Background
The lysosomal cysteine protease, Cathepsin K (CatK), is predominantly expressed in osteoclasts [
1] 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 [
2]. 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 [
6]. 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 [
9]. 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 [
10].
The preferred small animal model for evaluation of the efficacy of bone therapeutics is the ovariectomized (OVX) skeletally mature rat [
11]. 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 [
12]; 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 [IC
50 ]= 0.2 and 1 nM, respectively) but more than 500-fold less potent versus rat CatK (IC
50 = 112 nM) or mouse CatK (IC
50 = 108 nM) [
14]. 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 [
13]. 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) [
15]. 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) [
18]. 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 [
19]. 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.
Methods
Animals
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 [
22]. 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. IC
50 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 [
23]. 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 × 10
6 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)
2D
3 10 nM, and test compounds. The cultures were incubated for three days at 37°C in 5% CO
2. C-telopeptide of Type-I collagen (CTx-I) released into the medium was measured by the CROSSLAPS Elisa assay (Osteometer Biotech, Herlev, Denmark).
BMD analysis
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.
Statistical analysis
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.
Discussion
CatK is predominantly expressed in osteoclasts [
1], 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 [
6], 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 [
26].
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 [
15]. 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 [
15] and ODN in OVX NHPs [
32,
33].
Acknowledgments
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.
Competing interests
All authors were Merck’s employees during the execution of the studies as disclosed in this manuscript, and may own stock or stock options in the company. DK has received personal fees from Amgen, Bayer, Lexicon, Xradia and Arcarios.
Authors’ contributions
Study design: DK, RO; Assisting with study design: SL; Performing the experimental work: SL, BP; Performing the statistical analysis: DK, BP; Drafting of the manuscript: BP, RO, DK, LD, SL; All authors have read and approved the final manuscript.