RESUMO
Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody® (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy-cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1ß (10 ng/mL)) with or without M6495 (1000-0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.
Assuntos
Proteína ADAMTS5/antagonistas & inibidores , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/fisiopatologia , Anticorpos de Domínio Único/farmacologia , Proteína ADAMTS5/imunologia , Proteína ADAMTS5/metabolismo , Agrecanas/metabolismo , Animais , Cartilagem Articular/metabolismo , Bovinos , Técnicas de Cocultura , Colágeno Tipo II/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Oncostatina M/farmacologia , Técnicas de Cultura de Órgãos , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Albumina Sérica Humana/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Membrana Sinovial/citologiaRESUMO
We discovered a novel series of non-peptidic acylguanidine inhibitors of Cathepsin D as target for osteoarthritis. The initial HTS-hits were optimized by structure-based design using CatD X-ray structures resulting in single digit nanomolar potency in the biochemical CatD assay. However, the most potent analogues showed only micromolar activities in an ex vivo glycosaminoglycan (GAG) release assay in bovine cartilage together with low cellular permeability and suboptimal microsomal stability. This new scaffold can serve as a starting point for further optimization towards in vivo efficacy.
Assuntos
Catepsina D/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Catepsina D/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug. METHODS: Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. RESULTS: Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1ß (IL-1ß), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 µg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-µg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. CONCLUSION: R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.
Assuntos
Cartilagem Articular , Osteoartrite , Coelhos , Animais , Ovinos , Fator V/metabolismo , Fator V/uso terapêutico , Cartilagem Articular/patologia , Osteoartrite/metabolismo , Ligamento Cruzado Anterior/cirurgia , Ligamento Cruzado Anterior/metabolismo , Ligamento Cruzado Anterior/patologia , Diferenciação CelularRESUMO
Objectives: There is an unmet medical need for biomarkers in OA which can be applied in clinical drug development trials. The present study describes the development of a specific and robust assay measuring type II collagen degradation (T2CM) and discusses its potential as a noninvasive translational biomarker. Methods: A type II collagen specific neoepitope (T2CM) was identified by mass spectrometry and monoclonal antibodies were raised towards the epitope, employed in a chemiluminescence immunoassay. T2CM was assessed in bovine cartilage explants with or without MMP-13 inhibitor, and explant supernatants were analyzed by Western blot. T2CM was measured in plasma samples from one study (n â= â48 patients) where OA patients were referred to total knee replacement (TKR). Additionally, T2CM was quantified in serum from OA patients receiving salmon calcitonin treatment (sCT) (n â= â50) compared to placebo (n â= â57). Results: The T2CM assay was technically robust (13/4 â% inter/intra-variation) and specific for the type II collagen fragment cleaved by MMP-1 and -13. The MMP-13 inhibitor reduced the T2CM release from bovine cartilage explants receiving catabolic treatment. These results were confirmed by Western blot. In human end-stage OA patients (scheduled for TKR), the T2CM levels were elevated compared to moderate OA (p<0.004). The OA patients receiving sCT had lower levels of T2CM compared to placebo group after 1, 6, and 24 months of treatment (p â= â0.0285, p â= â0.0484, p â= â0.0035). Conclusions: To our knowledge, T2CM is the first technically robust serological biomarker assay which has shown biological relevance in ex vivo models and OA cohorts. This suggests that T2CM may have potential as a translational biomarker for cartilage degradation.