In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

Flavonoid-statin interactions causing myopathy and the possible significance of OATP transport, CYP450 metabolism and mevalonate synthesis.

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are a primary treatment for hyperlipidemic cardiovascular diseases which are a leading global cause of death. Statin therapy is life saving and discontinuation due to adverse events such as myotoxicity may lead to unfavourable outcomes. There is no known mechanism for statin-induced myotoxicity although it is theorized that it is due to inhibition of downstream products of the HMG-CoA pathway. It is known that drug-drug interactions with conventional medicines exacerbate the risk of statin-induced myotoxicity, though little attention has been paid to herb-drug interactions with complementary medicines. Flavonoids are a class of phytochemicals which can be purchased as high dose supplements. There is evidence that flavonoids can raise statin plasma levels, increasing the risk of statin-induced myopathy. This could be due to pharmacokinetic interactions involving hepatic cytochrome 450 (CYP450) metabolism and organic anion transporter (OATP) absorption. There is also the potential for flavonoids to directly and indirectly inhibit HMG-CoA reductase which could contraindicate statin-therapy. This review aims to discuss what is currently known about the potential for high dose flavonoids to interact with the hepatic CYP450 metabolism, OATP uptake of statins or their ability to interact with HMG-CoA reductase. Flavonoids of particular interest will be covered and the difficulties of examining herbal products will be discussed throughout.

Aidi injection altered the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and diethylnitrosamine-induced hepatocellular carcinoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aidi injection (ADI), a traditional chinese medicine preparation, is widely used in combination with chemotherapy for the treatment of various malignant tumors, such as hepatocellular carcinoma (HCC). Studies have shown that changes in cytochrome P450 (CYP450) activity in disease states would affect the metabolism of drugs in vivo, especially liver diseases. However, the changes of Aidi injection on the activities of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC states are still unknown. AIM OF THE STUDY: The cocktail probe drugs method was used to investigate the effects of ADI on the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC rats. MATERIALS AND METHODS: The HCC rats was induced by diethylnitrosamine (DEN). Then, both normal and HCC rats were randomly divided into 2 groups (n = 6). They were given saline or ADI (10 mL/kg/d, i.p) for 2 weeks, respectively. On the fifteenth day, cocktail probe mixing solution, including metoprolol (10 mg/kg), caffeine (1.0 mg/kg), omeprazole (2.0 mg/kg), midazolam (2.0 mg/kg), chlorzoxazone (4.0 mg/kg) and tolbutamide (0.5 mg/kg), was injected into tail vein of all rats in each group. The blood sample was obtained at specified time. After the protein is precipitated, six probe drugs are analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: Compared with control group, the activity of CYP3A2 and CYP2E1 was significantly lower in the ADI group. Compared with the model group, the activities of CYP1A2, CYP3A2, CYP2E1, and CYP2C11 enzymes in the ADI model group were significantly reduced. Additionally, the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 enzymes in model group was significantly lower than control group. CONCLUSIONS: ADI can inhibit a lot of CYP450 enzyme, so it may reduce the dosage of chemotherapeutic drugs to reach the required plasma concentration of chemotherapeutic drugs, which is of great significance for the combination of anti-tumor chemotherapeutic drugs and is worthy of further in-depth study and clinical attention.

Plumbagin and Plumbago indica Differentially Modulated Cytochrome P450 and Transporter Profiles in BeWo and HepG2 Cells.

<b>Background and Objective:</b> The medicinal herb <i>Plumbago indica</i> (PI) and its major constituent plumbagin have reported pharmacological properties but there is a lack of information about their herb-drug interactions. The effects of methanolic (PI-MeOH) and ethanolic (PI-EtOH) crude extracts of PI and plumbagin on the expression of cytochrome P450s (<i>CYP1A2</i>, <i>CYP2E1</i> and <i>CYP3A4</i>) and transporters (<i>ABCC1</i>, <i>ABCG2</i> and <i>SLC22A11</i>) were investigated in BeWo and HepG2 cells. <b>Materials and Methods:</b> BeWo or HepG2 cells were treated with 0.5-5 µM plumbagin or 25-500 µg mL¹ of PI-MeOH or PI-EtOH for 24 hrs. Total RNA was extracted and mRNA expression of CYPs and transporters were determined using RT-qPCR. <b>Results:</b> PI and plumbagin affected mRNA expression differently in the two tested cell types. In BeWo cells, all concentrations of PI-MeOH induced <i>CYP2E1</i>, 100 and 500 µg Ml¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i>, <i>CYP3A4 </i>and <i>ABCG2 </i>and 500 µg mL¹ PI-EtOH induced <i>ABCG2</i> expression. Plumbagin suppressed <i>CYP1A2</i> and induced <i>SLC22A11 </i>expression at the highest concentration, 5 µM. In HepG2 cells, 5 µM plumbagin and 500 µg Ml¹ PI-EtOH suppressed <i>CYP3A4 </i>expression and 500 µg mL¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i> and <i>CYP2E1 </i>expression. <i>ABCC1</i> expression was induced by all treatments while <i>ABCG2</i> and <i>SLC22A11 </i>were induced only by 500 µg mL¹ PI-MeOH and PI-EtOH. <b>Conclusion:</b> The use of PI or plumbagin supplements in large quantities or for long periods should be carefully considered due to the risk of herbal drug interactions via modulated expression of CYPs and transporters.

Evaluation of potential herb-drug interactions based on the effect of Suxiao Jiuxin Pill on CYP450 enzymes and transporters.

ETHNOPHARMACOLOGY RELEVANCE: Suxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters' activity related to drug metabolism is rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. MATERIALS AND METHODS: Human primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. RESULTS: The results showed that the SJP under the concentration of 1000 µg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 µg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 µg/mL, with the IC50 value of 21.9 µg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 µg/mL but did not affect human MDR1. CONCLUSIONS: Based on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.

Evaluation of the effects of notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF (CNS) on the activities of cytochrome P450 enzymes using a cocktail method in rats.

Notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF, namely CNS, are used for the treatment of cardiovascular diseases in clinic. This study developed a cocktail assay involving seven cytochrome P450 (CYP) enzymes to elucidate the effect of NS, SF, and CNS on CYP enzymes and to explore the synergistic effect of CNS in terms of CYP enzymes. Ultra-performance liquid chromatography-MS and reverse-transcription polymerase chain reaction were applied to detect the activities and mRNA expression levels of CYP enzymes. SF exhibited inhibitory effects on CYP1A2, 2B1, 2E1, and 2C11 and induction effects on CYP2C19 and 2D4. NS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C11, 2C19, and 2D4. CNS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C19, and 2D4 and inhibitory effects on CYP3A1 in vivo. Moreover, mRNA expression results were consistent with pharmacokinetic results. Potential herb-drug interactions should be studied closely when SF, NS, or CNS with clinical drugs are metabolized by CYP1A2, 2B1, 2E1, 2C11, 2C19, 2D4, and 3A1. CNS could change the inhibition or induction effects of CYP compared to the NS group, which might be one of the causes for the synergistic effects of the combination of NS and SF.

In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes.

CONTEXT: Peucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy. OBJECTIVE: The interaction between peucedanol and CYP450s was investigated. MATERIALS AND METHODS: Peucedanol (0, 2.5, 5, 10, 25, 50, and 100 µM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro. RESULTS: Peucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC50 values of 6.03, 13.57, and 7.58 µM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a Ki value of 4.07 µM and a competitive inhibitor of CYP1A2 and 2D6 with a Ki values of 3.39 and 6.77 µM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the Ki/Kinact value of 5.44/0.046 min/µM. DISCUSSION AND CONCLUSIONS: In vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.

Metabolite profiling and evaluation of CYP450 interaction potential of 'Trimada'- an Ayurvedic formulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Trimada is well-known polyherbal Ayurvedic formulation used in Indian Traditional medicine since ancient times. It consisted of three inebriant herbs including "Chitraka" (Plumbago zeylanica Linn. Family- Plumabaginaceae), "Musta" (Cyperus rotundus Linn. Family- Cyperaceae) and Vidanga (Embelia ribes Burm. F. Family- Myrsinaceae) in equal ratios as mentioned in Ayurveda. Trimada is traditionally used to increase the functioning of the digestive system and metabolism. Along with these, it also assists in the reduction of cholesterol as well as reduces stomach aches and chest pain. AIM OF THE STUDY: This study is aimed to identify the metabolites present in this polyherbal formulation. Further, the cytotoxicity and interaction potential of the formulation and individual herbs with Cytochrome P450 isozymes (CYP3A4, 2D6, 2C9, 1A2) was evaluated by MTT assay and CYP450 enzyme inhibition. The concentration of heavy metals was also determined. MATERIAL AND METHODS: Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was performed to detect and identify the phytoconstituents in the formulation. Cytotoxicity of the formulation was evaluated by MTT assay. CYP450 enzyme interaction potential of the individual herbs and the Trimada formulation was carried out through CYP-CO assay and fluorometric high throughput screening (HTS) assay for individual isozymes. The content of heavy metal in the formulation was quantified by Atomic Absorption Spectroscopy. RESULTS: Trimada formulation exhibited lower cytotoxicity to human liver carcinoma cell line (HepG2). CYP-CO assay revealed that the interaction potential of individual herbs and Trimada on the liver microsomes was found to be lesser than the standard inhibitor ketoconazole. Individual herbs and Trimada formulation displayed higher IC50 values than the respective standard inhibitors in the fluorimetric assay. UPLC-QTOF-MS analysis showed the presence of a number of active phytoconstituents including sesquiterpenes, phenolic acids, benzoquinones, triterpenes and flavonoids. The heavy metal concentration in the traditional medicinal herbal formulation was found within the approved limit. CONCLUSIONS: This study suggested that the individual herbs and Trimada formulation exhibited low cytotoxicity and contributes insignificant interaction with CYP450 isozymes. So, the formulation is considered to be safe for its therapeutic management without any potential drug interaction involving CYP 450 isozymes.

Application of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography for continuous extraction and separation of bioactive compounds from Citrus limon peel.

Drug discovery from complex mixtures, like Chinese herbs, is challenging and extensive false positives make it difficult to obtain compounds with anti-Alzheimer's activity. In this study, a continuous method comprised of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography was developed for the efficient, scaled-up extraction and separation of six bioactive compounds from Citrus limon peels: neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin. These active compounds were isolated and purified from the raw plant materials by two-dimensional countercurrent chromatography separation via two sets of an n-hexane/n-butanol/methanol/water solvent system: 0.23:1.00:0.25:1.13 and 0.47:1.00:0.38:1.46, v/v/v/v. The compounds were collected in yields of 0.22, 0.25, 0.10, 0.31, 0.29, and 0.28 mg/g, respectively, with purities of 95.79, 96.47, 97.69, 97.22, 98.11, and 98.82%, respectively. Subsequently, a simple and efficient in vitro method was developed for rapidly evaluating the acetylcholinesterase inhibitory activities of six bioactive components. Furthermore, the PC12 cell model and the in vitro metabolism of cytochromes P450 were employed to verify the monomers obtained from the continuous method. The results demonstrated that these six bioactive extracts from the C. limon peels were strong acetylcholinesterase inhibitors.

Quantitative analysis of PAH compounds in DWH crude oil and their effects on Caenorhabditis elegans germ cell apoptosis, associated with CYP450s upregulation.

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC-MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 µg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 µg/mL and 5 µg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.

Lycopene increases metabolic activity of rat liver CYP2B, CYP2D and CYP3A.

BACKGROUND: Lycopene as a naturally occurring carotenoid is a common part of the human diet. Several beneficial properties of lycopene have been identified, with the most studied being anti-cancer and antioxidant activity. However, no evidence of possible drug-drug or drug-food supplement interactions has been found. METHODS: We studied the in vivo effect of lycopene on the selected rat liver cytochromes P450 (CYPs): CYP1A2, CYP2B, CYP2C11, CYP2C6, CYP2D, and CYP3A. Lycopene was administered to rats intragastrically at doses of 4, 20, and 100 mg/kg/day for 10 consecutive days. Total protein content, P450 Content, and metabolic activity of selected CYPs were evaluated in the rat liver microsomal fraction. RESULTS: Increased CYP2B, CYP2D, and CYP3A metabolic activities were observed in animals treated with the lycopene dose of 100 mg/kg/day. The content of CYP3A1 protein was increased by the dose of 100 mg/kg/day and CYP3A2 protein was increased by all administered doses of lycopene. CONCLUSION: The results of our study indicate that lycopene increased the metabolic activity of enzymes that are orthologues to the most clinically important human enzymes involved in xenobiotic metabolism. The risk of pharmacokinetic interactions between lycopene dietary supplements and co-administered drugs should be evaluated.

Dried Leaf Artemisia Annua Improves Bioavailability of Artemisinin via Cytochrome P450 Inhibition and Enhances Artemisinin Efficacy Downstream.

Artemisia annua L. and artemisinin, have been used for millennia to treat malaria. We used human liver microsomes (HLM) and rats to compare hepatic metabolism, tissue distribution, and inflammation attenuation by dried leaves of A. annua (DLA) and pure artemisinin. For HLM assays, extracts, teas, and phytochemicals from DLA were tested and IC50 values for CYP2B6 and CYP3A4 were measured. For tissue distribution studies, artemisinin or DLA was orally delivered to rats, tissues harvested at 1 h, and blood, urine and feces over 8 h; all were analyzed for artemisinin and deoxyartemisinin by GC-MS. For inflammation, rats received an intraperitoneal injection of water or lipopolysaccharide (LPS) and 70 mg/kg oral artemisinin as pure drug or DLA. Serum was collected over 8 h and analyzed by ELISA for TNF-α, IL-6, and IL-10. DLA-delivered artemisinin distributed to tissues in higher concentrations in vivo, but elimination remained mostly unchanged. This seemed to be due to inhibition of first-pass metabolism by DLA phytochemicals, as demonstrated by HLM assays of DLA extracts, teas and phytochemicals. DLA was more effective than artemisinin in males at attenuating proinflammatory cytokine production; the data were less conclusive in females. These results suggest that the oral consumption of artemisinin as DLA enhances the bioavailability and anti-inflammatory potency of artemisinin.

Baihe Zhimu formula attenuates the efficacy of tamoxifen against breast cancer in mice through modulation of CYP450 enzymes.

BACKGROUND: Major depression is an important complication in patients with breast cancer, but is an underrecognized and undertreated condition in this population. The Baihe Zhimu Tang (BZ formula) is a traditional Chinese formula consisting of Lilium brownii var. viridulum Baker (L. brownii) and Anemarrhena asphodeloides (A. asphodeloides) Bunge that is used for the treatment of depression. However, the interaction between tamoxifen and BZ formula is frequently overlooked by traditional and alternative medical doctors. In the present study, the influence of BZ formula on the effectiveness of tamoxifen in breast cancer in mice and the effects of tamoxifen on the antidepressant effect of BZ formula and its major components mangiferin and timosaponin BII in mice were investigated. METHODS: Identification of the major components of BZ formula was performed using fast HPLC-tandem mass spectrometry (HPLC-MS/MS). The main flavonoids and saponins in A. asphodeloides were determined by HPLC-UV and HPLC-ELSD, separately. The antidepressant efficacy of BZ formula was evaluated using a mouse tail-suspension test. The effects of BZ formula on the antineoplastic activity of tamoxifen were performed in a mouse xenograft model of human breast cancer MCF-7 cells. P450 activity was determined using microsomal incubations by HPLC-MS/MS. Measurement of serum concentrations of tamoxifen and its metabolites was used by HPLC-MS/MS. RESULTS: BZ formula attenuated the effectiveness of tamoxifen treatment of breast cancer and reduced the concentrations of endoxifen and 4-OH-tamoxifen in tumor-bearing mice. Of two of the major components of BZ formula, the antidepressant effect of mangiferin, but not timosaponin BII, was significantly inhibited by tamoxifen in mice. BZ formula and its component mangiferin also significantly inhibited CYP450 enzyme activity in rat liver microsomes. CONCLUSION: BZ formula attenuated the effectiveness of tamoxifen in treatment of breast cancer in mice by influencing CYP450 enzymes. The present study laid a foundation for the treatment of patients with breast cancer and depression by BZ formula or other Chinese herbal formulas containing A. asphodeloides.

Epigallocatechin-3-Gallate Reduces Hepatic Oxidative Stress and Lowers CYP-Mediated Bioactivation and Toxicity of Acetaminophen in Rats.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. To investigate the effects of dietary EGCG on oxidative stress and the metabolism and toxicity of acetaminophen in the liver, rats were fed diets with (0.54%) or without EGCG supplementation for four weeks and were then injected intraperitoneally with acetaminophen (1 g/kg). The results showed that EGCG lowered hepatic oxidative stress and cytochrome P450 (CYP) 1A2, 2E1, and 3A, and UDP-glucurosyltransferase activities prior to acetaminophen injection. After acetaminophen challenge, the elevations in plasma alanine aminotransferase activity and histological changes in the liver were ameliorated by EGCG treatment. EGCG reduced acetaminophen-induced apoptosis by lowering the Bax/Bcl2 ratio in the liver. EGCG mildly increased autophagy by increasing the LC3B II/I ratio. Lower hepatic acetaminophen-glutathione and acetaminophen-protein adducts contents were observed after EGCG treatment. EGCG increased glutathione peroxidase and NAD(P)H quinone 1 oxidoreductase activities and reduced organic anion-transporting polypeptides 1a1 expression in the liver after acetaminophen treatment. Our results indicate that EGCG may reduce oxidative stress and lower the metabolism and toxicity of acetaminophen. The reductions in CYP-mediated acetaminophen bioactivation and uptake transporter, as well as enhanced antioxidant enzyme activity, may limit the accumulation of toxic products in the liver and thus lower hepatotoxicity.

Effect of bilberry extract (Vaccinium myrtillus L.) on drug-metabolizing enzymes in rats.

Vaccinium myrtillus L. (bilberry) fruit is a blue-colored berry with a high content of anthocyanins. These bioactive secondary metabolites are considered to play a major role in the health-promoting properties of bilberries. Our in vivo study was designed to assess the possible influence of bilberry extract on drug-metabolizing enzymes (DMEs). Rats were exposed to bilberry extract in drinking water at two concentrations (0.15 and 1.5 g/L). Selected DMEs were determined (mRNA expression and enzymatic activity) after 29 and 58 days in rat liver. In addition, a panel of antioxidant, physiological, biochemical and hematological parameters was studied; these parameters did not demonstrate any impact of bilberry extract on the health status of rats. A significant increase in activity was observed in cytochrome P450 (CYP) 2C11 (131% of control) and CYP2E1 (122% of control) after a 29-day administration, while the consumption of a higher concentration for a longer time led to a mild activity decrease. Slight changes were observed in some other DMEs, but they remained insignificant from a physiological perspective. According to our results, we conclude that the consumption of bilberries as a food supplement should not pose a risk of interacting with co-administered drugs based on their metabolism.

P450 enzymes-based metabolic interactions between monarch drugs and the other constituent herbs: A strategy to explore compatibility mechanism of Sangju-Yin.

BACKGROUND: Herbal compatibility of compound formulas can enhance therapeutic effects or reduce side effects of the monarch drugs, but majority of compatibility mechanisms are still unknown. Sangju-Yin, a well-known Chinese compound formula, is currently used to treat common cold in clinical. PURPOSE: In this study, we proposed a strategy to explore the compatibility mechanism of Sangju-Yin by investigating P450 enzymes-based metabolic interactions between monarch drugs and the other constituent herbs. METHODS: Under the guidance of traditional Chinese medicine theory, the constituent herbs of Sangju-Yin were divided into four groups, including monarch drugs, monarch drugs with addition of minister drugs, monarch drugs with addition of minister and adjuvant drugs, as well as the whole recipe, namely monarch drugs with addition of minister, adjuvant and conductant drugs. Their effects on rats in vivo P450 (CYP1A2, CYP2A3, CYP2C6, CYP2C11 and CYP3A1) activities after oral administration were evaluated using probe drug assay based on LC-MS/MS. Moreover, effects of the four groups of herbs on mRNA expression of P450 enzymes after oral administration, as well as in vitro P450 activities after co-incubation, were investigated to explore the underlying mechanisms. RESULTS: Comparing with monarch drugs, addition of different constituent herbs significantly enhanced CYP1A2 and CYP2C6 activities, and inhibited CYP2A3 and CYP3A1 activities, indicating their possible influences on plasma concentrations of active constituents in the monarch drugs. Mechanism study suggested that these herbs affected P450 activities by transcriptional regulation and/or direct interaction with the enzymes. CONCLUSION: This study clarified the compatibility mechanism of Sangju-Yin from the aspect of P450 enzymes-based metabolic interactions, which would benefit better understanding of the therapeutic basis of Sangju-Yin.

Critical pharmacokinetic and pharmacodynamic drug-herb interactions in rats between warfarin and pomegranate peel or guava leaves extracts.

BACKGROUND: In-depth information of potential drug-herb interactions between warfarin and herbal compounds with suspected anticoagulant blood thinning effects is needed to raise caution of concomitant administration. The current study aimed to investigate the impact of co-administration of pomegranate peel and guava leaves extracts, including their quality markers namely; ellagic acid and quercetin, respectively, on warfarin's in vivo dynamic activity and pharmacokinetic actions, in addition to potential in vitro cytochrome P450 enzymes (CYP) inhibition. METHODS: Influence of mentioned extracts and their key constituents on warfarin pharmacodynamic and kinetic actions and CYP activity were evaluated. The pharmacodynamic interactions were studied in Sprague Dawley rats through prothrombin time (PT) and International Normalized Ratio (INR) measurements, while pharmacokinetic interactions were detected in vivo using a validated HPLC method. Furthermore, potential involvement in CYP inhibition was also investigated in vitro on isolated primary rat hepatocytes. RESULTS: Preparations of pomegranate peel guava leaf extract, ellagic acid and quercetin in combination with warfarin were found to exert further significant increase on PT and INR values (p < 0.01) than when used alone (p < 0.05). Pomegranate peel extract showed insignificant effects on warfarin pharmacokinetics (p > 0.05), however, its constituent, namely, ellagic acid significantly increased warfarin Cmax (p < 0.05). Guava leaves extract and quercetin resulted in significant increase in warfarin Cmax when compared to control (p < 0.01). Furthermore, guava leaves extract showed a significant effect on changing the AUC, CL and Vz. Significant reduction in CYP2C8, 2C9, and 3A4 was seen upon concomitant use of warfarin with ellagic acid, guava leaves and quercetin, unlike pomegranate that insignificantly affected CYP activities. CONCLUSION: All combinations enhanced the anticoagulant activity of warfarin as the results of in vivo and in vitro studies were consistent. The current investigation confirmed serious drug herb interactions between warfarin and pomegranate peel or guava leaf extracts. Such results might conclude a high risk of bleeding from the co-administration of the investigated herbal drugs with warfarin therapy. In addition, the results raise attention to the blood-thinning effects of pomegranate peel and guava leaves when used alone.

Nonclinical pharmacokinetics and in vitro metabolism of H3B-6545, a novel selective ERα covalent antagonist (SERCA).

PURPOSE: H3B-6545, a novel selective estrogen receptor (ER)α covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα, is in clinical development for the treatment of metastatic breast cancer. Preclinical studies were conducted to characterize the pharmacokinetics and metabolism of H3B-6545 in rat and monkeys. METHODS: The clearance and metabolic profiles of H3B-6545 were studied using rat, monkey and human hepatocytes, and reaction phenotyping was done using recombinant human cytochrome P450 enzymes. Blood stability, protein binding, and permeability were also determined in vitro. Pharmacokinetics of H3B-6545 was assessed after both intravenous and oral dosing. A nonclinical PBPK model was developed to assess in vitro-in vivo correlation of clearance. RESULTS: H3B-6545 had a terminal elimination half-life of 2.4 h in rats and 4.0 h in monkeys and showed low to moderate bioavailability, in line with the in vitro permeability assessment. Plasma protein binding was similar across species, at 99.5-99.8%. Nine metabolites of H3B-6545 were identified in hepatocyte incubations, none of which were unique to humans. Formation of glutathione-related conjugate of H3B-6545 was minimal in vitro. H3B-6545, a CYP3A substrate, is expected to be mostly cleared via hepatic phase 1 metabolism. Hepatocyte clearance values were used to adequately model the time-concentration profiles in rat and monkey. CONCLUSIONS: We report on the absorption and metabolic fate and disposition of H3B-6545 in rats and dogs and illustrate that in vitro-in vivo correlation of clearance is possible for targeted covalent inhibitors, provided reactivity is not a predominant mechanism of clearance.

Profiling of hepatic metabolizing enzymes and nuclear receptors in rats with adjuvant arthritis by targeted proteomics.

Inflammatory conditions alter the expression and activity of factors influencing pharmacokinetics, such as metabolizing enzymes. The study examined alterations of hepatic protein levels of cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and nuclear receptors in rats with adjuvant-induced arthritis (AA rats), an inflammatory animal model, by liquid chromatography-tandem mass spectrometry-based targeted proteomics. The protein levels of CYP1A1, CYP1A2, CYP2A1, CYP2A3, CYP2C6, CYP2C12, CYP2D3, CYP2E1, CYP3A9, UGT1A1 and UGT1A2/3 in liver microsomes of AA rats were significantly lower than those in control rats. The protein levels of constitutive androstane receptor (CAR) and retinoid X receptor α (RXRα) in the cytoplasm and nucleus were also significantly decreased, to approximately 60% of the control levels. The decreased protein levels of CYP1A2, CYP2C6, CYP2D3, CYP2E1 and UGT1A1 were potentially associated with downregulation of CAR or RXRα expression in the nucleus.

Inhibitory effects of Danhong Injection and its major constituents on human cytochrome P450 enzymes in vitro.

Danhong Injection (DHI) as a Chinese patent medicine is mainly used to treat ischemic encephalopathy and coronary heart disease in combination with other chemotherapy. However, the information on DHI's potential drug interactions is limited. The goal of this work was to examine the potential P450-mediated metabolism drug interaction arising from DHI and its active components. The results showed that DHI inhibited CYP2C19, CYP2D6, CYP3A4, CYP2E1 and CYP2C9 with IC50 values of 1.26, 1.42, 1.63, 1.10 and 1.67% (v/v), respectively. Danshensu and rosmarinic acid inhibited CYP2E1 and CYP2C9 with IC50 values of 36.63 and 75.76 µm, and 34.42 and 76.89 µm, respectively. Salvianolic acid A and B inhibited CYP2D6, CYP2E1 and CYP2C9 with IC50 values of 33.79, 21.64 and 31.94 µm, and 45.47, 13.52 and 24.15 µm, respectively. The study provides some useful information for safe and effective use of DHI in clinical practice.