Anne Hulin

Efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in the treatment of vasculopathy associated with TMEM173-activating mutations in 3 children

Marie-Louise Frémond, MD, Mathieu Paul Rodero, PhD, Nadia Jeremiah, PhD, Alexandre Belot, MD, PhD, Eric Jeziorski, MD, Darragh Duffy, PhD, Didier Bessis, MD, Guilhem Cros, MD, Gillian I. Rice, PhD, Bruno Charbit, MSc, Anne Hulin, PharmD, PhD, Nihel Khoudour, MD, Consuelo Modesto Caballero, MD, Christine Bodemer, MD, PhD, Monique Fabre, MD, Laureline Berteloot, MD, Muriel Le Bourgeois, MD, Philippe Reix, MD, Thierry Walzer, PhD, Despina Moshous, MD, PhD, Stéphane Blanche, MD, PhD, Alain Fischer, MD, PhD, Brigitte Bader-Meunier, MD, Fréderic Rieux-Laucat, PhD, Yanick Joseph Crow, MD, PhD, Bénédicte Neven, MD, PhD

Evaluation of the Architect® tacrolimus assay in kidney, liver, and heart transplant recipients

The narrow therapeutic range of tacrolimus requires therapeutic drug monitoring to prevent transplant rejection and to minimize nephrotoxicity. The aim of this study was to evaluate the analytical performance of the tacrolimus chemiluminescent microparticle immunoassay (CMIA) in everyday practice comparatively with other methods. CMIA imprecision and accuracy were tested using low, medium, and high concentrations in control samples. The limits of quantification (LOQ) of CMIA and antibody-conjugated magnetic immunoassay (ACMIA) were evaluated using negative whole-blood samples containing 0.4-5.7 ng/ml of tacrolimus from a stock solution. CMIA was compared with ACMIA, enzyme multiplied immunoassay (EMIT), and liquid chromatography-tandem mass spectrometry (LC-MS/MS), using 176 samples from recipients (135 men and 41 women) of heart (n=19), kidney (n=107), or liver (n=50) transplants. CMIA total precision was 5.7%, 3.7% and 3.6% with the low-, medium-, and high-concentration controls, respectively; corresponding values for accuracy were 98%, 104%, and 104%. LOQ was 0.5 (95%CI, 0.22-1.38) with CMIA and 2.5 ng/ml with ACMIA. Linear regression results were as follows: CMIA=1.2LC-MS/MS+0.14 (r=0.98); CMIA=0.93EMIT+0.36 (r=0.975); CMIA=1.15ACMIA-0.25 (r=0.988); and, for tacrolimus concentrations in the 1-15 ng/ml range, of special interest as many transplant recipients are given low-dose tacrolimus, CMIA=1.05LC-MS/MS+0.38 (r=0.94). Two patients had falsely elevated tacrolimus concentrations due to interference in the ACMIA assay; one was a renal transplant recipient who stopped her treatment and had tacrolimus concentrations of 12.5 ng/ml by ACMIA and <0.5 ng/ml by CMIA; the other was an HIV-positive renal transplant recipient whose tacrolimus concentrations by ACMIA were 1.8-43.7-fold those by CMIA. Such interferences with ACMIA, which may be related to endogenous antibodies in the plasma, are likely to negatively impact patient care. In conclusion, the tacrolimus CMIA assay is suitable for routine laboratory use and does not suffer from the interferences seen with ACMIA in some patients.

Population pharmacokinetics and pharmacodynamics of hydroxyurea in sickle cell anemia patients, a basis for optimizing the dosing regimen

BackgroundHydroxyurea (HU) is the first approved pharmacological treatment of sickle cell anemia (SCA). The objectives of this study were to develop population pharmacokinetic(PK)-pharmacodynamic(PD) models for HU in order to characterize the exposure-efficacy relationships and their variability, compare two dosing regimens by simulations and develop some recommendations for monitoring the treatment.MethodsThe models were built using population modelling software NONMEM VII based on data from two clinical studies of SCA adult patients receiving 500-2000 mg of HU once daily. Fetal hemoglobin percentage (HbF%) and mean corpuscular volume (MCV) were used as biomarkers for response. A sequential modelling approach was applied. Models were evaluated using simulation-based techniques. Comparisons of two dosing regimens were performed by simulating 10000 patients in each arm during 12 months.ResultsThe PK profiles were described by a bicompartmental model. The median (and interindividual coefficient of variation (CV)) of clearance was 11.6 L/h (30%), the central volume was 45.3 L (35%). PK steady-state was reached in about 35 days. For a given dosing regimen, HU exposure varied approximately fivefold among patients. The dynamics of HbF% and MCV were described by turnover models with inhibition of elimination of response. In the studied range of drug exposures, the effect of HU on HbF% was at its maximum (median Imax was 0.57, CV was 27%); the effect on MCV was close to its maximum, with median value of 0.14 and CV of 49%. Simulations showed that 95% of the steady-state levels of HbF% and MCV need 26 months and 3 months to be reached, respectively. The CV of the steady-state value of HbF% was about 7 times larger than that of MCV. Simulations with two different dosing regimens showed that continuous dosing led to a stronger HbF% increase in some patients.ConclusionsThe high variability of response to HU was related in part to pharmacokinetics and to pharmacodynamics. The steady-state value of MCV at month 3 is not predictive of the HbF% value at month 26. Hence, HbF% level may be a better biomarker for monitoring HU treatment. Continuous dosing might be more advantageous in terms of HbF% for patients who have a strong response to HU.Trial RegistrationThe clinical studies whose data are analysed and reported in this work were not required to be registered in France at their time. Both studies were approved by local ethics committees (of Mondor Hospital and of Kremlin-Bicetre Hospital) and written informed consent was obtained from each patient.

Niveau de preuve du suivi thérapeutique pharmacologique du méthotrexate au décours de son administration à haute-dose

After HDMTX infusions, i.e. at a dose >1g/m(2), monitoring of serum MTX concentrations is a standard practice which helps reducing the incidence of toxicity in patients with decreased clearance by guiding dose adjustment of leucovorin. Therapeutic monitoring of methotrexate is based on iterative measurements of serum concentration until concentration is below a certain threshold. This practice is supported by numerous observations of exposure-related toxicity as well as some clinical studies comparing drug exposure between patients with and without toxicity. Despite no prospective clinical study has clearly demonstrated the usefulness of methotrexate TDM, historical comparison reveals a huge reduction of toxicity-related events over past decades. Systematic monitoring may also help identifying patients with abnormally high methotrexate concentrations, who require immediate treatment with carboxypeptidase.After HD-MTX infusions, i.e. at a dose>1 g/m2, monitoring of serum MTX concentrations is a standard practice which helps reducing the incidence of toxicity in patients with decreased clearance by guiding dose adjustment of leucovorin. Therapeutic monitoring of methotrexate is based on iterative measurements of serum concentration until concentration is below a certain threshold. This practice is supported by numerous observations of exposure-related toxicity as well as some clinical studies comparing drug exposure between patients with and without toxicity. Despite no prospective clinical study has clearly demonstrated the usefulness of methotrexate TDM, historical comparison reveals a huge reduction of toxicity-related events over past decades. Systematic monitoring may also help identifying patients with abnormally high methotrexate concentrations, who require immediate treatment with carboxypeptidase.

Simultaneous Determination of Eight β-Lactam Antibiotics, Amoxicillin, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Cloxacillin, Oxacillin, and Piperacillin, in Human Plasma by Using Ultra-High-Performance Liquid Chromatography with Ultraviolet Detection.

ABSTRACT A simple and rapid ultra-high-performance liquid chromatography (UHPLC) method using UV detection was developed for the simultaneous determination of eight β-lactam antibiotics in human plasma, including four penicillins, amoxicillin (AMX), cloxacillin (CLX), oxacillin (OXA), and piperacillin (PIP), and four cephalosporins, cefazolin (CFZ), cefepime (FEP), cefotaxime (CTX), and ceftazidime (CAZ). One hundred-microliter samples were spiked with thiopental as an internal standard, and proteins were precipitated by acetonitrile containing 0.1% formic acid. Separation was achieved on a pentafluorophenyl (PFP) column with a mobile phase composed of phosphoric acid (10 mM) and acetonitrile in gradient elution mode at a flow rate of 500 μl/min. Detection was performed at 230 nm for AMX, CLX, OXA, and PIP and 260 nm for CFZ, FEP, CTX, and CAZ. The total analysis time did not exceed 13 min. The method was found to be linear at concentrations ranging from 2 to 100 mg/liter for each compound, and all validation parameters fulfilled international requirements. Between- and within-run accuracy errors ranged from −5.2% to 11.4%, and precision was lower than 14.2%. This simple method requires small-volume samples and can easily be implemented in most clinical laboratories to promote the therapeutic drug monitoring of β-lactam antibiotics. The simultaneous determination of several antibiotics considerably reduces the time to results for clinicians, which may improve treatment efficiency, especially in critically ill patients.

Biological parameters predictive of percent dense red blood cell decrease under hydroxyurea

BackgroundDense red blood cells (DRBCs) are associated with chronic clinical manifestations of sickle-cell–disease (SCD). Hydroxyurea (HU) decreases the percent (%) DRBCs, thereby improving its therapeutic benefits, especially the prevention of SCD clinical complications, but parameters influencing %DRBCs remain unknown. The purpose of this study was to determine predictive biological parameters of %DRBC decline under HU.MethodsFactors affecting the %DRBC decrease in SCD patients HU-treated for ≥6xa0months were analyzed. Biological parameters and the %DRBCs were determined before starting HU and after ≥6xa0months of HU intake. Bivariate analyses evaluated the impact of each biological parameter variation on %DRBC changes under treatment. Multivariate analyses assessed the correlations between the decreased %DRBCs and biological parameters.ResultsThe %DRBCs declined by 40.95% after ≥6xa0months on HU. That decrease was associated with less hemolysis, however in several analyses on this group of patients we did not find a statistically significant correlation between decrease in %DRBCs and increase in HbF. Initial %DRBC values were the most relevant parameter to predict %DRBC decline.ConclusionOur results strengthen the known HU efficacy in SCD management statistically independently of the classical HbF biological response. Decreasing %DRBCs is essential to limiting chronic SCD symptoms related to DRBCs and predictive factors might help prevent those manifestations. The results of this study provide new perspectives on indication for HU use, i.e., to prevent SCD-induced organ damage.

Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human plasma using LC–MS/MS

HighlightsPrecise, accurate and sensitive method to quantify two multikinase inhibitors and their 3 active metabolites simultaneously.Using confirmation/quantification ion ratios criteria to improve specificity.Dynamic range of the concentrations carrying out some pharmacokinetics studies. Abstract A new liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of regorafenib (REGO), its two metabolites regorafenib‐M2 and regorafenib‐M5, sorafenib (SORA), and its N‐oxide metabolite in human plasma. Separation is achieved on an Hypersil Gold® column using a gradient elution of 10 mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3 mL/min. After addition of two internal standards and a protein precipitation, the supernatant is diluted two‐fold in a 0.1% (v/v) formic acid solution. Two selected reaction monitoring transitions are used, for each analyte, one for quantitation and the second one for confirmation. The standard curves are ranged from 50 to 5 000 ng/mL for REGO and its metabolites and 80 to 5 000 ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra‐ and inter‐day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of the analytes under various conditions. The method is usually used in clinical practice in order to improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for hepatocellular carcinoma.

Magnesium-deficiency does not alter calcineurin inhibitors activity in mice.

INTRODUCTIONnTacrolimus (TAC) and cyclosporin (CsA) are commonly responsible for hypomagnesemia that predisposes in turn for hypertension, renal impairment and encephalopathy.nnnOBJECTIVEnThe effects of TAC on Mg(2+)-homeostasis and of pre-existing Mg(2+)-deficiency on TAC immunosuppressive activity were compared to CsA in mice.nnnMETHODSnMg(2+) was quantified in plasma, erythrocytes, urine, feces, and femurs from mice treated with TAC 5mg/kg/day. Immunosuppression was assessed in splenocytes by mixed lymphocyte reaction, IL-2 quantification and CN activity determination.nnnRESULTSnPlasma and urine Mg(2+) levels in TAC-treated mice were significantly lower from day 7 until day 21 (p<0.05 versus control) and returned to control value at day 28. Mg(2+) levels were unchanged in erythrocyte, feces and femur. Inhibition of allogeneic proliferation, IL-2 production and CN activity were 68, 56 and 30% lower (p<0.01) after 7 days of TAC-treatment, and 72, 68 and 51% lower (p<0.01) after 7 days of CsA-treatment with a dose of 50mg/kg/day. Dietary-induced hypomagnesemia resulted in significant inhibition of CN activity (p<0.01) without alteration of IL-2 production or allogeneic proliferation. However, it did not alter the effects observed with CsA- or TAC-treatment on allogeneic proliferation, IL-2 production and CN activity.nnnCONCLUSIONnBy contrast with CsA, long-course TAC-treatment induced an early, but transient, and moderate hypomagnesemia without alteration of bone or erythrocyte stocks, intestinal absorption or renal function. Therefore, in clinical use, TAC should be preferred to CsA in patients with pathological or pharmacological conditions which favor Mg(2+)-deficiency. However, dietary-induced hypomagnesemia did not alter the immunosuppressive effects of TAC and CsA.

Plasma concentrations of atovaquone given to immunocompromised patients to prevent Pneumocystis jirovecii

Objectives: Atovaquone is one of the alternatives to trimethoprim/sulfamethoxazole for prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in immunocompromised patients. In volunteers, there was wide inter‐individual variability in atovaquone bioavailability. The aim of this study was to assess the plasma concentrations of atovaquone in immunocompromised patients under PCP prophylaxis. Methods: Adult haematology or HIV‐positive patients receiving atovaquone (750 mg oral suspension twice a day) for PCP prophylaxis were included. Plasma concentrations were assessed using UV‐HPLC, around 12 h after the evening dose (Cmin) and 1–5 h after the morning dose (Cmax). Results: A total of 82 measurements were performed in 33 patients. This included 19 HSCT recipients, 7 haematology non‐transplant patients and 7 HIV‐positive patients. The median Cmin (IQR) was 11.3 &mgr;g/mL (6.2–27.8) and the median Cmax was 13.4 &mgr;g/mL (6.0–28.3). The Cmin and Cmax of atovaquone were not different between HIV‐negative and HIV‐positive patients, or between HSCT and non‐HSCT patients. Atovaquone concentrations were not influenced by the co‐administration of valaciclovir (n = 20) or ciclosporin (n = 11), by gut graft‐versus‐host disease (n = 7) or by the intake of atovaquone with food. Nineteen of the 33 (58%) patients had Cmin <15 &mgr;g/mL, a threshold associated with a low rate of clinical response in PCP treatment. Conclusions: Atovaquone is poorly absorbed in more than half of immunocompromised patients and its bioavailability varies between individuals. These unpredictable variations raise the question of therapeutic drug monitoring, in order to identify patients with low concentrations and those who could benefit from regimen adaptation or from alternatives.

Niveau de preuve du suivi thérapeutique pharmacologique du cisplatine

Cisplatin is an anticancer agent widely used in clinical practice. Cisplatin undergoes irreversible protein binding in plasma and presents a major nephrotoxicity. Some studies determined unbound and bound platin concentrations using flameless atomic absorption spectrophotometry or chromatography. These studies showed a relationship between cisplatin exposition, notably its maximal concentration, and nephrotoxicity. However, the relationship between cisplatin exposition and its efficacy is not yet established. The population approach using a nonlinear mixed effects model showed a low variability of drug exposition parameters. In some cases, Bayesian adaptative dosing were proposed. However, some particular populations will have to be taken into account such as children, elderly patients, acute or chronic renal failure. The impact of cisplatin pharmacogenetics on its therapeutic monitoring need to be studied. Therapeutic cisplatin monitoring is today possibly useful measuring maximal concentration and using described ajusted-modelling. Some prospective multicentric validations are required.