title: In vitro metabolism of the alkaloid piplartine by rat liver microsomes.
abstract: Because piplartine (PPT) has demonstrated biological activities, such as cytotoxic, anxiolytic, antidepressant, antifungal and antiplatelet 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 μmol/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 μmol/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±0.3 μmol/mg mL⁻¹/min, h=2.5±0.4, S₅₀=44.7±0.3 μmol/L and CL(max)=0.054 μL/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.
authors.affiliation: Departamento de Física-Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Lucas Maciel Mauriz
authors.initials: LM
authors.lastname: Marques
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Eduardo Afonso
authors.initials: EA
authors.lastname: da Silva
authors.affiliation: Departamento de Física-Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Dayana Rubio
authors.initials: DR
authors.lastname: Gouvea
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Vessecchi
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Mônica Tallarico
authors.initials: MT
authors.lastname: Pupo
authors.affiliation: Departamento de Física-Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Norberto Peporine
authors.initials: NP
authors.lastname: Lopes
authors.affiliation: Instituto de Química, Universidade de São Paulo, 05508-000 São Paulo, São Paulo, Brazil.
authors.forename: Massuo Jorge
authors.initials: MJ
authors.lastname: Kato
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: deoliveira@usp.br.
authors.forename: Anderson Rodrigo Moraes
authors.initials: AR
authors.lastname: de Oliveira
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D010881:Piperidones / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010881:Piperidones; C498077:piperlongumine
keywords: Enzymatic kinetics; In vitro metabolism; Microsomes; Piplartine; Sigmoidal profile