RESUMEN
OBJECTIVE: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. DESIGN: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. RESULTS: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. CONCLUSION: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.
Asunto(s)
Agrecanos/metabolismo , Artritis Experimental/patología , Cartílago Articular/patología , Articulación de la Rodilla/patología , Osteoartritis/patología , Proteoglicanos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Agrecanos/farmacología , Animales , Artritis Experimental/tratamiento farmacológico , Western Blotting , Cartílago Articular/efectos de los fármacos , Inmunohistoquímica , Inyecciones Intraarticulares , Articulación de la Rodilla/efectos de los fármacos , Masculino , Osteoartritis/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.
Asunto(s)
Compuestos Heterocíclicos con 1 Anillo/síntesis química , Ácidos Hidroxámicos/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas ADAM , Proteína ADAM17 , Sitios de Unión , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Lipopolisacáridos/farmacología , Modelos Moleculares , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of =0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Ácidos Hidroxámicos/síntesis química , Lactamas/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas ADAM , Proteína ADAM17 , Administración Oral , Animales , Disponibilidad Biológica , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacocinética , Carbamatos/farmacología , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Lactamas/química , Lactamas/farmacocinética , Lactamas/farmacología , Masculino , Ratones , Estereoisomerismo , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.
Asunto(s)
Antiinflamatorios/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Animales , Butadienos/farmacología , Butadienos/uso terapéutico , Edema/prevención & control , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , Masculino , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Nitrilos/uso terapéutico , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.
Asunto(s)
Ácidos Hidroxámicos/química , Metaloendopeptidasas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Cinética , Lipopolisacáridos/metabolismo , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/clasificación , Modelos Químicos , Modelos MolecularesAsunto(s)
Compuestos de Bifenilo/uso terapéutico , Trasplante de Corazón/inmunología , Inmunosupresores/uso terapéutico , Trasplante Heterólogo/inmunología , Animales , Cricetinae , Ciclosporina/uso terapéutico , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Mesocricetus , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapéutico , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Relación Estructura-Actividad , Trasplante Homólogo/inmunologíaAsunto(s)
Ácidos Hidroxámicos/síntesis química , Lactamas/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Cristalografía por Rayos X , Humanos , Ácidos Hidroxámicos/sangre , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Lactamas/sangre , Lactamas/química , Lactamas/farmacología , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Activación de Linfocitos , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/sangre , Modelos Moleculares , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacologíaRESUMEN
Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.
Asunto(s)
Activación de Linfocitos , Proteínas Quinasas/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Complejo CD3/inmunología , División Celular/inmunología , Células Clonales , Interleucina-2/inmunología , Ratones , Ratones Endogámicos BALB C , Quinasas de Proteína Quinasa Activadas por Mitógenos , Linfocitos T/citologíaRESUMEN
Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.
Asunto(s)
Inmunosupresores/química , Inmunosupresores/farmacología , Compuestos de Bifenilo/química , Carbono/química , Prueba de Cultivo Mixto de Linfocitos , Relación Estructura-ActividadRESUMEN
The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.
Asunto(s)
Compuestos de Bifenilo/química , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Inmunosupresores/química , Inmunosupresores/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Prueba de Cultivo Mixto de Linfocitos , Oxidorreductasas/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.
Asunto(s)
Inflamación/enzimología , Riñón/anomalías , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Inhibidores de la Ciclooxigenasa , Modelos Animales de Enfermedad , Femenino , Fibrosis , Marcación de Gen , Cardiopatías/enzimología , Cardiopatías/genética , Infertilidad Femenina/enzimología , Infertilidad Femenina/genética , Inflamación/genética , Riñón/embriología , Riñón/enzimología , Hígado/embriología , Hígado/enzimología , Ratones , Ratones Noqueados , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/fisiologíaRESUMEN
Brequinar sodium (BQR) is a novel immunosuppressive agent that acts by inhibiting the activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine biosynthetic pathway. The activity of BQR as an immunosuppressive agent is believed to be inhibition of antigen-induced lymphocyte proliferation through inhibition of DNA and RNA synthesis. BQR, therefore, has a different mechanism of action than cyclosporine and may potentiate the immunosuppressive effects of cyclosporine. In this study, we determined the effect of BQR on peripheral blood mononuclear cell (PBMC) activation in a series of in vitro culture systems. In these studies, BQR inhibited PHA-stimulated activation in a dose-dependent fashion beginning at 10(-6) M. The immunosuppressive effect of BQR was similar in magnitude to cyclosporine. Proliferation assays suggested an additive immunosuppression by the combination of BQR and cyclosporine. Similar inhibition of CD2-stimulated or CD3-stimulated activation of PBMC was found. The mechanisms of action of BQR were complex. BQR inhibited interleukin 2 protein production in response to mitogen stimulation. Cell surface interleukin 2 receptor expression was inhibited by BQR. BQR inhibited cell cycle progression, preventing progression from G0/G1 into S and G2 + M phases. BQR had no effect on induction of transcripts for the interleukin 2 receptor, but markedly inhibited the production of transcripts for interleukin 2. Thus, our studies indicate that BQR exerts a potent immunosuppression on mitogen-induced PBMC activation through multiple mechanisms. Consequently, BQR may be an effective agent for immunosuppression in organ transplantation or inflammatory diseases.
Asunto(s)
Compuestos de Bifenilo/farmacología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , ADN/biosíntesis , Depresión Química , Humanos , Interleucina-2/genética , Trasplante de Órganos/fisiología , ARN Mensajero/genética , Receptores de Interleucina-2/fisiología , Transcripción Genética/efectos de los fármacosAsunto(s)
Compuestos de Bifenilo/farmacología , Inmunoglobulina A/efectos de los fármacos , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina M/efectos de los fármacos , Inmunosupresores/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Adulto , Compuestos de Bifenilo/administración & dosificación , Ciclosporina/administración & dosificación , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Inmunosupresores/administración & dosificación , Activación de Linfocitos/inmunología , Mitógenos de Phytolacca americana/farmacología , Tacrolimus/administración & dosificación , Tacrolimus/farmacologíaAsunto(s)
Compuestos de Bifenilo/farmacología , Ciclosporina/farmacología , Inmunosupresores/farmacología , Intestino Delgado/trasplante , Animales , Sinergismo Farmacológico , Quimioterapia Combinada , Rechazo de Injerto , Supervivencia de Injerto/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas BN , Ratas Endogámicas LewAsunto(s)
Compuestos de Bifenilo/farmacología , Inmunosupresores/farmacología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Dermatitis por Contacto/inmunología , Dinitrofluorobenceno , Epítopos/inmunología , Femenino , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/inmunología , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Ovinos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaAsunto(s)
Compuestos de Bifenilo/farmacología , Dihidroorotato Oxidasa/antagonistas & inhibidores , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Pirimidinas/metabolismo , Animales , Células Cultivadas , Concanavalina A/farmacología , Dihidroorotato Oxidasa/metabolismo , Humanos , Inmunidad/efectos de los fármacos , Inmunoensayo , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Linfocitos/inmunología , Nucleótidos/metabolismo , Ratas , Ratas Endogámicas Lew , Porcinos , Porcinos EnanosAsunto(s)
Compuestos de Bifenilo/farmacología , Inmunosupresores/farmacología , Linfocitos/inmunología , Administración Oral , Animales , Compuestos de Bifenilo/sangre , Compuestos de Bifenilo/farmacocinética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inmunosupresores/farmacocinética , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Macaca fascicularis , Timidina/metabolismo , Factores de Tiempo , TritioAsunto(s)
Compuestos de Bifenilo/uso terapéutico , Rechazo de Injerto/prevención & control , Inmunosupresores/uso terapéutico , Trasplante de Hígado/inmunología , Animales , Compuestos de Bifenilo/administración & dosificación , Esquema de Medicación , Inmunosupresores/administración & dosificación , Ratas , Factores de Tiempo , Trasplante HomólogoRESUMEN
Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. In these experiments, we have tested the ability of BQR to prevent the rejection of concordant cardiac xenografts. LEW inbred rats transplanted with heterotopic hamster hearts were treated orally with brequinar sodium as a single agent. The survival of the cardiac xenografts was significantly prolonged with a variety of treatment regimens. The most effective treatment was the daily oral administration of BQR at 3 mg/kg. At this level, the median graft survival was approximately 25 days. Four animals had hamster heart xenografts that functioned for more than 90 days. The prolonged survival of the xenografts was associated with relatively constant plasma drug levels of approximately 1 to 3 micrograms/ml and a marked suppression of IgM production. At rejection, there was a significant rise in IgM levels compared with those of recipients with stable xenografts. In vitro MLR responses were effectively inhibited by BQR, with an IC50 of 0.08 microgram/ml. The results of these experiments demonstrate that BQR is a new immunosuppressive agent that is highly effective as a single agent in prolonging the survival of hamster-to-rat cardiac xenografts. The prolonged xenograft survival is associated with effective suppression of rat antihamster antibody production, suggesting that brequinar sodium may be an important addition to multidrug immunosuppressive regimes designed to prevent B and T lymphocyte-mediated immune responses.
Asunto(s)
Compuestos de Bifenilo/farmacología , Trasplante de Corazón/inmunología , Inmunosupresores/farmacología , Trasplante Heterólogo , Animales , Compuestos de Bifenilo/sangre , Cricetinae , Rechazo de Injerto , Supervivencia de Injerto/efectos de los fármacos , Inmunoglobulina M/sangre , Mesocricetus , Ratas , Ratas Endogámicas LewRESUMEN
The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.