1. The potency and selectivity of the inhibitors were determined using recombinant human cathepsins K, S, L, H, F, V and B Functional reversibility of the inhibitors against cathepsin K was assessed Cellular inhibition of cathepsin K was monitored using a human osteoclast system as previously described Iip10 Accumulation, which reflects inhibition of cellular cathepsin S activity, was measured in a human EBV-B-cell line Conscious cynomolgus monkeys were dosed p.o. with cathepsin K inhibitor or corresponding vehicle between 7.00 a.m. and 9.00 a.m. Blood samples were drawn at various time points after dosing. compound levels and CTX-I. The C-terminal degradation product of collagen Plasma samples were collected for analysis of type I (CTX-I) in plasma was measured using a commercially available kit (CrossLaps, IDS Nordic A/S, Herlev, Denmark) Compound levels in plasma were determined using reverse–phase liquid chromatography and electrospray tandem mass spectrometry (LC-MS-MS) Abstract Cathepsin K Inhibitors for the Treatment of Bone Metastasis E. Lindström 1, L. Vrang 1, S. Sedig 1, Y. Terelius 1, B.-L. Sahlberg 1, K. Wikström 1, B. Samuelsson 1, T.J. Chambers 2, U. Grabowska 1 1 Medivir AB, Huddinge, Sweden; 2 St George’s, University of London, London, UK Bone metastasis is characterised by excessive bone turnover by osteoclasts. Cathepsin K is a lysosomal cysteine protease expressed abundantly in osteoclast cells. Numerous lines of evidence support a pivotal role for cathepsin K in bone degradation and the development of cathepsin K inhibitors is being pursued by many companies. Recently cathepsin K inhibitors have demonstrated efficacy in phase II trials, as measured by increased bone mineral density (BMD), in a dose dependent manner. Studies also show that inhibition of cathepsin K can decrease bone degradation without negatively impacting bone formation, differentiating this treatment from currently available anti-resorptives such as bisphosphonates. A rationale for this augmentation of bone formation may arise from a new mechanism of action wherein cathepsin K inhibitors lead to the intact release of matrix-derived growth factors and/or PTH spikes. Additionally, cathepsin K is produced by cancer cells to promote cancer cell invasion and cathepsin K inhibitors have been shown to reduce breast cancer-induced osteolysis and skeletal tumour burden in such diseases as bone metastasis and osteoporosis. Medivir has now developed a series of novel, highly potent, specific and non-nitrile warhead cathepsin K inhibitors. These inhibitors were additionally selected for their high potency in inhibiting bone resorption by human osteoclasts in-vitro. The pharmacodynamic effect of these inhibitors on attenuating bone resorption was evaluated in vivo in young male cynomolgus monkeys. Plasma levels of the C-terminal telopeptides of Type I collagen (CTX-I) was used as a collagenous bone resorption marker. Oral administration of compounds (3, 10 and 30 μmol /kg) to the animals resulted in a rapid reduction in CTX-I levels within 2 h to a maximum of 75-95% after 4-8 hours. This suppression of CTX-I was reversible with bone resorption marker returning to pre-treatment levels within 48 h. Bone metastases comprise cancer cells that separate from primary tumors and migrate to bone tissue where they settle and grow. The presence of cancer cells in bone disrupts the tight coupling between bone formation and bone resorption. A vicious cycle occurs where tumor growth triggers more bone resorption while more bone resorption liberates growth factors from bone which stimulate tumor growth. Cathepsin K inhibitors not only prevent bone resorption but also allow bone reformation to continue in contrast to approved and widely used bisphosphonates and other anti-resorptives such as denosumab which suppress bone formation. Besides the beneficial effects on the bone re-modelling cathepsin K inhibitors may assist in preventing the spread of cancer to bone. The current summary describes the pharmacology of two potent and highly selective cathepsin K inhibitors on markers of bone resorption in cynomolgus monkeys. Introduction Potency and Selectivity in vitro PK and Efficacy in vivo Exposure vs Effect Summary and Conclusions AssayMV074942MV076159 Cathepsin K1.60.75 Cathepsin S1400019000 Cathepsin L16001800 Cathepsin B12001300 Cathepsin H>10000 Cathepsin V17004000 Cathepsin F17002800 AssayMV074942MV076159 Human osteoclasts 4434 Iip10 accumulation 4800023000 All values are given as mean K i in nM TreatmentDose (μmol/kg) Max inhibition (%) Inhibition at 24h (%) Inhibition at 48h (%) Vehicle~5000 MV0749422878533 MV07615930886216 In vivo efficacy summary: The two inhibitors described are potent and highly selective inhibitors of human cathepsin K in vitro Advantageous lysosomotropic properties of these compounds lead to no loss of selectivity at the cellular level coupled with prolonged efficacy in an osteoclast cell-based assay The compounds are well-tolerated and inhibit circulating CTX-I levels by up to 88% in cynomolgus monkey in vivo Efficacy duration exceeds plasma exposure, likely due to a prolonged residence time in osteoclasts – the intended site of action The high potency and prolonged efficacy duration in vivo together with excellent selectivity renders these compounds attractive candidates for clinical development All values are given as mean IC 50 in nM Methods Human enzyme level  MV074942 and MV076159 display more than 1000-fold selectivity vs related cysteine proteases The inhibitors bind reversibly to cathepsin K enzyme with fast Koff Cellular level MV074942 and MV076159 display approx 1000- fold selectivity at cellular level After washout, MV076159 pre-treated osteoclasts take a longer time to recover than odanacatib pre- treated. This is likely due to the lysosomotropic nature of MV076159 and would predict enhanced efficacy MV074942 MV076159 Significant reductions of CTX-I are present 24h after dose of cathepsin K inhibitor, despite minimal plasma exposure at this time point Effects of inhibitors are fully reversible Degree of efficacy over 24h is significantly related to compound plasma exposure over 24h MV074942: r 2 : 0.71, p<0.001 MV076159: r 2 : 0.64, p<0.001