Hydroxylation In Human Liver Microsomes Research Articles

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

Modulation of CYP2C9 activity and hydrogen peroxide production by cytochrome b5

Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1′-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.

CYP2C8-mediated interaction between repaglinide and steviol acyl glucuronide: In vitro investigations using rat and human matrices and in vivo pharmacokinetic evaluation in rats

CYP2C8 is involved in the metabolic clearance of several important drugs and recent reports have shown that acyl glucuronides of gemfibrozil and clopidogrel are potent time-dependent inhibitors of CYP2C8 activity. In the present study, the inhibitory effect of steviol acyl glucuronide (SVAG), a circulating metabolite formed after the ingestion of rebaudioside A, was investigated using in vitro and in vivo systems. Results indicated that SVAG was a reversible but not a time-dependent inhibitor of CYP2C8-mediated paclitaxel 6α-hydroxylation. SVAG was also capable of inhibiting CYP2C8-mediated repaglinide 3′-hydroxylation in human liver microsomes and recombinant human CYP2C8, with Ki values of 15.8 μM and 11.6 μM, respectively. In contrast, SVAG did not exhibit inhibitory effect on CYP2C8 activity in rat liver microsomes. In addition, co-administration of rebaudioside A with repaglinide in rats did not lead to AUC and Cmax changes of repaglinide. Although mathematic prediction using a simplified mechanistic model revealed a moderate interaction potential between repaglinide and SVAG, cautions should be given to patients with hypoglycemia if repaglinide and rebaudioside A are used in combination for the blood sugar control.

Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms.

The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO. For all of the P450s tested, hydroxylation of d4IFO was improved and dechloroethylation was reduced as compared with nondeuterated IFO. Although the differences were not statistically significant, the trend favoring the 4'-hydroxylation pathway was noteworthy. CYP3A5 and CYP2C19 were the most efficient enzymes for catalyzing IFO hydroxylation. The importance of these enzymes in IFO metabolism has not been reported previously and warrants further investigation. The catalytic ability of the common polymorphisms of CYP2B6 and CYP2C9 for both reactions were tested with IFO and d4IFO. It was determined that the commonly expressed polymorphisms CYP2B6*4 and CYP2B6*6 had reduced catalytic ability for IFO compared with CYP2B6*1, whereas CYP2B6*7 and CYP2B6*9 had enhanced catalytic ability. As with the wild-type enzymes, d4IFO was more readily hydroxylated by the polymorphic variants than IFO, and d4IFO was not dechloroethylated by any of the polymorphic forms. We also assessed the use of specific inhibitors of P450 to favor hydroxylation in human liver microsomes. We were unable to separate the pathways with these experiments, suggesting that multiple P450s are responsible for catalyzing both metabolic pathways for IFO, which is not observed with the closely related drug cyclophosphamide.

Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction

The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug–drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5±0.3 and kinact value of 0.045±0.001min−1. Phenelzine inactivated CYP2B6 with KI and kinact values of 44.9±6.9μM and 0.085±0.003min−1 respectively. Inactivation of selegiline was characterized with KI and kinact values of 22.0±3.3 and 0.074±0.002min−1 respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index.

Relationship between Differential Hepatic microRNA Expression and Decreased Hepatic Cytochrome P450 3A Activity in Cirrhosis

Background and AimLiver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis.MethodsHepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity.ResultsHepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08).ConclusionThe strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.

Ligand-Based Design of a Potent and Selective Inhibitor of Cytochrome P450 2C19

A series of omeprazole-based analogues was synthesized and assessed for inhibitory activity against CYP2C19. The data was used to build a CYP2C19 inhibition pharmacophore model for the series. The model was employed to design additional analogues with inhibitory potency against CYP2C19. Upon identifying inhibitors of CYP2C19, ligand-based design shifted to attenuating the rapid clearance observed for many of the inhibitors. While most analogues underwent metabolism on their aliphatic side chain, metabolite identification indicated that for analogues such as compound 30 which contain a heterocycle adjacent to the sulfur moiety, metabolism primarily occurred on the benzimidazole moiety. Compound 30 exhibited improved metabolic stability (Cl(int) = 12.4 mL/min/nmol) and was selective in regard to inhibition of CYP2C19-catalyzed (S)-mephenytoin hydroxylation in human liver microsomes. Finally, representative compounds were docked into a homology model of CYP2C19 in an effort to understand the enzyme-ligand interactions that may lead to favorable inhibition or metabolism properties.

Drug Metabolism and Pharmacokinetics of 4-Substituted Methoxybenzoyl-aryl-thiazoles

Tubulins are some of the oldest and most extensively studied therapeutic targets for cancer. Although many tubulin polymerizing and depolymerizing agents are known, the search for improved agents continues. We screened a class of tubulins targeting small molecules and identified 4-(3,4,5-trimethoxybenzoyl)-2-phenyl-thiazole (SMART-H) as our lead compound. SMART-H inhibited the proliferation of a variety of cancer cells in vitro, at subnanomolar IC(50), and in vivo, in nude mice xenografts, with near 100% tumor growth inhibition. Metabolic stability studies with SMART-H in liver microsomes of four species (mouse, rat, dog, and human) revealed half-lives between <5 and 30 min, demonstrating an interspecies variability. The clearance predicted based on in vitro data correlated well with in vivo clearance obtained from mouse, rat, and dog in vivo pharmacokinetic studies. SMART-H underwent four major metabolic processes, including ketone reduction, demethylation, combination of ketone reduction and demethylation, and hydroxylation in human liver microsomes. Metabolite identification studies revealed that the ketone and the methoxy groups of SMART-H were most labile and that ketone reduction was the dominant metabolism reaction in human liver microsomes. We designed and tested four derivatives of SMART-H to improve the metabolic stability. The oxime and hydrazide derivatives, replacing the ketone site, demonstrated a 2- to 3-fold improved half-life in human liver microsomes, indicating that our prediction regarding metabolic stability of SMART-H can be extended by blocking ketone reduction. These studies led us to the next generation of SMART compounds with greater metabolic stability and higher pharmacologic potency.

Metabolism of Ramelteon in Human Liver Microsomes and Correlation with the Effect of Fluvoxamine on Ramelteon Pharmacokinetics

Ramelteon is a melatonin receptor agonist used as a treatment for insomnia. It is subject to a remarkably large drug-drug interaction (DDI) caused by fluvoxamine coadministration, resulting in a more than 100-fold increase in exposure. The objective of this study was to determine whether the DDI could be estimated using in vitro metabolism data. Ramelteon was shown to undergo hydroxylation in human liver microsomes to eight metabolites via six pathways. The main routes of metabolism included hydroxylation on the ethyl side chain and the benzylic position of the cyclopentyl ring, as assessed through enzyme kinetic measurements. Hydroxylation at the other benzylic position was observed in human intestinal microsomes. Ramelteon metabolism was catalyzed by CYP1A2, CYP2C19, and CYP3A4 as shown through the use of recombinant human cytochrome P450 enzymes and specific inhibitors. In liver, CYP1A2, CYP2C19, and CYP3A4 were estimated to contribute 49, 42, and 8.6%, respectively, whereas in intestine only CYP3A4 contributes. The in vitro data were used to estimate the magnitudes of DDI caused by ketoconazole, fluconazole, and fluvoxamine. The DDIs caused by the former were reliably estimated (1.82-fold estimated versus 1.82-fold actual for ketoconazole; 2.99-fold estimated versus 2.36-fold actual for fluconazole), whereas for fluvoxamine it was underestimated (11.4-fold estimated versus 128-fold actual). This suggests that there may be a limit on the magnitude of DDI that can be estimated from in vitro data. Nevertheless, the example of the fluvoxamine-ramelteon DDI offers a unique example wherein one drug can simultaneously inhibit multiple enzymatic pathways of a second drug.

Differential effect of Shenmai injection, a herbal preparation, on the cytochrome P450 3A-mediated 1′-hydroxylation and 4-hydroxylation of midazolam

Shenmai injection ( SMI), one of the most popular herbal preparations, is widely used for the treatment of coronary atherosclerotic cardiopathy and viral myocarditis. The purpose of this study was to investigate the effect of Shenmai injection ( SMI) on the CYP3A-mediated metabolism of midazolam (MDZ). The present study demonstrated that SMI could significantly inhibit MDZ 4-hydroxylation but activate its 1′-hydroxylation in human liver microsomes (HLMs), rat liver microsomes (RLM) and recombinant human CYP3A4 and CYP3A5. The opposing effect of SMI was characterized by the kinetic change of increasing V max/ K m for MDZ 1′-hydroxylation and decreasing V max/ K m for MDZ 4-hydroxylation in HLM and RLM. The presence of SMI enhanced the inhibition of ketoconazole on MDZ 4-hydroxylation but weakened or reversed its inhibition on MDZ 1′-hydroxylation in HLM. After single or multiple pretreatment with SMI, the ratios of AUC 4-OH MDZ/AUC MDZ in rats were significantly decreased, while the ratios of AUC 1′-OH MDZ/AUC MDZ were increased. Among the major components in SMI, total ginsenoside (TG), ophiopogon total saponins (OTS), ophiopogon total flavone (OTF), ginsenoside Rd, ophiopogonin D and ophiopogonone A exhibited significant inhibition on both 4-hydroxylation and 1′-hydroxylation of MDZ in HLM and RLM, while no activation on MDZ metabolism was observed in the presence of these major constituents alone or together. To further explore the responsible components, 3 mL of SMI was loaded on a solid phase extraction (SPE) C 18 cartridge and then separated by different concentrations of methanol. The fractions eluted with 60% and 90% methanol both showed significant activation on MDZ 1′-hydroxylation in HLM, but the fraction eluted with 30% methanol had no such effect. The results indicated that the activation of SMI on MDZ 1′-hydroxylation might be mainly resulted from the lipid-soluble components in SMI.

Role of NADPH-Cytochrome P450 Reductase and Cytochrome-b5/NADH-b5Reductase in Variability of CYP3A Activity in Human Liver Microsomes

NADPH-cytochrome P450 reductase (CPR) and cytochrome-b(5) (b(5)) together with NADH-b(5) reductase (b(5)R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes to the known interindividual variability in CYP3A activity. CPR and b(5) were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (>or=46 years) male donors (n=11) averaged 27% (P=0.034) and 41% (P=0.011) lower CPR levels than young (<or=45 years) male donors (n=21) for spectrophotometric and immunoblot values, respectively. Similarly, HLMs from elderly male donors averaged 43% (P=0.034) and 47% (P=0.011) lower b(5) levels than young male donors for spectrophotometric and immunoblot values, respectively. alpha-Lipoic acid and 6-propyl-2-thiouracil were evaluated for selectivity of inhibition of CPR and b(5)R activities, respectively, using recombinant enzymes and HLMs, as well as for effects on CYP3A-mediated triazolam hydroxylation in HLMs with either NADH or beta-NADPH. The results indicate that both compounds are relatively nonselective inhibitors of CPR and b(5)R activities. Finally, we used multivariate regression analysis and showed that variability in CPR or b(5) expression between HLMs does not contribute significantly to variability in CYP3A-mediated midazolam hydroxylation. Consequently, while aging is associated with decreased CPR and b(5) expression in human livers, this effect does not contribute to CYP3A variability.

Effects of Spice Constituents on P-Glycoprotein-Mediated Transport and CYP3A4-Mediated Metabolism in Vitro

The effects of eight components from six commonly consumed spices on P-glycoprotein (P-gp) transport and CYP3A4 metabolism were evaluated in vitro. P-gp-mediated [(3)H]digoxin fluxes across the L-MDR1 (LLC-PK1 cells transfected with human MDR1 gene) and Caco-2 (human colon carcinoma) cell monolayers showed a marked asymmetry compared with that in the LLC-PK1 (porcine kidney epithelial cells) cell monolayers. Curcumin (from turmeric) at 30 to 60 microM and 6-gingerol (from ginger) at 100 to 500 microM were observed to inhibit P-gp-mediated [(3)H]digoxin transport in L-MDR1 and Caco-2 cells. Effects of spices on midazolam (MDZ) 1'-hydroxylation and 4-hydroxylation of CYP3A4 activity were determined in pooled human liver microsomes (HLM). The following IC(50) values for effects of spices on MDZ 1'-hydroxylation in HLM were obtained: 29 microM for curcumin, 1.17 mM for allyl methyl disulfide (AMD) (from Chinese chive), 1.02 mM for 1,8-cineole (from coriander), and 1.28 mM for beta-caryophyllene (from curry leaf). CYP3A4-mediated 4-hydroxylation of MDZ was inhibited by curcumin at 30, 45, and 60 microM (4-hydroxy-MDZ formation was decreased to 52, 30, and 29%, respectively, compared with control), by 6-gingerol at 60, 100, and 500 microM (71, 68, and 38%), by AMD at 1 and 4 mM (29 and 14%), by d-limonene (from coriander) at 4 mM (65%), by 1,8-cineole at 0.5, 1, and 4 mM (74, 64, and 59%), and by citral (from lemongrass) at 1 mM (59%). Among the spices that showed inhibitory effect on MDZ metabolism in HLM, only AMD showed a preincubation time-dependent inhibitory effect on MDZ metabolism in HLM, suggesting the AMD as an irreversible CYP3A4 inhibitor.

Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1′-hydroxylation

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further.2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1′-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively.3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1′-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1′-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations.4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1′-hydroxylation, CYP3A4/5 play a more important role.

Inhibition of Cytochrome P450 3A4 by a Pyrimidineimidazole: Evidence for Complex Heme Interactions

PH-302 inhibits the inducible form of nitric oxide synthase (iNOS) by coordinating with the heme of the monomeric form and preventing formation of the active dimer. Inherent with the mechanism of pharmacology for this compound was the inhibition of cytochrome P450 3A4 (P450 3A4), observed from early ADME screening. Further investigation showed that PH-302 inhibited P450 3A4 competitively with a Ki of approximately 2.0 microM against both midazolam and testosterone hydroxylation in human liver microsomes. As expected, spectral binding analysis demonstrated that inhibition was a result of type II coordination to the P450 heme with the imidazole moiety of PH-302, although only 72% of the maximal absorbance difference was achievable with PH-302 compared to that of the smaller ligand imidazole. Time-dependent inhibition of P450 3A4 by PH-302 was also observed because of metabolite-inhibitory (MI) complex formation via metabolism of the methylenedioxyphenyl group. The profile for time-dependent inhibition in recombinant P450 3A4 was biphasic, and was kinetically characterized by a kinact of 0.08 min-1 and a Ki of 1.2 microM for the first phase (0-1.5 min) and a kinact of 0.06 min-1 and a Ki of 23.8 microM for the second phase (1.5-10 min). Spectral characterization of the PH-302 MI complex demonstrated that formation began to plateau within 3 min, consistent with the kinetic profile of inactivation by PH-302. Meanwhile, spectral evidence for the imidazole-derived type II difference spectrum of PH-302 was captured simultaneously with the formation of the MI complex. The presence of simultaneously operable type II coordination and rapidly saturable MI complex formation with heme by PH-302 indicates the presence of complex heme interactions with this unique molecule. Information from these mechanistic studies adds to our understanding of the nature of P450 3A4 inhibition by PH-302 and provides a potentially useful tool compound for future studies investigating binding interactions in this important drug-metabolizing enzyme.

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1′‐hydroxylation kinetics in human liver microsomes

The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.

Human Cytochrome P450 Enzymes of Importance for the Bioactivation of Methyleugenol to the Proximate Carcinogen 1‘-Hydroxymethyleugenol

In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1'-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19, and 2D6 are intrinsically able to 1'-hydroxylate methyleugenol. An additional experiment with Gentest microsomes, expressing the same individual enzymes to roughly average liver levels, indicated that P450 1A2, 2C9, 2C19, and 2D6 contribute to methyleugenol 1'-hydroxylation in the human liver. A study, in which correlations between methyleugenol 1'-hydroxylation in human liver microsomes from 15 individuals and the conversion of enzyme specific substrates by the same microsomes were investigated, showed that P450 1A2 and P450 2C9 are important enzymes in the bioactivation of methyleugenol. This was confirmed in an inhibition study in which pooled human liver microsomes were incubated with methyleugenol in the presence and absence of enzyme specific inhibitors. Kinetic studies revealed that at physiologically relevant concentrations of methyleugenol P450 1A2 is the most important enzyme for bioactivation of methyleugenol in the human liver showing an enzyme efficiency (kcat/Km) that is approximately 30, 50, and > 50 times higher than the enzyme efficiencies of, respectively, P450 2C9, 2C19, and 2D6. Only when relatively higher methyleugenol concentrations are present P450 2C9 and P450 2C19 might contribute as well to the bioactivation of methyleugenol in the human liver. A 5-fold difference in activities was found between the 15 human liver microsomes of the correlation study (range, 0.89-4.30 nmol min(-1) nmol P450(-1)). Therefore, interindividual differences might cause variation in sensitivity toward methyleugenol. Especially lifestyle factors such as smoking (induces P450 1A) and the use of barbiturates (induces P450 2C) can increase the susceptibility for adverse effects of methyleugenol.

CYP2C-catalyzed delta(9)-tetrahydrocannabinol metabolism: Kinetics, pharmacogenetics and interaction with phenytoin

delta(9) -Tetrahydrocannabinol (Δ 9-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-Δ 9-THC, which is oxidized further to 11-nor-9-carboxy-Δ 9-THC. The hydroxylation is thought to be mediated by CYP2C9. The present study was designed to address the kinetics and pharmacogenetics of CYP2C-mediated metabolism of (Δ 9)-THC by studying its metabolism in human liver microsomes and expressed enzymes. Expressed CYP2C9.1 exhibited high affinity for the hydroxylation of Δ 9-THC (apparent K m, 2 μM), similar to that observed in human liver microsomes (apparent K m, 0.8 μM). In contrast, the calculated intrinsic clearance (apparent V m/ K m) for CYP2C9.2 and CYP2C9.3 was approximately 30% that of the wild type, CYP2C9.1. Given the high affinity of CYP2C9 for the hydroxylation of Δ 9-THC, we evaluated the potential for an interaction between Δ 9-THC, 11-hydroxy-Δ 9-THC, or 11-nor-9-carboxy-Δ 9-THC and the CYP2C9 substrate, phenytoin. Surprisingly, Δ 9-THC increased the rate of phenytoin hydroxylation in human liver microsomes and expressed CYP2C9 enzyme. Similar increases in rate were observed with co-incubation of 11-hydroxy-Δ 9-THC and 11-nor-9-carboxy-Δ 9-THC with phenytoin. These in vitro data suggest the potential for an interaction from the concomitant administration of Δ 9-THC and phenytoin that could result in decreased phenytoin concentrations in vivo.

ATAZANAVIR: EFFECTS ON P-GLYCOPROTEIN TRANSPORT AND CYP3A METABOLISM IN VITRO

The effect of atazanavir on P-glycoprotein (P-gp) expression and activity, as well as its inhibitory potency against CYP3A activity, was evaluated in vitro. Induction of P-gp activity and expression was studied using LS180V cells. P-gp inhibition was studied using both LS180V cells and Caco-2 cells. P-gp activity was assessed by measuring P-gp-mediated rhodamine 123 (Rh123) transport, and P-gp expression was determined using SDS-polyacrylamide gel electrophoresis/Western blot analysis. CYP3A inhibition was tested using triazolam hydroxylation in human liver microsomes (HLM). Extended (3-day) exposure of LS180V cells to 30 microM atazanavir caused a 2.5-fold increase in immunoreactive P-gp expression as well as a concentration-dependent decrease of intracellular Rh123 to a mean 45% (S.D. 5.2%) of control. Acute exposure (2 h) of LS180V cells to atazanavir increased intracellular Rh123 concentrations up to 300% of control at 100 microM atazanavir. At 30 microM and above, acute atazanavir exposure reversed P-gp induction caused by 3-day pretreatment with 10 microM ritonavir. P-gp inhibition was also observed in Caco-2 cells, causing an effect comparable to that observed for the known P-gp inhibitor verapamil (50% of control). In HLM, atazanavir was an inhibitor of triazolam hydroxylation, with inhibitory potency greatly increased by preincubation. IC50 values with and without preincubation were 0.31 microM (S.D. 0.13) and 5.7 microM (S.D. 4.1), respectively. Thus, atazanavir is an inhibitor and inducer of P-gp as well as a potent inhibitor of CYP3A in vitro, suggesting a potential for atazanavir to cause drug-drug interactions in vivo.

375 HIV-1 protease inhibitor induces growth arrest and apoptosis of human prostate cancer cells in conjunction with blockade of androgen receptor, STAT3, and AKT signaling

Amprenavir (141W94, VX-478, KVX-478) is metabolized primarily by CYP3A4 (cytochrome P450 3A4) in recombinant systems and human liver microsomes (HLM). The effects of ketoconazole, terfenadine, astemizole, rifampicin, methadone, and rifabutin upon amprenavir metabolism were examined in vitro using HLM. Ketoconazole, terfenadine, and astemizole were observed to inhibit amprenavir depletion, consistent with their known specificity for CYP3A4. The HIV protease inhibitors, indinavir, saquinavir, ritonavir, and nelfinavir, were included in incubations containing amprenavir to examine the interactions of HIV protease inhibitors in vitro. The order of amprenavir metabolism inhibition in human liver microsomes was observed to be: ritonavir > indinavir > nelfinavir > saquinavir. The K value for amprenavir-mediated inhibition of testosterone hydroxylation in human liver microsomes was found to be approximately 0.5 μM. Studies suggest that amprenavir inhibits CYP3A4 to a greater extent than saquinavir, and to a much lesser extent than ritonavir. Amprenavir, nelfinavir, and indinavir appear to inhibit CYP3A4 to a moderate extent, suggesting a selected number of coadministration restrictions.