RESUMO
Quantitative measurement of process-related impurities is a critical safety requirement for the production of drug substances of vaccine and therapeutic biologics. A simple and sensitive HPLC method has been developed for separation and quantitation of residual valproic acid (VPA) used in the cell transfection procedure for the manufacturing of an influenza vaccine. The method is comprised of a modified Dole liquid phase extraction followed by a quick pre-column derivatization using 2-bromoacetophenone. Nonanoic acid (NNA) is used as the internal standard (IS) and the quantification is performed by reversed-phase liquid chromatography. This new method can accurately measure as low as 6.8 µg/mL (LOQ) residual VPA in the vaccine drug substance.
Assuntos
Contaminação de Medicamentos , Vacinas contra Influenza , Ácido Valproico/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Células HEK293 , Humanos , Vacinas contra Influenza/análise , Vacinas contra Influenza/química , Vacinas contra Influenza/normas , Limite de Detecção , Modelos Lineares , Extração Líquido-Líquido/métodos , Reprodutibilidade dos Testes , Cloreto de Sódio/química , Tecnologia Farmacêutica , Transfecção , Ácido Valproico/química , Ácido Valproico/isolamento & purificaçãoRESUMO
An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ilhotas Pancreáticas/embriologia , Modelos Animais , Organogênese/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Células COS , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Chlorocebus aethiops , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inibidores de Histona Desacetilases/isolamento & purificação , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/genética , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Transativadores/genética , Transativadores/metabolismo , Ácido Valproico/isolamento & purificação , Ácido Valproico/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
BACKGROUND: In 2006, there were 16 796 toxic exposures attributed to valproic acid (VPA), carbamazepine (CBZ) and phenytoin (PHT) reported to the US Toxic Exposure Surveillance System. Of these, 30% (5046) were treated in a health care facility with 12 cases resulting in death. These drugs are highly protein bound and poorly dialyzable; however, it has been suggested that albumin-supplemented dialysate may enhance dialytic clearance. We investigated whether the addition of albumin to dialysate affects dialytic clearance of VPA, CBZ and PHT. METHODS: VPA, CBZ and PHT were added to a bovine blood-based in vitro continuous hemodialysis circuit, which included a polysulfone or an AN69 hemodialyzer. VPA, CBZ and PHT clearances were calculated from spent dialysate and pre-dialyzer plasma concentrations. VPA, CBZ and PHT clearances with control (albumin-free) dialysate were compared to clearances achieved with 2.5% or 5% human albumin-containing dialysate. The influences of blood flow (180 and 270 mL/min) and dialysate flow (1, 2 and 4 L/h) on dialysis clearance were also assessed. RESULTS: The addition of 2.5% albumin to dialysate significantly enhanced dialytic clearance of VPA and CBZ, but not PHT. Use of 5% albumin dialysate further increased VPA and CBZ clearance. Overall, drug clearance was related directly to dialysate flow but independent of blood flow. CONCLUSION: Continuous hemodialysis with albumin-supplemented dialysate significantly enhanced VPA and CBZ, but not PHT, clearance compared to control dialysate. Continuous hemodialysis with albumin-supplemented dialysate may be a promising therapy to enhance dialytic clearance of selected highly protein-bound drugs.