RESUMEN
Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.
Asunto(s)
Osteoartritis , Animales , Huesos , Catepsina K , Catepsinas , Condrocitos , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/uso terapéutico , Perros , Osteoartritis/tratamiento farmacológico , OsteoclastosRESUMEN
The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.
Asunto(s)
Catepsina K/antagonistas & inhibidores , Osteoporosis/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Animales , Huesos/diagnóstico por imagen , Radioisótopos de Carbono/química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Femenino , Ligandos , Macaca mulatta , Unión Proteica , Conejos , Radiofármacos/efectos adversos , Radiofármacos/química , Distribución TisularRESUMEN
During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.
Asunto(s)
Catepsina K/antagonistas & inhibidores , Catepsina K/metabolismo , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Animales , Huesos/enzimología , Evaluación Preclínica de Medicamentos , Cinética , ConejosRESUMEN
For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.
Asunto(s)
Proteínas de la Cápside , Cápside , Humanos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Células HEK293 , Vectores Genéticos/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMEN
The trifluoroethylamine group found in cathepsin K inhibitors like odanacatib can be replaced by a difluoroethylamine group. This change increased the basicity of the nitrogen which positively impacted the log D. This translated into an improved oral bioavailability in pre-clinical species. Difluoroethylamine compounds exhibit a similar potency against cathepsin K and selectivity profile against other cathepsins when compared to trifluoroethylamine analogs.
Asunto(s)
Catepsina K/antagonistas & inhibidores , Etilaminas/química , Inhibidores de Proteasas/química , Administración Oral , Amidas/química , Animales , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Catepsina K/metabolismo , Perros , Etilaminas/síntesis química , Etilaminas/farmacocinética , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , RatasRESUMEN
MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/farmacocinética , Catepsina K/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/administración & dosificación , Inhibidores de Cisteína Proteinasa/farmacocinética , Descubrimiento de Drogas/métodos , Administración Oral , Animales , Disponibilidad Biológica , Compuestos de Bifenilo/química , Catepsina K/metabolismo , Inhibidores de Cisteína Proteinasa/química , Perros , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Macaca mulatta , Conejos , RatasRESUMEN
Cathepsin K (CatK) inhibitors exhibited chondroprotective and pain-reducing effects in animal models, however, improvements were relatively modest at dose levels achieving maximal suppression of CatK biomarkers in urine. In this report, a previously characterized CatK inhibitor (MK-1256) is utilized to explore the potential of reduced target engagement and/or suboptimal exposure (free drug) as limiting factors to the pharmacological potential of CatK inhibitors in the knee joint. Following oral administration of MK-1256 at a dose level achieving maximal inhibition of urinary biomarker (helical peptide) in dogs, full suppression of the biomarker in synovial fluid was observed. Subsequent tissue distribution studies conducted in dogs and rabbits revealed that MK-1256 levels in synovial fluid and cartilage were consistent with the free-drug hypothesis. Reasonable projection (within twofold) of drug levels in these tissues can be made based on plasma drug concentration with adjustments for binding factors. These results indicate that the previously observed efficacies in the animal models were not limited by compound distribution or target engagement in the knee tissues.
RESUMEN
Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.
Asunto(s)
Compuestos de Bifenilo/farmacología , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Animales , Azepinas/química , Azepinas/farmacología , Catepsina K , Colágeno/efectos de los fármacos , Colágeno/inmunología , Perros , Fibroblastos/efectos de los fármacos , Humanos , Modelos Biológicos , Estructura Molecular , Osteoporosis Posmenopáusica/tratamiento farmacológico , Piel/citología , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.
Asunto(s)
Catepsina K/sangre , Inhibidores de Cisteína Proteinasa/sangre , Adolescente , Adulto , Anciano , Animales , Biomarcadores/orina , Compuestos de Bifenilo/sangre , Compuestos de Bifenilo/farmacocinética , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/orina , Proteínas Sanguíneas/metabolismo , Catepsina K/antagonistas & inhibidores , Colágeno Tipo I/orina , Inhibidores de Cisteína Proteinasa/farmacocinética , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/orina , Perros , Femenino , Humanos , Macaca mulatta , Masculino , Persona de Mediana Edad , Péptidos/orina , Unión Proteica , Pirazoles/sangre , Pirazoles/farmacocinética , Pirazoles/farmacología , Pirazoles/orina , Conejos , Sulfonas/sangre , Sulfonas/farmacocinética , Sulfonas/farmacología , Sulfonas/orina , Adulto JovenRESUMEN
The macrophage colony stimulating factor receptor (cFms) and alpha(V)beta(3) integrin are both abundantly expressed and play critical roles in the differentiation, survival and migration of osteoclasts. We have previously demonstrated that cross-talk between cFms- and alpha(V)beta(3)-mediated signaling pathways regulated the cytoskeletal organization required for osteoclast migration. To investigate the nature of interaction between the two receptors, we sequentially used anion-exchange chromatography and immunoprecipitation to purify alpha(V)beta(3)-associated protein complexes. We have demonstrated that cFms stably associated with alpha(V)beta(3) in osteoclasts during adhesion, and that the association was induced by macrophage colony stimulating factor (M-CSF) stimulation. However, the kinetics of association of alpha(V)beta(3) and cFms did not correlate with the kinetics of tyrosine phosphorylation of cFms. Instead, maximally observed alpha(V)beta(3)/cFms association was after the peak of cFms tyrosine phosphorylation and correlated inversely with the total amount of cFms remaining. Furthermore, the complex containing cFms and alpha(V)beta(3) also contained a number of other signaling molecules including Pyk2, p130(Cas) and c-Cbl, known downstream regulators of the integrin-mediated signaling pathways in osteoclasts. In the presence of M-CSF, co-localization of alpha(V)beta(3) integrin and cFms was identified in the podosomal actin ring of the osteoclast during adhesion on glass. Interestingly, co-localization of both receptors was not found in the sealing zone, but in punctate structures associated with adhesion- or transcytosis-like structures in osteoclasts on bone. Taken together, we suggest that the association of alpha(V)beta(3) and cFms could be the result of signaling following tyrosine phosphorylation of cFms. The recruitment of cFms to alpha(V)beta(3) integrin may be an integral part of a larger signaling complex via which both of adhesion- and growth factor receptors coordinately regulate osteoclast adhesion, motility and membrane trafficking.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Factor Estimulante de Colonias de Macrófagos/fisiología , Osteoclastos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Células Cultivadas , Proteína Sustrato Asociada a CrK/metabolismo , Inmunoprecipitación , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Osteoclastos/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Osteoarthritis (OA) is a chronic joint disease characterized by cartilage destruction, subchondral bone sclerosis, and osteophyte formation. Subchondral bone stiffness has been proposed to initiate and/or contribute to cartilage deterioration in OA. The purpose of this study was to characterize subchondral bone remodeling, cartilage damage, and osteophytosis during the disease progression in two models of surgically induced OA. Rat knee joints were subjected either to anterior cruciate ligament transection (ACLT) alone or in combination with resection of medial menisci (ACLT + MMx). Histopathological changes in the surgical joints were compared with sham at 1, 2, 4, 6, and 10 weeks post-surgery. Using a modified Mankin scoring system, we demonstrate that articular cartilage damage occurs within 2 weeks post-surgery in both surgical models. Detectable cartilage surface damage and proteoglycan loss were observed as early as 1 week post-surgery. These were followed by the increases in vascular invasion into cartilage, in loss of chondrocyte number and in cell clustering. Histomorphometric analysis revealed subchondral bone loss in both models within 2 weeks post-surgery followed by significant increases in subchondral bone volume relative to sham up to 10 weeks post-surgery. Incidence of osteophyte formation was optimally observed in ACLT joints at 10 weeks and in ACLT + MMx joints at 6 weeks post-surgery. In summary, the two surgically induced rat OA models share many characteristics seen in human and other animal models of OA, including progressive articular cartilage degradation, subchondral bone sclerosis, and osteophyte formation. Moreover, increased subchondral bone resorption is associated with early development of cartilage lesions, which precedes significant cartilage thinning and subchondral bone sclerosis. Together, these findings support a role for bone remodeling in OA pathogenesis and suggest that these rat models are suitable for evaluating bone resorption inhibitors as potential disease-modifying pharmaco-therapies.
Asunto(s)
Ligamento Cruzado Anterior , Huesos , Calcinosis , Cartílago Articular , Osteoartritis , Animales , Masculino , Ratas , Ligamento Cruzado Anterior/patología , Ligamento Cruzado Anterior/cirugía , Huesos/patología , Remodelación Ósea , Cartílago Articular/irrigación sanguínea , Cartílago Articular/patología , Condrocitos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fémur/patología , Articulaciones/patología , Meniscos Tibiales/cirugía , Osteoartritis/etiología , Osteoartritis/patología , Ratas Sprague-Dawley , Osteofitosis Vertebral/patología , Tibia/anatomía & histología , Tibia/patología , Factores de TiempoRESUMEN
We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.
Asunto(s)
Benzamidas/síntesis química , Conservadores de la Densidad Ósea/síntesis química , Catepsinas/antagonistas & inhibidores , Nitrilos/síntesis química , Tiazoles/síntesis química , Administración Oral , Animales , Benzamidas/química , Benzamidas/farmacología , Disponibilidad Biológica , Biomarcadores/orina , Conservadores de la Densidad Ósea/química , Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/orina , Catepsina K , Catepsinas/química , Bovinos , Colágeno/antagonistas & inhibidores , Colágeno/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Cinética , Macaca mulatta , Modelos Moleculares , Estructura Molecular , Nitrilos/química , Nitrilos/farmacología , Conejos , Ratas , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
A novel series of nonpeptidic biaryl compounds was identified as potent and reversible inhibitors of cathepsin K. The P2-P3 amide bond of a known amino acetonitrile dipeptide 1 was replaced with a phenyl ring, thereby giving rise to this biaryl series that retained potency vs cathepsin K and showed an improved selectivity profile against other cathepsins. Structural modification within this series resulted in the identification of compound (R)-2, a potent human cathepsin K inhibitor (IC(50) = 3 nM) that is selective versus cathepsins B (IC(50) = 3950 nM), L (IC(50) = 3725 nM), and S (IC(50) = 2010 nM). In an in vitro assay involving rabbit osteoclasts and bovine bone, compound (R)-2 inhibited bone resorption with an IC(50) of 95 nM. It was shown that, unlike some peptidic nitrile inhibitors of cysteine proteases, the nitrile moiety of (R)-2 is not converted to the corresponding amide 3 by cathepsin K. This indicates that this class of nonpeptidic nitrile inhibitors is unlikely to be hydrolyzed by cysteine proteases. Furthermore, the inhibition of cathepsin K by compound (R)-2 was shown to be fully reversible and not observably time-dependent. To demonstrate the efficacy of compound (R)-2 in vivo, it was administered to ovariectomized (OVX) rhesus monkeys at 20 mg/kg, po once daily for 8 days, and a urinary marker of bone turnover, N-telopeptide of type I collagen (uNTx), was measured. During the eight-day dosing period, the mean reduction by compound (R)-2 in uNTx was 80% (p < 0.001). This demonstrates that inhibition of cathepsin K leads to an inhibition of this bone resorption marker in OVX rhesus monkeys and strongly suggests that inhibition of cathepsin K is a viable therapeutic approach for the treatment of osteoporosis.
Asunto(s)
Compuestos de Bifenilo/síntesis química , Catepsinas/antagonistas & inhibidores , Nitrilos/síntesis química , Piperazinas/síntesis química , Inhibidores de Proteasas/síntesis química , Animales , Biomarcadores/orina , Compuestos de Bifenilo/farmacocinética , Compuestos de Bifenilo/farmacología , Resorción Ósea/metabolismo , Huesos/efectos de los fármacos , Huesos/patología , Catepsina B/antagonistas & inhibidores , Catepsina B/química , Catepsina K , Catepsina L , Catepsinas/química , Bovinos , Colágeno/orina , Colágeno Tipo I , Cisteína Endopeptidasas , Humanos , Técnicas In Vitro , Macaca mulatta , Nitrilos/química , Nitrilos/farmacocinética , Nitrilos/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Ovariectomía , Péptidos/orina , Piperazinas/farmacocinética , Piperazinas/farmacología , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Conejos , Estereoisomerismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
3-(S)-Pyrimidin-5-yl-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5e) and 3-(S)-(methylpyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5f) were identified as potent and selective antagonists of the alpha(v)beta(3) receptor. These compounds have excellent in vitro profiles (IC(50) = 0.07 and 0.08 nM, respectively), significant unbound fractions in human plasma (6 and 4%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in an in vivo model of bone turnover following once-daily oral administration, these two compounds were selected for clinical development for the treatment of osteoporosis.
Asunto(s)
Integrinas/antagonistas & inhibidores , Naftiridinas/farmacología , Osteoporosis/tratamiento farmacológico , Receptores de Vitronectina/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Densidad Ósea/efectos de los fármacos , Resorción Ósea/tratamiento farmacológico , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Concentración 50 Inhibidora , Integrinas/metabolismo , Macaca mulatta , Modelos Moleculares , Naftiridinas/química , Naftiridinas/farmacocinética , Osteoporosis/prevención & control , Ovariectomía , Ratas , Ratas Sprague-Dawley , Receptores de Vitronectina/metabolismo , Relación Estructura-ActividadRESUMEN
Cathepsin K (CatK) is essential for osteoclast-mediated bone resorption. CatK expression is also detected in breast cancer cells that metastasize to bone. Here, the CatK inhibitor L-235 dosed in prevention (10, 30, and 100 mg/kg, p.o., b.i.d.) or treatment regimen (30 mg/kg) was compared with the bisphosphonate zoledronic acid (ZOL, 7.5 µg/kg/wk, s.c.) in the intratibial injection model of MDA-MB-231 breast carcinoma in nude rats. Progression of osteolysis, skeletal tumor burden, and local metastasis was evaluated by radiography through 42 days and ex vivo µCT and histology. IHC and RT-PCR confirmed the increases in CatK protein and mRNA levels in human breast cancer primary and metastatic tumors. In the experimental model of breast cancer bone metastasis, L-235 dosed in preventive mode resulted in a dose-related reduction of osteolysis of 72%, 75%, and 87% respectively, compared with ZOL by 86% versus intact. Similarly, L-235 significantly reduced intratibial tumor volume by 29%, 40%, and 63%, respectively, compared with 56% by ZOL versus vehicle. Efficacy of L-235 and ZOL on reduction of osteolytic lesions and tumor burden was comparable in treatment versus preventive regimens. All L-235 doses inhibited cortical disruption and extraskeletal tumor growth to a level comparable with ZOL. Assessment of local metastasis demonstrated that treatment with the CatK inhibitor was more effective than ZOL in reducing breast cancer invasion. These data support the role of CatK in breast cancer skeletal growth and metastasis and CatK inhibitors may represent a novel oral therapy for treatment of metastatic breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Catepsina K/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Animales , Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Catepsina K/genética , Catepsina K/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Clasificación del Tumor , Metástasis de la Neoplasia , Osteólisis , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate the disease modifying effects of cathepsin K (CatK) inhibitor L-006235 compared to alendronate (ALN) in two preclinical models of osteoarthritis (OA). METHODS: Skeletally mature rabbits underwent sham or anterior cruciate ligament transection (ACLT)-surgery and were treated with L-006235 (L-235, 10 mg/kg or 50 mg/kg, p.o., daily) or ALN (0.6 mg/kg, s.c., weekly) for 8-weeks. ACLT joint instability was also induced in CatK(-/-) versus wild type (wt) mice and treated for 16-weeks. Changes in cartilage degeneration, subchondral bone volume and osteophyte area were determined by histology and µ-CT. Collagen type I helical peptide (HP-I), a bone resorption marker and collagen type II C-telopeptide (CTX-II), a cartilage degradation marker were measured. RESULTS: L-235 (50 mg/kg) and ALN treatment resulted in significant chondroprotective effects, reducing CTX-II by 60% and the histological Mankin score for cartilage damage by 46% in the ACLT-rabbits. Both doses of L-235 were more potent than ALN in protecting against focal subchondral bone loss, and reducing HP-I by 70% compared to vehicle. L-235 (50 mg/kg) and ALN significantly reduced osteophyte formation in histomorphometric analysis by 55%. The Mankin score in ACLT-CatK(-/-) mice was ~2.5-fold lower than the ACLT-wt mice and was not different from sham-CatK(-/-). Osteophyte development was not different among the groups. CONCLUSION: Inhibition of CatK provides significant benefits in ACLT-model of OA, including: 1) protection of subchondral bone integrity, 2) protection against cartilage degradation and 3) reduced osteophytosis. Preclinical evidence supports the role of CatK as a potential therapeutic target for the treatment of OA.
Asunto(s)
Catepsina K/antagonistas & inhibidores , Osteoartritis/prevención & control , Alendronato/farmacología , Animales , Lesiones del Ligamento Cruzado Anterior , Benzamidas/farmacología , Biomarcadores/metabolismo , Conservadores de la Densidad Ósea/farmacología , Cartílago Articular/patología , Catepsina K/deficiencia , Catepsina K/genética , Colágeno Tipo I/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoartritis/enzimología , Osteoartritis/etiología , Osteoartritis/patología , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Conejos , Tiazoles/farmacologíaRESUMEN
Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiency-elevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L1 to L4) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L1 to L4 BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX-vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.
Asunto(s)
Compuestos de Bifenilo/farmacología , Conservadores de la Densidad Ósea/farmacología , Remodelación Ósea/efectos de los fármacos , Vértebras Lumbares/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Animales , Femenino , Macaca mulattaRESUMEN
Herein, we report on the identification of nonbasic, potent, and highly selective, nitrile-containing cathepsin K (Cat K) inhibitors that are built on our previously identified cyclohexanecarboxamide core structure. Subsequent to our initial investigations, we have found that incorporation of five-membered heterocycles as P2-P3 linkers allowed for the introduction of a methyl sulfone P3-substitutent that was not tolerated in inhibitors containing a six-membered aromatic P2-P3 linker. The combination of a five-membered N-methylpyrazole linker and a methyl sulfone in P3 yielded subnanomolar Cat K inhibitors that were minimally shifted (<10-fold) in our functional bone resorption assay. Issues that arose because of metabolic demethylation of the N-methylpyrazole were addressed through introduction of a 2,2,2-trifluoroethyl substituent. This culminated in the identification of 31 (MK-1256), a potent (Cat K IC 50 = 0.62 nM) and selective (>1100-fold selectivity vs Cat B, L, S, C, H, Z, and V, 110-fold vs Cat F) inhibitor of cathepsin K that is efficacious in a monkey model of osteoporosis.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/uso terapéutico , Nitrilos/química , Osteoporosis/tratamiento farmacológico , Osteoporosis/enzimología , Pirazoles/química , Pirazoles/uso terapéutico , Sulfonas/química , Sulfonas/uso terapéutico , Animales , Catepsina K , Catepsinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacocinética , Modelos Animales de Enfermedad , Perros , Femenino , Cinética , Macaca mulatta , Modelos Moleculares , Estructura Molecular , Pirazoles/metabolismo , Pirazoles/farmacocinética , Ratas , Relación Estructura-Actividad , Sulfonas/metabolismo , Sulfonas/farmacocinéticaRESUMEN
OBJECTIVE: The biologic changes associated with osteoarthritis (OA) are incompletely understood. The aim of this study was to elucidate the molecular mechanisms underlying OA progression in an STR/Ort murine model of spontaneous disease. METHODS: Global patterns of gene expression were assessed using microarray analysis of articular cartilage/subchondral bone from the tibial plateaus of STR/Ort mice at 3, 9, and 12 months of age. The age-dependent severity of osteophyte formation and extent of cartilage damage were determined in the corresponding femurs using microfocal computed tomography and the Mankin histologic scoring system. Pathway analysis was used to identify the functions of genes associated with OA progression, and changes in gene expression were confirmed using immunohistochemistry. RESULTS: Six hundred twenty-one genes were associated with both osteophyte formation and cartilage damage in the STR/Ort joints. Genes involved in the development/function of connective tissue and in lipid metabolism were most significantly enriched and regulated during disease progression. Genes directly interacting with peroxisome proliferator-activated receptor alpha (PPARalpha)/PPARgamma were down-regulated, whereas those genes involved with connective tissue remodeling were up-regulated during disease progression. Associations of down-regulation of myotubularin-related phosphatase 1 (a phosphoinositide 3-phosphatase involved in lipid signaling) and up-regulation of biglycan (a member of the small leucine-rich protein family known to modulate osteoblast differentiation and matrix mineralization) with OA progression were confirmed by immunohistochemistry. CONCLUSION: Since adipogenesis and osteogenesis are inversely related in the developing skeletal tissue, these results suggest that a shift in the differentiation of mesenchymal cells from adipogenesis toward osteogenesis is a component of the OA pathophysiologic processes occurring in the tibial plateau joints of STR/Ort mice.
Asunto(s)
Osteoartritis/genética , Osteoartritis/metabolismo , Adipogénesis , Animales , Expresión Génica , Ratones , Osteoartritis/patología , OsteogénesisRESUMEN
Further SAR study around the central 1,2-disubstituted phenyl of the previously disclosed Cat K inhibitor (-)-1 has demonstrated that the solvent exposed P2-P3 linker can be replaced by various 5- or 6-membered heteroaromatic rings. While some potency loss was observed in the 6-membered heteroaromatic series (IC(50)=1 nM for pyridine-linked 4 vs 0.5 nM for phenyl-linked (+/-)-1), several inhibitors showed a significantly decreased shift in the bone resorption functional assay (10-fold for pyridine 4 vs 53-fold for (-)-1). Though this shift was not reduced in the 5-membered heteroaromatic series, potency against Cat K was significantly improved for thiazole 9 (IC(50)=0.2 nM) as was the pharmacokinetic profile of N-methyl pyrazole 10 over our lead compound (-)-1.