Effects of replacement of factor VIII amino acids Asp519 and Glu665 with Val on plasma survival and efficacy in vivo.

Kosloski, Matthew P; Shetty, Krithika A; Wakabayashi, Hironao; Fay, Philip J; Balu-Iyer, Sathy V

Kosloski, Matthew P; Shetty, Krithika A; Wakabayashi, Hironao; Fay, Philip J; Balu-Iyer, Sathy V.

Afiliación

Kosloski MP; Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, 359 Kapoor Hall, Buffalo, New York, 14214, USA.

AAPS J ; 16(5): 1038-45, 2014 Sep.

Article en En | MEDLINE | ID: mdl-24934295

RESUMEN

Proteolytic cleavage of factor VIII (FVIII) to activated FVIIIa is required for participation in the coagulation cascade. The A2 domain is no longer covalently bound in the resulting activated heterotrimer and is highly unstable. Aspartic acid (D) 519 and glutamic acid (E) 665 at the A1-A2 and A2-A3 domain interfaces were identified as acidic residues in local hydrophobic pockets. Replacement with hydrophobic valine (V; D519V/E665V) improved the stability and activity of the mutant FVIII over the wild-type (WT) protein in several in vitro assays. In the current study, we examined the impact of mutations on secondary and tertiary structure as well as in vivo stability, pharmacokinetics (PK), efficacy, and immunogenicity in a murine model of Hemophilia A (HA). Biophysical characterization was performed with far-UV circular dichroism (CD) and fluorescence emission studies. PK and efficacy of FVIII was studied following i.v. bolus doses of 4, 10 and 40 IU/kg with chromogenic and tail clip assays. Immunogenicity was measured with the Bethesda assay and ELISA after a series of i.v. injections. Native secondary and tertiary structure was unaltered between variants. PK profiles were similar at higher doses, but at 4 IU/kg plasma survival of D519V/E665V was improved. Hemostasis at low concentrations was improved for the mutant. Immune response was similar between variants. Overall, these results demonstrate that stabilizing mutations in the A2 domain of FVIII can improve HA therapy in vivo.

Asunto(s)

Factor VIII/farmacología; Hemofilia A/tratamiento farmacológico; Hemostáticos/farmacología; Sustitución de Aminoácidos; Animales; Modelos Animales de Enfermedad; Estabilidad de Medicamentos; Factor VIII/administración & dosificación; Factor VIII/química; Factor VIII/genética; Factor VIII/inmunología; Factor VIII/farmacocinética; Hemofilia A/sangre; Hemofilia A/genética; Hemostasis/efectos de los fármacos; Hemostáticos/administración & dosificación; Hemostáticos/química; Hemostáticos/inmunología; Hemostáticos/farmacocinética; Interacciones Hidrofóbicas e Hidrofílicas; Inyecciones Intravenosas; Masculino; Ratones Endogámicos C57BL; Ratones Mutantes; Modelos Biológicos; Modelos Moleculares; Mutación; Ingeniería de Proteínas; Estabilidad Proteica; Estructura Secundaria de Proteína; Estructura Terciaria de Proteína; Relación Estructura-Actividad

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Factor VIII / Hemostáticos / Hemofilia A Tipo de estudio: Prognostic_studies Límite: Animals Idioma: En Revista: AAPS J Asunto de la revista: FARMACOLOGIA / TERAPIA POR MEDICAMENTOS Año: 2014 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

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