Pharmacokinetics Study of a New Isoxazole Derivative in Rats Using HPLC-MS/MS for Blood Sample Analysis

INTRODUCTION. Systemic exposure studies of a selective carbonic anhydrase II inhibitor, the isoxazole derivative 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (TFISA), require evaluating its pharmacokinetics in whole blood because the compound can accumulate in erythrocytes. Currently, no bioanalytical procedures have been developed to achieve this.

AIM. This study aimed to develop a bioanalytical procedure for the determination of TFISA and its metabolites (N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide) in the blood of laboratory animals and compare the pharmacokinetics of TFISA ophthalmic suspension in rats after a single ocular or intraperitoneal administration.

MATERIALS AND METHODS. The quantitative determination was performed by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) using rat and rabbit blood samples. The chromatographic separation used a Zorbax Eclipse Plus C18 column (150×3.0 mm, 3.5 µm) and a gradient elution system of 0.1% aqueous formic acid and methanol. The multiple reaction monitoring mass spectrometry mode was used for detection. The pharmacokinetics study was conducted in 2 groups of 6 Wistar rats (3 males and 3 females per group). Group 1 received an instillation of 1% TFISA ophthalmic suspension in each eye at a dose of 3.7 mg/kg. Group 2 received an intraperitoneal injection of the same product at the same dose. Blood samples were collected at baseline and at several intervals after administration.

RESULTS. The authors developed a bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals (rabbits and rats). This HPLC-MS/MS procedure was fully validated in accordance with the requirements of the EAEU legislation and the ICH M10 guideline. The analytical ranges in blood were 20–20000 for TFISA, 2–2000 for the N-hydroxy metabolite, and 0.1–100.0 ng/mL for the N-acetyl metabolite. The maximum blood levels after ocular instillation (mean±SD) were 8173±1491 for TFISA, 694±271 for the N-hydroxy metabolite, and 6.33±1.51 ng/mL for the N-acetyl metabolite. The half-lives for this route of administration were 58±10 (TFISA), 70±24 (N-hydroxy metabolite), and 14±3 h (N-acetyl metabolite). The bioavailability of TFISA was 90.18%. CONCLUSIONS. The developed and validated bioanalytical procedure for the determination of TFISA and its metabolites in the blood of laboratory animals has been successfully applied to samples of rat whole blood. According to the study of ophthalmic suspension pharmacokinetics, TFISA and its metabolites have long halflives and high bioavailability.

1. Popovic MM, Schlenker MB, Thiruchelvam D, Redelmeier DA. Serious adverse events of oral and topical carbonic anhydrase inhibitors. JAMA Ophthalmol. 2022;140(3):235–42. https://doi.org/10.1001/jamaophthalmol.2021.5977

2. Kurysheva NI. Carbonic anhydrase inhibitors in the treatment of glaucoma. Review. Part II. Ophthalmology in Russia. 2020;17(4):676–82 (In Russ.). https://doi.org/10.18008/1816-5095-2020-4-676-682

3. Khokhlov AL, Shetnev AA, Korsakov MK, Fedorov VN, Tyushina AN, Volkhin NN, Vdovichenko VP. Pharmacological properties of sulfonamide derivatives — new inhibitors of carbonic anhydrase. Bulletin of Experimental Biology and Medicine. 2023;175(2):166–70 (In Russ.). https://doi.org/10.47056/0365-9615-2023-175-2-166-170

4. Madrewar AB, Deshpande AS, Bhattacharya S. Mini-review on bioanalytical estimation of brinzolamide. Curr Pharm Anal. 2022;18(3):265–72. https://doi.org/10.2174/1573412917666210812103414

5. Lo Faro AF, Tini A, Gottardi M, Pirani F, Sirignano A, Giorgetti R, et al. Development and validation of a fast ultra-high-performance liquid chromatography tandem mass spectrometry method for determining carbonic anhydrase inhibitors and their metabolites in urine and hair. Drug Test Anal. 2021;13(8):1552–60. https://doi.org/10.1002/dta.3055

6. Khokhlov AL, Yaichkov II, Korsakov MK, Shetnev AA, Volkhin NN, Petukhov SS. 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. Res Results Pharmacol. 2023;9(4):53–64. https://doi.org/10.18413/rrpharmacology.9.10056

7. Yuan Y-S, Liao J-M, Kang C-M, Li B-L, Lei X-R, Yu K-W, et al. A simple and accurate LC–MS/MS method for monitoring cyclosporin A that is suitable for high throughput analysis. Exp Ther Med. 2023;26(1):342. https://doi.org/10.3892/etm.2023.12041

8. Yu K-W, Li B-L, Yuan Y-S, Liao J-M, Li W-K, Dong H, Ke P-F, et al. A modified LC-MS/MS method for the detection of whole blood tacrolimus and its clinical value in Chinese kidney transplant patients. Heliyon. 2022;8(8):e10214. https://doi.org/10.1016/j.heliyon.2022.e10214

9. Verheijen RB, Thijssen B, Atrafi F, Schellens JHM, Rosing H, de Vries N, et al. Validation and clinical application of an LC–MS/MS method for the quantification of everolimus using volumetric absorptive microsampling. J Chromatogr B Analyt Technol Biomed Life Sci. 2019;1104:234–9. https://doi.org/10.1016/j.jchromb.2018.11.030

10. Rajput AP, Edlabadkar AP. A coherent analytical review of indapamide. J Pharm Tech Res Manag. 2022;10(1):21–35. https://doi.org/10.15415/jptrm.2022.101004

11. Gopinath R, Narenderan ST, Kumar M. A sensitive liquid chromatography–tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: application to pharmacokinetic study. Biomed Chromatogr. 2022;34(6):e4822. https://doi.org/10.1002/bmc.4822

12. Raghuvanshi DS, Verma N. An iodine-mediated new avenue to sulfonylation employing N-hydroxy aryl sulfonamide as a sulfonylating agent. Org Biomol Chem. 2021;19:4760–7. https://doi.org/10.1039/d1ob00036e

13. Aladysheva ZhI, Belyaev VV, Beregovykh VV, Brkich GE, Greibo SV, Demina NB, et al. Industrial pharmacy: the way to create a product. Moscow: Russian Academy of Sciences; 2019 (In Russ.). EDN: KWJJUK

14. Gajdaj ЕА, Gajdaj DS. Genetic variety of laboratory mice and rats: history of occurrence, methods of obtaining and control. Laboratory Animals for Science. 2019;(4) (In Russ.). https://doi.org/10.29296/2618723X-2019-04-09