Effects of the Cathepsin K Inhibitor ONO-5334 and Concomitant Use of ONO-5334 with Methotrexate on Collagen-Induced Arthritis in Cynomolgus Monkeys.

We examined whether the cathepsin K inhibitor, ONO-5334, administered alone or in combination with methotrexate (MTX), could ameliorate joint destruction evoked by collagen-induced arthritis (CIA) in female cynomolgus monkeys. CIA was induced by immunizing with bovine type II collagen. ONO-5334 (30 mg/kg/day) was orally administered once daily and MTX (10 mg/body/day) twice weekly for 9 weeks. X-ray (evaluation of joint destruction) and swelling (inflammatory) scores of proximal interphalangeal (PIP), distal interphalangeal (DIP), and metacarpophalangeal (MP) joints were evaluated. Urinary concentrations of C-terminal telopeptide of type I collagen (CTX-I) and type II collagen (CTX-II) were measured. Arthritis, accompanied by bone and cartilage destruction, was successfully induced in this collagen-induced arthritis monkey model. ONO-5334 showed no suppressive effect on joint swelling, while the joint swelling scores in the MTX and combination (ONO-5334 + MTX) groups were less than 50% compared with the control group. ONO-5334 decreased X-ray score by a mean of 64% (p<0.05 vs the control group), and MTX also decreased in X-ray score by a mean of 46% but with no statistical significance. Combination of ONO-5334 and MTX further decreased the X-ray score by 28% over MTX group (74% reduction vs the control group, p<0.01). Maximum increase in CTX-I (10-fold) and CTX-II (7-fold) compared to baseline was observed at 7 and 3 weeks after the first sensitization, respectively. After treatment with ONO-5334 alone or in combination with MTX, concentrations were maintained near baseline for both markers. In conclusion, ONO-5334 prevented joint destruction but not joint inflammation in this monkey CIA model. Concomitant use of ONO-5334 with MTX reduced architectural joint destruction compared to MTX alone; therefore, ONO-5334 may help to prevent joint destruction in combination with MTX for the treatment of rheumatoid arthritis.

Cortical bone mineral density is increased by the cathepsin K inhibitor ONO-5334, which leads to a robust increase in bone strength: results from a 16-month study in ovariectomised cynomolgus monkeys.

This study evaluated the long-term effects of the cathepsin K inhibitor ONO-5334 on bone mass and strength in ovariectomised (OVX) cynomolgus monkeys. Animals were assigned to one of the following six groups: Sham (non-OVX), OVX control treated with vehicle, ONO-5334 1.2, 6 or 30 mg/kg/day, p.o., or alendronate (ALN) 0.05 mg/kg/2 weeks, i.v. for 16 months. Peripheral quantitative computed tomography (pQCT) analysis revealed that ONO-5334 increased not only trabecular bone mineral density (BMD) but also cortical BMD in the distal radius and the lumbar vertebra. ONO-5334 and ALN suppressed the deterioration of trabecular architecture by micro-CT analysis in the distal radius. Assessments of bone strength showed that ONO-5334 increased maximum load at the distal and midshaft radius. The linear regression lines between bone mass and strength in the lumbar vertebra were tended to be shifted towards increasing bone strength in the ONO-5334 6 and 30 mg/kg groups compared with the ALN groups. This indicated that bone strength was higher in the ONO-5334 groups than the ALN group, even though bone mineral content (BMC) and BMD were comparable. Subpopulation analysis revealed that, at similar integral BMC or BMD level, cortical bone mass for ONO-5334 was higher than for ALN; the opposite effects were observed for trabecular bone. In conclusion, ONO-5334 preferentially increased cortical bone, which may provide a greater contribution to bone strength. Since these results support a different mode of action for ONO-5334 compared with that of ALN, ONO-5334 may offer new therapeutic options to patients with osteoporosis.

Increased expression of CysLT2 receptors in the lung of asthmatic mice and role in allergic responses.

Compared with CysLT1 receptors, the functional role of CysLT2 receptors in asthma has not been clarified. The purpose of this study was to determine 1) whether CysLT2 receptors are expressed in the lung of mice and if expression increases in asthmatic mice, and 2) whether CysLT2 receptors are involved in allergic leukocyte infiltration into the lung and in the development of airway remodeling in asthmatic mice. BALB/c mice were sensitized with ovalbumin (OVA) + Al(OH)3, and intratracheally challenged with OVA 4 times. Lung tissue was isolated before and after the 4th OVA challenge for detection of CysLT2 receptors by immunohistochemistry and flow cytometry. The effect of a CysLT2 receptor antagonist BayCysLT2RA on multiple antigen challenge-induced leukocyte infiltration into the lung and the development of airway remodeling was evaluated. Even in non-challenged mice, CysLT2 receptors were expressed in bronchial smooth muscle. After multiple challenges, expression was also observed in leukocytes infiltrating into alveolar spaces. CysLT2R+ leukocytes included alveolar macrophages, conventional dendritic cells, and eosinophils. BayCysLT2RA significantly inhibited multiple antigen challenge-induced increases in eosinophils and mononuclear cells in the lung. The development of airway remodeling was tended to be suppressed by CysLT2 receptor antagonist. In conclusion, CysLT2 receptors were constitutively expressed in the lung, and expression was strengthened in asthmatic mice. Activation of CysLT2 receptors was functionally involved in allergic leukocyte infiltration into the lung. The CysLT2 receptor can be a molecular target for the development of new pharmacotherapies for asthma.

A new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model.

Although the effectiveness of CysLT1 receptor antagonists on asthma has been clinically established, the effects of CysLT2 receptor antagonists are still unclear. The purpose of this study was to develop a new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model using S-hexyl GSH, a γ-glutamyl transpeptidase (GTP) inhibitor, to suppress conversion of LTC4 to LTD4. Actively sensitized guinea pigs were challenged with OVA in the absence or presence of S-hexyl GSH, and survival rate following anaphylactic response was monitored. OVA-induced fatal anaphylaxis in the absence of S-hexyl GSH was almost completely inhibited by montelukast, a CysLT1 receptor antagonist, but not by the CysLT2 receptor antagonist BayCysLT2RA. However, under treatment with S-hexyl-GSH, the inhibitory effect of motelukast was dramatically diminished, whereas that of BayCysLT2RA was markedly increased. The dual CysLT1/2 receptor antagonist ONO-6950 effectively inhibited anaphylactic response in both S-hexyl GSH-treated and non-treated animals. LC/MS/MS analysis revealed that S-hexyl GSH treatment actually inhibited LTC4 metabolism in the blood and lung tissues. Using S-hexyl GSH, we developed a novel CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model that can be useful for not only screening both CysLT2 and CysLT1/2 receptors antagonists, but also for functional analysis of CysLT2 receptors.

CysLT2 receptor activation is involved in LTC4-induced lung air-trapping in guinea pigs.

CysLT1 receptors are known to be involved in the pathogenesis of asthma. However, the functional roles of CysLT2 receptors in this condition have not been determined. The purpose of this study is to develop an experimental model of CysLT2 receptor-mediated LTC4-induced lung air-trapping in guinea pigs and use this model to clarify the mechanism underlying response to such trapping. Because LTC4 is rapidly converted to LTD4 by γ-glutamyltranspeptidase (γ-GTP) under physiological conditions, S-hexyl GSH was used as a γ-GTP inhibitor. In anesthetized artificially ventilated guinea pigs with no S-hexyl GSH treatment, i.v. LTC4-induced bronchoconstriction was almost completely inhibited by montelukast, a CysLT1 receptor antagonist, but not by BayCysLT2RA, a CysLT2 receptor antagonist. The inhibitory effect of montelukast was diminished by treatment with S-hexyl GSH, whereas the effect of BayCysLT2RA was enhanced with increasing dose of S-hexyl GSH. Macroscopic and histological examination of lung tissue isolated from LTC4-/S-hexyl-GSH-treated guinea pigs revealed air-trapping expansion, particularly at the alveolar site. Inhaled LTC4 in conscious guinea pigs treated with S-hexyl GSH increased both airway resistance and airway hyperinflation. On the other hand, LTC4-induced air-trapping was only partially suppressed by treatment with the bronchodilator salmeterol. Although montelukast inhibition of LTC4-induced air-trapping was weak, treatment with BayCysLT2RA resulted in complete suppression of this air-trapping. Furthermore, BayCysLT2RA completely suppressed LTC4-induced airway vascular hyperpermeability. In conclusion, we found in this study that CysLT2 receptors mediate LTC4-induced bronchoconstriction and air-trapping in S-hexyl GSH-treated guinea pigs. It is therefore believed that CysLT2 receptors contribute to asthmatic response involving air-trapping.

Minodronic acid ameliorates vertebral bone strength by increasing bone mineral density in 9-month treatment of ovariectomized cynomolgus monkeys.

The effect of treatment for 9months with minodronic acid, a nitrogen-containing bisphosphonate, on vertebral mechanical strength was examined in ovariectomized (OVX) cynomolgus monkeys. Forty skeletally mature female monkeys were randomized into four OVX groups and one sham group (n=8) based on lumbar bone mineral density (BMD). OVX animals were treated orally with 15 and 150µg/kg QD of minodronic acid or 500µg/kg QD alendronate as a reference drug. Measurements of bone turnover markers and lumbar BMD were conducted at 0, 4 and 8months. Measurements of bone mechanical strength and minodronic acid concentration in vertebral bodies were also performed. OVX resulted in a decrease in lumbar BMD and an increase in bone turnover markers at 4 and 8months, compared to the sham group, and the ultimate load on the lumbar vertebra was decreased in OVX animals. Minodronic acid and alendronate prevented the OVX-induced increase in bone turnover markers and decrease in lumbar BMD. Minodronic acid at 150µg/kg increased the ultimate load on lumbar vertebra compared to untreated OVX animals. Regression analysis revealed that the ultimate load was correlated with lumbar BMD and bone mineral content (BMC), and most strongly with the increase in lumbar BMD and BMC over 8months. In a separate analysis within the sham-OVX controls and minodronic acid and alendronate treatment groups, the ultimate loads were also correlated with BMD and BMC. The load-BMD (BMC) correlation in the minodronic acid group showed a trend for a shift to a higher load from the basal relationship in the sham-OVX controls. These results indicate that treatment with minodronic acid for 9months increases vertebral mechanical strength in OVX monkeys, mainly by increasing BMD and BMC.

Effects of 16-month treatment with the cathepsin K inhibitor ONO-5334 on bone markers, mineral density, strength and histomorphometry in ovariectomized cynomolgus monkeys.

We examined the effects of ONO-5334, a cathepsin K inhibitor, on bone markers, BMD, strength and histomorphometry in ovariectomized (OVX) cynomolgus monkeys. ONO-5334 (1.2, 6 and 30mg/kg/day, p.o.), alendronate (0.05mg/kg/2weeks, i.v.), or vehicle was administered to OVX monkeys (all groups N=20) for 16months. A concurrent Sham group (N=20) was also treated with vehicle for 16months. OVX significantly increased bone resorption and formation markers and decreased BMD in lumbar vertebra, femoral neck, proximal tibia and distal radius. Alendronate suppressed these parameters to a level similar to that in the Sham-operated monkeys. ONO-5334 at doses 6 and 30mg/kg decreased bone resorption markers to a level roughly half of that in the Sham group, while keeping bone formation markers level above that in the Sham monkeys. Changes in DXA BMD confirmed that ONO-5334 at doses 6 and 30mg/kg increased BMD to a level greater than that in the Sham group in all examined sites. In the proximal tibia, in vivo pQCT analysis showed that ONO-5334 at doses 6 and 30mg/kg suppressed trabecular BMD loss to the sham level. However, ONO-5334 increased cortical BMD, cortical area and cortical thickness to a level greater than that in the Sham group, suggesting that ONO-5334 improves both cortical BMD and cortical geometry. Histomorphometric analysis revealed that ONO-5334 suppressed bone formation rate (BFR) at osteonal site in the midshaft femur but did not influence OVX-induced increase in BFR at either the periosteal or endocortical surfaces. Unlike alendronate, ONO-5334 increased osteoclasts surface (Oc.S/BS) and serum tartrate-resistant acid phosphatise 5b (TRAP5b) activity, highlighting the difference in the mode of action between these two drugs. Our results suggest that ONO-5334 has therapeutic potential not only in vertebral bones, but also in non-vertebral bones.

Combination therapy with ONO-KK1-300-01, a cathepsin K inhibitor, and parathyroid hormone results in additive beneficial effect on bone mineral density in ovariectomized rats.

This study examined the effects of a novel cathepsin K inhibitor, ONO-KK1-300-01 (KK1-300), used concurrently with parathyroid hormone (PTH) in ovariectomized (OVX) rats. KK1-300 (3 mg/kg, twice daily), alendronate (1 mg/kg, once daily) or vehicle were orally administered to OVX rats for 56 days, starting the day after ovariectomy, followed by combination treatment with or without PTH (3 µg/kg, subcutaneously three times a week) for another 28 days. OVX control animals exhibited a significant increase in both bone resorption (urinary deoxypyridinoline; DPD) and formation markers (serum osteocalcin) as well as microstructural changes associated with decreased bone mineral density (BMD). Combination treatment with KK1-300 and PTH significantly decreased urinary DPD and increased serum osteocalcin, indicating a sustained beneficial effect compared to the effect of each mono-therapy. On the other hand, combination therapy with alendronate and PTH weakened the PTH-induced increase in osteocalcin. In proximal tibia, combination treatment with KK1-300 and PTH increased BMD to a level significantly higher than that achieved following single treatment with KK1-300 or PTH alone. On the other hand, combination treatment with alendronate and PTH failed to produce any significant additive effect on BMD following single treatment with alendronate or PTH alone. Microstructural analysis revealed that the PTH-induced increase in bone formation (MS/BS and BFR/BS) was fully maintained following combination treatment with KK1-300 and PTH, but not following combination treatment with alendronate and PTH. These findings indicate that KK1-300, unlike alendronate, has an additive effect on the preventive action of PTH on bone loss in OVX rats.

Expression of CysLT2 receptors in asthma lung, and their possible role in bronchoconstriction.

BACKGROUND: The expression and functional role of CysLT2 receptors in asthma have not been clarified. In this study, we evaluated CysLT2 receptors expression, and effects of CysLT2-and CysLT1/2-receptor antagonists on antigen-induced bronchoconstriction using isolated lung tissues from both asthma and non-asthma subjects. METHODS: CysLT1 and CysLT2 receptors expression in asthma and non-asthma lung tissue preparations was examined in immunohistochemistry experiments, and their functional roles in antigen-induced bronchoconstriction were assessed using ONO-6950, a dual CysLT1/2-receptor antagonist, montelukast, a CysLT1 receptor antagonist, and BayCysLT2RA, a CysLT2 receptor-specific antagonist. RESULTS: CysLT1 receptors were expressed on the bronchial smooth muscle and epithelium, and on alveolar leukocytes in 5 in 5 non-asthma subjects and 2 in 2 asthma subjects. On the other hand, although degrees of CysLT2 receptors expression were variable among the 5 non-asthma subjects, the expression in the asthma lung was detected on bronchial smooth muscle, epithelium and alveolar leukocytes in 2 in 2 asthma subjects. In the non-asthma specimens, antagonism of CysLT2 receptors did not affect antigen-induced bronchial contractions, even after pretreatment with the CysLT1-receptor specific antagonist, montelukast. However, in the bronchus isolated from one of the 2 asthma subjects, antagonism of CysLT2 receptors suppressed contractions, and dual antagonism of CysLT1 and CysLT2 receptors resulted in additive inhibitory effect on anaphylactic contractions. CONCLUSIONS: CysLT2 receptors were expressed in lung specimens isolated from asthma subjects. Activation of CysLT2 receptors may contribute to antigen-induced bronchoconstriction in certain asthma population.

Effects of ONO-6950, a novel dual cysteinyl leukotriene 1 and 2 receptors antagonist, in a guinea pig model of asthma.

We assessed in this study the anti-asthmatic effects of ONO-6950, a novel cysteinyl leukotriene 1 (CysLT1) and 2 (CysLT2) receptors dual antagonist, in normal and S-hexyl glutathione (S-hexyl GSH)-treated guinea pigs, and compared these effects to those of montelukast, a CysLT1 selective receptor antagonist. Treatment with S-hexyl GSH reduced animals LTC4 metabolism, allowing practical evaluation of CysLT2 receptor-mediated airway response. ONO-6950 antagonized intracellular calcium signaling via human and guinea pig CysLT1 and CysLT2 receptors with IC50 values of 1.7 and 25 nM, respectively (human receptors) and 6.3 and 8.2 nM, respectively (guinea pig receptors). In normal guinea pigs, both ONO-6950 (1 or 0.3 mg/kg, p.o.) and the CysLT1 receptor antagonist montelukast (0.3 or 0.1 mg/kg, p.o.) fully attenuated CysLT1-mediated bronchoconstriction and airway vascular hyperpermeability induced by LTD4. On the other hand, in S-hexyl GSH-treated guinea pigs ONO-6950 at 3 mg/kg, p.o. or more almost completely inhibited bronchoconstriction and airway vascular hyperpermeability elicited by LTC4, while montelukast showed only partial or negligible inhibition of these airway responses. In ovalbumin sensitized guinea pigs, treatment with S-hexyl GSH on top of pyrilamine and indomethacin rendered antigen-induced bronchoconstriction sensitive to both CysLT1 and CysLT2 receptor antagonists. ONO-6950 strongly inhibited this asthmatic response to the level attained by combination therapy with montelukast and BayCysLT2RA, a selective CysLT2 receptor antagonist. These results clearly demonstrate that ONO-6950 is an orally active dual CysLT1/LT2 receptor antagonist that may provide a novel therapeutic option for patients with asthma.

Attenuation of Antiresorptive Action in Withdrawal of Minodronic Acid for Three Months After Treatment for Twelve Months in Ovariectomized Rats.

The purpose of this study was to evaluate the effects of withdrawal of minodronic acid (MIN) for 3 months after 12 months of treatment in ovariectomized (OVX) rat. OVX rats were orally treated with MIN (6, 30, and 150 µg/kg/day) for 12 months and necropsied on the day after the last dosing or following 3 months of withdrawal. Lumbar and femoral BMD were decreased in OVX controls. MIN dose-dependently increased BMD. Withdrawal eliminated the effect of MIN on BMD loss after treatment at 6 µg/kg, but not after treatment at 30 and 150 µg/kg. In MIN-treated rats, trabecular thinning occurred during withdrawal after treatment at 6 µg/kg, but the trabecular microstructure was maintained at 30 and 150 µg/kg. In a mechanical test of the femoral diaphysis, stiffness of in OVX controls was decreased but ultimate load was similar to that in sham after withdrawal. MIN increased ultimate load and stiffness, but endosteal length decreased after withdrawal. Suppression of bone turnover by MIN based on bone turnover markers and histomorphometric indices was attenuated by withdrawal after treatment at 6 and 30 µg/kg and partially at 150 µg/kg. The MIN concentration in the humerus decreased during withdrawal, and half-life at 30 µg/kg was shorter than that at 150 µg/kg. These results show that the antiresorptive action of MIN was dose-dependently attenuated by 3-month withdrawal in a rat OVX model. An absence of BMD increase was only observed at a low dose but decreases in antiresorptive activity occurred over a wide dose range.

IOP-Lowering Effect of ONO-9054, A Novel Dual Agonist of Prostanoid EP3 and FP Receptors, in Monkeys.

PURPOSE: The purpose of this study was to determine whether a better IOP reduction can be observed in conscious, normotensive monkeys treated with ONO-9054, a novel dual EP3 and FP receptor agonist, compared with prostaglandin F2α analogs. METHODS: The binding affinities and agonistic activities of ONO-AG-367, a carboxylic acid of ONO-9054, to prostanoid receptors were assessed. The IOP-lowering effect of ONO-9054 in monkeys was analyzed after a single (0.3, 3, or 30 µg/mL) or 7-day repeated (30 µg/mL, every day) topical ocular administration. Ophthalmologic and histopathologic evaluations of the eye were performed after 4-week ocular administration of ONO-9054 (30 µg/mL, twice a day) in monkeys. RESULTS: The ONO-AG-367 exhibited high affinity for both EP3 and FP receptors and potent agonist activity, with EC50 values of 28.6 nM for the EP3 receptor and 22.3 nM for the FP receptor. Single and repeated topical ocular administration of ONO-9054 caused IOP reductions in normotensive monkeys. The maximum IOP reductions on day 7 observed with ONO-9054 (7.3 ± 0.8 mm Hg) were significantly greater than those observed with latanoprost (50 µg/mL, 4.9 ± 0.4 mm Hg) or travoprost (40 µg/mL, 5.1 ± 0.6 mm Hg). In ophthalmologic and histopathologic evaluations, slight and transient mydriasis was occasionally observed and no histopathologic lesions attributable to ONO-9054 were noted. CONCLUSIONS: A more profound and longer-lasting reduction in IOP in normotensive monkeys can be observed with ONO-9054, which simultaneously stimulates both EP3 and FP receptors, compared with prostaglandin analogs.

Induction of creatine kinase release from cultured osteoclasts via the pharmacological action of aminobisphosphonates.

An increase of serum creatine kinase (CK) has been observed in clinical studies of nitrogen-containing aminobisphosphonates (N-BPs). Osteoclasts are thought to be the source of the CK, but there is no clear evidence for the hypothesis. In this study, CK release from rabbit osteoclasts induced by N-BPs was examined in an in vitro culture system. Rabbit bone-derived cells were cultured for 3 days on the N-BPs pretreated cortical bone slices. CK activity in the culture medium was measured at 3 days of culture. The CK activity was increased with all N-BPs at concentrations at which showed antiresorptive activity over 60% inhibition of C-terminal cross-linking telopeptide of type I collagen (CTX-1) release. The maximum induction of CK activity was 2.6 times the control level. The lowest N-BP concentration inducing CK release was 3 times lower than that required to decrease the osteoclast number. Bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, abrogated all N-BP actions, including CK release. Bone-derived cells except osteoclasts were insensitive to bafilomycin A1, suggesting that osteoclasts were the source of CK. Regarding the time course, CK release occurred after a 1 day lag time and increased steadily until day 3 of culture. These results show that CK release is induced by N-BPs from osteoclasts at concentrations at which N-BPs show antiresorptive activity over 60% inhibition of CTX-1 release in vitro. These findings explain the mechanism of the CK increase induced by clinical use of N-BPs.

Leukotriene C4 induces bronchoconstriction and airway vascular hyperpermeability via the cysteinyl leukotriene receptor 2 in S-hexyl glutathione-treated guinea pigs.

Cysteinyl leukotrienes act through G-protein-coupled receptors termed cysteinyl leukotriene 1 (CysLT1) and cysteinyl leukotriene 2 (CysLT2) receptors. However, little is known about the pathophysiological role of CysLT2 receptors in asthma. To elucidate the possible involvement of CysLT2 receptors in bronchoconstriction and airway vascular hyperpermeability, we have established a novel guinea pig model of asthma. In vitro study confirmed that CHO-K1 cells, expressing guinea pig CysLT2 and CysLT1 receptors are selectively stimulated by LTC4 and LTD4, respectively. However, when LTC4 was intravenously injected to guinea pigs, the resulting bronchoconstriction was fully abrogated by montelukast, a CysLT1 receptor antagonist, indicating rapid metabolism of LTC4 to LTD4 in the lung. We found that treatment with S-hexyl glutathione (S-hexyl GSH), an inhibitor of gamma-glutamyl transpeptidase, significantly increased LTC4 content and LTC4/(LTD4 plus LTE4) ratio in the lung. Under these circumstances, LTC4-induced bronchoconstriction became resistant to montelukast, but sensitive to Compound A, a CysLT2 receptor antagonist, depending on the dose of S-hexyl GSH. Combination with montelukast and Compound A completely abrogated this spasmogenic response. Additionally, we confirmed that LTC4 elicits airway vascular hyperpermeability via CysLT2 receptors in the presence of high dose of S-hexyl GSH as evidenced by complete inhibition of LTC4-induced hyperpermeability by Compound A, but not montelukast. These results suggest that CysLT2 receptors mediate bronchoconstriction and airway vascular hyperpermeability in guinea pigs and that the animal model used in this study may be useful to elucidate the functional role of CysLT2 receptors in various diseases, including asthma.

Effect of intermittent and daily regimens of minodronic Acid on bone metabolism in an ovariectomized rat model of osteoporosis.

The goal of the study was to compare the effects of minodronic acid on bone mineral density (BMD) and bone turnover in a rat ovariectomized (OVX) osteoporosis model, using two intermittent treatment regimens (weekly and 4 continuous days every 4 weeks) and a daily regimen. Female F344 rats (age 14 weeks) underwent ovariectomy or a sham operation. Minodronic acid was orally administered at 0.042, 0.21, and 1.05 mg/kg in the intermittent regimens, and at 0.03 and 0.15 mg/kg in the daily regimen for 12 weeks from the day after surgery. Minodronic acid dose-dependently ameliorated the decreases in areal BMD of the lumbar vertebrae and femur, and volumetric BMD of total and trabecular bone in the distal femur. Minodronic acid also suppressed the increase in urinary deoxypyridinoline levels and reduced serum osteocalcin levels. In bone histomorphometry, all three minodronic acid regimens suppressed OVX-induced increases in bone turnover at the tissue level and ameliorated all structural indices, except that an effect on trabecular thickness only occurred with daily treatment. In conclusion, minodronic acid administered weekly or for 4 continuous days every 4 weeks suppressed increased bone resorption and BMD to a similar extent to that of a similar total dose given daily in a rat OVX model.

Effects of eight-month treatment with ONO-5334, a cathepsin K inhibitor, on bone metabolism, strength and microstructure in ovariectomized cynomolgus monkeys.

This study examined the effect of ONO-5334, a cathepsin K inhibitor, on bone turnover, mineral density (BMD), mechanical strength and microstructure in ovariectomized (OVX) cynomolgus monkeys. Vehicle, ONO-5334 (3, 10 or 30 mg/kg) or alendronate (0.5 mg/kg) was orally administered for eight months to sham- and OVX-operated monkeys. ONO-5334 dose-dependently suppressed OVX-induced increase in bone turnover markers (urinary C-terminal cross-linking telopeptide of type I collagen (CTX) and serum osteocalcin). At the dose of 30 mg/kg, ONO-5334 maintained urinary CTX at nearly zero level and kept serum osteocalcin around the level of the sham animals. Marker levels in the alendronate-treated animals were similar to those in the sham animals throughout the study. ONO-5334 dose-dependently reversed the effect of OVX on vertebral BMD as measured by dual-energy X-ray absorptiometry (DXA) with improvement of bone mechanical strength. Both ONO-5334 and alendronate suppressed OVX-induced changes in vertebral microstructure and turnover state. In the femoral neck, peripheral quantitative computed tomography (pQCT) analysis showed that ONO-5334 increased total and cortical BMD. In particular, ONO-5334 significantly increased cortical BMD with improvement of bone mechanical strength. In microstructural analysis, alendronate suppressed OVX-induced increase in femoral mid-shaft osteonal bone formation rate (BFR) to a level below that recorded in the sham group, whereas ONO-5334 at 30 mg/kg did not suppress periosteal, osteonal and endocortical BFR. This finding supports the significant effect of ONO-5334 on cortical BMD and mechanical strength in the femoral neck. The results of this study suggest that ONO-5334 has good therapeutic potential for the treatment of osteoporosis.

ONO-5334, a cathepsin K inhibitor, improves bone strength by preferentially increasing cortical bone mass in ovariectomized rats.

This study compared the effects of ONO-5334, a cathepsin K inhibitor, with those of alendronate on bone mass and strength in ovariectomized rats. Ovariectomy resulted in significant elevation in urinary deoxypyridinoline and plasma C-terminal cross-linking telopeptide of type I collagen (CTX) 8 weeks after surgery. Peripheral quantitative computed tomography analysis showed that total, trabecular, and cortical bone mineral content (BMC) decreased in the proximal tibia, which was paralleled with a significant decline in bone strength. Treatment with ONO-5334 (0.12, 0.6, 3 or 15 mg/kg) once daily for 8 weeks dose-dependently restored the decrease in total BMC and bone mineral density (BMD) in the proximal tibia and suppressed urinary deoxypyridinoline and plasma CTX levels. Alendronate (1 mg/kg, once daily) also fully restored these bone mass parameters. Separate analysis of trabecular and cortical bones, however, showed that ONO-5334 only partially restored trabecular BMD and BMC at 15 mg/kg, whereas alendronate fully restored these parameters. On the other hand, ONO-5334 increased both cortical BMD and BMC with an effect more potent than that of alendronate. Bone geometric analysis indicated that ONO-5334 at 15 mg/kg decreased endosteal circumference without affecting periosteal circumference, resulting in marked increase in cortical thickness. Interestingly, the effects of ONO-5334 on bone strength parameters were more prominent than those of alendronate, although the two test compounds had a similar effect on total BMC. Taken together, our results indicate that ONO-5334 has pharmacological characteristics different from those of alendronate and may offer a unique therapy for patients with osteoporosis.

Effects of ONO-5334, a novel orally-active inhibitor of cathepsin K, on bone metabolism.

In the present study, we examined the in vitro and in vivo pharmacological effects of ONO-5334, a novel inhibitor of cathepsin K, on bone metabolism. In vitro experiments indicated that ONO-5334 is a potent inhibitor of cathepsin K with Ki value of 0.1 nM. Although this compound inhibited other cysteine proteases, such as cathepsin S, L and B, its inhibitory activity for these enzymes was 8 to 320 fold lower than that for cathepsin K. ONO-5334 also inhibited human osteoclasts bone resorption in vitro at a concentration more than 100 fold lower than that of alendronate, a bisphosphonate. While alendronate disrupted actin ring and induced pyknotic nuclei in osteoclasts, ONO-5334 did not have such effects, suggesting that this compound does not affect osteoclasts viability. In in vivo experiments, oral administration of ONO-5334 dose-dependently reduced plasma calcium level increased by parathyroid hormone related peptide in thyroparathyroidectomized rats. Furthermore, in vivo experiment using normal monkeys demonstrated that ONO-5334 decreases serum and urine C-telopeptide of type I collagen level, a bone resorption marker, soon after oral dosing. These levels were consistently decreased below pre-dose levels by repeated oral dosing with ONO-5334 for 7 days. ONO-5334 on the other hand did not affect bone formation markers, serum osteocalcin and bone specific alkaline phosphatase. These findings indicate that ONO-5334 is a specific inhibitor for cathepsin K and thus may be a novel therapeutic agent for metabolic bone diseases.

Inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters.

Neutrophil elastase (NE) has been recognized as a double-edge sword as it can be both host-defensive and pro-inflammatory depending on its tissue level and microenvironment. The present study was designed to examine whether inhibition of NE activity by sivelestat, a specific NE inhibitor with low cellular permeability, is beneficial for acute lung injury induced by Streptococcus pneumoniae in hamsters. Intratracheal inoculation of live S. pneumoniae (1.5 x 10(7) CFU/Lung) into hamsters caused acute lung injury characterized by an increase in lung alveolar permeability and leukocytes accumulation in the lungs. These pathophysiological changes, which were associated with elevation of NE activity in the bronchoalveolar lavage fluid (BALF), were transient but remained high 4-22 h post-bacterial inoculation. Intravenous infusion of sivelestat at 3mg/kg/h 0-22 h after bacterial inoculation reduced the increase in NE activity and lung alveolar permeability, as indicated by leakage of pre-injected human serum albumin into BALF, without affecting the number of leukocytes in BALF and lung interstitial tissue. Interestingly, the number of bacteria in BALF and lung interstitial tissue was also reduced by sivelestat to less than 10% of the control values. Furthermore, S. pneumoniae-induced decrease in the level of surfactant protein D (SP-D), a protein that protects against bacterial infection, was preserved by sivelestat treatment with a significant negative correlation between NE activity and SP-D content in BALF. These results suggest that sivelestat is beneficial in acute lung injury associated with lung bacterial infection and that this inhibitor rather decreases infection by preserving host defense system.

Neutrophil elastase activity in acute lung injury and respiratory distress syndrome.

BACKGROUND AND OBJECTIVE: Neutrophil elastase (NE) may play a key role in the development of acute lung injury (ALI) or ARDS. NE activity (NEA) was measured in patients with ALI treated with a selective NE inhibitor. METHODS: NEA and NE-alpha1-antitrypsin (NE-AT) complex were measured in plasma before, during and after the administration of the selective NE inhibitor, sivelestat, in 32 patients with a diagnosis of ALI or ARDS. NEA index (NEAI) was calculated as NEA/NE-AT. The sequential organ failure assessment (SOFA) score and the ratio PaO(2)/fraction of inspired oxygen (FiO(2)) were measured. RESULTS: NEA and NE-AT was raised in all patients. Sivelestat reduced NEAI and NEA (P < 0.01 for both) but not NE-AT and NEA, and NEAI returned to pretreatment levels. NEA correlated closely with NE-AT before, but not after treatment. No relationship was observed between these indices and SOFA score or PaO(2)/FiO(2) ratio. CONCLUSIONS: Sivelestat reduced NEA and NEAI in patients with ALI or ARDS suggesting NE inhibition. A larger study is needed to determine the relationship of NEA, NE-AT and NEAI with the outcome of ALI/ARDS.