In vitro metabolism of the alkaloid piplartine by rat liver microsomes

Author(s)

Mauriz Marques, Lucas Maciel ^[1] ; da Silva-Junior, Eduardo Afonso ^[2] ; Gouvea, Dayana Rubio ^[1] ; Vessecchi, Ricardo ^[3] ; Pupo, Monica Tallarico ^[2] ; Lopes, Norberto Peporine ^[1] ; Kato, Massuo Jorge ^[4] ; Moraes de Oliveira, Anderson Rodrigo ^[3]

Affiliation

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Fis Quim, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, BR-14040903 Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040901 Ribeirao Preto, SP - Brazil

^[4] Univ Sao Paulo, Inst Quim, BR-05508000 Sao Paulo - Brazil

• Document type: Journal article
• Source: Journal of Pharmaceutical and Biomedical Analysis; v. 95, p. 113-120, JUL 2014.
• Web of Science Citations: 25

Abstract

Because piplartine (PPT) has demonstrated biological activities, such as cytotoxic, anxiolytic, antidepressant, antifungal and antipiatelet activities, this molecule is a relevant drug candidate. The metabolic fate of drug candidates is an essential requirement in assessing their safety and efficacy. Based on this requirement, the biotransformation of PPT by cytochrome P450 enzymes (CYP) was investigated for the first time. To determine the in vitro enzymatic kinetic parameters, an HPLC method was developed and validated to quantify PPT. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile:water (40:60, v/v). The method exhibited a linear range of 2.4-157.7 mu mol/L, with the following calibration curve: y = 0.0934 (+/- 0.0010)x + 0.0027, r = 0.9975. The lower limit of quantitation was verified to be 2.4 mu mol/L, with an RSD below 7%. The precision and accuracy were assessed for both within-day and between-day determinations; neither relative standard (RSD%) deviations nor relative errors (RER) exceeded a value of 15%. The mean absolute recovery was 85%, with an RSD value below 6%. The enzymatic kinetic parameters revealed a sigmoidal profile, with V-max = 4.7 +/- 0.31 mu mol/mg mL(-1)/min, h = 2.5 +/- 0.4, S-50 = 44.7 +/- 0.3 mu mol/L and CLmax = 0.054 mu L/min/mg protein, indicating cooperativity behavior. Employing a mammalian model, PPT metabolism yielded two unreported monohydroxylated products (m/z 334). The identification and structural elucidation of the metabolites were performed by comparing their mass spectra with those spectra of the parent drug. For the first time, the in vitro metabolism studies employing microsomes were demonstrated to be a suitable tool for data regarding enzymatic kinetics and for the metabolites formed in the PPT mammalian metabolism. (C) 2014 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 11/17508-1 - Development and validation of chromatographic and electrophoretic methods for subsequent application in studies of in vitro metabolism and biotransformation
• Grantee: Anderson Rodrigo Moraes de Oliveira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 11/13560-9 - In vitro metabolism study of the piplartine alkaloid using rat liver microsomes
• Grantee: Lucas Maciel Mauriz Marques
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 09/51812-0 - Development of a platform for the study of in vitro and in vivo metabolism of natural products, a need for pre-clinical testing system
• Grantee: Norberto Peporine Lopes
• Support Opportunities: BIOTA-FAPESP Program - Thematic Grants