PHARMACOKINETIC STUDY OF DICLOPHENAC SODIUM IN RAT PLASMA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The aim of this study is to determine pharmacokinetics of diclofenac sodium in the experiment on rats by the method of high-performance liquid chromatography. Identification and quantitative determination of diclofenac sodium in the substance of diclofenac sodium were carried out by the method of high-performance liquid chromatography. For study conduct, a total of 94 male rats with body weight of 250-300 g were used. Experimental animals were subdivided into 15 groups. Diclofenac sodium was diluted in purified water and administered per os at the doses of ED50 and 1⁄2 ED50, equivalent to 8 mg/kg and 4 mg/kg of animal body weight, respectively. Animals were sacrificed in 15 min, 30 min, 60 min, 90 min, 120 min, 240 min, and 360 after drug administration. For extraction of diclofenac sodium from rat plasma samples, we used a method of solid-phase extraction, which had been modified due to microconcentrations of the active substance. Dependence of diclofenac sodium concentration on time and dose was studied by the method of high-performance liquid chromatography. In the result of the study, quantitative content of diclofenac sodium substance and suitability of the doses studied (4 mg/kg and 8 mg/kg) were confirmed. The study conducted revealed no dynamics of time, during which diclofenac sodium was present in systemic blood flow, on the dose. The results, which had been proved by chromatograms of diclofenac sodium and control sample (plasma without diclofenac) for each time interval, were obtained. First concentrations of diclofenac sodium in rat plasma are registered in rat plasma in 15 min after dose administration at the doses of 4 and 8 mg/kg. That is why this time index is recommended for further preclinical and clinical studies.


INTRODUCTION
Today, nonsteroidal anti-inflammatory drugs (NSAIDs) constitute one of the largest and clinically significant groups of drugs. In resent 30 years, its nomenclature has increased greatly and now it includes a great number of drugs that differ as for their peculiarities of action and use [3, 4,6]. Diclofenac sodium (DNa) remains "the gold standard" in treatment of inflammatory diseases of connective tissue and is most widely used in modern medicine and pharmacy. So, studies of its pharmacological activity characteristics depending on dose and time are an important and urgent problem.
The aim of this work is to determine pharmacokinetics of DNa in experiments on rats by the method of high-performance liquid chromatography for further preclinical and clinical studies. Identification and quantitative determination of DNa in the substance of DNa were carried out by most sensitive method of high-performance liquid chromatography [2,7,8,9,10]. This method was used to determine concentrations of the test substance at all stages of the study.

MATERIALS AND METHODS
The content of DNa in the substance (X), in per cent, was calculated according to the following formula: where: S х is the mean peak area of diclofenac sodium calculated from chromatograms of the test solution; S WRS is the mean peak area of diclofenac sodium calculated from chromatograms of the reference solution; m WRS is the weight of diclofenac sodium WRS, g; Р is the content of diclofenac sodium in the WRS, %; m х is the weight of diclofenac sodium substance, g; W is the loss on drying of diclofenac sodium substance, %.
Experimental studies were carried out at experimental biological clinics of the State Enterprise "Sytenko Institute of Spine and Joint Pathology of the National Academy of Medical Sciences of Ukraine". Animals used in the experiment: nonlinear white rats of the population of experimental biological clinics of the State Enterprise "Sytenko Institute of Spine and Joint Pathology of the National Academy of Medical Sciences of Ukraine".
For study conduct, 94 male rats with body weight of 250-300 g were selected; they were kept according to sanitary norms on a standard diet [2]. Work with animals was carried out according to Directive 86/609/ЕЕС on the protection of animals used for experimental and other scientific purposes.
Experimental animals were subdivided into 15 groups. An intact group (group 1) consisted of 10 rats, and groups 2-15 consisted of 6 rats each. Animals of group 1 received purified water in the volume of 0.5 mL per 100 g [2]. DNa was diluted in purified water and administered per os in the same volume at the doses of ED 50 and ½ ED 50 , equivalent to 8 mg/kg and 4 mg/kg of animal body weight, respectively [2,3,6].
Pharmacokinetic study of DNa was carried out according to the tactics under conditions indicated in Tab. 1. Animals were sacrificed in 15 min, 30 min, 60 min, 90 min, 120 min, 240 min, and 360 after drug administration.
Blood was sampled in the quantity of 7-10 ml into labeled test-tubes that were heparinized. Blood samples were centrifuged (3.000 rpm, 15 min), and plasma was obtained. The interval between blood sampling and its processing did not exceed 5 min. Before analysis, plasma samples were kept at -80 °С.
For extraction of DNa from rat plasma samples, a method of solid-phase extraction was used [10]. The method had been modified due to micro-concentrations of the active substance via sample concentration [1].
After determination of DNa concentration depending on the time of its administration, the following pharmacokinetic parameters were calculated by the noncompartmental method of statistical moments with the use of application software program M-ind on IBM PC Pentium III: C max (maximal concentration); T max (time to maximum drug concentration); Т 1/2 (half-life); Cl t (total clearance); AUC 0→t (area under the concentration-time curve within the limit of drug concentration observation); AUC 0→∞ (total area under the concentration-time curve within the limit of 0 to ∞); C max /AUC 0→∞ (coefficient that characterizes drug absorption rate).
Statistic processing of experimental data obtained was carried out with software STATISTICA (StatSoft Inc., the USA). Reliability of the results obtained was assessed at the level of significance not less than 95 % (р ≤ 0.05) [5].

RESULTS AND DISCUSSION Quantitative determination of diclofenac sodium in rat plasma
Comparative characteristic of diclofenac sodium substance Results of quantitative determination of DNa in substance samples are indicated in Tab. 2.
Chromatograms of DNa substance samples obtained under the same conditions are presented in Fig. 1, where the following is indicated: 1.

Assessment of pharmacokinetic parameters of diclofenac sodium
Profiles of averaged pharmacokinetic curves of dynamics of DNa rat plasma concentrations after single oral administra-tion of 4 mg/kg and 8 mg/kg and their comparative characteristic are presented in Fig. 2-4. DNa is registered in plasma already in 15 min after substance administration of both doses studies. At the same time, DNa concentration at this time is 4.88 ± 0.82 mcg/mL at administration of 4 mg/kg, and 17.45 ± 1.38 mcg/mL at administration of 8 mg/kg.      [66] Біохімія та фармакологія УКРАЇНСЬКИЙ БІОФАРМАЦЕВТИЧНИЙ ЖУРНАЛ, № 4 (45) 2016 ISSN 2311-715X After that, DNa plasma content increases rapidly and reaches its maximum within 60 min. At the same time, its concentration 14.72 ± 0.07 mcg/mL at administration of 4 mg/kg, and 47.41 ± 0.44 mcg/mL at administration of 8 mg/kg.
Starting with 90 min, there is noted a decrease in DNa plasma concentration, which is decreased 5.37-folds at administration of 4 mg/kg, and 1.68-folds at administration of 8 mg/kg when compared to initial product concentration. This indicated to the fact that DNa at the dose of ½ ED 50 is excreted from rat blood flow much more rapidly.
Starting with 120 min, there is noted too low level of DNa plasma concentration (be close to the limit of analytical method).
Pharmacokinetic parameters calculated are as follows: at administration of DNa 4 mg/kg, Cmax is 15.16 ± 0.24 mcg/mL, Tmax is 0.8 ± 0.06 h, T 1/2 is 0.36 ± 0.02 h, Cl t is 0.04 ± 0.00 L/h, MRT is 1.63 ± 0.04 h, AUC 0-t is 20.38 ± 0.54 mcg×h/mL, AUC 0→t is 20.93 ± 0.51 mcg×h/mL,  [67] UKRAINIAN     [68] Біохімія та фармакологія УКРАЇНСЬКИЙ БІОФАРМАЦЕВТИЧНИЙ ЖУРНАЛ, № 4 (45) 2016 ISSN 2311-715X CONCLUSIONS Dependence of diclofenac sodium concentration of dose and time has been determined in rat experiments by the method of high-performance liquid chromatography. According to study results, first concentrations of diclofenac sodium in rat plasma are registered in rat plasma in 15 min after dose administration at the doses of 4 and 8 mg/kg. That is why this time index is recommended for further preclinical and clinical studies.