ICH Topic S 8: "Immunotoxicity Studies for Human Pharmaceuticals"; Umsetzung in die Praxis

Transcription

1 ICH Topic S 8: "Immunotoxicity Studies for Human Pharmaceuticals"; Umsetzung in die Praxis Dr. Christoph Specht Scientific Director vivo Science GmbH Fabrikstraße Gronau Germany lab@vivoscience.de Fon: +49-(0)

2 Content 1. vivo Science GmbH General presentation 2. General toxicity testing (studies required prior phase I) 3. ICH-regulations: a network 4. Drug-induced immunotoxicity: The view of the regulatory agencies 5. Immunotoxicity testing: implementing the ICH topic S8 6. Statistics a word of caution

3 Location Gronau / Westfalia (near Enschede / NL) Germany

4 History 2001 founded as PARA BioScience GmbH 2004 GLP certificate granted (renewed 2016) 2006 merger with NewLab BioQuality AG as legally independent affiliate (GmbH) Image deleted 2007 GMP certificate granted (renewed 2015) 2008 private aquisition by Dr. Jürgen Schumacher and Stefan Fischer, independent company renamed to vivo Science GmbH

5 Company overview legal form site area no. of employees privately held, independent company, GmbH approx sqrm 18 (4 study directors, 8 technical officers, 2 ½ QA) Image deleted years of service 15 main sales market main customers quality status Europe, India pharma, biotechnology, chemical industry GLP, GMP

6 Facility Images deleted 5 animal labs (IVC[S2] / SPF / Standard) standard bio-lab biosafety level S2 lab radioisotopes lab histology lab 2 GxP archives (documents / materials & el. data)

7 Contract Research / Services Areas of testing Immunotoxicity testing [GLP] Immunogenicity testing [GLP] in-vivo safety tests [GLP] in-vivo assays [GMP]

8 Contract Research / Services Immunology [GLP] Cell-based and humoral immune response Immunotoxicity Immunogenicity Vaccination studies Potency tests Tumourigenicity studies Biosimilarity Toxicology [GLP] Toxicological in-vivo tests acc. to EMA or OECD (incl. DART) Testing of Medical Devices [GLP] Tests according to ISO Image deleted Assay validation [GLP] Method transfer, proof of concept Validation of Analytical Procedure [ICH Q2 (R1)] Viral Safety [GMP] Tests of cell substrates and cell-lines on adventitious viruses in vivo Potency Testing [GMP] in vivo potency testing of (recombinant) vaccines

9 Other in-vivo safety tests [GLP] EMA (Pharmaceuticals) (e.g.) CPMP/SWP/1042/99 Note for Guidance on Repeated Dose Toxicity OECD (Chemicals) (e.g.) species: mouse/rat 402 Acute Dermal Toxicity 407 Repeated Dose 28-day Oral Toxicity Study CPMP/SWP/465/95 Note for Guidance on 408 Repeated Dose 90-Day Oral Toxicity Study Preclinical Pharmacological and Toxicological 410 Repeated Dose Dermal Toxicity: 21/28-day Study Testing of Vaccines 414 Prenatal Developmental Toxicity Study CPMP/3097/02 Note for Guidance on Comparability of Medicinal Products containing 416 Two-Generation Reproduction Toxicity Study Biotechnology-derived Proteins as Drug Substance 420 Acute Oral Toxicity - Fixed Dose Method CPMP/SWP/2145/00 Note for Guidance on Non- 421 Reproduction/Developmental Toxicity Screening Test Clinical Local Tolerance Testing of Medicinal Products 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test 423 Acute Oral toxicity - Acute Toxic Class Method 425 Acute Oral Toxicity: Up-and-Down Procedure 443 Combined Chronic Toxicity/Carcinogenicity Studies (others on request)

10 Immunotoxicity testing [GLP] According to ICH - S8 Standard testing: species: mouse/rat Monitoring of clinical signs Haematology / Clinical chemistry General pathology / Histopathology Cell-mediated immunity Flow-cytometry of B and T cell subsets Mitogen stimulation assays for B and T cells Natural killer cell activity Quantitation of phagocytic ability Humoral-mediated immunity Humoral responses to T-dependent antigen (primary and secondary) Humoral responses to T-independent antigens (primary) Host resistance

11 General requirements for safety testing Test substance Quality GMP Batch, impurities Amount let's calculate for Formulation» 90 days Tox» Rat (+beagle dog)» Daily administration» High dose of 1000 mg/ kg/ d => 1 kg (+15 kg) material is derived from the production process for the clinical testing material Drug substance is in the (almost) final formulation used for the clinical trial Formulation must be neutral concerning modulation of immunogenicity Characterization Activity testing is established Stability (in formulation and in serum/plasma including freeze/ thaw cycles) Handling of the drug substance is planned and tested at the critical points: Any adhesion to materials used during the (non-) clinical trials Reasonable packing units are chosen Substance concentrations which secure minimal and maximal application volumes

12 General requirements for drug safety testing I STUDY TYPE Prior to Phase 1 REQUIREMENT Toxicity Studies Single-dose acute toxicity studies in two mammalian species required prior to Phase 1 Repeated dose studies in two mammalian species (one rodent, one non-rodent) are required Prior to Phase 1. The recommended duration of the repeated dose toxicity studies is usually related to the duration, the therapeutic indication, and scale of the proposed clinical trial. In principle, the duration of the animal toxicity studies conducted in two mammalian species (one nonrodent) should be equal to or exceed the duration of human clinical trials. Reproduction Toxicity Studies Reproduction toxicity studies should be conducted as is appropriate for the population that is to be exposed. Men may be included in Phase 1 trials prior to the conduct of the male fertility study since an evaluation of the male reproductive organs is performed in the repeated dose toxicity studies. Women not of childbearing potential (i.e., permanently sterilized, postmenopausal) may be included in clinical trials without reproduction toxicity studies provided the relevant repeated dose toxicity studies (which include an evaluation of the female reproductive organs) have been conducted. US: for women of child-bearing potential, reproduction toxicity studies are not required before Phase 1 with appropriate precautions (i.e., use of contraception, pregnancy testing). EU: reproduction toxicity are required prior to Phase 1 anytime women of child-bearing potential are to be enrolled. Japan: assessment of female fertility and embryo-fetal development should be completed prior to the inclusion of women of childbearing potential using birth control in any type of clinical trial.

13 General requirements for drug safety testing II STUDY TYPE Prior to Phase 1 Safety Pharmacology Studies Toxicokinetic and Pharmacokinetic Studies Genotoxicity Studies Local tolerance Studies Repeated Dose Toxicity Studies Assessment of effects on vital functions (central nervous, cardiovascular and respiratory system) needed prior to Phase 1. These evaluations may be conducted as additions to toxicity studies or as separate studies. Exposure data in animals should be evaluated prior to human clinical trials. Further information on ADME in animals should be made available to compare human and animal metabolic pathways. Appropriate information should usually be available by the time the Phase 1 Human Pharmacology studies have been completed. The following In vitro tests are generally needed prior to first human exposure: Test for gene mutation in bacteria. In vitro test with cytogenetic evaluation of chromosomal damage within mammalian cells or an in vitro mouse lymphoma tk assay. Local tolerance should be studied in animals using routes relevant to the proposed clinical administration prior to human exposure. The assessment of local tolerance may be a part of other toxicology studies. The recommended duration of the repeated dose toxicity studies is usually related to the duration, therapeutic indication, and scale of the proposed clinical trial (refer to Table 1 below).

14 A Regulatory network ICH International Council for Harmonisation (of Technical Requirements for Registration of Pharmaceuticals for Human Use) GLP regulation of FDA on non-clinical laboratory studies USA ICH Documents Q "Quality" Topics, Quality Assurance (Stability Testing, Impurity Testing) S "Safety" Topics, pre-clinical (Toxicity Testing, Immunotoxicity Testing, etc.) E "Efficacy" Topics, (Dose Response, Good Clinical Practices, etc.) M Multidisciplinary" Topics EU Committees for: Medicinal Products for Human Use (CHMP) Pharmacovigilance Risk Assessment Committee (PRAC) (Medicinal Products for Veterinary Use (CVMP)) Orphan Medicinal Products (COMP) Herbal Medicinal Products (HMPC) Advanced Therapies (CAT) Paediatric Committee (PDCO) Deutschland Arzneibücher

15 Drug-induced immunotoxicity: The view of the regulatory agencies Drug-induced immunotoxicity may occur as immunosuppression, immunogenicity/antigenicity, autoimmunity, hypersensitivity, adverse immunostimulation. Unintended (adverse) and intended (pharmacodynamic) effects, together with known drug class effects should be taken into consideration. Standard preclinical toxicity studies may be used to detect immunotoxicity. Initial preclinical immunotoxicity findings may be characterized by follow-up studies, providing a mechanistic basis of toxicity and informing risk-benefit determination. The development use of validated assays [ ] may provide more useful endpoints for drug immunotoxicity safety assessment Taken from: Overview on Immunotoxicology Testing- What it Predicts and What it Doesn t S. C. Kunder, Division of Special Pathogen and Immunologic Drug Products, Center for Drug Evaluation and Research, US Food and Drug Administration, Rockville, MD, USA. 2004

16 ICH Topic S 8 Note: EMA guidelines only help you to setup the Strategy but don't tell you how to perform it. EMA guidelines (non-clinical toxicology) Single and Repeat-Dose Toxicity Genotoxicity Carcinogenicity Reproductive and Dev.Toxicity Local Tolerance Other Toxicity

17 ICH Topic S 8: strategy 1. Standard Toxicity Studies, STS: 2. Additional Immunotoxicity Studies: 2.1 (Assay Characterization and Validation) 2.2 T-cell Dependent Antibody Response (TDAR) 2.3 Immunophenotyping 2.4 Natural Killer Cell Activity Assays 2.5 Host Resistance Studies 2.6 Macrophage/Neutrophil Function 2.7 Assays to Measure Cell-Mediated Immunity Many topics: How many assays?

18 Implementation of the ICH Topic S Assay Characterization and Validation In general, the immunotoxicity test selected should be widely used and have been demonstrated to be adequately sensitive and specific for known immunosuppressive agents. [...] Immunotoxicity studies are expected to be performed in compliance with Good Laboratory Practice (GLP). Use validated assays [ICH Q2(R1)], perform tests according to SOPs:...and many more: >120 SOPs at vivo Science

19 Implementation of the ICH Topic S 8 Study 1 (can be conducted as a part of a Standard Toxicity Study, STS Study 2 humoral and cellular immune responses (KLH) Study 3 Host resistance 2.3 Immunophenotyping Immunophenotyping is the identification and/or enumeration of leukocyte subsets using antibodies. Immunophenotyping is usually conducted by flow cytometric analysis or by immunohistochemistry. (T- B- NK-cells, CD4 +, CD8 + etc.) 2.4 Natural Killer Cell Activity Assays Natural killer (NK) cell activity assays [...] are ex vivo assays in which tissues (e.g. spleen) or blood are obtained from animals that have been treated with the test compound. Cell preparations are co-incubated with target cells that have been labeled. 2.6 Macrophage/Neutrophil Function These assays assess macrophage/neutrophil function of cells [...] obtained from animals treated with the test compound (ex vivo assay). 2.7 Assays to Measure Cell-Mediated Immunity Assays to measure cell-mediated immunity have not been as well established as those used for the antibody response. These are in vivo assays where antigens are used for sensitization. The endpoint is the ability of drugs to modulate the response to challenge [KLH]. 2.2 T-cell Dependent Antibody Response (TDAR) The TDAR should be performed using a recognized T-cell dependent antigen (e.g. sheep red blood cells (SRBC) or keyhole limpet hemocyanin (KLH)) that results in a robust antibody response. 2.5 Host Resistance Studies Host resistance studies involve challenging groups of mice or rats treated with the different doses of test compound with varying concentrations of a pathogen [...] or tumor cells. [...] tumor burden observed in vehicle versus test compound treated animals is used to determine if the test compound is able to alter host resistance.

20 Study 1: STS analyzing immune parameters Gross necropsy (PEC Spleen) Macrophage Function (opsonized SRBC) Phagocatosis rate [%] Phagocytic activity [Macrophages / SRBC] PI Design: 28d repeated dose toxicity study in outbred rats NK Activity (CFSE stained target cells; PI counter staining) Histopathology rats Administration (reflects situation in men: e.g. oral, s.c., i.v.; 3 dose groups + vehicle group) Health monitoring: Viability, general / detailed clinical signs (IRWIN test, grip strength, beam walk test) Food / water consumption, body weight Haematology (Blood) Immunophenotyping (flow cytometry) Carboxyfluorescein succinimidyl ester (CFSE) + : Target cells Propidium iodide (PI) + : Dead cells Urine analysis Clin. chem. T cells (CD3 + ) B cells (CD19 + ) NK cells (CD49b + ) NK T cells (CD3 + CD49b + )

21 Study 2 humoral / cellular immune responses Necropsy (Spleen) Design: 28d repeated dose study in inbred mice Administration (reflects situation in men: e.g. oral, s.c., i.v.; 3 dose groups + vehicle group with KLH; 1 dose group w/o KLH) T-cell proliferation ( 3 H-thymidine incorporation after stimilatin with KLH) mice Immunization with KLH Keyhole Limpet Hemocyanin, day 1, 15 and 21 Health monitoring: Viability, general clinical signs Serum KLH-specific IgG (ELISA)

22 Study 3: host resistance (to melanoma cells) Design: 21d (to 28d) repeated dose study in inbred mice Administration (reflects situation in men: e.g. oral, s.c., i.v.; 3 dose groups + vehicle group) Necropsy Tumor monitoring Determination of tumor volumes (Important: endpoint is not the death of the animal, but tumor volumes >1500mm 3, data generated) mice Inoculation of tumor cells (B16F10 melanoma; d1) Health monitoring: Viability, general clinical signs

23 Statistics a word of caution What does significantly different from mean? Decision tree for statistical analysis (schematic) Images deleted significance level (p 0,05) means below the 5% α-error means ( only ) up to 5% (1 of 20) of all comparisons will return an incorrect difference to the control group A toxicity-study consists of several hundred data (and the n is small) Rely on statistics, but not exclusively. Ask questions like: Are the effects dose-dependent? Do effects confirm each other (e.g. differences in organ-weights by histological examination; one liver enzyme by another)? Consider a bias (e.g. unblinded observers, time bias at cell preparation, cage placement etc.) at the design of the assay * choice of ANOVA and Post-Hoc Test depending on the number of variables in the data set

24 Certificates / Licenses April 2004 GLP inspection on toxicity studies and immunotoxicity and immunogenicity studies. Re-inspections successfully accomplished in 2008 and 2012 (and February 2016) June 2007 GMP inspection on in vivo analysis for adventitious viruses. /n vivo potency testing of (recombinant) vaccines Re-inspections successfully accomplished in 2009, 2012 and 2015

25 Certificates / Licenses Good Laboratory Practice acc. to Chemikaliengesetz (German Chemicals Act) Good Manufacturing Practice acc. to Arzneimittelgesetz (German Pharmaceuticals Act) Working acc. to Tierschutzgesetz (German Animal Welfare Act) Working acc. to Gentechnikgesetz S1 (German Genetic Engineering Act) Working acc. to Infektionsschutzgesetz S2 (Ger. Law on the Prevention and Control of Infectious Diseases) Working acc. to Strahlenschutzverordnung (German Radiation Protection Ordinance) Approved training provider for biology laboratory assistants * Chemikaliengesetz (German Chemicals Act) ** Arzneimittelgesetz (German Pharmaceuticals Act)

26 vivo Science GmbH Fabrikstraße Gronau Germany lab@vivoscience.de Fon: +49 (0) Thanks for your attention!