Effect of Routine Cytochrome P450 2D6 and 2C19 Genotyping on Antipsychotic Drug Persistence in Patients With Schizophrenia: A Randomized Clinical Trial.

Importance: Genetic polymorphism of genes encoding the drug metabolizing enzymes, cytochrome P450 2D6 and 2C19 (CYP2D6 and CYP2C19), is associated with treatment failure of and adverse reactions to psychotropic drugs. The clinical utility of routine CYP2D6 and CYP2C19 genotyping (CYP testing) is unclear. Objective: To estimate whether routine CYP testing effects the persistence of antipsychotic drug treatment. Design, Setting, and Participants: This single-masked, 3-group randomized clinical trial included patients aged 18 years or older who had been diagnosed within the schizophrenic spectrum (International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, F20-F29) and not previously genotyped. A total of 669 of 1406 potentially eligible patients from 12 psychiatric outpatient clinics in Denmark were approached between July 2008 and December 2009. Overall, 528 patients were genotyped and randomly allocated to 1 of 3 study groups or exclusion in a sequence of 1:1:1:3 using a predictive enrichment design, aiming to double the proportion of poor or ultrarapid metabolizers for CYP2D6 or CYP2C19. Outcome measurements were recorded at baseline and 1-year follow-up. Data analysis was performed in December 2012 and updated March 2019. Interventions: The trial included 2 intervention groups, where antipsychotic drug treatment was guided by either CYP test (CYP test-guided [CTG]) or structured clinical monitoring (SCM), in which adverse effects and factors influencing compliance were systematically recorded at least once quarterly, and 1 control group. Main Outcomes and Measures: Primary outcome was antipsychotic drug persistence, ie, days to first modification of the initial treatment. Secondary outcomes were number of drug and dose changes, adverse effects, and psychotic symptoms, ie, hallucinations and delusions. Results: A total of 528 participants were genotyped, and 311 (median [interquartile range {IQR} age, 41 [30-50] years; 139 [45%] women; median [IQR] duration of illness, 6 [3-13] years) were randomly allocated to 1 of 3 study groups. Overall, 61 participants (20%) were extreme metabolizers. There was no difference in antipsychotic drug persistence between the CTG group and the control group (hazard ratio [HR], 1.02; 95% CI, 0.71-1.45) or SCM and the control group (HR, 0.88; 95% CI, 0.61-1.26). Subanalyses among extreme metabolizers showed similar results (CTG: HR, 0.99; 95% CI, 0.48-2.03; SCM: HR, 0.93; 95% CI, 0.44-1.96). Conclusions and Relevance: The results of this randomized clinical trial do not support routine CYP testing in patients with schizophrenia. Trial Registration: ClinicalTrials.gov Identifier: NCT00707382.

Prognostic role of genetic polymorphisms of the interleukin-6 signaling pathway in patients with severe heart failure.

Heart failure (HF) is associated with perturbations of the interleukin-6 (IL-6) signaling pathway. A total of 559 Danish subjects with severe chronic HF enrolled in the previously reported Echocardiography and Heart Outcome Study were genotyped for three SNPs in IL6, nine in the IL-6 receptor gene (IL6R), and two in the IL-6 signal transducer gene (IL6ST). After a mean follow-up of 5.0 years, 5 SNPs in IL6R introns (rs12083537, rs6684439, rs4845622, rs4537545, and rs7529229) and a SNP in the IL6R coding region (rs2228145, also known as Asp358Ala) were associated with adverse outcomes, e.g., hazard ratios (HRs) for cardiovascular death and all-cause death 1.38 (CI: 1.09-1.76; P = 0.008) and 1.37 (CI: 1.10-1.70; P = 0.004) for rs6684439 heterozygotes, and 1.39 (CI: 1.09-1.77; P = 0.007) and 1.37 (CI: 1.10-1.70; P = 0.005) for rs4845622 heterozygotes, respectively. We conclude that SNPs in the IL-6 signaling pathway may be independent predictors of fatal outcomes in patients with severe HF.

Pharmacometabolomics Informs About Pharmacokinetic Profile of Methylphenidate.

Carboxylesterase 1 (CES1) metabolizes methylphenidate and other drugs. CES1 gene variation only partially explains pharmacokinetic (PK) variability. Biomarkers predicting the PKs of drugs metabolized by CES1 are needed. We identified lipids in plasma from 44 healthy subjects that correlated with CES1 activity as determined by PK parameters of methylphenidate including a ceramide (q value = 0.001) and a phosphatidylcholine (q value = 0.005). Carriers of the CES1 143E allele had decreased methylphenidate metabolism and altered concentration of this phosphatidylcholine (q value = 0.040) and several high polyunsaturated fatty acid lipids (PUFAs). The half-maximal inhibitory concentration (IC50 ) values of chenodeoxycholate and taurocholate were 13.55 and 19.51 µM, respectively, consistent with a physiological significance. In silico analysis suggested that bile acid inhibition of CES1 involved both binding to the active and superficial sites of the enzyme. We initiated identification of metabolites predicting PKs of drugs metabolized by CES1 and suggest lipids to regulate or be regulated by this enzyme.

Novel procedure with improved resolution and specificity for amplification and differentiation of variants of the gene encoding carboxylesterase 1.

Carboxylesterase 1 (CES1) is implicated in the metabolism of several commonly used drugs and other xenobiotics. The gene encoding this enzyme, CES1, is duplicated in some individuals. The original gene copy is called CES1A1. The duplicated version, CES1A2, is a hybrid of CES1A1 and the CES1-related pseudogene, CES1P1. Variants of CES1A2 with a weak and a strong promoter, respectively, have been reported. In addition, there are chimeric subtypes of CES1A1 that contain a segment of CES1P1. Collectively, this represents challenges to the genotyping of CES1 that previous procedures have had difficulties in solving, frequently leading to loss of specificity and inaccurate genotyping. Here, we report a novel and specific procedure that can selectively amplify CES1A1 and CES1A2 and accurately determine their variants. This procedure may be useful for personalization of treatments with drugs metabolized by CES1.

Pharmacodynamic Impact of Carboxylesterase 1 Gene Variants in Patients with Congestive Heart Failure Treated with Angiotensin-Converting Enzyme Inhibitors.

BACKGROUND: Variation in the carboxylesterase 1 gene (CES1) may contribute to the efficacy of ACEIs. Accordingly, we examined the impact of CES1 variants on plasma angiotensin II (ATII)/angiotensin I (ATI) ratio in patients with congestive heart failure (CHF) that underwent ACEI dose titrations. Five of these variants have previously been associated with drug response or increased CES1 expression, i.e., CES1 copy number variation, the variant of the duplicated CES1 gene with high transcriptional activity, rs71647871, rs2244613, and rs3815583. Additionally, nine variants, representatives of CES1Var, and three other CES1 variants were examined. METHODS: Patients with CHF, and clinical indication for ACEIs were categorized according to their CES1 genotype. Differences in mean plasma ATII/ATI ratios between genotype groups after ACEI dose titration, expressed as the least square mean (LSM) with 95% confidence intervals (CIs), were assessed by analysis of variance. RESULTS: A total of 200 patients were recruited and 127 patients (63.5%) completed the study. The mean duration of the CHF drug dose titration was 6.2 (SD 3.6) months. After ACEI dose titration, there was no difference in mean plasma ATII/ATI ratios between subjects with the investigated CES1 variants, and only one previously unexplored variation (rs2302722) qualified for further assessment. In the fully adjusted analysis of effects of rs2302722 on plasma ATII/ATI ratios, the difference in mean ATII/ATI ratio between the GG genotype and the minor allele carriers (GT and TT) was not significant, with a relative difference in LSMs of 0.67 (95% CI 0.43-1.07; P = 0.10). Results of analyses that only included enalapril-treated patients remained non-significant after Bonferroni correction for multiple parallel comparisons (difference in LSM 0.60 [95% CI 0.37-0.98], P = 0.045). CONCLUSION: These findings indicate that the included single variants of CES1 do not significantly influence plasma ATII/ATI ratios in CHF patients treated with ACEIs and are unlikely to be primary determinants of ACEI efficacy.

Prognostic impact of carboxylesterase 1 gene variants in patients with congestive heart failure treated with angiotensin-converting enzyme inhibitors.

OBJECTIVE: Most angiotensin-converting enzyme inhibitors (ACEIs) are prodrugs activated by carboxylesterase 1 (CES1). We investigated the prognostic importance of CES1 gene (CES1) copy number variation and the rs3815583 single-nucleotide polymorphism in CES1 among ACEI-treated patients with congestive heart failure (CHF). METHODS: Danish patients with chronic CHF enrolled in the previously reported Echocardiography and Heart Outcome Study were categorized according to their CES1 variants and followed up for up to 10 years. Risk for cardiovascular death and all-cause death was modeled by Cox proportional hazard analyses. RESULTS: A total of 491 ACEI-treated patients were included in the analyses. After a mean follow-up of 5.5 years, we found no difference in the risk for cardiovascular death and all-cause death between patients having three [hazard ratios (HRs) 1.06 (95% confidence interval (CI) 0.77-1.45) and 1.16 (95% CI 0.88-1.52)] or four [HRs 0.88 (95% CI 0.39-2.01) and 1.37 (95% CI 0.74-2.54)] CES1 copies and those with two copies, respectively. Similarly, no difference in the risk for cardiovascular and all-cause death was found for patients heterozygous [HRs 0.91 (95% CI 0.70-1.19) and 0.88 (95% CI 0.69-1.12)] or homozygous [HRs 0.58 (95% CI 0.30-1.15) and 0.82 (95% CI 0.48-1.39)] for the rs3815583 minor allele versus patients homozygous for the major allele. The active promoter of CES1A2 and the rs71647871 single-nucleotide polymorphism minor allele were detected at very low frequencies. CONCLUSION: This study did not support the use of CES1 copy number variation or rs3815583 as a predictor of fatal outcomes in ACEI-treated patients with CHF.

Identification of rare high-risk copy number variants affecting the dopamine transporter gene in mental disorders.

BACKGROUND: The dopamine transporter, also known as solute carrier 6A3 (SLC6A3), plays an important role in synaptic transmission by regulating the reuptake of dopamine in the synapses. In line with this, variations in the gene encoding this transporter have been linked to both schizophrenia and affective disorders. Recently, copy number variants (CNVs) in SLC6A3 have been identified in healthy subjects but so far, the implication of CNVs affecting this gene in psychiatric diseases has not been addressed. AIMS: In the present study, we aimed to investigate whether CNVs affecting SLC6A3 represent rare high-risk variants of psychiatric disorders. METHODS: We performed a systematic screening for CNVs affecting SLC6A3 in 761 healthy controls, 672 schizophrenia patients, and 194 patients with bipolar disorder in addition to 253 family members from six large pedigrees affected by mental disorders using single nucleotide polymorphism arrays and subsequent verification by real-time polymerase chain reaction. RESULTS: We identified two duplications and one deletion affecting SLC6A3 in the patients, while no such CNVs were identified in any of the controls. The identified CNVs were of different sizes and two affected several genes in addition to SLC6A3. CONCLUSION: Our findings suggest that rare high-risk CNVs affecting the gene encoding the dopamine transporter contribute to the pathogenesis of schizophrenia and affective disorders.

Synthetic cannabimimetic agents metabolized by carboxylesterases.

Synthetic cannabimimetic agents are a large group of diverse compounds which act as agonists at cannabinoid receptors. Since 2004, synthetic cannabinoids have been used recreationally, although several of the compounds have been shown to cause severe toxicity in humans. In this study, the metabolism of two indazole carboxamide derivatives, AB-PINACA and AB-FUBINACA, was investigated by using human liver microsomes (HLM). For both compounds, a major metabolic pathway was the enzymatic hydrolysis of the primary amide, resulting in the major metabolites AB-PINACA-COOH and AB-FUBINACA-COOH. Other major metabolic pathways were mono-hydroxylation of the N-pentyl chain in AB-PINACA and mono-hydroxylation of the 1-amino-3-methyl-1-oxobutane moiety in AB-FUBINACA. To identify the enzyme(s) responsible for the amide hydrolysis, incubations with recombinant carboxylesterases and human serum, as well as inhibition studies in HLM and human pulmonary microsomes (HPM) were performed. Carboxylesterase 1 (CES1) was identified as the major human hepatic and pulmonary enzyme responsible for the amide hydrolysis.We employed similar studies to identify the esterase(s) involved in the previously described hydrolytic metabolism of two quinolineindole synthetic cannabinoids, PB-22 and 5F-PB-22, as well as the closely related compound, BB-22. Our investigations again revealed CES1 to be the key enzyme catalyzing these reactions. The identified major metabolites of AB-PINACA and AB-FUBINACA are likely to be useful in documenting drug usage in forensic and clinical screening. Additionally, the identification of CES1 as the main enzyme hydrolyzing these compounds improves our knowledge in the emerging field of xenobiotic metabolism by esterases.

In vitro drug metabolism by human carboxylesterase 1: focus on angiotensin-converting enzyme inhibitors.

Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed angiotensin-converting enzyme inhibitors: enalapril and ramipril. Here, we studied recombinant human CES1- and CES2-mediated hydrolytic activation of the prodrug esters enalapril and ramipril, compared with the activation of the known substrate trandolapril. Enalapril, ramipril, and trandolapril were readily hydrolyzed by CES1, but not by CES2. Ramipril and trandolapril exhibited Michaelis-Menten kinetics, while enalapril demonstrated substrate inhibition kinetics. Intrinsic clearances were 1.061, 0.360, and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril, respectively. Additionally, we screened a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans, thus opening up possibilities for safer drug treatments.

Does pharmacogenetic testing for CYP450 2D6 and 2C19 among patients with diagnoses within the schizophrenic spectrum reduce treatment costs?

The effect of pharmacogenetic testing for CYP450 2D6 and 2C19 on treatment costs have not yet been documented. This study used Danish patient registers to calculate healthcare costs of treating patients with diagnoses within the schizophrenic spectrum for 1 year with or without pharmacogenetic testing for polymorphisms in the genes for the CYP2D6 and CYP2C19 enzymes. In a randomized, controlled trial, stratified with respect to metabolizer genotype, 104 patients were assigned to treatment based on pharmacogenetic testing and 103 patients to treatment as usual. Random exclusion of extensive and intermediate metabolizers was used to increase the frequency of extreme metabolizers (poor metabolizers and ultrarapid metabolizers for CYP2D6) to 20% in both groups. Cost differences were analysed at several levels including (i) overall healthcare expenditure, (ii) psychiatric hospital cost (iii) nonpsychiatric hospital cost, (iv) primary care spending and (v) pharmaceuticals. Statistically significant differences in costs of psychiatric care dependent on metabolizer status were found between intervention groups. Pharmacogenetic testing significantly reduced costs among the extreme metabolizers (poor metabolizers and ultrarapid metabolizers) to 28%. Use of primary care services and pharmaceuticals was also affected by the intervention.This study confirms earlier findings that extreme metabolizers (poor and ultrarapid metabolizers) incur higher costs than similar patients with a normal metabolizer genotype. However, this study shows that these excess costs can be reduced by pharmacogenetic testing. Pharmacogenetic testing for CYP2D6 and CYP2C19 could thus be considered as a means of curtailing high psychiatric treatment costs among extreme metabolizers.

Enantioselective determination of methylphenidate and ritalinic acid in whole blood from forensic cases using automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A chiral liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed and validated for quantifying methylphenidate and its major metabolite ritalinic acid in blood from forensic cases. Blood samples were prepared in a fully automated system by protein precipitation followed by solid-phase extraction. The LC-MS-MS method was linear in the range of 0.5 to 500 ng/g for the enantiomers of both analytes. For concentrations above the limit of quantification, coefficients of variation were 15% or less, and the accuracy was 89 to 94%. For 12 postmortem samples in which methylphenidate was not determined to be related to the cause of death, the femoral blood concentration of d-methylphenidate ranged from 5 to 58 ng/g, and from undetected to 48 ng/g for l-methylphenidate (median d/l-ratio 5.9). Ritalinic acid was present at concentrations 10-20 times higher with roughly equal amounts of the d- and l-forms. In blood from 10 living subjects that were not suspected of being intoxicated by methylphenidate, the concentration ranges and patterns were similar to those of the postmortem cases. Thus, methylphenidate does not appear to undergo significant postmortem redistribution.

Does the medication pattern reflect the CYP2D6 genotype in patients with diagnoses within the schizophrenic spectrum?

BACKGROUND: Cytochrome P450 2D6 enzyme (CYP2D6) is an important metabolic pathway for many antipsychotics. Its genetic polymorphism causes pharmacokinetic variability that might lead to adverse drug reactions or treatment failure unless countered by appropriate dose adjustments or shift to CYP2D6-independent antipsychotics. PURPOSE: To investigate the clinical impact of CYP2D6 genotype in patients with a diagnosis within the schizophrenic spectrum using medication pattern as proxy for therapeutic and side effect. METHODS: The study was conducted in patients genotyped during an inpatient stay (N = 576). Continuous antipsychotic, adjuvant, and anticholinergic drug regimens were registered retrospectively in a cross-sectional manner before genotyping. Antipsychotics were divided into CYP2D6 dependent and independent, and dose equivalents were calculated as chlorpromazine equivalents (CPZEq). RESULTS: Poor metabolizers and ultrarapid metabolizers were treated with significantly higher median CPZEq doses (625.8; inter quartile range [IQR], 460.4-926.7; and 550; IQR, 199.8-1049) than extensive metabolizers (EMs) and intermediate metabolizers (IMs) (384; IQR, 150-698; and 446; IQR, 150-800) (P = 0.018). Logistic regression showed no association between anticholinergic treatment and CYP2D6 genotype or concomitant treatment with CYP2D6 inhibitors (P = 0.79 and P = 0.46, respectively). CONCLUSIONS: Our results indicate that CYP2D6 genotype has no sufficient clinical impact that poor metabolizers and ultrarapid metabolizers are easily clinically identified with.

At-risk variant in TCF7L2 for type II diabetes increases risk of schizophrenia.

BACKGROUND: Schizophrenia is associated with increased risk of type II diabetes and metabolic disorders. However, it is unclear whether this comorbidity reflects shared genetic risk factors, at-risk lifestyle, or side effects of antipsychotic medication. METHODS: Eleven known risk variants of type II diabetes were genotyped in patients with schizophrenia in a sample of 410 Danish patients, each matched with two healthy control subjects on sex, birth year, and month. Replication was carried out in a large multinational European sample of 4089 patients with schizophrenia and 17,597 controls (SGENE+) using Mantel-Haenszel test. RESULTS: One type II diabetes at-risk allele located in TCF7L2, rs7903146 [T], was associated with schizophrenia in the discovery sample (p = .0052) and in the replication with an odds ratio of 1.07 (95% confidence interval 1.01-1.14, p = .033). CONCLUSION: The association reported here with a well-known diabetes variant suggests that the observed comorbidity is partially caused by genetic risk variants. This study also demonstrates how genetic studies can successfully examine an epidemiologically derived hypothesis of comorbidity.

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1.

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.

Maternally derived microduplications at 15q11-q13: implication of imprinted genes in psychotic illness.

OBJECTIVE: Rare copy number variants have been implicated in different neurodevelopmental disorders, with the same copy number variants often increasing risk of more than one of these phenotypes. In a discovery sample of 22 schizophrenia patients with an early onset of illness (10-15 years of age), the authors observed in one patient a maternally derived 15q11-q13 duplication overlapping the Prader-Willi/Angelman syndrome critical region. This prompted investigation of the role of 15q11-q13 duplications in psychotic illness. METHOD: The authors scanned 7,582 patients with schizophrenia or schizoaffective disorder and 41,370 comparison subjects without known psychiatric illness for copy number variants at 15q11-q13 and determined the parental origin of duplications using methylation-sensitive Southern hybridization analysis. RESULTS: Duplications were found in four case patients and five comparison subjects. All four case patients had maternally derived duplications (0.05%), while only three of the five comparison duplications were maternally derived (0.007%), resulting in a significant excess of maternally derived duplications in case patients (odds ratio=7.3). This excess is compatible with earlier observations that risk for psychosis in people with Prader-Willi syndrome caused by maternal uniparental disomy is much higher than in those caused by deletion of the paternal chromosome. CONCLUSIONS: These findings suggest that the presence of two maternal copies of a fragment of chromosome 15q11.2-q13.1 that overlaps with the Prader-Willi/Angelman syndrome critical region may be a rare risk factor for schizophrenia and other psychoses. Given that maternal duplications of this region are among the most consistent cytogenetic observations in autism, the findings provide further support for a shared genetic etiology between autism and psychosis.

No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma.

BACKGROUND: The response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the potential influence of different polymorphisms in the CYP enzymes on the outcome of treatment. METHODS: Data was analyzed from 348 patients undergoing high-dose treatment and stem cell support in Denmark in 1994 to 2004. Clinical information on relapse treatment in 243 individual patients was collected. The patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion), *6, and CYP2D6 gene duplication. RESULTS: In patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment. We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. CONCLUSION: There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome.

A large replication study and meta-analysis in European samples provides further support for association of AHI1 markers with schizophrenia.

The Abelson helper integration site 1 (AHI1) gene locus on chromosome 6q23 is among a group of candidate loci for schizophrenia susceptibility that were initially identified by linkage followed by linkage disequilibrium mapping, and subsequent replication of the association in an independent sample. Here, we present results of a replication study of AHI1 locus markers, previously implicated in schizophrenia, in a large European sample (in total 3907 affected and 7429 controls). Furthermore, we perform a meta-analysis of the implicated markers in 4496 affected and 18,920 controls. Both the replication study of new samples and the meta-analysis show evidence for significant overrepresentation of all tested alleles in patients compared with controls (meta-analysis; P = 8.2 x 10(-5)-1.7 x 10(-3), common OR = 1.09-1.11). The region contains two genes, AHI1 and C6orf217, and both genes-as well as the neighbouring phosphodiesterase 7B (PDE7B)-may be considered candidates for involvement in the genetic aetiology of schizophrenia.