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ENDOCRINE AND DIABETES

An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats

Dong-A Research Laboratories (M.K.K., H.D.K., J.H.P., J.I.L., J.S.Y., W.Y.K., S.Y.S., H.J.K., S.H.K., M.H.S.), Dong-A Pharmaceutical Co., Ltd., Gyeonggi-do, Republic of Korea; Yuhan Research Institute (C.H.L., J.Y.S., M.H.B., Y.A.S., Y.H., T.D.H., W.C., H.C., B.N.A.), Yuhan Corporation, Gyeonggi-do, Republic of Korea; and Department of Radiology (S.O.Y.), Eulji University Hospital, Daejeon, Republic of Korea.

Received February 13, 2006; accepted May 11, 2006.

	Abstract

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Human cathepsin K, a cysteine proteinase of the papain family, has been recognized as a potential drug target for the treatment of osteoporosis. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel antiresorptive drugs. Now, we report the pharmacological properties of OST-4077 [furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide] as a novel selective cathepsin K inhibitor. Human and rat cathepsin K were inhibited in vitro by OST-4077 with the IC₅₀ values of 11 and 427 nM, respectively. OST-4077 suppressed bone resorption induced by rabbit osteoclasts (IC₅₀, 37 nM) but did not affect bone mineralization or cellular alkaline phosphatase activity in MC3T3-E1 cells. Parathyroid hormone-induced bone resorption was inhibited in a dose-dependent manner in thyroparathyroidectomized rats gavaged with a single dose of OST-4077 (ED₅₀, 69 mg/kg). When given orally twice daily for 4 weeks to 3-month-old ovariectomized (OVX) rats, OST-4077 dose-dependently prevented bone loss, as monitored by bone densitometry, ash content, and urinary excretion of deoxypyridinoline. No change in serum osteocalcin in the OVX rats by OST-4077 suggested that bone formation might not be affected by the agent. In summary, OST-4077 selectively inhibited bone resorbing activities of osteoclasts and prevented bone loss induced by estrogen deficiency but did not affect bone formation. OST-4077, an orally active selective human cathepsin K inhibitor, may have the therapeutic potential for the treatment of diseases characterized by excessive bone loss including osteoporosis.

Evidence that cathepsin K is a major proteinase in humans can be found in a rare autosomal recessive condition known as pycnodysostosis, which is characterized by dwarfism, cranial anomalies, and increased bone mineral density (Gelb et al., 1996). Cathepsin K-deficient mice show osteopetrotic characteristics with increased trabeculation of the bone marrow space and no overt phenotypic abnormalities until the age of 10 months (Saftig et al., 1998). From these observations, cathepsin K has been focused on as a novel target for the treatment of osteoporosis.

In fact, the chemical inhibitors of cathepsin K have been shown to inhibit bone resorption in ovariectomized rats (Lark et al., 2002). Potent and efficacious inhibition of bone resorption markers by the chemical inhibitors have also been studied in nonhuman primates (Stroup et al., 2001). However, the cathepsin K inhibitors studied so far have been applied only parenterally. Furthermore, the effects of long term treatment with the agents on bone mineral density and bone turnover markers have not been examined in osteopenic animal models.

The researchers in our laboratories have synthesized a series of potential chemical inhibitors for human cathepsin K and examined their pharmacological efficacy. In this study, we describe the antiresorptive effects of OST-4077 in vitro and the long-term effects on bone loss in vivo. To our knowledge, this is the first report of an orally active cathepsin K inhibitor, which potently inhibits bone resorption in acute and chronic animal models.

	Materials and Methods

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Materials. Cloning and expression vectors for human and rat cathepsin K were pQE31 (QIAGEN GmbH, Hilden, Germany), pCR-TOPO vector, and pBlueBac4.5 (Invitrogen, Carlsbad, CA), respectively. Tissue culture media and supplements were purchased from Invitrogen. Bovine spleen cathepsin B was obtained from Sigma-Aldrich (St. Louis, MO). Human liver cathepsin L and bovine spleen cathepsin S were purchased from Calbiochem (La Jolla, CA). Recombinant human RANKL was purchased from Pepprotech (London, UK). Rat osteocalcin EIA kit was from Biomedical Technologies (Stoughton, MA). Serum CrossLaps One Step ELISA kit was purchased from Osteometer (Herlev, Denmark). Urinary deoxypyridinoline (DPD) EIA kit was purchased from Metra Biosystems (Mountain View, CA). Leukocyte acid phosphatase kit and other reagents without special mention were obtained from Sigma-Aldrich. A reference compound of cathepsin K inhibitor, SB357114 was synthesized at our laboratory.

Animals. This study was performed in accordance with the institutional Standard Procedure for Animal Care and Experiments of Dong-A Pharmaceutical and the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Sprague-Dawley rats were obtained from Orient Co., Ltd. (Seoul, Korea). After ovary removal, the animals were housed two heads per polycarbonate cage with wood shavings and were maintained under a controlled environment with temperature at 23 ± 2°C, relative humidity at 55 ± 5%, and a 12-h/12-h light/dark cycle throughout the experiment. The rats were given food and ultraviolet-sterilized tap water ad libitum.

Cloning, Expression, and Purification of Human Cathepsin K. Construction of human cathepsin K vector was carried out using the standard cloning procedure. The full-length cDNA of cathepsin K was obtained by PCR amplification with sense and antisense primers derived from the cDNA of MCF-7 human breast cancer cells (Littlewood-Evans et al., 1997). The PCR product was gel-purified and ligated into the pQE31 expression vector. The resulting construct encoding human cathepsin K gene was transformed into the Escherichia coli BL21 (DE3) pLysS expression cells. Transformed cells were grown in LB broth containing 100 µg/ml ampicillin for approximately 16 h, diluted 1/100 with the same medium, and grown to an A₆₀₀ of 1.0. Then, isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM, and the incubation was continued for another 3 h. Cells were collected and human cathepsin K protein was purified by Ni²⁺ affinity column chromatography. After renaturation, protein was used in enzyme inhibition assay of human cathepsin K (Bossard et al., 1996; Bromme et al., 1996; Linnevers et al., 1997).

Cloning and Expression of Rat Cathepsin K. The full-length cDNA encoding rat procathepsin K from a lung cDNA library (Takara, Shiga, Japan) was engineered for expression in baculovirus by PCR modification of the original cDNA cloned into the pCR-TOPO vector cloning vector. The oligonucleotide primers 5'-TGC-TGG-ATC-CAT-GCC-GGA-GGA-AAC-GCT-G-3' (containing a BamHI restriction site, underlined) and 5'-CTG-GTC-TAG-ATG-AAT-CAC-ATC-TTG-GGG-3' (containing an XbaI restriction site, underlined) were used for amplification of a 1-kb fragment. The PCR product was subcloned into the baculovirus vector, pBlueBac4.5, to create the plasmid pBlueBac-RCK (McQueney et al., 1998). To test the pBlueBac-RCK composite viruses for the expression of rat cathepsin K, Sf9 cells were infected at varying multiplicities of infection with pBlueBac-RCK, and the cell lysate and medium were collected every 24 to 96 h (Mitsudo et al., 2003). Collected crude proteins were used for the rat cathepsin K enzyme assay (Lark et al., 2002).

Cathepsin K Assays. The enzyme inhibition assay of human and rat cathepsin K was carried out according to the previously described method with minor modifications (Bromme et al., 1996). In brief, the substrate for both human and rat cathepsin K was 25 µM Z-Phe-Arg-AMC, and the composition of the final reaction buffer is comprised of the following buffers: 100 mM sodium acetate buffer, pH 5.5, containing 20 mM cysteine and 5 mM EDTA for human CK, and 50 mM MES buffer, pH 5.5, containing 4 mM DTT and 2 mM EDTA for rat CK, in the composition of the final 2% DMSO solution (Aibe et al., 1996). For the initiation of reaction, an enzyme solution was added to the substrate and inhibitor mixture, followed by incubation for 1 h at 37°C. Substrate hydrolysis was monitored with a SPECTRAFluor (Tecan, Salzburg, Austria) at excitation and emission wavelengths of 360 and 465 nm, respectively. The percentage inhibition of the reaction was calculated from a control reaction containing only vehicle.

Selectivity Assays. To measure enzyme activity of cathepsin B, L, and S, enzymatic assays were carried out in the respective assay buffers containing 1% DMSO. Each reaction buffer was a 45 mM phosphate buffer, pH 6.0, containing 0.9 mM DTT for cathepsin B, 50 mM sodium acetate, pH 5.5, containing 1 mM EDTA, 2 mM DTT for cathepsin L, and 100 mM phosphate buffer, pH 6.5, containing 5 mM EDTA, 5 mM DTT, and 0.01% Triton X-100 for cathepsin S. Inhibitory activities of compounds were determined using 90 µM Z-Arg-Arg-pNA for bovine cathepsin B and 5 and 15 µM of Z-Phe-Arg-AMC for human cathepsin L and bovine cathepsin S as a substrate. Before the addition of substrate, the inhibitors were preincubated at 25°C for 10 min with the enzyme to allow the establishment of the enzyme-inhibitor complex. Substrate was then added and incubated at 25°C for 1 h. The enzyme activity was measured from the increase of absorbance (405 nm) for p-nitroanilide or fluorescence (_ex 360 nm, _em 465 nm) for aminomethylcoumarin with a SPECTRAFlour (Tecan). All experiments were carried out in duplicate. IC₅₀ values were determined by four-parameter fits from the percentage of inhibition versus inhibitor concentration using the software program SigmaPlot 4.0 (SPSS, Inc., Chicago, IL).

Rabbit Bone Resorption Assay. Unfractionated bone cells were isolated from rabbit long bones as described previously (Kaneko et al., 2000). The femora and tibiae of 1-day-old rabbits were dissected out. After removal of adherent soft tissues, the bones were minced in -MEM, pH 6.8, containing 5% FBS for 5 min. The cells were dissociated from the bone fragments by brief vortexing, and the fragments were then allowed to sediment for 2 min. The cells in the supernatant were collected and used as unfractionated bone cells.

For the bone resorption assay, isolated bone cells (5 x 10⁵ cells) were plated and attached on a sterilized dentine slice (6 mm in diameter, 100-200 µm thickness) in -MEM supplemented with 5% FBS in each well of a 96-well plate. After 2-h incubation in humidified air at 37°C in 5% CO₂, the medium was replaced with either fresh media containing the test compound or vehicle. The cells were then cultured for 48 h under the same conditions; thereafter, the supernatant was harvested, and the CTx levels in the media were measured using an ELISA kit. The bone cells on dentine slices were fixed with 60% acetone in a citrate buffer for 30 s and washed with phosphate-buffered saline and stained for TRAP activity using a kit. All experiments were carried out in triplicate.

For the quantification of TRAP-positive multinucleated cells, isolated bone cells were plated in each well of a 96-well plate and treated with the same method as described above. The number of TRAP-positive multinucleated cells (>two nuclei per cell) in each well of a 96-well plate were measured using an Olympus IX 71 microscope (Olympus Co., Tokyo, Japan). Data were represented as mean ± S.E.M. from five determinants.

Human Bone Resorption Assay. Human peripheral blood was obtained from healthy adult volunteers and osteoclast-like cells were generated using slight modifications of previously described methods (Fujikawa et al., 2001; Massey et al., 2001). In brief, heparinized whole blood was collected and diluted 1:1 in -MEM, and layered over Ficoll-Paque. After centrifugation, isolated PBMCs (1 x 10⁵) were plated on dentine slices in a 96-well plate in a final volume of 200 µl of a medium containing reagents. Media contained 25 ng/ml M-CSF and test compounds throughout the experiments, 10 ng/ml TGF- for the first 3 days, and 30 ng/ml RANKL for days 3 to 19. Cultures were maintained at 37°C in 5% CO₂ humidified incubator. On day 3, 90% of the medium was removed and replaced with fresh medium containing cytokines and compounds. Thereafter, half of the medium was exchanged every 3 days. On day 19, whole media were removed, washed twice with fresh media, and replenished with fresh media containing cytokines and test compounds. After incubation for an additional 24 h, the conditioned media were harvested, and then the CTx level was measured. All experiments were carried out in triplicate. IC₅₀ values were calculated by the nonlinear regression method using Sigma Plot 4.0.

Bone Mineralization and Alkaline Phosphatase Assay. Bone mineralization was induced as described previously (Spinella-Jaegle et al., 2001). In brief, MC3T3-E1 mouse preosteoblast-like cells were plated in 24-well plates at a density of 1 x 10⁴/well and incubated in -MEM supplemented with 10% FBS. Bone mineralization was induced by the addition of 50 µg/ml ascorbic acid and 10 mM -glycerophosphate to the medium. Cells were either left untreated or treated with BMP-2 or OST-4077 for 19 days. At the end of each experiment, the cultures were rinsed with phosphate-buffered saline and fixed with 10% formalin. Fixed cultures were rinsed three times with distilled water, and stained with 2% alizarin red S for 5 min at room temperature with gentle agitation. Cultures were then washed three times with distilled water and photographed. For cellular alkaline phosphatase assay, on day 17, the medium was replenished with serum-deprived -MEM. After starved for 48 h, cell layers were detached and disrupted by an ultrasonicator (Sonics and Materials, Danbury, CT). Ten microliters of cytosolic supernatant was incubated with 100 µl of 2 mg/ml p-nitrophenyl phosphate in alkaline buffer for 15 min at 37°C. The reaction was terminated by adding 100 µl of ice-cold 0.5 N NaOH. The increase of released p-nitrophenol was measured at 405 nm using the SPECTRAFluor (Tecan). Total protein concentration of lysate was determined by the bicinchoninic acid method according to the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). The data were expressed as nanomoles of p-nitrophenol per milligram of protein.

PTH-Induced Bone Resorption in TPTX Rats. The TPTX rat model was used according to the previously described methods (Votta et al., 1997). Male Sprague-Dawley rats were allowed to acclimate for at least 7 days. The animals had free access to tap water and standard rat chow until the day of TPTX. Surgical TPTX was performed under anesthesia with pentothal sodium (40 mg/kg i.v.) on rats weighing 300 to 350 g. TPTX rats were allowed to recover under fasting conditions with free access to tap water. On the day following the surgery, femoral artery and vein were catheterized under ether anesthesia. The plasma calcium concentration was measured, and success of TPTX (plasma calcium < 7.5 mg/dl) was confirmed before animals were allocated to study groups. OST-4077 or 0.5% methylcellulose was orally administered to rats and 30 min later, rhPTH(1-34) or vehicle (0.025% BSA in saline) was infused at a rate of 0.3 nmol/kg/h. Plasma calcium levels were measured just before rhPTH(1-34) infusion and every 1 h for 6 h thereafter, using an automated clinical chemistry analyzer, Konelab 20i (Thermo Electron Corporation, Waltham, MA) according to manufacturer's instructions. ED₅₀ was mathematically calculated by detecting the compound-induced reduction of area under the curve (0-6 h) at several doses compared with the rhPTH(1-342)-treated group.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Chemical structures of OST-4077 and SB357114.

View larger version (15K): [in this window] [in a new window]   Fig. 2. A, OST-4077 () and SB357114 () inhibited human cathepsin K activities in a concentration-dependent manner. Rat cathepsin K inhibition () by OST-4077 was found at 39-fold higher concentration than human cathepsin K. Assay conditions are described under Materials and Methods. B, OST-4077 inhibited enzyme activities of cathepsin B (), cathepsin L (), and cathepsin S ().

Measurement of BMD and Bone Mineral Contents. Femora were isolated, and the adherent tissues were removed. BMD was measured by Lunar PIXImus II densitometer (GE Healthcare, Waukesha, WI) using dual energy X-ray absorptiometric technique. The coefficient of variation of the machine was 0.84%. Paired femora in crucibles were ashed using a furnace (600°C, 24 h) and weighed. Femur ash weights were divided by the length of femurs, measured from great trochanter to the external condyle (Rodan et al., 1993; Caselli et al., 1997). Data were expressed as milligrams per millimeters.

Bone Turnover Marker. The level of serum osteocalcin was measured by EIA specific for rat osteocalcin according to the manufacturer's instruction. The concentration of urinary total DPD was determined using EIA kit and normalized with the concentration of urinary creatinine concentration.

Statistical Analysis. Statistics were computed using the SigmaStat 5.0 software program (SPSS). The data were analyzed using one-way analysis of variance. When the analysis of variance performed over all groups indicated a significant (p < 0.05) difference among the groups, statistical differences between groups were subsequently evaluated with Student-Newman-Keuls multiple comparison test. A value of p < 0.05 was considered significant. The data are presented as the mean ± S.E.M.

	Results

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Cathepsin K Inhibition and Selectivity. OST-4077 is a potent inhibitor of human cathepsin K (IC₅₀, 11 nM), and the inhibitory activity against human CK is 39-fold more potent than against rat CK (Fig. 1). SB357114 showed concentration-dependent inhibition with an IC₅₀ of 3.5 nM (Fig. 2A). OST-4077 is more selective for inhibition of cathepsin K than cathepsin B (IC₅₀, 239 nM), L (IC₅₀, 981 nM), or S (IC₅₀, 196 nM) (Fig. 2B).

View larger version (63K): [in this window] [in a new window]   Fig. 3. After 48-h cultivation of unfractionated rabbit bone cells on dentine slices, TRAP-positive osteoclast-like cells were identified. Arrows, TRAP-positive multinucleated cells on dentine slices. Original magnification, x200. There were no significant changes in the number of TRAP-positive multinucleated cells between vehicle (A)- or 300 nM OST-4077 (B)-treated groups. OST-4077 and SB357114 inhibited osteoclast-mediated CTx release in a concentration-dependent manner (C). Data from five different experiments were expressed as mean ± S.E.M. of percent inhibition compared with vehicle control. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle control.

Inhibition of Bone Resorbing Activity by Human Osteoclasts. Treatment with hM-CSF and hTGF/hRANKL differentiated human PBMCs into TRAP-positive multinucleated osteoclast-like cells (Fig. 4, B and D). These cells on bone slices resorbed bone and left resorption pits on the surface (Fig. 4D). Concentration-dependent inhibition of the pit formation by OST-4077 was observed under microscopy (Fig. 4, E and F). OST-4077 and SB357114 inhibited the CTx release from dentine slices with IC₅₀ of 205 and 113 nM, respectively (Fig. 4G). The relative potency between OST-4077 and SB-357114 coincided with the results of human CK inhibition assay and rabbit bone resorption assay.

View larger version (62K): [in this window] [in a new window]   Fig. 4. TRAP-positive osteoclast-like cells on plastic plates (A and B) and dentine slices (C-F) in the absence (A and C) or presence (B and D-F) of hM-CSF and hTGF/hRANKL. Isolated human PBMCs were treated with or without compounds throughout the whole experimental period (20 days): 100 nM (E) and 1000 nM (F) OST-4077. Original magnifications, x200. Arrows, TRAP-positive multinucleated cells; arrowheads, traces that remained after bone resorption by multinucleated osteoclast-like cells. G, OST-4077 inhibited the CTx release by human PBMC-derived osteoclast-like cells in a concentration-dependent manner. The IC50 values of OST-4077 and SB357114 were calculated as 205 and 113 nM, respectively. The CTx level in the last 24-h-conditioned media were measured using Serum CrossLaps One Step ELISA kit. Data from five different experiments were expressed as mean ± S.E.M. of percent inhibition compared with vehicle control. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle control.

View larger version (59K): [in this window] [in a new window]   Fig. 5. MC3T3-E1 cells were cultured in mineralization medium containing 10% FBS and ascorbic acid for 19 days. Cells were treated with BMP-2 (A) or OST-4077 (B) at the indicated concentrations. The mineralized matrix was stained with Alizarin Red-S for calcium. Experiments were performed in triplicate. OST-4077 did not increase cellular ALP activities, an early marker of osteoblast differentiation (D). However, BMP-2 at 50 ng/ml significantly stimulated the expression of ALP (C). **, p < 0.01 versus vehicle control.

Antiresorptive Effects in TPTX Rats. In the preliminary pharmacokinetic study, OST-4077 showed a good oral availability (>80%) and favorable terminal half-life (>4 h) in rats. Because of its favorable pharmacokinetic characteristics as well as relatively potent inhibitory activity against rat CK, antiresorptive effects of OST-4077 were evaluated in the TPTX rat model. PTH infusion was started 0.5 h after oral treatment of OST-4077 considering T_max of 0.5 h. Dose-related inhibition of the calcemic response by rhPTH(1-34) was observed with an ED₅₀ of 69 mg/kg (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6. A, PTH-induced bone resorption was suppressed by OST-4077 in TPTX rats. PTH was infused 0.5 h after oral dosing of OST-4077 or vehicle. Blood samples were collected over the 6-h duration. Data are represented as mean ± S.E.M. of net change in plasma calcium level from each determinant (n = 7-10/group). , 0.5% methylcellulose; , PTH; , 10 mg/kg; , 30 mg/kg; , 100 mg/kg OST-4077. B, OST-4077 significantly reduced the PTH-induced rise in area under the curve (0-6 h) by plasma calcium levels in a dose-dependent manner. ###, p < 0.001 versus vehicle control; *, p < 0.05; ***, p < 0.001 versus PTH control.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Effects of OST-4077 on bone mass and turnover in 3-month-old OVX rats (n = 10/group). When orally administered by 10, 20, 50, and 100 mg/kg twice daily, OST-4077 preserved bone mineral density (A) and bone ash contents (B) in paired femora comparable with the level of sham control. Increased urinary DPD excretion by OVX was significantly inhibited at high doses of OST-4077 (C) without affecting the level of serum osteocalcin (D). #, p < 0.05 versus sham control; *, p < 0.05 versus OVX control.

	Discussion

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To ascertain whether the CK inhibitor cause any changes in osteoblasts, preosteoblast MC3T3-E1 cells were treated with OST-4077. In this study, OST-4077 did not induce any significant changes in bone-like nodule formation or ALP activities, whereas BMP-2 significantly increased these osteoblast differentiation markers. It is well documented that the CK is mainly localized within osteoclasts and is absent or at low frequency in other tissues (Drake et al., 1996). Therefore, these results mean that the pharmacological activities of selective cathepsin K inhibitors such as OST-4077 are restricted within osteoclasts.

It was reported that catalytic sites of CK are different between humans and rats (Tepel et al., 2000). Because of this species difference, in vivo elucidation of the pharmacological activities of CK inhibitor has been hampered. Although OST-4077 is a weak rat CK inhibitor with an IC₅₀ of 427 nM, its pharmacokinetic profile is favorable enough to reach approximately 20 µmol/ml C_max when administered at 50 or 100 mg/kg, orally. These characteristics enabled us to study the pharmacological activities of this CK inhibitor in vivo as well as in vitro.

Oral administration of OST-4077 partially prevented PTH-induced calcemic response in the TPTX rat model. Because the results are expressed as changes of plasma calcium concentration, these results mean that this selective CK inhibitor can partially prevent demineralization. This coincides with the results that peptide aldehyde CK inhibitors can prevent calcium release in a fetal rat long bone assay in vitro (Yasuma et al., 1998). In this acute bone resorption model, the inhibitory efficacy of OST-4077 is comparable with that of another CK inhibitor, SB357114, that showed a maximum inhibition of 57% at plasma level of 10 µmol/ml (Lark et al., 2002).

OST-4077 prevented bone resorption in the OVX rats, resulting in prevention of the OVX-induced bone loss. The compound inhibited OVX-induced decreases of BMD and mineral contents in femora dose-dependently. Furthermore, OST-4077 inhibited excretion of the bone resorption marker (urinary DPD) but did not affect the bone formation marker (serum osteocalcin). It is well known that bone formation and resorption are tightly coupled. However, these results indicate that this selective CK inhibitor reduced the bone resorption rate without modulating one of the bone formation markers in the OVX rats characterized by the increased bone turnover rate for at least 4 weeks. According to the previous evidence, alendronate as an antiresorptive, completely blocked bone resorption with bone formation rate at the periosteal surface still increased (Pennypacker et al., 2004). This indicates alendronate partly uncoupled the bone turnover. However, since data for the histomorphometrical change was not made, observations to these markers influenced by OST-4077 could not be interpreted directly as the explanation for the bone turnover uncoupling.

From these in vivo results, it appears that CK inhibitors can inhibit demineralization as well as matrix degradation as evaluated by urinary DPD. Because bone demineralization and protein matrix degradation are linked, inhibition of CK-mediated proteolysis would result in maintaining an intact mineralized matrix. Furthermore, CK knockout mice showed increased bone mass and strength (Pennypacker et al., 1999), suggesting that prevention of matrix degradation does not cause negative effects on bone integrity.

Taken together, these data suggests that a potent and selective CK inhibitor such as OST-4077 can be effective in preventing bone loss in vivo and that such a compound may be therapeutically beneficial for the treatment of diseases with accelerated bone loss such as postmenopausal osteoporosis.

	Footnotes

 
This work was supported by the Korea Health 21 R&D Project, Republic of Korea (Grant A010204).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.102798.

ABBREVIATIONS: OST-4077, furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide; RANKL, receptor activated nuclear factor B ligand; DPD, deoxypyridinoline; ELISA, enzyme-linked immunosorbent assay; SB357114, N-[3-methyl-1(S)-[N-[3-oxo-1-(pyridin-2-ylsulfonyl)perhydroazepin-4(S)-yl]carbamoyl]butyl]-1-benzofuran-2-carboxamide; PCR, polymerase chain reaction; CK, cathepsin K; DTT, dithiothreitol; MEM, minimum Eagle's medium; CTx, C-telopeptide of type 1 collagen; FBS, fetal bovine serum; TRAP, tartrate-resistant acid phosphatase; PBMC, peripheral blood mononuclear cell; TPTX, thyroparathyroidectomized; OVX, ovariectomized; BMD, bone mineral density; ALP, alkaline phosphatase; PTH, parathyroid hormone; rhPTH(1-34), recombinant human PTH fragment (1-34); BMP, bone morphogenetic protein; DMSO, dimethyl sulfoxide.

Address correspondence to: Dr. Moon-Ho Son, 47-5, Sanggal-Dong, Giheung-Gu, Yongin-Si, Gyeonggi-Do 446-905, Republic of Korea. E-mail: ssonmh{at}donga.co.kr

	References

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Aibe K, Yazawa H, Abe K, Teramura K, Kumegawa M, Kawashima H, and Honda K (1996) Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits. Biol Pharm Bull 19: 1026-1031.[Medline]

Bossard MJ, Tomaszek TA, Thompson SK, Amegadzie BY, Hanning CR, Jones C, Kurdyla JT, McNulty DE, Drake FH, Gowen M, et al. (1996) Proteolytic activity of human osteoclast cathepsin K. J Biol Chem 271: 12517-12524.[Abstract/Free Full Text]

Bromme D, Okamoto K, Wang BB, and Biroc S (1996) Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. J Biol Chem 271: 2126-2132.[Abstract/Free Full Text]

Caselli G, Mantovanini M, Gandolfi CA, Allegretti M, Fiorentino S, Pellegrini L, Melillo G, Bertini R, Sabbatini W, Anacardio R, et al. (1997) Tartronates: a new generation of drugs affecting bone metabolism. J Bone Miner Res 12: 972-981.[CrossRef][Medline]

Drake FH, Dodds RA, James IE, Connor JR, Debouck C, Richardson S, Lee-Rykaczewski E, Coleman L, Rieman D, Barthlow R, et al. (1996) Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 271: 12511-12516.[Abstract/Free Full Text]

Einhorn TA (1996) The bone organ system: form and function, in Osteoporosis (Marcus R, Feldman D, and Kelsey J eds) pp 3-22, Academic Press, New York.

Fujikawa Y, Sabokbar A, Neale SD, Itonaga I, Torisu T, and Athanasou NA (2001) The effect of macrophage-colony stimulating factor and other humoral factors (interleukin-1, -3, -6, and -11, tumor necrosis factor-, and granulocyte macrophage-colony stimulating factor) on human osteoclast formation from circulating cells. Bone (NY) 28: 261-267.

Gelb BD, Shi GP, Chapman HA, and Desnick RJ (1996) Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science (Wash DC) 273: 1236-1238.[Abstract]

Hou WS, Bromme D, Zhao Y, Mehler E, Dushey C, Weinstein H, Miranda CS, Fraga C, Greig F, Carey J, et al. (1999) Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis. J Clin Investig 103: 731-738.[Medline]

Kamiya T, Kobayashi Y, Kanaoka K, Nakashima T, Kato Y, Mizuno A, and Sakai H (1998) Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts. J Biochem (Tokyo) 123: 752-759.[Abstract/Free Full Text]

Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, Toyama Y, Yabe Y, Kumegawa M, and Hakeda Y (2000) Direct stimulation of osteoclastic bone resorption by bone morphogentic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone (NY) 27: 479-486.

Lark MW, Stroup GB, James IE, Dodds RA, Hwang SM, Blake SM, Lechowska BA, Hoffman SJ, Smith BR, Kapadia R, et al. (2002) A potent small molecule, nonpeptide inhibitor of cathepsin K (SB 331750) prevents bone matrix resorption in the ovariectomized rat. Bone (NY) 30: 746-753.

Linnevers CJ, McGrath ME, Armstrong R, Mistry FR, Barnes MG, Klaus JL, Palmer JT, Katz BA, and Bromme D (1997) Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex. Protein Sci 6: 919-921.[Medline]

Littlewood-Evans AJ, Bilbe G, Bowler WB, Farley D, Wlodarski B, Kokubo T, Inaoka T, Sloane J, Evans DB, and Gallagher JA (1997) The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma. Cancer Res 57: 5386-5390.[Abstract/Free Full Text]

Massey HM, Scopes J, Horton HA, and Flanagan AM (2001) Transforming Growth Factor-1(TGF-) stimulates the osteoclast-forming potential of peripheral blood hematopoietic precursors in a lymphocyte-rich microenvironment. Bone (NY) 28: 577-582.

McQueney MS, Feild J, Hanning CR, Brun K, Ramachandran K, Connor J, Drake F, Jones CS, and Amegadzie BY (1998) Cynomolgus monkey (Macaca fascicularis) cathepsin K: cloning, expression, purification, and activation. Protein Exp Purif 14: 387-394.[CrossRef][Medline]

Mitsudo K, Jayakumar A, Henderson Y, Frederick MJ, Kang Y, Wang M, El-Naggar AK, and Clayman GL (2003) Inhibition of serine proteinases plasmin, trypsin, subtilisin A, cathepsin G, and elastase by LEKTI: a kinetic analysis. Biochemistry 42: 3874-3881.[CrossRef][Medline]

Pennypacker BL, Shea M, Saftig P, Rodan SB, Rodan GA, and Kimmel DB (1999) Bone density and strength in cathepsin K null mice. J Bone Miner Res 14: S549.

Pennypacker BL, Cusick T, Masarachia P, and Kimmel DB (2004) Effect of alendronate in a lapine model of estrogen deficiency. J Bone Miner Res 19: S444.

Rodan GA, Seedor JG, and Balena R (1993) Preclinical pharmacology of alendronate. Osteoporos Int 3: S7-12.

Saftig P, Hunziker E, Wehmeyer O, Johnes S, Boyde A, Rommerskirch W, Moritz JD, Schu P, and Figura K (1998) Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci USA 95: 13453-13458.[Abstract/Free Full Text]

Spinella-Jaegle S, Roman-Roman S, Fauchen C, Dunn FW, Kawai S, Gallea S, Stoit V, Blanchet AM, Courtois B, Baron R, et al. (2001) Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone (NY) 29: 323-330.

Stroup GB, Lark MW, Veber DF, Bhattacharyya A, Blake S, Dare LC, Erhard KF, Hoffman SJ, James IE, Marquis RW, et al. (2001) Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate. J Bone Miner Res 16: 1739-1746.[CrossRef][Medline]

Tepel C, Bromme D, Herzog V, and Brix K (2000) Cathepsin K in thyroid epithelial cells: sequence, localization and possible function in extracellular proteolysis of thyroglobulin. J Cell Sci 113: 4487-4498.[Abstract]

Votta BJ, Levy MA, Badger A, Bradbeer J, Dodds RA, James IE, Thompson S, Bossard MJ, Carr T, Connor JR, et al. (1997) Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo. J Bone Miner Res 12: 1396-1406.[CrossRef][Medline]

Yasuma T, Oi S, Choh N, Nomura T, Furuyama N, Nishimura A, Fujisawa Y, and Sohda T (1998) Synthesis of peptide aldehyde derivatives as selective inhibitors of human cathepsin L and their inhibitory effect on bone resorption. J Med Chem 41: 4301-4308.[CrossRef][Medline]

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