Effect of the NFIB rs28379954 T>C polymorphism on CYP2D6-catalyzed metabolism of solanidine.

Cytochrome P450 2D6 (CYP2D6) is important for metabolism of 20%-25% of all clinically used drugs. Many known genetic variants contribute to the large interindividual variability in CYP2D6 metabolism, but much is still unexplained. We recently described that nuclear factor 1B (NFIB) regulates hepatic CYP2D6 expression with the minor allele of NFIB rs28379954 T>C significantly increasing CYP2D6-mediated risperidone metabolism. In this study, we investigated the effect of NFIB T>C on metabolism of solanidine, a dietary CYP2D6 substrate. Analyses of solanidine and metabolites (M414, M416, and M444) were performed by ultra-high performance liquid chromatography-high-resolution mass spectrometry in a cohort of 463 CYP2D6-genotyped patients of which with 58 (12.5%) carried NFIB TC (n = 56) or CC (n = 2). Increased metabolism of solanidine was found in CYP2D6 normal metabolizers (NMs; n = 258, 55.7%) carrying the NFIB C variant (n = 27, 5.8%) with 2.83- and 3.38-fold higher M416-to-solanidine (p = 0.039) and M444-to-solanidine (p = 0.046) ratios, respectively, whereas this effect was not significant among intermediate metabolizers (n = 166, 35.9%) (p ≥ 0.09). Importantly, no effect of the NFIB polymorphism on solanidine metabolism was seen in TC or CC carriers lacking CYP2D6 activity (poor metabolizers, n = 30, 6.5%, p ≥ 0.74). Furthermore, the NFIB polymorphism significantly explained variability in solanidine metabolism (M414 p = 0.013, M416 p = 0.020, and M416 and M444 p = 0.009) in multiple linear regression models for each metabolic ratio in the entire population, correcting for covariates (including CYP2D6 genotypes). Thus, the study confirms the effect of NFIB in regulating CYP2D6 activity, suggesting an about 200% increase in CYP2D6-mediated clearance in NMs being NFIB CT or CC carriers, comprising around 6% of Europeans.

Novel Identification of Cysteinyl Derivatives of Toxic Clozapine Nitrenium Ions and the Effect of Valproic Acid on Metabolite Formation: A Study Using Reprocessed High-Resolution Mass Spectra of Analyzed Therapeutic Drug Monitoring Samples.

BACKGROUND: Clozapine (CLZ) use is hampered by the risk of granulocyte toxicity, which is associated with the formation of nitrenium ions and the concurrent use of valproic acid (VPA). These highly reactive nitrenium ions cannot be measured in vivo. Instead, deactivated cysteinyl conjugates may potentially be detected. The aim of this study was to develop a novel method for identifying cysteinylated derivates of CLZ nitrenium ions to investigate the effect of VPA on their formation using therapeutic drug monitoring data. METHODS: A population comprising 93 VPA comedicated and 162 control patients from a therapeutic drug monitoring (TDM) service in Oslo, Norway, was included. Reprocessing of ultraperformance liquid chromatography high-resolution mass spectra (UHPLC-HR-MS) of previously analyzed TDM samples, combined with the assessment of MS/MS fragmentation patterns, was performed to identify the CLZ cysteinyl conjugates. Smoking, which induces CLZ metabolism, was assessed by detecting cotinine in the reprocessed mass spectra. RESULTS: By reprocessing the UHPLC-HR-MS files of the TDM analyses and reviewing the MS/MS fragment profiles, four cysteinyl conjugates of CLZ were identified. The formations of CLZ cysteinyl (CLZ-Cys 1+ ) and CLZ- N -oxide cysteinyl (CLZ-NOX-Cys 1+ ) were 1.5-fold ( P = 0.038) and 2.1-fold ( P < 0.001) higher in VPA-treated patients than those in the controls. In agreement with previous studies, a 45% reduction in N -desmethylclozapine formation was observed in VPA-treated patients ( P < 0.001). CONCLUSIONS: A novel method for detecting cysteinyl conjugates of CLZ was developed. Application of this method indicated that VPA significantly increased the formation of CLZ-Cys 1+ metabolites, which might explain the granulocyte toxicity reported after adding VPA to CLZ treatment. The developed method opens new avenues for investigating CLZ toxicity, e.g. by correlating cysteinyl conjugates and granulocyte counts in patients.

Evidence for solanidine as a dietary CYP2D6 biomarker: Significant correlation with risperidone metabolism.

AIMS: The extensive variability in cytochrome P450 2D6 (CYP2D6) metabolism is mainly caused by genetic polymorphisms. However, there is large, unexplained variability in CYP2D6 metabolism within CYP2D6 genotype subgroups. Solanidine, a dietary compound found in potatoes, is a promising phenotype biomarker predicting individual CYP2D6 metabolism. The aim of this study was to investigate the correlation between solanidine metabolism and the CYP2D6-mediated metabolism of risperidone in patients with known CYP2D6 genotypes. METHODS: The study included therapeutic drug monitoring (TDM) data from CYP2D6-genotyped patients treated with risperidone. Risperidone and 9-hydroxyrisperidone levels were determined during TDM, and reprocessing of the respective TDM full-scan high-resolution mass spectrometry files was applied for semi-quantitative measurements of solanidine and five metabolites (M402, M414, M416, M440 and M444). Spearman's tests determined the correlations between solanidine metabolic ratios (MRs) and the 9-hydroxyrisperidone-to-risperidone ratio. RESULTS: A total of 229 patients were included. Highly significant, positive correlationswere observed between all solanidine MRs and the 9-hydroxyrisperidone-to-risperidone ratio (ρ > 0.6, P < .0001). The strongest correlation was observed for the M444-to-solanidine MR in patients with functional CYP2D6 metabolism, i.e., genotype activity scores of 1 and 1.5 (ρ 0.72-0.77, P < .0001). CONCLUSION: The present study shows strong, positive correlations between solanidine metabolism and CYP2D6-mediated risperidone metabolism. The strong correlation within patients carrying CYP2D6 genotypes encoding functional CYP2D6 metabolism suggests that solanidine metabolism may predict individual CYP2D6 metabolism, and hence potentially improve personalized dosing of drugs metabolized by CYP2D6.

Prediction of CYP2D6 poor metabolizers by measurements of solanidine and metabolites-a study in 839 patients with known CYP2D6 genotype.

PURPOSE: Poor metabolizers (PMs) of the highly polymorphic enzyme CYP2D6 are usually at high risk of adverse effects during standard recommended dosing of CYP2D6-metabolized drugs. We studied if the metabolism of solanidine, a dietary compound found in potatoes, could serve as a biomarker predicting the CYP2D6 PM phenotype for precision dosing. METHODS: The study included 839 CYP2D6-genotyped patients who were randomized by a 4:1 ratio into test or validation cohorts. Full-scan high-resolution mass spectrometry data files of previously analyzed serum samples were reprocessed for identification and quantification of solanidine and seven metabolites. Metabolite-to-solanidine ratios (MRs) of the various solanidine metabolites were calculated prior to performing receiver operator characteristic (ROC) and multiple linear regression analyses on the test cohort. The MR thresholds obtained from the ROC analyses were tested for the prediction of CYP2D6 PMs in the validation cohort. RESULTS: In the test cohort, the M414-to-solanidine MR attained the highest sensitivity and specificity parameters from the ROC analyses (0.98 and 1.00) and highest explained variance from the linear models (R2 = 0.68). Below these thresholds, CYP2D6 PM predictions were tested in the validation cohort providing positive and negative predictive values of 100% for the MR of M414, while similar values for the other MRs ranged from 20.5 to 73.3% and 96.7 to 99.3%, respectively. CONCLUSION: The M414-to-solanidine MR is an excellent predictor of the CYP2D6 PM phenotype. By measuring solanidine and metabolites using liquid chromatography-mass spectrometry in patient serum samples, CYP2D6 PMs can easily be identified, hence facilitating the implementation of precision dosing in clinical practice.

Metabolite Profiling of Clozapine in Patients Switching Versus Maintaining Treatment: A Retrospective Pilot Study.

PURPOSE/BACKGROUND: Pharmacokinetics may be of relevance for the risk of clozapine discontinuation. We compared metabolite profiles, accounting for smoking habits, in patients switching versus maintaining clozapine treatment at therapeutic concentrations. METHODS/PROCEDURES: Adult patients with clozapine serum levels above 1070 nmol/L (350 ng/mL) were retrospectively included from a Norwegian therapeutic drug monitoring service during 2018-2020. Inclusion criteria were (1) known smoking habits, (2) blood sample drawn within 10 to 30 hours after last clozapine intake, and (3) detectable levels of N -desmethylclozapine, clozapine -N -oxide, clozapine-5 N -glucuronide, or clozapine- N + - glucuronide. Patients comedicated with cytochrome P450 enzyme inducers, inhibitors, or valproic acid were excluded. The high-resolution mass spectrometry assay enabled detection of 21 clozapine metabolites. Metabolite profiles were compared between patients switching treatment (switchers), measured as clozapine being replaced by another antipsychotic drug in blood samples, versus maintaining clozapine treatment (nonswitchers) during the study period. FINDINGS/RESULTS: Of the 84 patients fulfilling the study criteria, 7 patients (8.3%) were identified as clozapine switchers. After correcting for smoking habits, the clozapine-5 N -glucuronide/clozapine ratio was 69% lower ( P < 0.001), while the clozapine- N + -glucuronide/clozapine-5 N -glucuronide ratio was 143% higher ( P = 0.026), respectively, in switchers versus nonswitchers. The other metabolite ratios did not significantly differ between switchers and nonswitchers. IMPLICATIONS/CONCLUSIONS: The present study found a significantly reduced 5 N -glucuronidation phenotype in patients switching from clozapine at therapeutic serum concentrations (>1070 nmol/L) to other antipsychotic drugs. This may indicate that glucuronidation, as a potential detoxification mechanism, is related to clozapine tolerability. However, the causality of this observation needs to be investigated in future studies with larger patient populations.

Effects of a Novel UGT2B Haplotype and UGT1A4*3 Allele Variants on Glucuronidation of Clozapine In vivo.

BACKGROUND: Glucuronidation is an important metabolic pathway of clozapine (CLZ), but the impact of various uridine 5'diphospho-glucuronosyltransferases (UGT) polymorphisms on the exposure and metabolism of CLZ in vivo is unclear. OBJECTIVE: The objective of this study was to investigate the impact of UGT2B haplotype and UGT1A4*3 allele variants on the formation of CLZ glucuronide metabolites (5N- and N+-glucuronide) and CLZ exposure in patients' serum after adjusting for sex, age, and smoking habits. METHODS: The study was based on serum samples from CLZ-treated patients (n=79) subjected to routine therapeutic drug monitoring (TDM) at Diakonhjemmet Hospital, Oslo, Norway. From the same patients, the following UGT variants were genotyped using Real-Time PCR: UGT2B:GA haplotype (defined as UGT2B:GA; rs1513559A>G and rs416593T>A) and UGT1A4*3 (rs2011425T>G). Serum concentrations of CLZ 5N- and N+-glucuronide were measured by UPLC high-resolution mass spectrometry. RESULTS: None of the genotypes had significant impact on CLZ exposure (p>0.05). However, compared to UGT2B:AT/AT and UGT1A4*1/*1, the 5N-glucuronide exposure was reduced in UGT2B:GA/GA carriers (-75 %, p=0.03) while the exposure was non-significantly increased in UGT1A4*3 carriers (+100 %, p=0.14), respectively. The N+-glucuronide exposure was unchanged in UGT1A4*3 vs. noncarriers (p=0.28), but significantly reduced in heterozygous (-50 %, p=0.016) and homozygous carriers (-70 %, p=0.021) of UGT2B:GA compared to UGT2B:AT/AT carriers, respectively. CONCLUSION: The UGT2B:GA and UGT1A4*3 variants had no impact on CLZ exposure but were associated with differences and preferences in CLZ glucuronidation. The latter might be of potential relevance for CLZ tolerability since levels of the N+-glucuronide metabolite may reflect the generation and trapping of reactive metabolites involved in CLZ-induced toxicity.

Effect of Valproic Acid on the Metabolic Spectrum of Clozapine in Patients With Schizophrenia.

BACKGROUND: Valproic acid (VPA) is frequently used with clozapine (CLZ) as mood stabilizer and/or seizure prophylaxis. Valproic acid is known to reduce N-desmethylclozapine (N-DMC) but not CLZ levels. This leads to the hypothesis that VPA induces the CLZ metabolism via non-N-desmethylation pathways. Therefore, we aimed to investigate the effect of concurrent VPA use on the serum concentrations of a spectrum of CLZ metabolites in patients, adjusting for smoking. METHODS: In total, 288 patients with an overall number of 737 serum concentration measurements of CLZ and metabolites concurrently using VPA (cases, n = 22) or no interacting drugs (controls, n = 266) were included from a routine therapeutic drug monitoring service. Linear mixed model analyses were performed to compare the dose-adjusted concentrations (C/D) of CLZ, N-DMC, CLZ 5N/N+-glucuronides, and metabolite-to-parent ratios in cases versus controls. RESULTS: After adjusting for covariates, the N-DMC (-40%, P < 0.001) and N+-glucuronide C/Ds (-78%, P < 0.001) were reduced in cases versus controls, while the CLZ C/D was unchanged (P > 0.7). In contrast, the 5N-glucuronide C/D (+250%, P < 0.001) and 5N-glucuronide-to-CLZ ratios (+120%, P = 0.01) were increased in cases versus controls. CONCLUSIONS: Our findings show that complex changes in CLZ metabolism underly the pharmacokinetic interaction with VPA. The lower levels of N-DMC seem to be caused by VPA-mediated induction of CLZ 5N-glucuronide formation, subsequently leading to reduced substrate availability for N-desmethylation. Whether the changes in CLZ metabolism caused by VPA affects the clinical outcome warrants further investigation.

Primary human hepatocyte spheroids as an in vitro tool for investigating drug compounds with low clearance.

Characterizing the pharmacokinetic properties of drug candidates represents an essential task during drug development. In the past, liver microsomes and primary suspended hepatocytes have been extensively used for this purpose, but their relatively short stability limits the applicability of such in vitro systems for drug compounds with low metabolic turnover. In the present study, we used 3D primary human hepatocyte spheroids to predict the hepatic clearance of seven drugs with low to intermediate clearance in humans. Our results indicate that hepatocyte spheroids maintain their in vivo like phenotype during prolonged incubations allowing to monitor the depletion of parent drug for seven days. In contrast, attempts to increase the relative metabolic capacity by pooling hepatocyte spheroids resulted in an immediate fusion of the spheroids followed by hepatocellular de-differentiation processes, demonstrating limited applicability of the pooling approach for quantitative pharmacokinetic studies. The hepatic clearance values obtained from incubations with individual spheroids were in close correlation with the clinical reference data with six out of seven drug compounds being predicted within a three-fold deviation and average fold and absolute average fold errors of 0.57 and 1.74, respectively. In conclusion, the hepatocyte spheroid model enables accurate hepatic clearance predictions for slowly metabolized drug compounds and represents a valuable tool for determining the pharmacokinetic properties of new drug candidates as well as for mechanistic pharmacokinetic studies. Significance Statement Traditional in vitro systems often fail to predict the hepatic clearance of slowly metabolized drug compounds. The current study demonstrates the ability of primary human hepatocyte spheroids to provide accurate projections on the hepatic clearance of drug compounds with low and intermediate clearance.

Validation of a Population Pharmacokinetic Model of Vortioxetine Using Therapeutic Drug Monitoring Data.

INTRODUCTION: Vortioxetine is an antidepressant primarily metabolized by the polymorphic enzyme cytochrome P450 (CYP) 2D6. A population pharmacokinetic (popPK) model of vortioxetine and its CYP2D6-dependent metabolite was recently published. OBJECTIVE: The aim of the current study was to assess the predictive performance of the popPK model using vortioxetine concentration measurements from a clinical setting. Furthermore, the study aimed to evaluate the ability of different CYP2D6 phenotype classification systems to provide accurate concentration predictions. METHODS: Overall, 1388 patients receiving vortioxetine treatment were identified from a therapeutic drug monitoring (TDM) database in Oslo, Norway; 334 CYP2D6-genotyped patients with 502 serum concentrations of vortioxetine, analysed by a validated ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method, were retrospectively included. The performance of the vortioxetine popPK model was tested on the clinical data from the TDM database. RESULTS: Overall, the model had a good ability to predict vortioxetine concentrations measured in clinical practice, with a slight tendency to overpredict concentrations. Using simulation-based diagnostics, 76% of the prediction-corrected TDM concentrations were within the 90% prediction interval based on 1000 simulated data sets. Prediction-based diagnostics showed the best performance for CYP2D6 poor and ultrarapid metabolizers, with a median prediction error (MDPE) of 12% and 23%, respectively, while the poorest performance was observed for normal metabolizers, with an MDPE of 66%. In the comparison of different CYP2D6 phenotype classification systems, the use of differentiated activity scores for decreased function alleles did not improve the concentration predictions. Grouping the CYP2D6 genotypes into the four conventional phenotype groups provided predictions closest to the TDM measured concentrations. CONCLUSION: TDM data provide a unique insight into real-world clinical practice with vortioxetine. The tendency of the popPK model to overpredict vortioxetine concentrations measured in TDM may be attributed to several factors, including poor treatment compliance for some patients and, to a lesser extent, lack of information on patient characteristics and misspecified CYP2D6 alleles. To optimize personalized therapy with vortioxetine, real-world clinical data sets originating from different ethnicities need to be studied in the future.

Substantial Differences in Serum Concentrations of Psychoactive Drugs Measured in Samples Stored for 2 Days or More on Standard Serum Tubes Versus Serum Tubes Containing Gel Separators.

BACKGROUND: Drugs may potentially adsorb to blood collection tubes containing gel separators in the preanalytical phase of therapeutic drug monitoring. The aim of this study was to compare measured concentrations of 28 psychoactive drugs and 13 metabolites in spiked serum samples stored on standard (plain) tubes versus barrier gel tubes during a 2-6-day period at room temperature. METHODS: Drug-free ("blank") serum samples spiked with mixes of antidepressants, antipsychotics, or mood stabilizers (valproic acid and lamotrigine), including relevant metabolites, were transferred to tubes with and without gel, that is, BD Vacutainer SST II Advance gel tubes and BD Vacutainer Glass Serum Tubes (Becton-Dickinson Company, Plymouth, United Kingdom). Mean serum concentrations of the drugs or metabolites measured by ultra-high performance liquid chromatography-tandem mass spectrometry analyses of protein-precipitated samples were compared after storage on plain or gel tubes at 3 time points (day 0, day 2/48 hours, and day 6/144 hours) in room temperature. RESULTS: Mean serum concentrations of all antidepressants, except for one metabolite, and 13 of 18 antipsychotic drugs were significantly lower in gel tubes compared with plain tubes after 2 days of storage (2%-28% lower, P < 0.05). After 6 days of storage, mean serum concentrations of all antipsychotic drugs and antidepressants were significantly lower in gel tubes versus plain tubes (9%-49% lower, P < 0.02), except for amisulpride and O-desmethylvenlafaxine. Serum concentrations of the mood stabilizers were not significantly different in gel tubes compared with plain tubes (P > 0.1). There was a clear relationship between log P (partition coefficient) and residual serum concentrations during gel tube storage (r -0.50 and -0.42 at day 2 and day 6, respectively; P < 0.02). CONCLUSIONS: This study shows that storage on gel for more than 2 days significantly decreases the serum concentrations of antidepressant and antipsychotic drugs as compared to storage in plain tubes. Thus, using tubes with gel separators in the therapeutic drug monitoring of psychoactive drugs should be reconsidered.

Associations between Cytokine Levels and CYP3A4 Phenotype in Patients with Rheumatoid Arthritis.

Systemic inflammation has been linked to suppressed CYP3A4 activity. The aim of this study was to examine associations between levels of a broad selection of cytokines and CYP3A4 phenotype in patients with rheumatoid arthritis (RA). The study included 31 RA patients treated with tumor necrosis factor (TNF)-α inhibitors. CYP3A4 phenotype was measured as serum concentration of 4ß-hydroxycholesterol (4ßOHC) by ultra-performance liquid chromatography-tandem mass spectrometry in samples collected prior to and 3 months after initiation of treatment with TNF-α inhibitors. Serum levels of the following 21 cytokines were determined in the same samples using a bead-based multiplex immunoassay (Luminex technology): CCL2, CCL3, CXCL8, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon γ, interleukin (IL)-1ß, IL-1 receptor antagonist (ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-18, IL-23, and TNF-α Correlations between levels of cytokines and 4ßOHC were assessed by Spearman's rank correlation tests. Among the investigated cytokines, three were negatively correlated with CYP3A4 phenotype during treatment with TNF-α inhibitors: i.e., IL-1ra (r = -0.408, P = 0.023), IL-6 (r = -0.410, P = 0.022) and CXCL8 (r = -0.403, P = 0.025) (P ≥ 0.3 for all other cytokines). None of the analyzed cytokines were correlated with CYP3A4 phenotype prior to TNF-α inhibitor treatment (P > 0.1 for all cytokines). These findings suggest that immune responses associated with increased levels of IL-1ra, IL-6, and CXCL8 may suppress CYP3A4 metabolism. Further studies are required to evaluate these preliminary findings in different patient populations and also examine the possible molecular mechanisms behind our observations.

Comparison of CYP3A4-Inducing Capacity of Enzyme-Inducing Antiepileptic Drugs Using 4ß-Hydroxycholesterol as Biomarker.

BACKGROUND: Enzyme-inducing antiepileptic drugs (EIAEDs) are among the clinically most important inducers of cytochrome P450 (CYP) 3A4, but there is limited evidence regarding the comparative potency of each EIAED in raising CYP3A4 activity. The aim of this study was to estimate CYP3A4-inductive potency of EIAEDs by comparing CYP3A4 activity in patients treated with carbamazepine, phenobarbital, or phenytoin. METHODS: Residual serum samples from patients treated with EIAEDs or levetiracetam were collected from a therapeutic drug monitoring service for analysis of 4ß-hydroxycholesterol (4ßOHC), which is an indicator of CYP3A4 activity. The samples were collected between January and September 2016 at Diakonhjemmet Hospital, Oslo, Norway. Concentration of 4ßOHC, EIAEDs, and levetiracetam was measured by ultra-performance liquid chromatography tandem mass spectrometry. Kruskal-Wallis and Mann-Whitney tests were used for comparison of 4ßOHC levels between the subgroups. RESULTS: In total, 4ßOHC measurements for 343 and 339 patients treated with EIAEDs and levetiracetam, respectively, were included in the study. Compared with levetiracetam-treated patients, the median 4ßOHC concentration was 3.3-fold, 5.8-fold, and 6.9-fold higher in patients using phenobarbital, phenytoin, or carbamazepine, respectively (P < 0.0001). Phenytoin users (n = 65) and carbamazepine users (n = 225) had 1.8- and 2.1-fold higher median 4ßOHC concentration than phenobarbital users (n = 28), respectively (P ≤ 0.0001). CONCLUSIONS: This study shows that phenytoin and carbamazepine have approximately twice the CYP3A4-inducing potency of phenobarbital. The results indicate that 2-fold higher doses of CYP3A4-metabolized drugs may generally be required during concurrent treatment with phenytoin or carbamazepine compared with phenobarbital.