1. Safety Pharmacology Society Webinar Series: Safety Pharmacology Endpoints: Integration into Toxicology Studies Integrating functional CNS observations into toxicology studies: the CONS! Will Redfern, PhD Safety Assessment UK Alderley Park Cheshire United Kingdom September 20, 2012

2. Reasons for attrition of candidate drugs Kola & Landis (2004) Nature Reviews: Drug Discovery 3: 711-715. Meanwhile, ADME failures have been reduced by a ‘frontloading’ approach

3. Attrition due to inadequate safety – why? 3 ShortcomingImpactSolution? 1. Lack of early detection of safety signals ‘Doomed’ compounds enter in vivo tox phase Improve frontloaded screening: in silico and in vitro 2. Lack of detection of safety hazards preclinically ‘Doomed’ compounds enter clinical development Improve quality and increase information content of safety pharmacology and toxicology studies 3. Lack of confidence/knowledge/ precision in preclinical- clinical translation Defective risk assessment: ‘Doomed’ compounds may be let through, anticipating a large safety margin; ‘safe’ compounds may be stopped, anticipating an inadequate safety margin. Improve risk assessment and decision-making by better understanding of the translation of the preclinical signals to humans.

4. Attrition due to inadequate safety – why? 4 ShortcomingImpactSolution? 1. Lack of early detection of safety signals ‘Doomed’ compounds enter in vivo tox phase Improve frontloaded screening: in silico and in vitro 2. Lack of detection of safety hazards preclinically ‘Doomed’ compounds enter clinical development Improve quality and increase information content of safety pharmacology and toxicology studies 3. Lack of confidence/knowledge/ precision in preclinical- clinical translation Defective risk assessment: ‘Doomed’ compounds may be let through, anticipating a large safety margin; ‘safe’ compounds may be stopped, anticipating an inadequate safety margin. Improve risk assessment and decision-making by better understanding of the translation of the preclinical signals to humans.

5. Impact of adverse effects of drugs by organ function throughout the pharmaceutical life cycle Phase‘Nonclinical’Phase IPhase I-III Phase III/ Marketing Post- Marketing Information: Causes of attrition Serious ADRs Causes of attrition ADRs on labelSerious ADRs Withdrawal from sale Source:Car (2006) Sibille et al. (1998) Olson et al. (2000) BioPrint® (2006) Budnitz et al. (2006) Stevens & Baker (2008) Sample size:88 CDs stopped1,015 subjects82 CDs stopped1,138 drugs21,298 patients47 drugs Cardiovascular: 27%9%21%36%15%45% Hepatotoxicity: 8%7%21%13%0%32% Haematology/BM: 7%2%4%16%10%9% NERVOUS SYSTEM: 14%28%21%67%39%2% Immunotox; photosensitivity: 7%16%11%25%34%2% Gastrointestinal: 3%23%5%67%14%2% Reprotox: 13%0%1%10%0%2% Musculoskeletal: 4%0%1%28%3%2% Respiratory: 2%0% 32%8%2% Renal: 2%0%9%19%2%0% Genetic tox: 5%0% Carcinogenicity: 3%0% 1%0% Other: 0% 4%16%2% Adapted from Redfern WS et al. SOT 2010; 2011 1-9% 10-19% >20% 0%  The various toxicity domains have been ranked first by contribution to products withdrawn from sale, then by attrition during clinical development. No change in 10 years! Increased contribution from Nervous System AEs in 2010 2010 Update:

6. Impact of functional adverse effects on the nervous system on drug development during 2010: Source: DIA Daily January to December 2010

7. Impact of functional adverse effects on the nervous system on drug development during 2010: Source: DIA Daily January to December 2010 Impact of QT/TdP issues on drug development during 2010 by comparison:

8. Doing it in addition to standalone safety pharmacology studies Scientific drivers Doing it instead of standalone safety pharmacology studies Regulatory drivers Rationale: To provide early warning flags well ahead of the regulatory GLP SP core battery studies (by incorporating into early tox/MTD studies). To assess whether findings in acute SP studies persist, intensify, or diminish after repeated dosing, and to demonstrate recovery after cessation of dosing. To provide functional correlates of histopathological findings in previous tox studies. To assess potential effects that may only develop after prolonged exposure. Rationale: To opt for the minimum regulatory requirement for FTIM: ICHS6 (Biologics) ICHS9 (Oncology Products) FDA Guidance on Exploratory IND Studies by incorporating SP core battery assessments into the 1-month regulatory tox studies. Functional measurements in repeat-dose toxicity studies I’m OK with this. Let’s have more of it! I have reservations about this. This will be what I’m focusing on today.

9. Starting point... Clearly, adverse effects on the nervous system make a significant contribution to attrition of candidate drugs during clinical development. Therefore, the last thing we should do is reduce the quality of the preclinical CNS safety pharmacology assessment. So, do more ‘as well as’, and reduce the temptation to go for ‘instead of’*. *In other words, do include CNS safety pharmacology endpoints in repeat-dose toxicity studies as well as standalone single-dose safety pharmacology studies, rather than instead of.

10. Why not replace standalone CNS safety pharmacology studies with assessments in repeat-dose toxicity studies – what’s the big deal? 1.The laboratory conditions in toxicology holding rooms/procedure rooms are not optimal for obtaining high quality behavioural data (due to noise; disturbance etc.). 2.The phenomenon of tolerance means that the responses measured on Day X may be diminished compared to Day 1 (ie, first administration). 3.By Day X, what you may be measuring is not the pharmacological response to the compound, but the effects of overt toxicity (inappetance; weight loss; general malaise). 4.Circumventing ‘2’ and ‘3’ above by doing the assessments on Day 1 of dosing causes logistical difficulties.

11. Limitations of SP endpoints in tox studies The primary aim of a repeat-dose toxicity study is to expose animals to different levels of a test compound over a prolonged period, and to assess a standard list of in-life parameters (incl. clinical chemistry; body weights, food & water consumption; routine clinical observations; ophthalmoscopy; ECG, etc.), toxicokinetics, and post-mortem histological changes. Any additional functional measurements MUST NOT interfere with these aims or affect their outcome. The study design and laboratory conditions may be sub-optimal for obtaining high-quality functional data.

12. Safety pharmacology studiesGeneral toxicology studies Dosing staggered to accommodate functional measurements Animals dosed all in one session (usually a.m.) TK sample taken after key functional measurements TK sampling takes priority No necropsy to considerScheduled to accommodate necropsy slots Studies powered to detect the functional effectStudies adequate to detect histopathological effects Behavioural studies usually require young ratsSexually mature animals used Usually restricted to male animalsEqual numbers of both sexes used May require non-standard strains (e.g. pigmented rats) Restricted to standard strains Functional measurements may require pre- training of animals Rarely required Functional measurements require a quiet roomSometimes anything but! Equipment/software may not be fully GLP- compliant GLP sacrosanct Should be run by experienced safety pharmacologists and technicians fully au fait with safety pharmacology measurements and data interpretation Toxicology facilities may be geographically remote from available safety pharmacology expertise, or such expertise may not be available within the company. Differences in in-life environments (etc.)

13. Example of a custom-designed, fit-for-purpose in vivo safety pharmacology suite Features: Testing labs located remote from corridor noise (e.g., trundling of cage racks; loud conversations). Primary access to suite via single entry door, with warning to limit entry to essential visits and to minimise noise level. Staff requiring access to the other animals on the study can do so without disturbing the safety pharmacology observations/measurements. Entry to the testing labs restricted to staff involved in the observations/measurements. Designed to accommodate bulky test equipment, ergonomically. Lighting control with local (manual) override. CNS evaluations done here

14. Example of toxicology study holding rooms with ante room Drawbacks (for CNS safety pharmacology observations/measurements): Testing area adjacent to corridor noise (e.g., trundling of cage racks; loud conversations). Access from corridor directly into testing area. Staff requiring access to the other animals on the study disturb the safety pharmacology observations/measurements. Entry to the testing area unrestricted. Bulky test equipment may be difficult to accommodate ergonomically. Automated lighting control with no manual override. CNS evaluations done here

15. DrugTherapeutic target Effects Opiate analgesicsPainRapid tolerance to most effects develops on repeat-dosing BaclofenSpasticityTolerance develops to muscle relaxant effects due to down-regulation of GABA-B receptors BenzodiazepinesAnxietyTolerance develops to initial sedative effect L-DOPA; bromocriptineParkinson’sReduced efficacy SSRI’sDepressionReduced efficacy Haloperidol; chlorpromazine SchizophreniaReduced efficacy AnticonvulsantsEpilepsyReduced efficacy A DECREASE in response/clinical efficacy with repeat-dosing Development of tolerance with repeat-dosing ‘‘Some form of adaptive syndrome is the inevitable consequence of the reciprocal interaction between most or all classes of drugs and the organism’’. W Haefely (1986)

16. Pupillary light reflex in a repeat-dose toxicology study in rats: tolerance developing to a mydriatic effect Drug X µmol/kg po (n = 6 each) (slow) Redfern WS et al. (2007) A simple method for estimating pupil diameter in conscious rats and dogs during repeat-dose toxicity studies. J Pharmacol Toxicol Methods 56: e50. (No further dosing at high dose level)

17. Saliva production in a repeat-dose toxicology study in dogs: tolerance developing to a salivatory effect Salivation quantified by placing a pre-weighed gauze swab inside a jowl for 20 s; removed and re-weighed. First measurement was on Day 3 of study. (AZ in-house data)

18. Example of tolerance, increased response, and no change in response in the same study with the same compound! Day 1Day 2Day 3Effect Abnormal respiration 6/63/62/6Diminishing Decreased activity 6/6 Stable Increased scratching 0/6 3/6Delayed onset Effects of once-daily dosing with baclofen (10 mg/kg po) in the Irwin test in rats (3M; 3F) Conclusion: Change in magnitude of effect over repeated dosing is both pharmacology- and parameter-specific – and can’t be predicted in advance. AZ in-house data: courtesy of Lorna Ewart

19. Logistics for rodent studies… If you choose to go down this route (replacing the standalone safety pharmacology study), it is preferable to conduct functional measurements on Day 1 of the repeat-dose toxicity studies for the reasons outlined earlier (ie, you may miss an acute response that diminishes with repeat- dosing). But Day 1 of a tox study is usually mayhem, with timed TK bleeds etc. So, you could do the measurements on Day 2 of the repeat-dose study. However, you won’t get through all the Irwin tests (multiple time points) and whole-body plethysmography (WBP) measurements (4 hours’ recordings) on the vehicle and 3 dose levels (Irwin: 24 rats; WBP: 32 rats) in one day! So you could do (say) the Irwin tests on Day 2 and the WBP measurements on Day 3. Even then, you still won’t complete either of these evaluations in a single day. So you may have to stagger the start of the rodent 1-month study, e.g.: MONTUEWEDTHU Day 1 Start cohort 1Day 2 cohort 1: Irwin Day 3 cohort 1: WBPDay 4 cohort 1 Day 1 Start cohort 2Day 2 cohort 2: IrwinDay 3 cohort 2: WBP And you’ll have to reduce the standard number of time points in the Irwin test. Do you have enough quiet space to run Irwin and WBP simultaneously, close to the tox holding room…?

20. Conclusions Replacement of the ‘standalone’ CNS safety pharmacology study with ‘CNS safety pharmacology assessments’ in a repeat-dose tox study represents a dumbing-down of the preclinical CNS risk assessment. This would be like replacing the dog telemetry cardiovascular assessment with a ‘snapshot ECG’ in a tox study to assess QT risk. You wouldn’t do that, would you...?

21. Acknowledgements Colleagues at AstraZeneca Alderley Park: Sharon Storey; Helen Prior; Claire Grant; Louise Marks; Lorna Ewart; Kat Greenwood; Claire Barnard; Dave Simpson; Sally Robinson; Jean-Pierre Valentin.