RESUMO
BMS-986251 is a retinoid-related orphan receptor γt (RORγt) inverse agonist that was in development for the treatment of autoimmune diseases. RORγt is a nuclear hormone receptor and transcription factor that is involved in the differentiation and function of T helper 17 cells. RORγt-deficient (constitutive or conditional) mice develop thymic lymphomas with >50% mortality at 4 months, whereas heterozygous mice are normal. A 6-month study was conducted in rasH2-Tg hemizygous mice to assess the potential carcinogenicity of BMS-986251. BMS-986251 was administered once daily by oral gavage to groups of 27 mice/sex at doses of 0 (water control), 0 (vehicle control), 5, 25, or 75 mg/kg. The positive control, N-methyl-N-nitrosourea, was administered by a single intraperitoneal injection to 15 mice/sex at a dose of 75 mg/kg. There were no tumors attributed to BMS-986251 except for thymic lymphomas. Thymic lymphoma was observed in 1 male (3.7%) and 3 females (11.1%) at the mid dose, and 6 females (22.2%) at the high dose. No lymphomas were observed in the negative control groups whereas the incidence of lymphomas in the positive control group was 47-60%. The incidence of thymic lymphomas in the BMS-986251-treated groups was higher than published literature and test facility historical control data. Furthermore, increased thymic lymphoid cellularity (lymphoid hyperplasia) was observed at the mid dose in males and at all doses in females. Since lymphoid hyperplasia may represent a preneoplastic change, a no-effect dose for potential tumor induction was not identified in this study. These results led to the discontinuation of BMS-986251 and underscore the challenges in targeting RORγt for drug development.
Assuntos
Linfoma , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Animais , Testes de Carcinogenicidade , Feminino , Hiperplasia , Linfoma/induzido quimicamente , Linfoma/genética , Masculino , Camundongos , Camundongos TransgênicosRESUMO
PEGylation is considered a safe mechanism to enhance the pharmacokinetics (PK) and pharmacodynamics (PD) of biotherapeutics. Previous studies using PEGylation as a PK enhancement tool have reported benign PEG-related vacuolation in multiple tissues. This paper establishes a threshold for PEG burden beyond which there are alterations in tissue architecture that could potentially lead to dysfunction. As part of the nonclinical safety assessment of Compound A, a 12 kDa protein conjugated to a 40 kDa branched PEG molecule, monkeys were dosed subcutaneously twice weekly for 3 months at protein doses resulting in weekly PEG doses of 8, 24, 120, or 160 mg/kg. Consistent with previous reports with PEGylated biomolecules, Compound A administration resulted in intracellular vacuoles attributed to the PEG moiety in macrophages in numerous tissues and epithelial cells in the choroid plexus and kidney. Vacuolation occurred at all doses with dose-dependent severity and no evidence of recovery up to 2 months after dosing cessation. The vacuolation was considered nonadverse at PEG doses ≤120 mg/kg/week. However, at 160 mg/kg/week PEG, the vacuolation in choroid plexus, pituitary gland, kidney, and choroid of the eye was considered adverse due to significant alterations of tissue architecture that raised concern for the possibility of compromised tissue function. To our knowledge, this is the first report of potentially adverse cellular consequences of PEG accumulation in tissues other than kidney. Furthermore, the lack of reversibility of vacuolation coupled with the lack of a biomarker for intracellular PEG accumulation highlights a potential risk that should be weighed against the benefits of PK/PD enhancement for long-term administration of PEGylated compounds at high doses.
Assuntos
Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Polietilenoglicóis/toxicidade , Proteínas/toxicidade , Vacúolos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Composição de Medicamentos , Células Epiteliais/patologia , Feminino , Injeções Subcutâneas , Macaca fascicularis , Macrófagos/patologia , Masculino , Polietilenoglicóis/administração & dosagem , Proteínas/administração & dosagem , Medição de Risco , Fatores de Tempo , Vacúolos/patologiaRESUMO
Anomalies of renal development comprise abnormalities in the amount of renal tissue (agenesis and hypoplasia); anomalies of renal position, form, and orientation; and renal dysplasia. There are previous reports of canine renal dysplasia in different breeds but none in the Beagle breed. This is the first report of renal dysplasia in this breed of dog. Morphologic descriptions of the range of microscopic features observed in four cases of renal dysplasia from preclinical studies in laboratory Beagle dogs are presented (including persistent primitive mesenchyme, persistence of metanephric ducts, asynchronous differentiation of nephrons, and atypical tubular epithelium), along with a basis for the classification of the lesion.
Assuntos
Doenças do Cão/patologia , Nefropatias/veterinária , Rim/anormalidades , Animais , Doenças do Cão/genética , Cães , Feminino , Nefropatias/genética , Nefropatias/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Falência Renal Crônica/veterinária , MasculinoRESUMO
Therapeutic use of certain peroxisome proliferator-activated receptor (PPAR) alpha agonists (fibrates) for the treatment of dyslipidemia has infrequently been associated with the untoward side effect of myopathy. With interest in PPAR-delta as a therapeutic target, this study assessed whether a PPAR-delta agonist induced similar hepatic and skeletal muscle alterations as noted with some fibrates. PPAR-alpha null (KO) and corresponding wild-type (WT) mice were administered toxicological dosages of a potent PPAR-delta agonist tool ligand (GW0742; which also has weak PPAR-alpha agonist activity) or a potent PPAR-alpha agonist (WY-14,643) for 10 days. Increases in liver weights and clinical chemistry indicators of skeletal muscle damage and/or liver injury were more pronounced in WT mice compared with KO mice administered the PPAR-delta agonist. Likewise, the incidence and severity of skeletal myopathy were greater in WT mice given GW0742 compared with KO mice. Ultrastructural and immunohistochemical analyses revealed significant peroxisome proliferation in muscle and liver of WT mice treated with each agonist; however, KO animals showed little or no evidence of hepatic and muscle peroxisome proliferation. PMP-70 protein expression in liver was consistent with these results. The hepatomegaly, hepatic and skeletal muscle peroxisome proliferation, and skeletal myopathy induced by this PPAR-delta ligand was predominantly mediated by its cross-activation of PPAR-alpha, though PPAR-delta agonism contributed slightly to these effects.
Assuntos
Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR delta/agonistas , Peroxissomos/efeitos dos fármacos , Pirimidinas/toxicidade , Tiazóis/toxicidade , Animais , Relação Dose-Resposta a Droga , Hepatomegalia/induzido quimicamente , Hepatomegalia/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/metabolismo , Tamanho do Órgão , PPAR alfa/agonistas , PPAR alfa/deficiência , PPAR alfa/genética , PPAR delta/metabolismo , Peroxissomos/metabolismo , Peroxissomos/patologiaRESUMO
The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.
Assuntos
Anisóis/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Anisóis/farmacologia , Artrite Experimental/patologia , Células Cultivadas , Humanos , Masculino , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Endogâmicos Lew , Vírus do Sarcoma Felino/efeitos dos fármacos , Vírus do Sarcoma Felino/enzimologiaRESUMO
OBJECTIVE: The membrane-anchored metalloproteinase disintegrin ADAM15 is up-regulated in osteoarthritis and has been implicated in proteolysis and cell-matrix interactions. To address its role in cartilage metabolism, we performed an analysis of joint morphology in aging mice with a targeted inactivation of the ADAM15 gene (ADAM15(-/-)). In addition, a human chondrocyte cell line overexpressing ADAM15 was used to investigate the role of ADAM15 in an in vitro model of chondrocyte-matrix interactions. METHODS: Knee joint sections from 3-, 6-, and 12-14-month-old ADAM15(-/-) and wild-type (WT) 129/SvJ mice were examined for synovial hyperplasia, cartilage degradation, and osteophyte formation. Stable transfection of the human T/C28a4 chondrocyte cell line with full-length human ADAM15 complementary DNA led to the establishment of ADAM15-overexpressing chondrocytes that were further analyzed for their capability to adhere to and to survive on cartilage matrix molecules (fibronectin and types II and VI collagen) under conditions of serum starvation. ADAM15 expression was investigated by reverse transcription-polymerase chain reaction and Western blotting. RESULTS: Aging ADAM15(-/-) mice exhibited accelerated development of osteoarthritic lesions compared with WT mice, and the difference was statistically significant at age 12 months. The osteoarthritic changes preferentially affected male ADAM15(-/-) mice. ADAM15 overexpression in T/C28a4 cells led to the specific reinforcement of chondrocyte adhesion to cartilage types II and VI collagen, and this was associated with enhanced cell viability under conditions of serum starvation. CONCLUSION: The accelerated development of murine osteoarthritis in ADAM15 deficiency as well as the proadhesive and cell survival-promoting in vitro effect of ADAM15 overexpression suggest a homeostatic rather than a destructive role of ADAM15 in cartilage remodeling.
Assuntos
Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Homeostase , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Envelhecimento/fisiologia , Animais , Western Blotting , Cartilagem Articular/patologia , Adesão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular , Condrócitos/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Osteoartrite do Joelho/enzimologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Joelho de Quadrúpedes/metabolismo , Joelho de Quadrúpedes/patologia , Membrana Sinovial/enzimologia , Membrana Sinovial/patologia , TransfecçãoRESUMO
ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Metaloendopeptidases/genética , Metaloendopeptidases/fisiologia , Neovascularização Patológica , Células Tumorais Cultivadas , Proteínas ADAM , Alelos , Animais , Western Blotting , Encéfalo/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Endotélio Vascular/citologia , Citometria de Fluxo , Deleção de Genes , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Doenças Retinianas/patologia , Fatores de Tempo , Distribuição Tecidual , Veias Umbilicais/citologiaRESUMO
A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Formamidas/farmacologia , Hepatócitos/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Oxirredutases N-Desmetilantes/biossíntese , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteínas ADAM , Proteína ADAM17 , Administração Oral , Amidas/administração & dosagem , Amidas/farmacocinética , Aminopiridinas/administração & dosagem , Aminopiridinas/sangue , Aminopiridinas/farmacocinética , Animais , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Técnicas de Cultura de Células , Citocromo P-450 CYP3A , Dipeptídeos/administração & dosagem , Dipeptídeos/sangue , Dipeptídeos/farmacocinética , Relação Dose-Resposta a Droga , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática , Formamidas/química , Hepatócitos/metabolismo , Humanos , Masculino , Metaloproteinases da Matriz/administração & dosagem , Metaloproteinases da Matriz/farmacocinética , Oxirredutases N-Desmetilantes/efeitos dos fármacos , Receptor de Pregnano X , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Esteroides/efeitos dos fármacos , Tiazóis/administração & dosagem , Tiazóis/farmacocinéticaRESUMO
We describe and discuss microscopic findings in the brown adipose tissue (BAT) blood vessels of Han Wistar rats treated with GI262570X, a peroxisome proliferator-activated receptor-gamma agonist (PPAR-gamma agonist) by oral gavage for 28 days, 13 weeks, 27 weeks, and 2 years. Review of these studies revealed a consistent vascular change, consisting of multifocal fatty infiltration in the BAT of treated rats. A similar vascular change was not seen in other vessels or organs. Microscopically, fatty infiltration was characterized primarily by round, clear vacuoles within the tunica media and/or tunica adventitia of small and medium-sized arteries and arterioles. Occasionally, these vacuoles had peripherally located nuclei and morphologically resembled adipocytes, suggesting a well-characterized PPAR effect (ie, differentiation of stem cells or preadipocytes into mature adipocytes). However, administration of GI262570X up to 2 years failed to induce more severe or progressive lesions in the blood vessels of rat BAT and, in particular, did not result in induction of any atherosclerotic-like lesions or foam cell infiltration. At the longer exposure, there was an apparent reduction of severity and/or incidence, indicating a possible adaptive response. These results suggest that the possibility of generating atherosclerotic-like lesions through prolonged treatment of GI262570X (PPAR-gamma agonist) is highly unlikely in rats.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Hipoglicemiantes/toxicidade , Músculo Liso Vascular/efeitos dos fármacos , Oxazóis , Tecido Adiposo Marrom/irrigação sanguínea , Animais , Arteriosclerose/induzido quimicamente , Divisão Celular/efeitos dos fármacos , Feminino , Masculino , Músculo Liso Vascular/patologia , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Tempo , Fatores de Transcrição/fisiologia , Tirosina/análogos & derivadosRESUMO
MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.