June 2004 Bone Quality Sourced from NIH Consensus Development Panel on Osteoporosis. JAMA 285: ; 2001 Architecture Turnover Rate Damage Accumulation Degree of Mineralization Properties of the collagen/mineral matrix

June 2004 Bone Remodeling Process Resorption Cavities Bone Osteoclasts Lining Cells Osteoblasts Osteoid Lining Cells Mineralized Bone

June 2004 High Bone Turnover Leads to Development of Stress Risers and Perforations Stress Risers Perforations Bone Osteoclasts

Fracture Bone Strength Material Properties Remodeling Falls Shape & Architecture Exercise & Lifestyle Hormones Nutrition Bone Mass Postural Reflexes Soft Tissue Padding Reproduced with permission from Heaney RP. Bone 33: , 2003 Factors Leading to Osteoporotic Fracture: Role of Bone Remodeling June 2004

Consequences of an Imbalance in Bone Turnover Normal Bone Osteoporotic Bone Mechanism of Action Animation of Bone Remodeling Process, 2002, Eli Lilly & Company

Excessive suppression Increased mineralization AccumulationIncreased brittleness of microcracks Skeletal fragility Excessive suppression Increased mineralization AccumulationIncreased brittleness of microcracks Skeletal fragility There is a complex relationship between bone turnover and bone quality A decrease of bone turnover increases mineralization and permits filling of remodeling space Bone Turnover, Mineralization, and Bone Quality June 2004

Antiresorptive Agents Increase BMD by Decreasing Remodeling Space and/or Prolonging Mineralization Antiresorptive Agent Newly formed bone Increased Mineralization Remodeling space

June 2004 Rate of Bone Turnover Bone turnover is an essential physiological mechanism for repairing microdamage and replacing “old” bone by “new” bone. Healthy bone is a living tissue. Can excessive reduction in bone turnover be harmful for bone? How much suppression is too much? Clinical paradigm: Clinical question:

June 2004 Changes in Biochemical Markers Predict an Increase in Bone Mineral Density During Antiresorptive Therapy Treatment with antiresorptive agents produce greater proportional changes in bone turnover markers than in BMD Measurable changes in bone turnover markers tend to occur before changes in BMD There are significant correlations between changes in bone turnover markers and changes in BMD Sourced from Looker AC et al. Osteoporos Int 11: ; 2000

June 2004 Bone Turnover Markers Bone turnover markers are components of bone matrix or enzymes that are released from cells or matrix during the process of bone remodeling (resorption and formation). Bone turnover markers reflect but do not regulate bone remodeling dynamics.

June 2004 Relationship Between Changes in Bone Resorption Markers and Vertebral Fracture Risk VERT Study A decrease in urinary CTX and NTX at 3-6 months was associated with vertebral fracture risk at 3 years A decrease in urinary CTX >60% and of urinary NTX >40% gave little added benefit in fracture reduction Sourced from Eastell R et al. Osteoporos Int 13:520; 2002

June 2004 Raloxifene and Alendronate Reduce Bone Turnover in Women with Osteoporosis Sourced from Stepan JJ et al. J Bone Miner Res 17 (Suppl 1):S233; 2002 *p< 0.01 compared to premenopausal levels Mean Serum CTXMean Serum PINP ALN RLX * PINP (μg/L) ±1 SD CTX (ng/L) ±1 SD ALN RLX * Premenopausal † † The area between the dotted lines is + 2SD of the mean premenopausal value

June 2004 Effects of Raloxifene and Alendronate on Markers of Bone Resorption C-Telopeptide/Creatinine Ratio Placebo Raloxifene 60 mg/d Alendronate 10 mg/d Median % change at 1 Year *†*† * *†*† % of Women Below Lower Limit of Premenopausal Range (52 µg/mmol) at 1 Year * p <0.05 vs. PL † p <0.05 vs. RLX Sourced from Johnell O et al. J Clin Endocrinol Metab 87: , 2002

June 2004 Very low turnover leads to excessive mineralization and the accumulation of microdamage Very high turnover leads to accumulation of perforations and a negative bone balance Bone Turnover Effects Bone Quality

-6 Effects of Raloxifene on Trabecular and Cortical BMD Measured by Spinal vQCT MORE Trial - 2 Years June 2004 Genant H et al. J Bone Miner Res 18(Suppl 2); S383, 2003 vQCT Volumetric quantitative computed tomography DXA Dual x-ray absorptiometry

June 2004 Bone Quality Sourced from NIH Consensus Development Panel on Osteoporosis. JAMA 285:785-95, 2001 Architecture Turnover Rate Damage Accumulation Degree of Mineralization Properties of the Collagen/Mineral Matrix

Dogs Treated with High Doses of Bisphosphonates 2 Microcrack Surface Density (  m/mm 2 ) Mean ± SEM Placebo Risedronate Alendronate * ** *P<.05 vs placebo **P<.01 vs placebo 1 Reproduced with permission from Komatsubara S. J Bone Miner Res 18: , Reproduced with permission from Mashiba T et al. J Bone Miner Res 15: ; 2000 Bisphosphonates Increase Microcracks Risedronate 2 Alendronate 2 Incadronate 1 Beagle dogs treated 1 year with 6x the clinical dose Beagle dogs treated 3 years with 2.5 x the clinical dose June 2004

Reproduced with permission from Mashiba T et al. Bone 28: , 2001 Effects of Risedronate and Alendronate on Microcracks Microcrack in the right femoral neck cortex from a risedronate treated dog Microcracks in the third lumbar vertebral body from an alendronate treated dog RisedronateAlendronate

June 2004 Effect of Long-Term Bisphosphonate Treatment - Incadronate Reproduced with permission from Komatsubara S. J Bone Miner Res 18: , 2003

June 2004 Effects of Raloxifene on Microcracks in Monkey Vertebrae Microcrack Surface Density Sourced from Burr DB. Osteoporo Int 13, Suppl 3, S73-74; OvxCEERalox 1Ralox 2Sham * * * Crack Surface Density (Cr.S.Dn.) * p<0.05 CEE-conjugated equine estrogens Ralox 1 – 1 mg/kg Ralox 2 – 5 mg/kg

June 2004 Reproduced with permission from Seeman E. Advances in Osteoporotic Fracture Management 2: 2-8, 2002 and Fyhrie DP. Bone 15: , 1994 Microdamage in Human Trabecular and Cortical Bone

June 2004 Bone Quality Sourced from NIH Consensus Development Panel on Osteoporosis. JAMA 285:785-95, 2001 Architecture Turnover Rate Damage Accumulation Degree of Mineralization Properties of the Collagen/Mineral Matrix

June 2004 Old bone New bone ~50% mineralized Bone Mineralization of the Basic Multicellular Unit Sourced from Ott S. Advances in Osteoporotic Fracture Management 2: 48-54, 2003 Primary mineralization 3 months (3 months)

June Degree of Mineralization (%) - Degree of Mineralization (%) Old bone Ott S. Advances in Osteoporotic Fracture Management 2: 48-54, 2003 Bone Mineralization of the Basic Multicellular Unit -- Primary mineralization (3 months) Secondary mineralization (years) Bone fully mineralized

June 2004 Homogeneous vs. Heterogeneous Mineralization “ Microdamage progression is prevented by the roughness (or heterogeneity) of mineral densities and differing directions of mineralized collagen present.” “Cracks require energy to progress through bone, and when the mineral density is high and distribution of the tissue mineral density is homogeneous less energy (derived from deformation) is required for microdamage progression.” Seeman E. Advances in Osteoporotic Fracture Management 2: 2-8, 2002

June 2004 Homogeneous vs. Heterogeneous Mineralization Reproduced with permission from Boivin GY et al. Bone 27: ; 2000 Heterogeneous Homogeneous Low mineralization High Mineralization

June 2004 Heterogeneous Mineral Distribution in Iliac Bone Reproduced with permission from Boivin GY et al. Bone 27: ; 2000

*Stiffness **Toughness Reproduced with permission from Seeman E. Advances in Osteoporotic Fracture Management 2: 2-8; 2002 and Currey JD. J Biomechanics 12: ; 1979 The Relationship Between Mineralization and Bone Strength is Complex June Breaking Stress* Ash Density (%) kg/mm Ash Density (%) Modulus of Impact**

June 2004 Distributions of Mineralization Homogeneous Mineralization Heterogeneous Mineralization

June 2004 Alendronate Increases Bone Mineralization in Women with Osteoporosis Two YearsThree Years Reproduced with permission from Boivin GY et al. Bone 27: ;

June 2004 Baseline Degree of Mineralization (g mineralization/cm 3 bone) Total Iliac Bone - Raloxifene 60 mg/dTotal Iliac Bone - Placebo Endpoint Baseline % Distribution Raloxifene Treatment Induces a Normal Pattern of Bone Mineralization Sourced from Boivin G et al. J Clin Endocrinol Metab. 2003; 88: Two-year treatment with raloxifene results in a heterogeneous mineral distribution with a modest increase in mineralization Baseline

June 2004 Mineralization Distributions with Osteoporosis Agents: RLX Compared to Placebo Homogeneous bone Narrower curve higher peak Heterogeneous Bone Wider curve Baseline Mineralization Placebo – Ca VitD raloxifene Boivin GY et al. J Clin Endocrinol Metab 88: , 2003

June 2004 Teriparatide Treatment Forms New Not Fully Mineralized Bone Misof et al. J Clin Endocrinol Metab 88:1150-6, 2003

June 2004 Calcium Peak in Male and Female Patients Following Treatment with Intermittent PTH Misof et al. J Clin Endocrinol Metab 88:1150-6, Ca Peak [wt%] Ca Width [wt% Ca] CorticalCancellousCorticalCancellous BoneBoneBoneBone CorticalCancellousCorticalCancellous BoneBoneBoneBone Male PatientsFemale Patients Before PTH After PTD

June 2004 Overlaid Fluorescence Labeling Lines of Calcium Peak Widths Following Treatment with Intermittent PTH Misof et al. J Clin Endocrinol Metab 88:1150-6, 2003 Black Arrow– Ca Peak was 18.19% White Arrow- Interstitial bone Ca Peak was 23.05%