Vasoprotective Effects of Hyperoside against Cerebral Ischemia/Reperfusion Injury in Rats: Activation of Large-Conductance Ca2+-Activated K+ Channels.

Hyperoside (Hyp), a kind of Chinese herbal medicine, exerts multiple therapeutic effects on many diseases. However, the role and mechanisms of Hyp in vascular pathophysiology in ischemic stroke need to be further established. The study aimed to investigate the role of (large-conductance Ca2+-activated K+) BK channels on the vasoprotection of Hyp against cerebral ischemia and reperfusion (I/R) injury in rats. The concentration gradient of Hyp was pretreated in both the middle cerebral artery occlusion and reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) model of primary vascular smooth muscle cells (VSMCs) in rats. A series of indicators were detected, including neurological deficit score, infarct volume, malondialdehyde (MDA), superoxide dismutase (SOD), cerebral blood flow (CBF), cell viability, membrane potential, and BK channels α- and ß1-subunits expression. The results showed that Hyp significantly reduced infarct volume and ameliorated neurological dysfunction in I/R-injured rats. Besides, the effects of I/R-induced reduction of BK channels α- and ß1-subunits expression were significantly reversed by Hyp in endothelial-denudated cerebral basilar arteries. Furthermore, the protective effect against I/R-induced increases of MDA and reduction of SOD as well as CBF induced by Hyp was significantly reversed by iberiotoxin (IbTX). In OGD/R-injured VSMCs, downregulated cellular viability and BK channels ß1-subunits expression were remarkably reversed by Hyp. However, neither OGD/R nor Hyp affected BK channels α-subunits expression, and Hyp failed to induced hyperpolarization of VSMCs. Moreover, the protective effect against OGD/R-induced reduction of cell viability and SOD level and increases of MDA production induced by Hyp was significantly reversed by IbTX in VSMCs. The study indicates that Hyp has the therapeutic potential to improve vascular outcomes, and the mechanism is associated with suppressing oxidative stress and improving CBF through upregulating BK channels.

Metabolic Retroversion of Piperaquine (PQ) via Hepatic Cytochrome P450-Mediated N-Oxidation and Reduction: Not an Important Contributor to the Prolonged Elimination of PQ.

As a partner antimalarial with an extremely long elimination half-life (∼30 days), piperaquine (PQ) is mainly metabolized into a pharmacologically active N-oxide metabolite [piperaquine N-oxide (PN1)] in humans. In the present work, the metabolic retroversion of PQ and PN1, potentially associated with decreased clearance of PQ, was studied. The results showed that interconversion existed for PQ and its metabolite PN1. The N-oxidation of PQ to PN1 was mainly mediated by CYP3A4, and PN1 can rapidly reduce back to PQ via cytochrome P450 (P450)/flavin-containing monooxygenase enzymes. In accordance with these findings, the P450 nonselective inhibitor (1-ABT) or CYP3A4 inhibitor (ketoconazole) inhibited the N-oxidation pathway in liver microsomes (>90%), and the reduction metabolism was inhibited by 1-ABT (>90%) or methimazole (∼50%). Based on in vitro physiologic and enzyme kinetic studies, quantitative prediction of hepatic clearance (CLH) of PQ was performed, which indicated its negligible decreased elimination in humans in the presence of futile cycling, with the unbound CLH decreasing by 2.5% (0.069 l/h per kilogram); however, a minor decrease in unbound CLH (by 12.8%) was found in mice (0.024 l/h per kilogram). After an oral dose of PQ (or PN1) to mice, the parent form predominated in the blood circulation, and PN1 (or PQ) was detected as a major metabolite. Other factors probably associated with delayed elimination of PQ (intestinal metabolism and enterohepatic circulation) did not play a key role in PQ elimination. These data suggested that the metabolic interconversion of PQ and its N-oxide metabolite contributes to but may not significantly prolong its duration in humans. SIGNIFICANCE STATEMENT: This paper investigated the interconversion metabolism of piperaquine (PQ) and its N-oxide metabolite in vitro as well as in mice. The metabolic profiles of PQ were reestablished by this futile cycling, which contributes to but may not significantly prolong its elimination in humans. Enzyme phenotyping indicated a low possibility of interaction of PQ during artemisinin drug-based combination therapy treatment.

Simultaneous determination of five alkaloids from Rauvolfia vomitoria in rat plasma by LC-MS/MS: Application to a comparative pharmacokinetic study in normal and type 2 diabetic rats.

Rauvolfia vomitoria is widely distributed in the tropical regions of Africa and Asia, and has been used in traditional folk medicine in China. Indole alkaloids were found to be major bioactive components, while the effects of diabetes mellitus on the pharmacokinetic parameters of the components have not been reflected in vivo. In this study, an efficient and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of five ingredients of R. vomitoria in rats. Detection was implemented in multiple-reaction-monitoring mode with an electrospray positive-ionization source. Validation parameters were all in accordance with the current criterion. The established method was effectively employed to compare the pharmacokinetic behaviors of five alkaloids (reserpine, yohimbine, ajmaline, ajmalicine, and serpentine) between normal and type 2 diabetic rats. The single-dose pharmacokinetic parameters of the five alkaloids were determined in normal and diabetic rats after oral administration of 100 and 200 mg/kg body weight. The results indicated that diabetes mellitus significantly altered the pharmacokinetic characteristics of yohimbine, ajmaline, and ajmalicine after oral administration in rats. This is an attempt to provide some evidence for clinicians that may serve as a guide for the use of antidiabetic medicine in clinical practice.

The gender-related variability in the pharmacokinetics and antiplasmodial activity of naphthoquine in rodents.

BACKGROUND: Naphthoquine (NQ) is a suitable partner anti-malarial for the artemisinin-based combination therapy (ACT), which is recommended to be taken orally as a single-dose regimen. The metabolism of NQ was mainly mediated by CYP2D6, which is well-known to show gender-specific differences in its expression. In spite of its clinical use, there is limited information on the pharmacokinetics of NQ, and no data are available for females. In this study, the effect of gender on the pharmacokinetics and antiplasmodial efficacy of NQ in rodents was evaluated. The underlying factors leading to the potential gender difference, i.e., plasma protein binding and metabolic clearance, were also evaluated. METHODS: The pharmacokinetic profiles of NQ were investigated in healthy male or female rats after a single oral administration of NQ. The antiplasmodial efficacy of NQ was studied in male or female mice infected with Plasmodium yoelii. The recrudescence and survival time of infected mice were also recorded after drug treatment. Plasma protein binding of NQ was determined in pooled plasma collected from male or female mice, rat or human. In vitro metabolism experiments were performed in the liver microsomes of male or female mice, rat or human. RESULTS: The results showed that the gender of rats did not affect NQ exposure (AUC0-t and Cmax) significantly (P > 0.05). However, a significant (P < 0.05) longer t1/2 was found for NQ in male rats (192.1 ± 47.7), compared with female rats (143.9 ± 27.1). Slightly higher but not significant (P > 0.05) antiplasmodial activity was found for NQ in male mice (ED90, 1.10 mg/kg) infected with P. yoelii, compared with female mice (ED90, 1.67 mg/kg). The binding rates of NQ to plasma protein were similar in males and females. There was no metabolic difference for NQ in male and female mice, rat or human liver microsomes. CONCLUSIONS: These results indicated that the pharmacokinetic profiles of NQ were similar between male and female rats, except for a longer t1/2 in male rats. The difference was not associated with plasma protein binding or hepatic metabolic clearance. Equivalent antiplasmodial activity was found for NQ in male and female mice infected with P. yoelii. This study will be helpful for the rational design of clinical trials for NQ.

The effect of malaria-induced alteration of metabolism on piperaquine disposition in Plasmodium yoelii infected mice and predicted in malaria patients.

OBJECTIVES: Malaria-induced alteration of physiological parameters and pharmacokinetic properties of antimalarial drugs may be clinically relevant. Whether and how malaria alters the disposition of piperaquine (PQ) was investigated in this study. METHODS: The effect of malaria on drug metabolism-related enzymes and PQ pharmacokinetic profiles was studied in Plasmodium yoelii-infected mice in vitro/in vivo. Whether the malaria effect was clinically relevant for PQ was evaluated using a validated physiologically-based pharmacokinetic model with malaria-specific scalars obtained in mice. RESULTS: The infection led to a higher blood-to-plasma partitioning (Rbp) for PQ, which was concentration-dependent and correlated to parasitemia. No significant change in plasma protein binding was found for PQ. Drug metabolism-related genes (CYPs/UDP-glucuronosyltransferase/nuclear receptor, except for CYP2a5) were downregulated in infected mice, especially at the acute phase. The plasma oral clearances (CL/F) of three probe substrates for CYP enzymes were significantly decreased (by ≥35.9%) in mice even with moderate infection. The validated physiologically-based pharmacokinetic model indicated that the hepatic clearance (CLH) of PQ was the determinant of its simulated CL/F, which was predicted to slightly decrease (by ≤23.6%) in severely infected mice but not in malaria patients. The result fitted well with the plasma pharmacokinetics of PQ in infected mice and literature data on malaria patients. The blood clearance of PQ was much lower than its plasma clearance due to its high Rbp. CONCLUSIONS: The malaria-induced alteration of drug metabolism was substrate-dependent, and its impact on the disposition of PQ and maybe other long-acting aminoquinoline antimalarials was not expected to be clinically relevant.

The Effect of Retroconversion Metabolism of N-oxide Metabolites by Intestinal Microflora on Piperaquine Elimination in Mice, as well as in Humans Predicted Using a PBPK Model.

BACKGROUND: Piperaquine (PQ) and its pharmacologically active metabolite PQ N-oxide (PM1) can be metabolically interconverted via hepatic cytochrome P450 and FMO enzymes. OBJECTIVES: The reductive metabolism of PM1 and its further N-oxidation metabolite (PM2) by intestinal microflora was evaluated, and its role in PQ elimination was also investigated. METHODS: The hepatic and microbial reduction metabolism of PM1 and PM2 was studied in vitro. The reaction phenotyping experiments were performed using correlation analysis, selective chemical inhibition, and human recombinant CYP/FMO enzymes. The role of microbial reduction metabolism in PQ elimination was evaluated in mice pretreated with antibiotics. The effect of the reduction metabolism on PQ exposures in humans was predicted using a physiologically-based pharmacokinetic (PBPK) model. RESULTS: Both hepatic P450/FMOs enzymes and microbial nitroreductases (NTRs) contributed to the reduction metabolism of two PQ N-oxide metabolites. In vitro physiologic and enzyme kinetic studies of both N-oxides showed a comparable intrinsic clearance by the liver and intestinal microflora. Pretreatment with antibiotics did not lead to a significant (P > 0.05) change in PQ pharmacokinetics in mice after an oral dose. The predicted pharmacokinetic profiles of PQ in humans did not show an effect of metabolic recycling. CONCLUSION: Microbial NTRs and hepatic P450/FMO enzymes contributed to the reduction metabolism of PQ Noxide metabolites. The reduction metabolism by intestinal microflora did not affect PQ clearance, and the medical warning in patients with NTRs-related disease (e.g., hyperlipidemia) will not be clinically meaningful.

No Effect of PXR (8055C>T) Polymorphism on the Pharmacokinetic Profiles of Piperaquine in Healthy Chinese Subjects.

BACKGROUND: Significant inter-subject variability in pharmacokinetics and clinical outcomes has been observed for the antimalarial agent piperaquine (PQ). PQ is metabolized by CYP3A4, mainly regulated by the pregnane X receptor (PXR). CYP3A4(*1B) polymorphism did not affect PQ clearance. OBJECTIVES: The effect of PXR (8055C>T) polymorphism on the pharmacokinetic profiles of PQ was investigated. METHODS: The pharmacokinetic profiles of PQ and its major metabolite PQ N-oxide (PQM) were studied in healthy Chinese subjects after recommended oral doses of artemisinin-PQ. Twelve subjects were genotyped using PCRRFLP (six in each group with PXR 8055CC and 8055TT), and plasma concentrations were determined by a validated LC/MS/MS method. The dose-adjusted exposure (AUC and Cmax) to PQ or PQM was investigated, and the metabolic capability of PQ N-oxidation was determined by AUCPQM/AUCPQ. The antimalarial outcome of PQ was evaluated using its day 7 concentration. RESULTS: PQM formation was mediated by CYP3A4/3A5. Interindividual variability in dose-adjusted AUC of PQ and PQM was relatively low (%CV, <30.0%), whereas a larger inter-variability was observed for Cmax values (%CV, 68.1% for PQ). No polymorphic effect was found for PXR (C8055T) on the pharmacokinetic profiles of PQ or its Cday 7 concentrations. CONCLUSION: Both CYP3A4 and CYP3A5 were involved in PQ clearance. The genotypes of PXR (C8055T) may not contribute to the variability in PQ pharmacokinetics as well as antimalarial outcomes. There might be a low risk of variable exposures to PQ in malaria patients carrying mutated PXR (8055C>T) genes, which deserves further study, especially in a larger sample size.

The Effect of Gastric pH on the Pharmacokinetics-pharmacodynamics of Naphthoquine in Rodents, as well as in Human Predicted Using a PBPK Model.

BACKGROUND: Fixed-dose combination of artemisinin and naphthoquine (NQ) is a new artemisinin- based combination therapy for the treatment of uncomplicated Plasmodium falciparum. NQ absorption has been reported to be affected by food in humans. OBJECTIVES: The effect of gastric pH on NQ pharmacokinetics and antiplasmodial activity was investigated. METHODS: The pharmacokinetic profiles of NQ were studied in healthy rodents after an oral dose of NQ with or without gastric pH modulators, i.e., pentagastrin (stimulator) and famotidine (suppressant). The effect of gastric pH on NQ exposures in humans was predicted using a physiologically-based pharmacokinetic (PBPK) model. The effect of gastric pH on the antiplasmodial activity of NQ was evaluated in mice infected with Plasmodium yoelii. RESULTS: Neither pentagastrin nor famotidine affected NQ absorption (AUC0-t and Cmax) significantly (P > 0.05) in rodents. The predicted PK profiles of NQ in humans did not show an effect of gastric pH. Compared to pure NQ (ED90, 1.2 mg/kg), the combination with pentagastrin showed non-significantly (< 1.5-fold) higher antimalarial potency (ED90, 1.1 mg/kg). Correspondingly, the elevation of gastric pH (up to pH 5) by famotidine treatment resulted in a relatively weaker antimalarial potency for NQ (ED90, 1.4 mg/kg). Such a difference is within the acceptable range of variability in NQ pharmacokinetics and antiplasmodial activity. CONCLUSIONS: Although the food was found to significantly impact NQ pharmacokinetics, other factors except for gastric pH should account for the result, and the warning of careful use of NQ in patients with the acid-related disease is not expected to be clinically meaningful.

Metabolism is not a Major Contributor to the Toxicity of Piperaquine, a Long-acting Antimalarial Agent in Artemisinin-based Combination Therapy.

BACKGROUND: Hepatocellular damage has been reported for the antimalarial piperaquine (PQ) in the clinic after cumulative doses. OBJECTIVES: The role of metabolism in PQ toxicity was evaluated, and the mechanism mediating PQ hepatotoxicity was investigated. METHODS: The toxicity of PQ and its major metabolite (PQ N-oxide; M1) in mice was evaluated in terms of serum biochemical parameters. The role of metabolism in PQ toxicity was investigated in mice pretreated with an inhibitor of CYP450 (ABT) and/or FMO enzyme (MMI). The dose-dependent pharmacokinetics of PQ and M1 were studied in mice. Histopathological examination was performed to reveal the mechanism mediating PQ hepatotoxicity. RESULTS: Serum biochemical levels (ALT and BUN) increased significantly (P < 0.05) in mice after three-day oral doses of PQ (> 200 mg/kg/day), indicating hepatotoxicity and nephrotoxicity of PQ at a high dose. Weaker toxicity was observed for M1. Pretreatment with ABT and/or MMI did not increase PQ toxicity. PQ and M1 showed linear pharmacokinetics in mice after a single oral dose, and multiple oral doses led to their cumulative exposures. Histopathological examination showed that a high dose of PQ (> 200 mg/kg/day for three days) could induce hepatocyte apoptosis. The mRNA levels of targets in NF-κB and p53 pathways could be up-regulated by 2-30-fold in mice by PQ or M1. CONCLUSION: PQ metabolism led to detoxification of PQ, but there was a low possibility of altered toxicity induced by metabolism inhibition. The hepatotoxicity of PQ and its N-oxidation metabolite was partly mediated by NF-κB inflammatory pathway and p53 apoptosis pathway.

Rapid Profiling of the Marker Components in Artemisia annua L. and their Metabolites in Rats Using an Improved Liquid Chromatography-tandem Highresolution Mass Spectrometry-based Technology.

BACKGROUND: As parasite resistance to the main artemisinin drugs has emerged in Southern Asia, the traditional herb Artemisia annua L. (AAL) from which artemisinin (QHS) isolated was found to overcome resistance to QHS. However the component and metabolite profiles of AAL remain unclear. OBJECTIVE: In this study, component profiling of marker compounds in AAL (amorphane sesquiterpene lactones and flavonoids) was performed and their subsequent metabolism was investigated in rats. METHODS: For efficient component classification and structural characterization, an improved liquid chromatography- tandem high-resolution mass spectrometry (HRMS)-based analytical strategy was applied, i.e., background subtraction (BS) followed by ring-double-bond (RDB) filter in tandem with repeated BS processing. Structures of detected components/metabolites were characterized based on integrated information including their HRMSn patterns, RDB values, the established component/metabolite network, the biosynthesis pathways of AAL, and/or NMR data. RESULTS: A total of 38 amorphane sesquiterpene lactones and 35 flavonoids were found in AAL as prototype compounds, among which 26 components were previously undescribed. Major compounds were identified by comparing them with reference standards. Among 73 AAL prototypes administered, 38 were absorbed in the circulation as the prototype. Moreover, 20 metabolites of amorphane sesquiterpene lactones and 10 metabolites of flavonoids were detected in rats. The major metabolic pathways included oxidation, methylation, glucuronidation and sulfation. CONCLUSION: The component and metabolite network were established for marker components in AAL, which will be valuable to understand the synergistic antimalarial potency of QHS in A. annua L. The analytical strategy can also be applied to other herbal medicines.