Pharmacokinetic interactions between tacrolimus and Wuzhi capsule in liver transplant recipients: Genetic polymorphisms affect the drug interaction.

Wuzhi capsule (WZC), a commonly used Chinese patent medicine to treat various types of liver dysfunction in China, increases the exposure of tacrolimus (TAC) in liver transplant recipients. However, this interaction has inter-individual variability, and the underlying mechanism remains unclear. Current research indicates that CYP3A4/5 and drug transporters influence the disposal of both drugs. This study aims to evaluate the association between TAC dose-adjusted trough concentration (C/D) and specific genetic polymorphisms of CYP3A4/5, drug transporters and pregnane x receptor (PXR), and plasma levels of major WZC components, deoxyschisandrin and γ-schisandrin, in liver transplant patients receiving both TAC and WZC. Liquid chromatography-tandem-mass spectrometry was used to detect the plasma levels of deoxyschisandrin and γ-schisandrin, and nine polymorphisms related to metabolic enzymes, transporters and PXR were genotyped by sequencing. A linear mixed model was utilized to assess the impact of the interaction between genetic variations and WZC components on TAC lnC/D. Our results indicate a significant association of TAC lnC/D with the plasma levels of deoxyschisandrin and γ-schisandrin. Univariate analysis demonstrated three polymorphisms in the genes ABCB1 (rs2032582), ABCC2 (rs2273697), ABCC2 (rs3740066), and PXR (rs3842689) interact with both deoxyschisandrin and γ-schisandrin, influencing the TAC lnC/D. In multiple regression model analysis, the interactions between deoxyschisandrin and both ABCB1 (rs2032582) and ABCC2 (rs3740066), post-operative day (ß < 0.001, p < 0.001), proton pump inhibitor use (ß = -0.152, p = 0.008), body mass index (ß = 0.057, p < 0.001), and ABCC2 (rs717620, ß = -0.563, p = 0.041), were identified as significant factors of TAC lnC/D, accounting for 47.89% of the inter-individual variation. In summary, this study elucidates the influence of the interaction between ABCB1 and ABCC2 polymorphisms with WZC on TAC lnC/D. These findings offer a scientific basis for their clinical interaction, potentially aiding in the individualized management of TAC therapy in liver transplant patients.

Effects of WuZhi preparations on tacrolimus in pediatric and adult patients carrying the CYP3A5*1 allele of heart transplant during the early period after transplantation.

AIM: Wuzhi preparations (WZP) are commonly administrated with tacrolimus (TAC) in China to improve the liver function and increase the exposure of TAC. This study aims to investigate the effects of WZP on TAC in pediatric heart transplantation (HTx) patients carrying the CYP3A5*1 allele during the early period after transplantation and also make a comparison with these effects in adult recipients. METHODS: A total of 81 recipients with CYP3A5*1 allele were included and divided into the pediatric group (n = 29) and adult group (n = 52). The changes in TAC dose-corrected trough blood concentrations (C0 /D), dose requirement as well as intra-patient variability(IPV) of C0 /D after co-therapy with WZP were evaluated. RESULTS: The TAC C0 /D was significantly increased 1.7 and 1.8 times after co-administration of WZP in the pediatric and adult groups, respectively. We further analyzed the pediatric patients, found that no statistical difference was observed in TAC C0 /D before and after co-therapy with WZP in children <6 years old. The changes of C0 /D increased with the dose of the active ingredient (Schisantherin A) in adult patients, but not in pediatric patients. TAC IPV was reduced by 10.5% in pediatric patients and 4.8% in adult patients when co-administrated with WZP. Furthermore, after taking WZP, the AST and TB were dramatically lowered in pediatric recipients. CONCLUSION: Our study is the first attempt to demonstrate the effects of WZP on TAC in pediatric HTx recipients. By comparing these effects to those observed in adult recipients, valuable insights can be gained regarding the efficacy and potential benefits of WZP in the pediatric population.

Drug interaction potential of Ankaferd blood stopper® in human hepatocarcinoma cells.

BACKGROUND: Ankaferd blood stopper® (ABS) is an herbal extract consisting of mixtures of Alpinia officinarum, Gycyrrhiza glabra, Vitis vinifera, Thymus vulgaris, and Urtica dioica plants and has been used in recent years in Turkish medicine as a hemostatic agent. Despite its extensive usage, there is no information available about the drug interaction in HepG2 cells. The current work evaluated the effect of ABS on the expression of CYP1A1-1A2, CYP2E1, and CYP3A4 isozymes that are primarily involved in drug and carcinogen metabolism. METHODS: We selected HepG2 cells as in vitro cellular models of the human liver. The cells were treated with different concentrations of ABS [0.25%-40% (v/v)]. A crystal violet staining assay was used to determine the cytotoxicity of ABS. We examined drug-metabolizing enzymes, including 7-ethoxyresorufin O-deethylase (CYP1A1), 7-methoxyresorufin O-demethylase (CYP1A2), aniline 4-hydroxylase (CYP2E1), and erythromycin N-demethylase (CYP3A4), in vitro in HepG2 cells. The expression (mRNA, protein) levels of drug-metabolizing enzymes were analyzed by qPCR and Western blotting, respectively. RESULTS: The EC05 and EC10 values for ABS were 0.37% and 0.52% (v/v), respectively. Therefore, 0.37% and 0.52% (v/v) doses were used for the remaining portion of this study. Investigation of the expression and activity levels revealed that CYP1A1-1A2, CYP2E1, and CYP3A4 activities were not affected by ABS significantly, with qPCR and Western blot results corroborating this result. DISCUSSION: Our study found that the activity, mRNA, and protein expression levels of CYP isozymes did not change with the application of ABS, suggesting that when humans are exposed to ABS, there may not be any risk associated with clinical drug toxicity, cancer formation, and drug metabolism disorders in humans.

Panaxytriol upregulates CYP3A4 expression through the interaction between nuclear regulators and DNA response elements.

ETHNOPHARMACOLOGICAL RELEVANCE: Cytochrome P3A4 (CYP3A4) is a crucial drug-metabolizing enzyme, and its expression is regulated by the pregnane X receptor (PXR), constitutive androstane receptor (CAR), steroid receptor coactivator 1 (SRC-1), and acetyltransferase P300. Panaxytriol is a naturally derived active substance extracted from the roots of Panax ginseng C. A. Mey. which is widely used clinically. Our previous studies have shown that panaxytriol induces CYP3A4 expression through PXR activation, which is antagonized by high CAR expression. However, the underlying mechanism remains unclear. AIM OF THE STUDY: This study aimed to investigate the mechanism of panaxytriol in inducing CYP3A4 expression via interactions between nuclear regulators and DNA response elements. MATERIALS AND METHODS: Immunoprecipitation technique was used to assess the binding levels of PXR and CAR with the coactivators SRC-1 and P300 in HepG2 and Huh-7 cells. Furthermore, chromatin immunoprecipitation assay was used to investigate the PXR and CAR interaction with the CYP3A4 promoter response element ER-6/DR-3. RESULTS: The binding of PXR to SRC-1, P300, and the response elements ER-6 and DR-3 was improved with an increase in panaxytriol concentration (10-80 µM), and the binding affinity was further enhanced upon CAR silencing. The binding of CAR to SRC-1 and the response elements ER-6 and DR-3 was significantly higher at 80 µM panaxytriol, whereas no significant binding was observed between CAR and P300. CONCLUSION: Panaxytriol promoted the recruitment of PXR to SRC-1 and P300, binding to ER-6 and DR-3, and upregulating CYP3A4 expression. Furthermore, an interactive dialogue regulatory mechanism between PXR and CAR was observed.

Safety assessment of herbal supplement components targeting hepatotoxicity and CYP3A4 induction in cell-based assay using HepG2 cells.

Herbal supplements can cause hepatotoxicity and drug interactions via hepatic cytochrome P-450 (CYP) in some cases. However, there is no simple and stable cell-based assay to conduct a screening for hepatotoxicity and CYP induction. In the present study, we selected 14 components of the herbal supplement based on our previous reports and investigated the safety of the herbal supplement components focusing on toxicity and CYP3A4 induction in a cell-based assay using HepG2. The toxicity of the components was examined by lactate dehydrogenase (LDH) and cell proliferation assays. Then, the CYP3A4 induction of the components were examined by a reporter assay using reporter vectors of CYP3A4. The vector includes the CYP3A4 proximal promoter (CYP3A4PP) and the xenobiotic-responsive enhancer module (XREM) regions. Luteolin (LU) significantly increased LDH activity and decreased cell proliferation activity that suggests LU may cause toxicity in HepG2 cells. Quercetin (QU) increased the transcriptional activity of CYP3A4 (1.5-fold of control) in the reporter assay. However, the induction of QU was slightly in comparison to the validation of the transcriptional activity of CYP3A4 treated with CYP3A4 inducers. The CYP3A4 induction of QU may not involve CYP3A4PP but involves the XREM response. Throughout our results, the method in the present study may be useful for a safety assessment of herbal supplements, primarily focusing on hepatotoxicity and CYP3A4 induction. PRACTICAL APPLICATION: Even though there are problems with herbal supplements, studies related to toxicity are not actively carried out. The present methods may apply to the safety assessment for herbal supplements and be useful for the prevention and verification of health hazards caused by herbal supplements (the summary is shown in Figure S2).

Effects of breviscapine on cerebral ischemia-reperfusion injury and intestinal flora imbalance by regulating the TLR4/MyD88/NF-κB signaling pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Erigeron breviscapus (Vant.) Hand.-Mazz.,a Chinese herbal medicine with multiple pharmacological effects and clinical applications, has been traditionally used in the treatment of paralysis caused by stroke and joint pain from rheumatism by the Yi minority people of Southwest China for generations.However, its mechanism involves many factors and has not been fully clarified. AIM OF THE STUDY: Taking intestinal flora as the target, the protective effect of extract(breviscapine) of E. breviscapus on cerebral ischemia and its possible mechanism were discussed from the perspective of brain inflammatory pathway and intestinal CYP3A4, which depends on intestinal flora. MATERIALS AND METHODS: In this study, we first verified the binding ability between major active ingredient of Erigeron breviscapus and the core target TLR4 protein by molecular docking using Vina software.We established a rat model of cerebral ischemia-reperfusion injury in vivo.The neurological function of rats was scored by Bederson score table, the cerebral infarction volume was detected by TTC staining, and the serum NSE level was detected by ELASA. 16S rRNA sequencing was used to detect the intestinal flora of rats in each group.The expression levels of cerebral TLR4/MyD88/NF-κB and CYP3A4 mRNA and protein in different intestinal segments were detected by qRT-PCR and Western blot. RESULTS: Compared with the model group, the neurological injury score, infarct volume and serum NSE concentration of breviscapine low, medium and high dose groups and nimodipine groups decreased significantly. Meanwhile, breviscapine could significantly reduce the expression level of the TLR4/MyD88/NF-κB in brain tissue and CYP3A4 in different intestinal segments of rats with cerebral ischemia-reperfusion injury. In addition, breviscapine also significantly ameliorated intestinal flora dysbiosis of rats with cerebral ischemia-reperfusion injury. CONCLUSIONS: Breviscapine can protect rats from cerebral ischemia-reperfusion injury by regulating intestinal flora, inhibiting brain TLR4/MyD88/NF-κB inflammatory pathway and intestinal CYP3A4 expression.

The Pharmacogenetics of Cannabis in the Treatment of Chronic Pain.

BACKGROUND: The increase in the medical use of cannabis has revealed a number of beneficial effects, a variety of adverse side effects and great inter-individual variability. Association studies connecting consumption, addiction and side effects related to recreational cannabis use have led to the identification of several polymorphic genes that may play a role in the pharmacodynamics and pharmacokinetics of cannabis. METHOD: In total, 600 patients treated with cannabis were genotyped for several candidate polymorphic genes (single-nucleotide polymorphism; SNP), encoding receptors CNR1 and TRPV1; for the ABCB1 transporter; for biotransformation, bioactivation and biosynthesis; and CYP3A4, COMT and UGT2B7 conjugation. RESULTS: Three polymorphic genes (ABCB1, TRPV1 and UGT2B7) were identified as being significantly associated with decline in pain after treatment with cannabis. Patients simultaneously carrying the most favourable allele combinations showed a greater reduction (polygenic effect) in pain compared to those with a less favourable combination. Considering genotype combinations, we could group patients into good responders, intermediate responders and poor or non-responders. Results suggest that genetic makeup is, at the moment, a significant predictive factor of the variability in response to cannabis. CONCLUSIONS: This study proves, for the first time, that certain polymorphic candidate genes may be associated with cannabis effects, both in terms of pain management and side effects, including therapy dropout. SIGNIFICANCE: Our attention to pharmacogenetics began in 2008, with the publication of a first study on the association between genetic polymorphisms and morphine action in pain relief. The study we are presenting is the first observational study conducted on a large number of patients involving several polymorphic candidate genes. The data obtained suggest that genetic makeup can be a predictive factor in the response to cannabis therapy and that more extensive and planned studies are needed for the opening of new scenarios for the personalization of cannabis therapy.

Genetic polymorphisms of pharmacogenomic VIP variants in the Lahu population from Yunnan Province.

BACKGROUND: Pharmacogenomics has been widely used to study the very important pharmacogenetic (VIP) variants among populations, but information on pharmacogenomics in the Lahu population is limited. The purpose of this study was to determine the differences in the distribution of VIP variants between the Lahu and the other 26 populations. METHODS: We genotyped 55 VIP variants of 27 genes in the Lahu population from the PharmGKB database. χ2 test was used to compare the genotype and allele frequencies between the Lahu and the other 26 populations from the 1000 Genomes Project. RESULTS: The genotype and allele frequencies of single nucleotide polymorphisms (SNPs) on rs20417 (PTGS2), rs776746 (CYP3A5), rs2115819 (ALOX5), and rs3093105 (CYP4F2) were considerably different in the Lahu population compared with those in the other 26 populations. Besides, based on the PharmGKB database, we identified several VIP variants that may alter the drug metabolism of aspirin (PTGS2), tacrolimus (CYP3A5), montelukast (ALOX5), and vitamin E (CYP4F2). CONCLUSION: The results show that there are significant differences in the genotype frequency distribution between the Lahu and the other 26 populations. Our study supplements the pharmacogenomics information of the Lahu population and provides a theoretical basis for individualized medicine in Lahu.

Artemisia extracts differ from artemisinin effects on human hepatic CYP450s 2B6 and 3A4 in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese medicinal herb, Artemisia annua L., has been used for >2,000 yr as traditional tea infusions to treat a variety of infectious diseases including malaria, and its use is spreading globally (along with A. afra Jacq. ex Willd.) mainly through grassroots efforts. AIM OF THE STUDY: Artemisinin is more bioavailable delivered from the plant, Artemisia annua L. than the pure drug, but little is known about how delivery via a hot water infusion (tea) alters induction of hepatic CYP2B6 and CYP3A4 that metabolize artemisinin. MATERIALS AND METHODS: HepaRG cells were treated with 10 µM artemisinin or rifampicin (positive control), and teas (10 g/L) of A. annua SAM, and A. afra SEN and MAL with 1.6, 0.05 and 0 mg/g DW artemisinin in the leaves, respectively; qPCR and Western blots were used to measure CYP2B6 and CYP3A4 responses. Enzymatic activity of these P450s was measured using human liver microsomes and P450-Glo assays. RESULTS: All teas inhibited activity of CYP2B6 and CYP3A4. Artemisinin and the high artemisinin-containing tea infusion (SAM) induced CYP2B6 and CYP3A4 transcription, but artemisinin-deficient teas, MAL and SEN, did not. Artemisinin increased CYP2B6 and CYP3A4 protein levels, but none of the three teas did, indicating a post-transcription inhibition by all three teas. CONCLUSIONS: This study showed that Artemisia teas inhibit activity and artemisinin autoinduction of CYP2B6 and CYP3A4 post transcription, a response likely the effect of other phytochemicals in these teas. Results are important for understanding Artemisia tea posology.

Effect of repeated Shengmai-San administration on nifedipine pharmacokinetics and the risk/benefit under co-treatment.

Herbal interactions with nifedipine/felodipine through cytochrome P450 (CYP) 3A inhibition is significant in humans. Shengmai-San (SMS), a three-herbal formula of Chinese medicine, is commonly prescribed in Asia populations for cardiovascular disorders. This study aimed to elucidate the impact of SMS on nifedipine/felodipine treatment by the findings from rat pharmacokinetic study of nifedipine to the retrospective cohort study of patients with hypertension. The 3-week SMS treatment increased the systemic exposure to nifedipine by nearly two-fold and decreased nifedipine clearance by 39% in rats. Among the ingredients of SMS component herbs, schisandrin B, schisantherin A, and methylophiopogonanone A, inhibited the nifedipine oxidation (NFO) activities of rat hepatic and intestinal microsomes, as well as human CYP3A4. Methylophiopogonanone A was identified as a time-dependent inhibitor of CYP3A4. After 1:5 propensity score matching, 4,894 patients with nifedipine/felodipine use were analyzed. In patients receiving nifedipine/felodipine, the subgroup with concurrent SMS treatment had a higher incidence of headache (92.70 per 1,000 personyears) than the subgroup without SMS treatment (51.10 per 1,000 person-years). There was a positive association between headache incidence and cumulative doses of SMS (1-60 g SMS: hazard ratio (HR): 1.39; 95% confidence interval (CI): 1.11-1.74; >60 g SMS: HR: 1.97; 95% CI: 1.62-2.39; p < 0.0001). However, patients who had higher cumulative SMS doses had a lower risk of all-cause mortality (1-60 g SMS: HR: 0.67; 95% CI: 0.47-0.94; >60 g SMS: HR: 0.54; 95% CI: 0.37-0.79; p = 0.001). Results demonstrated increased rat plasma nifedipine levels after 3-week SMS treatment and increased headache incidence should be noted in nifedipine/felodipine-treated patients with prolonged SMS administration.

Cytochrome P450 inhibition activities of non-standardized botanical products.

ETHNOPHARMACOLOGICAL RELEVANCE: R-tab, H-tab and E-cap botanical products are used for the treatment of various ailments. R-tab is traditionally prescribed for improving urination, H-tab is for relieving piles, hemorrhoids, fissures, and rectal inflammation and E-cap is for regulating menstruation. AIMS OF THE STUDY: To extract the botanical products and determine their potential interaction with the cytochrome P450 (CYP1A2, CYP2D6 and CYP3A4) enzymes. MATERIALS AND METHODS: R-tab, H-tab and E-cap botanical products were first extracted using solvents and analyzed using HPLC and LC-MS/MS. The effects of methanol extracts on the cytochrome induction and inhibition activities were determined using a series of in vitro assays, including multiplex RT-qPCR, CYP activity assays (P450-Glo™) and LC-MS/MS-based assays. For the CYP induction assay, omeprazole, rifampicin and dexamethasone were used as CYP1A2, CYP2D6 and CYP3A4 inducers, respectively. Ketoconazole and acetaminophen were used as positive and negative controls for the CYP3A4 inhibition assay, whereas furafylline and ketoconazole were used as positive and negative controls for the CYP1A2 inhibition assay. RESULTS: All three botanical products did not show any significant induction in CYP1A2, CYP2D6 and CYP3A4 mRNA expression. By contrast, R-tab inhibited the mRNA expression of CYP1A2 significantly from the lowest concentration of 0.01 µg/mL, while, H-tab inhibited the mRNA expression of CYP1A2 and CYP3A4 from 0.1 µg/mL. Based on the P450 Glo assays, E-cap extract inhibited the metabolic activity of CYP1A2 with an IC50 value of 37.24 µg/mL. On the other hand, R-tab, H-tab and E-cap showed inhibitory effects on the CYP3A4 enzymatic activity with IC50 values of 17.42, 18.20 and 20.60 µg/mL, respectively. However, using the LC-MS/MS-based methods, the concentration-dependent effects of R-tab and H-tab extracts on the metabolism of testosterone appeared to be more prominent, with IC50 values of 51.90 and 56.90 µg/mL as compared with the rest of the results, which were all above 100 µg/mL CONCLUSION: The CYP3A4 mRNA and enzymatic activity were moderately inhibited by R-tab and H-tab. Methanol extract of botanical products in solid dosage forms can be evaluated for their herb-drug interaction risks using in vitro assays and may provide the minimum data required for safety labeling.

Herb-drug interaction between Shaoyao-Gancao-Fuzi decoction and tofacitinib via CYP450 enzymes.

ETHNOPHARMACOLOGY RELEVANCE: Shaoyao-Gancao-Fuzi decoction (SGFD), a well-known traditional Chinese medicine formula, was originally described in "Treatise on Febrile Diseases" and has been extensively used to dispel wind, eliminate dampness and treat paralysis. It is widely used for the treatment of rheumatoid arthritis in clinic. However, the effect of SGFD on the activity of cytochrome P450 enzymes (CYP450s) and the herb-drug interactions are rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SGFD on the activity of CYP450s and evaluate the potential herb-drug interactions between SGFD and tofacitinib, commonly used disease-modifying antirheumatic drug in rheumatoid arthritis. MATERIALS AND METHODS: The cocktail approach was employed to assess the effect of SGFD on the activity of CYP1A2, 3A4, 2A6, 2E1, and 2C9. The pharmacokinetic profile of oral administration of tofacitinib in rats after two weeks of treatment with SGFD was investigated. RT-qPCR and molecular docking were performed to unveil the underlying mechanism of the herb-drug interaction. RESULTS: SGFD had no effect on the activities of CYP2E1 and 2C9, had a weak effect on CYP2A6, and had activatory effect on CYP1A2. However, it had a dramatically inhibitory effect on the activity of CYP3A4. Simultaneously, the values of Cmax and AUC0-∞ of tofacitinib were obviously increased after treatment with SGFD for 14 days. The mechanism study manifested that SGFD significantly reduced the gene transcription of CYP3A. Molecular docking work confirmed that the inhibitory activity of glycyrrhetinic acid, glycyrrhizic acid and liquiritin, the main ingredients of SGFD, occurred by occupying the active sites of CYP3A4 and by making favorable interactions with its key residues. CONCLUSIONS: The system exposure of tofacitinib was increased by SGFD. SGFD could affect the activity and gene expression of the key metabolic enzyme CYP3A. These findings give a clear understanding to predict herb-drug interaction of SGFD for safe clinical use in future.

Glycyrrhiza uralensis promote the metabolism of toxic components of Aconitum carmichaeli by CYP3A and alleviate the development of chronic heart failure.

Aconitum, as "the first drug of choice for invigorating Yang and saving lives", has been widely used for the treatment of heart failure. However, toxic components of Aconitum can easily lead to serious arrhythmia, even death (Y. CT., 2009; Zhang XM., 2018). In this study, a High Performance Liquid Chromatography (HPLC) method for the determination of aconitine (AC), mesaconitine (MA) and hypaconitine (HA) was established; The effect of Glycyrrhiza on CYP3A1 / 2 mRNA expression was detected by RT-PCR; SD rats were given Aconitum and compatibility of Glycyrrhizae and Aconitum by gavage respectively, the blood concentration of toxic components were determined by LC-MS / MS; The CHF rat model was established by intraperitoneal injection of adriamycin (2.5 mg / kg), and were randomly divided into model, Aconitum, the compatibility of Glycyrrhizae and Aconitum and Captopril group, 5 mice/group. After 4 weeks of gavage, the corresponding indexes were detected by ELISA and HPLC. The results showed that Ketoconazole significantly inhibited the metabolites of AC, MA and HA; Glycyrrhiza induced CYP3A gene expression; The level of ALD in the compatibility of Glycyrrhizae and Aconitum group was significantly lower than that in Aconitum group. After intervention with the compatibility of Glycyrrhizae and Aconitum, ATP increased, ADP decreased significantly. In conclusion, we found Glycyrrhiza promoted the metabolism of toxic components of Aconitum by up regulating the expression of CYP3A, and reduced the content of BNP, Ang II and ALD, improved the energy metabolism disorder of myocardium, alleviated the development of CHF.

Inhibitory effects of traditional Chinese medicine colquhounia root tablet on the pharmacokinetics of tacrolimus in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tacrolimus (TAC) was widely used in various renal diseases while high recurrence rate and high expense restricted its applications. Traditional herbal medicine has become increasingly popular as an adjuvant therapy to minimize the adverse effects of TAC. Colquhounia root tablet (CRT), a prescribed drug prepared from the water extract of the peeled root of Tripterygium hypoglaucum (H. Lév.) Hutch., showed excellent anti-inflammatory, analgesic and immunosuppressive pharmacological properties. TAC used in combination with CRT was substantially more efficacious and safer than the monotherapy for the treatment of nephrotic syndrome. However, studies on their herb-drug interaction were scanty. AIM OF THE STUDY: The study was proposed to examine the effect of CRT on the pharmacokinetics of TAC in rats and identify the key natural constituents in CRT that affected the metabolism of TAC. MATERIALS AND METHODS: TAC was orally and intravenously administered to rats alone or in combination with CRT and the pharmacokinetic parameters of TAC were compared. After pretreatment with CRT for 15 d, the expressions of the drug-metabolizing enzymes (DMEs), drug transporters (DTs) and nuclear receptors (NRs) were determined by polymerase chain reaction and western blotting and compared with the control group. The hepatic microsomal incubation system was employed to confirm the inhibitory effects of CRT and its major components on rat cytochrome P450 (CYP) 3A2. The roles of the primary components in the regulation of human CYP3A4 and mouse P-gp activities were evaluated by using docking analysis. RESULTS: The blood concentrations of TAC were significantly increased in a dose- and pretreatment time-dependent manner after combined administration of CRT. The maximal effect was found at 300 mg/kg (43.70 ± 8.77 ng/mL and 141.45 ± 21.58 h·ng/mL) in a single dose run and the pharmacokinetic parameters gradually returned to the normal levels at 24 h interval of long-term CRT pretreatment. In contrast, CRT had no effect on the pharmacokinetics of intravenous TAC. Further study indicated that the mRNA and protein expressions of DMEs and DTs, such as CYP3A1, CYP3A2, P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 in rat intestine and liver were down-regulated, whereas the expressions of NRs like constitutive androstane receptor and pregnane X receptor were up-regulated after multiple oral doses of CRT. Molecular docking showed the binding potency of five CRT major constituents with both human CYP3A4 and mouse P-gp. Celastrol, wilforgine and wilforine were the strongest inhibitors towards midazolam metabolism in rat liver microsomes, with the 50% inhibition concentrations being at 8.33 µM, 22.18 µM and 22.22 µM, respectively. CONCLUSIONS: Our results revealed that co-dosing of CRT could lead to a significant increase in blood concentration of TAC and this effect could be ascribed to the resultant co-regulation of DMEs, DTs and NRs. Our study provided an experimental basis for the combination use of CRT and TAC in clinical practice.

Panaxytriol upregulates CYP3A4 expression based on the interaction of PXR, CAR, HSP90α, and RXRα.

BACKGROUND: Cytochrome P450 3A4 (CYP3A4) is one of the most important drug-metabolizing enzymes in the human body, mainly existing in the liver, small intestine, and kidney. Panaxytriol is one of the key active components in red ginseng and Shenmai injection. Our previous study demonstrated that panaxytriol regulates CYP3A4 expression mainly by activating pregnancy X receptor (PXR). At a high concentration of panaxytriol (80 µM), the constitutive androstane receptor (CAR) is also involved in the upregulation of CYP3A4. PURPOSE: This study investigated how the cofactors heat shock protein 90 alpha (HSP90α) and retinoid X receptor alpha (RXRα) interact with PXR and CAR to participate in the regulation of CYP3A4 by panaxytriol from the perspective of the PXR and CAR interaction. METHODS: The mRNA and protein expressions of PXR, CAR, CYP3A4, RXRα, and HSP90α in HepG2 cells and Huh-7 cells were detected by quantitative PCR and western blot analysis, respectively. The binding levels of PXR and CAR to RXRα and HSP90α were determined by co-immunoprecipitation analysis. The nuclear translocation of PXR and RXRα into HepG2 cells and human (hCAR)-silenced HepG2 cells were measured by immunofluorescence. RESULTS: In HepG2 cells and Huh-7 cells, panaxytriol (10-80 µM) upregulated CYP3A4 expression in a concentration-dependent manner by decreasing PXR binding to HSP90α and increasing PXR binding to RXRα. When hCAR was silenced, panaxytriol further enhanced CYP3A4 expression by strengthening PXR binding to RXRα, but it had no significant effect on the binding level of PXR and HSP90α. Additionally, at the high concentration of 80 µM panaxytriol, CAR binding to HSP90α was weakened while binding to RXRα was enhanced. CONCLUSION: Panaxytriol can upregulate CYP3A4 expression by promoting PXR dissociation from HSP90α and enhancing PXR binding to RXRα in HepG2 cells and Huh-7 cells. At high concentrations of panaxytriol, CAR also participates in the induction of CYP3A4 through a similar mechanism. However, in general, CAR antagonizes PXR binding to RXRα, thereby attenuating the upregulation of CYP3A4 by panaxytriol.

Probing PXR activation and modulation of CYP3A4 by Tinospora crispa and Tinospora sinensis.

ETHNOPHARMACOLOGICAL RELEVANCE: The two Tinospora species, T. crispa and T. sinensis, native to Southeast Asia, are integral components of various traditional preparations with structure-function claims to treat various disorders, including diabetes and inflammation. AIM OF THE STUDY: To assure the safety of the botanicals finished products, herb-drug interaction potential of T. crispa and T. sinensis was investigated by testing their extracts and compounds for in vitro activation of the pregnane X-receptor (PXR) and the modulation of CYP3A4 isozyme, selectively. MATERIALS AND METHODS: A total of sixteen fully characterized phytochemicals from T. crispa and T. sinensis were evaluated for PXR activation by luciferase reporter gene assay. CYP3A4 inhibition studies were carried out for eleven compounds. In addition, docking studies were performed to elucidate the possible binding modes to the PXR by the compounds using computational methods. RESULTS: Significant activation of PXR (2-fold) was observed for both extracts and non-polar fractions of T. crispa. Among the pure compounds, columbin showed highest activation of PXR (3-fold), which was comparable with the positive control, rifampicin. Vital interactions were predicted with docking simulation of PXR-columbin complex with critical amino acid residues (Trp-299) that are known for the activation of PXR. The methanolic extracts of T. crispa and T. sinensis also showed considerable CYP3A4 inhibition. CONCLUSION: T. crispa and T. sinensis, both demonstrated the potential to mediate herb-drug interaction through PXR activation and inhibition of CYP3A4 isozyme. Moreover, the elucidation of the potential to induce herb-drug interaction, by the phytochemicals of these Tinospora plants, thereby supports the need for further investigation to establish the clinical relevancy of these constituents for possible adverse interactions with pharmaceutical drugs.

Gene Expression Profiling of 1α,25(OH)2 D3 Treatment in 2D/3D Human Hepatocyte Models Reveals CYP3A4 Induction but Minor Changes in Other Xenobiotic-Metabolizing Genes.

SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

An insight on the nature of biochemical interactions between glycyrrhizin, myricetin and CYP3A4 isoform.

Drug interaction studies are imperative to gain insights into the beneficial or harmful effects of therapeutic and dietary agents. This study investigated the mechanism of modulatory roles of glycyrrhizin (GLH) and myricetin (MYC) on the human CYP3A4 isoform using in silico and in vitro methods. While MYC had concentration-dependent inhibitory effect on CYP3A4 (IC50 : 10.5 ± 0.55 µM) with characteristic Km and Vmax values of 1.13 µM and 1.54 nM/min, respectively, GLH exhibited no inhibitory effect on CYP3A4 activity in vitro. These observations are consistent with the results of in silico evaluations where the effect of MYC compared well with that of ketoconazole (a known CYP3A4 inhibitor) against CYP3A4. Overall, the established interactions between the study compounds and CYP3A4 could potentiate clinically vital drug-drug interactions and has lent credence to the mechanism of modulatory effect of MYC and GLH on CYP3A4 that could guide their safe use as therapeutic agents. PRACTICAL IMPLICATIONS: Myricetin (MYR) and glycyrrhizin (GLH) occur freely in commonly ingested foods and their supplements are recommended for the treatment of several debilitating diseases such as diabetes, cancer, and cardiovascular complications. This study provided an insight on the possible interactions that could be established between these compounds (MYR and GLH) and CYP3A4 when ingested and metabolized by the liver. The results suggested possibilities of potential clinical drug-drug interactions and advocates for their cautious use within the therapeutic dose in food supplements or medications to avoid probable liver damage.

Inhibition of CYP3A4 enhances aloe-emodin induced hepatocyte injury.

Aloe-emodin (AE) is a natural hydroxyanthraquinone derivative that was found in many medicinal plants and ethnic medicines. AE showed a wide array of pharmacological activities including anticancer, antifungal, laxative, antiviral, and antibacterial effects. However, increasing number of published studies have shown that AE may have some hepatotoxicity effects but the mechanism is not fully understood. Studies have shown that the liver injury induced by some free hydroxyanthraquinone compounds is associated with the inhibition of some metabolic enzymes. In this study, the CYP3A4 and CYP3A1 were found to be the main metabolic enzymes of AE in human and rat liver microsomes respectively. And AE was metabolized by liver microsomes to produce hydroxyl metabolites and rhein. When CYP3A4 was knocked down in L02 and HepaRG cells, the cytotoxicity of AE was increased significantly. Furthermore, AE increased the rates of apoptosis of L02 and HepaRG cells, accompanied by Ca2+ elevation, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) overproduction. The mRNA expression of heme oxygenase-1 in L02 and HepaRG cells increased significantly in the high-dose of AE (40 µmol/L) group, and the mRNA expression of quinone oxidoreductase-1 was activated by AE in all concentrations. Taken together, the inhibition of CYP3A4 enhances the hepatocyte injury of AE. AE can induce mitochondrial injury and the imbalance of oxidative stress of hepatocytes, which results in hepatocyte apoptosis.