A highly potent inhibitor of cathepsin K (relacatib) reduces biomarkers of bone resorption both in vitro and in an acute model of elevated bone turnover in vivo in monkeys.

Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.

Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

Changes in bone turnover following gonadotropin-releasing hormone (GnRH) agonist administration and estrogen treatment in cynomolgus monkeys: a short-term model for evaluation of antiresorptive therapy.

In this study we determine the early time course of estrogen deficiency-induced bone loss in the cynomolgus monkey and examine the potential of this method for evaluating antiresorptive therapies. In two groups of animals, estrogen deficiency was induced by the administration of a gonadotropin-releasing hormone agonist (GnRHa) and bone turnover was measured using biochemical markers. Two weeks after receiving GnRHa, serum estradiol decreased to below the detection limit in most animals and remained there through 6 months or until estrogen replacement started (months 4-6). Relative to untreated animals, urinary deoxypyridinoline (dPyr), as well as C- and N-telopeptides of type I collagen, were significantly elevated 4 weeks after receiving GnRHa. Serum osteocalcin increased in GnRHa-treated animals as early as week 4 and the level was significantly higher than in untreated control animals from weeks 8-24. Estradiol treatment returned all measures of bone turnover to control levels within 2 weeks. The use of biochemical markers as surrogates of bone turnover and loss was validated by measurement of bone mineral density (BMD), which showed a significant reduction at 6 months in estrogen-deficient animals. However, lumbar BMD in animals that received GnRHa and estradiol was similar to that in animals that had not received GnRHa. In conclusion, a monthly depot injection of GnRHa resulted in increased bone turnover due to estrogen deficiency, as early as 4 weeks after treatment. Estrogen administration returned bone turnover to control levels in 2 weeks. This method represents a valid model for evaluating antiresorptive agents in the short term in a nonhuman primate. Furthermore, the data suggest that changes in biochemical markers in response to antiresorptive therapy in humans may be detectable at much earlier timepoints than commonly used.

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Antisecretory mechanisms of peptide YY in rat distal colon.

Peptide YY (PYY) is a potent regulator of intestinal secretion. These studies investigated the role of Y1 and Y2 receptor subtypes in mediating the antisecretory effects of PYY on mucosa-submucosa preparations of rat distal colon. Addition of vasoactive intestinal peptide (VIP) to these tissues resulted in a 140 +/- 18% increase in basal short-circuit current (Isc) and the induction of Cl- secretion. VIP-stimulated increases in Isc were abolished by the addition of each of PYY, (Pro34)-PYY, a Y1 receptor-selective agonist, and PYY-(3-36), an endogenous Y2 receptor-selective ligand. However, when tissue neural transmission was blocked with tetrodotoxin, neither PYY nor its receptor subtype-selective analogs were able to inhibit VIP-stimulated increases in Isc. These results suggest that in the rat distal colon, the antisecretory actions of PYY are mediated through a combination of Y1 and Y2 receptor subtypes or through a novel receptor subtype that is unable to discriminate between (Pro34)-PYY and PYY-(3-36).