 9 9 - 10 10 - 11 11 - 12 12 - 13 13 - 14 14 - 15 15 - 16 16 - 17 17 - 18 18 - 19 19 - 20 ------------------------------------------------------------------------------------------------------------------------------------ 2176 P 138. 134. 116. 115. 83. 78. 77. 44. 43. 46. 70. 108. 130. 2177 P 180. 164. 168. 170. 181. 163. 180. 175. 164. 181. 182. 182. 169. 2178 P 159. 157. 155. 145. 156. 162. 114. 114. 184. 181. 182. 183. 184. 2179 P 182. 180. 184. 176. 181. 182. 182. 183. 184. 183. 182. 184. 182. 2180 P 182. 185. 182. 178. 181. 180. 185. 184. 49. 3. 0. 5. 15. ------------------------------------------------------------------------------------------------------------------------------------ DAYS 20 - 21 21 - 22 22 - 23 23 - 24 24 - 25 25 - 26 26 - 27 27 - 28 28 - 29 ------------------------------------------------------------------------------------------------------------------------------------ 2176 P 127. 151. 147. 126. 109. 93. 94. 95. 107. 2177 P 172. 180. 182. 158. 140. 117. 109. 113. 117. 2178 P 182. 181. 115. 106. 102. 96. 95. 106. 117. 2179 P 183. 181. 183. 185. 155. 113. 40. 38. 95. 2180 P 26. 125. 158. 170. 184. 182. 184. 185. 154. ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAYS = DAYS OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G).


Final Report Page 149 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 9 (PAGE 2): MATERNAL FOOD CONSUMPTION VALUES - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAYS 7 - 8 8 - 9 9 - 10 10 - 11 11 - 12 12 - 13 13 - 14 14 - 15 15 - 16 16 - 17 17 - 18 18 - 19 19 - 20 ------------------------------------------------------------------------------------------------------------------------------------ 2181 P 180. 183. 172. 173. 167. 180. 129. 57. 26. 3. 0. 1. 33. 2182 P 181. 180. 128. 104. 136. 185. 145. 156. 185. 180. 181. 183. 159. 2183 P 185. 183. 180. 179. 163. 183. 162. 92. 3. 62. 149. 160. 157. 2184 NP 181. 180. 182. 173. 181. 183. 141. 144. 157. 126. 41. 48. 44. 2185 P 182. 184. 183. 180. 180. 182. 184. 181. 183. 183. 183. 183. 184. ------------------------------------------------------------------------------------------------------------------------------------ DAYS 20 - 21 21 - 22 22 - 23 23 - 24 24 - 25 25 - 26 26 - 27 27 - 28 28 - 29 ------------------------------------------------------------------------------------------------------------------------------------ 2181 P 20. 13. 39. 45. 58. 106. 107. 116. 110. 2182 P 182. 169. 160. 156. 122. 111. 94. 108. 124. 2183 P 154. 183. 180. 150. 144. 105. 125. 124. 104. 2184 NP 116. 126. 135. 149. 109. 82. 105. 151. 141. 2185 P 155. 181. 123. 96. 73. 71. 45. 48. 70. ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAYS = DAYS OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G).


Final Report Page 150 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 9 (PAGE 3): MATERNAL FOOD CONSUMPTION VALUES - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAYS 7 - 8 8 - 9 9 - 10 10 - 11 11 - 12 12 - 13 13 - 14 14 - 15 15 - 16 16 - 17 17 - 18 18 - 19 19 - 20 ------------------------------------------------------------------------------------------------------------------------------------ 2186 P 183. 144. 142. 163. 150. 150. 143. 138. 151. 180. 161. 181. 166. 2187 NP 181. 180. 182. 182. 182. 184. 184. 154. 180. 72. 107. 136. 131. 2188 P 184. 183. 180. 184. 181. 185. 185. 180. 185. 184. 182. 182. 181. 2189 P 182. 184. 180. 181. 183. 180. 183. 180. 180. 184. 181. 181. 183. 2190 P 181. 185. 182. 181. 180. 182. 185. 180. 116. 184. 183. 183. 181. ------------------------------------------------------------------------------------------------------------------------------------ DAYS 20 - 21 21 - 22 22 - 23 23 - 24 24 - 25 25 - 26 26 - 27 27 - 28 28 - 29 ------------------------------------------------------------------------------------------------------------------------------------ 2186 P 176. 183. 147. 137. 118. 97. 101. 113. 121. 2187 NP 143. 152. 166. 154. 166. 184. 184. 181. 181. 2188 P 124. 180. 183. 179. 184. 182. 147. 160. 181. 2189 P 184. 180. 154. 146. 86. 54. 56. 62. 64. 2190 P 182. 180. 185. 184. 183. 182. 183. 180. 182. ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAYS = DAYS OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G).


Final Report Page 151 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 9 (PAGE 4): MATERNAL FOOD CONSUMPTION VALUES - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAYS 7 - 8 8 - 9 9 - 10 10 - 11 11 - 12 12 - 13 13 - 14 14 - 15 15 - 16 16 - 17 17 - 18 18 - 19 19 - 20 ------------------------------------------------------------------------------------------------------------------------------------ 2191 NP 118. 67. 79. 144. 138. 77. 48. 136. 123. 139. 150. 129. 100. 2192 P 184. 102. 181. 162. 147. 127. 102. 117. 115. 107. 175. 160. 163. 2193 P 182. 182. 180. 183. 183. 184. 185. 181. 185. 185. 180. 185. 182. 2194 P 115. 128. 138. 125. 126. 101. 83. 85. 180. 102. 128. 106. 100. 2195 P 184. 184. 145. 173. 182. 184. 131. 163. 182. 138. 170. 182. 166. ------------------------------------------------------------------------------------------------------------------------------------ DAYS 20 - 21 21 - 22 22 - 23 23 - 24 24 - 25 25 - 26 26 - 27 27 - 28 28 - 29 ------------------------------------------------------------------------------------------------------------------------------------ 2191 NP 99. 141. 138. 125. 157. 158. 129. 145. 154. 2192 P 152. 145. 112. 91. 75. 83. 82. 87. 69. 2193 P 180. 180. 183. 155. 140. 110. 82. 77. 84. 2194 P 102. 102. 77. 73. 81. 59. 80. 95. 82. 2195 P 182. 182. 185. 180. 183. 122. 119. 129. 113. ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAYS = DAYS OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G).


Final Report Page 152 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 9 (PAGE 5): MATERNAL FOOD CONSUMPTION VALUES - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAYS 7 - 8 8 - 9 9 - 10 10 - 11 11 - 12 12 - 13 13 - 14 14 - 15 15 - 16 16 - 17 17 - 18 18 - 19 19 - 20 ------------------------------------------------------------------------------------------------------------------------------------ 2196 P 180. 181. 185. 181. 182. 185. 182. 183. 184. 183. 183. 182. 180. 2197 P 147. 115. 107. 128. 130. 122. 122. 117. 101. 82. 93. 102. 99. 2198 P 182. 162. 158. 158. 182. 161. 139. 150. 110. 135. 166. 146. 147. 2199 P 156. 163. 155. 166. 165. 148. 135. 137. 122. 142. 159. 159. 164. 2200 P 108. 139. 129. 138. 128. 119. 100. 103. 98. 132. 139. 131. 100. ------------------------------------------------------------------------------------------------------------------------------------ DAYS 20 - 21 21 - 22 22 - 23 23 - 24 24 - 25 25 - 26 26 - 27 27 - 28 28 - 29 ------------------------------------------------------------------------------------------------------------------------------------ 2196 P 183. 181. 185. 182. 185. 165. 116. 22. 15. 2197 P 112. 111. 124. 118. 114. 109. 123. 137. 131. 2198 P 166. 93. 75. 59. 45. 50. 53. 72. 77. 2199 P 156. 153. 160. 134. 130. 131. 112. 125. 117. 2200 P 97. 70. 60. 33. 59. 68. 48. 94. 91. ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAYS = DAYS OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G).



Appendix 10 Necropsy Observations - Individual Data - Part B


Final Report Page 154 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 10 (PAGE 1): NECROPSY OBSERVATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ RABBIT DAY OF PREGNANCY DOSES NUMBER NECROPSY STATUS ADMINISTERED OBSERVATIONS ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2176 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2177 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2178 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2179 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2180 DG 29 P 13 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2181 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2182 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2183 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2184 DG 29 NP 13 ALL TISSUES APPEARED NORMAL. 2185 DG 29 P 13 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2186 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2187 DG 29 NP 13 ALL TISSUES APPEARED NORMAL. 2188 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2189 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2190 DG 29 P 13 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2191 DG 29 NP 13 ALL TISSUES APPEARED NORMAL. 2192 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2193 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2194 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2195 DG 29 P 13 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2196 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2197 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2198 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2199 DG 29 P 13 ALL TISSUES APPEARED NORMAL. 2200 DG 29 P 13 ALL TISSUES APPEARED NORMAL. ------------------------------------------------------------------------------------------------------------------------------------ DG = DAY OF PRESUMED GESTATION P = PREGNANT NP = NOT PREGNANT



Appendix 11 Caesarean-Sectioning Observations - Individual Data - Part B


Final Report Page 156 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 11 (PAGE 1): CAESAREAN-SECTIONING OBSERVATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ VIABLE FETUSES DEAD FETUSES EARLY RESORPTIONS LATE RESORPTIONS IMPLANTATION SITES CORPORA LUTEA ------------------------ ---------------- ---------------- ---------------- ---------------- ---------------- SEX RIGHT LEFT RIGHT LEFT RIGHT LEFT RIGHT LEFT RIGHT LEFT RIGHT LEFT RABBIT # M F HORN TOTAL HORN TOTAL HORN TOTAL HORN TOTAL HORN TOTAL OVARY TOTAL ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2176 5 7 5 7 12 0 0 0 0 0 0 0 0 0 5 7 12 5 8 13 2177 4 5 7 2 9 0 0 0 0 0 0 0 1 1 7 3 10 7 3 10 2178 2 5 4 3 7 0 0 0 0 0 0 0 0 0 4 3 7 4 4 8 2179 9 2 7 4 11 0 0 0 0 0 0 0 0 0 7 4 11 7 4 11 2180 3 4 2 5 7 0 0 0 0 0 0 0 0 0 2 5 7 2 5 7 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2181 5 6 5 6 11 0 0 0 0 0 0 1 1 2 6 7 13 6 8 14 2182 3 6 4 5 9 0 0 0 0 0 0 0 0 0 4 5 9 4 5 9 2183 4 4 6 2 8 0 0 0 0 0 0 0 0 0 6 2 8 6 3 9 2184 NOT PREGNANT 2185 5 5 6 4 10 0 0 0 0 0 0 0 0 0 6 4 10 6 5 11 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2186 3 3 3 3 6 0 0 0 0 0 0 0 0 0 3 3 6 4 4 8 2187 NOT PREGNANT 2188 5 3 3 5 8 0 0 0 0 0 0 0 0 0 3 5 8 4 5 9 2189 7 4 5 6 11 0 0 0 0 0 0 0 0 0 5 6 11 5 6 11 2190 2 0 2 0 2 0 0 0 0 0 0 0 0 0 2 0 2 3 2 5 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2191 NOT PREGNANT 2192 4 6 5 5 10 0 0 0 0 0 0 0 0 0 5 5 10 5 5 10 2193 5 2 5 2 7 0 0 0 0 0 0 0 0 0 5 2 7 5 2 7 2194 2 9 6 5 11 0 0 0 1 0 1 0 0 0 7 5 12 7 5 12 2195 3 3 3 3 6 0 0 0 0 0 0 0 0 0 3 3 6 3 3 6 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2196 0 8 3 5 8 0 0 0 0 1 1 0 0 0 3 6 9 4 6 10 2197 5 8 9 4 13 0 0 0 0 1 1 1 0 1 10 5 15 11 6 17 2198 4 7 7 4 11 0 0 0 0 0 0 0 0 0 7 4 11 7 5 12 2199 5 4 4 5 9 0 0 0 0 0 0 0 0 0 4 5 9 4 6 10 2200 3 5 4 4 8 0 0 0 0 0 0 0 0 0 4 4 8 4 4 8 ------------------------------------------------------------------------------------------------------------------------------------ M = MALE F = FEMALE PLACENTAE APPEARED NORMAL UNLESS NOTED OTHERWISE.



Appendix 12 Litter Observations (Caesarean-Delivered Fetuses) - Individual Data - Part B


Final Report Page 158 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 12 (PAGE 1): LITTER OBSERVATIONS (CAESAREAN-DELIVERED FETUSES) - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ NUMBER OF LIVE AVERAGE FETAL ------ CONCEPTUSES ------ FETUSES BODY WEIGHT (G) TOTAL RESORBED ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # MALE FEMALE TOTAL MALE FEMALE TOTAL a N N % ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2176 5 7 12 35.94 35.90 35.91 12 0 0.0 2177 4 5 9 44.26 38.57 41.10 10 1 10.0 2178 2 5 7 45.91 42.33 43.35 7 0 0.0 2179 9 2 11 42.14 36.34 41.09 11 0 0.0 2180 3 4 7 51.46 46.39 48.56 7 0 0.0 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2181 5 6 11 39.48 35.56 37.34 13 2 15.4 2182 3 6 9 45.93 40.70 42.44 9 0 0.0 2183 4 4 8 44.41 41.73 43.07 8 0 0.0 2184 NOT PREGNANT 2185 5 5 10 40.68 37.94 39.31 10 0 0.0 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2186 3 3 6 45.79 48.02 46.90 6 0 0.0 2187 NOT PREGNANT 2188 5 3 8 42.83 47.34 44.52 8 0 0.0 2189 7 4 11 42.22 42.41 42.29 11 0 0.0 2190 2 0 2 52.66 ----- 52.66 2 0 0.0 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2191 NOT PREGNANT 2192 4 6 10 35.04 38.25 36.96 10 0 0.0 2193 5 2 7 50.31 47.87 49.61 7 0 0.0 2194 2 9 11 44.13 38.87 39.82 12 1 8.3 2195 3 3 6 40.26 37.87 39.06 6 0 0.0 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2196 0 8 8 ----- 43.80 43.80 9 1 11.1 2197 5 8 13 35.58 33.50 34.30 15 2 13.3 2198 4 7 11 39.95 34.64 36.57 11 0 0.0 2199 5 4 9 37.34 32.95 35.39 9 0 0.0 2200 3 5 8 40.66 37.98 38.98 8 0 0.0 ------------------------------------------------------------------------------------------------------------------------------------ a. Sum of fetal weights/Number of live fetuses.



Appendix 13 Fetal Sex, Vital Status, Body Weight and Gross External Alterations - Individual Data -

Part B


Final Report Page 160 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 13 (PAGE 1): FETAL SEX, VITAL STATUS, BODY WEIGHT AND GROSS EXTERNAL ALTERATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ FETUS # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 5 CONTROL ARTICLE 0 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # CLs 2176 5/ 8 FA MA MA FA MA / FA FA FA MA FA MA FA 40.32 39.78 38.41 36.65 35.67 34.42 38.90 33.64 35.50 30.03 30.32 37.33 2177 7/ 3 MA MA FA MA FA FA FA / FL MA FA 47.10 43.85 44.50 44.61 36.61 33.60 38.45 3.56 41.50 39.68 2178 4/ 4 MA FA FA FA / FA MA FA 45.06 39.98 38.06 41.50 48.74 46.76 43.38 2179 7/ 4 MA MA MA FA FA MA MA / MA MA MA MA 48.26 37.74 45.67 38.79 33.88 32.56 38.38 49.64 45.43 39.65 41.98 2180 2/ 5 MA FA / MA FA MA FA FA 51.77 47.14 50.72 49.60 51.88 44.89 43.94 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # CLs 2181 6/ 8 MA FA FA FA FL MA / MA FA ML MA MA FA FA 44.40 41.05 38.54 34.69 28.09 37.65 37.63 32.54 10.24 38.62 39.10 29.60 36.92 2182 4/ 5 FA MA MA FA / FA FA FA FA MA 48.61 49.33 46.02 45.36 50.86 13.90 42.63 42.83 42.44 2183 6/ 3 FA MA MA MA FA FA / FA MA 43.04 43.45 43.74 46.01 38.16 39.64 46.07 44.45 2184 NOT PREGNANT 2185 6/ 5 MA FA FA MA MA FA / MA FA FA MA 45.14 43.56 40.78 39.66 32.52 36.71 45.45 36.30 32.35 40.61 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # CLs 2186 4/ 4 FA MA MA / FA FA MA 48.93 47.48 47.73 49.17 45.97 42.15 2187 NOT PREGNANT 2188 4/ 5 FA MA FA / MA FA MA MA MA 52.72 44.90 39.10 49.69 50.19 41.25 40.57 37.74 2189 5/ 6 MA MA MA FA MA / FA FA MA FA MA MA 41.66 44.47 44.65 45.92 40.61 46.43 37.67 43.16 39.61 40.36 40.64 2190 3/ 2 MA MA / 52.86 52.46 ------------------------------------------------------------------------------------------------------------------------------------ M = MALE F = FEMALE A = ALIVE E = EARLY RESORPTION L = LATE RESORPTION (AUTOLYSIS PRECLUDED FURTHER EVALUATION) "/" DENOTES POSITION OF CERVIX CLs = CORPORA LUTEA/OVARY FETAL BODY WEIGHTS WERE RECORDED IN GRAMS (G). FETUSES APPEARED NORMAL AT GROSS EXTERNAL EXAMINATION UNLESS NOTED OTHERWISE.


Final Report Page 161 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 13 (PAGE 2): FETAL SEX, VITAL STATUS, BODY WEIGHT AND GROSS EXTERNAL ALTERATIONS - INDIVIDUAL DATA - PART B ------------------------------------------------------------------------------------------------------------------------------------ FETUS # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # CLs 2191 NOT PREGNANT 2192a 5/ 5 FA FA FA MA MA / FA FA FA MA MA 39.78 37.85 36.06 37.23 41.60 43.38 37.08 35.34 31.16 30.16 2193 5/ 2 MA FA MA MA MA / FA MA 50.32 46.82 53.45 46.93 49.63 48.92 51.23 2194 7/ 5 FA FA MA FA FA MA E / FA FA FA FA FA 42.49 44.88 44.57 36.39 36.34 43.69 40.54 38.54 41.46 32.29 36.88 2195 3/ 3 FA FA MA / MA MA FA 38.37 39.99 34.66 42.66 43.45 35.26 ------------------------------------------------------------------------------------------------------------------------------------ GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # CLs 2196 4/ 6 FA FA FA / FA FA E FA FA FA 44.72 44.45 45.80 45.46 41.73 40.98 38.56 48.72 2197 11/ 6 FA MA MA MA FA FA ML FA FA MA / E FA FA MA FA 43.93 36.17 35.47 35.19 31.18 26.02 21.25 29.00 23.15 32.67 37.01 42.39 38.39 35.28 2198 7/ 5 MA FA FA MA FA FA FA / MA FA FA MA 44.55 37.89 36.01 34.59 29.40 23.90 30.88 44.18 42.67 41.73 36.49 2199 4/ 6 MA FA MA FA / MA MA MA FA FA 42.88 33.01 32.58 36.60 40.76 33.81 36.66 29.67 32.53 2200 4/ 4 MA FA FA FA / MA FA MA FA 40.95 36.80 39.60 37.01 41.56 42.57 39.47 33.92 ------------------------------------------------------------------------------------------------------------------------------------ M = MALE F = FEMALE A = ALIVE E = EARLY RESORPTION L = LATE RESORPTION (AUTOLYSIS PRECLUDED FURTHER EVALUATION) "/" DENOTES POSITION OF CERVIX CLs = CORPORA LUTEA/OVARY FETAL BODY WEIGHTS WERE RECORDED IN GRAMS (G). FETUSES APPEARED NORMAL AT GROSS EXTERNAL EXAMINATION UNLESS NOTED OTHERWISE. a. Fetus 2192-10 had gastroschisis (portion of liver protruded through abdominal opening) and a medially cleft snout.



Appendix 14 Individual Data for Toxicokinetic Rabbits - Part B


Final Report Page 163 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 14 (PAGE 1): INDIVIDUAL DATA FOR TOXICOKINETIC RABBITS - PART B ------------------------------------------------------------------------------------------------------------------------------------ CLINICAL OBSERVATIONS: ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # DESCRIPTION ------------------------------------------------------------------------------------------------------------------------------------ SATELLITE GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2130 NO ADVERSE FINDINGS 2131 DG( 16 ) SCANT FECES 2132 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ SATELLITE GROUP 7 RGN-352 10 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2133 DG( 12- 16) INJECTION SITE(S): PURPLE 2134 NO ADVERSE FINDINGS 2135 DG( 16- 18) SCANT FECES ------------------------------------------------------------------------------------------------------------------------------------ SATELLITE GROUP 8 RGN-352 30 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2136 NO ADVERSE FINDINGS 2137 NO ADVERSE FINDINGS 2138 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ SATELLITE GROUP 9 RGN-352 90 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ 2139 NO ADVERSE FINDINGS 2140 NO ADVERSE FINDINGS 2141 NO ADVERSE FINDINGS ------------------------------------------------------------------------------------------------------------------------------------ CLINICAL OBSERVATIONS APPEARED NORMAL UNLESS NOTED OTHERWISE DG = DAY OF GESTATION


Final Report Page 164 Testing Facility Study No. 20024505 PROTOCOL 20024505: A DOSAGE RANGE-FINDING EMBRYO-FETAL DEVELOPMENT STUDY OF THYMOSIN BETA 4 (FORMULATED AS RGN-352) BY INTRAVENOUS INJECTION IN RABBITS, INCLUDING A PRELIMINARY EVALUATION IN NON-PREGNANT RABBITS APPENDIX 14 (PAGE 2): INDIVIDUAL DATA FOR TOXICOKINETIC RABBITS - PART B ------------------------------------------------------------------------------------------------------------------------------------ MATERNAL BODY WEIGHTS: ------------------------------------------------------------------------------------------------------------------------------------ RABBIT # SATELLITE GROUP 6 RGN-352 3 MG/KG/DAY ------------------------------------------------------------------------------------------------------------------------------------ PREGNANCY STATUS DAY 0 7 8 9 10 11 12 13 14 15 16 17 18 ------------------------------------------------------------------------------------------------------------------------------------ 2130 P 3.85 4.15 4.14 4.15 4.16 4.17 4.18 4.18 4.18 4.20 4.28 4.26 4.28 2131 P 3.74 3.79 3.85 3.87 3.85 3.94 3.95 3.96 4.02 4.03 3.98 4.04 4.05 2132 P 3.09 3.41 3.41 3.39 3.45 3.47 3.53 3.67 3.70 3.75 3.73 3.72 3.73 ------------------------------------------------------------------------------------------------------------------------------------ DAY 19 20 ------------------------------------------------------------------------------------------------------------------------------------ 2130 P 4.30 4.30 2131 P 4.06 4.02 2132 P 3.76 3.77 ------------------------------------------------------------------------------------------------------------------------------------ P = PREGNANT DAY = DAY OF GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (G), ROUNDED TO THREE SIGNIFICANT DIGITS AND REPORTED IN KILOGRAMS (KG). ALL CALCULATIONS EXCEPT BODY WEIGHT AVERAGES ARE PERFORMED WITH THE UNROUNDED GRAM (G) VALUE. BODY WEIGHT AVERAGES ARE CALCULATED WITH THE ROUNDED KILOGRAM (KG) VALUE.



Appendix 15 Bioanalysis Report


FINAL REPORT

Study Phase: Bioanalysis

Test Site Phase Reference No. 142476

Test Facility Study No. 20024505

Analysis of Thymosin β4 in Rabbit Plasma (K2 EDTA) Samples from the Study Titled “A Dosage Range-finding Embryo-fetal Development Study of

Thymosin Beta 4 (Formulated as RGN-352) by Intravenous Injection in Rabbits, Including a Preliminary Evaluation in Non-pregnant Rabbits” by

Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS)


Rockville, MD 20850 USA

TEST FACILITY: Charles River Laboratories


Horsham, PA 19044 USA

TEST SITE: Charles River Laboratories

Preclinical Services, Montreal (PCS-MTL) 22022 Transcanadienne

Senneville, QC H9X 3R3 Canada

Page 1 of 111


Final Bioanalysis Report Page 2 Test Facility Study No. 20024505 Test Site Phase Reference No. 142476

TABLE OF CONTENTS

1. LIST OF TABLE S ..................................................................................................................3

2. LIST OF FIGURES .................................................................................................................3

3. LIST OF APPENDICES .........................................................................................................3

4. GLOSSARY OF TERMS ........................................................................................................4

5. COMPLIANCE STATEMENT ..............................................................................................5

6. QUALITY ASSURANCE STATEMENT ..............................................................................6

7. RESPONSIBLE PERSONNEL ...............................................................................................7

8. INTRODUCTION ...................................................................................................................7

9. EXPERIMENTAL METHOD ................................................................................................7

10. REFERENCE STANDARD, INTERNAL STANDARD, MATRIX AND METHODS .......8 10.1. Reference Standard, Internal Standard and Matrix ........................................................8 10.1.1. Reference Standard ........................................................................................................8 10.1.2. Internal Standard ............................................................................................................8 10.1.3. Blank Matrix ..................................................................................................................8 10.1.4. Reference Standard Characterization .............................................................................9 10.1.5. Reference Standard and Internal Standard Inventory and Disposition and Matrix

Inventory ........................................................................................................................9 10.2. Methods..........................................................................................................................9 10.2.1. Study Samples ................................................................................................................9 10.2.2. System Suitability ..........................................................................................................9 10.2.3. Incurred Sample Reanalysis ...........................................................................................9



13. RETENTION OF RECORDS ...............................................................................................10

14. RESULTS AND DISCUSSION ............................................................................................11 14.1. System Suitability ........................................................................................................11 14.2. Analytical Batch ...........................................................................................................11 14.3. Calibration Curve Parameter ........................................................................................11 14.4. Calibration Standards ...................................................................................................11 14.5. Quality Control Samples ..............................................................................................11 14.6. Study Samples ..............................................................................................................12 14.7. Incurred Sample Reanalysis .........................................................................................12



1. LIST OF TABLES

Table 1 Analytical Batch Summary ....................................................................................14

Table 2 Calibration Curve Parameter Summary .................................................................15

Table 3 Calibration Standard Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma ..........................................................................16

Table 4 Quality Control Sample Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma ..........................................................................17

Table 5 Dilution Integrity Quality Control Sample Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma ...................................18

Table 6 Concentrations of Thymosin β4 in Rabbit Plasma Samples ..................................19

Table 7 Study Sample Re-assay History of Thymosin β4 in Rabbit Plasma ......................24

Table 8 Incurred Sample Re-analysis of Thymosin β4 in Rabbit Plasma ..........................29

2. LIST OF FIGURES

Figure 1 Representative Calibration Curve (Theoretical Concentration 186 to 4655 ng/mL) ...................................................31


Appendix 1 Analytical Procedure ..............................................................................................32

Appendix 2 Reference Standard and Internal Standard Characterization .................................54

Appendix 3 Chromatograms ......................................................................................................66



4. GLOSSARY OF TERMS

Below is a Glossary of Terms detailing the equivalent nomenclature as defined by Organisation for Economic Co-operation and Development (OECD) and/or Food and Drug Administration (FDA). Terms are interchangeable throughout the text.

OECD FDA

Test Facility Testing Facility

Quality Assurance Programme Quality Assurance Unit

Non-clinical Health and Environmental Safety Study

Nonclinical Laboratory Study

Study Plan Protocol


Final Bioanalysis Report Test Facility Study No. 20024505

5. COMPLIANCE STATEMENT

Page 5 Test Site Phase Reference No. 142476

The Bioanalysis phase of this study performed by Charles River Laboratories Preclinical Services, Montreal (PCS-MTL) was performed in accordance with the OECD Principles of Good Laboratory Practice as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA) and Japan (MHL W).

This phase of the study was conducted in accordance with the procedures described herein. All deviations authorized/acknowledged by the Study Director are documented in the Study Records. The report represents an accurate and complete record of the results obtained for this study phase.

There were no deviations from the above regulations that affected the overall integrity of this study phase or the interpretation of the phase results and conclusions.

d Date: '5/ ~I "'VlJ --~~~------------------------------------------~ I Rob IT Jenkins, PhD Pro cipal InvestIgator


Final Bioanalysis Report Test Facility Study No. 20024505

6. QUALITY ASSURANCE STATEMENT

Page 6 Test Site Phase Reference No. 142476

QUALITY ASSURANCE STATEMENT

Protocol: 20024505 This phase has been audited by the Quality Assurance Programme in accordance with the applicable Good Laboratory Practice regulations. Reports were submitted in accordance with standard operating procedures as follows:

Date(s) of Audit

27-Aug-2012

QA INSPECTION DATES

Dates Findings Submitted to: Principal Study

Principal Investigator Study Director Phase(s) Audited Investigator Management Director Management

Procedural Statement 22-Jan-2013 22-Jan-2013 22-Jan-2013 22-Jan-2013

27-Aug-2012 - 28-Aug-2012 Data Review - Bioanalysis & 29-Aug-2012 29-Aug-2012 25-Jan-2013 25-Jan-2013 Immunology

27-Aug-2012 - 28-Aug-2012 Draft Phase Report - 29-Aug-201229-Aug-2012 22-Jan-2013 22-Jan-2013 Bioanalytical

27-Aug-2012 Report Preparation 29-Aug-201229-Aug-2012 22-Jan-2013 22-Jan-2013

27-Aug-2012 Data Review - Technical 29-Aug-2012 29-Aug-2012 04-Sep-2012 04-Sep-2012 Operations

17-Jan-2013 Final Phase Report - 17 -Jan-2013 17 -Jan-2013 22-Jan-2013 22-Jan-2013 Bioanalytical

17-Jan-2013 Report Preparation 17-Jan-2013 17-Jan-2013 22-Jan-2013 22-Jan-2013

Process-based inspections relevant to this study were conducted according to a predetermined schedule. The outcome of each inspection was reported to Management and, where relevant for processes seen as part of a study, the Study Director.

Facilities relevant to this study are included in Charles River's annual facility inspection programme. The outcome of each inspection is reported to Management.

Khatibzadeh-Davani, Nooshin Quality Assurance Auditor

31 LaA rfMlJ ... --.. --------G~.:.-----------------Date



7. RESPONSIBLE PERSONNEL Principal Investigator Bioanalysis (13 Sep 2012 to report finalization)

Robert Jenkins, PhD Charles River Laboratories, PCS-MTL

Principal Investigator Bioanalysis (03 Aug 2012 to 12 Sep 2012)

Nathalie Proulx, BSc Charles River Laboratories, PCS-MTL

8. INTRODUCTION This report describes the bioanalytical evaluation of Thymosin β4 in Rabbit Plasma (K2 EDTA) samples from Study No. 20024505 entitled “A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a Preliminary Evaluation in Non-pregnant Rabbits”, by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

For the work detailed in this report, the bioanalytical phase start date was 07 Aug 2012, and the bioanalytical phase completion date was 24 Aug 2012.

The objective of this study was to provide information for selection of dosages to be used in a subsequent embryo-fetal development study in rabbits and to provide a preliminary evaluation of the effects of Thymosin β4 (formulated as RGN-352) on pregnancy and embryo-fetal development.

The objective of this phase of the study was to determine the concentration of Thymosin β4 in Rabbit Plasma (K2 EDTA) samples.

The study was sponsored by RegeneRx Biopharmaceuticals, Inc., Rockville, MD. Valerie A. Sharper, MS, Charles River Laboratories, Preclinical Services, Pennsylvania (PCS-PA), Horsham, PA, served as the Study Director.

9. EXPERIMENTAL METHOD Samples were received on dry ice from Study No. 20024505, and were stored in a freezer set to maintain -80°C for up to 64 days prior to analysis.

The method of analysis used is documented in PCS-MTL Analytical Procedure AP.142476.PL.02 (Appendix 1), and was previously validated under Study No. 142474.



10. REFERENCE STANDARD, INTERNAL STANDARD, MATRIX AND METHODS

10.1. Reference Standard, Internal Standard and Matrix

10.1.1. Reference Standard

Identification: Thymosin β4

Physical Description: White powder

Lot Nos.: FTHYB40602B

Peptide Purity: 98.0%

Peptide Purity 0.980 used during analysis

Correction factors: 0.9125 used for retrospective recalculations

Retest Dates: May 2013

Handling Precautions: Standard laboratory precautions (NIOSH approved mask, gloves, safety glasses, lab coat)

Storage Conditions: Kept in a freezer set to maintain -20°C

Supplier: Bachem Inc. (Transferred from Charles River LaboratoriesPreclinical Services, Massachusetts)

10.1.2. Internal Standard

Identification: Leu6-Thymosin β4

Batch/Lot No.: 0701-035

Purity: Assumed 100%

Storage Conditions: Kept in a freezer set to maintain -20°C

An expiry date for the internal standard was not assigned by the Sponsor, therefore the stability of the provided compound is unknown. As the internal standard is only used to ratio the reference standard peak area against it, the stability of this compound is considered not to be vital to the integrity of the study. In addition, all indications are that the compound was stable for the period of use, as the instrument response for the internal standard remained comparable throughout.

10.1.3. Blank Matrix

Type: Plasma (double charcoal stripped and non-stripped)

Species: Rabbit

Strain: New-Zealand White

Anticoagulant: K2 EDTA



10.1.4. Reference Standard Characterization

The Sponsor/Supplier provided the documentation for the identity, strength, purity, composition, and stability for the Reference Standard. Certificates of Analysis (Appendix 2) or equivalent documentation were provided for inclusion in the Final Report.

10.1.5. Reference Standard and Internal Standard Inventory and Disposition and Matrix Inventory

Records of the receipt, distribution, and storage of the Reference Standard, Internal Standard and rabbit plasma (K2 EDTA) were maintained. All unused Sponsor-supplied Reference Standard and Internal Standard were used on subsequent studies for the Sponsor. Empty reference standard and Internal Standard containers were discarded.

10.2. Methods

10.2.1. Study Samples

Samples above the ULOQ on initial analysis were diluted with blank double charcoal stripped rabbit plasma (K2 EDTA) prior to re-analysis.

Remaining unused study samples were discarded prior to issuance of Final Study Report.

10.2.2. System Suitability

The reproducibility of the chromatographic system was determined by injecting an extracted calibration standard, at least in triplicate, at the beginning of the chromatographic run. To assess system stability, QC samples were injected at the end of each run.

A coefficient of variation (CV) of ≤ 5% with respect to peak area ratio for an extracted calibration standard injected at the beginning of the run, and QC samples injected at the end of each run meeting acceptance criteria, were considered acceptable.

10.2.3. Incurred Sample Reanalysis

Incurred samples reanalysis was conducted to evaluate the overall performance of the bioanalytical assay with actual samples from dosed animals, and not to confirm individual results. A minimum of 30 study samples, or 5-10% (10% for studies where the number of samples is less than 1000 samples and 5% of the number of samples exceeding 1000 samples) of total sample numbers excluding control groups, were required to be reanalyzed for the assessment of incurred sample reanalysis, therefore a total number of 32 samples were selected for analysis. For the analytical method to be considered as acceptable for incurred samples reanalysis, 67% of the reanalyzed samples should have results within 20% utilizing the following calculation:

%100)2/)((

)( xresultoriginalresultrepeatresultoriginalresultrepeat

+−



11. COMPUTERIZED SYSTEMS

The following critical computerized systems were used by the Test Site in the generation of this report (Text Table 1).

Text Table 1 Computerized Systems

System Name Version Description of Data Collected and/or Analyzed Analyst (AB Sciex) 1.4.1 Data collection

Microsoft Excel 2003/2007 Descriptive statistics

Watson Laboratory Information Management system (Thermo) 7.2.0.02 Regression analysis and descriptive statistics

12. STATISTICAL ANALYSIS

Statistical analysis (regression analysis) was performed and descriptive statistics such as arithmetic means, standard deviations, accuracy and precision were calculated.

13. RETENTION OF RECORDS

All study-specific raw data, documentation and Final Report generated from this study phase will be archived at the Testing Facility. Study materials will be retained for a period of 1 year following issue of the audited Draft Report.



14. RESULTS AND DISCUSSION

Thymosin β4 is a naturally occurring peptide. It is found in high concentrations in blood platelets, wound fluid and other tissues in the body. Thymosin β4 is not a growth factor, rather, it is a major actin regulating peptide. Thymosin β4 has been found to play an important role in protection, regeneration and remodeling of injured or damaged tissues. The gene for Thymosin β4 has also been found to be one of the first to be upregulated after a wound occurs.

Calibration standards and quality control samples were prepared in double charcoal stripped matrix. Results presented in this report represent the total measured concentration, as it would be impossible to separate the endogenous levels of Thymosin β4 from the amount present in the samples.

During the experimental phase of the study, an incorrect correction factor, based upon the peptide purity, was used for preparation of the stock solutions throughout the study. As a result, the back-calculated concentrations for all the assessments were recalculated using the appropriate correction factors listed in Section 10.1.1.

Due to this error, the nominal concentrations for the calibration curves and QC samples vary throughout the study. However, all recalculated nominal concentrations are within 15% of the original nominal concentrations documented in the analytical procedure used during the experimental phase of the study. Therefore, this error is not considered to have any impact on the integrity of the study.

A representative calibration curve is presented in Figure 1, and representative chromatograms are presented in Appendix 3.

14.1. System Suitability

The CV for the extracted calibration standard injected at the beginning of the run was ≤ 5%, and QC samples injected at the end of the runs met acceptance criteria on all occasions. Acceptance criteria with respect to system suitability were met.

14.2. Analytical Batch

A summary of the analytical batches is presented in Table 1.

14.3. Calibration Curve Parameter

Calibration curve parameter summary is presented in Table 2.

14.4. Calibration Standards

Calibration standard results and statistics are presented in Table 3.

14.5. Quality Control Samples

Quality control sample results and statistics are presented in Table 4.

The dilution integrity quality control sample results and statistics are presented in Table 5.



14.6. Study Samples

All study samples were analyzed and results are presented in Table 6. The study sample re-assay history results are presented in Table 7.

14.7. Incurred Sample Reanalysis

Incurred Sample Reanalysis (ISR) was successfully conducted in this study to support the method for determination of Thymosin β4 in rabbit plasma (K2 EDTA) by LC-MS/MS. Four samples were assessed during ISR after four freeze-thaw cycles which was outside the validated freeze-thaw matrix stability of three cycles. This had no impact on the ISR assessment since 75% of the reanalyzed ISR samples, not including these four samples, were within 20% of their original result and ISR acceptance criteria were met. The ISR results are presented in Table 8.



TABLES



Table 1 Analytical Batch Summary

Watson Run ID

Date of Extraction Description Status Comments

01 07 Aug 2012 Blank matrix screen (non charcoal

stripped and double charcoal stripped)

Pass -

02 10 Aug 2012 Samples analysis – animals 2130 to 2133 Pass -



05 15 Aug 2012 Repeat analysis of samples

> ULOQ in Watson Runs ID #02 to 04 – Gestation Day 7

Pass -


> ULOQ in Watson Runs ID #02 to 04 – Gestation Day 19

Pass -


> ULOQ in Watson Runs ID #05 and 06 – Gestation Day 7 and 19

Pass -

08 23 Aug 2012 Incurred samples reanalysis Pass -



Table 2 Calibration Curve Parameter Summary

Watson Run ID Slope Intercept R-squared 01 0.000534 0.00397 0.9969 02 0.000631 -0.0108 0.9993 03 0.000633 -0.0108 0.9994 04 0.000592 0.00320 0.9988 05 0.000627 0.000211 0.9983 06 0.000577 -0.0105 0.9981 07 0.000651 -0.00759 0.9961 08 0.000610 0.0112 0.9934



Table 3 Calibration Standard Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma

Watson Run ID

Concentration (ng/mL)

186 326 466 700 930 1395 2795 4190 4655 01 184 326 451 765 742a 1419 2732 4015 4642 02 183 336 457 713 955 1374 2780 4135 4629 03 186 330 455 696 944 1438 2730 4209 4614 04 182 331 470 714 935 1433 2832 3965 4577 05 192 312 463 666 958 1436 2751 4231 4722 06 189 331 449 677 904 1410 2899 4400 4517 07 193 313 474 628 918 1410 2914 4319 4727 08 178 341 472 698 987 1369 3037 4051 4134

Mean 186 328 461 695 918 1411 2834 4166 4570 S.D. 5.06 10.2 9.81 40.1 75.5 26.9 109 152 189

% CV 2.7 3.1 2.1 5.8 8.2 1.9 3.8 3.6 4.1 % Bias 0.0 0.5 -1.0 -0.8 -1.3 1.2 1.4 -0.6 -1.8

n 8 8 8 8 8 8 8 8 8 a Outside of acceptance criteria; result not included in the regression but included in the statistical calculations.



Table 4 Quality Control Sample Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma

Watson Run ID

Concentration (ng/mL) 560 1860 3725

01 598 2025 3368 619 1607 4155 543 1926 3465 494 1959 3689

02 592 1922 3717 560 2012 3534 605 1981 3856 615 1925 3629

03 539 1841 3680 591 1966 3694 618 1845 3549 548 1820 2595a

04 556 1913 3694 575 1751 3626 617 1883 3731 608 1853 3648

05 551 1832 3673 551 1976 3887 588 1958 3848 560 1919 3813

06 527 1860 3598 542 1996 3992 607 2002 3891 559 1963 3942

07 500 1907 4045 521 1951 3738 578 1994 3887 565 1910 3764

08 530 1893 4148 601 1906 3718 599 1889 3852 569 2152 3645

Mean 570 1917 3721 S.D. 34.9 93.9 273

% CV 6.1 4.9 7.3 % Bias 1.7 3.1 -0.1

n 32 32 32 a Outside of acceptance criteria; result included in the statistical calculations.



Table 5 Dilution Integrity Quality Control Sample Results and Statistics of Thymosin β4 in Double Charcoal Stripped Rabbit Plasma

Watson Run ID

Concentration (ng/mL) 372400 1398000 1401000

05a 383775 - - 404387 - - 388403 - - 410131 - - 388239 - - 382344 - -

Intra-run Mean 392880 - - Intra-run S.D. 11537 - -

Intra-run % CV 2.9 - - Intra-run % Bias 5.5 - -

06a 376633 - - 393389 - - 409547 - - 402760 - - 398052 - - 400886 - -

Intra-run Mean 396878 - - Intra-run S.D. 11265 - -

Intra-run % CV 2.8 - - Intra-run % Bias 6.6 - -

07b - 1375252 - - 1363958 - - 1408484 - - 1378936 - - 1453365 - - 1372485 -

Intra-run Mean - 1392080 - Intra-run S.D. - 33632 -

Intra-run % CV - 2.4 - Intra-run % Bias - -0.423 -

08b - - 1326464 - - 1374231 - - 1297382 - - 1491591 - - 1383306 - - 1266184

Intra-run Mean - - 1356526 Intra-run S.D. - - 79795

Intra-run % CV - - 5.9 Intra-run % Bias - - -3.2 Inter-run Mean 394879 1392080 1356526 Inter-run S.D. 11070 33632 79795

Inter-run % CV 2.8 2.4 5.9 Inter-run % Bias 6.0 -0.424 -3.2

Inter-run n 12 6 6 a Dilution factor of 100 was used (10 µL of QC + 990 µL of double charcoal stripped blank matrix). b Dilution factor of 400 was used (5 µL of QC + 1995 µL of double charcoal stripped blank matrix).



Table 6 Concentrations of Thymosin β4 in Rabbit Plasma Samples

Animal ID Group Gestation

Day Time Point

(Hour) Concentration

(ng/mL) Dilution Factor

2130 6 7 0 < LLOQ 1 2130 6 7 0.0833 45120 20 2130 6 7 0.167 27065 10 2130 6 7 0.333 16363 10 2130 6 7 1 7707 10 2130 6 7 2 3225 1 2130 6 7 6 < LLOQ 1 2130 6 7 12 < LLOQ 1 2130 6 19 0 < LLOQ 1 2130 6 19 0.0833 39415 20 2130 6 19 0.167 20206 10 2130 6 19 0.333 11517 10 2130 6 19 1 4017 1 2130 6 19 2 1489 1 2130 6 19 6 < LLOQ 1 2130 6 19 12 < LLOQ 1 2131 6 7 0 < LLOQ 1 2131 6 7 0.0833 23148 10 2131 6 7 0.167 16883 10 2131 6 7 0.333 10438 10 2131 6 7 1 3687 1 2131 6 7 2 1157 1 2131 6 7 6 < LLOQ 1 2131 6 7 12 < LLOQ 1 2131 6 19 0 < LLOQ 1 2131 6 19 0.0833 26136 10 2131 6 19 0.167 14092 10 2131 6 19 0.333 8431 10 2131 6 19 1 2694 1 2131 6 19 2 792 1 2131 6 19 6 < LLOQ 1 2131 6 19 12 < LLOQ 1 2132 6 7 0 < LLOQ 1 2132 6 7 0.0833 43972 20 2132 6 7 0.167 25931 10 2132 6 7 0.333 16710 10 2132 6 7 1 5976 10 2132 6 7 2 1723 1 2132 6 7 6 < LLOQ 1 2132 6 7 12 < LLOQ 1 2132 6 19 0 < LLOQ 1 2132 6 19 0.0833 39339 20 2132 6 19 0.167 20928 10 2132 6 19 0.333 11349 10 2132 6 19 1 3961 1



Table 6 Concentrations of Thymosin β4 in Rabbit Plasma Samples (Cont’d)


Day Time Point



2132 6 19 2 1241 1 2132 6 19 6 < LLOQ 1 2132 6 19 12 < LLOQ 1 2133 7 7 0 < LLOQ 1 2133 7 7 0.0833 149517 50 2133 7 7 0.167 93221 50 2133 7 7 0.333 61216 20 2133 7 7 1 23584 10 2133 7 7 2 6917 10 2133 7 7 6 222 1 2133 7 7 12 < LLOQ 1 2133 7 19 0 < LLOQ 1 2133 7 19 0.0833 108065 50 2133 7 19 0.167 61063 20 2133 7 19 0.333 41255 20 2133 7 19 1 14213 10 2133 7 19 2 4054 1 2133 7 19 6 213 1 2133 7 19 12 < LLOQ 1 2134 7 7 0 < LLOQ 1 2134 7 7 0.0833 125126 100 2134 7 7 0.167 79401 50 2134 7 7 0.333 55261 50 2134 7 7 1 24586 10 2134 7 7 2 7749 10 2134 7 7 6 < LLOQ 1 2134 7 7 12 < LLOQ 1 2134 7 19 0 < LLOQ 1 2134 7 19 0.0833 155314 100 2134 7 19 0.167 88332 50 2134 7 19 0.333 59810 50 2134 7 19 1 21801 10 2134 7 19 2 6005 10 2134 7 19 6 < LLOQ 1 2134 7 19 12 < LLOQ 1 2135 7 7 0 < LLOQ 1 2135 7 7 0.0833 137185 100 2135 7 7 0.167 96215 50 2135 7 7 0.333 63573 50 2135 7 7 1 25352 10 2135 7 7 2 7573 10 2135 7 7 6 < LLOQ 1 2135 7 7 12 < LLOQ 1 2135 7 19 0 < LLOQ 1 2135 7 19 0.0833 147533 100





Day Time Point



2135 7 19 0.167 73663 50 2135 7 19 0.333 48706 50 2135 7 19 1 20892 10 2135 7 19 2 6900 10 2135 7 19 6 203 1 2135 7 19 12 < LLOQ 1 2136 8 7 0 < LLOQ 1 2136 8 7 0.0833 436440 100 2136 8 7 0.167 222374 100 2136 8 7 0.333 166414 100 2136 8 7 1 66191 50 2136 8 7 2 21406 10 2136 8 7 6 406 1 2136 8 7 12 < LLOQ 1 2136 8 19 0 < LLOQ 1 2136 8 19 0.0833 363306 100 2136 8 19 0.167 152212 100 2136 8 19 0.333 99707 50 2136 8 19 1 38763 20 2136 8 19 2 11152 10 2136 8 19 6 333 1 2136 8 19 12 < LLOQ 1 2137 8 7 0 < LLOQ 1 2137 8 7 0.0833 387519 100 2137 8 7 0.167 275538 100 2137 8 7 0.333 196156 100 2137 8 7 1 92379 50 2137 8 7 2 28000 10 2137 8 7 6 478 1 2137 8 7 12 < LLOQ 1 2137 8 19 0 < LLOQ 1 2137 8 19 0.0833 291151 100 2137 8 19 0.167 199613 100 2137 8 19 0.333 122973 100 2137 8 19 1 50232 20 2137 8 19 2 14255 10 2137 8 19 6 356 1 2137 8 19 12 < LLOQ 1 2138 8 7 0 < LLOQ 1 2138 8 7 0.0833 363620 100 2138 8 7 0.167 209416 100 2138 8 7 0.333 145936 100 2138 8 7 1 59836 20 2138 8 7 2 17948 10 2138 8 7 6 306 1





Day Time Point



2138 8 7 12 < LLOQ 1 2138 8 19 0 < LLOQ 1 2138 8 19 0.0833 343560 100 2138 8 19 0.167 220928 100 2138 8 19 0.333 115594 100 2138 8 19 1 42670 20 2138 8 19 2 14013 10 2138 8 19 6 498 1 2138 8 19 12 < LLOQ 1 2139 9 7 0 < LLOQ 1 2139 9 7 0.0833 1088433 400 2139 9 7 0.167 729859 200 2139 9 7 0.333 439478 100 2139 9 7 1 179153 100 2139 9 7 2 57773 20 2139 9 7 6 1014 1 2139 9 7 12 < LLOQ 1 2139 9 19 0 < LLOQ 1 2139 9 19 0.0833 878406 400 2139 9 19 0.167 605610 200 2139 9 19 0.333 305019 100 2139 9 19 1 114938 50 2139 9 19 2 37644 20 2139 9 19 6 759 1 2139 9 19 12 < LLOQ 1 2140 9 7 0 < LLOQ 1 2140 9 7 0.0833 960986 400 2140 9 7 0.167 768130 200 2140 9 7 0.333 426199 100 2140 9 7 1 212074 100 2140 9 7 2 80539 50 2140 9 7 6 1627 1 2140 9 7 12 < LLOQ 1 2140 9 19 0 < LLOQ 1 2140 9 19 0.0833 1270118 400 2140 9 19 0.167 573640 200 2140 9 19 0.333 394632 100 2140 9 19 1 162201 50 2140 9 19 2 56211 20 2140 9 19 6 1660 1 2140 9 19 12 < LLOQ 1 2141 9 7 0 < LLOQ 1





Day Time Point



2141 9 7 0.0833 1129465 400 2141 9 7 0.1667 743724 200 2141 9 7 0.333 446895 100 2141 9 7 1 201499 100 2141 9 7 2 48466 20 2141 9 7 6 728 1 2141 9 7 12 < LLOQ 1 2141 9 19 0 < LLOQ 1 2141 9 19 0.0833 1109972 400 2141 9 19 0.167 588943 200 2141 9 19 0.333 295163 100 2141 9 19 1 98574 50 2141 9 19 2 29998 20 2141 9 19 6 640 1 2141 9 19 12 < LLOQ 1

LLOQ Lower limit of quantitation (theoretical concentration 200 ng/mL). a Repeat result, refer to Table 7.



Table 7 Study Sample Re-assay History of Thymosin β4 in Rabbit Plasma

Animal ID

Gestation Day

Time Point (Hour)

Original Conc.

(ng/mL) Original Run No.

Reason for Re-assay

Re-assay Conc.

(ng/mL) Re-assay Run No.

Reported Conc.

(ng/mL)

Reason for Reported

Conc.

2130 7 1 > ULOQ 2 1 7707 5 7707 1 2130 7 0.0833 > ULOQ 2 1 45120 5 45120 1 2130 7 0.167 > ULOQ 2 1 27065 5 27065 1 2130 7 0.333 > ULOQ 2 1 16363 5 16363 1 2130 19 0.0833 > ULOQ 2 1 39415 6 39415 1 2130 19 0.167 > ULOQ 2 1 20206 6 20206 1 2130 19 0.333 > ULOQ 2 1 11517 6 11517 1 2131 7 0.0833 > ULOQ 2 1 23148 5 23148 1 2131 7 0.167 > ULOQ 2 1 16883 5 16883 1 2131 7 0.333 > ULOQ 2 1 10438 5 10438 1 2131 19 0.0833 > ULOQ 2 1 26136 6 26136 1 2131 19 0.167 > ULOQ 2 1 14092 6 14092 1 2131 19 0.333 > ULOQ 2 1 8431 6 8431 1 2132 7 1 > ULOQ 2 1 5976 5 5976 1 2132 7 0.0833 > ULOQ 2 1 43972 5 43972 1 2132 7 0.167 > ULOQ 2 1 25931 5 25931 1 2132 7 0.333 > ULOQ 2 1 16710 5 16710 1 2132 19 0.0833 > ULOQ 2 1 39339 6 39339 1 2132 19 0.167 > ULOQ 2 1 20928 6 20928 1 2132 19 0.333 > ULOQ 2 1 11349 6 11349 1 2133 7 1 > ULOQ 2 1 23584 5 23584 1 2133 7 2 > ULOQ 2 1 6917 5 6917 1 2133 7 0.0833 > ULOQ 2 1 149517 5 149517 1 2133 7 0.167 > ULOQ 2 1 93221 5 93221 1 2133 7 0.333 > ULOQ 2 1 61216 5 61216 1 2133 19 1 > ULOQ 2 1 14213 6 14213 1 2133 19 0.0833 > ULOQ 2 1 108065 6 108065 1 2133 19 0.167 > ULOQ 2 1 61063 6 61063 1 2133 19 0.333 > ULOQ 2 1 41255 6 41255 1



Table 7 Study Sample Re-assay History of Thymosin β4 in Rabbit Plasma (Cont'd)

Animal ID

Gestation Day

Time Point (Hour)

Original Conc.


Reason for Re-assay

Re-assay Conc.


Reported Conc.

(ng/mL)

Reason for Reported

Conc.

2134 7 1 > ULOQ 3 1 24586 5 24586 1 2134 7 2 > ULOQ 3 1 7749 5 7749 1 2134 7 0.0833 > ULOQ 3 1 125126 5 125126 1 2134 7 0.167 > ULOQ 3 1 79401 5 79401 1 2134 7 0.333 > ULOQ 3 1 55261 5 55261 1 2134 19 1 > ULOQ 3 1 21801 6 21801 1 2134 19 2 > ULOQ 3 1 6005 6 6005 1 2134 19 0.0833 > ULOQ 3 1 155314 6 155314 1 2134 19 0.167 > ULOQ 3 1 88332 6 88332 1 2134 19 0.333 > ULOQ 3 1 59810 6 59810 1 2135 7 1 > ULOQ 3 1 25352 5 25352 1 2135 7 2 > ULOQ 3 1 7573 5 7573 1 2135 7 0.0833 > ULOQ 3 1 137185 5 137185 1 2135 7 0.167 > ULOQ 3 1 96215 5 96215 1 2135 7 0.333 > ULOQ 3 1 63573 5 63573 1 2135 19 1 > ULOQ 3 1 20892 6 20892 1 2135 19 2 > ULOQ 3 1 6900 6 6900 1 2135 19 0.0833 > ULOQ 3 1 147533 6 147533 1 2135 19 0.167 > ULOQ 3 1 73663 6 73663 1 2135 19 0.333 > ULOQ 3 1 48706 6 48706 1 2136 7 1 > ULOQ 3 1 66191 5 66191 1 2136 7 2 > ULOQ 3 1 21406 5 21406 1 2136 7 0.0833 > ULOQ 3 1 436440 5 436440 1 2136 7 0.167 > ULOQ 3 1 222374 5 222374 1 2136 7 0.333 > ULOQ 3 1 166414 5 166414 1 2136 19 1 > ULOQ 3 1 38763 6 38763 1 2136 19 2 > ULOQ 3 1 11152 6 11152 1 2136 19 0.0833 > ULOQ 3 1 363306 6 363306 1 2136 19 0.167 > ULOQ 3 1 152212 6 152212 1 2136 19 0.333 > ULOQ 3 1 99707 6 99707 1




Animal ID

Gestation Day

Time Point (Hour)

Original Conc.


Reason for Re-assay

Re-assay Conc.


Reported Conc.

(ng/mL)

Reason for Reported

Conc.

2137 7 1 > ULOQ 3 1 92379 5 92379 1 2137 7 2 > ULOQ 3 1 28000 5 28000 1 2137 7 0.0833 > ULOQ 3 1 387519 5 387519 1 2137 7 0.167 > ULOQ 3 1 275538 5 275538 1 2137 7 0.333 > ULOQ 3 1 196156 5 196156 1 2137 19 1 > ULOQ 3 1 50232 6 50232 1 2137 19 2 > ULOQ 3 1 14255 6 14255 1 2137 19 0.0833 > ULOQ 3 1 291151 6 291151 1 2137 19 0.167 > ULOQ 3 1 199613 6 199613 1 2137 19 0.333 > ULOQ 3 1 122973 6 122973 1 2138 7 1 > ULOQ 4 1 59836 5 59836 1 2138 7 2 > ULOQ 4 1 17948 5 17948 1 2138 7 0.0833 > ULOQ 4 1 363620 5 363620 1 2138 7 0.167 > ULOQ 4 1 209416 5 209416 1 2138 7 0.333 > ULOQ 4 1 145936 5 145936 1 2138 19 1 > ULOQ 4 1 42670 6 42670 1 2138 19 2 > ULOQ 4 1 14013 6 14013 1 2138 19 0.0833 > ULOQ 4 1 343560 6 343560 1 2138 19 0.167 > ULOQ 4 1 220928 6 220928 1 2138 19 0.333 > ULOQ 4 1 115594 6 115594 1 2139 7 1 > ULOQ 4 1 179153 5 179153 1 2139 7 2 > ULOQ 4 1 57773 5 57773 1

2139 7 0.0833 > ULOQ 4 2 > 372400a, 1088433 5, 7 1088433 1

2139 7 0.167 > ULOQ 4 2 > 372400a, 729859 5, 7 729859 1

2139 7 0.333 > ULOQ 4 1 439478 5 439478 1 2139 19 1 > ULOQ 4 1 114938 6 114938 1 2139 19 2 > ULOQ 4 1 37644 6 37644 1




Animal ID

Gestation Day

Time Point (Hour)

Original Conc.


Reason for Re-assay

Re-assay Conc.


Reported Conc.

(ng/mL)

Reason for Reported

Conc.

2139 19 0.0833 > ULOQ 4 2 > 372400a, 878406 6, 7 878406 1

2139 19 0.167 > ULOQ 4 2 > 372400a, 605610 6, 7 605610 1

2139 19 0.333 > ULOQ 4 1 305019 6 305019 1 2140 7 1 > ULOQ 4 1 212074 5 212074 1 2140 7 2 > ULOQ 4 1 80539 5 80539 1

2140 7 0.0833 > ULOQ 4 2 > 372400a, 960986 5, 7 960986 1

2140 7 0.167 > ULOQ 4 2 > 372400a, 768130 5, 7 768130 1

2140 7 0.333 > ULOQ 4 1 426199 5 426199 1 2140 19 1 > ULOQ 4 1 162201 6 162201 1 2140 19 2 > ULOQ 4 1 56211 6 56211 1

2140 19 0.0833 > ULOQ 4 2 > 372400a, 1270118 6, 7 1270118 1

2140 19 0.167 > ULOQ 4 2 > 372400a, 573640 6, 7 573640 1

2140 19 0.333 > ULOQ 4 1 394632 6 394632 1 2141 7 1 > ULOQ 4 1 201499 5 201499 1 2141 7 2 > ULOQ 4 1 48466 5 48466 1

2141 7 0.0833 > ULOQ 4 2 > 372400a, 1129465 5, 7 1129465 1

2141 7 0.167 > ULOQ 4 2 > 372400a, 743724 5, 7 743724 1

2141 7 0.333 > ULOQ 4 1 446895 5 446895 1 2141 19 1 > ULOQ 4 1 98574 6 98574 1 2141 19 2 > ULOQ 4 1 29998 6 29998 1

2141 19 0.0833 > ULOQ 4 2 > 372400a, 1109972 6, 7 1109972 1




Animal ID

Gestation Day

Time Point (Hour)

Original Conc.

(ng/mL)

Original Run No.

Reason for Re-assay

Re-assay Conc.


Reported Conc.

(ng/mL)

Reason for Reported

Conc.

2141 19 0.167 > ULOQ 4 2 > 372400a, 588943 6, 7 588943 1

2141 19 0.333 > ULOQ 4 1 295163 6 295163 1 ULOQ Upper limit of quantitation (5000 ng/mL). a Sample analyzed with a dilution factor of 100. Reason For Re-assay: 1. Sample results > ULOQ when analyzed without dilution. 2. Initial sample results > ULOQ when analyzed without dilution; Repeat analysis with dilution still > ULOQ, therefore sample was re-assayed with a higher

dilution factor. Reason For Reported Conc.: 1. Re-assay results within the calibration curve range.



Table 8 Incurred Sample Re-analysis of Thymosin β4 in Rabbit Plasma

Animal ID Group

Gestation Day

Time Point (Hour)

Original Conc.

(ng/mL)

Reassay Conc.

(ng/mL)

Percent Difference

(%)a

2130 6 7 0.0833 45120 43163 -4.4 2130 6 7 0.333 16363 18643 13.0 2130 6 19 0.0833 39415 37318 -5.5 2130 6 19 0.333 11517 11629 1.0 2131 6 7 0.0833 23148 22347 -3.5 2131 6 7 0.333 10438 12861 20.8 2131 6 19 0.0833 26136 22708 -14.0 2131 6 19 0.333 8431 7880 -6.8 2133 7 7 0.0833 149517 117440 -24.0 2133 7 7 0.333 61216 61993 1.3 2133 7 19 0.0833 108065 95593 -12.2 2133 7 19 0.333 41255 36743 -11.6 2134 7 7 0.0833 125126 109080 -13.7 2134 7 7 0.333 55261 48580 -12.9 2134 7 19 0.0833 155314 170605 9.4 2134 7 19 0.333 59810 46947 -24.1 2136 8 7 0.0833 436440 381135 -13.5 2136 8 7 0.333 166414 163243 -1.9 2136 8 19 0.0833 363306 347030 -4.6 2136 8 19 0.333 99707 93181 -6.8 2137 8 7 0.0833 387519 359441 -7.5 2137 8 7 0.333 196156 142330 -31.8 2137 8 19 0.0833 291151 242332 -18.3 2137 8 19 0.333 122973 121622 -1.1 2139 9 7 0.0833 1088433b 948587 -13.7 2139 9 7 0.333 439478 417638 -5.1 2139 9 19 0.0833 878406 b 1017282 14.7 2139 9 19 0.333 305019 272796 -11.2 2140 9 7 0.0833 960986 b 831672 -14.4 2140 9 7 0.333 426199 410114 -3.8 2140 9 19 0.0833 1270118 b 1377634 8.1 2140 9 19 0.333 394632 390641 -1.0

a Calculation: % Difference = ((Re-assay result - original result)/((Re-assay result + original result)/2))*100. b Assayed outside validated freeze-thaw matrix stability of 3 cycles.



FIGURE



Figure 1 Representative Calibration Curve (Theoretical Concentration 186 to 4655 ng/mL)



Appendix 1 Analytical Procedure


ANALYTICAL PROCEDURE AP.142476.PL.02

PROCEDURE: Determination of Thymosin ~4 in rabbit plasma (New-Zealand White; K2 EDTA) by LC-MS/MS

RANGE: REFERENCE STANDARD: INTERNAL STANDARD:

200 to 5000 ng/mL Thymosin ~4 (Lot No. FTHYB40602B; Peptide purity: 98.0%) Leu6-Thymosin ~4 (Lot No. 0701-035; Correction factor 1.00)

All volumes and weighings in this procedure may be modified, as long as proportions and final concentrations remain the same.

The use of the term approximately for temperatures in the following sections and appendices, is used to stipulate that the target temperature, as stated, was achieved within the acceptable limits.

To optimize the response, the MS/MS instrument parameters may be changed. Such changes will be documented in the raw data

All other changes to the AP will also be noted, with their reason(s), in the raw data

Peptide Purity Correction factor: 0.980 used during experimental phase:

Correction factor: 0.9125 used for retrospective recalculations

Verified by: /~..?-k ~. Date: c2 J J ~ n..... t..-c. / J

Approved by: __ ~",'-+-____________ _

Authorized t ~ ~~

Page 1 of 7

Test Facility Study No. 20024505 Test Site Phase Reference No. 142476Final Bioanalysis Report Page 33



1. PREPARATION OF REAGENT SOLUTIONS

All mobile phase and autosampler components must be a minimum of LC-MS grade. For all other solutions, components should be LC-MS grade when available or a minimum of HPLC grade.

All glassware (graduated cylinder, container, volumetric pipette, etc) should be rinsed prior to use.

LC Mobile Phase A (5.00 mM Ammonium Acetate in Water:Formic Acid (99:1, vlv))

Dissolve 0.385 9 of ammonium acetate (MW = 77.08) in 1000 mL of water. Add 10 mL of formic acid. Store at room temperature. Expiration : 1 month.

LC Mobile Phase B (5.00 mM Ammonium Acetate in Acetonitrile:Water: Formic Acid (98:1:1 , v/vlv))

Dissolve 0.385 9 of ammonium acetate (MW = 77.08) in 10 mL of water. Add 1000 mL of acetonitrile Add 10 mL of formic acid. Store at room temperature. Expiration: 1 month.

Autosampler Wash (10.0 mM Ammonium Acetate in Water:Methanol:sodium hypochlorite (93:6: 1, v/v/v))

Dissolve 0.771 9 of ammonium acetate (MW = 77.08) in 930 mL of water. Add 60 mL of methanol Add 10 mL of sodium hypochlorite solution Store at room temperature. Expiration: 1 month.

Dilution Solution (Water:Acetonitrile:Formic Acid (94:5:1, v/vlv))

Add 1 0 mL of formic acid to 940 mL of water Add 50 mL of acetonitrile Store at room temperature.

Expiration: 1 month .

Buffer Solution (50.0 mM Ammonium Acetate in Water)

Dissolve 3.86 9 of ammonium acetate (MW = 77.08) in 1000 mL of water. Store at room temperature.

Expiration: 1 month.

Wash Solution (50.0 mM Ammonium Acetate:Methanol (95:5, v/v))

Add 475 mL of Buffer Solution to 25 mL of methanol Store at room temperature. Expiration: 1 month .

100 mM ammonium formate Solution Add 3.20g of ammonium formate (MW = 63.06) to 500 mL of water Store at room temperature. Expiration: 1 month.

Page 2 of 7




Elution Solution (Methanol:100 mM ammonium formate solution:Formic Acid (78:18:4, v/vlv))

Add 90 mL of 100mM ammonium formate solution to 390 mL of methanol Add 20 mL of formic acid Store at room temperature. Expiration: 1 month.

Reconstitution Solution (Water:Methanol:Formic Acid (89:9:2, v/v/v))

Add 445 mL of water to 45 mL of methanol. Add 10 mL of formic acid Store at room temperature. Expiration: 1 month

2. STANDARD AND QUALITY CONTROL STOCK SOLUTIONS PREPARATION

For weighing, refer to the microbalance (6 figures balance) procedure in the appropriate SOP.

2.1 Standard and Quality Control Stock Solutions of Thymosin 134 (STD/QC STK, theoretical concentration - 1.00 mg/mL)

Following the 6 figures balance procedure, weigh in duplicate, at least 3.100 mg of Thymosin-134 (recording to the nearest 0.001 mg). Transfer to a polypropylene tube and dissolve with the appropriate volume of water to obtain a concentration of 1.00 mg/mL using the formula below:

Water Volume (mL) = Amount weighed (mgl x correction factor concentration (mg/mL)

Vortex at approximately 21 DC until completely dissolved. Transfer to an appropriate polypropylene container and store at approximately -20 DC, dark. Expiration: 72 days

The stocks solutions should be vortexed for approximately 30 seconds before use (once they have reach room temperature if frozen solutions are used).

3. INTERNAL STANDARD STOCK SOLUTIONS PREPARATION

3.1 Internal Standard Stock Solution of Leu6-Thymosin 134 (IS STK, theoretical concentration - 1.00 mg/mL)

Following the 6 figures balance procedure, weigh in singlet, at least 1.00 mg of Leu6-Thymosin 134 (recording to the nearest 0.001 mg). Transfer to a polypropylene tube and dissolve with the appropriate volume water to obtain a concentration of 1.00 mg/mL using the formula below:

Water Volume (mL) = Amount weighed (mgl x correction factor concentration (mg/mL)

Vortex at approximately 21 DC until completely dissolved. Transfer to an appropriate polypropylene container and store at approximately -20 DC, dark. Expiration: N/AP, to be assessed in each individual run for potential interference via the STO A.

The stocks solutions should be vortexed for approximately 30 seconds before use (once they have reach room temperature if frozen solutions are used).

Page 3 of7




4. WORKING SOLUTIONS PREPARATION

The stocks and intermediate solutions should be vortexed for approximately 30 seconds before use (once they have reach room temperature if frozen solutions are used).

4.1 Standard and QC Working Solution

Follow the dilution scheme in the table below to prepare standard and quality control working solutions. Use an appropriate polypropylene screw cap tubes.

Note that if a stock solution check has been performed, then the same stock solution can be used to prepare STD/QC working solutions.

Store at approximately -20°C, dark. Expiration: 8 days

Table 1. Preparation of Standard and QC Working Solutions

Stock Solution Dilution Working Stock Solution Volume Solution Final Volume

Solution Ref. Ref. (J.lL) Volume (J.lL) (J.lL)

W-J STD STK 300 2700 3000

W-I STD STK 270 2730 3000

W-H STD STK 180 2820 3000

W-G STD STK 90 2910 3000

W-F STD STK 60 2940 3000

W-E STD STK 45 2955 3000

W-D STD STK 30 2970 3000

W-C STD STK 21 2979 3000

W-B STD STK 12 2988 3000

W-QCC QCSTK 240 2760 3000

W-QCB QCSTK 120 2880 3000

W-QCA QCSTK 36 2964 3000

4.2 Internal Standard Working Solution (ISWS, Theoretical Concentration - 8.00 Ilg/mL)

Theoretical Concentration

(ng/mL)

100000

90000

60000

30000

20000

15000

10000

7000

4000

80000

40000

12000

Pipette 400 flL of IS STK into a polypropylene tube containing 49.6 mL of Dilution Solution and mix well Expiration: N/AP, to be assessed in each individual run for potential interference via the STD A. approximately -20°C, dark

Page 4 of 7




5 BLANK, STANDARD AND QUALITY CONTROLS PREPARATION

Matrix: Double charcoal stripped rabbit plasma Strain: New-Zealand White Anticoagulant: K2 EDT A

Allow the plasma to thaw unassisted to room temperature. Vortex and then centrifuge the plasma prior to use at -1800 rpm/4°C/10 minutes, if required.

Follow the spiking scheme in table 2 below to prepare standard and quality control samples in an appropriate polypropylene container.

Expiration: To be prepared fresh on day of use

The working solutions should be vortexed for approximately 30 seconds before use (once they have reach room temperature if frozen solutions are used).

Table 2. Preparation of Standard and Quality Control Samples

W-S WSVolume Matrix Volume Theoretical STD/QC Ref. Concentration Ref. (Ill) (Ill) (ng/ml)

STDJ W-J 5 95 5000

STDI W-I 5 95 4500

STDH W-H 5 95 3000

STDG W-G 5 95 1500

STDF W-F 5 95 1000

STD E W-E 5 95 750

STD 0 W-D 5 95 500

STDC W-C 5 95 350

STD B W-B 5 95 200

STDA Dilution Solution 5 95 -DBLK Dilution Solution 5 95 -

QCC W-QCC 5 95 4000

QCB W-QC B 5 95 2000

QCA W-QCA 5 95 600

PREPARATION OF SYSTEM SUITABILITY TEST (SST)

Prepare additional sample in triplicate (spiked in double charcoal stripped rabbit plasma, equivalent to the STD C concentration) and pool final extracts in order to verify the system suitability and sensitivity.

Page 5 of7



ANALYTICAL PROCEDURE AP .142476.PL.02

Matrix: rabbit plasma (non-stripped) Strain: New-Zealand White Anticoagulant: K2 EDTA

Allow the plasma to thaw unassisted to room temperature. Vortex and then centrifuge the plasma prior to use at -1800 rpm/4°C/10 minutes, if required.

Follow the spiking scheme in table 3 below to prepare samples in an appropriate polypropylene container.

Expiration: To be prepared fresh on day of use

The stock solutions should be vortexed for approximately 30 seconds before use (once they have reach room temperature if frozen solutions are used).

Table 3. Preparation of NORM Blank and NORM Quality Control Samples

STK STKVolume Matrix Volume Theoretical STDIQC Ref. Concentration Ref. btl) btl) (ng/ml) NORM STDA Dilution Solution 5 95 -NORM DBLK Dilution Solution 5 95 -

DI QC* TBD TBD TBD TBD

*If 01 QC samples are to be extracted, refer to the Study Note for preparation instruction.

6 EXTRACTION PROCEDURES

Refer to Appendix 1.

7 INJECTION PARAMETERS

Refer to Appendix 2.

7.1 Typical Injection Sequence

Inject SST sample at least in triplicate to assess system suitability Calculate the % CV and ensure that the criteria have been met before further injections (%CV $ 5) Inject samples as per Watson sequence Store extracted samples in a refrigerator set to maintain 4°C after injection, dark

Page 6 of7




8 CALCULATIONS

Generate a standard curve using a linear regression weighted by 1/concentration2. (1 reported to 4 significant figures.

REASONS FOR UPDATE o N/Ap (if first version)

This procedure was copied from AP.142474.PL.02 with the following updates: Format updates made throughout.

• All reference to the QC LLOQ samples was removed for this procedure. • Page 1: Procedure clarified to include the use of non-stripped rat plasma. • Page 1: Reference standard and internal standard lot number and purity updated to reflect new lot

to be used. • Page 1 : clarification added for the use of the term approximate for temperatures.

Page 3, Section 2: microbalance procedure removed from AP, as the SOP is to be followed. • Page 3, Section 2.1 : expiration updated to reflect proven stability.

Page 4, Section 4.1: expiration updated to reflect proven stability, Table 1 final volume updated based on stock and dilution solutions volume used. Pages 5, Section 5: matrix clarified for the preparation of standard and quality control samples.

• Pages 6, Section 5: updated to include section for the preparation of NORM DBLK, NORM STD A and NORM QC samples in non-stripped matrix. Page 6, Section 5: matrix to be used clarified for SST preparation. Appendix 1: updated to differentiate between validation a'nd sample analysis procedures Appendix 1, page 1: Prompt added/clarified for blank matrix. Appendix 1, page 4: Timer I D prompt added to steps 8 and 11. Appendix 1, page 5: Vortex ID prompt added to steps 16. Appendix 2, page 1: Pressure entry for the Shimadzu pumps removed as the prompt is already on page 5.

Version 01 to 02: Page 1: Lot numbers of both reference standards added; peptide purity of both lots added and correction factor removed . Page 7: Section 8, clarification of (1 reported to 4 significant figures

Page 7 of 7




APPENDIX 1

Watson run 10: _____________ _ Study note #: __________ _

Entered by/Date: ____________ _

DESCRIPTION DETAILS ENTERED BY

Matrix:

Species:

Anticoagulant:

Blank matrix lot #*:

Blank matrix taken out Initial:

(Double charcoal stripped matrix) *If pool is listed, refer to appropriate Date:

pool matrix log form for individual lot

numbers.

Storage location:

o N/Ap

Matrix:

Species:

Anticoagulant:

Blank matrix lot #*:

Blank matrix taken out Initial:

(non-stripped matrix) *If pool is listed, refer to appropriate Date:

pool matrix log form for individual lot

numbers.

Storage location:

o N/Ap

Study/Ref. #

from Watson run #:

Prep. date: Initial: Working solutions taken out

Storage location: Date:

o N/Ap

Page 1 of 7





Study/Ref. # from Watson run #:

Prep. date: Initial: ISWS taken out

Storage location: Date:

o N/Ap (if prepared fresh)

Any comments and/or observations:

Page 2 of?




STEP DESCRIPTION DETAILS ENTERED BY # Vortex ID:

Allow blank matrix to thaw at room Initial: 1.

temperature and vortex to ensure mix. D N/Ap Date:

Centrifuge ID:

Centrifuge blank matrix (set at 4°C. Initial: 2.

1800 rpm for 10 minutes), if required. D N/Ap (if centrifugation is not Date:

required)

Vortex ID: Vortex working solutions for at least

30 seconds before use (once they D N/Ap (if frozen double blank Initial:

3. have reach room temperature if frozen Date:

solutions are used). (DBLK), single blank (STD A),

STD and QC samples are used)

Pipette ID for 5 jJL:

Pipette 10 for 95 jJL:

Solution used for DBLK and

STD A preparation:

Prepare double blank (DBLK), single Solution ID:

blank (STD A), standards and QC Study/Ref. #: Initial: 4.

samples into polypropylene tubes as Solution lot #: Date:

per table 2 of AP.

Vortex ID:

D N/Ap (if frozen double blank

(DBLK), single blank (STD A),

STD and QC samples are used)

Allow study samples to thaw D Step performed Initial:

5. unassisted at room temperature. Date:

Page 3 of 7




STEP DESCRIPTION DETAILS ENTERED BY # Solution 10:

Pipette 10:

Volume:

Prepare 01 QC samples as per study Pipette 10 for rabbit plasma note,

(non-stripped): 6,

Vortex to ensure mix, Volume of rabbit plasma (non-

stripped):

Vortex 10:

Initial:

o N/Ap Oate:

Pipette 10 for 5 iJL:

Pipette 10 for 95 iJL:

Solution 10: If 01 QC are required , prepare NORM

Study/Ref. #: 7,

OBLK and NORM STD A samples into Solution lot #:

polypropylene tubes as per table 3 of

AP, Vortex ID :

o N/Ap (if DI QC are not

required)

Vortex to ensure mix, Blank double charcoal stripped

rabbit plasma lot #: For study samples, aliquot 100 iJL of

study sample or apply the appropriate o N/Ap (if no dilution applied) dilution factor as per Study Note, Initial:

8, Pipette ID: Date:

If applicable, for DI QC, apply the Vortex ID: appropriate dilution factor as per Study

Note, o N/Ap (if no study samplesl

DI QC are extracted)

Page 4 of 7




STEP DESCRIPTION DETAILS ENTERED BY # Pipette 10:

Solution 10: Add 20 ~L of Dilution Solution to OBLK Initial:

9. Study/Ref. #: and NORM OBLK sample(s). Date:

Solution lot #:

Pipette 10: Add 20 iJL of ISWS to all STO, QC,

Study/Ref. # Initial: 10. SST, NORM STO A, NORM 01 QC

Watson run Id#: Date: and study samples.

Prepared on:

Pipette 10:

Add 1 mL of Buffer Solution to all Solution 10: Initial: 11 .

tubes. Study/Ref. #: Date:

Solution lot #:

Vortex all samples on the multi-tube Vortex 10: Initial: 12.

vortexer for 5 minutes. Timer 10: Date:

Pipette 10:

Condition the 96-well extraction plate Solution 10:

(Waters Accell Plus QMA 100 mg) Supplier: Initial : 13.

using 1 mL of methanol, using gentle Solution lot #: Date:

vacuum. 96-Wells Extraction Plate Lot #:

Condition the 96-well extraction plate Pipette 10:

(Waters Accell Plus QMA 100 mg) Solution 10: Initial: 14.

using 1 mL of Buffer Solution , using Study/Ref. #: Date:

gentle vacuum. Solution lot #:

Transfer the sample to the 96-well

extraction plate and allow to stand for Pipette 10: Initial: 15.

5 minutes before applying gentle Timer 10: Date:

vacuum.

Wash the 96-well extraction plate Pipette 10:

(Waters Accell Plus QMA 100 mg) Solution 10: Initial: 16.

using 1 mL of Wash Solution, using Study/Ref. #: Date:

gentle vacuum. Solution lot #:

Page 5 of 7




STEP DESCRIPTION DETAILS ENTERED BY # Wash the 96-well extraction plate Pipette ID:

(Waters Accell Plus QMA 100 mg) Solution ID: Initial: 17.

using 1 ml of MeOH, using gentle Supplier: Date:

vacuum. Solution lot #:

Pipette ID:

Elute using 600 iJl of Elution Solution , Solution ID: Initial : 18.

using gentle vacuum. Study/Ref. #: Date:

Solution lot #:

Using the previously cleaned Evaporator ID:

evaporator, evaporate sample at 0 Initial:

Temperature setting -40°C, for both the upper and lower 0

Date: Nitrogen flow set

19. portion, under the gentle stream of

nitrogen. Nitrogen flow should be set at

50 Umin for the top manifold and Approximate duration: Initial:

25 Umin for the bottom manifold. Timer ID: Date:

Evaporate to dryness.

Pipette ID:

Reconstitute the samples using 100 iJl Solution ID: Initial:

20. of Reconstitution Solution, vortex the Study/Ref. #: Date:

plate gently to mix. Solution lot #:

Vortexer ID:

Storage location: Store plate at -4°C at the end of Initial:

21. Plate stored at (time): extraction. Date:

Clock ID:

Page 6 of 7





Storage location : Initial:

Blank matrix stored back after use Date: o N/Ap

Storage location: Initial :

Working solutions stored back after use Date: o N/Ap (if discarded after use)

Storage location : Initial:

ISWS stored back after use Date: o N/Ap (if discarded after use)


Reviewed by/Date: __________ _

Approved bylDate: __________ _

Page 7 of 7




APPENDIX 2

Watson run 10: _____________ _ Study note #: __________ _


Refer to Study Note # ___ _ for injection/acquisition parameters D N/AP

Plate(s)Nial(s) (Watson run Id# ______ _ transferred in autosampler Id _______ _

) were removed from previous location and to be injected.


Svstem Confiauration

Sciex API 4000 Mass Spectrometer Alias # AI-

Two Shimadzu LC-10 ADvp Pumps Al- AI-

Shimadzu SIL-HTC Autosampler AI-

PEE~ Tubing throughout system, with 10/00 0.005" /1/16" (red) Post-Injector D

Entered by/Date:

Verified by/Date:

LC Conditions

Column 10: Jupiter C18 Analytical Column' Dimension: 50 X 4.6 mm; 5 ~m; 300A C-

Supplier: Phenomenex

Pre-filter Titanium Pre-Column 0.5 flm Filter D Column Heater 10#

Column Set at 35°C D Temperature

Entered by/Date:

Verified by/Date: .. • Prior to first use, the HPLC column should be conditioned as per manufacturer instructions

Page 1 of 7




Solutions

Study/Ref. #:

LC Mobile Phase A 5.00 mM Ammonium Acetate in Solution lot #: Water: Formic Acid (99: 1, v/v)

Exp. Date:

5.00 mM Ammonium Acetate in Study/Ref. #:

LC Mobile Phase B Acetonitrile:Water: Formic Acid Solution lot #: (98: 1: 1, v/v/v) Exp. Date:

Study/Ref. #: Autosampler Wash 10.0 mM Ammonium Acetate in

Water: Methanol : sodium Solution lot #: Solution hypochlorite (93:6: 1, v/v/v) Exp. Date:

Entered by/Date:

Verified by/Date:

Page 2 of7




Monitoring ions and respective parameters

Compound Name Thymosin /34 Leu6-Thymosin /34

01 Mass (amu) 828.3 825.3

03 Mass (amu) 129.2 129.2

Retention time (min) 4.3 4.4

Dwell time (msec) 200 200

Declustering Potential (V) 45 60

Entrance Potential (V) 10 10

Collision Energy (eV) 90 90

Collision Exit Potential (V) 9 9

Verified by/Date:

MS Conditions Polarity Positive

Ion Source Electrospray (ESI)

Scan Type Multiple Reaction Monitoring (MRM)

Resolution 01 Unit

Resolution 03 Unit

MR Pause (msec) Set at 5.007

Curtain Gas Flow (N2) Set at 25psi

CAD Gas (N2) Set at 8 dacs

Ion Spray Voltage Set at 5500 V

Source Temperature Set at 550°C

Ion Source Gas 1 (GS1) Set at 70 psi

Ion Source Gas 2 (GS2) Set at 70 psi

Probe Position Set at horizontal = 5 ; vertical = 7.5

Acquisition Delay o seconds

Acquisition Period 7.2 minutes

Interface heater On

Verified by/Date:

Page 3 of 7




Shimadzu Time Program

Time Module Event Parameter (min) 0.01 Pumps %8 5 0.20 Pumps %8 5 1.00 Pumps %8 10 1.20 Pumps %8 10 1.50 Autosampler Rinse -4.80 Pumps %8 35 5.00 Pumps %8 50 5.20 Autosampler Rinse -5.80 Pumps %8 10 6.00 Pumps %8 10 6.20 Pumps %8 5 7.20 System Controller Stop -

EnteredNerified by/Date:

Binary Gradient Total Flow: 0.50 mL/min D

Entered by/Date:

Shimadzu Sil-HTC Autosampler Properties

Injection Volume (Ill) Set at 5 III

Rinse Volume (J.ll) Set at 400 III

Needle Stroke (mm) Set at 49 mm

Rinse Speed (ilL/sec) 35 [JL/seconds

Sampling Speed (ilL/sec) 5 ilL/seconds Purge Time (min) 1 min

Rinse Dip Time (sec) 5 seconds

Rinse Mode Before and After Aspiration

Cooler Enabled Yes Cooler Temperature (OC) Set at 4·C

Verified by/Date:

Page 4 of7




System suitability

Sequence submitted back-to-back with Watson run Id# __ from Study/Ref # 0 Entered by/Date: N/Ap

Shimadzu LC-10ADvp pumps

Pressure: Bar/Psi

Inject SST at least in triplicate to assess system suitability

Data file name:

Chromatography suitable Yes 0 NoD

Retention times suitable Yes 0 NoD

Instrument response suitable Yes 0 NoD

If one or more of the above parameters is not acceptable, specify what action was taken to solve

the issue:

0 Entered by/Date:

N/Ap

Calculate the % CV and ensure that the criteria have been met before further injections

(%CV,,; 5)

System suitability acceptable Yes 0 NoD

If no, specify what action was taken to solve the issue:

Entered by/Date:

Processed Data saved under filename:

SST Data processed bylDate:

Page 5 of 7




Watson Batch

I D,ta file "me

If problems were encountered before the samples injections are completed and system suitability was to

be performed complete the following' 0 N/Ap Entered by/Date'

Problem encountered:

Shimadzu LC-10ADvp pumps

Pressure: Bar/Psi

I nject SST at least in triplicate to assess system suitability

Data file name:

Chromatography suitable YesD NoD

Retention times suitable Yes 0 NoD

Instrument response suitable Yes 0 NoD

If one or more of the above parameters is not acceptable, specify what action was taken to solve the

issue:

Entered bylDate:

Calculate the % CV and ensure that the criteria have been met before further injections (%CV " 5).

System suitability acceptable Yes 0 NoD

The difference between the two system suitabilities should be within 10%. (not required if batch is

restarted from the beginning)

Difference acceptable Yes 0 NoD N/ApD

If no, specify what action was taken to solve the issue:

Entered by/Date:

SST Data processed bylDate:

Processed Data saved under filename:

Page 6 of 7




Watson Batch (for Re-injection) o N/Ap Entered by/Date: ________ _

I D,ta" "me

Following injection, plate(s) was removed from autosampler and transferred in the following location: ________________ _

o (N/Ap, if stored at room temperature)

Entered by/Date:

Processed Data saved under filename: _______ _

Baseline processed by/Date: ___________ _


Reviewed by/Date: __________ _

Approved by/Date: __________ _ Page? of?



Test Facility Study No. 20024505 Test Site Phase Reference No. 142476

Appendix 2 Reference Standard and Internal Standard Characterization

Final Bioanalysis Report Page 54


/SACHEn7/ Certificate of Analysis

RETEST DATE EXTENSION

PRODUCT: Thymosin ~4 Reference Standard (25 mg net peptide vials)

LOT NUMBER: FTHYB40602B

NEW RETEST DATE: May 2012

PEPTIDE PURITY: 98.2 % (See attached HPLC trace)

PURITY RETESTED: May2011

QUALIT

Retest date extensions apply only to bulk dry powder materials stored at recommended s~orage conditions. Extensions are based upon results from tests perfonned on materials stored at Sachem. Inc. under conditions specifically established for in-house stability studies.

BACHEM. Inc. 3132 Kashiwa Street Torrance, CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telefax (310) 530-2426



Bachem QC Report TMHPLC.T1iYB4

"'." , ...... Ph ...

214 ""'. P .... , <> ~

PeakRHultt

c ..... pOLndHmM " "'IalInRT ......... _. PIIt.auabuUJP

NlA 60400 rTin . 0.283 0.3109 0.003% 94804.000 NJA 12.250 min 0.542 31.2642 0.323 % 2QO.12.ooo

NlA 12.700 mn 0.552 02_ 0.003% 23386.000 NJA 13.750 mln 0.'" 5.8426 0.060 % 22357.000 NlA 15.887 min 0.70'2 "'77 0.010% 32296.000 NlA 17.~7m1n o.m 1.<1338 0.015% 17440.000 NlA t9,060 min 0.843 4.8387 0.050 " NIA NlA 19.317 frin 0.855 16.2387 0.151% 5i48.000

NJA 20.598 min 0.911 5.0385 0.052% 1858.000

~IIIIII' .... " IWIIII¥IAT .... 11 ...... 1 MO' PIale.,.l>trUSP

NJA 21.020 rnn 0..,. 12.4069 0.128% 7685.000 NtA 21.788 min 0.063 .. .6026 0 .... % 8349.000

THY-84 22.6Il0mn 1.000 9515.1SM 98.1£18% 7279.000 NlA 2<4.606 min 1.089 7.11163 D.rm", NlA NJA 25.787 min 1.140 19.4839 0.201 % 6666.000 NJA 26.563 min 1.175 1.2275 0.075% 2701 .000 NIA 28A67mn " .. 0._ 0.010% 19924.000 NJA 29.651 min 1.321 236,. 0.024 % 8958.000 NJA 30.867 min 1.3116 1.3683 0.014% 11743.000 NJA 32.400 mn 1.434 2.8514 0.027 % 13885.000 NlA 39.483 min 1.747 1.3288 0.014 " 30894.000 NtA 45.060m~ 1 .... 21035 0.022% 91~.OOO

NlA 411.Bl1 mIn 2.012 02820 0.034% 51n4.ooo NJA 47.550 rnn 2104 1._ 0.Ot8% NlA NJA 49.317 min 2.t82 .8852 0.030 % 18&l3.000

8~ ·'I1I~h Sample Name: FTHY840601,,(O.2O"C. 48 month. ~ectCn: 1 of 1 Print Data:05l2Sl2Oll : 12;42:67

.... 'IiIOIuf ... UIP

NlA 19.212 1.334 3.015 5.871 3.841

NlA NlA

0 ...

Pu"'~aI>LIIP

0.204 0.780 0.829

NlA NJA

0.487

1.335 1.338

0.848 1.385 7.OS1 7.437 2.S7O

NlA NJA

• "'tTHYll4·

,,1"'0'_ 1.094 1.166 0 .... 0,862 1.017 1.532 0.500 1.455 0.602

AO OS/7CJ.'

Page 1 of4

T"~r_

0.951

0.792

1.786 1620.887

0.927 1.183

1.608 0 .... 0.878 1.067 0 .... 0.789 0.887

17.814 1.1>33

~ O"'ICJn

Page-~ Cf4



Bachem QC Report TMHPLC.THYB4

Full Seale Cfv'omltogram

_8_~1"'" SMIp1e Namlt: FTHVB4080~2O'C. 48 months In~on; 10f I PtlntDall:05l2.5l2011 : lz.:.4l'.57

AD 05(/$/11

Page 3 of4



'I SAC HE n1 7 OVER 30 YEARS OF COMMITMENT TO THE SCIENTIFIC COMMUNITY

CERTIFICATE OF CONFORMIIT

I hereby certify that the production, testing, and the documentation thereof,

of Lot

FTHYB40602B

conform with the requirements for Current Good Manufacturing Practice as specified in the International Conference on Harmo nisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)

Q7A Good Manufacturing Practice Guidance for

Active Pharmaceutical Ingredients

Date: fv/avd-J 81 J 2001-

Jackie Little Quality Assurance Department

BACH EM, Inc. 3132 Kashiwa Street Torrance, CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telelax (310) 530-2426



'/ SACHEm 7 OVER" YEARS OF COMMITMENT TO THE SCIENTIFIC COMMUNITY

RAW MATERIAL SOURCE STATEMENT

Product: Thymosin P4

Product Code: FBC0098

Lot Number: FTHYB40602B

Thymosin P4 lot FTHYB40602B manufactured by Bachem California is a synthetic peptide manufactured using synthetic amino acid derivatives, as well as solvents and reagents of non-human and non-animal sources. Supplies of synthetic amino acid derivatives are also manufactured using amino acids of non-human and non-animal sources.

BACHEM, Inc.

Jackie Little Senior Director Quality Assurance / Regulatory Affairs

3132 Kashiwa Street Torrance, CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telefax (310) 530-2426

Date: Mavch 3/) 200'1-



/SRCHEn7/ Certificate of Analysis

RETEST DATE EXTENSION



NEW RETEST DATE: May 2011

PEPTIDE PURITY: 98.0 % (See attached HPLC trace)

PURITY RETESTED: May 2010

DATE

'DATE

Retest date extensions apply only to bulk dry powder materials stored at recommended storage conditions. Ex.tensions are based upon results from tests perfonned on materials stored at Sachem, Inc, under conditions specifically established for in·house stability studies.

BACH EM, Inc. 3132 Kashiwa Street Torrance, CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telefax (310) 530-2426



Bachem QC Report TMHPLC.THYB4

Samp/eNam&: mlYB40602A@-2t)OC.36 monlha Acqdsltlon Date:

Sample Conc(mg/mL}: FTHYS40602B@-2O'lC,36monthS(2.171rng/ 21711JLH20) PrfntDate:

Samp/eType: s.m¢. Method:

Row (mlJrnln): 0.600 Sequ~:

Vtalno.: 3 MobilePheseA:. Injection 10f 1 Mobile Phase B:

Injection Volume (ul) 10.0 Mobile Phase C:

Wav&lenglh (nm): 214 Mobile Phase D:

Autosarnpler Tray Temp (C): 8 Column NOrM Run llrne (min); 72.000 Corumn Temp (0)

Instrument Name: Waters01841 Software

""''''' NavindraDeoram Version Lab Book Reference N[).7·133 Gta!flent:

Peak Results

Comp«lndN;uno "' RtI'I'IIv&RI Pu1cArea Area,!. PI~~ nuMbtr USP

NlA 2.550 min 0.108 0.2764 0.003 % 3239.000

NlA 6.550mln 0.278 0.3419 0.003% 13346.000 NlA 12.4SOm!n 0.'" 35.2085 0.347% 20715.000

NlA 13.967 min 0.593 7.1296 0.070% 21740.000 NlA 15.617 mln 0.664 0.2198 0.002% 25430.000 NlA 16.283m!n 0.692 2.1430 0.027% 11759.000 NlA 1S.176m/n 0.772 1._ 0.013% 14070.000

NJA 18.532mln 0.787 0.4582 0,005% NlA

NlA 19.0CIQ min 0 .... 0.3775 0.004% 22041.000

CompOUfldNama "' R'lItUveRT PnkArta Area % 1'1111 numbtr USP

NlA 19.767 min 0.840 20.4754 0.202 % 10865.000 NlA 20.301 min "'63 2.7807 0.027% 695.000 NlA 21.296 min 0.905 9.8560 0.097% 6431.000 WA 21.698m!n 0.930 14.6902 0.145% 4668.000 NlA 22.606 mIn 0.961 65.0076 0.542% 7675.000

ThymoslnB-4 23.533 min 1.000 9950.6515 97.992% 7494.000

NlA 25.625 mIn 1.000 7.6160 0.Q75% NlA NlA 26.931 min 1.144 34.3706 0.336 % 3450.000 NlA 27.783 min 1.181 7.2471 0.071 % 132.000 NJA 45.867 min 1.949 1.9949 0.020% 131040.000

NJA 47,350m!n 2.012. 1.2993 0.013% 126248.000

NJA 47.579mkl 2.022 0.3915 0.004% NlA

Sample Name: [email protected] Injection: 1 of 1 Pllnt Dale:O$lU1I20fO; 11:34;39

05/19}2010: 00:19:14

0511912010: 11:34:39

mHPlC.THY84 (01841) NS'N Stab_FTH840602B_051810_01841 ~

Reprocessed HPlC H20lotT-35338 Acetonitrilb LoIT-35201 Melhanollot T--35349 500mM Ammonium Phosphate lol THYBA· 021 ""~J, THY ... Putosphere Stat RP18e} 60

eerily NOS for PilannaceU1fea1 ONQC _SIJ,.02.03.572 Refe( to TMHPLC.THYB4

P<!u .... o:vtlOI'lUSl>

NlA 19.705

20.601 4.184 4.282

1.344 3.119

NlA NlA

Ptol.c-soMlon USP

1.198 0.279 0.481

0.510 0.614

0.875 NlA NlA

0.146 3.555

2.853

NlA

T;lI!Itog:F_

1.266

1.364 1. ... 0.822 0.978

1.493 OS ..

1.224

0.722

~/,q/lfl Page 1 of4

Tllr;"gf.ct<>r

0,916 2.148 0.740

0.834 0.820

1.74S 1528.501

0.981 5.096

0,842

0"'" 441.261

1Jlf~IIO Page2of4



Sachem QC Report TMHPlC.THYB4

Full Scale Chromatogram

Samp!e Name: FTHYB40602A@-2O"C.36months lnje<:oon: 1 of 1 Print Da!e:05l1912010: 11:34:39

~/lqltO Page 3 of4



/SRCHEn7/ Certificate of Analysis Page 1 of2


LOT NUMBER: FTHYB40602B RETEST DATE: May 2008 (May be extended on the basis of stability data.)

PRODUCT NUMBER: CBC0094 DATE OF VIALING: May 2007

MOLECULAR WEIGHT: 4963.55

STRUCTURE: Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH .

TEST

APPEARANCE:

MASS SPECTRAL ANALYSIS:

SPECIFIC OPTICAL ROTATION: [~lD-"(c = 0.1, water)

PEPTIDE PURlTY BY RP-HPLC:

AMINO ACID ANALYSIS:

BACHEM, Inc. 3132 Kashiwa Street Torrance. CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telefax (310) 530-2426

SPECIFICATION

White to off-white powder; no visible extraneous materials

4963.6 ± 2.0 Da (average mass)

RepOlt (corrected for anhydrous, acetic acid-fl'ee substance)

:2: 97%; no individual related substance;?: 1.0%

Ala: 1.8 to 2.2 Asx: 3.6t04.4 Glx: 9.9 to 12.1 Gly: 0.9 to 1.1 IIe: 1.8 to 2.2 Leu: 1.8 to 2.2 Lys: 8.1 to 9.9 Met: 0.9 to 1.1 Phe: 0.9to 1.1 Pro: 2.7 to 3.3 Ser: 2.8 to 4.4 11R. MiS 9.9

RESULTS

White powder; no visible extraneous materials

4963.3 Da

TMHPLC.THYB4 (System II): 98.1 % Largest impurity: 0.40 % (RRT 0.56)

Ala: 1.9 Asx: 4.0 Glx: 11.2 Gly: 0.9 IIe: 1.9 Leu: 2.0 Lys: 9.1 Met: 1.0 Phe: 1.0 Pro: 3.1 Ser: 3.5 ;P' 22



·/SRCHEm!

Certificate of Analysis


ADDITIONAL TESTS

SEQUENCING: (MS-MS)

CIDRAL AMINQ ACID ANALYSIS:

Ref. Spec: FTHYB41v03 and Protocol TP07-010

BACHEM, Inc. 3132 Kashiwa Street Torrance, CA 90505 Telephone (310) 517-1858 Toll Free (888) 422-2436 Telefax (310) 530-2426

Page 2 of2

SPECIFICATION RESULTS

Amino acid sequence confinned . Sequence confitmed

Report Alanine 0.12% D-enantiomer Threonine > 99.70% L-Threonine

<0.10% D-Threonine <0.10% L-allo Threonine <0.10% D-allo Threonine

Isoleucine > 99.70% L-Isoleucine <0.10% D-Isoleucine <0;10% L-allo Isoleucine <0.10% D-a116' Isoleucine

Proline <0.10% D-enantiomer Leucine 0.44% D-enantiomer Serine 0.97% D-enantiomer Aspartic Acid 0.26% D-enantiomer Methionine 0.23% D-enantiomer Phenylalanine 0.10% D-enantiomer Glutamic Acid 0.36% D-enantiomer Lysine 0.16% D-enantiomer

I DATE



Retest Date Extension Certificate Lot number

Product

Product number

Molecular formula (net)

Molecular mass (average)

Date of vialing

Date of relest

New relest date

Specification

Storage condition

Tests

Appearance

Peptide purity by RP-HPLC

FTHYB40602B Thymosin P4 (Reference Standard (25 mg net peptide vials) Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-lIe-Glu-Lys-Phe-Asp-Lys-Ser-lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH CBC0094

4963.55

May 2007

May 25,2012

May 2013

FTHYB4/v06

-20 ± 2°C

Specifications

White to off-white powder; no visible extraneous material

;;:: 97%; no individual related substance;;:: 1.0%

Results

White powder. No visible extraneous material

98.0% Largest impurity: 0.5% (0.97 RRT) Total im urities: 2.0%

This retest dote extension certificate is based on Sachem Americas, Inc. stability study results generated for the above mentioned lot.

I hereby certify that the above information is authentic and accurate.

I hereby certify that the retest analysis of this lot was performed in compliance with Good Manufacturing Practice and is appropriate for use in clinical trials as specified in the International Conference on Harmonisation (ICHI Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients,

/SRCHEn7/

Sachem Americas, Inc, 3132 Kashiwa Street Torrance, CA 90505 USA Tel 1-310-539-4171 Fax 1-310-539-9428

Retest Date Extension Certificate page 1 of 1



Test Facility Study No. 20024505 Test Site Phase Reference No. 142476

Appendix 3 Chromatograms

Final Bioanalysis Report Page 66


Sample Name: "2 001 142476 DBLK 1 (inj 1) 1" Sample ID: "1" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000000 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 3:05:39 PM Modified: Yes

1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

Inte

nsity

, cps

5.69

7.03

6.09

5.61

6.44

5.49

5.41

5.28

5.226.56

4.984.35

0.511.27

Sample Name: "2 001 142476 DBLK 1 (inj 1) 1" Sample ID: "1" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 3:05:39 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 9.90 cpsArea Threshold: 49.52 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.49 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.49 minArea: 3.93684e+002 countsHeight: 1.25e+002 cpsStart Time: 4.43 minEnd Time: 4.59 min

1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

Inte

nsity

, cps

7.16

5.68

7.09

5.58

5.99

5.49

5.41

4.49

Sample Name: "2 002 142476 NORM DBLK 1" Sample ID: "2" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Inte

nsity

, cps

7.14

7.04

6.44

6.284.13

5.84

5.67

3.12

Sample Name: "2 002 142476 NORM DBLK 1" Sample ID: "2" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 2 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 3:13:32 PM Modified: Yes

1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

1350

1400

1450

1500

1550

1600

1650

1700

1750

1800

1850

1900

Inte

nsity

, cps

4.39

7.09

4.48

4.18

Results Name: 142476-02A.rdb

Page 1 of 45



Sample Name: "2 003 142476 STD A 1" Sample ID: "3" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Inte

nsity

, cps

4.49

7.17

7.03

6.235.70

Sample Name: "2 003 142476 STD A 1" Sample ID: "3" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 3 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 3:21:30 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.48 minUse Relative RT: No Int. Type: Valley Retention Time: 4.49 minArea: 1.11344e+005 countsHeight: 4.41e+004 cpsStart Time: 4.36 minEnd Time: 4.63 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.49

7.16

Sample Name: "2 004 142476 NORM STD A 1" Sample ID: "4" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

Inte

nsity

, cps

Sample Name: "2 004 142476 NORM STD A 1" Sample ID: "4" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.50

4.40

7.16


Page 2 of 45



Sample Name: "2 005 142476 STD B 1 1" Sample ID: "5" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 5 Sample Type: Standard Concentration: 200.000 ng/mL Calculated Conc: 196.572782 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 3:37:27 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.37 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.38 minArea: 1.04917e+004 countsHeight: 3.98e+003 cpsStart Time: 4.27 minEnd Time: 4.46 min

1 2 3 4 5 6 7Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

3000

3100

3200

3300

3400

3500

3600

3700

3800

3900

Inte

nsity

, cps

4.38

Sample Name: "2 005 142476 STD B 1 1" Sample ID: "5" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 5 Sample Type: Standard Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 3:37:27 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.50 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.50 minArea: 1.00355e+005 countsHeight: 3.55e+004 cpsStart Time: 4.42 minEnd Time: 4.61 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.50

7.175.71

Sample Name: "2 006 142476 STD C 1 1" Sample ID: "6" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 6 Sample Type: Standard Concentration: 350.000 ng/mL Calculated Conc: 360.616263 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 3:45:28 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.38 minUse Relative RT: No Int. Type: Valley Retention Time: 4.38 minArea: 2.20876e+004 countsHeight: 7.86e+003 cpsStart Time: 4.32 minEnd Time: 4.46 min

1 2 3 4 5 6 7Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

4600

4800

5000

5200

5400

5600

5800

6000

6200

6400

6600

6800

7000

7200

7400

7600

7800

Inte

nsity

, cps

4.38

Sample Name: "2 006 142476 STD C 1 1" Sample ID: "6" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.50


Page 3 of 45



Sample Name: "2 007 142476 STD D 1 1" Sample ID: "7" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

Inte

nsity

, cps

4.39

Sample Name: "2 007 142476 STD D 1 1" Sample ID: "7" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 7 Sample Type: Standard Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 3:53:24 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.52 minUse Relative RT: No Int. Type: Valley Retention Time: 4.52 minArea: 1.08706e+005 countsHeight: 4.16e+004 cpsStart Time: 4.36 minEnd Time: 4.61 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.52

Sample Name: "2 008 142476 STD E 1 1" Sample ID: "8" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.6e4

1.7e4

1.7e4

1.8e4

1.8e4

1.9e4

Inte

nsity

, cps

4.39

Sample Name: "2 008 142476 STD E 1 1" Sample ID: "8" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.52


Page 4 of 45



Sample Name: "2 009 142476 STD F 1 1" Sample ID: "9" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

Inte

nsity

, cps

4.39

Sample Name: "2 009 142476 STD F 1 1" Sample ID: "9" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 9 Sample Type: Standard Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 4:09:18 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.52 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.52 minArea: 1.05721e+005 countsHeight: 3.93e+004 cpsStart Time: 4.42 minEnd Time: 4.60 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.52

Sample Name: "2 010 142476 STD G 1 1" Sample ID: "10" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.40

Sample Name: "2 010 142476 STD G 1 1" Sample ID: "10" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

4.0e4

Inte

nsity

, cps

4.52


Page 5 of 45



Sample Name: "2 011 142476 STD H 1 1" Sample ID: "11" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

6.0e4

6.2e4

6.4e4

6.6e4

Inte

nsity

, cps

4.41

Sample Name: "2 011 142476 STD H 1 1" Sample ID: "11" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.53

Sample Name: "2 012 142476 STD I 1 1" Sample ID: "12" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

Inte

nsity

, cps

4.40

Sample Name: "2 012 142476 STD I 1 1" Sample ID: "12" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 12 Sample Type: Standard Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 4:33:12 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.53 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.53 minArea: 1.09019e+005 countsHeight: 3.97e+004 cpsStart Time: 4.43 minEnd Time: 4.61 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.53


Page 6 of 45



Sample Name: "2 013 142476 STD J 1 1" Sample ID: "13" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 13 Sample Type: Standard Concentration: 5000.00 ng/mL Calculated Conc: 4970.189750 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 4:41:12 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.40 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.40 minArea: 3.10749e+005 countsHeight: 1.12e+005 cpsStart Time: 4.34 minEnd Time: 4.49 min

1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

Inte

nsity

, cps

4.40

Sample Name: "2 013 142476 STD J 1 1" Sample ID: "13" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps

4.54

Sample Name: "2 014 142476 DBLK 1 (inj 2) 1" Sample ID: "14" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 14 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.000000 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 4:49:08 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.42 minArea: 4.94167e+002 countsHeight: 1.68e+002 cpsStart Time: 4.34 minEnd Time: 4.48 min

1 2 3 4 5 6 7Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

Inte

nsity

, cps

Sample Name: "2 014 142476 DBLK 1 (inj 2) 1" Sample ID: "14" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 14 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 4:49:08 PM Modified: Yes

1 2 3 4 5 6 7Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

620

640

660

680

Inte

nsity

, cps

7.17

7.09

6.44

4.555.54

0.551.64

4.282.52


Page 7 of 45



Sample Name: "2 015 142476 QC A 1 1" Sample ID: "15" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 15 Sample Type: QC Concentration: 600.000 ng/mL Calculated Conc: 635.525130 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 4:57:05 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.42 minUse Relative RT: No Int. Type: Valley Retention Time: 4.42 minArea: 3.99876e+004 countsHeight: 1.48e+004 cpsStart Time: 4.35 minEnd Time: 4.50 min

1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

Inte

nsity

, cps

4.42

4.55

7.046.49

Sample Name: "2 015 142476 QC A 1 1" Sample ID: "15" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 15 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 4:57:05 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.54 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.54 minArea: 1.10368e+005 countsHeight: 3.96e+004 cpsStart Time: 4.40 minEnd Time: 4.67 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.54

Sample Name: "2 016 142476 QC B 1 1" Sample ID: "16" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

Inte

nsity

, cps

4.42

Sample Name: "2 016 142476 QC B 1 1" Sample ID: "16" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.55


Page 8 of 45



Sample Name: "2 017 142476 QC C 1 1" Sample ID: "17" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 17 Sample Type: QC Concentration: 4000.00 ng/mL Calculated Conc: 3990.959858 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 5:13:01 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.43 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.43 minArea: 2.48269e+005 countsHeight: 9.62e+004 cpsStart Time: 4.35 minEnd Time: 4.50 min

1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

Inte

nsity

, cps

4.43

Sample Name: "2 017 142476 QC C 1 1" Sample ID: "17" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 17 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 5:13:01 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.56 minUse Relative RT: No Int. Type: Valley Retention Time: 4.56 minArea: 1.06428e+005 countsHeight: 4.03e+004 cpsStart Time: 4.43 minEnd Time: 4.70 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

4.0e4

Inte

nsity

, cps

4.56

Sample Name: "2 018 142476 142476-01-001 2130 A GD 7 Predose PL-1 1" Sample ID: "18" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

Inte

nsity

, cps

Sample Name: "2 018 142476 142476-01-001 2130 A GD 7 Predose PL-1 1" Sample ID: "18" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

Inte

nsity

, cps

4.55

4.45 7.12


Page 9 of 45



Sample Name: "2 019 142476 142476-01-002 2130 A GD 7 5min PL-1 1" Sample ID: "19" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 19 Sample Type: Unknown Concentration: N/A Calculated Conc: 37499.093667 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 5:28:52 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.43 minArea: 1.37243e+006 countsHeight: 5.26e+005 cpsStart Time: 4.37 minEnd Time: 4.53 min

2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

Inte

nsity

, cps

4.43

Sample Name: "2 019 142476 142476-01-002 2130 A GD 7 5min PL-1 1" Sample ID: "19" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 19 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 5:28:52 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Valley Retention Time: 4.56 minArea: 6.23556e+004 countsHeight: 2.21e+004 cpsStart Time: 4.50 minEnd Time: 4.75 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

Inte

nsity

, cps


Sample Index: 20 Sample Type: Unknown Concentration: N/A Calculated Conc: 26700.002777 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 5:36:50 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.44 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.44 minArea: 1.48396e+006 countsHeight: 5.92e+005 cpsStart Time: 4.32 minEnd Time: 4.50 min

2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

5.6e5

5.8e5

Inte

nsity

, cps

4.44

4.56



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.57


Page 10 of 45





2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

3.1e5

Inte

nsity

, cps

4.44



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.57

Sample Name: "2 022 142476 142476-01-005 2130 A GD 7 1hr PL-1 1" Sample ID: "22" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

1.0e5

1.1e5

1.1e5

1.2e5

1.2e5

1.3e5

1.3e5

1.4e5

1.4e5

1.5e5

1.5e5

Inte

nsity

, cps

4.44

4.56

Sample Name: "2 022 142476 142476-01-005 2130 A GD 7 1hr PL-1 1" Sample ID: "22" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps

4.57


Page 11 of 45





1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps

4.45



1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.6e4

Inte

nsity

, cps

4.59


Sample Index: 24 Sample Type: Unknown Concentration: N/A Calculated Conc: 81.199457 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 6:08:40 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.45 minUse Relative RT: No Int. Type: Valley Retention Time: 4.45 minArea: 3.56801e+003 countsHeight: 1.29e+003 cpsStart Time: 4.35 minEnd Time: 4.52 min

1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

Inte

nsity

, cps

4.45

4.59



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

Inte

nsity

, cps

4.58

4.48


Page 12 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.58

4.49



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

Inte

nsity

, cps

4.58

4.49

7.076.36


Page 13 of 45





2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

5.6e5

5.8e5

6.0e5

6.2e5

6.4e5

6.6e5

Inte

nsity

, cps

4.46



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

Inte

nsity

, cps



2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

3.1e5

3.2e5

3.3e5

Inte

nsity

, cps

4.46



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

Inte

nsity

, cps

4.59


Page 14 of 45





2 4 6Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

1.0e5

1.1e5

1.1e5

1.2e5

1.2e5

1.3e5

1.3e5

1.4e5

1.4e5

1.5e5

1.5e5

1.6e5

1.6e5

1.7e5

1.7e5

1.8e5

1.8e5

1.9e5

1.9e5

2.0e5

2.0e5

Inte

nsity

, cps

4.47



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

Inte

nsity

, cps

4.60



1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Inte

nsity

, cps

4.48

4.61



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps

4.61


Page 15 of 45





1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

Inte

nsity

, cps

4.48



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.62



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.61

4.51


Page 16 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

Inte

nsity

, cps

4.63

4.525.73



1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

Inte

nsity

, cps

4.50



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.63


Page 17 of 45





1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

Inte

nsity

, cps

4.50



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

Inte

nsity

, cps

4.63



1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

Inte

nsity

, cps

4.51



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.64


Page 18 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.64

4.557.10



2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

Inte

nsity

, cps

4.51

4.64


Sample Index: 38 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 8:00:23 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.64 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.64 minArea: 1.24095e+005 countsHeight: 4.66e+004 cpsStart Time: 4.59 minEnd Time: 4.72 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

Inte

nsity

, cps

4.64


Page 19 of 45





2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

3.1e5

3.2e5

3.3e5

3.4e5

3.5e5

Inte

nsity

, cps

4.52



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

Inte

nsity

, cps

4.65



2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

Inte

nsity

, cps

4.53

4.65



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.65


Page 20 of 45





1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

6.0e4

6.2e4

6.4e4

6.6e4

6.8e4

7.0e4

7.2e4

7.4e4

7.6e4

7.8e4

8.0e4

Inte

nsity

, cps

4.54



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.6e4

1.7e4

1.7e4

1.8e4

Inte

nsity

, cps

4.54



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

Inte

nsity

, cps

4.66

4.57


Page 21 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps

4.67

4.57



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.68

4.58


Page 22 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

Inte

nsity

, cps

4.69

4.597.09



2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

Inte

nsity

, cps

4.56

4.68


Sample Index: 46 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 9:04:10 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.70 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.69 minArea: 8.55133e+004 countsHeight: 3.02e+004 cpsStart Time: 4.63 minEnd Time: 4.77 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

Inte

nsity

, cps

4.69


Page 23 of 45





2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

Inte

nsity

, cps

4.56

4.69



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.69



1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

Inte

nsity

, cps

4.58



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

Inte

nsity

, cps

4.71


Page 24 of 45





1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

5.6e4

5.8e4

Inte

nsity

, cps

4.58

4.71



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.71



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

Inte

nsity

, cps

4.58



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.70


Page 25 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

1350

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.72

4.63



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.71

4.61


Page 26 of 45





1 2 3 4 5 6 7Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

620

640

660

680

700

720

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

Inte

nsity

, cps

4.72

4.617.126.24



2 4 6Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

Inte

nsity

, cps

4.60



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

Inte

nsity

, cps


Page 27 of 45





2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

5.6e5

5.8e5

6.0e5

6.2e5

6.4e5

6.6e5

Inte

nsity

, cps

4.62


Sample Index: 55 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 10:15:55 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.74 minArea: 1.19555e+005 countsHeight: 4.32e+004 cpsStart Time: 4.69 minEnd Time: 4.83 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.74


Sample Index: 56 Sample Type: Unknown Concentration: N/A Calculated Conc: 15657.208413 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 10:23:52 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.62 minArea: 8.23215e+005 countsHeight: 3.04e+005 cpsStart Time: 4.45 minEnd Time: 4.69 min

2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

2.6e5

2.7e5

2.8e5

2.9e5

3.0e5

Inte

nsity

, cps

4.62

4.74



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.74


Page 28 of 45





1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

1.15e5

Inte

nsity

, cps

4.63

4.74



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

Inte

nsity

, cps

4.75


Sample Index: 58 Sample Type: Unknown Concentration: N/A Calculated Conc: 1850.416109 ng/mL Acq. Date: 10/Aug/2012 Acq. Time: 10:39:47 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Valley Retention Time: 4.64 minArea: 3.67829e+004 countsHeight: 1.45e+004 cpsStart Time: 4.52 minEnd Time: 4.73 min

1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

Inte

nsity

, cps

4.64



1 2 3 4 5 6 7Time, min

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

1.15e4

1.20e4

1.25e4

Inte

nsity

, cps

4.77

6.64


Page 29 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

1350

Inte

nsity

, cps

4.77

7.10



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps

4.77

4.67



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

Inte

nsity

, cps

4.78

4.687.136.65


Page 30 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

Inte

nsity

, cps

4.76

4.66

7.11



2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

5.6e5

5.8e5

6.0e5

Inte

nsity

, cps

4.64

4.76



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

Inte

nsity

, cps

4.76


Page 31 of 45





2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

4.0e4

Inte

nsity

, cps

4.78



2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

Inte

nsity

, cps

4.66

4.78



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

Inte

nsity

, cps

4.78


Page 32 of 45





1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Inte

nsity

, cps

4.65

4.77


Sample Index: 65 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/Aug/2012 Acq. Time: 11:35:29 PM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.77 minArea: 1.12821e+005 countsHeight: 4.14e+004 cpsStart Time: 4.72 minEnd Time: 4.87 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.77



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps

4.78

4.69


Page 33 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.79

4.69



1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.80

4.70

7.13


Page 34 of 45




Sample Index: 69 Sample Type: QC Concentration: 600.000 ng/mL Calculated Conc: 650.194103 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 12:07:25 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Valley Retention Time: 4.67 minArea: 4.36115e+004 countsHeight: 1.74e+004 cpsStart Time: 4.57 minEnd Time: 4.74 min

1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.6e4

1.7e4

1.7e4

Inte

nsity

, cps

4.67


Sample Index: 69 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 12:07:25 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.79 minArea: 1.17574e+005 countsHeight: 4.60e+004 cpsStart Time: 4.61 minEnd Time: 4.94 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

Inte

nsity

, cps

4.79



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e4

Inte

nsity

, cps

4.66


Sample Index: 70 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 12:15:23 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Valley Retention Time: 4.79 minArea: 1.17003e+005 countsHeight: 4.13e+004 cpsStart Time: 4.67 minEnd Time: 4.98 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

Inte

nsity

, cps

4.79


Page 35 of 45




Sample Index: 71 Sample Type: QC Concentration: 4000.00 ng/mL Calculated Conc: 4139.892259 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 12:23:19 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.66 minArea: 2.71582e+005 countsHeight: 9.64e+004 cpsStart Time: 4.57 minEnd Time: 4.74 min

1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

4.0e4

Inte

nsity

, cps

4.79

Sample Name: "2 072 142476 142476-01-049 2133 B GD 7 Predose PL-1 1" Sample ID: "72" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 72 Sample Type: Unknown Concentration: N/A Calculated Conc: 38.781902 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 12:31:17 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Valley Retention Time: 4.66 minArea: 1.01443e+003 countsHeight: 2.71e+002 cpsStart Time: 4.61 minEnd Time: 4.74 min

1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

Inte

nsity

, cps

Sample Name: "2 072 142476 142476-01-049 2133 B GD 7 Predose PL-1 1" Sample ID: "72" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 72 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 12:31:17 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Valley Retention Time: 4.79 minArea: 8.53463e+004 countsHeight: 3.15e+004 cpsStart Time: 4.74 minEnd Time: 4.96 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

Inte

nsity

, cps

4.79

4.70 7.146.64


Page 36 of 45



Sample Name: "2 073 142476 142476-01-050 2133 B GD 7 5min PL-1 1" Sample ID: "73" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 73 Sample Type: Unknown Concentration: N/A Calculated Conc: 118035.306830 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 12:39:15 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.67 minArea: 5.19772e+006 countsHeight: 1.89e+006 cpsStart Time: 4.61 minEnd Time: 4.77 min

2 4 6Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.5e5

1.0e6

1.1e6

1.1e6

1.2e6

1.2e6

1.3e6

1.3e6

1.4e6

1.4e6

1.5e6

1.5e6

1.6e6

1.6e6

1.7e6

1.7e6

1.8e6

1.8e6

1.9e6

Inte

nsity

, cps

4.67

Sample Name: "2 073 142476 142476-01-050 2133 B GD 7 5min PL-1 1" Sample ID: "73" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

Inte

nsity

, cps



2 4 6Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.5e5

1.0e6

1.1e6

1.1e6

1.2e6

1.2e6

1.3e6

1.3e6

1.4e6

1.4e6

1.5e6

1.5e6

1.6e6

1.6e6

1.7e6

1.7e6

1.8e6

1.8e6

1.9e6

1.9e6

2.0e6

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps


Page 37 of 45





2 4 6Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

Inte

nsity

, cps

Sample Name: "2 076 142476 142476-01-053 2133 B GD 7 1hr PL-1 1" Sample ID: "76" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""

Sample Index: 76 Sample Type: Unknown Concentration: N/A Calculated Conc: 24366.224209 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 1:03:06 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.66 minArea: 1.40484e+006 countsHeight: 5.49e+005 cpsStart Time: 4.59 minEnd Time: 4.72 min

2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

Inte

nsity

, cps

4.66

Sample Name: "2 076 142476 142476-01-053 2133 B GD 7 1hr PL-1 1" Sample ID: "76" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.79


Page 38 of 45





1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

1.0e5

1.1e5

1.1e5

1.2e5

1.2e5

1.3e5

1.3e5

Inte

nsity

, cps

4.68

4.80


Sample Index: 77 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 1:11:06 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.80 minArea: 1.19316e+005 countsHeight: 4.38e+004 cpsStart Time: 4.74 minEnd Time: 4.88 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Inte

nsity

, cps

4.80



1 2 3 4 5 6 7Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

4600

Inte

nsity

, cps

4.67



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps

4.80

4.70

6.20


Page 39 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps

4.78

4.69

Sample Name: "2 080 142476 142476-01-057 2133 B GD 19 Predose PL-1 1" Sample ID: "80" File: "142476-02A.wiff"Peak Name: "Thymosin B4" Mass(es): "828.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Inte

nsity

, cps

Sample Name: "2 080 142476 142476-01-057 2133 B GD 19 Predose PL-1 1" Sample ID: "80" File: "142476-02A.wiff"Peak Name: "Leu6-Thymosin B4(IS)" Mass(es): "825.3/129.2 amu"Comment: "none" Annotation: ""


1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

Inte

nsity

, cps

4.79

4.71

7.11


Page 40 of 45





2 4 6Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.5e5

1.0e6

1.1e6

1.1e6

1.2e6

1.2e6

1.3e6

1.3e6

1.4e6

1.4e6

1.5e6

1.5e6

1.6e6

1.6e6

1.7e6

1.7e6

1.8e6

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

Inte

nsity

, cps



2 4 6Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

Inte

nsity

, cps


Page 41 of 45





2 4 6Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

5.0e5

5.2e5

5.4e5

5.6e5

5.8e5

6.0e5

6.2e5

6.4e5

6.6e5

6.8e5

7.0e5

Inte

nsity

, cps

4.67



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

Inte

nsity

, cps



2 4 6Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

2.5e5

Inte

nsity

, cps

4.66

4.78



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

Inte

nsity

, cps

4.78


Page 42 of 45





1 2 3 4 5 6 7Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

Inte

nsity

, cps

4.66

4.78



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

Inte

nsity

, cps

4.78



1 2 3 4 5 6 7Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200

4400

4600

4800

Inte

nsity

, cps

4.68



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.80

4.716.65


Page 43 of 45





1 2 3 4 5 6 7Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

1300

Inte

nsity

, cps



1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

3.9e4

Inte

nsity

, cps

4.78

4.69



1 2 3 4 5 6 7Time, min

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

5500.0

6000.0

6500.0

7000.0

7500.0

8000.0

8500.0

9000.0

9500.0

1.0e4

1.1e4

1.1e4

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

Inte

nsity

, cps

4.66


Sample Index: 88 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 2:38:42 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.78 minArea: 1.08313e+005 countsHeight: 3.87e+004 cpsStart Time: 4.66 minEnd Time: 4.90 min

1 2 3 4 5 6 7Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

2.4e4

2.5e4

2.6e4

2.7e4

2.8e4

2.9e4

3.0e4

3.1e4

3.2e4

3.3e4

3.4e4

3.5e4

3.6e4

3.7e4

3.8e4

Inte

nsity

, cps

4.78


Page 44 of 45





1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

Inte

nsity

, cps

4.66



1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

Inte

nsity

, cps

4.78


Sample Index: 90 Sample Type: QC Concentration: 4000.00 ng/mL Calculated Conc: 3896.871947 ng/mL Acq. Date: 11/Aug/2012 Acq. Time: 2:54:40 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 24.37 cpsArea Threshold: 121.83 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.66 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.66 minArea: 2.82592e+005 countsHeight: 1.04e+005 cpsStart Time: 4.59 minEnd Time: 4.74 min

1 2 3 4 5 6 7Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Inte

nsity

, cps

4.66


Sample Index: 90 Sample Type: QC Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 11/Aug/2012 Acq. Time: 2:54:40 AM Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 2 Noise Threshold: 2.68 cpsArea Threshold: 13.40 cps,Num. Smooths: 0 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 4.78 minUse Relative RT: No Int. Type: Exponential Skim Retention Time: 4.78 minArea: 1.24081e+005 countsHeight: 4.65e+004 cpsStart Time: 4.69 minEnd Time: 4.88 min

1 2 3 4 5 6 7Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

Inte

nsity

, cps

4.78


Page 45 of 45




Appendix 16 Toxicokinetic Report


Toxicokinetic Report: 12-RS-032TK A Dosage Range-finding Embryo-fetal Development Study of

Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a Preliminary Evaluation in

Non-Pregnant Rabbits

Author

Andrew M. Vick, PhD

Seventh Wave Completion Date

February 15, 2013

Performing Laboratory (Toxicokinetic Analysis)

Seventh Wave Laboratories LLC 743 Spirit 40 Park Drive, Suite 209

Chesterfield, MO 63005

Testing Facility and Study Number

Charles River Laboratories


Horsham, PA 19044 Charles River Laboratories Study Number: 20024505

Sponsor

RegeneRx Biopharmaceuticals, Inc. 15245 Shady Grove Road, Suite 470

Rockville, MD 20850


Toxicokinetic Report: February 15, 2013 Seventh Wave Reference Number: 12-RS-032TK

Charles River Laboratories Study Number: 20024505 Page 5 of 17

TABLE OF CONTENTS Section Page

TITLE PAGE.......................................................................................................................1

REGULATORY COMPLIANCE STATEMENT...............................................................2

SIGNATURE OF APPROVAL...........................................................................................3

SEVENTH WAVE QUALITY ASSURANCE STATEMENT..........................................4

TABLE OF CONTENTS.....................................................................................................5

1.0 GENERAL INFORMATION .....................................................................................6

2.0 OBJECTIVES..............................................................................................................6

3.0 METHODS..................................................................................................................6

3.1 Study Design and Toxicokinetic Sample Collection ..........................................6

Table A. Study Design........................................................................................................6

Table B. Toxicokinetic Sample Collection Schedule (Targeted Times) ............................7

3.2 Relevant Toxicokinetic Study Events .................................................................7

3.3 Toxicokinetic Calculations .................................................................................7

4.0 RESULTS....................................................................................................................8

4.1 Tables and Figures ..............................................................................................8

4.2 Single- and Repeat-Dose Toxicokinetics of Tß4 ................................................8

5.0 CONCLUSIONS .........................................................................................................9

6.0 ABBREVIATIONS.....................................................................................................9

7.0 ARCHIVES .................................................................................................................9

8.0 TABLES AND FIGURES.........................................................................................10

Table 1. Group Mean Tß4 Plasma Concentration (µg/mL) versus Time Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.).................10

Table 2. Group Mean Tß4 Toxicokinetic Summary Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.) ........................................................12

Figure 1. Tß4 Concentration versus Time—Dose Comparison, Gestation Day 7............14

Figure 2. Tß4 Concentration versus Time—Dose Comparison, Gestation Day 19..........15

Figure 3. Tß4 Cmax versus Dose—Gestation Day Comparison ........................................16

Figure 4. Tß4 AUCall versus Dose—Gestation Day Comparison.....................................17



Charles River Laboratories Study Number: 20024505 Page 6 of 17 1.0 GENERAL INFORMATION Audited plasma Thymosin beta 4 (Tß4) bioanalytical data from Groups 6 through 9, as received from Charles River Laboratories Preclinical Services (Senneville QC, Canada), were analyzed using WinNonlin, version 5.2.1 software (Pharsight Inc., Mountain View, CA) and Microsoft Office Excel 2003 maintained at Seventh Wave. The TK data analysis was conducted by Dr. Andrew M. Vick. Use of Excel was limited to receipt of raw data and transfer into WinNonlin for TK data analysis. 2.0 OBJECTIVES The objectives of this study were to provide information for selection of dosages to be used in a subsequent embryo-fetal development study in rabbits and to provide a preliminary evaluation of the effects of the Injectable formulation of Tß4 (RGN-352) on pregnancy and embryo-fetal development. This study evaluated ICH Harmonised Tripartite Guideline stages C to D of the reproductive process. In addition, the TK characteristics of RGN-352 were determined. The objective of this TK data analysis was to characterize Tß4 TK at the test article doses administered to mated female rabbits during the course of this study. 3.0 METHODS 3.1 Study Design and Toxicokinetic Sample Collection A total of 12 mated female rabbits were assigned to dose groups as shown in the following tables: Table A. Study Design

Group No.

Test Material Dosage Level

(mg/kg)


Dosage Volume (mL/kg)

Number of Main Study

Animals

Number of TK Animals

5 Control Article 0 0 0.9 5 NA 6 3 100 0.03 5 3 7 10 100 0.1 5 3 8 30 100 0.3 5 3 9

RGN-352

90 100 0.9 5 3 NA = Not Applicable According to the Protocol and Protocol Amendments, RGN-352 was administered to Groups 6, 7, 8, and 9 at 3, 10, 30, and 90 mg/kg, respectively, via intravenous (IV) bolus injection via the marginal ear vein on Days 7 through 19 of presumed gestation (DGs 7 through 19). Doses were adjusted based on the most recently recorded body weight and administered at approximately the same time each day.



Charles River Laboratories Study Number: 20024505 Page 7 of 17 Blood samples for TK data analysis were collected from TK animals pre-dose and also postdose according to Table B: Table B. Toxicokinetic Sample Collection Schedule (Targeted Times)

Sample Collection Time Points (Approximate Time Postdose) on Days 7 and 19 of

Presumed Gestation Group

No. No. of

Females 5 m 10 m 20 m 1 h 2 h 6 h 12 h

6 3 X X X X X X X 7 3 X X X X X X X 8 3 X X X X X X X 9 3 X X X X X X X

X = Blood collected m = minute; h = hour Plasma samples were analyzed by Charles River for concentrations of Tß4 using LC–MS/MS under Analytical Procedure 142476.PL.xx (where 'xx' denotes the version number), using a validated analytical procedure (Study No. 142474). The results were used for the generation of this TK report. 3.2 Relevant Toxicokinetic Study Events

All animals on study were confirmed pregnant. There were no deviations or study-related observations that impact TK analysis or interpretation for this study. 3.3 Toxicokinetic Calculations

Toxicokinetic analysis was performed on the individual animal plasma concentration versus time data for Tß4 (DGs 7 and 19) using WinNonlin noncompartmental analysis (linear trapezoidal rule for AUC calculation). Nominal dose values and sampling times were used for calculations. For the purpose of TK analysis, predose values for Groups 6 through 9 were excluded from analysis on DGs 7 and 19, since they were all BLQ (<0.200 µg/mL). Any postdose concentrations reported as BLQ were set equal to zero. User-defined selection criteria were used in the determination of Lambda z, the elimination rate constant upon which AUCINFobs, HLλz, CL, and Vd were based. Individual animal and group mean plasma Tß4 concentration data are presented with standard deviation (SD) and percent coefficient of variation (CV%). Individual and group mean TK parameters were also reported with descriptive statistics. For abbreviations, please go to Section 6.0.



Charles River Laboratories Study Number: 20024505 Page 8 of 17 4.0 RESULTS

4.1 Tables and Figures

Individual subject and group mean Tß4 plasma concentration versus time data, sorted by DG, Group, and Dose are presented in Table 1.

Mean log Tß4 plasma concentration versus time data are plotted in Figures 1 and 2 for all dose levels on DG 7 and DG 19, respectively.

Group mean Tß4 TK parameters, sorted by DG, Group, and Dose are presented in Table 2.

Mean plasma Tß4 Cmax and AUCall are plotted versus dose of RGN-352 in Figures 3 and 4, respectively.

4.2 Single- and Repeat-Dose Toxicokinetics of Tß4

Of note, predose samples collected on DGs 7 and 19 from the 3, 10, 30, and 90 mg/kg dose groups were all BLQ (Table 1).

Evidence of systemic plasma exposure to Tß4 was observed in all RGN-352-treated TK rabbits (Group 6 through 9) following both single and repeat IV administration. Peak plasma concentrations of Tß4 following IV administration were observed at the first time point (0.083 hour) for both DG 7 and DG 19 TK collections (Table 1 and Figures 1 and 2).

Following IV bolus injection, Tß4 plasma concentrations exhibited a polyphasic pattern of decay, characterized by a rapid distribution phase followed by a log-linear elimination phase. Although occasional differences were noted, mean CL, Vd, and HLλz values appeared to be dose-independent between the 3 and 90 mg/kg dose levels for DGs 7 and 19. Group mean CL and Vd values on DG 19 ranged from 128 to 185 mL/h/kg and 133 to 167 mL/kg, respectively, and these values were 14–41% and 16-59% higher, respectively, than those obtained on DG 7. No clear change in HLλz values was noted following repeat-dosing of Tß4 (relative to DG 7), with group mean values ranging from 0.531 to 0.751 hours across DGs 7 and 19 (Table 2).

Tß4 exposure (as assessed by mean Cmax and AUCall) following IV administration increased in a dose-related fashion over the dose range evaluated on both DG 7 and DG 19. Over the dose range evaluated, the observed increase in exposure was reasonably proportional to dose on both TK collection days (Table 2 and Figures 3 and 4).

o On DG 7, mean Tß4 Cmax and AUCall values increased 28.3- and 30.7-fold over a 30-fold dose range.

o On DG 19, mean Tß4 Cmax and AUCall values increased 31.1- and 33.0-fold over a 30-fold dose range.

Mean AUCall values on DG 19 for Tß4 were decreased 26%, 14%, 30%, and 20% compared to AUCall values on DG 7 for the 3, 10, 30, and 90 mg/kg dose groups, respectively. Based on the data; there was no clear or consistent evidence of accumulation of Tß4 in plasma with repeated IV dosing of RGN-352 over the study duration evaluated (Table 2).



Charles River Laboratories Study Number: 20024505 Page 9 of 17 5.0 CONCLUSIONS

This report describes the plasma TK of Tß4 when RGN-352 was administered via IV bolus injection on DGs 7 through 19 to rabbits. RGN-352 was administered at 3, 10, 30, and 90 mg/kg by IV injection to animals in Groups 6 through 9, respectively. Group 5 was the control group and was not included in this TK analysis. Evidence of systemic plasma exposure to Tß4 was observed in all RGN-352-treated TK rabbits following both single and repeat IV administration. Tß4 exposure following IV administration increased with increasing dose and was reasonably proportional to dose on both TK collection days. Mean AUCall values on DG 19 for Tß4 were decreased 26%, 14%, 30%, and 20% compared to AUCall values on DG 7 for the 3, 10, 30, and 90 mg/kg dose groups, respectively. Based on the data; there was no clear or consistent evidence of accumulation of Tß4 in plasma with repeated IV dosing of RGN-352 over the study duration evaluated.

6.0 ABBREVIATIONS AUCall Area under the curve between the time of dose and the last time point AUCINFobs Total AUC up to the last measurable concentration plus the AUC extrapolated

from the last measurable concentration (Clast at tlast) to infinity: AUC0-tlast + Clast/Lambda z

AUClast Area under the curve between the time of dose and the last measurable concentration

BLQ Below limit of quantitation CL Total body clearance Cmax Maximum observed concentration, occurring at Tmax CV% Percent Coefficient of Variation DG Presumed Day of Gestation HLλz Half-life associated with the terminal (log-linear) elimination phase N Number NA Not Applicable NC Not Calculable SD Standard Deviation SE Standard Error Vd Volume of distribution based on the terminal phase 7.0 ARCHIVES The final TK report will be forwarded to the Study Director. All raw data and supporting documents will be temporarily retained at Seventh Wave Laboratories LLC, 743 Spirit 40 Park Drive, Chesterfield, MO 63005 until issuance of the final TK report. Once the report is issued, all raw data and supporting documents will be transferred to Charles River Laboratories, Preclinical Services, Pennsylvania (PCS-PA), 905 Sheehy Drive, Building A, Horsham, PA 19044.



Charles River Laboratories Study Number: 20024505 Page 10 of 17 8.0 TABLES AND FIGURES

Table 1. Group Mean Tß4 Plasma Concentration (µg/mL) versus Time Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.)

Time (h)

DG Group Dose

(mg/kg) Rabbit

No. 0.00 0.083 0.167 0.333 1 2 6 12 2130 0.00 45.1 27.1 16.4 7.71 3.22 0.00 0.00 2131 0.00 23.1 16.9 10.4 3.69 1.16 0.00 0.00 2132 0.00 44.0 25.9 16.7 5.98 1.72 0.00 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 37.4 23.3 14.5 5.79 2.03 0.00 0.00

SD 0.00 12.4 5.58 3.52 2.02 1.07 0.00 0.00

6 3

CV% NC 33.1 24.0 24.3 34.8 52.5 NC NC 2133 0.00 150 93.2 61.2 23.6 6.92 0.222 0.00 2134 0.00 125 79.4 55.3 24.6 7.75 0.00 0.00 2135 0.00 137 96.2 63.6 25.4 7.57 0.00 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 137 89.6 60.0 24.5 7.41 0.0739 0.00

SD 0.00 12.2 8.97 4.28 0.887 0.438 0.128 0.00

7 10

CV% NC 8.9 10.0 7.1 3.6 5.9 173.2 NC 2136 0.00 436 222 166 66.2 21.4 0.406 0.00 2137 0.00 388 276 196 92.4 28.0 0.478 0.00 2138 0.00 364 209 146 59.8 17.9 0.306 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 396 236 170 72.8 22.5 0.396 0.00

SD 0.00 37.1 35.0 25.3 17.2 5.11 0.0863 0.00

8 30

CV% NC 9.4 14.9 14.9 23.7 22.7 21.8 NC 2139 0.00 1090 730 439 179 57.8 1.01 0.00 2140 0.00 961 768 426 212 80.5 1.63 0.00 2141 0.00 1130 744 447 201 48.5 0.728 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 1060 747 438 198 62.3 1.12 0.00

SD 0.00 87.9 19.4 10.5 16.8 16.5 0.460 0.00

7

9 90

CV% NC 8.3 2.6 2.4 8.5 26.5 40.9 NC Variable = Concentration (µg/mL)




Table 1. Group Mean Tß4 Plasma Concentration (µg/mL) versus Time Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.) (continued)

Time (h)

DG Group Dose

(mg/kg) Rabbit

No. 0.00 0.083 0.167 0.333 1 2 6 12 2130 0.00 39.4 20.2 11.5 4.02 1.49 0.00 0.00 2131 0.00 26.1 14.1 8.43 2.69 0.792 0.00 0.00 2132 0.00 39.3 20.9 11.3 3.96 1.24 0.00 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 35.0 18.4 10.4 3.56 1.17 0.00 0.00

SD 0.00 7.64 3.76 1.74 0.748 0.354 0.00 0.00

6 3

CV% NC 21.9 20.4 16.6 21.0 30.1 NC NC 2133 0.00 108 61.1 41.3 14.2 4.05 0.213 0.00 2134 0.00 155 88.3 59.8 21.8 6.01 0.00 0.00 2135 0.00 148 73.7 48.7 20.9 6.90 0.203 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 137 74.4 49.9 19.0 5.65 0.139 0.00

SD 0.00 25.3 13.6 9.34 4.14 1.46 0.120 0.00

7 10

CV% NC 18.5 18.4 18.7 21.8 25.7 86.7 NC 2136 0.00 363 152 99.7 38.8 11.2 0.333 0.00 2137 0.00 291 200 123 50.2 14.3 0.356 0.00 2138 0.00 344 221 116 42.7 14.0 0.498 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 333 191 113 43.9 13.1 0.396 0.00

SD 0.00 37.3 35.2 11.9 5.83 1.73 0.0896 0.00

8 30

CV% NC 11.2 18.4 10.5 13.3 13.1 22.6 NC 2139 0.00 878 606 305 115 37.6 0.759 0.00 2140 0.00 1270 574 395 162 56.2 1.66 0.00 2141 0.00 1110 589 295 98.6 30.0 0.640 0.00

N 3 3 3 3 3 3 3 3 Mean 0.00 1090 589 332 125 41.3 1.02 0.00

SD 0.00 197 16.0 54.8 33.0 13.5 0.557 0.00

19

9 90

CV% NC 18.1 2.7 16.5 26.4 32.7 54.6 NC Variable = Concentration (µg/mL)




Table 2. Group Mean Tß4 Toxicokinetic Summary Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.)

DG Group Dose

(mg/kg) Rabbit

No. Cmax

(µg/mL) AUClast

(µg*h/mL)AUCall

(µg*h/mL)AUCINFobs (µg*h/mL)

CL (mL/h/kg)

Vd (mL/kg)

HLλz

(h) 2130 45.1 25.1 31.6 28.5 105 109 0.717 2131 23.1 13.4 15.7 14.2 211 161 0.530 2132 44.0 22.8 26.3 24.1 125 92.0 0.512

N 3 3 3 3 3 3 3 Mean 37.4 20.4 24.5 22.3 147 121 0.587

SD 12.4 6.23 8.10 7.28 56.0 36.0 0.114

6 3

CV% 33.1 30.5 33.0 32.7 38.2 29.8 19.4 2133 150 97.0 97.7 97.2 103 113 0.760 2134 125 76.0 91.5 82.5 121 103 0.589 2135 137 83.0 98.2 89.0 112 88.4 0.545

N 3 3 3 3 3 3 3 Mean 137 85.3 95.8 89.6 112 101 0.631

SD 12.2 10.7 3.74 7.37 9.16 12.2 0.113

7 10

CV% 8.9 12.6 3.9 8.2 8.2 12.0 17.9 2136 436 279 280 279 107 106 0.686 2137 388 319 321 320 93.9 90.0 0.665 2138 364 239 240 239 125 120 0.664

N 3 3 3 3 3 3 3 Mean 396 279 280 279 109 105 0.671

SD 37.1 40.1 40.3 40.2 15.8 15.0 0.0124

8 30

CV% 9.4 14.4 14.4 14.4 14.5 14.3 1.8 2139 1090 728 731 729 123 120 0.674 2140 961 785 790 787 114 117 0.711 2141 1130 735 737 736 122 111 0.628

N 3 3 3 3 3 3 3 Mean 1060 750 753 751 120 116 0.671

SD 87.9 30.9 32.2 31.4 4.91 4.74 0.0416

7

9 90

CV% 8.3 4.1 4.3 4.2 4.1 4.1 6.2




Table 2. Group Mean Tß4 Toxicokinetic Summary Data; Sorted by Gestation Day (DG), Group, Dose, and Rabbit Number (No.) (continued)

DG Group Dose

(mg/kg) Rabbit

No. Cmax

(µg/mL) AUClast

(µg*h/mL)AUCall

(µg*h/mL)AUCINFobs (µg*h/mL)

CL (mL/h/kg)

Vd (mL/kg)

HLλz

(h) 2130 39.4 17.9 20.9 19.1 157 130 0.573 2131 26.1 12.1 13.7 12.7 237 169 0.494 2132 39.3 17.6 20.1 18.6 162 123 0.527

N 3 3 3 3 3 3 3 Mean 35.0 15.9 18.2 16.8 185 140 0.531

SD 7.64 3.27 3.94 3.58 44.8 24.6 0.040

6 3

CV% 21.9 20.6 21.6 21.3 24.2 17.6 7.5 2133 108 64.2 64.8 64.5 155 191 0.855 2134 155 81.5 93.5 85.8 117 84.9 0.505 2135 148 89.2 89.8 89.4 112 122 0.759

N 3 3 3 3 3 3 3 Mean 137 78.3 82.7 79.9 128 133 0.706

SD 25.3 12.8 15.6 13.5 23.8 54.0 0.181

7 10

CV% 18.5 16.3 18.8 16.9 18.6 40.6 25.6 2136 363 188 189 188 159 171 0.745 2137 291 196 197 197 152 157 0.714 2138 344 198 200 199 151 173 0.793

N 3 3 3 3 3 3 3 Mean 333 194 195 195 154 167 0.751

SD 37.3 5.56 5.76 5.64 4.54 8.60 0.0398

8 30

CV% 11.2 2.9 2.9 2.9 2.9 5.1 5.3 2139 878 520 523 521 173 173 0.696 2140 1270 738 743 740 122 134 0.765 2141 1110 535 537 535 168 169 0.697

N 3 3 3 3 3 3 3 Mean 1090 598 601 599 154 159 0.719

SD 197 122 123 122 28.2 21.5 0.0394

19

9 90

CV% 18.1 20.4 20.5 20.4 18.3 13.5 5.5




Figure 1. Tß4 Concentration versus Time—Dose Comparison, Gestation Day 7

* Plasma Tß4 levels were BLQ (defined as zero for calculations) at the 6 h time point for the 3 mg/kg dose group on DG 7.




Figure 2. Tß4 Concentration versus Time—Dose Comparison, Gestation Day 19

* Plasma Tß4 levels were BLQ (defined as zero for calculations) at the 6 h time point for the 3 mg/kg dose group on DG 7.




Figure 3. Tß4 Cmax versus Dose—Gestation Day Comparison




Figure 4. Tß4 AUCall versus Dose—Gestation Day Comparison



Appendix 17 Historical Control Data


SUMMARY OF REPRODUCTIVE INDICES NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION

PERIOD: JANUARY 2010 - JANUARY 2012

FULL STUDIES DOSAGE-RANGE STUDIESNUMBER OF STUDIES: 29 12

NUMBER OF RABBITS: TESTED 623 73 PREGNANT 589 66 FOUND DEAD 5* 2** ABORTED 3 0 DELIVERED PREMATURELY 1 0

NUMBER OF RABBITS PREGNANT AT CAESAREAN-SECTIONING: 580 63

NUMBER OF RABBITS WITH SINGLE CONCEPTUS LITTER: LIVE 1 0 RESORBED 1 0 ABORTED 0 0

RANGE/STUDY RANGE/STUDY MEAN or % MEAN or % MEAN or % MEAN or %

% PREGNANT 94.9 (80.0-100) 91.6 (66.7-100)

AVERAGE NO. CORPORA LUTEA 9.6 (8.3-10.4) 8.8 (7.7-9.8)

AVERAGE NO. IMPLANTATIONS 9.0 (8.2-10.0) 8.4 (7.3-9.5)

AVERAGE % PREIMPLANTATION LOSS 5.7 (1.6-15.2) 4.4 (0-15.0)

AVERAGE LITTER SIZE

AVERAGE NO. LIVE FETUSES 8.6 (7.5-9.6) 8.0 (7.2-9.0)

AVERAGE NO. DEAD FETUSES 0.0 --- 0.0 ---

AVERAGE NO. RESORPTIONS 0.4 (0.1-0.7) 0.4 (0-0.8)

AVERAGE NO. EARLY RESORPTIONS 0.2 (0-0.6) 0.3 (0-0.6)

* Four were unscheduled euthanasia** Intubation accidents


SUMMARY OF REPRODUCTIVE INDICES NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION

FULL STUDIES DOSAGE-RANGE STUDIES

RANGE/STUDY RANGE/STUDY MEAN or % MEAN or % MEAN or % MEAN or %

AVERAGE NO. LATE RESORPTIONS 0.1 (0-0.3) 0.1 (0-0.5)

AVERAGE % POSTIMPLANTATION LOSS 4.0 (1.1-9.0) 4.7 (0-10.3)

AVERAGE % DOES WITH ANY RESORPTIONS 27.9 (10.0-50.0) 30.2 (0-75.0)

AVERAGE % DOES WITH ALL CONCEPTUSES RESORBED 0.0 --- 0.0 ---

AVERAGE % DOES WITH ONE OR MORE LIVE FETUSES 100.0 --- 100.0 ---

AVERAGE SEX RATIO (% MALES/LITTER) 49.2 (40.8-61.2) 56.3 (39.8-72.5) AVERAGE FETAL BODY WEIGHT (G) 42.16 (38.82-45.32) 42.02 (36.83-47.02)

AVERAGE FOR MALES (G) 42.54 (39.63-46.26) 42.59 (37.14-47.54)

AVERAGE FOR FEMALES (G) 41.58 (38.25-44.56) 41.16 (36.40-46.83) AVERAGE % DEAD OR RESORBED CONCEPTUSES/LITTER 4.0 (1.1-9.0) 4.7 (0-10.3)


SUMMARY OF MATERNAL NECROPSY OBSERVATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF]

DAY 29 CAESAREAN-SECTION OR NATURAL DELIVERY

PERIOD: JANUARY 2010 - JANUARY 2012TOTAL NO. STUDIES 41TOTAL NO. DOES 696NO. PREGNANT 655NO. DIED 7*NO. ABORTED 3NO. DELIVERED PREMATURELY 1NO. DOES WITH 100% RESORPTIONS 0

RANGE / STUDY GROSS LESIONS N % N %EPIGLOTTIS

Red area proximal to epiglottis 1 0.14 0-1 (0-4.2)

ESOPHAGUS/TRACHEAPerforation (found dead or euthanized) 2 0.29 0-1 (0-16.7)

THORACIC CAVITYContained red fluid (found dead) 1 0.14 0-1 (0-16.7)

HEARTPale and/or white 1 0.14 0-1 (0-20.0)Large 1 0.14 0-1 (0-20.0)White band surrounded ventricles 1 0.14 0-1 (0-20.0)

LUNGSDiscolored and/or rough 5 0.72 0-2 (0-16.7)

LIVERDiscolored 3 0.43 0-1 (0-20.0)Median lobe was misshapen 1 0.14 0-1 (0-5.0)All lobes thick 1 0.14 0-1 (0-20.0)Extra lobe 2 0.29 0-1 (0-5.0)Bifurcation - median lobe 1 0.14 0-1 (0-2.5)

GALLBLADDERAbsent 2 0.29 0-1 (0-5.6)

* Four were unscheduled euthanasia and two were intubation accidents


SUMMARY OF MATERNAL NECROPSY OBSERVATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF]

DAY 29 CAESAREAN-SECTION OR NATURAL DELIVERY

RANGE / STUDY GROSS LESIONS N % N %

STOMACHRed substance present 1 0.14 0-1 (0-2.5)Mucosal surface of cardiac region, edematous; mucosal surface of fundic and pyloric regions, numerous red foci 1 0.14 0-1 (0-4.2)

SPLEENAccessory spleen 6 0.86 0-2 (0-10.0)Constricted area 1 0.14 0-1 (0-5.0)Misshapen 2 0.29 0-1 (0-5.0)Rough 1 0.14 0-1 (0-20.0)Raised area 1 0.14 0-1 (0-5.0)Small 1 0.14 0-1 (0-20.0)

PANCREASLymph node(s), red or dark red 1 0.14 0-1 (0-5.0)

KIDNEY(S)Pelvis, slight dilation 1 0.14 0-1 (0-12.5)

URETERSNumerous calculi 1 0.14 0-1 (0-12.5)

OVARYLeft, small 1 0.14 0-1 (0-20.0)

UTERUSHorn(s), reduced to a ligament 6 0.86 0-2 (0-25.0)Left horn; clear, fluid-filled cyst 1 0.14 0-1 (0-5.0)

ADIPOSE TISSUEAbdominal adipose, friable 2 0.29 0-1 (0-5.0)Mass present, caudal to right ovary, tan and firm 1 0.14 0-1 (0-5.0)


LITTER AND FETAL ALTERATIONS SUMMARY NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION


NUMBER OF STUDIES: 30

NUMBER OF LITTERS EVALUATED: 572

NUMBER OF FETUSES EVALUATED (LIVE): 4918

RANGE/STUDY MEAN or % MEAN or %

LITTERS WITH FETUSES WITHANY ALTERATION OBSERVED % 38.4 (10.0-66.7)

FETUSES WITH ANYALTERATION OBSERVED % 6.7 (1.7-14.6)

% FETUSES WITH ANYALTERATION/LITTER MEAN 6.6 (1.6-13.9)


SUMMARY OF FETAL GROSS EXTERNAL ALTERATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION

PERIOD: JANUARY 2010 - JANUARY 2012FULL DOSAGE-RANGE

STUDIES STUDIES

NO. OF STUDIES 29 12NO. LITTERS EXAMINED 580 63NO. LIVE FETUSES EXAMINED 5004 504

ALTERATION RANGE/STUDY RANGE/ STUDYN % N % N % N %

HEADTongue protrudes L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Domed L 1 0.17 0-1 (0-5.3) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Exencephaly L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Ears low set L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Acrania L 2 0.34 0-1 (0-5.3) 0 --- --- ---

F 2 0.04 0-1 (0-0.6) 0 --- --- ---

EYEOne or both lids open L 1 0.17 0-1 (0-4.2) 1 1.59 0-1 (0-14.3)

F 1 0.02 0-1 (0-0.5) 1 0.20 0-1 (0-1.6)

PALATECleft L 0 --- --- --- 1 1.59 0-1 (0-25.0)

F 0 --- --- --- 1 0.20 0-1 (0-3.1)

SNOUTShort L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Cleft L 0 --- --- --- 2 3.17 0-1 (0-25.0)

F 0 --- --- --- 2 0.40 0-1 (0-3.1)

BODY Umbilical hernia L 4 0.69 0-1 (0-6.2) 1 1.59 0-1 (0-14.3)

F 4 0.08 0-1 (0-0.8) 1 0.20 0-1 (0-1.6)

L: LITTER INCIDENCEF: FETAL INCIDENCE


SUMMARY OF FETAL GROSS EXTERNAL ALTERATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION

FULL DOSAGE-RANGESTUDIES STUDIES

ALTERATION RANGE/STUDY RANGE/ STUDYN % N % N % N %

BODY (CONT.) Gastroschisis L 2 0.34 0-1 (0-5.3) 0 --- --- ---

F 2 0.04 0-1 (0-0.6) 0 --- --- ---Trunk short L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Spina bifida L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Thoracogastroschisis L 1 0.17 0-1 (0-4.2) 0 --- --- ---

F 1 0.02 0-1 (0-0.5) 0 --- --- ---Skin thin L 1 0.17 0-1 (0-5.0) 0 --- --- ---

F 1 0.02 0-1 (0-0.6) 0 --- --- ---

FORE AND/OR HINDLIMB(S)

Paw(s), Flexed/Rotated L 3 0.52 0-1 (0-5.3) 0 --- --- ---F 3 0.06 0-1 (0-0.6) 0 --- --- ---

Limb(s), Flexed L 2 0.34 0-1 (0-5.3) 0 --- --- ---F 2 0.04 0-1 (0-0.6) 0 --- --- ---

Limb(s), Rotated L 2 0.34 0-1 (0-5.3) 0 --- --- ---F 2 0.04 0-1 (0-0.6) 0 --- --- ---

Limb(s), Short L 1 0.17 0-1 (0-4.2) 0 --- --- ---F 1 0.02 0-1 (0-0.5) 0 --- --- ---

Digit(s), Absent L 2 0.34 0-1 (0-5.3) 0 --- --- ---F 2 0.04 0-1 (0-0.6) 0 --- --- ---

Digit(s), Short L 2 0.34 0-1 (0-5.3) 0 --- --- ---F 2 0.04 0-1 (0-0.6) 0 --- --- ---

TAILBent L 0 --- --- --- 1 1.59 0-1 (0-25.0)

F 0 --- --- --- 1 0.20 0-1 (0-2.8)Short L 0 --- --- --- 1 1.59 0-1 (0-25.0)

F 0 --- --- --- 1 0.20 0-1 (0-2.8)



SUMMARY OF FETAL SOFT TISSUE ALTERATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION


NO. STUDIES 34NO. LITTERS EXAMINED 607NO. FETUSES EXAMINED 5219

ALTERATION RANGE/STUDYN % N %

BRAINSlight ventricular dilation L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Moderate ventricular dilation L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)

THYMUSRed L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)

EYE(S)Circumcorneal hemorrhage L 3 0.49 0-2 (0-10.5)

F 3 0.06 0-2 (0-1.2)Microphthalmia L 3 0.49 0-2 (0-10.5)

F 3 0.06 0-2 (0-1.2)

LUNGSOne or more lobes, partial or L 16 2.64 0-4 (0-15.8) complete agenesis F 22 0.42 0-7 (0-2.2)Small L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)Protrudes through thoracic opening L 1 0.16 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)Lobes fused L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

DIAPHRAGMDiaphragm absent L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

HEARTInterventricular septal defect L 4 0.66 0-1 (0-5.3)

F 4 0.08 0-1 (0-0.6)





HEART (CONT.)Two semilunar valves present L 1 0.16 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)Semilunar valves absent L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Protrudes through thoracic opening L 1 0.16 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)Atrium absent L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Septum absent L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

VESSELSPersistent truncus arteriosus L 5 0.82 0-1 (0-5.3)

F 5 0.10 0-1 (0-0.6)Left carotid artery arises from L 2 0.33 0-1 (0-25.0) the innominate artery F 2 0.04 0-1 (0-3.1)Innominate artery absent L 4 0.66 0-1 (0-5.3)

F 4 0.08 0-1 (0-0.6)Aorta passes dorsal to the L 1 0.16 0-1 (0-5.0) trachea and esophagus F 1 0.02 0-1 (0-0.6)Constricted pulmonary L 1 0.16 0-1 (0-5.6)

F 1 0.02 0-1 (0-0.6)Left subclavian arises from the L 1 0.16 0-1 (0-5.0) pulmonary artery F 1 0.02 0-1 (0-0.6)Right subclavian artery arises to left L 1 0.16 0-1 (0-25.0) of left subclavian F 1 0.02 0-1 (0-3.1)Right subclavian artery passes L 1 0.16 0-1 (0-25.0) dorsal to trachea and esophagus F 1 0.02 0-1 (0-3.1)Aorta descends to the right L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Subclavian absent L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Carotid arteries absent L 2 0.33 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6)Aorta distended L 2 0.33 0-1 (0-5.6)

F 2 0.04 0-1 (0-0.6)





LIVERDiscolored L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.5)Protrudes through umbilicus or L 5 0.82 0-1 (0-6.2) abdominal wall F 5 0.10 0-1 (0-0.8)Misshapen L 3 0.49 0-1 (0-5.3)

F 3 0.06 0-1 (0-0.6)Adhesion L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.5)Extra lobe L 1 0.16 0-1 (0-2.9)

F 1 0.02 0-1 (0-0.3)Thick L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)Cysts L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

GALLBLADDERAbsent L 2 0.33 0-1 (0-5.0)

F 2 0.04 0-1 (0-0.6)

KIDNEY(S)Low set L 2 0.33 0-1 (0-5.0)

F 2 0.04 0-1 (0-0.6)Absent L 2 0.33 0-1 (0-25.0)

F 2 0.04 0-1 (0-3.1)Misshapen L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)Protrude through abdominal L 1 0.16 0-1 (0-4.2) opening F 1 0.02 0-1 (0-0.5)Pelvis, marked dilation L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)High set L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

ADRENALSFused L 1 0.16 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)





STOMACHProtrudes through abdominal L 2 0.33 0-1 (0-5.0) opening F 2 0.04 0-1 (0-0.6)

SPLEENProtrudes through abdominal L 2 0.33 0-1 (0-5.0) opening F 2 0.04 0-1 (0-0.6)Small L 1 0.16 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)

PANCREASProtrudes through abdominal L 2 0.33 0-1 (0-5.0) opening F 2 0.04 0-1 (0-0.6)

INTESTINESProtrudes through umbilicus L 6 0.99 0-1 (0-14.3) or abdominal wall F 6 0.12 0-1 (0-1.6)

BLADDERProtrudes through umbilicus L 1 0.16 0-1 (0-4.2) or abdominal wall F 1 0.02 0-1 (0-0.5)

URETERSAbsent L 2 0.33 0-1 (0-25.0)

F 2 0.04 0-1 (0-3.1)

GENITALIATestes, high set L 1 0.16 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)



SUMMARY OF FETAL SKELETAL ALTERATIONS NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION

PERIOD: JANUARY 2010 - JANUARY 2012NO. STUDIES 31NO. LITTERS EXAMINED 591NO. FETUSES EXAMINED 5087

ALTERATIONS RANGE/ STUDY N % N %

SKULLSummarization of all irregular L 42 7.11 0-4 (0-33.3) ossification of skull F 56 1.10 0-6 (0-4.6)

Anterior Fontanelle : Irregularly shaped L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)

Frontals : Interfrontals present L 3 0.51 0-1 (0-5.0)

F 3 0.06 0-1 (0-0.6) : Intrafrontals present L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : Fused L 3 0.51 0-1 (0-5.3)

F 3 0.06 0-1 (0-0.6)

Parietals : Contained an interparietal L 1 0.17 0-1 (0-4.0)

F 1 0.02 0-1 (0-0.5) : Contained an intraparietal L 3 0.51 0-1 (0-5.0)

F 3 0.06 0-1 (0-0.6)

Interparietals : Incompletely ossified L 3 0.51 0-1 (0-6.2)

F 4 0.08 0-2 (0-1.5) : Not ossified L 1 0.17 0-1 (0-6.2)

F 1 0.02 0-1 (0-0.8) : Small L 1 0.17 0-1 (0-4.8)

F 1 0.02 0-1 (0-0.6)

Nasals : Contained an internasal L 10 1.69 0-2 (0-10.5)

F 10 0.20 0-2 (0-1.2) : Contained an intranasal L 5 0.85 0-2 (0-10.0)

F 6 0.12 0-2 (0-1.1)




RANGE/ STUDY ALTERATIONS N % N %

SKULL (CONT.)Nasals (cont.) : Suture, displaced L 24 4.06 0-3 (0-16.7)

F 25 0.49 0-3 (0-2.3) : Suture, irregular L 2 0.34 0-2 (0-10.5)

F 2 0.04 0-2 (0-1.3) : Fused L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : Short L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)

Nasal/Frontal: sutures, irregular L 21 3.55 0-3 (0-20.0) and/or misaligned F 27 0.53 0-5 (0-2.5)Premaxillae: Short L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)Maxillae: Short L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)Eye socket: Small L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Close-set L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)Basioccipitals: Incompletely L 1 0.17 0-1 (0-6.7) ossified F 1 0.02 0-1 (0-0.8)Skull: Incompletely or not L 1 0.17 0-1 (0-4.2) ossified F 1 0.02 0-1 (0-0.5) : Small L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Absent L 2 0.34 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6)

HYOID

Ala(e), angulated L 84 14.21 0-8 (0-40.0) F 114 2.24 0-10 (0-5.7)

Ala(e), incompletely or not L 2 0.34 0-1 (0-5.3) ossified F 2 0.04 0-1 (0-0.6)Absent L 2 0.34 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6)Ala, small L 2 0.34 0-1 (0-6.2)

F 2 0.04 0-1 (0-0.8)





VERTEBRAECervical : Arches and/or Centra, L 2 0.34 0-1 (0-5.3) fused F 2 0.04 0-1 (0-0.6) : Arch, incompletely ossified L 3 0.51 0-1 (0-6.7)

F 3 0.06 0-1 (0-0.8) : Arch, irrregularly shaped L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Centrum, misaligned L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : Centrum, not ossified L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Centrum, small L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Centrum, unilateral L 1 0.17 0-1 (0-6.7) ossification F 1 0.02 0-1 (0-0.8) : Extra ossification L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.5) : 6 present L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)

Thoracic : Hemivertebra L 3 0.51 0-2 (0-10.5)

F 3 0.06 0-2 (0-1.1) : Arches and/or Centra, L 7 1.18 0-2 (0-10.5) fused F 7 0.14 0-2 (0-1.1) : Centrum, unilateral L 3 0.51 0-1 (0-6.7) ossification F 3 0.06 0-1 (0-0.8) : Centrum, bifid L 6 1.02 0-2 (0-5.9)

F 6 0.12 0-2 (0-0.7) : Centrum, irregularly shaped L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Arch, small L 3 0.51 0-1 (0-5.6)

F 3 0.06 0-1 (0-0.7) : Arch, open L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : 11 present L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

Lumbar : Hemivertebra L 2 0.34 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6)





VERTEBRAE (CONT.)

Lumbar (cont.) : Arches and/or Centra, L 3 0.51 0-1 (0-5.3) fused F 3 0.06 0-1 (0-0.6) : Centrum, bifid L 2 0.34 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6) : Arch, open L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Arch, incompletely L 1 0.17 0-1 (0-4.2) ossified F 1 0.02 0-1 (0-0.5)

Sacral : Arch, incompletely L 1 0.17 0-1 (0-4.2) ossified F 1 0.02 0-1 (0-0.5)

Caudal : One or more misaligned L 20 3.38 0-2 (0-13.3)

F 20 0.39 0-2 (0-1.6) : Fused L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Small L 2 0.34 0-1 (0-5.6)

F 2 0.04 0-1 (0-0.6) : Incompletely ossified L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : 13 present L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) : Irregularly shaped L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

RIBS Cervical rib(s) present L 4 0.68 0-2 (0-10.5)

F 5 0.10 0-3 (0-1.8) Two or more, fused L 4 0.68 0-1 (0-6.7)

F 4 0.08 0-1 (0-0.8) One or more, split L 2 0.34 0-1 (0-5.3)

F 2 0.04 0-1 (0-0.6) One or more, thickened areas L 8 1.35 0-1 (0-6.7)

F 9 0.18 0-2 (0-1.1) Thin L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)





RIBS (CONT.) Flat L 3 0.51 0-1 (0-5.3)

F 3 0.06 0-1 (0-0.6) Wavy L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5) Proximate L 4 0.68 0-1 (0-5.9)

F 4 0.08 0-1 (0-0.6) Short L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) Extra L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) 11 present L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) Irregularly shaped L 2 0.34 0-1 (0-5.6)

F 2 0.04 0-1 (0-0.7) Two segments L 1 0.17 0-1 (0-5.6)

F 1 0.02 0-1 (0-0.7)

STERNEBRAE Two or more, fused L 45 7.61 0-5 (0-26.3)

F 56 1.10 0-5 (0-3.2) One or more asymmetric L 8 1.35 0-2 (0-10.0)

F 8 0.16 0-2 (0-1.1) One or more, incompletely L 9 1.52 0-2 (0-10.0) or not ossified F 9 0.18 0-2 (0-1.1) Large L 2 0.34 0-1 (0-5.0)

F 2 0.04 0-1 (0-0.6) Irregularly shaped/variation in L 3 0.51 0-1 (0-6.2) shape F 3 0.06 0-1 (0-0.8) 1 present L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

MANUBRIUM Fused L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6) Irregularly shaped L 10 1.69 0-2 (0-10.0)

F 10 0.20 0-2 (0-1.1) Small L 5 0.85 0-2 (0-13.3)

F 5 0.10 0-2 (0-1.6) Absent L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)





MANUBRIUM (CONT.) Not ossified L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)

XIPHOID Irregularly shaped L 5 0.85 0-1 (0-5.3)

F 5 0.10 0-1 (0-0.6) Fused L 1 0.17 0-1 (0-2.9)

F 1 0.02 0-1 (0-0.3) Large L 2 0.34 0-1 (0-5.6)

F 2 0.04 0-1 (0-0.6) Absent L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

SCAPULAE Ala(e): thin L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) Ala(e): wavy L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6)

CLAVICULAE Irregularly shaped L 1 0.17 0-1 (0-5.0)

F 1 0.02 0-1 (0-0.6)

PELVIS

Pubis(es): incompletely or not L 6 1.02 0-2 (0-10.5) ossified F 6 0.12 0-2 (0-1.2) Ilium, small L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)

FORELIMB(S)

: Phalanx, misaligned L 2 0.34 0-1 (0-5.3)F 2 0.04 0-1 (0-0.6)

: Phalanx, absent L 4 0.68 0-1 (0-5.9)F 4 0.08 0-1 (0-0.6)

: Phalanx, not ossified L 2 0.34 0-1 (0-5.3)F 2 0.04 0-1 (0-0.6)

: Phalanx, small L 1 0.17 0-1 (0-5.9)F 1 0.02 0-1 (0-0.6)





FORELIMB(S) (CONT.) : Digit(s), absent L 3 0.51 0-1 (0-5.9)

F 3 0.06 0-1 (0-0.6) : Digits, 4 present L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Metacarpals, absent L 2 0.34 0-1 (0-5.9)

F 2 0.04 0-1 (0-0.6) : Metacarpals, not ossified L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : Metacarpals, 4 present L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5)

HINDLIMB(S)

: Phalanx or Phalanges, not L 1 0.17 0-1 (0-5.3) ossified F 1 0.02 0-1 (0-0.6) : Phalanx, absent L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Phalanx, small L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.6) : Limb, small L 1 0.17 0-1 (0-4.2)

F 1 0.02 0-1 (0-0.5) : Tibia, short L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5) : Fibula, bent L 1 0.17 0-1 (0-5.3)

F 1 0.02 0-1 (0-0.5)



SUMMARY OF FETAL OSSIFICATION SITE AVERAGES NEW ZEALAND WHITE RABBITS [Hra:(NZW)SPF] DAY 29 CAESAREAN-SECTION


NO. STUDIES: 31NO. LITTERS EXAMINED: 591NO. FETUSES EXAMINED: 5087

FETUS / LITTER

SKELETAL AVERAGES MEAN RANGE/STUDY

HYOID 1.00 (0.98-1.00)

VERTEBRAE CERVICAL 7.00 --- THORACIC 12.52 (12.40-12.66) LUMBAR 6.48 (6.34-6.60) SACRAL 3.00 --- CAUDAL 16.67 (16.43-16.84)

RIBS (Pairs) 12.45 (12.34-12.58)

STERNUM MANUBRIUM 1.00 (0.99-1.00) STERNAL CENTERS 3.91 (3.74-3.97) XIPHOID 0.95 (0.89-1.00)

FOREPAWS (Calculated as average per limb) CARPALS 0.00 --- METACARPALS 4.96 (4.90-5.00) DIGITS 5.00 --- PHALANGES 13.90 (13.80-13.97)

HINDPAWS (Calculated as average per limb) TARSALS 2.00 (1.99-2.00) METATARSALS 4.00 --- DIGITS 4.00 --- PHALANGES 12.00 (11.99-12.00)



Appendix 18 Tabulated Summary of the Common Technical Document


The Common Technical Document —Safety__________________________________________________________________________________

2.6.7.6 Repeat-Dose Toxicity Nonpivotal Studies Test Article: Thymosin beta 4 (Tβ4) formulated as RGN-352 (Tβ4 Injectable Solution)

Species/ Strain

Method of Administration (Vehicle/ Formulation)

Duration of Dosing

Doses (mg/kg)

Gender and No. per Group

NOAELa (mg/kg)

Noteworthy Findings

Study Number

Rabbit/ New Zealand White

Intravenous injection (aqueous solution)

5 days 0, 10, 30, 90 F: 3 90 none 20024505

_____________ a. No Observed Adverse Effect Level.


The Common Technical Document — Safety____________________________________________________________________________________________

2.6.7.13 Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development

Report Title: A Dosage Range-finding Embryo-fetal Development Study of Thymosin beta 4 (formulated as RGN-352) by Intravenous Injection in Rabbits, Including a Preliminary Evaluation in Non-Pregnant Rabbits

Test Article: Thymosin beta 4 (Tβ4) formulated as RGN-352 (Tβ4 Injectable Solution)

Design similar to ICH 4.1.3? Yes Species/Strain: Rabbit/ New Zealand White Initial Age: 6 ½ months

Duration of Dosing: DG 7 through 19 Study No. 20024505 Location in CTD: Vol.! Page !

Date of First Dose: 20 Jun 2012 Day of Mating: DG 0

Special Features: Toxicokinetics Day of C-Section: DG 29 No Observed Adverse Effect Level:

F0 Females: 90 mg/kg/day F1 Litters: 90 mg/kg/day

Method of Administration: Intravenous injection Vehicle/Formulation: Aqueous solution

GLP Compliance: Yes

Daily Dose (mg/kg) 0 (Control) 3 10 30 90 Does: Toxicokinetics: AUCall (µgh/mL) DG 7 n/a 24.5 95.8 280 753 DG 19 n/a 18.2 82.7 195 601 No. Pregnant 5 4 4 4 5 No. Died or Sacrificed Moribund 0 0 0 0 0 No. Aborted or with Total Resorption of Litter 0 0 0 0 0 Clinical Observations - - - - - Necropsy Observations - - - - - Body Weight DG 20 (%)a 3.97 kg -3.5 +2.8 -1.5 -0.3 Food Consumption DG 7-20 (%)a 144.8 g/day +1.4 +20.8 +5.2 -0.5

Mean No. Corpora Lutea 9.8 10.8 8.2 8.8 11.4 Mean No. Implantations 9.4 10.0 6.8 8.8 10.4 Mean % Preimplantation Loss 4.0 6.8 24.0 0.0 8.0

- No noteworthy findings, DG = Day of presumed gestation a. At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown.


The Common Technical Document —Safety____________________________________________________________________________________________ 2.6.7.13 Reproductive and Developmental Toxicity -

Effects on Embryo-fetal Development (Continued) Study No. 20024505

Daily Dose (mg/kg) 0 (Control) 3 10 30 90 Litters: No. Litters Evaluated 5 4 4 4 5 No. Live Fetuses 46 38 27 34 49 Mean No. Resorptions 0.2 0.5 0.0 0.2 0.6 No. of Litters with Dead Fetuses 0 0 0 0 0 Mean % Postimplantation Loss 2.0 3.8 0.0 2.1 4.9 Mean Fetal Body Weight (g) 42.00 40.54 46.59 41.36 37.81 Fetal Sex Ratios (% males) 47.9 44.7 69.0 44.9 33.6 Fetal Anomalies:

Gross External - - - - - Total Affected Fetuses (Litters) 0 (0) 0 (0) 0 (0) 1 (1) 0 (0)

- No noteworthy findings


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