L-873724

Effect of cathepsin k inhibitor basicity on in vivo off-target activities

Cathepsin K is a lysosomal cysteine protease that is a pharmacological target for the treatment of osteoporosis. Previous studies showed that basic, lipophilic cathepsin K inhibitors are lysosomotropic and have greater activities in cell-based assays against cathepsin K, as well as the physiologically important lysosomal cysteine cathepsins B, L, and S, than expected based on their potencies against these isolated enzymes. Long-term administration of the basic cathepsin K inhibitors N-(1-(((cyanomethyl)amino)carbonyl)cyclohexyl)-4-(2-(4-methyl-piperazin-1-yl)-1,3-thiazol-4-yl)benzamide (L-006235) and balicatib to rats at a supratherapeutic dose of 500 mg/kg/day for 4 weeks resulted in increased tissue protein levels of cathepsin B and L but had no effect on cathepsin B and L message. This is attributed to the inhibitor engagement of these off-target enzymes and their stabilization to proteolytic degradation. No such increase in these tissue cathepsins was detected at the same dose of N-(cyanomethyl)-N(2)-{(1S)-2,2,2-trifluoro-1-[4'-methylsulfonyl)biphenyl-4-yl]ethyl}-l-leucinamide (L-873724), a potent nonbasic cathepsin K inhibitor with a similar off-target profile, although all three inhibitors provided similar plasma exposures. Using an activity-based probe, (125)I-BIL-DMK, in vivo inhibition of cathepsins B, L, and S was detected in tissues of mice given a single oral dose of L-006235 and balicatib, but not in mice given L-873724. In each case, similar tissue levels were achieved by all three compounds, thereby demonstrating the in vivo cathepsin selectivity of L-873724. In conclusion, basic cathepsin K inhibitors demonstrate increased off-target cysteine cathepsin activities than their nonbasic analogs and potentially have a greater risk of adverse effects associated with inhibition of these cathepsins.

The consequences of lysosomotropism on the design of selective cathepsin K inhibitors

Many drug candidates contain a basic functional group that results in lysosomotropism--the accumulation of drug in the acidic lysosomes of a cell. When evaluating inhibitors of lysosomal enzymes, such as the cathepsins, this physical property can have a dramatic impact on the functional selectivity of the test compounds. A basic P3 substituent in cathepsin K inhibitors provides a means of achieving potent and selective enzyme inhibition. To evaluate the whole-cell selectivity of the basic cathepsin K inhibitor L-006235, we identified the irreversible pan-selective cathepsin probe BIL-DMK and used it to design whole-cell enzyme-occupancy assays. These cell-based assays showed a dramatic reduction in selectivity against cathepsins B, L, and S relative to the selectivities observed in enzyme assays. Two-photon confocal fluorescence microscopy showed punctated subcellular localization of L-006235, which colocalized with BODIPY-labelled Lysotracker, consistent with compound lysosomotropism. To address this potential problem, a series of potent cathepsin K inhibitors was developed by replacing the P2--P3 amide bond with a metabolically stable trifluoroethylamine moiety. X-ray crystallography has identified the binding of this functional group to active-site residues in cathepsin K. This modification resulted in increased potency and selectivity that allowed the removal of the basic P3 substituent. The resulting nonbasic inhibitor L-873724 is a 0.2 nM inhibitor of cathepsin K with cathepsin B, L, and S potencies that were not shifted between purified enzyme and whole-cell assays; thus indicating that this compound is not lysosomotropic. L-873724 exhibits excellent pharmacokinetics and is orally active in a monkey model of osteoporosis at 3 mg kg(-1) q.d.

Identification of a potent and selective non-basic cathepsin K inhibitor

Based on our previous study with trifluoroethylamine as a P2-P3 amide isostere of cathepsin K inhibitor, further optimization led to identification of compound 22 (L-873724) as a potent and selective non-basic cathepsin K inhibitor. This compound showed excellent pharmacokinetics and efficacy in an ovariectomized (OVX) rhesus monkey model. The volumes of distribution close to unity were consistent with this compound not being lysosomotropic, which is a characteristic of basic cathepsin K inhibitors.

Cathepsin K inhibitors increase distal femoral bone mineral density in rapidly growing rabbits

Background: Selective and reversible inhibitors of human Cathepsin K (CatK), including odanacatib (ODN), have been developed as potential therapeutics for the treatment of osteoporosis. Inhibitors of human CatK show significantly less potency for the rodent enzymes compared with that for the human or rabbit enzymes; thus the Schenk model in growing rabbit was developed as a screening assay for the in vivo activity of CatK inhibitors in blocking bone resorption.
Methods: In this study, the efficacy of the selective inhibitors L-833905, L-006235, L-873724, and L-1037536 (ODN) of human CatK in the rapidly growing rabbit 'Schenk' model (age seven weeks) was compared to vehicle, using the bisphosphonate, alendronate (ALN), as a positive control, to assess inhibition of bone resorption. An enzyme inhibition assay (EIA) and an in vitro bone resorption assay using rabbit osteoclasts on bovine cortical bone slices were performed to evaluate the potency of these CatK inhibitors. Bone mineral density of the distal femur (DFBMD) was measured after ten days of treatment using ex vivo DXA densitometry.
Results: Results of the EIA using rabbit CatK and the rabbit bone resorption assay showed that three of the four compounds (L-006235, L-873724, and ODN) had similar potencies in the reduction of collagen degradation. L-833905 appeared to be a weaker inhibitor of CatK. Taking into account the respective in vitro potencies and pharmacokinetic profiles via oral administration, the efficacy of these four CatK inhibitors was demonstrated in a dose-related manner in the growing rabbit. Significant increases in DFBMD in animals dosed with the CatK inhibitors compared to vehicle were seen.
Conclusions: Efficacy of the CatK inhibitors in the Schenk rabbit correlated well with that in the in vitro rabbit bone resorption assay and in the ovariectomized rabbit model as previously published. Hence, these studies validated the rabbit Schenk assay as a rapid and reliable in vivo model for prioritizing human CatK inhibitors as potential therapeutic agents.

Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools

Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC(50) values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [(14)C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH(4)Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.