Inhibitory effects of citrus fruits on cytochrome P450 3A (CYP3A) activity in humans

The capacities of citrus fruits to inhibit midazolam 1'-hydroxylase activity of cytochrome P450 3A (CYP3A) expressed in human liver microsomes were evaluated. Eight citrus fruits such as ama-natsu, banpeiyu, Dekopon, hassaku, hyuga-natsu, completely matured kinkan (Tamatama), takaoka-buntan and unshu-mikan were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and grapefruit juice (white, Tropicana-Kirin). The addition of a fruit juice prepared from banpeiyu, hassaku, takaoka-buntan or Tamatama caused the inhibition of the microsomal CYP3A activity. The inhibition depended on the amount of a fruit juice added to the incubation mixture (2.5 and 5.0%, v/v). The fruit juice from banpeiyu showed the most potent inhibition of CYP3A. The addition of a banpeiyu juice (5.0%, v/v) resulted in the inhibition of midazolam 1'-hydroxylase activity to about 20% of control without a fruit juice. The elongation of the preincubation period of a fruit juice from banpeiyu (5.0%, v/v) with the microsomal fraction (5 to 15 min) led to the enhancement of the CYP3A inhibition (5% of control). Thus, we discovered ingredients of banpeiyu to be inhibitor(s) or mechanism-based inhibitor(s) of human CYP3A activity, but the inhibitory effects of them were somewhat lower than those of grapefruit.     

Inhibitory effects of fruit juices on CYP3A activity.

There have been very limited reports on the effects of commercial fruit juices on human CYP3A activity. Therefore, the inhibitory effects of readily available commercial fruit juices on midazolam 1'-hydroxylase activity, a marker of CYP3A, were evaluated in pooled human liver microsomes. The fruit juices investigated were black raspberry, black mulberry, plum, and wild grape. White grapefruit, pomegranate, and orange juice were used as positive and negative controls. The black mulberry juice showed the most potent inhibition of CYP3A except for grapefruit juice. The inhibition depended on the amount of a fruit juice added to the incubation mixture. The inhibitory potential of human CYP3A was in the order: grapefruit > black mulberry > wild grape > pomegranate > black raspberry. The IC(50) values of all fruit juices tested were reduced after preincubation with microsomes in the presence of the NADPH-generating system, suggesting that a mechanism-based inhibitory component was present in these fruit juices, as in the case of grapefruit. The results suggest that, like grapefruit juice, commercial fruit juices also have the potential to inhibit CYP3A-catalzyed midazolam 1'-hydroxylation. Therefore, in vivo studies investigating the interactions between fruit juices such as black mulberry and wild grape and CYP3A substrates are necessary to determine whether inhibition of CYP3A activity by fruit juices is clinically relevant.     

Inhibition of human liver cytochrome P450 by star fruit juice.

PURPOSE: To examine the inhibitory effects of star fruit (Averrhoa carambola) juice towards seven major cytochrome P450 (CYP) isoforms and NADPH-cytochrome P450 reductase (CPR). METHODS: The inhibitory effects of star fruit juice (0.5 to 5%, v/v) against the activities of seven CYP isoforms including CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4 and CPR were examined in human liver microsomes. To identify time-dependent inhibition, star fruit juice (2.5%, v/v) was preincubated with microsomes and a NADPH-generating system for 0-15 min, and then the extent of inhibition towards seven CYP isoforms were examined. RESULTS: Star fruit juice (5.0%, v/v) was found to inhibit all the activities of CYP isoforms tested by more than 70%. Based on the half inhibition values (%, v/v), the inhibitory effects towards different CYP isoforms were in the following order: CYP2A6 (0.9) > CYP1A2 (1.4) > CYP2D6 (1.6) > CYP2E1 (2.0) > CYP2C8 (2.2) > CYP2C9 (3.0) > CYP3A4 (3.2). Time-dependent inhibition was not observed towards any of the tested CYP isoforms. In addition, star fruit juice was found not to inhibit the activity of CPR. CONCLUSIONS: Star fruit juice inhibited the seven CYP isoforms tested, with the strongest inhibitory effect against CYP2A6 and the least towards CYP3A4.     

Transient inhibition of cyp3a in rats by star fruit juice.

Star fruit juice is a potent in vitro inhibitor of CYP3A; however, few reports are available on the inhibition of CYP3A activities by star fruit juice in vivo. Therefore, in this study, we investigated the CYP3A-mediated star fruit-drug interaction in vivo. The effect of star fruit juice on carbamazepine pharmacokinetics was examined in rats. In comparison with water, the area under the concentration-time curve (AUC) of carbamazepine was approximately 1.3-fold greater when star fruit juice (2 ml) was orally administered 1 h before the oral administration of carbamazepine (50 mg/kg). In contrast, the elimination half-life of carbamazepine and the AUC ratio of carbamazepine 10,11-epoxide to carbamazepine were not altered by the administration of star fruit juice. These results suggest that star fruit juice impairs the function of enteric CYP3A, but not of hepatic CYP3A. In addition, we evaluated the time course of recovery of CYP3A activity that was reduced after the treatment with star fruit juice. The inhibition by star fruit juice was recovered within approximately 24 h. These data suggest that the effect of star fruit juice is mainly reversible and transient. Thus, we discovered that star fruit juice alters the carbamazepine pharmacokinetics in rats.     

Pomegranate juice does not impair clearance of oral or intravenous midazolam, a probe for cytochrome P450-3A activity: comparison with grapefruit juice

The effect of pomegranate juice (PJ) or grapefruit juice (GFJ) on CYP3A activity was studied in vitro and in healthy human volunteers. In human liver microsomes, the mean 50% inhibitory concentrations (IC(50)) for PJ and GFJ versus CYP3A (triazolam alpha-hydroxylation) were 0.61% and 0.55%, (v/v) respectively, without preincubation of inhibitor with microsomes. After preincubation, the IC(50) for PJ increased to 0.97% (P < .05), whereas the IC(50) for GFJ decreased to 0.41% (P < .05), suggesting mechanism-based inhibition by GFJ but not PJ. Pretreatment of volunteer subjects (n = 13) with PJ (8 oz) did not alter the elimination half-life, volume of distribution, or clearance of intravenous midazolam (2 mg). Administration of PJ also did not affect C(max), total area under the curve (AUC), or clearance of oral midazolam (6 mg). However, GFJ (8 oz) increased midazolam C(max) and AUC by a factor of 1.3 and 1.5, respectively, and reduced oral clearance to 72% of control values. Thus, PJ does not alter clearance of intravenous or oral midazolam, whereas GFJ impairs clearance and elevates plasma levels of oral midazolam.     

Mechanism-based inhibition of recombinant human cytochrome P450 3A4 by tomato juice extract

This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6β-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6β-hydroxylation activity by 19.3-26.2% with 0-min preincubation. Residual activity was strongly reduced by 69.9-83.5% at 20-min preincubation, a reduction similar to that of grapefruit juice extract, known to contain constituents of mechanism-based inhibitors. One juice extract (tomato juice C) showed irreversible dose- and preincubation time-dependent and partial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP3A4 activity. Furthermore, we examined whether the CYP3A4 inhibitory effect of tomato juice was substrate dependent by examining midazolam 1'-hydroxylation activity and nifedipine oxidation activity, in addition to testosterone 6β-hydroxylation activity. Tomato juice showed a potent inhibitory effect on nifedipine oxidation activity, which was comparable to that on testosterone 6β-hydroxylation activity; however, it showed a weak inhibitory effect on midazolam 1'-hydroxylation activity. We conclude that tomato juice contains one or more mechanism-based and competitive inhibitor(s) of CYP3A4. Additionally, significant CYP3A4 inhibitory activity did not result from lycopene, a major compound in tomato. Although the active compound was uncertain, a strong CYP3A4 inhibitory activity was observed in other solanaceous plants, i.e., potato, eggplant, sweet pepper, and capsicum. Therefore, responsible compounds in tomato are likely commonly shared among solanaceous vegetables.     

Effects of pomegranate juice on human cytochrome p450 3A (CYP3A) and carbamazepine pharmacokinetics in rats.

In this study, we investigated whether components of pomegranate could inhibit CYP3A-mediated drug metabolism. The ability of pomegranate to inhibit the carbamazepine 10,11-epoxidase activity of CYP3A was examined using human liver microsomes, and pomegranate juice was shown to be a potent inhibitor of human CYP3A. Addition of 25 microl (5.0% v/v) of pomegranate juice resulted in almost complete inhibition of the carbamazepine 10,11-epoxidase activity of human CYP3A (1.8%). The inhibition potency of pomegranate juice was similar to that of grapefruit juice. In addition, we investigated the in vivo interaction between pomegranate juice and carbamazepine pharmacokinetics using rats. In comparison with water, the area under the concentration-time curve (AUC) of carbamazepine was approximately 1.5-fold higher when pomegranate juice (2 ml) was orally injected 1 h before the oral administration of the carbamazepine (50 mg/kg). On the other hand, the elimination half-life of carbamazepine and the AUC ratio of carbamazepine 10,11-epoxide to carbamazepine were not altered by the injection of pomegranate juice. These data suggest that pomegranate juice component(s) impairs the function of enteric but not hepatic CYP3A. Thus, we discovered that a component(s) of pomegranate inhibits the human CYP3A-mediated metabolism of carbamazepine. Furthermore, pomegranate juice alters the carbamazepine pharmacokinetics in rats.     

Role of furanocoumarin derivatives on grapefruit juice-mediated inhibition of human CYP3A activity.

With juices of grapefruit and related fruits, possible relationships between contents of six different furanocoumarins and extents of inhibition of microsomal CYP3A activity have been studied in vitro. Microsomal CYP3A-mediated testosterone 6beta-hydroxylation was inhibited by the addition of a fruit juice (2.5%, v/v) from eight different grapefruit sources, two sweeties, three pomelos, and one sour orange, whereas no clear inhibition was observed with two sweet orange juices. The inhibitory component in grapefruit juice resides mainly in the precipitate rather than in the supernatant after centrifugation. Higher amounts of (R)-6',7'-dihydroxybergamottin (DHB) were distributed in the supernatant, whereas GF-I-1, GF-I-2, GF-I-4, and the newly isolated GF-I-5 and GF-I-6 were detected predominantly in the precipitate. Mixing of five representative furanocoumarins at their detectable levels in grapefruit juice reproduced roughly the inhibitory potencies of grapefruit juice, but omission of any of the components resulted in decreased potencies. These results suggested that all the major furanocoumarins contributed to the CYP3A inhibitory properties of grapefruit juice. Furthermore, all six furanocoumarins showed stronger CYP3A inhibitory potencies after preincubation in the presence of NADPH, suggesting that both competitive and mechanism-based inhibition occur in a grapefruit juices-drug interaction.     

Urinary metabolic markers reflect on hepatic,not intestinal,CYP3A activity in healthy subjects

Intestinal cytochrome P450 3A (CYP3A) plays an important role in oral drug metabolism, but only endogenous metabolic markers for measuring hepatic CYP3A activity were identified. Our study evaluated whether hepatic CYP3A markers reflected intestinal CYP3A activity. An open-label, three-period, six-treatment, one-sequence clinical trial was performed in 16 healthy Korean males. In the control phase, all subjects received a single dose of intravenous (IV) and oral midazolam (1 mg and 5 mg, respectively). Clarithromycin (500 mg) was administered twice daily for 4 days to inhibit hepatic and intestinal CYP3A, and 500 mL of grapefruit juice was given to inhibit intestinal CYP3A. Clarithromycin significantly inhibited total CYP3A activity, and the clearance of IV and apparent clearance of oral midazolam decreased by 0.15- and 0.32-fold, respectively. Grapefruit juice only reduced the apparent clearance of oral midazolam by 0.84-fold, which indicates a slight inhibition of intestinal CYP3A activity. Urinary markers, including 6β–OH–cortisol/cortisol and 6β–OH–cortisone/cortisone, were significantly decreased 0.5-fold after clarithromycin administration but not after grapefruit juice. The fold changes in 6β–OH–cortisol/cortisol and 6β–OH–cortisone/cortisone did not correlate to changes in intestinal availability but did correlate to hepatic availability. In conclusion, endogenous metabolic markers are only useful to measure hepatic, but not intestinal, CYP3A activity.     

Assessment of oral midazolam limited sampling strategies to predict area under the concentration time curve (AUC) during cytochrome P450 (CYP) 3A baseline, inhibition and induction or activation

A previous study reported a 2- and 3-timepoint limited sampling strategy (LSS) model accurately predicted oral midazolam area under the concentration time curve (AUC), and thus cytochrome P450 (CYP) 3A activity. Objective: This study evaluated whether the LSS models predict midazolam AUC during CYP3A baseline, inhibition and induction/activation. Materials and methods: Plasma midazolam concentrations from 106 healthy adults from 6 published studies were obtained where oral midazolam was co-administered alone or with ketoconazole, double-strength grapefruit juice, Ginkgo biloba extract, pleconaril, or rifampin. Observed and predicted midazolam AUCs were determined. Bias and precision of the LSS models were determined. Results: Contrasting results were observed for the 2- and 3-timepoint LSS models in accurately predicting midazolam AUC during baseline CYP3A conditions. With the exception of 1 study (single dose, double-strength grapefruit juice), the 2- and 3-timepoint LSS models did not accurately predict midazolam AUC during conditions of CYP3A inhibition and induction/activation. Conclusion: The previously reported 2- and 3-timepoint oral midazolam LSS models are not applicable to the evaluated conditions of CYP3A baseline, inhibition, and induction/ activation.     

In vitro and in vivo CYP3A64 induction and inhibition studies in rhesus monkeys: a preclinical approach for CYP3A-mediated drug interaction studies.

In this study, induction and inhibition of rhesus monkey CYP3A64 versus human CYP3A4 were characterized in vitro, and the corresponding pharmacokinetic consequences were evaluated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced CYP3A64 mRNA (EC50 = 0.5 microM; Emax = 6-fold) and midazolam (MDZ) 1'-hydroxylase activity (EC50 = 0.2 microM; Emax = 2-fold). Compound A (N-[2(R)-hydroxy-1(S)-indanyl-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-b]pyridin-5-yl)-methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethylpentanamide), a known potent and mechanism-based inhibitor of CYP3A4, strongly inhibited the formation of 1'-hydroxy MDZ by recombinant CYP3A64 in a concentration- and time-dependent manner (KI = 0.25 microM; k(inact) = 0.4 min(-1)). Similar corresponding results also were obtained with human CYP3A4 in the presence of rifampin or compound A. In rhesus monkeys, MDZ exhibited a relatively high metabolic clearance (primarily via 1'-hydroxylation followed by glucuronidation) and a low hepatic availability (Fh = 16%). Consistent with the induction of hepatic metabolism of a high-clearance compound, pretreatment with rifampin (18 mg/kg p.o. for 5 days) did not significantly affect the i.v. kinetics of MDZ, but caused a pronounced reduction (approximately 10-fold) in the systemic exposure to MDZ and, consequently, its Fh following intrahepatic portal vein administration (i.pv.) of MDZ. A comparable extent of the pharmacokinetic interaction also was obtained after a 1.8 mg/kg rifampin dose. Also consistent with the in vitro CYP3A64 inhibition finding, compound A (6 mg/kg i.v.) markedly increased (10-fold) the i.pv. administered MDZ exposure. At the doses studied, plasma concentrations of rifampin or compound A reached or exceeded their respective in vitro EC50 or KI values. These findings suggest the potential applicability of the in vitro-in vivo relationship approach in rhesus monkeys for studying CYP3A-mediated interactions in humans.     

Drug interactions with calcium channel blockers: possible involvement of metabolite-intermediate complexation with CYP3A.

The inhibitory effects of six commonly used calcium channel blockers on three major cytochrome P-450 activities were examined and characterized in human liver microsomes. All six compounds reversibly inhibited CYP2D6 (bufuralol 1'-hydroxylation) and CYP2C9 (tolbutamide methyl hydroxylation) activities. The IC(50) values for the inhibition of CYP2D6 and CYP2C9 for nicardipine were 3 to 9 microM, whereas those for all others ranged from 14 to >150 microM. Except for nifedipine, all calcium channel blockers showed increased inhibitory potency toward CYP3A activities (testosterone 6beta-hydroxylation and midazolam 1'-hydroxylation) after 30-min preincubation with NADPH. IC(50) values for the inhibition of testosterone 6beta-hydroxylase obtained in the NADPH-preincubation experiment for nicardipine (1 microM), verapamil (2 microM), and diltiazem (5 microM) were within 10-fold, whereas those for amlodipine (5 microM) and felodipine (13 microM) were >200-fold of their respective plasma concentrations reported after therapeutic doses. Similar results also were obtained based on midazolam 1'-hydroxylase activity. Unlike the observations with mibefradil, a potent irreversible inhibitor of CYP3A, the NADPH-dependent inhibition of CYP3A activity by nicardipine and verapamil was completely reversible on dialysis, whereas that by diltiazem was partially restored (80%). Additional experiments revealed that nicardipine, verapamil, and diltiazem formed cytochrome P-450-iron (II)-metabolite complex in both human liver microsomes and recombinant CYP3A4. Nicardipine yielded a higher extent of complex formation ( approximately 30% at 100 microM), and was a much faster-acting inhibitor (maximal inhibition rate constant approximately 2 min(-1)) as compared with verapamil and diltiazem. These present findings that the CYP3A inhibition caused by nicardipine, verapamil, and diltiazem is, at least in part, quasi-irreversible provide a rational basis for pharmacokinetically significant interactions reported when they were coadministered with agents that are cleared primarily by CYP3A-mediated pathways.     

Biotransformation of alprazolam by members of the human cytochrome P4503A subfamily.

1. To aid in the prediction of drug interactions with alprazolam, the human CYP involved in the 1'- and 4-hydroxylation of alprazolam were characterized using human liver microsomes, expressed enzymes and selective chemical inhibitors. 2. The formation of 4-hydroxyalprazolam and 1'-hydroxyalprazolam at an alprazolam concentration of 62.5 microM were reduced by the prototypic CYP3A inhibitor, troleandomycin (50 microM), by 97 and 9900 respectively. Only microsomes from B-lymphoblastoid cells expressing CYP3A4 were capable of catalysing the 1'- and 4-hydroxylation of alprazolam. 3. The formation rates of 1'-hydroxyalprazolam and 4-hydroxyalprazolam at an alprazolam concentration of 1 mM were significantly correlated (n = 19, r = 0.95, p<0.01) indicating that the same enzyme(s) mediated these biotransformations. A significant (p<0.01) correlation was observed between alprazolam 4- and 1'-hydroxylase activity and CYP3A-mediated midazolam 4-hydroxylase, midazolam 1'-hydroxylase, dextromethorphan N-demethylase and erythromycin N-demethylase activities. 4. In conclusion, in adult human liver the CYP3A subfamily members are the principal enzymes involved in the 1'- and 4-hydroxylation of alprazolam. Thus, clinically significant drug drug interactions between alprazolam and other CYP3A substrates are to be expected.     

Metabolic characterization of the major human small intestinal cytochrome p450s.

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. CYP3A4 is the major form of CYP expressed in enterocytes and CYP2C is also expressed at a significant level. In this study, we further characterized the expression of CYP3A4 and CYP2C in human enterocytes and their interindividual variations by examining the metabolic activities from 10 individuals. CYP3A4 in human jejunum microsomes, as determined by 6beta-testosterone hydroxylase activity, varied from 0.36 to 2.46 nmol/min/mg. The apparent average K(m) and V(max) values from two representative individuals were 54 microM and 3.2 nmol/min/mg, respectively. CYP2C9 and CYP2C19 in human jejunum microsomes, as determined by diclofenac 4'-hydroxylase and mephenytoin 4'-hydroxylase activities, varied over an 18-fold range (7.3-129 pmol/min/mg) and 17-fold range (0.8-13.1 pmol/min/mg), respectively. The mean apparent K(m) for diclofenac 4'-hydroxylase was 9.9 microM , whereas the apparent mean K(m) for S-mephenytoin 4'-hydroxylase was 79.3 microM . The mean intrinsic clearance (V(max)/K(m)) was approximately 130-fold greater for diclofenac 4'-hydroxylase than for mephenytoin 4'-hydroxylase. The metabolic activities of CYP2C9 and CYP2C19 were confirmed by inhibition by sulfaphenazole for CYP2C9 and ticlopidine for CYP2C19. In addition, CYP2C9 activities did not correlate with CYP3A4 activities, while CYP2C19 activities had a significant but poor correlation with those of CYP3A4. Thus the major CYP activities in human enterocytes have large interindividual variabilities that are not strongly related.     

Use of midazolam urinary metabolic ratios for cytochrome P450 3A (CYP3A) phenotyping.

Midazolam (MDZ) total clearance (ClT) is widely used for cytochrome P450 3A (CYP3A) phenotyping, but requires up to eight blood samples. This study was conducted to compare the use of midazolam ClT to use of a midazolam urinary metabolic ratio for CYP3A phenotyping. Ten male and 10 female subjects received i.v. midazolam 0.025 mg/kg eight times over a 4-month period at approximately 2-week intervals. The first six phenotyping measures were used to estimate baseline CYP3A activity, then subjects received the moderate CYP3A inhibitor fluvoxamine 150 mg/day for the last 4 weeks (two phenotyping visits) of the study. Serial blood samples were obtained for calculation of ClT. Urine was collected for 6 h following each midazolam dose. Midazolam, 1'-hydroxymidazolam (1-OHMDZ), and 4-hydroxymidazolam were measured in plasma and urine by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Analysis of 148 samples from 20 subjects revealed a weak overall correlation between the urinary ratio of 1-OHMDZ/MDZ to midazolam ClT of r(s) = 0.372 (P = 0.0001). There was no correlation when examining either baseline samples or fluvoxamine-inhibited samples alone (r(s) = 0.101, P = 0.289 and r(s) = 0.266, P = 0.123, respectively). The median (range) urinary ratio decreased significantly with fluvoxamine [219 (141-409) versus 127 (50-464); P = 0.005] and to a similar extent to the midazolam ClT (-33.6% versus -42.4%, respectively; P > 0.05). Median urinary recovery of the i.v. midazolam dose varied between 1.4% and 53% and was significantly lower in samples collected while patients were receiving fluvoxamine (34.3% versus 23.1%; P= 0.0004). Based on these results, although this midazolam urinary ratio was not very reflective of baseline CYP3A activity, it may be a useful indicator of CYP3A inhibition.     

Diazinon is activated by CYP2C19 in human liver.

Phosphorothioate compounds are used throughout the world as agricultural and domestic pesticides. Here, the activation of the phosphorothioate diazinon to diazoxon in human liver is described. In an initial study using three human liver microsomal samples, K(m) for diazoxon formation varied markedly (31, 208, and 660 microM; V(max) 1125, 685, and 1028 pmol/min/mg protein, respectively), suggesting the involvement of more than one P450 enzyme. A wide variation in activity was found using 50 microM diazinon as substrate, (11-648 pmol/min/mg protein, n = 15), whereas, with 500 microM, variation was less (164-978 pmol/min/mg protein). Among eight P450-catalyzed reactions, the putative high-affinity component (50 microM diazinon) correlated with S-mephenytoin 4'-hydroxylase activity (r = 0.686, p < 0.01), suggesting the involvement of CYP2C19. The putative low-affinity component (500 microM diazinon) correlated with both S-mephenytoin 4'-hydroxylase (r = 0.714; p < 0.005) and high-affinity phenacetin O-deethylase activity (r = 0.625; p < 0.05). This activity was partially inhibited by furafylline, troleandomycin, and ketoconazole. These data suggest contributions from CYP2C19, CYP1A2, and CYP3A4. None of the inhibitors affected the high-affinity component. Of seven heterologously expressed human P450 enzymes, CYP2C19 activated diazinon (500 microM) at the fastest rate, followed by CYP3A4, CYP1A2, and CYP2C9. Both hepatic microsomal S-mephenytoin 4'-hydroxylase and high-affinity phenacetin O-deethylase activities were strongly inhibited by diazinon (IC50 < 2.5 microM), while no effect was seen on midazolam 1'-hydroxylase activity. These data indicate that CYP2C19 is the major enzyme involved in diazinon activation in human liver, while other enzymes including CYP1A2 may play a more minor role.     

柑橘类果汁对大鼠细胞色素P450 3A的抑制与果汁中呋喃香豆素组成的相关性

目的：呋喃香豆素类(补骨脂素类)是导致葡萄柚汁与药物相互作用的重要成分。本研究拟调查该类化合物在其它柑橘类果汁中的存在，并比较这些果汁对细胞色素P450(CYP)3A活性的抑制强度。方法：用梯度高压液相色谱法测定柑橘类的鲜榨果汁中6种呋喃香豆素化合物的组成与含量。以雄性Sprague-Dawley大鼠肝微粒体的睾酮613．羟化活性测试每种果汁对CYP3A的抑制作用。结果：与葡萄柚汁比较，Jaffa sweetie果汁中呋喃香豆素的组成与含量基本相同，对CYP3A的抑制强度也相当；日本红柚、溪蜜柚和金柚的果汁中呋喃香豆素组成相似但含量较低，对CYP3A的抑制强度也较弱；常山胡柚、甜橙与宽皮橘果汁中呋喃香豆素种类少且含量极低。且多数对CYP3A无抑制作用。结论：根据呋喃香豆素组成可以预测柑橘类果汁因为抑制CYP3A引起的药物相互作用；琯溪蜜柚及近缘品种的果汁可能导致类似于葡萄柚汁的药物相互作用，有必要在临床上加以验证。     

Exposure-dependent inhibition of intestinal and hepatic CYP3A4 in vivo by grapefruit juice

Consumption of typical quantities of grapefruit juice (GFJ) increases the oral bioavailability of several CYP3A4 substrates without affecting their elimination, consistent with selective inhibition of intestinal but not hepatic CYP3A4. However, increases in the AUCs of CYP3A4 substrates recently associated with the consumption of large amounts of GFJ were similar to those observed with potent inhibitors of hepatic CYP3A4. The current study compared the effects of consuming large quantities and more typical amounts of GFJ on the activity of hepatic and intestinal cytochrome P450 3A4 in vivo, employing the erythromycin breath test (EBT) and oral midazolam pharmacokinetics. This was a two-phase, randomized, placebo-controlled crossover study, with each phase conducted with a separate panel of subjects. In Phase I, 8 male volunteers were randomized to the order of receiving one glass (240 mL) of water (placebo) or double-strength (DS) GFJ tid for 2 days and then 90, 60, and 30 minutes prior to administration of probe drugs on the 3rd day. In Phase II, 16 male volunteers were randomized to the order of receiving one glass of (1) single-strength (SS) GFJ, (2) DS GFJ, and (3) water (placebo). All treatments were administered in a fasted state. There was at least a 7-day washout period between treatments. Probe drugs, administered 30 minutes or 1 hour following each treatment in Phase I or II, respectively, consisted of oral midazolam (2 mg) coadministered with IV [14G N-methyl] erythromycin (0.03 mg). The EBT was performed 20 minutes following erythromycin administration. Blood was collected during the 24 hours following probe drug administration for the analysis of midazolam pharmacokinetics. In Phase I, consumption of one glass of DS GFJ tid for 3 days increased the Cmax of midazolam 3-fold, the AUC 6-fold, and the t1/2 2-fold and decreased the amount of exhaled 14CO2 in all 8 subjects, with a mean decrease in EBT of 18%. In Phase II, consumption of one glass of DS GFJ significantly increased the AUC and Cmax of midazolam approximately 2-fold without a significant effect on the t1/2 of midazolam or the EBT. The effects of consuming one glass of SS GFJ on midazolam pharmacokinetics and the EBT were not significantly different from those of one glass of DS GFJ. It was concluded that consumption of one glass of DS GFJ tid for 3 days significantly increased the AUC, Cmax, and t1/2 of midazolam and reduced EBT values, reflecting inhibition of both hepatic and intestinal CYP3A4. In contrast, consumption of one glass of SS or DS GFJ increased midazolam AUC and Cmax, with little effect on the midazolam t1/2 and EBT values, reflecting preferential inhibition of intestinal CYP3A4. Alterations of midazolam AUC and Cmax induced by nine glasses of DS GFJ were significantly greater than those produced by one glass of SS or DS GFJ. These data suggest that GFJ inhibits intestinal and hepatic CYP3A4 in an exposure-dependent fashion and that patients taking medications that are CYP3A4 substrates are at risk for developing drug-related adverse events if they consume large amounts of grapefruit juice.     

Drug-metabolizing enzyme inhibition by ketoconazole does not reduce interindividual variability of CYP3A activity as measured by oral midazolam.

Variable interindividual expression of cytochrome P450 3A presents a challenge in dosing drugs. The use of potent inhibitors of CYP3A such as ketoconazole has been explored to reduce the clearance of CYP3A substrates, thereby resulting in smaller dose requirements; however, the impact of CYP3A inhibition on interindividual variability has not been well characterized. Our objective was to examine the effect of ketoconazole inhibition on CYP3A metabolic variability as measured by the CYP3A biomarker oral midazolam. A single dose of midazolam (0.075 mg/kg) was administered to 19 healthy Caucasian adults (38.7 +/- 8.8 years, nine male/10 female) at baseline and concurrently with ketoconazole (400 mg daily for 10 days) on day 6 or 9 of ketoconazole. Plasma samples were collected over 6 to 30 h. A paired t test and percent coefficient of variation (CV%) were used to evaluate differences in midazolam clearance and interindividual variability during both phases. Monte Carlo simulation was performed to determine probability distribution of area under the concentration-time curves (AUCs). Midazolam apparent oral clearance decreased by 89% (p < 0.0001) during inhibition. Cmax increased from 23 ng/ml (95% CI 19-29 ng/ml) to 55 ng/ml (95% CI 46-66 ng/ml), p < 0.0001. CV% increased from 41 to 58% from baseline to ketoconazole inhibition. AUCs [median (range)] were 0.20 mg . min/ml (0.05-0.81 mg . min/ml) and 1.94 mg . min/ml (0.25-25.4 mg . min/ml) at baseline and inhibition phase, with CV% of 41 and 61%, respectively. Ketoconazole decreased CYP3A activity but did not reduce interindividual variability. Use of a CYP3A inhibitor to standardize dosing of CYP3A substrates may not be feasible in clinical practice.     

Mechanism-based inactivation of human liver microsomal CYP3A4 by rutaecarpine and limonin from Evodia fruit extract

Evodia fruit (Evodiae Fructus) is used as a herbal medicine prepared from the matured fruit of the Evodia rutaecarpa Bentham or Evodia officinalis Dode, of the Rutaceae plant family. An extract of Evodia fruit in the presence of NADPH was shown to inhibit human liver microsomal erythromycin N-demethylation activity, mediated by cytochrome P450 3A4 (CYP3A4), in a preincubation-time dependent manner. The present study was conducted to identify components of Evodia fruit extract having preincubation-time dependent inhibitory effects on CYP3A4 by analyzing human liver microsomal erythromycin N-demethylation activity. Rutaecarpine, a major component of Evodia fruit, and limonin caused the most dramatic decrease in residual CYP3A4 activity (IC50 before and after 20 min preincubation with: rutaecarpine, >100 microM and 1.4 microM; limonin, 23.5 microM and 1.8 microM, respectively). Furthermore, rutaecarpine and limonin were identified as mechanism-based inhibitors of CYP3A4 from the following observations: 1) The inhibitory effects of rutaecarpine and limonin on CYP3A4 activity were dependent on the preincubation time, 2) The inhibition required NADPH, 3) The inhibition was depressed in the presence of the competitive CYP3A4 inhibitor, ketoconazole, 4) Dialysis resulted in no recovery of CYP3A4 activity. The kinetic parameters for inactivation k(inact) and K(I) were: 0.387 min-1 and 107.7 microM for rutaecarpine, 0.266 min-1 and 23.2 microM for limonin, respectively. These results indicate that rutaecarpine and limonin are mechanism-based inhibitors of CYP3A4.