RESUMO
The degradation of klerval (I) was studied as a function of pH. The extent and routes of degradation were found to be pH-dependent. Under strongly acidic conditions (pH<2), the drug predominantly undergoes specific acid-catalyzed hydrolysis of the side-chain amide bond yielding II8), the drug undergoes specific base-catalyzed hyrolysis yielding II and epimerization generating D-epimer. The epimerization appears to occur via the succinimide intermediate in neutral pH region. With increasing pH, however, the epimerization rate increases due to direct epimerization of the peptides.
Assuntos
Oligopeptídeos/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Oligopeptídeos/análise , Estereoisomerismo , Fatores de TempoRESUMO
The integrin receptor recognition sequence Arg-Gly-Asp was successfully used as a template from which to develop a series of potent, selective, orally active, peptide-based fibrinogen receptor antagonists with a long duration of action. Simple modifications centered on the Arg and Gly residues quickly led to a modified peptide (1) with significantly enhanced ability to inhibit in vitro platelet aggregation. Substitution of the guanidino group in 1 by piperidine provided 3, which showed not only a further increase in potency but also a modest degree of oral efficacy. Finally, exploration of the nature of the C-terminal amino acid, with respect to its side-chain functionality and the carboxy terminus, yielded a group of molecules that showed excellent in vitro potency for inhibiting platelet aggregation, excellent integrin selectivity, a high level of oral efficacy, and an extended duration of action.
Assuntos
Oligopeptídeos , Piperidinas , Inibidores da Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Administração Oral , Animais , Plaquetas/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Cães , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Masculino , Oligopeptídeos/administração & dosagem , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Piperidinas/administração & dosagem , Piperidinas/síntese química , Piperidinas/metabolismo , Piperidinas/farmacologia , Ativação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Relação Estrutura-Atividade , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacosRESUMO
Salmon calcitonin (sCT), a 32-amino-acid peptide, is the active component in many pharmaceuticals used for the management of bone diseases. The degradation pathways of sCT were determined, and the structures of the major degradation products were identified. Aqueous solutions of sCT at pH values of 3, 4, 5, and 6 were degraded, and the major degradation products were detected using reversed phase and size-exclusion high-performance liquid chromatography (HPLC). The degradation rate and pathways of sCT are strongly dependent on pH in the pH range between 3 and 6. The major degradation products were isolated by semipreparative HPLC and identified using a variety of spectroscopic and bioanalytical techniques. The results show that sCT can undergo hydrolyses resulting in cleavage of the 1-2 amide bond and deamidation of the Gln14 and Gln20 residues, sulfide exchange that leads to an unusual trisulfide derivative, and dimerization to reducible and nonreducible dimers. The mechanisms for the pathways can be rationalized from known degradation pathways of peptide and proteins.
Assuntos
Calcitonina/química , Sequência de Aminoácidos , Animais , Calcitonina/farmacologia , Cálcio/sangue , Dimerização , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Ratos , ÁguaRESUMO
PURPOSE: The purpose of this work was to study the photostability of the antiproliferative tyrphostin drug compounds RG 13022(I) and RG 14620(II) as a part of preformulation program. METHODS: The compounds were exposed to cool white fluorescent light in solution as well as in the solid state and analyzed by HPLC. The degradation products were isolated chromatographically and their structures determined by spectroscopic methods. X-ray crystallographic analyses of the above compounds and their solid state degradation products were carried out to understand the mechanism of photodegradation. RESULTS: The compounds were found to undergo efficient photochemical transformations in solution as well as in the solid state. The degradation in the solution was due to the photoisomerization into their E-isomers (III and IV). The solid state photodegradation products were [2 + 2]-cycloaddition products (V and VI). The stereochemistry of the photocycloaddition products was indicative of the crystal packing of their monomeric precursors. The photocycloaddition product of RG 13022 possesses the head-to-tail linkage as expected from the head-to-tail packing of RG 13022 molecules in the crystal. The photocycloaddition product of RG 14620, however, was found to involve head-to-head linkage in agreement with the head-to-head crystal packing of RG 14620. CONCLUSIONS: Drug compounds containing open chain olefinic double bonds could be sensitive to mild condition of light in the solid state if the distance between the two double bonds in the crystal approaches 4.2 degrees A and they have suitable UV absorption characteristics. Attractive interactions between chlorine atoms have significant influence in controlling the crystal packing of chlorinated aromatic compounds.
Assuntos
Antineoplásicos/química , Nitrilas/química , Piridinas/química , Tirfostinas , Alcenos/química , Antineoplásicos/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Estabilidade de Medicamentos , Conformação Molecular , Peso Molecular , Nitrilas/efeitos da radiação , Fotoquímica , Piridinas/efeitos da radiação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.
Assuntos
Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Fator de von Willebrand/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Humanos , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/farmacologiaRESUMO
A highly sensitive LC procedure was developed that utilizes fluorescence derivatization and detection coupled with size exclusion chromatography for the analysis of salmon calcitonin in salmon calcitonin aerosols. The LC procedure uses fluorescamine derivatization to label the primary amino groups of the peptide. The derivatization procedure is completely automated by an autosampler capable of pre-column mixing. Size exclusion chromatography is performed using a Supelco G2000 SWXL column. The method can be used to assay the amount of salmon calcitonin delivered per actuation of an aerosol unit. The procedure is simple, accurate, and precise and can detect as little as 2 ng ml-1 concentrations of salmon calcitonin.