Françoise Bressolle

Validation of liquid chromatographic and gas chromatographic methods. Applications to pharmacokinetics.

Validations of analytical methods are important for the generation of data for bioavailability, bioequivalence and pharmacokinetic studies. It is essential to use well defined and fully validated analytical methods to obtain reliable results that can be satisfactorily interpreted. This manuscript is intended to provide guiding principles for the evaluation of a methods overall performance. For this purpose, all of the variables of the method are considered, including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The criteria considered are as follows: stability, selectivity, limits of quantification and of detection, accuracy, precision, linearity, recovery and ruggedness. Models used for analytical calibration curves are explained in term of validity and limitations, along with a presentation of the most common statistical considerations used to validate the model. Appropriate means of testing precision and accuracy, the most important factors in assessing method quality, are presented. Other issues, such as re-validation, cross-validation, partial sample volume, endogenous drugs and biological matrix of limited availability, are also discussed.

Cisplatin-induced renal toxicity and toxicity-modulating strategies: a review

Cisplatin, or cis-diamminedichloroplatinum(II) (CDDP), is an antineoplastic agent developed in 1965 by Rosenberg et al. [70], who were studying the effects of electrolysis products from a platinum electrode on growing cells. Cisplatin was clinically tested in 1972 by Hill et al. [40]. In spite of its good antineoplastic activity against ovarian, lung, bladder, breast, head and neck.~ and testicular cancer, its clinical use was rapidly limited due to unexpected and very severe renal toxicity. Acute and cumulative renal toxicity associated with histological damage has been shown in both animal and human studies. Several theories concerning the pathophysiological mechanism behind this toxicity have been suggested [13, 59]. Since the therapeutic efficacy of cisplatin seems to be proportional to the delivered dose [80], there has been a continuous search for biological and pharmacological strategies to protect the renal function and thus permit the administration of high quantities of the drug; these strategies include modification of administration modes, development of new galenic forms, and the use of chemoprotectors, among others. Additionally, other platinum analogs with less nephrotoxicity have been studied, but these agents have less antitumor activity than cisplatin or have other inherent toxicities restricting their use [78].

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.

Pharmacodynamics and Pharmacokinetics of Thiopental

Thiopental is an ultra short-acting barbiturate which remains the standard against which other induction agents are judged; it is also indicated for the therapy of brain hypoxic-ischaemia injuries and status epilepticus. Aspects of drug distribution that govern the onset and end of drug effect have been intensively studied to determine which parameters (in patient characteristics, diseases and administration modalities) influence effective dose and concentrations in individual patients. Thiopental has been used as a reference for pharmacokinetic and/or pharmacodynamic models in the study of rapid and short acting effect drugs. In anaesthesiology the pharmacokinetics of thiopental are described as linear; when doses and duration of treatment increase, nonlinear pharmacokinetics occur because of the saturation and/or the induction of the metabolism.

Clinical Pharmacokinetics During Continuous Haemofiltration

SummaryContinuous haemofiltration is an extracorporeal technique that is increasingly used to remove fluid, electrolytes, and other waste products from the blood supply of critically ill patients with acute renal failure. Continuous arteriovenous haemofiltration (CAVH), where the blood exits the body from an artery and re-enters through a vein, is widely used. Continuous venovenous haemofiltration (CVVH), where blood both exits and enters through a vein by way of a mechanical pump, avoids problems that result from the variable ultrafiltration rate found during CAVH. Continuous arteriovenous or venovenous haemodiafiltration (CAVHD or CVVHD) combine continuous haemofiltration and haemodialysis.All methods involve ultrafiltration of the patient’s blood through a filter that is highly permeable to water and small molecules. Drug elimination by haemofiltration depends mainly on the rate of ultrafiltration, the drug protein binding and the sieving coefficient of the membrane. Because patients undergoing continuous haemofiltration have impaired renal function, dosage reduction is often recommended so that adverse drug reactions are avoided. In contrast, if drug removal by haemofiltration is significant, dosage supplementation may be required to ensure therapeutic efficacy of the drug. Therefore, knowledge of the impact of continuous haemofiltration on drug elimination and the pharmacokinetic profile of drugs is essential to good clinical management.The currently available information on the clinical pharmacokinetic aspects of drug therapy during continuous haemofiltration are summarised. Drugs commonly associated with haemofiltration therapy are tabulated with updated pharmacokinetics and drug-monitoring information.

In vitro and in vivo anti-tumoral activities of imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline derivatives.

Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized, and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies were assessed against melanoma (A375, M4Be, RPMI-7591), colon (LS174T), breast (MCF7), and lymphoma (Raji) human cancer cell lines. In vivo studies were carried out in M4Be xenografted athymic mice. EAPB0103, EAPB0201, EAPB0202, and EAPB0203 showed significant in vitro activities against A375 compared to fotemustine and imiquimod used as references. These compounds were 6-110 and 2-45 times more active than fotemustine and imiquimod, respectively. EAPB0203 bearing phenethyl as substituent at position 1 and methylamine at position 4 showed the highest activity. EAPB0203 has also a more potent cytotoxic activity than imiquimod and fotemustine in M4Be and RPMI-7591 and interesting cytotoxic activity in other tumor cell lines tested. In vivo, EAPB0203 treatment schedules caused a significant decrease in tumor size compared to vehicle control and fotemustine treatments.

Drugs for Increasing Oxygen Transport and Their Potential Use in Doping

Blood oxygenation is a fundamental factor in optimising muscular activity. Enhancement of oxygen delivery to tissues is associated with a substantial improvement in athletic performance, particularly in endurance sports. Progress in medical research has led to the identification of new chemicals for the treatment of severe anaemia. Effective and promising molecules have been created and sometimes used for doping purposes. The aim of this review is to present methods, and drugs, known to be (or that might be) used by athletes to increase oxygen transport in an attempt to improve endurance capacity. These methods and drugs include: (i) blood transfusion; (ii) endogenous stimulation of red blood cell production at altitude, or using hypoxic rooms, erythropoietins (EPOs), EPO gene therapy or EPO mimetics; (iii) allosteric effectors of haemoglobin; and (iv) blood substitutes such as modified haemoglobin solutions and perfluorochemicals. Often, new chemicals are used before safety tests have been completed and athletes are taking great health risks. Such new chemicals have also created the need for new instrumental strategies in doping control laboratories, but not all of these chemicals are detectable. Further progress in analytical research is necessary.

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC-MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)-alpha-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5( 4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid-liquid extraction with diethyl ether followed by a solid-liquid extraction on C18 cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and alpha-bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X2; weighted calibration curve obtained in the range 5-250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.

Prostatic Tissual Distribution of Alfuzosin in Patients with Benign Prostatic Hyperplasia Following Repeated Oral Administration

OBJECTIVES The blood and prostatic concentrations of alfuzosin were determined in patients with benign prostatic hyperplasia (BPH). METHODS 12 patients scheduled for BPH surgery were treated with alfuzosin 5 mg twice daily prior to surgery in an open trial. Seven doses were given over a 4-day period. Blood samples were drawn before the first and the last intake (day 3). On day 4 (surgery day), a blood and prostate tissue sample were taken simultaneously 12 hours after the last drug intake. RESULTS Mean trough blood levels were 6.0+/-4.6 ng/ml and 5.8+/-3.7 ng/ml on day 3 and day 4, respectively, indicating a stable alfuzosin concentration. The mean prostate concentration on day 4 was 12.3+/-5.6 ng/g. Alfuzosin prostate and blood concentrations at 12 hours post dosing on day 4 were significantly correlated (r=0.804, p=0.0016); the prostate-blood ratio was 2.4+/-0.7. CONCLUSIONS Oral administration of alfuzosin leads to a high diffusion of the drug into the prostate of BPH patients.

Circadian rhythm of 5-fluorouracil population pharmacokinetics in patients with metastatic colorectal cancer.

Purpose: The purpose of this work was to estimate the population pharmacokinetic parameters of 5-fluorouracil (5-FU) in patients with advanced colorectal cancer using circadian change kinetics. Methods: Eighty-five patients (32 females, 53 males) were enrolled onto this study. All patients received folinic acid (200 mg/m2) by intravenous infusion over 2 h followed by a 5-FU loading dose (400 mg/m2) and then continuous infusion (600 mg/m2) for 22 h. This whole regimen was repeated on day 2 and was given on a 14-day cycle. Plasma 5-FU determinations were performed by high-performance liquid chromatography with ultraviolet absorbance detection. Pharmacokinetic analyses were performed using the NONMEM computer program through the Visual-NM graphical interface. An open one-compartment pharmacokinetic model with zero-order input rate was used to describe the kinetics of 5-FU; moreover, circadian time-dependent changes in 5-FU concentrations were taken into account in the model. The circadian model was defined as the sum of two cyclic components; the amplitude of the first cyclic component (over 24 h) was about 30% of the average clearance and the amplitude of the second cyclic component (over 12 h) was about 50% of the amplitude of the first component. The acrophase (peak) times of the first and the second periodic component were 04 h 12 m and 00 h 25 m, respectively. The potential sources of variability on the population parameters (65 patients) were investigated using patients sex, body area, age, body weight, height, liver enzymes and serum creatinine as covariables. Results: Only the estimated clearance circadian changes were different for the two sexes. The population parameter estimates of mean clearance (CLmean) and initial volume of distribution (V), were as follows: the male subgroup showed a CLmean value twice larger (125 l/h) than the value observed in the female subgroup (65 l/h), and V = 21 l. A validation group of 20 additional patients was used to evaluate the predictive performances of the population parameters. The individual pharmacokinetic parameters were computed by means of a Bayesian fitting procedure. From the resulting individualized parameter values, concentrations of 5-FU in the plasma were calculated. To evaluate the performance of the Bayesian estimation, the experimental concentrations were compared with the predicted ones. Conclusion: In conclusion, a chronomodulated delivery schedule of 5-FU should be performed, using a perfusion rate inversely proportional to the circadian variations of clearance in order to maintain stable 5-FU plasma levels. Such a treatment schedule may result in increased effectiveness of the treatment and decreased occurrence of drug-associated side-effects. The present study develops a complete procedure to efficiently estimate 5-FU clearance in order to optimize dosage regimens in individual patients.