Introduction. 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide (R004) is a prospective new molecule for the treatment of rheumatoid arthritis. This compound is at the stage of preclinical research, during which its pharmacokinetics must be studied along with efficacy and safety. In this case, it is necessary to establish the main pharmacokinetic parameters of the drug and its metabolites in blood plasma after a single administration.Aim. Evaluation of linearity of pharmacokinetics and relative bioavailability of 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide and its main metabolites in plasma after a single administration of the substance to rats.Materials and methods. Substance R004 was administered to rats once orally at dosages of 10 mg/kg, 20 mg/kg and 40 mg/kg and intraperitoneally at a dosage of 10 mg/kg. The study was carried out using 24 Wistar rats: 6 rats were used for each of the dosage. Blood samples were collected before administration and 30 min, 1 h, 1 h, 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h after administration of the drug. The obtained plasma was stabilized with a 250 mM ammonium acetate solution to prevent hydrolysis of R004. Plasma samples were prepared by means proteins precipitation by acetonitrile solution of internal standards. Measurement of the concentration of R004 and its metabolites 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid (M1) and 4-methoxy-3-(trifluoromethyl)aniline (M2) was performed using HPLC-MS/MS. A non-compartment approach was applied for evaluation pharmacokinetic parameters.Results and discussion. The developed bioanalytical method has been fully validated in accordance with the requirements of the EAEU guideline for conducting bioequivalence study and ICH M10 guideline. The analytical range of determination in plasma of R004 was 2–2000 ng/ml, M1 and M2 – 1–1000 ng/ml. The dependence of the parameters C_max and AUC_0–t of the studied compound and its metabolites on the administered dose was linear. Thus, the value of the correlation coefficient of C_max R004 was 0.9995, and value of the correlation coefficient of AUC_0–t was 0.9991. The presence of enterohepatic recirculation R004 has been determined. The relative bioavailability of R004 was 21.26%.Conclusion. The developed method was successfully applied for the pharmacokinetic research. R004 and its metabolites have linear pharmacokinetics in case of oral administration.

Introduction: 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (ODASA) is a new pharmacologically activecompound, which is capable of reducing intraocular pressure by inhibiting carbonic anhydrase II. It is necessary to calculate the pharmacokinetic parameters of this compound and its metabolites in blood plasma of laboratory animals during a preclinical study. Aim: Evaluation of pharmacokinetic parameters of ODASA and its metabolites in rat bloodplasma after instillation of ocular suspension and its intraperitoneal administration. Materials and Methods: The study was conducted on 2 groups of Wistar rats, each including 6 individuals. The firstgroup underwent instillation of 1% ocular suspension of ODASA of 20 µL into each eye (1.6 mg/kg). The drug was injected intraperitoneally at the same dose to the second group. Blood sampling was performed before the administration,0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72 h, 96h, 120h, 144h, 192h, 240h, 288h after the administration. Plasma was immediately stabilized by 10% solution of ascorbic acid and frozen to a temperature no higher than -70°C. The sampleswere analyzed using the HPLC-MS/MS method. Chromatographic separation was performed on a Kinetex Phenyl Hexylcolumn (50*4.6 mm, 2.6 microns) in a gradient mode. Detection was carried out in the MRM mode using electrosprayionization. Results: The developed method was validated in the range of 2-2000 ng/mL for ODASA and 4-[5-(hydroxymethyl)-1,3,4-oxadiazole-2-yl]-benzenesulfonamide (M1) and 0.5 -500.0 ng/mL for N-hydroxy-4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (M2). The size of the maximum plasma concentration after instillation of ODASA into eyes was 349.85±62.50 ng/mL, in M1 – 30.91±6.00 ng/mL and in M2 – 2.70±0.62 ng/mL (M±SEM). The half-life time of ODASA after ocular administration was 46.4±3.8 h, M1 – 70.0±14.3 h, and M2 - 36.5±15.2 h (M±SEM). The relative bioavailability of ODASA compared with injection was 81.03%. Conclusion: The performed validation of the method guaranteed the accuracy of the obtained results. The activesubstance has a high relative bioavailability after instillation into eyes. M1 is a major metabolite, and M2 is a minormetabolite. The studied compounds had a long half-life.

INTRODUCTION. The administration of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (TFISA) into the eyes can reduce intraocular pressure. Currently, this novel selective carbonic anhydrase II inhibitor is at the preclinical research stage. The excretion of TFISA and its metabolites has not been studied yet. AIM. This study aimed to calculate the parameters of urinary and faecal excretion of TFISA and its major metabolites in rats. MATERIALS AND METHODS. The study was performed in 6 Wistar rats (3 males and 3 females). The rats received an instillation of 1% TFISA ophthalmic suspension in each eye at a dose of 3.7 mg/kg. Stool samples were taken prior to TFISA administration and at multiple intervals up to 360 h afterwards. Excreta were collected using metabolic cages. The study used high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) to measure the concentrations of TFISA, N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M2), and N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M1) with its degradation product 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonic acid (M3) in the excreta. RESULTS. The study involved full validation of bioanalytical procedures for the determination of TFISA and its metabolites in rat excreta. The analytical ranges were 10–10,000 ng/mL for the urinary levels of TFISA, M2, and M3; 1–1,000 ng/mL for the urinary levels of M1; 10–4,000 ng/g for the faecal levels of TFISA; 5–2,000 ng/g for the faecal levels of M2 and M3; and 1–1000 ng/g for the faecal levels of M1. Out of the total amount eliminated, 45.7±2.0% of unchanged TFISA, 38.7±2.7% of TFISA in the form of M1 and its degradation product M3, and 4.0±0.6% of TFISA in the form of M2 were excreted in urine, while 8.2±1.0% of unchanged TFISA and 3.3±0.2% of the N-hydroxy metabolite M3 were excreted in faeces. TFISA elimination continued for up to 336 h after administration. CONCLUSION. The developed and successfully validated bioanalytical procedures were used to study the excretion of TFISA and its metabolites in rats after single-dose administration of the ophthalmic TFISA suspension. According to the results, TFISA is predominantly excreted unchanged in urine. In addition, TFISA is eliminated as its N-hydroxy metabolite, which almost completely degrades to the sulfonic acid derivative during the sampling process. The N-acetyl metabolite of TFISA is minor and is exclusively excreted in urine.

Introduction: The 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (ODASA) is new antiglaucoma drug. The compounds similar in structure are able to accumulate in red blood cells. Therefore, it is necessary to evaluate the pharmacokinetic parameters of ODASA and its metabolite in whole blood of laboratory animals. Aim: Calculation of pharmacokineticparameters of ODASA and its metabolites in rat whole blood after ocular instillation andintraperitoneal injection of the drug. Materials and Methods: The 1% ocular suspension of ODASA was instilled into each eye in a volume of 20 µL to a group of 6 Wistar rats. The drug was also administrated by intraperitoneal injection at a dose of 1.6 mg/kg to another group of 6 Wistar rats. Sampling was carried out before administration and at 16 points for 288 hours after administration. HPLC-MS/MS was used for quantification of ODASA and its metabolites 4-[5-(hydroxymethyl)-1,3,4-oxadiazole-2-yl]-benzenesulfonamide (M1) and N-hydroxy-4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (M2). Results: The high concentration level of ODASA and M1 was observed: maximum blood concentration (Cmax) of ODASA after eye instillation was 10326±532 ng/mL, Cmax of M1 was 3722±505 ng/mL(M±SEM). The Cmax value of M2 was lower – 49.9±9.4 ng/mL (M±SEM). The half-life time of the studied compounds was more than 69 h. M1 has the greatest affinity for red blood cells. The value of the area under the “concentration-time” curve (AUC0-t) of M1 in bloodwas more than 150 times higher than AUC0-t of M1 in plasma. Conclusion: The ODASA and its metabolites have long half-life time and high exposition in blood in comparison with that in plasma, which proves their accumulation in red blood cells.

Introduction. The new pharmacologically active substance 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide (R004) is a new PAR-2 receptor inhibitor for treatment of rheumatoid arthritis. This compound is at the stage of preclinical trail. The distribution study of R004 and its metabolites by organs and tissues has not been performed before. Aim. The investigation of distribution of R004 and its metabolites in rat organs after a single oral administration of the active substance. Materials and methods. The study was carried out on 50 male Wistar rats. Substance of R004 was administered orally at a therapeutic dosage of 10 mg/kg. Sampling of liver, kidneys, heart, lungs, spleen, brain, skin and muscles was performed 1, 2, 3, 4, 6, 8, 10, 12, 16, 24 h after administration of the drug. Organs were immediately homogenized using acetonitrile and frozen. Further sample preparation was carried out by addition an acetonitrile solution of internal standards to the homogenates. Quantification of R004 and its metabolites 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid (M1) and 4-methoxy-3-(trifluoromethyl)aniline (M2) was performed by HPLC-MS/MS. Results and discussion. The developed method for quantification of R004, M1 and M2 in organs has been successfully validated in the analytical ranges of 2–2000 ng/g for R004 and 1–1000 ng/g for M1 and M2. R004 is distributed by all studied biological objects. The tissue bioavailability of R004 decreases in the following sequence: liver > skin > kidneys > lungs > muscles > heart > spleen > brain. M1 and M2 was detected in large quantities in liver and kidneys. Metabolites penetrated into the heart, lungs and spleen in much lower concentrations. In skin tissues only M2 was identified. M1 and M2 were not found in brain and muscles. Conclusions. The validated bioanalytical method has been successfully used for distribution study of R004 substance. The drug is well distributed throughout the studied organs and has a high tissue bioavailability. The highest concentrations of metabolites were observed in liver and kidneys.

Introduction. The new selective inhibitor of PAR-2 receptors, 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-N-[4-methoxy-3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2-oxazole-5-carboxamide (R004), is at the stage of preclinical trail. The excretion of R004 and its metabolites has not been studied before.Aim. Investigation of excretion of R004 and its metabolites in urine and feces after a single oral and intraperitoneal administration of substance.Materials and methods. The study was carried out on 2 groups of 6 Wistar rats. The R004 substance was administered orally to the first group at a dosage of 10 mg/kg, to the second group intraperitoneally at a dosage of 10 mg/kg. Biomaterial sampling was carried out with using metabolic cages. Urine was collected before administration of the drug and in the intervals of 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120 h after administration. Feces were collected before administration of the drug and in the intervals of 0–12, 12–24, 24–48, 48–72, 72–96, 96–120 h. The samples were analyzed using HPLC-MS/MS.Results and discussion. The analytical range of the urine quantification method for R004 and 4-methoxy-3-(trifluoromethyl) aniline (М2) was 5–2000 ng/ml, and 3-(2-butyl-5-chloro-1H-imidazole-4-yl)-4,5-dihydro-1,2-oxazole-5-carboxylic acid (M1) – 100–40 000 ng/ml. In feces concentrations of R004 were measured in the range of 0.5–500.0 µg/g, M1 – 4–4000 ng/g, M2 – 40–40 000 ng/g. The main part of the drug and metabolites was excreted within 48 h after administration. Complete elimination was achieved after 96 h. R004 is excreted unchanged only with feces: 94.83 ± 0.78 % after oral administration and 67.04 ± 0.59 % after intraperitoneal administration (M + m). The metabolite M1 is mainly excreted by renal route, the metabolite M2 is mainly excreted through intestine.Conclusion. Bioanalytical methods for determination of R004, M1 and M2 in urine and feces have been successfully validated. Most part of the R004 is eliminated unchanged by enteric route. M1 is excreted mainly in urine, M2 – mainly in feces.