Methodical approaches to bioassay of substances containing unstable functional groups

This article describes the method development approaches for bioassay of substances containing unstable functional groups and forming unstable metabolites using the example of mycophenolic acid, methyldopa and mebeverine metabolite

Development of quantification methods of a new selective carbonic anhydrase II inhibitor in plasma and blood and study of the pharmacokinetics of its ophthalmic suspension in rats

The developed methods have been fully validated according to the requirements of Russian and internatonal guidelines and have been successfully used for pharmacokinetic research. It was found that a content of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and its main metabolite in whole blood is significantly higher than in plasm

Identification and synthesis of metabolites of the new antiglaucoma drug

The determination of biotransformation products is an essential part of the preclinical trial of original medicines. These studies have not been conducted before for the new drug 5-[5- (trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide. Identification and synthesis of metabolite substances are necessary for subsequent tests of bioavailability, linearity of pharmacokinetics, accumulation, distribution and excretio

APPROACHES TO THE DEVELOPMENT OF BIOANALYTICAL METHODS FOR DETERMINATION OF UNSTABLE SUBSTANCES IN BIOLOGICAL FLUIDS

The approaches to bioanalytical method development for determination of substances which contain unstable functional groups in the structure are described. The oxidation and the hydrolysis are main causes of the decomposition of substances in biological fluids. Phenolic hydroxyls contain drugs were selected as examples of oxidable compounds, glucuronides of drugs were selected as examples of hydrolysable compounds. Determination of mycophenolic acid, which contains one phenolic hydroxyl and metabolized by formation of glucuronides, in plasma was performed using high performance liquid chromatography with mass-spectrometry and tandem mass-spectrometry detection. Methyldopa, which contains two phenolic hydroxyls, in stabilized plasma was assayed by high performance liquid chromatography – tandem mass-spectrometry in the range of 0.02–3.00 μg/ml. Concentrations of desmethyl mebeverine acid, which contains in the structure one phenolic hydroxyl and metabolized by formation of phenolic glucuronide, was measured simultaneously with mebeverine acid in the range of 10–2000 ng/ml. The influence of the ion source conversion of glucuronides on the quantitative determination of the substances was studied in the initial part of methods development. The next, selection of anticoagulants based on the study of short-term stability and freeze/thaw stability of the analytes and back conversion of their glucuronides was performed. The combination of anticoagulant K3EDTA and the antioxidant solution containing a mixture of ascorbic acid, sodium sulfite and sodium hydrogen carbonate in the concentrations of 5.0 %, 0.2 % and 2.4 %, respectively, was used to prevent degradation of methyldopa

Исследование сравнительной фармакокинетики таблетированных форм микофеноловой кислоты

In a single-dose, two-treatment, two-period, two-sequence crossover study with a 14-days washout period was carried out the bioequivalence study of two tablet coated formulation of mycophenolic acid that given to 48 volunteers in equal doses (dosage 360 mg). There were calculated the followed parameters: AUC0-t, Cmax, Tmax, Cmax/AUC. 90% confidence interval for ratio of geometric mean AUC0-t values was 98,97% - 111,49% and one for ratio of geometric mean Cmax was 121,27% - 153,94%. The upper limit of the confidence interval of Cmax valuesa goes beyond the permissible range according to the protocol study (75-133%). It is not possible to state bioequivalence of drugs. Possible causes of discrepancies of pharmacokinetic parameters were analyzed.В рамках открытого, рандомизированного, перекрёстного исследования с 14-дневным периодом отмывки, с двумя последовательностями была изучена биоэквивалентность двух таблетированных форм микофеноловой кислоты на 48 добровольцах (дозировка 360 мг). Для анализируемых препаратов рассчитаны следующие фармакокинетические параметры: AUC0-t, Cmax, Tmax, Cmax/AUC. 90% доверительные интервалы для отношения геометрических средних значений параметров AUC0-t и Cmax микофеноловой кислоты составили 98,97-111,49% и 121,27-153,94% соответственно. Верхняя граница доверительного интервала, соответствующего параметру Cmax, выходит за рамки допустимого согласно протоколу исследования диапазона (75-133%), что не позволило констатировать биоэквивалентность исследуемых препаратов. Также были проанализированы возможные причины расхождений фармакокинетических параметров

Исследование фармакокинетики мебеверина в форме капсул с пролонгированным высвобождением

Pharmacokinetic study of prolonged release capsules of mebeverine was carried out on 24 volunteers. It is known that the drug substance is completely metabolized due to first-pass effect. Therefore, pharmacokinetic parameters of the main metabolites -mebeverine acid and desmethyl mebeverine acid were measured. Bioanalytical method was developed to measurement of concentrations of these metabolites in blood plasma by using HPLC-MS/MS.Было проведено исследование фармакокинетики мебеверина в форме капсул с пролонгированным высвобождением на 24 здоровых добровольцах. Известно, что данное лекарственное вещество полностью метаболизируется на пресистемном этапе. Поэтому измерялись фармакокинетические параметры только его основных метаболитов - мебевериновой кислоты и деметилированной мебевериновой кислоты. Для определения концентрации данных метаболитов в плазме крови разработана биоаналитическая методика с использованием высокоэффективной жидкостной хроматографии с масс-спектрометрическим детектированием

METHODS OF STABILIZATION OF DRUG SUBSTANCES AND THEIR METABOLITES IN BIOLOGICAL FLUIDS DURING BIOANALYTICAL STUDY (REVIEW)

This article describes main methods of preventing degradation of chemically unstable drugs and their metabolites in biological fluids during storage after collection from patients. It is necessary for accurate quantitative determination of these compounds

Methodical approaches to bioassay of substances containing unstable functional groups

This article describes the method development approaches for bioassay of substances containing unstable functional groups and forming unstable metabolites using the example of mycophenolic acid, methyldopa and mebeverine metabolite

Development of quantification methods of a new selective carbonic anhydrase II inhibitor in plasma and blood and study of the pharmacokinetics of its ophthalmic suspension in rats

Introduction: Development of new bioanalytical methods is required for studying the systemic exposure of new selective inhibitor of carbonic anhydrase II, 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide, and its N-hydroxymetabolite in plasma and in whole blood. The results of the experiment with a single administration of an ophthalmic suspension of the drug are necessary to optimize the subsequent design of a full pharmacokinetic study. Materials and Methods: HPLC-MS/MS method was used to measure a concentration of analytes in plasma and whole blood. Chromatographic separation was performed on the Poroshell 120EC-C18 column (50*3.0 mm, 2.7 µm). Pharmacokinetics was studied on 6 Wistar rats weighing 287.50±18.64 g (Mean±SD). Each animal was instilled with 40 µL of the ophthalmic suspension in concentration of 2% in each eye. Blood samples were collected before administration of the drug and 30 min, 1 h, 1 h 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after administration. Non-compartment approach was used for the evaluation of pharmacokinetic parameters. Results and Discussion: The protein precipitation was chosen for a sample preparation of biological fluids. A solution of ascorbic acid in concentration of 10% was added to plasma, and a solution of sodium thiosulfate in concentration of 10% was added to blood to prevent the degradation of N-hydroxymetabolite of the drug. The analytical range of determination of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and its N-hydroxyderivative in blood was 50-10000 ng/mL and 5-1000 ng/mL, respectively, in plasma – 10-2000 ng/mL and 1-200 ng/mL, respectively. The maximum plasma concentration of the studied drug was 264.32±68.47 ng/mL (Mean±SD) 1.92±0.92 h (Mean±SD) after administration, and its metabolite was 10.43±1.79 ng/mL 2.17±1.13 h after administration. The maximum concentration of the drug in blood reached 8705.23±1301.84 ng/mL (Mean±SD) 1.17±0.52 h (Mean±SD) after administration, and the maximum concentration of N-hydroxymetabolite reached 230.00±69.54 ng/mL (Mean±SD) 1.33±0.41 h (Mean± SD) after administration. Conclusion: The developed methods have been fully validated according to the requirements of Russian and internatonal guidelines and have been successfully used for pharmacokinetic research. It was found that a content of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and its main metabolite in whole blood is significantly higher than in plasma