Metabolic profile of the antitumor agent piplartine by Cytochrome P450 enzymes, in vitro study and prediction of pharmacokinetic parameters

Author(s)

Fernanda de Lima Moreira

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-02-21

Examining board members

Anderson Rodrigo Moraes de Oliveira; Thiago Barth; Jonas Augusto Rizzato Paschoal; Rosangela Gonçalves Peccinini

• Advisor: Anderson Rodrigo Moraes de Oliveira

Abstract

Piplartine (PPT) or Piperlongumine is a naturally occurring alkaloid found in species of Pipereaceae family. Due its high potency and selectivity of inhibition of several cancer cell lines, PPT has been investigated as a potential drug candidate. In this context, studies related with toxicity and safety should be performed, including the role of the Cytochrome P450 (CYP450) enzymes in PPT metabolism. This family of enzymes is responsible for the biotransformation of 75% of the drugs in the market. The preclinical studies that aim to evaluate the drug metabolism can be performed by employing in vitro models as a tool for prediction of in vivo pharmacokinetic characteristics. Therefore, the aim of this work was to evaluate the metabolic profile of PPT after metabolism by CYP450 enzymes employing in vitro studies with human liver microsomes (HLM) and the ensuing prediction of pharmacokinetic parameters. These studies embraced the kinetic parameters determination, inhibition ability of PPT over the most important CYP450 isoforms, structural elucidation of the produced metabolites after metabolism reaction and, finally, the enzymatic phenotyping study. The general procedure for in vitro metabolism studies consisted of the use of chromatographic techniques coupled to different detectors/analyzers, such as diode array, mass spectrometry and nuclear magnetic resonance. The metabolism was evaluated measuring the rate of disappearance of the PPT from de microsomal medium. After method validation for PPT quantification and determination of initial velocity conditions, the enzymatic kinetics with a sigmoidal profile indicating a metabolism of PPT by enzymes with multiple active sites and/or metabolism by multiple CYP450 enzymes was observed. The following parameters were calculated: Vmax = 5.5 ± 0.5 nmol/mg protein/min, S50 = 127.7 ?mol/L, and Hill coefficient of 3.0. The intrinsic clearance was 22.68 ?L min -1 mg -1. The unbound fraction of PPT on plasmatic and microsomal proteins was 0.07 and 0.76, respectively. The predicted in vivo clearance was 19.79 mL min -1 kg -1, the hepatic clearance was 1.89 mL min -1 kg -1 and the hepatic extraction was 0.09. Among 4 isoforms evaluated, CYP3A, CYP2C9, CYP2D6 and CYP1A2, a potential natural product-drug interaction for only CYP1A2 isoenzyme by PPT was observed. PPT showed to be a competitive and dosedependent inhibitor of CYP1A2, showing a Ki value of 1.5 ?mol L-1. The ratio [I]/Ki of 9.1 predicts an important in vivo interaction. Furthermore, a time-dependent inhibition of CYP1A2 with a KI of 8 ?mol L-1 and a kinact of 0.014 min-1 by PPT was demonstrated. The dose-, time- and NADPH-dependent inhibition confirms an inhibition based on mechanism through an irreversible bond. Based on results obtained from the mass spectrometry analysis and from the nuclear magnetic resonance analysis, four metabolites were identified and characterized. The metabolites characterized were: M1 (product of a demethylation in the 3,4,5-trimethoxyphenyl portion, M2 (derived from an epoxidation between C3 and C4 on the lactone ring), M3 (product of a simple oxidation on C5 of lactone ring), and finally M4 (derived from a dihydrodiol reaction between C3 and C4). The metabolite M4 is produced later (after 40 min of reaction) and probably is a secondary metabolite produced from M2 through a dihydrodiol reaction. The phenotyping study demonstrated that the main isoforms involved in PPT metabolism are CYP1A2 (production of M1) and CYP3A4 (production of M2 and M3). The recombinant isoforms study demonstrated that several isoforms (CYP2C19, CYP2C8, CYP2D6, CYP2B6 and CYP2E1) catalyze the production of M4. In summary, a wide view about the metabolism of the promising drug candidate PPT by CYP450 enzymes was accomplished. These results, certainly, will be a useful guide for further clinical studies of PPT (AU)

• FAPESP's process: 13/04188-4 - In vitro metabolism study and enzymatic inhibition of natural product piplartine employing human liver microsomes and supersomes.
• Grantee: Fernanda de Lima Moreira
• Support Opportunities: Scholarships in Brazil - Doctorate