Biopharmaceutical Pre-clinical Safety Evaluation – What’s the Difference?

1. Biopharmaceutical Pre-clinical Safety Evaluation – What’s the Difference?

2. Biopharmaceutical Pre-clinical Safety Evaluation – What’s the Difference? • Introduction/Historical Background and overview of registered products • Special Considerations with respect to pre-clinical safety package • Changes in ICH S6 Guideline (R1) will be highlighted • How these special considerations impact study types • Case Studies • Conclusion

3. Purpose of Nonclinical Development Program Safety Evaluation Hazard Identification Dose Response Analysis Risk Communication Hazard Characterisation Risk Evaluation HAZARD VERSUS RISK

4. In the 1970s, scientific advances in technology led to: • The cloning of the genes that coded for a number of proteins • Advanced cell culture techniques that enabled production of large quantities of recombinant proteins • Kohler and Milstein developed the hybridoma technology

5. Erythropoietin Graphic from www.dshs-koeln.de/biochemie/rubriken/00_home/00_epo2.html

6. Overview of Marketed Biopharmaceuticals Over 600 product monographs (mid-September 2011) • Recombinant and other proteins • Growth hormone, insulin (analogues) and erythropoietin • Blood products • Albumin, thrombolytics, fibrinolytics and clotting factors • Cytokines and growth factors • Interferons, interleukins and colony-stimulating factors • Monoclonal antibodies and related products • Mouse, chimeric or humanized; whole molecule or fragment; single chain or bispecific; and naked or conjugated • Modified human proteins • Fusion (IgFc), polyethyleneglycol (PEG)ylation, liposome encapsulation and drug–toxin • Vaccines, including bio-defense vaccines and other products, e.g., vaccine adjuvants, antisense drugs

7. A few successes… Muromonab Slide courtesy of Dr Dempster

8. Fab Fc region: Fc binding Complement activation Monoclonal Antibody HumanisedMonoclonal antibody Graphics from www.biology.arizona.eduand www.harmonystudy.com From http://pim.medicine.dal.ca/atb.jpg

9. Differences between Conventional Drugs and Biotechnologically-derived Pharmaceuticals • Usually synthetic, organic compounds • Usually protein or carbohydrate-based • Well defined structures and relatively stable • Complex physiochemical characteristics and heat sensitive • MW < 500 • MW >>500 can be 30000 to 150000 • Typically orally available • Typically given parenterally • Metabolized • Catabolized

10. Tissue Cross Reactivity • Ex vivo immunohistochemical assay using range of human tissues • Confirm on-target binding in human tissues • May identify off-target binding in human tissues • Case by Case approach on IHC using tissues from relevant preclinical species • Literature review suggests a difference in expression or unexpected toxicity noted in preclinical toxicology study

11. Two Primary Differences between Biopharmaceuticals and NCEs • Toxicity Type • Exaggerated pharmacodynamic responses • 2 U/kg dose of insulin produces significant hypoglycaemia in dogs • Biological toxicity • Vascular leak syndrome and hepatocyte necrosis result of IL-2 stimulated lymphocytes • Intrinsic toxicity • Few published examples Therefore is imperative to evaluate toxicity inpharmacologically relevant species

12. Two Primary Differences between Biopharmaceuticals and NCEs • Potential Immunogenicity • Need to assess for potential immune response to the biopharmaceutical preclinically and clinically • Preclinical: assists in the interpretation of the study findings; immunogenicity noted in animals is not always indicative of immunogenicity noted in patients • Antibodies to Monoclonal antibodies have been seen in clinical trials even with completely human monoclonal antibodies • Need to classify type of antibody (i.e. neutralising vs non-neutralising) • Neutralising antibodies could be reason for change in either efficacy or pharmacokinetic profile • Decreased response to human recombinant erythropoietin

13. Toxicity Testing (1) • Repeat Dose Studies • Ideally in two relevant preclinical species (rodent and non rodent) • If the study findings in the two preclinical species is the same then only need to conduct LT toxicity study in one species • Dosing less frequent due to longer T1/2 • Often weekly vs daily Half- life Data of Selected FDA-Approved Biotech Drugs as Reported in the Prescribing Information Key: IV – intravenous; SC – subcutaneous Tang et al (2004) Journal of Pharmaceutical Sciences; 93(2) 2184-2204

14. Toxicity Testing (2) • Dose regimen and route of administration • Should mimic the clinical route of administration • Need to take immunogenicity into consideration particularly with respect to dosing frequency In both studies neutralising ADA detected following 3rd weekly dose Weekly dosing may not be appropriate for LT study

15. Safety Pharmacology • Safety Pharmacology • Importance of species relevance • Can often be included in repeat dose toxicity study; for example ECGs and neurobehavioral assessment are often measured during a repeat dose study

16. What about in vitro Cardiovascular Evaluation (hERG)? It is predicted that certain drugs bind to the central cavity of hERG such as colfilium, ibutilide and analogues of colfilium. The central cavity would most likely be inaccessible to large molecular weight compounds such as proteins. Ref: Perry et al, 2006 Mol Pharmacol 69(2):509-19.

17. This example illustrates the binding of several toxins to an external binding site of hERG. Although these are peptides, these toxins have specific binding motifs that are unlikely to be present in most proteins. Zhang et al, 2003, Biophys J 84(5):3022-36.

18. Fertility • For products where rodents are a relevant species, an assessment of fertility can be made in a rodent species • ICH M3 (R2) should also be followed for the timing of data on fertility for products where rodents are relevant species • When the NHP is the only relevant species, the potential for effects on male and female fertility can be assessed by standard histopathological evaluation and assessment of menstrual cyclicity in repeat dose toxicity studies of at least 3 months duration using sexually mature NHPs • If there is a specific cause for concern, specialized assessments such as sperm count, sperm morphology/motility, testicular volume, and male or female reproductive hormone levels should be evaluated in the repeat dose toxicity study • It is recognized that mating studies are not practical for NHPs.

19. Reproductive/Developmental Toxicity • Points to take into consideration: • Species relevance • If the rodent or rabbit is a relevant species and embryo-fetal exposure is demonstrated, see ICH M3 (R2) for timing of reproductive toxicity studies • Potential need to alter study design to take due to immunogenicity • Special case of monoclonal antibodies – when placental transfer of IgG occurs

20. Transfer of IgG to the Conceptus in Humans Greater than 50% cellular degradation of IgG is known to occur during transit In rodents immunoglobulins detectable in the yolk sack during organogenesis

21. Study Design Considerations Fertility and early embryonic development Embryofetal development Pre and post-natal development

22. Enhanced Pre & Post-Natal Study Overview Milk 180 270 140 90 28 Treatment 20 Mating Delivery - infant blood - maternal blood Pregnancy - ultrasound- Fetal growth • Infant • -immunophenotyping • Growth and behavior • External malformations Items in red: Growth & morphology endpoints traditionally assessed in EFD study Duration of Dosing incorporates duration of EFD to delivery • Infant • clinical pathology • - immunophenotyping - TDAR- Immunoglobulin • Behavior • Xray skeletal assessment • Opthalmology Day0 • Infant • - clinical pathology • behavior • - learning test • - immunophenotyping • - TDAR • NK cell activity • lymphocyte proliferation • Growth & development • Visceral morphology- Histopathologyimmunohistochemistry Post natal phase duration & endpoints designed to address specific mAb concerns eg ontogeny of immune system, CNS development etc

23. Exposure data from PPN study with Golimumab (CNTO 148): fully human monoclonal IgG1 antibody against human TNF-a Concentrations of golimumab in maternal serum and breast milk and in neonatal serum. Dams were dosed from gestation day 50 through lactation day 33. Martin et al, American Journal of Reproductive Immunology 58 (2007) 138–149

24. Genotoxicity • Genotoxic Potential • NO unless products contains organic linkers

25. Carcinogenicity • Species relevance and antibodies would normally preclude standard assays so may need to design special studies • When an assessment is warranted, the sponsor should design a strategy to address carcinogenicity • This strategy could be based on: • Review of relevant data from a variety of sources. The data sources can include published data (e.g. information from transgenic, knock-out or animal disease models, human genetic diseases), information on class effects, detailed information on target biology, in vitro data, data from chronic toxicity studies and clinical data

26. Carcinogenicity (cont.) • The product specific assessment of carcinogenic potential is used to communicate risk and provide input to the risk management plan along with labelling proposals, clinical monitoring, post-marketing surveillance, or a combination of these approaches. • In some cases, the available information can be considered sufficient to address carcinogenic potential and inform clinical risk without warranting additional nonclinical studies. • For example, immunomodulators and growth factors pose a potential carcinogenic risk which can best be evaluated by post-marketing clinical surveillance rather than further nonclinical studies. • However, when the weight of evidence is unclear, the sponsor can propose additional studies that could mitigate the mechanism-based concern

27. Carcinogenicity (cont.) • For products where there is insufficient knowledge about specific product characteristics and mode of action in relation to carcinogenic potential, a more extensive assessment might be appropriate (e.g., understanding of target biology related to potential carcinogenic concern, inclusion of additional endpoints in toxicity studies) • If the weight of evidence from this more extensive assessment does not suggest carcinogenic potential, no additional nonclinical testing is recommended. Alternatively, if the weight of evidence suggests a concern about carcinogenic potential, then the sponsor can propose additional nonclinical studies that could mitigate the concern, or the label should reflect the concern. • Rodent bioassays (or short-term carcinogenicity studies) with homologous products are generally of limited value to assess carcinogenic potential of the clinical candidate.

28. Examples of Carcinogenicity Assessments Completed For Marketed Biopharmaceuticals Vahle et al, 2010 Toxicologic Pathology

29. ICH S6: Preclinical safety evaluation of biotechnology-derived pharmaceuticals • Endorse flexible, case-by-case, science-based approach • Importance of species specificity as primary toxicity is exaggerated pharmacodynamics • Potential impact of immunogenicity • Genotoxicity studies generally not indicated • Six months usually sufficient for chronic treatment • Reproductive toxicology study not required prior to registration (ICH M3 [R2]) • Standard carcinogenicity studies generally considered inappropriate but …..

30. NCE Genotoxicity studies MTD/DRF – rodent MTD/DRF – non-rodent Safety Pharmacology studies (rat CNS, non-rodent ECG, rodent respiratory) 1 month repeat dose rodent 1 month repeat dose non-rodent Biopharmaceutical Tissue cross reactivity study (monoclonal antibody only) Sometimes need DRF study Repeat dose in relevant species (1 or 2) with ECG/CNS incorporated Support for FTIH

31. Toxicology Determine ‘No Adverse Effect Level’ (NOAEL) Convert NOAEL to a ‘Human Equivalent Dose’ (HED) Adjust for anticipated exposure in man Adjust for inter-species differences in affinity/potency Apply safety factor (10-fold) Pharmacology Estimate human starting dose – justify based on pharmacology Adjust for anticipated exposure in man Include anticipated duration of effect Adjust for inter-species differences in affinity/potency FTIH Starting Dose • Maximum Recommended Starting Dose • Define anticipated safety window based on NOAEL and Pharmacology • Use appropriate safety factor based on potential risk

32. Safety Margins – NOAEL/MABEL Why start with the highest dose you think is safe…better to start with the lowest dose you think is active MABEL – Minimum Anticipated Biological Effect Level Unacceptable Toxicity Therapeutic Range Consider healthy animals vs patients with e.g. compromised blood picture, hence more suitable to consider a biological approach i.e. MABEL NOAEL Effect Minimum effective Dose (MED) Dose or Exposure

33. Monoclonal AntibodyAnti-cytokine (RA) • Support FTIH Clinical trial • Tissue cross reactivity - human, primates • Four week monkey study with recovery, including immune function testing and safety pharmacology endpoints • Support for Phase IIb/III and registration (> 1month dosing) • Chronic toxicity in monkeys (26 weeks) • Reproductive toxicity • Host resistance assays (mouse influenza model) • Carcinogenicity addressed using data from KO mice, plus a B16 mouse melanoma study

34. HormoneInsulin Analogue Case Study • Repeat dose studies in rats and dogs (both pharmacologically relevant species and used for insulin) • Duration up to 52 weeks • Safety pharmacology study • Glucose clamping • Reproductive toxicity in rats and rabbits - with human insulin as comparator • No carcinogenicity study (replacement therapy), but in vitro studies performed to assess mitogenic potential vs. human insulin

35. HormoneInsulin Analogue Case Study (cont.) • Non-clinical experience with one of the analogues (Asp B10) • Rat mammary tumours (trials terminated) • Subsequent investigation revealed tumours due to unintended IGF-1 activity • AspB10 now used as a comparator for the safety evaluation of newer analogues Important to conduct appropriate biology studies to provide useful answers prior to toxicity studies

36. When a Phase I goes wrong…………. Phase I TGN1412 clinical trials conducted by PAREXEL at Northwick Park Hospital, London, resulted in hospitalization of six volunteers in March 2006

37. Summary Timeline of the Main Events after Infusion of TGN1412

38. TGN1412: Mode of Action Nature, Vol 440; 2006

39. Toxicology NOAEL 50.0 mg/kg HED 16.0 mg/kg- adjust for anticipated exposure in man(not done) - adjust for inter-species differences in affinity / potency (not done) Apply >10-fold safety factor 1.6 mg/kg increased to 160-fold: 0.1 mg/kg Pharmacology MABEL- justify based on pharmacology- adjust for anticipated exposure in man - include anticipated duration of effect - adjust for inter-species differences in affinity / potency in-vitro T-cell proliferation (0.1 g/mL) murine parent to TGN1412 (5.11A1) ref 3 = ~0.003 mg/Kg in maninitial 10% receptor occupancy ~0.001 mg/kg in man TGN1412: Dose calculation “Maximum Recommended Starting Dose”- define anticipated safety window based on NOAEL and MABEL - appropriate safety factor based on potential risk0.001 mg/kg

40. Lessons from TGN1412 • Species selection • Non-human primate (NHP) not always the most relevant species • In vitro data show that NHP cells and human cells responded differently to TGN1412 • Data from rat surrogate model appeared to be closer to human but dose response and relevant potency would have been helpful • Do not rely solely on NOAEL • Need to consider biology - agonism versus antagonism • All of preclinical data should be assessed as a package

41. Conclusions • It is important to understand the limitations of conventional toxicity study models when used for biopharmaceutical testing • Understand the biology of the molecule you are assessing, and what the key safety concerns are for the target patient population • Design a safety testing programme in relevant, biologically responsive species incorporating appropriate parameters • Also need to be aware of potency as well in relation to human • Avoid unnecessary/irrelevant studies • Each product is different - ‘case-by-case’ approach • Do not rely solely on NOAEL for clinical dose selection but think of pharmacology and toxicity data as a continuum