RESUMO
Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Benzamidas/uso terapêutico , Dermatite de Contato/tratamento farmacológico , Edema/tratamento farmacológico , Indazóis/uso terapêutico , Receptores de Glucocorticoides/metabolismo , Animais , Anti-Inflamatórios/sangue , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Benzamidas/sangue , Benzamidas/farmacocinética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Colágeno , Citocinas/sangue , Cães , Feminino , Células HeLa , Humanos , Indazóis/sangue , Indazóis/farmacocinética , Indazóis/farmacologia , Insulina , Lipopolissacarídeos , Masculino , Metilprednisolona/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
The effects of nitrogen-containing bisphosphonates (N-BPs) on osteoclasts (Ocs) may differ with dose and regimen. N-BPs reduce Oc bone resorption by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), an effect counteracted by geranylgeraniol (GGOH), which restores geranylgeranylation downstream of FPPS. We assessed GGOH effects on inhibition of bone resorption by the N-BPs alendronate (ALN), ibandronate (IBN), and zoledronate (ZOL) in an assay of rabbit Oc resorption of bovine cortical bone. GGOH blocked inhibition of resorption at low, but not high, N-BP concentrations, with a 14- to 20-fold increase in IC50 values for each N-BP. In vivo, growing male rats were administered doses calculated to mimic bioavailable exposures in daily (ALN, IBN), weekly (ALN), monthly (IBN), and yearly (ZOL) clinical regimens. Tibiae were harvested at 48 h, and metaphyses were analyzed. With lower ALN and IBN doses, Oc numbers rose by 26-48 %, morphology was normal, and there was no increase in apoptotic Ocs. In contrast, with higher IBN and ZOL doses, bone-associated Ocs were generally rounded in appearance and numbers of nuclei/Oc versus vehicle increased 42 and 31 %, respectively (P < 0.05). With ZOL, there was no rise in Oc number, but there was a 6.5-fold increase in apoptotic Ocs versus vehicle and a ≥13.5-fold increase versus lower-dose ALN or IBN (P < 0.05). With higher-dose IBN there was no rise in Oc number but 7- and 14-fold increases in Oc apoptosis versus low-dose ALN and IBN (P < 0.02). These results suggest that different mechanisms may come into play across the dosing spectrum of N-BPs.
Assuntos
Reabsorção Óssea/metabolismo , Difosfonatos/administração & dosagem , Osteoclastos/efeitos dos fármacos , Animais , Bovinos , Difosfonatos/química , Relação Dose-Resposta a Droga , Masculino , Nitrogênio/administração & dosagem , CoelhosRESUMO
Odanacatib (ODN) has been developed as a selective inhibitor of cathepsin K, the major cysteine protease in osteoclasts. In adult rhesus monkeys, treatment with ODN prevents ovariectomy-induced bone loss in lumbar vertebrae and hip. In this study, we evaluate the effects of ODN on bone mineralization density distribution (BMDD) by quantitative backscattered electron imaging in vertebral spongiosa, distal femoral metaphyseal and cortical shaft from monkeys (aged 16-23 years), treated with vehicle (n=5) or ODN (6 mg/kg, n=4 or 30 mg/kg, n=4, PO daily) for 21 months. Dual-energy X-ray absorptiometry was measured in a subset of distal femoral samples. In lumbar vertebrae there was a shift to higher mineralization in samples from ODN-treated groups, compared to vehicle: CaMean (+4%), CaPeak (+3%), CaWidth (-9%), CaLow (-28%) in the 6 mg/kg group and CaMean (+5.1%, p<0.023), CaPeak (+3.4%, p<0.046), CaWidth (-15.7%, p=0.06) and CaLow (-38.2%, p<0.034) in the 30 mg/kg group. In distal femoral metaphyseal cancellous bone, there was a clear tendency toward a dose-dependent increase in matrix mineralization, as in the spine. However, primary and osteonal bone of the distal cortical diaphyses showed no significant change in BMDD, whereas bone mineral density was significantly increased after treatment. In ovariectomized monkeys, this study shows that ODN treatment increased trabecular BMDD, consistent with its previously reported ability to reduce cancellous remodeling. Here, ODN also showed no changes in BMDD in cortical bone sites, consistent with its actions on maintaining endocortical and stimulating periosteal bone formation.
Assuntos
Compostos de Bifenilo/farmacologia , Conservadores da Densidade Óssea/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Absorciometria de Fóton , Animais , Densidade Óssea/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Macaca mulatta , Ovariectomia , Vértebras Torácicas/efeitos dos fármacosRESUMO
Parathyroid hormone (PTH) is the anabolic standard of care for patients with severe osteoporosis. The CaSR allosteric antagonist JTT-305/MK-5442, a PTH secretagogue, could offer an oral osteoanabolic treatment alternative for postmenopausal women with osteoporosis. Here we disclose the pharmacokinetic profile of JTT-305/MK-5442 and its activity on bone remodeling in ovariectomized (OVX) osteopenic rats. Daily treatments (0.3 to 2.4 mg/kg/d) for 12 weeks resulted in plateaued BMD increases (3.8 to 5.3%) at axial and appendicular skeletal sites. However, treatment effects were not statistically significant, in agreement with effects seen in animals treated with low dose PTH (1-84) (5 µg/kg/d). In a consecutive study we tested JTT-305/MK-5442 effects on bone formation in OVX-rats challenged with combined alendronate (ALN) treatment paradigms. At 7 month, JTT-305/MK-5442 treatment significantly increased BMD in lumbar vertebrae (LV), while no change in BMD was observed in femora or tibiae. ALN add-on co-treatment produced incremental increases in LV, distal femur (DF) and proximal tibia (PT) BMD over the respective ALN control. Histological analyses confirmed modest increases in mineralized surface (MS/BS) and bone formation rate (30.5±1.9%) on trabecular surfaces by JTT-305/MK-5442. As expected, ALN administration profoundly reduced bone formation, however, JTT-305/MK-5442 significantly stimulated MS/BS and BFR in ALN treated groups. In summary, JTT-305/MK-5442 acts as a PTH secretagogue in the osteopenic OVX-rat, eliciting consistent, though modest effects on remediation of BMD due to estrogen depletion. Induction of bone formation by JTT-305/MK-5442 at trabecular bone surfaces appears to be resilient to ALN-mediated suppression of bone formation. This study provides for the first time, a mechanistic evaluation of combination treatment of a PTH secretagogue with ALN.
Assuntos
Alendronato/administração & dosagem , Benzoatos/administração & dosagem , Conservadores da Densidade Óssea/administração & dosagem , Doenças Ósseas Metabólicas/tratamento farmacológico , Propanolaminas/administração & dosagem , Receptores de Detecção de Cálcio/antagonistas & inibidores , Animais , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Remodelação Óssea/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Estrogênios/deficiência , Feminino , Ovariectomia , Hormônio Paratireóideo/sangue , Ratos , Ratos Sprague-DawleyRESUMO
Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Osteogênese/imunologia , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/terapia , Animais , Densidade Óssea/imunologia , Células CHO , Cricetinae , Cricetulus , Feminino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoporose Pós-Menopausa/patologiaRESUMO
Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Especificidade de Anticorpos , Densidade Óssea/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Densidade Óssea/imunologia , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/imunologia , Doenças Ósseas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteínas Relacionadas a Receptor de LDL/imunologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Macaca fascicularis , Macaca mulatta , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/imunologiaRESUMO
Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.
Assuntos
Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Animais , Benzoquinonas , Células COS , Chlorocebus aethiops , Quinases Ciclina-Dependentes/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Feminino , Fulvestranto , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 1 da Matriz/genética , Tamanho do Órgão/efeitos dos fármacos , Fosforilação , Regiões Promotoras Genéticas , Quinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Serina/metabolismo , Útero/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).
Assuntos
Cromanos/química , Cromanos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Modelos Químicos , Estrutura Molecular , Tamanho do Órgão , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Útero/efeitos dos fármacosAssuntos
Hepatite Viral Humana/complicações , Hepatite Viral Humana/patologia , Herpes Simples/complicações , Herpes Simples/patologia , Falência Hepática/complicações , Insuficiência Renal/complicações , Evolução Fatal , Feminino , Hepatite Viral Humana/congênito , Herpes Simples/congênito , Humanos , Hipotermia/complicações , Recém-Nascido , Fígado/patologiaRESUMO
3(S)-(6-methoxypyridin-3-yl)-3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]-naphthyridin-2-yl)propyl]imidazolidin-1-yl]propionic acid 6 was identified as a potent and selective antagonist of the alpha(v)beta(3) receptor. This compound has an excellent in vitro profile (IC(50) = 0.08 nM), a significant unbound fraction in human plasma (12%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in three in vivo models of bone turnover, the compound was selected for clinical development. To support the ongoing metabolism and safety studies, a novel strategy was employed in which a series of oxidized derivatives of 6 were prepared by exposure of 6 (or the methyl ester) to chemical oxidizing agents. These products proved useful in the identification of active metabolites generated by either in vitro or in vivo metabolism.