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0Research Results in Pharmacology
Research Results in Pharmacology 9(4): 53-64 UDC: 615.015; 543.51+543.54 DOI 10.18413/rrpharmacology.9.10056
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
Alexander L. Khokhlov1, Ilya I. Yaichkov1-2, Mikhail K. Korsakov2, Anton A. Shetnev2, Nikita N. Volkhin2, Sergey S. Petukhov1-2
1 Yaroslavl State Medical University, 5 Revolutsiormaya St., Yaroslavl 150000 Russia
2 Yaroslavl State Pedagogical University named after K.D. Ushinsky, 11/2 Technoparkovaya St., Yaroslavl 150030Russia Corresponding author: Ilya I. Yaichkov (i.yaichkovxbyspu.org)
Academic editor: Tatyana Avtina ♦ Received 15 June 2023 ♦ Accepted 01 October 2023 ♦ Published 22 November 2023
Citation: Khokhlov AL, Yaichkov II, Korsakov MK, Shetnev AA, Volkhin NN, Petukhov SS (2023) 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. Research Results in Pharmacology 9(4): 53-64. https://doi.org/10.18413/ rrpharmacology. 9.10056
Introduction: Development of new bioanalytical methods is required for studying the systemic exposure of new selective inhibitor of carbonic anhydrase II, 4-(2-methyl-l,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 (im). Pharmacokinetics was studied on 6 Wistar rats weighing 287.50±18.64 g (Mean±SD). Each animal was instilled with 40 (iL 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-metliyl-l,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.
Abstract
Copyright Khokhlov AL. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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-l,3-oxazole-5-yl)-benzenesulfonamide and its main metabolite in whole blood is significantly higher than in plasma.
Graphical Abstract
4-(2-mcthyl-l,3-oxazolc-5-yl)-benzenesulfonamide (The Drug - OXSA)
Ar-hydroxy-4-(2-methyl-l,3-oxazol-5-yl)-benzenesu^ (Metabolite-OXSA-Ml)
Selection of HPLC-MS/MS-analysis and sample preparation conditions
!
KiEDTA-Plasma j +10% ascorbic acid (1:5 v/v) KSDTA-Blood } +10%Na2S2O3 (1:1 v/v)
OXSA-M1 Stabilator selection
Validation of both methods
Instillation of 2% ophthalmic suspension in
Storage (no higher than -70°C)
OXSA
OXSA-M1
(n=6)
each eye Blood and Plasma sample collection
Sample preparation and HPLC-MS/MS analysis
Administration of the drug
Pharmacokinetic results
Keywords
HPLC-MS/MS, stabilization, pharmacokinetics, selective carbonic anhydrase II inhibitor
Introduction
Glaucoma is one of the main causes of irreversible blindness. Increased intraocular pressure (IOP) is the important symptom and the initial element in the pathogenesis of the disease. One of the ways of decreasing IOP in glaucoma is reduction of the secretion of intraocular fluid by inhibiting carbonic anhydrase II of a ciliary body of the eye (Strakhov et al. 2023). The new selective carbonic anhydrase II inhibitor (iCAII) 4-(2-methyl-l,3-oxazole-5-yl)-benzenesulfonamide (OXSA) (Fig. 1A) demonstrates pharmacological activity to this isoform in picomolar concentrations. The drug was developed in the form of a ophthalmic suspension in concentration of 2% (Ferraroni et al. 2017). OXSA undergoes biotransformation with formation the single metabolite of N-hydroxy-4-(2-methyl-l,3-oxazole-5-yl)-benzenesulfonamide (OXSA-M1) (Fig. IB).
The pharmacokinetic study is a mandatory part of the preclinical investigation of any drug including ophthalmic
drug forms. The initial stage of the study is researching the systemic exposure of the drug and its metabolites in plasma or serum with a single administration (Mironov 2012). The obtained results may be used for determination of the time intervals of excreta collection in the elimination study, correction of time points of organ and tissue collection in the distribution study, as well as calculation of the analytical range of bioanalytical methods for quantification of the analytes in these biological objects. Previously developed drugs of the group iCAII such as dorzolamide (Lo Faro et al. 2021; Kintz et al. 2022) and brinzolamide (Foivas et al. 2016; Madrewar et al. 2022) are able to accumulate in red blood cells. Therefore, additional calculation of pharmacokinetic parameters of OXSA and OXSA-M1 in blood is necessary for full evaluation of systemic exposure of the substances. Bioanalytical quantification methods of the drug and its metabolite in biological fluids have not been developed. The most rapid and selective method for bioassay is HPLC-MS/MS (Khokhlov et al. 2018).
Figure 1. Structures of 4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide (A), its metabolite #-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide (B) and sulfamethazine (C)
Materials and Methods
Design of bioanalytical part
Studied compounds
Substances 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide produced by M.V. Dorogov Pharmaceutical Technology Transfer Center of Yaroslavl State Pedagogical University (YSPU) named after K.D. Ushinsky were used as standards.
Reagents
Sulfamethazine (Sulf) (CAS 57-68-1) was applied as an internal standard (Sigma-Aldrich (S6256-25G)) (Fig. 1C). Methanol for LC-MS (CAS 67-56-1; Merck LLC, LiChrosolv hypergrade for LC-MS 1060352500) and formic acid for LC-MS (Thermo Fisher Scientific LLC, Optima LC-MS-Grade A117) were used for mobile phase preparation. Ascorbic acid (CAS 50-81-7, chemically pure grade), sodium sulfite (CAS 7757-83-7, pure for analysis grade), sodium thiosulfate pentahydrate (CAS 10102-17-7, pure for analysis grade), sodium metabisulfite (CAS 7681-57-4, pure for analysis grade), ammonium acetate (CAS 631-61-8, HPLC-Grade) were investigated as stabilizers for OXSA-M1.
Analytical equipment
The development and validation of the methods, as well as the analysis of the rat samples were carried out on a HPLC-MS/MS system, including a liquid chromatograph "Agilent 1260 Infinity" (Germany) (pump G1312B, autosampler G1329B with thermostat G1330B, column thermostat G1316A) and a tandem mass spectrometric detector AB Sciex QTRAP5500 (Singapore) (device control -software "Analyst 1.6.2" (USA), chromatogram processing - software MultiQuant 3.0.5"(USA)).
Validation parameters
Validation of the methods was carried out according to the requirements of Russian (On Approval of the Rules for Conducting Bioequivalence Studies on Medicines in the Eurasian Economic Union. Decision of the Council of the Eurasian Economic Commission № 85, 2016; Mironov A.N. (ed), 2014) and international (ICH Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis 2022) guidelines. The concentration levels of calibration (K1-K8) and control samples (LLOQ, LQC, MQC, HQC), as well as samples for evaluating the effect of dilution are presented in Table 1.
Table 1. The concentration of calibration samples and QC samples
Concentration, ng/ml
Designation Plasma Blood
OXSA OXSA-M1 OXSA OXSA-M1
K1 (LLOQ) 10 1 50 5
K2 50 5 250 25
K3 100 10 500 50
K4 250 25 1000 100
K5 500 50 2500 250
K6 1000 100 5000 500
K7 1500 150 7500 750
K8 2000 200 10000 1000
LQC 30 3 150 15
MQC 750 75 3500 350
HQC 1600 160 8000 800
Dil 3200 320 16000 1600
Note: OXSA - 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide; OXSA-M1 - N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide.
Design of pharmacokinetic part
Animals
The pharmacokinetics of an ophthalmic suspension of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide in a concentration of 2% was studied on 6 Wistar rats weighing 287.50±18.64 g (Mean±SD) obtained from SMK Stezar LLC (Russian Federation). The study group included 3 males and 3 females. The animals were previously catheterized into the right jugular vein. The study was approved by the Ethics Committee of YSPU named after K.D. Ushinsky (Minutes №1 of 10 September 2023).
Dosing of the drug and sample collection
The administration was carried out by instillation of 40 ^L of ophthalmic suspension of the drug in a concentration of 2% into each eye. Blood samples collection was performed before the administration of OXSA (blank sample) 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, 72 h after administration in an amount of 0,2 mL. K3EDTA was used as an anticoagulant. Each sample was divided into 2 parts: 30 ^l of sodium thiosulfate solution 10% was added to 30 ^l of whole blood and frozen to a temperature no higher than -70°C. The remaining part was centrifuged for 10 min at 2500 rpm. The plasma was separated,
stabilized by an ascorbic acid solution 10% and frozen. Statistical analysis
Non-compartment approach was used for the evaluation of pharmacokinetic parameters of OXSA and OXSA-M1. The following pharmacokinetic constants were calculated using R software v. 3.3.2 with package Bear v. 2.7.7: maximum drug concentration in plasma and blood (Cmax), time-to-peak concentration in plasma and blood (Tmax), area under the pharmacokinetic «concentration - time» curve from zero to the last blood sampling procedure (AUCo-t), area under the pharmacokinetic curve from time zero to infinity (AUCo-»), terminal elimination rate constant (Kel), half-life elimination of the drug (T1/2), and average drug retention time in blood and plasma (mean resident time (MRT)). Descriptive statistics (mean, standard deviation (SD), coefficient of variation (CV), standard error of the mean (SEM)) were calculated using Statsoft Statistica 10.0.1011 software. Pharmacokinetic curves were plotted using Microsoft Excel 2016.
Results and Discussion
The parameters of chromatography and mass spectrometric detection were selected at the initial stage of development of the bioanalytical methods. Chromatographic separation was carried out on a Poroshell 12EC-C18 column (50*3.0 mm, 2.7 ^m) with a Zorbax Eclipse Plus C18 pre-column (12.5*2.1 mm, 5.0 ^m) at a thermostat temperature of 40°C. The gradient elution parameters are presented in Table 2. Mass spectrometric detection was performed in MRM mode with positive polarity (Table 3).
Table 2. The parameters of gradient elution of OXSA and its metabolite Mobile phase: 0,1% formic acid aqueous solution - A, methanol - B
Time, min Flow rate, pL/min A, % B, %
0.00 500 75 25
0.50 500 75 25
2.00 500 20 80
4.00 500 20 80
4.10 500 75 25
7.00 500 75 25
Protein precipitation was used for sample preparation: 125 ^L of sulfamethazine solution in methanol (IS solution) was added to 25 ^L of plasma, and 500 ^L of IS solution was added to 25 ^L of blood. Mixtures were vortexed and centrifuged for 5 min at 10000 rpm. The supernatant was injected in HPLC-MS/MS-system. It was found during the method development that N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide in samples of biological objects underwent degradation with a formation of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonic acid (Fig. 2). Thus, OXSA-M1 is practically absent in plasma samples after 24 hours of storage at room temperature and 3 freeze/thaw cycles (Fig. 3). Its concentration in blood during storage also decreases below the minimum permissible 85% of the nominal value (Fig.4). Besides, OXSA-M1 is more stable in whole blood than in thawed hemolyzed blood.
Figure 2. Reaction of oxidative degradation of N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-enzene-1-sulfonamide in plasma and blood.
A stabilizer solution was selected by studying short-term stability for 24 hours at room temperature (STS), stability after 3 freeze/thaw cycles (FTS), and stability of the analyte in prepared samples in an autosampler (ASS) to prevent the decomposition of N-hydroxymetabolite of OXSA in plasma (Khokhlov et al. 2018). Initially, an aqueous solution of ammonium acetate 250 mM with pH=3.8 was added to the plasma sample with HQC concentration level (according to levels in Table 1) in a volume ratio of 1:5 and 1:2 (stabilizer: plasma). Acidification of the sample made it possible to stabilize only the prepared samples in the autosampler and also to slow down the degradation of OXSA-M1 in STS and FTS tests (Fig. 3). Preliminary tests of STS and FTS using whole blood and thawed hemolyzed blood showed that the studied metabolite was less exposed to degradation compared to plasma (Fig. 4). This may indicate that the formation of 4-(2-methyl - 1,3-oxazol-5-yl)-benzenesulfonic acid occurs due to the oxidation of the N-hydroxysulfonamide group. Therefore the effect of antioxidant solutions on the decomposition process of OXSA-M1 was subsequently studied.
Table 3. The parameters of mass spectrometry detection of OXSA and its metabolite OXSA-M1
№ Analyte ESI SRM-transition DP EP CE CXP
Voltage, V Q1 Q3
1 OXSA 5500 239 159 150 10 40 10
2 OXSA (Control) 5500 239 117 150 10 40 10
3 OXSA-M1 5500 255 159 120 10 40 10
4 OXSA-M1 (Control) 5500 255 117 120 10 40 10
5 Sulf (IS) 5500 279 124 120 10 60 10
Nos 1,3 were used for quantification, Nos 2,4 were used for proof of correctness of identification.
Note: OXSA - 4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; OXSA-M1 - N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; Sulf - sulfamethazine; ESI - electrospray ionization; Q1 - m/z of parent ion; Q3- m/z of product ion; DP - declustering potential; EP-entrance potential; CE - collision energy; CXP - collision cell exit potential.
Figure 3. The results of preliminary stability investigation of N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide in plasma. Note: STS short-term stability; FTS - stability after 3 freeze/thaw cycles; ASS - autosampler stability.
Solutions of ascorbic acid, sodium sulfite, sodium thiosulfate, sodium metabisulfite in concentrations of 5 and 10% were added to plasma at the rate of 20 pL of solution per 100 pL of plasma (1:5, v/v) for the investigation. There were 2 replicates of each kind of stabilized samples. The results of preliminary FTS, STS, ASS tests were within the acceptable range of 85-115% of the nominal value only after using the solution of ascorbic acid at a concentration of 10% (Fig. 3). Therefore, this stabilizer was chosen for subsequent studies. The chromatographic peak OXSA-M1 was absent in samples with the addition of sodium sulfite solution. Therefore these results are not presented in Figure 3.
The antioxidant solutions listed above and 250mM ammonium acetate solution were also added to hemolyzed thawed blood to stabilize OXSA-M1 at the rate of 50 pL of solution per 100 pL of blood (1:2, v/v).
STS test was performed using an ice bath. Blood samples, which were stabilized by solutions of ascorbic acid 10%, sodium sulfite and sodium metabisulfite, ammonium acetate, thickened during storage. It did not allow the sample preparation. Therefore, these results are not presented in Figure 4. FTS and STS test results were close to the acceptable concentration range after usage of 10% sodium thiosulfate solution. Subsequent tests were carried out with the addition of Na2S2O3 solutions to blood at concentrations of 10% and 20% in a volumetric ratio of 1:1. Also 25 pL of a 5% formic acid solution was added to the prepared methanol deproteinizates after mixing to prevent the formation of sulfonic acid. The obtained results met the established requirements after applying these stabilizers, as well as acidification of the prepared samples (Fig. 4). A 10% Na2S2O3 solution in the ratio of 1:1 (v/v) was selected for subsequent tests,
because the signal-to-noise ratio of the chromatographic peaks of OXSA and OXSA-M1 was approximately 2 times higher than after usage of a 20% Na2S2O3 solution.
Thus, the preparation of blood samples was carried out as follows: 500 ^L of IS solution was added to 25 ^L of stabilized blood. The mixture was vortexed and stabilized by 25 ^L of a 5% formic acid aqueous solution, re-vortexed and centrifuged for 5 min at 10000 rpm. The supernatant was separated and analyzed. The method of plasma sample preparation of ascorbic acid stabilized plasma samples did not differ from the initial method.
The full validation of the methods was carried out
after choosing the optimal conditions of sample storage. The analytical range of quantification of OXSA in blood was 50-10000 ng/mL, and in plasma - 10-2000 ng/mL. Concentrations of OXSA-M1 were measured in the range of 5-1000 ng/mL in blood and 1-200 ng/mL in plasma. The dependence of the "analyte/internal standard" peak area ratio from the concentration of the studied compounds in blood and plasma was linear. There was no indicated chromatographic peaks in regions of the analytes and internal standard retention time on main and control MRM-traces of chromatograms of blank samples of the studied biological fluids (Figs 5-6).
Figure 4. The results of preliminary stability investigation of N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide in blood. Note: STS -short-term stability; FTS - stability after 3 freeze/thaw cycles; ASS - autosampler stability.
Figure 6. Examples of chromatograms of blank blood sample (A), LLOQ blood sample (B)
The average value of the relative error in the study of within-run and between-run accuracy of the developed methods, as well as reinjection reproducibility was in the range of 80.0-120.0% for the LLOQ concentration level, and in the range of 85.0-115.0% for the LQC, MQC, HQC concentration levels (Table 4). The coefficient of variation of the concentrations measured in these tests did not exceed 20% at the LLOQ concentration level and 15% at the LQC, MQC, HQC concentration levels. Twofold dilution of blood and plasma samples with Dil concentration level (according to levels in Table 1) by a blank matrix did not affect the accuracy of the determination of analytes in these objects. The matrix effect was studied in two ways on samples with
LQC and HQC levels (according to levels in Table 1) prepared using plasma and blood obtained from 6 different rats: by evaluation of accuracy and precision of the measured concentrations (Table 4, Batch 1) (ICH Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis, 2022), as well as by calculation the CV of the normalized matrix factor (NMF) (On Approval of the Rules for Conducting Bioequivalence Studies on Medicines in the Eurasian Economic Union, Decision of the Council of the Eurasian Economic Commission № 85. 2016; Mironov 2014). The results of both tests of the developed methods met the established requirements. There was no carry-over of analytes from the previous sample in both matrices.
Table 4. The results of validation of developed methods
Plasma
Blood
Parameter
OXSA
OXSA-M1
OXSA
OXSA-M1
Selectivity
Interference in the area of analyte retention times in blank samples did not exceed 20% of the LLOQ level and in the area of internal standard retention times did not exceed 5% of the peak area*
LLOQ 10 ng/mL 1 ng/mL 50 ng/mL 5 ng/mL
Calibration range (linear dependence) 10-2000 ng/mL 1-200 ng/mL 50-10000 ng/mL 5-1000 ng/mL
Accuracy and precision Acc., % CV, % Acc., % CV, % Acc., % CV, % Acc., % CV, %
LLOQ 85.82 6.54 92.52 8.21 96.90 13.79 115.67 6.78
Batch 1* (n=6**) LQC MQC 90.13 94.64 8.85 5.15 93.56 91.00 11.76 7.14 105.97 99.87 6.07 4.23 103.67 90.45 7.23 6.94
HQC 90.57 7.08 90.0 8.44 89.94 4.32 92.37 5.38
LLOQ 95.65 8.96 110.21 9.96 98.90 11.95 97.33 16.11
Batch 2 (n=6**) LQC MQC 98.36 98.65 6.61 5.01 109.71 98.87 6.64 5.40 101.76 96.23 7.10 7.52 109.44 97.76 12.08 5.55
HQC 93.54 6.18 95.45 4.12 94.62 7.13 87.47 4.70
LLOQ 106.56 7.62 104.78 13.13 103.73 13.28 114.33 7.12
Batch 3 (n=6**) LQC MQC 98.88 92.31 6.74 4.60 96.58 91.67 7.32 5.24 87.77 101.72 7.60 2.65 104.78 102.26 3.72 4.88
HQC 95.74 1.82 95.09 2.08 107.24 14.07 100.52 4.30
LLOQ 95.06 10.39 102.92 12.74 99.84 12.59 109.11 12.42
Inter-batch accuracy and precision (n=18**) LQC MQC 95.94 94.92 8.66 5.32 100.37 93.84 10.80 6.83 98.50 99.27 10.39 5.36 105.96 96.82 8.39 7.49
HQC 93.25 5.92 93.51 5.78 97.27 12.22 93.45 7.44
LLOQ 87.46 14.58 96.74 11.89 100.23 9.97 102.33 9.20
Reinjection LQC 92.22 9.44 95.45 11.04 102.69 5.28 100.44 9.53
reproducibility MQC 98.61 6.76 99.83 5.09 99.68 7.91 93.86 4.19
HQC 93.51 6.87 96.00 8.21 105.82 7.60 86.39 4.09
Dilution integrity (n=6) 2-fold 98.25 9.18 98.68 8.22 107.19 3.17 105.60 2.69
Matrix effect (CV NMF, %)
LQC HQC
7.37 7.49
4.90 8.28
5.84 3.19
4.54 4.05
* The selectivity evaluation was performed at Batch 1; Matrix effect evaluation accordance to ICH M10 was performed at Batch 1; **number of samples at each concentration level;
Note: OXSA - 4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; OXSA-M1 - N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; Acc. - relative accuracy; LLOQ - lower limit of quantification; LQC - low concentration quality control samples; MQC - middle concentration quality control samples; HQC - high concentration quality control samples; CV NMF - coefficient of variation of the normalized matrix factor.
The performed validation tests of short-term stability, stability after 3 freeze/thaw cycles, stability in prepared samples in the autosampler, long-term stability in the freezer at a temperature no higher than -70°С (LTS) meet the acceptance criteria (Table 5). Thus, the selected antioxidant solutions prevent the decomposition of OXSA-M1 during
the storage of plasma and blood samples. Blood and plasma samples obtained during the pharmacokinetic study were analyzed at the next stage using the developed methods. The obtained values of the pharmacokinetic parameters of the studied substances are presented in Table 6, and the pharmacokinetic profiles are shown in Figure 7.
Table 5. The results of evaluation of stability of OXSA and OXSA-M1 in plasma and blood
Plasma
Parameter
OXSA
OXSA-M1
* number of samples at each concentration level
Blood
OXSA
OXSA-M1
STS (24 h, room temperature- for plasma; ice bath - for blood) (n=6*) LQC HQC 95.71 99.01 100.04 97.60 106.44 98.74 92.56 87.93
% from initial FTS (n=6*) (3 cycles) LQC HQC 96.18 94.76 100.16 93.02 104.94 103.75 93.56 90.68
concentration ASS (48 h at +4°C) (n=6*) LQC HQC 95.71 99.01 87.25 100.36 107.28 98.68 100.22 89.74
LTS (28 days at temp. no higher than -70° C) (n=6*) LQC HQC 98.70 97.71 101.56 95.18 107.84 103.22 93.89 88.64
Note: OXSA - 4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; OXSA-M1 - N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzene-1-sulfonamide; LQC - low concentration quality control samples; HQC - high concentration quality control samples; STS - short-term stability; FTS -stability after 3 freeze/thaw cycles; ASS - autosampler stability; LTS - long-term stability.
Table 6. The pharmacokinetic parameters of 4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide and its N-hydroxymetabolite in plasma and blood
Parameter Cmax, ng/ml Tmax, h AUC0-t, ng*h/mL AUC0-», ng*h/mL Т1/2, h Kel, h-1 MRT, h
Mean 264.32 1.92 982.06 1113.38 4.25 0.1760 4.09
OXSA SD 68.47 0.92 281.55 287.32 1.35 0.0502 0.41
(n=6) CV 25.90 47.87 28.67 25.81 31.84 28.49 10.07
SEM 27.95 0.37 114.94 117.30 0.55 0.0205 0.17
то Plasma
Mean 10.43 2.17 45.07 54.26 3.79 0.1942 4.17
OXSA-M1 SD 1.79 1.13 8.95 10.79 1.12 0.0468 0.54
(n=6) CV 17.16 41.83 19.86 19.89 29.52 24.11 12.96
SEM 0.73 0.34 3.65 4.41 0.46 0.0191 0.22
Mean 8705.23 1.17 57243.17 62829.17 26.02 0.0342 13.11
OXSA SD 1301.84 0.52 15124.99 17844.11 10.85 0.0238 2.45
(n=6) CV 14.95 44.26 26.42 28.40 41.68 69.67 18.71
SEM 531.47 0.21 6174.75 7284.83 4.43 0.0097 1.00
Blood
Mean 230.00 1.33 1439.43 1577.36 16.66 0.0787 10.18
OXSA-M1 SD 69.54 0.41 396.97 444.47 13.07 0.0763 3.88
(n=6) CV 30.23 30.62 27.58 28.18 78.47 96.85 38.07
SEM 28.39 0.17 162.06 181.45 5.34 0.0311 1.58
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D
Figure 7. Averaged Pharmacokinetic profiles of 4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide in plasma (A) and in blood (B) and Averaged Pharmacokinetic profiles of N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide in plasma (C) and in blood (D) (error intervals: ±SD)
OXSA and its metabolite have monoexponential pharmacokinetic profiles in plasma. The concentration of OXSA increases to a maximum value of 264.32±68.47 ng/ mL (Mean±SD) 1.92±0.92 h (Mean±SD) after administration. Cmax of OXSA-M1 is 10.43±1.79 ng/mL (Mean±SD) which reaches 2.17±1.13 h (Mean±SD) after instillation. It is significantly lower than Cmax of OXSA. The concentration of analytes in plasma begins to decrease rapidly after 4-hour point (Fig. 7 A,C) and its analytical signal on chromatograms becomes below the lower limit of quantification of the method after 12-hour point (LLOQ -Table 1). The plasma half-life of OXSA is 4.25±1.35 h (Mean±SD) and of OXSA-M1 - 3.79±1.12 h (Mean±SD).
The content of these substances in blood is significantly higher than in plasma. Thus, Cmax of OXSA in blood is 8705.23±1301.84 ng/mL (Mean±SD), and Cmax of its metabolite is 230.00±69.54 ng/mL (Mean±SD). The time-to-peak concentration of OXSA in blood comes in 1.17±0.52 hours after administration the drug and in 1.33 ± 0.41 hours for OXSA-M1. Amount of analytes in blood also rapidly decreases after 4-hour point (Fig. 7 B,D). However, the half-life of OXSA and OXSA-M1 in blood is longer than in plasma - 26.02±10.85 hours (Mean±SD) and 16.66±13.07 hours (Mean±SD), respectively. It is due to the deposition of these compounds in red blood cells.
The systemic exposure of the single metabolite OXSA-M1 in both biological fluids is significantly lower compared to OXSA. Probably, most part of active substance is eliminated unchanged. There was no additional increase in a concentration of analytes at the late points of 12-72 h of the pharmacokinetic curve. It indicates the absence of enterohepatic recirculation of these substances. Therefore, there is no necessity to study the bile excretion of OXSA and OXSA-M1.
Thus, the selected analytical ranges of methods are
sufficient for subsequent tests with multiple ocular administration of the drug, as well as checking the hypothesis of linearity of pharmacokinetics. Twofold dilution of blood samples may be required with an increase in the volume of the installed drops. It will increase the upper limit of the quantitative determination of OXSA to 20000 ng/mL.
The high value of the upper limit of the quantification (ULOQ) of methods for OXSA of 10000 ng/mL should be chosen in the subsequent study of the distribution of the drug and its metabolite in tissues and urinary excretion, taking into account their high concentrations in blood. The ULOQ level can be reduced to 500 ng/mL, and the lower limit of quantitative determination can be reduced to 0.5 ng/mL for OXSA-M1 in order to detect trace concentrations of this analyte in samples 24, 48 and 72 hours after administration.
Conclusion
The developed methods for the quantitative determination of 4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide and N-hydroxy-4-(2-methyl-1,3-oxazol-5-yl)-benzenesulfonamide in plasma and blood samples have been fully validated in parameters of selectivity, calibration curve and range, within-run and between-run accuracy and precision, dilution integrity, carry-over, reinjection reproducibility, and stability. Stabilization of plasma and blood samples with 10% ascorbic acid solution and 10% sodium thiosulfate solution, respectively, guarantee the trueness of measurement of concentration of OXSA-M1. The methods have been successfully applied in the pharmacokinetic study with a single administration of ophthalmic suspension of OXSA. It was found that the analytes accumulate in red blood cells. Thus, the concentration of the drug in blood reaches high values of
8705.23±1301.84 ng/mL (Mean±SD). The selected sample collection period of up to 72 hours after administration is sufficient for a complete description of the pharmacokinetic properties of the studied substances in plasma and blood.
Conflict of Interests
The authors declare the absence of a conflict of interests.
Funding
The study was carried out at the expense of the grant of the Ministry of Education of the Russian Federation "Development of an innovative drug for the treatment of open-angle glaucoma by selective inhibition of carbonic anhydrase II" № 073-00077-21-02 (register number: 730000®.99.1.EB 10AA00006).
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Author Contributions
■ Alexander L. Khokhlov, Doctor Habil. of Medical Sciences, Professor, Member of The Russian Academy of Sciences, Head of the Department of Pharmacology and Clinical Pharmacology, rector of Yaroslavl State Medical University; e-mail: al460935@yandex.ru; ORCID ID https://orcid.org/0000-0002-0032-0341. The author's contribution: formulation and development of the aim and objectives; development of design of pharmacokinetic study; analysis and interpretation of the obtained data; critical review of the draft copy and provision of valuable comments.
Ilya I. Yaichkov, Candidate of Pharmaceutical Sciences, research fellow of the Department of Analytical Development and Quality Control of M.V. Dorogov Pharmaceutical Technology Transfer Center of Yaroslavl State Pedagogical University named after K.D. Ushinsky; research fellow of the Institute of Pharmacy of Yaroslavl State Medical University; e-mail: i.yaichkov@yspu.org; ORCID ID https://orcid.org/ 0000-0002-0066-7388. The author's contribution: concept development; development of design of pharmacokinetic study; development and validation of bioanalytical methods; analysis of blood and plasma samples; analysis and interpretation of the obtained data; writing the bioanalytical part and editing the manuscript.
■ Mikhail K. Korsakov, Doctor Habil.of Chemical Sciences, Professor of the Department of Chemistry, Theory and Methods of Teaching Chemistry, Head of The Center of Transfer of Pharmaceutical Technology named after M.V. Dorogov of Yaroslavl State Pedagogical University named after K.D. Ushinsky; e-mail: m.korsakov@yspu.org; ORCID ID https://orcid.org/0000-0003-0913-2571. The author's contribution: formulation and development of the aim and objectives; analysis and interpretation of the obtained data; critical review of the draft copy and provision of valuable comments.
■ Anton A. Shetnev, Candidate of Chemical Sciences, Head of the Department of Pharmaceutical Development of M.V. Dorogov Pharmaceutical Technology Transfer Center of Yaroslavl State Pedagogical University named after K.D. Ushinsky; e-mail: a.shetnev@list.ru; ORCID ID https://orcid.org/0000-0002-4389-461X. The author's contribution: formulation and development of the aim and objectives; development of synthesis technology of the drug and its metabolite; writing the synthesis part and interpretation of the obtained data.
■ Nikita N. Volkhin, junior research fellow of the Department of Pharmacological Studies of M.V. Dorogov Pharmaceutical Technology Transfer Center of Yaroslavl State Pedagogical University named after K.D. Ushinsky; e-mail: nnvolkhin@ysmu.ru; ORCID ID https://orcid.org/0000-0002-4275-9037. The author's contribution: development of design of pharmacokinetic study; blood and plasma sample collection;analysis and interpretation of the data obtained.
■ Sergey S. Petukhov, engineer of the Department of Pharmacological Studies of M.V. Dorogov Pharmaceutical Technology Transfer Center of Yaroslavl State Pedagogical University named after K.D. Ushinsky; junior research fellow of the Institute of Pharmacy of Yaroslavl State Medical University; e-mail: sspp465@mail.ru; ORCID ID https://orcid.org/0009-0007-8435-7689. The author's contribution: development of design of pharmacokinetic study; blood and plasma sample collection; analysis and interpretation of the obtained data.
