Arnaud Capron

1199G>A and 2677G>T/A polymorphisms of ABCB1 independently affect tacrolimus concentration in hepatic tissue after liver transplantation.

Objective Tacrolimus is an immunosuppressive drug widely used in hepatic transplantation to avoid graft rejection. Its pharmacokinetics is characterized by a large interindividual variability requiring the use of therapeutic drug monitoring in daily clinical practice. Some genetic polymorphisms in biotransformation enzymes or transporter proteins, such as CYP3A5 and P-glycoprotein (ABCB1), in donors and/or recipients, appear as important determinants of the Tac blood pharmacokinetics. A recent study has shown that Tac hepatic tissue concentrations vary greatly among patients and are well correlated with graft outcome. The aim of our study was to investigate the effect of genetic polymorphisms in biotransformation enzymes (CYP3A5 and CYP3A7) or in their regulatory protein pregnane X receptor as well as in transporter proteins (ABCB1 and OATP-C) on Tac pharmacokinetics in liver transplant patients and more specifically on Tac hepatic concentrations. Methods One hundred and fifty liver donors were genotyped for 13 different polymorphisms. Tac blood and hepatic concentrations were compared according to hepatic genotypes. Results and conclusion We confirmed that Tac dose requirement (on the basis of blood therapeutic drug monitoring) was higher among patients expressing hepatic CYP3A5 (at least one CYP3A5*1 allele) compared with patients who did not (CYP3A5*3/*3). Hepatic expression of CYP3A5, however, did not seem to influence Tac hepatic concentrations. In contrast, ABCB1 genetic polymorphisms significantly influenced Tac hepatic concentrations, whereas their impact on blood concentrations seemed negligible. Among these ABCB1 polymorphisms, the 1199G>A and 2677G>T/A single nucleotide polymorphisms seemed to reduce the activity of P-gp on Tac. As Tac hepatic concentrations have been significantly related to the graft outcome, it might be interesting, in the future, to genotype donors for ABCB1 polymorphisms to better individualize the Tac immunosuppressive therapy in hepatic transplantation.

CYP3A5 and ABCB1 polymorphisms influence tacrolimus concentrations in peripheral blood mononuclear cells after renal transplantation.

AIMS This prospective study investigated the effect of genetic polymorphisms in a biotransformation enzyme (CYP3A5) and a transporter protein (ABCB1) on tacrolimus (Tac) whole blood concentrations in renal transplantation, and more specifically on peripheral blood mononuclear cell (PBMC) drug concentrations, after renal transplantation. MATERIALS & METHODS A total of 96 renal transplant recipients were genotyped for the exon 11 (1199G>A), 21 (3435C>T) and 26 (2677G>T/A) polymorphisms in the ABCB1 gene and for the intron 3 polymorphism in the CYP3A5 gene. Tac blood and PBMC concentrations were determined at day 7 after transplantation and at steady state, and then compared with recipient genotypes. RESULTS & CONCLUSION The ABCB1 1199G>A, 3435C>T and 2677G>T/A SNPs, appeared to reduce the activity of P-glycoprotein towards Tac, increasing Tac PBMC concentrations. The impact of ABCB1 genetic polymorphisms on Tac blood concentrations was negligible. As increased Tac intracellular concentrations might in turn enhance immunosuppressive status and prevention or rejection, ABCB1 recipient genotyping might be useful to better individualize the Tac immunosuppressive therapy in renal transplantation.

Correlation of tacrolimus levels in peripheral blood mononuclear cells with histological staging of rejection after liver transplantation: preliminary results of a prospective study

Therapeutic drug monitoring of tacrolimus (TAC) is characterized by a complex relationship between trough blood TAC concentrations and therapeutic efficacy. This prospective study evaluates the predictive value of intrahepatic, peripheral blood mononuclear cells (PBMCs) and blood TAC concentrations during the early postliver transplantation (LT) period. In a cohort of 90 adult liver recipients under TAC‐based monotherapy, liver biopsies were performed at day 7 post‐LT, and PBMCs TAC concentrations were measured at day 1, 3, 5, and 7 post‐LT. Both intrahepatic and PBMCs TAC concentrations were determined. All biopsies were graded following the Banff scoring. Intrahepatic, and day 3, 5, 7 PBMCs concentrations correlated very well with day 7 liver Banff rejection scores (P < 0.05). Clinical rejection was characterized by significantly lower mean TAC PBMCs concentrations at day 5 and 7 (P < 0.05) and tended to be associated to lower mean intrahepatic TAC concentrations at day 7 (P = 0.059). Intrahepatic TAC concentrations at day 7 significantly correlated with TAC PBMCs concentrations from day 5 post‐LT (P < 0.05). TAC PBMCs concentrations might be reliable markers of immunosuppression efficacy during the early phase after LT. This finding could represent an additional tool to individualize more precisely early immunosuppressive schemes after liver transplantation.

Impact of CYP3A4*22 allele on tacrolimus pharmacokinetics in early period after renal transplantation: toward updated genotype-based dosage guidelines

Background: Tacrolimus (Tac) metabolism is mainly mediated by the cytochrome P450 3A (CYP3A) subfamily. Recently, it has been reported that kidney transplant recipients carrying the CYP3A4*22 decrease-of-function allele require lower Tac doses and are more at risk of Tac overexposure than CYP3A4*1/*1 patients. This effect was shown to be independent of the CYP3A5*3 allelic status. However, the pharmacokinetic (PK) parameters assessed in previous studies were limited on single time point whole blood trough concentrations (C0) during routine follow-up of the patient after transplantation. Methods: Our study investigates the impact of the CYP3A4*22 allele on Tac PK [C0, area under the time vs concentration curve (AUC0–12h), apparent clearance (Cl/F), Cmax, and dose requirement], time to achieve target C0, and creatinine clearance (CrCl) in 96 kidney transplant recipients considering the 2 first weeks after the graft. All patients were genotyped for both the CYP3A4*22 and the CYP3A5*3 polymorphisms. Results: CYP3A4*22 carriers had higher Tac C0 during the first week with significant longer exposures to C0 > 15 ng/mL. These patients showed reduced Tac Cl/F but higher dose-adjusted AUC0–12h and Cmax and were at increased risk of C0 > 20 ng/mL. These effects were independent from CYP3A5*3 genotype: clustering patients according to both CYP3A4*22 and CYP3A5*3 allelic status did increase the predictive value of the genotype to explain interindividual differences in Tac PK. During the second week after transplantation, CrCl was on average 9.5 mL/min higher for CYP3A4*22 carriers compared with CYP3A4*1/*1 patients (P = 0.007), suggesting that Tac overexposure in CYP3A4*22 carriers might provide a renal function benefit. Conclusions: Our study confirms the decreased CYP3A4 activity toward Tac for CYP3A4*22 carriers early after transplantation and provides evidence for refining genotype-based dosage by adding the CYP3A4*22 genotype information to the CYP3A5*3 allelic status.

Evaluation of Adherence Should Become an Integral Part of Assessment of Patients With Apparently Treatment-Resistant Hypertension.

Since the publication of the first Symplicity studies in 2009 to 2010, renal sympathetic denervation gained acceptance as a novel treatment of drug-resistant hypertension. The latter has been defined as a blood pressure (BP) >140/90 mm Hg, despite appropriate lifestyle measures plus a diuretic and 2 other antihypertensive drugs belonging to different classes at adequate doses.1 According to the US definition, patients with controlled BP on ≥4 antihypertensive drugs are also considered as resistant hypertensives.2 However, a substantial proportion of patients with apparently resistant hypertension are in fact poorly adherents to drug treatment. The highly variable BP response to renal denervation (RDN)3–5 prompted to a more rigorous evaluation of eligible patients, with the goal to exclude false resistant hypertension, because of poor adherence to drug treatment.6–8 In particular, several publications documented a high proportion of low drug adherence in patients with apparently resistant hypertension (23%–66%), using witnessed drug intake9 or plasma/urine drug determinations10–18 (Figure 1). Figure 1. Proportion of poor or nonadherence according to drug monitoring in different cohorts of patients with apparently resistant hypertension. Black indicates total nonadherence, whereas gray indicates partial adherence. Partial adherence was defined as the presence of at least one undetectable drug10–12,14,16–18 or as the presence of fewer medications than prescribed.13,15 Furthermore, RDN studies shed the light on the dynamic character of drug adherence. Inclusion in RDN trials may influence drug adherence in various, unpredictable directions.6 In some patients, close follow-up and massive attention devoted to them may lead to improved adherence to lifestyle measures and drug treatment, particularly in the RDN arm (Hawthorne effect). Other patients may stop their medications after RDN according to their perception that the intervention cured their …

Validation of a Liquid Chromatography-Mass Spectrometric Assay for Tacrolimus in Peripheral Blood Mononuclear Cells.

As a potential alternative to whole-blood tacrolimus (TAC) monitoring, a sensitive and selective method was developed for quantifying this immunosuppressant in human peripheral blood mononuclear cell population (PBMCs). These cells, expected to be a more specific biological matrix than whole blood to reflect pharmacological efficacy, could be promising for TAC therapeutic drug monitoring (TDM). The assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs are isolated from 7 mL whole blood by centrifugation over Ficoll gradient density and washed twice with phosphate-buffered saline at 4°C. Harvested cells were suspended within 1.5 mL of phosphate-buffered saline. Cell counts were performed to express and normalize TAC amount per 106 cells. TAC was extracted by a liquid-liquid extraction in basic medium (NH4OH) with 1-chlorobutane, and ascomycin was used as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in methanol (MeOH). Compounds were eluted on a C18 column by a mixture of acetonitrile/water (90/10, vol/vol) containing 0.1% formic acid and 2 mmol/L of ammonium acetate. TAC and internal standard were monitored by detecting specific ammoniated product ions using multiple reaction monitoring acquisition mode in electrospray positive ionization. This method was fully validated in the range of 0.01-5 ng/mL. Limit of detection and of quantification are 0.005 and 0.01 ng/mL, respectively. Intra-assay and interassay recoveries ranged from 89.2% to 114.3% and 85.3% to 103.9%, respectively. Intra-assay and interassay imprecisions ranged from 9.3% to 12% and 10.7% to 12.2%, respectively, across the analytical range. Mean TAC extraction efficiency was 80.9% ± 8.3%. Matrix effects were minimal with <8% ion suppression. This method is currently applied in clinical research protocols and allows the measurement of small intracellular amounts of TAC down to 0.006 ng per 106 PBMCs in kidney-transplanted recipients. This method could be a new potential tool for TAC TDM, providing new perspectives for optimizing immunosuppressive therapy. Further studies should be conducted to fully evaluate the benefit of intracellular TAC concentrations in refinement of TDM strategies for TAC to ensure optimal clinical outcomes.

Validation of a liquid chromatography-mass spectrometric assay for tacrolimus in liver biopsies after hepatic transplantation: correlation with histopathologic staging of rejection.

The aims of this work were both to validate a sensitive and specific method to quantify tacrolimus (TAC) in liver biopsies after hepatic transplantation and to evaluate the predictive value of either tissue or blood TAC concentrations for rejection in 146 adult patients under a TAC-based immunosuppression. Trough blood levels were monitored daily during the hospital stay by immunoassay. Liver biopsies were routinely performed at day 7 posttransplantation. The tissue assay was developed by liquid chromatography-mass spectrometry. The limit of quantification was 5 pg/mg, with intra- and interassay precision ranging from 3.9% to 14.3% and 4.7% to 15.9%, respectively. The extraction efficiency was approximately 80%. TAC found in liver biopsies ranged from less than 5 up to 387 pg/mg. Blood TAC levels ranged from 2.7 to 19.3 ng/mL. Tissue levels displayed excellent correlation with liver histopathologic BANFF rejection score, whereas blood levels did not. Clinically significant rejections (BANFF scores ≥6) were characterized by mean TAC tissue and blood concentration of 13.1 pg/mg and 7.6 ng/mL, respectively, whereas these mean values became, respectively, 74.9 pg/mg (P < 0.05) and 7.1 ng/mL (not significant) for nonclinically significant rejection episodes (BANFF <6). In this study, hepatic tissue TAC concentrations were distributed in a wider range and displayed a significantly better correlation with the severity of the organ rejection than predose blood levels. A tissue TAC concentration less than 30 pg/mg is 89% sensitive and 98% specific to discriminate clinically significant cellular rejection. Further studies are required to better understand the factors affecting TAC distribution within liver tissue (such as carrier proteins and cytochrome genetic polymorphism, liver function, age, hepatic blood flow, type of organ transplanted, time posttransplantation) and to define its value in the treatment of liver allograft rejection.

Ultra-high performance liquid chromatography tandem mass spectrometric method for the determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots--development, validation and clinical application during breast cancer adjuvant therapy.

A LC-MSMS method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots samples was developed and validated. The method employs an ultrasound-assisted liquid extraction and a reversed phase separation in an Acquity(®) C18 column (150×2.1 mm, 1.7 µm). Mobile phase was a mixture of formic acid 0.1% (v/v) pH 2.7 and acetonitrile (gradient from 60:40 to 50:50, v/v). Total analytical run time was 8 min. Precision assays showed CV % lower than 10.75% and accuracy in the range 94.5 to 110.3%. Mean analytes recoveries from DBS ranged from 40% to 92%. The method was successfully applied to 91 paired clinical DBS and plasma samples. Dried blood spots concentrations were highly correlated to plasma, with rs>0.83 (P<0.01). Median estimated plasma concentrations after hematocrit and partition factor adjustment were: TAM 123.3 ng mL(-1); NDT 267.9 ng mL(-1), EDF 10.0 ng mL(-1) and HTF 1.3 ng mL(-1,) representing in average 98 to 104% of the actually measured concentrations. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold related to better prognosis (5.9 ng mL(-1)). The procedure has adequate analytical performance and can be an efficient tool to optimize adjuvant breast cancer treatment, especially in resource limited settings.

ABCB1 1199G>A genetic polymorphism (Rs2229109) influences the intracellular accumulation of tacrolimus in HEK293 and K562 recombinant cell lines.

Objective ATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs. Method Two recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments. Results Tacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine. Conclusion ABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response.

The spectrum of acute heart failure after venlafaxine overdose

Abstract Objective. Venlafaxine is a bicyclic antidepressant that may be associated with severe cardiotoxicity following large overdose. The purpose of this short case series is to present different patterns of venlafaxine-related cardiotoxicity and to discuss the potential mechanisms. Case series. Between January 2010 and July 2011, four patients were admitted to an ICU with acute left ventricular failure following large venlafaxine overdoses. The age of the four female patients ranged from 35 to 65 years. None of them had no history of cardiovascular disease. The amount of venlafaxine ingested by history ranged from 3150 to 13500 mg (extended-release preparation in two cases). The peak serum venlafaxine concentration was between 2153.3 and 9950 ng/ml. Three patients died and one recovered rapidly. The initial ECG revealed only mild abnormalities in two cases. In two patients, at least one ECG recording demonstrated a widening of QRS interval. In three patients, echocardiography disclosed a left ejection fraction of 15%–18%. Two patients presented a severe serotonin syndrome, with major rhabdomyolysis. Seizures were noted in two cases, including one patient with status epilepticus. Three patients were mechanically ventilated. The causes of death were refractory hypoxemia, malignant arrhythmias, and cardiogenic shock, respectively. Discussion. Severe and diffuse left ventricular dysfunction may be observed after large venlafaxine overdoses and this is not always associated with severe cardiac conduction function abnormalities. The mechanisms underlying venlafaxine-related cardiac failure with preserved normal cardiac conduction are discussed. A possible explanation may be a catecholamine-induced myocardial damage in relationship with the inhibition of norepinephrine (and dopamine) reuptake.