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

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

Biomolekylære markører som kan bidra til individualisert immundempende behandling hos nyretransplanterte

Etter en allogen nyretransplantasjon er pasientene avhengige av livslang immundempende behandling for å forebygge avstøtning av transplantatet. På grunn av farmakokinetisk variasjon mellom individer er det innført konsentrasjonsmålinger i fullblod eller plasma for å tilpasse doseringen til den enkelte pasient. Nyere forskning viser at det også er stor variasjon i den molekylære responsen mellom individer. Det er derfor potensial til ytterligere individualisering av den immundempende behandlingen ved å måle biomolekylære farmakodynamiske responsmarkører hos pasientene. Denne oppgaven utgjorde et delprosjekt hvor det ble utført farmakokinetiske og farmakodynamiske undersøkelser hos nyretransplanterte pasienter. Hensikten var å se hvordan responsmarkører i immunceller kan relateres til eksponering og effekt av immundempende legemidler hos nyretransplanterte. Responsmarkørenes legemiddelselektivitet skulle i tillegg undersøkes med immunceller fra friske personer. Immuncellers metabolske aktivitet, personers legemiddelsensitivitet, inosin monofosfat dehydrogenase (IMPDH)-aktivitet, nivå av purinbaser og cytokinproduksjon skulle vurderes som potensielle biomarkører. Det ble tatt blodprøve fra nyretransplanterte pasienter før transplantasjon (pre-Tx) og 6-9 dager, 5-7 uker og 1 år etter transplantasjon (post-Tx). Post-Tx ble prøvene tatt rett før og 1,5 time etter dosering av immundempende legemidler på morgenen. Fullblod og isolerte mononukleære celler i perifert blod (PBMC) ble inkubert i 72 timer med og uten ex vivo-stimulering. Metabolsk aktivitet i PBMC ble kvantifisert etter tilsetning av tetrazoliumsaltet WST-1. Legemiddelsensitivitet ble målt hos friske og hos pasienter pre-Tx med metabolsk aktivitet som markør. IMPDH-aktivitet og nivå av purinbaser i PBMC ble kvantifisert ved hjelp av væskekromatografi koblet til tandem massespektrometri (LC-MS/MS). Cytokinproduksjon i stimulert fullblod ble kvantifisert ved hjelp av et multiplex-assay basert på fluorescensmerkede antistoffer. Metabolsk aktivitet og cytokinproduksjon av interleukin (IL)-6, og IL-17 fremstod som mulige generelle markører for immundempende legemidler. Kalsineurinhemmere hadde den mest fremtredende effekten på IL-2 og tumor nekrosefaktor (TNF)-α, og produksjonen av IL-10 ble sterkest hemmet av mTOR- og kalsineurinhemmere. IMPDH-aktivitet, guanin og adenin i stimulerte PBMC fremstod som selektive markører for mykofenolats effekt. I forsøk hos friske var mykofenolat en 2,6 ganger sterkere hemmer av IMPDH-aktivitet i stimulerte PBMC enn i non-stimulerte. Det bør utføres flere forsøk hvor høyere konsentrasjoner av immundempende legemidler tilsettes PBMC og fullblod fra friske for å bekrefte resultatene. To av 17 pasienter fikk avstøtning under studieforløpet. Avstøtningene inntraff i løpet av første uke post-Tx. I tillegg ble det påvist subklinisk avstøtning ved biopsi 1 år post-Tx hos en av disse to pasientene. Det ble påvist viremi (polyoma eller cytomegalovirus) hos tre pasienter 5-10 uker post-Tx. Aktiveringsgrad (basert på metabolsk aktivitet), IMPDH-aktivitet, guanin og adenin i stimulerte PBMC kan potensielt både predikere og reflektere avstøtningsepisoder og viremier. Høy relativ cytokinproduksjon (t1,5 i forhold til t0) av IL-2, IL-10, IL-17 og TNF-α kan potensielt reflektere pågående avstøtningsepisode. Legemiddelresponskurver ble konstruert for takrolimus, mykofenolat og everolimus i forkant av transplantasjon, men responskurvevariabler viste ingen sammenheng med avstøtning eller viremi i det begrensede datamaterialet. Det er nødvendig med data fra flere pasienter for å trekke konklusjoner om sammenheng mellom biomarkørene og kliniske endepunkter. Når data fra hele studiepopulasjonen foreligger, bør biomarkørene på nytt korreleres til legemiddeleksponering og klinisk utfall. Potensielle biomarkører bør velges ut og undersøkes nærmere i en større studie av nyretransplanterte, for å demonstrere eventuell klinisk nytteverdi av farmakodynamisk legemiddelmonitorering

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

The Human N-Alpha-Acetyltransferase 40 (hNaa40p/ hNatD) Is Conserved from Yeast and N-Terminally Acetylates Histones H2A and H4

Protein Na-terminal acetylation (Nt-acetylation) is considered one of the most common protein modification in eukaryotes, and 80-90% of all soluble human proteins are modified in this way, with functional implications ranging from altered protein function and stability to translocation potency amongst others. Nt-acetylation is catalyzed by N-terminal acetyltransferases (NATs), and in yeast five NAT types are identified and denoted NatA-NatE. Higher eukaryotes additionally express NatF. Except for NatD, human orthologues for all yeast NATs are identified. yNatD is defined as the catalytic unit Naa40p (Nat4) which co-translationally Nt-acetylates histones H2A and H4. In this study we identified and characterized hNaa40p/hNatD, the human orthologue of the yeast Naa40p. An in vitro proteome-derived peptide library Nt-acetylation assay indicated that recombinant hNaa40p acetylates N-termini starting with the consensus sequence Ser-Gly-Gly-Gly-Lys-, strongly resembling the N-termini of the human histones H2A and H4. This was confirmed as recombinant hNaa40p Nt-acetylated the oligopeptides derived from the N-termini of both histones. In contrast, a synthetically Nt-acetylated H4 N-terminal peptide with all lysines being non-acetylated, was not significantly acetylated by hNaa40p, indicating that hNaa40p catalyzed H4 Na- acetylation and not H4 lysine Ne-acetylation. Also, immunoprecipitated hNaa40p specifically Nt-acetylated H4 in vitro. Heterologous expression of hNaa40p in a yeast naa40-D strain restored Nt-acetylation of yeast histone H4, but not H2A in vivo, probably reflecting the fact that the N-terminal sequences of human H2A and H4 are highly similar to each other and to yeast H4 while the N-terminal sequence of yeast H2A differs. Thus, Naa40p seems to have co-evolved with the human H2A sequence. Finally, a partial co-sedimentation with ribosomes indicates that hNaa40p co-translationally acetylates H2A and H4. Combined, our results strongly suggest that human Naa40p/NatD is conserved from yeast. Thus, the NATs of all classes of N-terminally acetylated proteins in humans now appear to be accounted for

The CYP3A biomarker 4β-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 4β‐hydroxycholesterol (4βOHC) for tacrolimus dose individualization early after kidney transplantation. Methods: Data were obtained from 79 adult kidney transplant recipients who contributed a total of 625 4βOHC measurements and 1999 tacrolimus whole blood concentrations during the first 2 months after transplantation. The relationships between 4βOHC 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 4βOHC levels and tacrolimus CL/Fplasma the first week (r = 0.19 [95% CI −0.03‐0.40]) or between 4βOHC 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 4βOHC 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). 4βOHC 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: 4βOHC does not appear to have a clinical potential to improve individualization of tacrolimus doses early after kidney transplantation. This research has been accepted and published in the British Journal of Clinical Pharmacology. © 2017 Wile

Elevated 4β‐hydroxycholesterol/cholesterol ratio in anorexianervosa patients

Recent studies have shown that the cytochrome P450 (CYP) 3A phenotype marker 4β‐hydroxycholesterol/cholesterol (4βOHC/C) ratio is negatively correlated with body weight in healthy volunteers, and that obese patients have lower 4βOHC levels than healthy controls. However, 4βOHC/C ratio in underweight patients has yet to be reported. The aim of this study was to examine potential differences in CYP3A activity between underweight patients with anorexia nervosa and normal‐weight volunteers by measuring plasma 4βOHC/C ratio. Furthermore, we wished to describe any association between body mass index (BMI) and 4βOHC/C ratio in underweight patients. A total of 20 underweight patients and 16 normal‐weight volunteers were included in the study, all females. Underweight patients had a median 4βOHC/C ratio (molar ratio × 10−5) of 2.52 (range, 0.90–11.3) compared to 1.29 (0.56–2.09) in normal‐weight subjects (Mann‐Whitney P = 0.0005). 4βOHC/C ratio was negatively correlated with BMI in underweight patients (r = −0.56, P = 0.011), and in the whole study population (r = −0.67, P < 0.0001). This suggests that the negative correlation between 4βOHC/C and BMI, which has previously been reported between 4βOHC/C and body weight in healthy volunteers, extends to underweight patients. The findings indicate that CYP3A activity increases with decreasing BMI, resulting in higher CYP3A activity in underweight patients compared to normal‐weight subjects. The potential clinical relevance of this needs to be studied further by comparing pharmacokinetics of drugs subjected to CYP3A‐mediated metabolism in underweight vs. normal‐weight individuals

Structures and Role of the Intermediate Phases on the Crystallization of BaTiO3 from an Aqueous Synthesis Route

Carbonate formation is a prevailing challenge in synthesis of BaTiO3, especially through wet chemical synthesis routes. In this work, we report the phase evolution during thermal annealing of an aqueous BaTiO3 precursor solution, with a particular focus on the structures and role of intermediate phases forming prior to BaTiO3 nucleation. In situ infrared spectroscopy, in situ X-ray total scattering, and transmission electron microscopy were used to reveal the decomposition, pyrolysis, and crystallization reactions occurring during thermal processing. Our results show that the intermediate phases consist of nanosized calcite-like BaCO3 and BaTi4O9 phases and that the intimate mixing of these along with their metastability ensures complete decomposition to form BaTiO3 above 600 °C. We demonstrate that the stability of the intermediate phases is dependent on the processing atmosphere, where especially enhanced CO2 levels is detrimental for the formation of phase pure BaTiO3