Experimental study of the nonsteroidal anti-inflammatory drugs application under using low-intensity infrared laser radiation

Topicality. The combined application of nonsteroidal anti-inflammatory drugs (NSAIDs) and low-intensity infrared laser radiation (LIIRLR) for treatment of osteoarthrosis has remained an actual task. Aim to study the NSAIDs usage under LIIRLR application in experiments in rats. Materials and methods. The time for LIIRLR application (15 min after drug administration) was chosen according to our previous studies on diclofenac sodium (DNa) pharmacokinetics in rats blood plasma. To study the NSAIDs application at the influence of LIIRLR, white beedless male rats (n=15) of 250-300 g were used in the experiments. The animals were divided into 3 groups. Rats of group the 1were received purified water (per os) and exposed to LIIRLR. Rats of group the 2 were received DNa at a dose of ED50 (per os, 8 mg/kg). Rats of group the 3 were influenced of LIIRLR and in 15 min received DNa (per os, 8 mg/kg). For DNa extraction from rat plasma samples, the method of solid-phase extraction was used. Concentration of DNa was determined by method of high-performance liquid chromatography (HPLC). Results and discussion. It was established that at the combined use LIIRLR and DNa the drug concentration in the rats blood serum was 1.7 fold higher in comparison to DNa application alone. Conclusions. It is proved, that the method of combine application of LIIRLRand NSAIDs (in 15 min) was more effective than the use of NSAIDs alone.


INTRODUCTION
Diseases of musculoskeletal system (MSS) and connective tissue are widespread in many countries of the world; they lead to decreased working capacity, worsening of the quality of life, social disadaptation, and, in the consequence, disability. Osteoarthrosis (OA) is leading as for incidence among this category of diseases. At least 20 % of world population suffer from it. That is why its treatment is a medical and social problem that is urgent all over the world [1,2].
There are various drugs and methods of treatment for MSS and connective tissue diseases. Nonsteroidal antiinflammatory drugs (NSAIDs) constitute one of the central positions in therapy of these diseases and are most widely used in modern medicine, clinical practice, and pharmacy [2,4].
Diclofenac sodium (DNa) remains the reference drug of NSAIDs group [1,3,4]. However, in spite of a great number of treatment schemes used, pharmacological correction of MSS and connective tissue diseases is an important and urgent task, which calls for new methods of treatment [1,2].
It has been found that physical factors can potentiate drugs' action. Due to this fact, use of low-intensity infrared laser radiation (LIIRLR) attracts the attention of scientists. This method of treatment has analgesic, anti-inflammatory, pain alleviating, regenerating, desensitization, immune-corrective, hypocholesterolemic, bactericidal, and bacteriostatic effects; it also improves local blood circulation [1,4,5].
Thus, the possibility of combined use of NSAIDs and LIIRLR for treatment of MSS and connective tissue diseases is an urgent and well-grounded task; it will allow to improve the results of treatment of OA patients.
The aim of this work is to study NSAIDs use along with LIIRLR in the experiment in rats.
Identification and quantitative determination of DNa in the substance of DNa were carried out by most sen-sitive method of high-performance liquid chromatography (HPLC) [6,7]. This method was used to determine concentrations of the test substance at all stages of the study.
Animals The time of LIIRLR use and the time of animal sacrifice have been determined in our previous studies on pharmacokinetics of DNa in rat plasma by method of high-performance liquid chromatography (HPLC) [8].
LIIRLR was carried out in 15 min after drug administration with laser therapeutic unit "Mustang" set as follows: wavelength is 0.89 µm, pulse power is 7-8 W, pulse frequency is 3.000 Hz, duration of the session is 3 min 42 sec, radiation dose is 0.3 J (calculated by specialists of National Research Center "Metrology Institute", Kharkiv, Ukraine). The apparatus was used by contact along the posterior surface of the rat knee joint with preliminary removed hair [9].
To study concomitant use of NSAIDs and LIIRLR, we selected 15 male rats with body weight of 250-300 g, which were kept according to according to sanitary norms on a standard diet [10]. Experimental animals were subdivided into 3 groups, 5 rats per group. DNa was diluted in purified water and administered in the same volume at a dose of ED 50 (per os, 8 mg/kg of animal body weight). Animals of group 1 received purified water (per os, 0.5 mL per 100 g of animal body weight) and were exposed to LIIRLR. Animals of group 2 received per os DNa at a dose of ED 50 [10]. Animals of group 3 were subjected to the influence of LIIRLR and in 15 min received DNa (per os, 8 mg/kg). Animals were sacrificed in 60 min after drug introduction [8].
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.
[32] Бiохiмiя та фармакологiя Український біофармацевтичний журнал, № 1 (48) 2017 For extraction of DNa from rat plasma samples, a method of solid-phase extraction was used [7]. Cartridges Supel™-Select HLB SPE (Supelco) 30 mg/1 ml were used for work. The method had been modified due to microconcentrations of the active substance during previous stages of the complex study [11].
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) [12].

RESULTS AND DISCUSSION
Confirmation and determination of quantitative determination of DNa (manufactured by BCPP) in rat plasma were carried out in comparison to DNa WRS manufactured by "Amoli Organics Ltd". Chromatograms of DNa substance samples obtained under the same conditions are presented in Fig. 1.
Retention time of DNa in the chromatogram of the test sample of the solution (5.673 min) corresponds to that of DNa in the chromatogram of DNa WRS (5.673 min), so the sample provided is DNa substance. In the result of the studies conducted, quantitative content of DNa and suitability of the dose studied. Actual dose of DNa during study conduct is 8 mg/kg.
Chromatogram of the control solution (rat's blood plasma), which were exposed to LIIRLR is shown in Fig. 2.
When studying plasma concentration of DNa in animals of group 2, which received DNa per os (8 mg/kg), it was determined to be 2.36 ± 1.10 µg/mL. Plasma concentration of DNa in animals of group 3 subjected to the influence of LIIRLR and in 15 min received DNa per os (8 mg/kg) was 4.08 ± 1.96 µg/mL. Typical chromatograms of the test solution and reference solution are presented in Fig. 3, 4.
Thus, quantitative content of DNa in the substance of DNa and suitability of the dose studied (8 mg/kg) were confirmed. It was established that under the scheme of the combined use of LIIRLR and DNa (per os, in 15 min) in comparison to the group of animals with DNa (per os) administration, the concentration of drug in blood plasma of rats was increased in 1.7 times. This shows the effectiveness of the combined use LIIRLR of and NSAIDs.
The results obtained indicate to the fact that concomitant use of NSAIDs and LIIRLR potentiate the ac- tion of drugs. This allows to decrease the dose, influence intake and duration of NSAIDs' action in the organism, decrease the frequency of their administration, and is a subject for further study.

CONCLUSIONS
Use of NSAIDs along with LIIRLR has been studied in experiments on rats. It is proved, that the method of com-bine application of LIIRLR and NSAIDs (per os, in 15 min) was more effective than the use of NSAIDs alone. The scheme of concomitant indication of LIIRLR and NSAIDs studied will be used for further preclinical and clinical studies, as well as for improvement of the quality of treatment in osteoarthrosis patients.
Conflicts of Interest: authors have no conflict of interest to declare.