RESUMO
Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.
Assuntos
Apoptose/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Agonistas da Guanilil Ciclase C/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Nefrite/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Linhagem Celular , Citocinas/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Enalapril/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Nefrite/metabolismo , Nefrite/patologia , Fosforilação , Ratos Zucker , Transdução de Sinais , Proteína Smad3/metabolismoRESUMO
Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.
Assuntos
Interleucina-6/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Desenho de Fármacos , Meia-Vida , Humanos , Hibridomas , Interleucina-6/química , Interleucina-6/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-6/química , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Células U937RESUMO
BACKGROUND & AIMS: Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. METHODS: We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 µg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. RESULTS: In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). CONCLUSIONS: We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.
Assuntos
Dor Abdominal/prevenção & controle , Colo/inervação , GMP Cíclico/fisiologia , Guanilato Ciclase/fisiologia , Nociceptores/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Dor Abdominal/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CACO-2 , Linhagem Celular , Colo/efeitos dos fármacos , Colo/patologia , Modelos Animais de Doenças , Método Duplo-Cego , Feminino , Humanos , Síndrome do Intestino Irritável/induzido quimicamente , Síndrome do Intestino Irritável/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Peptídeos Natriuréticos/farmacologia , Nociceptores/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/fisiologia , Receptores de Peptídeos/fisiologia , Resultado do Tratamento , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
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.