Molecular dynamics simulation and in vitro evaluation of herb-drug interactions involving dietary polyphenols and CDK inhibitors in breast cancer chemotherapy.

Dietary polyphenols such as quercetin and curcumin have been extensively administered to patients with cancer in the form of herbal supplements. They may have a synergistic anticancer effect; however, a risk of pharmacokinetic interactions with selective CDK-4/6 inhibitors that are metabolized by the CYP3A4 enzyme exists. Considering these pharmacokinetic aspects, the current study examined the effects of curcumin and quercetin on human CYP3A4 to ascertain CYP3A4-mediated herb-drug interactions with CDK inhibitors. In this study, using in silico methods and CYP3A4 inhibition kinetics in human liver microsomes and recombinant CYP3A4 enzymes, the effects of concentration-dependent inhibition of CYP3A4 by quercetin and curcumin on CDK inhibitors metabolism were examined. Based on our in-silico docking findings, curcumin and quercetin were considerably bound to CYP3A4 protein and displace CDK inhibitors from the CYP3A4 substrate binding domain. The IC50 values of curcumin and quercetin were 16.10 and 0.05 µM, respectively, for CYP3A4-mediated 1'-hydroxylation of midazolam. The dietary polyphenols prolonged the in vitro half-life of palbociclib and ribociclib by 6.4-fold and decreased their intrinsic microsomal clearance by approximately 4.6 times. Our findings indicate that curcumin and quercetin effectively cause herb-drug interactions and should be cautiously used to avoid therapeutic failure.

Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.

Beneficial effects of Wuzhi Capsule on tacrolimus blood concentrations in liver transplant patients with different donor-recipient CYP3A5 genotypes.

WHAT IS KNOWN AND OBJECTIVE: Tacrolimus (Tac) is an immunosuppressant that is widely used to prevent allograft rejection in patients after liver transplantation. Its metabolism mainly depends on the cytochrome P450 3A5 (CYP3A5), which has genetic polymorphisms. Recently, a Chinese herbal medicine known as Wuzhi Capsule (WZC) was shown to increase Tac blood concentrations by inhibiting the activity of CYP3A in animal studies in rats. To date, it remains unexplored whether WZC can be efficiently used to enhance the blood concentration of Tac in liver transplant patients with different donor-recipient CYP3A5 genotypes. METHODS: A total of 185 liver transplant patients were enrolled and two-way ANOVA was carried out, then they were divided into four groups according to the combinations of donor-recipient CYP3A5 phenotypes. WZC was given to patients when the dose of Tac was ≥4 mg, and the dose-adjusted C0 (C0 /D) of Tac measured twice in succession was ≤1 ng/ml/mg. The blood trough concentration of Tac (C0 ), C0 /D, and dose- and body weight-adjusted C0 (C0 /D/W) was analysed on days 7 and 14 after liver transplantation. RESULTS: The genotypes of donor and recipient or WZC had significant effects on C0, C0/D and C0/D/W. There were significant differences in the Tac blood concentrations between the groups. The recipient expression (*1)/donor expression (*1) (R+/D+) group had the lowest C0 , C0 /D and C0 /D/W among the four groups. Furthermore, a larger proportion of patients in the CYP3A5 expression groups required Tac dose adjustment to achieve a therapeutic effect and were given Tac with WZC. Notably, the use of WZC significantly increased the blood concentrations of Tac in the CYP3A5 expression groups and greater increases in the C0 /D and C0 /D/W were significantly associated with higher doses of WZC in the CYP3A5 expression groups. What is more, WZC reduced the hospitalization cost of patients to a certain extent. WHAT IS NEW AND CONCLUSION: WZC significantly increased the C0 , C0 /D and C0 /D/W in the CYP3A5 expression groups and reduced the hospitalization expenses of patients to a certain extent. What is more, greater increases in the C0 /D and C0 /D/W were significantly associated with higher doses of WZC.

The pharmacokinetic study on the interaction between nobiletin and anemarsaponin BII in vivo and in vitro.

CONTEXT: The interaction between nobiletin and anemarsaponin BII could affect the pharmacological activity of these two drugs during their combination. OBJECTIVE: The co-administration of nobiletin and anemarsaponin BII was investigated to explore the interaction and the potential mechanism. MATERIALS AND METHODS: Male Sprague-Dawley rats were only orally administrated with 50 mg/kg nobiletin as the control and another six rats were pre-treated with 100 mg/kg anemarsaponin BII for 7 d followed by the administration of nobiletin. The transport and metabolic stability of nobiletin were evaluated in vitro, and the effect of anemarsaponin BII on the activity of CYP3A4 was also assessed to explore the potential mechanism underlying the interaction. RESULTS: The increasing Cmax (2309.67 ± 68.06 µg/L vs. 1767.67 ± 68.86 µg/L), AUC (28.84 ± 1.34 mg/L × h vs. 19.57 ± 2.76 mg/L × h), prolonged t1/2 (9.80 ± 2.33 h vs. 6.24 ± 1.53 h), and decreased clearance rate (1.46 ± 0.26 vs. 2.42 ± 0.40) of nobilein was observed in rats. Anemarsaponin BII significantly enhanced the metabolic stability of nobiletin in rat liver microsomes (half-life increased from 31.56 min to 39.44 min) and suppressed the transport of nobiletin in Caco-2 cells (efflux rate decreased from 1.57 ± 0.04 to 1.30 ± 0.03). The inhibitory effect of anemarsaponin BII on CYP3A4 was also found with an IC50 value of 10.23 µM. DISCUSSION AND CONCLUSIONS: The interaction between anemarsaponin BII and nobiletin was induced by the inhibition of CYP3A4, which should draw special attention in their clinical co-administration.

Saikosaponins and the deglycosylated metabolites exert liver meridian guiding effect through PXR/CYP3A4 inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Bupleuri (RB), traditionally used to treat inflammatory disorders and infectious diseases, represents one of the most successful and widely used herbal drugs in Asia over the past 2000 years. Being realized the role in regulating metabolism and controlling Yin/Yang, RB is not only chosen specifically for treating liver meridian and the corresponding organs, but also believed to have liver meridian guiding property and help potentiate the therapeutic effects of liver. However, the ingredients in RB with liver meridian guiding property and the underly mechanism have not been comprehensively investigated. AIM OF STUDY: Considering the important role of CYP3A4 in first-pass metabolism and the liver exposure of drugs, the present study aimed to determine whether saikosaponins (SSs) and the corresponding saikogenins (SGs) have a role in inhibiting the catalytic activity of CYP3A4 in human liver microsomes and HepG2 hepatoma cells and whether they could suppress CYP3A4 expression by PXR-mediated pathways in HepG2 hepatoma cells. MATERIALS AND METHODS: The effect of SSs and SGs on CYP3A4-mediated midazolam1'-hydroxylation activities in pooled human liver microsomes (HLMs) was first studied. Dose-dependent experiments were performed to obtain the half inhibit concentration (IC50) values. HepG2 cells were used to assay catalytic activity of CYP3A4, reporter function, mRNA levels, and protein expression. The inhibitory effects of SSa and SSd on CYP3A4 activity are negligible, while the corresponding SGs (SGF and SGG) have obvious inhibitory effects on CYP3A4 activity, with IC50 values of 0.45 and 1.30 µM. The similar results were obtained from testing CYP3A4 catalytic activity in HepG2 cells, which correlated well with the suppression of the mRNA and protein levels of CYP3A4. Time-dependent testing of CYP3A4 mRNA and protein levels, as well as co-transfection experiments using the CYP3A4 promoter luciferase plasmid, further confirmed that SSs and SGs could inhibit the expression of CYP3A4 at the transcription level. Furthermore, PXR protein expression decreased in a concentration- and time-dependent manner after cells were exposed to SSs and SGs. PXR overexpression and RNA interference experiments further showed that SSs and SGs down-regulate the catalytic activity and expression of CYP3A4 in HepG2 may be mainly through PXR-dependent manner. CONCLUSION: SSs and SGs inhibit the catalytic activity and expression of CYP3A4 in a PXR-dependent manner, which may be highly related to the liver meridian guiding property of RB.

The inhibitory effect of 225 frequently-used traditional Chinese medicines for CYP3A4 metabolic enzyme by isoform-specific probe.

Traditional Chinese medicines (TCMs), have been widely used for the prevention, treatment, and cure of various diseases for thousands of years in China and Asian countries. It is usually applied either alone or in combination with synthetic drugs or other herbs to be more effective. However, the evaluation of TCMs against the main phase I metabolic enzyme CYP3A4 in vitro was limited. In the present study, a high throughput method based on an isoform-specific probe was applied to evaluate the inhibitory effect of 225 frequently-used TCMs on CYP3A4 activity. The results showed that 25 TCM herbs possessed inhibition effect with residual activity below 50%, and four TCMs (Curcumae Rhizoma, Piperis Longi Fructus, Dalbergiae Odoriferae Lignum, Arisaematis Rhizoma Preparatum) had fairly strong inhibition effect with residual activity below 20%. In an attempt to validate the results obtained from isoform-specific probe, the Curcumae Rhizoma with lowest residual activity was further tested to screen main bioactive constituents which possessed significant inhibitive effect. The crude extract of Curcumae Rhizoma was fractionated to investigate the inhibition effect of each fraction, the results showed that fractions 9-13 exhibited obvious inhibitory effect, and the main constituent (curdione) was identified with standard reference. The molecular docking results verified that the inhibiting effect of curdione could be explained that curdione was interacted with 7 amino acid residues to generate the hydrophobic interaction, and also interacted with imidazole to form hydrogen bond. It is anticipated that the results could be used as reference data to avoid drug-drug interaction and guide the clinical application of TCM or prescriptions.

Effects of Shengmai San on key enzymes involved in hepatic and intestinal drug metabolism in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. AIM OF THE STUDY: The studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. MATERIALS AND METHODS: The SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. RESULTS: Schisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. CONCLUSIONS: These findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.

Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis containing supplements.

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.

Buprenorphine-cannabis interaction in patients undergoing opioid maintenance therapy.

Buprenorphine is a partial µ-opioid agonist widely used for opioid maintenance therapy (OMT). It is mainly metabolized to pharmacologically active norbuprenorphine by the cytochrome P450 (CYP) isozyme 3A4. This may give rise to drug-drug interactions under combinations with inhibitors or inducers of CYP3A4. Cannabis is a potential inhibitor of CYP3A4, and there is a large degree of concomitant cannabis use among OMT patients. We performed a retrospective analysis on liver healthy OMT patients substituted with buprenorphine, either with (n = 15) or without (n = 17) concomitant use of cannabis. Patients with additional illicit drugs or medications affecting CYP3A were excluded. Measured blood concentrations of buprenorphine and norbuprenorphine were compared between the two groups. Cannabis users and non-users received similar doses, but users had 2.7-fold higher concentrations of buprenorphine (p < 0.01) and 1.4-fold for norbuprenorphine (1.4-fold, p = 0.07). Moreover, the metabolite-to-parent drug ratio was 0.98 in non-users and 0.38 in users (p = 0.02). Female gender did not produce significant effects. These findings indicate that cannabis use decreases the formation of norbuprenorphine and elevates buprenorphine and norbuprenorphine concentrations in blood most probably by inhibition of CYP3A4. The pharmacokinetic interaction may give rise to enhanced or altered opioid activity and risk of intoxications. Physicians should inform patients about this risk and supervise cannabis users by regular control of buprenorphine blood levels, i.e., by therapeutic drug monitoring.

Components characterisation of Berchemia lineata (L.) DC. by UPLC-QTOF-MS/MS and its metabolism with human liver microsomes.

Investigations were performed on the determination of the main components in Berchemia lineata (L.) DC. (BL) and its metabolism with human liver microsomes (HLM). A total of 35 compounds were detected in BL extracts and 25 of them including 6 naphthopyrones, 10 flavonoids, 2 phenolic acids, 2 phenols, 4 fatty acids and 1 quinone were unambiguously or tentatively identified by UPLC-QTOF-MS/MS. Among them, naphthopyrones were first identified in BL extracts and labelled in chromatography. In addition, the weak inhibitory effects of BL extracts (IC50＝149.25 µg/mL) and rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glu-copyranside (the main component of BL extracts, M0; IC50＝82.14 µM) on CYP3A4 were also proved using testosterone as specific probe drug. The main metabolic pathway of M0 by HLM was hydroxylation in its aglycone, the metabolite was tentatively identified as 10-hydroxy-rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glucopyranside. Components characterisation and the metabolism with HLM could help the further development and application of BL.

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism.

BACKGROUND: Styrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions. PURPOSE: This study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered. STUDY DESIGN: The inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated. METHODS: The inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis. RESULTS: In vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6ß-hydroxylation. CONCLUSION: Styrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.

Discovery and Structural Optimization of 4-(Aminomethyl)benzamides as Potent Entry Inhibitors of Ebola and Marburg Virus Infections.

The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33, and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses. Representative compounds (20, 32, and 35) have shown good metabolic stability in plasma and liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.

Inhibitory Effect of Lygodium Root on the Cytochrome P450 3A Enzyme in vitro and in vivo.

PURPOSE: The aim of the present study was to investigate the interactions of the main components of Lygodium root (ie, p-coumaric acid, acacetin, apigenin, buddleoside and Diosmetin-7-O-ß-D-glucopyranoside) with cytochrome P450 3A enzyme activity both in vitro and in vivo. METHODS: In vitro inhibition of drugs was assessed by incubating rat liver microsomes (RLMs) with a typical P450 3A enzyme substrate, midazolam, to determine their 50% inhibitory concentration (IC50) values. For the in vivo study, healthy male Sprague Dawley rats were consecutively administered acacetin or apigenin for 7 days at the dosage of 5 mg/kg after being randomly divided into 3 groups: Group A (control group), Group B (acacetin group) and Group C (apigenin group). RESULTS: Among the five main components of Lygodium root, only acacetin and apigenin showed inhibitory effects on the cytochrome P450 3A enzyme in vitro. The IC50 values of acacetin and apigenin were 58.46 µM and 8.20 µM, respectively. Additionally, the in vivo analysis results revealed that acacetin and apigenin could systemically inhibit midazolam metabolism in rats. The Tmax, AUC(0-t) and Cmax of midazolam in group B and group C were significantly increased (P<0.05), accompanied by a significant decrease in Vz/F and CLz/F (P<0.05). CONCLUSION: Acacetin and apigenin could inhibit the activity of the cytochrome P450 3A enzyme in vitro and in vivo, indicating that herbal drug interactions might occur when taking Lygodium root and midazolam synchronously.

Evaluation of Erythromycin as a Tool to Assess CYP3A Contribution of Low Clearance Compounds in a Long-Term Hepatocyte Culture.

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 µM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 µM of ERY, 75% by 15 µM of ERY, 89% by 30 µM of ERY, and 94% by 60 µM of ERY. ERY (30 µM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.

Application of Azamulin to Determine the Contribution of CYP3A4/5 to Drug Metabolic Clearance Using Human Hepatocytes.

Early determination of CYP3A4/5 contribution to the clearance of new chemical entities is critical to inform on the risk of drug-drug interactions with CYP3A inhibitors and inducers. Several in vitro approaches (recombinant P450 enzymes, correlation analysis, chemical and antibody inhibition in human liver microsomes) are available, but they are usually labor-intensive and/or suffer from specific limitations. In the present study, we have validated the use of azamulin as a specific CYP3A inhibitor in human hepatocytes. Azamulin (3 µM) was found to significantly inhibit CYP3A4/5 (>90%), whereas other P450 enzymes were not affected (less than 20% inhibition). Because human hepatocytes were used as a test system, the effect of azamulin on other key drug-metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, flavin monooxygenase, and sulfotransferase) was also investigated. Apart from some UGTs showing minor inhibition (∼20%-30%), none of these non-P450 enzymes were inhibited by azamulin. Use of CYP3A5-genotyped human hepatocyte batches in combination with CYP3cide demonstrated that azamulin (at 3 µM) inhibits both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocyte assay. Results showed that the effect of azamulin on the in vitro intrinsic clearance of these known CYP3A4/5 substrates was predictive of the in vivo CYP3A4/5 contribution. Overall, the study showed that human hepatocytes treated with azamulin provide a fast and accurate estimation of CYP3A4/5 contribution in metabolic clearance of new chemical entities. SIGNIFICANCE STATEMENT: Accurate estimation of CYP3A4/5 contribution in drug clearance is essential to anticipate risk of drug-drug interactions and select the appropriate candidate for clinical development. The present study validated the use of azamulin as selective CYP3A4/5 inhibitor in suspended human hepatocytes and demonstrated that this novel approach provides a direct and accurate determination of the contribution of CYP3A4/5 (fraction metabolized by CYP3A4/5) in the metabolic clearance of new chemical entities.

Effect of High-Dose Esomeprazole on CYP1A2, CYP2C19, and CYP3A4 Activities in Humans: Evidence for Substantial and Long-lasting Inhibition of CYP2C19.

In vitro, esomeprazole is a time-dependent inhibitor of CYP2C19. Additionally, racemic omeprazole induces CYP1A2 and omeprazole and its metabolites inhibit CYP3A4 in vitro. In this 5-phase study, 10 healthy volunteers ingested 20 mg pantoprazole, 0.5 mg midazolam, and 50 mg caffeine as respective index substrates for CYP2C19, 3A4, and 1A2 before and 1, 25, 49 (pantoprazole only), and 73 hours after an 8-day pretreatment with 80 mg esomeprazole twice daily. The area under the plasma concentration-time curve (AUC) of R-pantoprazole increased 4.92-fold (90% confidence interval (CI) 3.55-6.82), 2.31-fold (90% CI 1.85-2.88), and 1.33-fold (90% CI 1.06-1.68) at the 1-hour, 25-hour, and 73-hour phases, respectively, consistent with a substantial and persistent inhibition of CYP2C19. The AUC of midazolam increased up to 1.44-fold (90% CI 1.22-1.72) and the paraxanthine/caffeine metabolic ratio up to 1.19-fold (90% CI 1.04-1.36), when the index substrates were taken 1 hour after esomeprazole. Based on the recovery of R-pantoprazole oral clearance, the turnover half-life of CYP2C19 was estimated to average 53 hours. Pharmacokinetic simulation based on the observed concentrations of esomeprazole and its metabolites as well as their published CYP2C19 inhibitory constants was well in line with the observed changes in R-pantoprazole pharmacokinetics during the course of the study. Extrapolations assuming linear pharmacokinetics of esomeprazole suggested weak to moderate inhibition at 20 and 40 mg twice daily dosing. In conclusion, high-dose esomeprazole can cause strong inhibition of CYP2C19, but only weakly inhibits CYP3A4 and leads to minor induction of CYP1A2. The enzymatic activity of CYP2C19 recovers gradually in ~ 3-4 days after discontinuation of esomeprazole treatment.

Evaluation of the Effects of Maytenus ilicifolia on the Activities of Cytochrome P450 3A and P-glycoprotein.

BACKGROUND: Maytenus ilicifolia is a Brazilian popular medicine commonly used to treat ulcer and gastritis. Despite the absence of toxicity regarding its consumption, possible interactions when co-administrated with conventional drugs, are unknown. OBJECTIVE: This study aimed to evaluate the effects of M. ilicifolia extracts on Cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) activities. METHODS: The extracts were obtained by infusion (MI) or turbo-extraction using hydro-acetonic solvent (MT70). The content of polyphenols in each extract was determined. To assess the modulation of M. ilicifolia on P-gp activity, the uptake of fexofenadine (FEX) by Caco-2 cells was investigated in the absence or presence of MI or MT70. The effect on CYP3A activity was evaluated by the co-administration of midazolam (MDZ) with each extract in male Wistar rats. The pharmacokinetic parameters of the drug were determined and compared with those from the control group. The content of total phenolic compounds, tannins, and flavonoids on MT70 extract was about double of that found in MI. RESULTS: In the presence of the extracts, the uptake of the P-gp marker (FEX) by Caco-2 cells increased from 1.7 ± 0.4 ng.mg-1 protein (control) to 3.5 ± 0.2 ng.mg-1 protein (MI) and 4.4 ± 0.5 ng.mg-1 protein (MT70), respectively. When orally co-administrated with MDZ (substrate of CYP3A), the extracts augmented the AUC(0-∞) (Control: 911.7 ± 215.7 ng.h.mL-1; MI: 1947 ± 554.3 ng.h.mL-1; MT70: 2219.0 ± 506.3 ng.h.mL-1) and the Cmax (Control: 407.7 ± 90.4 ng.mL-1; MI: 1770.5 ± 764.5 ng.mL-1; MT70: 1987.2 ± 544.9 ng.mL-1) of the drug in rats indicating a 50% reduction of the oral Cl. No effect was observed when midazolam was given intravenously. CONCLUSION: The results suggest that M. ilicifolia can inhibit the intestinal metabolism and transport of drugs mediated by CYP3A and P-gp, respectively, however, the involvement of other transporters and the clinical relevance of such interaction still need to be clarified.

Effect of CYP3A4 on liver injury induced by triptolide.

Triptolide (TP), one of the main bioactive diterpenes of the herbal medicine Tripterygium wilfordii Hook F, is used for the treatment of autoimmune diseases in the clinic and is accompanied by severe hepatotoxicity. CYP3A4 has been reported to be responsible for TP metabolism, but the mechanism remains unclear. The present study applied a UPLC-QTOF-MS-based metabolomics analysis to characterize the effect of CYP3A4 on TP-induced hepatotoxicity. The metabolites carnitines, lysophosphatidylcholines (LPCs) and a serious of amino acids were found to be closely related to liver damage indexes in TP-treated female mice. Metabolomics analysis further revealed that the CYP3A4 inducer dexamethasone improved the level of LPCs and amino acids, and defended against oxidative stress. On the contrary, pretreatment with the CYP3A4 inhibitor ketoconazole increased liver damage with most metabolites being markedly altered, especially carnitines. Among these metabolites, except for LPC18:2, LPC20:1 and arginine, dexamethasone and ketoconazole both affected oxidative stress induced by TP. The current study provides new mechanistic insights into the metabolic alterations, leading to understanding of the role of CYP3A4 in hepatotoxicity induced by TP.

Role of kaempferol to increase bioavailability and pharmacokinetics of nifedipine in rats.

Herein, the purpose of this study is to evaluate the effects of kaempferol on bioavailability and pharmacokinetics of nifedipine and its metabolite dehydronifedipine in rats. The experimental design is based on with or without kaempferol in the oral and intravenous administration of nifedipine in rats. Moreover, the pharmacokinetic parameters including nifedipine and dehydronifedipine were evaluated in rats.The in vitro studies ofkaempferol were investigated on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity. Kaempferol reduced a 50% inhibitory concentration (IC50) of 8.6 µmol·L-1 on CYP3A4 enzyme activity. Moreover, kaempferol clearly improved the cell internalization of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. Depending on increased concentrations of kaempferol, the areas under the plasma concentration-time curve (AUC0-∞) and the peak concentration (Cmax) of nifedipine were increased after oral and intravenous administration. Moreover, the absolute bioavailability (AB) and relative bioavailability (RB) of nifedipine in the presence of kaempferol was significantly higher than those of the control group after oral and intravenous administration. Improvement of bioavailability of nifedipine by kaempferol may be mainly because of the inhibition of the P-gp-mediated efflux transporter in the small intestine and CYP3A4-mediated metabolism in the small intestine or liver, or both.

Isolation, analysis and in vitro assessment of CYP3A4 inhibition by methylxanthines extracted from Pu-erh and Bancha tea leaves.

Methylxanthines, purine alkaloids found in plants, are found in beverages (coffee, tea, cocoa) and foods (chocolate and other cocoa-containing foods) commonly consumed worldwide. Members of this family include caffeine, theophylline and theobromine. Methylxanthines have a variety of pharmacological effects, and caffeine and theophylline are used as pharmaceuticals. Methylxanthines are metabolized in the liver predominantly by the enzyme CYP1A2. Their co-administration with CYP1A2 inhibitors may lead to pharmacokinetic interactions. Little is known about the possible drug interactions between caffeine and substrates of other CYP450 enzymes. In our study, methylxanthine fractions inhibited CYP3A4 in a concentration-dependent manner. Concomitant consumption of green tea with CYP3A4 substrates could increase the possibility of interactions, and this requires further clarification. The inhibition of CYP3A4 is not only due to the presence of catechin derivatives but methylxanthines may also contribute to this effect.