Identification and tissue distribution of two differentially spliced variants of the rat carnitine transporter OCTN2

AbstractIn this paper we show that the only known Na+ dependent transporter of carnitine in mammals, organic cation transporter number 2 (OCTN2), is subject to differential splicing. Cloning of OCTN2 in different rat tissues identified two splicing variants. We have developed a real time quantitative polymerase chain reaction method for quantification of these splice variants. Both splice variants could be detected in all tissues examined with a relative abundance of 0.1–1% of the full length transcript. We also draw attention to the previously described mutations in clinical examples of primary carnitine deficiency in humans where the described mutations appear to be those of a splicing or mis-splicing event

Metformin, Sulfonylureas, or Other Antidiabetes Drugs and the Risk of Lactic Acidosis or Hypoglycemia

OBJECTIVE: Lactic acidosis has been associated with use of metformin. Hypoglycemia is a major concern using sulfonylureas. The aim of this study was to compare the risk of lactic acidosis and hypoglycemia among patients with type 2 diabetes using oral antidiabetes drugs. RESEARCH DESIGN AND METHODS: This study is a nested case-control analysis using the U.K.-based General Practice Research Database to identify patients with type 2 diabetes who used oral antidiabetes drugs. Within the study population, all incident cases of lactic acidosis and hypoglycemia were identified, and hypoglycemia case subjects were matched to up to four control patients based on age, sex, practice, and calendar time. RESULTS: Among the study population of 50,048 type 2 diabetic subjects, six cases of lactic acidosis during current use of oral antidiabetes drugs were identified, yielding a crude incidence rate of 3.3 cases per 100,000 person-years among metformin users and 4.8 cases per 100,000 person-years among users of sulfonylureas. Relevant comorbidities known as risk factors for lactic acidosis could be identified in all case subjects. A total of 2,025 case subjects with hypoglycemia and 7,278 matched control subjects were identified. Use of sulfonylureas was associated with a materially elevated risk of hypoglycemia. The adjusted odds ratio for current use of sulfonylureas was 2.79 (95% CI 2.23–3.50) compared with current metformin use. CONCLUSIONS: Lactic acidosis during current use of oral antidiabetes drugs was very rare and was associated with concurrent comorbidity. Hypoglycemic episodes were substantially more common among sulfonylurea users than among users of metformin.Merck SA, Lyon, Franc

Mechanisms of hepatocellular toxicity associated with new psychoactive synthetic cathinones

Synthetic cathinones are a new class of psychostimulant substances. Rarely, they can cause liver injury but associated mechanisms are not completely elucidated. In order to increase our knowledge about mechanisms of hepatotoxicity, we investigated the effect of five frequently used cathinones on two human cell lines. Bupropion was included as structurally related drug used therapeutically. In HepG2 cells, bupropion, MDPV, mephedrone and naphyrone depleted the cellular ATP content at lower concentrations (0.2-1mM) than cytotoxicity occurred (0.5-2mM), suggesting mitochondrial toxicity. In comparison, methedrone and methylone depleted the cellular ATP pool and induced cytotoxicity at similar concentrations (≥2mM). In HepaRG cells, cytotoxicity and ATP depletion could also be demonstrated, but cytochrome P450 induction did not increase the toxicity of the compounds investigated. The mitochondrial membrane potential was decreased in HepG2 cells by bupropion, MDPV and naphyrone, confirming mitochondrial toxicity. Bupropion, but not the other compounds, uncoupled oxidative phosphorylation. Bupropion, MDPV, mephedrone and naphyrone inhibited complex I and II of the electron transport chain, naphyrone also complex III. All four mitochondrial toxicants were associated with increased mitochondrial ROS and increased lactate production, which was accompanied by a decrease in the cellular total GSH pool for naphyrone and MDPV. In conclusion, bupropion, MDPV, mephedrone and naphyrone are mitochondrial toxicants impairing the function of the electron transport chain and depleting cellular ATP stores. Since liver injury is rare in users of these drugs, affected persons must have susceptibility factors rendering them more sensitive for these drugs

Determination of −3858G→A and −164C→A genetic polymorphisms of CYP1A2 in blood and saliva by rapid allelic discrimination: large difference in the prevalence of the −3858G→A mutation between Caucasians and Asians

Introduction: Two mutations in CYP1A2, −164C→A (allele CYP1A2*F) and −3858G→A (allele CYP1A2*C), affecting the inducibility of the enzyme, have been published. The aim of this study was to develop a high throughput allelic discrimination assay for these mutations in both saliva and blood and to determine their frequency in Caucasians. Methods: An allelic discrimination assay, based on the fluorogenic 5′-nuclease activity (TaqMan), was developed for the two mutations. Genomic DNA extracted from 17 saliva and 100 blood samples from Caucasians was analysed. Results and conclusions: For the −164C→A mutation, we found an allelic frequency of 68% in the Caucasian population, comparable with data published for Asians and Caucasians. For the −3858G→A mutation, the allele frequency was only 2% in Caucasians, a much lower value than the ~25% reported in Asians (P<0.001). The presented allelic discrimination allows fast and accurate detection of these two mutations. Genotype calls were 100% identical for DNA from saliva and blood. Saliva is easily accessible and represents an excellent alternative to the traditionally used venous blood for genotypin

Recognition and management of potential drug-drug interactions in patients on internal medicine wards

Introduction: Our aim was to study and possibly improve the clinical management of potential drug-drug interactions (pDDIs) in hospitalized patients by specific interventions. Methods: During the initial period, inpatients on three medical wards were screened for major and moderate pDDIs using the interaction screening program Pharmavista. During the second period, patients at discharge were screened similarly. After assessment of the detected pDDIs for clinical relevance, written recommendations and/or information about the pDDIs were sent to the treating physicians. Feedback from the physicians and implementation of the recommendations were analyzed. Results: During the initial period, 502 inpatients were exposed to 567 pDDIs, of which 419 (74%) were judged to be clinically relevant. Three hundred and forty-nine substantiated recommendations and 70 simple information leaflets were handed out to the physicians. Eighty percent (278 of 349) of the recommendations were accepted and implemented. During the second period, 792 patients at hospital discharge were exposed to 392 pDDIs, of which 258 (66%) were judged to be clinically relevant. Two hundred and forty-seven substantiated recommendations and 11 simple information leaflets were sent to the physicians. Seventy-three percent (180 of 247) of the recommendations were accepted. At hospital discharge, 47 of 71 interventions recommending checkable medication changes were implemented. One year after hospital discharge, 11 of 13 checked medication changes were still in place. Conclusions: Clinically relevant pDDIs are common in patients on medical wards, and their management can be influenced by providing substantiated recommendations to physicians. Most changes in medication following such recommendations are still in place 1year after discharg

Cytochrome P450 Enzymes Involved in Metoprolol Metabolism and Use of Metoprolol as a CYP2D6 Phenotyping Probe Drug

Metoprolol is used for phenotyping of cytochrome P450 (CYP) 2D6, a CYP isoform considered not to be inducible by inducers of the CYP2C, CYP2B, and CYP3A families such as rifampicin. While assessing CYP2D6 activity under basal conditions and after pre-treatment with rifampicin in vivo, we surprisingly observed a drop in the metoprolol/α-OH-metoprolol clearance ratio, suggesting CYP2D6 induction. To study this problem, we performed in vitro investigations using HepaRG cells and primary human hepatocytes (before and after treatment with 20 μM rifampicin), human liver microsomes, and CYP3A4-overexpressing supersomes. While mRNA expression levels of CYP3A4 showed a 15- to 30-fold increase in both cell models, mRNA of CYP2D6 was not affected by rifampicin. 1′-OH-midazolam formation (reflecting CYP3A4 activity) increased by a factor of 5–8 in both cell models, while the formation of α-OH-metoprolol increased by a factor of 6 in HepaRG cells and of 1.4 in primary human hepatocytes. Inhibition studies using human liver microsomes showed that CYP3A4, 2B6, and 2C9 together contributed 19.0 ± 2.6% (mean ± 95%CI) to O-demethylation, 4.0 ± 0.7% to α-hydroxylation, and 7.6 ± 1.7% to N-dealkylation of metoprolol. In supersomes overexpressing CYP3A4, metoprolol was α-hydroxylated in a reaction inhibited by the CYP3A4-specific inhibitor ketoconazole, but not by the CYP2D6-specific inhibitor quinidine. We conclude that metoprolol is not exclusively metabolized by CYP2D6. CYP3A4, 2B6, and 2C9, which are inducible by rifampicin, contribute to α-hydroxylation, O-demethylation, and N-dealkylation of metoprolol. This contribution is larger after CYP induction by rifampicin but is too small to compromise the usability of metoprolol α-hydroxylation for CYP2D6 phenotyping

Effect of l-carnitine on the kinetics of carnitine, acylcarnitines and butyrobetaine in long-term haemodialysis

Background. The current study was performed to investigate the kinetics of carnitine, individual acylcarnitines and butyrobetaine in patients on haemodialysis. Methods. Eight stable long-term haemodialysis patients were studied under basal conditions (no carnitine supplementation) and 3 weeks after intravenous supplementation with l-carnitine (10 or 20 mg/kg body weight) after each haemodialysis session. The kinetic studies included serial determinations of carnitine and metabolites just before, during or between haemodialysis sessions. Analysis was performed by liquid chromatography-tandem mass spectrometry. Results. Before haemodialysis, the plasma concentrations were (µmol/l) 15.1±0.6 (mean±SEM) for carnitine, 5.9±0.7 for acetylcarnitine, 0.66±0.04 for propionylcarnitine and 0.98±0.08 for butyrobetaine (basal conditions) or 142±23 for carnitine, 69±12 for acetylcarnitine, 6.0±1.1 for propionylcarnitine and 2.6±0.3 for butyrobetaine (carnitine 20 mg/kg). During haemodialysis, the plasma concentrations dropped by ∼80% for all compounds determined, with extraction coefficients ranging from 0.65 to 0.86. In patients supplemented with 20 mg/kg carnitine, the amount of carnitine removed by haemodialysis equalled 42% of the dose administered, consisting of 2.08 mmol carnitine, 1.03 mmol acetylcarnitine and 0.051 mmol propionylcarnitine. Between the haemodialysis sessions, carnitine, acylcarnitines and butyrobetaine reached apparent steady-state concentrations within 1 day both under basal conditions and after supplementation. Conclusions. Patients on haemodialysis have reduced carnitine, acylcarnitine and butyrobetaine plasma levels, which can be increased by supplementing carnitine. Propionylcarnitine, an important constituent of the acylcarnitine pool, can be removed by haemodialysis. Removal of potentially toxic acyl-groups may represent a mechanism for a beneficial effect of carnitine in these patient

Potential drug-drug interactions and adverse drug reactions in patients with liver cirrhosis

Background and aims: Patients with liver cirrhosis may be at risk for potential drug-drug interactions (pDDIs) and/or adverse drug reactions (ADRs) due to the severity of their disease and comorbidities associated with polypharmacy. Methods: We performed a cross-sectional retrospective study including 400 cirrhotic patients and assessed diagnoses, medication patterns, pDDIs, and ADRs at hospital admission. Results: The median (range) age of the patients was 60 (21-88) years; 68.5% were male. They had a total of 2,415 diagnoses, resulting in 6 (1-10) diagnoses per patient. Frequent were diagnoses of the digestive system (28.4%), circulatory system (14.2%), blood and blood-forming organs (8.7%), and psychiatric disorders (7.5%); 60.7% of the diagnoses were not liver-associated. The median number of drugs per patient was 5 (0-18), whereof 3 (0-16) were predominantly hepatically eliminated. Drugs were primarily indicated for gastrointestinal, cardiovascular, or nervous system disorders, reflecting the prevalent diagnoses. In 112 (28%) patients, 200 ADRs were detected, mainly associated with spironolactone, torasemide, furosemide, and ibuprofen. In 86 (21.5%) patients, 132 pDDIs were detected. Seven of these pDDIs were the direct cause of 15 ADRs, whereof 3 resulted in hospital admission. Patients with ADRs were older, had more comorbidities, were treated with more drugs, and had a worse renal function and more pDDIs than patients without ADRs. Conclusions: Pharmacotherapy is complex in cirrhotic patients. Hepatologists should know the principles of dose adjustment in cirrhosis and renal failure, but also the most important pDDIs of the drugs used to treat liver disease and comorbidities in this populatio

Mechanisms of Hepatocellular Toxicity Associated with Dronedarone—A Comparison to Amiodarone

Dronedarone is a new antiarrhythmic drug with an amiodarone-like benzofuran structure. Shortly after its introduction, dronedarone became implicated in causing severe liver injury. Amiodarone is a well-known mitochondrial toxicant. The aim of our study was to investigate mechanisms of hepatotoxicity of dronedarone in vitro and to compare them with amiodarone. We used isolated rat liver mitochondria, primary human hepatocytes, and the human hepatoma cell line HepG2, which were exposed acutely or up to 24h. After exposure of primary hepatocytes or HepG2 cells for 24h, dronedarone and amiodarone caused cytotoxicity and apoptosis starting at 20 and 50µM, respectively. The cellular ATP content started to decrease at 20µM for both drugs, suggesting mitochondrial toxicity. Inhibition of the respiratory chain required concentrations of ~10µM and was caused by an impairment of complexes I and II for both drugs. In parallel, mitochondrial accumulation of reactive oxygen species (ROS) was observed. In isolated rat liver mitochondria, acute treatment with dronedarone decreased the mitochondrial membrane potential, inhibited complex I, and uncoupled the respiratory chain. Furthermore, in acutely treated rat liver mitochondria and in HepG2 cells exposed for 24h, dronedarone started to inhibit mitochondrial β-oxidation at 10µM and amiodarone at 20µM. Similar to amiodarone, dronedarone is an uncoupler and an inhibitor of the mitochondrial respiratory chain and of β-oxidation both acutely and after exposure for 24h. Inhibition of mitochondrial function leads to accumulation of ROS and fatty acids, eventually leading to apoptosis and/or necrosis of hepatocytes. Mitochondrial toxicity may be an explanation for hepatotoxicity of dronedarone in viv

Characteristics of Presentations to the Emergency Department Following Attempted Suicide with Drugs.

A relatively high proportion of attempted suicides employ self-poisoning with medication. Data from emergency department presentations can help to identify possible risk drug classes and provide a basis for preventive measures. This retrospective analysis included cases presenting at the emergency department of the University Hospital of Bern, Switzerland, from May 2012 to August 2016, after attempted suicide with drugs. We excluded attempted suicides with only alcohol or other non-medical substances. During the study period, there were 488 cases (466 patients) of attempted suicide with medical substances. The median patient age was 33 years (range 16-93) and 354 (73%) cases were female. The most commonly involved substances/drug classes were benzo-diazepines (n = 167, 34%), neuroleptics (n = 114, 23%) and paracetamol (n = 111, 23%). A total of 231 (47%) cases employed only a single substance. Common symptoms included somnolence (n = 245, 50%), tachycardia (n = 119, 24%) and nausea/vomiting (n = 76, 16%). In most cases, the poisoning was of minor severity (n = 231, 47%) and the patients were admitted to a psychiatric hospital (n = 264, 54%). Important preventive measures may include careful monitoring for suicidal behaviour when prescribing psychotropic drugs, in addition to restrictions in pack size. Efforts should also be made to enhance the awareness of health professionals qualified to prescribe or supply paracetamol