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What is known and objective The reasons of clopidogrel ( CLP) resistance are still unclear. The response to CLP may be influenced by both genetic and non-genetic factors. Among genetic factors, common polymorphisms in the gene coding glycoprotein-P (P-gp, MDR1 and ABCB1) are considered as potential determinants of the efficacy of CLP treatment. The aim of this study was to evaluate the influence of ABCB1 3435C>T genetic polymorphism on the pharmacokinetics and pharmacodynamics of CLP and its metabolites: diastereoisomers of thiol metabolite (the inactive H3 and the active H4) and inactive carboxylic derivative. Methods The study group included 42 patients undergoing elective coronary angiography and percutaneous coronary intervention. The plasma concentrations of CLP and its metabolites were measured by a validated HPLC- MS/ MS method. Whole-blood aggregation was determined with Multiplate analyzer. For evaluation of ABCB1 3435C>T polymorphism, PCR- RFLP method was applied. Results and discussion It was found that Exposition to the unchanged CLP, measured by AUC0- t of the drug, was significantly lower ( P = 0·012) in TT homozygotes comparing to that observed in CC and CT genotypes, although no correlation was found between platelet aggregation and ABCB1 genetic polymorphism. What is new and conclusion Our findings show that the presence of 3435C>T allele has an impact on CLP pharmacokinetics but not on the drug pharmacodynamics. Therefore, the 3435C>T genotype may not be the primary determinant influencing the pharmacokinetics of the active H4 metabolite and antiplatelet effect of the drug. [ABSTRACT FROM AUTHOR]

Purpose: P2Y 12 receptor inhibitors are drugs that decrease the risk of stent thrombosis and lower the long-term risk of non-stent-related myocardial infarction and stroke. They inhibit the binding of adenosine diphosphate (ADP) to the P2Y 12 receptor and effectively reduce platelet reactivity. However, considerable variability in the pharmacodynamics response contributes to a failure of antiplatelet therapy; this phenomenon is especially notorious for older drugs, such as clopidogrel. Some genetic polymorphisms associated with these drugs' metabolic pathway, especially in the CYP2C19 gene, can significantly decrease antiplatelet efficacy. There are few reports on the variability stemming from the target of this drug class that is the P2Y 12 receptor itself., Results and Conclusion: This review summarizes the results of research that focus on the influence of P2Y 12 genetic polymorphisms on the pharmacodynamics and the efficacy of P2Y 12 inhibitors. We found that the conclusions of the studies are unequivocal, and despite several strong candidates, such as G52T (rs6809699) or T744C (rs2046934), they may not be independent predictors of the inadequate response to the drug. Most probably, P2Y 12 genetic polymorphisms contribute to the effect exerted by other gene variants (such as CYP2C19*2/*3/*17), drug interactions, or patient habits, such as smoking. Also, epigenetic modifications, such as methylation or miRNA levels, may play a role in the efficacy of antiplatelet treatment., (© 2022. The Author(s).)

Aim: This work was designed to investigate the associations between vitamin D metabolites, VDR gene polymorphisms and echocardiographic markers in a population of patients with cardiovascular disease. Methods: Echocardiographic markers for 42 patients were determined with tissue Doppler techniques. PCR-restriction fragment length polymorphism analysis identified genetic variants ApaI , TaqI , BsmI and FokI . A validated UHPLC-MS/MS method determined vitamin D metabolites. Results: Patients with the ApaI -GT genotype exhibited a lower pressure gradient across the aortic valve than ApaI -TT carriers. BMI, ApaI -GT, TaqI -TC, aortic arch diameter and maximal pressure gradient were significant univariate predictors of hypertension. Conclusion: A potential link exists between VDR gene polymorphisms and cardiovascular function.

Background: Vitamin D is a fat-soluble vitamin, and it is a potential key factor in maintaining a healthy status. Various observational studies have reported the association between vitamin D deficiency and an elevated risk of osteoporosis, cardiovascular disease, diabetes mellitus, and certain types of cancer. The number of studies that investigates the genetic determinants of vitamin D hydroxy metabolism has been growing. Still, its association with the genetic variants remains unclear, particularly those genes related to vitamin D metabolism., Aims: This work is a comprehensive review of available evidence of the effect of genetic variants on vitamin D metabolism and their impact on vitamin D status in the human body, disorders including coronavirus disease 2019 infection, and its importance for clinical investigators and public health., Results: Genome-wide association studies and candidate gene studies show that genetic factors are influencing the circulating levels of vitamin D. These genetic changes are implicated in various pathways of vitamin D, such as metabolism and transport. It is also involved in the formation of the ternary complex (vitamin D receptor - retinoid receptor - transcription factor II B)., Conclusion: Linkage studies may fail to identify replicated genetic architecture of vitD metabolism. Genome-wide association studies and the candidate gene approach have shown reproducible influences of gene control on vitD status., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25-hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC-MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid-liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triple-quadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (<20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: Efficacy of treosulfan, used in the treatment of marrow disorders, depends on the activity of its monoepoxy-(EBDM) and diepoxy compounds. The study aimed to describe the pharmacokinetics of treosulfan and EBDM in the rat plasma and brain by means of mixed-effects modelling., Methods: The study had a one-animal-per-sample design and included ninty-six 10-week-old Wistar rats of both sexes. Treosulfan and EBDM concentrations in the brain and plasma were measured by an HPLC-MS/MS method. The population pharmacokinetic model was established in NONMEM software with a first-order estimation method with interaction., Results: One-compartment pharmacokinetic model best described changes in the concentrations of treosulfan in plasma, and EBDM concentrations in plasma and in the brain. Treosulfan concentrations in the brain followed a two-compartment model. Both treosulfan and EBDM poorly penetrated the blood-brain barrier (ratio of influx and efflux clearances through the blood-brain barrier was 0.120 and 0.317 for treosulfan and EBDM, respectively). Treosulfan plasma clearance was significantly lower in male rats than in females (0.273 L/h/kg vs 0.419 L/h/kg)., Conclusions: The developed population pharmacokinetic model is the first that allows the prediction of treosulfan and EBDM concentrations in rat plasma and brain. These results provide directions for future studies on treosulfan regarding the contribution of transport proteins or the development of a physiological-based model.

The tyrosine kinase inhibitor sorafenib is the first-line treatment for patients with hepatocellular carcinoma (HCC), in which hyperlipidemia and type 2 diabetes mellitus (T2DM) may often coexist. Protein transporters like organic cation (OCT) and multidrug and toxin extrusion (MATE) are involved in the response to sorafenib, as well as in that to the anti-diabetic drug metformin or atorvastatin, used in hyperlipidemia. Changes in the activity of these transporters may lead to pharmacokinetic interactions, which are of clinical significance. The study aimed to assess the sorafenib-metformin and sorafenib-atorvastatin interactions in rats. The rats were divided into five groups (eight animals in each) that received sorafenib and atorvastatin (I SOR+AT ), sorafenib and metformin (II SOR+MET ), sorafenib (III SOR ), atorvastatin (IV AT ), and metformin (V MET ). Atorvastatin significantly increased the maximum plasma concentration (C max ) and the area under the plasma concentration-time curve (AUC) of sorafenib by 134.4% ( p < 0.0001) and 66.6% ( p < 0.0001), respectively. Sorafenib, in turn, caused a significant increase in the AUC of atorvastatin by 94.0% ( p = 0.0038) and its metabolites 2-hydroxy atorvastatin ( p = 0.0239) and 4-hydroxy atorvastatin ( p = 0.0002) by 55.3% and 209.4%, respectively. Metformin significantly decreased the AUC of sorafenib ( p = 0.0065). The AUC ratio (II SOR+MET group/III SOR group) for sorafenib was equal to 0.6. Sorafenib did not statistically significantly influence the exposure to metformin. The pharmacokinetic interactions observed in this study may be of clinical relevance in HCC patients with coexistent hyperlipidemia or T2DM.

Purpose: Impaired antiplatelet effect of clopidogrel (CLP) can result from drug-drug interactions and genetic polymorphisms of drug-metabolizing enzymes. The aim of the study was to evaluate the effect of genetic polymorphisms of ABCB1 and the selected cytochrome P450 isoenzymes on the pharmacodynamics and pharmacokinetics of CLP and its metabolites in patients co-treated with atorvastatin or rosuvastatin., Methods: The study involved 50 patients after coronary angiography/angioplasty treated with CLP and atorvastatin (n = 25) or rosuvastatin (n = 25) for at least 6 months. Plasma concentrations of CLP, diastereoisomers of thiol metabolite (inactive H3 and active H4), and inactive CLP carboxylic acid metabolite were measured by UPLC-MS/MS method. Identification of the CYP2C19*2, CYP2C19*17, CYP3A4*1G, CYP1A2*1F, and ABCB1 C3435T genetic polymorphisms was performed by PCR-RFLP, while platelet reactivity units (PRU) were tested using the VerifyNow P2Y12 assay., Results: There were significant differences in the pharmacokinetic parameters of the H4 active metabolite of CLP in the atorvastatin and rosuvastatin group divided according to their CYP2C19 genotype. There were no significant associations between CYP3A4, CYP1A2, and ABCB1 genotypes and pharmacokinetic parameters in either statin groups. In the multivariate analysis, CYP2C19*2 genotype and non-genetic factors including BMI, age, and diabetes significantly affected platelet reactivity in the studied groups of patients (P < 0.01). In the atorvastatin group, CYP2C19*2, CYP3A4*1G, and ABCB1 C3435T TT genotypes were independent determinants of PRU values (P < 0.01)., Conclusion: The CYP2C19*2 allele is the primary determinant of the exposition to the H4 active metabolite of clopidogrel and platelet reactivity in patients co-treated with atorvastatin or rosuvastatin.

Possible interaction between clopidogrel and CYP3A4-metabolised atorvastatin or non-CYP3A4-metabolised rosuvastatin was investigated based on pharmacokinetic parameters of clopidogrel and its metabolites as well as the platelet reactivity test in patients undergoing coronary angiography/angioplasty. The study involved 50 patients (62.7 ± 7.8 years old) who underwent coronary angiography/angioplasty and were treated with clopidogrel and atorvastatin or rosuvastatin during the six months after the procedure. The P2Y12 reaction units (PRU) and pharmacokinetic parameters of clopidogrel, diastereoisomers of thiol metabolite (inactive H3 and active H4), and inactive carboxylic metabolite were measured 12-18 h and six months after the coronary angiography/angioplasty. There were no significant differences in concentrations of clopidogrel and its metabolites including the H4 active metabolite in plasma of patients co-treated with clopidogrel and atorvastatin or rosuvastatin. The use of statins did not affect the pharmacokinetic parameters of the studied compounds. A significant correlation was found between the C max and AUC 0-t of the active H4 isomer and platelet aggregation in a group of patients treated with rosuvastatin but not in the atorvastatin group. No significant differences in PRU values were observed between the atorvastatin and rosuvastatin groups at the beginning of the study (171.4 ± 54.3 vs 146.3 ± 48.1 PRU, p = 0.192) as well as at six months (173.7 ± 45.8 vs 157.3 ± 54.9 PRU, p = 0.562). However, in a small group of patients, who were discharged from atorvastatin to rosuvastatin, an increase in the PRU values accompanied by a decreased AUC of the H4 active isomer was observed. The study confirmed that the systemic exposure to clopidogrel and its active H4 isomer of thiol metabolite, as well as the antiplatelet effect of the drug, were not negatively affected by co-administration of atorvastatin as compared with rosuvastatin., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A ,,sine qua non" requirement for a generic formulation to be admitted for a medical use is to provide bioavailability studies in healthy subjects. Therefore, those studies were performed for 150 mg moclobemide tablets (Jelfa, Poland) versus 150 mg Aurorix (Hoffmann la Roche) reference tablets. An open-label, two-phase crossover study was conducted with 10 healthy subjects. Pharmacokinetic parameters (AUC, ke, t 1/2 , C max , t max , t lag , V/f, Cl/f) obtained at the same time for moclobemide were supposed to be confronted with the literature data available for healthy volunteers. The plasma moclobemide levels as a function of time were calculated according to either an open one-compartment body model with lag time of absorption or non-compartmental method for calculation of bioavailability using Phoenix WinNonlin 8.0 software. For those reasons a suitable HPLC method was worked out. Carbamazepine was proposed as an internal standard and ammonia as well as Na₂HPO₄ as alkalizing agents for the mobile phase and the liquid-liquid extraction of moclobemide from human blood plasma, respectively. Basic pharmacokinetic parameters of moclobemide obtained in the paper are essentially equal to the literature data for the healthy subjects. However, bioavailability parameters (AUC 0→t , AUC 0→∞, C max , t max ) were greater for moclobemide tablets (Jelfa) if compared to Aurorix tablets (Roche) by more than 20 %. Furthermore, the extent of bioavailability (110.6 %) for the generic moclobemide tablets if compared to Aurorix tablets is not significantly different. It seems to us that the number of subjects should be increased from 10 to 24 to help to clarify that inconsequence.

A combination of antiplatelet drugs with high-intensity statin therapy is a standard in patients with coronary events. Concomitant treatment with ticagrelor, a moderate CYP3A4 inhibitor, and CYP3A4-metabolized statins such as atorvastatin, might lead to an increased risk of muscle-related adverse events. Therefore, investigation of concentrations of these compounds in clinical samples is necessary. For this purpose, an LC-MS/MS method was developed for simultaneous determination of ticagrelor and its active metabolite (AR-C124910XX), as well as 2-hydroxyatorvastatin, which is the main metabolite of atorvastatin. Protein precipitation was used for sample preparation and afterwards the analytes were separated on a Kinetex XB-C18 column with an isocratic elution (water and acetonitrile with 0.1% formic acid, 57:43, v/v). Detection was performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was fully validated according to the EMA's recommendations. Determination was possible within ranges: 1.25-2000 ng/mL for ticagrelor, 1.25-1000 ng/mL for its AR-C124910XX, 1.25-50 ng/mL for atorvastatin and 1.14-45.73 for 2-hydroxyatorvastatin. Within and between-run accuracy, expressed as a relative error, was within 0.05-10.56% for all analytes, while within and between-run precision, expressed as coefficient of variation, was within 0.61-9.91%. Ticagrelor, atorvastatin and their main metabolites were found to be stable in acetonitrile stock solutions, and in plasma samples stored for 24 h at room temperature, 1 month at -25 °C, after 3 cycles of freezing and thawing, and in processed samples stored as a dry residue for 24 h at 4 °C and for 24 h in autosampler at room temperature. This simple and rapid method allowed simultaneous determination of the analytes for the first time. The procedure was applied for the pharmacokinetic study of ticagrelor, its active metabolite AR-C124910XX, and 2-hydroxyatorvastatin in patients simultaneously treated with ticagrelor and atorvastatin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The analysis of steroids in biological matrices is challenging. One can apply immunoassay as well as gas and liquid chromatography with various types of detection, depending on the available equipment and the experience of the analyst. The question is how the methods are interchangeable between themselves. Doubts were reported having compared immunoassays and chromatography-mass spectrometry, but there are scarce data on chromatographic methods with detection types other than mass spectrometry., Methods: Here, we present the detailed comparison of two liquid chromatographic methods for the determination of free urinary cortisol and cortisone: one with fluorescence detection (high-performance liquid chromatography [HPLC-FLD]) and the other with tandem mass spectrometry (HPLC-MS/MS). The comparison was made with 199 human urine samples. The data analysis included Passing-Bablok and Deming regression, Bland-Altman test, Wilcoxon test, mountain plot and Lin's concordance correlation coefficient., Results: The validation data indicated that both methods met the requirements of the European Medicines Agency. However, the statistical analysis revealed the systematic bias between the two assays. The Passing-Bablok and the Deming tests showed that the HPLC-FLD method overestimated results for cortisol and underestimated measurements for cortisone. The Bland-Altman analysis estimated the mean differences between the methods: 18.8 nmol/L for cortisol and -16.9 nmol/L for cortisone measurement., Conclusions: Both methods' results led to the same conclusion in observational studies, but the techniques are not interchangeable. The literature data, the observations from the clinical setting and our experience clearly indicate that the future of steroid measurements will belong to chromatography coupled with mass spectrometry.

Treosulfan (TREO), a structural analog of busulfan, is currently studied as a myeloablative agent in conditioning regimens before hematopoietic stem cell transplantation in pediatric patients. High exposure to TREO (>1650 mg∗h/mL) might be related to early toxicity, especially skin toxicity and mucositis. The aim of the present study was to investigate a potential relationship between exposure to TREO and its monoepoxytransformer (S,S-EBDM), as well as variability of the pharmacokinetics of these entities by means of a population pharmacokinetic approach with a non-linear mixed-effects analysis. The study included data from 14 children with malignant and non-malignant diseases treated with TREO in daily doses 10-14 g/m 2 . The parent-metabolite population pharmacokinetic model was developed in NONMEM 7.3 software. Upon the constructed model, an extensive simulation was performed to assess the correlation between exposure to TREO and S,S-EBDM. It was found that TREO and S,S-EBDM pharmacokinetics was best described with 2-compartmental and 1-compartmental linear models, respectively. The vast majority (>65%) of TREO was transformed to S,S-EBDM. Overall, a considerable interpatient variability of pharmacokinetic parameters was observed, especially the clearance of S,S-EBDM. A weak correlation was found between the exposure to TREO and S,S-EBDM (r = 0.1681, p < 0.0001). Also, patients with an exposure to TREO above 1650 mg∗h/mL were most likely to have also a high exposure to S,S-EBDM (35.38 μM∗h vs. 43.14 μM∗h, p < 0.0001). In summary, a parent-metabolite population pharmacokinetic model for TREO and S,S-EBDM was developed for the first time. It was shown that there is a weak correlation between exposure to TREO and S,S-EBDM. Therefore therapeutic drug monitoring of not only prodrug but also its active epoxide might be needed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The introduction of ticagrelor, one of the first directly-acting oral antiplatelet drugs, provided new possibilities in the prevention of thrombotic events in patients with acute coronary syndromes (ACS). Current guidelines recommend ticagrelor in dual antiplatelet therapy with aspirin over clopidogrel for prevention of stent thrombosis in patients with ACS. Moreover, in the management of ACS, lipid-lowering treatment with high-intensity statin therapy is advised for secondary prevention of cardiovascular events over the long term. Despite the apparent advantages of combined antiplatelet and lipid-lowering treatments, a possible interaction between statins and ticagrelor may lead to myopathy and rhabdomyolysis. In this review, relevant information was gathered on the ticagrelor-statin interaction that might lead to this life-threatening condition. This review focuses on the most widely used statins-simvastatin, atorvastatin, and rosuvastatin. Possible mechanisms of this interaction are discussed, including CYP3A4 isoenzymes, organic anion transporter polypeptide (OATPs), P-glycoprotein and glucuronidation. PubMed database was searched for relevant case reports and all data gathered from the introduction of ticagrelor to March 2018 are presented and discussed. In summary, co-administration of statins and ticagrelor was found to be relatively safe in routinely prescribed doses. However, caution should be exercised, especially in elder populations.

Currently, there is an urgent need to establish the optimal dosing of TREO in conditioning prior to hematopoietic stem cell transplantation, especially in children. For that purpose, pharmacokinetic analyses are ongoing within clinical phase II and III trials. In this paper, HPLC methods for determination of prodrug treosulfan and/or its biologically active epoxides in human plasma or serum are reviewed for the first time, including the spectrum of analytes being quantified, detection type, and derivatization methodology. The major focus is addressed to the stability of TREO and its monoepoxide related with different strategies of patients' blood processing, e.g. blood pH lowering to different values, no pH adjustment; centrifugation of blood immediately after collection or within a few hours later. This issue is crucially important for the robust bioanalysis because the epoxytransformation of TREO is a nonenzymatic, highly pH and temperature-dependent reaction. In-depth analysis of the literature results demonstrates that some methodologies of blood treatment could produce the systematic underestimation of TREO concentrations. Consequently, the drug clearance and volume of distribution will be overestimated, which might false the association of the drug exposure with the regimen-related toxicity and clinical outcomes. The paper indicates the deficiencies of the blood processing strategies and offers hints for their refinement. The provided information ought to be important in the current investigations of the personalized TREO pharmacokinetics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Ticagrelor is a first drug of a new chemical class cyclopentyltriazolopyrimidines. It is an antiplatelet agent with a unique mechanism of action, allowing a direct and reversible competitive inhibition of P2Y 12 receptor. According to newest guidelines, it is recommended for prevention of thrombotic events in patients with acute coronary syndromes. Moreover, it is preferred over clopidogrel, an older generation antiplatelet drug, and therefore gains more interest in modern cardiology and vascular medicine., Areas Covered: This review is a comprehensive and thorough summary of the most important findings on ticagrelor. Pharmacokinetics, pharmacogenetics, drug-drug interactions, adverse effects, efficacy in specific patient populations and off-label properties of ticagrelor are discussed in this paper. Moreover, the results from pivotal clinical trials are presented., Expert Opinion: Introduction of ticagrelor, a first directly-acting and reversible P2Y 12 inhibitor, gave some new possibilities as the efficacy of older drugs was often insufficient. Despite some drawbacks, such as a risk of bleeding events or dyspnea, a rapid onset of action, consistency in the antiplatelet effect and reports on pleiotropic properties make this drug a promising candidate for a first-choice antiplatelet agent in patients with acute coronary events.

Purpose: There is an increasing interest in use of treosulfan (TREO), a structural analogue of busulfan, as an agent in conditioning regimens prior to hematopoietic stem cell transplantation (HSCT), both in pediatric and adult populations. The aim of this study was to develop a population pharmacokinetic model and to establish limited sampling strategies (LSSs) enabling accurate estimation of exposure to this drug., Methods: The study included 15 pediatric patients with malignant and non-malignant diseases, undergoing conditioning regimens prior to HSCT including TREO administered as a 1 h or 2 h infusion at daily doses of 10, 12, or 14 g/m 2 . A population pharmacokinetic model was developed by means of non-linear mixed-effect modeling approach in Monolix® software. Multivariate regression analysis and Bayesian method were used to develop 2- and 3-point strategies for estimation of exposure to TREO., Results: Pharmacokinetics of TREO was best described with a two-compartmental linear model with proportional residual error. Following sampling schedules allowed accurate estimation of exposure to TREO: 1 h and 6 h or 1 h, 2 h, and 6 h for a TREO dose 12 g/m 2 in a 1 h infusion, or at 2 h and 6 h or 2 h, 4 h, and 8 h for a TREO dose of 12 g/m 2 and 14 g/m 2 in a 2 h infusion., Conclusions: A two-compartmental population pharmacokinetic model of TREO was developed and successfully used to establish 2- and 3-point LSSs for accurate and precise estimation of TREO AUC 0→∞ .

Purpose: A high interindividual variability is observed in the pharmacokinetics of clopidogrel, a widely used antiplatelet drug. In the present study, a joint parent-metabolite population pharmacokinetic model was developed to adequately describe observed concentrations of clopidogrel and its active thiol metabolite (H4)., Methods: The study included 63 patients undergoing elective coronarography or percutaneous coronary intervention. The population pharmacokinetic model was developed in the NONMEM 7.3 software, and first-order conditional estimation method with interaction was applied. Also, the influence of covariates was evaluated (age, weight, body mass index (BMI), obesity defined as BMI ≥ 30 kg/m 2 , sex, diabetes mellitus, co-administration of PPI or statins, presence of CYP2C19*2, CYP2C19*17, CYP3A4*1G alleles, and ABCB1 3435 TT genotype)., Results: It was found that the only significant covariate was the presence of CYP2C19*2 allele, which had an impact on lower conversion of clopidogrel to H4. As a result, predicted area under the time-concentration curve values was lower in carriers of this allele, with median 5.94 ng h/ml (interquartile range 3.92-12.51 [ng∙h/ml]) vs. 12.70 ng h/ml in non-carriers (interquartile range, 7.00-19.39 [ng∙h/ml]), respectively (p = 0.004)., Conclusions: Developed model predicts that the only significant covariate influencing the observed concentrations and therefore the exposure to the active H4 metabolite is the presence of CYP2C19*2 allele.

For the recent years, the application of treosulfan (TREO)-based conditioning prior to hematopoietic stem cell transplantation (HSCT) has been increasing as an alternative to busulfan-based therapy, especially for patients presenting high risk of developing hepato-, pulmo-, and neurotoxicity. So far, the penetration of TREO and its epoxy-derivatives into central nervous system and aqueous humor of the eye has been investigated. However, lacking knowledge on the compounds distribution into the other key tissues precludes comprehensive understanding and assessment of TREO clinical efficacy and toxicity. In this paper, the disposition of TREO and its active monoepoxide (S,S-EBDM) in a bone marrow, liver, lungs, brain, and quadriceps femoris was studied in an animal model. Male and female adult Wistar rats (n=48/48) received an intraperitoneal injection of TREO at the dose of 500mg/kg b.w. Concentrations of TREO and S,S-EBDM in tissues were determined with a validated HPLC-MS/MS method. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis. Mean values of the maximal concentrations of TREO and S,S-EBDM in the organs were sex-independent and ranged from 61 to 1650μM and 25-105μM, respectively. No quantifiable levels of S,S-EBDM were found in the liver. Average tissue/plasma area under the curve (AUC) ratio for unbound TREO increased in the sequence: brain (0.10)

A prodrug treosulfan (T) undergoes a pH-dependent activation to epoxide derivatives. The process seems to involve an intramolecular Williamson reaction (IWR) but clear kinetic evidence is lacking. Moreover, a cis-diol system present in the T structure is expected to promote complexation with boric acid. As a result, the prodrug epoxidation would be inhibited; however, this phenomenon has not been investigated. In this article, the effect of pH on the kinetics of T conversion to its monoepoxide was studied from a mechanistic point of view. Also, the influence of boric acid on the reaction kinetics was examined. The rate constants observed for the activation of T (k obs ) in acetate, phosphate, and carbonate buffers satisfied the equation logk obs  = -7.48 + 0.96 pH. The reaction was inhibited in the excess of boric acid over T, and the k obs decreased with increasing borate buffer concentration. The experimental results were consistent with the inhibition model that included the formation of a tetrahedral, anionic T-boric acid monoester. To conclude, in nonborate buffers, the T activation to (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate follows IWR mechanism. A borate buffer changes the reaction kinetics and complicates kinetic analysis., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide (S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been investigated in animal models and the obtained results presented clinical relevance. In this paper, a selective and rapid HPLC-ESI-MS/MS method was elaborated and validated for the studies of disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two analytes and codeine, internal standard (IS), were isolated from 50μL of plasma and 100μL of supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93μM. The HPLC-MS/MS method was adequately precise and accurate within and between runs. The IS-normalized matrix effect differed among the tissues and was the most pronounced in a liver homogenate supernatant (approximately 0.55 for TREO and 0.35 for S,S-EBDM). Stability of the analytes in experimental samples was also established. The validated method for the first time enabled determination of TREO and S,S-EBDM in the six life-important tissues in rats following administration of the prodrug., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: One of the hypotheses concerning the etiology of gestational hypertension (GH) and pre-eclampsia (PE) assumes that they develop as a result of placenta malfunctioning at the early stage of pregnancy. Placental dysfunction is also associated with the decreased activity of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which in normal pregnancy protects the fetus from the excess of maternal cortisol., Objective: The aim of the study was to analyze the sequence of HSD11B2, a gene that encodes 11β-HSD2, searching for mutations and haplotypes associated with the increased risk of GH or PE. Those may serve as potential genetic markers of GH and PE., Methods: The study was performed in case-control structure and included pregnant women (in third trimester) diagnosed with: GH, PE or being normotensive (control group). The research comprised DNA sequencing of HSD11B2, followed by restriction analysis (PCR-RFLP). The linkage disequilibrium analysis and haplotype-based case-control analysis were performed., Results: Six sequence variations were observed. Four mutations were indicated in the coding region of HSD11B2 and the other two in 3'-UTR. Two SNPs: c.468C > A and c.534G > A were found to be in total disequilibrium., Conclusions: High variability in HSD11B2 sequence was indicated in the study population, but the relevance of observed SNPs to GH or PE development was not confirmed.

Dual antiplatelet therapy with clopidogrel is a regimen used before and after drug-eluting stent (DES) implantation. Point-of-care platelet reactivity assays are easy-to-use methods to determine adequate response to the drug. The aim of this study was a comparison of the two platelet reactivity assays: Multiplate ® and VerifyNow ® and an identification of factors potentially influencing the results of these tests, including common genetic polymorphisms. The study included 39 patients receiving 75 mg clopidogrel daily before angioplasty with DES implantation. Platelet reactivity was measured with Multiplate and P2Y 12 VerifyNow assays. Genetic polymorphisms of CYP2C19*2, ABCB1 3435C > T, and CYP3A4*1G were determined with PCR-RFLP method and CYP2C19*17 was determined by means of an allele-specific PCR. Agreement between Multiplate and VerifyNow assays was poor (Cohen's κ = 0.056, p = .273). Hematocrit significantly negatively correlated with VerifyNow assayed platelet reactivity (r = -.487, p = .002). Female sex was significantly associated with higher VerifyNow assay results after adjustment to hematocrit (253.2 ± 47.6 PRU vs. 195.9 ± 56.9 PRU, p = .013) and the prevalence of high-on-treatment platelet reactivity (OR: 8.50; 95% CI 1.13-77.60, p = .024). Reactivity measured with Multiplate was lower in women (82.3 vs. 175.6 AU·min, p = .037) and in patients who received calcium channel blockers (74.7 vs. 191.7 AU·min, p = .002). None of the studied polymorphisms significantly influenced platelet aggregation measurements. In conclusion, different aspects modify between-patient variability of the Multiplate and VerifyNow assays and agreement between those two assays was poor.

Background: Etoposide (VP-16), a podophyllotoxin derivative, is used in conditioning regimens before allogeneic hematopoietic stem cell transplantation in children with acute lymphoblastic leukemia. The aim of this study was to develop a limited sampling strategy (LSS) suitable for the prediction of exposure to VP-16 defined as area under time-concentration curve (AUC)., Methods: The study included 28 pediatric patients with acute lymphoblastic leukemia, who were administered a 4-hour infusion of 60 mg/kg VP-16. VP-16 concentrations were determined in samples collected 4-124 hours after the beginning of infusion. On obtaining the pharmacokinetic (PK) profiles, a population PK model was developed in NONMEM (ICON Development Solutions, Hanover, MD) with first-order conditional estimation with interaction algorithm. LSSs were chosen by means of a multivariate regression analysis and cross-validated with a leave-one-out approach. Predictive performance of LSSs was assessed by calculating relative prediction error (PE), mean PE, mean absolute PE, and root mean squared PE for model-predicted and observed AUC., Results: VP-16 PKs was best described by a 2-compartment first-order model, and a large variability in the PK parameters was observed. A 3-sample strategy allowed the estimation of VP-16 with highest accuracy and precision (mean relative PE = 0.18%, 95% confidence interval, 1.73%-2.09%; mean absolute relative PE = 3.47%, 95% confidence interval, 2.28%-4.66%; root mean squared PE = 4.43%). The final equation was AUC = 6.85 × C6 h + 3.88 × C12 h + 46.11 × C28 h + 282.0 (adjusted R = 0.9540)., Conclusions: In conclusion, developed LSS allows accurate and precise estimation of VP-16 AUC and might be useful for therapeutic drug monitoring.

Background and Objectives: Resistance to the antiplatelet treatment with clopidogrel has both genetic and non-genetic causes. Polymorphic variants of cytochrome P450 3A4 isoenzyme involved in the bioactivation of clopidogrel might have an influence on responsiveness to the drug. The aim of this study was to evaluate the influence of CYP3A4*1G (IVS10+12G>A, rs2242480) on the pharmacokinetics and pharmacodynamics of clopidogrel., Methods: CYP3A4*1G polymorphism was determined in a group of 82 patients undergoing percutaneous coronary intervention and taking 75 mg of clopidogrel daily. Concentrations of clopidogrel and its metabolites, inactive carboxylic acid derivative and two diastereoisomers of active thiol metabolite: H3 and H4, were determined by a validated HPLC-MS/MS method. Pharmacodynamic effect was measured by an impedance method with a Multiplate analyzer. Moreover, an effect of factors, such as CYP2C19 phenotype, age, gender, body mass index and interactions with drugs metabolized by CYP3A4 were also investigated., Results: In the studied group allele frequencies were: wt-0.921, *1G-0.079. Pharmacokinetic parameters of clopidogrel and its metabolites were not significantly different in carriers of *1G allele, comparing to wt/wt homozygotes. Platelet aggregation was higher in heterozygotes than in wt/wt carriers; however, the difference was not statistically significant (p = 0.484). In a multivariate analysis, which included age, body mass index, co-morbidities and coadministered drugs, CYP3A4*1G was not a predictor of values of H3 and H4 pharmacokinetic parameters and platelet aggregation., Conclusion: CYP3A4*1G might not be a significant contributor to the variability in pharmacokinetic and pharmacodynamic response to clopidogrel therapy., Competing Interests: Compliance with Ethical Standards The study was supported by Polish Ministry of Science and Higher Education, Grant Number NN 405 419739 and Poznan University of Medical Sciences Grant Number 502-14-03306413-09628. The study protocol was approved by the local Ethical Committee at Poznan University of Medical Sciences. All performed procedures were in accordance with the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Conflict of interest Authors declare no conflict of interest. Informed consent Informed consent was obtained from all individual participants included in the study.

Resistance to an anti-platelet agent clopidogrel (CLP) and the growing number of products with the drug cause the need for comparison of their quality to assure patients safe and effective treatment. Therefore, the aim of the study was to compare in vitro dissolution kinetics of CLP immediate-release tablets, commonly used in anti-platelet therapy in Poland. For analysis of CLP in samples obtained from dissolution test a capillary zone electrophoresis (CZE) method was elaborated and validated. Separation of CLP and ticlopidine, used as an internal standard, was performed in silica capillary filled with phosphate buffer of pH 2.5, at the applied voltage of 20 kV. The CZE method fulfilled the validation requirements for determination of drugs in pharmaceutical matrices and was successfully applied for analysis of CLP dissolved from the tablets. Dissolution profiles were prepared for each product and mean dose fractions of CLP dissolved from tablets at 30 min were calculated. Kinetic parameters of the CLP dissolution from the studied products were compared. Analysis of variance (ANOVA) did not reveal differences between CLP fractions dissolved at 30 min time point from the tested drug products. However, ANOVA with Tukey multiple comparison test revealed significant differences in first-order dissolution rate constants and t₀.₅ values (times at which 50% of drug is dissolved) of CLP among tested tablets. It was concluded that the studied CLP products met the acceptance criteria regarding dissolution test but differed with each other in dissolution kinetics.

Pro-drug treosulfan (TREO) is currently evaluated in randomized phase III clinical trials as a conditioning agent prior to HSCT. In the present paper pharmacokinetics of both TREO and its biologically active monoepoxide (S,S-EBDM) was investigated in pediatric patients for the first time. The studies were carried out in 16 children (median age 7.5 years) undergoing TREO-based preparative regimen prior to HSCT, who received 10, 12 or 14 g/m(2) of the drug as a 1h or 2h intravenous infusion. Plasma concentrations of TREO as well as S,S-EBDM were determined using the validated HPLC-MS/MS method. The changes in S,S-EBDM concentration over time followed TREO levels. The area under the curve (AUC) of TREO was 100-fold higher than AUC of S,S-EBDM. No statistically significant dependency of the dose-normalized AUC of either TREO or S,S-EBDM on the patients' age and body surface area was stated. Moreover, plasma C(max) as well as AUC of S,S-EBDM demonstrated linear correlation with the C(max) and AUC of TREO, respectively. The biological half-lives of TREO and S,S-EBDM were similar. This indicates that S,S-EBDM was completely eliminated from the patients' blood within relatively short time, comparable to TREO., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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). We concluded that the CYP2C19*2 genotype is the primary determinant of the antiplatelet response to clopidogrel therapy., (© 2014, The American College of Clinical Pharmacology.)

Background and Objective: Approximately 5-40 % of patients treated with clopidogrel do not display an adequate antiplatelet response. Clopidogrel resistance may be caused by insufficient drug absorption or impaired metabolic activation of the drug. The aim of this study was to evaluate the pharmacokinetics of clopidogrel and its metabolites in plasma samples from patients treated with high and low doses of clopidogrel, to obtain a possible explanation for antiplatelet resistance., Methods: The study included patients receiving either a single 300 mg loading dose of clopidogrel (n = 17) or a 75 mg dose (n = 45) for at least 7 days before sample collection. The concentrations of clopidogrel and its metabolites-the inactive H3 and the pharmacologically active H4 isomers of the thiol metabolite and the inactive carboxylic acid metabolite-in plasma samples (stabilized with 2-bromo-3'-methoxyacetophenone) from three patients after 300 mg and from 41 patients after 75 mg of the drug were determined using a validated high-performance liquid chromatography method with tandem mass spectrometry. The non-stabilized samples from the remaining patients were analysed using a validated capillary electrophoresis method. The calculated concentrations were used to determine the pharmacokinetic parameters of the analytes. The pharmacodynamic response to clopidogrel treatment, expressed as adenosine diphosphate-induced platelet aggregation, was measured using a Multiplate analyser., Results: The pharmacokinetic parameter values for the H3 and H4 isomers determined in the studied group of patients treated with clopidogrel 75 mg (maximum plasma concentration [C max] 5.29 ± 5.54 and 7.13 ± 6.32 ng/mL for H3 and H4, respectively; area under the plasma concentration-time curve from time zero to time t [AUC t ] 7.37 ± 6.71 and 11.30 ± 9.58 ng·h/mL for H3 and H4, respectively) were lower than those reported in healthy volunteers, according to the literature data. Platelet aggregation measured with a Multiplate analyser ranged between 37 and 747 AU·min. A significant correlation was found between the C max of the active H4 isomer and platelet aggregation (p = 0.025)., Conclusion: The C max of the active H4 isomer and platelet aggregation measured by the Multiplate analyser may serve as markers of the patient response to clopidogrel therapy.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Pharmacokinetics of ketoprofen (KTP) enantiomers has been studied in patients with rheumatoid arthritis (RA) following administration of a single oral dose of 100 mg rac-KTP during multidrug therapy taking into consideration the genotype of RA patients Concentrations of (-)-R and (+)-S enantiomers of KTP in plasma, urine and synovial fluid samples were determined using a validated HPCE method. The genotype of the patients was analyzed using PCR-RFLP method to determine the polymorphic variants of genes coding CYP2C8 and CYP2C9 isoenzymes. The levels of KTP enantiomers in synovial fluid at 4 h following administration were insignificantly greater [(-)-R = 1.34 ± 0.91 mg/L; (+)-S = 1.38 ± 0.91 mg/L] than in plasma [(-)-R = 1.15 ± 0.95 mg/L; (+)-S = 1.22 ± 0.95 mg/L]. The values of AUC(0-∞) were 11.89 ± 5.00 and 10.92 ± 4.10 mg h/L for (-)-R and (+)-S enantiomer, respectively, and were lower compared with data obtained in healthy volunteers following administration of the same dose of rac-KTP. But, no statistically significant differences were observed also for C (max), Cl, V (d), t (0.5) and MRT of KTP enantiomers. The total percentage of unchanged KTP eliminated with urine of RA patients was in the range of 30-50% of the administered dose. Though RA patients represented the same wild genotype, quite significant variabilities (Cl((-)-R ) = 2.37-13.50 L/h and Cl((+)-S ) = 2.44-9.90 L/h) existed in the pharmacokinetics parameters of KTP. We concluded that KTP data obtained from healthy volunteers cannot be sufficient to predict disposition of KTP enantiomers in RA patients, especially when undergoing long-term multidrug therapy.

Fast and reproducible Capillary Zone Electrophoresis (CZE) method for the quantification of (+)-S clopidogrel carboxylic acid metabolite in human fluids was elaborated for the first time. Optimal buffer and CZE conditions were established to obtain the complete separation of clopidogrel, its metabolite and piroxicam (internal standard), during one analytical run. Finally, resolution of the analytes was obtained in an uncoated silica capillary filled with a phosphate buffer of pH 2.5. The analytes were isolated from plasma and urine samples using solid phase extraction (SPE). Validation of the CZE method was carried out. The calibration curve of clopidogrel was linear in the range of 0.5-10.0mg/L in plasma and urine, whereas for (+)-S carboxylic acid metabolite linearity was confirmed in the range of 0.25-20.0mg/L in plasma and 0.25-10.0mg/L in urine. Intra- and inter-day precision and accuracy were repeatable. LOD and LOQ were also estimated. SPE recovery of the analytes from plasma and urine was comparable and greater than 80%. The validated method was successfully applied in pharmacokinetic investigations of (+)-S carboxylic acid metabolite of clopidogrel following the oral administration of clopidogrel to patients prior to percutaneous coronary intervention., (Copyright 2010 Elsevier B.V. All rights reserved.)

A stereospecific CE method was elaborated for the quantification of ibuprofen enantiomers and their major phase I metabolites: 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen in plasma and urine. Optimal temperature and pH of BGE were established to obtain complete separation of eight ibuprofen chiral compounds and (+)-S indobufen, applied as an internal standard, during one analytical run. After isolation from biological matrices using SPE on an octadecyl stationary phase, the analytes were separated and resolved up to 10 min in a silica capillary filled with BGE, consisting of heptakis 2,3,6-tri-O-methyl-beta-CD in triethanolamine-phosphate buffer, pH 5.0. Complete enantioseparation of the all analytes confirmed specificity of the method. The calibration curves were linear in the range of 0.1-25.0 mg/L for IBP enantiomers and their chiral metabolites in 0.5 mL of plasma and 1.0-200.0 mg/L in 0.05 mL of urine. Following SPE procedure, recovery of the chiral analytes from the two media was in the ranges of 82-87%, 90-95% and 70-76% for ibuprofen, 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen enantiomers, respectively. The validated method was successfully applied in pharmacokinetic investigations of IBP enantiomers as well as free chiral metabolites in reference to the genetic polymorphism of CYP450 2C isoenzymes.

Studies were performed on the effect of ibuprofen racemate ionisation extent on the pharmacokinetics of its enantiomers following administration in suppositories to rabbits. The suppositories, containing 146.3 mg ibuprofen in acidic form (IBP) or 250 mg ibuprofen lysinate (IBPL), equivalent to the above IBP dose, were prepared using lipophilic Witepsol H-15 as a base and administered to rabbits in a crossover design. Compared with IBP, administration of IBPL was followed by faster absorption and elimination of R and S enantiomers. However, significant differences at alpha=0.05 were observed only at the stage of elimination. AUC was markedly higher following administration of suppositories containing IBP than following suppositories with IBPL and this pertained to both R and S enantiomers. Evident inversion of R into S form was noted 30 min following IBPL administration and 1 h after IBP administration. Ionisation extent only insignificantly affected the scope of chiral inversion of ibuprofen R into S form (AUC(S-IBP)/AUC(R-IBP)=1.66, AUC(S-IBPL)/AUC(R-IBPL)=1.57). No presystemic inversion of R into S was observed in rabbits following administration of IBP or IBPL in suppositories. IBP enantiomers were isolated from 0.5 ml serum using solid phase extraction in C(18) columns and were quantified by HPLC using the chiral Whelk O1 column and UV detector (lambda=264 nm).

A rapid and selective liquid chromatography (LC) with solid phase extraction (SPE) to quantify indobufen (INDB) enantiomers is described. The INDB enantiomers and internal standard (racemic flurbiprofen) are extracted from a small volume of acidified serum (0.2 ml) by means of SPE using cartridges with octadecyl chemically bound phase and analysed on reversed phase (RP), C18 column with ultraviolet detection at 275 nm. Recovery of both INDB enantiomers was in the range 92.1-94.3%. The mobile phase is composed of acetonitrile-potassium dihydrogen phosphate (pH 4.75; 10 mM) (38:62, v/v). The linear range of the standards curves was from 0.25 to 25.00 microg ml(-1) in serum for both enantiomers. The limit of quantification and detection for both INDB enantiomers in serum were 0.25 microg ml(-1) (CV < or = 10%), and 0.1 microg ml(-1), respectively. Both intra- and inter-day accuracy and precision of the calibration curves were determined and their CV values did not exceed 10%. The validated method has been successfully applied for chiral pharmacokinetics studies of INDB from tablets and intramuscular injections in man.

The influence of administration route (oral or intramuscular (i.m.)) on the pharmacokinetics of total indobufen (INDB) enantiomers was studied in healthy volunteers after a 200 mg dose as a single oral tablet or i.m. injection. Enantiospecific reversed phase (RP) HPLC with UV detection was used for the determination of INDB enantiomers in serum. INDB enantiomers were isolated from serum by solid phase extraction (SPE) procedure using C(18) columns. INDB enantiomers were converted to their L-leucinamide diastereoisomers and separated on a C(18) HPLC column. INDB from i.m. injections is absorbed faster (t(max) = 0.6-0.9 h) than from tablets (t(max) = 1.3-1.8 h). The area under curve (AUC) after administration of the tablet was slightly higher than after i.m. injection. The pharmacokinetic behaviour of (+)-S- and (-)-R-INDB after administration of the tablet was different from i.m. injection of racemic INDB. The (+)-S-enantiomer is more rapidly eliminated than its (-)-R-antipode. Statistically significant differences also occurred between enantiomers in AUC, first order elimination rate constant (k), clearance (Cl). The ratio AUC(R):AUC(S) was similar for the tablet (1.57-1.62) and i.m. injection (1.59-1.62). It was concluded that the formulation and extent of ionisation of rac-INDB (acid or sodium salt of INDB) do not significantly influence its stereoselective pharmacokinetics., (Copyright 2000 Wiley-Liss, Inc.)

Ibuprofen (IBP) was used to demonstrate that freshly isolated rat hepatocytes offer a suitable model to investigate the oxidative metabolism of antiinflammatory 2-arylpropionic acids (profens). The formation of two major oxidative metabolites of IBP (metabolite A--hydroxyibuprofen and metabolite B--carboxyibuprofen) was observed with the use of rat hepatocytes. The incubation of ibuprofen with a suspension of rat hepatocytes in Hanks' buffer during 60 min. resulted in a decrease of racemic IBP and both R(-)-IBP and S(+)-IBP concentration coincided with the appearance of its major racemic metabolites (metabolite A and metabolite B). The relative abundance of the above IBP metabolites produced by hepatocytes was consistent with their quantitative profiles in vivo in rat. The results confirm the value of isolated hepatocytes as a predictive model for the in vivo metabolism pattern of profens.

Concentration of ibuprofen in rabbit blood was found to be considerably higher after application of Imbun-500 suppositories, containing 500 mg of ibuprofen lysine salt equivalent to 292.6 mg of free ibuprofen, than after application of ibuprofen dragees containing 400 mg of the free drug. Parameters characterizing biological availability of ibuprofen in Imbun-500 suppositories (AUC, Cmax, tmax) were determined. Ibuprofen lysine salt proved to be considerably more easily absorbed from suppositories than ibuprofen from dragees of either home or foreign production.