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General Manager of Inspection and Control of Drugs and ProductsAntônio Carlos da Costa Bezerra

Inspection Coordination in Bioequivalence CentersCláudia Franklin de Oliveira

E-mail: [email protected]


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Brazilian Sanitary Surveillance Agency

Manual for Good Bioavailability and

BioequivalencePractices

Volume I

Brasília2002


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Rights reserved for Brazilian Sanitary Surveillance AgencySEPN 515, Edifício Ômega, Bloco B, Brasília (DF), CEP 70770-502.Internet: www.anvisa.gov.br

Copyright © 2002. Brazilian Sanitary Surveillance Agency.The total or partial reproduction of this work is permitted provided that the source is mentioned.

First edition - 2002

ISBN: 85-88233-06-1

Brazilian Sanitary Surveillance AgencyPerformed by: Inspection Coordination in Bioequivalence Centers -General Office of Inspection and Control of Drugs and ProductsGeneral Coordination: Cláudia Franklin de Oliveira / Inspection Coordination inBioequivalence CentersRevision: Karla de Araújo Ferreira / Inspection Coordination in Bioequivalence Centers

Divulgation: Divulgation UnitCovers: João Carlos de Souza Machado / Multimedia Communication Office

Graphic Art, composition and printing: Dupligráfica Editora Ltda./DF

Printed in Brazil

Manual de boas práticas em biodisponibilidade: bioequivalência/AgênciaNacional de Vigilância Sanitária. Gerência-Geral de Inspeção eControle de Medicamentos e Produtos. Brasília: ANVISA, 2002.

2 v.QV38

1. Equivalência terapêutica. 2. Bioequivalência. 3. Disponibilidade

biológica. 4 . Medicamentos. I. Agência Nacional de Vigilância Sanitária.Gerência-Geral de Inspeção e Controle de Medicamentos e Produtos.


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PRESENTATION

The Brazilian Sanitary Surveillance Agency, embedded of its institucional mission, elaborated the

present work with the intention to foment discussion, education and the conduction of clinicalstudies in Brazil.

Concerning the bioavailability/bioequivalence studies, this compendium is part of a bigger context which is intended to enclose the clinical research in its amplest aspect: non clinical studies andclinical studies of phase I, phase II, phase III and IV.

In presenting this work , The Brazilian Sanitary Surveillance Agency would thank the contributionof all the professionals directly and indirectly involved in its consolidation, and it is available for

critics and suggestions that may contribute for future editions, as well as, for the subsequent modules.

Dr. Gonzalo Vecina Neto


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PREFACE

A number of clinical trials are being presently conducted in Brazil aiming to evaluate the

Bioavailability/Bioequivalence of pharmaceutical products. As of June 2001, Anvisa, through theInspection Coordination in Bioequivalence Centers – bound to the General Office of Inspectionand Control of Drugs and Products – started evaluating such centers by means of periodicalinspections in order to assure the quality of the trials.

During the inspection activities, originally for orientation purposes, the coordination observed theneed to clarify some aspects that still posed technical questions to the centers, especially concerning the standardization of analytical methods, statistical analysis of the trials, storage of biologicalsamples, confinement of volunteers and stability studies of drugs and others.

When such requirement was identified and in an attempt to avoid impairing the quality of the workscarried out, the coordination stimulated the creation of discussion nuclei intended to wear out theexplanation of all aspects related to the conduction of the studies and the integration of theirphases. Such nuclei counted on the participation of 40 experts from the Pharmaceuticals, Medicine,Statistics and Chemistry fields.

This “Manual for Good Bioavailability/Bioequivalence Practices” was created under such contextand comprises six main topics presented in a didactic manner, trying to overwhelm the centers’difficulties and, as a result, complement the guidelines provided for in the Brazilian health legislationfor conducting trials.

The working nuclei started the discussions in September 2001. Each nucleus was dedicated to oneof the three phases of the process – Clinical, Analytical and Statistical Phases – under the coordinationof Dra. Cláudia Franklin de Oliveira and the inestimable cooperation of professor Sílvia Storpirts.During the subsequent months, several meetings were held so as to promote technical debates andreach a consensus concerning theses questions. Consequently, each group prepared a set of relevanttopics to be included in the manual. All such topics were then subjected to careful researches andthe results reported so as to allow a good understanding by the target population. The complete

manual took 11 months to be finished. This process involved the participation of about 50professionals in total from several fields of knowledge, including researchers from public universities,technicians from Anvisa and manufacturers of laboratory instruments and equipment. Anvisa isthankful to all such associates for having contributed to such an excellent job.

The final structure comprises two volumes with three modules each. The first volume technically details each phase of the Bioavailability/Bioequivalence studies in the natural sequence of conduction:Module 1: Clinical Phase, Module 2: Analytical Phase and Module 3: Statistical Phase. The second

volume covers the important aspects concerning laboratory instrumentation and equipment used

to carry out the analytical phase and regarded as critical for the process. In that volume, Module 1relates to the Principles and Operation of Micropipettes, Module 2 refers to Water for Chemical


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Instrumental Analyses and the contents of Module 3 relates to the Ultraviolet VisibleSpectrophotometry, Liquid Chromatography (LC), Gas Chromatography (GC), Chromatography Systems Connected to Mass Detectors and Checking of Analytical Instruments Performance.

It is worth emphasizing the international novelty character of gathering information from severalsubjects in a single compendium aiming at synthesizing all the aspects involving the GoodBioavailability/Bioequivalence Practices.

It is evident that the major purpose of this work is to improve the quality of the Bioavailability/Bioequivalence assays carried out in Brazil and, as a result, contribute to the quality of generic drugsavailable in the market by providing widely studied and carefully prepared technical subsides. Withthis in view, we hope to contribute to capacitating the Bioavailability/Bioequivalence Centers, inaddition to develop study monitors for the domestic pharmaceutical industry and help in theformation of knowledge multiplier agents at the Brazilian universities.

The preparation of this manual was only possible thanks to the crucial work of several persons. I would like to apologize in advance for possibly forgetting any name. The editors José Pedrazzoli Júnior (USF/Unifag) and João Antônio Saraiva Fittipaldi (Pfizer) were essential, as well as thecooperators Fernanda Maria Villaça Boueri (Anvisa), Eliana Regina Marques Zlochevsky (Anvisa),Cláudia Simone Costa da Cunha (Ministry of Health) and Beatriz Helena Carvalho Tess (Ministry of Health), for the module Clinical Phase; the editors Cláudia Franklin de Oliveira (GGIMP/Anvisa),Rui Oliveira Macedo (UFPA), Flávio Leite (T&E Analítica) and Pedro Eduardo Froehlich (UFRGS),and the cooperators Pedro de Lima Filho (GGMEG/SP), Davi Pereira de Santana (UFPE), Rafael

Eliseo Barrientos Astigarraga (Cartesius), Silvana Calafatti de Castro (Unifag), Thaís Reis Machado, Jaime Oliveira Ilha (Cartesius), Itapuan Abimael Silva (Anvisa), Karen Noffs Brisolla (Anvisa), MarceloCláudio Pereira (Anvisa), for the module Analytical Phase; the editors Arminda Lucia Siqueira(UFMG), Chang Chiann (GGMEG/SP), Cicilia Yuko Wada (Unicamp), Karla de Araújo Ferreira(Anvisa) and Gilberto Bernasconi (USF/Unifag), and the cooperators Reinaldo Charnet (Unicamp)and Renato Almeida Lopes (Anvisa), for the module Statistical Phase; the editors Melissa M. Silva(Nova Analítica) and Walter Pereira (Nova Analítica), Principles and Operation of Micropipettes;the editor José Muradian Filho (Millipore), Water for Clinical Analyses; the editors Ivan Jonaitis(Agilent), Renato Garcia Peres (Flowscience), Ricardo Lira (Flowscience), Renato Gouveia, José

Aparecido Soares (Varian), Josué D.M. Neto (Sync Brazil) Juarez Araújo Filho (Sync Brazil), AlexandreRosolia (Waters), Adauto Silva (Varian) and Reinaldo Castanheira (Agilent), AnalyticalInstrumentation; and the coordination team composed by Cláudia Franklin de Oliveira (GGIMP/

Anvisa), Marcelo Cláudio Pereira (GGIMP/Anvisa), Max Weber Marques Pereira (GGIMP/Anvisa),Karla de Araújo Ferreira (GGIMP/Anvisa), Karen Noffs Brisolla (GGIMP/Anvisa), Itapuan Abimaelda Silva (GGIMP/ Anvisa) and Renato Almeida Lopes (GGIMP/Anvisa).

Dr. Gonzalo Vecina Neto


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TECHNICAL STAFF

Volume I – Module 1 – Clinical Step

Editors:

• João Antônio Saraiva Fittipaldi - Pfizer

• José Pedrazzoli Júnior - UNIFAG - USF

Cooperators:

• Beatriz Helena Carvalho Tess - Ministério da Saúde

• Cláudia Simone Costa da Cunha - Ministério da Saúde

• Eliana Regina Marques Zlochevsky - ANVISA

• Fernanda Maria Villaça Boueri - ANVISA

• Gilberto de Nucci - USP

Coordination:

• Cláudia Franklin de Oliveira - ANVISA

• Itapuan Abimael da Silva - ANVISA

• Karen de Aquino Noffs Brisolla - ANVISA

• Karla de Araújo Ferreira - ANVISA

• Marcelo Cláudio Pereira - ANVISA

• Max Weber Marques Pereira - ANVISA

• Renato Almeida Lopes - ANVISA

Logistic Support:

• Daniela Salles de A. B. Corrêa - ANVISA

• Diva Sales Freitas - ANVISA


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SUMMARY

1. CLINICAL RESEARCH ............................................................................................................ 5

1.1. Background .............................................................................................................................. 51.2. New drugs ................................................................................................................................ 7

1.3. Bioavailabil ity/Bioequivalence ............................................................................................ 8

1.4. Clinical studies ........................................................................................................................ 9

1.5. Safety aspects.......................................................................................................................... 10

1.5.1. Adverse events ............................................................................................................ 10

1.5.2. Serious adverse events ............................................................................................... 11

1.5.3. Abnormal laboratory test results ........................................................................... 13

1.6. Responsibilit ies ...................................................................................................................... 13

1.6.1. Sponsor ......................................................................................................................... 13

1.6.2. Principal investigator ................................................................................................ 14

2. DRUG BIOAVAILABILITY/BIOEQUIVALENCE STUDIES – CLINICAL STEP ..16

2.1. Introduct ion ........................................................................................................................... 16

2.2. Facilities .................................................................................................................................. 16

2.3. Volunteer recruitment and selection ............................................................................... 17

2.4. Volunteer hospitalization and discharge ......................................................................... 17

2.5. Administration of the drug and biologic material collection .................................... 18

2.6. Biologic sample handling .................................................................................................... 19

2.7. Documentation ..................................................................................................................... 19

2.7.1. Research protocol ...................................................................................................... 19

2.7.2. Clinical protocol ........................................................................................................ 19

2.7.3. Informed consent form ............................................................................................ 21

2.7.4. Clinical chart .............................................................................................................. 22

2.8. Responsibilit ies ...................................................................................................................... 23

2.8.1. Sponsor ......................................................................................................................... 232.8.2. Principal investigator ................................................................................................ 24

3. REFERENCES .............................................................................................................................. 25


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Volume I / Módule 1 - Clinical Step

1. CLINICAL RESEARCH

1.1. Background

When analyzing the requirements for conduction of research in human beings, we have the falseimpression that most of them are just unnecessary burocracy and rules existing with the sole puroposeof delay the scientific investigation process.

A brief understanding of the origin of this regulation shows unequivocally their importance andthe need for continued research in bioethics.

Until 1906, there were no rules regarding the commerce of drugs or food. Exhibitors going fromtown to town advertised and sold wonderful drugs, without any scientific evidence of efficacy and

safety.

In 1906, Upton Sinclair publishes the book “The Jungle” describing in a realistic way the terriblehygiene conditions in Chicago slaughterhouses. This publication caused an urge of indignationfrom North-American population, which reached the Congress, leading to the approval of the Actknown as “Pure Food and Drug Act”. This Act creates FDA (“Food and Drug Administration”),

which would be the Agency responsible for the regulation of production, transport and sale of drug and foodstuff products in the United States. Unfortunately in this first moment, the Actrequired only a correct labeling of the products, not mentioning in any way the efficacy and safety

conditions.

A new breakthrough in this field only happened after an episode occurring in 1937. In this year,Sulfanilamide (Prontosil) used, since 1932, for treatment of streptococcal infections was launchedas a syrup, countaining diethyleneglycol as solvent. Although tested regarding aspect, taste andodor, its safety was not evaluated before launching. It was responsible for the death of 105 individuals(34 children and 71 adults) and diethyleneglycol was incriminated. This tragedy caused the AmericanCongress to approve in 1938 the “Food Drug and Cosmetic Act”, in which pharmaceutical productmanufacturers should have to show scientific evidences of the safety of the drugs before releasing them to sale.

After the end of WWII, atrocities performed by German physicians against human beings “inbehalf of science” were made known. Among them, experiments of “vaccination against typhus”,

with healthy subjects being infected, the massive “non-surgical sterilizations” of Jewish women by means of formaline injection into the uterine cavity, as well as experiences of “prolonged hypothermiaand depressurization”.

As a reaction to these abuses, the Nürembeg Code was prepared in 1947, establishing parameters tobe followed, with the intention to avoid such occurences. This Code was the first to introduce the

concept of consent by research subjects. Unfortunately, only after another tragedy the researchregulations were improved.


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In 1957 “thalidomide” is launched as a somniferous. It was tested in approximately 300 patients without toxicity. In 1962, after reports of numerous cases of phocomelia it was discontinued.In the same year, the Kefauver-Harris amendment was approved, requiring scientific evidencesof efficacy and safety before drug tests in humans.

In 1964, the World Medical Association approved in Helsinki a document with principles forprotection of biomedical research subjects. Concepts of researcher responsibilities, ethics committeesand informed consent form were introduced. With regular reviews (1975, 1983, 1989 and 2000), theDeclaration of Helsinki is currently the universal document that regulates scientific and technologicaldevelopment involving human beings.

In 1977, FDA publishes the first guidelines for clinical research intending to guarantee data quality and clinical research subject protection. Between 1977 and 1981, new guidelines on Good ClinicalPractices were published. In 1988, a consolidated Code of Good Clinical Practices (GCP) is published

by the FDA.

Good Clinical Practices were also adopted in other countries:

1985 – Japan and Canada1986 – UK 1991 – Australia and EU1995 – Publication of the Code of Good Clinical Practices (GCP) by WHO.

In 1996, the International Conference on Harmonization (ICH) with posterior amendments servedas a basis for the conduction of clinical trials, according to similar norms and rules in differentcountries and in accordance with scientific and ethical standards.

In Brazil, the implementation of rules defining research in human beings started with Resolutionnumber 01/88, through an initiative from Professor Elisaldo Carlini. In October 1996, it was revokedby resolution number 196/MS/CNS, being afterwards complemented by Resolution number 251/97. Through these Resolutions, the Ministry of Health defined guidelines and rules to protect subjectsenrolled in research.

Resolution 196/96, was based on the four milestones of bioethics, autonomy, non-maleficence,beneficence and justice, and brought to the scientific community, as well as to Brazilian society,reflexions on ethical aspects of research involving human beings and establishes guidelines for theimplementation of a ethical review system in research, comprised by Research Ethics Committees(CEP) and by the National Commission on Research Ethics (CONEP), of the National HealthCouncil.

Therefore, Resolutions196/96 and 251/97, give researchers ethical guidelines to conduct studiesinvolving human beings.


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These Resolutions establish that all researches enrolling human beings should be appraised by a Research Ethics Committee (CEP), accredited by CONEP (National Commission onResearch Ethics). They also state that every institution carrying out researches shouldimplement a CEP [Research Ethics Committee]. Fall a CEP [Research Ethics Committee]

may not be implemented, the institution or principal researcher should submit the project forthe appraisal of a CEP [Research Ethics Committee] belonging to other institution, among those accredited by CONEP.

Considering the importance of bioavailability/bioequivalence studies for the implementation of ageneric drug policy in Brazil, researchers should be aware of guidelines ethics established by theMinistry of Health in this area.

1.2. New drugs

When animal studies suggest that a new molecule may be useful, i.e., it is effective and safe whencorrelated with its effects, it is usually indicated for studies involving human beings. New drugs areintroduced in the medical practice through clinical pharmacology studies, in which an increasing number of patients or healthy volunteers is evaluated, until an adequate quantity of data is obtainedand a formal therapeutic study may be justified.

Therefore, studies involving human beings may be classified as:

– therapeutic: in which enrolled subjects may have a direct benefit. – non-therapeutic: in which enrolled individuals will have no benefits

During its existence, a new drug, is considered to have 3 life periods. The first of them is before itsapproval for public use, when pre-clinical and clinical studies are carried out, to evaluate efficacy and safety. The second comprises the life span of its patent, in which a marketing exclusivity is still

valid. The last period beginns just after the patents expires and other companies may market theproduct, as a similar or as a generic drug. Thus, several companies may manufacture and marketdifferent formulations of a same active substance, with similar qualities and performances, so as the

interchangeability among different formulations, when given in equivalent doses, presents the samesafety and efficacy.

Scientific and technological basis make possible the regular and reproducible production of formulations with the same pharmaceutical characteristics, also providing evidences for their safety,efficacy and interchangeability.

The interchangeability evidence is given through pharmaceutical equivalence and/or bioequivalence.Bioavailability/bioequivalence studies may be requested after the drug registration is granted, eitherby regulatory agencies and/or industry.


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1.3. Bioavailability/Bioequivalence

We may consider that bioavailability era was started as early as 1945, with the first publication of the

concept of biological availability. The development of analytical techniques, during the 1960’s, madepossible the development of methods sensitive enough to allow quantification of drugs or metabolites,initially in the urine and afterwards in the plasma, making possible the evaluation and t comparisonof bioavailability of different formulations, as well as demonstration that significant differencesamong them could occur.

After the legislation of compulsory drug registration in 1969, which facilitated the introduction of generic drugs in Canadian market, the “Drugs Directorate” of the “Canadian Federal Departmentof Health and Welfare” begann to use bioequivalence as a measure to approve the registration of adrug during the 1970’s. This program, along with other information, was analyzed by the FDA,

agency that published the first guidelines for the performance of bioequivalence studies in 1977. The application of these guidelines was enhanced by the “Drug Price Competition and Patent Term Restoration Act” of 1984, which granted the FDA powers to authorize the approval of generic drugs without clinical evidences of safety or efficacy, provided that the drug is bioequivalentto the innovating product.

The performance of routine bioavailability and bioequivalence studies in Brazil may be related tothe Generic Act number 9787/99. The pioneer was Dr. Gilberto de Nucci, in the last decade,through the implementation of the Miguel Servet Unit in the Department of Pharmacology of the

Medical Sciences School of Unicamp. A number of the professionals who work in the clinicalpharmacology area in Brazil spent much of their academic education under guidance of ProfessorDr de Nucci.

The term bioavailability is a contraction of the term biological availability. Bioavailability and may be considered as the rate and extension in which an active molecule is absorbed and becomesavailable at the drug action site. Considering that the quantity of drug contained in the biologicalfluid is in balance with the action site, the bioavailability is determined through the measure of theconcentration of the active ingredient of the drug in whole blood, serum or other adequate biological

fluid, as a function of the time.

The main pharmacokinetic parameters used for the bioavailability evaluation are:

– Peak concentration – Cmax; – Time for achieving peak concentration – Tmax; – Area under the curve – AUC.

These measures are obtained from biological fluid concentration versus time curves, obtainedduring bioequivalence/bioavailability study.


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Absolute bioavailability

It is the fraction effectively absorbed after extravascular administration of a drug, whencompared to the administration of the same drug intravenously, which has, by definition, a

100%bioavailability.

Relative bioavailability (Bioequivalence)

Bioequivalence between drugs, administered by the same extra vascular route, may be evaluatedcomparing pharmacokinetic parameters related to bioavailability, i.e., to the quantity absorbed andto the rate of the absorption process. Two products may be compared with one of them being considered as the reference.

Bioequivalent drugs are pharmaceutical equivalents (same pharmaceutical formulation and quantity

of the same active ingredient) that, when given in the same molar dose, in the same conditions, donot present significant statistical differences regarding bioavailability.

A generic drug is a drug similar to a reference product that intends to be interchangeable with it,usually produced after the expiration or patent waiver protection or other exclusivity rights, after itsefficacy, safety and quality have been evidenced, and designated by a DCB [Brazilian CommonName] or, if not available, by a DCI [International Common Name].

1.4. Clinical studies

Clinical studies using drugs in human beings are conventionally divided into 4 phases:

– Phase I

It is the first study conducted in human beings, involving approximately 20 to 50 subjects, thesebeing usually healthy volunteers, according to the class of the drug to be evaluated. They are clinicalpharmacology studies, aiming to evaluate safety characteristics and pharmacological profile.

– Phase II These studies are the first administration of the drug to patients, involving approximately 50 to 300subjects. Their purpose is to study the therapeutic potential and side effects of the drug, in additionto establishing dose-response relationships in order to use them in more definitive therapeutic assays.

– Phase III

They are multicenter therapeutic studies, involving at least 250 subjects (usually this number may reach 3000/4000 patients), evaluating efficacy and safety and comparing them with placebo or withalready available drugs, with same therapeutic purpose. In this phase, the type and the profile of themost frequent adverse reactions are explored.


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– Phase IV

Usually are post-marketing surveillance studies r to establish therapeutic value, appearance of new adverse reactions and/or confirmation of the frequency of the appearance of adversereactions already known, and treatment strategies. They are carried out based on the

characteristics from which the drug and/or pharmaceutical specialty has been authorized.

Thus, among studies aiming to identify initial safety and efficacy parameters, bioavailability studiesare included, when absorption, distribution, metabolism and elimination data, as well as adverseeffect data, are not totally known. In these studies, a strict medical monitoring during the entireinvestigative phase is required. Bioequivalence studies, on the other hand, have as the main purpose,to obtain evidences from a pharmacokinetic point of view, that a test formulation is not differentfrom a given reference formulation. These studies are usually carried out as a basis for the registrationapplication of a generic drug. They are usually carried out in health volunteers, i.e., are non-therapeuticstudies, since pharmacokinetic and pharmacodynamic characteristics of the drug are already known,

having a lower risk than initial bioavailability studies.

1.5. Safety aspects

1.5.1. Adverse events

The concept of adverse event in clinical trials and its correct and prompt report, when necessary,are essential for the conduction of clinical studies.

All adverse events, observed or voluntarily reported, regardless of treatment group or suspicion of a causal relationship with the study drug, should be recorded in the section(s) corresponding to

Adverse Events, necessarily present in the Clinical Chart.

Events related to adverse drug effects, illnesses starting during the study or exacerbations of preexisting illnesses should also be recorded.

An exacerbation of a preexisting illness, including the study illness, is defined as a manifestation of

the illness (sign or symptom) that indicates its significant worsening when compared to the severity observed at the beginning of the observation period. It may include worsening, increase in thefrequency or new signs and symptoms.

An exacerbation of a preexistent illness should be considered if the patient/subject requires additionaldrugs or non-drug therapies, concurrently to the treatment of this illness during the study.

An insufficient clinical response or a lack of response, benefit, efficacy, therapeutic effect orpharmacological action should not be recorded as an adverse event. The investigator shoulddistinguish between preexistent exacerbation of the illness and lack of therapeutic efficacy.


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Additionally, clinically significant changes in the physical exam such as abnormal findings inobjective tests (e.g., laboratory, X-ray, ECG) should also be recorded as adverse events.

Any abnormal test result that is confirmed as an error does not need to be reported as an adverse

event.

The investigator should effort in order to obtain adequate information for all adverse events, so asto determine its course and to evaluate if they meet the criteria for serious adverse events, whichrequiring prompt report. For all adverse events, the investigator should obtain sufficient informationin order to determine its causality (verify whether the cause of the event is the study drug or otherillness). The investigator should evaluate the causality of the adverse event and indicate it in theClinical Chart. The follow-up of an adverse event after the discontinuation of the treatment isrequired if the it, or a sequelae, persists. A follow-up is required until the resolution of the adverseevent, or sequelae, or until it is stabilized at an acceptable level by the investigator.

1.5.2. Serious adverse events

All serious adverse events (as defined below), regardless of the treatment group or suspicionconcerning the causal relationship with the drug, should be reported to the Research EthicsCommittee that approved the protocol, in addition to its recording in the Clinical Chart.

A serious adverse event is any adverse event that:

• Results in death;• Is a life-threat for the volunteer/patient;• Results in hospitalization or extension of it;• Results in significant or persisting disability;• Results in congenital anomalies or birth malformations.

Important medical events may not result in death, life-threat for the patient or require hospitalization,but may be considered serious adverse events when, based on an adequate medical judgment, may jeopardize the patient/subject and require clinical or surgical intervention in order to avoid the

occurrence of one of the outcomes listed above.

Examples of these include allergic bronchospasm requiring intensive treatment in emergency roomor at home, blood discrasias or seizures that do not result in hospitalization or the development of drug addiction or abuse.

Regardless the abovementioned criteria, any additional adverse event considered as serious by theinvestigator should be reported and included in the clinical chart.

Initial hospitalization is defined as any hospital admission (even if it lasts less than 24 hours). Forchronic or long-term hospitalizations, the admission also includes the transfer, within the hospitalitself, for an intensive care unit.


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In some occasions, hospital admission may not be considered as an adverse event.For instance:

• Admission for treatment of a preexistent condition not associated with the development of a

new adverse event or with a worsening of a preexistent condition;• Social admission (e.g., the subject has no place to sleep);• Administrative admission (e.g., annual clinical exam);• Protocol-specified admission during a clinical study (e.g., for the performance of a procedure

requested by the study protocol);• Optional admission not associated to the precipitating clinical adverse event (e.g., for an elective

cosmetic surgery).

However, in the event of a hospitalization due to an unknown event, this should be considered as aserious adverse event.

However, the following cases are not considered hospital admissions:

• Emergency room or emergency unit assistance;• Outpatient/external procedures carried out at the same say;• Short-term observation units;• Rehabilitation units;• Nursing homes;• Specialized nursing clinics;

• Nursing homes and health clinics;• Phase I Clinical Research Units.

Extension of hospitalization is defined as any extension of the hospitalization beyond theexpected/required period regarding the original reason for the initial admission, as determined bythe principal investigator or by the responsible clinician. For clinical study protocol-specifiedhospitalization, the extension is defined as any extension of the hospitalization period beyond therequired by protocol.

Pre-planned surgical treatments or procedures should be recorded in the initial documentationduring the protocol period and for each individual subject.

Disability is the substantial loss of ability of a person to normally perform daily life tasks.

Any serious adverse event or death should be immediately reported, regardless of the circumstancesor the suspicion of a causal relationship with the drug, in the event it occurs or is known by theinvestigator at any time during the study up to the last follow-up visit required by the protocol or upto 30 days after the last administration of the study drug, whichever occurs last. Any serious adverseevent occurring at any moment after study end should be immediately reported in the event of asuspicion of a causal relationship with the study drug. The only exception to these report-requiredprocedures refers to serious adverse events occurring during the washout period, in which


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placebo is being solely given, or no active study drug is being given, also as any ground drug specified by the protocol.

For all adverse events, the investigator is required to provide the most complete information as

possible. Usually, this includes the description of the adverse event, detailed enough to allow thecomplete clinical evaluation of the case, and evaluation regardless a possible causal relationship with the study drug. Information should be given on other possible causes for the event, including concomitant drugs and illnesses. The investigator’s evaluation regarding the causality should also beprovided. In the event the causality is unknown and the investigator does not know if the study drug relates to the event or not, then it should be related to the study drug. In the event theinvestigator’s causality evaluation is “unknown, however, not related to the study drug”, this shouldbe clearly documented in the study records. In the event of death, an abstract of the necropsy findings should be filed with the study documents. The investigator should assure that the informationreported and that the information contained in the Clinical Records are accurate and consistent.

1.5.3. Abnormal laboratory test results

The results of all laboratory exams required by the protocol should be recorded in the ClinicalChart. All clinically important abnormal laboratory results occurring during the study should berevaluated at adequate time intervals until they return to baseline values, to an acceptable levelaccording to the investigator or until a diagnosis that explains said changes is made.

The criteria to determine whether an abnormal test result should be reported as an adverse event

are the following:

• When the test result is accompanied by an associated symptom;• When the test result requires an additional diagnostic exam or medical/surgical intervention;• When the test result leads to a change in the study drug dose or study discontinuation, introduction

of a significant concomitant drug treatment or other therapy;• When the test results leads to any of the outcomes included in the definition of serious adverse

event;• When the test result is considered by the principal investigator or by the sponsor as an adverse

event.

1.6. Responsibilities

1.6.1. Sponsor

The Sponsor is responsible for providing the Principal Investigator and his group with the necessary information for the understanding of the study procedures, as well as agreeing with the Researcherrequirements.

It is a Sponsor’s responsibility the choise of a Research Facility as well as the Principal Investigator.


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The Sponsor must assure that the study is conducted under Good Clinical Practices and localrules, confirming the approval of the study protocol by a CEP [Research Ethics Committee],before its start.

All drugs required by the study, in accordance with the protocol, should be provided free of chargesto the research subjects, and the benefits resulting from the project should be assured, regarding social return, access to the procedures, products and research agents (Resolution 196/96CNS).

Other obligations:

• To provide treatment for adverse events;• Compensation for damages;• Report of adverse events for the Regulatory Agencies;• Final study report;

• Signature of the agreement and protocol with the investigator;• To monitor the study;• To control the quality;• To verify the access of the monitors to the data;• To supervise the performance of the research;• To file documents;• To assure access to the test drug, in the case of its superiority when compared to the control

treatment (Resolution 251/97, CNS).

1.6.2. Principal investigator

The principal investigator should be qualified by his education, training and experience in order tobe responsible for the correct conduction of the studies, as well as presenting all the qualificationsspecified by the legislation in force. It is recommended that said qualifications be documented.

It is a responsibility of the principal investigator to assure that the rights and welfare of the researchsubjects are guaranteed, correctly and opportunely obtaining the signature of the informed consentform.

Other responsibilities of the principal investigator:

• Supervise or personally conduct the research under Good Clinical Practice;• Assure that all study subjects have been informed of their obligations pursuant to the research

protocol;• Assure that research subjects are given the drugs without charges;• Provide resources, staff and facilities required for the timely conclusion of the study;• Knowledge of the product, protocol and the contents of the investigator’s brochure;• Evaluation of the adverse events and their course;• Be aware of medical decisions and adverse event treatment;• Protocol compliance;


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• Adequate use of the research drug;• Data collection, recording and report;• Signature of the agreement and of the protocol;• Adverse event report;

• Maintenance of the file with the required documents;• Facilitate the access of documents to the monitor or for audits;• To assign responsibilities for the designees, or to authorize the conduction of study phases in

third centers. Said authorization should be formally documented and submitted to the competentagencies;

• Guarantee the approval by CEP [Research Ethics Committee] and the necessary reviews;• Interim and final reports to CEP [Research Ethics Committee].


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2.1. Introduction

The clinical phase is comprehended from volunteer selection up to hospital discharge and the lastfollow-up return.

When a bioavailability/bioequivalence center is assembled to carry out clinical step, it shouldbe reminded that the participants of such studies are not entitled to direct therapeutic benefitsfrom their participation. Therefore, all possible measures should be taken in order to minimizerisks related to drug administration or hospitalization, in addition to providing the mostcomfortable situation as possible to the subjects.

When organizing a clinical research unit to carry out bioavailability/bioequivalence studies, it shouldbe taken into consideration that such center is not only comprised of a physical facilitiy with aspecific legislation, but mainly by the interaction of a staff that is, by definition, multidisciplinary.Considering the multidisciplinarity of the professional staff required for the performance of theclinical step of the studies, as well as the importance of this phase for the following steps (analyticaland statistical) and final outcome of the study, it is mandatory that all professionals involved have ageneral knowledge about the analytical and statistical steps, since their participation may influencethe final outcome of the study.

2.2. Facilities

The center shall have its legal situation duly registered, with a legal designee in charge, presenting safety conditions in accordance with the legislation in force, as well as the operational authorizationissued by ANVISA,. The principal investigator, responsible for the conduction of the studies shouldbe defined, since he is not necessarily required to be the legal representative.

The facilities should be mainly for clinical research. Volunteers shall not be confined at the sameroom with patients.

The rooms or infirmaries should be well ventilated and lightened, in addition to the minimum spacerequired by the competent sanitary legislation for the number of beds installed. Restrooms shouldbe in satisfactory hygiene conditions and be enough for the maximum of volunteers to be hospitalized.

The set of rooms or infirmaries should be provided with a nursing station nearby. The supportpersonnel should be provided with a locker room, living room and restrooms in a sufficient numberand in satisfactory conditions.

2. DRUG BIOAVAILABILITY/BIOEQUIVALENCE STUDIES –

CLINICAL STEP


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The unit intended to run clinical steps should be provided with an Intensive Care Unit (ICU)located in easy access area from the hospitalization location. In the event the center is notprovided with its own ICU, a mobile ICU should be made available for the volunteers during the entire confinement period, as well as a support institution with an ICU should be aware

of the performance of the clinical study, agreeing with the referral and reception of volunteers,if necessary. This consent should be duly documented.

The inpatient ward should be provided with an emergency cart, containing a bag valve mask,laryngoscope, orotracheal intubation cannulae, defibrillator, disposable syringes and drugs necessary to carry out procedures of cardiorrespiratory arrest reversion or for the treatment of events thatmay potentially cause a cardiorrespiratory arrest. Said items should be available during the entireperiod concerning the Step. It is also required that the hospitalization is provided with an emergency electrical generator, as well as essential electric equipment to be plugged into voltage stabilizers andno breaks.

2.3. Volunteer recruitment and selection

Volunteers may spontaneously come to a bioavailability/bioequivalence clinical center or be recruitedby advertisements, provided that these are previously approved by a CEP [Research EthicsCommittee]. After being informed on the nature of the study and signing the informed consentform, the volunteer should be submited to medical evaluation and complementary exams, according to the legislation in force and the research protocol.

In the event any disease is diagnosed during the selection process, the subject should be submittedto a health service for the correct follow-up. These data should be duly documented in the clinicalchart of the study.

The entire volunteer recruitment and selection should be duly described, known and followed by allprofessionals involved.

2.4. Volunteer hospitalization and discharge

The volunteer selected for a bioavailability/bioequivalence assay should be hospitalized the nightbefore starting the study, being reevaluated by a physician at the time of admission, preferably at theclinical unit, where they will have their vital signs (pulse, blood pressure and temperature) evaluatedto reduce a possible interference that may occur in the pharmacokinetics of the drug to be given,due to the ingestion of alcoholic beverages, sleep deprivation or intense physical activity within thehours previous to the study, as well as identifying occurrences of possible exclusion factors betweenselection and hospitalization.


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The procedures regarding volunteer hospitalization should be standardized in a specificdocument and followed by those in charge of admission and release. The equipment used inthe evaluation and pre-hospitalization (sphygmomanometer, stethoscope and thermometer)should be in good use conditions. It is recommended that when admitted, the volunteer receives

written information of the events to be carried out during the confinement period, with, atleast, the scheduled times for the drug administration, meals, blood sampling and release.

After the end of the drug administration and biologic material collection phases foreseen by theprotocol, the volunteers shall be mandatorily subject of a new medical evaluation and complementary exams foreseen by the study protocol for diagnose of possible intercurrences that may have beencaused by the participation in the clinical study.

2.5. Administration of the drug and biologic material collection

The volunteers should fast for at least 8 hours before drug administration. Immediately before thestart of the drug administration, a peripheral vein should be catheterized and maintained for theperiod required in order to blood sampling.

Sampling should be carried out according to the pharmacokinetic profile of the drug to be studiedand research protocol, the first collection (baseline collection) being mandatorily performed beforethe drug is administered (ca. thirty minutes).

All drug administration, sample collection, preparation and storage procedures should be recordedin a specific document, with an identification of the person in charge of it. The timepoints determinedby the protocol for sample collection should be strictly followed and any deviation recorded in thecorresponding chart.

The drug should be given as described in the protocol under medical supervision, by a nurse,pharmacist or a qualified professional. A physician should supervise the volunteers during the hoursafter the administration of the drug, in a period enough to assure medical support to fortuitousadverse events. Vital signs from volunteers should be controlled during all confinement period.

Complementary measures should be taken in the event the research protocol determines them.Fortuitous intercurrences should be duly documented.

The clinical unit should keep an on-duty physician during the entire hospitalization period, whoshould be aware of the performance of a bioavailability/bioequivalence study, if not directly involvedon it. In this case, the physician in charge of the clinical step should be easy to locate and availablefor the resolution of possible intercurrences. The occurrence of adverse events, their intensity, as

well as the measures taken for their reversion, should be duly recorded in an adequate document, with the identification and signature of the person in charge for the assistance of the volunteer, as well as the principal researcher. The occurrence of severe adverse events should be documentedand reported to the competent authorities.


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During the hospitalization period, the diet of the volunteers should be supervised by a nutritionist,and defined to not interfere with drug analysis and pharmacokinetics .

2.6. Biologic sample handling

Biologic samples should be prepared for storage in a standardized manner and following drug characteristics and analytical methodology (e.g., plasma, whole blood or serum, sensitivity totemperature or light, among others). The freezers used should have their temperature monitored toassure sample integrity. All procedures should be standardized, performed by qualified professionals,and all personnel and steps involved shall be identified and recorded.

2.7. Documentation

All research projects intending to access bioavailability of a drug compared to a reference product,should comprise a research protocol, with all the documents recommended by Resolutions numbers196/96 and 84/02 and their Guidelines, and be submitted for the appraisal of a CEP [ResearchEthics Committee].

Every study should meet good clinical practice and effective local rules, be carried out in accordance with the study protocol.

2.7.1. Research protocol

The research protocol, in its most encompassing manner, according to Resolution 196/96 item II Terms and Definitions, is the assemble of documents that contains the description of the researchin its fundamental aspects, information regarding research subjects, qualification of the researchersand all personnel involved.

The contents of a research protocol may vary according to the type of study, being that Resolution196/96 exhibits the information that should be contained therein.

2.7.2. Clinical protocol

By definition, the clinical protocol is the document that describes the objectives, design, methodology,statistical considerations and organization of a clinical study. The protocols also contain the history for the clinical study and its rationale.

Another source of information for prepararing a clinical protocol is the International Conferenceon Harmonization (ICH). According to the ICH, the sections that may be contained in a protocolare:


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• General information:o Protocol title, number and date;o Confidentiality paragraph;o Name and address of the sponsor and monitor (in the event he is not the sponsor);o

Name and address of the clinical laboratories;o Names and titles of the investigators and co-investigators;

• Information on the study design:o Study type and design;o Primary and secondary objectives;o Description of the randomization and blinding procedures;o Study treatments and dosages regimen, including package and label information;

• Information on study population:o Number of patients;o Duration of subject participation;o Informed consent form;o Maintenance of the randomization codes and procedures for code break (double-blind

study);• Selection and discontinuation:

o Inclusion criteria;o Exclusion criteria;o Patient discontinuation criteria;

when and how to discontinue; type of data to be collected;

subject replacement; discontinued subject follow-up;

o Subject treatment; names of all products, doses, dose escalation, administration routes, treatment

period and follow-up period; allowed and forbidden treatments/medications, before and during the study;

o Compliance monitoring methods;• Efficacy and safety evaluations:

o Specification of the efficacy and safety parameters;o

Methods and timepoints to evaluate, record and analyze these parameters;o Procedures for the recording and reporting of adverse events and intercurrent illnesses;o Type and duration of subject follow-up after the occurrence of an adverse event;

• Conduction of the study:o Monitoring frequencies;o Audits;o Data and records maintenance;o Publication policy;o Procedures to monitor subject compliance;

• Others:o Clinical laboratory parameters;o Concomitant therapy;


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o Documentation of investigator’s approval and date (signature page);o Funding and insurance;o Direct access;o Ethics.

All documents requested for the protocol are essential tools for the ethical appraisal by the membersof CEP [Research Ethics Committee]. Among them, special attention should be given to the InformedConsent Form (ICF) to be signed by the research volunteer.

2.7.3. Informed consent form

Resolution 196/96 , item IV – Informed consent form, encompasses all aspects related to the IFC,to which the investigator should give attention when preparing it. Special attention should be givento the obtaining process of the IFC, that, in addition to being just the process of obtaining a

signature, is a real educational process for the research subject.

The informed consent form should provide study subject with all the information required for his/her decision to participate in the research. The study subject should be free to make questions,clarifying any doubt that he may have, as well as have an adequate time to reflect on his participation.

The informed consent form should also:

• Meet Resolution 196/96/GCP/ Declaration of Helsinki;

• Be approved by an Ethics Committee before its use;• Be revised whenever new information arise;• Be written in accessible language for the study subject;• Not lead the subject to waive any of his legal rights;• A copy of the signed informed consent form should be given to the subject;• Be written in accessible language;• State that the study involves research;• State that the participation of the subject in the study is voluntary;• Provide the rationale, objectives and procedures that will be used in the research, including all

invasive procedures;• State the possible discomforts and risks and the expected benefits. In the event there is noclinical benefit intended for the subject, he should be aware of this fact;

• Existent alternative methods, as well as their potential benefits and risks;• The responsibilities of the subject participating in the study;• Experimental aspects of the study;• The follow-up and assistance manner, as well as their responsible;• The expected duration of the participation of the subject in the study;• The approximate number of subjects involved in the study;


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• The assurance of clarifications on the methodology, before and after the course of theresearch, informing the possibility of inclusion in a control or placebo group and thelikelihood inclusion in each arm;

• The person to be contacted in the event of additional clarifications regarding the study and

rights of the subjects involved. Who to be contacted in the event of fortuitous damages arisenfrom the research;• Compensation manner from the expenses arisen from the participation in the research;• Indemnification manners in the event of fortuitous damages arisen from the research;• That monitor(s), auditor(s), Research Ethics Committees and regulatory authorities will be granted

direct access to the source medical records of the subject in order to check clinical study procedures and/or their data, without violating their confidentiality concerning applicable lawsand/or rules and that, when signing the Informed consent form, the subject or his legalrepresentative authorize this access;

• The records that identify the subject will be kept confidential in accordance with the applicable

laws and rules, not becoming of public use. In the event the study results are published, theidentity of the subject will be confidentially kept;

• The subject or his legal representative will be informed in the event any new information thatmay be relevant for the subject’s decision arises, regarding his participation in the study;

• Provide protection for “vulnerable populations”.

As examples of vulnerable populations, we have:

• Children;

• Army members;• Employees of hospitals and laboratories;• Medical students;• Ethnical minorities;• Nursing home residents;• Mentally challenged people;• Patients with incurable illnesses or in emergency situations;• Unemployed, beggars, homeless. refugees, nomads, underaged, etc.

It is very important to highlight, among the various items above, that obtaining the informed consentform should take place before any procedure is carried out with the research subject, as well asrequiring that the clarification to research subjects be in an accessible language.

Informed consent form should be dated and signed by the study participant, as well as by theperson in charge of its application.

2.7.4. Clinical chart

A clinical study chart, containing the information required for the study and participant identificationshall exist. This document contains the data related to the subject’s follow-up. These data areconsidered confidential, preserving the volunteer’s identity.


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It is necessary that the Clinical protocol and its respective Informed consent form are approved by a Research Ethics Committee (CEP) registered by CONEP (National Commission on ResearchEthics) before its beginning.

Any amendment to this clinical protocol or Informed consent form that may occur during thecourse of the study should also be submitted to CEP [Research Ethics Committee] for its approval.In the event the amendment aims to preserve the safety of the study subjects, it may be immediately implemented, being concomitantly submitted to CEP [Research Ethics Committee] to be duly approved. In the event of an amendment of the Informed consent form, research subjects who arestill participating of it should also sign the new version of the consent.

The characteristics of the Research Ethics Committee are described in Resolution 196/96 item VII – Research Ethics Committee – CEP.

The Committee should have at least 7 members, and not more than half of its members can pertainto the same professional category. Its contribution will have a multi- and transdisciplinary characteristic, including persons of both genders and the participation of health, exact, social andhuman sciences professionals, such as judges, theologists, sociologists, philosophers, bioethicistsand at least a member of the society representing the users of the institution. They may occur a

variation it its composition, depending on the specificities of the institution and the research linesto be analyzed.

The Research Ethics Committee may or may not pertain to the Institution in which the study is

being carried out, and it may use ad hoc consultants for specific analyses. In the event the institutiondoes not have a CEP [Research Ethics Committee], an independent CEP [Research EthicsCommittee] or from other institution may be used, provided that this CEP [Research EthicsCommittee] is registered by CONEP.

Members of CEP [Research Ethics Committee] shall not participate of the ballot of projects in which they are involved.

The abovementioned documents, source or copies, should be duly filed with other study documents

and be available at request by the sponsor and/or regulatory agency.

All documents related to the clinical phaseof a bioavailability/bioequivalence study shall be retainedfor at least 15 years, and be available for possible inspections conducted by the regulatory authorities.

2.8. Responsibilities

2.8.1. Sponsor

• Study monitoring.• Quality control.


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• Supervision of the research conduction.• Document filing.• Financial and logistic subvention.

2.8.2. Principal investigator

The main investigator is responsible before ANVISA for all study phases, even those not carriedout at his/her facility, for the accuracy of data presented, as well as for the experimental designpresented in the research protocol.


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3. REFERENCES

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Goldman DS. Principles of the Good Laboratory Practice Regulations Applied to Pharmacokinetics.

In Welling PG e Tse FLS (eds). Pharmacokinetics. Regulatory-Industrial-Academic Perspectives.Second Edition Marcel Dekker, Inc, New York, Basel, Honk Kong, 1995, pgs 1-20.

Gomes, GR. Manual de Investigación Clínica. Editorial ICIC, 1999, 25-210.

Hutchinson, D. The Trial Investigator’s GCP Handbook: a pratical guide to ICH requerements.Brookwood Medical Publications Ltd., 1997, 4-47.

Hutchinson, D. Which documents, why? A guide to essential clinical trial documentation forinvestigators. Brookwood Medical Publications Ltd., 1997, 3-36.

McGilveray IJ. Progress in Harmonization of Bioavailability and Bioequivalence Standards. In André J. Jackson (ed). Generics and Bioequivalence. CRC Press, Boca Raton, Ann Arbor, London, Tokyo,1994, pgs 187-192.

Narang PK. Issues in bioequivalence: An industrial Scientist´s Perspective. In André J. Jackson (ed).Generics and Bioequivalence. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1994, pgs 179-186.

Shargel L, Yu ABC. Applied Biopharmaceutics and Pharmacokinetics. Fourth Edition. Appleton

& Lange , USA, 1999.


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TECHNICAL STAFF

Volume I - Module 2 – Analytical Step

Editors:

• Flávio Leite - PUC - Campinas


• Pedro Eduardo Froehlich - UFRS

• Rui Oliveira Macedo – UFPB

Cooperators:

• Davi Pereira de Santana - UFPE• Itapuan Abimael Silva - ANVISA

• Jaime Oliveira Ilha - Cartesius

• Karen Noffs Brisolla - ANVISA


• Max Weber Pereira - ANVISA

• Pedro de Lima Filho - ANVISA

• Rafael Eliseo Barrientos Astigarraga - Cartesius

• Silvana Calafatti de Castro - UNIFAG• Thaís Reis Machado

Coordination:


• Itapuan Abimael da Silva - ANVISA

• Karen de Aquino Noffs Brisolla - ANVISA

• Karla de Araújo Ferreira - ANVISA


• Max Weber Marques Pereira - ANVISA

• Renato Almeida Lopes - ANVISA

Logistic Support:

• Daniela Salles de A. B. Corrêa - ANVISA

• Diva Sales Freitas - ANVISA


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1 . THEORETICAL BACKGROUND – MATERIALS AND REAGENTS.................. 5

1.1. Introduction .............................................................................................................................. 5

1.2. Reference chemical substances (SQR) and internal standard ........................................ 6

1.2.1. Commercially available SQRs ................................................................................... 6

1.2.2. Non-comercially available SQR................................................................................ 6

1.2.3. Authorized analytical laboratory (AAL) ................................................................ 7

1.3. Metabolites ................................................................................................................................ 7

1.4. Internal standards ..................................................................................................................... 7

1.5. Storage and handling ............................................................................................................... 7

1.6. Reagents and solvents ............................................................................................................. 8

1.6.1. Storage ............................................................................................................................. 81.6.2. Chromatographic grade water ................................................................................... 8

1.7. Glassware ................................................................................................................................... 8

1.8. Scales ........................................................................................................................................... 9

1.8.1. Instalation ....................................................................................................................... 9

1.8.2. Maintenance and preservation ................................................................................. 10

1.9. Control led temperature equipment .................................................................................. 10

1.10. Sampling tubes and analysis ................................................................................................ 10

2. BIOANALYTICAL METHOD .................................................................................................. 11

2.1. Introduction ............................................................................................................................ 11

2.2. Pre-validation .......................................................................................................................... 11

2.2.1. Completeness, accuracy and recovery ................................................................... 11

2.2.2. Linearity and quantification limits ......................................................................... 12

2.2.3. Specificity/selectivity ................................................................................................. 12

2.3. Drug stability in biological fluids ...................................................................................... 13

2.3.1. Introduction ................................................................................................................. 132.3.2. Literature review ........................................................................................................ 13

2.3.3. Degradation kinetics .................................................................................................. 14

2.3.4. Analytical problems related to stability ................................................................ 18

2.4. Samples storage ....................................................................................................................... 18

2.5. Method validation .................................................................................................................. 19

2.5.1. Introduction ................................................................................................................. 19

2.5.2. Bioanalytical method ................................................................................................. 20

2.5.3. Validation tools ........................................................................................................... 20

2.5.4. Glossary ........................................................................................................................ 23

2.6. Operational flow chart of bioequivalence analytical step ............................................ 26

SUMMARY


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3. RECOMMENDATIONS FOR THE ANALYTICAL STEP ........................................ 28

3.1. Reference materials ................................................................................................................ 28

3.1.1. Reagents and solvents ................................................................................................ 28

3.1.2. Scales and calibration weights .................................................................................. 28

3.1.3. Thermometers and other temperature checking devices .................................. 283.1.4. pHmeter ........................................................................................................................ 28

3.1.5. Centrifuge ..................................................................................................................... 28

3.1.6. Sample evaporation system ...................................................................................... 29

3.1.7. Glassware ...................................................................................................................... 29

3.1.8. Pippetes ......................................................................................................................... 29

3.2. Sample receiving ..................................................................................................................... 29

3.3. Stability study ......................................................................................................................... 30

3.3.1. Short-term stability .................................................................................................... 303.3.2. Intermediate-term stability ....................................................................................... 31

3.3.3. Long-term stability ..................................................................................................... 31

3.4. Validation ................................................................................................................................ 32

3.4.1. Selectivity ..................................................................................................................... 32

3.4.2. Recovery ....................................................................................................................... 32

3.4.3. Limits ............................................................................................................................. 33

3.4.4. Linearity ........................................................................................................................ 34

3.4.5. Accuracy ....................................................................................................................... 343.4.6. Completeness ............................................................................................................... 36

3.4.7. Validation acceptance ................................................................................................ 36

3.5. Application of the validated method in the study ......................................................... 36

4. REFERENCES ............................................................................................................................... 38


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1.1. Introduction

The use of chemicals with high purity grade is essential to assure the quality of the analytical data(GARFIELD, 1997; CROSBY et al., 1997).

As the chemical reagents used, the use of SQR is essential for the correct quantification of the drug and/or its metabolites.

For a better understanding of this material, some definitions are important:

Primary standard: according to SKOOG & WEST (1979), a substance should show the following characteristics:

– it should show an elevated purity grade, that should be determined; – it should be stable; – it should not be hygroscopic or efluorescent; – it should be easy to be obtained and preferably at a low cost; – it should present a relatively high molecular weight.

As examples of primary standard we have potassium dichromate, sodium carbonate and potassium

biphthalate.

This latter definition does not seem to be the most adequate for drugs, since most of them do notmeet the conditions described by Skoog & West.

The definitions below are based on publications of the National Institute of Standards and The Technology (NIST) and North-American Pharmacopoeia (USP 25) and accepted by AOAC(Association of Officials Analytical Chemists), as summarized by GARFIELD (1997):

Certified reference material (CRM): material with one or more properties certified by valid technicalprocedures, followed or traceable by a certificate, or other type of documentation issued by a certifying agency.

Standard reference material (SRM): material produced by NIST. SRMs are certified regarding their specific physical-chemical properties and followed by certificates that report results and indicatethe use of the material.

USP Reference Standards (USP Reference Standards): purified or highly pure drugs distributedby USP after recommendation of the USP Reference Standards Committee. The selection of theraw material lots used in the preparation of these standards is based on the critical characteristics of

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1. THEORETICAL BACKGROUND - MATERIALS AND REAGENTS


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each drug, analyzed by three or more laboratories, among USP, FDA, academic and privatelaboratories. (USP 25)

Working standard or secondary standard: prepared from the analysis of a lot of an adequate

purity material against a standard or SQR-certified one, using official methodology and keeping ananalysis record. When this working standard is used in the analysis of a sample, the SQR from which it has been prepared should be mentioned.

In addition to USP, various others pharmacopoeias, such as the European or British, have theirstandards. These institutions refer to their standards as chemical reference substance (from English“chemical reference substance”, or simply “CRS”). In addition to those, chemical and biologicstandards of several drugs are distributed by the World Health Organization (WHO).

A similar procedure has been recently adopted by the Brazilian Pharmacopoeia (F.Bras.). In 2000,

the Reference Material Commission has been appointed (Administrative Ruling 733, DOU [FederalOfficial Gazette] 201-E, of 10/18/2000), that has started the production of the first referencechemical substances (SQR) of Brazilian Pharmacopoeia.

According to Resolution – RDC number 56, of February 26, 2002, D.O. [Federal Official Gazette]of 02/27/2002, after being made available, the SQR from F. Bras. are the official ones throughoutthe Brazilian territory and should be mandatorily used regarding those mentioned above.

1.2. Reference chemical substances (SQR) and internal standard

The purity grade of chemical substances used as the reference in clinical studies may affect thequality of the results. The term SQR refers to the standards of the study drug and its metabolite(s),

when applicable. They are duly certified substances with a high purity grade. They may comprisetwo types:

1.2.1. Commercially available SQRs

Whenever available, SQRs from Brazilian Pharmacopoeia or those provided by other institutions/companies nationally or internationally known should be used, provided that their traceability ispossible.

1.2.2. Non-commercially available SQR

They should be obtained from pharmaceutical grade substances, accompanied by its respective lotcertificate of analysis and in a quantity enough for the production of a working standard, which willbe used in the studies as a reference. This working standard may only be manufactured by an

Authorized Analytical Laboratory (AAL), that should keep the analytical records. There are twopossibilities for the development of a analysis schedule for the quantification of the raw material asa working standard:


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– There is a pharmacopoeia monograph available

In this case, AAL should perform all assays foreseen by the monograph and issue a certificate of analysis, with the contents obtained in the dosage assay being adopted as the purity value for the

working standard;

Note: In the event the monograph is not available in the last edition of the Brazilian Pharmacopoeia,use preferably the most recent editions of the European, British, American Pharmacopoeias or

Administrative Rulings issued by INMETRO.

– There is no pharmacopoeia monograph

Studies with chemical substances will be allowed, as long as their certification is evidenced.

1.2.3. Authorized analytical laboratory (AAL)

AAL are REBLAS laboratories and Bioequivalence Analytical Centers, as long as they show evidencedtechnical ability for the development of the analytical methodology indicated.

1.3. Metabolites

In the case of metabolites, the analytical center should prove, through the certificate of analysis of

the supplier or assays carried out at the center itself, that those present a defined purity grade andadequate to be used as working standard.

1.4. Internal standards

The internal standards used should have an analytical grade (p.a.) or above, so as they do not interfere with the analysis.

1.5. Storage and handling

SQRs should be stored according to the instructions of the supplier. Usually, they should be storedin a cool place, away from light and with a low humidity, always kept in sealed vials.

During receiving, a custody chain should be opened for each vial received, in which the use of theSQR is controlled by recording the mass used for each purpose, initialized by the person who usedit. The certificates of analysis of the substance should be kept along with the custody chain. Thedestination given to the remaining mass of SQR after the expiration date is over should be alsorecorded.


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1.6. Reagents and solvents

The reagents and solvents used in the studies should not interfere with the results. This should bechecked through proper procedures.

Supplier controlling procedures should be established so as to assure that solvents and reagentspurchased have the desired quality. It is recommended that the suppliers have certificates of analysis,as well as documented evidences in order to assure their reliability.

1.6.1. Storage

Substance, reagents, solvents and solutions storage areas should be adequate.

1.6.2. Chromatographic grade water

They should present a quality consistent with the use of HPLC. They may be:

– deionized – distilled – bidistilled – ultra-pure

The purity of the water should be evidenced through proper tests.

1.7. Glassware

The accurate volume measure is as important in several analytical methods as the measure of themass. For such, it is necessary to consider some essential items for the accurate measurement of agiven volume, such as maintenance of the measuring instruments, quality of the instruments andregular calibration.

The volume marks are made by manufacturers with the well-cleansed volumetric equipment. Ananalogous cleansing level should be kept whether these marks are to be reliably used. Only cleanglass surfaces keep a uniform liquid film. Dust or oil disrupts this film. Therefore, the existence of film disruptions is an indication of a “dirty” surface.

The volume occupied by a given liquid mass varies according to the temperature, as it varies according to the container in which the liquid is placed during the measurement. However, most of the volumemeasuring equipment is made of glass, which fortunately has a low expansion coefficient.Consequently, volume variations due to temperature of a glass container should not be consideredin analytical chemistry assays. Volumetric measurements should take as a reference any standardtemperature; this reference point is usually 20°C.


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1.8. Scales

Scales (according to Administrative Ruling 236, of December 22, 1994, of Inmetro).Standard weights (according to Administrative Ruling 233, of December 22, 1994, of Inmetro).

1.8.1. Installation

Analytical scales should be installed in a proper place, leveled, free of air currents, in an exclusivebench and stable. Whenever possible, they should be placed in a controlled temperature room.

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The expansion coefficient for organic liquids may require corrections for temperature differencesof 1°C or even less, which makes extremely important room temperature in laboratories.

Generally, analytical procedures are carried out at a temperature that varies from 15 to 25°C. (British

Pharmacopoeia, 2000).

Volumetric glassware may be individually calibrated by INMETRO or a laboratory certified by INMETRO. However, a “Class A” glassware meets international standards established by theInternational Organisation for Standardization. (British Pharmacopoeia, 2000; GARFIELD, 1997;USP 25).

The laboratory should regularly check the volumes dispensed by volumetric glassware, using the water mass for such. For pipettes, the flow time should be observed, it is usually printed on theglass, and finally make the tip of the pipette touch the container wall into which the volume is being

transferred, so as the entire contents flow. (USP 2002)

The accuracy of “Class A” is as follows:


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1.8.2. Maintenance and preservation

The scale should be immediately cleaned after each use. There should be a scale maintenance andpreservation program, including regular calibrations (at least annually) with all information recorded

in a logbook.

For the analytical scales used in laboratories (Class I), the value of the actual checking division (d)should be 0.1 mg or lower. (Administrative Ruling 236, of December 22, 1994, of Inmetro).

The minimum load (min) of the scale should not be less than 100 x d.

Ex.: Analytical scale with a capacity for 200 g and sensitivity of 0.1 mg.

min = 100 x 0.1 = 10.0 mg

However, according to USP 25 (2002), the uncertainty of weighting (systematic + random error)should not exceed 0.1% of the weighted mass.

In case of electronic scales not provided with a self-calibration system, the measurement should beperformed daily at the start of the works, and the records should be properly stored. The weightsused should be annually certified.

1.9. Controlled temperature equipment

The refrigerator and the freezer should have their temperatures daily checked and recorded in theuse book. A maximum and minimum thermometer should be provided, and the minimum andmaximum temperatures of the period should be recorded. The most adequate place for thethermometer to be located is in the internal central part of the equipment. In the event the temperaturereading is carried out by thermal pairs, these should be annually calibrated before RBC. Thereshould be a SOP for refrigerator/freezer, describing their use, maintenance, cleaning anddecontamination.

In case of equipment that make automatic temperature records, they should allow a daily temperaturecheck and the printed or recorded data will be stored for control.

1.10. Sampling tubes and analysis

They may be made of high-density polypropylene or polyethylene, and they should not be reused. The use of glass tubes should be avoided, since they may break during storage or transport. Whenchanging the supplier and/or the type of the material, carry out a recovery and a blank test in orderto check whether there is any interference of the material with the results of the analysis.

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2.1. Introduction

The performance of a literature research is the first step for the search of the bioanalytical method.Once the method exists, it should be tested for its reproducibility. In the event there is no bioanalyticalmethod for a given drug, the analytical center should develop a method that responds satisfactorily to the study aimed.

The previous performance of the steps required for the development of the analytical method forbioequivalence studies assures to the analytical center and its contractor that the services hired willbe carried out at

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