Hepatocyte Growth Factor Enhances Antineoplastic Effect of 5-Fluorouracil by Increasing UPP1 Expression in HepG2 Cells.

Aberrant activation of hepatocyte growth factor (HGF) and its receptor c-Met axis promotes tumor growth. Therefore, many clinical trials have been conducted. A phase 3 trial investigating a monoclonal antibody targeting HGF in combination with fluoropyrimidine-based chemotherapy had to be terminated prematurely; however, the reason behind the failure remains poorly defined. In this study, we investigated the influence of HGF on the antineoplastic effects of 5-fluorouracil (5-FU), a fluoropyrimidine, in HepG2 cells. HGF suppressed the proliferative activity of cells concomitantly treated with 5-FU more robustly as compared to that of cells treated with 5-FU alone, and markedly increased the expression of uridine phosphorylase 1 (UPP1). Intracellular concentration of 5-fluorouridine, an initial anabolite of 5-FU catalyzed by UPP1, was increased by HGF. Interestingly, erlotinib enhanced HGF-induced increase in UPP1 mRNA; in contrast, gefitinib suppressed it. Furthermore, erlotinib suppressed HGF-increased phosphorylation of the epidermal growth factor receptor at the Tyr1173 site involved in downregulation of extracellular signal-regulated kinase (Erk) activation, and enhanced the HGF-increased phosphorylation of Erk. Collectively, these findings suggest that inhibition of the HGF/c-Met axis diminishes the effects of fluoropyrimidine through downregulation of UPP1 expression. Therefore, extreme caution must be exercised in terms of patient safety while offering chemotherapy comprising fluoropyrimidine concomitantly with inhibitors of the HGF/c-Met axis.

Role of FK506 Binding Protein on Tacrolimus Distribution in Red Blood Cells.

PURPOSE: Tacrolimus is distributed mainly in red blood cells (RBCs) after transfer into blood. This study aimed to evaluate the effect of FK506-binding proteins (FKBPs) on RBC distribution of tacrolimus in a physiological environment. METHODS: Human RBCs were isolated from fresh blood samples from healthy volunteers. The effect of FKBPs on each process of the RBC distribution of tacrolimus was evaluated in vitro. Effect of intracellular FKBPs was assessed by inhibition experiment with rapamycin, which competitively inhibits the binding of tacrolimus to FKBPs. Effect of extracellular FKBPs was examined by pre-exposure of RBCs to FKBP and preincubation of tacrolimus with FKBP. RESULTS: Pretreatment with rapamycin significantly reduced the rate of tacrolimus distribution in RBCs in a concentration-dependent manner. Pre-exposure of RBCs to FKBP12 followed by exposure to tacrolimus significantly decreased tacrolimus distribution in RBCs in a concentration-dependent manner. In addition, preincubation of tacrolimus with FKBP12 significantly reduced the rate of tacrolimus distribution in RBCs. CONCLUSIONS: FKBP played an important role in the distribution of tacrolimus in RBCs. The effect of intracellular and extracellular FKBPs on RBC distribution of tacrolimus in circulating blood was substantial. FKBP was shown as a potential biomarker for predicting the pharmacokinetics and pharmacodynamics of tacrolimus.

Caffeine increases the antitumor effect of Cisplatin in human hepatocellular carcinoma cells.

Caffeine is thought to increase the antitumor effect of cisplatin or DNA-damaging agents because it is known that caffeine inhibits DNA repair. Caffeine-assisted chemotherapy has been used in the treatment of osteosarcomas. In addition, there are several reports about combination chemotherapy with caffeine for certain malignancies other than osteosarcomas. However, there are no reports that show the utility of combination chemotherapy with caffeine for hepatocellular carcinoma (HCC). We examined the combined effects of caffeine and cisplatin in human HCC cell lines, and screened for a more effective administration method of caffeine in vitro. Human HCC cell lines (HepG2, HLF, HuH-7, and Li-7) were exposed to caffeine (0-0.5 mM) and cisplatin (0-1.2 µg/mL) for 72 h, either alone or in combination. Cell numbers were measured by WST-8 assay, and cell apoptosis was determined by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) binding assay. As a result, caffeine increased the antitumor effect of cisplatin on cell proliferation and cell apoptosis in the HCC cell lines. Moreover, this effect was dependent on the amount of exposure to caffeine. These results suggest that caffeine-assisted chemotherapy is useful for HCC treatment.

Hepatic fibrosis does not affect the pharmacokinetics of 5-fluorouracil in rats.

In the case of cancer chemotherapy for hepatocellular carcinoma, 5-fluorouracil (5-FU) is used widely, and has typically been given by intrahepatic arterial (i.a.) infusion to increase treatment efficacy and to reduce systemic toxicity. 5-Fluorouracil is eliminated primarily by the liver, and so its use in patients with hepatic disease can be difficult. This study investigated the effect of hepatic fibrosis on the pharmacokinetics of 5-FU in rats. Experimental hepatic fibrosis was induced by carbon tetrachloride treatment. 5-fluorouracil was infused for 15 min into the hepatic artery or the saphenous vein of the rats at a dose of 1.25 mg/kg. There were no significant differences in the plasma concentration and AUC of 5-FU between hepatic disease rats and their controls after both intravenous and intraarterial injection. This result is probably attributed to the fact that there were no significant differences in hepatic blood flow and dihydropyrimidine dehydrogenase (DPD; an initial and rate-limiting enzyme in 5-FU catabolism) activity between hepatic disease rats and their controls, because the total clearance of 5-FU after intravenous and intraarterial administration is mainly limited by hepatic blood flow and DPD activity, respectively. In conclusion, the pharmacokinetics of 5-FU is not affected by hepatic fibrosis, unlike that of many hepatically metabolized drugs.

Hepatocyte growth factor increases uptake of estradiol 17beta-D-glucuronide and Oatp1 protein level in rat hepatocytes.

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17beta-d-glucuronide ([(3)H]E(2)17betaG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [(3)H]E(2)17betaG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E(2)17betaG. However, inhibition by taurocholic acid, a Na(+)-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E(2)17betaG. Further, the increase was not altered by replacing Na(+) in the medium with Li(+). In the inhibition study, the increased [(3)H]E(2)17betaG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

Relationship between UGT1A1*27 and UGT1A1*7 polymorphisms and irinotecan-related toxicities in patients with lung cancer.

BACKGROUND: The objective of this study was to evaluate the effects of gene polymorphisms, including UGT1A1*7, *27, and *29, on the safety of irinotecan therapy. METHODS: The eligibility criteria were: lung cancer patients scheduled to undergo irinotecan therapy, aged ≥ 20 years, with a performance status of 0-2. Thirty-one patients were enrolled and their blood was collected and used to examine the frequency of UGT1A1*6, *7, *27, *28, and *29 polymorphisms and the concentrations of irinotecan, SN-38, and SN-38G after irinotecan therapy. RESULTS: The patients' characteristics were as follows: male/female 25/6, median age 71 years (range 55-84), stage IIB/IIIA/IIIB/IV 2/6/11/12, and adenocarcinoma/squamous cell carcinoma/small cell carcinoma/other 14/10/3/4, respectively. The -/-, *6/-, *7/-, *27/-, *28/-, and *29/- UGT1A1 gene polymorphisms were observed in 10 (32%), 10 (32%), 2 (6%), 2 (6%), 7 (23%), and 0 (0%) cases, respectively. The UGT1A1*27 polymorphism occurred separately from the UGT1A1*28 polymorphism. The lowest leukocyte counts of the patients with the UGT1A1*27 and UGT1A1*6 gene polymorphisms were lower than those observed in the wild-type patients. SN-38 tended to remain in the blood for a prolonged period after the infusion of irinotecan in patients with UGT1A1*27 or UGT1A1*28 polymorphisms. No severe myelotoxicity was seen in the patients with UGT1A1*7. CONCLUSION: UGT1A1*27 can occur separately from UGT1A1*28 and is related to leukopenia during irinotecan treatment. UGT1A1*7 is less relevant to irinotecan-induced toxicities, and UGT1A1*29 seems to have little clinical impact.

Adsorption of irinotecan onto oral adsorbent AST-120 (Kremezin) for preventing delayed diarrhea.

PURPOSE: One of the significant dose-limiting toxicities of irinotecan hydrochloride (CPT-11) is severe diarrhea due to impairment of the intestinal membrane induced by the excreted CPT-11 and its metabolites. AST-120 (Kremezin) is a prominent oral adsorbent that consists of porous spherical carbonic particles. To evaluate whether Kremezin can prevent the diarrhea induced by CPT-11, we investigated the adsorption characteristics of CPT-11 and its metabolites onto Kremezin in vitro and in vivo. METHODS: For in vitro studies, Kremezin was added to each solution containing one of the camptothecin drugs (CPT-11, SN-38, and SN-38-glucuronide), and adsorption activities were determined under various conditions. For in vivo studies, CPT-11 was consecutively administered, and the occurrence of diarrhea was compared between Kremezin-treated and non-treated rats. RESULTS: Kremezin drastically adsorbed the camptothecin drugs in vitro, and the adsorption percentages of the camptothecin drugs for 60 min were more than 85%. In addition, the frequency of diarrhea in Kremezin-treated rats decreased by approximately half of that in the non-treated rats. CONCLUSION: Kremezin showed potent adsorption capacities for the camptothecin drugs and mitigated the symptoms of diarrhea in rats. These results suggest that Kremezin is useful to prevent the diarrhea in clinical CPT-11 chemotherapy.

Hepatocyte growth factor suppresses the anticancer effect of irinotecan by decreasing the level of active metabolite in HepG2 cells.

In the liver, carboxylesterase (CES) converts irinotecan (CPT-11) to its active metabolite SN-38, which exerts anticancer effects. SN-38 is metabolized to an inactive metabolite SN-38 glucuronide by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1). Therefore, single nucleotide polymorphisms (SNPs) of the UGT1A1 gene are responsible for the severe adverse effects associated with the disruption of SN-38 metabolism. However, despite having SNPs of the UGT1A1 gene, many patients metabolize SN-38 sufficiently to avoid severe adverse effects. Among these patients, we found individuals with elevated serum concentrations of hepatocyte growth factor (HGF). The aim of this study was to evaluate whether HGF alters the metabolism of CPT-11, resulting in a reduction in the anticancer effect of CPT11. The cytotoxicity of CPT-11 and SN-38 was evaluated in HepG2 cells pretreated with HGF. Furthermore, we explored the level of expression and mechanisms of activity of CES and UGT1A1. HGF suppressed the cytotoxicity of CPT-11 by decreasing intracellular SN-38 levels that resulted from a decrease in CES2 and an increase in UGT1A1. Furthermore, this HGF-induced suppression was improved by pretreatment with an inhibitor of HGF receptor c-Met, and the improvement was synergistically potentiated by epidermal growth factor receptor (EGFR) inhibitors. Moreover, HGF induced phosphorylation of signal transducer and activator of transcription 3 and transactivated EGFR. These results suggest that HGF is a possible causative agent of acquired clinical resistance in chemotherapy with CPT-11 and could be useful as a predictor of clinical resistance. Additional treatment using c-Met and/or EGFR inhibitors could be a novel strategy to overcome resistance.

Transcriptional up-regulation of cell surface Na V 1.7 sodium channels by insulin-like growth factor-1 via inhibition of glycogen synthase kinase-3ß in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion.

Insulin-like growth factor-1 (IGF-1) plays important roles in the regulation of neuronal development. The electrical activity of Na(+) channels is crucial for the regulation of synaptic formation and maintenance/repair of neuronal circuits. Here, we examined the effects of chronic IGF-1 treatment on cell surface expression and function of Na(+) channels. In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, chronic IGF-1 treatment increased cell surface [(3)H]saxitoxin binding by 31%, without altering the Kd value. In cells treated with IGF-1, veratridine-induced (22)Na(+) influx, and subsequent (45)Ca(2+) influx and catecholamine secretion were augmented by 35%, 33%, 31%, respectively. Pharmacological properties of Na(+) channels characterized by neurotoxins were similar between nontreated and IGF-1-treated cells. IGF-1-induced up-regulation of [(3)H]saxitoxin binding was prevented by phosphatydil inositol-3 kinase inhibitors (LY204002 or wortmannin), or Akt inhibitor (Akt inhibitor IV). Glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl, valproic acid, SB216763 or SB415286) also increased cell surface [(3)H]saxitoxin binding by ∼ 33%, whereas simultaneous treatment of IGF-1 with GSK-3 inhibitors did not produce additive increasing effect on [(3)H]saxitoxin binding. IGF-1 (100 nM) increased Ser(437)-phosphorylated Akt and Ser(9)-phosphorylated GSK-3ß, and inhibited GSK-3ß activity. Treatment with IGF-1, LiCl or SB216763 increased protein level of Na(+) channel α-subunit; it was prevented by cycloheximide. Either treatment increased α-subunit mRNA level by ∼ 48% and accelerated α-subunit gene transcription by ∼ 30% without altering α-subunit mRNA stability. Thus, inhibition of GSK-3ß caused by IGF-1 up-regulates cell surface expression of functional Na(+) channels via acceleration of α-subunit gene transcription.

Effect of acute hepatic failure on the hepatic first-pass effect of 5-fluorouracil in rats.

OBJECTIVES: In cancer chemotherapy for hepatocellular carcinoma, 5-fluorouracil is widely used and has typically been given by intrahepatic arterial (i.a.) infusion to increase treatment efficacy and reduce systemic toxicity. 5-Fluorouracil is eliminated primarily by the liver and so the hepatic first-pass effect after intrahepatic arterial administration of 5-fluorouracil may be lower in patients with hepatic failure, and systemic toxicity may not be reduced. In this study, we have investigated the effect of acute hepatic failure on the first-pass effect of 5-fluorouracil in rats. METHODS: Experimental acute hepatic failure was induced by treatment with carbon tetrachloride (CCl4). 5-Fluorouracil was infused for 15 min into the hepatic artery or the saphenous vein of rats at a dose of 1.25 mg/kg. KEY FINDINGS: Hepatic availability in 50% CCl4-treated (severe hepatic failure) rats was higher than in controls. CONCLUSIONS: The hepatic first-pass effect after intrahepatic arterial administration of 5-fluorouracil was lower in severe hepatic failure. Therefore, the reducing effect of the systemic toxicity after intrahepatic arterial administration may be lower in severe hepatic failure.

Inhibition of carrier-mediated uptake of epirubicin reduces cytotoxicity in primary culture of rat hepatocytes.

Epirubicin, an antineoplastic drug, is considered to be taken up by tumor cells via a common carrier by facilitated diffusion and is then pumped out in an energy-dependent manner because epirubicin is a substrate for P-glycoprotein (P-gp). However, this study investigated the details of the influx mechanism of epirubicin and demonstrated that epirubicin uptake was mediated by active carrier systems in addition to facilitated diffusion in the primary culture of rat hepatocytes. The uptake of epirubicin gradually increased in a saturated manner when the concentrations were between 1 x 10(-7) M and 1 x 10(-6) M. In contrast, the uptake increased progressively in a linear manner when the concentration was high (greater than 1 x 10(-6) M). The uptake of epirubicin at a clinical concentration (7.5 x 10(-7) M) was significantly reduced at 4 degrees C and significantly inhibited when pretreated with metabolic inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and sodium azide) by nearly 25%. Furthermore, an organic anion transporter inhibitor, namely, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS); organic anion transport substrates, namely, para-aminohippurate (PAH), taurocholic acid and estradiol 17-beta-D-glucuronide; and organic cation transporter inhibitors, namely, verapamil and tetraethylammonium significantly reduced the uptake of epirubicin. Furthermore, pretreatment with verapamil and PAH significantly prevented epirubicin-induced reduction of proliferative activity in rat hepatocytes. These results indicated that the uptake of epirubicin was induced, at least in part, by the active transport protein in rat hepatocytes; the inhibition of the probable transport protein protected the intact normal cells from the injury induced by the cytotoxicity of epirubicin.

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.

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.

Inhibitory effects of amino-acid fluids on drug binding to site II of human serum albumin in vitro.

The effects of amino-acid fluids on ligand binding to human serum albumin (HSA) were investigated by fluorescence and ultrafiltration techniques. Warfarin and dansylsarcosine were used as the site marker fluorescence probes for site I and site II of HSA, respectively. Amino-acid fluids specifically decreased the fluorescence intensity induced by dansylsarcosine-HSA binding without any effects on that induced by warfarin-HSA binding. The ultrafiltration technique clarified that the free fraction of the site II drug, diazepam, in human serum was increased in the presence of amino-acid fluids, while no effect was observed in the free fraction of the site I drug, warfarin. The potencies of the effect on binding to site II, observed by fluorescence and ultrafiltration techniques, correlated well with the L-tryptophan contents in amino-acid fluids or with those in L-tryptophan solutions. Based on the comparison between the effects of amino-acid fluids and L-tryptophan solutions, we confirmed that L-tryptophan in amino-acid fluids specifically inhibits drug binding to site II of HSA.

Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

Effect of P-glycoprotein modulator, cyclosporin A, on the gastrointestinal excretion of irinotecan and its metabolite SN-38 in rats.

PURPOSE: The purpose of this work was to investigate the role of the hepatic and intestinal P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) on both biliary excretion and intestinal exsorption of irinotecan hydrochloride (CPT-11) and its metabolite, SN-38, in the lactone and carboxylate forms. Cyclosporin A (CsA) was used to modulate P-gp and cMOAT/MRP2. METHODS: The transcellular transport of CPT-11 and SN-38 was examined by using LLC-PK1 derivative cell lines transfected with murine mdrla both in the absence or in the presence of CsA. The excretions of the compounds through the biliary and intestinal membrane routes were investigated by in situ perfusion technique. RESULTS: Basolateral-to-apical transport of CPT-11 lactone in L-mdr1a cells was significantly decreased by CsA (10 microM). The transcellular transport of SN-38 lactone showed similar behaviors as those of CPT-11 lactone. The biliary excretion and the intestinal exsorption of both forms of CPT-11 and SN-38 were significantly inhibited when the drug was co-administered with CsA. CONCLUSIONS: The transports of CPT-11 and SN-38 via the biliary route seem to be essentially related with cMOAT/MRP2, whereas those of both compounds via the intestinal membrane seem to be related with P-gp.

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.