Genetically engineered bacterial cells expressing human cytochrome P450 (CYP) have been developed as new tools to predict the metabolism and toxicity of drugs in humans. There are various host cells for the heterologous expression of a form of CYP. Among them, bacterial cells such as Escherichia coli (E. coli) have advantages with regard to ease of use and high yield of protein. CYP protein could be first expressed by the modification of the N-terminal amino acid sequence in E. coli cells in 1991. Since then, many forms of human CYP have been successfully expressed in E. coli cells. Since the E. coli cells do not possess endogeneous electron transport systems to support the full catalytic activity of CYP, E. coli strains co-expressing both human CYP and NADPH-cytochrome P450 reductase (OR) have been established. Each form of CYP expressed in the E. coli cells efficiently catalyzed the oxidation of a representative substrate at an efficient rate, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. According to the studies performed so far, the modification of the N-terminal amino acid sequence of CYP did not seem to affect the catalytic properties of CYP. The human CYP expressed in the E. coli cells were applicable for studies to determine a metabolic pathway(s) of drugs and to estimate kinetic parameters of drug metabolism by human CYP. Drug-drug interactions caused by inhibition of the metabolism of drugs by human CYP could also be examined by in vitro inhibition studies with CYP expressed in the E. coli cells. Recently, human CYP was co-expressed with the OR in Salmonella typhimurium (S. typhimurium) cells used for mutation assay (Ames test) by applying the technology for the expression of human CYP and the OR in E. coli cells, to evaluate whether chemicals including drugs are metabolically activated by human CYP and show mutagenicity. These strains of bacteria are considered as useful tools to study the metabolism and the toxicity of drugs in humans.
Irinotecan hydrochloride (CPT-11) is an anticancer agent with unpredictable bouts of diarrhea as a dose-limiting toxic side-effect. Since the biliary excretion of its active metabolite (SN-38) and SN-38 glucuronide (SN38-Glu), which are mediated by the multidrug resistance associated protein-2 (MRP2/ABCC2), has been proposed to be related to this gastrointestinal toxicity, we have attempted here to examine the potential of various therapeutic agents to interact with the biliary excretion in order to identify MRP2 inhibitors to prevent this toxicity. The inhibition constants (Ki) of 26 compounds were examined for the transport of a typical MRP2 substrate in isolated canalicular membrane vesicles. Of these, 13 compounds inhibited the transport with Ki values from 0.0461 to 281 μM. Three inhibitors (probenecid, sulfobromophthalein and glycyrrhizin) were also found to inhibit the biliary excretion of SN-38 and SN38-Glu in rats in vivo, and the degrees of inhibition were compatible with the estimated values based on the ratios of Ki and unbound concentrations in circulating plasma. A similar estimation of the potential inhibitory effect in human was also examined by considering both the Ki of each therapeutic agent and its unbound concentration both in circulating plasma and the inlet to the liver. The predicted degrees of inhibition by most compounds were minimal whereas approximately 75% inhibition was predicted for probenecid. Thus, probenecid may be a candidate which can be used clinically to inhibit the biliary excretion of CPT-11 metabolites, whereas an interaction between most of the other compounds and MRP2 is more unlikely.
To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as γ-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [3H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [14C]benzoic acid was saturable, and the Km value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [14C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [3H]dehydroepiandrosterone sulfate, [3H]estrone sulfate and [3H]estradiol 17β-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.
Troglitazone is an insulin sensitizer and also known as an agonist of peroxisome proliferator-activated receptor-γ (PPARγ). In the present study, we have studied the influence of troglitazone on CYP3A form expressions in HepG2 cells for a model of human tissue. Interestingly, constitutively expressed forms of cytochrome P450, CYP3A5 and CYP3A7, were suppressed by the pretreatment of troglitazone but not of the related thiazolidinediones, pioglitazone and rosiglitazone in this cell line. A major liver CYP3A form, CYP3A4, was not detected in this cell line with and without troglitazone treatment. The troglitazone-mediated suppressions of CYP3A5 and CYP3A7 were found to be independent of expression levels of nuclear transcriptional factors, PXR, RXRα and PPARγ. These results suggest that the selectively suppressive effects of troglitazone on CYP3A5 and CYP3A7 expressions may be caused by a novel pathway.
Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2° strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6β-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.
Pretreatment of adult male C57BL/6 mice with propranolol (PL, 100 mg/kg, p.o., once a day for five days) significantly increased PL N-deisopropylase activity and decreased PL 7-hydroxylase activity in liver microsomes, whereas PL 4- and 5-hydroxylase activities remained unchanged. In the present study, we have examined the mechanism for the elevation of the oxidation of PL side chain. Immunoblot analysis using polyclonal antibodies raised against rat liver CYP enzymes such as CYP1A1, -2B2, -2C11, -2D2, -2E1 and 3A2 showed that, compared with the vehicle-treated control, the levels of two protein bands (54 KD and 52 KD) were increased by the pretreatment. Both proteins immunochemically cross-reacted with the antibodies against rat CYP1A1, and from their molecular weights, the 54 KD and 52 KD proteins were deduced to be CYP1A1 and 1A2, respectively. Computer-assisted scanning analysis revealed that the levels of CYP1A1 and CYP1A2 proteins were increased 1.8 and 1.2 times, respectively, over those of control microsomes. PL N-deisopropylase activity correlated well with ethoxyresorufin O-deethylase (r=0.828) and phenacetin O-deethylase (r=0.851) activities in the same microsomal fractions. These results show that repeated oral administration of PL in mice induces mainly CYP1A1 and also CYP1A2 to some extent, which contrasts from our previous results in rats in which CYP1A2 only was induced with PL pretreatment [Narimatsu et al., Chemico-Biol. Interact., 101, 207-224 (1996)].
The metabolic profile of M17055, a novel diuretic, after administration to experimental animals and after incubation with human liver microsomes was investigated. 1. Extensive metabolism was observed in rats and monkeys and the structures of six metabolites (RU1, RU2, and RU3 from rat urine or liver perfusate; MU1, MU2 and MU3 from monkey urine) were assumed or identified. The clear species difference of metabolism was revealed between rats and a monkey with different structures of the isolated metabolites. 2. When these metabolites were quantified using radioactive material, RU3, RU1 and MU3 were considered to be major metabolites in rat urine, rat bile and monkey urine respectively, while in a dog, unchanged drug was observed as the major component indicating only little metabolism occurred in dog, when administered intravenously. 3. RU1 and RU2 were also generated from [14C]M17055 after incubation with human liver microsomes, suggesting that the metabolic pathway of M17055 in humans involves that observed in rats. 4. [14C]M17055 metabolism in human liver microsomes was inhibited by CYP2C8/9 and CYP3A4/5 inhibitors, and also by the antibodies that recognize CYP2C8/9/19 and CYP3A4. Significant correlations were observed between the rate of [14C]M17055 metabolism and the activity of testosterone 6β-hydroxylation or tolbutamide methyl-hydroxylation. cDNA-expressed CYP3A4 and CYP2C9 could catalyze the metabolism of [14C]M17055. These results suggest that the metabolism of M17055 in human liver microsomes is catalyzed mainly by CYP3A4 and CYP2C9.
To clarify the cause of the canine individual variability in plasma concentration after oral administration of GTS-21, we evaluated in vitro the metabolism to 4-OH-GTS-21 in liver microsomes of the same individuals from in vivo pharmacokinetic study. First, we applied to the Michaelis-Menten kinetic parameters to a dispersion model, and compared hepatic availability (FH) and hepatic clearance (CLH) values from in vitro with bioavailability (F), hepatic plasma flow (QPH), and plasma clearance (CLP) values from in vivo. The ratios of CLH to QPH were ranged 0.74 to 0.94, suggesting that GTS-21 is a hepatic plasma flow-limiting drug. A significant correlation of FH and F in the four dogs (r=0.995, p=0.005) indicates that the variability is predominantly caused by GTS-21 O4-demethylase activity. Second, we specified the cytochrome P450 (CYP) enzymes that are involved with the metabolism by chemical inhibition. α-Naphthoflavone, furafylline, quinidine, quinine, and troleandomycin significantly inhibited GTS-21 O4-demethylase activity. Thus CYP1A, CYP2D15, and CYP3A12 were involved with O4-demethylation. The variability in control activity decreased on addition of α-naphthoflavone and furafylline. Third, we quantified the contents of CYP1A and CYP3A12 by enzyme-linked immunosorbent assay. The content of CYP1A was consistent with GTS-21 O4-demethylase activity. We concluded that canine liver CYP1A causes the individual variability in GTS-21 plasma concentration after oral administration.
Recently, we established a system for assessing ischemia/reperfusion injury, specifically the opening of tight junctions (TJ), caused by reoxygenation following the induction of lipid peroxidation by tertiary-butylhydroperoxide (t-BuOOH), using the human intestinal epithelial cell line Caco-2 in order to focus on the barrier function of the epithelium independent of the vascular compartment. In the present study, we attempted to identify factors involved in the structural changes induced by reoxygenation using 0.5 mM t-BuOOH in Caco-2 cell monolayers. Glutathione (GSH) and N-acetylcystein, a precursor of GSH, inhibited the opening of TJ evoked by reoxygenation following the induction of lipid peroxidation by 0.5 mM of t-BuOOH. Tiron, as a cell permeable superoxide anion scavenger and deferoxamine, an iron-chelating agent ameliorated the opening in a dose-dependent manner. Also, Tiron suppressed the apical-to-basal and basal-to-apical permeability of the increased Rhodamine123 by reoxygenation in a concentration-dependent manner. These results collectively suggest that superoxide anion and iron ions play an important role or contribute to structural changes such as the opening of TJ induced by reoxygenation following the induction of lipid peroxidation by 0.5 mM t-BuOOH.