Rheumatoid arthritis (RA) is an incurable immune-mediated inflammatory disease with a multifactorial etiology, which affects up to 1.0% of the world’s population [1], with its prevalence being increased with age. According to official statistics, more than 300 thousand patients with RA were registered in Russia in 2017 [2]. Its progression leads to joint deformities, destruction of cartilage and bone tissue and subsequent disability.

Currently, treat-to-target (T2T) has been established as a guiding principle for the treatment of RA [2, 3]. It does not recommend any specific drugs, but only gives general treatment recommendations. The primary therapeutic goal for RA is to achieve remission [2]. For this purpose, several classes of drugs are used such as disease-modifying anti-rheumatic drug (DMARDs), which constitute an extensive group of synthetic and biological drugs that have a common ability to influence the pathogenetic mechanisms of RA; glucocorticoids, which are recommended to be used in combination with DMARDs, and nonsteroidal anti-inflammatory drugs used to relieve acute and chronic pain [4].

Treatment of rheumatoid arthritis is a long process, and the drugs used to treat it are far from being safe and significantly effective. In average, 20–50% of patients are forced to discontinue therapy due to lack of efficacy or intolerance to prescribed medications [5]. Therefore, there is an urgent need to develop new pharmacological targets, which would increase the effectiveness and reduce the toxicity of the drugs used.

One of the promising therapeutic targets includes proteinase-activated receptors (PARs). They belong to the class of G-protein-coupled receptors (GPCRs). Irreversible activation by proteases is the specific feature of these receptors. Discovered in the 1990s, 4 forms of PARs (PAR1, PAR2, PAR3 and PAR4) are expressed on the cell membrane of almost all organs and systems and regulate numerous physiological functions, including contraction of smooth muscle cells, sensitivity to pain, release of lipid mediators, cytokines, and neuropeptides. Activation of PAR2 is associated with clinical manifestations in the form of inflammation, swelling, and pain. They are expressed in immune cells of both the innate and adaptive immune systems and play an important role in the development of a wide range of diseases [6]. Their activation contributes to the occurrence of inflammation, fibrosis and proliferation of connective tissue. Experiments have demonstrated that inhibition of PAR2 activity prevents the development of RA pathogenesis and positively modifies the course of the disease [6]. The drugs that inhibit PAR2 are searched in the following directions: indirect blockade of PAR2 activity; creation of monoclonal antibodies; search for PAR2 inhibitors among peptide compounds; and synthesis of low molecular weight inhibitory compounds. The last direction is the most perspective one [7].

During 2016–2019, synthesis of low molecular weight PAR-2 antagonists related to 4,5-dihydroisoxazole-5-carboxamide amide derivatives was done. The work was based on preliminary mathematical prediction of pharmacologically significant properties of a large number of multinucleated derivatives of imidazole, isoxazole and oxazole with a wide structural diversity. The antagonistic activity of the compounds with respect to PAR-2 was evaluated in vitro using the CHO cell line with high expression of human PAR-2 [8]. Five most active P AR-2 antagonists, derivatives of 4.5- dihydroisoxazol-5-carboxamide, the structure of which is shown in the figure, were investigated in vivo on a formaldehyde edema model in various dosages. It would allow to select the most active compound for testing on a model of chronic autoimmune inflammation [9].

The aim of the study was to determine the potential therapeutic efficacy of a promising compound from a number of 3-imidazole-substituted-4,5-dihydroisoxazole carboxylic acid amide derivatives on a model of autoimmune inflammation in rats.

MATERIALS AND METHODS

A single subcutaneous injection of pristane into the base of the rat tail in a volume of 0.1 ml [10], caused chronic inflammation followed by pathological changes seen in white rats at early stages of RA: a chronic recurrent process, typical histological signs of joint damage, as well as development of specific serological immune abnormalities can be observed [10].

The experiment was carried out on 50 white mongrel rats with a mean weight of 180–220 g, kept at a temperature of 22 ± 2 °C, humidity of 55 ± 5%, in a 12/12-hour light cycle, with unlimited access to food and water. All animals were divided into 5 groups with 10 rats in every group: intact animals in the first group; control animals (rats were injected with pristane and saline solution) in the second group; rats of the third group were injected pristane + diclofenac sodium at a dose of 5 mg/ kg [11]; rats of the fourth group were injected with pristane + dexamethasone 1 mg/kg [10]; animals of the fifth group were injected with pristane + R004 10 mg/kg. The duration of the experiment was 28 days; on a daily basis, dexamethasone and physiological solution, diclofenac sodium and R004 were administered s/c and intragastrically respectively. Dynamic examination of the animals was carried out 5 times: at baseline and on the 7th, 14th, 21st and 28th days of the experiment. The following parameters were studied: the volume of the affected foot using digital anhydrous plethysmometer PH1901 (Russia), the width of the articular slits using digital X-ray of the lower extremities (on day 28 only) [12], rectal temperature, general blood test, biochemical values (total protein, albumins, globulins, glucose, triglycerides, total cholesterol, AlAT, AsAT, total bilirubin, GGT, alkaline phosphatase, creatinine, urea).

The results were statistically processed using the Biostat program. Every value determined in various experiments was 6–10. The Student’s t-test (in the presence of a normal distribution) and the Mann-Whitney U-test was applied to the comparison of the values between groups, whereas the Student’s T-test with a Bonferroni adjustment was used in case of multiple testing. Reliability of intra-group differences was determined by the Student’s paired t-test. The differences were considered significant at p < 0.05 [13].

THE RESULTS OF THE SURVEY AND THEIR DISCUSSION

Changes of rectal temperature and paw volume of animals, as well as the width of the interarticular gap of their feet are the investigated values that most specifically reflect the process of inflammation in rats.

In the intact group, there was an unreliable change in the volume of feet throughout the experiment (tab. 1). At 14 days after pristane injection to white rats, the volume of the right foot and the average value of both feet were significantly increased from baseline by 5.5% and 3.8% respectively. On the 21st day of the experiment, the volume of both feet significantly increased from baseline: by 7.6% for the right one, by 3.1% for the left one; on the 28th day, the volume of the right foot and both feet remained significantly increased by 5.2% and 2.7%, respectively. In relation to healthy animals, foot volume in rats was significantly increased by 6.5–11.2% on days 14–28 of pristane inflammation.

When treating rats with diclofenac sodium, a significant increase in foot volume (right and average) by 5.6% and 4.7% from baseline occurred only on the 21st day of the experiment.

On day 28, the volume of feet in rats became normal from baseline, but the right foot became significantly more voluminous (+12%) than in intact animals. No increased foot volume was seen in rats treated with dexamethasone. Starting from the 7th day of the experiment and until the end, there was a significant decrease in foot volume by 9.4–13.4% from baseline and by 9.3– 16.6% in relation to the control group. No significant increase in foot volume was seen when R004 was administered to animals, and, moreover, the paw volume of treated rats was significantly lower by 7.5–18.7% than in the control group and, at the same time, did not differ from those in the intact group (tab. 2).

The experiment showed that when administered to animals, chronic inflammation is developed, which is accompanied by swelling of paws. Diclofenac sodium reduced (but did not completely prevent) severity of the inflammatory process; its therapeutic effect was more pronounced in the first three weeks of the developing process, and then slightly decreased (against the background of its administration, there was a significant increase in the volume of the rat’s right paw on day 21 and a significant increase in its volume relative to intact animals on day 28). Dexamethasone completely blocked the inflammatory edema significantly reducing the volume of rat paws both in comparison with the baseline and in relation to intact control on days 4 and 14 of the experiment. This may be evidence of dexamethasone myopathy due to impaired synthesis of muscle proteins. Treatment of rates with R004 was optimal: in animals, there was no significant increase of foot edema, both when compared with baseline and in relation to healthy rats.

Modeling of autoimmune inflammation with pristane caused damage to the joint tissue of white rats, one of the manifestations of which was an X-ray change in the width of the articular gap (tab. 2). It significantly decreased by 11.1% in the prematarsalmetatarsal joints and by 11.3% in the metatarsal-phalangeal joints; in the interphalangeal joint, the narrowing was observed as a trend (–11.5%). In this case, diclofenac sodium did not exert any therapeutic activity: the width of the articular gap in the metatarsal-phalangeal and metatarsal-phalangeal joints changed almost to the same extent as in the control group (–12.7% and –13.2%), and in the premaxillary-metatarsal joint, there was even a deterioration: the width of the interarticular gap was significantly decreased not only in relation to intact animals (–20%), but also in relation to control ones as well (–9.7%). Dexamethasone also had no therapeutic activity: the width of the articular gap significantly decreased by 16.4% in the prematarsal-metatarsal joint and by 18.7% in the metatarsal-phalangeal joints, and in the interphalangeal joint, narrowing of the articular gap was observed as a trend

(–11.4%). Administration of R004 reduced the degree of narrowing of the articular gap significantly by 9.8% in relation to healthy animals, this occurred in the pre-metatarsal joint only. At the same time, changes in the width of the articular gap in the metatarsophalangeal joint did not reach the level of reliability (–9.5%), and they were absent in the interphalangeal joint. It was significantly wider by 10.8% in relation to the control group and by 11.9% in relation to the dexamethasone group.

Thus, experience has shown that with pristane inflammation, damage to the articular surfaces occurs. Diclofenac sodium and dexamethasone proved to be ineffective; R004 partially prevented the damaging effect of pristane on articular tissue.

Measurement of rectal temperature in white rats turned out to be a less informative indicator: its changes in all the studied groups did not actually differ from those of intact animals.

Blood test in animals allowed measurement in dynamics: before the initial injection of pristane and on the 28th day of the experiment. In the intact control group, an elevation in the white blood cell count was observed during the experiment, but only eosinophils and monocytes were significantly increased by 51% and 80% respectively. After administration of pristane, the qualitative picture did not change for most of the studied values, only two values significantly differed from those in the intact control group: there was a 2.5-fold increase in neutrophil count from baseline (p 0.05), and lymphocyte count decreased by 35% (tab. 3).

While using diclofenac sodium, there was an even greater increase in neutrophil count (4 times compared to baseline, p 0.05), whereas lymphocyte count in blood decreased almost twice (p 0.05). Additionally, basophils were no longer detected in blood. When using dexamethasone, there was a tendency to a low number of neutrophils (their content increased in other groups), the content of neutrophils increased 4 times, but the level of lymphocytes dropped 20 times. Basophils and eosinophils were not detected in blood any longer. R004 leveled drug-induced blood changes: none of the investigated values was changed in a reliable way. Qualitative differences between animals of this group and intact rats consisted in a tendency to a decreased number of basophils and lymphocytes, and significant quantitative differences were determined for lymphocytes only: WBC content in R004 group in relation to intact animals was 36.2% lower though the baseline content was almost the same (tab. 3).

Thus, a significantly high neutrophils and low lymphocytes in the control group of rats indicate that the immune system was affected. Unlike with reference drugs, therapeutic use of R004 neutralizes these processes to some extent. With diclofenac sodium, these changes were aggravated (monocytes and basophils were added to the drop in lymphocyte content), and dexamethasone itself had a significant immunosuppressive and lymphotoxic activity, contributing to an even greater change in the investigated parameters.

Analyzing the data obtained with respect to red blood, it can be noted that the administration of pristane produced practically no negative effect on its values.

Metabolism of proteins, fats and carbohydrates. Pristane causes a disturbed balance of protein metabolism. Despite the fact that the change in blood albumin and globulin is trending, a significant decrease in the albumin/globulin index (AHI) by 13.9% indicates a shift in protein synthesis towards globulins, which structurally include antibodies that are essential in autoimmune inflammation. A significant decrease in glucose by 9.7% and cholesterol by 23.6% (decrease in the rate of synthesis of steroid hormones and repair of cell membranes) was observed as well. Against the background of a decrease in glucose concentration and a tendency to increase the TG content, the carbohydrate-fat index (CFI) significantly decreased by 30% (the ratio of glucose and triglycerides). It shows the body’s shift to “fatty” energy, which is more consumable in terms of oxygen and energy substrates (tab. 4).

Diclofenac sodium caused a uniform decrease in the synthesis of both albumins and globulins, which may be associated with the impaired liver function. A further decrease in the CFI by 37.4% compared to intact animals occurred, demonstrating an increase in the lipid component of energy balance. With dexamethasone, dissonance increased in favor of globulin synthesis (a significant decrease in AHI by 19.5% in relation to healthy animals) and a sharp decrease in CFI by almost 5 times, showing the body’s shift to “lipid” energy (the effect of high doses of glucocorticoids). The therapeutic administration of R004 stops changes in metabolism of proteins, fats and carbohydrates that occur against the background of pristane inflammation.

Functional biochemistry of the liver and kidneys. When pristane was administered, there was a significant increase of AsAT activity by 35.7% and creatinine content by 14% (tab. 4). It means that the liver and kidneys display an interest in developing inflammatory process. There was only a significant increase in blood creatinine (+9.8%) with R004, but to a lesser extent than for the control group. When using diclofenac sodium in sick animals, only blood creatinine (+9.9%) and urea (+25.4%) significantly increased, indicating at a possible kidney damage. The tendency to an increased activity of blood transaminases and alkaline phosphatase, as well as to an increased concentration of bilirubin, demonstrates liver function strain. Dexamethasone showed a significant increase in the activity of AlAT by almost 2 times, AsAT by 1.6 times, GGT by 1.8 times and alkaline phosphatase by 3.1 times (the latter p > 0.05), demonstrating cytolysis of hepatocytes and developing cholestasis. A significant increase of blood levels of urea shows a decrease in the reabsorption function of the kidneys.

Thus, the experiment showed that with pristane inflammation, liver and kidney damage is possible. R004 weakens this process, whereas diclofenac sodium and dexamethasone enhance it.

CONCLUSIONS

1. R004 in the treatment of chronic inflammation in rats prevented significant development of edema of feet, damage to small joints, and specific changes in white blood count, and according to biochemical blood test led to normalization of liver and kidney functions and energy metabolism.
2. R004 turned out to be more effective and safer than the comparator drugs such as diclofenac sodium and dexamethasone.