Elsevier

Biochemical Pharmacology

Volume 90, Issue 2, 15 July 2014, Pages 166-178
Biochemical Pharmacology

Metabolic profiling of praziquantel enantiomers

https://doi.org/10.1016/j.bcp.2014.05.001Get rights and content

Abstract

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24 h after dosing, indicating that the major metabolic reactions encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity toward the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in substrate location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Introduction

Schistosomiasis, the second most prevalent parasitic disease after malaria, has been estimated to affect 207 million people [1]. Praziquantel (PZQ) is the least expensive, easiest to use and most readily available drug among all the currently available schistosomicides [2]. However, its utilization is limited by many disadvantages, including decreased susceptibility of Schistosoma mansoni toward PZQ [3]. PZQ has poor metabolic stability (t1/2 = 1–3 h), and undergoes the extensive first-pass metabolism [4]. Mono- and di-oxidized PZQ were identified to be the main metabolites using liver microsomes [4] and isolated rat hepatocytes [5]. However, the complete metabolite profile of PZQ remains unclear, including the identity of the phase II metabolites of PZQ. Drug-metabolizing enzymes involved in the elimination of PZQ in rat liver microsomes have been investigated, revealing that cytochromes P450 (CYP) CYP1A2, CYP2E1, CYP2C9 and CYP2D6 probably do not significantly contribute to phase I metabolism of PZQ in rat [6]. Others found that CYP1A2, CYP2C19 and CYP3A4/5 were responsible for the metabolism of PZQ by the comparison of the consumption of PZQ among different recombinant CYPs [7]. Inhibitors of CYPs excluded the involvement of CYP1A and CYP2C [6], but the complex in vivo factors (e.g. drug–drug interactions) and the specificity of these inhibitors might complicate interpretation of these results. Additionally, both inhibitors and inducers of CYP3A can affect the in vivo exposure of PZQ [8], [9], indicating the involvement of CYP3A in the metabolism of PZQ. Taken together, the metabolic pathway of PZQ still remains unclear, and needs to be further clarified, including the complete metabolic profile and the involvement of specific enzymes.

PZQ is a chiral compound marketed as a racemic mixture (Fig. 1). The major pharmacological activity is ascribed to (R)-PZQ, but no evidence has been obtained to exclude the efficacy of (S)-PZQ [10], [11], [12]. The same activity was obtained with the single administration of 20 mg/kg of (R)-PZQ and 40 mg/kg of the racemic PZQ [(R,S)-PZQ], while fewer side effects were observed in the (R)-PZQ treatment group [10]. The difference in metabolic elimination of the two enantiomers has been studied and systemic levels of (S)-PZQ were reported to be higher than those of (R)-PZQ [13]. In isolated rat hepatocytes, (R)-PZQ produced cis- and trans-4-hydroxy-PZQ, whereas (S)-PZQ produced additional unknown metabolites [5]. However, details of the metabolic differences between (R)- and (S)-PZQ remain unclear.

Metabolomics has been evolving as an important tool to study drug metabolism, and has been applied in the study of several drugs, such as the cancer chemotherapeutic drug N,N′,N″-triethylenethiophosphoramide (ThioTEPA) [14] and areca nut alkaloid arecoline used in the treatment of parasitic worms [15]. Compared with traditional metabolite identification methods, metabolomics can yield the full spectrum of metabolites, even those with relatively low abundance, which facilitates the elucidation of novel metabolic pathways [16]. Importantly, metabolomics-based metabolic mapping has been employed to detect reactive metabolites produced at very low abundance [17], [18]. In the present study, ultraperformance chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS)-based metabolomics was used to (i) elucidate the complete metabolic pathway of PZQ, including identification of all phase I and phase II metabolites, and (ii) comparison of differences in the metabolic pathways between (R)-PZQ and (S)-PZQ. The possible mechanism for these differences is discussed.

Section snippets

Chemicals and reagents

(R,S)-PZQ, (R)-PZQ, (S)-PZQ and 4-hydroxyl-PZQ were synthesized Marine College, Shandong University at Weihai. The enantiomeric excess values of (R)-PZQ and (S)-PZQ were >99% by HPLC. β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH) was purchased from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were of the highest grade commercially available.

In vivo treatment of mice with (R,S)-PZQ, (R)-PZQ and (S)-PZQ and sample preparation

To investigate in vivo metabolic behavior of PZQ, eighteen 6- to 8-week-old male 129/Sv mice were divided into

Metabolic profiling of PZQ in mice

As shown in the scores scatter plot, urine samples (Fig. 2A) and feces samples (unpublished data) from the PZQ-treated Sv/129 mice were significantly separated from the control vehicle-treated group. Loading scatter analysis of the samples suggested some potential PZQ metabolites and their fragments contributed to the separation (Fig. 2B). The relative abundance of each metabolite in urine was determined (Fig. 2C) and the chemical structures of these metabolites further identified based on the

Discussion

It has been long recognized that two enantiomers of a chiral drug commonly possess different pharmacological and toxicological properties [20], [21], [22]. About one in four of all therapeutic agents are administered to humans as mixtures of enantiomers pharmacological activity and toxicity may exhibit difference between two the enantiomers. Stereoselective metabolism of the racemic mixtures may further complicate the therapeutic role and the adverse effects encountered with drugs [23]. During

Conflicts of interest

None.

Authors contributions

The experiments were conceived and designed by HNW, ZZF, FJG. The experiments were performed by HNW, ZZF. The data were analyzed by HNW, ZZF, KZ. The reagent/materials/analysis tools were contributed by DQS, KWK, YZ, CYH. The paper was written by HNW, ZZF. The manuscript was reviewed by HNW, ZZF, DQS, JRI, FJG.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (No. 81102504 and 81202586), the National High-Tech Program of China (863 Project No. 2012AA020306), Shandong Natural Science Foundation of China (No. BS2013YY054 and ZR2010HL023), and the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (1ZIABC005562-24).

References (40)

  • N. Mano et al.

    Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids

    J Pharm Sci

    (2003)
  • M.J. Bailey et al.

    Acyl glucuronide reactivity in perspective: biological consequences

    Chem Biol Interact

    (2003)
  • M.B. Sanchez et al.

    Genetic factors associated with drug-resistance of epilepsy: relevance of stratification by patient age and aetiology of epilepsy

    Seizure: J Brit Epilepsy Assoc

    (2010)
  • D.S. Jenkins-Holick et al.

    Schistosomiasis

    Urol Nurs

    (2013)
  • S.D. Melman et al.

    Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni

    PLoS Negl Trop Dis

    (2009)
  • X.Q. Li et al.

    Identification of human cytochrome P(450)s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data

    Eur J Clin Pharmacol

    (2003)
  • W. Ridtitid et al.

    Rifampin markedly decreases plasma concentrations of praziquantel in healthy volunteers

    Clin Pharmacol Ther

    (2002)
  • W. Ridtitid et al.

    Pharmacokinetic interaction between ketoconazole and praziquantel in healthy volunteers

    J Clin Pharm Therapeut

    (2007)
  • M.H. Wu et al.

    Comparison of the therapeutic efficacy and side effects of a single dose of levo-praziquantel with mixed isomer praziquantel in 278 cases of schistosomiasis japonica

    Am J Trop Med Hyg

    (1991)
  • S.H. Xiao et al.

    Comparative in vitro and in vivo activity of racemic praziquantel and its levorotated isomer on Schistosoma mansoni

    J Infect Dis

    (1989)
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