Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI)

Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC 3.4.14.1) is a novel putative drug target for the treatment of inflammatory diseases. Using 1 as a starting point (IC50>10 microM), we have improved potency by more than 500-fold and successfully identified novel inhibitors of DPPI via screening of a one-bead-two-compounds library of semicarbazide derivatives. Selected compounds were shown to inhibit intracellular DPPI in RBL-2H3 cells. These compounds were further characterized for adverse effects on HepG2 cells (cytotoxicity and viability) and their metabolic stability in rat liver microsomes was estimated. One of the most potent inhibitors, 8 (IC50=31+/-3 nM; Ki=45+/-2 nM, competitive inhibition), is selective for DPPI over other cysteine and serine proteases, has a half-life of 24 min in rat liver microsomes, shows approximately 50% inhibition of intracellular DPPI at 20 microM and is noncytotoxic.     

Synthesis and structure-activity relationships of selective norepinephrine reuptake inhibitors (sNRI) with improved pharmaceutical characteristics

The design synthesis and SAR of a series of chiral ring-constrained norepinephrine reuptake inhibitors with improved physicochemical properties is described. Typical compounds are potent (IC(50)s<10 nM), selective against the other monoamine transporters, weak CYP2D6 inhibitors (IC(50)s>1 microM) and stable to oxidation by human liver microsomes. In addition, the compounds exhibit a favorable polarity profile.     

Identification of novel enzyme-prodrug combinations for use in cytochrome P450-based gene therapy for cancer

Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.     

Characterization of hepatic drug-metabolizing activities of Bama miniature pigs (Sus scrofa domestica): comparison with human enzyme analogs

We used various substrates and selective inhibitors of human cytochrome P450 (CYP) isozymes as probes to study the metabolism of liver microsomes from Chinese Bama miniature pigs. Nifedipine oxidation (NOD) and testosterone 6beta-hydroxylation (6beta-OHT) activities were similar between human liver microsomes and those from Bama miniature pigs. However, compared with those from humans, liver microsomes from Bama miniature pigs showed decreased phenacetin O-deethylation, coumarin 7-hydroxylation, and chlorzoxazone 6-hydroxylation activities, whereas dextromethorphan O-demethylation activity was increased. Ketoconazole selectively inhibited NOD and 6beta-OHT activities in microsomes from Bama pigs, and 8-methoxypsoralen and tranylcypromine inhibited coumarin 7-hydroxylation in pig microsomes. However, furafylline and quinidine failed to selectively inhibit phenacetin O-deethylation and dextromethorphan O-demethylation in microsomes from Bama pigs, whereas chlormethiazole more efficiently inhibited coumarin 7-hydroxylation activity than chlorzoxazone 6-hydroxylation in pig microsomes. Our results suggest that liver microsomes from Chinese Bama miniature pigs are similar to those from humans in regard to metabolism of nifedipine and testosterone (both are probe substrates for human CYP3A4). In addition, chemical inhibitors used as specific probes for human P450 enzymes did not always show the same selectivity toward corresponding enzyme activities in liver microsomes from Bama pigs. However, ketoconazole (a potent inhibitor of human CYP3A4) could be used as a selective inhibitor probe for the NOD and 6beta-OHT activities in liver microsomes from Chinese Bama miniature pigs.     

Use of electrospray ionization liquid chromatography-mass spectrometry to study the role of CYP2D^ in the in vitro metabolism of 5-hydroxytryptamine receptor antagonists

An electrospray ionization liquid chromatographic-mass spectrometric (ESI-LC-MS) method has been developed to study the involvement of the cytochrome P450 isoenzyme CYP2D6 in the in vitro metabolism of the indole containing 5-hydroxytryptamine (5-HT₃) receptor antagonists tropisetron, ondansetron and dolasetron in human liver microsomes. Compounds were eluted using linear gradients of acetonitrile-20 mM ammonium acetate, solvent A, (10:90, v/v) (ph 6.0) and solvent B, (60:40, v/v) (pH 6.0) and a Nucleosil C₄ column. Microsomal incubations were analysed using selected ion monitoring of the molecular ion of parent drug and the molecular ion of hydroxylated metabolites. The involvement of CYP2D6 in drug metabolism was assessed by inhibition studies using quinidine (5 μM), a specific inhibitor of human CYP2D6, as well as by incubating compounds with microsomes prepared from celss transfected with cDNA encoding human CYP2D6. Results showed that the oxidation of all three compounds involved CYP2D6, but only that of tropisetron was inhibited by over 90% in the presence of quinidine. The present method can be applied to pre-clinical compounds, at an early stage of drug discovery, to assess the involvement of CYP2D6 in their metabolism and to screen for those compounds where CYP2D6 is the only isoenzyme implicated in the formation of major metabolites.     

Ontogeny of hepatic microsomal 3-hydroxylation of quinine in Adélie Penguins

Ontogeny of hepatic cytochrome P450 (CYP) content and of quinine 3-hydroxylation, a biomarker of human CYP3A activity, was investigated in Adélie Penguins (Pygoscelis adeliae) by comparing liver microsomes from chicks aged 13-28 days (n=10) with those from adults. The total CYP content in chick microsomes was significantly lower than in adults and correlated with the age of the chicks (r=0.894, P<0.001). Kinetic parameters (mean+/-S.D.) for quinine 3-hydroxylation in chick microsomes were lower than the corresponding values in adult penguins (Km, 122+/-27 vs. 160+/-73 microM; Vmax, 119+/-35 vs. 160+/-72 pmol/min/mg protein) but the differences were not significant, and neither Km nor Vmax correlated with age of the chicks. The pattern of inhibition of quinine 3-hydroxylation by specific human CYP inhibitors suggests that the CYP enzyme mediating quinine 3-hydroxylation in penguin chicks is similar to human CYP3A, but is a different isoform from that in adult penguins. The results imply that Adélie Penguin chicks may have a different susceptibility to environmental pollutants from adult penguins.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.

Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

Some 1,2-diphenylethane derivatives as inhibitors of retinoic acid--metabolising enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50 = 52.1 microM; ketoconazole, 2.8 microM) and the RA-induced enzyme (100 microM, 65.9% inhibition; ketoconazole, 20 microM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

In vitro inhibition of human liver drug metabolizing enzymes by second generation antihistamines

Cetirizine, terfenadine, loratadine, astemizole and mizolastine were compared for their ability to inhibit marker activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and for some glucuronidation isoenzymes in human liver microsomes. The most pronounced effects were observed with terfenadine, astemizole and loratadine which inhibited CYP3A4-mediated testosterone 6beta-hydroxylation (IC50 of 23, 21 and 32 microM, respectively) and CYP2D6-mediated dextromethorphan O-demethylation (IC50 of 18, 36 and 15 microM, respectively). In addition, loratadine markedly inhibited the CYP2C19 marker activity, (S)-mephenytoin 4-hydroxylation (Ki of 0.17 microM). Furthermore, loratadine activated the CYP2C9-catalyzed tolbutamide hydroxylation (ca. 3-fold increase at 30 microM) and inhibited some glucuronidation enzymes. Mizolastine appeared to be a relatively weak and unspecific inhibitor of CYP2E1, CYP2C9, CYP2D6 and CYP3A4 (IC50Ss in the 100 micromolar range). Cetirizine demonstrated no effect on the investigated activities. A comparison of the inhibitory potencies of cetirizine, terfenadine, loratidine, astemizole and mizolastine with their corresponding plasma concentrations in humans suggests that these antihistamines are not likely to interfere with the metabolic clearance of coadministered drugs, with the exception of loratidine, which appears to inhibit CYP2C19 with sufficient potency to warrant additional investigation.     

Optimizing higher throughput methods to assess drug-drug interactions for CYP1A2, CYP2C9, CYP2C19, CYP2D6, rCYP2D6, and CYP3A4 in vitro using a single point IC(50)

Drug-drug interactions involving cytochrome P(450) (CYP) are an important factor in whether a new chemical entity will survive through to the development stage. Therefore, the identification of this potential as early as possible in vitro could save considerable future unnecessary investment. In vitro CYP interaction screening data generated for CYP2C9, CYP2D6, and CYP3A4 were initially analyzed to determine the correlation of IC(50) from 10- and 3-point determinations. A high correlation (r = 0.99) prompted the further assessment of predicting the IC(50) by a single value of percent inhibition at either 10, 3, or 1 microM. Statistical analysis of the initial proprietary compounds showed that there was a strong linear relationship between log IC(50) and percent inhibition at 3 microM, and that it was possible to predict a compound's IC(50) by the percent inhibition value obtained at 3 microM. Additional data for CYP1A2, CYP2C19, and the recombinant CYP2D6 were later obtained and used together with the initial data to demonstrate that a single statistical model could be applicable across different CYPs and different in vitro microsomal systems. Ultimately, the data for all five CYPs and the recombinant CYP2D6 were used to build a statistical model for predicting the IC(50) with a single point. The 95% prediction boundary for the region of interest was about +/- 0.37 on log(10) scale, comparable to the variability of in vitro determinations for positive control IC(50) data. The use of a single inhibitor concentration would enable determination of more IC(50) values on a 96-well plate and result in more economical use of compounds, human liver or expressed enzyme microsomes, substrates, and reagents. This approach would offer the opportunity to increase screening for CYP-mediated drug-drug interactions, which may be important given the challenges provided by the generation of orders of magnitude more new chemical entities in the field of combinatorial chemistry. In addition, the algorithmic approach we propose would obviously be applicable for other in vitro bioactivity and therapeutic target enzyme and receptor screens.     

In vitro metabolism and inductive or inhibitive effect of DL111 on rat cytochrome P4501A enzyme

In vitro metabolism and the inductive or inhibitive effect of DL111, a non-hormonal early pregnancy-terminating agent, toward cytochrome P450 (CYP) enzymes in rat liver microsomes were studied. In vitro metabolism of DL111 was performed in different rat liver microsomes (pretreated with phenobarbital (PB), dexamethasone (Dex), beta-naphthoflavone (BNF), DL111, respectively) and the catalytic abilities of these microsomes for DL111 were compared with control group. DL111 was well metabolized in microsomes pretreated with beta-naphthoflavone and itself. The K(m) and V(max) was 41.76 +/- 3.26 microM and 15.34 +/- 1.03 nM min(-1) mg(-1) protein for beta-naphthoflavone group, 48.17 +/- 6.06 microM and 17.54 +/- 1.79 nM min(-1)mg(-1) protein for DL111 group, 77.81 +/- 4.73 microM and 3.087 +/- 0.202 nM min(-1)mg(-1) protein for control group, respectively. The rats were pretreated intraperitoneally with the same daily dose of DL111 for different days. The DL111-pretreated microsomal enzymatic activities were evaluated by measuring the metabolic abilities for specific substrates of various enzymes. The results showed that DL111 had the same inductive function as beta-naphthoflavone (the specific inducer of CYP1A) toward rat liver microsomes. The inhibitive effect of DL111 on CYP1A was investigated by coincubating DL111 with the specific substrates of CYP1A-ethoxyresorufin or phenacetin in the microsome induced by beta-naphthoflavone, and the inhibitive level was compared with fluvoxamine (Flu), the specific inhibitor of CYP1A. DL111 inhibited significantly the metabolism of phenacetin and ethoxyresorufin with the inhibition constant (K(i)) 6.836 +/- 0.10 and 1.222 +/- 0.230 microM, respectively and its inhibition potential on CYP1A was higher than fluvoxamine.     

Some 1,2-Diphenylethane Derivatives as Inhibitors of Retinoic Acid—Metabolising Enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50=52.1?μM; ketoconazole, 2.8?μM) and the RA-induced enzyme (100?μM, 65.9% inhibition; ketoconazole, 20?μM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.