The study of distribution of a new 4,5-dihydroisoxazole-5-carboxamide derivative in rats

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.

1. Korsakov M. K., Fedorov V. N., Smirnov N. A., Shetnev A. A., Leonova O. V., Volkhin N. N., Andreyev A. I. Screening of anti-inflammatory activity of 4.5-dihydroisoxazol-5-carboxamide (PAR-2 inhibitors) based on formaldehyde oedema model among white lab rats. Research Results in Pharmacology. 2023;9(4):105–111. DOI: 10.18413/rrpharmacology.9.10061.

2. Khokhlov A. L., Yaichkov I. I., Alexeev M. A., Korsakov M. K., Shetnev A. A., Ivanovskiy S. A., Volkhin N. N., Petukhov S. S., Vasilyeva E. A. Identification and synthesis of metabolites of the new 4.5-dihydroisoxazol-5-carboxamide derivate. Research Results in Pharmacology. 2024;10(2):83–95. DOI: 10.18413/rrpharmacology.10.482.

3. Yaichkov I. I., Korsakov M. K., Volkhin N. N., Petukhov S. S., Tyushina A. N., Zaykova V. E., Lasaraynz O. E. Pharmacokinetic study of a new 4,5-dihydroisoxazole-5-carboxamide derivative in rats. Drug development & registration. 2024;13(4):238–250. (In Russ.) DOI: 10.33380/2305-2066-2024-13-4-1876.

4. Liang D., Wu Z., Liu Y., Li C., Li X., Yang B., Xie H., Sun H. HPLC-MS/MS-Mediated Analysis of the Pharmacokinetics, Bioavailability, and Tissue Distribution of Schisandrol B in Rats. International Journal of Analytical Chemistry. 2021;2021:8862291. DOI: 10.1155/2021/8862291.

5. Hu H., Xiao H., Bao H., Li M., Xue C., Li Y. T., Wang G., Chen S., Huang Y., Zheng L., Wang A., Li Y. J., Gong Z. P. Tissue Distribution Comparison of Six Active Ingredients from an Eucommiae Cortex Extract between Normal and Spontaneously Hypertensive Rats. Evidence-Based Complementary and Alternative Medicine. 2020;2020:1–16. DOI: 10.1155/2020/2049059.

6. Körmöczi T., Szabó Í., Farkas E., Penke B., Janáky T., Ilisz I., Berkecz R. Heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry for determination of N,N-dimethyltryptamine in rat plasma and brain; Method development and application Journal of Pharmaceutical and Biomedical Analysis. 2020;191:113615. DOI: 10.1016/j.jpba.2020.113615.

7. Kusuma Kumari G., Krishnamurthy P. T., Ravi Kiran Ammu V. V. V., Vishwanath K., Narenderan S. T., Babu B., Krishnaveni N. Development and validation of a sensitive LC-MS/MS method for pioglitazone: application towards pharmacokinetic and tissue distribution study in rats. RSC Advances. 2021;11:11437–11443. DOI: 10.1039/d1ra01126j.

8. Liu J., Pang Y., Li W., Sun J., He Y., Guo Y., Dong J. Impact of hepatic impairment and renal failure on the pharmacokinetics of linezolid and its metabolites: contribution of hepatic metabolism and renal excretion. Antimicrobial Agents and Chemotherapy. 2025;69(5):e0189224. DOI: 10.1128/aac.01892-24.

9. Ramöller I. K., Abbate M. T. A., Vora L. K., Hutton A. R. J., Peng K., Volpe-Zanutto F., Tekko I. A., Moffatt K., Paredes A. J., McCarthy H. O., Donnelly R. F. HPLC-MS method for simultaneous quantification of the antiretroviral agents rilpivirine and cabotegravir in rat plasma and tissues. Journal of Pharmaceutical and Biomedical Analysis. 2022;213:114698. DOI: 10.1016/j.jpba.2022.114698.

10. Rosebooma I. C., Thijssena B., Rosinga H., Alvesb F., Mondalc D., Teunissend M. B. M., Beijnena J. H., Dorloa T. P. C. Development and validation of an HPLC-MS/MS method for the quantification of the anti-leishmanial drug miltefosine in human skin tissue. Journal of Pharmaceutical and Biomedical Analysis. 2022;207:114402. DOI: 10.1016/j.jpba.2021.114402.

11. Sheng Y.-H., Siemiątkowska A., Kosicka-Noworzyń K., Brunetti L., Kagan L. A validated LC-MS/MS method for simultaneous quantitation of piperacillin, cefazolin, and cefoxitin in rat plasma and twelve tissues. Journal of Pharmaceutical and Biomedical Analysis. 2024;248:116259. DOI: 10.1016/j.jpba.2024.116259.

12. Yin W., Al-Wabli R. I., Attwa M. W., Rahman A. F. M. M., Kadi A. A. Detection and characterization of simvastatin and its metabolites in rat tissues and biological fluids using MALDI high resolution mass spectrometry approach. Scientific Reports. 2022;12(1):4757. DOI: 10.1038/s41598-022-08804-x.