RESUMO
We investigated anti-colitic effects of N-(2-mercaptopropionyl)-glycine (NMPG), a diffusible antioxidant, in TNBS-induced rat colitis model and a potential molecular mechanism underlying the pharmacologic effect of the antioxidant. NMPG alleviated colonic injury and effectively lowered myeloperoxidase activity. Moreover, NMPG substantially attenuated expression of pro-inflammatory mediators in the inflamed colon. NMPG induced hypoxia-inducible factor-1α (HIF-1α) in human colon carcinoma cells, leading to elevated secretion of vascular endothelial growth factor (VEGF), a target gene product of HIF-1 involved in ulcer healing of gastrointestinal mucosa. NMPG induction of HIF-1α occurred by inhibiting HIF prolyl hydroxylase-2 (HPH-2), an enzyme that plays a major role in negatively regulating HIF-1α protein stability. In in vitro Von Hippel-Lindau protein binding assay, the inhibitory effect of NMPG on HPH-2 was attenuated by escalating dose of ascorbate but not 2-ketoglutarate, cofactors of the enzyme. Consistent with this, cell-permeable ascorbate significantly attenuated NMPG induction of HIF-1α in cells. Our data suggest that NMPG is an anti-colitic antioxidant that exerts its pharmacologic effects at least partly through activation of an ulcer healing pathway, HIF-1-VEGF.
Assuntos
Antioxidantes/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Colite/tratamento farmacológico , Colite/enzimologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolil Hidroxilases/metabolismo , Tiopronina/uso terapêutico , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Colite/patologia , Difusão , Ativação Enzimática/efeitos dos fármacos , Células HCT116 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Tiopronina/farmacologia , Ácido Trinitrobenzenossulfônico , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.
Assuntos
Peptídeos Penetradores de Células/química , Colo/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/metabolismo , NF-kappa B/antagonistas & inibidores , Peptídeos/química , Peptídeos/uso terapêutico , Administração Oral , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Síndrome do Intestino Irritável/induzido quimicamente , Masculino , Ratos , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
N-aromatic acyl-amino acid conjugates possess a colon-targeted property, implying that such conjugates are stable and are not absorbable until reaching the large intestine in which they are microbially converted (hydrolysed) to the parent drugs that are therapeutically active. To investigate the structural effect of N-aromatic acyl-amino acid conjugates on the large intestinal deconjugation, the hydrolysis of various N-aromatic acyl-amino acid conjugates was examined in the cecal contents. On incubation of conjugates with glycine, D or/and L forms of alanine or phenylalanine in the cecal contents, the conjugates with D amino acids were not hydrolysed. The other conjugates are susceptible to the hydrolysis, the rates of which decreased as the size of the substituent on the 2-position of the amino acids increased. The conjugates with alkyl analogs (2-4 carbons) of glycine and taurine were resistant to the hydrolysis, while taurine- and glycine-conjugates were hydrolysed effectively. The hydrolysis of N-aromatic acyl-glycine conjugates was enhanced by para-substitution of electron withdrawing groups on the aromatic acyl moiety and vice versa for electron-donating groups. While a methyl, methoxy or chloro group on the ortho-position retarded the hydrolysis, a hydroxyl group on the position accelerated it. Our data may provide useful information for the design of a colon-specific prodrug with controlled conversion rate in the large intestine.
Assuntos
Alanina/análogos & derivados , Aminoácidos Aromáticos/metabolismo , Ceco/metabolismo , Pró-Fármacos/metabolismo , Alanina/síntese química , Alanina/metabolismo , Aminoácidos Aromáticos/análise , Aminoácidos Aromáticos/química , Animais , Antimetabólitos Antineoplásicos/análise , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/metabolismo , Colo/metabolismo , Preparações de Ação Retardada , Fluoruracila/análogos & derivados , Fluoruracila/metabolismo , Conteúdo Gastrointestinal/química , Concentração de Íons de Hidrogênio , Masculino , Terapia de Alvo Molecular , Pró-Fármacos/análise , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Glycogen synthase kinase-3ß (GSK3ß), a multi-functional kinase, is a promising therapeutic target for the treatment of inflammation. Inhibitory κB kinase (IKK)-activated GSK3ß inhibitory peptide (IAGIP) was designed as an inflammation-responsive anti-colitic therapeutic. To optimize therapeutic efficiency, IAGIP was tested using two different drug delivery techniques: colon-targeted delivery and cell-permeable peptide modification. In cell-based experiments, in response to tumor necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated activation of IKK, cell-permeable IAGIP (CTP-IAGIP) inhibited GSK3ß, leading to increased production of anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of TNF- and LPS-induced NFκB activity. Oral gavage of CTP-IAGIP loaded in the colon-targeted capsule attenuated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and lowered the expression levels of NFκB-regulated proteins in the inflamed colons. CTP-IAGIP further induced IL-10 production in the inflamed colonic tissues; however, the levels of IL-10 were not affected in the normal colonic tissue or colonic tissue in which inflammation had subsided. Collectively, our data suggest that IAGIP administered using the aforementioned drug delivery techniques is an orally active anti-colitic drug selectively responding to inflammation.
Assuntos
Colite , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Peptídeos , Ratos , Ácido TrinitrobenzenossulfônicoRESUMO
Hypoxia-inducible factor prolyl hydroxylases (HPHs) are responsible for hydroxylation of proline residues in hypoxia-inducible factor-α (HIF-α), resulting in von Hippel-Lindau (VHL)-mediated proteasome degradation of the hydroxylated proteins. Pharmacological inhibition of the enzyme leads to stabilization of HIF-α proteins and consequent activation of HIF, which provides therapeutic benefit for a variety of tissues undergoing ischemic stress. In an effort to develop a new assay for measuring HPH activity, we designed a fusion protein, VHL ß-domain-luciferase. Recombinant fusion protein with a glutathione S-transferase (GST) tag was purified from Escherichia coli. GST-VHL ß-domain-luciferase with C-terminal deletion (GVbL-CD) was obtained as a major product and found to have luciferase activity. In a GVbL-CD capture assay using HIF peptide-bound beads, at least a 13-fold increase in luciferase activity was elicited for HIF peptide with hydroxyproline compared with unhydroxylated HIF peptide. HPH inhibitory activities of known HPH inhibitors or HIF-1α inducers were assessed using this assay, whose results were in good agreement with those obtained from conventional methods. The competitive effect of 2-ketoglutarate on dimethyloxalylglycine-mediated HPH inhibition was assessed very well in the new assay. Taken together, the VHL ß-domain protein with luciferase activity is of use for HPH activity assay.
Assuntos
Técnicas Biossensoriais/métodos , Dioxigenases/metabolismo , Ensaios Enzimáticos/métodos , Hidroxiprolina/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Aminoácidos Dicarboxílicos/química , Linhagem Celular Tumoral , Dioxigenases/antagonistas & inibidores , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia , Ácidos Cetoglutáricos/química , Luciferases/análise , Luciferases/genética , Proteínas Nucleares/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Manganese (II), a transition metal, causes pulmonary inflammation upon environmental or occupational inhalation in excess. We investigated a potential molecular mechanism underlying manganese-induced pulmonary inflammation. Manganese (II) delayed HIF-1alpha protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1alpha hydroxylation and subsequent von Hippel-Lindau(VHL)-dependent HIF-1alpha degradation. HPH inhibition by manganese (II) was neutralized significantly by elevated dose of iron. Consistent with this, the induction of cellular HIF-1alpha protein by manganese (II) was abolished by pretreatment with iron. Manganese (II) induced the HIF-1 target gene involved in pulmonary inflammation, vascular endothelial growth factor (VEGF), in lung carcinoma cell lines.The induction of VEGF was dependent on HIF-1. Manganese-induced VEGF promoted tube formation of HUVEC. Taken together, these data suggest that HIF-1 may be a potential mediator of manganese-induced pulmonary inflammation.
Assuntos
Cloretos/toxicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Mediadores da Inflamação/toxicidade , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Compostos de Manganês , Pneumonia/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway.
Assuntos
Cinamatos/química , Cinamatos/farmacologia , Colite/tratamento farmacológico , Depsídeos/química , Depsídeos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Animais , Ácido Ascórbico/farmacologia , Ácidos Carboxílicos/química , Linhagem Celular Tumoral , Cinamatos/uso terapêutico , Coenzimas/fisiologia , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Depsídeos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Ésteres , Humanos , Hidroxilação/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Ácidos Cetoglutáricos/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ácido Trinitrobenzenossulfônico/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ácido RosmarínicoRESUMO
The clinical usefulness of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, for treatment of inflammatory bowel disease (IBD) is controversial in terms of efficacy and toxicity. To overcome these problems, colon-specific drug delivery was adopted, which generally confers therapeutic and toxicological advantages of drugs for treatment of colonic diseases. N-succinylaspart-1-yl celecoxib (SA1C), a colon-specific prodrug of celecoxib, was administered orally to rats with experimental colitis, and the anti-colitic effects and a molecular mechanism were investigated and compared to those of conventional celecoxib. SA1C, which delivered a much greater amount of celecoxib to the inflamed colon, alleviated the colonic injury, lowered myeloperoxidase activity in the inflamed colonic tissues and was much more effective than conventional celecoxib. SA1C but not conventional celecoxib significantly attenuated expression of NFκB target gene products in the inflamed tissues. Consistent with this, SA1C effectively prevented nuclear accumulation of p65 in the inflamed tissues. Moreover, while conventional celecoxib lowered the serum level of 6-keto-PGF1α, an inverse indicator of cardiovascular toxicity, SA1C did not change its serum level. Our data suggest that colonic delivery of celecoxib is a feasible strategy for treatment of IBD with improved therapeutic and toxicological properties.
Assuntos
Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ácido Trinitrobenzenossulfônico/efeitos adversos , 6-Cetoprostaglandina F1 alfa/sangue , Animais , Celecoxib , Linhagem Celular , Colite/sangue , Colite/metabolismo , Colo/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pró-Fármacos/metabolismo , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Ratos , Sulfonamidas/metabolismo , Sulfonamidas/uso terapêuticoRESUMO
To investigate the mechanisms underlying the biological activity of piceatannol (PCT), a hydroxystilbene natural product that has anti-colitic properties, we examined whether PCT could modulate hypoxia-inducible factor (HIF)-1 activity in human colon carcinoma cells. PCT induced HIF-1α protein, leading to induction of its target gene products, vascular endothelial growth factor and heme oxygenase-1, which are involved in amelioration of colitis. PCT induction of HIF-1α resulted from HIF-1α protein stabilization, which occurred through inhibition of HIF-prolyl hydroxylase-2 (HPH-2). PCT inhibition of HPH-2 was reversed by addition of ascorbate, a cofactor of HPH-2, but not the cosubstrate, 2-ketoglutarate, to the reaction mixture of an in vitro von Hippel-Lindau (VHL) capture assay, and pretreatment with ascorbate abrogated PCT induction of cellular HIF-1α. Moreover, PCT prevented hydroxylation of cellular HIF-1α and attenuated coimmunoprecipitation of Flag-VHL protein and HA-HIF-1α over-expressed in human embryonic kidney 293 cells. Structural analysis using derivatives of PCT revealed that the catechol moiety in PCT was required for the stabilization of HIF-1α protein. Taken together, PCT activation of HIF-1 resulting from inhibition of HPH-2 may be a molecular mechanism for an anti-colitic effect of the natural product.
Assuntos
Heme Oxigenase-1/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Estilbenos/farmacologia , Ácido Ascórbico/farmacologia , Linhagem Celular Tumoral , Colite/tratamento farmacológico , Colite/patologia , Neoplasias do Colo/metabolismo , Células HEK293 , Heme Oxigenase-1/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Caffeic acid phenethyl ester (CAPE) is a polyphenolic natural product that possesses numerous biological activities including anti-inflammatory effects. CAPE-mediated nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2) activation is likely responsible for some of its biological effects. CAPE was chemically modified to yield CAPE analogues that were subjected to experiments examining cellular Nrf2 activity. CAPE and the CAPE analogue with a catechol moiety, but not the other analogues, activated the Nrf2 pathway. In addition, only biotin-labeled CAPE analogues with the catechol moiety precipitated Kelch-like ECH associated protein 1 (Keap1) when incubated with cell lysates and streptavidin agarose beads. Sodium hypochlorite (NaOCl) oxidation of the catechol moiety in CAPE produced an oxidized, electrophilic form of CAPE (Oxi-CAPE) and greatly enhanced the ability of CAPE to activate Nrf2 and to bind to Keap1. Rectal administration of CAPE ameliorated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and activated the Nrf2 pathway in the inflamed colon, and incubation of CAPE in the lumen of the inflamed distal colon generated Oxi-CAPE. However, these biological effects and chemical change of CAPE were not observed in the normal colon. Our data suggest that CAPE requires the catechol moiety for the oxidation-enhanced activation of the Nrf2 pathway and has potential as a pathologically targeted Nrf2-activating agent that is exclusively activated in pathological states with oxidative stress such as colonic inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Cafeicos/farmacologia , Colite/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Álcool Feniletílico/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Western Blotting , Ácidos Cafeicos/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/química , Álcool Feniletílico/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-AtividadeRESUMO
To develop a more potent NFκB inhibitor from salicylic acid which is known to inhibit activity of NFκB, a transcription factor regulating genes involved in immunity, inflammation and tumorigenesis, derivatives of salicylic acid (SA) where the 5 position, carboxyl or hydroxyl group was modified were treated in HCT116 cells transfected with an NFκB dependent luciferase gene and LPS-stimulated RAW264.7 cells. Amidation of the carboxylic group or substitution of chlorine at the 5 position increased the ability of SA to suppress the expression of NFκB dependent luciferase and inducible nitric oxide synthase, a product of an NFκB target gene. Moreover, simultaneous amidation and chlorination of SA (5-chlorosalicylamide; 5-CSAM) conferred an additive NFκB inhibitory activity on SA. To further enhance the inhibitory activity, N-modification was imposed on 5-CSAM. N-(5-chlorosalicyloyl)phenethylamine (5-CSPA), N-(5-chlorosalicyloyl)3-phenylpropylamine (5-CSPPA) and N-(5-chlorosalicyloyl)4-hydroxyphenylethylamine (5-CSHPA) showed greater potencies for inhibiting NFκB activity than other derivatives. Their IC(50)s' in the luciferase assay measured 15µM (5-CSPA), 17µM (5-CSPPA) and 91µM (5-CSHPA). Rectal administration of 5-CSPA ameliorated TNBS-induced rat colitis, which was more effective than a conventional drug, 5-aminosalicylic acid. These data may provide useful information for development of a therapeutic agent for treatment of diseases where NFκB plays a critical role in the pathogenic progresses.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Colite/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Salicilatos/síntese química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL2/análise , Quimiocina CXCL2/metabolismo , Células HCT116 , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Estrutura Molecular , NF-kappa B/metabolismo , Ratos , Salicilatos/química , Salicilatos/farmacologia , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVES: The systemic side effects of glucocorticoids have prevented their long-term use for treatment of inflammatory bowel disease. Colon-specific delivery of glucocorticoids has been adopted as a strategy to circumvent the toxicological trouble. Glucocorticoids delivered to the large intestine might undergo metabolisms by colonic microflora, which should affect therapeutic availability at the target site. It was investigated whether the susceptibility of glucocorticoids to the colonic metabolisms and pharmacologic intervention in the metabolism could modulate the therapeutic availability of colon-targeted glucocorticoids. METHODS: Various glucocorticoids and their derivatives, glucocorticoid 21-sulfate sodium compounds, were incubated in the cecal contents in the presence or absence of reduction inhibitors and the change in the levels of the drugs was monitored. KEY FINDINGS: The accumulation profiles of the corresponding glucocorticoids liberated from glucocorticoid 21-sulfate sodium compounds vary, depending on the metabolic susceptibility of glucocorticoids. Reduction inhibitors prevented the cecal metabolisms of glucocorticoids, which was most prominent for prednisolone (PD) and methylprednisolone (MP). Moreover, reduction inhibitors increased the accumulated amount of MP and PD released from PD- and MP-21-sulfate sodium in the cecal contents. CONCLUSIONS: Our data provide information useful for selection of a glucocorticoid and a pharmacologic strategy for the design of an efficient colon-specific glucocorticoid prodrug.
Assuntos
Colo/metabolismo , Glucocorticoides/farmacocinética , Animais , Carbenoxolona/farmacologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Ácido Glicirrízico/farmacologia , Absorção Intestinal , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Prolyl 4-hydroxylases (P4Hs), such as collagen prolyl-4-hydroxylases (CPHs) and hypoxia inducible factor prolyl-4-hydroxylases (HPHs), have recently been recognized as promising drug targets for the treatment of fibrotic and ischemic diseases. CPHs and HPHs catalyze identical metabolic reactions, yet lead to quite different physiological consequences, collagen synthesis and the regulation of oxygen homeostasis. Selective modulation of the two enzymes should provide a therapeutic benefit upon pharmacotherapy. In an in vitro VHL capture assay, hydroxylation of the 19mer HIF peptide (corresponding to HIF-1α residues 556-574) by HPH-2 was effectively prevented by nitric oxide (NO) donors, (±)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The NO donors also caused inhibition of HPHs and accumulation of nonhydroxylated HIF-1α protein in A549 human lung adenocarcinoma cells. Hyperoxia (100% O(2)) attenuated both NO donor-induced accumulation of HIF-1α and inhibition of HPH-mediated hydroxylation. In the presence of a proteasome inhibitor, MG132, the hyperoxia-mediated degradation of HIF-1α was deterred and hydroxylated HIF-1α was detected. SNAP, while being an effective inhibitor of proline 4-hydroxylation of HIF-1α by HPH-2, did not diminish proline hydroxylation of collagen by CPHs. Our data suggest that NO inhibits HPH-2 via competing with dioxygen and that the discriminative effect of NO on CPHs and HPH-2 is attributable to the difference in the affinity of the two enzymes toward dioxygen.
Assuntos
Colágeno/metabolismo , Hiperóxia/enzimologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/fisiologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Sequência de Aminoácidos , Células Cultivadas , Humanos , Dados de Sequência Molecular , Pró-Colágeno-Prolina Dioxigenase/fisiologia , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated potential molecular mechanisms underlying CAPE-mediated nuclear factor kappa beta (NFkappaB) inhibition and analyzed structure of CAPE for its biological effect. CAPE attenuated expression of NFkappaB dependent luciferase stimulated with TNF-alpha or LPS and suppressed LPS-mediated induction of iNOS, a target gene product of NFkappaB. In HCT116 cells, CAPE interfered with TNF-alpha dependent IkappaBalpha degradation and subsequent nuclear accumulation of p65, which occurred by direct inhibition of inhibitory protein kappaB kinase (IKK). CAPE increased the expression of Nrf2-dependent luciferase and heme oxygenase-1, a target gene of Nrf2, and elevated the nuclear level of Nrf2 protein, indicating that CAPE activated the Nrf2 pathway. In HCT116 cells with stable expression of Nrf2 shRNA, CAPE elicited a reduced inhibitory effect on TNF-alpha-activated NFsmall ka, CyrillicB compared to scramble RNA expressing control cells. On the other hand, the NFkappaB inhibitory effect of CAPE was diminished by removal or modification of the Michael reaction acceptor, catechol or phenethyl moiety in CAPE. These data suggest that CAPE inhibits TNF-alpha-dependent NFkappaB activation via direct inhibition of IKK as well as activation of Nrf2 pathway, in which the functional groups in CAPE may be involved.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Cafeicos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/antagonistas & inibidores , Álcool Feniletílico/análogos & derivados , Animais , Anti-Inflamatórios não Esteroides/química , Ácidos Cafeicos/química , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Citosol/metabolismo , Genes Reporter , Humanos , Immunoblotting , Lipopolissacarídeos/farmacologia , Luciferases/genética , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Álcool Feniletílico/química , Álcool Feniletílico/farmacologia , Plasmídeos , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) alpha protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1alpha protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel-Lindau-dependent HIF-1alpha degradation. CAPE inhibition of HPH and induction of HIF-1alpha protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (-C=C-) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH.
Assuntos
Ácidos Cafeicos/farmacologia , Álcool Feniletílico/análogos & derivados , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Células HEK293 , Heme Oxigenase-1/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Álcool Feniletílico/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Relação Estrutura-Atividade , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Metronidazole (MTZ) is a drug of choice for protozoal infections such as luminal amoebiasis. We designed and synthesized N-nicotinoyl-2-{2-(2-methyl-5-nitroimidazol-1-yl)ethyloxy}-D,L-glycine (NMG) as a colon-specific prodrug of MTZ. The synthetic yield of NMG was about 34%. The apparent partition coefficient of MTZ was greatly reduced by the chemical modification. While (bio)chemically stable in the contents of the upper intestine, NMG was rapidly cleaved to liberate MTZ on incubation with the cecal contents of rats. MTZ metabolized quickly in the cecal contents at least partly by a microbial nitroreductase, suggesting that the metabolism of MTZ is relevant to its bioactivation leading to amoebicidal action. The systemic absorption, analyzed by the blood concentration and urinary recovery of NMG, was very low after oral administration of NMG. In parallel with this, whereas MTZ disappeared mostly during the transit of the proximal small intestine, a substantial amount of NMG remained in the small intestine moving down to the large intestine where it metabolized rapidly. Moreover, comparing systemic absorption of MTZ after oral administration of NMG or MTZ, NMG markedly reduced the systemic absorption. These results suggest that NMG is a potential colon-specific prodrug of MTZ which improves therapeutic and toxicological properties.
Assuntos
Colo/efeitos dos fármacos , Metronidazol/análogos & derivados , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Administração Oral , Animais , Antiprotozoários/química , Antiprotozoários/metabolismo , Antiprotozoários/farmacocinética , Antiprotozoários/farmacologia , Ceco/metabolismo , Colo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Glicina/análogos & derivados , Concentração de Íons de Hidrogênio , Absorção Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Metronidazol/química , Metronidazol/metabolismo , Metronidazol/farmacocinética , Metronidazol/farmacologia , Estrutura Molecular , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Ratos , Ratos Sprague-DawleyRESUMO
Previously, we reported that oral administration of taurine conjugated 5-aminosalicylic acid, a colon-specific prodrug of 5-aminosalicylic acid (5-ASA), is effective in ameliorating experimental colitis and taurine elicits an additive anti-inflammatory effect upon cotreatment with 5-ASA. To explore a molecular mechanism for the anti-inflammatory property of the prodrug, we investigated the effect of the conjugate on IL-1beta-mediated NFkappaB activation. In human colon carcinoma Caco-2 and HCT116 cells, NFkappaB activity was accessed by a luciferase reporter assay and IL-6 secretion. Protein levels were determined by Western blotting. IL-6 levels were monitored by an Elisa kit. Treatment with either 5-ASA or taurine chloramine (TauCl) inhibited IL-1beta-mediated NFkappaB dependent luciferase expression and IL-6 secretion. In HCT116 cells, the inhibitory effect by TauCl or 5-ASA was through preventing IL-1beta-induced IkappaB kinase activation and subsequently interfering with IkappaBalpha degradation and p65 nuclear accumulation. Furthermore, combined TauCl/5-ASA treatment interfered additively with the activation process, leading to additive inhibitory effect on IL-1beta-mediated NFkappaB activation. Our results suggest that the anti-inflammatory effect of the prodrug on experimental colitis is attributed to the inhibition of the IL-1beta-mediated NFkappaB activation and the taurine effect is through TauCl potentiating the ability of 5-ASA to inhibit IL-1beta dependent NFkappaB activation.
Assuntos
Colite/tratamento farmacológico , Interleucina-1beta/metabolismo , Mesalamina/química , Mesalamina/farmacologia , NF-kappa B/metabolismo , Taurina/análogos & derivados , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular Tumoral , Colite/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Mesalamina/uso terapêutico , Pró-Fármacos/metabolismo , Taurina/química , Taurina/farmacologiaRESUMO
PURPOSE: We investigated in vivo-colon targetability and therapeutic properties of DS against experimental rat colitis. METHODS: The systemic absorption and colonic delivery of D after oral administration of DS was analyzed by examining the concentration of drugs in the GI tract, plasma, urine and feces. Therapeutic activity of DS was determined using a TNBS-induced rat colitis model. Adrenal suppression by DS administration was evaluated by monitoring the concentration of ACTH and corticosterone in the plasma. RESULTS: DS administered orally was delivered efficiently to the large intestine resulting in D accumulation at the target site. In addition, DS was not detectable in the plasma and was detected very low in the urine after DS administration. The fecal and urinary recovery of D (after DS administration) was much greater and less than that after D administration, suggesting that DS should exhibit enhanced therapeutic activity and reduced systemic side effects. Consistent with this notion, DS was more effective than D in healing rat colitis. Moreover, oral administration of either D or DS reduced the plasma corticosterone and ACTH levels from the normal levels, which is significantly greater for D. CONCLUSION: DS is a promising colon specific prodrug that improves therapeutic properties of D.
Assuntos
Anti-Inflamatórios/farmacologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Dexametasona/farmacologia , Fármacos Gastrointestinais/farmacologia , Pró-Fármacos/farmacologia , Sulfatos/farmacologia , Administração Oral , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/sangue , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Dexametasona/administração & dosagem , Dexametasona/análogos & derivados , Dexametasona/farmacocinética , Modelos Animais de Doenças , Fezes/química , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/farmacocinética , Hidrocortisona/sangue , Absorção Intestinal , Masculino , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Ratos , Ratos Sprague-Dawley , Sulfatos/administração & dosagem , Sulfatos/farmacocinética , Ácido TrinitrobenzenossulfônicoRESUMO
Methylprednisolone (MP) is one of the most widely used corticosteroids for the treatment of inflammatory bowel disease (IBD). However, systemic adverse effects of MP limit its availability for the disease. In present study, sulfate-conjugated methylprednisolone (MPS) was evaluated in vivo as a colon-targeted prodrug of MP and its therapeutic properties against 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis were investigated. Upon oral administration, a large fraction of MPS reached the large intestine, where MPS was converted to MP implying that MPS would deliver MP effectively to the large intestine. The fecal recovery of MP (after MPS administration) was much greater than that after MP administration and the urinary recovery of MP (after MPS administration) was much less than that after MP administration, suggesting that MPS should exhibit enhanced therapeutic activity and reduced systemic adverse effects. Consistent with this notion, MPS was more effective than MP in ameliorating rat colitis. Moreover, the adverse effects of MPS on adrenal function and thymus were much lower than those of MP. Taken together, MPS may be therapeutically superior to MP in IBD treatment.