Combined effect of CYP2C19 and CYP2D6 genotypes on escitalopram serum concentration and its metabolic ratio in a European patient population.

AIMS: The aim of the present study was to investigate the impact of CYP2D6 genotype on exposure and metabolism of escitalopram in patients stratified by CYP2C19 genotype in a large real-world population. METHODS: Patients were included from a therapeutic drug monitoring service if they had measured serum concentration of escitalopram and the metabolite, N-desmethyl escitalopram, and performed CYP2C19 and CYP2D6 genotyping. Patients were divided into 16 combined genotype-predicted phenotype subgroups (poor [PM], intermediate [IM], normal [NM] and ultrarapid metabolizers [UM]) of CYP2C19/CYP2D6. The concentration-to-dose (CD) ratio and metabolite-to-parent ratio (metabolic ratio) of escitalopram were compared across subgroups using the Kruskal-Wallis test followed by Dunn's test with CYP2D6 NMs as the reference group. RESULTS: A total of 5067 patients were included in the study. A stepwise increase in escitalopram CD ratio by decreasing CYP2D6 activity was observed in all CYP2C19 subgroups, except for in CYP2C19 UMs. The percentage differences in escitalopram CD ratio between CYP2D6 PMs and NMs were 24% in CYP2C19 NMs (P < .001), 28% in CYP2C19 IMs (P < .001) and 31% in CYP2C19 PMs (P = .04). As for the CD ratio, CYP2D6 genotype effect on metabolic ratio increased stepwise by decreasing CYP2C19 metabolism. CONCLUSIONS: CYP2D6 genotype is of significant importance for the individual variation in escitalopram pharmacokinetics. The most relevant increase in escitalopram concentration is seen in individuals with decreased and/or absent CYP2C19 activity. By combining CYP2C19 and CYP2D6 genotypes, the optimal dose for patients may be predicted with greater precision than for CYP2C19 genotype alone.

Age of onset for increased dose-adjusted serum concentrations of antidepressants and association with sex and genotype: An observational study of 34,777 individuals.

PURPOSE: The aim of this study was to examine the age of onset for increased dose-adjusted serum concentrations (C/D ratio) of common antidepressant drugs and to explore the potential association with sex and CYP2C19/CYP2D6 genotype. METHODS: Serum concentrations and prescribed daily doses for citalopram, escitalopram, sertraline, venlafaxine and mirtazapine, and CYP genotypes, were obtained from a therapeutic drug monitoring (TDM) service. Segmented linear regression analysis was used to examine the relationship between age and antidepressant log C/D ratio in (i) all individuals, (ii) men and women, and (iii) CYP2D6/CYP2C19 normal metabolizers (NMs) and CYP2D6/CYP2C19 intermediate or poor metabolizers (IMs/PMs). RESULTS: A total of 34,777 individuals were included in the study; CYP genotype was available for 21.3%. An increase in C/D ratio started at 44‒55 years of age. Thereafter, the increase progressed more rapidly for citalopram and escitalopram than for venlafaxine and mirtazapine. A doubled C/D ratio was estimated to occur at 79 (citalopram), 81 (escitalopram), 86 (venlafaxine), and 90 years (mirtazapine). For sertraline, only modest changes in C/D ratio were observed. For escitalopram and venlafaxine, the observed increase in C/D ratio started earlier in women than in men. The results regarding CYP genotype were inconclusive. CONCLUSION: The age-related increase in C/D ratio starts in middle-aged adults and progresses up to more than twofold higher C/D ratio in the oldest old. Sertraline seems to be less prone to age-related changes in C/D ratio than the other antidepressants.

Dose-dependent effect of lamotrigine on quetiapine serum concentration in patients using instant release tablets.

PURPOSE: Lamotrigine was previously reported to reduce serum concentration of quetiapine. The aim of this study was to investigate whether lamotrigine dose or quetiapine formulation was of importance for the drug interaction. METHODS: Patients combining lamotrigine with quetiapine (cases) were included retrospectively from a routine therapeutic drug monitoring (TDM) service, as were a control group of patients using quetiapine without any interacting drugs. The case and control groups were divided into groups using immediate release (IR) and extended release (XR) quetiapine. The case group was further split into high-dose (> 200 mg/day) and low-dose (≤ 200 mg/day) lamotrigine users. Quetiapine concentration-to-dose (C/D) ratio and metabolite-to-parent ratio (MPR) were compared between the control group and dose-separated case groups using ANOVA test and t-tests. RESULTS: In total, 406 patients were included. The mean C/D ratio of IR quetiapine was 46% lower in the high-dose lamotrigine group compared with the control group (P < 0.001), while no interaction effect was present in the low dose lamotrigine group (P = 0.7). Regardless of lamotrigine dose, there was no difference in quetiapine C/D ratio for patients using the XR formulation (P = 0.4). The quetiapine MPR was unaffected regardless of formulation and lamotrigine dose (P ≥ 0.06). CONCLUSION: The effect of lamotrigine in reducing quetiapine concentration is only significant for patients using quetiapine IR tablets who are treated with lamotrigine doses > 200 mg/day. Because of high variability in the interaction effect, TDM of quetiapine should be recommended during co-prescription of high-dose lamotrigine.

Impact of CYP2D6 Genotype on Paroxetine Serum Concentration.

BACKGROUND: Paroxetine is a selective serotonin reuptake inhibitor metabolized by cytochrome P450 (CYP)2D6. Only small-scale studies have reported the impact of CYP2D6 genotype on paroxetine exposure, and international guidelines differ in their recommendations on whether paroxetine should be administered according to CYP2D6 genotype. To clarify this issue, the aim of the present study was to investigate the impact of CYP2D6 genotype on paroxetine serum concentration in a large population of patients after adjusting for CYP2C19 genotype, age, and sex. METHODS: Patients from a therapeutic drug monitoring database with records on their paroxetine serum concentrations and CYP2D6 and CYP2C19 genotyping between 2010 and 2021 were included in the study. The impact of CYP2D6 and CYP2C19 genotypes, age, and sex on the paroxetine concentration-to-dose (C/D) ratio was investigated by multiple linear regression analysis. Patients treated with relevant CYP inhibitors or inducers were excluded. RESULTS: In total, 304 patients were included in the study: 17 CYP2D6 poor metabolizers (PMs), 114 intermediate metabolizers (IMs), 168 extensive metabolizers (EMs), and 5 ultrarapid metabolizers. Multiple linear regression analysis showed that CYP2D6 IMs and PMs had 2.2-fold and 3.8-fold higher paroxetine C/D-ratios than extensive metabolizers, respectively ( P < 0.001). Patients who were CYP2C19 IMs (n = 70) or PMs (n = 13) had 1.6-fold higher paroxetine C/D ratio than extensive metabolizers ( P = 0.04). An age ≥65 years was associated with a 2.9-fold increased C/D ratio ( P < 0.001), whereas sex was not significantly associated with paroxetine exposure. CONCLUSIONS: The present study showed that CYP2D6 genotype is of significant importance for paroxetine dose adjustments. For CYP2D6 PMs, 25% of the regular paroxetine starting dose may be sufficient, whereas CYP2D6 IMs could receive 50% of the regular dosage. This well-powered study shows that the guidelines should consider the importance of CYP2D6 genotype for personalized dosing of paroxetine.

Identification of Escitalopram Metabolic Ratios as Potential Biomarkers for Predicting CYP2C19 Poor Metabolizers.

BACKGROUND: Escitalopram is metabolized by CYP2C19 to N-desmethyl escitalopram and escitalopram propionic acid. The primary aims of this study were to investigate the impact of the CYP2C19 phenotype on metabolic ratios of escitalopram in vivo and propose a biomarker for the CYP2C19 phenotype in patients treated with escitalopram. METHODS: Median steady-state serum metabolite/parent drug ratio of N-desmethyl escitalopram and escitalopram propionic acid was investigated across CYP2C19 genotype-translated phenotype groups. The receiver operator characteristics method and the area-under-the-receiver-operator-characteristics curve was used to determine the best suited metabolic ratio for detecting CYP2C19 poor metabolizers (PMs). RESULTS: A total of 441 patients were included in the study. The N-desmethyl escitalopram/escitalopram ratio was 67% and 44% lower in CYP2C19 PMs and intermediate metabolizers (IMs), respectively, than normal metabolizers. Furthermore, the ability of the ratio to predict CYP2C19 PMs was 92%. A metabolic ratio of <0.24 was detected in 8 of 8 PMs in the study, indicating that it is a promising biomarker of reduced CYP2C19 activity. The escitalopram propionic acid/escitalopram ratio was 77% and 48% lower in CYP2C19 PMs and IMs, respectively; however, the ability of the ratio to detect CYP2C19 PMs was only 87%. CONCLUSIONS: These findings suggest that DECT/ECT reflects CYP2C19 activity, and a metabolic ratio of <0.24 strongly predicts CYP2C19 PM phenotype. The ratio could be a valuable alternative to genotyping in personalized dosing of escitalopram and possibly other CYP2C19 substrates. The escitalopram propionic acid/escitalopram ratio was also associated with CYP2C19 activity; however, the ratio was inferior to the DECT/ECT at predicting PMs.

Correlations between 4ß-hydroxycholesterol and hepatic and intestinal CYP3A4: protein expression, microsomal ex vivo activity, and in vivo activity in patients with a wide body weight range.

PURPOSE: Variability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4ß-hydroxycholesterol (4ßOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4ßOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range. METHODS: The patients (n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4ßOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam. RESULTS: 4ßOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (ρ = 0.53, p < 0.001), and hepatic CYP3A4 concentrations (ρ = 0.30, p = 0.027), but not with intestinal CYP3A4 concentrations (ρ = 0.18, p = 0.28) or intestinal microsomal CYP3A4 activity (ρ = 0.15, p = 0.53). 4ßOHC concentrations correlated weakly with midazolam absolute bioavailability (ρ = - 0.23, p = 0.027) and apparent oral clearance (ρ = 0.28, p = 0.008), but not with systemic clearance (ρ = - 0.03, p = 0.81). CONCLUSION: These findings suggest that 4ßOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4ßOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs. TRIAL REGISTRATION: Clinical. TRIALS: gov identifier: NCT02386917.

Dose-Dependent Inhibition of CYP2D6 by Bupropion in Patients With Depression.

PURPOSE: The aim of this study was to investigate the potential dose-dependent CYP2D6 inhibition by bupropion (BUP) in patients with depression. METHODS: Patients combining BUP with venlafaxine were included from a therapeutic drug monitoring (TDM) database at the Diakonhjemmet Hospital (Oslo, Norway). The O/N-desmethylvenlafaxine metabolic ratio measured in TDM samples was used as a biomarker for CYP2D6 phenotype and was compared between patients treated with BUP 150 mg/d and 300 mg/d or greater. In addition, reference groups of venlafaxine-treated patients genotyped as CYP2D6 poor metabolizers (PMs, no CYP2D6 activity) and normal metabolizers (NMs, fully functional CYP2D6 activity) were included. FINDINGS: A total of 221 patients were included in the study. The median O/N-desmethylvenlafaxine metabolic ratio was significantly higher in patients treated with BUP 150 mg/d (n = 59) versus 300 mg/d or greater (n = 34, 1.77 vs 0.96, P < 0.001). In CYP2D6 NMs (n = 62) and PMs (n = 66), the median metabolic ratios were 40.55 and 0.48, respectively. For patients treated with BUP 150 mg/d, 11 (19%) of the 59 patients were phenoconverted to PMs, whereas this was the case for 17 (50%) of the 34 patients treated with BUP 300 mg/d or greater. CONCLUSIONS: Bupropion exhibits a clear dose-dependent CYP2D6 inhibitory effect during treatment of patients with depression. This finding is of clinical relevance when adjusting dosing of CYP2D6 substrates during comedication with BUP. Half of the patients treated with high-dose BUP are converted to CYP2D6 PM phenotype. Because of the variability in CYP2D6 inhibition, TDM of CYP2D6 substrates should be considered to provide individualized dose adjustments during comedication with BUP.

Pharmacodynamic assessment of mycophenolic acid in resting and activated target cell population during the first year after renal transplantation.

AIMS: To explore the pharmacodynamics of mycophenolic acid (MPA) through inosine monophosphate dehydrogenase (IMPDH) capacity measurement and purine levels in peripheral blood mononuclear cells (PBMC) longitudinally during the first year after renal transplantation (TX). METHODS: PBMC were isolated from renal recipients 0-4 days prior to and 6-9 days, 5-7 weeks and 1 year after TX (before and 1.5 hours after dose). IMPDH capacity and purine (guanine and adenine) levels were measured in stimulated and nonstimulated PBMC. RESULTS: Twenty-nine patients completed the follow-up period, of whom 24 received MPA. In stimulated PBMC, the IMPDH capacity (pmol 10-6 cells min-1 ) was median (interquartile range) 127 (95.8-147) before TX and thereafter 44.9 (19.2-93.2) predose and 12.1 (4.64-23.6) 1.5 hours postdose across study days after TX. The corresponding IMPDH capacity in nonstimulated PBMC was 5.71 (3.79-6.93), 3.35 (2.31-5.62) and 2.71 (1.38-4.08), respectively. Predose IMPDH capacity in nonstimulated PBMC increased with time, reaching pre-TX values at 1 year. In stimulated PBMC, both purines were reduced before (median 39% reduction across days after TX) and after (69% reduction) dose compared to before TX. No alteration in the purine levels was observed in nonstimulated PBMC. Patients needing dose reductions during the first year had lower pre-dose IMPDH capacity in nonstimulated PBMC (1.87 vs 3.00 pmol 10-6 cells min-1 , P = .049) at 6-9 days. CONCLUSION: The inhibitory effect of MPA was stronger in stimulated PBMC. Nonstimulated PBMC became less sensitive to MPA during the first year after TX. Early IMPDH capacity appeared to be predictive of dose reductions.

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.

Recovery of CYP3A Phenotype after Kidney Transplantation.

End-stage renal disease impairs drug metabolism via cytochrome P450 CYP3A; however, it is unclear whether CYP3A activity recovers after kidney transplantation. Therefore, the aim of this study was to evaluate the change in CYP3A activity measured as 4ß-hydroxycholesterol (4ßOHC) concentration after kidney transplantation. In total, data from 58 renal transplant recipients with 550 prospective 4ßOHC measurements were included in the study. One sample per patient was collected before transplantation, and 2-12 samples per patient were collected 1-82 days after transplantation. The measured pretransplant 4ßOHC concentrations ranged by >7-fold, with a median value of 22.8 ng/ml. Linear mixed-model analysis identified a 0.16-ng/ml increase in 4ßOHC concentration per day after transplantation (P < 0.001), indicating a regain in CYP3A activity. Increasing estimated glomerular filtration rate after transplantation was associated with increasing 4ßOHC concentration (P < 0.001), supporting that CYP3A activity increases with recovering uremia. In conclusion, this study indicates that CYP3A activity is regained subsequent to kidney transplantation.

The CYP3A biomarker 4ß-hydroxycholesterol does not improve tacrolimus dose predictions early after kidney transplantation.

AIMS: Tacrolimus is a cornerstone in modern immunosuppressive therapy after kidney transplantation. Tacrolimus dosing is challenged by considerable pharmacokinetic variability, both between patients and over time after transplantation, partly due to variability in cytochrome P450 3A (CYP3A) activity. The aim of this study was to assess the value of the endogenous CYP3A marker 4ß-hydroxycholesterol (4ßOHC) for tacrolimus dose individualization early after kidney transplantation. METHODS: Data were obtained from 79 adult kidney transplant recipients who contributed a total of 625 4ßOHC measurements and 1999 tacrolimus whole blood concentrations during the first 2 months after transplantation. The relationships between 4ßOHC levels and individual estimates of tacrolimus apparent plasma clearance (CL/Fplasma ) at different time points after transplantation were investigated using scatterplots and population pharmacokinetic modelling. RESULTS: There was no significant correlation between pre-transplant 4ßOHC levels and tacrolimus CL/Fplasma the first week (r = 0.19 [95% CI -0.03-0.40]) or between 4ßOHC and tacrolimus CL/Fplasma 1 week (r = 0.20 [-0.11-0.47]), 4 weeks (r = 0.21 [-0.07-0.46]) or 2 months (r = 0.24 [-0.03-0.48]) after transplantation (P ≥ 0.06). In the population analysis, time-varying 4ßOHC was not a statistically significant covariate on tacrolimus CL/Fplasma , neither in terms of absolute values (P = 0.11) nor in terms of changes from baseline (P = 0.17). 4ßOHC values increased between 1 week and 2 months after transplantation (median change +57% [IQR +22-83%], P < 0.001), indicating increasing CYP3A activity. Contradictorily, tacrolimus CL/Fplasma decreased over the same period (median change -13% [IQR -3 to -26%], P < 0.001). CONCLUSIONS: 4ßOHC does not appear to have a clinical potential to improve individualization of tacrolimus doses early after kidney transplantation.

Impact of genetic and nongenetic factors on interindividual variability in 4ß-hydroxycholesterol concentration.

PURPOSE: Individual variability in the endogenous CYP3A metabolite 4ß-hydroxycholesterol (4ßOHC) is substantial, but to which extent this is determined by genetic and nongenetic factors remains unclear. The aim of the study was to evaluate the explanatory power of candidate genetic variants and key nongenetic factors on individual variability in 4ßOHC levels in a large naturalistic patient population. METHODS: We measured 4ßOHC concentration in serum samples from 655 patients and used multiple linear regression analysis to estimate the quantitative effects of CYP3A4*22, CYP3A5*3, and POR*28 variant alleles, comedication with CYP3A inducers, inhibitors and substrates, sex, and age on individual 4ßOHC levels. RESULTS: 4ßOHC concentration ranged >100-fold in the population, and the multiple linear regression model explained about one fourth of the variability (R 2 = 0.23). Only comedication with inducers or inhibitors, sex, and POR genotype were significantly associated with individual variability in 4ßOHC level. The estimated quantitative effects on 4ßOHC levels were greatest for inducer comedication (+>313%, P < 0.001), inhibitor comedication (-34%, P = 0.021), and female sex (+30%, P < 0.001), while only a modestly elevated 4ßOHC level was observed in carriers vs. noncarriers of POR*28 (+11%, P = 0.023). CONCLUSIONS: These findings suggest that the CYP3A4*22, CYP3A5*3, and POR*28 variant alleles are of limited importance for overall individual variability in 4ßOHC levels compared to nongenetic factors.

N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.

Cotranslational N-terminal (Nt-) acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently, there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study, we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala, or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.

Effect of CYP2D6 genotype on duloxetine serum concentration.

Duloxetine is metabolized by cytochrome P450 (CYP)1A2 and CYP2D6. The aim of this study was to investigate the effect of the CYP2D6 genotype on duloxetine serum concentration adjusting for age and sex. Patients were included retrospectively from a therapeutic drug monitoring service. Multiple linear regression analysis was used to investigate the effect of CYP2D6 genotype, age and sex on the duloxetine concentration-to-dose (C/D) ratio. In total, 269 patients were included and assigned to the following genotype-predicted phenotype subgroups: CYP2D6 poor metabolizers (PMs, n = 23), intermediate metabolizers (IMs, n = 121), normal metabolizers (NMs, n = 120) and ultrarapid metabolizers (UMs, n = 5). Multiple linear regression analysis revealed a 95% higher duloxetine C/D ratio in PMs compared with NMs (p = 0.009). Patients ≥65 years had a 56% higher C/D ratio than younger patients (p = 0.01), while women had a 46% higher C/D ratio than men (p = 0.04). In conclusion, the CYP2D6 PM phenotype is associated with a twofold higher concentration at recommended dosing compared with the NM phenotype. CYP2D6 PM females above 65 years are at particular risk of high duloxetine levels as they may obtain a threefold higher C/D ratio compared with younger, male NMs.

Access to a tailored mobile application enhances medication adherence among young users of antidepressants.

Introduction: Patients' adherence to antidepressants is generally reported to be poor. This study examined whether users of selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) enhance medication adherence following access to a mobile application (app) tailored for this patient group. The study addresses the implementation phase of medication adherence. Methods: The study was a single group pre-post intervention design. Data were collected using the validated OsloMet Adherence-to-medication Survey tool (OMAS-37) before and after app access. Pre-app access survey (Survey 1) was conducted via social media and online newspapers, encompassing 445 SSRI/SNRI users aged 18 years and above. Post-app access survey (Survey 2) was sent to 103 SSRI/SNRI users from Survey 1. Wilcoxon Signed Rank Test compared pre- and post-intervention adherence measurements. Pearson's chi-square tests and Fisher's exact tests compared study population categories. Results: Forty-two SSRI/SNRI users, median age 26 (IQR 17), 93% identifying as female, used the app while using the same antidepressant during the 2-month period between gaining access to the app and Survey 2. There was a statistically significant reduction in non-adherence score post-app access (z = 3.57, n = 42, p < 0.001) with medium effect size (r = 0.39), indicating enhanced adherence. Total non-adherence score decreased by 39% from pre-to post-access, and there was a 12% decrease in users scoring equivalent with poor adherence (score <2) post-access. Twenty-nine of 37 non-adherence causes improved, with three showing statistical significance. Of 42 responders, 50% (n = 21) indicated using the app one to two times, while 50% (n = 21) more than three times. Approximately 69% (n = 28) found it useful, and 43% (n = 18) felt safer in their use of antidepressants after access to the app. No significant preference was observed for the app over alternative sources of information. Discussion: Enhanced medication adherence was observed among antidepressant users following access to the tailored app. Further studies are warranted to evaluate the app applicability to a broader range of antidepressants users or other patient groups, encompassing those in the initiation phase of medication adherence. The app is intended as an easily accessible supplement to the information and advice provided by prescribing physicians and dispensing pharmacists.

Pro-arrhythmic effect of escitalopram and citalopram at serum concentrations commonly observed in older patients - a study based on a cohort of 19,742 patients.

BACKGROUND: For a decade, patients have been advised against using high citalopram- and escitalopram-doses due to risk for ventricular arrhythmia and cardiac arrest. Still, these drugs are widely used to treat depression and anxiety especially in older patients. It is unclear why they are cardiotoxic and at what serum concentrations patients are at risk for arrhythmias. Thus, how many patients that are at risk for iatrogenic cardiac arrest is unknown. METHODS: We studied the arrhythmogenic effects of citalopram, escitalopram and their metabolites on human cardiomyocytes. Concentrations showing pro-arrhythmic activity were compared with observed drug and metabolite serum concentrations in a cohort of 19,742 patients (age 12-105 years) using escitalopram or citalopram in Norway (2010-2019). As arrhythmia-risk is related to maximum serum concentration, this was simulated for different age-groups from the escitalopram patient material. FINDINGS: Therapeutic concentrations of both citalopram and escitalopram but not their metabolites showed pro-arrhythmic changes in the human cardiac action potential. Due to age-dependent reduction of drug clearance, the proportion of patients above threshold for arrhythmia-risk increased with age. 20% of patients >65 years were predicted to reach potentially pro-arrhythmic concentrations, following intake of 10 mg escitalopram. INTERPRETATION: All patients that are using escitalopram or citalopram and have genetic disposition for acquired long-QT syndrome, are >65 years, are using additional pro-arrhythmic drugs or have predisposition for arrhythmias, should be monitored with therapeutic drug monitoring (TDM) to avoid exposure to potentially cardiotoxic concentrations. Serum concentrations should be kept below 100 nM, to reduce arrhythmia-risk. FUNDING: This study was funded by The Research Council of Norway (project number: 324062).