Novel selective type II carbonic anhydrase inhibitor 4-(5-methyl1,3,4-oxadiazole-2-yl)-benzenesulfonamide (ODASA) (fig. 1A) is a promising new molecule for treating open-angle glaucoma. The compound is undergoing preclinical testing. It has been shown to lower intraocular pressure after topical ocular dosing. ODASA reduces the risk of any systemic adverse drug reactions. Its effect can last up to 24 hours [1]. 1% ophthalmic suspension of ODASA has been currently engineered. In rats, relative bioavailability (RB) is over 80% when the preparation is instilled in the eye compared to an intraperitoneal administration. ODASA enters the systemic circulation and is then hydroxylated. The main reaction involves the methyl group on the 1,3,4-oxadiazole ring, leading to the formation of 4-[5-(hydroxymethyl)-1, 4-oxadiazole-2-yl]-benzenesulfonamide (M1) (fig. 1B). The minor reaction affects the sulfonamide group, forming the metabolite N-hydroxy4-(5-methyl-1,3,4-oxadiazol-2-yl)-benzenesulfonamide (M2) (fig. 1C) [2].

According to regulatory requirements, the study of pharmacokinetics and bioavailability should be conducted in two species (one non-rodent) [3]. In this case, rabbits are commonly used. They are the most affordable lab animal species [4–7]. Beagle dogs [8–9], minipigs [10–11], and monkeys [12–14] are less frequently used as a second species. ODASA shows limited solubility in water, which makes its intravenous administration difficult and study of absolute bioavailability impossible. Thus, RB is calculated following intraperitoneal injection.

High-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) is the most common method for analyzing biological samples in pharmacokinetic studies. An express method has been developed for quantitative determination of ODASA and its hydroxylated derivatives in laboratory animal blood plasma. Protein precipitation was used to prepare samples. The minor metabolite N-hydroxy4-(5-methyl-1,3,4-oxadiazol-2-yl)-benzenesulfonamide is chemically unstable in biological objects. Therefore, stabilization with a 10% ascorbic acid solution is necessary after plasma sampling [2].

Thus, the work is aimed at calculation of pharmacokinetic parameters and relative bioavailability of ocular suspension of 4-(5-methyl-1,3,4-oxadiazol-2-yl)-benzenesulfonamide in rabbits by analyzing samples with HPLC-MS/MS.

MATERIALS AND METHODS

The study was conducted using 3–4-month-old Soviet Chinchilla male rabbits obtained from the SMK Stezar nursery. It was a parallel group study investigating RB. The design was used because of deposition of ODASA within erythrocytes and its long half-life [2]. According to the ethical principles, the sample size was minimally allowable for pharmacokinetic studies [3, 15]. A vivarium housed animals in comfortable conditions using individual cages. Access to water and mixed feed was available free-choice, except for 4 hours before administration and 2 hours after administration of the investigational preparation. Blood sampling from the lab animals was quick and relatively painless. Thus, no anesthesia was used. This study was approved by the Independent Ethics Committee of the Federal State Budgetary Educational Institution of Higher Education Yaroslavl State Medical University of the Ministry of Health of the Russian Federation, Protocol No. 2 dated 04/20/2025.

About 40 µl of 1% ocular suspension of ODASA was instilled into each eye (EI) of animals from the first group, including 6 rabbits weighing 2.84 ± 0.05 kg (M± SEM). It corresponded to a dose of 0.28 mg/kg. 6 other animals with a weight of 3.13 ± 0.05 kg (M±SEM) received an equivalent dose of intraperitoneal (IP) injections of the investigational drug. A 0.2 mL blood sample was drawn from a vein using an insulin syringe as in other similar studies [4–6]. Samples were obtained prior to administration of ODASA and at 30 min, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 192 hours, 240 hours, and 288 hours following the administration. K₃EDTA was chosen as an anticoagulant. The plasma obtained after centrifugation was stabilized with a 10% ascorbic acid solution in a 1:2 volume ratio (ascorbic acid solution: plasma). The samples were stored at or below — 70 °C till analysis.

A validated bioanalytical technique was used to quantitatively measure the concentration of ODASA and its metabolites. The solution of methanol containing 4-(3-methyl6-oxo-5,6-dihydropyridazine-1(4H)-yl)-benzenesulfonamide, an internal standard, was added to precipitate the proteins and prepare the samples. To do that, a 100 µl reagent was combined with 20 µl of stabilized plasma. The mixture was stirred and centrifuged for 5 min at 10,000 rpm. The supernatant was transferred to micro-inserts and analyzed. A HPLC-MS/MS system was used, including Agilent 1260 Infinity chromatograph (Germany) and AB Sciex QTRAP5500 mass spectrometer (Singapore). Kinetex Phenyl-Hexyl column (50*4.6 mm, 2.6 microns) and Phenyl SecurityGuard Ultra Catridge pre-column (4.6 mm, 2.6 microns) were used for chromatographic separation. The Multiple Reaction Monitoring (MRM) mode was employed to detect the analytes and the internal standard [2]. The analytical range of plasma concentrations measured was 2–2000 ng/ml for ODASA and M1, and 0.5–500.0 ng/ml for M2.

Non-compartmental analysis was used to determine the pharmacokinetic parameters. Maximum plasma concentration (C_max), time to maximum plasma concentration (T_max), the area under the pharmacokinetic curve capturing drug exposure from time zero to the last measurable concentration (AUC_0-t), the area under the curve from zero to infinity (AUC_0-∞), half-life (Т_1/2), mean residence time (MRT), apparent volume of distribution (Vd/F), and apparent clearance (Cl/F) were calculated for ODASA and its metabolites using R v. 3.3.2 (Bear v. 2.7.7) package software.

The rate of conversion (R(M)) of the active substance into a metabolite was calculated using the formula:

formula

where AUC_0-∞ (M) is AUC_0-∞ of plasma metabolite; AUC_0-∞ (Drug) is AUC_0-∞ of ODASA.

THE RESULTS OF THE STUDY AND THEIR DISCUSSION

ODASA peak plasma concentration was 58.6 ± 7.2 ng / ml (M ± SEM) within 1 h after therapeutic administration (table). After 48 hours, the content of the active substance in this biological fluid was below the lower limit of quantitation (LLOQ) (fig. 2A). Following eyedrop instillation, RB for ODASA was 30.76% as compared to IP administration, which is 2.5-fold lower than that in rats. In both modes of administration, the half-life of ODASA was over 6 times shorter than that in rats [2].

Following eyedrop instillation, T_max of main metabolite M1 occurred later than that of ODASA and M2. It was detected in plasma over the time range from 0 to 120 hours (fig. 2B). Its T_1/2 was about 1.5 times shorter than in rats [2]. The peak concentration of the N-hydroxy derivative was reached from 1 hour to 2 hours after instillation, just like in the case of the active substance (fig. 2C). Following eyedrop instillation, T_1/2 of M2 was about 10 hours. It is approximately 3 times shorter than in rats [2]. The compound concentrations in plasma samples were lower than the LLOQ at 24 hours following instillation in the eye and at 48 hours following intraperitoneal administration.

In experiments with rabbits, ODASA and its metabolites had high apparent volume of distribution (table). They were significantly higher than the actual volume of circulating blood. It means that the investigational agents penetrate well into the organs and tissues of animals.

While using the therapeutic method of administration, the rate of conversion of ODASA to M1 was 2.122 ± 0.292, whereas the rate of conversion of ODASA to M2 was 0.106 ± 0.017 (M ± SEM). This is approximately 10 times higher than the values of R(M) of these hydroxylation products of the active substance calculated in rats. The difference can be explained by a lower preparation dose per unit of body weight and lower plasma concentrations of ODASA. The active centers of microsomal enzymes were less saturated, and ODASA biotransformation occurred much faster. For this reason, clearance values of ODASA in rabbits after EI are over 40 times higher than in rats [2].

Thus, ODASA was well absorbed into the systemic circulation of rabbits following topical eyedrop instillation. High relative bioavailability of the active agent was also detected in the pharmacokinetic study in rats. If the preparation proceeds to phase 1 of clinical studies, testing its pharmacokinetics in healthy volunteers will be obligatory.

CONCLUSIONS

1. ODASA can enter the systemic circulation of rabbits if instilled into the eyes.
2. Relative bioavailability of ODASA after instillation into the eyes was 30.76% compared with intraperitoneal administration.
3. The rate at which ODASA and its metabolites are eliminated differs across species. It is faster in rabbits than in rats.
4. As ODASA is absorbed after topical application in animals, the list of phase I clinical trials should include the study of pharmacokinetics.