Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib

Abstract

1.?Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown.

2.?We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined.

3.?A single-enzyme Michaelis–Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n?=?5) and recombinant CYP2C8 kinetic data (median?±?SD K_i?=?139?±?61?µM and 149?µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median?±?SD K_m?=?6?±?2 versus 11?±?2?µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (K_m?=?4?µM) and single-enzyme weak autoinhibition (K_i?=?449?µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47–75%, compared to 0–30% for CYP3A4 inhibitors.

4.?In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

The inhibitory effects of five alkaloids on the substrate transport mediated through human organic anion and cation transporters

1.?Human solute carrier transporters (SLCs) are important membrane proteins mediate the cellular transport of many endogenous and exogenous substances. Organic anion/cation transporters (OATs/OCTs) and organic anion transporting polypeptides (OATPs) are essential SLCs involved in drug influx. Drug–drug/herb interactions through competing for specific SLCs often lead to unsatisfied therapeutic outcomes and/or unwanted side effects. In this study, we comprehensively investigated the inhibitory effects of five clinically relevant alkaloids (dendrobine, matrine, oxymatrine, tryptanthrin and chelerythrine) on the substrate transport through several OATs/OCTs and OATPs.

2.?We performed transport functional assay and kinetic analysis on the HEK-293 cells over-expressing each SLC gene.

3.?Our data showed tryptanthrin significantly inhibited the transport activity of OAT3 (IC₅₀?=?0.93?±?0.22?μM, K_i?=?0.43?μM); chelerythrine acted as a potent inhibitor to the substrate transport mediated through OATP1A2 (IC₅₀?=?0.63?±?0.43?μM, K_i?=?0.60?μM), OCT1 (IC₅₀?=?13.60?±?2.81?μM) and OCT2 (IC₅₀?=10.80?±?1.16?μM).

4.?Our study suggested tryptanthrin and chelerythrine could potently impact on the drug transport via specific OATs/OCTs. Therefore, the co-administration of these alkaloids with drugs could have clinical consequences due to drug–drug/herb interactions. Precautions should be warranted in the multi-drug therapies involving these alkaloids.     

Effect of natural analogues of trans-resveratrol on cytochromes P4501A2 and 2E1 catalytic activities

The aim was to assess the inhibitory effect of a series of naturally occurring trans-resveratrol analogues on cytochromes P450, namely CYP1A2 and CYP2E1, in vitro in order to analyse any structure–activity relationships. 3,5-Dimethoxy-4′-hydroxy-trans-stilbene (pterostilbene), 3,4′,5-trimethoxy-trans-stilbene (TMS), 3,4′-dihydroxy-5-methoxy-trans-stilbene (3,4′-DH-5-MS) and 3,5-dihydroxy-4′-methoxy-trans-stilbene (3,5-DH-4′-MS) inhibited the activity of CYP1A2, with K_i?=?0.39, 0.79, 0.94 and 1.04?µM, respectively. Piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene) was the least potent inhibitor of CYP1A2 with a K_i?=?9.67?µM. Piceatannol and TMS in the concentration range 1–100?µM did not inhibit CYP2E1 activity. The activity of this enzyme likewise was not significantly influenced by pterostilbene and 3,5-DH-4′-MS with IC₅₀?>?100?µM, whereas 3,4′-DH-5-MS appeared to be a moderately potent, competitive inhibitor of CYP2E1 (K_i?=?42.6?µM). Structure–activity relationship analysis leads to the conclusion that the substitution of hydroxy groups of resveratrol with methoxy groups increases the inhibition of CYP1A2, yet the number and position of methylation are not essential. However, the 4′-hydroxy group in trans-resveratrol and its analogues may play an important role in the interaction with a binding site of CYP2E1.     

In vitro glucuronidation of aprepitant: a moderate inhibitor of UGT2B7

Abstract

1.?Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.

2.?Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%) and UGT1A8 (6%) and K_ms were 161.6?±?15.6, 69.4?±?1.9 and 197.1?±?28.2?µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (r_s?=?0.77, p?<?0.0001 for both substrates; n?=?44), and with the UGT1A3 substrate thyroxine (r_s?=?0.58, p?<?0.0001; n?=?44).

3.?We found aprepitant to be a moderate inhibitor of UGT2B7 with a K_i of ～10?µM for 4-MU, morphine and zidovudine. Our results suggest that aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine.     

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1.?The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.

2.?The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with K_i of 35.95?±?6.96 and 60.56?±?3.53?μmol/l and CYP3A4 (inhibited by genistein with K_i of 23.25?±?5.85?μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (K_i of 57.69?±?2.36?μmol/l) and equol (K_i of 38.47?±?2.32?μmol/l).

3.?Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.     

Inhibition of human liver microsomal cytochrome P450 activities by adefovir and tenofovir

Adefovir (PMEA) and tenofovir (PMPA) and their prodrugs, adefovir dipivoxil (bisPOM-PMEA) and tenofovir disoproxil (bisPOC-PMPA), were subjected to a detailed study of their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The inhibition of marker enzyme activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 was examined with high-performance liquid chromatography (HPLC) or spectroscopic (fluorescence, luminescence) detection. Adefovir and adefovir dipivoxil did not significantly influence activities of most CYP enzymes. The activity of CYP3A4 was inhibited by adefovir dipivoxil at concentrations over 100?µM. Adefovir and its prodrug inhibited CYP2C9 at concentrations below 100?µM; inhibition by adefovir was of the uncompetitive (at the lower inhibitor concentrations) or of the competitive nature with a K_i?=?420?µM. Tenofovir and tenofovir disoproxil influenced the activity of CYP2C9, and competitive inhibition was found with K_i?=?580 and 395?µM, respectively. Tenofovir disoproxil was shown to inhibit microsomal CYP2E1 activities by a mixed-type inhibition with K_i values at about 140?µM. The results indicate the possibility of an influence of the compounds tested on the respective CYP activities when used at high doses.     

Metabolism and related human risk factors for hepatic damage by usnic acid containing nutritional supplements

Usnic acid is a component of nutritional supplements promoted for weight loss that have been associated with liver-related adverse events including mild hepatic toxicity, chemical hepatitis, and liver failure requiring transplant. To determine if metabolism factors might have had a role in defining individual susceptibility to hepatotoxicity, in vitro metabolism studies were undertaken using human plasma, hepatocytes, and liver subcellular fractions. Usnic acid was metabolized to form three monohydroxylated metabolites and two regio-isomeric glucuronide conjugates of the parent drug. Oxidative metabolism was mainly by cytochrome P450 (CYP) 1A2 and glucuronidation was carried out by uridine diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A3. In human hepatocytes, usnic acid at 20 µM was not an inducer of CYP1A2, CYP2B6, or CYP3A4 relative to positive controls omeprazole, phenobarbital, and rifampicin, respectively. Usnic acid was a relatively weak inhibitor of CYP2D6 and a potent inhibitor of CYP2C19 (the concentration eliciting 50% inhibition (IC₅₀)?=?9 nM) and CYP2C9 (IC₅₀?=?94 nM), with less potent inhibition of CYP2C8 (IC₅₀?=?1.9 µM) and CYP2C18 (IC₅₀?=?6.3 µM). Pre-incubation of microsomes with usnic acid did not afford any evidence of time-dependent inhibition of CYP2C19, although evidence of slight time-dependent inhibition of CYP2C9 (K_I?=?2.79 µM and K_inact?=?0.022 min^?1) was obtained. In vitro data were used with SimCYP^Rto model potential drug interactions. Based on usnic acid doses in case reports of 450 mg to >1 g day^?1, these in vitro data indicate that usnic acid has significant potential to interact with other medications. Individual characteristics such as CYP1A induction status, co-administration of CYP1A2 inhibitors, UGT1A1 polymorphisms, and related hyperbilirubinaemias, or co-administration of low therapeutic index CYP2C substrates could work alone or in consort with other idiosyncrasy risk factors to increase the risk of adverse events and/or hepatotoxicity. Thus, usnic acid in nutritional supplements might be involved as both victim and/or perpetrator in clinically significant drug–drug interactions.     

Comparative study of selective in vitro and in silico BACE1 inhibitory potential of glycyrrhizin together with its metabolites, 18α- and 18β-glycyrrhetinic acid,isolated from <Emphasis Type="Italic">Hizikia fusiformis</Emphasis>

Hizikia fusiformis (Harvey) Okamura is a brown seaweed widely used in Korea and Japan, and it contains different therapeutically active constituents. In the present study, we investigated the activities of glycyrrhizin isolated from H. fusiformis, including its metabolites, 18α- and 18β-glycyrrhetinic acid against Alzheimer’s disease (AD) via acetyl and butyrylcholinesterase and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition. Among these three compounds, 18β-glycyrrhetinic acid (IC₅₀?=?8.93?±?0.69 µM) demonstrated two fold potent activity against BACE1 compared to the positive control, quercetin (IC₅₀?=?20.18?±?0.79 µM). Additionally, glycyrrhizin with an IC₅₀ value of 20.12?±?1.87 µM showed similarity to quercetin, while 18α-glycyrrhetinic acid showed moderate activity (IC₅₀?=?104.35?±?2.84 µM). A kinetic study revealed that glycyrrhizin and 18β-glycyrrhetinic acid were non-competitive and competitive inhibitiors of BACE1, demonstrated via K _i values of 16.92 and 10.91 µM, respectively. Molecular docking simulation studies evidently revealed strong binding energy of these compounds for BACE1, indicating their high affinity and capacity for tighter binding to the active site of the enzyme. These data suggest that glycyrrhizin isolated from the edible seaweed, H. fusiformis and its metabolite, 18β-glycyrrhetinic acid demonstrated selective inhibitory activity against BACE1 to alleviate AD.     

Retrospective use of PBPK modelling to understand a clinical drug–drug interaction between dextromethorphan and GSK1034702

1.?In a clinical trial, a strong drug–drug interaction (DDI) was observed between dextromethorphan (DM, the object or victim drug) and GSK1034702 (the precipitant or perpetrator drug), following single and repeat doses. This study determined the inhibition parameters of GSK1034702 in vitro and applied PBPK modelling approaches to simulate the clinical observations and provide mechanistic hypotheses to understand the DDI.

2.?In vitro assays were conducted to determine the inhibition parameters of human CYP2D6 by GSK1034702. PBPK models were populated with the in vitro parameters and DDI simulations conducted and compared to the observed data from a clinical study with DM and GSK1034702.

3.?GSK1034702 was a potent direct and metabolism-dependent inhibitor of human CYP2D6, with inhibition parameters of: IC_50?=_?1.6?μM, K_inact?=?3.7?h^?1 and K_I?=?0.8?μM. Incorporating these data into PBPK models predicted a DDI after repeat, but not single, 5?mg doses of GSK1034702.

4.?The DDI observed with repeat administration of GSK1034702 (5?mg) can be attributed to metabolism-dependent inhibition of CYP2D6. Further, in vitro data were generated and several potential mechanisms proposed to explain the interaction observed following a single dose of GSK1034702.     

Epigallocatechin-3-gallate decreases the transport and metabolism of simvastatin in rats

1.?This study aimed to investigate the potential impact of epigallocatechin-3-gallate (EGCG) on the pharmacokinetic behaviors of simvastatin and its metabolite simvastatin acid and explored the possible role of metabolizing enzymes and transporters of this food–drug interaction.

2.?Female SD rats were intravenously administered with EGCG (5?mg/kg), ketoconazole (10?mg/kg) and rifampin (10?mg/kg), followed by intravenous administration of 2?mg/kg simvastatin. In vitro, the effects of EGCG on Cytochrome P450 enzymes (CYP450) and organic anion transporting polypeptides (OATPs) were studied using human hepatic microsomes and human embryonic kidney 293 (HEK293) cells overexpressing OATP1B1 or OATP1B3. The results showed that areas under concentration–time (AUC) curves of simvastatin and simvastatin acid increased by 2.21- and 1.4-fold while the clearance was reduced by 2.29- and 1.4-fold, respectively, when co-administered with EGCG. In vitro experiments suggested the inhibitory effect of EGCG on CYP enzymes (IC₅₀: 18.37?±?1.36?μM, 26.08?±?1.51?μM for simvastatin and simvastatin acid, respectively). Simvastatin transport by OATP1B1 and OATP1B3 was also inhibited by EGCG (IC₅₀: 8.68?±?1.27?μM and 22.67?±?1.42?μM, respectively).

3.?The presently reported novel food–drug interaction between EGCG and simvastatin involves the inhibition of not only CYP450 enzymes but also OATPs by EGCG.     

Xanthine oxidase/tyrosinase inhibiting,antioxidant, and antifungal oxindole alkaloids from Isatis costata

Phytochemical investigations on the ethyl acetate soluble fraction of the whole plant of Isatis costata Linn. (Brassicaseae) led to the isolation of the oxindole alkaloids costinones A (1), B (2), isatinones A (3), B (4), indirubin (5), and trisindoline (6). Compounds 1–6 displayed significant to moderate inhibition against xanthine oxidase enzyme with IC₅₀ values ranging from 90.3?±?0.06 to 179.6?±?0.04 µM, whereas the standard inhibitor of xanthine oxidase (allopurinol) had an IC₅₀ value of 7.4?±?0.07 µM. Compounds 1 (IC₅₀ 7.21?±?0.05 µM), 2 (IC₅₀ 9.40?±?0.03 µM), 3 (IC₅₀ 11.51?±?0.07 µM), 4 (IC₅₀ 12.53?±?0.06 µM), 5 (IC₅₀ 14.29?±?0.09 µM), and 6 (IC₅₀ 17.34?±?0.04 µM) exhibited pronounced activities when compared with the standard tyrosinase inhibitor l-mimosine (IC₅₀ 3.70?±?0.03 µM), along with DPPH radical scavenging activity with IC₅₀ 226, 270, 300, 320, 401, and 431 µM, respectively. The crude extract and compounds 1, 2, 5, and 6 showed significant antifungal activity against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.     

In vitro inhibition and induction of human cytochrome P450 enzymes by SIPI5357, a potential antidepressant

This study investigated the in vitro drug–drug interaction potential of SIPI5357, an arylalkanol‐piperazine derivative used in the treatment of depression. Drug–drug interaction occurs via inhibition or induction of enzymes involved in their metabolism. In human liver microsomes, SIPI5357 showed the strongest inhibition of CYP2D6, followed by CYP3A4 (testosterone) and CYP2C8. Inhibition was observed in a concentration‐dependent manner, with IC₅₀ values of 18.45 µM, 36.63 µM (CYP3A4/testosterone), 89.23 µM, respectively. SIPI5357 was predicted not to cause significant metabolic drug–drug interaction via inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2E1 or CYP3A4 (midazolam) because the IC₅₀ values for these enzymes were both >100 µM (200 times maximum plasma concentration [C_max]). SIPI5357 showed a mixed model inhibition of CYP2D6 (K_i = 11.12 µM). The value of [I]/K_i for CYP2D6 inhibition by SIPI5357 is below the FDA cut‐off value of 0.1; it is therefore reasonable to assume that SIPI5357 will not cause significant CYP2D6 inhibition. However, positive controls (50 µM omeprazole and 25 µM rifampin) caused the anticipated CYP induction, but the highest concentration of SIPI5357 (5 µM; 10 times plasma C_max) had a minimal effect on CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that SIPI5357 is not an inducer of these enzymes. However, significant induction of CYP2B6 was observed at 0.5 µM and 5 µM. In conclusion, SIPI5357 might cause drug–drug interaction via induction of CYP2B6. The in vivo drug–drug interaction potential deserves further investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug,in human liver microsomes

The metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3′,4′-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent K_m and V_max values of 120.0?±?13.4?µM and 3.2?±?0.1?pmol/min/pmol?CYP, respectively (mean?±?standard deviation). CYP2D6 catalysed the demethylenation with apparent K_m and V_max values of 13.5?±?1.5?µM and 1.3?±?0.1 pmol/min/pmol?CYP, respectively. HLM exhibited a clear biphasic profile with an apparent K_m,1 value of 7.6?±?9.0 and a V_max,1 value of 11.1?±?3.6?pmol/min/mg?protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4′-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3?µM) significantly (p?相似文献   

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1.?The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.

2.?100?μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC₅₀) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00?μM, and the inhibition kinetic parameters (K_i) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11?μM, respectively.

3.?Metabolism of morusin exhibited significant species differences. The quantities of M₁ from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The K_m values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83?μM, and V_max for these species were 0.09, 1.23, 1.43, 0.15, and 0.75?nmol/min/mg, respectively. CL_int (intrinsic clearance) values (V_max/K_m) for morusin obeyed the following order: monkey?>?rat?>?minipig?>?dog?>?human. CL_H (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12?mL/min/kg body weight, respectively.

4.?This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.     

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.

2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (K_m/K_i ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of K_m/K_i ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (K_m/K_i ratio of 8.0 versus 4.0).

3. The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.

4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (K_i?=?0.37?μM; K_m/K_i ratio about 73). Inhibition was not enhanced at parathion concentrations near the K_i by a preincubation step with NADPH.

5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (K_s?=?7.7?μM, ΔA_max?=?2.2?×?10^?2?a.u.?nmol CYP^?1; ΔA_max/K_s 2.86?×?10³?a.u. nmol?CYP^?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.

6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

     

Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes

Abstract

1.?Herbal supplements widely used in the US were screened for the potential to inhibit CYP2C8 activity in human liver microsomes. The herbal extracts screened were garlic, echinacea, saw palmetto, valerian, black cohosh and cranberry. N-desethylamodiaquine (DEAQ) and hydroxypioglitazone metabolite formation were used as indices of CYP2C8 activity.

2.?All herbal extracts showed inhibition of CYP2C8 activity for at least one of three concentrations tested. A volume per dose index (VDI) was calculated to determine the volume in which a dose should be diluted to obtain IC₅₀ equivalent concentration. Cranberry and saw palmetto had a VDI value >5.0?l per dose unit, suggesting a potential for interaction.

3.?Inhibition curves were constructed and the IC₅₀ (mean?±?SE) values were 24.7?±?2.7?μg/ml for cranberry and 15.4?±?1.7?μg/ml for saw palmetto.

4.?The results suggest a potential for cranberry or saw palmetto extracts to inhibit CYP2C8 activity. Clinical studies are needed to evaluate the significance of this interaction.     

The role of human cytochrome P450 enzymes in metabolism of acrylamide in vitro

Abstract

Objective: Acrylamide (AA), a probable human carcinogen, is present in fried and baked starch-rich food. In vivo, the substance is partly biotransformed to glycidamide (GA), which may account for carcinogenic effects. Existing data suggest an important but not exclusive contribution of CYP2E1 to GA formation. The aim of this project was to derive respective enzyme kinetic parameters for CYP2E1 and to assess a possible role of other important human CYPs for this reaction in vitro.

Methods: AA (0.2–20?mM) was incubated with human liver microsomes (HLM) and human cytochrome P450 enzymes (supersomes?). GA was quantified by a specific LC-MS/MS method. Enzyme kinetic parameters were estimated assuming a single binding site. Furthermore, inhibition experiments were performed with diethyldithiocarbamate (DDC), a potent inhibitor of CYP2E1.

Results: The mean?±?SD maximum formation rate (V_max) and Michaelis–Menten constant (K_m) for GA formation in HLM were 199?±?36?pmol GA/mg protein/min and 3.3?±?0.5?mM, respectively. In AA incubations with supersomes?, only for CYP2E1 measurable GA formation was detected in all tested AA concentrations (V_max and K_m were 5.4?nmol GA/nmol CYP2E1/min and 1.3?mM, respectively). Inhibition constant (IC₅₀) of DDC was 3.1?±?0.5?µM for HLM and 1.2?±?0.2?µM for CYP2E1 supersomes?. Therefore, relevant participation of CYPs other than CYP2E1 in the metabolism of AA to GA in humans does not seem likely.

Conclusion: Our results confirm the major role of CYP2E1 in GA formation from AA, albeit with low affinity and low capacity. Further studies are needed to identify other pathways of GA formation.     

Assessment of drug–drug interaction potential and PBPK modeling of CC-223, a potent inhibitor of the mammalian target of rapamycin kinase

1.?CC-223 was studied in vitro for metabolism and drug–drug interactions (DDI), and in clinic for interaction with ketoconazole.

2.?In vitro, human metabolites of CC-223 included O-desmethyl CC-223 (M1), keto (M2), N-oxide (M3) and imine (M13), with M1 being the most prominent metabolite.

3.?CC-223 was metabolized by CYP2C9 and CYP3A, while metabolism of M1 was mediated by CYP2C8 and CYP3A. Ketoconazole increased CC-223 and M1 exposure by 60–70% in healthy volunteers.

4.?CC-223 (IC₅₀?≥?27?µM) and M1 (IC₅₀?≥?46?µM) were inhibitors of CYP2C9 and CYP2C19 in human liver microsomes. CC-223 and M1 were moderate inducers of CYP3A in human hepatocytes.

5.?CC-223 was a substrate of BCRP, and M1 was a substrate of P-gp and BCRP. CC-223 was an inhibitor of P-gp (IC₅₀?=?3.67?µM) and BCRP (IC₅₀?=?11.7?µM), but at a clinically relevant concentration showed no inhibition of other transporters examined. M1 is a weak inhibitor of P-gp and BCRP.

6.?PBPK model of CC-223 and M1 was developed and verified using clinical results. Model based predictions of DDI with ketoconazole were in agreement with observed results enabling prospective predictions of DDIs between CC-223 and CYP3A4 inhibitors.     

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1.?5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.

2.?It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.

3.?The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.

4.?Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, K_I?=?10.8?±?2.14?μM) and testosterone 6β-hydroxylation (CYP3A4, K_I = 9.1?±?2.82?μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.

5.?In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.