Human Cytochrome P450 Isozymes Research Articles

In vitro evaluation of the inhibition potential of echinacoside on human cytochrome P450 isozymes

BackgroundEchinacoside (ECH) possesses a wide range of biological activity. This present study analyzes the effect of ECH on cytochrome P450 isozymes (CYPs) activities of human liver microsomes.MethodsThe effect of ECH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), chlorzoxazone (2E1), S-mephenytoin (2C19), testosterone (3A4), coumarin (2A6), diclofenac (2C9), paclitaxel (2C8), and dextromethorphan (2D6). The IC50 values for CYP1A2, CYP2E1, CYP2C19, and CYP3A4 isoforms were examined to express the strength of inhibition. Further, the inhibition of CYPs was checked for time-dependent or not, and then fitted with competitive or non-competitive inhibition models. The corresponding parameters were also obtained.ResultsECH caused inhibitions on CYP1A2, CYP2E1, CYP2C19 and CYP3A4 enzyme activities in HLMs with IC50 of 21.23, 19.15, 8.70 and 55.42 μM, respectively. The obtained results showed that the inhibition of ECH on CYP3A4 was time-dependent with the KI/Kinact value of 6.63/0.066 min− 1·μM− 1. Moreover, ECH inhibited the activity of CYP1A2 and CYP2E1 via non-competitive manners (Ki = 10.90 μM and Ki = 14.40 μM, respectively), while ECH attenuated the CYP2C19 activity via a competitive manner (Ki = 4.41 μM).ConclusionsThe results of this study indicate that ECH inhibits CYP1A2, CYP2E1, CYP2C19 and CYP3A4 activities in vitro. In vivo and clinical studies are warranted to verify the relevance of these interactions.

Bulbine natalensis (currently Bulbine latifolia) and select bulbine knipholones modulate the activity of AhR, CYP1A2, CYP2B6, and P-gp.

Bulbine natalensis, an emerging medicinal herb on the global market with androgenic properties, is often formulated in dietary supplements that promote perceived sexual enhancement. However, to date, comprehensive safety studies of B. natalensis are lacking, particularly those related to its herb-drug interaction potential. The purpose of this study was to assess the inductive and inhibitory effects of extracts and pure compounds of B. natalensis on human cytochrome P-450 isozymes in vitro. Our findings demonstrated that both water and methanolic extracts of B. natalensis as well as knipholone, bulbine-knipholone, and 6'-O-methylknipholone dose-dependently increased mRNA expression encoded by CYP2B6, CYP1A2, and ABCB1 genes. Functional analyses showed that water (60 to 2.20 µg/mL) and methanolic (30 to 3.75 µg/mL) extracts and knipholones (10 to 0.33 µM) increased CYP2B6 and CYP1A2 activity in a dose-dependent manner. Additionally, water extract (60 µg/mL), methanolic extract (30 µg/mL), and knipholone (10 µM) caused activation of the aryl hydrocarbon receptor up to 11.1 ± 0.7, 8.9 ± 0.6, and 7.1 ± 2.0-fold, respectively. Furthermore, inhibition studies revealed that methanolic extract attenuated the activity of metabolically active CYP1A2 (IC50, 22.6 ± 0.4 µg/mL) and CYP2B6 (IC50, 34.2 ± 6.6 µg/mL) proteins, whereas water extracts had no inhibitory effect on either isoform. These findings suggest that chronic consumption of B. natalensis may affect normal homeostasis of select CYPs with subsequent risks for HDIs when concomitantly ingested with conventional medications that are substrates of CYP2B6 and CYP1A2. However, more in-depth translational studies are required to validate our current findings and their clinical relevance.

Identification of human cytochrome P450 isozymes involved in the oxidative metabolism of carfentanil

Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. In the present study, the human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of carfentanil were investigated. Using UHPLC-HRMS, Michaelis-Menten kinetics of formation for three major metabolites norcarfentanil (M1), pharmaceutical active metabolite 4-[(1-oxopropyl)phenylamino]-1-(2-hydroxyl-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M11), and 4-[(1-oxopropyl)phenylamino]-1-(2-oxo-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M15) were determined. Isozymes catalyzing the formation of the low abundant, highly active metabolite 1-[2-(2-hydroxylphenyl)ethyl]-4-[(1-oxopropyl)phenylamino]-4-piperidinecarboxylic acid methyl ester (M13) were also identified. Selective P450 inhibition studies with pooled human liver microsomes (HLMs) and recombinant CYP isozymes suggested that metabolites M1, M11, and M15 were predominantly formed by isozyme CYP3A5, followed by CYP3A4. Isozymes CYP2C8 and CYP2C9 also made contributions but to a much lesser extent. Highly potent metabolite M13 was predominantly formed by isozyme CYP2C9, followed by CYP2C8. These findings indicate that CYP3A5, CYP3A4, CYP2C8 and CYP2C9 play a major role in the transformation of carfentanil to M1 (norcarfentanil), M11, M13 and M15 through N-dealkylation of piperidine ring, hydroxylation of phenethyl group and ketone formation on phenethyl linker by human liver micrsomes.

Multiple circulating alkaloids and saponins from intravenous Kang-Ai injection inhibit human cytochrome P450 and UDP-glucuronosyltransferase isozymes: potential drug\u2013drug interactions

BackgroundKang-Ai injection is widely used as an adjuvant therapy drug for many cancers, leukopenia, and chronic hepatitis B. Circulating alkaloids and saponins are believed to be responsible for therapeutic effects. However, their pharmacokinetics (PK) and excretion in vivo and the risk of drug–drug interactions (DDI) through inhibiting human cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes remain unclear.MethodsPK and excretion of circulating compounds were investigated in rats using a validated ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS) method. Further, the inhibitory effects of nine major compounds against eleven CYP and UGT isozymes were assayed using well-accepted specific substrate for each enzyme.ResultsAfter dosing, 9 alkaloids were found with Cmax and t1/2 values of 0.17–422.70 μmol/L and 1.78–4.33 h, respectively. Additionally, 28 saponins exhibited considerable systemic exposure with t1/2 values of 0.63–7.22 h, whereas other trace saponins could be negligible or undetected. Besides, over 90% of alkaloids were excreted through hepatobiliary and renal excretion. Likewise, astragalosides and protopanaxatriol (PPT) type ginsenosides also involved in hepatobiliary and/or renal excretion. Protopanaxadiol (PPD) type ginsenosides were mainly excreted to urine. Furthermore, PPD-type ginsenosides were extensively bound (fu-plasma approximately 1%), whereas astragalosides and PPT-type ginsenosides displayed fu-plasma values of 12.35% and 60.23–87.36%, respectively. Moreover, matrine, oxymatrine, astragaloside IV, ginsenoside Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside Rb1 exhibited no inhibition or weak inhibition against several common CYP and UGT enzymes IC50 values between 8.81 and 92.21 μM. Through kinetic modeling, their inhibition mechanisms towards those CYP and UGT isozymes were explored with obtained Ki values. In vitro-in vivo extrapolation showed the inhibition of systemic clearance for CYP or UGT substrates seemed impossible due to [I]/Ki no more than 0.1.ConclusionsWe summarized the PK behaviors, excretion characteristics and protein binding rates of circulating alkaloids, astragalosides and ginsenosides after intravenous Kang-Ai injection. Furthermore, weak inhibition or no inhibition towards these CYP and UGT activities could not trigger harmful DDI when Kang-Ai injection is co-administered with clinical drugs primarily cleared by these CYP or UGT isozymes.

Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data.2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes.3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study.4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.

Safety assessment of Gyejibokryeong-hwan water extract: Study of acute and subacute toxicity, and influence on drug metabolizing enzymes

Ethnopharmacological relevanceGyejibokryeong-hwan is a traditional herbal medicine and is reported to have various pharmacological actions. Despite many reports of previous studies, there is limited scientific evidence concerning its safety and few drug-metabolism profiles to support the continued therapeutic application of Gyejibokryeong-hwan. Aim of the studyThe purpose of the present study was to investigate the acute and subacute toxicity profile of a Gyejibokryeong-hwan water extract (GBHW) in vivo, and its effects on the activities of drug-metabolizing enzymes in vitro. Materials and methodsAcute and subacute toxicity was evaluated by giving GBHW to rats. In a study of acute toxicity, the rats were given GBHW by single oral gavage administration at 0 and 5000 mg/kg. In a study of subacute toxicity, rats were given GBHW by oral gavage at 0, 1000, 2000, and 5000 mg/kg/day daily for 28 days. The activities of the major human microsomal cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) isozymes were investigated using fluorescence- and luminescence-based enzyme assays in vitro, respectively. ResultsGBHW did not cause any mortality in the study of acute toxicity. In the study of subacute toxicity, GBHW at more than 2000 mg/kg/day was observed with minor changes in the absolute and relative organ weight, hematology, serum biochemistry and urinalysis parameters in rats of either sex. However, these changes were not considered to be important toxicologically. GBHW moderately inhibited the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, and UGT1A1. ConclusionsOur present data suggest that GBHW does not cause toxicologically important adverse events at doses up to 2000 mg/kg/day in the 4-week repeated dose toxicity study and provide valuable information concerning its potential to interact with conventional medicine.

Effect of inter-individual variability in human liver cytochrome P450 isozymes on cyclophosphamide-induced micronucleus formation

We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 μg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system.The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.

Inhibitory potential of three Yin-tonification herbal formulas on the activities of human major cytochrome P450 and UDP- glucuronosyltransferases isozymes in vitro

OBJECTIVETo investigate the influence of Yin-tonification herbal formulas Jaeumganghwa-tang (Ziyin Jianghuo Tang, JEGHT), Ssanghwa-tang (Shuanghe Tang, SHT) and Yukmijihwang-tang (Liuwei Di huang Tang, YMJHT) on the activities of human major cytochrome P450 (CYP450s) and UDP-glucuronosyltransferases isozymes (UGTs) in vitro. METHODSThe activities of CYP450s (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) and UGTs (UGT1A1, UGT1A4 and UGT2B7) were assessed using in vitro fluorescence- and luminescence-based enzyme assays, respectively. The effects of herbal formulas on the activities of CYP450s and UGTs were presented as IC50 values. RESULTSJEGHT showed the potent inhibition of the CYP2D6 activity, with weak inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. SHT inhibited the activities of CYP1A2 and CYP2E1, whereas the negligible inhibition of the activities of CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7 through SHT was observed. YMJHT inhibited CYP2E1 activity, with a negligible inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. CONCLUSIONThese findings provide information about the potential interactions between three Yin-tonification herbal formulas and conventional drugs.

An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software.

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes.

Effects of traditional herbal formulae on human CYP450 isozymes.

To assess the effects of traditional herbal formulae Sijunzi Decoction (, Sagunja-tang, SJZD), Siwu Decoction (, Samul-tang, SWD), Bawu Decoction (, Palmul-tang, BWD) and Shiquan Dabu Decoction (, Sipjeondaebo-tang, SDD) on the activities of human cytochrome P450 (CYP450), a drug-metabolizing enzyme. Herbal formula water extracts were filtered and lyophilized after the powder extracts were dissolved in distilled water. The activities of major human CYP450 isozymes (CYP3A4, CYP2C19, CYP2D6 and CYP2E1) were measured using in vitro fluorescence-based enzyme assays. The inhibitory effects of the herbal formulas on the activities of CYP450 were characterized as half maximal inhibition concentration (IC50) values. All the tested herbal formulae inhibited CYP2C19 activity (IC50: SJZD, 83.28 μg/mL; SWD, 235.54 μg/mL; BWD, 166.82 μg/mL; SDD, 178.19 μg/mL); SJZD (IC50 = 196.46 μg/mL), SWD (IC50 = 333.42 μg/mL) and SDD (IC50 = 163.42 μg/mL) inhibited CYP2E1-mediated metabolism; whereas BWD exhibited comparatively weak inhibition of CYP2E1 (IC50 = 501.78 μg/mL). None of the four herbal formulas significantly affected CYP3A4 or CYP2D6. These results suggest that SJZD, SWD, BWD and SDD could potentially inhibit the metabolism of co-administered synthetic drugs whose primary route of elimination is via CYP2C19. In addition, clinically relevant pharmacokinetic interactions could occur when SJZD, SWD or SDD is co-administered with drugs metabolized by CYP2E1. Our findings provide information for the safety and effective clinical use of these four classic herbal formulas.

In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography–high resolution mass spectrometry (LC–HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N-dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N-pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse.

Identification and in silico prediction of metabolites of the model compound, tebufenozide by human CYP3A4 and CYP2C19

The metabolites of tebufenozide, a model compound, formed by the yeast-expressed human CYP3A4 and CYP2C19 were identified to clarify the substrate recognition mechanism of the human cytochrome P450 (CYP) isozymes. We then determined whether tebufenozide metabolites may be predicted in silico. Hydrogen abstraction energies were calculated with the density functional theory method B3LYP/6-31G∗. A docking simulation was performed using FRED software. Several alkyl sites of tebufenozide were hydroxylated by CYP3A4 whereas only one site was modified by CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen abstraction energy estimation, respectively.

Toxicokinetics of lefetamine and derived diphenylethylamine designer drugs-Contribution of human cytochrome P450 isozymes to their main phase I metabolic steps.

Lefetamine was first marketed in the 1940s as an opioid analgesic. Since withdrawal symptoms were observed during treatment, it became a controlled substance. Its N-ethyl and N-iso-propyl derivatives appeared on the illicit drug market in 2008. Metabolism studies for lefetamine and these derivatives showed that N-dealkylation was the initial step for all three substances in rats. The involvement of the ten most important human cytochrome P450 (CYP) isozymes in this N-dealkylation should be studied now. Studies showed the involvement of CYP1A2, CYP2B6, CYP2C19, and CYP3A4 in N-dealkylation of all three compounds and additionally CYP2D6 for lefetamine and NEDPA. All kinetic profiles followed classic Michaelis-Menten kinetics. Using the relative activity factor approach, the following net clearances were calculated: for lefetamine 8% by CYP1A2, 72% by CYP2B6, 2% by CYP2C19, 1% by CYP2D6, and 17% by CYP3A4; for NEDPA 27% by CYP1A2, 30% by CYP2B6, 23% by CYP2C19, 4% by CYP2D6, and 17% by CYP3A4; for NPDPA 18% by CYP1A2, 24% by CYP2B6, 28% by CYP2C19, and 30% by CYP3A4. In addition, calculated data were compared to chemical inhibition studies in human liver microsomes. Due to the involvement of at least four enzymes in the initial metabolic steps, the risk of CYP-related drug-drug or drug-food interactions should be low.

Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations.

Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV–vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the structural diversity of the isozymes. These findings may represent additional mechanisms for the differential inhibitory effects arising from the coincubated AuNPs on the metabolic activities of the hepatic CYP isozymes.

Identification of human cytochrome P450 isozymes involved in the metabolism of naftopidil enantiomers in vitro.

Naftopidil (NAF) is a chiral compound with two enantiomers (R(+)-NAF and S(-)-NAF) and is used as a racemic mixture in clinical practice. This study aims to investigate the metabolism of NAF enantiomers in pooled human liver microsomes (HLMs) and cytochrome P450 isozymes (CYPs) involved in their metabolism. Metabolism studies were conducted in vitro using HLMs. Specific chemical inhibitors and recombinant human CYPs were used to confirm that the CYPs contributed to the metabolism of NAF enantiomers. Three metabolites were found and characterized in the HLMs incubations from R(+)-NAF and S(-)-NAF, respectively. The major metabolic pathways of R(+)-NAF and S(-)-NAF were demethylation and hydroxylation. CYP2C9 and CYP2C19 inhibitors strongly inhibited R(+)-NAF metabolism, and CYP1A2, CYP2C8, CYP2D6 and CYP3A4/5 inhibitors moderately inhibited R(+)-NAF metabolism. CYP2C9 inhibitors strongly inhibited S(-)-NAF metabolism, and CYP2C8, CYP2C19 and CYP3A4/5 inhibitors moderately inhibited S(-)-NAF metabolism. Consistent with the results of chemical inhibitors experiments, recombinant human CYP2C9 and CYP2C19 contributed greatly to R(+)-NAF metabolism, and CYP2C9 contributed greatly to S(-)-NAF metabolism. Both R(+)-NAF and S(-)-NAF are metabolized to three metabolites in HLMs. CYP2C9 plays the most important role in the demethylation and hydroxylation of both NAF enantiomers, CYP2C19 is another major CYP isoform that is involved in R(+)-NAF metabolism.

In vitro metabolism and metabolic effects of ajulemic acid, a synthetic cannabinoid agonist.

Ajulemic acid is a synthetic analog of Δ8-THC-11-oic acid, the terminal metabolite of Δ8-THC. Unlike Δ9-THC, the psychoactive principle of Cannabis, it shows potent anti-inflammatory action and has minimal CNS cannabimimetic activity. Its in vitro metabolism by hepatocytes from rats, dogs, cynomolgus monkeys and humans was studied and the results are reported here. Five metabolites, M1 to M5, were observed in human hepatocyte incubations. One metabolite, M5, a glucuronide, was observed in the chromatogram of canine hepatocyte incubations. In monkey hepatocyte incubations, M5 was observed in the chromatograms of both the 120 and 240 min samples, trace metabolite M1 (side-chain hydroxyl) was observed in the 120 min samples, and trace metabolite M4 (side-chain dehydrogenation) was observed in the 240 min samples. No metabolites were found in the rat hepatocyte incubations. Unchanged amounts of ajulemic acid detected after the 2-h incubation were 103%, 90%, 86%, and 83% for rat, dog, monkey, and human hepatocytes, respectively. Additional studies were done to ascertain if ajulemic acid can inhibit the activities of five principal human cytochrome P450 isozymes; CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5. In contrast to the phytocannabinoids Δ9-THC and CBD, no significant inhibition of cytochrome activity was observed. These data further support the conclusions reached in earlier reports on ajulemic acid's high margin of safety and suggest that it undergoes minimal metabolism and is not likely to interfere with the normal metabolism of drugs or endogenous substances.

In vitro evaluation of the effects of traditional herbal formulas on human Cytochrome P450 isozymes

The aim of this study was to assess influence of Sagunja‐tang (SGT; Sizunji‐tang, Shikunshi‐to), Samul‐tang (SMT; Siwu‐tang, Shimotsu‐to), Palmul‐tang (PMT; Bawu‐tang) and Sipjeondaebo‐tang (SJT; Shiquandabu‐tang, Juzentaiho‐to) on the activities of human cytochrome P450 (CYP450), a drug‐metabolizing enzyme. These popular herbal formulas have been used in Asian countries for the prevention and treatment of various diseases. Herbal formulas usually consist of mixtures of crud drugs, and the chance of herb–drug interactions may increase when they are coadministered with conventional drugs. In this study, we established in vitro fluorescence‐based enzyme assays for major human CYP450 isozymes (CYP3A4, CYP2C19, CYP2D6 and CYP2E1) using Vivid® CYP450 Screening Kits. Among these four CYP450 isozymes, CYP2C19 was inhibited by all the tested herbal formulas. In contrast, SGT, SMT and SJT inhibited CYP2E1‐mediated metabolism; and PMT exhibited comparatively weak inhibition of CYP2E1. However, none of the four herbal formulas significantly affected CYP3A4 or CYP2D6. These results suggest that SGT, SMT, PMT and SJT could potentially inhibit the metabolism of coadministered drugs of which primary route of elimination is via CYP2C19. In addition, clinically relevant pharmacokinetic interactions could occur when SGT, SMT or SJT is coadministered with drugs metabolized by CYP2E1. Overall, our findings provide information for the safe and effective clinical use of these four herbal formulas.

Enzyme kinetic and molecular docking studies for the inhibitions of miltirone on major human cytochrome P450 isozymes

Previous studies have shown that major tanshinones isolated from Danshen (Salvia miltiorrhiza) inhibited human and rat CYP450 enzymes-mediated metabolism of model probe substrates, with potential in causing herb–drug interactions. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC50=1.73μM) and CYP2C9 (IC50=8.61μM), and weak inhibition on CYP2D6 (IC50=30.20μM) and CYP3A4 (IC50=33.88μM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (Ki=1.48μM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with Ki values of 3.17μM, 24.25μM and 35.09μM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of these human CYP450 isoforms, and provided some information on structure–activity relationships for the CYPs inhibition by tanshinones. Taken together, CYPs inhibitions of miltirone were weaker than dihydrotanshinone, but stronger than cryptotanshinone, tanshinone I and tanshinone IIA.

Discovery of a Potent HIV Integrase Inhibitor That Leads to a Prodrug with Significant anti-HIV Activity

Worldwide research efforts in drug discovery involving HIV integrase have produced only one compound, raltegravir, that has been approved for clinical use in HIV/AIDS. As resistance, toxicity and drug-drug interactions are recurring issues with all classes of anti-HIV drugs, the discovery of novel integrase inhibitors remains a significant scientific challenge. We have designed a lead HIV-1 strand transfer (ST) inhibitor (IC(50) 70 nM), strategically assembled on a pyridinone scaffold. A focused structure-activity investigation of this parent compound led to a significantly more potent ST inhibitor, 2 (IC(50) 6 ± 3 nM). Compound 2 exhibits good stability in pooled human liver microsomes. It also displays a notably favorable profile with respect to key human cytochrome P450 (CYP) isozymes and human UDP glucuronosyl transferases (UGTs). The prodrug of inhibitor 2, i.e., compound 10, was found to possess remarkable anti-HIV-1 activity in cell culture (EC(50) 9 ± 4 nM, CC(50) 135 ± 7 μM, therapeutic index = 15,000).

Predictive Models for Cytochrome P450 Isozymes Based on Quantitative High Throughput Screening Data

The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over 17,000 compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 data sets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85, and 0.87 for 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline or recognizing the toxic potential of environmental chemicals.