RESUMO
MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.
Assuntos
GMP Cíclico/metabolismo , Eletrólitos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptídeos/farmacologia , Propionatos/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/fisiologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Cinética , Ratos , Ratos Sprague-Dawley , Receptores de EnterotoxinaRESUMO
Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.
Assuntos
Constipação Intestinal/tratamento farmacológico , Síndrome do Intestino Irritável/tratamento farmacológico , Peptídeos/farmacologia , Alquilação , Animais , Área Sob a Curva , Disponibilidade Biológica , Biotransformação , Constipação Intestinal/complicações , AMP Cíclico/metabolismo , Fezes/química , Feminino , Trânsito Gastrointestinal/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Síndrome do Intestino Irritável/complicações , Masculino , Peptídeo Hidrolases/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyRESUMO
Activation of guanylate cyclase-C (GC-C) expressed predominantly on intestinal epithelial cells by guanylin, uroguanylin or the closely related GC-C agonist peptide, linaclotide, stimulates generation, and release of cyclic guanosine-3',5'-monophosphate (cGMP). Evidence that the visceral analgesic effects of linaclotide are mediated by a novel, GC-C-dependent peripheral sensory mechanism was first demonstrated in animal models of visceral pain. Subsequent studies with uroguanylin or linaclotide have confirmed the activation of a GC-C/cGMP pathway leading to increased submucosal cGMP mediated by cGMP efflux pumps, which modulates intestinal nociceptor function resulting in peripheral analgesia. These effects can be reproduced by the addition of exogenous cGMP and support a role for GC-C/cGMP signaling in the regulation of visceral sensation, a physiological function that has not previously been linked to the GC-C/cGMP pathway. Notably, targeting the GC-C/cGMP pathway for treatment of gastrointestinal pain and abdominal sensory symptoms has now been validated in the clinic. In 2012, linaclotide was approved in the United States and European Union for the treatment of adult patients with irritable bowel syndrome with constipation.
RESUMO
The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.
Assuntos
Guanilato Ciclase/metabolismo , Peptídeos Natriuréticos/metabolismo , Transdução de Sinais/fisiologia , Dor Visceral/metabolismo , Acetilcolina/farmacologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Adenocarcinoma/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/complicações , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias Colorretais/patologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica , Feminino , Gastroenteropatias/complicações , Gastroenteropatias/etiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hiperalgesia/fisiopatologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Morfina/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Peptídeos Natriuréticos/uso terapêutico , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Peroxidase/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Restrição Física , Ácido Trinitrobenzenossulfônico/toxicidade , Dor Visceral/tratamento farmacológico , Dor Visceral/etiologiaRESUMO
Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC50:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 µg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.
Assuntos
Trânsito Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Laxantes/farmacologia , Peptídeos/farmacologia , Receptores Acoplados a Guanilato Ciclase/agonistas , Receptores de Peptídeos/agonistas , Animais , Ligação Competitiva , Disponibilidade Biológica , Linhagem Celular , Células Cultivadas , Constipação Intestinal/tratamento farmacológico , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/citologia , Secreções Intestinais/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Laxantes/química , Laxantes/metabolismo , Laxantes/farmacocinética , Masculino , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacocinética , Conformação Proteica , Estabilidade Proteica , Ratos , Receptores de EnterotoxinaRESUMO
AIMS: Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). MAIN METHODS: Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. KEY FINDINGS: Linaclotide inhibited in vitro [(125)I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [(125)I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3',5'-monophosphate and accelerated gastrointestinal transit. SIGNIFICANCE: Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.
Assuntos
Fármacos Gastrointestinais/farmacologia , Peptídeos/farmacologia , Receptores de Peptídeos/agonistas , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Feminino , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/farmacocinética , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trânsito Gastrointestinal/efeitos dos fármacos , Guanilato Ciclase/genética , Mucosa Intestinal/metabolismo , Secreções Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/genéticaRESUMO
Candida albicans remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in C. albicans pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a C. albicans tet-NRG1 strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Doxiciclina/farmacologia , Hifas/efeitos dos fármacos , Animais , Antifúngicos/uso terapêutico , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase/microbiologia , Candidíase/mortalidade , Candidíase/patologia , Proteínas de Ligação a DNA/genética , Doxiciclina/uso terapêutico , Feminino , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Hifas/crescimento & desenvolvimento , Rim/microbiologia , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , VirulênciaRESUMO
Streptococcus pneumoniae is an important human pathogen capable of causing serious infections. NADH oxidase, a factor necessary for infection, was previously identified as part of a signature-tagged mutagenesis screen of a S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nox gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined nox deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10(5)-fold in the murine respiratory tract infection model and at least 10(4)-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of S. pneumoniae infection. NADH oxidase converts O(2) to H(2)O. If O(2) is not fully reduced, it can form superoxide anion (O2(-)) and hydrogen peroxide (H(2)O(2)), both of which can be toxic to cells. Bacterial cell extracts from the allelic-replacement mutant were found to lack NADH oxidase activity and the mutant was unable to grow exponentially under conditions of vigorous aeration. In contrast, the mutant displayed normal growth characteristics under conditions of limited aeration. The S. pneumoniae nox gene was cloned and expressed in E. coli. The purified His-tagged NADH oxidase was shown to oxidize NADH with a K:(m) of 32 microM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.
Assuntos
Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Infecções Pneumocócicas/fisiopatologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , Alelos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Otite Média/microbiologia , Otite Média/fisiopatologia , Filogenia , Infecções Pneumocócicas/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/fisiopatologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , VirulênciaRESUMO
Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.