Jean Cumps

Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction.

Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%.

Metyrapone-reduced Cytochrome-p-450 Complex - a Specific Method for the Determination of the Phenobarbital Inducible Form of Rat Hepatic-microsomal Cytochrome-p-450

Abstract Using homogeneous cytochrome P-450, we have shown that the well-known metyrapone-dithionite reduced cytochrome P-450 complex is specific for the cytochrome P-450b induced by phenobarbital. A linear relationship was observed between the absorbance of metyrapone-reduced cytochrome P-450 complex and the one of CO-reduced cytochrome P-450 complex, the usual method for the determination of cytochrome P-450. A method has been proposed for the specific determination of the cytochrome P-450b.

Limited sampling models and Bayesian estimation for mycophenolic acid area under the curve prediction in stable renal transplant patients co-medicated with ciclosporin or sirolimus.

Background and ObjectiveMycophenolate mofetil is a prodrug of mycophenolic acid (MPA), an immunosuppressive agent used in combination with corticosteroids and calcineurin inhibitors or sirolimus for the prevention of acute rejection after solid organ transplantation. Although MPA has a rather narrow therapeutic window and its pharmacokinetics show considerable intra- and interindividual variability, dosing guidelines recommend a standard dosage regimen of 0.5–1.0g twice daily in adult renal, liver and cardiac transplant recipients. The main objective ofthe present study was to develop a method to predict the MPA area under the plasma concentration-time curve during one 12-hour dosing interval (AUC12) by using multiple linear regression models and maximum aposteriori (MAP) Bayesian estimation methods in patients co-medicated with ciclosporin or sirolimus, aiming to individualize the dosage regimen of mycophenolate mofetil.Patients and MethodsPharmacokinetic profiles of MPA and mycophenolic acid glucuronide (MPAG), the main metabolite of MPA, were obtained from 40 stable adult renal allograft recipients on three different occasions: the day before switching from ciclosporin to sirolimus co-medication (±7.4 months posttransplantation; period I) and at 60 days and 270 days after the switch (periods II and III). Blood samples for determination of MPA and MPAG concentrations in plasma were taken at 0 hours (pre-dose) and at 0.33, 0.66, 1.25, 2, 4, 6, 8 and 12 hours after oral intake ofmycophenolate mofetil. The MPA AUC12 was calculated by the trapezoidal method (the observed AUC12). Patients were randomly divided into (i) a model-building test group (n = 27); and (ii) a model-validation group (n = 13). Multiple linear regression models were developed, based on three sampling times after drug administration. Subsequently, a population pharmacokinetic model describing MPA and MPAG plasma concentrations was developed using nonlinear mixed-effects modelling and a Bayesian estimator based on the population pharmacokinetic model was used to predict the MPA AUC12 based on three sampling times taken within 2 hours following dosing.ResultsFifty-two percent ofthe observed AUC12 values (three periods) in the 40 patients receiving a fixed dose of mycophenolate mofetil 750mg twice daily were outside the recommended therapeutic range (30–60 mg • h/mL). The failure of the standard dose to yield an AUC12 value within the therapeutic range was especially pronounced during the first study period. Of the multiple linear regression models that were tested, the equation based on the 0-hour (pre-dose), 0.66- and 2-hour sampling times showed the best predictive performance in the validation group: r2 = 0.79, relative root mean square error (rRMSE)= 14% and mean relative prediction error (MRPE) = 0.9%. The pharmacokinetics of MPA and MPAG were best described by a two-compartment model with first-order absorption and elimination for MPA, plus a compartment for MPAG, also including a gastrointestinal compartment and enterohepatic cycling in the case of sirolimus co-medication. The ratio of aminotransferase liver enzymes (AST and ALT) and the glomerular filtration rate significantly influenced MPA glucuronidation and MPAG renal excretion, respectively. Bayesian estimation ofthe MPA AUC12 based on 0-, 1.25- and 2-hour sampling times predicted the observed AUC12 values ofthe patients in the validation group, with the following predictive performance characteristics: r2 = 0.93, rRMSE= 12.4% and MRPE = -0.4%.ConclusionUse ofthe developed multiple linear regression equation and Bayesian estimator, both based on only three blood sampling times within 2 hours following a dose of mycophenolate mofetil, allowed an accurate prediction ofa patient’s MPA AUC12 for therapeutic drug monitoring and dose individualization. These findings should be validated in a randomized prospective trial.

C-terminal FGF23 is a strong predictor of survival in systolic heart failure

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the regulation of phosphate and calcium metabolism. We have evaluated the levels of C-terminal FGF23 (Ct-FGF23) in 73 patients presenting heart failure with reduced ejection fraction (HF-REF) and assess their potential predictive value for long-term survival through a 6 years follow-up. Ct-FGF23 levels were markedly increased in HF-REF. In univariate proportional hazard model, survival was related to glomerular filtration rate (eGFR), intact parathyroid hormone (PTH), B-type natriuretic peptides (BNP) and Ct-FGF23. In a multivariate analysis including age, EF, PTH, BNP, Ct-FGF23, calcium, phosphorus and eGFR levels, Ct-FGF23 is the strongest predictor of long term CV death.

Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.

The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Kis for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).

Concentration-dependent plasma protein binding of flurbiprofen in the rat: an in vivo microdialysis study.

AbstractPurpose. The in vivo plasma protein binding and pharmacokinetics of flurbiprofen were studied in awake, unrestrained rats using intravenous microdialysis sampling. Methods. Flurbiprofen (20 mg/kg) was administered i.v. to 2 groups of 6 rats: in both groups sampling was carried out by microdialysis, but in the second group an addional 10 blood samples were withdrawn via a jugular cannula. In vitro and ex vivo (following i.v. administration of flurbiprofen 20 mg/kg to another group of 13 rats) plasma protein binding of the drug was determined by equilibrium dialysis. Results. The area under the unbound plasma concentration-time profile of flurbiprofen (AUCU), determined by microdialysis sampling, was somewhat smaller (−19%, p = 0.066) in the rats undergoing simultaneous serial blood sampling (2.21 ± 0.36 (µg.h/ml) as compared to the rats undergoing microdialysis sampling only (2.73 ± 0.60 µg.h/ml). Comparison of total and unbound concentrations of flurbiprofen showed an in vivo plasma binding varying between 99.5% at low and 98.0% at high total flurbiprofen plasma concentrations. Plasma binding of flurbiprofen determined in vitro over the same concentration range was higher (99.5–99.9%) but also concentration-dependent. Plasma binding of flurbiprofen determined ex vivo, on the other hand, corresponded well with the in vivo binding. Conclusions. Monitoring the fraction of drug unbound in blood of an individual rat throughout a pharmacokinetic experiment has now become possible by using simultaneous sampling of blood and intravenous microdialysates.

Corrections for heteroscedasticity in window evolving factor analysis

Window evolving factor analysis (WEFA) is a powerful technique for checking peak purity in liquid chromatography with diode array detection (LC-DAD). However, practical application of the technique can be limited by instrumental and experimental non-idealities. One of the problem sources is heteroscedasticity. In this work, we propose two new data transformation procedures and one technique for directly adjusting the log-eigenvalue profiles, which eliminate most of the heteroscedastic effect in the WEFA plots. The pretreatment and the adjustment techniques can be used in combination to obtain even better results. The performance of the techniques are demonstrated on simulated and actual data.

Evaluation of the formalin test to assess the analgesic activity of diflunisal in the rat.

The formalin test was evaluated to assess the analgesic activity of diflunisal in the rat. Fifty microliters of a 5% formalin solution was injected into the hindpaw of rats and two distinct nociceptive behaviors, i.e. flinching/shaking and licking/biting of the injected paw, were recorded over 120 min. The effect of factors such as age of the animal, time of injection (morning vs. afternoon), site of injection (right vs. left hind paw) were evaluated. Both nociceptive behaviors exhibited a biphasic time course. Rats weighing 210-220 grams showed a more intense response compared to older rats weighing 240-250 or 270-280 grams. The nociceptive behavior response was affected by the time of formalin injection and was more pronounced in the morning. Diflunisal (100 mg/kg, i.v. infusion over 3 min) caused a significant delay in the flinching/shaking response vs. time curve, whereas the licking/biting response was significantly inhibited. When carried out under carefully controlled conditions, the formalin test may be useful to study the analgesic effect of diflunisal in the rat. It seems to be less sensitive, however, than other commonly used nociceptive tests.

Empirical models for dosage optimization of four beta-lactams in critically ill septic patients based on therapeutic drug monitoring of amikacin.

OBJECTIVES The study aims to develop empirical models able to predict the pharmacokinetics (PK) of four beta-lactams using the amikacin (AMK) therapeutic drug monitoring (TDM), in order to optimize their dosage regimens. DESIGN AND METHODS 69 critically ill septic patients were included. All received a first dose of AMK combined with piperacillin/tazobactam, ceftazidime, cefepime or meropenem. A multivariate analysis was performed to predict the beta-lactam PK using AMK PK parameters estimated from TDM and using pathophysiological variables. RESULTS An optimal prediction model was identified for each PK parameter of each beta-lactam. The best predictor of each model was one of the AMK PK parameters estimated from TDM. Other variables included colloid solution, renal and hepatic biomarkers, age and body weight. CONCLUSION PK of the four beta-lactams could be easily and rapidly predicted in critically ill septic patients using the AMK TDM. These predictions could improve the beta-lactam dosages in clinical practice.

Airway ion transport impacts on disease presentation and severity in cystic fibrosis

OBJECTIVES Abnormal airway ion transport is a feature of cystic fibrosis. The aim of this study was to investigate whether distinct components of ion transport are associated with the clinical expression and severity of the disease. DESIGN AND METHODS Univariate and multivariate analyses were used to study interaction effects between nasal potential difference parameters and clinical status, recorded at stable conditions, in 75 F508del homozygous young adults. RESULTS All patients demonstrated increased sodium and reduced chloride conductances. Less sodium transport abnormalities were related to better respiratory function and nutrition. Presentation with digestive symptoms at diagnosis was associated with lower chloride conductance. With an accuracy of 85% good nutritional status was linked to more preserved lung function, increasing age and more preserved chloride conductance. CONCLUSIONS Ion transport abnormalities have distinct clinical outcomes. Sodium conductance relates to respiratory function and nutrition; chloride conductance to nutrition and presentation with digestive symptoms at diagnosis.