The potency and selectivity of the inhibitors were determined using recombinant human cathepsins K, S, L, H, F, V and B

1
Cathepsin K Inhibitors for the Treatment of Bone
Metastasis E. Lindström1, L. Vrang1, S. Sedig1,
Y. Terelius1, B.-L. Sahlberg1, K. Wikström1, B.
Samuelsson1, T.J. Chambers2, U.
Grabowska1 1Medivir AB, Huddinge, Sweden 2St
Georges, University of London, London, UK
Abstract
Potency and Selectivity in vitro
MV076159
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.
Human enzyme level
Assay MV074942 MV076159
Cathepsin K 1.6 0.75
Cathepsin S 14000 19000
Cathepsin L 1600 1800
Cathepsin B 1200 1300
Cathepsin H gt10000 gt10000
Cathepsin V 1700 4000
Cathepsin F 1700 2800
In vivo efficacy summary
Treatment Dose (µmol/kg) Max inhibition () Inhibition at 24h () Inhibition at 48h ()
Vehicle 50 0 0
MV074942 28 78 53 3
MV076159 30 88 62 16
All values are given as mean Ki in nM

• MV074942 and MV076159 display more than
  1000-fold selectivity vs related cysteine
  proteases
• The inhibitors bind reversibly to cathepsin K
  enzyme with fast Koff

• 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

Cellular level
Assay MV074942 MV076159
Human osteoclasts 44 34
Iip10 accumulation 48000 23000
Exposure vs Effect
All values are given as mean IC50 in nM
MV074942 and MV076159 display approx 1000-fold
selectivity at cellular level
Introduction
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.
Degree of efficacy over 24h is significantly
related to compound plasma exposure over 24h
MV074942 r2 0.71, plt0.001 MV076159 r2 0.64,
plt0.001
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
Methods
Summary and Conclusions

• 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
  reversephase liquid chromatography and
  electrospray tandem mass spectrometry (LC-MS-MS)

• 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

PK and Efficacy in vivo
MV074942