RESUMO
A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.
Assuntos
Apolipoproteínas B/genética , Ornitina/química , Peptídeos/administração & dosagem , Fenilalanina/química , Polímeros/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Feminino , Fígado/metabolismo , Peso Molecular , Peptídeos/química , Polímeros/química , RNA Mensageiro/genética , RNA Interferente Pequeno/química , Ratos Sprague-DawleyRESUMO
The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.
Assuntos
Materiais Biocompatíveis/síntese química , Portadores de Fármacos/síntese química , Fígado/metabolismo , Nylons/síntese química , Peptídeos/síntese química , RNA Interferente Pequeno/administração & dosagem , Animais , Autorradiografia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacocinética , Materiais Biocompatíveis/toxicidade , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/toxicidade , Desenho de Fármacos , Estabilidade de Medicamentos , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/diagnóstico por imagem , Macaca mulatta , Nylons/química , Nylons/farmacocinética , Nylons/toxicidade , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/toxicidade , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/toxicidade , Cintilografia , Ratos Sprague-Dawley , Especificidade da Espécie , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (K(i) = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.