Optimizing higher throughput methods to assess drug-drug interactions for CYP1A2, CYP2C9, CYP2C19, CYP2D6, rCYP2D6, and CYP3A4 in vitro using a single point IC(50)

Drug-drug interactions involving cytochrome P(450) (CYP) are an important factor in whether a new chemical entity will survive through to the development stage. Therefore, the identification of this potential as early as possible in vitro could save considerable future unnecessary investment. In vitro CYP interaction screening data generated for CYP2C9, CYP2D6, and CYP3A4 were initially analyzed to determine the correlation of IC(50) from 10- and 3-point determinations. A high correlation (r = 0.99) prompted the further assessment of predicting the IC(50) by a single value of percent inhibition at either 10, 3, or 1 microM. Statistical analysis of the initial proprietary compounds showed that there was a strong linear relationship between log IC(50) and percent inhibition at 3 microM, and that it was possible to predict a compound's IC(50) by the percent inhibition value obtained at 3 microM. Additional data for CYP1A2, CYP2C19, and the recombinant CYP2D6 were later obtained and used together with the initial data to demonstrate that a single statistical model could be applicable across different CYPs and different in vitro microsomal systems. Ultimately, the data for all five CYPs and the recombinant CYP2D6 were used to build a statistical model for predicting the IC(50) with a single point. The 95% prediction boundary for the region of interest was about +/- 0.37 on log(10) scale, comparable to the variability of in vitro determinations for positive control IC(50) data. The use of a single inhibitor concentration would enable determination of more IC(50) values on a 96-well plate and result in more economical use of compounds, human liver or expressed enzyme microsomes, substrates, and reagents. This approach would offer the opportunity to increase screening for CYP-mediated drug-drug interactions, which may be important given the challenges provided by the generation of orders of magnitude more new chemical entities in the field of combinatorial chemistry. In addition, the algorithmic approach we propose would obviously be applicable for other in vitro bioactivity and therapeutic target enzyme and receptor screens.     

Inhibitory Effects of the Monoamine Oxidase Inhibitor Tranylcypromine on the Cytochrome P450 Enzymes CYP2C19, CYP2C9, and CYP2D6

1. The inhibitory effects of tranylcypromine, a nonselective irreversible inhibitor of monoamine oxidase (MAO), on three cytochrome P450 (CYP) enzymes, namely CYP2C9, CYP2C19, and CYP2D6, have been evaluated in vitro. 2. The studies were conducted using cDNA-expressed human CYP enzymes and probe substrates. 3. A range of substrate concentrations was coincubated with a range of tranylcypromine concentrations in the presence of each of the CYP enzymes at 37 degrees C for a predetermined period of time. Product concentrations were quantified by HPLC with UV detection. 4. The results demonstrated that tranylcypromine is a competitive inhibitor of CYP2C19 (Ki = 32 microM) and CYP2D6 (Ki = 367 microM) and a noncompetitive inhibitor of CYP2C9 (Ki = 56 microM). 5. None of these inhibitory effects are considered clinically significant at usual therapeutic doses. However, in certain situations such as high dose tranylcypromine therapy, or in poor metabolizers of CYP2C19 substrates, clinically significant interactions might occur, particularly when tranylcypromine is coadministered with drugs with a narrow therapeutic index.     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Interethnic and intraethnic variability of CYP2C8 and CYP2C9 polymorphisms in healthy individuals

Cytochrome P450 (CYP) superfamily members CYP2C8 and CYP2C9 are polymorphically expressed enzymes that are involved in the metabolic inactivation of several drugs, including, among others, antiepileptics, NSAIDs, oral hypoglycemics, and anticoagulants. Many of these drugs have a narrow therapeutic index, and growing evidence indicates a prominent role of CYP2C8 and CYP2C9 polymorphisms in the therapeutic efficacy and in the development of adverse effects among patients treated with drugs that are CYP2C8 or CYP2C9 substrates. In this review, we summarize present knowledge on human variability in the frequency of variant CYP2C8 and CYP2C9 alleles. Besides an expected interethnic variability in allele frequencies, a large intraethnic variability exists. Among Asian subjects, for example, statistically significant differences (p < 0.0001) in CYP2C9*3 allele frequencies between Chinese and Japanese individuals have been reported. In addition, individuals from East Asia present different allele frequencies for CYP2C9*2 and CYP2C9*3 compared with South Asian subjects (p < 0.0001). Among Caucasian Europeans, statistically significant differences for the frequency of CYP2C8*3, CYP2C9*2, and CYP2C9*3 exist (p < 0.0001). This indicates that Asian individuals or Caucasian European individuals cannot be considered as homogeneous groups regarding CYP2C8 or CYP2C9 allele frequencies. Caucasian American subjects also show a large variability in allele frequencies, which is likely to be related to ethnic ancestry. A higher frequency of variant CYP2C8 and CYP2C9 alleles is expected among Caucasian Americans with South European ancestry than in individuals with North European ancestry. The findings summarized in this review suggest that among individuals with Asian or European ancestry, intraethnic differences in the risk of developing adverse effects with drugs that are CYP2C8 or CYP2C9 substrates are to be expected. In addition, the observed intraethnic variability reinforces the need for proper selection of control subjects and points against the use of surrogate control groups for studies involving association of CYP2C8 or CYP2C9 alleles with adverse drug reactions or spontaneous diseases.     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

Lycopene attenuates alcoholic apoptosis in HepG2 cells expressing CYP2E1

To test the hypothesis that ethanol-induced hepatic apoptosis is secondary to the oxidative stress generated by cytochrome P4502E1 (CYP2E1), we assessed the effects of the carotenoid lycopene, a potent antioxidant extracted from tomatoes, on oxidative stress and apoptosis in HepG2 cells overexpressing CYP2E1 (2E1 cells). These were exposed for 5 days to 100mM ethanol and 10 microM lycopene or an equal volume of placebo (vehicle). Ethanol significantly increased apoptosis measured by flow cytometry and by TUNEL assay. This was accompanied by an ethanol-induced oxidative stress: hydrogen peroxide production was significantly increased and mitochondrial GSH was strikingly decreased. Both were restored by lycopene, with a significant decrease in apoptosis. The placebo had no protective effect. In conclusion, Lycopene opposes the ethanol-induced oxidative stress and apoptosis in 2E1 cells. The parallelism between these effects suggests a causal link. Furthermore, these beneficial effects and the innocuity of lycopene now justify an in vivo trial.     

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases.

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital-induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well-defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48-h treatment of the hepatocytes with 2-aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose-dependent increases in both basal and phenobarbital-induced CYP2A5 catalytic activity (assayed as coumarin 7-hydroxylation), the maximal induction being 60-fold greater than the control value upon cotreatment with 1.5 mM phenobarbital and 10 mM 2-aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O-deethylase) and CYP1A1/2 (ethoxyresorufin O-deethylase) activities were blocked by 2-aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K-252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital-induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.     

Chimeric cDNA expression and site directed mutagenesis studies of cytochrome P450s CYP2A1 and CYP2A2

Construction of chimeras and site directed mutagenesis were used to study the regioselectivity and kinetics of testosterone hydroxylation by the cytochrome P450s CYP2A1 and CYP2A2. Although these enzymes exhibit 88% sequence similarity, they catalyze very different regioselective hydroxylations of testosterone. Active chimeras inwhich the first 355 amino acids do not correspond to a single enzyme show broad radioselectivity, whereas the specificity of the parent enzyme is obtained if the first 355 amino acids are unchanged. Therefore, the region between amino acids 275 and 355 is important in maintaining regioselectivity. Single point mutants were constructed for the 13 amino acid differences in this region. For 26 single point and 2 double mutants all active mutants have the same regioselectivity as the parent enzymes. However, kinetic analysis of the CYP2A1 mutants showed that 4 single point mutants and 1 double mutant had kinetic parameters very different from the parent enzyme. All of these substitutions are associated with the conserved dioxygen binding region of the putative I helix predicted from the crystal structure of P450_cam. Deuterium isotope effects were used to determine any changes in the rate of reduction and to estimate the relative amount of excess water formation. Changes in reduction rates are not sufficient to account for the differences in V_max values. Therefore, it is likely that the amount of hydrogen peroxide formed is a primary determinant of V_max.     

Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-β/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-β1-induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy.     

Phosphorylation/Dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital.

The effects of several protein kinase activators and protein phosphatase inhibitors on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes were investigated. Insulin, epidermal growth factor, interleukin 6, cAMP, phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, vanadate, and okadaic acid were found to suppress the induction of CYP2B1/2B2 at mRNA and protein levels in hepatocytes. cAMP and vanadate completely suppressed the induction of CYP2B1/2B2 gene expression in both rat hepatocytes and liver. The addition of genistein to vanadate-treated hepatocytes partially recovered the induction of CYP2B1/2B1 gene expression by PB. These results of the present study demonstrate that phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1/2B2 gene expression by PB.     

High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.     

Characterization of CYP1A2, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in South Brazilians

Potential causes of variability in drug response include intrinsic factors such as ethnicity and genetic differences in the expression of enzymes that metabolize drugs, such as those from Cytochrome P450 (CYPs) superfamily. Pharmacogenetic studies search for genetic differences between populations since relevant alleles occur with varying frequencies among different ethnic populations. The Brazilian population is one of the most heterogeneous in the world, resulting from multiethnic admixture of Amerindians, Europeans, and Africans across centuries. Since the knowledge of CYP allele frequency distributions is relevant to pharmacogenetic strategies and these data are scarce in the Brazilian population, this study aimed to describe genotype and allele distributions of 15 single nucleotide polymorphisms (SNPs) at CYP 1A2, 2C19, 3A4, and 3A5 genes in African and European descents from South Brazil. A sample of 179 healthy individuals of European and African ancestry was genotyped by the MassARRAY SNP genotyping system. CYP3A5*3, CYP1A2*1F, CYP3A4*1B, and CYP2C19*2 were the most frequent alleles found in our sample. Significant differences in genotype and allelic distribution between African and European descents were observed for CYP3A4 and CYP3A5 genes. CYP3A4*1B was observed in higher frequency in African descents (0.379) than in European descents (0.098), and European descents showed higher frequency of CYP3A5*3 (0.810) than African descents (0.523). Our results indicate that only a few polymorphisms would have impact in pharmacogenetic testing in South Brazilians. Further studies with larger sample sizes are required also among other Brazilian regions.     

Changes of midazolam pharmacokinetics in Wistar rats treated with lipopolysaccharide: relationship between total CYP and CYP3A2

It has been reported that infection interferes with drug metabolism, resulting in changes in pharmacokinetics. In this study, we investigated the effects of lipopolysaccharide (LPS) on hepatic total cytochrome P450 (CYP), CYP3A2, and CYP2C11 contents in a transient, LPS-induced, endotoxemia model of rats. In addition, to assess the effects on CYP3A2 activities, the pharmacokinetics of midazolam (CYP3A2 substrate) and 1-OH-midazolam (metabolite of midazolam) were investigated. Hepatic total CYP contents were significantly low until day 3 (P < 0.05) but returned to the control level on day 5. Hepatic CYP3A2 contents were significantly decreased on day 1 until day 5 (P < 0.05) but returned to the control level on day 7. Hepatic CYP2C11 contents were continuously low until day 7, and lowest on day 3. The AUC of 1-OH-midazolam was significantly decreased on day 1 after LPS administration (P < 0.01). In conclusion, LPS (5 mg/kg) challenge decreased hepatic total CYP, CYP3A2, and CYP2C11 contents and also decreased the activities of hepatic CYP3A2. It took at least 7 days for hepatic total CYP and CYP3A2 to recover to control levels, and it was suggested that the changes of hepatic total CYP contents might correlate with those of hepatic CYP3A2 contents and activities. Additionally, it is shown that their changes might reflect the recovery process from inflammation.     

Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19

Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug–drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.     

Soy protein isolate induces CYP3A1 and CYP3A2 in prepubertal rats

Feeding soy diets has been shown to induce cytochrome P450s in gene family CYP3A in Sprague-Dawley rat liver. We compared expression of CYP3A enzymes on postnatal Day 33 (PND33) rats fed casein or soy protein isolate (SPI+)-based AIN-93G diets continuously from gestational Day 4 through PND33 or the diets were switched on PND15 (n = 3-6 litters) to examine the potential imprinting effects of soy on drug metabolism. In addition rats were fed casein, SPI+, SPI+ stripped of phytochemicals (SPI-), or casein diets supplemented with the soy-associated isoflavones genistein or daidzein from weaning through PND33 to examine the hypothesis that the isoflavones are responsible for CYP3A induction by soy feeding. Feeding SPI either continuously or from weaning induced hepatic CYP3A1 and CYP3A2 mRNA, apoprotein, and CYP3A-dependent testosterone 6beta-hydroxylase activity in liver microsomes 2- to 5-fold (P < 0.05). CYP3A mRNA expression was also elevated 2- to 3-fold in the jejunum of SPI-fed rats (P < 0.05). CYP3A was not induced in livers of rats switched to casein from soy at weaning. Induction of CYP3A1 also did not occur in rats fed SPI-, but CYP3A2 mRNA and apoprotein were induced (P < 0.05) in females fed SPI-. Offspring weaned onto genistein-supplemented diets had no elevation of CYP3A mRNAs or apoproteins. Weaning onto daidzein diets increased CYP3A2 mRNA and apoprotein expression in male rats (P < 0.05). These data suggest that early soy consumption may increase the metabolism of a wide variety of CYP3A substrates, but that soy does not imprint the expression of CYP3A enzymes. Effects on CYP3A1 expression appear to be primarily due to phytochemical components of SPI other than isoflavones. In contrast, consumption of soy protein and daidzein appear to be associated with the induction of CYP3A2.