Franciszek K. Główka

Pharmacokinetics of high-dose i.v. treosulfan in children undergoing treosulfan-based preparative regimen for allogeneic haematopoietic SCT

Pharmacokinetic studies of high-dose treosulfan were carried out in seven paediatric patients (age range: 2–15 years) undergoing treosulfan-based conditioning regimen prior to allogeneic haematopoietic SCT. Treosulfan was administered intravenously in a daily dose of 10, 12 or 14 g/m2 within 2 h. Five out of seven patients received 12 g/m2. The plasma concentrations of treosulfan and its quantity eliminated with urine were determined using a validated HPLC method with refractometric detection. Pharmacokinetic parameters were evaluated following first dose using a two-compartment disposition model. These studies demonstrated a dose-dependent increase of area under the concentration (AUC) and maximum concentrationplasma (Cmax), but there was variability of these parameters. Rapid clearance of tresoulfan was observed, especially in 10 and 12 g/m2 doses. Terminal half-life (t0.5) of treosulfan was in the range of 1.71–2.15 h, but the mean percent of parent drug eliminated with urine was 30%, range 16.3–45.4% of the total dose eliminated during the first 12 h after administration. The results of this study confirmed the linear pharmacokinetics of treosulfan, as used in children. However, variability of pharmacokinetic results observed in children studied demonstrates the need for pharmacokinetic evaluation in each paediatric patient undergoing the treosulfan-based preparative regimen, including those using different doses. This approach could enable further reduction of the risk of early and late organ toxicity related to high-dose treosulfan in paediatric patients.

HPLC-MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples.

A fast and reproducible HPLC-MS/MS method was developed for the simultaneous determination of clopidogrel (CLP), its carboxylic acid derivative (CLPM), derivatized thiol metabolite isomers MP-H3 and the active MP-H4 in incurred human plasma. CLP, CLPM, MP-H3 and MP-H4 isomers together with the internal standard piroxicam were extracted from plasma samples using a simple protein precipitation with acetonitrile. The analytes were separated on HPLC Zorbax Plus C18 column via gradient elution with water and acetonitrile, both containing 0.1% (v/v) formic acid. Detection of the analytes were performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. Calibration curves of the analytes prepared in 250μL plasma were found to be linear in ranges: 0.25-5.00ng/mL for CLP, 0.25-50.00ng/mL for MP-H3 and MP-H4 isomers and 50-10,000ng/mL for CLPM. The lower limit of quantitation was 0.25ng/mL for CLP, MP-H3, MP-H4 and 50.00ng/mL for CLPM. Intra- and inter-assay precision, expressed as relative standard deviation, was ≤18.1% for CLP, ≤15.2% for CLPM, ≤10.1% for MP-H3 and ≤19.9% for MP-H4. Intra- and inter-day accuracy of the method, expressed as relative error, was ≤16%. The analytes were stable in samples stored for 6h in autosampler, in plasma samples for 24h at room temperature and for 3 months at -25°C. Resolution of CLP, CLPM and MP-H3 and MP-H4 isomers of thiol metabolite during one analytical run was reported in patient plasma. The HPLC-MS/MS method was applied for pharmacokinetic studies of CLP and its metabolites in patients treated with daily dose of 75mg CLP.

Genetic and non-genetic factors affecting the response to clopidogrel therapy

Introduction: Clopidogrel (CLP) is a second-generation thienopyridine that prevents platelet aggregation by inhibiting the adenosine diphosphate receptor located on the platelet surface. The use of CLP in combination with aspirin has become standard treatment in patients with acute coronary syndromes and stent implantation. Data suggests that a significant percentage of individuals treated with CLP do not receive the expected therapeutic benefit because of a decreased platelet inhibition. The clinical consequences of an inadequate platelet response are cardiovascular complications, which can lead to acute myocardial infarction, stroke and death. The mechanism underlying CLP resistance is multifactorial and includes genetic polymorphisms and non-genetic causes (such as drug–drug interactions, co-morbidities, age). Areas covered: This article reviews the so-far accumulated evidence on the role of genetic polymorphisms and non-genetic factors, as determinants of the antiplatelet response to CLP. Pharmacodynamic and clinical aspects of the CLP nonresponsiveness are also presented. Relevant papers were identified by an extensive PubMed search using appropriate keywords. Expert opinion: Impaired platelet inhibition in CLP poor responders is a real problem, as it leads to serious clinical consequences. Therefore, prediction models that include pharmacogenetic knowledge and non-genetic risk factors of low response to the drug are needed in the individualization of antithrombotic therapy. Alternative antiplatelet strategies that should be considered to overcome this problem include dose modification, adjunctive antiplatelet drug usage, and use of newer agents.

High-dose treosulfan in conditioning prior to hematopoietic stem cell transplantation.

Importance of the field: Despite the marked development in the field of preparative regimens prior to hematopoietic stem cell transplantation (HSCT) over the last decade, the search for a superior conditioning agent is still continuing. In view of the literature reports, treosulfan (TREO), a structural analog of busulfan (BU), appears to be a promising candidate in terms of myeloablative, immunosuppressive and antimalignancy effects as well as low organ toxicity. Areas covered in this review: The article focuses on pharmacological activity, pharmacokinetics and toxicity of TREO. Compressed description of the drug-based conditioning prior to HSCT is also presented. Finally, TREO and BU characteristics are compared. Specific information of TREO concerning pediatric and adult patients is provided throughout the whole paper. The data refer predominantly to the publications, in majority from the last 10 years. What the reader will gain: According to our best knowledge, the paper is the first such comprehensive review on TREO, especially in terms of its application in pediatric HSCT. Take home message: TREO offers a great potential as a conditioning agent prior to HSCT but further investigations of the drug are warranted to clearly verify its advantages. However, we expect TREO to be registered as a novel conditioning agent in the near future.

Pharmacokinetic studies of enantiomers of ibuprofen and its chiral metabolites in humans with different variants of genes coding CYP2C8 and CYP2C9 isoenzymes

The pharmacokinetics of ibuprofen enantiomers and its chiral metabolites, namely (R,S)-29-hydroxyibuprofen and (RR,RS,SR,SS)-29-carboxyibuprofen, was studied in healthy volunteers carrying different alleles coding cytochrome P450 (CYP) 4502C isoenzymes. Following administration of  400 mg of racemic ibuprofen, enantiomers of the parent compound and their metabolites were isolated from plasma and urine samples using solid-phase extraction and were quantified by the validated capillary zone electrophoresis method. The levels of the analytes in biological fluids were used to calculate their pharmacokinetic parameters in subjects with different variants of CYP2C8 and CYP2C9 isoenzymes. The analysis of each subject’s genotype was carried out using polymerase chain reaction-restriction fragment length polymorphism. Impaired metabolism of ibuprofen enantiomers was associated with the presence of CYP2C8*3, CYP2C9*2 and CYP2C9*3 alleles. The greatest effect of mutated alleles on pharmacokinetics was observed in a subject with a CYP2C8*1/*3, CYP2C9*1/*2 genotype. This subject appeared to have lower value of clearance, greater area under the curve (AUC) and longer time t0.5 in comparison with the wild-type.

HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids

11Beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11beta-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard--prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C(18) cartridges. The enzymatic hydrolysis of conjugated steroids was provided using beta-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0-1000.0 ng mL(-1), for allo-THF and THE + allo-THE 10.0-1000.0 ng mL(-1). LOD (S/N=3:1) for all analytes amounted 3.0 ng mL(-1). Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0-12.1% and 9.2-14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0-174.5 ng mL(-1) and 17.4-35.9 ng mL(-1), respectively. Free urinary steroids were in the ranges: 12.0-54.1 microg/24 h (UFF) and 37.8-76.2 microg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11beta-HSD2 activity in man.

The influence of genetic polymorphism of Cyp2c19 isoenzyme on the pharmacokinetics of clopidogrel and its metabolites in patients with cardiovascular diseases

An extensive investigation on pharmacokinetics of clopidogrel and its metabolites as well as pharmacodynamics of the drug was performed in patients with cardiovascular disease carrying various alleles coding CYP2C19 isoenzyme. The influence of non‐genetic factors on the clopidogrel response was also studied. Plasma concentrations of clopidogrel, its carboxylic metabolite, and diastereoisomers of a thiol metabolite (the inactive H3 and the active H4) following an administration of 75 mg of the drug were determined in three groups of patients divided with respect to their CYP2C19 genotype: ultrametabolizers, extensive metabolizers, and intermediate metabolizers. The mean peak plasma concentration of H4 in intermediate metabolizers was 3.1‐ and 2.8‐fold lower than that of ultrametabolizers (P = 0.055) and extensive metabolizers (P = 0.026), respectively. The mean H4 area under the curve (AUC0–24 h) for intermediate metabolizers were significantly lower than that for ultrametabolizers (P = 0.046). Intermediate metabolizers exhibited a significantly higher platelet aggregation than ultrametabolizers and extensive metabolizers (P = 0.035). A multivariate analysis showed that the effect of CYP2C19*2 allele on an ADP‐induced platelet aggregation was better pronounced in the presence of non‐genetic risk factors (P = 0.008).

Determination of ketoprofen enantiomers in human serum by capillary zone electrophoresis: man pharmacokinetic studies after administration of rac-ketoprofen tablets

A rapid and stereospecific capillary zone electrophoresis (CZE) method to quantify ketoprofen (KTP) enantiomers was developed. The KTP enantiomers and (+)-S-naproxen [(+)-S-NPX] as an internal standard (IS) were extracted with methylene chloride from serum acidified. Recovery of both enantiomers was in the range of 85-91%. The enantiomers were determined using a background electrolyte (BGE), consisting of 0.05 M heptakis 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbetaCD) in a phosphate-triethanolamine buffer, which filled a fused silica capillary of 75 micrometer i.d. The linear range of calibration curves was between 0.25 and 50 mg l(-1), with detection limit of 0.1 mg l(-1) (signal-to-noise baseline ratio (S/N) >4). Intra- and interday precision and accuracy of the calibration curves, expressed by the coefficient of variation (CV), did not exceed 15.0%. The validated method has been successfully applied for pharmacokinetic studies of KTP enantiomers from tablets with rac-KTP in man.

Activation of Prodrug Treosulfan at pH 7.4 and 37°C Accompanied by Hydrolysis of Its Active Epoxides: Kinetic Studies with Clinical Relevance

Treosulfan (TREO), originally registered for treatment of ovarian cancer, is currently being investigated for conditioning prior to hematopoietic stem cell transplantation. TREO is a prodrug, which undergoes a pH- and temperature-dependent two-step conversion to active monoepoxide [S,S-EBDM, (2S,3S)-1,2-epoxybutane-3,4-diol-4-methanesulfonate] and diepoxide [S,S-DEB, (2S,3S)-1,2:3,4-diepoxybutane]. In this paper, the kinetics of the nonenzymatic transformation of TREO at pH 7.4 and 37°C were studied for the first time including the effects of the TREO concentration, buffer concentration, ionic strength, and the presence of NaCl. Transformation of TREO was well described by a kinetic model, which included first-order reactions for TREO activation, that is, TREO → S,S-EBDM → S,S-DEB, and pseudo-first-order reactions for the hydrolytic decomposition of S,S-EBDM and S,S-DEB. In contrast to the two-step activation of TREO, the hydrolysis of epoxides was influenced by electrolytes. In phosphate-buffered saline, decomposition of S,S-EBDM and S,S-DEB (mean half-lives 25.7 and 15.4 h) proceeded much slower than their formation (mean half-lives 1.5 and 3.5 h). In conclusion, the kinetics of the nonenzymatic transformation of TREO in the presence of plasma electrolytes cannot contribute to the very low levels of S,S-EBDM and S,S-DEB observed in patient plasma. The results also indicate that elimination of TREO proceeds primarily via conversion to S,S-EBDM.

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of protein-free pro-drug treosulfan and its biologically active monoepoxy-transformer in plasma and brain tissue

For the first time a high performance liquid chromatography method with tandem mass spectrometry detection (HPLC-MS/MS) was developed for simultaneous determination of a pro-drug treosulfan (TREO) and its active monoepoxide (S,S-EBDM) in biological matrices. Small volumes of rat plasma (50 μL) and the brain homogenate supernatant (100 μL), equivalent to 0.02 g of brain tissue, were required for the analysis. Protein-free TREO, S,S-EBDM and acetaminophen, internal standard (IS), were isolated from the samples by ultrafiltration. Complete resolution of the analytes and the IS was accomplished on Zorbax Eclipse column using an isocratic elution with a mobile phase composed of ammonium formate - formic acid buffer pH 4.0 and acetonitrile. Detection was performed on a triple-quadrupole MS via multiple-reaction-monitoring following electrospray ionization. The developed method was fully validated according to the current guidelines of the European Medicines Agency. Calibration curves were linear in ranges: TREO 0.2-5720 μM and S,S-EBDM 0.9-175 μM for plasma, and TREO 0.2-29 μM and S,S-EBDM 0.4-44 μM for the brain homogenate supernatant. The limits of quantitation of TREO and S,S-EBDM in the studied matrices were much lower in comparison to the previously used bioanalytical methods. The HPLC-MS/MS method was adequately precise (coefficient of variation≤12.2%), accurate (relative error≤8.6%), and provided no carry-over, acceptable matrix effect as well as dilution integrity. The analytes were stable in acidified plasma and the brain homogenate supernatant samples for 4 h at room temperature, for 4 months at-80°C as well as within two cycles of freezing and thawing, and demonstrated 18-24h autosampler stability. The validated method enabled determination of low concentrations of TREO and S,S-EBDM in incurred brain samples of the rats treated with TREO, which constitutes a novel bioanalytical application.