Analysis of the Immune Response to PEGylated Nanoparticles By

Analysis of the Immune Response to PEGylated Nanoparticles

By

Ken Simmons

A dissertation submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

(Pharmaceutical Sciences)

at the

UNIVERSITY OF WISCONSIN - MADISON

2012

Date of final oral examination: 06/01/12

The dissertation is approved by the following members of the Final Oral Committee:

Sandro Mecozzi

Ralph Albrecht

Glen Kwon

Mark Cook

Robert Pearce

i

Acknowledgments

First, I would like to thank my two advisors, Dr. Sandro Mecozzi and Dr. Ralph Albrecht.

It was a great experience working in collaboration with these two labs. Thank-you for all your

help. You were both essential in putting this project together. I would also like to thank my

committee members, Dr. Glen Kwon, Dr. Robert Pearce, and Dr. Mark Cook for their advice and

additional guidance on these projects. Special recognition is needed for Dr. Becky Johnson for

her role in the in vivo dog studies and for her contributions to this thesis. In addition, thanks to

all the lab members from both the Mecozzi and Albrecht labs for your help along the way.

Thanks to Elham Nejati for synthesizing many of the particles discussed in this dissertation and

to Jonathan Fast for guidance in the early stages of this project as well as his contributions to this

thesis.

Finally, I would like to thank my parents and the rest of my family for their help and

support for this time in Madison.

Most importantly, thanks to my wife, Betsie. My time with you has been the best in my

life; thanks for everything.

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Table of Contents Acknowledgements ............................................................................................................... i

Table of Contents ................................................................................................................. ii

List of tables and figures ..................................................................................................... vi

Abstract .............................................................................................................................. vii

Chapter 1: Introduction and Background ............................................................ 1

Introduction ........................................................................................................................ 2

Thesis Approach and Outline .............................................................................................. 2

Background (in vivo canine studies) ................................................................................... 4

Gel and Coombs Classification of Hypersensitivities .......................................................... 7

CARPA................................................................................................................................ 10

Immunogenicity and PEGylated Drugs ............................................................................. 14

CARPA and FDA Approved PEGylated Agents ................................................................... 14

Antibody Generation to PEGylated Agents....................................................................... 15

CARPA in Preclinical Studies ............................................................................................. 16

PEGylated Micelles in Clinical Trials .................................................................................. 17

Chapter 1 References ........................................................................................................ 19

Chapter 2: Histamine Release Associated with Intravenous Delivery of a Fluorocarbon-Based Sevoflurane Emulsion in Canines ...................................... 23

Preface .............................................................................................................................. 24

Abstract ............................................................................................................................. 25

Introduction ...................................................................................................................... 26

Materials and Methods ..................................................................................................... 27

Sevoflurane (20% v/v) Emulsion Preparation ................................................................... 27

Animals ............................................................................................................................. 28

Pilot Studies ...................................................................................................................... 28

Part I: Cardiovascular Measurements ............................................................................... 29

Part II: Component Analysis .............................................................................................. 30

Histamine Analysis ............................................................................................................ 31

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Complement Analysis ....................................................................................................... 31

Statistical Analysis ............................................................................................................. 32

Results ............................................................................................................................... 33

Emulsion Sizing ................................................................................................................. 33

Pilot Studies ...................................................................................................................... 33

Part I: Cardiovascular Effects ............................................................................................ 34

Part II: Component Analysis .............................................................................................. 35

Complement Analysis ....................................................................................................... 36

Discussion.......................................................................................................................... 39

Conclusion ......................................................................................................................... 42 Acknowledgments............................................................................................................. 43


Chapter 3: Disadvantages of Using Hemolytic Assays to test PEGylated Block Copolymers for Complement Activation During Drug Development .................. 46

Abstract ............................................................................................................................. 47

Introduction ...................................................................................................................... 48


Polymer Preparations, Blood, and Serum Solutions ......................................................... 51

Modified Complement Hemolytic Assay .......................................................................... 52

Alternative Method Complement Hemolytic Assay ......................................................... 54

Results ............................................................................................................................... 55

Discussion.......................................................................................................................... 61

Conclusion ......................................................................................................................... 65

Acknowledgements ........................................................................................................... 66


Chapter 4: Enhanced Allergic-Type Response to PEG-Fluorocarbon Polymer Used for Intravenous Delivery of Sevoflurane Upon Repeat Injection in Canines ....... 71

Abstract ............................................................................................................................. 72

Introduction ...................................................................................................................... 74


Animals ............................................................................................................................. 75

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Study Design ..................................................................................................................... 75

Emulsion Preparation ....................................................................................................... 77

Polymer Solution Preparation........................................................................................... 77


Statistical Analysis ............................................................................................................. 78

Results ............................................................................................................................... 79

Discussion.......................................................................................................................... 82

Conclusion ......................................................................................................................... 84


Chapter 5: Antibody Generation Associated with Intravenous Delivery of PEG-fluorocarbon polymers and other PEGylated Nanoparticles in Dogs ................. 87

Abstract ............................................................................................................................. 88

Introduction ...................................................................................................................... 90


Dog Study Design .............................................................................................................. 91

Preparation of Polymeric Micelle and Dendrimer Solutions ............................................ 92

Blood Processing and Collection ....................................................................................... 92


Nanoparticle-Specific IgM ELISA ....................................................................................... 93

Results ............................................................................................................................... 94

Discussion........................................................................................................................ 100

Conclusion ....................................................................................................................... 103

Chapter 5 References ...................................................................................................... 104

Chapter 6: Analysis of the Immune Response to a Fluorocarbon-Based Sevoflurane Emulsion in Monkeys .................................................................. 106

Abstract ........................................................................................................................... 107

Introduction .................................................................................................................... 108

Materials and Methods ................................................................................................... 108

Study Design ................................................................................................................... 108

Blood Processing and Collection ..................................................................................... 109

Histamine Analysis .......................................................................................................... 109

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Anti-F13M5 IgM ELISA .................................................................................................... 110

Results ............................................................................................................................. 110

Discussion........................................................................................................................ 111

Conclusion ....................................................................................................................... 112


Chapter 7: Conclusions and Future Directions ................................................. 114

Conclusions ..................................................................................................................... 115

Future Studies ................................................................................................................. 116

Mechanism of HSR to F13M1 ......................................................................................... 116

Development of Intravenous Sevoflurane ...................................................................... 119

Clinical Development of PEGylated Nanoparticles ......................................................... 120


Appendix………………………………………………………………………………………………………………………….…123

vi

List of Tables and Figures

Figures

Figure 1.1: The structure of F13M5 ........ …………………………………………………………4 Figure 1.2: The complement cascade .................................... …………………………………....11 Figure 2.1: The structure of F13M5, H13M5, H19M5……………..…………………………....27

Figure 2.2: Heart rate values and areterial blood pressures after administration of sevoflurane emulsion in

dogs……………………..…………………………….…………………………………………..35

Figure 2.3: Histamine analysis (F13M5).......………..…………………………………………..36

Figure 2.4: C3a and C5a (in vitro incubation of F13M5 in human serum)……….................….38

Figure 3.1: The structure of F13M5, mPEG , Pluronic F-68………..………………………..….50

Figure 3.2: Guinea Pig RBC Complement Hemolytic Assay (F13M5 and mPEG5K)……...…..55

Figure 3.3: Sheep RBC Complement Hemolytic Assay (F13M5 and mPEG5K)….................…57

Figure 3.4: Sheep and Guinea Pig Hemolytic Assays (Pluronic F-68)…..……………...……….59

Figure 3.5: Sheep RBC Complement Hemolytic Assay (Alternate Method - F13M5 and

mPEG5K)………………………………..………..…………………………….………………....60

Figure 3.6: Guinea Pig RBC Complement Hemolytic Assay (Alternate Method - F13M5, mPEG 550,

mPEG 5K)………………….......………..…………………………………………………………61

Figure 4.1: The structure of F13M1, G1F13M1, G3F13M1………..…………………………….74

Figure 4.2: Histamine analysis (F13M1, G1F13M1, and G3F13M1)…………………….………79

Figure 4.3: Histamine analysis (Submicellar F13M1).…………………………………………....81

Figure 4.4: Histamine analysis (Micellar solutions of F13M1 or G1F13M1 in saline)…………...82

Figure 5.1: Structure of F13M1 and G1F13M1…………………………………………………...90

Figure 5.2: Structure of mPEG1K-DSPE, PAMAM G3 dendrimer, and PEGylated PAMAM G3

dendrimer……………………………………………………………………………………….…..91

Figure 5.3: IgM generation (F13M1 vs. Saline)....…………………………….……………….….95

Figure 5.4: IgM generation (F13M1 micellar solution)…..………………………………………..96

Figure 5.5: IgM generation (G1F13M1)……………..……………………………………….…....97

Figure 5.6: Histamine analysis (mPEG1K-DSPE)…….……………………….…………….….....98

Figure 5.7: IgM generation (mPEG1K-DSPE)….…..……………………………….………….....98

Figure 5.8: Histamine analysis (PEGylated dendrimers)….………..……………………………...99

Figure 5.9: IgM generation(PEGylated dendrimers)………….……………….……......................99

Figure 6.1: Histamine analysis (F13M1 Micellar Solution and Full Emulsion -

monkeys)…………………………………………………………………………….......................110

Figure 6.2: IgM generation (F13M1 - monkeys)……………..…………………………………...111

Figure A.1: IgG generation (F13M1 - dogs)…………………………………………....................124

Tables

Table 1.1: Full emulsion injections (F13M5 in dogs)…………………….………...……………..…5

Table 1.2: Injection of emulsion components (emulsion without sevoflurane, F13M5 micellar solution,

and mPEG 5K solution)…………………………..……………………………………………….…..6

Table 1.3: Gel and Coombs classification of hypersensitivity reactions…………........................…..7

Table 1.4: Similarities and differences between CARPA and IgE mediated allergic

reactions…………………………………………………………………………………………...….12

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Abstract

Hypersensitivity reactions (HSRs) are a major problem in the field of medicine, with

anaphylaxis estimated to cause 500-1000 deaths per year in the US [1]. Recently, there has been

discussion about rapid-onset HSRs to liposomes and micellar solvents. These reactions, which

can occur in up to 45% of patients, are thought to be due to complement activation in a form of

pseudoallergy known as complement activation related pseudoallergy (CARPA) [2]. Though

complement activation has been linked to HSRs involving liposomes and solvents, questions

remain about the exact mechanism of action in certain cases. Specifically, what role does

polyethylene-glycol play in some HSRs to PEGylated nano systems?

This thesis examined (pseudo)allergy to PEGylated nanoparticles by investigating the

HSR elicited by a mPEG-fluorocarbon-based sevoflurane emulsion in dogs. In particular, both

direct complement activation and antibody-based mechanisms were explored as the cause of the

immune response. By examining the mechanism behind the HSR associated with this molecule,

guidelines can be developed to diminish immunogenicity associated with intravenous

sevoflurane. The mPEG-fluorocarbon polymer used in our emulsion (F13M1) did not cause

direct complement activation in human serum. A noted characteristic of other PEGylated

particles associated with CARPA is a diminishing or absent HSR upon repeat injection. Unlike

these particles, the HSR to F13M1 appears to strengthen, suggesting an antibody-based

mechanism. Since these antibodies are likely to the PEG moiety, modifications to F13M1 were

made in order to “mask” the PEG surface. Antibody generation and histamine release to these

particles, and other PEGylated nanoparticles, were characterized in a dog model to further

explore the mechanism. Results suggest that other factors, such as size and micelle stability, may

viii

also play a role in the HSR. Additional studies in monkeys suggest that F13M1 may not be as

immunogenic in this species. In the end, these findings can be used to guide development of

intravenous sevoflurane and direct immunogenicity studies for other PEGylated carriers.

References

1. Lee JK, Vadas P. Anaphylaxis: mechanisms and management. Clin Exp Allergy 2011, 41,

923-38.

2. Szebeni, J. Complement activation-related pseudoallergy: A new class of drug induced acute

immune toxicity. Toxicology 2005, 216, 106.

1

Chapter 1:

Introduction and Background

2

Introduction

Polyethylene glycol (PEG) has been incorporated into many nanodrug formulations to

improve everything from kinetics, to solubility, to protein adsorption [1, 2]. It has even been

stated that PEG adds a “stealth” effect to nanoparticles [3]. Yet, hypersensitivity reactions which

may involve PEG are an adverse effect of some of these nanoparticles [4,5]. It is possible that

these immune reactions could be attributed to complement activation related pseudoallergy

(CARPA) or classical Type I IgE mediated hypersensitivity. To date, there has been no

comprehensive study to pinpoint this mechanism.

The overall goal of this dissertation is to examine the role of PEG and PEGylated

nanoparticles in hypersensitivity reactions. The overlying hypothesis is that PEG can activate

complement or generate antibodies that can lead to hypersensitivity reactions to these

nanoparticles.

Thesis Approach and Outline

This dissertation, a biology-based study on hypersensitivity reactions, actually had its

beginnings in a chemistry lab working on polymers for drug delivery. During in vivo canine

testing of a PEG-fluorocarbon polymer used for intravenous delivery of sevoflurane, an

unexpected hypersensitivity was observed. This allergic-type reaction was generated by a

polymer that was predicted to be non-immunogenic. Similar cases of hypersensitivity have been

reported in the literature [5]. Hence, it has become important to understand nanoparticle-

associated hypersensitivities. This dissertation investigates hypersensitivities associated with this

PEG-fluorocarbon polymer (F13M5), as well as other PEGylated block copolymers.

3

Chapter 2 presents the canine studies, as well as in vitro studies examining the polymer’s

direct effects on human complement.

Chapter 3 looks at the variability in results obtained when using F13M5 and other

PEGylated block copolymers in hemolytic assays designed to identify complement activation

prior to drug development. The analysis shows that some polymers may have multiple effects on

the complement system and/or RBC membranes, suggesting that investigators should use caution

when using similar methodology.

Chapter 4 involves in vivo studies in canines. This study used modified versions of the

PEG-fluorocarbon polymer in an attempt to diminish or eliminate the ensuing allergic-type

response. In addition, the study was designed to distinguish between direct complement

activation and antibody generation as possible mechanisms involved in the adverse reaction. A

strengthening of the immune response upon repeat injection was observed, suggesting antibody

involvement as the mechanism behind the hypersensitivity.

Chapter 5 characterizes the antibody-based response described in Chapter 4. In addition,

antibody generation to other PEGylated block copolymers is described. Our primary polymer

(F13M1) generates an IgM response, but does not appear to substantially effect IgG. The current

hypothesis is that classical complement activation, caused by IgM generated to our polymer, is

the cause of the hypersensitivity. Nevertheless, an IgE mediated response is not ruled out and

further investigation into both these mechanisms is needed.

Chapter 6 examines the effect of PEGylated particles on the immune system of

nonhuman primates. Rhesus macaques were injected with both F13M1 micellar solutions and

4

full emulsions of the polymer with sevoflurane. Though no immune response was observed, this

chapter details the results including histamine and antibody analyses.

Chapter 7 concludes the current studies to date and outlines upcoming studies that will

contribute further guidelines for the future of nanoparticle drug development.

Background (in vivo canine studies)

This project has a basis in research dealing with an injectable delivery system for the

general anesthetic, sevoflurane. An emulsion of sevoflurane was produced that consisted of the

drug, a diblock copolymer (F13M5), and the stabilizing agent, perfluoroctyl bromide. F13M5

consists of an mPEG of molecular weight 5000 (M5), attached to a chain of thirteen

fluorocarbons (F13). The structure of F13M5 is shown in Figure 1.1. The polymer itself makes

20 nm micelles in solution.

Figure 1.1: The structure of F13M5 (n~113)

In vivo studies were designed to test the physiological parameters associated with

delivery of sevoflurane via this method. However, the full emulsion elicited an allergic response

within minutes when injected into canines, characterized by hives and itching. When individual

components of the emulsion were injected, the polymer alone gave a similar allergic reaction.

Charts tabulating the allergic response to the polymer, as well as the full emulsion, are seen in

tables 1.1 and 1.2 (Jonathan Fast, personal communication, 2008).

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Table 1.1: Full emulsion injections. The parentheses in the injections column indicate the composition of the full emulsion in the format (% sevoflurane v/v / % PFOB v/v / F13M5 mg/mL). (Jonathan Fast, personal communication, 2008).

In table 1.1, increasingly larger doses of the emulsion were injected into the dogs to

assess the formulation’s efficacy. If the dog was “intubatable,” it meant that the dog was fully

anesthetized and a breathing apparatus was able to be inserted down the dog’s throat. It is

important to note that during the first round of injections (5/11/07), an allergic response was not

expected so symptoms characteristic of allergy were not closely monitored for. Excitement

during this round was to be expected, as it is consistent with low doses of anesthetic. One dog

vomited, which is sometimes associated with sevoflurane administration, but also could have

been a sign of an allergic response. In hindsight, the veterinarian in charge of the studies

recounted pinkness and vasodilation which, again, can be seen with general anesthetic

administration and can also be a symptom of an allergic response. In summary, while evidence

6

exists that there could have been an allergic response upon first administration, it was not

entirely clear at the time. Upon second administration (5/21/07), clear signs of an allergic

reaction were apparent. After the canines experienced pruritis (itching), urticaria (hives), and

swelling, the studies progressed from evaluating the formulation’s efficacy to testing which

component of the emulsion was responsible for the immune response.

Day Dog Injection Volume Result

5/21/2007 ZXX5

Emulsion without sevoflurane (0/10/25) 0.41 mL/kg Vasodilated, pruritic, urticaria

5/30/2007 EAV5

Emulsion without sevoflurane (0/10/25) 0.41 mL/kg

Similar to above (allergic-type rxn)

5/30/2007 WKX5 F13M5 (25 mg/mL) 0.41 mL/kg Similar to above (allergic-type rxn)

10/3/2007 EAV5 F13M5 (15 mg/mL) 0.41 mL/kg Similar to above (allergic-type rxn)

10/3/2007 WKX5 F13M5 (15 mg/mL) 0.41 mL/kg Similar to above (allergic-type rxn)

10/9/2007 ZXX5 mPEG 5K (13.2 mg/mL) 0.41 mL/kg No reaction

10/9/2007 EAV5 mPEG 5K (13.2 mg/mL) 0.41 mL/kg No reaction

10/9/2007 WKX5 mPEG 5K (13.2 mg/mL) 0.41 mL/kg No reaction Table 1.2: Injection of emulsion components. The parentheses in the injections column indicate the composition of the emulsion in the format (% sevoflurane v/v / % PFOB v/v / F13M5 mg/mL). (Jonathan Fast, personal communication, 2008).

Table 1.2 tabulates these studies. The emulsion, without the addition of sevoflurane,

produced a similar allergic response as did micellar solutions of F13M5. Of particular note is that

solutions of mPEG 5000, the hydrophilic block used in F13M5, did not produce a reaction.

Hydrophilic molecules are more likely to be immunogenic than those that are not, and since the

fluorocarbon section of the polymer is not expected to interact with antibodies, it is postulated

that the structural configuration of the micelle in vivo could be a determining factor whether or

not an immune response is generated. Regardless, these preliminary studies indicate that there

still many unknowns in the mechanism of this allergic response.

7

Much of the focus in the following chapters is on deciphering the mechanism of the

immune response seen in the aforementioned studies. Due to the immediate nature of this

hypersensitivity, both complement activation related pseudoallergy (CARPA) and Type 1

classical IgE mediated hypersensitivity, are two likely candidates for this mechanism.

Gel and Coombs Classification of Hypersensitivities

When deciding what hypersensitivity reactions could account for the effect seen in the

dog studies, it is important to take the Gel and Coombs classification system into consideration.

This system is reproduced below in Table 1.3 [6].

Table 1.3: Gel and Coombs classification of the 4 types of hypersensitivities. Replicated from [6].

Type 1 is a possible candidate for the immune response seen to our polymer. Known as

an immediate hypersensitivity, Type 1 can develop minutes to hours after antigen exposure [7].

It should be noted that this allergy should require multiple exposures to the allergen. Upon first

8

exposure, B cells recognize the incoming antigen. With the help of TH2 cells, isotype switching

occurs and IgE is created. IgE then binds to the surface of mast cells and basophils. During

subsequent exposures, IgE binds the antigen. Binding to two IgE molecules on the mast cell

surface cross-links those antibodies, leading to an intracellular process which ultimately ends

with degranulation and release of histamine and other factors that lead to the symptoms of an

allergic response.

Type 2 is another antibody-mediated response. In this form of hypersensitivity, IgM and

IgG bind to antigens located on a cell surface. Complement is then activated. At the end of the

complement pathway, the membrane attack complex is generated. This complex, made from

multiple components in the complement pathway, forms pores in the affected cell, thereby

killing it. In addition, complement can form split products that bind to the surface of the cell

labeling it for degradation by phagocytes. IgM and IgG can also activate phagocytes themselves,

promoting further cell killing. Type 2 happens on a variable timescale; it can be an immediate or

a delayed reaction.

Type 3 involves deposition of antibody-antigen immune complexes on tissue cells which

usually takes 1-3 weeks [7]. Complement activation by these immune complexes attracts

phagocytes. Since the complexes are deposited on a surface such as the basement membrane

(fibers underlining the epithelial cells) and are not free in the bloodstream, phagocytosis is

hindered causing the release of lytic enzymes. These lytic enzymes, as well as complement’s

membrane attack complex, can contribute to tissue damage and further incite the immune

response [6].

9

An important aspect of Type 4 is that it is a cell-mediated, rather than an antibody-

mediated, response. It is directed towards clearing intracellular pathogens, rather than free

pathogens in the bloodstream. Generally, TH 1 cells recognize antigen bound to MHC molecules

on macrophage cells, causing the TH 1 cells to release cytokines that, in turn, activate and recruit

macrophages and additional inflammatory cells. Amplification occurs, and sometimes, the

macrophages merge to create multinucleated giant cells. These granuloma cells release large

amounts of lytic enzymes and can cause severe tissue damage. Termed delayed-type

hypersensitivity, Type 4 responses require prior contact with the antigen before the reaction fully

develops. Then, the reaction generally reaches a peak 48-72 hours after second exposure of the

antigen [6].

One important factor to consider when choosing likely candidates for the canine allergic

response to F13M5 is the reaction time before the onset of the dogs’ symptoms. All dogs in our

studies responded within minutes, classifying their responses as immediate hypersensitivities.

Only Type 1 and 2 are observed within minutes after introduction of the antigen, eliminating

Types 3 and 4 as possibilities. While Type 1 remains a possible response, Type 2 is also not a

likely cause due to the difference in symptoms classically associated with Type 2

hypersensitivities and the observed response in dogs. Cell destruction associated with Type 2

hypersensitivity typically results in anemia and related symptoms rather than the anaphylaxis-

type symptoms seen in our experiments. Thus, Type 1 hypersensitivity is perhaps the most

likely mechanism to account for the canine response to the F13M5 polymer. However, another

form of complement activation also must be considered. This pseudoallergy, not accounted for

10

in the Gel and Coombs classification, is termed “complement activation related pseudoallergy”

(CARPA).

CARPA

CARPA is an unfortunate byproduct of the complement system. Complement was

important in the elimination of pathogens from the body and is one of its first defenses against

intruders. An incoming pathogen can trigger one of the activator proteins of complement, which

sets off the proteolytic complement cascade. This cascade generates split products which can

initiate an inflammatory response and clear the pathogen from the body. Figure 1.2 shows the

cascade [6]. It is made up of three pathways. The classical, alternative, and lectin pathways

have different triggers but eventually converge into formation of the membrane attack complex

(MAC - a pore forming complex used to lyse certain pathogens). The classical pathway is

antibody-dependent, and typically begins with activation via IgM/IgG-pathogen complexes. On

the other hand, the alternative and lectin pathways are normally antibody-independent. The

alternative pathway is activated by a variety of substances including some bacteria, fungal and

yeast cell walls, pure carbohydrates, and tumor cells [6]. Lastly, the lectin pathway is initiated

by sugar groups on the surface of bacteria, fungi, and viruses.

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Figure 1.2: The complement cascade [6]

CARPA occurs if an allergen, or in this case a nanoparticle, activates the complement

cascade. The allergic reaction is generated through production of C3a, C5a, and C4a. These

split products are known as anaphylatoxins; they can bind to mast cells and basophils causing

them to degranulate. This releases a variety of inflammatory mediators, such as histamine, that

can lead to the classical symptoms of an allergic reaction (hives, itching). This effect on mast

cells/basophils is identical to that seen in Type 1 hypersensitivity. Indeed, there are many

similarities between CARPA and Type 1 that would make them difficult to distinguish from one

another in a clinical setting. A table comparing the two reactions is found in Table 1.4 [5].

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IgE-mediated Type I CARPA

Common symptoms

Angioedema, asthma attack, bronchospasm, chest pain, chill, choking, confusion, conjunctivitis, coughing, death,

dermatitis, diaphoresis, dispnoea, edema, erythema, feeling of imminent death, fever, flush, headache, hypertension,

hypotension, hypoxemia, low back pain, lumbar pain, metabolic acidosis, nausea, pruritus, rash, rhinitis, shock, skin

eruptions, sneezing, tachypnea, tingling sensations, urticaria, wheezing

Unique symptoms

Reaction arises after repeated exposure to allergen Reaction arises at first treatment (no prior exposure to

allergen)

Reaction is stronger after repeated exposures Reaction is milder or absent upon repeated exposures

Reaction does not cease without treatment Spontaneous resolution

Reaction rate is low (<2%) High reaction rate (up to 45%), average 7%, severe 2%

Table 1.4: Similarities and differences between CARPA and IgE mediated allergic reactions [5].

As shown in Table 1.4, though CARPA and Type 1 can lead to the same physical

manifestations, there are some unique differences in their symptoms. The biggest difference is

that an IgE response needs repeated exposure to the allergen, whereas CARPA arises at the first

treatment. In addition, CARPA is sometimes subject to tachyphylaxis [8,9]. In contrast to Table

1.4, not only did the hypersensitivity reaction (HSR) to F13M5 seem to appear upon first

exposure to the polymer, it also appeared to strengthen upon repeat injection. The combination

of these two observations seemingly contraindicates both IgE and CARPA as the cause of the

allergic-response. But, upon further examination neither CARPA nor IgE can be completely

ruled out as the mechanism.

13

In rare cases, CARPA can appear on second or third exposure [8]. In this case, it’s

possible the lower dose given to the dogs upon first injection minimized the HSR (Table 1.1). In

theory, it is also possible that CARPA could get stronger after repeat exposure. CARPA is

unique compared to the Gel and Coombs hypersensitivities in that the mechanism for activation

can vary between three separate pathways. Direct activation by the nanoparticle could occur

through the alternative or lectin pathway. In contrast, the classical pathway could lead to indirect

activation of complement through the presence of nanoparticle-specific IgM/IgG antibodies. If

IgM titers rise upon repeat exposure to a nanoparticle, it is theoretically possible for CARPA to

strengthen depending on when the particle is next administered. To my knowledge, this has not

yet been observed in a clinical setting, but this possibility is further explored in Chapter 4 and 5.

Just as CARPA cannot be discounted, neither can Type 1 hypersensitivity. Though it

appeared that the allergic response was seen upon first exposure, this was only determined by

retrospective analysis. In addition, if the HSR was present during the initial injection, it is

possible the dogs had already generated antibodies that could cross react with F13M5 due to

exposure to other PEGylated block copolymers in their bedding or food. This is just conjecture,

but it displays how complicated the mechanism of this HSR can be and the possibility that it

might not fit perfectly in Table 1.4. Despite the noted differences in the F13M5 HSR, CARPA

and Type 1 still remain likely candidates to explain the hypersensitivity. In fact, there are many

examples linking complement activation and antibody generation to similar PEGylated systems;

a number of these systems are also linked to HSRs.

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Immunogenicity and PEGylated Drugs

CARPA and FDA Approved PEGylated Agents

A variety of PEGylated drugs are already approved for use. Some of these drugs have

been linked to HSRs. The PEG-phospholipid liposomal formulation of doxorubicin, Doxil®,

may be the most well-known example of a PEGylated system linked to CARPA. Doxil been

shown to activate complement in human serum in vitro [10]. Szebeni and colleagues noted that

the minimum dose of Doxil to activate the complement in vitro was 50-100 ug/mL, which was

10-20 times the peak amount of Doxil found in patient’s plasma [10]. They hypothesized that

since complement activation occurs in a matter of seconds, it may be more appropriate to analyze

the concentration in the infusion liquid (80-400ug/mL) versus the peak plasma concentration (4-

8ug/mL). Doxil was also proven to activate complement in vivo [11]. Khan and colleagues

noted that complement activation sometimes occurred in their patients without presence of an

HSR, indicating that complement activation may not be the only factor in this hypersensitivity

[11]. It is also unknown what role PEG plays in Doxil-mediated complement activation. The

PEGylated liposome used in Doxil is linked to a less severe allergic-type response in pigs than

the full formulation, indicating that doxorubicin itself may have some role in the pseudoallergy,

even if the liposome is still capable of producing a response [12].

Another anticancer agent linked to CARPA is Taxol®. Taxol is a formulation of

paclitaxel dissolved in Cremophor EL and ethanol. Cremophor EL (CrEL) is a polyethoxylated

castor oil used in Taxol as well as formulations for other drugs and vitamins. CrEL in ethanol

was found to activate complement independent of paclitaxel in human serum [13]. Szebeni et al

hypothesized that the hydroxyl-rich surface of CrEL could lead to alternative pathway activation.

15

In addition, they postulated that naturally occurring anti-cholesterol antibodies could cross-react

with CrEL and activate complement via the classical pathway [13]. Szebeni and colleagues later

suggested that large aggregates of >30kDA CrEL may be required to cause complement

activation [14]. Dogs also display a strong HSR to Taxol and CrEL [15-17], though

accompanying complement activation has not been investigated in this species.

It is important to note that oral and dermal administration of PEGylated formulations has

also led to HSRs [18]. To my knowledge, CARPA has not been investigated as the mechanism

to these responses. These examples are not discussed in detail in this thesis. Instead, the focus on

HSRs will remain on intravenous administration of liposomal/micellar PEGylated systems.

Antibody Generation to PEGylated Agents

Taxol, Cremophor EL, and other PEGylated solvent systems described throughout this

thesis provided incentive to look at CARPA as the mechanism of the HSR to F13M5. Links

between CARPA and PEGylated particles exist, but this does not rule out Type 1

hypersensitivity as the mechanism of the HSR. In fact, antibodies have been produced upon

injection of PEGylated nanoparticles into animals. These antibodies have been associated with

faster clearing of those particles upon repeat injection [19,20]. Most of these studies have not

linked Anti-PEG antibodies to HSRs. Nevertheless, there are examples correlating either

increased morbidity or HSRs to anti-PEG antibodies in mice [20,21]. Moreover, studies have

shown anti-PEG antibodies exist in about 25% of human blood donors [22-25]. Antibodies

generated to PEG have been of the IgM or IgG subtype. This seems reasonable since PEG is a

polymer and is most likely a thymus-independent antigen which would not undergo extensive

16

isotype switching. No anti-PEG IgE antibodies have been found, but the possibility of these

antibodies still exists and Type 1 hypersensitivity will be considered in this thesis.

CARPA in Preclinical Studies

Liposomes have consistently been linked to CARPA after injection into pigs [8]. A

variety of factors appear to play a role in liposome-directed activation of complement. Some of

these factors include include increasing liposomal size (70-300 nm range), positive or negative

surface charge, presence of aggregates, and endotoxin contamination [8]. Liposomes do not

have to contain PEG to activate complement. In some cases, it is thought that complement

activation by PEGylated liposomes is actually due to the hydrophobic portion of the added PEG

diblock copolymer. For instance, the negatively charged phosphodiester moiety of mPEG-

dipalmitoylphosphatidylcholine was thought to activate complement in one example of a

PEGylated liposome [26]. Similar non-PEGylated liposomes did not activate complement [26].

In addition, methylation of the acidic moiety on the phospholipid portion of the copolymer

eliminated complement activation [26]. Furthermore, Szebeni et al showed that PEGylation of

liposomes by negatively-charged 2KPEG-DSPE (N-carbamyl-poly(ethylene glycol methyl

ether)-1,2-distearoyl-sn-glycerol-3-phosphoethanolaminetriethyl ammonium salt) activated

complement whereas addition of a neutral 2KPEG-DS (3-methoxy polyethylene glycol-

oxycarbonyl 3-amino-1,2-propandiol distearoyl ester) did not [12]. These two cases highlight

the importance of the hydrophobic portion of PEG copolymers. Yet, they do not explain the

pseudoallergy to F13M5 since the fluorocarbon used in the polymer is considered to be

chemically inert.

17

Complement activation has been linked to other PEGylated particles besides liposomes.

In fact, PEG itself has been linked to complement activation in vitro [27]. Increasing chain

length correlated with stronger complement activation. Since anti-MASP 2 antibodies halted

complement activation by PEG, it was suggested complement was activated through the lectin

pathway (MASP2 is an initiator protein of that pathway). PEG was also found to affect the

alternative pathway [27]. In addition, PEGylated single-walled carbon nanotubes were also

linked to complement activation, through the lectin pathway [28].

PEGylated Micelles in Clinical Trials

This thesis should help explain how PEGylated delivery systems are associated with

immunotoxicity. This is especially important since there are a number of PEGylated micellar

systems undergoing clinical trials. Some of these systems have already been associated with

HSRs, which demonstrates the importance of understanding nanoparticle hypersensitivities.

Genexol-PM is a PEG-PDLLA (poly(D,L-lactic acid)) micellar delivery system for

paclitaxel that has undergone extensive clinical research. No HSRs were recorded in phase 1

studies [29,30], but later studies did display hypersensitivities [31,32]. It was suggested that

paclitaxel itself may be the cause of the HSR [31]. This may be true, but HSRs to the PEG-PLA

polymer should not be discounted. To my knowledge, the mechanism for this hypersensitivity

has not been investigated.

Similar systems have also reported HSRs in recent clinical trials. NC-6004 utilizes a

PEG-poly(γ-benzyl L-glutamate) polymer for encapsulating cisplatin, another anticancer agent.

18

Hypersensitivity reactions were seen in 6/17 patients in phase 1 trials [33]. Another example is

NK012, a PEG-polyglutamate polymer conjugated to 7-ethyl-10-hydroxy-campothecin (SN-38).

In phase 1 trials, 3/24 patients experienced infusion-related reactions for this anticancer agent

[34]. A final example is NK105, a PEG-poly(aspartate) formulation for paclitaxel. No HSRs

were reported in phase 2 trials [35], but 1 patient who received a high dose experienced an HSR

in phase 1 [36].

Just as with Genexol-PM, it is entirely possible that the HSRs seen in the aforementioned

cases do not involve PEG. And, it is important to note that some systems, such as SP1049C

(pluronic polymer bound doxorubicin) have not been linked with HSRs in phase 1 or 2 trials

[37,38]. Yet, these cases illustrate two important points: There are many PEGylated

formulations undergoing clinical studies, and HSRs with undefined mechanisms are associated

with these particles. As more PEGylated formulations become commercially-available, it’s

possible that HSRs will be found at a greater rate than what can be determined by the small

sample sizes found in these trials. With this in mind, the need for a greater understanding of the

HSRs associated with these particles is apparent. The rest of this thesis will attempt to further

define this mechanism.

19

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most effective constituent. Agents and Actions 1977, 7, 63-67.

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19. Wang X, Ishida T, Kiwada H. Anti-PEG IgM elicited by injection of liposomes is involved

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20. Judge A,McClintock K, Phelps JR, Maclachlan I. Hypersensitivity and loss of disease site

targeting caused by antibody responses to PEGylated liposomes. Mol Ther. 2006;13, 328–337.

21. Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. Immunogenicity

and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and

nucleic acid. The Journal of Pharmacology and Experimental Therapeutics 2005, 312, 1020-

1026.

22. Armstrong JK, Hempel G, Koling S, Chan LS, Fisher T, Meiselman HJ, Garratty G.

Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute

21

lymphoblastic leukemia patients. Cancer 2007, 110, 103-111.

23. Leger RM, Arndt P, Garratty G, Armstrong JK, MeiselmanHJ, Fisher TC. Normal donor

sera can contain antibodies to polyethylene glycol (PEG). Transfusion 2001, 41, 29S–30S.

24. Armstrong JK, Leger R, Wenby RB, Meiselman HJ, Garratty G, Fisher TC. Occurrence of

an antibody to poly(ethylene glycol) in normal donors. Blood 2003, 102, 556a.

25. Garratty G. Progress in modulating the RBC membrane to produce transfusable

universal/stealth donor RBC. Trans Med Rev 2004, 18, 245–256.

26. Moghimi SM, Hamad I, Andresen TL, Jorgensen K, Szebeni J. Methylation of the

phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents

PEGylated liposome-mediated complement activation and anaphylatoxin production. The

FASEB Journal 2006, 20, 2057-2067.

27. Hamad I, Hunter AC, Szebeni J, Moghimi SM. Poly(ethylene-glycol)s generate

complement activation products in human serum through increased alternative pathway

turnover and a MASP-2-dependent process. Molecular Immunology 2008, 46, 225.

28. Hamad I, Hunter AC, Rutt KJ, Liu Z, Dai H, Moghimi SM. Complement activation by

PEGylated single-walled carbon nanotubes is independent of C1q and alternative pathway

turnover. Molecular Immunology 2008, 45, 3797.

29. Kim TY, Kim DW, Chung JY, Shin SG, Kim SC, Heo DS, Kim NK, Bang YJ. Phase I and

pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle-formulated

paclitaxel, in patients with advanced malignancies. Clin Cancer Res 2004, 10, 3708–3716.

30. Tan E, Leong SS, Lim WT, Toh CK, Chowbay B. Weekly administration of a cremophor-

free, polymeric micelle formulation of paclitaxel to Asian patients with advanced solid tumor:

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31. Lee KS, Chung HC, Im SA, Park YH, Kim CS, Kim SB, Rha SY, Lee MY, Ro J.

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32. Kim DW, Kim SY, Kim HK, Kim SW, Shin SW, Kim JS, Park K, Lee MY, Hero DS.

Multicenter phase II trial of Genexol-PM, a novel Cremophor free, polymeric micelle

formulation of paclitaxel, with cisplatin in patients with advanced non-small-cell lung cancer.

Ann Oncol 2007, 18, 2009–14.

33. Plummer R, Wilson RH, Calvert H, Boddy AV, Griffin M, Sludden J, Tilby MJ, Eatock

M, Pearson DG, Ottley CJ, Matsumura Y, Kataoka K, Nishiya T. A phase 1 clinical study of

22

cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid tumors. British

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A, Matsumoto S, Takanashi M, Matsumura Y. Phase 1 study of NK012, a novel SN-38-

incorporating micellar nanoparticle, in adult patients with solid tumors. Clin Cancer Res 2010,

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Hamaguchi T, Shimada Y, Matsumura Y, Ikeda R. Phase II study of NK105, a paclitaxel-

incorporating micellar nanoparticle, for previously treated advanced or recurrent gastric

cancer. [published online ahead of print July 5 2011]. Invest New Drugs 2011.

36. Hamaguchi T, Kato K, Yasui H, Morizane C, Ikeda M, Ueno H, Muro K, Yamada Y,

Okusaka T, Shirao K, Shimada Y, Nakahama H, Matsumura Y. A phase 1 and

pharmacokinetic study of NK105, a paclitaxel-incorporating micellar nanoparticle

formulation. British Journal of Cancer 2007, 97, 170-176.

37. Danson S, Ferry D, Alakhov V, Margison J, Kerr D, Jowle D, Brampton M, Halbert G,

Ranson M. Phase 1 dose escalation and pharmacokinetic study of pluronic polymer-bound

doxorubicin (SP1049C) in patients with advanced cancer. British Journal of Cancer 2004, 90,

2085-2091.

38. Valle JW, Armstrong A, Newman C, Alakhov V, Pietrzynski G, Brewer J, Campbell S,

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glycoprotein-targeting pluronics, in patients with advanced adenocarcinoma of the esophagus

and gastroesophageal junction. Invest New Drugs 2011, 29, 1029-1037.

23

* The material in this chapter has been published: Johnson RA, Simmons KT, Fast JP, Schroeder CA, Pearce

RA, Albrecht RM, Mecozzi S. Histamine release associated with intravenous delivery of a fluorocarbon-based

sevoflurane emulsion in canines. J Pharm Sci 2011, 100, 2685-2692.

Chapter 2: Histamine Release Associated with Intravenous Delivery of a

Fluorocarbon-Based Sevoflurane Emulsion in Canines*

24

Preface

Though I am not first author on this paper, it is an essential addition to this thesis. The

studies described within this chapter give further explanation to the “background in vivo canine

studies” previously described in Chapter 1. I wrote this paper in conjunction with Dr. Rebecca

Johnson, the veterinarian in charge of these studies. Specifically, I wrote the parts of the chapter

dealing with the discussion of the immune response. This includes the abstract, parts of

materials and methods, and the discussion after the first two paragraphs. It is important to note

that the in vivo studies took place before I joined the group. In terms of the in vitro studies

described in this chapter, I am responsible for Figure 2.4, in which the emulsion and its

components were checked for complement activation in human serum. I would like to thank

everyone involved in this study. Most importantly, I would like to thank Dr. Rebecca Johnson

for her important contributions to this study and to this chapter of my dissertation.

25

Abstract

The purpose of this study was to evaluate the effectiveness of a novel fluorocarbon-based

sevoflurane emulsion in dogs previously shown to produce short-term rodent anesthesia. On the

basis of an unexpected allergic-type clinical reaction, we also tested the hypothesis that this type

of formulation causes histamine release and complement activation. Physiological parameters,

plasma histamine levels (radioimmunoassay), and complement activation (enzyme

immunoassay) were quantified in response to emulsion components, including F13M5 (the

emulsion’s fluorocarbon-based polymer) and methoxy poly(ethylene glycol) 5000 (the polymer’s

hydrophilic block). Although the emulsion produced general anesthesia in dogs, they also

experienced hypotension and clinical signs suggestive of an allergic-like response (i.e.,

vasodilation, urticaria, and pruritus upon recovery). Emulsions lacking sevoflurane failed to

induce anesthesia but did elicit the allergic response. Plasma histamine levels were significantly

increased following injection of micellar solutions of F13M5. Direct complement activation by

the emulsion or its components was weak or absent. An allergic response leading to histamine

release, likely initiated by the F13M5 component via an immunoglobulin pathway, is associated

with an intravenous fluorocarbon-based emulsion of sevoflurane. Subsequently, its usefulness in

medicine in its present formulation is limited.

26

Introduction

General anesthesia induced by volatile inhalants (e.g., sevoflurane, isoflurane, and

desflurane) and delivered via precision anesthetic vaporizers offers many distinct advantages

over intravenously (i.v.) administered drugs, including the elimination of the inhalant mainly via

the patient’s lungs rather than metabolism and excretion. However, when anesthetic drugs are

delivered by inhalation, there is a significant delay in the onset of anesthesia, as the

concentrations in the anesthetic breathing system, lung (alveoli), blood, and brain rise slowly

over time. To avoid this delay in anesthetic onset and action, i.v. delivered halogenated volatile

anesthetics have been developed. Injection of pure liquid forms of halogenated anesthetics

causes significant pulmonary damage and death in animals and human [1–3]. In contrast, lipid

emulsions based on Intralipid (a phospholipid-stabilized soybean oil emulsion, Baxter, Deerfield,

IL) have been found to be safe and effective for the delivery of isoflurane and sevoflurane [4–6],

and may be beneficial in the treatment of local anesthetic toxicity [7,8]. Because fluorinated

volatile anesthetics do not mix well with classical lipids [9], the maximum concentration of

sevoflurane in Intralipid emulsions is only 3.5% [6], a level that is inadequate for the efficient

induction and maintenance of general anesthesia.

To increase the amount of sevoflurane that can be delivered IV, novel emulsions using

semifluorinated surfactants have been developed and used successfully to produce sevoflurane

anesthesia [10]. The fluoropolymer used in this emulsion, F13M5 (Fig. 2.1), surrounds

nanodroplets composed of sevoflurane and the stabilizing agent perfluoroctyl bromide. This

nanoemulsion, which is stable in solution but releases rapidly upon IV administration, can

27

contain up to 30% of sevoflurane. This represents a powerful

technology for inducing general anesthesia by IV

administration of an “inhaled agent.”

Although previous experiments conducted in rats

demonstrated that this novel formulation is effective,

extremely fast, and safe [10], cardiorespiratory effects were

not measured, and efficacy and safety were tested only in

this one species. Therefore, we sought to test whether the i.v.

fluoropolymer-based sevoflurane emulsion would produce

dose-dependent general anesthesia, and if so, to measure the

associated cardiovascular effects in dogs. However, an

unexpected allergic-type reaction was observed in all dogs

during these initial studies. Therefore, we further test

whether injection of the fluoropolymer-based sevoflurane

emulsion and its constituents was associated with histamine release and/or complement

activation.

Materials and Methods

Sevoflurane (20%, v/v) Emulsion Preparation

Two hundred ninety-eight milligrams of F13M5 [molecular weight (MW) = 5668 g/mol]

was dissolved in 11.9 mL of normal saline. Perfluorooctyl bromide [PFOB (1.7 mL)] and

sevoflurane (3.4 mL) were added. PFOB was supplied by SynQuest Laboratories, Inc. (Alachua,

Figure 2.1: The structure of the

flouropolymer used in the emulsion,

F13M5. The nomenclature stands for a

13-chain fluorocarbon (F13) attached to a

methoxy poly(ethylene glycol) of average

molecular weight 5000 (M5) (n∼113).

Shown below are two similar hydrocarbon

polymers, H13M5 (hydrocarbon-based

structural analogue of F13M5) and

H19M5 (equivalent critical micelle

concentration to

F13M5).

28

Florida), and sevoflurane was manufactured by Abbott Labs (Chicago, Illinois). This mixture

was subjected to high-speed homogenization (Power Gen 500; Fisher Scientific, Hampton, New

Hampshire) at 22,500 rpm for 1 min. A microfluidizer (model 110 S; Microfluidics Corp.,

Newton, Massachusetts) was used to make the final emulsion at 5000 psi for 1 min. Larger

particles were eliminated with a 0.45-micrometer nylon syringe filter (Microliter Analytical

Supplies, Inc., Suwanee, Georgia).

An emulsion without sevoflurane was also made. This solution contained all of the

components of the original emulsion except sevoflurane, which was replaced with additional

saline. The steps to create the emulsion were the same as above. On the day of injection, the

emulsions were sized by dynamic light scattering.

Animals

All studies were approved by the University of Wisconsin Animal Care and Use

Committee. Three healthy, adult male Beagles (Ridglan Laboratories, Mt. Horeb, WI) were

fasted overnight before each study period.

Pilot Studies

In pilot studies directed at determining an effective dose, cephalic i.v. catheters were

placed for drug delivery. Three doses of the fluoropolymer-based 20% sevoflurane emulsion

were tested: 0.041, 0.123, and 0.41 mL/kg, based on previously published rat studies. Dog 1

received all three doses in ascending order. On the basis of the observations from Dog 1, Dog 2

received only the 0.041 and 0.41 mL/kg doses, and Dog 3 only received the 0.41 mL/kg dose.

29

Injections within each dog were administered in a single session and were separated by 10–15

min when appropriate. Dogs were observed for sedation or loss of reflexes (e.g., righting, pedal,

swallow, and corneal reflexes) and the unconsciousness indicative of general anesthesia, and for

any unexpected side effects.

Part I: Cardiovascular Measurements

Ten days after the pilot studies, dogs were instrumented to investigate the cardiovascular

effects of the fluoropolymer-based sevoflurane emulsion. Topical 4% lidocaine cream was

placed over the cranial tibial artery for 20 min to facilitate catheter placement. Cephalic i.v.

catheters were placed for drug delivery. Dogs were instrumented with electrocardiography

(ECG) to monitor heart rhythm, pulse oximetry to monitor heart rate (HR), and hemoglobin

saturation (SpO2), and the cranial tibial arterial line was, connected to a transducer to measure

systolic (SAP), diastolic (DAP), and mean (MAP) arterial bloodpressures (Cardiocap 5; Datex-

Ohmeda, Madison, Wisconsin). Awake baseline measurements of HR, arterial blood pressures,

and SpO2 were made for 5 min. Dogs 1 and 3 then received the injectable sevoflurane emulsion

at 0.41 mL/kg. All physiologic parameters were monitored every 10 s throughout the injection,

during general anesthesia, and for 30 min into the recovery period (S/5TM Collect; Datex-

Ohmeda–GE Healthcare, Helsinki, Finland). Because the first two dogs exhibited profound

hypotension with clinical signs of hypersensitivity reactions at 0.41 mL/kg (see Results section),

further doses were not tested and Dog 2 was not administered the fluoropolymer-based

sevoflurane emulsion.

30

Following a second 10-day washout period, dogs were instrumented and monitored as

above. Dog 1 was subsequently injected with an emulsion that did not contain sevoflurane (0.41

mL/kg; 10%, v/v, PFOB and 25 mg/mL F13M5). Dogs 2 and 3 received F13M5 alone (25

mg/mL) in 0.9% saline.

Part II: Component Analysis

On the basis of results obtained in Part I, the same three dogs were used to investigate the

effects of individual chemical components of the emulsion. A cephalic venous catheter was again

placed for drug delivery and blood sampling. Dogs were instrumented with ECG and pulse

oximetry to measure HR, rhythm, and SpO2, and an oscillometric blood pressure monitor was

placed on the front limb above the carpus with a cuff approximately 40% of the limb diameter

(Cardiocap 5; Datex-Ohmeda). Dogs received in random order 0.41 mL/kg of (1) 0.9% saline

(negative control); (2) 2.64 mM methoxy poly(ethylene glycol) (mPEG; MW = 5000 g/mol) in

saline; (3) 2.64 mM F13M5, the fluorocarbon polymer used in the emulsion; (4)

2.64mMH13M5, a hydrocarbon-based structural analog of F13M5; and (5) 2.64 mM H19M5, a

hydrocarbon-based polymer with an equivalent critical micelle concentration to F13M5.

Injections of Cremophor EL (25 mg/mL, BASF Corp., Florham Park, NJ) were used as a positive

control. Each dog received each treatment with at least an intervening 4-day washout period. HR,

respiratory rate, SpO2, SAP, DAP, and MAP were recorded 5 min before the injection (baseline)

and every minute thereafter for 30 min. At that time, treatment for hypersensitivity reactions was

administered to dogs when necessary (diphenhydramine 0.5 mg/kg i.v., dexamethasone SP 0.025

31

mg/kg i.v.). Blood samples were taken for the analysis of histamine levels during the baseline

period and 10–15 min following injection.

Histamine Analysis

Five milliliters of whole blood was taken from each dog just prior to and 10–15 min

following i.v. administration of 0.41 mL/kg of (1) 0.9% saline (negative control), (2) mPEG in

saline, (3) F13M5, (4) H13M5, (5) H19M5, and (6) Cremophor EL (positive control). Samples

were placed in tubes containing 50 µg/mL of lepirudin and 10 mM EDTA, and cold centrifuged

at 2053 g for 15 min. Plasma was removed for subsequent histamine analysis by a commercial

laboratory (Antech Diagnostics GLP, Morrisville, North Carolina) via radioimmunoassay

(Immunotech Laboratories, Glendale, California).

Complement Analysis

Pooled complement-preserved human serum (Innovative Research, Novi, Michigan) was

combined with components of the emulsion to test for complement activation in vitro.

Complement activation was analyzed by generation of C3a and C5a via enzyme immunoassay

(EIA) kits (Quidel, Santa Clara, California). Serum was pooled from 15 donors to account for

possible differences in complement protein concentrations

and reactivities. The emulsion (with and without sevoflurane), F13M5 (2.64 mM), and mPEG

5000 (Sigma–Aldrich, St Louis, Missouri) (2.64 mM) were tested for their ability to activate

complement. Each substance was tested in duplicate wells and in multiple EIAs. Dextran sulfate

(0.1 mg/mL,MW>500,000) (Sigma–Aldrich) was used as a positive control for complement

32

activation and 0.9% saline was the negative control. One hundred microlitres of each reagent was

added to 100 µL of serum and heated in 37°C water bath. Incubation times were 15 min for C3a

and 15–30 min for C5a. Each combination was transferred to ice and subsequently diluted in ice-

cold, EIA kit- specific buffer to stop any remaining complement activity. Generation of C3a and

C5a products were then analyzed according to manufacturer’s protocol.

The emulsions used for complement analysis were sized by a NICOMP 380 ZLS Particle

Sizer (Particle Sizing Systems, Santa Barbara, California) and the micellar solutions of F13M5

were sized by a Zetasizer Nano ZS (Malvern Instruments, Westborough, Massachusetts).

Complement studies were completed within 7 days of micellar solution preparation.

Statistical Analysis

In Part I, HR, SpO2, SAP, DAP, and MAP were averaged over 5 min bins and reported

with their respective standard errors for each dog following the fluoropolymer based sevoflurane

emulsion injection in the figures. In Part II, histamine levels are reported as means ± SE. A two-

way analysis of variance with repeated measures using time point (before or after treatment) and

treatment (saline, mPEG, F13M5, H13M5, H19M5, and Cremophor EL) as factors was used to

determine differences between groups with a Student–Newman–Keuls post-hoc test. A p value

of less than 0.05 was considered significant. Complement activation was also analyzed in Part II.

Generation

of complement split products are reported as a fold difference in concentration of C3a or C5a

compared with the negative control (saline). Ninety five percent confidence limits using the

Student’s t distribution are shown in Figure 2.4 as the error bars for each test substance.

33

Results

Emulsion Sizing

For the pilot and cardiovascular studies, the 20% sevoflurane emulsion was found to be

191.0 ± 29.2 nm; the emulsion without sevoflurane was 195.9 ± 41.3 nm. The emulsions used for

the complement analyses with sevoflurane ranged in size from 158.9 ±48.4 to 325.7 ± 35.4 nm;

the emulsions without sevoflurane ranged from 246.9 ± 96.2 to 311 ± 86.0 nm. All emulsions

were sized within 1 day of complement analysis and used within 11 days of preparation. The

micellar solutions of F13M5 used in complement analysis contained particles of approximately

19 nm.

Pilot Studies

Three i.v. doses of the fluoropolymer-based 20% sevoflurane emulsion were used to

determine efficacy [0.041, 0.123, and 0.41 mL/kg, delivered in ascending doses to each dog were

appropriate (see Methods section)]. Only the highest volume produced general anesthesia with

unconsciousness, areflexia, and no purposeful movement in the first two dogs and was

subsequently administered to the third dog (data not shown). Thus, this dose was used in Part I

and Part II of these investigations. At 0.41 mL/kg, dogs had subjectively weak femoral pulses

(based on palpation) and hyperemic, vasodilated mucous membranes and conjunctiva (although

these clinical signs were not subjectively as profound as those seen in subsequent studies; see

Part I: Cardiovascular Effects section). However, cardiovascular parameters were not quantified

during these studies.

34

Part I: Cardiovascular Effects

To assess the cardiovascular effects of the sevoflurane emulsion, blood pressure by

arterial catheter, ECG, and pulse oximetry were monitored, and sevoflurane was injected IV.

Injection of 0.41 mL/kg sevoflurane emulsion in the first two study dogs did not measurably alter

SpO2 or HR from baseline levels (data not shown). However, within 10 min following i.v.

sevoflurane administration, SAP, DAP, and MAP had decreased substantially from average

baseline values of 121, 80, and 97 mmHg, respectively, to minimum values of 36, 33, and 34

mmHg, respectively (Figs. 2.2a–2.2c). In Dog 1, the MAP remained below the levels defined as

hypotensive (<60 mmHg) for 20 min, whereas Dog 2 remained hypotensive until the study was

terminated (30 min; Fig. 2.2c). HR changes were variable (Fig. 2.2d). Both dogs exhibited signs

of hypersensitivity, including erythema of the skin, urticaria, vasodilation of the capillary beds in

the conjunctiva and pinna, and pruritus upon anesthetic recovery. Because the hypotension and

associated physical examination findings were so profound (i.e., MAP of 23 mmHg in one dog),

Part I was discontinued using the sevoflurane emulsion.

Because this allergic-type response was seen when dogs received the full sevoflurane

emulsion, Dog 1 was subsequently administered emulsion without sevoflurane to evaluate the

effects of the sevoflurane itself versus the other components of the emulsion, and Dogs 2 and 3

received F13M5 in saline alone. As expected, none of these three dogs showed signs of

anesthesia (because no sevoflurane was injected), but all

three experienced extreme pruritus and it was exceedingly difficult to maintain proper

instrumentation and data collection. Therefore, specific quantification of data during these

35

injections is not reported. These dogs were quickly treated with diphenhydramine, as described

in Methods section, with rapid resolution of symptoms.

Figure 2.2: Individual arterial blood pressure and heart rate values from Dog 1 and Dog 2 before and after intravenous

administration of the novel sevoflurane emulsion. The emulsion was injected at time 0. Systolic, diastolic, and mean

arterial pressures were markedly decreased from baseline values following injection for at least 20 min. However, heart

rate trends did not show a predictable pattern following injection in Dog 1 and Dog 2.

Part II: Component Analysis

Dogs were injected separately with F13M5, mPEG 5000, H13M5, and H19M5 (two

similar hydrocarbon-based polymers, Fig. 2.1), and a positive control, Cremophor EL. To

correlate possible histamine release with clinical signs, plasma histamine levels were measured

before and after IV administration of these components as described in Methods section.

Preinjection histamine levels were not significantly different between treatment groups (all p >

36

0.05, Fig. 2.3). However, histamine levels were significantly increased from baseline levels

following injection of Cremophor EL and F13M5 (all p < 0.05, Fig. 2.3).

Figure 2.3: Plasma histamine levels at baseline and following intravenous administration of the emulsion

components. There were no significantly different histamine levels during baseline (all p > 0.05).

However, following injection of Cremophor EL (positive control) and F13M5, plasma histamine levels

were significantly elevated from baseline and were significantly different from saline injection (∗ both p <

0.05). When not visible, error bars are within data set.

Complement Analysis

During Part II, serum was not collected from the dogs to check for complement activity

ex vivo. Still, complement activation by a component of the emulsion could have played a role in

the mechanisms underlying the hypersensitivity response. To check for this possibility, human

serum was mixed with components of the emulsion in vitro and analyzed for C3a and C5a via

EIA. Human serum was used instead of canine serum because no suitable antibodies for canine

complement split products were available. C3a and C5a were monitored because these two split

products anaphylatoxins would be the complement proteins most likely to lead to mast cell

37

degeneration and a pseudoallergic response. Neither the emulsion, nor its constituents, activated

complement to any substantial extent (Fig. 2.4). Rather, F13M5, mPEG 5000, or emulsion

without sevoflurane led to a smaller generation of complement split products than saline alone.

The emulsion itself led to a statistically significant, but nevertheless slight, increase in C3a. Only

the positive control, dextran sulfate, showed a strong change in C3a and C5a generation

compared with saline.

38

Figure 2.4: Enzyme immunoassay (EIAs) tested the generation of complement split products C5a (a) and C3a (b). Mean

fold difference was used to compare the emulsion and its separate components to controls. The bold line at “1” represents

the baseline, when saline is added to serum. Dextran sulfate (0.1mg/mL) is a potent activator of complement and the

positive control. “n” designates the number of EIAs used in the average of each component. Each EIA tested the

components in duplicate. The error bars are displayed as 95% confidence intervals based on the Student’s t distribution.

Only dextran sulfate shows strong complement activation. It should be noted that the values for dextran sulfate

sometimes fell out of the range of the standard curve generated in the EIA. Therefore, the values for dextran sulfate are

extrapolated.

39

Discussion

Intravenous volatile anesthetics have the potential to considerably change the way

general anesthesia is performed in human and veterinary medicine because they allow for rapid

alterations in anesthetic planes without prolonged hemodynamic changes as the inhalants are

eliminated rapidly from the body through the lungs. In addition, more rapid titration of drug

levels via i.v. delivery of inhalant agents would allow for the use of volatile anesthetics where

delivery via inhalation is difficult, such as in the MRI suite where magnetized metal is not

feasible, in the field or at the farm for veterinary procedures, or in humane societies, or rural

areas where the cost of anesthesia machines prohibits their use in veterinary patients.

We show here that a sevoflurane emulsion that was previously found to produce general

anesthesia in rats, also induces general anesthesia in dogs. However, it also results in profound

hypotension and histamine release. The cardiovascular effects of the emulsion were only tested

in two dogs due to the profound hypotension seen. Although no statistical analysis could be

performed on only two dogs, there was a clear, clinically significant decrease in blood pressure,

with variable effects on HR. Indeed, a mean blood pressure of less than 60 mmHg in dogs is

defined as hypotension, and the mean blood pressure fell from an average of 97 mmHg to a

minimum of 34 mmHg 10 min following injection. Blood pressure is a complex cardiovascular

parameter and is determined by the systemic vascular resistance and cardiac output. Cardiac

output is determined by the product of HR and cardiac stroke volume. Because histamine release

is associated with decreases in systemic vascular resistance and vasodilation, this is the most

likely cause for the hypotension seen with emulsion injection. Although it does not appear that

40

sevoflurane injection meaningfully altered HR, we cannot rule out hypotensive effects of the

sevoflurane emulsion through direct effects on cardiac output via changes in stroke volume.

The utility of these emulsions in veterinary practice is predicated on elimination of any

allergic-like responses associated with their use. Therefore, a clear understanding of the

hypersensitivity seen in these dog studies is needed. We began investigations into the underlying

mechanisms via injection of the individual components of the emulsion in order to determine

which components were responsible for this hypersensitivity. The emulsion (with and without

sevoflurane) and the fluorocarbon polymer F13M5 were all associated with a similar allergic-

type response upon injection, and F13M5 injection was associated with histamine release, most

likely from mast cell/basophil degranulation. Presumably, micelles composed of either H13M5

or H19M5 did not induce any immune response due to their quick dissociation in blood. We

speculate that the ability of fluoropolymers to form micellar structures that can be stable in blood

for up to several days (unpublished observations), rather than the characteristics of the

monomers, is related to their immunogenicity.

Two possible mechanisms for this histamine release are complement activation-related

pseudoallergy (CARPA) and classical Immunoglobulin E (IgE)-mediated hypersensitivity.

CARPA is an IgE-independent mechanism of allergy that is associated with other PEGylated

solvent systems and nanoparticles, including Cremophor EL and pluronics, which appear to

activate complement and cause a pseudoallergic response [11]. Mild clinical signs (weak pulses,

vasodilation of capillary beds) that may have been indicative of an allergic-type response were

observed during our pilot studies directed at finding an effective dose to produce anesthesia in

the three dogs. However, these effects were not as profound as those seen during subsequent

41

studies, and they were not quantified because dogs were not instrumented for cardiovascular

monitoring. These signs were observed upon first injection of the emulsion, without prior

sensitization. Because of this, and the similarity of F13M5 to other particles associated with

CARPA, direct complement activation was the initial focus of our studies.

However, Figure 2.4 suggests that, at least in the case of human serum, no direct

complement activation by the emulsion or its components is apparent. This would tend to rule

out CARPA as the cause for the hypersensitivity. Nevertheless, it is possible that differences in

the complement proteins between humans and canines could explain the lack of effect seen in

these EIAs. An additional consideration for this EIA is the pooled human serum itself. One of the

15 patients could have a high concentration of complement-inhibiting proteins, or some other

variation, that would inhibit complement activation by the polymer in the serum. Variation in

donor serum reactivity could mean that other serum samples could show complement activation,

especially if activation by this emulsion is rare in humans.

An alternative explanation for the hypersensitivity reaction is that an antibody-mediated

response occurred. This possibility is supported by the observation that the allergic-type response

was enhanced upon repeat injection of the emulsion. In this scenario, an increase in antibody titer

would have occurred as a result of antigen exposure each time the emulsion was injected. A

traditional explanation for the observed immune response would be an IgE-mediated reaction. To

our knowledge, IgE has yet to be linked to PEGylated particles. Nevertheless, this remains a

possibility because IgE is the antibody involved in a typical type I immediate hypersensitivity.

Alternatively, IgM or IgG may have played a role in this hypersensitivity. Anti-PEG IgM

and IgG have been discovered in instances of repeat administration of PEGylated particles to

42

various animals [12–14]. This leads to faster clearance of the particle from the body, a process

called “accelerated blood clearance phenomenon.” Another group of studies showed that anti-

PEG antibodies (IgM and IgG) were found in up to 25% of healthy blood donors [15–18]. Thus,

it is possible that the dogs had endogenous anti-PEG antibodies that reacted with F13M5

nanoparticles, potentially due to incidental exposure to these or cross-reactivity to similar

polymer antigens at some time during the animals’ lifetimes. If these dogs had endogenous IgM,

a complement-mediated response could still have occurred through classical pathway activation

that would not be detected by our experimental methods. Alternatively, endogenous IgG could

have led to degranulation of basophils and release of platelet-activating factor [19]. Although it

is unknown whether this pathway of anaphylaxis exists in dogs, this mechanism has been

suspected in at least one case of allergy to PEGylated liposomes in mice [14]. Current studies in

our laboratory are directed toward evaluating the possible roles of the different types of

antibodies in the hypersensitivity seen in dogs exposed to the emulsion.

Conclusion

In conclusion, our data suggest that injectable sevoflurane emulsions have the potential to

be useful in human and veterinary medicine because they do produce general anesthesia.

However, our formulation was also associated with profound hypotension and histamine release

in dogs, by a mechanism that likely involves immunoglobulin-mediated hypersensitivity.

Understanding the basis of this allergic-type response requires further investigation.

43

Acknowledgments

This project was supported in part by the UW Institute for Clinical and Translational

Research funded through a National Institutes of Health (NIH) Clinical and Translational

Science Award (CTSA), 1UL1RR025011, and the STEP program administered by the Wisconsin

Alumni Research Foundation. R.A. Johnson was supported by grant 1UL1RR025011 from the

CTSA program of the National Center for Research Resources, NIH.

44


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H, Mizushima Y, Mizushima T. Accelerated blood clearance phenomenon upon repeated

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46

* The material presented in this chapter will be the object of a manuscript (in preparation).

Chapter 3: Disadvantages of Using Hemolytic Assays to test PEGylated Block

Copolymers for Complement Activation During Drug Development*

47

Abstract

Background: Polyethylene glycol (PEG) is a hydrophilic polymer that can improve the

functionality of nanoparticle drug delivery systems. Immediate allergic-type responses involving

complement activation can be a problem with some of these systems. Reports of these

pseudoallergic events may cause drug developers to screen their nanoparticles for complement

activation with CH50 assays prior to in vivo use. The CH50 assay measures total complement

activity of a serum sample by finding the serum dilution able to lyse 50% of the assay’s blood

cells (usually antibody-sensitized sheep RBC) through a complement pathway. This study reports

on the difficulties experienced in assessing complement activation by a fluorocarbon-mPEG

polymer (F13M5), Pluronic F-68 (PF68), and mPEG (methoxy polyethylene glycol) using a

similar hemolytic assay.

Methods: F13M5, mPEG (5000 and 550), and PF68 were analyzed with two separate hemolytic

assays designed to test for complement activation. Polymers were incubated with serum and

then with either antibody-sensitized sheep red blood cells (Ab-SRBCs) or guinea pig red blood

cells (GpRBCs) to analyze each polymer’s effect on complement mediated lysis.

Results: mPEG 5000, but not 550, enhanced complement-mediated lysis in both assays. PF68

also enhanced lysis. F13M5 exhibited highly variable results in the SRBC assay, sometimes

increasing lysis and other times decreasing it or having no effect. In the GpRBC assay, F13M5

consistently decreased lysis.

Conclusions: PEG has been known to enhance complement mediated lysis. mPEG is capable of

this effect, which may mask complement activation by PEGylated nanoparticles like PF68.

48

F13M5 also demonstrates the difficulty in testing PEGylated block copolymers with hemolytic

assays, as they may have competing effects that vary with RBC membrane structure.

Investigators should use caution when attempting hemolytic assays with PEGylated

nanoparticles.

Introduction

The use of polyethylene glycol (PEG) in drug development has grown rapidly in recent

years. Addition of this non-ionic, hydrophilic polymer via direct conjugation to the therapeutic

agent or as part of a nanoscale carrier system can have many benefits. Among them, PEG has

been shown to increase solubility, prolong retention times, and lower toxicity in many drug

formulations [1,2]. Hypersensitivity reactions (HSRs) remain a concern in the use of PEG for

drug delivery despite PEG’s many benefits. Some of these adverse reactions are thought to

involve complement activation and have been termed complement activation related

pseudoallergy (CARPA) [3]. While there is no definitive link that PEG is the cause of CARPA,

instances of PEGylated formulations causing these allergic-type responses have been reported in

the literature. One example is Fluosol DA, an artificial blood substitute. There is some research

suggesting that hypersensitivity to Fluosol DA is due to complement activation by one of its

components, poloxamer 188, a copolymer consisting of PEG and polypropylene oxide [4,5].

Cremophor EL, a PEGylated solvent used in drug formulations with paclitaxel, cyclosporine, and

various other agents is also thought to cause complement activation and an ensuing

pseudoallergic response [6,7]. Delivery systems using PEGylated liposomes are also known to

cause CARPA, including the anti-cancer formulation Doxil® [8,9]. Preliminary studies have

49

indicated that imaging agents for Crohn’s Disease, Tc-99m-PEG-liposomes, may also be

potentially involved in CARPA [10]. Finally, in vitro studies on both PEGylated carbon

nanotubes, [11] and most notably, PEG itself, [12] have shown complement activation in human

serum. Again, though there is no direct proof that PEG is the cause of the in vivo reactions, these

recent findings may provide sufficient incentive for developers of PEGylated drugs to test their

formulations for complement activation in vitro before expanding to in vivo studies and risking

potential HSRs.

Considering this, it is essential to have an inexpensive, accurate, and rapid way to test

PEGylated compounds for complement activation in vitro. ELISAs are commercially available

to test for generation of complement split products, but these assays are very expensive. An

alternative to the ELISA is the CH50 assay, a complement consumption assay long used to assess

clinical complement levels and to test substances for in vitro activation of complement [13-15].

The assay utilizes complement’s ability to lyse blood cells via the pore-forming membrane attack

complex (MAC). Serum is incubated with antibody-sensitized sheep red blood cells

(AbSRBCs); the antibodies are activators of the complement pathway. The MAC formed at the

end of complement activation lyses the blood cell, releasing hemoglobin, therefore changing the

color of the solution which can then be read in a spectrophotometer. A patient’s complement

activity can be assessed by determining the dilution of complement able to lyse 50% of the blood

cells. Alternatively, a substance can be tested for complement activation by incubating it with

complement serum before the serum is incubated with AbSRBCs. The remaining complement

activity of the serum can be analyzed to determine what effect the test substance had on

50

complement. Traditionally, a decrease in the serum’s ability to lyse RBCs indicates that the test

substance activated complement itself and depleted the serum’s complement activity.

Our laboratory recently developed an intravenous delivery system for sevoflurane using a

PEG-fluorocarbon polymer, F13M5 [16]. Due to observed hypersensitivity reactions in dogs, it

was originally hypothesized that this polymer may be able to activate complement [17]. A

modified hemolytic assay similar to the CH50 was employed to test if F13M5 did in fact activate

complement. Over the course of

several years multiple versions of the

assay were employed, using different

species of RBCs. In the end, F13M5,

poloxamer 188, and mPEG (550 and

5000) (Figure 3.1) were tested in

versions of the assay using either

sheep (SRBC) or guinea pig blood

cells (GpRBC). When F13M5 was

analyzed with the SRBC assay, the

outcome varied greatly. In early

versions of the assay (termed the

“alternative method” throughout this

paper), F13M5 would enhance lysis, whereas in later versions, it would decrease lysis or have no

significant effect. In contrast, the same polymer always decreased lysis when guinea pig blood

cells were used. Another PEGylated polymer, Pluronic F-68 (poloxamer 188) was also tested in

Figure 3.1: Structures of the polymers used in the hemolytic assays.

F13M5 (n≈113) is a thirteen chain fluorocarbon attached to a mPEG

of MW 5000. mPEG 5000 as well as mPEG 550 was tested in these

studies. Pluronic F-68 (x,z=75, y=30) has an average MW of 8350.

51

our hemolytic assay. Since previous reports indicate that this polymer causes complement

activation, a decrease in lysis would be predicted in this hemolytic assay. Nevertheless,

poloxamer 188 enhanced lysis no matter which species of RBCs were used. mPEG 5000, the

PEG block used in F13M5, was also analyzed. mPEG 5000, but not the smaller molecular

weight mPEG 550, significantly enhanced complement-mediated lysis in SRBC and GpRBC

assays. The overall results indicated that some PEGylated block copolymers may interact with

RBCs differently depending on the type of blood cell used, making it difficult to gauge the

polymer’s effect on complement. In addition, intrinsic properties of the polymer may “mask”

effects on complement activation. Overall, this investigation lead to some concerns in using

hemolytic assays to test PEGylated block copolymers for complement activation and the results

contained herein may serve as a warning to others trying to test their PEGylated compounds in a

similar manner.


Polymer Preparations, Blood, and Serum Solutions

Guinea pig red blood cells (GpRBC) or sheep red blood cells (SRBC) were used in these

assays. Whole blood in alsever’s solution was obtained from Colorado Serum Company

(Denver, CO). The blood was concentrated and washed according to previously published

methods [13]. Briefly, blood was washed 1x in gelatin veronal buffer with EDTA and 3x in

gelatin veronal buffer with Ca and Mg (GVB++), both obtained from Boston Bioproducts

(Ashland, MA). GpRBC and SRBC were concentrated to approximately 1x109

cells/mL. The

same reference was also followed to sensitize SRBC with antibodies [13]. Hemolysin was

52

obtained from Sigma-Aldrich (Item S1389, St. Louis, MO). Due to the addition of hemolysin,

SRBCs were used at ½ the concentration of GpRBCs in these assays.

Human serum was used for most of the experiments. Pooled, 15 donor human

complement was obtained from Innovative Research (Novi, MI). For the GpRBC assay using

the “alternative method” a separate sample of pooled human complement was used, also

obtained from Innovative Research. For the example of the SRBC assay using the “alternative

method,” canine (beagle) complement from Innovative Research was used. All sera was kept at -

80°C until use and repeated freeze/thaw cycles were avoided.

F13M5, mPEG 5K (Sigma-Aldrich), mPEG 550 (Sigma-Aldrich), and Pluronic F-68

(Sigma-Aldrich) solutions were made by dissolving the appropriate concentration in 0.9% saline

and mixing with a magnetic stirrer. Solutions were filtered with 0.45 um nylon syringe filter

(Microliter Analytical Supplies, Suwanee, GA). In some trials using the later “alternative

method” F13M5 solutions were made by the solvent evaporation method in methanol. The mode

of preparation for F13M5 solutions was found to have no impact on the outcome of the

hemolytic assays. F13M5 micellar solutions were sized by dynamic light scattering after

preparation and are traditionally around 19 nm.

Dextran sulfate (MW≥500,000) was used a positive control for the hemolytic assays by

mixing 0.1 mg/mL in saline. It was obtained from Sigma (Item #D8906).

Modified Complement Hemolytic Assay

To test our polymer for complement activation, a modified hemolytic assay, similar to the

CH50 test, was performed. The difference between this assay and a traditional CH50 is that the

53

CH50 tests multiple dilutions of serum to find the “CH50” unit, or the reciprocal of the dilution

of serum able to lyse 50% of the blood cells. Our assay does not calculate a CH50, instead it

examines what effect a specific concentration of polymer has on the complement hemolytic

activity of a dilution of serum predetermined to lyse around 50% of RBCs. This allows multiple

concentrations of polymer to be examined in a less complex setup, without requiring dilutions of

both serum and polymer.

The assay is setup by making a serial dilution of a polymer solution in a V-bottom

microwell plate (Corning Incorporated, Corning, NY). Most assays were set up with 6 replicate

wells for each testing combination. In this way, the plate was split in half, allowing F13M5 to be

tested on one half and mPEG 5k to be tested in the other. To follow the setup step by step, 200 ul

of a saline solution of polymer are added to Row B of the plate. Rows C-F are filled with 100 ul

of pure saline. Using a pipette, 100 ul of the polymer from Row B is transferred and mixed to

Row C, D, E, and F. Row F is mixed, then 100 ul is discarded. Wells A1-A6 test what percent

of RBCs the serum will lyse without polymer added; 100 ul saline is added to dilute the serum to

the proper concentration. Wells G1-G6 test the amount of hemoglobin in the RBCs by lysing

them completely with water; 200 ul of water is added. Wells H1-H6 test a positive control.

Dextran sulfate, a strong activator of complement is used, with 100 ul added to each of these

wells. The remaining wells are set up to see what percent of RBCs lyse spontaneously without

serum in the presence of either buffer or polymer. This analyzes both the health of the RBCs and

the hemolytic activity of the polymer. 100 ul of either saline, F13M5, or mPEG 5K are added to

these wells and combined with 100 ul of GVB++ (Boston Bioproducts, Ashland, MA) instead of

serum. A blank row comprised of 100 ul saline and 200 ul GVB++ is also used. Once the

54

experiment is set up, 100 ul of serum diluted in GVB++ is added to all wells except those used

for total lysis, blanks, and background lysis measurements. The plate is then incubated for 30

minutes at 37°C. Then, depending on the assay, 100 ul of GpRBCs or Ab-SRBCs in GVB++ are

added to all wells and the plate is incubated for 1 hour at 37°C. After incubation, the plate is

centrifuged at 1250 x g at 4°C for 5 minutes and 150 ul of the supernatant is transferred to a flat

bottom microplate. The solution is read in a plate reader (EL800, Biotek, Winooski, VT) at 541

nm.

To calculate the percent lysis of RBCs in each test, the sample readings were averaged

and divided by the average of the total lysis readings. Each test using this method with human

complement and mPEG 5K, F13M5, and Pluronic F-68 was run multiple times. It was run 3

times for Pluronics F-68 with both GpRBC and Ab-SRBC. In addition, it was run 8 times for

F13M5/mPEG 5k with Ab-SRBC and 4 times with GpRBC. An example graph was chosen from

the runs when the normal complement lysis was closest to 50%, as this level of lysis was

predicted to result in the most sensitive assay. It should be noted that all results from these runs

were very similar except for the F13M5/Ab-SRBC combination, of which multiple examples are

shown and discussed. Each bar graph is presented with 95% confidence intervals based on the

student’s t distribution. Differences are termed “statistically significant” if confidence intervals

do not overlap.

Alternative Method Complement Hemolytic Assay

To showcase the variability in results obtained from the F13M5/Ab-SRBC combination,

some results are shown from previous versions the assay. There are only slight differences in the

55

two assays. In the alternative method, serial dilutions of the polymer are not used. Instead, 100

ul of test substance was incubated for 30 minutes at 37°C with 100 ul of diluted serum in a flat-

bottom plate. 100 ul of RBCs in GVB++ was then added, and incubated for 1 hour at 37°C. The

plate was then centrifuged and 150 ul of the supernatant was transferred to another well for

reading at 541 nm. Total lysis was calculated by adding 2 mL of water to 1 mL of blood in a

separate tube, shaking until all cells were lysed, then adding to the plate. Samples were usually

replicated in 5 wells. Example graphs were selected in the same manner as above and presented

with 95% confidence intervals.

Results

Figure 3.2: All concentrations of F13M5 significantly decreased complement-mediated lysis in the GpRBC assay with

human serum. mPEG 5000 enhanced lysis. Bars in all graphs are shown with 95% confidence intervals.

56

Two versions of a modified hemolytic assay were used to test our polymer, F13M5, and

mPEG 5000 for complement activation. In the GpRBC version of the assay, F13M5 consistently

led to a decrease in complement mediated lysis of the blood cells. (Fig 3.2) All concentrations of

polymer (7.047 mM to 0.441 mM) led to strong, significant decrease in lysis not in just this

example graph, but all four times this assay was attempted. Figure 3.3 shows the effect of

F13M5 on the Ab-SRBC version of the assay. Highly variable results were obtained using sheep

blood and F13M5, as shown by Fig 3.3a and 3.3b. 3.3a shows that, for the most part, the

polymer did not affect lysis. The highest concentration of polymer (7.057 mM) did show a

significant enhancement of lysis in Figure 3.3a. In contrast, 3.3b demonstrates the polymer (to a

0.882 mM concentration) significantly decreased lysis. The Ab-SRBC modified assay was

attempted 8 times in total. In 4 of the trials, F13M5 had no effect on complement-mediated lysis,

whereas in the other 4, F13M5 decreased lysis with overall results generally resembling the

examples shown in Figure 3.3.

57

Figure 3.3: Results were highly variable when F13M5 was analyzed with antibody-sensitized sheep red blood cells and

human serum. “a” and “b” are two examples using of results using the identical methods. In “a,” most concentrations of

F13M5 had no effect on lysis, whereas in “b,” most concentrations decreased lysis. The effect of mPEG 5000

(enhancement of lysis) remained consistent in this assay.

In contrast to the variability displayed by F13M5, the effect of mPEG 5000 on

complement-mediated lysis remained generally consistent throughout both versions of the assay.

In Figure 3.2, it is shown that mPEG 5k significantly enhanced complement-mediated lysis to a

concentration of 1.76 mM in the guinea pig assay. Throughout the other trials, mPEG 5k seemed

58

to enhance lysis clearly at the 7.057 mM and 3.53 mM concentrations, and sometimes at the 1.76

mM. Figure 3.3b shows a similar effect of mPEG 5k on the sheep version of the version.

Generally, the highest two concentrations of mPEG 5k enhanced lysis, though in Figure 3.3b,

even the lowest concentration had a significant effect.

To test if a polymer with a high mass percent of PEG produced a similar effect to mPEG

5k in these assays, the effect of Pluronic F-68 on complement mediated lysis was also analyzed.

Figure 3.4a and 3.4b displays both the Ab-SRBC and GpRBC versions. PF68 significantly

enhanced complement-mediated lysis in each of the assays. Figure 3.4 shows the lowest

concentrations to significantly enhance lysis were 0.441 mM and 0.882 mM in the sheep and

guinea pig versions, respectively.

59

Figure 3.4: In general, Pluronic F-68 enhanced lysis in both assay versions (Ab-SRBC and GpRBC) with human serum.

To further illustrate the variability in the F13M5/Ab-SRBC version of the assay, prior

results using a hemolytic assay with slight differences in the protocol is shown in Figure 3.5.

This “alternative method” demonstrated that F13M5 greatly enhanced complement-mediated

60

lysis of sheep red blood cells. It should be noted that this assay was mainly attempted using

canine serum, but the same effect was seen in one run of the same protocol with human serum.

Figure 3.5: To further illustrate the variability when F13M5 was tested with SRBC, results using an older method of the

assay are displayed. Canine serum was used, although results were confirmed in one run of the same methods with human

serum. Though changes in the protocol were minimal, F13M5 now showed an enhancement of lysis, in contrast to the

results obtained in Figure 3.3. mPEG 5000 still showed an increase in lysis.

The “alternative method” was also attempted using guinea pig blood cells and human

serum. A representative graph is shown in Figure 3.6. This combination shows very similar

results to those obtained using the method in Figure 3.2; F13M5 led to a significant decrease in

lysis using GpRBCs.

61

Figure 3.6: Despite using an older method (identical to Figure 3.5), results for F13M5 in the GpRBC/human serum assay

remained consistent with Figure 3.2. F13M5 led to a decrease in lysis and mPEG 5000 enhanced lysis. mPEG 550 was

also analyzed and showed no effect on complement-mediated lysis.

Figure 3.6 also displays the effect of mPEG 550. The smaller molecular weight mPEG

had no effect on complement-mediated lysis. This result was confirmed two additional times

with the “alternative method” and canine serum (once with GpRBCs and once with Ab-SRBCs).

Discussion The “alternative method” described in this paper was the earliest version of our modified

hemolytic assay. It was originally designed to test PEGylated nanoparticles for complement

activation with canine serum. ELISAs were the other option, but they are expensive, and at the

time, were not widely compatible with porcine or canine serum, the two species that have shown

the most reactivity to CARPA [18]. Since humans display variable reactivity to these

hypersensitivities, basing an in vitro hemolytic assay off a highly reactive animal was thought to

be a useful tool for modeling the small percentage of humans that would react strongly to

62

PEGylated particles in the general population. The earliest versions of the assay tested F13M5

with canine serum and noted an enhancement of complement-mediated lysis by F13M5.

GpRBCs then replaced Ab-SRBCs. The rationale for this was that they would be easier to use

since they didn’t need antibodies to efficiently activate canine complement and were noted to be

a good activator of the canine complement alternative pathway [19]. Since the results with

GpRBCs were trending in a different direction than the results with Ab-SRBCs, the serum source

was changed to humans to see if that was the source for the variability between the two assays.

When the results didn’t change, it became apparent that F13M5 had different interactions with

the two species’ RBCs. Before reporting of this phenomenon, the method was finalized to

include a concentration gradient for the polymer (Figs 3.2-3.4). Slight changes to the protocol

introduced further variability into the F13M5/SRBC version of the assay. Along with F13M5,

poloxamer 188 was tested with the new assay methods to observe how an additional PEGylated

block copolymer known to cause complement activation, reacted in these tests. The results

indicate some difficulties in using hemolytic assays to test nanoparticles for complement

activation that may be, in part, due to the PEG moiety.

PEG has been known to enhance complement-mediated lysis of RBCs [20]. In 1949,

McVickar incubated 4% carbowax-4000 (PEG 4000) with Ab-SRBCs and complement to find

out that PEG does enhance complement-mediated lysis. Our results with mPEG 5000 show a

similar effect. Different than McVickar’s method, our method incubated mPEG 5000 with the

complement source for 30 minutes before adding RBCs. Even with this additional incubation

time, our assay was not able to show any complement-activating effect of mPEG, which would

have been demonstrated by a decrease in hemolysis. It is unknown if mPEG itself is able

63

activate complement. But, recent work displayed that PEG (without a methyl group) is able to

directly activate complement [12]. Hamad et al demonstrated that PEG 4240 (5mM) and PEG

8350 (1.2mM) led to significant increases in complement split product C3a-desarg via ELISA. It

is unknown if the final concentration of mPEG 5000 is high enough in our studies to demonstrate

a similar effect (concentrations are depicted before incubation with diluted serum), but even at

higher concentrations, it seems unlikely that our hemolytic assay would be able to demonstrate

any direct complement-activating properties of PEG due to the competing effect of PEG-

enhanced lysis.

To our knowledge, the mechanism for PEG-enhanced complement-mediated lysis has not

been described. PEG has already been known to increase the number of hemolytic foci on RBCs

when cultured with complement-preserved plasma [21]. With this in mind, one possibility for

this mechanism is an increase in the number of MACs following PEG amplification of the entire

complement pathway. Or, PEG could enhance the effectiveness of the MAC, possibly by

increasing the interaction between the terminal complex and the RBC membrane. Further studies

are required to decipher the mechanism, but our studies have shown that PEG-enhanced lysis is

applicable to mPEGs, apparent in assays using sheep as well as guinea pig RBCs, and is

dependent on the molecular weight of PEG.

Pluronic F-68 was chosen for these studies to test if our hemolytic assays could detect

complement activation by a PEGylated block copolymer already linked to CARPA. The

enhancement of lysis by Pluronic F-68 in Figure 3.4 is likely due to the PEG moiety. So, if

complement activation did occur, it is likely PEG would mask the effect of the polymer. It

should be noted that PF68 was able to cause complement activation in vitro when it was tested

64

via ELISA [5]. Interestingly, there are some cases where PF68 has been tested in CH50

hemolytic assays. PF68 activated complement in one [4] and had no effect in the other [22].

Though the disparity in results for PF68 in CH50 assays could be solely due to the reactivity of

the serum samples, it is possible that the effects of PF68 (complement activation) and PEG

(hemolysis enhancement) can compete to different degrees in separate batches of PF68, leading

to variability in results.

Contrary to mPEG and PF68, F13M5 gave extremely variable results in these two assays.

The mechanism of F13M5’s effect on complement-mediated lysis appears to be very complex.

The polymer reacted differently even with slight changes to the protocol in the SRBC assay

(Figure 3.3 vs. Figure 3.5). In truth, it is difficult to justify the “alternative method” as a

different protocol when compared to the new method. Besides the addition of multiple dilutions

of polymer, the only substantial change is the shape of the well when the polymer was incubated

with the serum, yet the difference in results is substantial. Different serum samples were used in

the old and new versions, and that may contribute to the variability in the results. But, it is

important to consider that F13M5 continued to show variable results even when the same serum

sample was used in the same assay (Figure 3.3) or when the same sample was used with different

RBCs (Figures 3.2 and 3.3). And, different serum samples showed consistent results in the old

and new versions of the GpRBC assay (Figures 3.2 and 3.6). It is possible that these results can

be explained with competing effects of the polymer that differ in strength depending on the

species of RBC. Throughout this report, it has been shown that PEG can enhance hemolysis.

Recent work has demonstrated that fluorinated amphiphiles can actually inhibit staphylococcal

α-hemolysin pores into lipid bilayers [23]. Staphylococcal α-toxins are channel-forming proteins

65

similar to complement’s membrane attack complex [24]. If our fluoropolymer has similar

inhibitory properties, it is possible that the inhibitory and enhancing effects of F13M5 are

competing against each other and leading to the variable results obtained throughout the assays.

The alternative hypothesis is that PEG sometimes masks a complement-activating effect of the

polymer. While this remains a possibility, complement activation by F13M5 was not apparent

when the polymer was tested via ELISA using the same serum sample used in Figures 3.2 and

3.3 [17]. With both hypotheses, there also must be some difference between the membrane

structure of SRBCs and GpRBCs that contributes to the effects. Further studies are required to

identify the exact mechanism and the structural differences between GpRBCs and SRBCs that

contribute to the results, but F13M5 remains a prime example of a PEGylated block copolymer

that is difficult to analyze via a hemolytic assay.

Conclusion

There is growing concern that nanoparticle drug delivery systems may cause allergic-type

reactions through complement activation in a process known as CARPA. The CH50 assay may

be a potential method to investigate complement activation by these particles in vitro before in

vivo studies. This study examines the use of a modified hemolytic assay to investigate possible

complement-activating properties of a novel polymer, F13M5, which has been linked to an

allergic-type response. In addition to F13M5, mPEG (MW 5000 and 550) and Pluronic F-68

were also analyzed in a variety of hemolytic assays using different species of RBCs. It was found

that mPEG 5000, but not 550, led to an enhancement of lysis that may be a concern for

investigators using similar assays to screen PEGylated particles for CARPA. PF68, though it has

already been linked to complement activation via other methods, showed an enhancement of

66

lysis presumably due to the presence of the PEG moiety, further illustrating the concern over

using hemolytic assays with particles containing polyethylene glycols. F13M5 had highly

variable results within the same assay and when guinea pig blood cells were used in place of

sheep blood cells. The case of F13M5 demonstrates that some PEGylated block copolymers

may have competing effects, in part due to the PEG moiety, that react in a complex manner when

exposed to different RBC membranes, making it impossible to tell the polymer’s true effect on

complement.

In the end, researchers should use caution when testing PEGylated block copolymers in

similar hemolytic assays and consider confirming their results with an alternative method. The

intent here is not to say that CH50 assays can’t be used to screen PEGylated particles, or that all

prior studies using this combination are invalid. CH50 assays with alternative methods then

those explained here, especially those involving removal of the polymer before incubation with

RBCs, could prove to be a valuable tool in screening for CARPA with novel PEGylated

compounds. Nevertheless, investigators should be aware of the potential hazards of this

combination and take caution when analyzing results from such a study.

Acknowledgments This project was supported in part by the UW Institute for Clinical and Translational

Research funded through a National Institutes of Health (NIH) Clinical and Translational

Science Award (CTSA), 1UL1 RR025011, and the STEP program administered by the

Wisconsin Alumni Research Foundation.

67

Special thanks go to Dr. Jonathan Fast for his part in design of the F13M5 polymer, as

well as Elham Nejati for synthesis of F13M5.

68


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* The material presented in this chapter will be the object of a manuscript (in preparation).

Chapter 4: Enhanced Allergic-Type Response to PEG-Fluorocarbon Polymer Used for

Intravenous Delivery of Sevoflurane Upon Repeat Injection in Canines*

72

Abstract

Background: We previously developed an intravenous delivery method for the general

anesthetic, sevoflurane, by mixing the drug with a polyethylene glycol (PEG)-fluorocarbon

polymer and perfluorooctyl bromide (PFOB). This formulation elicited an allergic-like response

in canines, thought to be due to the PEG portion of our polymer. This study tests three new

formulations with modified polymers designed to eliminate the allergic-like response. One

polymer has a smaller PEG, whereas the other two have glucose molecules attached to the PEG

in order to change the polymer’s surface configuration. In addition, the study is designed to

differentiate between common pathways of hypersensitivity, by testing if any observed immune

response strengthens upon repeat exposure to our formulations.

Methods: 8 beagles were injected with either saline, or one of the new formulations. Three new

formulations were tested. One used a methoxy poly(ethylene glycol)-fluorocarbon polymer with

a 1K mPEG (F13M1). The other two formulations used polymers with either 1 or 3 glucose

moieties attached to the PEG (G1F13M1 and G3F13M1, respectively). Injections were repeated

after a two week period. Dogs were observed for allergic-like symptoms and blood was drawn

pre and post injection to test for histamine levels via ELISA as a measure of the strength of the

allergic-type response. In addition to the full emulsions, saline-based polymer solutions were

also injected into the dogs.

Results: All new formulations generated adverse reactions. The G3F13M1 elicited the strongest

allergic-like reaction of the new polymers. Repeat injection of the F13M1 emulsion led to a

73

significantly stronger allergic-like response based on histamine analysis. Micellar solutions of

the F13M1 and G1F13M1 polymers also led to significantly greater histamine release over

baseline levels. A submicellar solution of F13M1 did not produce symptoms of an allergic-like

response.

Conclusion: Strengthening of the immune response upon repeat injection of the F13M1

emulsion suggests the hypersensitivity was antibody-mediated. Future studies will investigate

this possibility, which could have important ramifications for the use of PEG in drug delivery.

Moreover, addition of glucose groups to our polymer resulted in highly immunogenic

formulations. This suggests that similar use of glucose moieties should be avoided in

intravenous drug delivery.

74

Introduction

Intravenous

sevoflurane delivery has many

potential advantages. Besides

an increased safety profile, IV

sevoflurane would have a

wider range of use since it

would be possible to induce

general anesthesia with only a

syringe and drug. To create an

IV version of this drug, our lab

has emulsified sevoflurane

with a mPEG-fluorocarbon

polymer, and a FDA-approved

stabilizing agent, PFOB [1]. This formulation has induced general anesthesia in both rats and

dogs. Unfortunately, an allergic-type response was also induced in dogs, halting further studies

in other animal models [2]. The hypersensitivity was attributed to injection of the polymer alone.

At this point in the development of IV sevoflurane, it is essential to decipher the mechanism

behind the allergic-type reaction.

Due to the chemically inert nature of the fluorocarbon in our polymer, the PEG moiety is

thought to be responsible for the allergic-type response. Though PEG is sometimes considered a

“stealth” molecule, there are examples of other PEGylated particles in the literature which can

Figure 4.1: The structures of F13M1, G1F13M1, and G3F13M1. F13M1 is a thirteen chain fluorocarbon attached to a methoxy-polyethylene glycol of MW 1K. The other polymers have glucose groups attached to the PEG of the original polymer. (n≈23)

75

induce an allergic-type response [3-5]. The purpose of this study was to modify the PEG moiety

in our formulation to see if it would eliminate that response. One modification used a smaller

PEG (1K), whereas the others attach a glucose molecule to the PEG in order to “mask” the

poly(ethylene) glycol surface (Figure 4.1). In addition, our study was designed to determine

some key traits of the allergic response. Namely, our formulations were injected twice; the

second time was two weeks into the experiment. This would determine if the immune response

would strengthen over time, which would be characteristic of an antibody-mediated reaction,

opposed to another form of pseudoallergy, such as direct complement activation or mast

cell/basophil degranulation. In the end, these studies were designed to both test new formulations

and characterize the mechanism behind the allergic-like response. These findings may prove

valuable in the synthesis of new, non-immunogenic formulations.

Material and Methods

Animals

8 adult beagles were obtained from Covance (Madison, WI). All studies were approved

by the University of Wisconsin Animal Care and Use Committee.

Study Design

8 beagles were divided into 4 treatment groups. 3 beagles (Dog B, Dog S, Dog X) were

injected with 0.9% saline (0.51 mL/kg). 3 beagles (Dog 15, Dog O, Dog W) were injected with

the M1F13 emulsions (0.6 mL/kg). 2 beagles (Dog 71, Dog 2) were injected with the G1F13M1

emulsions (0.48 mL/kg). 1 beagle (Dog I) was injected with the G3F13M1 emulsion (0.48

mL/kg). Since Dog I experienced a serious allergic-type response following exposure to the G3

76

emulsion, studies using this polymer were discontinued. Repeat injections were not attempted

for the G3 emulsion. Blood was withdrawn 5 minutes prior to injection as well as 25 minutes

after injection. Serum was prepared from the whole blood and stored at -80⁰C until histamine

analysis.

Dogs were non-invasively instrumented to investigate the cardiovascular effects of the

sevoflurane emulsions. Cephalic i.v. catheters were placed for drug delivery. Dogs were

instrumented with electrocardiography (ECG) to monitor heart rhythm, pulse oximetry to

monitor heart rate (HR), and hemoglobin saturation (SpO2), and an oscillometric blood pressure

monitor attached to a cuff (~40% of the limb circumference) placed on the metatarsal region was

used to measure systolic (SAP), diastolic (DAP), and mean (MAP) arterial blood pressures

(Cardell Veterinary Monitor, #9402, Sharn Veterinary, Inc., Tampa, FL). Awake baseline

measurements of HR, arterial blood pressures, and SpO2 were made for 5 min. Followed by

measurements every 5 minutes throughout the procedure.

When signs suggestive of histamine release were noted, diphenhydramine (1 mg/kg

slowly IV) and famotidine (1 mg/kg slowly IV) was administered along with a 0.9% NaCl bolus

(10 ml/kg IV) to help maintain blood pressure.

Two weeks later, the injections were repeated. All saline and M1F13 dogs received a

repeat administration of the same substance. Since the G1F13M1 formulation did not mask the

allergic-like response on its first injection, it was decided that it should not be injected a second

time. Instead, a micellar solution of the polymer alone was injected into the 2 dogs that

previously received the full G1F13M1 emulsion to determine if the polymer itself was

responsible for all the symptoms seen in the hypersensitivity.

77

In later experiments, a sub-micellar concentration of F13M1 was injected into three dogs

(Dog DDE, CMN, CNZ) to see if it generated an allergic response.

Drawing from previous studies, it was suspected that the F13M1 polymer alone could

also cause an allergic-type response [2]. But, it was not clear if the polymer itself could elicit an

allergic-type reaction without previous exposure of the animal to the full emulsion. A 7mM

solution of F13M1 (0.6 mL/kg) was injected into Dog B, which previously received only saline

injections, to determine if this prior sensitization was necessary.

Emulsion Preparation

Emulsions with sevoflurane were prepared in a manner similar to that previously reported

[2]. The F13M1 formulation used 212.06 milligrams of F13M1, 1.7 mL PFOB, 3.4 mL

sevoflurane. The G1F13M1 formulation used 229.67 milligrams of G1F13M1, 1.7 mL PFOB,

3.4 mL sevoflurane. The G3F13M1 formulation used 320.11 milligrams of G3F13M1, 1.7 mL

PFOB, 3.4 mL sevoflurane. All emulsions were filtered with a 0.45 µm nylon syringe filter.

Polymer Solution Preparation

The G1F13M1 solution (13.47 mg/mL) was prepared in normal saline and filtered with a

0.45 µm nylon syringe filter. The micellar solution of F13M1 (12.47 mg/mL) injected into Dog

B was also made in saline and filtered as above.

Finally, the concentration of submicellar F13M1 was 5 x 10-9

mM (100x lower than the

CMC). It was injected at 0.6 mL/kg and was filtered at 0.2 µm with a nylon syringe filter.

78

Histamine Analysis

Change in histamine concentration between pre and post injection serum samples was

used as a measure of the change in strength of the allergic response between the first and second

injections. Histamine concentration was measured by ELISA (Neogen Corporation, Lansing,

MI, Product # 409010). 3 separate ELISAs were completed per sample at the same time. Each

sample was tested in duplicate wells as per manufacturer’s recommendation. The fold change in

histamine concentration was calculated between pre and post serum samples by averaging the

fold change measured in each of the 3 separate ELISAs. Additional ELISAs were conducted

(results not shown) that demonstrated the polymers themselves (F13M1, G1F13M1, G3F13M1)

did not interfere with assay results at the concentrations used in vivo.

For Figure 4.3 (submicellar F13M1), 1 ELISA was performed (samples were tested in

duplicate).

Statistical Analysis

3 separate ELISAs were carried out for each sample. Each sample was tested in

duplicate wells per ELISA. The fold change between pre and post samples was calculated per

ELISA by averaging the concentrations of the 2 wells and finding the ratio between the pre and

post samples. Those three numbers were then averaged to find the final fold change across all

ELISAs. Findings were termed “statistically significant” using 95% confidence intervals based

on the student’s t-distribution. Five degrees of freedom were used corresponding to the six wells

tested per sample. Since 1 ELISA was performed for Figure 4.3 (submicellar F13M1), the graph

is presented with fixed 10% error bars representing the inter-assay precision noted in the

manufacturer’s instructions.

79

Results

Figure 4.2. Part a.) displays the fold change in histamine release for the first and second injections for the saline control

and F13M1 emulsion. All bars are displayed with 95% confidence intervals. All F13M1 dogs exhibited a significant

increase on the 2nd injection, suggesting the allergic-type response was antibody-mediated. Saline dogs did not see any

change in histamine concentration. Part b.) displays the fold change in histamine release for the G3F13M1 dog and the

G1F13M1 dogs on first injection of the emulsion and reprints the bars for the saline dogs in part a.). Both dogs treated

with the G1F13M1 emulsion had a significant change compared to the saline dogs, and both dogs also had a larger fold

change in histamine concentration than those treated with F13M1. The dog treated with G3F13M1 had the largest fold

change in histamine compared to any other group.

80

Histamine release was used as a biological marker for the strength of the immune

response induced by our formulations. Figure 4.2 shows histamine release correlated with

injection of the G3F13M1, G1F13M1 and F13M1 formulations. The G3 formulation was highly

immunogenic, and resulted in about a 14 fold change in histamine levels. The G1 polymer gave a

strong immune response, and led to an over fivefold change in histamine concentrations for both

dogs. Both dogs had a significant change in histamine concentration compared to baseline (1 –

no change) and saline dogs. Figure 4.2a shows that, at least for the first injection, none of the

F13M1 dogs had a significant change in histamine release compared to the saline dogs. It should

be noted that even though Figure 4.2 does not display any significant differences for the F13M1

dogs on the first injection, they all did show signs of an allergic-like response. Dog 15 was the

only F13M1 dog from the first injection to display a significant increase over baseline.

The histamine release data correlated well with clinical observations on the strength of

the allergic-type response, at least between the G3F13M1, G1F13M1, and F13M1 groups. The

G3 dog displayed the strongest allergic-type response with the G1 dogs and finally the F13M1

dogs displaying progressively decreasing severity. The G3 dog experienced profound, immediate

erythema and swelling, followed by severe GI symptoms after exposure. Vomiting and diarrhea

were present in the G1 polymer dogs, in addition to other signs of histamine release such as

erythema and scleral injection. The F13M1 dogs had milder signs compared to the G1 group and

did not exhibit any GI symptoms on first exposure.

There were some differences between observational data and histamine release data

within the F13M1 group. For instance, Dog 15 exhibited the most histamine release in Figure

4.2a, but was also the only dog that did not need treatment for his symptoms. The differences

81

between our observational notes and the ELISA for the first round of injections are suspected to

be due to the relatively mild nature of the immune response in combination with genetic

variations in the dogs’ reactivity to histamine release.

Figure 4.2a also displays data for the second round of F13M1 injections. All of the

F13M1 dogs had a significant increase in histamine release compared to their first round of

injections.

Figure 4.3: A submicellar concentration of F13M1 was also injected into 3 dogs. The dogs did not exhibit any signs of an allergic-like response and did not show changes in histamine concentration. This graph is presented with fixed 10% error bars.

Figure 4.3 shows that submicellar concentrations of F13M1 do not lead to histamine

release. On the other hand, Figure 4.4 displays that micellar solutions of F13M1 and G1F13M1

did lead to a significant release of histamine compared to baseline levels.

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Figure 4.4: Micellar solutions of both F13M1 and G1F13M1 produced allergic-type reactions in dogs and were associated with changes in histamine concentration. This graph is presented with 95% confidence intervals.

Discussion

The first goal of this study was to make modifications to our original polymer to

eliminate our formulation’s immunogenicity. Unfortunately, the G3/G1 and the F13M1

polymers remained immunogenic. Attaching glucose groups to the PEG moiety in our polymer

was meant to block interaction of immune factors with PEG, leading to diminished

immunogenicity. Unfortunately, it appears that intravenously delivering clusters of glucose

molecules in this configuration is highly immunogenic, at least in canines. In fact, the 3-glucose

version of the polymer elicited a more severe allergic-type response than the corresponding

versions with either one (G1M1F13), or no (M1F13), glucose groups attached. Based on

confidence intervals, the histamine release associated with the G3 emulsion was significantly

greater than the G1 emulsion, indicating that the polymer becomes more immunogenic as more

83

glucose groups are added. Though the mechanism remains unknown, it is apparent that clusters

of glucose moieties introduce a severe hypersensitivity in dogs. While covering the PEG surface

may decrease immunogenicity with other functional groups, it appears a glucose surface is

strongly immunogenic itself, and is not suitable for this method of drug delivery.

The second polymer was identical to the original polymer, except for the smaller (1K)

mPEG moiety. It was thought that the smaller PEG could lessen, or possibly eliminate, the

immune response. Smaller PEGs have been shown to cause less complement activation in vitro

compared to larger molecular weight PEGs [6]. In addition, it is known that smaller molecules

are less likely to be antigenic. Despite this, F13M1 still retained immunogenicity.

Retrospectively, this polymer is likely less immunogenic than its larger molecular weight

predecessor based solely on observational data. Nevertheless, as the “inhaled” version of

sevoflurane is virtually non-immunogenic, it is essential to completely eliminate all allergic-type

responses associated with our polymers.

The most important discovery of this study is the enhancement of the immune response

seen between the first and second round of injections (Figure 4.2a). This suggests the

hypersensitivity seen to the F13M1 polymer is antibody-mediated. Classically, an IgE mediated

hypersensitivity would be suspected. But, it is also possible the hypersensitivity could be caused

by IgM/IgG complement activation. In fact, there have been a number of instances where anti-

PEG IgM has been found upon repeat injection of other PEGylated nanoparticles for drug

delivery systems [7-11]. Theoretically, if our polymer induced generation of anti-PEG IgM, two

weeks would give enough time for antibody titer to rise and the immune response to strengthen.

Ongoing studies in our lab are investigating this possibility.

84

Figure 4.3 displays that the polymers themselves, not just the full emulsions, can produce

an allergic-type response. While it is true that our previous research demonstrated this for the

original polymer, it should be noted that in those studies, the polymer itself was only exposed to

the dogs after they had already been exposed to the full emulsion. In this case, the F13M1

micellar solution was administered to a dog which had previously received only saline. Since the

F13M1 polymer could generate an allergic-like response upon first exposure, it can now be

certain that exposure to the full emulsion is not required as a “priming step” for subsequent

responses to the emulsion’s individual components. Furthermore, Figure 4.3 displays that

submicellar concentrations of F13M1 did not elicit an adverse reaction. So far, it is unclear

whether the micelle structure is necessary to generate the allergic response, or if the

concentration of polymer is too low when it is below the critical micelle concentration to affect

the immune system.

Conclusion

Developing a safe method to deliver sevoflurane intravenously would have a positive

effect relative to the field of anesthesiology. We developed three new polymers designed to

reduce or eliminate the allergic-like response associated with the original formulation. Although

the polymers still proved to be immunogenic, we obtained crucial data which demonstrates that

the allergic response strengthens after repeat exposure to our polymer. This suggests the

mechanism of this allergy is antibody-mediated. Further studies will investigate this possibility,

which could have significant ramifications for our formulation, and possibly on other drug

delivery formulations which use PEG-based polymers.

85


1. Fast JP, Perkins MG, Pearce RA, Mecozzi S. Fluoropolymer-based Emulsions for the

Intravenous Delivery of Sevoflurane. Anesthesiology 2008, 109, 651-656.

2. Johnson RA, Simmons KT, Fast JP, Schroeder CA, Pearce RA, Albrecht RM, Mecozzi S.

Histamine release associated with intravenous delivery of a fluorocarbon-based


3. Szebeni J. Complement activation-related pseudoallergy: A new class of drug induced

acute immune toxicity. Toxicology 2005, 216, 106–121.

4. Brouwers AH, De Jong DJ, Dams ET, Oyen WJ, Boerman OC, Laverman P, Naber TH,

Storm G, Corstens FH. Tc-99m-PEG-Liposomes for the Evaluation of Colitis in Crohn’s

Disease. J Drug Target 2000, 8, 225-233.

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turnover and a MASP-2-dependent process. Mol Immunol 2008, 46, 225-232.

7. Ishida T, Kiwada H. Accelerated blood clearance (ABC) phenomenon upon repeated

injection of PEGylated liposomes. Int J Pharm 2008, 354, 56–62.

8. Ishihara T, Takeda M, Sakamoto H, Kimoto A, Kobayashi C, Takasaki N, Yuki K,

Tanaka K, Takenaga M, Igarashi R, Maeda T, Yamakawa N, Okamoto Y, Otsuka M,

Ishida T, Kiwada H, Mizushima Y, Mizushima T. Accelerated blood clearance

phenomenon upon repeated injection of PEG-modified PLA-nanoparticles. Pharm Res

2009, 26, 2270–2279.

9. Judge A,McClintock K, Phelps JR, Maclachlan I. Hypersensitivity and loss of disease site

targeting caused by antibody responses to PEGylated liposomes. Mol Ther 2006, 13,

328–337.

10. Ishihara T, Maeda T, Sakamoto H, Takasaki N, Shigyo M, Ishida T, Kiwada H,

Mizushima Y, Mizushima T. Evasion of the accelerated blood clearance phenomenon by

coating of nanoparticles with various hydrophilic polymers. Biomacromolecules 2010,

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11. Koide H, Asai T, Hatanaka K, Akai S, Ishii T, Kenjo E, Ishida T, Kiwada H, Tsukada H,

Oku N. T cell-independent B cell response is responsible for ABC phenomenon induced

by repeated injection of PEGylated liposomes. Int J Pharm 2010, 392, 218-223.

87

*The material presented in this chapter will be the object of a manuscript (in preparation).

Chapter 5: Antibody Generation Associated with Intravenous Delivery of PEG-

fluorocarbon polymers and other PEGylated Nanoparticles in Dogs*

88

Abstract

Background: It was previously reported that injection of a novel intravenous delivery system for

sevoflurane causes hypersensitivity reactions (HSRs) in dogs. This HSR appears to strengthen

upon repeat injection, suggesting the presence of an antibody-mediated pathway. Similar

allergic-like reactions are elicited by injection of the methoxy poly(ethylene glycol)-fluorocarbon

polymer (F13M1) used in the emulsion. It was hypothesized that antibodies specific for F13M1

may be a factor in the HSR. Further analyses of a previous study were completed to test for

polymer-specific antibodies after repeat injection of our emulsion in canines. In addition, a PEG-

phospholipid polymer and PEGylated dendrimers (Poly(amido amine), generation 3) were

injected. The immune response to these PEGylated nanoparticles (presence/absence of HSR and

particle-specific antibodies) was measured.

Methods: Dogs were injected on days 0 and 14 of the experiment. Serum or EDTA plasma was

drawn approximately every 5 days throughout the study to test for particle-specific IgM

generation via ELISA. Histamine was measured (via ELISA) as a marker of the strength of the

allergic-type response to the PEG-phospholipid polymer and the PEGylated dendrimer.

Results: Injection of the full emulsion and the polymer solutions were correlated with a rise in

polymer-specific antibodies. The additional PEGylated nanoparticles (PEG-phospholipid and

PEGylated dendrimer) were not linked with HSRs. However, these injections also elicited

particle-specific antibodies.

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Conclusions: A rise in F13M1-specific IgM titers is seen before the second injection of the full

emulsion in dogs. This may suggest that the strengthening of the immune response is due to IgM

mediated complement activation. However, further studies are required as other mechanisms,

such as type 1 IgE mediated hypersensitivity, may also be involved. Additional studies are

needed to determine how PEGylated nanoparticles cause HSRs in dogs. The absence of allergic-

type reactions to PEGylated dendrimers and PEG-phospholipids may suggest that micelle

stability and particle size are factors in determining what will elicit an HSR.

90

Introduction

Intravenous delivery systems for sevoflurane, a general anesthetic, have been previously

described [1, 2]. Solubilization of sevoflurane in blood is achieved by emulsification with an

mPEG-fluorocarbon polymer (F13M1) and a stabilizing agent, perfluorooctyl bromide. The

PEG-fluorocarbon polymers used in these emulsions have been linked to allergic-type responses

in canines [3,4]. Micellar systems are known to elicit allergic-like responses in dogs [5]. In fact,

canines are utilized as an animal model for complement activation related pseudoallergy

(CARPA) [5]. Recently, it has been shown that the hypersensitivity reaction (HSR) to one of our

emulsions (using the F13M1 polymer) strengthens upon repeat injection in dogs [4]. This could

be indicative of an antibody-mediated allergic-like response. Though this is not typical of

CARPA [6,7], it is thought the HSR may be

due to rising of polymer-specific IgM titers

and enhanced complement activation upon

repeat injection. To investigate this

possibility, further analyses for antibody

generation to the F13M1 polymer in the

previous study [4] have been completed. Antibody generation in response to both full emulsions

(with sevoflurane) and micellar solutions of two of our fluorocarbon polymers (F13M1 and

G1F13M1 – Figure 1) are presented in this paper.

Figure 5.1: Structure of F13M1 and G1F13M1 (n≈23)

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Additional studies were

performed to further characterize

the immune response to

PEGylated nanoparticles in dogs.

A micellar solution of a

mPEG(1K)-DSPE polymer

(Figure 2) was injected to

determine if all micelles with

surface mPEG moieties cause

HSRs in dogs. Since the stability

of the mPEG1K-DSPE micelle is

unknown in vivo, a PEGylated

dendrimer (Figure 2) was also

tested. In theory, the covalently bound dendrimer creates a PEGylated surface that would mimic

that of a stable micelle and it cannot be dissociated in blood. Both PEGylated nanoparticles were

injected twice over a two week period and plasma samples were withdrawn to analyze both

histamine and antibody levels.


Dog Study Design

Since this study contains additional analysis of a previously published study, the design

of the in vivo canine study can be seen elsewhere [4]. Though the mPEG-DSPE and the

Figure 5.2: The structure of mPEG1K-DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] is presented in (a). An unconjugated poly(amidoamine) (PAMAM) dendrimer. G3 represents “generation 3.” c.) The dendrimer used in these studies is a PEGylated PAMAM G3 dendrimer. It contains 14 mPEG 2K groups.

92

PEGylated dendrimer study have not been described previously, they were carried out according

to the same protocol [4]. Dogs were injected on day 0 and day 14 of the experiment.

Preparation of Polymeric Micelle and Dendrimer Solutions

Descriptions of F13M1 and G1F13M1 solutions (full emulsions and micellar solutions)

can be found in the previous protocol [4]. Synthesis of these polymers were also described

previously [2]. mPEG1K-DSPE (Avanti Polar Lipids Inc, Alabaster, AL) was prepared at 12.53

mg/mL in normal saline and injected intravenously at a dose of 0.45 mL/kg. The PEGylated

dendrimer (PAMAM G3) was prepared at 8.315 mg/mL and injected intravenously at 0.6 mL/kg.

Further information on the synthesis of the PEGylated dendrimer can be found elsewhere [2].

All solutions were filtered with a 0.45 µm nylon syringe filter.

Blood Processing and Collection

Serum was processed according to the previous protocol for DSPE and PEG-

fluorocarbon dogs. For the dogs injected with dendrimers, plasma was used instead of serum.

EDTA whole blood was kept on ice then centrifuged (1500 g, 15min, 4ºC), aliquoted, and stored

at -80°C until analysis. Serum or plasma was collected pre and post (25 minutes) injection for

histamine analysis. Additionally, samples were collected approximately every 5-7 days for two

weeks to one month (depending on the test substance) for antibody analysis.

Histamine Analysis

Histamine was analyzed via ELISA (Neogen Corporation, Lansing, MI, Product #

409010). Results were processed as a fold change in histamine concentration between post and

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pre injection samples. For the PEG-DSPE data, 3 ELISAs were run at the same time. The final

fold change was calculated by averaging the fold change in each of the 3 tests. Samples were run

in duplicate wells as per manufacturer’s recommendation. DSPE findings were termed

“statistically significant” using 95% confidence intervals based on the student’s t-distribution.

Five degrees of freedom were used corresponding to the six wells tested per sample. For the

PEGylated dendrimer data, 1 ELISA was used to determine the fold change and the graphs are

presented with standard deviations instead of confidence intervals.

Nanoparticle-Specific IgM ELISA

ELISAs were designed to measure IgM generation specific for the particles used in this

experiment, by coating the particular substance on a microwell plate and incubating it with

sera/plasma withdrawn from the dogs. For the F13M1 experiments, a similar polymer (F13M5)

with a larger mPEG (MW 5K) was coated on the microwell plates. The step by step protocol is

as follows: F13M5 (10 ug/well), G1F13M1 (3.4 ug/well), mPEG1K-DSPE (3.2 ug/well), or

PEGylated dendrimers (mPEG2k-PAMAM G3) (10ug/well) in methanol was added to a 96 well

polystyrene plate (Corning Incorporated, Corning, NY, Product #9017) and air dried for 3 hours

in a fume hood. 350 ul of 1% BSA blocking buffer in PBS (Thermo Fisher Scientific Inc,

Rockford, IL, Product #37525) was added to each well for 30 minutes. 100 ul of sera diluted

(1/50) in BSA blocking buffer with added 0.05% Tween 20 (Thermo Fisher Scientific Inc,

Rockford, IL, Product # 28320) was added to each well for 1 hour then washed 3x with ELISA

wash buffer (Thermo Fisher Scientific Inc, Rockford, IL, Product ## N503). 100 ul of anti-Dog

IgM with HRP (Novus Biologicals, Littleton, CO, Product #NB7321) diluted (1/100,000) in

BSA blocking buffer with 0.05% Tween 20 added to wells for 1 hour. Plates were again washed

94

3x with ELISA Wash Buffer. 100 ul of 1-step Ultra TMB (Thermo Fisher Scientific Inc,

Rockford, IL, Product #34028) was added to each well for 15 minutes and the reaction was

stopped with 2M sulfuric acid. Plates were analyzed in a plate reader (EL800, Biotek, Winooski,

VT) at 450 nm.

The F13M1, G1F13M1, and mPEG1K-DSPE IgM data was repeated in 3 ELISAs.

Samples were tested in three wells per ELISA. An example ELISA is shown with standard

deviations displayed as error bars. 1 ELISA was performed for the PEGylated dendrimer data.

Results

Figure 5.3 displays IgM generation after injection of the F13M1 sevoflurane emulsion

compared to saline controls. All F13M1 dogs generated IgM specific for F13M5. As expected,

the saline dogs developed no antibody response.

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Figure 5.3: Either the full F13M1 emulsion or saline were repeatedly injected into three dogs. Injections occurred on days 0 and 14 of the experiment. All dogs were tested for antibodies reactive to F13M5 (a similar polymer to F13M1 except with a larger (MW 5K) mPEG. F13M5 was used because it was predicted that it may bind the microwell plate better than the F13M1 polymer.

Dog W generated the largest peak concentration of reactive antibodies, followed by Dog

15 and Dog O. Antibody generation profiles were slightly different among the F13M1 dogs.

Peak concentrations were reached approximately 5-10 days after injection. Levels did not return

to baseline until after day 20 for Dog W and Dog 15. It is important to note that the F13M1 dogs

developed HSRs upon injection of the formulation, with the reactions strengthening upon the

second injection [4].

One dog that was previously treated only with saline was injected with a micellar solution

of F13M1. This dog also produced polymer-specific IgM (Figure 5.4).

96

Figure 5.4: Dog B was treated with an F13M1 micellar solution and produced an IgM response. The full emulsion is not needed to see an antibody response.

The previous study reported that Dog B also exhibited an HSR to the F13M1 micellar solution

injection [4].

G1F13M1 dogs were treated with the full emulsion the first week, and a micellar solution

of G1F13M1 the next week [4]. Allergic-like responses were seen to both injections [4].

Antibody generation to G1F13M1 is plotted in Figure 5.5.

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Figure 5.5: Dog 2 and dog 71 generated polymer specific IgM to G1F13M1 injections. Both dogs were injected with the full G1F13M1 emulsion on day 0 and a micellar solution of G1F13M1 on day 14.

The dogs treated with G1F13M1 generated two peaks of IgM specific for the polymer.

In addition to our PEG-fluorocarbon polymers, additional PEGylated nanoparticles were

tested for their immunogenicity in a canine model. A micellar solution of mPEG1K-DSPE was

injected on days 0 and 14. This formulation did not elicit an HSR. Histamine concentration was

analyzed as a biological marker for the strength of any potential HSR. The results from this

experiment are reproduced in Figure 5.6. As expected, no substantial changes in histamine

concentration were observed.

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Figure 5.6: Dogs were injected with a micellar solution of mPEG1K-DSPE (on days 0 and 14) and measured for a change in histamine concentration for each injection. Neither injection resulted in a significant change in histamine concentration compared to baseline.

The DSPE dogs were also tested for antibody generation in response to their injections (Figure

5.7). Both dogs treated with this polymer generated IgM antibodies specific for mPEG1K-

DSPE. Antibodies concentrations had not returned to baseline after 1 month of monitoring.

Figure 5.7: Dog S and X, previously treated only with saline, were also analyzed for an antibody response against the mPEG1K-DSPE polymer. Both generated antibodies after the initial injection.

99

Lastly, PEGylated dendrimers were also injected. No HSR or significant increase in

histamine was observed (Figure 5.8). Figure 5.9 analyzed IgM generation in response to the

dendrimer injections. Only one dog had a substantial change in particle-specific IgM

concentration following injection of the dendrimers.

Figure 5.8: PEGylated dendrimers (PAMAM G3) were injected into 3 dogs on days 0 and 14 of the experiment. No substantial effects on histamine concentration were observed.

Figure 5.9: IgM generation in response to injection of PEGylated dendrimers. Two out of three dogs displayed little (if any) antibody generation. Only dog CNZ had a substantial change in concentration. Data points are displayed with standard deviations.

100

Discussion

The focus of this paper was to further characterize the immune response to our PEG-

fluorocarbon polymer. Figure 5.3 displays that a rise in IgM is associated with a strengthening

of the allergic-type response upon second injection. Classically, sensitization to an immune

response would be explained by a rise in IgE and a Type 1 hypersensitivity. While this remains

a definite possibility, it is also possible IgM-mediated complement activation plays a role in the

HSR. The concentration of polymer-specific IgM is higher for the dogs by the time of second

injection. It is suspected that our polymer does not activate complement directly [3], but anti-

F13M1 IgM could activate complement via the classical pathway. Theoretically, the HSR could

strengthen as antibody titers rise after the first injection. In addition, low levels of polymer-

specific IgM that could not be quantified by this assay could account for the mild HSR seen on

the first injection. In addition, Figure 5.5 displays the presence of anti-G1F13M1 IgM on day 0

of the experiment. It’s possible that the existence of measurable G1 IgM contributed to the

severity of the HSR elicited to the G1 polymer compared to the F13M1 polymer. Linking of

complement activation to the HSR and the ensuing rise in IgM is needed to validate these

theories.

In most cases of CARPA, a milder or absent immune response is expected upon

reinjection [6,7] and a strengthening of the immune response would indicate an IgE-mediated

hypersensitivity. Boosting of the IgG response upon second injection was not apparent in our

analysis of the F13M1 data (see Appendix). Because of this, and the polymeric nature of our

antigen, we suspect F13M1 is a thymus independent (TI) antigen. This suggests limited isotype

101

switching in the immune response [8] which would make an IgE reaction appear unlikely. Koide

et al recently demonstrated that PEGylated liposomes used for encapsulation of doxorubicin are

likely thymus-independent antigens [9]. Though our polymer differs from this formulation, it

does have some similarities (PEG), further suggesting that our antigen may be thymus

independent. Nevertheless, studies tracking total IgE levels throughout this experiment remained

inconclusive and future studies should also continue to investigate IgE as the mechanism of this

HSR.

Additional points can be made concerning the antibody generation profile of our PEG-

fluorocarbon polymers. Figure 5.4 demonstrated that the micellar solution is sufficient to

produce an immune response. This dog did appear to produce a smaller concentration of

antibodies than those treated with the full emulsion, but considering the small sample size and

the variability in magnitude of the response to the full emulsion in Figure 5.3, no statistical

meaning can be taken from this occurrence.

Figure 5.3 also suggests that the full emulsion of F13M1 produced B cell tolerance

towards the second injection. If F13M1 is considered a thymus independent antigen, an

additional peak of IgM might be expected after the second injection [10]. However, the ability to

induce tolerance is also a noted characteristic of TI antigens [11]. F13M1’s failure to produce a

second peak is especially interesting considering Figure 5.5. The G1F13M1 polymer induced

two peaks of IgM associated with its injections on day 0 of the full emulsion, and on day 14, of

the micellar solution. There are multiple explanations for the two peaks seen with the G1

polymer. Some TI antigens display cyclical appearance of IgM even after one exposure,

depending on the persistence of the antigen and the ability of the antibodies to inhibit their own

102

generation [12]. Alternatively, the G1 polymer might not have induced the tolerance seen with

F13M1. Lastly, it should be emphasized that two different delivery methods were used in the G1

injections. The injection of the full emulsion and then the micellar solution might have allowed

the polymer to be presented in different ways such that alternate epitopes were exposed on

F13M1, thereby generating two separate peaks of antibodies. Though all of these mechanisms

are possible, further investigation is required to explain the antibody generation profiles to

G1F13M1 and F13M1.

Other PEGylated particles were also tested to further analyze this immune response.

Micellar solutions of an mPEG1K-DSPE polymer did not induce an immune response. But, it

should be noted that the dose of injection for these PEG-phospholipids (0.45 mL/kg) was less

than that of the F13M1 micelle (0.6 mL/kg). Interestingly, mPEG-DSPE did lead to IgM

production. If IgM is involved in the HSR, this indicates that it isn’t the only factor in

determining an HSR to a PEGylated particle. Indeed, it has been shown that injection of

PEGylated particles into rats [13,14] and rabbits [15] and the formation of anti-PEG antibodies

doesn’t necessarily cause an HSR, though it should be noted that these animals are less

susceptible to HSRs from these types of formulations than dogs [5].

Because of the result with the phospholipid polymer, it was hypothesized that

development of a HSR from a PEGylated particle in dogs may be due to the stability of the

micelle. PEG-fluorocarbon micelles are expected to be more stable than micelles of PEG-

phospholipids in vivo due to the chemical inertness of the fluorocarbon compared to the DSPE.

To mimic the PEG surface of a stable micelle, PEGylated dendrimers were synthesized [2] to test

their ability to generate an HSR in dogs. The dendrimers did not cause an HSR, but there are

103

many other differences between this particle and our fluorocarbon polymers that could explain

the lack of this response. First, the size of the dendrimers was only 7 nm, which is substantially

smaller than a F13M1 micelle. This could mean that the dendrimers are filtered from blood

circulation by the kidney too quickly to cause an allergic-like response. Finally, though the

dendrimers were predicted to have a covered PEG surface, there is no way to definitively

confirm their structure. Taking all this account, it is still unclear what effect a stable PEG

surface plays in the generation of this HSR.

Conclusion

This article associates IgM generation with injection of a PEG-fluorocarbon based

sevoflurane emulsion in dogs. It is hypothesized that this IgM generation leads to the enhanced

allergic-like response seen to our formulations upon repeat injection [4], but further investigation

into both this possibility and Type 1 IgE hypersensitivity is needed.

Further explorations analyzed the immune response to PEG-phospholipid and PEGylated

dendrimers. Neither particle elicited an HSR in dogs, indicating the need for a better

understanding of how some PEGylated particles elicit hypersensitivity in this animal.

104


1. Fast JP, Perkins MG, Pearce RA, Mecozzi S. Fluoropolymer-based emulsions for the

intravenous delivery of sevoflurane. Anesthesiology 2008, 109, 651–656.

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Delivery of Sevoflurane: Synthesis, Physicochemical Characterization, and

Immunogenicity Studies” (PhD dissertation, University of Wisconsin – Madison, 2012).

3. Johnson RA, Simmons KT, Fast JP, Schroeder CA, Pearce RA, Albrecht RM, Mecozzi S.

Histamine release associated with intravenous delivery of a fluorocarbon-based


4. Simmons K, Nejati E, Johnson R, Rejaei D, Pearce R, Albrecht R, Mecozzi S. “Enhanced

allergic-type response to PEG-fluorocarbon polymer used for intravenous delivery of

sevoflurane upon repeat injection in canines.” University of Wisconsin – Madison.

Unpublished Manuscript. Submitted for publication.




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10, 37–47.

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*The material presented in this chapter will be the object of a manuscript (in preparation).

Chapter 6:

Analysis of the Immune Response to a Fluorocarbon-

Based Sevoflurane Emulsion in Monkeys*

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Abstract

Background: An intravenous delivery system for the general anesthetic, sevoflurane, has been

developed. Solubilization of sevoflurane in blood is achieved via emulsification with a mPEG-

fluorocarbon polymer (F13M1) and a stabilizing agent, perfluorooctyl bromide. This emulsion

has been linked to hypersensitivity reactions (HSRs) in dogs. However, the immunotoxicology

of this formulation is unknown in other species. Rhesus macaques were injected with the

emulsion to assess its effects on the immune response of nonhuman primates.

Methods: Three rhesus macaques were injected with an F13M1 solution and the full emulsion.

The monkeys were observed for an HSR. EDTA plasma samples were drawn before and after

injection to analyze histamine release via ELISA. Seven days later, the injections were repeated

and the macaques were analyzed for F13M1-specific antibodies.

Results: No HSRs or substantial changes in histamine concentrations were observed. Two out

of three monkeys developed IgM specific for F13M1.

Conclusions: The formulation did not elicit HSRs in these three monkeys. However, the sample

size is too small to fully determine its safety in this species. It was previously suggested that the

HSR seen in dogs may involve F13M1-specific IgM. This remains a possibility, yet it should be

noted that 2 out of 3 monkeys did develop F13M1-specific IgM and did not develop an HSR

upon second administration. Another mechanism may be involved in the HSR, however, it is

also possible that IgM generation is just one factor in determining whether or not an allergic-type

reaction is elicited by this formulation in a susceptible individual.

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Introduction

The previous chapters described that the F13M1-sevoflurane emulsion produced allergic-

like reactions in dogs. We are assuming that the HSR seen in dogs would represent a small

portion of the human population that would react in a similar manner. This is based off the idea

that dogs are a good animal model for CARPA and similar micellar based drug delivery systems

[1]. Nevertheless, the possibility remained that the F13M1 emulsion would be immunogenic in

all primates. Before the emulsion is considered for human trials, it was essential to test the

formulation in a non-human primate to see if any extreme immunotoxicity was observed. To

achieve this, the F13M1 emulsion was tested in 3 rhesus macaques. In addition, the efficacy of

the emulsion was also tested. Anesthesia was achieved in this model, but further explanation of

the physiological data will not be presented here and this chapter will deal primarily with the

immunotoxicity of F13M1 in monkeys.

Similar to the dog studies, the monkey studies were analyzed for histamine release and

antibody generation. The polymer alone, and the full emulsion, were injected to observe if they

elicited an HSRs in the macaques. The experiment was later repeated to test if sensitization was

required for the emergence of an allergic-like response.


Study Design

In order to test the immunotoxicity of F13M1 and the sevoflurane emulsion, our

formulations were injected into 3 rhesus macaques. On the first injection day, a bolus dose (0.41

mL/kg) of F13M1 solution (12 mg/mL) in normal saline was administered to the monkeys while

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they were observed for an allergic-like response. Blood was drawn prior to and 20 minutes after

injection.

After administration of the F13M1 solution, a bolus dose of the full emulsion (0.41

mL/kg) was injected. After 20 minutes of observation for immunotoxic and anesthetic effects,

blood was drawn again.

The macaques were treated with the F13M1 solution and the full emulsion again, 7 days

after the first experiment. Blood was drawn before injection of the F13M1 solution. Since no

allergic-like reactions were seen, no more blood was drawn during the course of the experiments.

The focus of the experiments then shifted towards studying the efficacy and delivery methods of

the full emulsion, which will not be discussed in this chapter.

Blood Processing and Collection

EDTA plasma was collected (1500 g, 15min, 4ºC), aliquoted into polypropylene tubes,

and stored at -80 ºC until analysis.

Histamine Analysis

Histamine was measured via ELISA (Neogen Corporation, Lansing, MI, Product #

409010). Pre and post samples for both the F13M1 solution and the full emulsion were analyzed

for each monkey. A fold change in histamine concentration for the two injections was calculated.

The ELISA was run 1 time. The procedure was carried out according to manufacturer’s

recommendations (samples were run in duplicate wells).

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Anti-F13M5 IgM ELISA

The antibody analysis was run according to the procedure listed in chapter 5. F13M5 was

coated on a microwell plate to test for IgM generated to the F13M1 emulsion. The secondary

antibody was specific for monkey IgM (Acris Antibodies, San Diego, CA, Product

#AP31440HR-N). Two timepoints were analyzed in this assay. Plasma was used prior to the first

injection of the F13M1 solution, and prior to the start of the second set of experiments (one week

after first exposure to the formulation). 1 ELISA was run for this experiment. Bars are presented

with standard deviations.

Results

All the monkeys responded well to treatment of both the polymer alone and the full

emulsion. No obvious signs of hypersensitivity were recorded. The histamine analysis (Figure

6.1) confirms the lack of HSR.

Figure 6.1: Neither the F13M1 emulsion or the polymer solution induced substantial changes in histamine concentration after injection. Values are displayed with 10% error bars, corresponding to the intra-assay precision for the ELISA as listed in the manufacturer’s instructions.

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IgM generation in response to the injections was also recorded (Figure 6.2). Two of the

monkeys demonstrated a rise in polymer-specific IgM concentration seven days after injection of

the emulsion.

Figure 6.2: Antibody generation after F13M1 injection. Two out of three monkeys produced an antibody response to the polymer. Bars are presented with standard deviations.

Discussion

Our formulations did not produce HSRs when tested in rhesus macaques. Unfortunately,

these studies could only detect if F13M1 was strongly immunogenic in other animals besides

dogs. The sample size is too small to detect if the formulation would be safe in most monkeys.

Most importantly, it is difficult to use this experiment to predict how the F13M1 emulsion would

react in humans, specifically because there are no comprehensive studies analyzing the HSR

response rate to nanoparticle systems between the two species.

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Antibody generation to the polymer was apparent in two out of three monkeys. If IgM is

involved in the HSR observed in dogs, differences in other factors must account for the

presence/absence of the allergic-type response demonstrated between the two species.

Conclusion

The F13M1 emulsion did not prove to be highly toxic in any rhesus exposed to the

formulation. This is an indication that the allergic-type reactions seen in dogs may not appear at

high rates in other species, but further characterization of the immune response is needed before

the sevoflurane emulsion is tested in humans.

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Chapter 7:

Conclusions and Future Directions

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Conclusions

The ultimate goal of this dissertation was to determine the mechanism of the HSR to

F13M1 and apply what was learned from this study to guide design of non-immunogenic

PEGylated carriers. The HSR’s complexity made the pathway difficult to elucidate. Questions

remain about the mechanism. What is the role of anti-polymer antibodies? Additionally, new

questions arose over the course of the research. Is micelle stability a factor in generation of these

HSRs? Despite all the remaining questions, important discoveries were made characterizing the

reaction to F13M1. These findings can guide development of intravenous sevoflurane and direct

research with respect to the immunogenicity of other PEGylated carriers.

The HSR to F13M1 differs from other allergic-type reactions previously attributed to

CARPA, in that it appears to strengthen upon repeated injections of the polymer. This

phenomenon was correlated with a rise in histamine at the second injection and antibody

generation upon first exposure to the formulation. In addition, F13M1 was not linked to direct

complement activation in vitro. Though further exploration of the pathway is needed, this finding

is significant in that previous (pseudo)allergies to PEGylated particles diminished upon repeat

injection. The current study suggests that antibody generation to polymers may contribute to

HSRs. Moreover, the reaction of canines to F13M1 and their lack of reaction to corresponding

PEG-hydrocarbon and PEG-DSPE polymers suggests the importance of additional

characteristics of PEG copolymers, such as micellar stability, in generation of an allergic-like

response. If antibodies to PEG are involved in the HSR, it also appears likely that antibodies

specific for PEG clusters (like that found on a micelle surface), instead of single PEG molecules,

are important in generating the allergic-type response. Antibodies specific for single PEGs likely

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have a minimal role in this HSR due to the fact that PEG solutions do not cause allergic-like

responses in canines. Finally, exploration into in vitro hemolytic assays for complement

activation indicates that this method may not be optimal for PEGylated copolymers because

interactions with the polymers and the test system might interfere with results. Taking all of

these findings into account, future studies and insight into the direction of this research and what

it means for clinical development of these systems is described in the following sections.

Future Studies

Mechanism of HSR to F13M1

This thesis presents some key findings into the mechanism of allergy to F13M1, but

further exploration is needed to fully decipher the pathway. One current hypothesis is that

polymer-specific IgM, increased by the time of the second injection, enables a greater degree of

complement activation and allows an enhancement of the allergic-type reaction. Essential

evidence to validate this theory is the measurement of complement activation from pre and post

samples from each round of injections.

Some attempts were made to study complement levels prior to completion of this thesis.

Unfortunately, while the dog may be a good model for screening micellar systems for HSRs, it is

not a good model for deciphering the mechanism of those reactions. Unlike humans, which have

a variety of ELISA kits for complement activation split products, dogs only have a few options

available. ELISA kits for dog C5b-9 (a terminal complex) (TSZ Elisa, Framingham, MA) and

C3a (an anaphylatoxin) (Uscn Life Science Inc, Wuhan, China) were used to analyze the

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samples. The standard curves generated with these kits exhibited variability implying that

accurate detection of low levels of complement activation may not be possible.

Therefore, an alternative method to assess complement activation is needed. A CH50

assay for measurement of total complement activity might work with these samples [1].

Suggesting use of a CH50 assay might seem like a contradiction to data presented in Chapter 3

of this thesis, but an important difference is that addition/incubation of the polymer will not be

required. The small amount of F13M1 already found in the dog’s serum (approximately 0.012

mg/mL based on 1L blood volume) from injection may not affect the outcome of the results.

This would have to be confirmed by spiking a saline dog’s samples with an equivalent amount of

F13M1 to determine if the outcome is altered.

An alternative hypothesis to IgM enhanced complement activation is the formation of

polymer-specific IgE. Finding specific IgE in a dog is a harder task than finding complement

activation. Attempts were made to chart total IgE over the course of the study via ELISA, but

the kit produced inconclusive (highly variable) results. Previous attempts to create an ELISA for

polymer-specific IgE produced too much background to determine if anti-F13M5 IgE existed in

a dog that had anti-F13M5 IgM but had not been previously intentionally exposed to the

polymer. It may be possible to repeat this assay with the new set of samples, since these dogs

may have higher concentrations of IgE after being repeatedly exposed to the polymer. Further

optimization of the assay (changing the microwell plate, well coating concentration, and buffers)

may also be required to find IgE.

Further studies into the mechanism of this HSR should also investigate micellar size and

stability. Additional studies with PEGylated dendrimers could analyze both of these factors.

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Dendrimers [2,3] are a useful nanoparticle for this type of experiment because their size can be

controlled. This experiment could use PEGylated dendrimers of progressively higher generations

and correlate them with an HSR in vivo. Larger dendrimers may mimic the surface of a stable

micelle better than the PAMAM G3 dendrimers discussed in Chapter 5. In addition, using

multiple generations could determine how important the size of the molecule is in the HSR. The

injection concentrations could then be varied to further assess the importance of the surface area

to volume ratios of the nanoparticles. The length of the PEG conjugated to the dendrimer could

also be changed. In the end, this series of studies could prove useful in determining how a wide

range of attributes contribute to the generation of HSRs by PEGylated nanoparticles.

In this thesis, submicellar concentrations of F13M1 were found to be non-immunogenic.

However, it was not known if the concentration was too low to cause an allergic response. For

new studies, injection of different mPEG—fluorocarbon polymers that cannot form micelles

could be used as a control. Using this or a similar control could show that PEG-fluorocarbons

are non-immunogenic when they are not in an aggregated form.

Future studies should put less emphasis on deciphering the mechanism to the glucose-

functionalized polymers. This thesis shows that functionalizing the PEG surface with glucose

creates highly immunogenic polymers, so future studies will most likely focus on further

modifying the original polymer, F13M1. Nevertheless, it would be interesting to explore the

mechanism behind G1 and G3F13M1. The current hypothesis is that these polymers may be able

to activate complement directly (in addition to an antibody-based mechanism), since the exposed

OH groups on the polymer surface could activate the alternative pathway of complement.

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Incubation of the glucose-functionalized polymers in human serum as was done in Chapter 2

with F13M5 may be an experiment that would support alternative pathway activation.

Development of Intravenous Sevoflurane

More investigation is needed for some results contained within this dissertation. So far, it

appears that the best formulation for sevoflurane administration may be contradictory to what is

desired in most micellar drug delivery systems. There is some evidence that the stability of the

micelle may play a role in generation of the HSR. The fluorocarbon polymers used in this

formulation are predicted to be more stable than their hydrocarbon counterparts. Though the

fluorocarbons are necessary to emulsify sevoflurane, it’s possible the stability contributes to the

generation of HSRs. The ideal polymer for this formulation would be small so it could be

eliminated quickly and would also be unable to form aggregates.

If it is not possible to generate such a polymer that would work for this system, other

modifications could be made to the original F13M1 polymer. The mPEG included should be as

small as possible. Alternatively, the PEG surface could be masked with a molecule known to

inhibit complement, such as sialic acid which is known to protect host cells from complement

activation [4].

Another modification would be to utilize mixed micelles in the sevoflurane emulsion.

This could eliminate repeating epitopes that may exist on the micelle surface. Other fluorous

copolymers with different hydrophilic blocks could be included in the formulation along with

F13M1, lowering the concentration of the mPEG-fluorocarbon polymer. Cyclodextrins [5] have

already been synthesized for this purpose, but a variety of other hydrophilic blocks [6] could also

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be investigated. In addition to making this formulation less immunogenic, mixed micelles could

also be used to investigate the role of poly(ethylene glycol) surface density in HSRs to

PEGylated particles.

Clinical Development of PEGylated Nanoparticles

It is apparent that F13M1 may have a different mechanism of action than PEGylated

particles which have been associated with CARPA. The potential diversity of nanoparticle-

based HSRs is easy to imagine given the variability found (type of nanoparticle, drug, delivery

method) in each particular formulation. This thesis suggests an antibody-based mechanism for

F13M1 which may be applicable to similar PEG micellar systems. Yet, direct complement

activation, direct mast cell/basophil degranulation, or a variety of other potential mechanisms

could be attributed to less similar PEGylated systems.

Considering this, it may be more cost-effective to design a screening method for patients

likely to exhibit these HSRs rather than deciphering the exact mechanism for every reaction. A

starting point to design an effective screening method could be the animal model used for

F13M1, the dog. Skin tests, ocular challenges, and pulmonary provocation tests have been

described as alternative methods to confirm allergy in dogs [7]. HSRs elicited by intravenous

administration of F13M1 could be correlated with HSRs elicited by F13M1 in the skin or eye

exam. The method could be expanded to similar polymers and confirmed as one enters clinical

trials.

An alternative screening method may be desired that would test the likelihood of an HSR

to a whole formulation (drug and delivery vehicle) instead of just the delivery vehicle. It is

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likely that immunogenetic screening could be a valuable tool for this in the future. Analyzing

the genetic code of those that have exhibited strong HSRs, regardless of the mechanism, could

identify numerous protein variations (sensitivity of complement proteins to activation, sensitivity

of histamine receptors, sensitivity of basophils to degranulation, etc) that could result in a higher

likelihood of HSRs to all kinds of antigens.

The most convenient, realistic method to screen patients for potential HSRs may be to

analyze serum concentration of antibodies or complement that may serve as biomarkers for

allergic-type responses to nanoparticles. Investigations into the mechanism behind particular

HSRs to these particles may be the best way to find these biomarkers, as well as to ultimately

guide design of non-immunogenic delivery systems. Finally, it should be easier to determine the

mechanism of action for these HSRs in humans than in animal models. There are numerous

examples of PEGylated particles currently undergoing clinical development (see Chapter 1).

This thesis suggests anti-polymer antibodies may play role in the HSR to F13M1. Other

particles have been linked to complement activation. Knowledge of these findings should guide

subsequent preparation for assessing potential HSRs to PEGylated nanoparticles during the

clinical trial stage. Assessing concentrations of IgM and IgE specific for the delivery vehicle, and

testing for complement activation prior to and after administration of these test substances (or

having the applicable serum samples available) during clinical trials would be the best way to

ultimately make both design and administration of these delivery vehicles safer.

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1. Giclas PC. Classical pathway evaluation. Current Protocols in Immunology 2001,

Chapter 13, Unit 13.1.

2. Xu Q, Wang CH, Pack DW. Polymeric carriers for gene delivery: chitosan and

poly(amidoamine) dendrimers. Current Pharmaceutical Design 2010, 16, 2350-2368.

3. Mishra V, Gupta U, Jain NK. Surface-engineered dendrimers: a solution for toxicity

issues. Journal of Biomaterials Science 2009, 20, 141-166.

4. Rother K, Till GO, Hänsch GM. The Complement System. Germany: Springer, 1998.

5. Elham Nejati, “Glucosylated Derivatives of Semifluorinated Surfactants for Intravenous

Delivery of Sevoflurane: Synthesis, Physicochemical Characterization, and

Immunogenicity Studies” (PhD dissertation, University of Wisconsin – Madison, 2012).

6. Knop K, Hoogenboom R, Fischer D, Schubert US. Poly(ethylene glycol) in drug

delivery: pros and cons as well as potential alternatives. Angew Chem Int Ed 2010, 49,

6288-6308.

7. Zemann B, Griot-Wenk M, Marti E, Mayer P, Eder C, Nefzger M, Schneider H, de Weck

A, Liehl E. Allergic pulmonary and ocular tissue responses in the absence of serum IgE

antibodies (IgE) in an allergic dog model. Veterinary Immunology and

Immunopathology 2002, 87, 373-378.

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Appendix

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Appendix 1 – IgG Analysis of F13M1 and G1F13M1

Figure A.1: IgG analysis for the F13M1 injections. Procedure for this ELISA follows the same procedure in chapter 5 for the IgM analysis, except that anti-canine IgG-HRP (Product #NB7341, Novus Biologicals, Littleton, CO) was used as the secondary antibody. This antibody cross-reacts with light chains of other immunoglobulins. The cross-reaction of this antibody with Dog IgM is the hypothesis for why this graph resembles figure 5.3. No boosting of the immune response is seen with the second injection, supporting the hypothesis that F13M1 is a thymus-independent antigen.

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