Cathepsin K inhibitors increase distal femoral bone mineral density in rapidly growing rabbits
© Pennypacker et al.; licensee BioMed Central Ltd. 2013
Received: 12 July 2013
Accepted: 14 November 2013
Published: 9 December 2013
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.
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 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.
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.
KeywordsOdanacatib Cathepsin K inhibitor Osteoclast Antiresorptives DXA Rabbit Schenk assay Postmenopausal osteoporosis
The lysosomal cysteine protease, Cathepsin K (CatK), is predominantly expressed in osteoclasts  and has an important role in the degradation of the demineralized collagen matrix components of bone (predominantly Type-I collagen) at neutral and acidic pH . Genetic confirmation for a role of CatK in bone resorption exists in the form of pycnodysostosis, a rare human genetic disease linked to several loss-of-function mutations in the CatK gene characterized by high bone mineral density (BMD), acroosteolysis of the distal phalanges, short stature, and skull deformities with delayed suture closure [3–5]. Mice with gene deletion of CatK show impaired osteoclastic resorption which leads to osteopetrosis . These mice display higher bone mass in both cortical and trabecular bone, greater trabecular and cortical thickness, and normal bone strength [7, 8]. Mice overexpressing CatK show a decrease in trabecular bone volume of the distal femoral metaphyses and accelerated bone turnover . Based on its substrate preference, cellular distribution, and genetic evidence, CatK is likely to play an important role in bone resorption. The selective inhibitor of CatK, odanacatib (ODN), is currently in development for the treatment of osteoporosis .
The preferred small animal model for evaluation of the efficacy of bone therapeutics is the ovariectomized (OVX) skeletally mature rat . The utility of the OVX rat is limited by significant interspecies sequence variation (88% homology) in key residues within the active site of human and rat CatK enzymes ; inhibitors of human CatK have significantly lower potency against the rodent CatK enzyme to be reliably tested for bone effects in the rat or mouse model. In contrast, rabbit CatK shares 96% sequence identity and 99% similarity with its human counterpart with only two amino acid differences in the active site [12, 13]. Therefore, the selection of rabbit as the preclinical species for in vivo screening assay for CatK inhibitors was due to the species differences in response to this class of bone resorption inhibitors. The differences in homologies between rat, rabbit and human CatK are derived from inhibitor potencies. For example, ODN is highly potent versus human and rabbit CatK (half maximal inhibitory concentration [IC50 ]= 0.2 and 1 nM, respectively) but more than 500-fold less potent versus rat CatK (IC50 = 112 nM) or mouse CatK (IC50 = 108 nM) . Our work with the adult OVX rabbit shows that it is a relevant in vivo bone model for testing selective inhibitors of the human CatK enzyme . However, the estrogen-deficient OVX model in skeletally mature rabbits requires approximately six months to achieve predictable and measurable bone loss by dual energy x-ray absorptiometry (DXA) . Due to the long duration of the adult OVX rabbit model and the relatively large size of adult rabbits (3.5 kg), the use of the adult OVX rabbit model for quick in vivo screening and selection of compounds with limited drug quantity in the early pre-clinical phase is impractical.
A rapidly growing rabbit model has been developed for in vivo prioritization of CatK inhibitors before testing in the OVX rabbit assay. Development of this growing rabbit model is based upon the growing rat model used for testing the anti-resorptive efficacy of the bisphosphonates [16–18]. This model is often referred to as the “rat Schenk assay.” This assay relies upon inhibiting the process of bone resorption in rapidly growing animals at the periosteal surface (the “funnel region”) of the metaphysis, and the aspect of metaphyseal trabeculae in the marrow cavity that is opposite to the nearby epiphyseal growth cartilage. Inhibiting the removal of calcified cartilage by resorption in the primary spongiosa is also an important target. In early work, treatment of growing rats treated with the first-generation bisphosphonates, etidronate or clodronate, for ten days resulted in higher trabecular bone volume in the proximal tibial metaphysis of treated compared to untreated rats [16, 17]. A more recent experiment showed increased trabecular bone volume following seven days of subcutaneous (SC) alendronate (ALN) (0.010 mg/kg/d) . Furthermore, a more recent study using weanling rats showed an increase in distal femoral metaphyseal BMD following six weeks of once-weekly treatment with ALN . A higher growth rate of the distal and proximal femur, the proximal tibia, and the proximal and distal radius has been observed in the rapidly growing rabbit than that typically observed with other species or in more mature animals [20, 21].
The primary objective of the current study was to determine if the rapidly growing rabbit (“rabbit Schenk assay”) could be used to quantify and prioritize CatK inhibitors according to their respective potencies in inhibiting bone resorption in vivo. The model was first characterized by dose titrating ALN and determining areal BMD of the distal femur (DFBMD) using ex vivo DXA. Next, the efficacy of four relatively potent CatK inhibitors with varied chemical structures, in vitro potency, and in vivo pharmacokinetic profiles was assayed in a similar fashion.
Six- to seven-week-old female New Zealand White (NZW) rabbits (Covance Research Products, Denver, PA, USA), weighing approximately 1.35–1.5 kg were received. The rabbits were kept in wire-bottomed cages under standard laboratory conditions with lighting set for 12 h light, 12 h dark per 24 h, a constant temperature of 21 ± 3°C, relative humidity 50 ± 20%, and 10–15 air changes per hour. Rabbits received a standard pelleted diet (High Fiber Lab Rabbit Diet 5326, PMI Nutrition International, Brentwood, MO, USA), with water ad libitum. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committees at Merck Frosst Center for Therapeutic Research (Montreal, Quebec, Canada) and Merck Research Laboratories (West Point, PA, USA).
Dose-ranging ALN in growing rabbits
To characterize the dose of ALN required to show increases in DFBMD, dose-ranging studies were conducted using ALN, administered at doses of 0, 50, 100, 200, and 500 μg/kg (SC, once daily). Seven-week-old rabbits were weight-randomized into groups (n = 8-11 per group). ALN was prepared in deionized water and pH adjusted to 7.2. The injection volume was 0.3 mL per rabbit. Rabbits were treated daily for 10 consecutive days. Rabbits were reweighed on Day 6 and dosing volume was adjusted accordingly. At necropsy, the right femur was excised and stored in 70% ethanol.
CatK inhibitor screening
Rabbits were treated once daily by oral gavage for 10 consecutive days with vehicle (1% carboxymethylcellulose or 0.5%/0.2% carboxymethylcellulose/SDS), CatK inhibitors, or ALN (100 μg/kg, SC). Several studies were conducted, with each study including vehicle (n = 11–14), multiple dose levels of a CatK inhibitor (n = 13–14 per group), and ALN (n = 8–9). On Day 1, two rabbits from each group receiving CatK inhibitors were bled via the central ear artery (0.5 mL each) at 0, 0.25, 1, 3, 6, 8, and 24 h after dosing. Plasma concentrations of CatK inhibitors were determined in all samples. On Day 11, rabbits were euthanized. Femurs were removed and stored in 70% ethanol for BMD analysis.
Enzyme inhibition assay (EIA)
Enzyme activity assays were carried out using rabbit CatK as previously described . Briefly, the assay was carried out in 2-(N-morpholino) ethanesulfonic acid 50 mM pH 5.5 containing dithiothreitol 2.5 mM, ethylenediaminetetraacetic acid 2.5 mM, and 10% dimethyl sulfoxide. Prior to the addition of substrate, different concentrations of the inhibitor ranging from 100 μM to 0.2 nM were pre-incubated for 15 min with each enzyme (0.2–1 nM) to allow the formation of the enzyme-inhibitor complex. Substrate was then added and the enzyme activity measured from the increase of fluorescence at 460 nm 355 nm. The final volume of the reaction was 100 μL. Assays were performed in 96-well plate format and the plate was read using a Spectramax (Molecular Devices) plate reader. The percentage inhibition of the reaction was calculated from a control reaction containing only vehicle. IC50 curves were generated by fitting percentage inhibition values to a four-parameter logistic model (SOFTMAX PRO, Molecular Devices, Sunnyvale, CA, USA).
Bone resorption assay
The bone resorption assay is a functional in vitro assay that measures Type-I collagen degradation after a three-day incubation of rabbit osteoclasts cultured on bovine bone with varying concentrations of test compound, as previously described . Briefly, long bones isolated from a 10-day-old NZW rabbit were finely minced in alpha-minimal essential medium (α-MEM) (Gibco BRL; Gaithersburg, MD, USA) containing penicillin/streptomycin, pH 7.1 to obtain a cell suspension and 1 × 106 cells were seeded onto each 6 mm diameter × 0.22 mm thick bovine bone slice in the same medium containing 2% fetal bovine serum (FBS). After 4 h, the medium was replaced with α-MEM, 2% FBS, 1,25(OH)2D3 10 nM, and test compounds. The cultures were incubated for three days at 37°C in 5% CO2. C-telopeptide of Type-I collagen (CTx-I) released into the medium was measured by the CROSSLAPS Elisa assay (Osteometer Biotech, Herlev, Denmark).
Whole right femurs with muscles removed, were immersed in two inches of water in an acrylic box, and positioned with both distal condyles resting on the bottom of the box. The distal 5 cm of the femur was scanned using small animal software in high resolution mode on a Hologic QDR 4500 fan-beam bone densitometer (DXA; Hologic, Inc., Waltham, MA, USA). The distal 3 cm of the femur was analyzed. A region of interest (ROI) beginning one line distal to the distal edge of the femur and centered 70 lines wide and extending 60 lines proximally in the long axis of the bone was applied. Bone mineral content (BMC) and bone area (BAr) were output by DXA software. BMD was calculated as BMC/BAr.
Histological examination of distal femur
Following ex vivo DXA scanning, the distal one-third of the femur was cut mid-sagitally and then dehydrated, without prior decalcification, in increasing concentrations of ethanol. The right portion was embedded in 80% methylmethacrylate/20% dibutyl phthalate. Parasagittal sections (6 μm) were cut on a Reichert-Jung Polycut sledge microtome (Nussloch, Germany) and mounted on glass slides. A Masson’s trichrome stain was performed to view calcified tissue.
For dose-ranging ALN and CatK inhibitor L-833905 studies, DFBMD differences of treatment groups compared to vehicle were analyzed by Kruskal-Wallis non-parametric analysis of variance (ANOVA) with Student-Neuman-Keuls post hoc testing. Differences were considered significant when p ≤ 0.05. All comparisons were made using CRUNCH software (JanDel Corp.; San Jose, CA, USA). For all other studies of CatK inhibitors, statistical computation of DFBMD data was performed using Statview (SAS Institute, Inc., Cary, NC). Differences among treatment groups were tested by one-way ANOVA. If significant differences were indicated by ANOVA, comparison between group means was tested by Fisher’s partial least-squares difference for post hoc analysis. Differences were considered significant when p ≤ 0.05.
Characterization of the rabbit Schenk model in growing rabbits
Effect of 10 days' ALN treatment on DFBMD in growing rabbits
ALN dose (μg/kg)
% BMD gain
241.4 ± 9.2
277.8 ± 8.4**
270.5 ± 7.9
313.7 ± 3.9***
242.0 ± 3.4
281.7 ± 6.4***
241.6 ± 5.2
291.2 ± 9.4***
Importantly, these results, that tested doses of ALN that varied by an order of magnitude, established the “rabbit Schenk assay” as one that could assess anti-resorptive efficacy, by testing an established osteoporosis treatment agent that had been previously tested in the rat Schenk assay . ALN 100 μg/kg/day was selected as a reliable positive control for subsequent studies that tested CatK inhibitors in growing rabbits.
Evaluation of in vivo efficacy of potent CatK inhibitors
Selection of CatK inhibitors based on in vitro potencies
[Cmin]/bone res IC50
Rabbit bone resorption assay
33 ± 2
149 ± 20
5 ± 1
13 ± 1
23 ± 6
Pharmacokinetic evaluation of CatK inhibitors in growing rabbits
Doses of the respective CatK inhibitors for the rabbit Schenk assays were selected on the basis of trough plasma levels (Cmin) following oral dosing in the same species. As indicated in Table 2, the mid-dose of all inhibitors, except L-833905, provided trough plasma levels that remained at or above the rabbit bone resorption IC50 for the duration of the study (i.e. Cmin at 24 h following oral dose ≥ bone resorption IC50 or [Cmin]/bone resorption IC50 ≥ 1). For both doses of L-833905 and the lowest dose of L-873724, the plasma levels dropped below the bone resorption IC50 at 24 h (Table 2). Although ODN was slightly less potent compared to L-006235 and L-873724 in the bone resorption assay, lower doses were selected due to its longer rabbit plasma half-life which thereby provided higher Cmin plasma levels at 24 h post-dosing.
Effects of CatK inhibitors on distal femoral BMD (DFBMD)
Distal femur BMD (mg/cm 2 ) in three rabbit Schenk assays
274.3 ± 4.5
282.5 ± 5.5
301.8 ± 4.9**
314.0 ± 9.1**
263.3 ± 4.5
281.7 ± 4.9**
293.5 ± 2.8***
303.5 ± 5.4***
300.5 ± 4.6***
285.5 ± 5.4
301.2 ± 3.7
310.5 ± 6.5**
327.7 ± 7.5***
352.8 ± 9.4***
CatK is predominantly expressed in osteoclasts , and has an important role in the degradation of the collagen matrix components of bone (predominantly Type-I collagen) at acidic pH. Based on human genetics [3–5], experimental genetics in mice , substrate preference, and cellular distribution, the pivotal role of CatK in osteoclastic bone resorption has been demonstrated. These findings have led to the development of pharmacologic inhibitors of CatK to treat diseases characterized by high bone turnover such as osteoporosis.
In this report, four CatK inhibitors, L-833905, L-006235, L-873724, and ODN, were evaluated for their effects on DFBMD in the rapidly growing rabbit. Based on their respective pharmacokinetic profiles in rabbits and their in vitro profiles, these compounds were chosen to evaluate in vivo anti-resorptive efficacy. These four compounds, when administered daily for 10 consecutive days to growing rabbits, significantly increased DFBMD versus vehicle treatment in a dose-dependent fashion, showing that inhibitors of the CatK enzyme inhibit bone resorption in vivo, as predicted by their respective potencies in the EIA and the in vitro bone resorption assay, and their pharmacokinetic profile in rabbits.
From the EIA and functional cell-based assay, L-006235, L-873724, and ODN were selected as potent inhibitors of CatK-mediated activity of rabbit osteoclasts in the degradation of Type-I collagen in vitro. L-833905 was a 6–30-fold less potent inhibitor of CatK than the above three compounds. The rank order of potencies of these inhibitors in the bone resorption assay tracked well with that in the EIA. The shift in potencies between the two assays may reflect the degree of protein binding of the inhibitors and high fractional inhibition of CatK required to inhibit bone resorption in osteoclasts [13, 25]. Moreover, the potencies of these compounds were also dependent on their ability to penetrate and exit the resorption lacunae and lysosomes of osteoclasts during resorption .
Based on our historical database, inhibitors of the human CatK enzyme are about two orders of magnitude less active in inhibition versus the rat CatK enzyme. The development of a potent and selective inhibitor of rat CatK versus other rat cathepsins has been shown to be challenging [27, 28]. However, human CatK inhibitors are generally effective in the rabbit, usually displaying only approximately 5-fold less potency toward the rabbit enzyme. These findings are supported by the higher amino acid sequence homology between human and rabbit enzymes. These fundamental interspecies differences in CatK led us to select the growing rabbit as a relatively small size animal model, which can be used to rapidly identify in vivo anti-resorptive activity of numerous CatK inhibitors prior to the evaluation of selected candidates in the long-term OVX model of postmenopausal osteoporosis in the adult rabbit or non-human primate (NHP).
The growing rabbit has several advantages, including a higher bone growth rate than that typically observed with other laboratory large species such as dog, pig or monkey, which have been used previously to evaluate therapeutic agents for osteoporosis [20, 21]. We previously described the development of the adult OVX rabbit assay to assess the efficacy of CatK inhibitors on preventing estrogen deficiency bone loss in the lumbar vertebrae and distal femur . However, the adult OVX rabbit model has many limitations which preclude its use for routine drug screening, including limited availability of skeletally mature aged rabbits, requirements for surgical manipulation, long study duration and a large body weight that requires preparation of large quantities of agents. An NHP in vivo screening assay examining markers of bone resorption, (e.g. serum CTx, collagen Type-I N-telopeptides) [27, 29] also has disadvantages, including availability of trained personnel, long washout periods, high demand for drug quantity, high cost, and limited numbers of skeletally mature NHPs available for drug screening purposes.
Thus, a short-term reliable in vivo screening assay to quickly assess potencies of compounds for further optimization was highly valuable for a drug screening program. The current study is the first to report the use of the rapidly growing rabbit as an assay for in vivo activity of bone resorption inhibitors, with similar fundamental characteristics to that previously used in growing rats [16–18, 30, 31]. Unlike the adult OVX rabbit, this model requires only 10 days of dosing. Considering that the assay itself is 18 times shorter in duration and the weight of the animals during the assay is 40% of those used in the adult OVX rabbit model, the total requirement for compound is 40-fold less than that needed for an adult OVX rabbit study. Efficacy of the bone resorption inhibitors is assessed by ex vivo DXA of the distal femur, a region that contains an active epiphyseal growth cartilage in growing rabbits. During longitudinal growth, the structure and density of metaphyseal trabecular bone relies on a well-controlled balance between calcified cartilage formation in the zone of cell hypertrophy of the epiphyseal growth cartilage, bone deposition in the primary and secondary spongiosa, and the removal of both calcified cartilage and bone in both the primary and secondary spongiosa. Inhibition of calcified tissue resorption during growth without effects on chondrocyte activity in the epiphyseal growth cartilage results in a density increase in the primary and secondary spongiosa that is characterized by higher trabecular number. The higher trabecular number is due to an increase in the number of persisting calcified cartilage septa upon which new bone tissue is deposited. Bisphosphonates increase metaphyseal trabecular bone volume and trabecular number in the proximal tibial metaphysis of the growing rat [16–18, 30, 31]. In addition, when non-decalcified histologic sections are used, the rat Schenk assay becomes useful for screening for the existence of mineralization defects [16, 17].
The results of these rabbit Schenk studies suggest that potent and orally active CatK inhibitors are effective as bone resorption inhibitors in vivo. This assay can be used to quickly rank order potencies of the CatK inhibitors prior to their evaluation in estrogen deficiency-related bone-loss studies. In fact, efficacy of the CatK inhibitors L-006235 and ODN were further demonstrated in adult OVX rabbits  and ODN in OVX NHPs [32, 33].
The rabbit Schenk assay is a valid, consistent in vivo screen for two classes of bone resorption inhibitors, CatK inhibitors and bisphosphonates. The current data confirm the efficacy of four different selective CatK inhibitors and ALN in reducing bone resorption in vivo in the rapidly growing rabbits. The results for CatK inhibitors were well-correlated with those in the in vitro bone resorption assay. The Schenk assay results testing L-006235 and odanacatib predict the outcome of tests of the same agents on estrogen deficiency-induced bone loss in both skeletally mature rabbit  and monkey models . Therefore, while the OVX animal model is the standard method for assessing efficacy and long-term effects of anti-osteoporosis agents on bone quality, we demonstrated that the rabbit Schenk assay can serve as a rapid, cost-effective, and reliable test for evaluation of numerous bone resorption inhibitors prior to the evaluation of a few selected candidates in the long-term OVX-model of the same species or non-human primates.
Analysis of variance
Area under the curve
Bone mineral content
Bone mineral density
Maximum concentration after dosing (peak plasma levels)
Minimum concentration after dosing (trough plasma levels)
C-telopeptide of Type-I collagen
Distal femur bone mineral density
Dual energy x-ray absorptiometry
Enzyme inhibition assay
Fetal bovine serum
Half maximal inhibitory concentration
Limit of detection
Alpha-minimal essential medium
New Zealand White
Region of interest
Sodium dodecyl sulfate.
This study was sponsored by Merck & Co., Inc. We thank Denise Graham, PhD, who provided medical writing support on behalf of Complete Medical Communications, funded by Merck & Co., Inc. Further medical writing assistance was provided by Boyd Scott, PhD, of Merck & Co., Inc. We thank David Percival, PhD, and Gregg Wesolowski for generating the published values of these compounds in the EIA and bone resorption assay. We also thank Denis Normandin, Simon Wong, and Karen Ortega, previously from Merck Frosst Therapeutics for their technical assistance with tissue harvesting and animal dosing.
- Brömme D, Okamoto K: Human cathepsin O2, a novel cysteine protease highly expressed in osteoclastomas and ovary molecular cloning, sequencing and tissue distribution. Biol Chem Hoppe Seyler. 1995, 376: 379-384. 10.1515/bchm3.1995.376.6.379.View ArticlePubMedGoogle Scholar
- Kafienah W, Brömme D, Buttle DJ, Croucher LJ, Hollander AP: Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J. 1998, 331 (Pt 3): 727-732.View ArticlePubMedPubMed CentralGoogle Scholar
- Gelb BD, Shi GP, Chapman HA, Desnick RJ: Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science. 1996, 273: 1236-1238. 10.1126/science.273.5279.1236.View ArticlePubMedGoogle Scholar
- Ho N, Punturieri A, Wilkin D, Szabo J, Johnson M, Whaley J, Davis J, Clark A, Weiss S, Francomano C: Mutations of CTSK result in pycnodysostosis via a reduction in cathepsin K protein. J Bone Miner Res. 1999, 14: 1649-1653. 10.1359/jbmr.19126.96.36.1999.View ArticlePubMedGoogle Scholar
- Johnson MR, Polymeropoulos MH, Vos HL, De Luna RI O, Francomano CA: A nonsense mutation in the cathepsin K gene observed in a family with pycnodysostosis. Genome Res. 1996, 6: 1050-1055. 10.1101/gr.6.11.1050.View ArticlePubMedGoogle Scholar
- Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, Von Figura K: Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci USA. 1998, 95: 13453-13458. 10.1073/pnas.95.23.13453.View ArticlePubMedPubMed CentralGoogle Scholar
- Gowen M, Lazner F, Dodds R, Kapadia R, Feild J, Tavaria M, Bertoncello I, Drake F, Zavarselk S, Tellis I, Hertzog P, Debouck C, Kola I: Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization. J Bone Miner Res. 1999, 14: 1654-1663. 10.1359/jbmr.19188.8.131.524.View ArticlePubMedGoogle Scholar
- Pennypacker B, Shea M, Liu Q, Masarachia P, Saftig P, Rodan S, Rodan G, Kimmel D: Bone density, strength, and formation in adult cathepsin K (−/−) mice. Bone. 2009, 44: 199-207. 10.1016/j.bone.2008.08.130.View ArticlePubMedGoogle Scholar
- Kiviranta R, Morko J, Uusitalo H, Aro HT, Vuorio E, Rantakokko J: Accelerated turnover of metaphyseal trabecular bone in mice overexpressing cathepsin K. J Bone Miner Res. 2001, 16: 1444-1452. 10.1359/jbmr.2001.16.8.1444.View ArticlePubMedGoogle Scholar
- Boonen S, Rosenberg E, Claessens F, Vanderschueren D, Papapoulos S: Inhibition of cathepsin K for treatment of osteoporosis. Curr Osteoporos Rep. 2012, 10: 73-79. 10.1007/s11914-011-0085-9.View ArticlePubMedGoogle Scholar
- Thompson DD, Simmons HA, Pirie CM, Ke HZ: FDA Guidelines and animal models for osteoporosis. Bone. 1995, 17: 125S-133S.View ArticlePubMedGoogle Scholar
- Stroup GB, Lark MW, Veber DF, Bhattacharyya A, Blake S, Dare LC, Erhard KF, Hoffman SJ, James IE, Marquis RW, Ru Y, Vasko-Moser JA, Smith BR, Tomaszek T, Gowen M: Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate. J Bone Miner Res. 2001, 16: 1739-1746. 10.1359/jbmr.2001.16.10.1739.View ArticlePubMedGoogle Scholar
- Falgueyret JP, Desmarais S, Oballa R, Black WC, Cromlish W, Khougaz K, Lamontagne S, Massé F, Riendeau D, Toulmond S, Percival MD: Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity. J Med Chem. 2005, 48: 7535-7543. 10.1021/jm0504961.View ArticlePubMedGoogle Scholar
- Desmarais S, Masse F, Percival MD: Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools. Biol Chem. 2009, 390: 941-948.View ArticlePubMedGoogle Scholar
- Pennypacker BL, Duong Le T, Cusick TE, Masarachia PJ, Gentile MA, Gauthier JY, Black WC, Scott BB, Samadfam R, Smith SY, Kimmel DB: Cathepsin K inhibitors prevent bone loss in estrogen-deficient rabbits. J Bone Miner Res. 2011, 26: 252-262. 10.1002/jbmr.223.View ArticlePubMedGoogle Scholar
- Schenk R, Merz WA, Muhlbauer R, Russell RG, Fleisch H: Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res. 1973, 11: 196-214. 10.1007/BF02547219.View ArticlePubMedGoogle Scholar
- Miller SC, Jee WS: Ethane-1-hydroxy-1, 1-diphosphonate (EHDP). Effects on growth and modeling of the rat tibia. Calcif Tissue Res. 1975, 18: 215-231. 10.1007/BF02546241.View ArticlePubMedGoogle Scholar
- Schenk R, Eggli P, Fleisch H, Rosini S: Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int. 1986, 38: 342-349. 10.1007/BF02555748.View ArticlePubMedGoogle Scholar
- Spadaro JA, Damron TA, Horton JA, Margulies BS, Murray GM, Clemente DA, Strauss JA: Density and structural changes in the bone of growing rats after weekly alendronate administration with and without a methotrexate challenge. J Orthop Res. 2006, 24: 936-944. 10.1002/jor.20145.View ArticlePubMedGoogle Scholar
- Kuhn JL, DeLacey JH, Leenellett EE: Relationship between bone growth rate and hypertrophic chondrocyte volume in New Zealand white rabbits of varying ages. J Orthop Res. 1996, 14: 706-711. 10.1002/jor.1100140505.View ArticlePubMedGoogle Scholar
- Lerner AL, Kuhn JL: Characterization of regional and age-related variations in the growth of the rabbit distal femur. J Orthop Res. 1997, 15: 353-361. 10.1002/jor.1100150307.View ArticlePubMedGoogle Scholar
- Falgueyret JP, Black WC, Cromlish W, Desmarais S, Lamontagne S, Mellon C, Riendeau D, Rodan S, Tawa P, Wesolowski G, Bass KE, Venkatraman S, Percival MD: An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells. Anal Biochem. 2004, 335: 218-227. 10.1016/j.ab.2004.09.005.View ArticlePubMedGoogle Scholar
- Falgueyret JP, Oballa RM, Okamoto O, Wesolowski G, Aubin Y, Rydzewski RM, Prasit P, Riendeau D, Rodan SB, Percival MD: Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and L. J Med Chem. 2001, 44: 94-104. 10.1021/jm0003440.View ArticlePubMedGoogle Scholar
- Robichaud J, Oballa R, Prasit P, Falgueyret JP, Percival MD, Wesolowski G, Rodan SB, Kimmel D, Johnson C, Bryant C, Venkatraman S, Setti E, Mendonca R, Palmer JT: A novel class of nonpeptidic biaryl inhibitors of human cathepsin K. J Med Chem. 2003, 46: 3709-3727. 10.1021/jm0301078.View ArticlePubMedGoogle Scholar
- Gauthier JY, Chauret N, Cromlish W, Desmarais S, Duong Le T, Falgueyret JP, Kimmel DB, Lamontagne S, Léger S, LeRiche T, Li CS, Massé F, McKay DJ, Nicoll-Griffith DA, Oballa RM, Palmer JT, Percival MD, Riendeau D, Robichaud J, Rodan GA, Rodan SB, Seto C, Therien M, Truong VL, Venuti MC, Wesolowski G, Young RN, Zamboni R, Black WC: The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K. Bioorg Med Chem Lett. 2008, 18: 923-928. 10.1016/j.bmcl.2007.12.047.View ArticlePubMedGoogle Scholar
- Leung P, Pickarski M, Zhuo Y, Masarachia PJ, Duong LT: The effects of the cathepsin K inhibitor odanacatib on osteoclastic bone resorption and vesicular trafficking. Bone. 2011, 49: 623-635. 10.1016/j.bone.2011.06.014.View ArticlePubMedGoogle Scholar
- Kumar S, Dare L, Vasko-Moser JA, James IE, Blake SM, Rickard DJ, Hwang SM, Tomaszek T, Yamashita DS, Marquis RW, Oh H, Jeong JU, Veber DF, Gowen M, Lark MW, Stroup G: 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. Bone. 2007, 40: 122-131. 10.1016/j.bone.2006.07.015.View ArticlePubMedGoogle Scholar
- Ochi Y, Yamada H, Mori H, Nakanishi Y, Nishikawa S, Kayasuga R, Kawada N, Kunishige A, Hashimoto Y, Tanaka M, Sugitani M, Kawabata K: Effects of ONO-5334, a novel orally-active inhibitor of cathepsin K, on bone metabolism. Bone. 2011, 49: 1351-1356. 10.1016/j.bone.2011.09.041.View ArticlePubMedGoogle Scholar
- Stroup GB, Hoffman SJ, Vasko-Moser JA, Lechowska BA, Jenkins EL, Dare LC, Gowen M: 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. Bone. 2001, 28: 532-537. 10.1016/S8756-3282(01)00421-5.View ArticlePubMedGoogle Scholar
- Sietsema WK, Ebetino FH, Salvagno AM, Bevan JA: Antiresorptive dose–response relationships across three generations of bisphosphonates. Drugs Exp Clin Res. 1989, 15: 389-396.PubMedGoogle Scholar
- Muhlbauer RC, Bauss F, Schenk R, Janner M, Bosies E, Strein K, Fleisch H: BM 21.0955, a potent new bisphosphonate to inhibit bone resorption. J Bone Miner Res. 1991, 6: 1003-1011.View ArticlePubMedGoogle Scholar
- Masarachia PJ, Pennypacker BL, Pickarski M, Scott KR, Wesolowski GA, Smith SY, Samadfam R, Goetzmann JE, Scott BB, Kimmel DB, Duong IT: Odanacatib reduces bone turnover and increases bone mass in the lumbar spine of skeletally mature ovariectomized rhesus monkeys. J Bone Miner Res. 2012, 27: 509-523. 10.1002/jbmr.1475.View ArticlePubMedGoogle Scholar
- Cusick T, Chen CM, Pennypacker BL, Pickarski M, Kimmel DB, Scott BB, Duong IT: Odanacatib treatment increases hip bone mass and cortical thickness by preserving endocortical bone formation and stimulating periosteal bone formation in the ovariectomized adult rhesus monkey. J Bone Miner Res. 2012, 27: 524-537. 10.1002/jbmr.1477.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/344/prepub
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.