Dextromethorphan and debrisoquine metabolism and polymorphism of the gene for cytochrome P450 isozyme 2D50 in Thoroughbreds.

OBJECTIVE To characterize polymorphisms of the gene for cytochrome P450 isozyme 2D50 (CYP2D50) and the disposition of 2 CYP2D50 probe drugs, dextromethorphan and debrisoquine, in horses. ANIMALS 23 healthy horses (22 Thoroughbreds and 1 Standardbred). PROCEDURES Single-nucleotide polymorphisms (SNPs) in CYP2D50 were identified. Disposition of dextromethorphan (2 mg/kg) and debrisoquine (0.2 mg/kg) were determined after oral (dextromethorphan) or nasogastric (debrisoquine) administration to the horses. Metabolic ratios of plasma dextromethorphan and total dextrorphan (dextrorphan plus dextrorphan-O-ß-glucuronide) and 4-hydroxydebrisoquine concentrations were calculated on the basis of the area under the plasma concentration-versus-time curve extrapolated to infinity for the parent drug divided by that for the corresponding metabolite. Pharmacokinetic data were used to categorize horses into the phenotypic drug-metabolism categories poor, extensive, and ultrarapid. Disposition patterns were compared among categories, and relationships between SNPs and metabolism categories were explored. RESULTS Gene sequencing identified 51 SNPs, including 27 nonsynonymous SNPs. Debrisoquine was minimally detected after oral administration. Disposition of dextromethorphan varied markedly among horses. Metabolic ratios for dextromethorphan ranged from 0.03 to 0.46 (mean, 0.12). On the basis of these data, 1 horse was characterized as a poor metabolizer, 18 were characterized as extensive metabolizers, and 3 were characterized as ultrarapid metabolizers. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that CYP2D50 is polymorphic and that the disposition of the probe drug varies markedly in horses. The polymorphisms may be related to rates of drug metabolism. Additional research involving more horses of various breeds is needed to fully explore the functional implication of polymorphisms in CYP2D50.

Characterization of the CYP2D6 drug metabolizing phenotypes of the Chilean mestizo population through polymorphism analyses.

We tested the influence of four polymorphisms and gene duplication in CYP2D6 on in vivo enzyme activity in a Chilean mestizo population in order to identify the most relevant genetic profiles that account for observed phenotypes in this ethnic group. CYP2D6*2 (2850C>T), *3 (2549A>del), *4 (1846G>A), *17 (1023C>T) and gene duplication were determined by PCR-RFLP or PCRL in a group of 321 healthy volunteers. Individuals with different variant alleles were phenotyped by determining debrisoquine 4-hydroxylase activity as a metabolic ratio (MR) using a validated HPLC assay. Minor allele frequencies were 0.41, 0.01, 0.12 and 0.00 for CYP2D6*2, *3, *4 and *17 variants, respectively, and the duplication frequency was 0.003. Genotype analysis correlated with phenotypes in 18 of 23 subjects (78.3%). 11 subjects were extensive metabolizers (EM), 8 were intermediate metabolizers (IM), 2 were poor metabolizers (PM) and 2 were ultra-rapid metabolizers (UM) which is fairly coincident with expected phenotypes metabolic ratios ranged from 0.11 to 126.41. The influence of CYP2D6*3 was particularly notable, although only heterozygote carriers were present in our population. Individuals homozygous for *4 were always PM. As expected, the only subject with gene duplication was UM. In conclusion, there was a clear effect of genotype on observed CYP2D6 activity. Classification of EM, PM and UM through genotyping was useful to characterize CYP2D6 phenotype in the Chilean mestizo population.

Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities.

INTRODUCTION: Chimeric mice with humanized livers were recently established by transplanting human hepatocytes. This mouse model that is repopulated with functional human hepatocytes could be a useful tool for investigating human hepatic cell biology and drug metabolism and for other preclinical applications. Successfully transplanting human hepatocytes into mice requires that recipient mice with liver failure do not reject these human cells and provide a suitable microenvironment (supportive niche) to promote human donor cell expansion and differentiation. To overcome the limitations of current mouse models, we used Alb-TRECK/SCID mice for in vivo human immature hepatocyte differentiation and humanized liver generation. METHODS: 1.5 µg/kg diphtheria toxin was administrated into 8-week-old Alb-TRECK/SCID mice, and the degree of liver damage was assessed by serum aspartate aminotransferase activity levels. Forty-eight hours later, mice livers were sampled for histological analyses, and the human donor cells were then transplanted into mice livers on the same day. Chimeric rate and survival rate after cell transplantation was evaluated. Expressions of human hepatic-related genes were detected. A human albumin enzyme-linked immunosorbent assay was performed after 50 days of transplantation. On day 60 after transplantation, drug metabolism was examined in mice. RESULTS: Both human primary fetal liver cells and hepatic stem cells were successfully repopulated in the livers of Alb-TRECK/SCID mice that developed lethal fulminant hepatic failure after administering diphtheria toxin; the repopulation rate in some mice was nearly 100%. Compared with human primary fetal liver cells, human hepatic stem cell transplantation rescued Alb-TRECK/SCID mice with lethal fulminant hepatic failure, and human hepatic stem cell-derived humanized livers secreted more human albumin into mouse sera and also functioned as a "human liver" that could metabolize the drugs ketoprofen and debrisoquine. CONCLUSION: Our model of a humanized liver in Alb-TRECK/SCID mice may provide for functional applications such as drug metabolism, drug to drug interactions, and promote other in vivo and in vitro studies.

Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies.

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin. METHODS: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions. RESULTS: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site. MAJOR CONCLUSION: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model. GENERAL SIGNIFICANCE: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.

Molecular insight into affinities of drugs and their metabolites to lipid bilayers.

The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in the human body. Here, we analyze partitioning of substrates of cytochrome P450s (caffeine, chlorzoxazone, coumarin, ibuprofen, and debrisoquine) and their metabolites (paraxanthine, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, 3-hydroxyibuprofen, and 4-hydroxydebrisoquine) on two model membranes: dioleoylphosphatidylcholine (DOPC) and palmitoyloleoylphophatidylglycerol (POPG). We calculated the free energy profiles of these molecules and the distribution coefficients on the model membranes. The drugs were usually located deeper in the membrane than the corresponding metabolites and also had a higher affinity to the membranes. Moreover, the behavior of the molecules on the membranes differed, as they seemed to have a higher affinity to the DOPC membrane than to POPG, implying they have different modes of action in human (mostly PC) and bacterial (mostly PG) cells. As the xenobiotics need to pass through lipid membranes on their way through the body and the effect of some drugs might depend on their accumulation on membranes, we believe that detailed information of penetration phenomenon is important for understanding the overall metabolism of xenobiotics.

The prototypic pharmacogenetic drug debrisoquine is a substrate of the genetically polymorphic organic cation transporter OCT1.

Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug-drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake. Debrisoquine showed low carrier-independent membrane permeability (P(e) of 0.01×10⁻6 cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP+ (IC50 of 6.2 ± 0.8 µM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis-Menten kinetics (K(M) of 5.9 ± 1.5 µM and V(max) of 41.9 ± 4.5pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V(max) by 48, 63 and 91%, respectively, but did not affect the K(M). The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake. In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.

Debrisoquine metabolism and CYP2D expression in marmoset liver microsomes.

The objective of this study was to define CYP2D enzymes in marmoset (Callithrix jacchus) liver microsomes, both at the activity level using debrisoquine as the model substrate and at the protein level using antibodies raised to human CYP2D6. Marmoset liver microsomes were incubated with [(14)C]debrisoquine, and the structure of the generated metabolites was determined using liquid chromatography-tandem mass spectrometry and NMR. Marmoset liver microsomes were very effective in hydroxylating debrisoquine at various positions. Although 4-hydroxydebrisoquine was formed, in contrast to rat and human it was only a minor metabolite. Debrisoquine was more extensively hydroxylated in the 7, 5, 6, and 8 positions. In addition to the monohydroxylated metabolites, a dihydroxy metabolite, namely 6,7-dihydroxydebrisoquine, was identified. Finally, metabolites that had undergone ring opening were also detected but were not investigated further. Antibodies to CYP2D6 immunoreacted with protein in marmoset and human but not rat hepatic microsomes. In conclusion, we demonstrate that marmoset liver microsomes are effective in hydroxylating debrisoquine at various positions and that they contain a protein that is immunorelated to human CYP2D6.

CYP2D6 genotype and debrisoquine hydroxylation phenotype in Cubans and Nicaraguans.

CYP2D6 genotype and debrisoquine metabolic ratio (MR) were analyzed in 133 Nicaraguan Mestizos (NMs) and 260 Cubans divided into Cuban Mestizos (CMs) and White Cubans (WCs). The frequencies of poor metabolizers (MR12.6) were 6% in NMs, 3.9% in CMs and 5.3% in WCs. The frequencies of ultrarapid metabolizers (MR0.1) were 0% in NMs, 2.3% in CMs and 5.3% in WCs. Mean (±s.d.) MR among extensive metabolizers (MR<12.6) was higher in NMs (1.5±1.6; n=118) than in CMs (1.0±1.3; n=124; P<0.001) and WCs (0.7±1.0; n=124; P<0.001). MR correlated with the 'activity score' of CYP2D6 genotypes (P<0.05; r=-0.55). Mean MR was higher among NMs than WCs and CMs for groups classified as 1 (P<0.05) or 2 (P<0.01) 'activity score'. In addition, mean (±s.d.) MR was higher among subjects carrying CYP2D6*17 than in CYP2D6 wt/wt (P<0.001). The CYP2D6*10 allele was higher in NMs (3.1%) than in CMs (0.8%; P<0.05) and WCs (0.4%; P<0.05). CYP2D6*17 allele was higher in CMs (10.2%) than WC (2.7%; P<0.005) and NMs (0%). Thus, the variability in CYP2D6 phenotypes found may be related to differences in allele frequency among groups (that is, CYP2D6*10 and *17 highest in NMs and CMs, respectively). However, the influence of environmental factors or alleles different than those studied here cannot be ruled out.

Assessment of chimeric mice with humanized liver as a tool for predicting circulating human metabolites.

The ability to predict circulating human metabolites of a candidate drug before first-in-man studies are carried out would provide a clear advantage in drug development. A recent report demonstrated that while in vitro studies using human liver preparations reliably predict primary human metabolites in plasma, the predictability of secondary metabolites, formed by multiple reactions, was low, with total success rates of < or =65%. Here, we assess the use of chimeric mice with humanized liver as an animal model for the prediction of human metabolism in vivo. Metabolism studies with debrisoquine and (S)-warfarin demonstrated significantly higher concentrations of their primary human abundant metabolites in serum or plasma in chimeric mice than in control mice. Humanized chimeric mice were also capable of producing human-specific metabolites of several in-house compounds which were generated through more than one metabolism reaction. This model is closer to in vivo human physiology and therefore appears to have an advantage over in vitro systems in predicting complex metabolites in human plasma. However, prediction of human metabolites failed for other compounds which were highly metabolized in mice. Although requiring careful consideration of compound suitability, this model represents a potential tool for predicting human metabolites in combination with conventional in vitro systems.

Pharmacogenetics of debrisoquine and its use as a marker for CYP2D6 hydroxylation capacity.

Debrisoquine hydroxylation polymorphism is by far the most thoroughly studied genetic polymorphism of the CYP2D6 drug-metabolizing enzyme. Debrisoquine hydroxylation phenotype has been the most used test in humans to evaluate CYP2D6 activity. Two debrisoquine hydroxylation phenotypes have been described: poor and extensive metabolizers. A group with a very low debrisoquine metabolic ratio within the extensive metabolizers, named ultrarapid metabolizers, has also been distinguished. This CYP2D6 variability can be for a large part alternatively determined by genotyping, which appears to be of clinical importance given CYP2D6 involvement in the metabolism of a large number of commonly prescribed drugs. CYP2D6 pharmacogenetics may then become a useful tool to predict drug-related side effects, interactions or therapeutic failures. However, a number of reasons appear to have made research into this field lag behind. The present review focuses on the relevance of genetics and environmental factors for determining debrisoquine hydroxylation phenotype, as well as the relevance of CYP2D6 genetic polymorphism in psychiatric patients treated with antipsychotic drugs.

Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver.

Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from equine liver and expressed in a baculovirus expression system. The nucleotide sequence of CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the enzyme was characterized using dextromethorphan and debrisoquine, two isoform selective substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15. Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6. Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial interspecies variability in metabolism of substrates by CYP2D orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.

Chimeric mice with humanized liver.

Recently, chimeric mice with humanized liver were established by transplanting human hepatocytes into an urokinase-type plasminogen activator(+/+)/severe combined immunodeficient transgenic mouse line. The replacement with human hepatocytes is more than 80-90% and is higher than any other chimeric mouse reported previously. In drug development, the liver is one of the most important organs because it is mainly involved in the pharmacokinetics of drugs and is frequently damaged by many drugs due to the accumulation of drugs and/or metabolites. The pharmacokinetics could affect the efficacy and toxicity of a drug, and thus prediction of the human pharmacokinetics is important for developing new drugs without adverse reactions and toxicity. Extrapolation from experimental animals or in vitro studies to the human in vivo pharmacokinetics is still difficult. To date, human hepatocytes and liver microsomes are recognized as better tools and are frequently used to estimate the human pharmacokinetics. We thought that chimeric mice with humanized liver could become a new tool for estimating the human toxicity and pharmacokinetics. At first, metabolism, which plays an essential role in pharmacokinetics, was investigated in the chimeric mice. In the liver of the chimeric mice, human drug metabolizing enzymes were found to be expressed and to reflect the capacities and genetic polymorphism of the donor. In an in vivo study on metabolism, human specific metabolites could be detected in the serum of the chimeric mice indicating that the chimeric mice could be used as an in vivo model to address human metabolism. These results suggested that the chimeric mice could overcome the species differences in drug metabolism and be used to evaluate drug toxicity due to genetic polymorphism. The reasons for drug interaction are often enzyme induction and inhibition. By the treatment with a typical inducer of cytochrome P450 (P450), which is the central drug-metabolizing enzyme, P450s expressed in the liver of the chimeric mice were found to possess induction potencies. After the treatment with a specific inhibitor of human P450, the area under the curve of the P450 metabolite was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be useful for assessing drug interactions in vivo. Moreover, drug excretion was determined to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and humans but in the feces in control uPA(-/-)/SCID mice. Drug transporters expressed in the liver of the chimeric mice were also humanized. In this review, studies of the chimeric mice with humanized liver, particularly on metabolism and excretion, are summarized and the possibility of using the chimeric mice is proposed for the advanced prediction of human pharmacokinetics and toxicity.

Metabolism of antidepressant and neuroleptic drugs by cytochrome p450s: clinical and interethnic aspects.

Early after the introduction of the classical tricyclic antidepressants and neuroleptics, it was shown that the plasma concentrations of these drugs varied between patients given the same dose. This variation is to a major extent due to the variation in the activity of cytochrome P450 (CYP) enzymes (cf. review by Bertilsson et al.1) During recent year(s), the different CYP enzymes catalyzing the metabolism of these drugs have been identified and the clinical relevance has also been identified. This brief review highlights the clinical importance and ethnic differences in the metabolism of these drugs.

Comparative metabolic capabilities and inhibitory profiles of CYP2D6.1, CYP2D6.10, and CYP2D6.17.

Polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene are a major cause of pharmacokinetic variability in human. Although the poor metabolizer phenotype is known to be caused by two null alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remains mostly unexplained. Thus, the goal of this study was to examine the intrinsic enzymatic differences that exist among the several active CYP2D6 allelic variants. The relative catalytic activities (enzyme kinetics) of three functionally active human CYP2D6 allelic variants, CYP2D6.1, CYP2D6.10, and CYP2D6.17, were systematically investigated for their ability to metabolize a structurally diverse set of clinically important CYP2D6-metabolized drugs [atomoxetine, bufuralol, codeine, debrisoquine, dextromethorphan, (S)-fluoxetine, nortriptyline, and tramadol] and the effects of various CYP2D6-inhibitors [cocaine, (S)-fluoxetine, (S)-norfluoxetine, imipramine, quinidine, and thioridazine] on these three variants. The most significant difference observed was a consistent but substrate-dependent decease in the catalytic efficiencies of cDNA-expressed CYP2D6.10 and CYP2D6.17 compared with CYP2D6.1, yielding 1.32 to 27.9 and 7.33 to 80.4% of the efficiency of CYP2D6.1, respectively. The most important finding from this study is that there are mixed effects on the functionally reduced allelic variants in enzyme-substrate affinity or enzyme-inhibitor affinity, which is lower, higher, or comparable to that for CYP2D6.1. Considering the rather high frequencies of CYP2D6*10 and CYP2D6*17 alleles for Asians and African Americans, respectively, these data provide further insight into ethnic differences in CYP2D6-mediated drug metabolism. However, as with all in vitro to in vivo extrapolations, caution should be applied to the clinical consequences.

Estudio farmacogenético del polimorfismo metabólico de la debrisoquina (CYP2D6) en la población cubana en relación con la española / Study of debrisoquine hydroxylation polymorphism (CYP2D6) in the Cuban population compared to Spaniards

Fundamento y objetivo: La capacidad metabólica del CYP2D6 presenta polimorfismo genético. A partir del cálculo del índice metabólico (IM) urinario de un fármaco test como la debrisoquina se ha demostrado la existencia de 2 fenotipos metabólicos: metabolizadores lentos y rápidos, entre los que hay un subgrupo de ultrarrápidos. El objetivo de este trabajo ha sido evaluar las diferencias interétnicas del polimorfismo metabólico de hidroxilación de debrisoquina en la población cubana, y compararla con la española. Pacientes y método: El IM se evaluó en 260 voluntarios sanos cubanos (divididos en blancos y mestizos) que se compararon con 925 voluntarios sanos españoles. Resultados: La frecuencia de metabolizadores lentos fue similar entre cubanos y españoles (un 4,6 y un 4,9%, respectivamente). Sin embargo, la prevalencia de metabolizadores ultrarrápidos fue menor en cubanos (3,8%) que en españoles (5,2%). El IM en metabolizadores rápidos fue mayor (p < 0,05) en los mestizos que en blancos cubanos. Conclusiones: Se observan diferencias interétnicas en el polimorfismo genético del CYP2D6 en la población cubana y en la frecuencia de metabolizadores ultrarrápidos respecto a los españoles. Estas diferencias podrían condicionar en la población cubana una variabilidad interindividual e interétnica en la respuesta a los fármacos sustratos del CYP2D6

Background and objective: CYP2D6 metabolic capacity shows genetic polymorphism. Two metabolic phenotypes, poor and extensive, can be determined by the ratio of debrisoquine to its metabolite in urine (MR). A subgroup of ultrarapids has been also described. We analyzed the inter-ethnic differences on the polymorphic hydroxylation of debrisoquine in a Cuban population in comparison to Spaniards. Patients and method: MR in a Cuban population of 260 white and mestizo healthy volunteers was studied and compared to 925 Spanish healthy volunteers. Results: The frequency of poor metabolizer in Cubans (4.6%) was almost identical to that found in Spaniards (4.9%). However, ultrarapids were lower in Cubans (3.8%) than in Spaniards (5.2%). MR in Cuban-mestizo extensive metabolizers was higher than in white (p < 0.05). Conclusions: Interethnic differences on debrisoquine hydroxylation have been demonstrated in a Cuban population. Furthermore, differences on the frequency of ultrarapids between Cubans and Spaniards have been shown. These results could explain inter-individual and interethnic differences on drug response such as side effects or therapeutic failures among Cuban patients receiving treatment with CYP2D6 substrates

Genotyping of the cytochrome P450 2D6 4469 C>T polymorphism using SimpleProbes.

BACKGROUND: Genotyping of human cytochrome P450s is a pharmacogenetic approach to diagnosing inherited deficiencies in drug metabolizing enzymes that influence therapeutic responses. The P450 CYP2D6 (debrisoquine hydroxylase) metabolizes numerous antidepressants and neuroleptic agents and there is evidence of a relationship between gene polymorphism and variant therapeutic response. Polymorphism in CYP2D6 causes poor, intermediate, efficient or ultrarapid metabolization of substrate drugs affecting pharmacokinetic parameters and requiring dose adjustments. Predictive genotyping for broader clinical application is reliant on fast, technically simple analyses. A new genotyping method was explored. It identifies the single nucleotide polymorphism (SNP) 4469 C>T (NCBI access no. M33388) with one fluorescent hybridization probe (SimpleProbes; SP) using the LightCycler (LC). This SNP is found in 21 alleles, comprising 30% in Caucasian populations and encoding enzymes with poor, intermediate or efficient activity. The remaining 65 known alleles either harbour a C in position 4469 or are deletion mutants. METHODS: Comparative detection of C>T polymorphism was done using a well-established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and PCR followed by melting-point (T(m)) analysis with an SP covering the SNP position in 144 samples encompassing alleles *2 and *41 with a T, alleles *1,*3, *4, *6, *9, *10, *15 with a C and the deletion mutant allele *5. RESULTS: C>T polymorphism was detected with complete concordance. T(m) of SP/target heteroduplex complexes for C was: T(m) 67, 89 degrees C to 68, 62 degrees C and for T: T(m) 60, 70 degrees C to 61, 51 degrees C. CONCLUSION: By one-step SP methodology it proved possible within 2 h to identify an SNP in genotypes comprising >90% in Caucasian populations.

Evaluation of probe drugs and pharmacokinetic metrics for CYP2D6 phenotyping.

INTRODUCTION: Cytochrome P450 2D6 (CYP2D6) is one of the most important enzymes catalyzing biotransformation of xenobiotics in the human liver. This enzyme's activity shows a high degree of interindividual variability caused in part by its genetic polymorphism, the so-called debrisoquine/sparteine polymorphism. The genetic component influencing CYP2D6 activity can be determined by genotyping. However, genotyping alone is not sufficient to accurately predict an individual's actual CYP2D6 activity, as this is also influenced by other factors. For the determination of the exact actual enzymatic activity ("phenotyping"), adequate probe drugs have to be administered prior to measurements of these compounds and/or their metabolites in body fluids. PROBE DRUGS: Debrisoquine, sparteine, metoprolol or dextromethorphan represent well-established probe drugs while tramadol has been recently investigated for this purpose. The enzymatic activity is reflected by various pharmacokinetic metrics such as the partial clearance of a parent compound to the respective CYP2D6-mediated metabolite or metabolic ratios. Appropriate metrics need to fulfill pre-defined validation criteria. METHODS: In this review, we have compiled a list of such criteria useful to select the best metrics to reflect CYP2D6 activity. A comprehensive Medline search for reports on CYP2D6 phenotyping trials with the above mentioned probe drugs was carried out. CONCLUSION: Application of the validation criteria suggests that dextromethorphan and debrisoquine are the best CYP2D6 phenotyping drugs, with debrisoquine having the problem of very limited availability as a therapeutic drug. However, the assessment of the best dextromethorphan CYP2D6 phenotyping metric/procedure is still ongoing.

Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the "cocktail" approach.

Phenotyping for drug metabolizing enzymes and transporters is used to assess quantitatively the effect of an intervention (e.g., drug therapy, diet) or a condition (e.g., genetic polymorphism, disease) on their activity. Appropriate selection of test drug and metric is essential to obtain results applicable for other substrates of the respective enzyme/transporter. The following phenotyping metrics are recommended based on the level of validation and on practicability: CYP1A2, paraxanthine/caffeine in plasma 6 h after 150 mg caffeine; CYP2C9, tolbutamide plasma concentration 24 h after 125 mg tolbutamide; CYP2C19, urinary excretion of 4'-OH-mephenytoin 0-12 h after 50 mg mephenytoin; CYP2D6, urinary molar ratio debrisoquine/4-OH-debrisoquine 0-8 h after 10 mg debrisoquine; and CYP3A4, plasma clearance of midazolam after 2 mg midazolam (all drugs given orally).

Liver disease selectively modulates cytochrome P450--mediated metabolism.

BACKGROUND: The liver plays a significant role in drug metabolism; thus it would be expected that liver disease may have a detrimental effect on the activity of cytochrome P450 (CYP) enzymes. The extent to which the presence and severity of liver disease affect the activity of different individual drug-metabolizing enzymes is still not well characterized. The purpose of this study was to assess the effect of liver disease on multiple CYP enzymes by use of a validated cocktail approach. METHODS: The participants in this investigation were 20 patients with different etiologies and severity of liver disease and 20 age-, sex-, and weight-matched healthy volunteers. Liver disease severity was categorized by use of the Child-Pugh score. All participants received a cocktail of 4 oral drugs simultaneously, caffeine, mephenytoin, debrisoquin (INN, debrisoquine), and chlorzoxazone, as in vivo probes of the drug-metabolizing enzymes CYP1A2, CYP2C19, CYP2D6, and CYP2E1, respectively. The primary end points were measurements of specific CYP metabolism indexes for each enzyme. RESULTS: Mephenytoin metabolism was significantly decreased in both patients with mild liver disease (Child-Pugh score of 5/6) (-63% [95% confidence interval (CI), -86% to -40%]; P = .0003) and patients with moderate to severe liver disease (Child-Pugh score >6) (-80% [95% CI, -95% to -64%]; P = .0003). In comparison with control subjects, the caffeine metabolic ratio was 69% lower (95% CI, -85% to -54%; median, 0.14 versus 0.62; P = .0003), the debrisoquin recovery ratio was 71% lower (95% CI, -96% to -47%; median, 0.10 versus 0.65; P = .012), and the chlorzoxazone metabolic ratio was 60% lower (95% CI, -91% to -29%; median, 0.21 versus 0.83; P = .0111) in patients with moderate to severe liver disease. All 4 drugs showed significant negative relationships with the Child-Pugh score. CONCLUSIONS: CYP enzyme activity is differentially affected by the presence of liver disease. We propose that the data can be explained by the "sequential progressive model of hepatic dysfunction," whereby liver disease severity has a differential effect on the metabolic activity of specific CYP enzymes.