RESUMO
BACKGROUND: Zenpep (APT-1008) is a pancreatic enzyme product for the treatment of exocrine pancreatic insufficiency (EPI) associated with cystic fibrosis (CF). METHODS: Zenpep and Kreon, both containing 25,000 lipase units, were compared in a randomised, double-blind, crossover, non-inferiority study for CF-associated EPI in patients aged ≥12years. Patients on a standardised diet and stabilised treatment were randomised to two treatment sequences: Zenpep/Kreon or Kreon/Zenpep. The primary efficacy endpoint was the coefficient of fat absorption over 72h (CFA-72h). RESULTS: 96 patients (mean age 19.2years, 60.4% males) were randomised with 83 completers of both sequences comprising the efficacy population. Zenpep demonstrated non-inferiority and equivalence to Kreon in fat absorption (LS mean CFA-72h: Zenpep, 84.1% [SE 1.1] vs. Kreon, 85.3% [SE 1.1]; p=0.297). Safety and tolerability were similar. CONCLUSIONS: Zenpep is comparable with Kreon in efficacy and safety for the treatment of adolescents and adults with CF-associated EPI. NCT01641393.
Assuntos
Fibrose Cística , Terapia de Reposição de Enzimas/métodos , Insuficiência Pancreática Exócrina , Pâncreas/enzimologia , Pancrelipase , Adolescente , Adulto , Fibrose Cística/complicações , Fibrose Cística/diagnóstico , Fibrose Cística/fisiopatologia , Fibrose Cística/terapia , Método Duplo-Cego , Monitoramento de Medicamentos , Insuficiência Pancreática Exócrina/diagnóstico , Insuficiência Pancreática Exócrina/etiologia , Insuficiência Pancreática Exócrina/terapia , Feminino , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/efeitos adversos , Humanos , Absorção Intestinal/efeitos dos fármacos , Masculino , Testes de Função Pancreática/métodos , Pancrelipase/administração & dosagem , Pancrelipase/efeitos adversos , Resultado do TratamentoRESUMO
Interleukin-17 (IL-17) has been previously reported to induce stromal cells to produce a number of hematopoietic and proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF). Here, we have evaluated the mechanisms responsible for the augmentation of G-CSF gene expression by IL-17, using the murine 3T3 fibroblast cell line. Treatment of 3T3 cells, but not primary bone marrow-derived macrophages or murine monocyte/macrophage cell lines, resulted in increased steady-state G-CSF mRNA levels within 2-4 h and augmented G-CSF protein production. The combination of IL-17 and LPS enhanced G-CSF expression in an additive fashion. Stability studies revealed that IL-17 stabilized G-CSF mRNA levels, with a t1/2 of 4 h, compared to a t1/2 of less than 2 h in medium or LPS-treated cells. Induction of G-CSF expression in 3T3 cells by IL-17 did not appear to require tyrosine kinase activation or de novo protein synthesis. These studies indicate that post-transcriptional mechanisms play an important role in IL-17-induced G-CSF expression in fibroblasts and suggest that IL-17 may be useful for further delineating mechanisms of G-CSF gene regulation.
Assuntos
Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/genética , Interleucinas/farmacologia , Células 3T3 , Animais , Linhagem Celular , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Interleucina-17 , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Mitógenos/farmacologia , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
We have identified a small molecular weight compound, SCH 14988, which specifically stimulates in vitro granulocyte-colony stimulating factor (G-CSF) production from activated human peripheral blood mononuclear cells and monocytes but not other cytokines or CSFs with hematoregulatory activity. In vivo administration of SCH 14988 to mice rendered neutropenic by cyclophosphamide treatment resulted in the accelerated recovery of the peripheral neutrophil compartment. This activity correlated with increased in vivo G-CSF levels and stimulation of marrow granulopoiesis, and was comparable to that of exogenously administered recombinant human G-CSF. No alterations to other leukocyte populations in peripheral blood, spleen, or the peritoneal cavity were observed. These findings suggest that SCH 14988 may be clinically useful to enhance neutrophil granulopoiesis, as well as to study the mechanisms involved in G-CSF gene regulation.
Assuntos
Ciclofosfamida/toxicidade , Citocinas/biossíntese , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/farmacologia , Leucócitos Mononucleares/imunologia , Monócitos/fisiologia , Naftiridinas/farmacologia , Neutropenia/terapia , Neutrófilos/fisiologia , Animais , Medula Óssea/patologia , Células Cultivadas , Granulócitos/efeitos dos fármacos , Granulócitos/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Naftiridinas/uso terapêutico , Neutropenia/induzido quimicamente , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal conditions, 2-3 mg of GST-CSBP2 could be obtained per liter of infected cell culture. The fusion protein was easily purified from the soluble fraction of the total cell lysate under nondenaturing conditions by using a glutathione-Sepharose 4B affinity resin. As expected, the purified GST-CSBP2 fusion protein was approximately 68 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and reacted with antibodies directed toward either the GST or the CSBP amino terminus. To obtain activated CSBP2, SF9 cells were coinfected with two recombinant baculovirus vectors: one that directed the synthesis of the GST-CSBP2 fusion protein and a second vector that directed the synthesis of a constitutively active form of the CSBP activating kinase, MKK3. Coexpression of GST-CSBP2 kinase with the MKK3 activator increased GST-CSBP2 activity 8- to 10-fold based on the ability of GST-CSBP2 to phosphorylate the substrate, myelin basic protein (MBP), and the ATF2 transcription factor, in vitro. Moreover, activated GST-CSBP2 was capable of activating a bacterially derived mitogen-activated protein kinase-activating protein kinase 2 in vitro. The activity of insect-derived GST-CSBP2 was also inhibited by the CSBP inhibitor, SB202190. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of this kinase.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/isolamento & purificação , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Clonagem Molecular , Citocinas/antagonistas & inibidores , DNA Complementar/genética , Ativação Enzimática , Vetores Genéticos/síntese química , Glutationa Transferase/genética , Humanos , MAP Quinase Quinase 3 , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera/citologia , Spodoptera/enzimologia , Spodoptera/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
SCH 40120 is a potent acute anti-inflammatory agent under development for topical treatment of dermal inflammatory and allergic disorders such as atopic dermatitis, contact dermatitis and psoriasis. In order to support percutaneous absorption studies, a competitive enzyme immunoassay (EIA) was developed to determine SCH 40120 in unextracted plasma samples. SCH 38280, a carboxylated analogue of SCH 40120, was used as the hapten and conjugated with bovine thyroglobulin (Thy). The hapten-Thy conjugate was used as the immunogen to immunize rabbits for antibody production. The hapten was also coupled to horseradish peroxidase (HRP) to form SCH 38280-HRP, which was used as the tracer. The EIA can detect SCH 40120 concentrations as low as 50 pg ml-1 of plasma, and can reliably quantitate SCH 40120 in plasma samples from 100 pg ml-1 to 10 ng ml-1 with good linearity, accuracy and precision. A variety of structurally related compounds and potential metabolites did not significantly cross-react with the antibodies, except for a few analogues. The availability of this sensitive assay makes it possible to evaluate the pharmacokinetics of SCH 40120 in man.