1. CONTRIBUTION OF PRECLINICAL STUDIES TO EVALUATION OF OSTEOPOROSIS THERAPY Gideon A Rodan MD PhD Merck Research Laboratories Bone Biology and Osteoporosis Research

2. PRECLINICAL INFORMATION Hypothesis: Preclinical studies can reduce the burden of proof required of clinical trials, by providing information on Safety (general and bone) Efficacy Mechanism (pharmacological activity and adverse events)

3. Historical Perspective and Current Osteoporosis Guidelines Pre 1994: Increased BMD in 2 year PBO- controlled trials plus preclinical evidence for bone safety/quality Reasons for change: –No fracture reduction during third year with etidronate treatment, hence three year studies –No fracture reduction during fluoride treatment, in spite of increased BMD, hence fracture endpoint

4. ETIDRONATE PRECLINICAL STUDIES Spontaneous fractures in dogs (Flora et al) Impaired fracture healing in dogs (Nunnemaker et al) Narrow efficacy/safety window (MRL study)

5. Control Bisphosphonate Microradiograph Growth Cartilage Epiphysis Diaphysis Metaphysis Schenk Assay

9. 25 20 15 10 5 5 0 0 0 0 0.01 0.1 1 1 10 100 20 10 0 0 Efficacy Cn -BV/TV (%) Dose mg P/kg/day S.C. Safety OV/BV (%) Bone Volume ( Cn - - BV/TV) Osteoid Volume (OV/BV) 15 5 5 0.001 1 Dose Response for Inhibition of Resorption and of Mineralization by Alendronate in Schenk Assay

10. 35 30 25 20 15 10 5 5 0 0 0 0 0.01 0.1 1 1 10 100 60 50 40 30 20 10 0 0 Efficacy Cn -BV/TV (%) Dose mg P/kg/day S.C. Safety OV/TV (%) Bone Volume ( Cn - - BV/TV) Osteoid Volume (OV/BV) Dose Response for Inhibition of Resorption and Mineralization by Etidronate in Schenk Assay

11. FLUORIDE PRECLINICAL STUDIES Bone strength increase is not commensurate with bone mass increases (Mosekilde et al. CTI 1987, 40:318-322) Abnormal mineralization by x-ray scattering (Fratzl et al JBMR 1994, 9:1541- 1549) MRL study (Lafage et al, JCI 1995, 95:2127-2133)

12. Correlation of Vertebral Bone Mass and Bone Strength In Alendronate Treated Animals 30 27.5 25 22.5 20 17.5 15 12.5 10 7.5 5 0.90.951.01.051.11.151.21.251.31.35 Bone Mineral Density L2-L4 (g/cm 2 ) Ultimate Strength (MPa) Non-OVX OVX+VEH OVX+ALN 0.05 mg/kg IV OVX+ALN 0.25 mg/kg IV r=0.9 p(x 2 )=0.0034 JCI, 92, 2577 (1993) Baboons 2 Years 350 250 200 150 100 50 30 Ash Weight (mg) Ultimate Load (N) Non-OVX OVX+VEH OVX+ALN 1.8 mg/kg SC OVX+ALN 18 mg/kg SC CTI, 53, 283(1993) Rats 1 Year 300 3540455055606570 Similar Findings in Normal Minipigs (1 Yr), Rats ( 2 Yrs), and Dogs (3 Yrs) Oral Dosing.

13. Comparison of Alendronate and NaF Effects on Bone Strength vs. Bone Mass 1600 1400 1200 1000 800 600 400 200 20253035404550 Bone Volume/Tissue Volume % Failure Load (N) Alendronate 1600 1400 1200 1000 800 600 400 200 20253035404550 Bone Volume/Tissue Volume % Sodium Fluoride JCI, 95, 2127 (1995)

14. Bone Strength decreases with increased NaF content 1150 JCI, 95, 2127, (1995) 1050 950 850 750 1.61.82.02.2 Bone Fluoride Content (mg/g bone ash) L4 Core Ultimate Strength (MPa) N.B. In clinical trials NaF increased BMD w/o reducing fractures

15. PRECLINICAL MODELS FOR BONE SAFETY CONCLUSIONS Bone measurements (histology and strength) in animal models at multiples (5x?) of the therapeutic dose detected deleterious effects of etidronate and fluoride, and could be sensitive enough to evaluate the bone safety of prospective OP therapies. Recommendation Use bones from long term toxicology studies to evaluate bone safety (histology and strength).

16. PRECLINICAL MODELS FOR EFFICACY Estrogen-deficiency bone loss (cancellous>cortical) occurs in most mammals including humans, rodents, primates and other species (in sheep, dogs, rabbits, apparently seasonal). Agents that increase BMD and bone strength in preclinical models reduced fracture risk in humans: bisphosphonates, estrogens, SERMs, PTH. However, quantitative relationships would have to be determined in clinical trials.

17. Recommended Principles for Preclinical Efficacy Studies Use adult animals - to eliminate confounding effect of growth. Use any species documented to lose an easily quantifiable amount of bone following oophorectomy, cancellous or cortical. Use several parameters and accepted methodology (DXA, histomorphometry, QCT, mechanical testing, biochemical markers), look for internal consistency. Use multiple doses (2-3).

18. Recommended Principles for Preclinical Efficacy Studies (Cont.) For prevention registration document the prevention of bone loss. For treatment registration document the restoration of lost bone (treatment of osteopenia). Follow bone retention after cessation of therapy.

19. Mechanism Studies Provide important insights for defining the necessary safety and efficacy studies. Safety: For agents binding to the mineral, effects on mineralization and mineral structure (BPs, F). For bone forming agents, woven vs. lamellar bone, tumors, ectopic ossification… Efficacy: At the tissue level all resorption inhibitors act similarly (suppression of bone turnover). No known mechanistic difference between cancellous and cortical bone resorption.

20. INHIBITORS OF RESORPTION vs. FORMATION STIMULATORS Inhibitors of bone resorption retain existing normal bone and bone structure and can produce a positive bone balance. Unless they alter bone/mineral structure (e.g. etidronate) they should be totally safe for bone. Formation stimulators engender production of new bone (e.g. fluoride), which could be “woven”, normal structure should be confirmed by histology.

21. SUMMARY AND CONCLUSIONS Preclinical studies –Can validate the “bone safety” of osteoporosis therapeutic agents and potentially predict if increases in bone mass will be associated with increases in bone strength. –Can test the efficacy of prospective therapeutic agents in animal models of estrogen-deficiency bone loss, and potentially other types of bone loss. –Could, accordingly, help the design of clinical trials.

22. SUMMARY AND CONCLUSIONS Topics in current preclinical guidelines which can be revisited: Multiple species (cortical remodeling) for efficacy studies. Duration of efficacy studies vs. use of long- term toxicology animals for bone safety. Different criteria for different resorption inhibitors.