Deficient hydroxylation of debrlsoquine Is an autosomal recessive trait that affects =7% of the Caucasian population. These individuals (poor metabolizers) carry deficient CYP2D6 gene variants and have an impaired metabolsm of several commonly used drugs. The opposite phenomenon also exists, and certain individuals metabolize the drugs very rapidly, resulting in subtherapeutic plasma concentrations at normal doses. In the present study, we have investigated the molecular genetic basis for ultrarapid metabolism of debrisoquine. Restriction fragment length polymorphism analysis of the CYP2D locus in two families with very rapid metabolism of debrisoquine [metabolic ratio (MR) for debrisoquine = 0.01-0.11 revealed the variant CYP2D6 gene CYP2D6L. EcoRI RFLP and Xba I pulsed-field gel electrophoresis analyses showed that this gene had been amplifled 12-fold in three members (father and his two children) of one ofthe families, and two copies were present among members of the other family. The CYP2D6L gene had an open reading frame and carried two mutations causing amino acid substitutions: one in exon 6, yielding an Arg-296 --Cys exchange and one in exon 9 causing Ser-486 --Thr. The MR of subjects carrying one copy of the CYP2D6L gene did not signicantiy differ from that of those with the wild-ype gene, indicating that the structural alterations were not of importance for the catalytic properties of the gene product. Examination of the MR among subjects carrying wild-type CYP2D6, CYP2D6L, or deficient alleles revealed a relationship between the number of active genes and MR. The data show the principle of inherited amplification of an active gene. Furthermore, the finding of a specific haplotype with two or more active CYP2D6 genes allows genotyping for ultrarapid drug metabolizers. This genotyping could be of predictive value for individualized and more efficient drug therapy.

Patients who are involuntary admitted to psychiatric care are extremely vulnerable as a consequence of the control from others, and of the personal limitations due to a psychiatric disease that can influence their own control of their lives. This group of patients are seldom asked about their experiences of being cared for. In this study five involuntary hospitalized psychiatric patients narrated their experience of being subjected to involuntary psychiatric care. The aim of the study was to obtain a deeper understanding of this experience. The interview text was analysed by means of a phenomenological hermeneutic method. The result of the analysis gave a complex picture of both support and violation. On the one hand experiences of not being seen or heard, of loss of liberty and of violation of integrity were found. On the other hand, there were experiences of respect and caring and opportunities to take responsibility for oneself were offered. Being treated involuntarily in psychiatric care was interpreted as a balancing act between good opportunities and great losses.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:To study mechanisms behind the interindividual variability in CYP3A expression and the relative contribution of the different CYP3A enzymes to the overall CYP3A activity, we have analyzed CYP3A4, CYP3A5, CYP3A43, and PXR mRNA and CYP3A4 and CYP3A5 protein expression, catalytic activity, and polymorphism in the CYP3A5 gene in a panel of 46 Caucasian human livers. Protein quantification was performed by Western blotting using enzymespecific antibodies directed to the C termini of CYP3A4 or CYP3A5, and carrier protein-coupled peptides as standards. The mRNA levels were determined by quantitative real-time PCR. CYP3A activity was measured by analysis of the rate of testosterone 6␤-hydroxylation. A correlation existed between all CYP3A and PXR mRNA transcripts measured. The interindividual variability in CYP3A4 and CYP3A5 mRNA expression was higher than that of CYP3A protein and activity. The CYP3A5 protein was expressed at quantifiable levels in 5 (10.9%) of the livers. Four of those were heterozygous for the CYP3A5*1 allele and had CYP3A5 protein at a mean level of 17% of that of total CYP3A, whereas one liver sample was from a CYP3A5*3 homozygote individual having lower amounts of CYP3A5. In total, CYP3A5 only contributed 2% of the overall CYP3A protein among all samples. In conclusion, our data indicate that CYP3A4, CYP3A5, CYP3A43, and PXR hepatic mRNA expression correlate, indicating common regulatory features, and that the contribution of CYP3A5 to hepatic drug metabolism in Caucasians is insignificant.Interindividual variation in the activity and levels of expression of CYP3A is considerable. This variation is manifested by differences greater than 10-fold in the in vivo metabolism of drugs that are substrates for CYP3As (Kleinbloesem et al

Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health concern as reflected in its widespread distribution in the general population. Yet, treatment options are scarce which is at least in part due to lack of reliable human in vitro disease models. Here, we report a human hepatic 3D spheroid system cultured under defined chemical conditions that has the potential to mimic steatotic conditions in a reversible manner, useful for identification of novel drug treatment conditions. Primary human hepatocytes (PHH) from different donors were cultured as spheroid microtissues in physiological in vivo -like culture conditions. Hepatic steatosis was induced over the course of three weeks in culture by supplementing the culture medium with pathophysiological concentrations of free fatty acids, carbohydrates and insulin. Effects of steatosis in the 3D system were evaluated on transcriptional, metabolomic and lipidomic levels. Free fatty acids on one hand as well as a combination of insulin and monosaccharides, promoted lipid accumulation in hepatocytes and increased expression of lipogenic genes, such as fatty acid synthase. This milieu also promoted development of insulin resistance within 2 weeks as manifested by an increase in gluconeogenic and insulin resistance markers, which are observed in type 2 diabetes mellitus and metabolic syndrome. Induced steatosis was reversible after withdrawal of lipogenic substrates and a further reduction in cellular fat content was observed following treatment with different antisteatotic compounds, such as metformin, glucagon, olaparib and antioxidants. Taken together, these results demonstrate that the 3D hepatic spheroids can serve as a valuable, HTS compatible model for the study of liver steatosis and facilitate translational discovery of novel drug targets.

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Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. ; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 mm), triacetyloleandomycin (33±15% at 10 mm), and ketoconazole (51±28% at 2 mm) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 mm) and ketoconazole (51±13% at 2 mm), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K m and V max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.

Individual susceptibility to environmental, chemical, and drug toxicity is to some extent determined by polymorphism in drug-metabolizing enzymes, in particular the cytochromes P450 (CYPs). This polymorphism is in particular translated into risk differences concerning drugs metabolized by the highly polymorphic enzymes CYP2C9, CYP2C19, and CYP2D6, whereas CYP enzymes active in procarcinogen activation are relatively well conserved without important functional polymorphisms. Examples of drug toxicities that can be predicted by P450 polymorphism include those exerted by codeine, tramadol, warfarin, acenocoumarol, and clopidogrel. The polymorphic CYP2A6 has a role in nicotine metabolism and smoking behavior. Besides this genetic variation, genome-wide association studies now allow for the identification of an increasing number of predictive genetic biomarkers among, e.g., human leukocyte antigens and to some extent drug transporters that provide useful information regarding the choice of the drug and drug dosage in order to avoid toxicity. The translation of this information into the clinical practice has been slow; however, an increasing number of pharmacogenomic drug labels are assigned, where the predictive genotyping before drug treatment can be mandatory, recommended, or only for informational purposes. In this review, we provide an update of the field with emphasis on CYP polymorphism.

The role of the polymorphic cytochrome P450 2D6 (CYP2D6) in the metabolism of risperidone to its major active metabolite, 9-hydroxyrisperidone (9-OH-risperidone), has been documented after single oral doses of the drug. In this study, the influence of the CYP2D6 polymorphism on the steady-state plasma concentrations of risperidone and 9-OH-risperidone was investigated. Thirty-seven schizophrenic patients on monotherapy with risperidone, 4-8 mg/day, were genotyped by RFLP and PCR for the major functional variants of the CYP2D6 gene. Steady state plasma levels of risperidone and 9-OH-risperidone were analysed by HPLC. Based on the genotype analysis, three patients were classified as ultrarapid metabolizers (UM) with an extra functional CYP2D6 gene, 16 were homozygous extensive metabolizers (EM), 15 heterozygous EM and three poor metabolizers (PM). The median steady-state plasma concentration-to-dose (C/D) ratios of risperidone were 0.6, 1.1, 9.7 and 17.4 nmol/l per mg in UM, homozygous EM, heterozygous EM and PM, respectively, with statistically significant differences between PM and the other genotypes (P < 0.02). The C/D of 9-OH-risperidone also varied widely but was not related to the genotype. The risperidone/9-OH-risperidone ratio was strongly associated with the CYP2D6 genotype, with the highest ratios in PM (median 0.79). Heterozygous EM also had significantly higher ratios than homozygous EM (median value 0.23 versus 0.04; P < 0.01) or UM (median 0.03; P < 0.02). No significant differences were found in the C/D of the sum of the plasma concentrations of risperidone and 9-OH-risperidone between the genotype groups. In conclusion, the steady-state plasma concentrations of risperidone and the risperidone/9-OH-risperidone ratio are highly dependent on the CYP2D6 genotype. However, as risperidone and 9-OH-risperidone are considered to have similar pharmacological activity, the lack of relationship between the genotype and the sum of risperidone and 9-OH-risperidone indicates that the CYP2D6 polymorphism may be of limited importance for the clinical outcome of the treatment.