Appendix I

lelfINTERNATIONAL CONFERENCE ONHARMONISATION OF TECHNICALREQUIREMENTS FOR REGISTRATION OFPHARMACEUTICALS FOR HUMAN USE

ICH Harmonised Tripartite Guideline

PRECLINICAL SAFETY EVALUATION OFBIOTECHNOLOGY-DERIVED

PHARMACEUTICALS

Recommended for Adoption atStep 4 of the ICH Process on 16July 1997 by the ICH Steering

Committee

This Guideline has been developed by the appropriateICH Expert Working Group and has been subject toconsultation by the regulatory parties, in accordance withthe ICH Process. At Step 4 of the Process the final draft isrecommended for adoption to the regulatory bodies of theEuropean Union, Japan and USA.

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© Copyright ICH Secretariat (c/o IFPMA)

Copying is permitted, with reference to source,but material in this publication may not be usedin any documentation or electronic media whichis offered for sale, without the prior permissionof the copyright owner.

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This document is reproduced with the permission of theICH Secretariat

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PRECLINICAL SAFETY EVALUATION OF

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ICH Harmonised Tripartite Guideline

Having reached Step 4 of the ICH Process at the ICH Steering Committeemeeting on 16 July 1997, this guideline is recommended for adoption to

the three regulatory parties to ICH

TABLE OF CONTENTS

1. Introduction 1721.1 Background 1721.2 Objectives 1721.3 Scope 173

2. Specification of the Test MateriaI.. 1733. Preclinical Safety Testing 1743.1 General principles 1743.2 Biological activity/pharmacodynamics 1753.3 Animal species/model selection 1763.4 Number/gender of animals 1783.5 Administration/dose selection 1783.6 Immunogenicity 179

4. Specific Considerations 1804.1 Safety pharmacology 1804.2 Exposure assessment 181

4.2.1 Pharmacokinetics and toxicokinetics 1814.2.2 Assays 1824.2.3 Metabolism 182

4.3 Single dose toxicity studies 1824.4 Repeated dose toxicity studies 1834.5 Immunotoxicity studies 1834.6 Reproductive performance and 184

developmental toxicity studies4.7 Genotoxicity studies 1844.8 Carcinogenicity studies 1854.9 Local tolerance studies 186

Notes 187

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1. INTRODUCTION

1.1 Background

Biotechnology-derived pharmaceuticals (biopharmaceuticals)were initially developed in the early 1980s. The first marketingauthorisations were granted later in the decade. Several guidelinesand points-to-consider documents have been issued by variousregulatory agencies regarding safety assessment of these products.Review of such documents, which are available from regulatoryauthorities, may provide useful background in developing newbiopharmaceuticals.

Considerable experience has now been gathered with submissionof applications for biopharmaceuticals. Critical review of this ex­perience has been the basis for development of this guidance thatis intended to provide general principles for designing scientifi­cally acceptable preclinical safety evaluation programs.

1.2 Objectives

Regulatory standards for biotechnology-derived pharmaceuticalshave generally been comparable among the European Union,Japan and United States. All regions have adopted a flexible, case­by-case, science-based approach to preclinical safety evaluationneeded to support clinical development and marketing authorisa­tion. In this rapidly evolving scientific area, there is a need forcommon understanding and continuing dialogue among theregions.

The primary goals of preclinical safety evaluation are: (1) to iden­tify an initial safe dose and subsequent dose escalation schemes inhumans; (2) to identify potential target organs for toxicity and forthe study of whether such toxicity is reversible; and (3) to identifysafety parameters for clinical monitoring. Adherence to the principles

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presented in this document is intended to improve the quality andconsistency of the preclinical safety data supporting the develop­ment of biopharmaceuticals.

1.3 Scope

This guidance is intended primarily to recommend a basic frame­work for the preclinical safety evaluation of biotechnology-derivedpharmaceuticals. It applies to products derived from characterisedcells through the use of a variety of expression systems includingbacteria, yeast, insect, plant, and mammalian cells. The intendedindicationsmay include in vivo diagnostic, therapeutic, or prophylac­tic uses. The active substances include proteins and peptides, theirderivatives and products of which they are components; they couldbe derived from cell cultures or produced using recombinant DNAtechnology including production by transgenic plants and animals.Examples include but are not limited to: cytokines, plasminogenactivators, recombinant plasma factors, growth factors, fusion proteins,enzymes, receptors, hormones, and monoclonal antibodies.

The principles outlined in this guidance may also be applicable torecombinant DNA protein vaccines, chemically synthesised pep­tides, plasma derived products, endogenous proteins extractedfrom human tissue, and oligonucleotide drugs.

This document does not cover antibiotics, allergenic extracts,heparin, vitamins, cellular blood components, conventional bacter­ial or viral vaccines, DNA vaccines, or cellular and gene therapies.

2. SPECIFICATION OF THE TEST MATERIAL

Safety concerns may arise from the presence of impurities or con­taminants. It is preferable to rely on purification processes to re­move impurities and contaminants rather than to establish apreclinical testing program for their qualification. In all cases, theproduct should be sufficiently characterised to allow an appropri­ate design of preclinical safety studies.

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There are potential risks associated with host cell contaminantsderived from bacteria, yeast, insect, plants, and mammalian cells.The presence of cellular host contaminants can result in allergicreactions and other immunopathological effects. The adverseeffects associated with nucleic acid contaminants are theoreticalbut include potential integration into the host genome. For prod­ucts derived from insect, plant and mammalian cells, or transgenicplants and animals there may be an additional risk of viral infec­tions.

In general, the product that is used in the definitive pharmacologyand toxicology studies should be comparable to the productproposed for the initial clinical studies. However, it is appreciatedthat during the course of development programs, changes nor­mally occur in the manufacturing process in order to improveproduct quality and yields. The potential impact of such changesfor extrapolation of the animal findings to humans should be con­sidered.

The comparability of the test material during a developmentprogram should be demonstrated when a new or modified manu­facturing process or other significant changes in the product orformulation are made in an ongoing development program. Com­parability can be evaluated on the basis of biochemical and bio­logical characterisation (i.e., identity, purity, stability, and potency).In some cases additional studies may be needed (i.e., pharmacoki­netics, pharmacodynamics and/or safety). The scientific rationalefor the approach taken should be provided.

3. PRECLINICAL SAFETYTESTING

3.1 General principles

The objectives of the preclinical safety studies are to define phar­macological and toxicological effects not only prior to initiation ofhuman studies but throughout clinical development. Both in vitroand in vivo studies can contribute to this characterisation. Biopharma­ceuticals that are structurally and pharmacologically comparable

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to a product for which there is wide experience in clinical practicemay need less extensive toxicity testing.

Preclinical safety testing should consider:

(1) selection of the relevant animal species;(2) age;(3) physiological state;(4) the manner of delivery, including dose, route of administra­tion/ and treatment regimen; and

(5) stability of the test material under the conditions of use.

Toxicity studies are expected to be performed in compliance withGood Laboratory Practice (GLP); however, it is recognised thatsome studies employing specialised test systems which are oftenneeded for biopharmaceuticals, may not be able to comply fullywith GLP. Areas of non-compliance should be identified and theirsignificance evaluated relative to the overall safety assessment. Insome cases, lack of full GLP compliance does not necessarily meanthat the data from these studies cannot be used to support clinicaltrials and marketing authorisations.

Conventional approaches to toxicity testing of pharmaceuticalsmay not be appropriate for biopharmaceuticals due to the uniqueand diverse structural and biological properties of the latter thatmay include species specificity, immunogenicity, and unpredictedpleiotropic activities.

3.2 Biological activity! pharmacodynamics

Biological activity may be evaluated using in vitro assays to deter­mine which effects of the product may be related to clinical activ­ity. The use of cell lines and!or primary cell cultures can be usefulto examine the direct effects on cellular phenotype and prolifera­tion. Due to the species specificity of many biotechnology-derivedpharmaceuticals, it is important to select relevant animal speciesfor toxicity testing. In vitro cell lines derived from mammalian cellscan be used to predict specific aspects of in vivo activity and to

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assess quantitatively the relative sensitivity of various species (in­cluding human) to the biopharmaceutical. Such studies may bedesigned to determine, for example, receptor occupancy, receptoraffinity, and/or pharmacological effects, and to assist in the selec­tion of an appropriate animal species for further in vivo pharma­cology and toxicology studies. The combined results from in vitroand in vivo studies assist in the extrapolation of the findings tohumans. In vivo studies to assess pharmacological activity, includ­ing defining mechanism(s) of action, are often used to support therationale of the proposed use of the product in clinical studies.

For monoclonal antibodies, the immunological properties of theantibody should be described in detail, including its antigenicspecificity, complement binding, and any unintentional reactivityand/or cytotoxicity towards human tissues distinct from the in­tended target. Such cross-reactivity studies should be carried outby appropriate immunohistochemical procedures using a range ofhuman tissues.

3.3 Animal species/model selection

The biological activity together with species and/or tissue specific­ity of many biotechnology-derived pharmaceuticals often precludestandard toxicity testing designs in commonly used species (e.g.,rats and dogs). Safety evaluation programs should include the useof relevant species. A relevant species is one in which the testmaterial is pharmacologically active due to the expression of thereceptor or an epitope (in the case of monoclonal antibodies). Avariety of techniques (e.g., immunochemical or functional tests)can be used to identify a relevant species. Knowledge of recep­tor / epitope distribution can provide greater understanding of po­tential in vivo toxicity.

Relevant animal species for testing of monoclonal antibodies arethose that express the desired epitope and demonstrate a similartissue cross-reactivity profile as for human tissues. This wouldoptimise the ability to evaluate toxicity arising from the binding to

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the epitope and any unintentional tissue cross-reactivity. An ani­mal species which does not express the desired epitope may stillbe of some relevance for assessing toxicity if comparable uninten­tional tissue cross-reactivity to humans is demonstrated.

Safety evaluation programs should normally include two relevantspecies. However, in certain justified cases one relevant speciesmay suffice (e.g., when only one relevant species can be identifiedor where the biological activity of the biopharmaceutical is wellunderstood). In addition even where two species may be necessaryto characterise toxicity in short term studies, it may be possible tojustify the use of only one species for subsequent long term toxic­ity studies (e.g., if the toxicity profile in the two species is compa­rable in the short term).

Toxicity studies in non-relevant species may be misleading and arediscouraged. When no relevant species exists, the use of relevanttransgenic animals expressing the human receptor or the use ofhomologous proteins should be considered. The informationgained from use of a transgenic animal model expressing thehuman receptor is optimised when the interaction of the product andthe humanised receptor has similar physiological consequences tothose expected in humans. While useful information may also begained from the use of homologous proteins, it should be notedthat the production process, range of impurities/contaminants,pharmacokinetics, and exact pharmacological mechanism(s) maydiffer between the homologous form and the product intended forclinical use. Where it is not possible to use transgenic animal mod­els or homologous proteins, it may still be prudent to assess someaspects of potential toxicity in a limited toxicity evaluation in asingle species, e.g., a repeated dose toxicity study of ~14 daysduration that includes an evaluation of important functional end­points (e.g., cardiovascular and respiratory).

In recent years, there has been much progress in the developmentof animal models that are thought to be similar to the humandisease. These animal models include induced and spontaneousmodels of disease, gene knockout(s), and transgenic animals.

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These models may provide further insight, not only in determin­ing the pharmacological action of the product, pharmacokinetics,and dosimetry, but may also be useful in the determination ofsafety (e.g., evaluation of undesirable promotion of disease pro­gression). In certain cases, studies performed in animal models ofdisease may be used as an acceptable alternative to toxicity studiesin normal animals (Note 1). The scientific justification for the use ofthese animal models of disease to support safety should beprovided.

3.4 Number! gender of animals

The number of animals used per dose has a direct bearing on theability to detect toxicity. A small sample size may lead to failure toobserve toxic events due to observed frequency alone regardless ofseverity. The limitations that are imposed by sample size, as oftenis the case for non-human primate studies, may be in part compen­sated by increasing the frequency and duration of monitoring.Both genders should generally be used or justification given forspecific omissions.

3.5 Administration/dose selection

The route and frequency of administration should be as close aspossible to that proposed for clinical use. Consideration should begiven to pharmacokinetics and bioavailability of the product in thespecies being used, and the volume which can be safely andhumanely administered to the test animals. For example, the fre­quency of administration in laboratory animals may be increasedcompared to the proposed schedule for the human clinical studiesin order to compensate for faster clearance rates or low solubilityof the active ingredient. In these cases, the level of exposure of thetest animal relative to the clinical exposure should be defined.Consideration should also be given to the effects of volume, con­centration, formulation, and site of administration. The use ofroutes of administration other than those used clinically may be

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acceptable if the route must be modified due to limited bioavail­ability, limitations due to the route of administration, or tosize/physiology of the animal species.

Dosage levels should be selected to provide information on a dose­response relationship, including a toxic dose and a no observedadverse effect level (NOAEL). For some classes of products withlittle to no toxicity it may not be possible to define a specific maxi­mum dose. In these cases, a scientific justification of the rationalefor the dose selection and projected multiples of human exposureshould be provided. To justify high dose selection, considerationshould be given to the expected pharmacological!physiologicaleffects, availability of suitable test material, and the intended clini­cal use. Where a product has a lower affinity to or potency in thecells of the selected species than in human cells, testing of higherdoses may be important. The multiples of the human dose that areneeded to determine adequate safety margins may vary with eachclass of biotechnology-derived pharmaceutical and its clinical in­dication(s).

3.6 Immunogenicity

Many biotechnology-derived pharmaceuticals intended for humanare immunogenic in animals. Therefore, measurement of antibod­ies associated with administration of these types of productsshould be performed when conducting repeated dose toxicitystudies in order to aid in the interpretation of these studies. Anti­body responses should be characterised (e.g., titer, number of re­sponding animals, neutralising or non-neutralising), and theirappearance should be correlated with any pharmacologicaland/or toxicological changes. Specifically, the effects of antibodyformation on pharmacokinetic/ pharmacodynamic parameters, in­cidence and/or severity of adverse effects, complement activation,or the emergence of new toxic effects should be considered wheninterpreting the data. Attention should also be paid to the evalu­ation of possible pathological changes related to immune complexformation and deposition.

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The detection of antibodies should not be the sole criterion for theearly termination of a preclinical safety study or modification in theduration of the study design unless the immune response neutralisesthe pharmacological and/or toxicological effects of the biopharma­ceutical in a large proportion of the animals. In most cases, theimmune response to biopharmaceuticals is variable, like thatobserved in humans. If the interpretation of the data from the safetystudy is not compromised by these issues, then no special signifi­cance should be ascribed to the antibody response.

The induction of antibody formation in animals is not predictive ofa potential for antibody formation in humans. Humans maydevelop serum antibodies against humanised proteins, andfrequently the therapeutic response persists in their presence. Theoccurrence of severe anaphylactic responses to recombinant pro­teins is rare in humans. In this regard, the results of guinea piganaphylaxis tests, which are generally positive for protein prod­ucts, are not predictive for reactions in humans; therefore, suchstudies are considered of little value for the routine evaluation ofthese types of products.

4. SPECIFIC CONSIDERATIONS

4.1 Safety pharmacology

It is important to investigate the potential for undesirable pharma­cological activity in appropriate animal models and, where necessary,to incorporate particular monitoring for these activities in the toxicitystudies and/or clinical studies. Safety pharmacology studies meas­ure functional indices of potential toxicity. These functional indicesmay be investigated in separate studies or incorporated in the designof toxicity studies. The aimof the safety pharmacology studies shouldbe to reveal any functional effects on the major physiological systems(e.g., cardiovascular, respiratory, renal, and central nervous systems).Investigations may also include the use of isolated organs or othertest systems not involving intact animals. All of these studies mayallow for a mechanistically-based explanation of specific organ

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toxicities, which should be considered carefully with respect tohuman use and indication(s).

4.2 Exposure assessment

4.2.1 Phannacokinetics and toxicokinetics

It is difficult to establish uniform guidelines for pharmacokineticstudies for biotechnology-derived pharmaceuticals. Single andmultiple dose pharmacokinetics, toxicokinetics, and tissue distri­bution studies in relevant species are useful; however, routinestudies that attempt to assess mass balance are not useful. Differ­ences in pharmacokinetics among animal species may have a sig­nificant impact on the predictiveness of animal studies or on theassessment of dose response relationships in toxicity studies. Al­terations in the pharmacokinetic profile due to immune-mediatedclearance mechanisms may affect the kinetic profiles and the inter­pretation of the toxicity data. For some products there may also beinherent, significant delays in the expression of pharmacodynamiceffects relative to the pharmacokinetic profile (e.g., cytokines) orthere may be prolonged expression of pharmacodynamic effectsrelative to plasma levels.

Pharmacokinetic studies should, whenever possible, utilisepreparations that are representative of that intended for toxicitytesting and clinical use, and employ a route of administration thatis relevant to the anticipated clinical studies. Patterns of absorptionmay be influenced by formulation, concentration, site, and/or vol­ume. Whenever possible, systemic exposure should be monitoredduring the toxicity studies.

When using radiolabeled proteins, it is important to show that theradiolabeled test material maintains activity and biologicalproperties equivalent to that of the unlabeled material. Tissueconcentrations of radioactivity and/or autoradiography datausing radiolabeled proteins may be difficult to interpret due torapid in vivo metabolism or unstable radiolabeled linkage. Careshould be taken in the interpretation of studies using radioactive

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tracers incorporated into specific amino acids because of recyclingof amino acids into non-drug related proteins/peptides.

Some information on absorption, disposition and clearance in rele­vant animal models should be available prior to clinical studies inorder to predict margins of safety based upon exposure and dose.

4.2.2 Assays

The use of one or more assay methods should be addressed on acase-by-case basis and the scientific rationale should be provided.One validated method is usually considered sufficient. For exam­ple, quantitation of TCA-precipitable radioactivity followingadministration of a radiolabeled protein may provide adequateinformation, but a specific assay for the analyte is preferred.Ideally the assay methods should be the same for animals andhumans. The possible influence of plasma binding proteinsand/or antibodies in plasmal serum on the assay performanceshould be determined.

4.2.3 Metabolism

The expected consequence of metabolism of biotechnology­derived pharmaceuticals is the degradation to small peptides andindividual amino acids. Therefore, the metabolic pathways aregenerally understood. Classical biotransformation studies as per­formed for pharmaceuticals are not needed.

Understanding the behaviour of the biopharmaceutical in thebiologic matrix, (e.g., plasma, serum, cerebral spinal fluid) and thepossible influence of binding proteins is important for under­standing the pharmacodynamic effect.

4.3 Single dose toxicity studies

Single dose studies may generate useful data to describe the rela­tionship of dose to systemic and/or local toxicity. These data canbe used to select doses for repeated dose toxicity studies. Informa­tion on dose-response relationships may be gathered through the

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conduct of a single dose toxicity study, as a component of pharma­cology or animal model efficacy studies. The incorporation ofsafety pharmacology parameters in the design of these studiesshould be considered.

4.4 Repeated dose toxicity studies

For consideration of the selection of animal species for repeated dosestudies see section 3.3. The route and dosing regimen (e.g., dailyversus intermittent dosing) should reflect the intended clinical use orexposure. When feasible, these studies should include toxicokinetics.

A recovery period should generally be included in study designsto determine the reversal or potential worsening of pharmacologi­cal/toxicological effects, and/or potential delayed toxic effects.For biopharmaceuticals that induce prolonged pharmacologi­cal/toxicological effects, recovery group animals should be moni­tored until reversibility is demonstrated. The duration of repeateddose studies should be based on the intended duration of clinicalexposure and disease indication. This duration of animal dosinghas generally been 1-3 months for most biotechnology-derivedpharmaceuticals. For biopharmaceuticals intended for short-termuse (e.g., $ 7 days) and for acute life-threatening diseases, repeateddose studies up to two weeks duration have been consideredadequate to support clinical studies as well as marketing authori­sation. For those biopharmaceuticals intended for chronic indica­tions, studies of 6 months duration have generally beenappropriate although in some cases shorter or longer durationshave supported marketing authorisations. For biopharmaceuticalsintended for chronic use, the duration of long term toxicity studiesshould be scientifically justified.

4.5 Immunotoxicity studies

One aspect of immunotoxicological evaluation includes assess­ment of potential immunogenicity (see section 3.6). Many biotech­nology-derived pharmaceuticals are intended to stimulate or

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suppress the immune system and therefore may affect not onlyhumoral but also cell-mediated immunity. Inflammatory reactionsat the injection site may be indicative of a stimulatory response. Itis important, however, to recognise that simple injection traumaand/or specific toxic effects caused by the formulation vehiclemay also result in toxic changes at the injection site. In addition,the expression of surface antigens on target cells may be altered,which has implications for autoimmune potential. Immunotoxi­cological testing strategies may require screening studies followedby mechanistic studies to clarify such issues. Routine tiered testingapproaches or standard testing batteries, however, are not recom­mended for biotechnology-derived pharmaceuticals.

4.6 Reproductive performance and developmental toxicity studies

The need for reproductive/ developmental toxicity studies is de­pendent upon the product, clinical indication and intended patientpopulation (Note 2). The specific study design and dosing schedulemay be modified based on issues related to species specificity,immunogenicity, biological activity and/or a long eliminationhalf-life. For example, concerns regarding potential developmentalimmunotoxicity, which may apply particularly to certain mono­clonal antibodies with prolonged immunological effects, could beaddressed in a study design modified to assess immune functionof the neonate.

4.7 Genotoxicity studies

The range and type of genotoxicity studies routinely conductedfor pharmaceuticals are not applicable to biotechnology-derivedpharmaceuticals and therefore are not needed. Moreover, theadministration of large quantities of peptides/proteins may yielduninterpretable results. It is not expected that these substanceswould interact directly with DNA or other chromosomal material(Note 3).

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Studies in available and relevant systems, including newly devel­oped systems, should be performed in those cases where there iscause for concern about the product ( e.g., because of the presenceof an organic linker molecule in a conjugated protein product).The use of standard genotoxicity studies for assessing the geno­toxic potential of process contaminants is not considered appropri­ate. If performed for this purpose, however, the rationale shouldbe provided.

4.8 Carcinogenicity studies

Standard carcinogenicity bioassays are generally inappropriate forbiotechnology-derived pharmaceuticals. However, product­specific assessment of carcinogenic potential may still be neededdepending upon duration of clinical dosing, patient populationand/or biological activity of the product (e.g., growth factors,immunosuppressive agents, etc.) When there is a concern aboutcarcinogenic potential a variety of approaches may be consideredto evaluate risk.

Products that may have the potential to support or induce prolif­eration of transformed cells and clonal expansion possibly leadingto neoplasia should be evaluated with respect to receptor expres­sion in various malignant and normal human cells that are poten­tially relevant to the patient population under study. The ability ofthe product to stimulate growth of normal or malignant cells ex­pressing the receptor should be determined. When in vitro datagive cause for concern about carcinogenic potential, further stud­ies in relevant animal models may be needed. Incorporation ofsensitive indices of cellular proliferation in long term repeateddose toxicity studies may provide useful information.

In those cases where the product is biologically active and non-im­munogenic in rodents and other studies have not provided suffi­cient information to allow an assessment of carcinogenic potentialthen the utility of a single rodent species should be considered.Careful consideration should be given to the selection of doses.

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The use of a combination of pharmacokinetic and pharmacody­namic endpoints with consideration of comparative receptor char­acteristics and intended human exposures represents the mostscientifically based approach for defining the appropriate doses.The rationale for the selection of doses should be provided.

4.9 Local tolerance studies

Local tolerance should be evaluated. The formulation intended formarketing should be tested; however, in certain justified cases, thetesting of representative formulations may be acceptable. In somecases, the potential adverse effects of the product can be evaluatedin single or repeated dose toxicity studies thus obviating the needfor separate local tolerance studies.

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Notes

Note 1 Animal models of disease may be useful in defining toxicityendpoints, selection of clinical indications, and determinationof appropriate formulations, route of administration, andtreatment regimen. It should be noted that with these modelsof disease there is often a paucity of historical data for use as areference when evaluating study results. Therefore, the collec­tion of concurrent control and baseline data is critical to opti­mise study design.

Note 2 There may be extensive public information available regard­ing potential reproductive and/or developmental effects of aparticular class of compounds (e.g., interferons) where theonly relevant species is the non-human primate. In such cases,mechanistic studies indicating that similar effects are likely tobe caused by a new but related molecule, may obviate theneed for formal reproductive/developmental toxicity studies.In each case, the scientific basis for assessing the potential forpossible effects on reproduction/development should be pro­vided.

Note 3 With some biopharmaceuticals there is a potential concernabout accumulation of spontaneously mutated cells (e.g., viafacilitating a selective advantage of proliferation) leading tocarcinogenicity. The standard battery of genotoxicity tests isnot designed to detect these conditions. Alternative in vitro orin vivo models to address such concerns may have to bedeveloped and evaluated.

187

Appendix II - List of Participants

Dr Timothy Anderson....Senior Director, ToxicologyHoffmann-La Roche Inc, USA

Miss Gabby AshtonResearch AssistantCMR International, UK

Dr Jakob BackManager, Toxicology Project &Planning

Novo Nordisk A/S, Denmark

Dr Debra BarrettResearch Manager, ToxicologyPreclinical DevelopmentRW Johnson PharmaceuticalResearch Institute, USA

Dr Gerd BodeHead, Toxicology, Safety &MolecularHoechst Marion Roussel, France

Dr Peter BugelskiDirector of Cell and MolecularToxicologySmithKline BeechamPharmaceuticals, UK

t Meeting Session Chairman.. Syndicate Group Chairman....Syndicate Group Rapporteur

Dr Joy Cavagnaro"formerly Senior Pharmacologist/Director, Quality AssuranceCenter for Biologics Evaluation &Research, FDA, USA(now Vice President, RegulatoryAffairsHuman Genome Sciences Inc.USA)

Professor Jean-Roger ClaudeHead of the Laboratory ofToxicologyUniversite Rene Descartes, France

Dr John CurdSenior Director of ClinicalResearchGenentech Inc/ USA

Dr Thomas J DavidsonAssociate Director, BiologicsEvaluation Department, DrugSafety EvaluationBristol-Myers Squibb CompanyUSA

Dr Harold DavisDirector of ToxicologyAmgen Inc, USA

189

Safety Evaluation ofBiotechnologically-derived Pharmaceuticals

Professor Anthony Dayan"Director, DH Department ofToxicologySt Bartholomew/s & RoyalLondon School of Medicine &Dentistry, UK

Dr Roland De CosterDirector of Safety PharmacologyJanssen Research FoundationBelgium

Dr Felix de la Iglesia"Vice President, Pathology &Experimental ToxicologyParke-Davis PharmaceuticalResearch Division, USA

Dr Eric de WaalPreclinical Assessor of theMedicines Evaluation BoardNational Institute for PublicHealth, The Netherlands

Dr Maggie DempsterInternational Project ToxicologistZeneca Pharmaceuticals, UK

Dr Joachim DennerHead of Section, RecombinantProteins, Department of MedicalBiotechnologyPaul-Ehrlich Institute, Germany

Dr Michael DoratoDirector, ToxicologyLilly Research Laboratories, USA

Dr Carl EistrupHead of Department, Drug SafetyDepartment, InternationalRelationsNovo Nordisk A/S, Denmark

Dr Gordon FindlayResearch AssociateCMR International, UK

Dr Leena GajjarRegulatory Affairs AssociateChiron SpA, Italy

Dr Andrew GalazkaCorporate Vice President, MedicalAffairsAres Serono SA, Switzerland

Dr James Green""Director, Preclinical DevelopmentBiogen Inc, USA

Dr Susan GriffithsProject Leader, R&D StrategiesCMR International, UK

Dr Peter Harris""Medical DirectorTherexsys, UK

Dr Wolfgang HeimannHead of PathologyKnoll AG, Germany

Dr Flemming HojelseHead of ToxicologyLundbeck A/S, Denmark

Dr Tohru InoueChairman, Division of Cellular &Molecular ToxicologyNational Institute of HealthSciences, Japan

Dr Helen JacksonHead, Experimental TherapeuticsCambridge Antibody TechnologyUK

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Appendix II - List ofParticipants

Dr Dale JohnsonDirector of ToxicologyChiron Corporation, USA

Dr Jeff KiplingDirector, Department of Science &TechnologyABPI, UK

Dr John Lipaniformerly Group Director,RheumatologySmithKline BeechamPharmaceuticals, USA(now Vice President, ClinicalDevelopment at Abgenix Inc.,USA)

Dr Jack LipmanInternational Project ToxicologistHoffmann-La Roche Inc., USA

Dr Cyndy LumleyAssociate DirectorCMR International, UK

Dr Neil McAuslaneResearch ManagerCMR International, UK

Dr James MacDonaldSenior Vice President, Drug Safety& MetabolismSchering-Plough ResearchInstitute, USA

Ms Janice MacdonaldDirector of Regulatory AffairsAthena Neurosciences (Europe)Ltd, UK

Dr Bernhard MatterHead of Compliance GLP/GCPAuditingNovartis Pharma AG, Switzerland

Dr Marjorie MohlerResearch FellowRW Johnson PharmaceuticalResearch Institute, USA

Dr Gwyn Morgant

Vice President, Safety AssessmentSmithKline BeechamPharmaceuticals, USA

Dr Douglas MortonVice President, Preclinical SafetyEvaluationLilly Research Laboratories, USA

DrWolfgang NeumannHead, Department of ToxicologyBoehringer Ingelheim KGGermany

Dr Klaus OlejniczakPreclinical AssessorInstitute for Drugs and MedicalDevices, Germany

Dr Anthony PhillipsWorld Wide Director ofBiotechnology ProductDevelopmentGlaxo Wellcome Research &Development, UK

Dr Anne PilaroToxicologistCenter for Biologics Evaluation &Research, FDA, USA

Dr Mary PrevoVice President, Environmental &Product SafetyAlza Corporation, USA

Dr John Purves*Sector Head for CentralisedApplications: List AEMEA,UK

191

Safety Evaluation of Biotechnologically-derived Pharmaceuticals

Dr Hansjorg RonnebergerHead of Department ofPharmacologyjToxicologyBehringwerke AG, Germany

Dr Sigrid RosenerToxicologistInstitut fur ToxikologieMerck KGaA, Germany

Dr Christer SafholmDirector of Non-RodentToxicologyAstra AB, Sweden

M David C ScalestWorldwide Director of Bioanalysis& Drug MetabolismGlaxo Wellcome Research andDevelopment, UK

Ms Mercedes SerabianToxicologistCenter for Biologics Evaluation &Research, FDA, USA

Dr Jennifer Sims**Principal Scientific OfficerMedicines Control Agency, UK

Dr Per SjobergChairman of CPMPWorkingParty on Safety, Head ofToxicologyMedical Products Agency, Sweden

Professor Lewis SmithtDirectorMRC Toxicology Unit, UK

Dr Sandra SnookPathologist IISearle R&D, USA

Dr Mark SopwithDirector of MedicineCelltech Therapeutics Ltd, UK

Dr Per SpindlerHead of Preclinical MedicinesEvaluationDanish Medicines AgencyDenmark

Dr Frank van MeelManager Preclinical ProjectsNV Organon, The Netherlands

Professor Giuseppe Vicarit

Chairman of CPMPBiotechnology Working Party,(former DirectorInstituto Superiore di Sanita, Italy)

Dr Eckhard von KeutzDirector, Institut furToxikologie-PharmaBayer AG, Germany

Professor Stuart WalkerDirectorCMR International, UK

Dr Michael WoodHead of Pharmacology &ToxicologyBritish Biotech PharmaceuticalsUK

Dr Ruprecht ZierzHead of Hematology &ImmunotoxicologyExperimental ToxicologySchering AG, Germany

192

Index

absorption, lymphatic, of IL-2,89-90, see also ADME

adenoviral vectors for gene therapy,123

ADME (absorption, distribution,metabolism, excretion) studies,13-14

gene therapy products, 116, 125ICH guidance on, confusing, 48administration, 178-9duration, 12ICH guidelines on, 178-9multiple cycles of, gene therapyproducts,120,121,163

route, see routeschedule/frequency, see dosingschedule/ frequency

allometric scaling, IFN-y/IL-2, 80, 89Amgen Company, Neupogen®, 72analogues of proteins, structural,

design of non-clinical safetyevaluation programmes, 73-4

analysis of pre-clinical safetyevaluation programmes,case-by-case approach, 35-6

animal(s), number and gender of,178

animal models, 2,176-8of disease, 2,111-12,177-8,187industry survey on conduct ofstudies with, 26-7

ICH guidelines on selection of,176-8, 187

IFNs/ILs and margin of safetyderived from, 95

MAb and choice of various,111-12,154

non-traditional/new/ alternativesto, 11, 27-8, 76, see also specificalternatives

species used, see individual speciesantibodiesmonoclonal, see monoclonalantibodies

monoclonal antibody binding toother, 107

to products, 179-80ICH guidelines on, 179-80toIFNs/ILs, 79,82-6,95

anticoagulants on Japanese market,61

anti-idiotype responses, 107arcitumomab,41arthritis, animal models, 3assay methods in ICH guidelines,

182autoimmune disease, animal

models, 3

basic FCF, 72Betaferon, see interferon-p-1bbiological activity, 175-6ICH guidelines on, 175-6of IFNs/ILs, neutralisation, 95biotechnology-derived

pharmaceuticals, definition,32-3

body weight, IL-2 clearance and,interspecies scaling, 88

CAMPATH-1H, clinical trials, 4carcinogenicity, 13, 185-6CSFs/growth factors/hormones,133, 136-7

gene therapy products, 164ICH guidelines on, 48, 185-6, 187IFNs/ILs, 80,97, 141, 147-8industry survey of conduct ofstudies, 25-6

insulin lispro, 74

193

Safety Evaluation of Biotechnologically-derived Pharnwceuticals

MAbs, 156-7anti-eD4,4anti-TNF,4,153

CBER responsibilities, 31CD4,MAbtomurine, clinical trials, 4primatised,5clinical trials, 4species differences between animalmodels, 3

CEA-Scan,41cellular host contaminants, ICH

guidelines, 174Center for Biologics Evaluation and

Research, responsibilities, 31Centralised Procedure, 40Centre for Medicines Research

International survey ofcompany strategies fordesigning non-clinical safetyevaluation programmes, 17-29

methodology/ objectives, 19-20response, 20-1clearance of IFNs/ILs, 144, see also

elimination; excretioninterspecies scaling, 86-9clinical trialsCAMPATH-1H,4MAb, see monoclonal antibodiesTNF, 4clinician's view on design of

non-clinical safety evaluationprogrammes, 106

Clinton, President, on regulatoryreform initiatives, 34-5

CMR International, see Centre forMedicines ResearchInternational

collagen-induced arthritis, 3colony-stimulating factors, 71-2,

133-8designing non-clinical safetyevaluation programmes, 71-2,133-8

industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23

reproductive toxicology studies, 24species selection, 22, 23receptor, transgene studies, 56Committee for Proprietary

Medicinal Products, 39-49review procedure, 41-3company strategies for designing

non-clinical safety evaluationprogrammes, 17-29

computer information onanimal-human proteinhomology, 10

contaminants, see impurities andcontaminants

CPMP, see Committee forProprietary Medicinal Products

CSFs, see colony-stimulating factorscystic fibrosis transmembrane

conductance regulator gene,viral-induced inflammationlimiting therapy with, 123

cytochrome P450 enzymes, hepatic,IFN-mediated regulation, 144

cytokines, see also specific cytokineson Japanese market, 61receptors, transgene studies, 56-9

delayed adverse effects, seelong-term adverse effects

design of non-clinical safetyevaluation programmes

clinician's view, 106company strategies, 17-29CSFs, 71-2, 133-8gene therapy products, 115-28,159-67

growth factors, 71-2,133-8hormones,65-78,133-8IFNs/ILs, 79-101, 139-49MAbs, see monoclonal antibodiesregulatory authorities, 21, 34-6toxicologist's view, 7-15developmental (fetal!neonatal)

toxicity, 184gene therapy products, 164ICH guidelines on, 184, 187IFNs/ILs, 141, 146

194

Index

MAbs, 153, 156distribution, tissue, see also ADMEgene/gene product/gene vector,116, 125, 163

IFNs/ILs, 145DNA in lipid 'sandwiches', 123-4dog 9,10,46,47,69-73, 143dose, see also duration; multiple

(repeat)-dose toxicity studies;single-dose administration

first, in gene therapy studies, 162-3selectionICH guidelines on, 178-9MAb,109-10

dosing schedule/frequency, 11, 178-9IL-2 and influence of, 90-3MAb,111duration (of repeat-/multiple-dose

toxicity study), 12CMR International survey ofindustry, 23

erythropoietin, 71gene therapy, 159, 163-4growth hormone (biosynthetichuman), 70

insulin (biosynthetic human), 69

EC, List A products approved by, 40,41

Eli Lilly and Co.growth hormone (biosynthetichuman), 70, 71

insulinbiosynthetic human, 69structural analogue (insulinlispro),73

elimination, CSFs/growthfactors/hormones, 133, see alsoclearance; excretion

EMEA,40endpoints with gene therapy

products, 126enzyme pro-drug therapy,

gene-directed, 164EPAR,40epitope (of MAb)binding, 110, 153

signal transduction with, 106-7expression, 103, 108, 112, 152, 154,155

turnover, 110erythropoietin, 10, 71, 72design of non-clinical safetyevaluation programmes,71,72

Europe, regulatory systems, 39-49European Agency for Evaluation of

Medicinal Products, 40European Commission, List A

products approved by, 40, 41European Public Assessment

Report, 40excipients, novel, 108excretion, CSFs/growth

factors/hormones, 137-8, seealso ADME; clearance;elimination

exposure assessment, 181-2ICH guidelines on, 181-2IFNs/ILs, 144expression, gene therapy products,

115,125-6expression vectors, GM-eSF a/~

receptor, 56, 59

factor VIla, 41Farmitalia Carlo Erba, bFGF, 72Fc portion of MAb,

pharmacodynamic effects, 153FDA, see Food and Drug

Administrationfetal toxicity, see developmental

toxicityFGF, basic, 72fibroblast growth factor, basic, 72follitrophin-a,41follitrophin-~ (Puregon), 41safety studies supporting Europeanmarketing application, 45-7

Food and Drug Administration(regulatory assessment), 31-8

differences from Europe, 49guidance document on somaticcell and gene therapy, 165, 167

195

Safety Evaluation ofBiotechnologically-derived Pharmaceuticals

gene-directed enzyme pro-drugtherapy, 164

gene therapy products, 115-28,159-67

change of gene, 165-6designing non-clinical safetyevaluation programmes,115-28, 159-67

nature of gene, 122Genentech Co., growth hormone

(biosynthetic human), 70, 71genotoxicity, 13, 77, 184-5CSFs!growth factors!hormones,133,135

gene therapy products, 122, 164ICH guidelines on, 48,184-5,187IF~s!ILs, 141, 146-7industry survey of conduct ofstudies, 25-6

insulin (biosynthetic human), 70MAbs, 156-7germ cell, genes (therapeutic)

present!expressed in, 126, 163GM-CSF, see granulocyte!

macrophage-CSFGonal-F,41Good Laboratory Practice, 175granulocyte!macrophage-CSFalp receptor transgene studies, 56animal species selection, 71-2growth factors, 71-2, 133-8designing non-clinical safetyevaluation programmes, 71-2,133-8

industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23reproductive toxicology studies,24species selection, 22, 23on Japanese market, 61growth hormone(s) (in general), on

Japanese market, 61growth hormone (GH), human, 70-1immunogenicity,76haemopoietic factors on Japanese

market, 61

hepatic clearance!enzymes, see liverhepatitis vaccines, 41homologous proteins, 11, 68-72designing non-clinical safetyevaluation programmes, 67,68-72

IF~s!ILs, 147-8industry survey of conduct ofstudies with, 26-7

hormones,67-78,133-8designing non-clinical safetyevaluation programmes, 67-78,133-8

industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23reproductive toxicology studies,24species selection, 22, 23on Japanese market, 61Humalog®, 41, 73-4Humatrope®,71hyperimmunisation studies, 76

ICH, see International Conference onHarmonisation

igovomab, 41immune complex formationwith IF~s!ILs,95with MAbs, 106immune response

(immunogenicity!immunotoxicity etc.), 7, 75-6,179-80,183-4

gene therapy products, 122, 124-5ICH guidelines on, 176, 179-80,183-4

IF~s!ILs,95,140, 145-6MAbs, ISS, 176Immunex-Hoechst Company,

Leukine®, 72immunoglobulin G transport

mechanism with MAbs, 153impurities and contaminantsICH guidelines on presence of,173-4

MAb preparations, 107-8

196

Index

in vitro methods as alternativemodels, 11

in vitro tissue binding, MAbs, 152-3Indimacis-125, 41inflammationanimal models, 3viral vector-induced, 123insertional mutagenesis, 166insulin(s)biosynthetic humandesigning non-clinical safetyevaluation programmes, 68-70on Japanese market, 61immunogenicity studies intransgenic mice, 76

structural analogues (insulin lisproetc.), 41, 73-4

interferon(s), 79-101, 139-49antibodies to, 79, 82-6, 95biological effects vs. systemicexposure, timeframe, 86

clearance, see clearancedesigning non-clinical safetyevaluation programmes,79-101, 139-49

industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23reproductive toxicology studies,24species selection, 22, 23on Japanese market, 61

proposed scheme for safetyevaluation, 96, 97

toxicity of, 84interferon-a-2a, 83, 84interferon-a-2b, 83, 84interferon-~, 83prevention of VSV infection by, 82

interferon-~-la, 84interferon-~-lb (Betaferon), 41, 83, 84safety studies supportingEuropean marketingapplication, 44-5

interferon-y, 83, 84homologous (in mouse), 147pharmacokinetics, 88-9

interleukin(s),79-101antibodies to, 79, 82-6, 95biological effects vs. systemicexposure, timeframe, 86

clearance, see clearancedesigning non-clinical safetyevaluation programmes,79-101,139-49

industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23reproductive toxicology studies,24species selection, 22, 23on Japanese market, 61

proposed scheme for safetyevaluation, 96, 97

toxicity of, 84interleukin-l, 83, 84species used in assessment of, 143interleukin-la,84interleukin-2, 83, 85pharmacokinetics in humans,prediction, 88

receptors, 93receptors, biolocalisation, anddistribution in IL-2 toxicity, 92

route of administration, influence,89-93

species used in assessment of,143-4

interleukin-3, 83, 85receptors, transgene studies, 59interleukin-4, 83, 85interleukin-5, 83interleukin-6, 83, 85interleukin-7, 83interleukin-8,83interleukin-10,83interleukin-11,83interleukin-12, 83, 85homologous (in mouse), 147interleukin-15,83International Conference on

Harmonisation guidelines, 29,38, 169-87

background, 172

197

Safety Evaluation ofBiotechnologically-derived Pharmaceuticals

companies using, 21contentious points, 48on definition ofbiotechnology-derivedpharmaceuticals, 32-3

FDA and, 36, 37MAbs and, 151, 152, 176objectives, 172-3preclinical safety testing, 174-80scope, 173specification of test material, 173-4table of contents, 171interspecies scaling 13, 86, 87

Japan, 51-63Ministry of Health and Welfare, 60

kidney, IFN/IL clearance, 86-9

Leukine®,72leukocyte receptors, IL-2, 93Leukoscan, 41lipid-DNA 'sandwiches', 123-4liposomal gene delivery systems,

123-4List A products, approved, 40, 41liverIFN clearance, 86-9IFN-mediated regulation of P450enzymes, 144

local tolerance studies, ICHguidelines, 186

long-term/delayed adverseeffects/toxicity (of proteinagents), 4, 5-6

MAbs, 153lymphatic absorption of IL-2, 89-90

melanoma, cutaneous, MAbdetecting, 41

metabolism, 182, see also ADMECSFs/growth factors/hormones,137-8

in ICH guidelines, 182Ministry of Health and Welfare,

Japanese, 60

monoclonal antibodies, 103-14,151-7, see also specific antibodies

clinical trials, 4approach to opening, 113, 114design of non-clinical safetyevaluation programmes, 151-7issues unique to MAbs, 152-3EC approved List A, 40, 41ICH guidelines on, 151, 152, 176industry surveycarcinogenicity studies, 25repeat-dose studies, 22, 23reproductive toxicology studies,24species selection, 22

on Japanese market, 61mechanisms producing toxicity,106-8

solubility 110monooxygenases, hepatic P450,

IFN-mediated regulation, 144mouse 23,24,44,45,59,69,72,82,

88,136,143,147,155multiple cycles of administration,

gene therapy products, 120,121,163

multiple (repeat)-dose toxicitystudies, 12, 183

duration, see durationgene therapy products, 120, 121,163-4

ICH guidelines on, 183IFNs/ILs,97,144MAbs, 111, 155survey of industry on, 22-3mutagenesis, insertional, 166mutagenicity, IFNs/ILs, 80, 97

Neupogen®, 72neutralisation of biological activity

of IFNs/ILs, 95Novaseven, 41Novo Company, biosynthetic

human insulin, 69

P450 enzymes, hepatic,IFN-mediated regulation, 144

198

Index

peptide fragments, designingnon-clinical safety evaluationprogrammes, 74-5

pharmaceutical(s),biotechnology-derived,definition, 32-3

pharmaceutical company strategiesfor designing non-clinicalsafety evaluation programmes,17-29

pharmacodynamicsFc portion of MAb and effect on,153

ICH guidelines on, 175-6pharmacokinetics, 181-2, see also

ADME and specificpharmacokinetic parameters

evaluation, 13-14in ICH guidelines, 48,181-2IF~s/ILs,86-9,140, 144MAb, 109, 155, 181-2pharmacology, safety, ICH

guidelines on, 180-1, see alsosafety pharmacology

pig model, IL-2 studies, 90Points to Consider in Human Somatic

Cell and Gene Therapy, 165, 167porcine model, IL-2 studies, 90primates (non-human) 9, 10, 13,22,

23,44,45,60,72,75,83,88,89,94, 112, 143, 146, 147, 154, 156,162, 178, 187

pro-drug activation by geneexpressing an enzyme, 164

proteinscomputer information onanimal-human homology, 10

homologous, see homologousproteins

structural analogues, design ofnon-clinical safety evaluationpro~ammes,73-4

Protropin ,71Puregon, see follitrophin-~

Rapilysin, see Reteplase

rabbit 12, 22, 24, 58, 72, 73, 82, 88, 90rat3,22,23,24,44,45,69-75,87,88,

125, 136, 143receptor, for IL-2 on leucocytes, 93receptor-mediated clearance of IL-2,

87receptor-mediated toxicity, 54-5transgenic animal studies, 55-9regulatory systems, 31-63on design of non-clinical safetyevaluation programmes, 21,34-6

Europe, 39-49gene therapy products, 126Japan, 51-63MAb development and interactionwith,104

US, see United Statesrenal clearance, IF~s/ILs, 86-9repeat-dose toxicity studies, see

multiple (repeat)-dose toxicitystudies

reproductive organs, genes(therapeutic)presentlexpressed in, 126, 163

reproductive toxicology studies, 184,see also germ cell line alterations

gene therapy products, 164ICH guidelines on, 184, 187IF~s/ILs, 141, 146industry survey on conduct of, 24MAbs, 156Reteplase (Rapilysin), 41safety studies supportingEuropean marketingapplication, 47-8

review procedure of CPMP, 41-3risk assessment, gene therapy

products, 115, 117-18route of administration, 11-12gene therapy products, 120change, 166intended in clinical practice, 125ICH guidelines on, 178-9IL-2,89-93

199

Safety Evaluation ofBiotechnologically-derived Pharmaceuticals

safety pharmacology 9,12,44,46,47,67,96,142,144,145,155,180

short-term adverse effects/toxicityof protein agents, 4, 5

signal transduction with MAbs,106-7

single-dose administrationin animals, 182-3gene therapy products, 119-20,121, 162-3ICH guidelines on, 182-3MAbs, 111, 155in clinical studies, multiple dosetoxicity studies preceding, 12

species of animalsdifferences between, 3IFN/ILstudies, 81-2, 143-4number of species tested, 94scaling between, 80specificity,81-2selection, 7, 9-10, 55-9,176-8gene therapy products, 116, 124-5GM-eSF, 71-2ICH guidelines, 176-8insulin (biosynthetic human), 69MAb studies, 108-9, 154-5survey of industry on, 22

sulesomab, 41systemic exposure 81, 86, 88, 89, 90,

93, 120,144,181

Tecnemab K1, 41TNF, see tumour necrosis factor

tolerance, local, ICH guidelines onevaluation of, 186

toxicokinetics in ICH guidelines,181-2

toxicologist's view on design ofnon-clinical safety evaluationprogrammes, 7-15

transgenic animals (mice etc.), 4, 11,56,76

industry survey on conduct ofstudies with, 26-7

insulin studies, 76receptor-mediated toxicity, 55-9Tritanrix-HB,41tumorigenicity, see carcinogenicitytumour necrosis factor (TNF), MAb

to, clinical trials, 4Twinrix adult, 41Twinrix paediatric, 41

United States regulatory systems,31-8

differences from Europe, 49

vaccines, EC approved List A, 40, 41vectors (for genes)expression, GM-eSF a./~ receptor,56,59

for gene therapy, 122-4, 125, 160,161-2, 165-6change of, 165-6

vesicular stomatitis virus, IFN-~preventing infection by, 82

viral vectors for gene therapy, 122-4,125,162

200