Teratogenicity of cyclophosphamide in a coupled microsomal activating/embryo culture system

Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.     

Rat conceptus development in vitro: Comparative effects of alkylating agents

Conceptuses removed from the rats in the eleventh day of gestation were cultured in vitro for 2 days. Growth and differentiation of the major organs of the embryo in vitro resembled those developed in vivo. Embryonic development and organogenesis were markedly affected when the alkylating agents TEM (2,4,6-triethylenimino-1,3,5-triazine) and nitrogen mustard (mechlorethamine hydrochloride) were added to the culture medium. At concentrations of 1 and 5 μg/ml medium, these teratogens were highly embryotoxic and affected both growth and differentiation. DNA and protein content of embryos and yolk sacs was reduced significantly (p < 0.001) from the controls. Development of conceptuses in the culture medium that contained cyclophosphamide (0.35 mm) alone apparently were normal. However, addition of cyclophosphamide (0.35 mm), microsomes (0.5 mg protein/ml), and NADPH (1 mm) to the culture medium induced marked deleterious effects on the conceptus growth and differentiation. DNA and protein contents were significantly (p < 0.001) reduced by the combined treatment indicative of formation of reactive metabolites and their interference with macro-molecular biosynthesis. Aminopyrine which has not been shown to be teratogenic, in equimolar amounts to cyclophosphamide, under identical conditions of culture, did not affect conceptus development.     

Comparative evaluation of the teratogenicity of genistein and genistin using rat whole embryo culture and limbud micromass culture methods

Genistein (GEN) is one kind of phytoestrogen. Several studies have demonstrated the teratogenic potential of GEN in vitro by postimplantation rat whole embryo culture (WEC) assay, but GEN showed no teratogenic effects in vivo even at a dose up to 1000 mg/kg bw/day. The mechanism of such discrepancy is still unclear. Because more than 80% of total genistein (free plus glycoside form) in circulation is its glycoside metabolite, genistin (GIN), we thus hypothesize that genistin is non-teratogenic. To prove this hypothesis, rat whole embryo culture (WEC) and limbud micromass culture methods were applied to compare the teratogenic effects of GEN and GIN on developing embryos in vitro. In WEC assay, we found that the development of embryos was affected by GEN treatment dose-dependently, while GIN-treated embryos displayed slight developmental defects only at the highest dose (222 μM). In micromass culture assay, the IC50 of cell proliferation and differentiation for GEN were 15.6 and 37.2 μM, respectively, while neither was influenced by GIN treatment up to 111 μM. Collectively, our study indicated that GEN showed no teratogenic effects in vivo probably due to its transformation to the non-teratogenic metabolite, GIN.     

Teratogenicity induced in cultured rat embryos by the serum of procarbazine treated rats

Male rats were injected with single intraperitoneal doses of 50–200 mg/kg of procarbazine hydrochloride (P). Their undiluted serum was then used as the culture medium for 9.5-day-old embryos to evaluate its teratogenic and toxic potential in vitro. A 48-h exposure to this medium resulted in a variety of dysmorphogenic effects. Somite, limb bud and otic vesicle deformities were seen at all dose levels, and neural tube and optic vesicle abnormalities were frequently observed in the 150 and 200 mg/kg groups. Embryonic growth and differentiation were only moderately affected at any dose levels.In a second set of experiments, embryos were directly exposed to 100–150 μg/ml P combined with a liver enzymatic activating system. No abnormalities were detected but toxicity occurred within narrow concentration ranges; 125 μg/ml affected growth and differentiation slightly, and 150 μg/ml suppressed the embryonic development severely. Exposure to 125 μg/ml P without the liver enzymatic activating system had no effect on embryonic growth and differentiation, suggesting that the metablites responsible for the toxic effect were generated by the liver enzymatic preparation.Our results indicate that stable metabolites of P, responsible for its teratogenic action, are formed only within body compartments, whereas the formation of these metabolites does not take place in vitro in the presence of hepatic enzyme preparations.     

Modulation of acrylonitrile-induced embryotoxicity in vitro by glutathione depletion

The effects of glutathione (GSH) depletion on the embryotoxicity of acrylonitrile were assessed in vitro using the rat whole-embryo culture system. Day 10 rat embryos were cultured in rat serum medium for 6 h in the presence of 250 Ml-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH synthesis, to deplete GSH in both embryo and visceral yolk sac. Following pretreatment, conceptuses were cultured for an additional 21 h in the presence of 152, 228, or 304 M acrylonitrile. At the end of the culture period, conceptuses were assessed for survival, growth and development, malformations, and the protein and glutathione content of embryos and yolk sacs were assayed. Acrylonitrile alone produced concentrationrelated and statistically significant decreases in yolk sac diameter, crown-rump length, head length and number of somite pairs, as well as in embryonic and yolk sac proteins. The chemical also caused dysmorphogenesis of the brain and of the caudal extremity, and a concentration-related and statistically significant increase in GSH content in the yolk sac. Pretreatment with BSO significantly enhanced the embryotoxic effects of acrylonitrile. The conceptuses displayed further decreases in functional yolk sac circulation, yolk sac diameter, crown-rump and head length, when compared to either acrylonitrile or BSO alone. The incidence of caudal malformations and the severity of brain malformations produced by acrylonitrile were also increased. Marked decreases in embryonic and yolk sac GSH contents were observed after exposure to BSO alone or in combination with acrylonitrile. Thus, depletion in embryonic and yolk sac GSH by BSO enhanced teratogenic and growth retarding effects of acrylonitrile in vitro, suggesting that GSH plays a critical role in modulating acrylonitrileelicited embryotoxicity.     

A coupled microsomal-activating/embryo culture system: Toxicity of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH)

An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decreases in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1.0 mM NADPH alone, or 0.2 mM NADPH in the presence of microsomes and a glucose-6-phosphate dehydrogenase-based NADPH-generating system, greatly reduced embryo and yolk sac growth in vitro. The toxicity of NADPH was not due to lipid peroxidation. Only minor decreases in final yolk sac protein levels occurred when embryos were grown in media containing male rat microsomes and 1.0 mM NADPH. The protective effect of rat hepatic microsomes on NADPH toxicity does not seem to have been due to the oxidation of NADPH to the less toxic NADP. Although cyclophosphamide alone was not toxic to rat embryos cultured in vitro, in the coupled microsomal-activating/embryo culture system, cyclophosphamide reduced yolk sac and embryo growth and caused abnormal embryonic differentiation. The uses of the coupled microsomal-activating/embryo culture system to study mechanisms in anomalous development, as well as its possible use in embryo toxicity and teratogenicity testing, are discussed.     

Increased sister-chromatid exchange frequency in preimplantation mouse embryos after maternal cyclophosphamide treatment before implantation

The effect of a toxic agent in vivo on sister-chromatid exchange (SCE) frequency of preimplantation mouse embryos and bone marrow cells was determined using combined in vivo treatment and in vitro culture in the presence of 5-bromo-2-desoxyuridine (BrdU) for differential staining of the chromatids. In mice exposed to cyclophosphamide (CPA) on day 2 of pregnancy SCE frequency was increased dose-dependently both in embryos and bone marrow cells 1 h after treatment. It returned to control values in bone marrow cells obtained 24 h after exposure but was still significantly increased in the embryos. A closer time-related evaluation of SCE on day 2 of gestation showed a significant increase in SCE in bone marrow cells and in embryos obtained 20-60 min after CPA treatment. Furthermore, SCE frequency was the most sensitive toxicological endpoint to detect embryotoxic effects of CPA treatment before implantation, since it was significantly increased in embryos exposed to 5 mg/kg CPA on day 2 of pregnancy while embryolethality at term and both cytogenetical (structural chromosomal aberrations, micronuclei) and developmental parameters (cell number, differentiation in culture) before implantation did not indicate any toxic effect.     

Relative potency of albendazole and its sulfoxide metabolite in two in vitro tests for developmental toxicity: The rat whole embryo culture and the mouse embryonic stem cell test

The benzimidazole carbamate albendazole (ABZ), a potent anthelmintic, is a teratogenic compound in rats. At present it is unclear to which degree this effect is caused by the parent compound or its major metabolite, albendazole sulfoxide (ASO). Both substances were studied separately and in combinations to mimic incomplete bioactivation in two in vitro tests: mouse embryonic stem cell test (EST) and rat whole embryo culture (WEC). In both assays, ABZ and mixtures with ASO induced detrimental effects at lower concentrations compared to ASO alone. While ABZ caused half-maximal effects on cardiomyocyte differentiation at a mean concentration of 0.26μM (EST) and dysmorphogenic development of rat embryos at 3.7μM (WEC), effective concentrations of ASO were similar in both assays (10-13μM). By using WEC and EST we demonstrate that ABZ exhibits stronger inherent embryotoxic potency although ASO might be the proximate teratogen in vivo because of higher plasma concentrations.     

Affinity of Drugs for Cytochrome P-450 Determined by Inhibition of p-Nitrophenetole O-Deethylation by Rat Liver Microsomes

Abstract: The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent K_m for oxygen was 0.07 μM. Furthermore, cytochrome b₅ seemed to be involved in the formation of p-nitrophenol. The effect on p-nitrophenol formation of drugs known to be involved in drug interaction in clinical practice was studied. There was a competitive inhibition by phenytoin (inhibitor constant, K_i, 30 μM), disulfiram (K_i, 2 μM) and chloramphenicol (K_i, 20 μM), whereas a mixed-type inhibition by isoniazid was observed (K_is, 1,3 mM and K_ii, 10,6 mM).     

Effect of neuroleptics on cytochrome P450 2C11 (CYP2C11) in rat liver

The aim of the present study was to investigate the influence of classic and atypical neuroleptics on the activity of cytochrome P450 2C11 (CYP2C11), measured as a rate of testosterone 2α- and 16α-hydroxylation. The reaction was studied in control liver microsomes in the presence of neuroleptics, as well as in the microsomes of rats treated intraperitoneally (ip) with pharmacological doses of the drugs (promazine, levomepromazine, thioridazine and perazine 10 mg/kg; chlorpromazine 3 mg/kg; haloperidol 0.3 mg/kg; risperidone 0.1 mg/kg; sertindole 0.05 mg/kg) for one day or two weeks (twice a day), in the absence of the neuroleptics in vitro. The investigated neuroleptics added to control liver microsomes produced some inhibitory effects on CYP2C11 activity, which were moderate (thioridazine: K(i) = 55), modest (sertindole and perazine: K(i) = 76 and 94 μM, respectively) or week (promazine, levomepromazine, haloperidol and chlorpromazine: K(i) = 285, 280, 223 and 157 μM, respectively). Risperidone had the weakest inhibitory effect on the CYP2C11 activity (K(i) = 641 μM). One-day exposure of rats to the neuroleptics did not significantly change the activity of CYP2C11 in liver microsomes. Of the neuroleptics studied, only chronic treatment with levomepromazine, perazine and thioridazine diminished CYP2C11 activity; those effects were positively correlated with the observed decreases in the protein level of the enzyme. The in vivo inhibition of CYP2C11 by chronic treatment with the three phenothiazines suggests their influence on the enzyme regulation. A possible mechanism of CYP2C11 regulation by the neuroleptics and its pharmacological significance are discussed.     

Monitoring of organ formation in rat embryos after in vitro exposure to azathioprine, mercaptopurine, methotrexate or cyclosporin A

Rat embryos in the organ formation phase (days 9.5-11.5 post coitum) were cultivated in pure rat serum in the presence of an Aroclor 1254 pretreated liver microsomal preparation (S9-mix). Various concentrations of the immunosuppressive drugs azathioprine (AZ), 6-mercaptopurine (MP), methotrexate (MTX) or cyclosporin A (CS-A) were added at the beginning of the culture period. Forty-eight hours later, malformations were observed in the AZ, MP and MTX treated embryos at concentrations as low as 1 microgram/ml, 1.8 micrograms/ml and 0.05 microgram/ml, respectively. This indicates that these drugs have a direct effect on embryonic development. They selectively affected the rhombencephalic and telencephalic brain regions. Other malformations were seen in the caudal trunk, the heart and forelimb regions, and in the vesicular structures. It is suggested that the similarity of the pharmacological action of these drugs, that is, the DNA de novo synthesis inhibition, was the cause of the comparable types of malformations observed. Higher AZ, MP and MTX concentrations caused concentration-dependent increases in the types and incidences of malformations, as well as inhibited overall growth and differentiation. CS-A, a new type of immunosuppressant agent, had no effect on the morphogenetic events at the concentrations tested. These results are generally in agreement with the literature data, indicating that AZ, MP and MTX induce malformations in whole-animal systems, whereas CY-A does not. When AZ and MTX were assayed in the rat species in vivo, on the other hand, embryolethalities and retardations, but few malformations, were observed. The possibility of controlled exposure in vitro may, therefore, offer the advantage that clearer distinctions between embryolethal and teratogenic effects can be made.     

Cooperativity of α-naphthoflavone in cytochrome P450 3A-dependent drug oxidation activities in hepatic and intestinal microsomes from mouse and human

1. The effects of several CYP3A substrates (α-naphthoflavone (αNF), terfenadine, midazolam, erythromycin) on nifedipine oxidation and testosterone 6-β-hydroxylation activities were investigated in hepatic and intestinal microsomes from mouse and human. 2. αNF (10 μM) and terfenadine (100 μM) inhibited nifedipine oxidation activities (at substrate concentration of 100 μM) in mouse hepatic microsomes to ～50%, but not in mouse intestinal microsomes. αNF (30 μM) stimulated nifedipine oxidation activities in mouse and human intestinal microsomes and in human hepatic microsomes to ～1.3-1.8-fold. Inhibitory potencies (50% inhibition concentration, IC₅₀) of midazolam and erythromycin for nifedipine oxidations were calculated to be ～90 μM in human intestinal microsomes. In contrast, testosterone (100 μM) stimulated the nifedipine oxidation activities ～1.5-fold in hepatic and intestinal microsomes from mouse and human. 3. αNF showed different effects on the kinetic parameters including the Hill coefficients of nifedipine oxidation and testosterone 6-β-hydroxylation catalysed by hepatic and intestinal microsomes from mouse and human. Cooperativity in nifedipine oxidation was increased by the addition of αNF to pooled human hepatic microsomes, but little effects of αNF could be observed in individual human intestinal microsomes. 4. These results suggest that CYP3A enzymes in liver and intestine might have different characteristics and that observations from hepatic microsomes should not be directly applicable to intestine metabolism in some cases. Studies of drug-drug interactions of CYP3A substrates are recommended to be performed using intestinal samples.     

Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc.

Rat liver microsomes were incubated in the presence of zinc and the rate of NADPH oxidation and related metabolism of aniline and ethylmorphine by appropriate oxidases were studied. A competitive mechanism of the inhibition of NADPH oxidation by zinc was found, with V_max = 10.3 nmoles NADP/min/mg of protein and K_i amounting to 7.22 μM zinc. In microsomes dialyzed against EDTA, addition of Mn²⁺ but not of Mg²⁺ enhanced the rate of NADPH oxidation. A complex relation of Zn²⁺ and Mn²⁺ in liver microsomes was found, the data not obeying the rigorous treatment for enzyme kinetics. The activity of aniline hydroxylase and ethylmorphine-N-demethylase was inhibited by zinc; 50 per cent inhibition was reached at 60 and 55 μM Zn²⁺ respectively. Another microsomal enzyme, glucose 6-phosphatase, independent of NADPH, was not affected by zinc. The content and spectral characteristics of cytochrome P-450 were not affected by zinc. It is concluded that Zn²⁺ inhibits oxidation of NADPH and prevents this pyridine nucleotide from functioning in the microsomal electron transport system. The possibility that Zn²⁺ may interfere with other ions or enzymes involved in microsomal electron transport cannot be excluded.     

Oxidative deamination of cyclohexylamine and its homologs by rabbit liver microsomes.

Cyclohexylamine (CHA) and its homologs, cyclopentylamine (CPA) and cycloheptylamine (CHPA), which formed the type II spectral changes in hepatic microsomes, were deaminated to the corresponding ketones by rabbit liver microsomes in the presence of NADPH and molecular oxygen. The alicyclic ketones were then reduced to the alcohols, of which average percentages in the deaminated products were approximately 75 (CHA), 3 (CPA) and 14 (CHPA). The apparent K_m's for these amines were 5.0 mM (CHA), 4.2 mM (CPA) and 2.1 mM (CHPA), and V_max's were 11.0 (CHA), 42.1 (CPA) and 16.4 (CHPA) nmoles/mg protein/30 min. The activity of deamination of these alicyclic primary amines was dependent on both NADPH and oxygen, and inhibited by carbon monoxide, SKF 525A, metyrapone, potassium cyanide and mercuric chloride. These experiments indicate that the deamination of the alicyclic primary amines is catalyzed by a microsomal cytochrome P-450-dependent monooxygenase system in the rabbit liver. Cyclohexanone oxime and other oximes were also identified from the incubation mixtures, and these oximes are suggested as possible intermediates of microsomal deamination of alicyclic primary amines.     

Inhibition of NADPH-cytochrome P450 reductase by tannic acid in rat liver microsomes and primary hepatocytes: methodological artifacts and application to ischemia-reperfusion injury

Tannic acid (TA) inhibits nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) activity, which is measured by reduction of cytochrome c, in rat liver microsomes (RLMs). In the current study, we noticed that TA directly reduces cytochrome c in the absence of microsomes, thus confounding the CPR activity assay. A method is presented that measures CPR activity in the presence of TA by subtracting the cytochrome c reduction in the absence of NADPH (TA effect) from that in the presence of NADPH (TA plus CPR effect). The method was used to determine the inhibitory effect of TA in RLMs, recombinant CPR enzyme, and primary hepatocytes. Additionally, application of TA in a study of role of CPR in a primary rat hepatocyte model of ischemia-reperfusion (IR) was investigated. TA showed concentration-dependent, complete inhibition of CPR with half maximal inhibitory concentration (IC(50) ) values of 58.2 μM in RLMs and 54.6 and 275 μM in primary rat hepatocytes in the absence and presence of serum in the medium, respectively. Additionally, inhibition of CPR by TA was associated with a significant reduction in reactive oxygen species and cell death after IR injury. These data may be useful in future studies using TA as an inhibitor of CPR in microsomes and primary hepatocytes.     

Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo

Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC??) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC?? > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids.     

Behavioral and immunohistological assessment of painful neuropathy induced by a single oxaliplatin injection in the rat

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.     

Characterization of human liver cytochrome P450 enzymes involved in the metabolism of rutaecarpine

Rutaecarpine has recently been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition. The incubation of rutaecarpine with human liver microsomes in the presence of NADPH generated six isobaric mono-hydroxylated metabolites. The specific cytochrome P450 (CYP) isozymes responsible for rutaecarpine metabolites were identified using the combination of chemical inhibition, immuno-inhibition and metabolism by cDNA expressed CYP enzymes. The results suggested that CYP3A4 might play major roles in the metabolism of rutaecarpine in human liver microsomes. The production of M1, M2, M3, M4 and M6 formed in human liver microsomes was inhibited by ketoconazole, a selective CYP3A4 inhibitor, and anti-CYP3A4 antibody. CYP1A2 and CYP2C9 played minor roles in the metabolism of rutaecarpine. These results were confirmed in microsomes derived from cDNA expressed lymphoblastoid cells. CYP3A4 microsome clearly formed M1, M2, M3 and M6. CYP1A2 and CYP2C9 microsomes comparably formed M5.     

Influence of antidepressant drugs on chlorpromazine metabolism in human liver--an in vitro study

The aim of the present study was to investigate the possible effects of antidepressant drugs (fluvoxamine, imipramine) on the metabolism of the aliphatic-type phenothiazine neuroleptic chlorpromazine in the human liver. The experiment was performed in vitro using human liver microsomes. The kinetic analysis of chlorpromazine metabolism carried out in the absence or presence of antidepressants showed that fluvoxamine potently inhibited chlorpromazine 5-sulfoxidation (K(i) = 2.8 μM), mono-N-demethylation (K(i) = 1.4 μM) and di-N-demethylation (K(i) = 1.1 μM) via a competitive mechanism at therapeutic antidepressant concentrations. Imipramine moderately diminished the rate of chlorpromazine 5-sulfoxidation (K(i) = 8.7 μM, competitive inhibition), mono-N-demethylation (K(i) = 16.0 μM, non-competitive inhibition) and di-N-demethylation (K(i) = 13.5 μM mixed inhibition). Considering the serious side-effects of chlorpromazine and some of its metabolites, metabolic interactions between this neuroleptic and antidepressant drugs (especially the chlorpromazine-fluvoxamine interaction) may be of pharmacological and clinical importance.     

Evaluation of Six Proton Pump Inhibitors As Inhibitors of Various Human Cytochromes P450: Focus on Cytochrome P450 2C19

Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC(50) values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC(50) = ～8 μM) and esomeprazole with CYP2C8 (IC(50) = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC(50) ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC(50) (IC(50) ratio, ≤2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC(50) = 0.73 μM) and esomeprazole (IC(50) = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC(50) = ～7.0 μM). Rabeprazole and pantoprazole (IC(50) = ≥25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC(50) ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.