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De la physiologie osseuse à l’inhibition du RANKL: nouvelle approche ciblée dans l’ostéoporose post-ménopausique T Thomas – INSERM U1080, CHU de St-Etienne.

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Présentation au sujet: "De la physiologie osseuse à l’inhibition du RANKL: nouvelle approche ciblée dans l’ostéoporose post-ménopausique T Thomas – INSERM U1080, CHU de St-Etienne."— Transcription de la présentation:

1 De la physiologie osseuse à l’inhibition du RANKL: nouvelle approche ciblée dans l’ostéoporose post-ménopausique T Thomas – INSERM U1080, CHU de St-Etienne

2 La physiologie osseuse: la découverte de la voie OPG/RANK/RANKL dans le remodelage osseux
2

3 Le RANK Ligand est un médiateur essentiel de la formation, fonction et survie des ostéoclastes
CFU-M M-CSF RANKL RANK Ostéoclaste avant fusion Ostéoclaste multinucléé - Hormones - Facteurs de croissance - Cytokines Ostéoclaste activé Ostéoblastes In the presence of low levels of macrophage colony-stimulating factor (M-CSF), RANK Ligand is an essential mediator for osteoclast formation, function, and survival in both cortical and trabecular bone throughout the skeleton.1-6 The RANK Ligand polypeptide is a type II transmembrane protein found on the surface of expressing cells as well as in a proteolytically released (cleaved) soluble form1,4 RANK Ligand is expressed (both in a transmembrane and soluble form) from the osteoblast lineage cells.1,3 Additionally, RANK Ligand is expressed in various cell types including, but not limited to: lymphoid cells,7 basal epithelial, luminal epithelial and stromal cells from normal prostate8 and non-malignant breast tissue,9 including mammary epithelial cells,10 tumor cells including breast cancer cells,9 prostate cancer cells and subsequent bone metastases;8 dendritic cells, activated T-cells and B-cells,11 osteoclasts, mesenchymal cells, proliferative chondrocytes, early osteocytes and periosteal cells; synovial tissue,12 cardiomyocytes, vascular smooth muscle cells, endothelial cells,13 and the GI tract.14 The clinical significance of these findings is unknown RANK Ligand subsequently binds to its receptor, RANK, on immature and mature osteoclasts, which leads to maturation of pre-fusion osteoclasts to multinucleated osteoclasts and finally to activated osteoclasts1 RANK is another member of the TNF receptor family and is expressed on osteoclasts and osteoclast progenitors.11,15 In addition, RANK has been observed on cartilage cells (chondrocytes), mammary gland epithelial cells, and trophoblast cells1,10,15 1. Boyle WJ, et al. Nature. 2003;423: 2. Fuller K, et al. J Exp Med. 1998;188: 3. Lacey DL, et al. Am J Pathol. 2000;157: 4. Lacey DL, et al. Cell. 1998;93: 5. Yasuda H, et al. Proc Natl Acad Sci USA. 1998;95: 6. Hofbauer LC, Schoppet M. JAMA. 2004;292: 7. Kong Y-Y, et al. Nature. 1999;397: 8. Brown JM, et al. Urology. 2001;57: 9. Van Poznak C, et al. J Clin Pathol. 2006;59:56-63. 10. Fata JE, et al. Cell :103:41-50. 11. Anderson DM, et al. Nature. 1997;390: 12. Crotti TN, et al. Ann Rheum Dis. 2002;61: 13. Ueland T, et al. Circulation. 2005;111: 14. Moschen AR, et al. Gut. 2005;54: 15. Hsu H, et al. Proc Natl Acad Sci USA. 1999:96: Formation osseuse CFU-M=colony forming unit macrophage M-CSF=macrophage colony stimulating factor Résorption osseuse Adaptée de Boyle WJ, et al. Nature. 2003;423: 3

4 Survie des ostéoclastes Heures après changement du milieu de culture 2
Le RANK Ligand est essentiel à la différenciation, fonction et survie des ostéoclastes différenciation et fonction des ostéoclastes survie des ostéoclastes M-CSF (200 ng/ml) M-CSF (200 ng/ml) + sRANKL (100 ng/ml) Coloration TRAP Lacunes de Résorption Culture de cellules sanguines humaines 1 120 100 80 60 40 20 Survie des ostéoclastes (% de baseline) Heures après changement du milieu de culture 2 6 12 24 Control RANK Ligand M-CSF M-CSF + RANK Ligand 1) Human PBMCs were cultured in 48-well plates ( cells per well) with or without human M-CSF and mouse sRANKL in the presence of dexamethasone (1027 M). After culture for 7 days, adherent cells were stained for TRAP. Lower panels, Human PBMCs were cultured in 48-well plates ( cells per well) in which a dentine slice had been placed. Cultures were treated with human M-CSF and mouse sRANKL in the presence of dexamethasone (1027 M). After culture for 10 days, resorption pits formed on the slices were stained with Mayer’s hematoxylin. Bar mm. 354 SUDA ET AL. Vol. 20, No. 3 Downloaded from edrv.endojournals.org at Amgen Inc on November 24, 2009 human peripheral blood mononuclear cells (PBMCs) 2) Schéma: Osteoclasts were developed in vitro from bone-marrow cells using media supplemented with CSF-1 and/or RANK Ligand.1 Differentiated osteoclasts were counted.1 The combination of CSF-1 plus RANK Ligand maintained osteoclast numbers at starting levels throughout the 24-hour observation period.1 1 Suda et al, Endocrine Reviews 1999;20(3): 345–357; 2 D’après Lacey DL et al. Am J Path 2000; 157: 435–448 4

5 L’absence du RANK Ligand augmente la densité osseuse
Phénotype normal Absence de RANK Ligand1 Les souris ayant une délétion du gène RANK Ligand présentent une ostéopétrose sévère, caractérisée par des os longs, des corps vertébraux et des côtes radio-opaques.1 Les souris ayant une délétion du gène RANK présentent elles aussi un phénotype ostéopétrotique sévère.2 DMO accrue 1 Kong YY et al. Nature 1999; 397: 315–323; Références 1 Kong YY et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999; 397: 315–323 2 Li J et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 2000; 97: 1566–1571 5

6 * L’excès de RANK Ligand entraine une raréfaction du tissu osseux 0,12
0,02 0,04 0,06 0,08 0,10 0,12 BV/TV Trabéculaire 0,4 * 2 Dose de RANK Ligand (mg/kg) [RANK Ligand (mg/kg) soluble 2 fois/j pendant 10 jours] *p<0,05 Souris âgées de 10 semaines Soluble RANK Ligand Reduces Trabecular Bone in the Mouse Tibia 10 week old mice Twice-daily SC injections of soluble human RANK Ligand 0.4 mg/kg/day or 2.0 mg/kg/day, for 10 days Yuan et al. J Bone Miner Res 2005; 20(Suppl. 1): S161–162 6

7 L’ostéoprotégérine, OPG, est l’inhibiteur naturel endogène du RANK Ligand
CFU-M M-CSF Ostéoclaste avant fusion RANKL RANK OPG - Hormones - Facteurs de croissance - Cytokines Formation, Fonction, et Survie des Ostéoclastes Inhibées The body naturally produces a protein called osteoprotegerin (OPG) to neutralize the effects of RANK Ligand and defend against bone loss.1,2 When RANK Ligand is bound and neutralized by osteoprotegerin (OPG), osteoclasts cannot form3,4, function3 or survive.5 OPG, a natural endogenous inhibitor of RANK Ligand, acts as a decoy receptor by binding with RANK Ligand, thereby inhibiting osteoclastogenesis and the survival of pre-existing osteoclasts1,2,6,7 OPG, a member of the tumor necrosis factor (TNF) receptor family, binds to and neutralizes the effects of RANK Ligand, thereby inhibiting bone resorption1,2,6,7 OPG is an important inhibitor of the terminal differentiation and function of osteoclasts1,2,6,7 Boyle WJ, et al. Nature. 2003;423: Simonet WS, et al. Cell. 1997;89: Fuller K, et al. J Exp Med. 1998;188: Yasuda H, et al. Proc Natl Acad Sci USA. 1998;95: Lacey DL, et al. Am J Pathol. 2000;158: Lacey DL, et al. Cell. 1998;93: Bekker PJ, et al. J Bone Miner Res. 2001;16: Ostéoblastes Résorption osseuse inhibée Formation osseuse CFU-M=colony forming unit macrophage M-CSF=macrophage colony stimulating factor Adaptée de Boyle WJ, et al. Nature. 2003;423: 7

8 L‘OPG est un régulateur de la densité osseuse
Augmentation de la DMO Excès d’OPG X Diminution de la DMO OPG absent Normal Key Point OPG levels regulate bone density in mice.4 Supplementary Information Preclinical studies demonstrate the critical role of RANK Ligand and OPG in bone remodelling.1-5 The radiographs depict the femur in a normal mouse (left), an OPG-knockout mouse (middle), and a transgenic mouse overexpressing OPG (right).4 In the OPG knockout mouse (left )4: Decreased bone density (osteoporosis) develops due to increased osteoclast activity in the absence of OPG.4 These mice develop spontaneous fractures early in life.2 The severity of osteoporosis in OPG knockout mice demonstrates that the skeleton is unable to compensate for the lack of endogenous OPG.2 This finding highlights the essential role for OPG in protecting bone.2 In the OPG transgenic mouse (right)4: Increased bone density (osteopetrosis) is observed as a result of impaired osteoclast formation and hence reduced resorption.1,4 Bones from these mice have a normal shape, but bone mineral density is dramatically increased.1 1. Simonet WS, et al. Cell 1997;89: 2. Bucay N, et al. Genes Dev 1998;12: 3. Kong Y-Y, et al. Nature 1999;397: 4. Bolon B, et al. Arthritis Rheum 2002;46: 5. Blair JM, et al. Nat Clin Pract Oncol 2006;3:41-49. Pas de changement de la DMO Bucay N et al. Genes Dev : 1260–1268 Bolon B, et al. Arthritis Rheum. 2002; 46: Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. 8

9 L'OPG inhibe l’action du RANK Ligand
M-CSF M-CSF + RANK Ligand RANK Ligand (ng/ml) 500 100 OPG (ng/ml) Nombre d’ostéoclastes différenciés (A405) 0.0 2.000 3.000 4.000 1.000 0.000 0.10 1.00 10.00 100.00 To assay osteoclast differentiation, osteoclasts were developed from mouse bone-marrow cells cultured with colony stimulating factor-1 (CSF-1) and various concentrations of RANK Ligand and OPG. Osteoclast differentiation was measured by TRAP solution assay. A405 denotes absorbance at 405 nm.1 RANK Ligand increased TRAP activity in a dose-dependent manner.1 OPG inhibited this effect in a dose-dependent manner.1 Osteoclast development from mouse bone marrow cells cultured with CSF-1 (30 ng/ml) and various concentrations of OPGL [158–316] and OPG as measured by TRAP solution assay. A405 denotes absorbance at 405 nm. Data are presented as mean 6 SD, n 5 3. The right panels show representative photomicrographs (203 objective) of TRAP-stained cultures treated with CSF-1 alone (30 ng/ml), or CSF-1 and RANK Ligand [158–316] (100 ng/ml). Culture de cellules de moelle osseuse marquées TRAP+ TRAP, tartrate-resistant acid phosphatase Lacey DL et al. Cell 1998; 93: 165–176 Li X, et al. J Bone Miner Res. 2006;21(suppl 1):S304. Reference 1 Lacey DL et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165–176 9

10 l’expression du RANK Ligand
La régulation de la résorption osseuse dépend de l’équilibre RANKL/OPG, protéines sécrétées par l’ostéoblaste Régulations de l’expression du RANK Ligand Régulations de l’expression de l’OPG + - IL-1 TNF- PTH PTHrP Glucocorticoïdes Vitamine D PGE2 IL-11 IL-6 RANK Ligand OPG PTH PTHrP Glucocorticoïdes PGE2 Ostéoblastes + TNF- IL-1 Vitamine D IL-11 Oestrogènes Alterations of the RANK Ligand/OPG ratio are critical in the pathogenesis of bone diseases that result in increased bone resorption: Unopposed RANK Ligand (i.e. an elevated RANK Ligand/OPG ratio) within the skeleton promotes bone loss Restoring a balanced RANK Ligand/OPG ratio or inhibiting RANK Ligand decreases osteoclast activation and bone resorption.1–3 In many diseases involving increased bone resorption, RANK Ligand expression is upregulated by osteoclastogenic factors (growth factors, hormones, cytokines), while OPG expression is simultaneously downregulated.3 1 Hofbauer LC et al. JAMA 2004; 292: 490–495; 2 Lacey DL et al. Cell 1998; 93: 165–176; 3 Boyle WJ et al. Nature 2003; 423: 337–342 References 1 Hofbauer LC et al. Clinical implications of the osteoprotegerin/RANK Ligand/RANK system for bone and vascular diseases. JAMA 2004; 292: 490–495 2 Lacey DL et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165–176 3 Boyle WJ et al. Osteoclast differentiation and activation. Nature 2003; 423: 337–342 10

11 Région d’analyse (coupe frontale) Images micro-CT représentatives de:
Dans le modèle de rat ovariectomisée, l’OPG prévient la perte osseuse corticale et trabéculaire Région d’analyse (coupe frontale) Images 3D Images micro-CT représentatives de: SHAM + VEH OVX + VEH OVX + OPG La 5ième vertèbre lombaire Le fémur distal Key Point Inhibition of RANK Ligand with OPG reduced bone resorption and produced increases in both trabecular and cortical bone density and strength in ovariectomised rats1. Supplementary Information Ovariectomised rats represent the preferred preclinical model of postmenopausal decline in oestrogen levels in human females1. Oestrogen loss increased available RANK Ligand and hence bone resorption, but could be countered by administration of OPG-Fc which inhibits the RANK pathway1. Reduced bone loss was observed throughout the skeleton1. The arrest of bone loss at the femoral neck was particularly important, because this mixed cortical and trabecular bone is a common site for debilitating fragility fractures in postmenopausal subjects1,2. 1. Ominsky M.S. JBMR 2008; 23(5): 2. Lindsay R and Cosman F, Treatment of the Postmenopausal Woman: Basic and Clinical Aspects. 2nd ed. 1999: Le col fémoral Ominsky M.S. JBMR 2008; 23(5): 11

12 La biothérapie: une thérapeutique ciblée
Approche traditionnelle dans la découverte des médicaments Créer une liste de produits chimiques Evaluer leurs activités Identifier les composants à haute activité Peu spécifique de la cible Agent principal Approche de biothérapie Mécanisme d’action ciblé Key Point Traditional medications and biopharmaceuticals differ in how they are identified and developed.1 Supplemental Information Traditional approaches to drug discovery are grounded in medicinal chemistry and use high-throughput screening of randomly generated compounds to identify potential drug candidates.1-4 In general, these small molecule drugs have less specificity for desired targets than biopharmaceuticals.1-3 With biotechnology, specific targets whose function or dysfunction are involved in disease, are identified, and protein- or peptide-based therapies are designed to specifically interact with these targets.1 Avidor Y et al. Urology 2002; 59: 643–651. Emerging biotechnology brings change to pharmaceutical industry. Accessed 10 August 2007. Bleicher KH et al. Nat Rev Drug Discov 2003; 2: 369–378. Macarron R. Drug Discov Today 2006; 11: 277–279. Définir le schéma physiologique impliqué dans la pathologie Identifier la protéine à cibler Fabriquer la thérapie protidique ciblant la voie impliquée Avidor Y, et al. South Med J 2003; 96: 1174–1186; Avidor Y, et al. Urology 2002; 59: 643–651; Bleicher KH, et al. Nature Rev Drug Discov 2003; 2: 369–378 12

13 Propriétés pharmacologiques du denosumab
DENse OS hUman Monoclonal AntiBody Anticorps monoclonal totalement humain; Isotype IgG2 Haute affinité pour le RANK ligand humain Haute spécificité pour le RANK ligand Pas d’interaction détectable avec le TNF-α, TNF-β, TRAIL, ou CD40L Pas d’anticorps neutralisant détectés dans les essais cliniques à ce jour denosumab 100% Protéine murine 34% 5 - 10% Potentiel d’immunogénicité Haut Murin Humanisé Chimérique Faible Totalement Humain Protéine humaine Denosumab, an investigational fully human monoclonal antibody (IgG2), binds with high affinity and specificity to human RANK Ligand, an essential mediator of osteoclast activity.1-3 No neutralizing antibodies have been detected in clinical trials to date.1,3 Binding of denosumab to RANK Ligand was investigated in an in vitro study using flow cytometry and enzyme-linked immunosorbent assay (ELISA). Binding affinity was measured using BIAcore and a kinetic exclusion assay. Denosumab bound both soluble and membrane-bound forms of human RANK Ligand. This binding was inhibited by excess human RANK Ligand, but not by TNF-α, TNF-β, TRAIL, or CD40 ligand. The dissociation constants of denosumab were calculated to be 9.5 x M and 3 x M using the BIAcore and kinetic exclusion assay, respectively.2 No neutralizing antibodies have been detected in clinical trials to date. In a phase 1, double-blind study, 49 healthy postmenopausal women were randomized to receive a single dose of denosumab 0.01, 0.03, 0.1, 0.3, 1.0, or 3.0 mg/kg or placebo. No anti-denosumab antibodies were detected in subjects enrolled in this study.1 In a phase 2 study, 412 postmenopausal women with low bone mineral density (BMD) were randomized to receive denosumab SC either every 3 months (6, 14, or 30 mg) or every 6 months (14, 60, 100, or 210 mg), open-label alendronate (70 mg orally once weekly), or placebo. Denosumab-binding antibodies were observed in 2 subjects—one at 1 month and the other at 12 months. These antibodies were not neutralizing and were not detected in subsequent samples in either patient.3 Note: The graphic in the slide is a ribbon model depiction of denosumab. Bekker PJ, et al. J Bone Miner Res. 2004;19: Elliott R, et al. Osteoporos Int. 2007;18:S54. Abstract P149. McClung MR, et al. N Engl J Med. 2006;354: Ig = immunoglobulin; TNF = tumor necrosis factor; TRAIL = TNF-α–related apoptosis-inducing ligand. Bekker PJ, et al. J Bone Miner Res. 2004;19: Elliott R, et al. Osteoporos Int. 2007;18:S54. Abstract P149. McClung MR, et al. N Engl J Med. 2006;354: Data on file, Amgen. 13 13

14 Product Blue Template_Logo_SMS_v1b.PPT
Denosumab in OVX monkeys Trabecular turnover parameters were negatively correlated with bone strength in OVX monkeys Mineralising surface (L2 vertebrae) 30 * 20 Mineralising surface / bone surface (%) 10 ^ ^ * * Dempster DW, Lindsay R. Lancet 1993;341: Peak load (L3-L4 vertebral body) Sham OVX Dmab Dmab 8 Eroded surface (L2 vertebrae) Sham OVX Dmab Dmab 1000 2000 3000 4000 * ^ Peak load (N) 6 Eroded surface (%) Denosumab also increased strength endpoints at the femur neck (tested in shear) and diaphysis (tested in 3-pt bending). Both doses of denosumab were associated with significantly greater mean peak load for femur neck (19% to 34%), while peak load at the femur diaphysis was increased a non-significant 10% Stiffness was significantly increased at the diaphysis by ~15% in both denosumab groups, while 50 mg/kg denosumab increased stiffness at the femur neck by 26%. No significant changes were found in energy to failure (which represents area under the load-displacement curve). 4 ^ * ^ 2 * Sham OVX Dmab Dmab Data: Mean ± SE *p < 0.05 vs OVX-vehicle ^p < 0.05 vs Sham-vehicle Ominsky MS, et al. J Bone Miner Res 2007: (Suppl 1):Abstract 1082. 14

15 Biopsies des côtes (mois 6)
Remodelage intra-cortical et porosité Modèle de singe cynomolgus ovariectomisée SHAM Control OVX Control OVX + 25 mg/kg Dmab OVX + 50 mg/kg Dmab Biopsies des côtes (mois 6) 210 160 * * 110 /yr) 2 60 m * Haversian BFR / BS m / 10 3 ^ ^ m * * m 3 ^ ( ^ * 2 * ^ ^ 1 * * Mois 6 Mois 12 Diaphyse Tibia SHAM- Veh OVX- Veh Biopsies des côtes 5 Moyenne ± SE *P< 0.05 vs. OVX-Veh ^P< 0.05 vs. Sham-Veh 4 Denosumab also reduced intracortical bone formation rate to levels below OVX and sham controls, resulting in significantly lower cortical porosity. The 6-month rib biopsy images demonstrate the increase in cortical porosity and haversian bone formation with ovariectomy, which were reduced by denosumab It is interesting to note the rapid increase in cortical porosity in rib biopsies within 6 months of OVX. While cortical remodeling has been suggested to be related to the deliberate repair of microdamage, it is hard to imagine from these data that such “targeted” remodeling is a substantial component of the overall cortical remodeling response. These are ~10 year old animals that had years to repair any existing microdamage, and yet there is a marked increase in intracortical remodeling within 6 months of ovariectomy. It seems more likely that this intracortical remodeling response is a metabolically-driven process resulting from estrogen ablation. 3 * * Cortical Porosity (%) * * 2 ^ * ^ ^ ^ * * * 1 25mg/kg Dmab 50mg/kg Dmab Mois 6 Mois 12 Diaphyse Tibia Biopsies des côtes Ominsky MS, et al. J Bone Miner Res. 2006;21(suppl 1):S72 ;

16 Product Blue Template_Logo_SMS_v1b.PPT
Gain osseux cortical sous Denosumab lié à des modifications de l’aire corticale et de la vBMD Modèle de singe cynomolgus ovariectomisée Radial Cortical Area 3 6 9 12 15 -4 -3 -2 -1 1 * ^ Months of Treatment % Change from Baseline Tibial Cortical Area 3 6 9 12 15 -10 -8 -6 -4 -2 2 * ^ Months of Treatment SHAM Control OVX Control OVX + 25 mg/kg DMAB OVX + 50 mg/kg DMAB pQCT Tibial Diaphysis Radial Cortical vBMD 3 6 9 12 15 -5 -4 -3 -2 -1 1 * ^ Months of Treatment % Change from Baseline Tibial Cortical vBMD 3 6 9 12 15 -6 -5 -4 -3 -2 -1 1 2 * ^ Months of Treatment The decreases in cortical bone mass can be broken down into loss of cortical area and loss of cortical BMD (BMC = Area*BMD). Here we see that ovariectomy led to decreases in both cortical area and BMD at both sites. The loss of BMC in the sham group appeared to be primarily due to loss of cortical area and not BMD (which would reflect a change in intracortical turnover). Finally, denosumab led to slight (1-2%) increases from baseline in both cortical area and BMD by study’s end. Data: Mean ± SE *P< 0.05 vs. OVX-Veh ^P< 0.05 vs. Sham-Veh Ominsky et al. J Bone Miner Res 2007 (Suppl 1) abstract 1082 16

17 Femur neck peak load (N)
Product Blue Template_Logo_SMS_v1b.PPT Denosumab in OVX monkeys Denosumab increased femur neck strength in OVX monkeys at 16 months Sham OVX Dmab 25 mg/kg Dmab 50 mg/kg ^ * 1000 1500 2000 2500 2000 * r2 = 0.34 1500 Femur neck peak load (N) Femur neck peak load (N) 1000 500 Denosumab also increased strength endpoints at the femur neck (tested in shear) and diaphysis (tested in 3-pt bending). Both doses of denosumab were associated with significantly greater mean peak load for femur neck (19% to 34%), while peak load at the femur diaphysis was increased a non-significant 10% Stiffness was significantly increased at the diaphysis by ~15% in both denosumab groups, while 50 mg/kg denosumab increased stiffness at the femur neck by 26%. No significant changes were found in energy to failure (which represents area under the load-displacement curve). Sham OVX Dmab Dmab 10 20 30 Tibia haversian labelled perimeter (%) Data: Mean ± SE *p < 0.05 vs OVX-vehicle ^p < 0.05 vs Sham-vehicle Ominsky MS, et al. J Bone Miner Res 2007: (Suppl 1):Abstract 1082. 17

18 Alendronate to denosumab in OVX monkeys Transition from alendronate to denosumab further reduced biochemical markers of bone turnover Vehicle -Vehicle Vehicle -Dmab Resorption marker ALN -ALN Formation marker ALN -Dmab Dmab -Dmab 4 160 140 3 120 100 Serum CTx (ng/ml) Serum BSAP (U/L) * ^ * 2 80 * 60 * ^ * 1 40 * ^ ^ * * ^ 20 * ^ * ^ 3 6 9 12 3 6 9 12 Treatment (months) Treatment (months) *p < 0.05 vs vehicle ^p < 0.05 vs ALN Ominsky MS, et al. ECTS Congress 2009; Poster P483.

19 Mineralising surfaces Peak load : L5-L6 vertebral trabecular core
Alendronate to denosumab in OVX monkeys Denosumab further reduced bone remodelling with a superior increase in vertebral bone strength Mineralising surfaces (12 months) Vehicle ALN ALN-Dmab 5 10 15 20 VEH-Dmab Dmab Peak load : L5-L6 vertebral trabecular core MS/ BS (%) * 100 200 300 Peak load (N) Vehicle Vehicle to Dmab ALN ALN to Dmab Dmab * ^ * * * * * * * ^ ^ * * Osteoclast surfaces (12 months) 0.0 0.5 1.0 1.5 2.0 Oc.S/ BS (%) ^ ^ ^ ^ ^ ^ * * * * * * * * * Iliac Crest L2 Vertebrae Proximal Tibia Mean ± SEM; *p < 0.05 vs vehicle; ^p < 0.05 vs ALN Ominsky MS, et al. ECTS Congress 2009; Poster P483.

20 Inhibition du RANK Ligand dans l’ostéoporose post-ménopausique Nouvelle perspective
20

21 Un nouveau mécanisme d’action: L’inhibition sélective du RANK Ligand
OPG denosumab BP = bisphosphonates Précurseurs de l’ostéoclaste Blocage du RANK Ligand Les BPs se lient à l’os sur les sites de résorption BP BP BP BP Os X Désorganisation du cytosquelette Perte de la bordure en brosse Accélération de l’apoptose Alteration du trafic vésiculaire Os Le denosumab inhibe la formation des ostéoclastes, leur fonction et leur survie Les BPs entraînent la perte de la fonction de résorption des ostéoclastes, mais non leur disparition… 21

22 Quel impact en pratique clinique
Quel impact en pratique clinique? DMO - Marqueurs du remodelage osseux - Tolérance DECIDE (Determining Efficacy: Comparison of Initiating Denosumab versus Alendronate)1 Essai clinique de phase 3 chez des patientes initiant le tt anti-OP - Denosumab 60 mg sc / 6 mois - Alendronate 70 mg hebdomadaire STAND (Study of Transitioning from Alendronate to Denosumab)2 : Essai clinique de phase 3 comparant - La transition de l’Alendronate hebdomadaire vers Denosumab - vs la poursuite de l’Alendronate Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print]

23 Schéma des études multicentriques, randomisées en double-aveugle, double placebo
DECIDE1 Femmes ménopausées, “naïves” de Tt anti-OP T-score ≤ – 2.0 en site lombaire ou fémoral total Denosumab 60 mg injection SC / 6 M Placebo Oral hebdomadaire Placebo injection SC / 6 M Alendronate 70 mg Oral /sem Apports en Calcium et Vitamine D R A N D O M I S T F de E C G 12 mois STAND2 Femmes ménopausées Tt par ALN [ 70 mg/sem ] x ≥6 mois T-score ≤ –2.0 et ≥ – 4.0 en site lombaire ou fémoral total Objectif principal: variation DMO hanche totale à 12 M Study Design This was an international, multi-center, randomized, double-blind, active-controlled, double-dummy, parallel group study. All subjects received branded alendronate 70 mg QW during a 1-month run-in period before randomization (Figure). Subjects were randomly assigned to continue receiving branded alendronate therapy or to receive subcutaneous denosumab 60 mg every 6 months (Q6M) and were followed for 12 months. Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 23

24 Caractéristiques des patientes
DECIDE1 STAND2 Moyenne (DS) ou Médiane (Q1, Q3) Alendronate 70 mg / Sem (N = 595) Denosumab 60 mg / 6 M (N = 594) 70 mg / Sem (N = 251) 60 mg / 6 M (N = 253) Age, ans 64.6 (8.3) 64.1 (8.6) 68.2 (7.7) 66.9 (7.8) Années depuis ménopause 17.8 (9.8) 16.5 (10.2) 19.9 (9.9) 18.8 (9.2) T-score hanche totale –1.69 (0.81) –1.75 (0.79) –1.81 (0.74) –1.79 (0.82) T-score lombaire –2.57 (0.75) –2.62 (0.79) –2.64 (0.75) CTX-I sérique (ng/mL) 0.61 (0.47, 0.82) 0.66 (0.49, 0.87) 0.21 (0.13, 0.32) 0.19 (0.13, 0.29) Ancienneté sous BPs N/A 34.5 (15.5, 60.0) 36.0 (16.0, 60.0) Ont terminé l’essai, n (%) 553 (92.9) 561 (94.4) 238 (94.8) 243 (96.0) Baseline demographics and clinical characteristics were similar between treatment groups. The median time on prior bisphosphonate therapy was 36 months. Baseline serum levels of biochemical markers of bone turnover were reflective of prior bisphosphonate therapy. 95% of subjects completed the study, and rates of completion were balanced between groups. Extra information: Prior osteoporosis medication use: Oral BP – 67 (11%) ALN subjects; 75 (13%) Dmab subjects; 142 (12%) all subjects 1 ALN subject did report prior use of an IV BP; 2 ALN subjects reported prior use of PTH or PTH derivatives Other OP medication use reported included: calcitriol, SERM, HRT, estrogens, or calcitonin (similar numbers in each treatment group) BPs : Bisphosphonates Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 24

25 % variation DMO LS Mean (95% CI)
Pourcentages de variation de la DMO à 12 mois aux sites étudiés: objectif principal Alendronate 70 mg hebdomadaire Denosumab 60 mg / 6 mois DECIDE1 STAND2 7 7 1.1%* 0.6%* 1.0%* Rachis Lombaire 1/3 Radius Col Fémoral Trochanter *P ≤ 0.001 0.9%* Hanche Totale *P ≤ 0.001 6 6 0.7% ** 1.0% ** 1.1% ** 1.2%* Rachis Lombaire 1/3 Radius Col Fémoral Trochanter **P ≤ 0.7% ** 1.0% ** 1.1% ** 1.2%* Rachis Lombaire 1/3 Radius Col Fémoral Trochanter **P ≤ 5 5 1.0%* 4 4 % variation DMO LS Mean (95% CI) 3 3 Results from superiority analyses When adjusted for prior bisphosphonate exposure: Significantly greater increase in BMD at the total hip among subjects with history of bisphosphonate treatment (n =140; 73 denosumab, 67 alendronate) who received denosumab than alendronate (3.5% denosumab vs 1.3% alendronate; p < ) 2 2 1 1 Hanche Totale Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 25

26 Variation des CTX sériques à 12 mois: objectif secondaire
DECIDE1 STAND2 0.9 Alendronate Denosumab 0.8 0.7 0.6 0.5 Serum CTX-I (ng/mL) 0.4 0.3 0.2 Denosumab significantly reduced serum CTX-I levels (P < ) at all measured time points compared with alendronate. Reductions were rapid and sustained through month 3, with an attenuation of the reduction at the end of the dosing interval at month 6. A similar pattern was observed following the second dose of denosumab. In the continued alendronate group, median serum CTX-I levels remained near baseline throughout the study, consistent with the expected pattern for ongoing alendronate therapy. PINP reductions with denosumab were significantly greater than with alendronate beginning at month 1 with maximal steady-state reduction observed by month 3 and with attenuation of reduction noted at the end of the dosing interval. Ligne hachurée : limite inférieure des valeurs mesurées en préménopause (Université de Sheffield , Bone Marker Laboratory) – Valeurs Médianes Barres = variation interquartile *P ≤ 0.01 0.1 * * * * * * * * * BL 1 3 6 9 12 BL 1 3 6 9 12 Mois Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 26

27 Inhibition du RANK Ligand dans l’ostéoporose post-ménopausique Action anti-fracturaire
McClung MR, Lewiecki EM, Cohen SB, et al. Denosumab in postmenopausal women with low bone mineral density. N Engl J Med. 2006;354: 27

28 Variation uNTx/créatinine
Etude chez les femmes ménopausées : diminution rapide, dose-dépendante et réversible du NTx urinaire Phase 1 Placebo (n = 12) Denosumab 0.3 mg/kg (n = 6) Denosumab 1.0 mg/kg (n = 6) Denosumab 3.0 mg/kg (n = 7) 50 NTx urinaire Variation uNTx/créatinine ( % médian + ET) –50 Denosumab produced dose-dependent, rapid (within 12 hours; the first time point measured) and sustained (up to 9 months) reductions from baseline in bone turnover as assessed by urinary NTx (uNTx)/creatinine.1 Serum CTx and uNTx are products of the proteolytic process of bone resorption brought about by osteoclasts, and thus serve as biochemical markers of bone resorption.2-4 The maximum mean decrease in uNTx/creatinine was approximately 80%, and duration of effect was at least 6 months at higher doses (1 and 3 mg/kg denosumab) in healthy postmenopausal women. The effect, however, is reversible, as indicated by a return of NTx levels towards baseline when denosumab is cleared from the circulation.1 Bekker PJ, et al. J Bone Miner Res. 2004;19: Cormier C. Curr Opin Rheumatol. 1995;7: Khosla S, et al. J Clin Endocrinol Metab. 1997;82: Chailurkit L, et al. Clinical Chemistry. 2001;47: –100 1 2 3 4 5 6 7 8 9 Mois d’étude NTx = N-télopeptide; uNTx = NTx urinaire Bekker PJ, et al. J Bone Miner Res. 2004;19: 28

29 Variation de DMO (% LS Mean ± SE)
Effet sur la Densité Minérale Osseuse (DMO) après 4 ans d’administration continue de denosumab (60 mg / 6M) Phase 2 Placebo 60 mg / 6M Rachis lombaire Mois d’étude -6 -4 -2 2 4 12 24 36 48 6 8 Hanche totale * -5 -3 -1 1 3 -6 -4 -2 2 4 12 24 36 48 1/3 distal du radius * 14 Traitement continu 60 mg / 6M Traitement continu 60 mg / 6M Traitement continu 60 mg / 6M 12 * 10.3% 6.1% 10 1.7% CSR TBA Numbers in abstract are different than on the slide. 8 6 + 9.6 % vs placebo + 6.4 % vs placebo Variation de DMO (% LS Mean ± SE) +12.7 % vs placebo 4 Lewiecki EM, et al. J Bone Miner Res. 2007; 5C 2 After 48 months, continuous denosumab treatment was also associated with significant increases from baseline in total hip and distal 1/3 radius BMD compared with placebo.1 The mean percent changes in total hip and distal 1/3 radius BMD from baseline were statistically significant (P < 0.001) at months 24, 36, and 48 for continuous denosumab 60 mg SC q6mo compared with placebo. At 48 months, the mean percent change in total hip BMD from baseline was 6.1% for continuous denosumab 60 mg SC q6mo compared with –3.5% for placebo (between-group difference: 9.6%, P < 0.001). At 48 months, the mean percent change in distal 1/3 radius BMD from baseline was 1.7% for continuous denosumab 60 mg SC q6mo compared with –4.7% for placebo (between-group difference: 6.4%, P < 0.001). -2 - 3.5% - 4.7% - 2.4% CSR TBA Numbers in abstract are different than on the slide. -4 12 24 36 48 *P < groupe denosumab vs placebo au mois 48. Miller PD, et al. Bone. 2008; 43(2):222-9 29 Miller P, et al. ASBMR Abstract 1205 and oral presentation.

30 Variation des marqueurs
Variations de marqueurs du remodelage osseux après arrêt et reprise du traitement Phase 2 210 mg / 6M Alendronate Placebo 30 mg / 3M 60 mg / 6M (reprise) CTx sérique BSAP -100 -75 -50 -25 25 50 75 100 125 -100 -75 -50 -25 25 50 75 100 125 *P < au mois 36 et P = 0.05 au mois 48 vs placebo Reprise de traitement 60 mg / 6M Arrêt de traitement *P = au mois 36 vs placebo †P = 0.01 vs placebo. Reprise de traitement 60 mg / 6M Arrêt de traitement * * CSR; TBA * * * Variation des marqueurs (% Médian [Q1, Q3]) Bone turnover markers significantly increased after discontinuation of denosumab 210 mg q6mo compared with placebo.1 Patients on denosumab 210 mg q6mo were discontinued from therapy at 24 months. Twelve months after the discontinuation of denosumab, bone turnover marker levels were higher than those observed at 24 months after discontinuation. At 36 months the levels of CTx (P < 0.001) and BSAP (P = 0.008) were significantly higher in patients who had received denosumab 210 mg q6mo than patients in the placebo arm. At 48 months, levels of CTx, although reduced, were significantly (P = 0.05) higher than placebo, while there was not significant difference in BSAP levels. Miller P, et al. ASMBR Abstract F CSR; TBA 12 18 21 24 36 48 12 18 21 24 36 48 Mois Mois 30 mg / 3M dernière dose à 21 mois 210 mg / 6M dernière dose à 18 mois Miller PD, et al. Bone. 2008; 43(2):222-9 30 Miller P, et al. ASBMR Abstract 1205 and oral presentation.

31 FREEDOM: phase 3, étude pivot de prévention fracturaire
Fracture REduction Evaluation of Denosumab in Osteoporosis every 6 Months Critères d’inclusion Femmes post-ménopausiques avec ostéoporose (-4.0< T-score <-2.5) 60 à 90 ans Critères d’exclusion Toute fracture vertébrale sévère ou >2 fractures vertébrales modérées R A N D O M I S T Denosumab 60mg SC / 6M + calcium (≥1000 mg) et vitamine D (≥400 IU) N = 7808 36 mois Placebo Critères d’analyse principaux: Incidence de nouvelles fractures vertébrales Profil de tolérance du denosumab Critères d’analyse secondaires: Temps jusqu’à la 1ère fracture non-vertébrale Temps jusqu’à la 1ère fracture de hanche Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65

32 Caractéristiques des patientes Phase 3: FREEDOM – Etude pivot anti-fracturaire
Placebo Denosumab Randomisées* 3906 3902 Age, années 72.3 (5.2) T-score rachis lombaire -2.8 (0.7) T-score hanche totale -1.9 (0.8) T-score col fémoral -2.2 (0.7) Fractures vertébrales prévalentes 23.4% 23.8% Prior osteoporosis medication use: Oral BP – 67 (11%) ALN subjects; 75 (13%) Dmab subjects; 142 (12%) all subjects 1 ALN subject did report prior use of an IV BP; 2 ALN subjects reported prior use of PTH or PTH derivatives Other OP medication use reported included: calcitriol, SERM, HRT, estrogens, or calcitonin (similar numbers in each treatment group) *Patientes inclues dans l’analyse d’efficacité; les valeurs sont des moyennes (déviation standard) ou pourcentages Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 32

33 Réduction du risque de nouvelles fractures vertébrales après 3 ans de traitement par le denosumab Phase 3: FREEDOM – Etude pivot anti-fracturaire Placebo 68% (59%, 74%) P < 0,0001 Denosumab 60 mg / 6M 9 8 8.0% 7 7,2% 6 6.5% 5 Incidence fracturaire (%) 4 3 2 2,3% 1 1.2% 0.7% Fractures vertébrales Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65

34 Réduction du risque de fractures après 3 ans de traitement par le denosumab Phase 3: FREEDOM – Etude pivot anti-fracturaire 20% (5%, 33%) P = 0,0106 Placebo 68% (59%, 74%) P < 0,0001 Denosumab 60 mg / 6M 9 8 8,0% 7 7,2% 6 6,5% 5 Incidence fracturaire (%) 4 40% (3%, 63%) P = 0,0362 3 2 2,3% 1 1,2% 0,7% Fractures vertébrales Fractures non-vertébrales Hanche Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65

35 FREEDOM Effet du denosumab sur le risque de fractures cliniques sur 3 ans1
RRR: 30% [19 ; 41]*** Placebo 10 RRR: 35% Denosumab 10,2% 1,2% 9 Risque absolu 2,9% [22 ; 45]*** [1,6 ; 4,2] 8 8,0% RRR: 20% Risque absolu 2,7% 7 7,2% 20 [3 ; 34]* [1,6 ; 3,9] 6 6,4% Risque absolu 1,5% [1,2 ; 2,2] 5 5,3% 5,2% Proportion de femmes avec une fracture 4 RRR: 69% 3 [53 ; 80]*** 2,6% 2 Risque absolu 1,8% 1,2% [1,2 ; 2,4] 1 0,8% Toute fracture clinique(1) Fracture vertébrale clinique Fracture non vertébrale Majeure(3) Fracture ostéoporotique majeure(4) *p ≤ 0,05; **p = 0,0106 (critère d'évaluation secondaire inclus dans l'ajustement de la multiplicité), ***p ≤ 0,0001 (1) Inclut les fractures vertébrales cliniques et les fractures non vertébrales. (2) Exclut les fractures des vertèbres, du crâne, du visage, de la mandibule, du métacarpe et des phalanges des doigts et des orteils. (3) Inclut le pelvis, le fémur distal, le tibia proximal, les côtes, l'humérus proximal, l'avant-bras et la hanche. (4) Inclut les fractures vertébrales cliniques, de la hanche, de l'avant-bras et de l'humérus, selon la définition de l'OMS. 1. Résumé des Caractéristiques Produit

36 Denosumab et risque de nouvelles fractures vertébrales année par année Phase 3: FREEDOM – Etude pivot anti-fracturaire 78% P < 0.001 65% P < 0.001 Placebo 3,5% Denosumab 3,0% 3,1% 3,1% 61% P < 0.001 2,5% 2,0% 2,2% Incidence (%) 1,5% 1,0% 1,1% 0,9% At month 36 of the study, treatment with 60 mg of denosumab every six months significantly reduced the risk of new vertebral fractures by 68% versus placebo (2.3% denosumab versus 7.2% placebo; P < 0.001).1 The FREEDOM trial was designed with > 99% power to detect a 45% reduction in the incidence of new vertebral fractures.1 Statistical analysis was based on the assumption of 4% per year vertebral fracture rate in the placebo group.1 The last known fracture status of patients who were lost to follow-up or withdrew before having a fracture event was carried forward in the analysis.1 The reduction in risk was similar during each year of the trial, 61%, 78%, and 65% for years 1–3, respectively.1 Similar reductions were observed for1: New clinical vertebral fractures, with a 69% reduction over 3 years (P < 0.001) Multiple new vertebral fractures, with a 61% reduction over 3 years (P < 0.001) A central imaging center was utilized to assess annual lateral spine radiographs for new vertebral fractures based on a semiquantitative grading scale.1 The criteria for a new vertebral fracture was an increase of at least one grade in a vertebral body that was normal at baseline.1 All fractures diagnosed based on clinical symptoms were confirmed by radiographs.1 Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): 0,5% 0,7% 0,0% Année 1 Année 2 Année 3 Analyse en Intention de Traitement Le pourcentage de nouvelles fractures vertébrales a été calculé sur la base du nombre de patientes avec une radiographie rachis à l’inclusion et au moins un examen après. Adapté de Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 36 36

37 Risque de fractures de hanche réduit de 40% (95% IC: 0.37, 0.97)
Denosumab et temps jusqu’à la première fracture de hanche sur 36 mois Phase 3: FREEDOM – Etude pivot anti-fracturaire Cumulative Incidence (%) Month 6 12 24 0.0 18 30 36 0.4 0.8 1.2 0.7%* 1.2% Placebo Denosumab 60 mg / 6M 40% Treatment with denosumab significantly reduced hip fracture risk by 40% (0.7% denosumab versus 1.2% placebo, P = 0.04).1 The FREEDOM trial was designed with 91% power to detect a 40% reduction in the risk of hip fractures.1 Statistical analysis was based on the assumption of 1% hip fracture rate over 3 years in the placebo group.1 This Kaplan-Meier curve reflects point estimates based on patients who have not had a hip fracture or who did not leave the study before the time point measured.1 Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): . Nombre de patientes à risque Placebo, n 3,906 3,799 3,672 3,538 3,430 3,311 3,221 Denosumab, n 3,902 3,796 3,676 3,566 3,477 3,397 Risque de fractures de hanche réduit de 40% (95% IC: 0.37, 0.97) *P = 0.04 Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 37 37

38 Analyses supplémentaires des groupes à risque fracturaire plus élevé Phase 3: FREEDOM – Etude pivot anti-fracturaire Analyses pré-spécifiées chez les patientes ayant ≥ 2 des 3 critères suivant (45% de la population totale): Age > 70 ans T-score ≤ -3,0 au rachis lombaire, hanche totale ou col fémoral Fracture vertébrale prévalente à l’inclusion Analyses en post-hoc Chez les patientes avec ≥ 2 fractures vertébrales prévalentes et/ou ≥ 1 fracture vertébrale prévalente modérée ou sévère Chez les patientes avec un T-score au col fémoral à l’inclusion ≤ -2,5 Chez les patientes ≥ 75 ans Boonen S, et al. ASBMR 2009

39 Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente
Réduction du risque de nouvelles fractures vertébrales chez les patientes à risque Phase 3: FREEDOM – Etude pivot anti-fracturaire Placebo Groupe pré-spécifié: Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente Denosumab ≥ 2 fractures vertébrales prévalentes et/ou ≥ 1 fracture vertébrale prévalente modérée ou sévère 18 16 55% (31%, 71%) P = 0,0002 14 65% (53%, 74%) P < 0,0001 12 68% (59%, 74%) P < 0,0001 10 72% (63%, 79%) P < 0,0001 Incidence fracturaire (%) 71% (55%, 81%) P < 0,0001 8 6 4 2 Population totale Risque haut Risque bas Risque haut Risque bas Interaction, p: 0,1774 0,6041 Boonen S, et al. ASBMR 2009

40 Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente
Réduction du risque des fractures de hanche chez les patientes à risque Phase 3: FREEDOM – Etude pivot anti-fracturaire Placebo Groupe pré-spécifié: Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente Denosumab T-score col fémoral ≤ -2,5 à l’inclusion Age ≥ 75 ans 3 47% (8%, 70%) P = 0,0227 2.5 48% (9%, 71%) P = 0,0208 62% (22%, 82%) P = 0,0065 2 40% (3%, 63%) P = 0,0362 Incidence fracturaire (%) 1.5 6% (-85%, 53%) P = 0,8490 1 43% (-89%, 76%) P = 0,4478 17% (-171%, 75%) P = 0,7538 0.5 Population totale Risque haut Risque bas Risque haut Risque bas Risque haut Risque bas Interaction, p: 0,0714 0,5074 0,6601

41 Efficacité du denosumab en fonction des facteurs de risque Phase 3: FREEDOM – Etude pivot anti-fracturaire Réduction des fractures selon le niveau de remodelage Fractures vertébrales Fractures non vertébrales 14 p = 0,06 11 p < 0,0001 12 p = 0,89 p = 0,37 p = 0,06 10 9,7 % p = 0,0009 p = 0,0002 9,9 %  26 % 10  2 %  15 % 8 7,5 % 7,2 % 7,3 % 7,5 % p = 0,0002 7,2 %  30 % 8 6,7 % 6,9 % 6,7 % Incidence à 36 mois (%) Incidence à 36 mois (%) 6 5,2 % 6 4,9 %  51 %  55 %  86 %  64 % 4 4 3,2 % 3,1 % 2 1,4 % 1,8 % 2 891 884 898 917 895 937 914 887 937 944 950 951 938 972 956 936 < 0,381 0,381-0,536 0,537-0,717 > 0,718 < 0,381 0,381-0,536 0,537-0,717 > 0,718 Quartiles du CTX à l’inclusion (ng/ml) Quartiles du CTX à l’inclusion (ng/ml) Placebo Denosumab ASBMR D’après Cummings S. et al., San Francisco, États-Unis, abstr. FR0385 et Eastell R. et al., Sheeld, Royaume-Uni, abstr. FR03857, actualisés

42 Tolérance (1) Phase 3: FREEDOM – Etude pivot anti-fracturaire
Placebo (n = 3 876) Denosumab 60 mg / 6M (n = 3 886) Valeur P Tous les EI 3 607 (93,1) 3 605 (92,8) 0,91 EI graves 972 (25,1) 1 004 (25,8) 0,61 Décès 90 (2,3) 70 (1,8) 0,08 EI à l’origine de la sortie de l’étude 81 (2,1) 93 (2,4) 0,39 EI entraînant l’arrêt du médicament 202 (5,2) 192 (4,9) 0,55 There were no significant differences between patients who received denosumab and those who received placebo in the total incidence of adverse events, serious adverse events, deaths, or discontinuation of the study or the study treatment due to adverse events.1 Opportunistic infections were reported in four patients in the denosumab arm and three patients in the placebo arm.1 Deaths occurred in 1.8% of the denosumab patients (n = 70) and 2.3% of the placebo patients (n = 90), P = Adverse events were coded by physicians at the study sites using the Medical Dictionary for Regulatory Activities (MedDRA) system.1 The safety analysis included all patients who received at least one dose of the study drug.1 Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): EI = Effets indésirables Adapté de Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 42 42

43 Tolérance (2) Phase 3: FREEDOM – Etude pivot anti-fracturaire
Placebo (n = 3 876) Denosumab 60 mg / 6M (n = 3 886) EI Infection 2 108 (54,4) 2 055 (52,9) Néoplasies 166 (4,3) 187 (4,8) Réaction au site d’injection 26 (0,7) 33 (0,8) Hypocalcémie clinique 3 (0,1) 0 (0) Consolidation fracturaire retardée 4 (0,1) 2 (0,05) Fracture fémorale atypique Fracture humérale (non union) 1 (0,03) Ostéonécrose de la mâchoire (ONJ) Effets indésirables avec une incidence  2% et un P  0,05 Eczema 65 (1,7) 118 (3,0) Chute* 219 (5,7) 175 (4,5) Flatulence 53 (1,4) 84 (2,2) Opportunistic infections were reported in four patients in the denosumab arm and three patients in the placebo arm.1 Although clinical symptoms of hypocalcemia were observed in only three patients in the placebo arm, serum calcium levels decreased below 8.0 mg/dL in four patients in the denosumab arm and five patients in the placebo arm.1 No cases of osteonecrosis of the jaw (defined as an area of exposed bone in the maxillofacial region that did not heal within 8 weeks after diagnosis) were seen in either study group.1 No cases of neutralizing antibodies to denosumab were reported.1 In order to adjust to multiple comparisons of many adverse events, the following prespecified criteria were used1: Adverse events reported for at least 2% of patients and for which P ≤ 0.05 The data shown above does not include all1: 58 MedDRA preferred terms of adverse events occurring at an incidence of at least 2% in either treatment arm Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): *Excluant les chutes survenant le jour de la fracture Adapté de Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 43 43

44 Tolérance (3) Phase 3: FREEDOM – Etude pivot anti-fracturaire
Placebo (n = 3 876) Denosumab 60 mg / 6M (n = 3 886) Valeur P EI graves Néoplasie 125 (3,2) 144 (3,7) 0,28 Infection 133 (3,4) 159 (4,1) 0,14 Evénement Cardiovasculaire 178 (4,6) 186 (4,8) 0,74 AVC 54 (1,4) 56 (1,4) 0,89 Coronaropathie 39 (1,0) 47 (1,2) 0,41 Vasculaire périphérique 30 (0,8) 31 (0,8) 0,93 Fibrillation auriculaire 29 (0,7) 0,98 EI graves avec une incidence  0.1% et un P  0.01 Cellulites (incluant érysipèles) 1 (< 0,1) 12 (0,3) 0,002 Commotion cérébrale 11 (0,3) 0,004 No statistical difference was evident when the overall incidence of cellulitis adverse events were considered (denosumab 1.2%, n = 47; placebo 0.9%, n = 36).1 In order to adjust to multiple comparisons of many adverse events, the following prespecified criteria were used1: Serious adverse events reported for at least 0.1% of patients and for which P ≤ 0.01 The data shown above does not include all1: 152 MedDRA preferred terms of serious adverse events occurring at an incidence of at least 0.1% in either treatment arm Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): Adapté de Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 44 44

45 Conclusions Le denosumab agit en inhibant spécifiquement le RANKL, régulateur de la différenciation, fonction et survie des ostéoclastes Une injection sous-cutanée tous les 6 mois pendant 3 ans de denosumab a réduit le risque de nouvelles fractures vertébrales de 68%, celui des fractures de hanche de 40% et celui des fractures périphériques de 20% Le denosumab a été bien toléré Le denosumab représente une nouvelle voie prometteuse dans le traitement de l’ostéoporose post-ménopausique

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47 Variation vs baseline (%)
Prévention de perte osseuse au radius: os trabéculaire et cortical Modèle de singe cynomolgus ovariectomisée Sham-Veh OVX-Veh OVX-25 mg/kg denosumab OVX-50 mg/kg denosumab DMO (1/3 distal du radius) vBMC total (métaphyse du radius) vBMC cortical (diaphyse du radius) 5 5 5 *^ * * *^ *^ *^ Variation vs baseline (%) –5 –5 –5 * –10 –10 –10 Denosumab prevented loss of both trabecular and cortical bone at the radius in aged OVX monkeys.1 As shown in the left graph, at month 16, areal BMD at the distal third of the radius was significantly greater in aged OVX cynomolgus monkeys treated with denosumab (25 and 50 mg/kg) compared with OVX-V monkeys (P < 0.05).1 As shown in the middle graph, at month 16, total volumetric BMC of the radial metaphysis was significantly greater in aged OVX cynomolgus monkeys treated with denosumab (25 and 50 mg/kg) compared with OVX-V and sham-V monkeys (P < 0.05).1 As shown in the right graph, at month 16, cortical volumetric BMC of the radial diaphysis was significantly greater in aged OVX cynomolgus monkeys treated with denosumab (25 and 50 mg/kg) compared with OVX-V and sham-V monkeys (P < 0.05).1 This hypothesis has not been proven in humans. Potential Question: Why did animals in the sham groups lose bone during the study? Answer: The negative changes in densitometry were not consistent across all endpoints. The majority of apparent bone loss at certain endpoints appeared to occur during the first 4 months after sham surgery. This suggests there may have been changes caused by the sham surgery, increased animal handling, or animal stress during acclimation. The animals used in this study were considered to be “semi-feral” which means they had been caught within the past year. Although the animals were acclimatized for 2 months, their frequent change in diet, combined with multiple visits to the animal facility may have left them in a higher turnover state than normal. In fact, biomarkers of bone turnover decreased over the course of the 16-month study after month 3. Changes in densitometric measures in sham-operated monkeys are not unusual; gains or losses in spine BMD have been reported in similar studies.2,3 Ominsky MS, et al. J Bone Miner Res. 2006;21(suppl 1):S72. Abstract 1272 and presentation. Brommage R. J Musculoskel Neuro Inter. 2001;1(4): Jerome CP, Peterson PE. Bone. 2001;29(1):1-6. –15 –15 –15 Après 16 mois de traitement DMO: reflète la densité minérale osseuse surfacique mesurée par un scanner DEXA vBMC: reflète le contenu minéral osseux par unité de volume mesurée par pQCT n = 14–20/groupe. *P < 0.05 vs OVX-V; ^P < 0.05 vs sham-V. Ominsky MS, et al. J Bone Miner Res. 2006;21(suppl 1):S72. Abstract 1272 et oral.

48 Denosumab Is Being Investigated in an Extensive Clinical Development Program
Postmenopausal Osteoporosis (Key Registration Studies) Additional Studies Oncology Treatment of PMO (n = 7808) Dmab vs Alendronate (n = 1189) Breast SRE (n = 1400) Prevention of PMO (n = 332) Pilot Dmab vs Alendronate (n = 247) Prostate SRE (n = 1700) Phase 2: PMO (n = 412) Bisphosphonate Transition (n = 504) Metastasis Prevention (n = 1266) Phase 2: Rheumatoid Arthritis (n = 210) Solid Tumor SRE (n = 1576) >8,500 subjects Multiple Myeloma (n = 100) >2,000 subjects Giant Cell Tumor (n = 25) POSSIBLE US (n=6000) HALT Breast (n = 252) POSSIBLE EU (n=3000) HALT Prostate (n = 1468) DAPS US (n=250) >7,500 subjects 48

49 HALT Prostate Cancer Study Design
HALT Study HALT Prostate Cancer Study Design International, multi-center, randomized, double-blind, placebo-controlled study n = 734 S C R E N I G R A N D O M I Z T Denosumab SC 60 mg Q6M Study End N = 1468 Primary End-point LS- BMD All subjects received daily calcium (≥ 1 g) and vitamin D (≥ 400 IU) supplementation n = 734 Placebo Key Inclusion Criteria: Men with non-metastatic, prostate cancer receiving continuous ADT or post orchiectomy Stratified by Age (<70 yr vs ≥ 70 yr) Men <70 years old were required to have a history of osteoporotic fracture or baseline BMD T‑score < ‑1.0 at the lumbar spine, total hip, or femoral neck Prior ADT duration (≤ 6 mo vs >6 mo) 24 months 36 months Key Exclusion criteria: Concurrent systemic anti-neoplastic therapy or radiotherapy PSA 5 ng/mL after being on ADT 1 month BMD T-score at lumbar spine, total hip, or femoral neck <-4.0 Smith, MR et al. N Engl J Med Aug 20;361(8): 49

50 Baseline Characteristics
HALT Study Characteristic Placebo (N = 734) Denosumab (N = 734) Mean age, years (SD) 75.5 (7.1) 75.3 (7.0) <70, n (%) 125 (17.0) 124 (16.9) ≥70, n (%) 609 (83.0) 610 (83.1) White, n (%) 615 (83.8) Prevalent vertebral fracture, n (%) 174 (23.7) 155 (21.1) Calculated median ADT duration at study entry, months 20.4 20.8 ADT  6 months, n (%) 23.8 ADT > 6 months, n (%) 76.2 Median PSA, ng/mL 0.15 0.13 Median total testosterone, (nmol/L) 0.28 Median sCTX (ng/mL) 0.61 0.62 Smith, MR et al. N Engl J Med Aug 20;361(8): 50

51 Baseline T-Scores at Key Sites
HALT Study Baseline T-Scores at Key Sites Mean T-score (SD) Placebo (N = 734) Denosumab Lumbar Spine -0.41 ± 1.80 -0.31 ± 1.78 Femoral Neck -1.42 ± 0.91 -1.41 ± 0.86 Total Hip -0.88 ± 1.03 -0.87 ± 1.00 <15% of subjects were osteoporotic at any site Smith, MR et al. N Engl J Med Aug 20;361(8): 51

52 Effect of Denosumab on Lumbar Spine BMD Over 36 Months
HALT Study Effect of Denosumab on Lumbar Spine BMD Over 36 Months 10 Placebo (N = 734) Denosumab (N = 734) 8 * 6 4 Percentage Change From Baseline (± 95% CI) 6.7% difference at 24 months 2 -2 -4 BL 1 3 6 12 24 36 Study Month * P< vs Placebo Smith, MR et al. N Engl J Med Aug 20;361(8): 52

53 Effect of Denosumab on BMD at the Total Hip and Femoral Neck
HALT Study Effect of Denosumab on BMD at the Total Hip and Femoral Neck 4.8% difference at 24 months * Placebo (N = 734) Denosumab (N = 734) -6 -5 -4 -3 -2 -1 1 2 3 4 5 BL 6 12 24 36 Percentage Change From Baseline (±95% CI) Study Month Total Hip BMD -6 -5 -4 -3 -2 -1 1 2 3 4 5 BL 6 12 24 36 Percentage Change From Baseline (±95% CI) Study Month Femoral Neck BMD Placebo (N = 734) Denosumab (N = 734) 3.9% difference at 24 months * * P < vs Placebo Smith, MR et al. N Engl J Med Aug 20;361(8): 53

54 Effect of Denosumab on New Vertebral Fracture
HALT Study 62% Reduction in Subject Incidence of New Vertebral Fracture Over 36 Months (Adjusted P = ) Placebo (N = 673) Denosumab (N = 679) Month 12 Month 24 Month 36 RR 0.38 RR 0.31 P = 0.006 6 P = 0.004 RR 0.15 P = 0.004 4 Percentage of Subjects (%) 2 1.9% 0.3% 3.3% 1.0% 3.9% 1.5% Subject incidence 13 2 22 7 26 10 Smith, MR et al. N Engl J Med Aug 20;361(8): 54

55 Summary of Adverse Events Over 36 Months
HALT Study Summary of Adverse Events Over 36 Months Placebo (N = 725) n (%) Denosumab (N = 731) n (%) Adverse events regardless of relationship All 627 (86.5) 638 (87.3) Serious 222 (30.6) 253 (34.6) Fatal 46 (6.3) 44 (6.0) Leading to study discontinuation 44 (6.1) 51 (7.0) Leading to investigational product discontinuation 47 (6.5) 49 (6.7) CTCAE Grade 3, 4, or 5 244 (33.7) 269 (36.8) Adverse events considered potentially related to investigational product 65 (9.0) 62 (8.5) 4 (0.6) 3 (0.4) 1 (0.1) 0 (0.0) 5 (0.7) 2 (0.3) 7 (1.0) Smith, MR et al. N Engl J Med Aug 20;361(8): 55

56 Tolérance (1) DECIDE1 STAND2 Effets Alendronate 70 mg / Sem (N = 586)
Denosumab 60 mg / 6M (N = 593) 70 mg / Sem (N = 249) 60 mg / 6M (N = 253) Tout effet secondaire (%) 82 81 79 78 Effets secondaires conduisant à l’arrêt du traitement (%) 2 1 0.8 Différents effets d’intérêt (%) Fractures * 3 4 Tr. gastro-intestinaux 29 28 24 23 Infections 35 37 44 Tumeurs (bénignes ou malignes) Incidence of adverse events and serious adverse events was balanced between groups. No subjects reported adverse events of hypocalcemia, and mean serum calcium concentrations remained in the normal range in both groups. One subject in the denosumab group experienced an asymptomatic, transient decrease in serum calcium to 7.9 mg/dL. No other subjects in the denosumab group and no subjects in the placebo group had serum calcium levels below 8.0 mg/dL. Serious adverse events of infections and neoplasms were balanced between treatment groups. Adverse events of fractures were reported in 8 denosumab subjects and 4 alendronate subjects. The study was not designed to evaluate differences in fracture rates between the groups. Toutes les valeurs de p sont non significatives * STAND : 5 fractures cliniques observées pendant la phase de recrutement Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 56

57 Tolérance (2) DECIDE1 STAND2 Effets Alendronate 70 mg / Sem (N = 586)
Denosumab 60 mg / 6 M (N = 593) 70 mg / Sem (N = 249) 60 mg / 6 M (N = 253) Effets secondaires graves (%) 6 Décès (%) 0.2 0.4 Principaux effets secondaires graves (%) Infections 1 2 Tumeurs (bénignes ou malignes) Incidence of adverse events and serious adverse events was balanced between groups. No subjects reported adverse events of hypocalcemia, and mean serum calcium concentrations remained in the normal range in both groups. One subject in the denosumab group experienced an asymptomatic, transient decrease in serum calcium to 7.9 mg/dL. No other subjects in the denosumab group and no subjects in the placebo group had serum calcium levels below 8.0 mg/dL. Serious adverse events of infections and neoplasms were balanced between treatment groups. Adverse events of fractures were reported in 8 denosumab subjects and 4 alendronate subjects. The study was not designed to evaluate differences in fracture rates between the groups. Valeurs de p non significatives pour tous les items Brown JP et al. JBMR 2009 ; 24 : Kendler DL, et al. J Bone Miner Res Jul 13. [Epub ahead of print] 57

58 Densité Minérale Osseuse (DMO) à l’arrêt du traitement par le denosumab et après réintroduction du traitement Phase 2 210 mg / 6M Alendronate Placebo 30 mg / 3M 60 mg / 6M (reprise) Rachis lombaire Hanche totale CSR; TBA 14 8 Reprise de Traitement 60 mg / 6M Arrêt de traitement Reprise de traitement 60 mg / 6M Arrêt de traitement 12 6 10 8 4 6 2 Variation de DMO (% LS Mean ± SE) 4 2 Denosumab discontinuation was associated with lumbar spine and total hip BMD decrease to values near baseline. BMD changes remained higher in patients who received denosumab than in patients who had not been treated (placebo). The mean percent change in lumbar spine BMD was significantly different than placebo at 36 months (P = 0.013) and 48 months (P = 0.001). The mean percent change in total hip BMD was significantly different than placebo at 48 months (P = 0.015). In patients who discontinued alendronate, a slight decline of BMD was observed at the lumbar spine and decreases to near baseline at the total hip. -2 Miller P, et al. ASMBR Abstract E -2 -4 -4 -6 -6 CSR; TBA 12 12 24 48 18 21 24 36 48 18 21 36 Mois Mois 30 mg / 3M dernière dose à 21 mois 210 mg / 6M dernière dose à 18 mois Miller PD, et al. Bone. 2008; 43(2):222-9 58 Miller P, et al. ASBMR Abstract 1205 and oral presentation.

59 Risque des Fx non-vertébrales réduit de 20% (95% IC: 0.67, 0.95)
Denosumab et temps jusqu’à la première fracture non-vertébrale sur 36 mois Phase 3: FREEDOM – Etude pivot anti-fracturaire Placebo Denosumab 60 mg / 6M Cumulative Incidence (%) Month 6 12 24 2 4 8 18 30 36 6.5%* 8.0% 20% Treatment with denosumab significantly reduced nonvertebral fracture risk by 20% (6.5% denosumab versus 8.0% placebo, P = 0.01).1 The FREEDOM trial was designed with > 99% power to detect a 40% reduction in the incidence of nonvertebral fractures.1 Statistical analysis was based on the assumption of 3.3% per year nonvertebral fracture rate in the placebo group.1 The last known fracture status of patients who were lost to follow-up or withdrew before having a fracture event was carried forward in the analysis.1 This Kaplan-Meier curve reflects point estimates based on patients who have not had a nonvertebral fracture or who did not leave the study before the time point measured.1 Fractures not typically associated with decreased bone mineral density (eg, severe trauma, fractures of the skull/face/mandible/metacarpals/fingers/toes, pathologic fractures) were excluded in 28 patients (15 from the placebo arm and 13 from the denosumab arm).1 Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med Aug 20;361(8): Nombre de patientes à risque Placebo, n 3,906 3,750 3,578 3,410 3,264 3,121 3,009 Denosumab, n 3,902 3,759 3,594 3,453 3,337 3,228 3,130 Risque des Fx non-vertébrales réduit de 20% (95% IC: 0.67, 0.95) *P = 0.01 Cummings SR, et al. N Engl J Med Aug 20;361(8):756-65 59 59

60 Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente
Réduction du risque des fractures non-vertébrales chez les patientes à risque Phase 3: FREEDOM – Etude pivot anti-fracturaire Placebo Groupe pré-spécifié: Age > 70 ans T-score ≤ -3,0 Fx vertébrale prévalente Denosumab T-score col fémoral ≤ -2,5 à l’inclusion 14 35% (17%, 49%) P = 0,0006 12% (-11%, 30%) P = 0,2901 12 20% (5%, 33%) P = 0,0106 32% (11%, 47%) P = 0,0037 10 3% (-23%, 24%) P = 0,7903 8 Incidence fracturaire (%) 6 4 2 Population totale Risque haut Risque bas Risque haut Risque bas Interaction, p: 0,1525 0,0229 Boonen S, et al. ASBMR 2009


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