Effect of cyclooxygenase inhibitors, ethylmethylhydroxypyridine succinate and their combinations in a collagen-induced arthritis model in BALB/c mice

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective agents for the treatment of pain and inflammation and are indicated for a wide range of pathological conditions, including osteoarthritis, rheumatoid arthritis, spondyloarthritis, gout, and other metabolic arthropathies. NSAIDs are characterized by dose-dependent, class-specific adverse effects related to their mechanism of action—inhibition of cyclooxygenase (COX) isoforms 1 and 2—which include gastrointestinal, cardiovascular, and nephrotoxic adverse events that limit their use [1].

During inflammation, enzymatic and non-enzymatic destruction of membrane phospholipids, lipoproteins, and glycolipids occurs, leading to the release of higher fatty acids (including arachidonic acid) and free lipids. Increased production of arachidonic acid significantly enhances the formation of prostaglandins (via COX) and leukotrienes (via 5-lipoxygenase). Inflammation also intensifies free radical lipid peroxidation (LPO), the products of which increase membrane permeability, resulting in cytolysis and dysregulation of tissue processes [2]. The ability of NSAIDs to induce mitochondrial dysfunction with excessive formation of reactive oxygen species is considered a mechanism underlying their damaging effects on the gastrointestinal tract and cardiovascular system [3, 4]. Therefore, one approach to improving the efficacy and safety of NSAIDs is their use in combination with antioxidant and membrane-protective agents.

Previously, we have shown that ethylmethylhydroxypyridine succinate (EMHPS), which possesses antioxidant and membrane-stabilizing properties [5], enhances the effects of diclofenac sodium and etoricoxib: the antiexudative effect in a carrageenan-induced edema model in mice and rats [6–8], and the analgesic effect in models of acute visceral pain in mice, acute somatic pain induced by formalin and carrageenan, and postoperative pain in rats [7, 9, 10]. Along with increasing anti-inflammatory and analgesic efficacy, the combination of NSAIDs with EMHPS also improves the safety profile of NSAIDs. For example, EMHPS administered with etoricoxib prevents etoricoxib-induced elevation of blood pressure [11], and when administered with diclofenac sodium or etoricoxib, it attenuates the ulcerogenic effect of NSAIDs in a rat model of gastotoxicity [12].

The aim of this study was to evaluate the effects of EMHPS, the non-selective COX inhibitor diclofenac sodium, the selective COX-2 inhibitor etoricoxib, and combinations of these NSAIDs with EMHPS during repeated oral administration in a collagen-induced arthritis (CIA) model in BALB/c mice.

Materials and Methods

Animals. The study was performed on sexually mature male BALB/c mice weighing 21–26 g (n = 53) obtained from the Stolbovaya branch of the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency (Moscow region, Russia). Animals were kept under standard vivarium conditions with free access to food and water under a 12-hour light cycle. The organization and conduct of the work complied with GOST 33216-2014 “Guidelines for housing and care of laboratory animals. Rules for housing and care of laboratory rodents and rabbits” and GOST 33215-2014 “Guidelines for housing and care of laboratory animals. Rules for facility design and procedure organization.” The experiment was approved by the Biomedical Ethics Committee of the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies (Protocol No. 01 dated January 18, 2023).

Collagen-induced arthritis (CIA) is one of the most well-studied models of rheumatoid arthritis [13]. Animals with this model develop an autoimmune process with exudative inflammation in the early stages, the severity of which is reduced by NSAIDs. The efficacy of EMHPS, diclofenac sodium, etoricoxib, and their combinations in the CIA model in BALB/c mice was assessed by their ability to reduce the severity of pathological symptoms: CIA severity, hind paw edema, hyperalgesia, and motor deficits. In the blood serum of CIA mice, the concentration of the collagen degradation product hydroxyproline (oxyproline) and the activity of the antioxidant enzymes catalase and glutathione peroxidase were measured.

CIA was induced in BALB/c mice according to [14]. Immunization was performed twice: the first immunization was carried out by intradermal injection into the base of the tail of 100 μg of bovine type II collagen emulsified with complete Freund’s adjuvant (CFA) in a volume of 50 μL. Prior to the second immunization, animals that had received the intradermal injection of bovine type II collagen with CFA were randomized into groups based on the presence of developing paw edema and body weight. The second immunization was performed on day 21 after the first, using the same method.

Animals were randomized into the following groups:

1. Control – mice that received intradermal injections of saline (50 μL) into the base of the tail on the days of immunization and daily oral administration of saline from day 21 of the experiment for 21 days, n = 10.

2. Collagen-induced arthritis (CIA) – mice that received intradermal collagen injections and daily oral saline from day 21, n = 7.

3. EMHPS 25 mg/kg – mice with CIA that received oral EMHPS at a dose of 25 mg/kg, n = 7.

4. Diclofenac sodium 1 mg/kg – mice with CIA that received daily oral diclofenac sodium at a dose of 1 mg/kg from day 21, n = 7.

5. Etoricoxib 1 mg/kg – mice with CIA that received daily oral etoricoxib at a dose of 1 mg/kg from day 21, n = 7.

6. Diclofenac sodium 1 mg/kg + EMHPS 25 mg/kg – mice with CIA that received daily oral diclofenac sodium (1 mg/kg) and EMHPS (25 mg/kg) from day 21, n = 8.

7. Etoricoxib 1 mg/kg + EMHPS 25 mg/kg – mice with CIA that received daily oral etoricoxib (1 mg/kg) and EMHPS (25 mg/kg) from day 21, n = 7.

The choice of NSAID and EMHPS doses for the combination was based on the results of previous experiments [8].

Arthritis severity was assessed visually for each paw individually, and the total score per mouse was calculated [15]: 0 – no visible changes; 0.5 – redness and swelling of one toe joint; 1 – redness and mild swelling of the foot pad, tarsal joints, or 2–5 toes; 2 – redness and swelling of 2 joints; 3 – redness and swelling of the entire paw; 4 – reduced swelling and joint deformities. Thus, the maximum arthritis score per animal was 16 points.

In BALB/c mice with CIA, hind paw edema was evaluated by measuring the increase in paw diameter in the metatarsal region (mm) using an electronic caliper on day 21 after the first immunization (before the second immunization with bovine type II collagen) and on days 7, 14, and 21 after the second immunization with bovine type II collagen.

On days 7, 14, and 21 after the second immunization with bovine type II collagen, mechanical hyperalgesia was assessed in BALB/c mice using von Frey filaments ranging from 0.06 to 23.96 g (Aesthesio®, Ugo Basile, Italy). Mice were placed individually in plastic chambers (20×10×14 cm) on an elevated perforated grid platform and allowed to acclimate for 15 min in a resting state. Von Frey filaments were applied perpendicularly to various areas of the plantar surface of the hind paws, avoiding the toes and tarsal joint (stimulus duration 2 s). Each filament was tested 5 times with an interval of 3 s. The minimal threshold that elicited paw withdrawal was determined [16].

Motor deficit in BALB/c mice with CIA was assessed by evaluating coordination in the Rotarod test (Rota Rod, Ugo Basile, Italy) [17]. Animals were adapted to the procedure before the second immunization with bovine type II collagen by placing them on the rotating drum three times at a speed of 5 rpm. Motor performance was tested at a constant drum rotation speed of 10 rpm, and the latency to fall (seconds) was recorded on days 7 and 14 after the second immunization.

On day 42 after the first injection of bovine type II collagen, BALB/c mice were euthanized (decapitation), and blood serum was collected for biochemical analysis.

The concentration of oxyproline was determined using a colorimetric method [18]. Catalase activity was measured according to [19], and glutathione peroxidase activity according to [20].

Statistical analysis. Statistical analysis was performed using GraphPad Prism 8 for Windows. Normality of distribution was tested using the Shapiro–Wilk test. Since the distribution of parameters in the groups deviated from normal, the nonparametric Kruskal–Wallis test followed by the Mann–Whitney test without/with Benjamini–Hochberg correction was used for independent group comparisons. Results in tables are presented as median (Q1; Q3). Differences between groups were considered statistically significant at p ≤ 0.05.

Results and Discussion

On day 3 after the second injection of bovine type II collagen, signs of inflammation were recorded in the CIA group: except for one animal, all others showed at least redness and swelling of two joints on one hind paw. Paw edema in the CIA group continued to increase until day 14 after the second injection. The median arthritis severity in this group on day 14 after the second injection was 10 points and remained at this level until day 17, after which paw edema decreased and joint deformation became more pronounced (Table 1). On day 21 after the second injection (experiment day 42), 5 animals in the CIA group showed severe joint deformation of the hind paws.

Administration of etoricoxib (1 mg/kg) or EMHPS (25 mg/kg) to BALB/c mice for 3 days after the second injection delayed the manifestation of arthritis symptoms, as evidenced by a reduction in severity (to 0 points). However, during subsequent monitoring, no reduction in arthritis symptoms compared to the CIA group was observed in mice treated with etoricoxib or EMHPS. Assessment of arthritis severity revealed no reduction in animals treated with diclofenac sodium (1 mg/kg) throughout the observation period. In contrast, the combination of diclofenac sodium (1 mg/kg) and EMHPS (25 mg/kg) significantly reduced arthritis severity on day 3 after the second injection. The combination of etoricoxib and EMHPS was even more effective: it significantly reduced CIA severity in BALB/c mice from day 3 to day 17 after the second injection (Table 1).

Table 1. Effect of ethylmethylhydroxypyridine succinate, diclofenac sodium, etoricoxib, and combinations of NSAIDs with EMHPS on the severity of collagen-induced arthritis in BALB/c mice (median (Q1; Q3))

Notes: * – p ≤ 0.05 compared to the CIA group; # – p ≤ 0.05 compared to the CIA + EMHPS 25 mg/kg group, Mann–Whitney test with Benjamini–Hochberg correction; CIA – collagen-induced arthritis; EMHPS – ethylmethylhydroxypyridine succinate.

Seven days after the second injection of bovine type II collagen, a significant increase in hind paw diameter in the metatarsal region was recorded in the CIA group: the median edema of the left paw increased by 22.7%, and of the right paw by 28.6% compared to the corresponding values in the control group. From day 14 after the second injection, hind paw edema decreased. On day 14, only a significant increase in the median diameter of the left metatarsus (by 14.3%) was observed. On day 21, no significant differences in hind paw diameters between the CIA and Control groups were found (Table 2).

On day 7 of drug administration, diclofenac sodium, etoricoxib, and their combinations with EMHPS significantly reduced the severity of exudative inflammation in the right paw of CIA animals by 11.1–18.5%. No significant effect on hind paw metatarsal diameter was observed on days 14 and 21 after the second injection (Table 2).

Table 2. Effect of ethylmethylhydroxypyridine succinate, diclofenac sodium, etoricoxib, and combinations of NSAIDs with EMHPS on tarsal edema in BALB/c mice with collagen-induced arthritis (median (Q1; Q3))

Notes: * – p ≤ 0.05 compared to the CIA group, Mann–Whitney test with Benjamini–Hochberg correction; CIA – collagen-induced arthritis; EMHPS – ethylmethylhydroxypyridine succinate.

Throughout the observation period (21 days after the second injection), BALB/c mice with CIA exhibited hyperalgesia upon stimulation of the plantar paw surface with von Frey filaments, and its severity did not decrease over time. Diclofenac sodium, etoricoxib, and EMHPS alone did not significantly affect pain sensitivity. However, the combination of diclofenac sodium (1 mg/kg) or etoricoxib (1 mg/kg) with EMHPS (25 mg/kg) significantly increased the pain threshold on day 7 of recording compared to the untreated CIA group. The most pronounced analgesic effect was observed with the combination of diclofenac sodium and EMHPS. This combination significantly increased the pain threshold compared to groups treated with EMHPS or diclofenac sodium alone. On days 14 and 21, the combinations of NSAIDs with EMHPS had no significant effect on pain sensitivity in the von Frey test (Table 3).

Table 3. Effect of ethylmethylhydroxypyridine succinate, diclofenac sodium, etoricoxib, and combinations of NSAIDs with EMHPS on the pain threshold in BALB/c mice with collagen-induced arthritis in the von Frey test (median (Q1; Q3))

Notes: * – p < 0.05 compared to the Control group; # – p ≤ 0.04 compared to the CIA group, Mann–Whitney test with Benjamini–Hochberg correction; & – p ≤ 0.04 compared to the Diclofenac 1 mg/kg + EMHPS 25 mg/kg group, Mann–Whitney test with Benjamini–Hochberg correction; CIA – collagen-induced arthritis; EMHPS – ethylmethylhydroxypyridine succinate.

On day 7 after the second injection, animals in the CIA group exhibited motor deficit, as evidenced by a 3-fold decrease in the latency to fall from the Rotarod compared to the control group. In CIA mice treated with NSAIDs (1 mg/kg), EMHPS (25 mg/kg), or their combinations, the latency to fall did not differ significantly from that in non-pathological animals. A significant increase in this parameter compared to the CIA group was observed with the combination of etoricoxib and EMHPS, which restored coordination in CIA mice almost to the level of controls. By day 14 after the second injection, motor coordination in the CIA group had recovered to control levels, and EMHPS or its combinations with NSAIDs did not affect coordination. In contrast, diclofenac sodium and etoricoxib alone reduced coordination, as shown by a significant decrease in latency to fall (2.3- and 2.7-fold, respectively) compared to the control/CIA group (Table 4).

Table 4. Effect of ethylmethylhydroxypyridine succinate, diclofenac sodium, etoricoxib, and combinations of NSAIDs with EMHPS on the time spent on the rotating rod in BALB/c mice with collagen-induced arthritis (median (Q1; Q3))

Notes: * – p < 0.05 compared to the Control group; # – p < 0.05 compared to the Collagen Arthritis group, Mann–Whitney test; CIA – collagen-induced arthritis; EMHPS – ethylmethylhydroxypyridine succinate.

On day 42 of the pathological process, serum oxyproline concentration in CIA mice was increased by 21.1% compared to controls. EMHPS reduced the elevated oxyproline level by 25.7%, whereas etoricoxib increased it by a further 54.0%. Coadministration of etoricoxib with EMHPS prevented this effect of the selective COX-2 inhibitor. Serum oxyproline levels in CIA mice were lower after treatment with diclofenac alone or in combination with EMHPS (Table 5).

On day 42, serum catalase activity in CIA mice was decreased 1.9-fold compared to controls, while glutathione peroxidase activity was increased 2.1-fold. Repeated administration of EMHPS, diclofenac, etoricoxib, or their combinations prevented the decrease in serum catalase activity. Glutathione peroxidase activity in treated CIA groups did not differ significantly from that in non-pathological animals (Table 5).

Table 5. Effect of ethylmethylhydroxypyridine succinate, diclofenac sodium, etoricoxib, and combinations of NSAIDs with EMHPS on serum oxyproline concentration, catalase activity, and glutathione peroxidase activity in BALB/c mice with collagen-induced arthritis (median (Q1; Q3))

Notes: * – p < 0.05 compared to the Control group; # – p < 0.05 compared to the CIA group, Mann–Whitney test with Benjamini–Hochberg correction; ## – p < 0.05 compared to the CIA group, Mann–Whitney test; CIA – collagen-induced arthritis; EMHPS – ethylmethylhydroxypyridine succinate.

The results of this experiment demonstrate that two intradermal injections of bovine type II collagen emulsified with CFA into the base of the tail of BALB/c mice induced a chronic inflammatory process. Exudative inflammation, manifested by hind paw edema, was observed from day 3 to day 17 after the booster injection (maximum edema on days 4–17). Thereafter, paw edema decreased, and joint deformation and destruction became more prominent, accompanied by increased serum oxyproline levels [21]. Along with increased serum oxyproline, a functional imbalance of the antioxidant enzymes catalase and glutathione peroxidase was observed on day 42 of the pathological process. Catalase activity, the first line of antioxidant defense that prevents hydrogen peroxide accumulation by oxidizing it to water and molecular oxygen [22], was significantly reduced, while glutathione peroxidase activity was compensatorily increased. Glutathione peroxidase detoxifies hydroperoxides and prevents the formation of fatty acid hydroperoxides by reducing hydrogen peroxide to water and molecular oxygen [23]. The compensatory increase in glutathione peroxidase activity alongside reduced catalase activity in CIA mice indicates a concurrent protective process aimed at counteracting the intensification of free radical lipid peroxidation. Oxidative stress is known to damage chondrocytes [24]; therefore, the elevated serum oxyproline levels in CIA mice may result from the cytotoxic effects of LPO products, which increase during inflammation.

Diclofenac sodium and etoricoxib were administered orally at a low daily dose of 1 mg/kg for 21 days, because daily repeated administration of these NSAIDs at higher doses (5 and 10 mg/kg), at which they exhibit antiexudative and analgesic effects [7–10], leads to severe adverse effects, including body weight loss and deterioration of the animals’ condition, even death (in the case of diclofenac sodium) [6, 11]. Etoricoxib at 1 mg/kg administered on days 21–24 after the first collagen injection delayed the development of arthritis symptoms in BALB/c mice. Both etoricoxib and diclofenac sodium at 1 mg/kg reduced hind paw edema on day 7 after the booster injection.

EMHPS at 25 mg/kg delayed the onset of arthritis symptoms when administered on days 21–24 after the first collagen injection and enhanced the effects of NSAIDs. This was evidenced by a longer period of reduced arthritis severity in animals receiving NSAID+EMHPS combinations compared to the untreated CIA group. Specifically, the effect of etoricoxib was enhanced, extending the period of reduced arthritis severity (p ≤ 0.05) to 17 days, compared to only 3 days with etoricoxib alone. The combination of diclofenac sodium and EMHPS delayed the manifestation of arthritis symptoms for 3 days of daily administration, whereas diclofenac sodium alone had no such effect. The NSAID+EMHPS combinations significantly increased the pain threshold in CIA mice compared to the untreated CIA group on day 7 of administration, whereas NSAIDs alone at 1 mg/kg did not reduce hyperalgesia. Moreover, the pain threshold in animals receiving diclofenac sodium + EMHPS was significantly higher than in those receiving diclofenac sodium alone. Notably, mice with CIA treated with NSAIDs alone exhibited locomotor deficits, which were not observed in animals receiving NSAID+EMHPS combinations.

The reduced imbalance of antioxidant enzyme activities in NSAID-, EMHPS-, or combination-treated CIA mice compared to the untreated CIA group is likely related to the reduction in CIA symptom severity. The ability of EMHPS to enhance the efficacy of NSAIDs is attributed to its membrane-protective and antioxidant actions, as well as its effect on phospholipase A2 activity, since EMHPS has been shown to reduce phospholipase A2 activity in canine pancreatitis [25]. The complex cytoprotective mechanism of EMHPS may also account for its chondroprotective effect, as inferred from its ability to reduce the elevated serum oxyproline level in CIA mice on day 21 after the booster injection. Unlike EMHPS, diclofenac sodium did not significantly correct this parameter, while etoricoxib significantly increased serum oxyproline. Etoricoxib is known to affect the microstructure and biomechanical properties of subchondral bone and to promote bone loss in a mouse model of early-stage osteoarthritis [26]. The combination of EMHPS with NSAIDs favorably affected serum oxyproline levels: EMHPS with diclofenac sodium significantly reduced the collagen degradation marker, and with etoricoxib it prevented the etoricoxib-induced increase in serum oxyproline.

Conclusion

Thus, the combined use of EMHPS with diclofenac sodium and etoricoxib enhances the efficacy and safety of these NSAIDs in the collagen-induced arthritis model in BALB/c mice. These findings support further investigation of the effects of EMHPS in combination with low-dose NSAIDs in arthritis. Reducing the therapeutic doses of NSAIDs by co-administering EMHPS may ensure both efficacy and greater safety, as NSAID-related adverse events reduce patients’ quality of life and may even pose a health threat in certain patient populations.