Breaking the sensitivity limitations of cytochrome P450 oxidation product: dansyl chloride derivatisation of 4-OH mephenytoin, a CYP2C19 metabolite and its application to in vitro CYP inhibition assay.

A rapid selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of derivatised cytochrome P450-2C19 oxidation product (dansyl-4-OH mephenytoin) and its underivatised form (4-OH mephenytoin). Samples were anaysed on C18 column (Waters Xbridge, 50 mm×4.6 mm, 3.5 µm particle size) with the mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A gradient method with a short run time of 2.5 min and 3.5 min was developed for the analysis of dansyl-4-OH mephenytoin and 4-OH mephenytoin, respectively. The standard curve was linear (r(2)=0.9972 for 4-OH mephenytoin; r(2)=0.9946 for dansyl-4-OH mephenytoin) over the concentration range of 0.16 to 40 ng/mL for both derivatised and underivatised forms. The CV (%) and relative error (RE) for inter and intraassay at three QC levels for dansyl-4-OH mephenytoin was 0.97-5.85% and -9.80 to 2.51%, respectively. Whereas, for 4-OH mephenytoin the CV (%) and RE (%) at three QC levels was 0.82-3.47% and -6.69 to -0.01%, respectively. The developed method was validated for various parameters such as linearity, precision & accuracy, extraction recovery, matrix effect, autosampler stability and was proved to be consistent across three QC levels with overall CV (%) less than 15. Dansylation helped in increasing the sensitivity of hydroxy mephenytoin by 100-200 fold. Given the simplicity involved in derivatisation process, we believe that this novel methodology will change the current approaches used for the enhancing the detection sensitivity of 4-OH mephenytoin.

How did phenobarbital's chemical structure affect the development of subsequent antiepileptic drugs (AEDs)?

Phenobarbital has been in clinical use as an antiepileptic drug (AED) since 1912. The initial clinical success of phenobarbital and other barbiturates affected the design of subsequent AEDs (e.g., phenytoin, primidone, ethosuximide), developed between 1938 and 1962, the chemical structures of which resemble that of phenobarbital. However, the empirical discovery of carbamazepine (1962) and the serendipitous discovery of valproic acid (1967) led to subsequent AEDs having chemical structures that are diverse and completely different from that of phenobarbital. Sixteen AEDs were introduced between 1990 and 2012. Most of these AEDs were developed empirically, using mechanism-unbiased anticonvulsant animal models. The empirical nature of the discovery of these AEDs, coupled with their multiple mechanisms of action, explains their diverse chemical structures. The antiepileptic market is therefore crowded. Future design of new AEDs must have a potential for treating nonepileptic central nervous system (CNS) disorders (e.g., bipolar disorder, neuropathic pain, migraine prophylaxis, or restless legs syndrome). The barbiturates were once used as sedative-hypnotic drugs, but have been largely replaced in this role by the much safer benzodiazepines. In contrast, phenobarbital is still used worldwide in epilepsy. Nevertheless, the development of nonsedating phenobarbital derivatives will answer a clinical unmet need and might make this old AED more attractive.

Identification of new CYP2C19 variants exhibiting decreased enzyme activity in the metabolism of S-mephenytoin and omeprazole.

Although many cases of interindividual variation in the metabolism of CYP2C19 drugs are explained by the CYP2C19*2, *3, and *17, a wide range of metabolic variation still occurs in people who do not carry these genetic variants. The objectives of this study were to identify new genetic variants and to characterize functional consequences of these variants in metabolism of CYP2C19 substrates. In total, 21 single-nucleotide polymorphisms including three new coding variants, V394M, E405K, and D256N, were identified by direct DNA sequencing in 50 randomly selected subjects and in individuals who exhibited an outlier phenotype response in the omeprazole study. Recombinant proteins produced from the coding variants V394M, E405K, and D256N were prepared by using an Escherichia coli expression system and purified. Metabolism of S-mephenytoin and omeprazole by V394M was comparable with that of the wild-type protein. E405K showed a moderate decrease in metabolism of the substrates. However, D256N exhibited a significantly decreased activity in S-mephenytoin metabolism, resulting in 50 and 76% decreases in V(max) and intrinsic clearance, respectively, compared with the wild type. This variant also exhibited a significant decrease in omeprazole metabolism in vivo. CYP2C19 D256N and E405K were assigned as CYP2C19*26 and *2D, respectively, by the Cytochrome P450 Nomenclature Committee. In summary, this report characterizes the allele frequency and haplotype distribution of CYP2C19 in a Korean population and provides functional analysis of new coding variants of the CYP2C19 gene. Our findings suggest that individuals carrying CYP2C19*26 would have lower activity for metabolizing CYP2C19 substrate drugs.

CYP2C19 inhibition: the impact of substrate probe selection on in vitro inhibition profiles.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical part of the drug discovery process. Factors such as nonspecific binding, atypical kinetics, poor effector solubility, and varying ratios of accessory proteins may alter the kinetic behavior of an enzyme and subsequently confound the extrapolation of in vitro data to the human situation. The architecture of the P450 active site and the presence of multiple binding regions within the active site may also confound in vitro-in vivo extrapolation, as inhibition profiles may be dependent on a specific inhibitor-substrate interaction. In these studies, the inhibition profiles of a set of 24 inhibitors were paneled against the CYP2C19 substrate probes (S)-mephenytoin, (R)-omeprazole, (S)-omeprazole, and (S)-fluoxetine, on the basis of their inclusion in recent U.S. Food and Drug Administration guidance for in vitro drug-drug interactions with CYP2C19. (S)-Mephenytoin was inhibited an average of 5.6-fold more potently than (R)- or (S)-omeprazole and 9.2-fold more potently than (S)-fluoxetine. Hierarchical clustering of the inhibition data suggested three substrate probe groupings, with (S)-mephenytoin exhibiting the largest difference from the rest of the substrate probes, (S)-fluoxetine exhibiting less difference from (S)-mephenytoin and the omeprazoles and (R)- and (S)-omeprazole exhibiting minimal differences from each other. Predictions of in vivo inhibition potency based on the in vitro data suggest that most drug-drug interactions will be identified by either (S)-mephenytoin or omeprazole, although the expected magnitude of the interaction may vary depending on the chosen substrate probe.

Functional characterization of two novel CYP2C19 variants (CYP2C19*18 and CYP2C19*19) found in a Japanese population.

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.

Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry.

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.

CYP2B6, CYP3A4, and CYP2C19 are responsible for the in vitro N-demethylation of meperidine in human liver microsomes.

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors.

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K(i) towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(-)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K(i) values of < 250nM--at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.

Studies of binding modes of (S)-mephenytoin to wild types and mutants of cytochrome P450 2C19 and 2C9 using homology modeling and computational docking.

PURPOSE: This study investigated the structural features of CYP2C19 complexed with (S)-mephenytoin, using computational methods. In addition to wild-type CYP2C19 proteins (1A and 1B), which have selective 4-hydroxylase activities of (S)-mephenytoin, CYP2C19 mutants were also studied, together with a wild type and artificial mutants of CYP2C19. METHODS: Three-dimensional structures of wild-type and mutant proteins of CYP2C19 and CYP2C9 were estimated from homology modeling using the crystal structure of rabbit CYP2C5 as a reference. The binding mode of (S)-mephenytoin to CYP2C19 was investigated using computational docking. RESULTS: The results reproduced the specific bindings between (S)-mephenytoin and the wild types of CYP2C19. Our findings suggest that Asp293 of CYP2C19 plays an important role in the binding of (S)-mephenytoin, which was surrounded by Vall13 and Ala297, and points the phenyl ring at the heme iron. In addition the wild types of CYP2C19, the computational docking studies also accounted for the experimental activities of CYP2C19 mutants, and wild-type and mutant CYP2C19 proteins. CONCLUSIONS: These results confirm that the predicted three-dimensional structure of the CYP2C19-(S)-mephenytoin complex is reasonable, and that this strategy is useful for investigating complex structures. Virtual screening for drug discovery can also be carried out using these methods.

(+)-N-3-Benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital: new potent and selective in vitro inhibitors of CYP2C19.

Highly potent and selective CYP2C19 inhibitors are not currently available. In the present study, N-3-benzyl derivatives of nirvanol and phenobarbital were synthesized, their respective (+)- and (-)-enantiomers resolved chromatographically, and inhibitor potencies determined for these compounds toward CYP2C19 and other human liver cytochromes P450 (P450s). (-)-N-3-Benzyl-phenobarbital and (+)-N-3-benzyl-nirvanol were found to be highly potent, competitive inhibitors of recombinant CYP2C19, exhibiting K(i) values of 79 and 250 nM, respectively, whereas their antipodes were 20- to 60-fold less potent. In human liver preparations, (-)-N-3-benzyl-phenobarbital and (+)-N-3-benzyl-nirvanol inhibited (S)-mephenytoin 4'-hydroxylase activity, a marker for native microsomal CYP2C19, with K(i) values ranging from 71 to 94 nM and 210 to 280 nM, respectively. At single substrate concentrations of 0.3 microM [(-)-N-3-benzyl-phenobarbital] and 1 microM [(+)-N-3-benzyl-nirvanol] that were used to examine inhibition of a panel of cDNA-expressed P450 isoforms, neither CYP1A2, 2A6, 2C8, 2C9, 2D6, 2E1, nor 3A4 activities were decreased by greater than 16%. In contrast, CYP2C19 activity was inhibited approximately 80% under these conditions. Therefore, (+)-N-3-benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital represent new, highly potent and selective inhibitors of CYP2C19 that are likely to prove generally useful for screening purposes during early phases of drug metabolism studies with new chemical entities.

Phenotype analysis of cytochrome P450 2C19 in Chinese subjects with mephenytoin S/R enantiomeric ratio in urine measured by chiral GC.

A chiral gas chromatographic method with FID was developed for the determination of S- and R-mephenytoin in human urine. The assay is linear from 25 to 800 ng/mL for each enantiomer and the limit of detection and limit of quantitation were 12 and 25ng/mL for each enantiomer, respectively. The method affords average recoveries of 74.41 +/- 3.93% and 73.78 +/- 3.02% for S- and R-mephenytoin, respectively. The method allows the phenotype study of CYP2C19 in Chinese subjects. The phenotype pattern of 90 Chinese volunteers was determined, in which 26 volunteers received phenotyping and genotyping tests. The results of phenotype analysis showed that the interindividual variation was marked. The mephenytoin S/R enantiomeric ratios in urine of 11 volunteers were > or = 0.95 and identified as poor metabolizers. The frequency of poor metabolizers was 12.2% in the Chinese subjects tested. A good relationship between phenotype and genotype analysis of CYP2C19 was observed.

Mephenytoin overdose--phenytoin poisoning incognito? Case report and mephenytoin/phenytoin comparison.

BACKGROUND: Unlike phenytoin, overdose from mephenytoin (CAS No. 50-12-4) is rare. A review of mephenytoin shows a number of differences from phenytoin, including structural, metabolic, pharmacodynamic, and toxicologic effects. Mephenytoin metabolism is also characterized by genetic polymorphism, which can result in prolonged elimination. Routine blood testing for phenytoin may show no interference from mephenytoin. Mephenytoin levels are not readily available nor clinically useful when they do become available; urine toxicology screen may be positive for barbiturates. CASE REPORT: A 26-year-old female overdosed on approximately 12 g of mephenytoin and an unknown amount of valproic acid. She became comatose, developing pulmonary aspiration and pancreatitis with fever. Her Intensive Care Unit treatment was prolonged with slow resolution over 10 days. A review of mephenytoin and comparison to phenytoin overdose is provided in the context of this case report.

Homology modeling and substrate binding study of human CYP2C18 and CYP2C19 enzymes.

It is well established that the variable binding-site architecture and composition of the P450 metabolizing heme proteins are major modulators of substrate and product specificity. Even the three closely related human liver isozymes, CYP2C9, CYP2C18, and CYP2C19, do not share all substrates and do not always lead to the same preferred hydroxylation products. The lack of knowledge of their three-dimensional (3D) structures has hindered efforts to understand the differences in their specificities. Building on previous work for the CYP2C9 enzyme, 3D models of CYP2C18 and 2C19 have been constructed and validated by computational methods developed and tested in our laboratory. They were used to characterize explicit enzyme-substrate complexes using the isoform-specific substrates progesterone and (S)-mephenytoin for 2C19 and 2-[2,3-dichloro-4-(3-hydroxypropyloxy)benzoyl]thiophene for 2C18. The results allowed both common and unique binding-site residues to be identified in each model. The calculated preferred hydroxylation site was obtained for each substrate and was found to be consistent with experimental observation. Comparisons were made among the 2C9, 2C18, and 2C19 model binding sites to investigate the subtle differences among them. These models can be used as structure-based guides for mutagenesis studies and screening of potential pharmaceuticals or toxins.

[Determination of R- and S-mephenytoin in human urine by gas chromatography].

The levels of R- and S-mephenytoin in human urine were determined by chiral capillary gas chromatography with nitrogen-phosphorus detector. The conditions of the chromatography and detection included a chiral capillary column (Chirasil-Val, 25 m x 0.25 mm i.d., Alltech), column temperature (T) 190 degrees C, injector T 220 degrees C, detector T 240 degrees C, and the flow-rates of 0.85, 3.5 and 120 mL/min for nitrogen, hydrogen and air respectively. Based on the above conditions, the satisfactory separation of R- and S-mephenytoin was gained, and other interference from urine sample was not found. The linear curves for both tested compounds ranged between 12.5 and 2500 microg/L, with a minimum detectable concentration of about 6 microg/L. Because of its simplicity, rapidity, sensitivity and accuracy, this method has been extensively used for testing metabolic ability of S-mephenytoin polymorphic oxidation in the Chinese populations and for the determination of the activity of hepatic drug-metabolizing enzyme CYP2C19 using mephenytoin S/R ratio of the urine samples of subjects as an indicator.

Stereoselective disposition of mianserin is related to debrisoquin hydroxylation polymorphism.

The pharmacokinetics of mianserin and its main metabolite desmethylmianserin were studied in poor and extensive metabolizers of debrisoquin and of S-mephenytoin after a single oral dose of racemic mianserin. The debrisoquin metabolic ratio (MR) correlated significantly with area under the serum concentration-time curves (AUC) for (+/-)-mianserin and (+/-)-desmethylmianserin. Enantioselective high-performance liquid chromatographic analysis of mianserin showed that debrisoquin MR was related to AUC(0-12) for S(+)-mianserin (rs = 0.87; p = 0.001; n = 15) but not for R(-)-mianserin. The ratio between the AUC(0-12) for S(+)-mianserin and that for R(-)-mianserin was higher in poor metabolizers than in extensive metabolizers. Two extremely rapid extensive metabolizer subjects had the lowest mianserin S/R ratios. No differences in the pharmacokinetics of mianserin or desmethylmianserin were found between extensive metabolizers and poor metabolizers of S-mephenytoin. The study shows that the elimination of both mianserin and its main metabolite desmethylmianserin is dependent on CYP2D6 activity. Furthermore, the CYP2D6-dependent elimination of mianserin shows marked enantioselectivity for the more active S(+)-enantiomer of mianserin.

A methodological investigation on the estimation of the S-mephenytoin hydroxylation phenotype using the urinary S/R ratio.

After a single oral dose of racemic mephenytoin the S/R ratio in urine can be used to phenotype extensive (EM) and poor metabolizers (PM) of S-mephenytoin. We confirmed the increased S/R ratio by storage time in EM because of the hydrolysis of a conjugate of S-mephenytoin excreted in EM, but not in PM. The S/R ratio in the 0-8 h urine increased 8- to 127-fold (from 0.22 +/- 0.16 to 9.9 +/- 11.3) after acid treatment of urine from 30 EM, but there was no effect of acid in that of 12 PM. We suggest that the phenotype of mephenytoin in combination with debrisoquine can be determined in the 0-8 h urine by estimating the mephenytoin S/R ratio before and after acid treatment.

Drug-metal interactions: spectroscopic studies of copper hydantoin complexes.

The preparation and spectral properties of copper(II) complexes of two hydantoins are reported. Complexes of the general formula Cu(hyd)2(py)2, where hyd = phenytoin or nirvanol; and py = pyridine were prepared and characterized by infrared and ESR. Spectral data show that the copper atom is bound to the nitrogen atom of the hydantoin anion and to the nitrogen atom of the pyridine molecule to form 2:2:1 hydantoin:pyridine:copper complexes. The ESR data indicate that both complexes have tetragonal symmetry (g11 greater than g perpendicular greater than g e) with the unpaired electron in the d x2-y2 orbital.