Metabolism of Piperaquine to Its Antiplasmodial Metabolites and Their Pharmacokinetic Profiles in Healthy Volunteers.

As a partner antimalarial for artemisinin drug-based combination therapy (ACT), piperaquine (PQ) can be metabolized into two major metabolites, including piperaquine N-oxide (M1) and piperaquine N,N-dioxide (M2). To better understand the antimalarial potency of PQ, the antimalarial activity of the PQ metabolites (M1 and M2) was studied in vitro (in Plasmodium falciparum strains Pf3D7 and PfDd2) and in vivo (in the murine species Plasmodium yoelii) in this study. The recrudescence and survival time of infected mice were also recorded after drug treatment. The pharmacokinetic profiles of PQ and its two metabolites (M1 and M2) were investigated in healthy subjects after oral doses of two widely used ACT regimens, i.e., dihydroartemisinin plus piperaquine phosphate (Duo-Cotecxin) and artemisinin plus piperaquine (Artequick). Remarkable antiplasmodial activity was found for PQ (50% growth-inhibitory concentration [IC50], 4.5 nM against Pf3D7 and 6.9 nM against PfDd2; 90% effective dose [ED90], 1.3 mg/kg of body weight), M1 (IC50, 25.5 nM against Pf3D7 and 38.7 nM against PfDd2; ED90, 1.3 mg/kg), and M2 (IC50, 31.2 nM against Pf3D7 and 33.8 nM against PfDd2; ED90, 2.9 mg/kg). Compared with PQ, M1 showed comparable efficacy in terms of recrudescence and survival time and M2 had relatively weaker antimalarial potency. PQ and its two metabolites displayed a long elimination half-life (∼11 days for PQ, ∼9 days for M1, and ∼4 days for M2), and they accumulated after repeated administrations. The contribution of the two PQ metabolites to the efficacy of piperaquine as a partner drug of ACT for the treatment of malaria should be considered for PQ dose optimization.

The application of a novel high-resolution mass spectrometry-based analytical strategy to rapid metabolite profiling of a dual drug combination in humans.

Metabolite profiling of combination drugs in complex matrix is a big challenge. Development of an effective data mining technique for simultaneously extracting metabolites of one parent drug from both background matrix and combined drug-related signals could be a solution. This study presented a novel high resolution mass spectrometry (HRMS)-based data-mining strategy to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was verapamil-irbesartan (VER-IRB), which is widely used in clinic to treat hypertension. First, mass defect filter (MDF), as a targeted data mining tool, worked effectively except for those metabolites with similar MDF values. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, was able to recover all relevant metabolites of VER-IRB from the full-scan MS dataset except for trace metabolites buried in the background noise and/or combined drug-related signals. Third, the novel ring double bond (RDB; valence values of elements in structure) filter, could show rich structural information in more sensitive full-scan MS chromatograms; however, it had a low capability to remove background noise and was difficult to differentiate the metabolites with RDB coverage. Fourth, an integrated strategy, i.e., untargeted BS followed by RDB, was effective for metabolite identification of VER and IRB, which have different RDB values. Majority of matrix signals were firstly removed using BS. Metabolite ions for each parent drug were then isolated from remaining background matrix and combined drug-related signals by imposing of preset RDB values/ranges around the parent drug and selected core substructures. In parallel, MDF was used to recover potential metabolites with similar RDB. As a result, a total of 74 metabolites were found for VER-IRB in human plasma and urine, among which ten metabolites have not been previously reported in human. The results demonstrated that the combination of accurate mass-based multiple data-mining techniques, i.e., untargeted background subtraction followed by ring double bond filtering in parallel with targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drug.

Simultaneous determination of piperaquine and its N-oxidated metabolite in rat plasma using LC-MS/MS.

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N-oxidated metabolite (PQ-M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C18 column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple-quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0-400.0 ng/mL for PQ and 1.0-50.0 ng/mL for PQ-M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ-M, respectively, using 40 µL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co-administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ-M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography-high-resolution mass spectrometry in combination with multiple data-mining tools in tandem.

Artemisinin-based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid-phase extraction. Data processes were carried out using multiple data-mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1-M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N-oxidation (M1, M2) and carboxylic products (M3). The subsequent N-oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N-dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC-HRMS(n) in combination with multiple data-mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans.

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo.

Effect of CAR polymorphism on the pharmacokinetics of artemisinin in healthy Chinese subjects.

Repeated pretreatment with the antimalarial drug artemisinin (QHS) could lead to reduced exposure to the parent drug, which is mainly mediated by auto-induction of CYP2B6 activity. CYP2B6 is most sensitive to the inductive effect of constitutive androstane receptor (CAR), which can be activated by QHS. CYP2B6 polymorphism has no influence on pharmacokinetics of QHS derivatives. This study aimed to investigate the effect of CAR (C540T) polymorphism on the auto-induction metabolism-mediated pharmacokinetics of QHS. Healthy Chinese subjects (six in each group with the genotypes of CAR 540C/C, 540C/T and 540T/T; all carrying the CYP2B6*1*1 genotype) received a recommended two-day oral doses of QHS-piperaquine (PQ) to assess the pharmacokinetics of QHS and its metabolite deoxyartemisinin (DQHS). The exposures to QHS and DQHS were significantly lower (p < 0.05) in subjects homozygous for the CAR 540T/T genotype than those with the 540C/C genotype after the repeated dose. QHS did not show different induction clearance in subjects homozygous for the 540C/C genotype (1.3-fold), compared with those carrying the heterozygous 540C/T (2.1-fold) or homozygous 540T/T (1.7-fold) genotype. In conclusion, the CAR (C540T) genotype contributed to the interindividual variability of QHS pharmacokinetics, and the dose regimen for QHS deserves further evaluation especially in specific populations.

Oligosaccharides composition in eight food legumes species as detected by high-resolution mass spectrometry.

BACKGROUND: As probiotics, soy oligosaccharides have become popular as healthy foods to reduce disease risk. However, comprehensive information about oligosaccharides in different food legumes is limited. RESULTS: In this study, eight oligosaccharides were well detected and quantified in different varieties of eight legume species using high-resolution mass spectrometry. It was determined that species could be distinguished by total content of oligosaccharides and their distribution modes. Among the studied species, Vigna unguiculata is a better resource of non-digestible oligosaccharides, while Vicia faba and black soybean (Glycine max) are at a disadvantage. Normally, stachyose predominates in non-digestible oligosaccharides, except in mung bean and broad bean, where verbascose predominates. For mung bean and green soybean, the seed coat should be taken into account for oligosaccharide consumption. The developed high-resolution mass spectrometry method greatly simplified the sample preparation process and permitted the identification of oligosaccharides without reference compounds. CONCLUSION: This work involved extensive sample collecting and provided useful information for consumers. The developed method may be useful for rapid quantification of oligosaccharides in related foods.

Rapid determination of oligopeptides and amino acids in soybean protein hydrolysates using high-resolution mass spectrometry.

INTRODUCTION: Soybean protein hydrolysates (SPHs), especially oligopeptides, have shown a variety of functional properties, including immunomodulatory and anti-oxidant effects. Soybean protein hydrolysate products have been used as functional ingredients in food, sports nutrition or clinical nutrition. However, the mixture is mostly undefined due to its complex nature, containing peptides and minor amino acids as well as small proteins. OBJECTIVES: To develop a specific and efficient method for the identification and structural characterisation of oligopeptides in SPHs, and to determine free amino acids in SPHs in the same analytical run, for evaluation of the chemical profile of SPH products. METHODS: Accurate mass spectrometry (MS) datasets of SPH samples were recorded on a high-performance liquid chromatography (HPLC) tandem high-resolution (HR) MS system. Potential oligopeptides were tentatively characterised based on their elemental compositions and ring double bond equivalent (RDBE) values, as well as HRMS/MS data. The analytical method to determine amino acids was evaluated in terms of linearity, precision, apparent recovery and limits of detection and quantitation. RESULTS: In total, 186 oligopeptides spanning the mass range of m/z 200-1500 and three major free amino acids could be determined in SPH samples in a single sample injection. Ninety-nine oligopeptides were tentatively characterised. The sensitive and specific instrumental performances also permitted the determination of 19 amino acids with a limit of quantitation of ≤ 0.1 µg/mL. CONCLUSION: The HPLC-HRMS technique has proven to be an advantageous tool for the rapid characterisation of oligopeptides and determination of amino acids in soybean protein hydrolysates.

The effect of UGTs polymorphism on the auto-induction phase II metabolism-mediated pharmacokinetics of dihydroartemisinin in healthy Chinese subjects after oral administration of a fixed combination of dihydroartemisinin-piperaquine.

BACKGROUND: Dihydroartemisinin (DHA) is a component of artemisinin-based combination therapy (ACT), which is widely recommended for treatment of uncomplicated falciparum malaria. DHA is also the main metabolite of artemether and artesunate, both of which are used in ACT. Due to auto-induction metabolism, declining plasma concentrations after the repeated dosing have been reported for artemisinin (Qing-hao-su) and artemether. This study was designed to evaluate the potential auto-induction metabolism of DHA in healthy Chinese adults after multiple oral doses of DHA. The polymorphic effects of UGT1A9 (I399C>T) and UGT2B7*2 (802C>T), the major enzymes involved in the metabolism of DHA, on the pharmacokinetic profiles of DHA and its metabolite was also studied. METHODS: Sixteen healthy Chinese subjects (four I399TT/802CC, four I399CC/802TT, four I399TT/802TT and four I399CT/802CT) received four recommended oral doses of Artekin, an ACT containing DHA (80 mg/dose) and piperaquine (PQ; 640 mg/dose), at 0, 6, 24 and 32 h. Plasma samples were analysed for DHA and its metabolite using a validated liquid chromatography tandem mass spectrometric (LC-MS) method. RESULTS: DHA and its glucuronidated metabolite DHA-Glu were detected in human plasma after oral administration of DHA-PQ. Compared with the first dose, the AUC0-t of the parent drug DHA decreased significantly (P<0.01) with increased oral clearance (CL/F) after each repeated dose of DHA-PQ, whereas its metabolite DHA-Glu did not change (P>0.05) in AUC(0-t) or C(max). The phase II metabolic capability, calculated by the AUC(0-t) ratio of DHA-Glu to the parent drug DHA, increased 1.5-fold (90% CI, 1.3-1.7), 1.2-fold (90% CI, 1.1-1.3) and 1.7-fold (90% CI, 1.5-1.8) after the second, third and fourth dose, respectively. No polymorphic effect was found for UGT1A9 (I399C>T) and UGT2B7*2 (802C>T) on the pharmacokinetic profiles of DHA and its metabolite DHA-Glu. CONCLUSIONS: The auto-induction phase II metabolism of DHA was present in healthy Chinese subjects after the recommended two-day oral doses of DHA-PQ (Artekin). The metabolic capability could recover after a 12-h dosing interval, which suggested that the alternative common three-day regimen (once daily) for DHA-PQ could probably lead to higher bioavailability of DHA. The polymorphism of UGT1A9 (I399C>T) and UGT2B7*2 (802C>T) may not be a concern during the treatment with DHA.

Auto-induction of phase I and phase II metabolism of artemisinin in healthy Chinese subjects after oral administration of a new artemisinin-piperaquine fixed combination.

BACKGROUND: Artequick is a relatively inexpensive artemisinin (Qing-hao-su; QHS)-based combination therapy (ACT) that contains QHS and piperaquine (PQ), which has not been widely used because of the decreased concentration level of QHS after repeated oral administrations for five to seven days as a monotherapy. This study was designed to evaluate the potential auto-induction metabolism of QHS in healthy Chinese adults after a two-day oral administration of QHS-PQ. The effect of QHS-PQ on the activity of the CYP2B6 and CYP3A4 was also investigated. METHODS: Fourteen healthy Chinese subjects received two-day oral doses of QHS-PQ (Artequick). A two-drug cocktail consisting of bupropion and midazolam was used to assess the activities of CYP2B6 and CYP3A, respectively. Plasma samples were analysed for QHS and its phase I/II metabolites, probe drugs and their metabolites, using a validated liquid chromatography tandem mass spectrometric (LC-MS) method. RESULTS: Four major phase I metabolites of QHS (M1-M3 and deoxy-QHS) and two subsequent phase II metabolites (M4-M5) were detected in human plasma after oral administrations of QHS-PQ. The AUC0-t of the QHS and its phase I metabolites decreased significantly (P < 0.05) with increased oral clearance (CL/F) after two-day oral doses of QHS-PQ, whereas its phase II metabolites exhibited higher AUC (P < 0.01). The phase I metabolic capability, calculated by the AUC0-t ratio of all phase I metabolites to QHS, increased 1.5-fold after the repeated dose (P < 0.01), and the phase II metabolic capability increased 1.5-fold for M4 and 3.0-fold for M5. The enzyme activity of CYP2B6 and CYP3A4 increased 2.1-fold and 3.2-fold, respectively, after two-day oral doses of QHS-PQ. CONCLUSIONS: The auto-induction of both phase I and phase II metabolism of QHS was present in healthy Chinese subjects after a recommended two-day oral dose of QHS-PQ. The auto-induction metabolism also existed for phase I metabolites of QHS. The enzyme activity of CYP2B6 and CYP3A4 was induced after the two-day oral doses of QHS-PQ. Based on these results, the alternative common three-day regimen for QHS-PQ could probably lead to lower bioavailability of QHS and higher potential of drug-drug interaction caused by the induction of drug-metabolizing enzymes.