Synergistic Antinociceptive Effects of Ketorolac and Ascorbic Acid in a Formalin-Induced Pain Model

Abstract

Pain is a widespread global issue and one of the most common disabling conditions in daily life. A wide range of medications are available to reduce or eliminate pain, with nonsteroidal anti-inflammatory drugs (NSAIDs) being among those most commonly used. Additionally, new analgesic approaches, such as antioxidants (Ascorbic Acid), have been explored for their potential to relieve acute pain after surgery, cancer-related pain, and chronic pain not related to cancer with fewer adverse effects. Furthermore, the use of pharmacological combinations is an alternative treatment strategy to obtain a higher efficacy using lower drug concentrations, at which side effects are minimal. Background/Objectives: The aim of this study was to evaluate the pharmacological synergism of ketorolac and ascorbic acid in an inflammatory pain model. Methods: The individual and combined effects of ketorolac and ascorbic acid were evaluated in a formalin-induced pain model in mice. Four experimental groups were established: control (vehicle), ketorolac (KET), ascorbic acid (AA), and combination (KET/AA). Results: The combination of ketorolac and ascorbic acid produced a greater antinociceptive effect compared to the vehicle and individual treatments in the formalin model. Notably, even the lowest dose of the combination (KET 6.26/AA 3.21 µg/paw) exhibited a stronger effect than the maximum doses of each individual treatment KET (100 µg/paw) and AA (100 µg/paw). The effective concentration that produced 30% of antinociception (EC₃₀) for the tested treatments were determined, and an isobologram analysis confirmed the presence of a synergistic interaction in these combinations. Conclusions: These findings suggest that the combination of ketorolac and ascorbic acid produces a synergistic antinociceptive effect in the formalin-induced pain model. The enhanced efficacy of the combination indicates a potential therapeutic advantage in pain management by reducing the required dosage of each compound while maintaining or improving analgesic effects.

1. Introduction

Pain is an unpleasant sensory and emotional experience, generally associated with real or potential tissue damage [1]. This phenomenon is common in daily life, and although we prefer to avoid it, it can manifest suddenly and temporarily or persist for prolonged periods. In either case, pain can become a debilitating condition, limiting daily activities and significantly affecting the quality of life. From acute injuries to chronic diseases and neoplasms, pain affects millions of people worldwide, highlighting the importance of proper management. Some studies [2,3,4] have identified the most common types of pain, including lumbar pain, sciatica, headaches, and neuropathic pain associated with chronic degenerative diseases. Additionally, other types of musculoskeletal pain, especially in the joints, are also common. The aforementioned conditions exert a direct influence on the quotidian existence of the individuals impacted, markedly diminishing their overall quality of life. Pain often begins as nociceptive, but if not properly managed, it may evolve into other types of pain. This progression underscores the need to investigate the molecular mechanisms involved and to develop effective treatments to mitigate or eliminate this discomfort.

In this context, non-steroidal anti-inflammatory drugs (NSAIDs), including both non-selective and selective inhibitors of cyclooxygenase 2 (COX-2), are among the most widely prescribed drugs worldwide [5]. These drugs are commonly used to treat fever, pain, and inflammation in conditions such as rheumatoid arthritis and osteoarthritis. However, their action on arachidonic acid-derived prostanoids and the inhibition of COX can have both beneficial and adverse effects. Prostaglandin H2 (PGH2), the product of COX activity, is converted into biologically active prostanoids that play a crucial role in inflammation and pain [6]. A widely used NSAID, ketorolac, is employed for the relief of nociceptive pain, primarily of inflammatory origin, and has been shown to be highly effective. Its mechanism of action involves inhibiting COX, thereby interfering with prostanoid synthesis. However, prolonged use can lead to adverse effects, particularly in the gastric mucosa, due to the inhibition of COX-1, which is responsible for protecting the gastric mucosa by synthesizing protective prostanoids [5].

Given the potential to combine different drugs to enhance analgesic effects and reduce the adverse effects of prolonged treatments, several studies have explored drug synergy. For instance, it has been found that ketorolac exhibits synergistic effects when combined with resveratrol [7] or B vitamins [8,9].

Additionally, some molecules, such as ascorbic acid (vitamin C), have demonstrated analgesic effects by stabilizing and reducing reactive oxygen species, decreasing levels of pro-inflammatory markers such as C-reactive protein and pro-inflammatory cytokines, e.g., tumor necrosis factor, interferon, and interleukin, and protecting cells and tissues from oxidative damage. Moreover, ascorbic acid is involved in collagen synthesis, promoting the recovery of damaged tissues [10]. AA either as monotherapy or in combination with other antioxidants has been shown to temper the need for painkillers after surgery. The analgesic benefits have also been demonstrated in the contexts of acute pain after surgery, cancer-related pain, and chronic pain not related to cancer [11,12,13]; for example, it has been shown that using doses of 2 g per day orally or 50 mg/kg/day intravenously of AA decreases the incidence of chronic pain syndromes [14]. In patients with osteoarthritis, a decrease in pain has been found using lower doses of 1 g/day for two weeks and in rheumatoid arthritis, using infusions used twice a week [15]. Recent research has shown a positive impact on the reduction in cancer-related pain, decreasing the requirement for analgesics and improving the quality of life of patients supplemented with high doses of AA (4 g/day). In addition, there is a significant decrease in pain secondary to bone metastasis and in episodes of breakthrough pain, using doses of 5 g intravenously every week or 500 to 4000 milligrams orally [16]. Also, a meta-analysis concluded that antioxidant supplementation with a combination of vitamins C and E was associated with a higher proportion of patients with endometriosis reporting a reduction in chronic pelvic pain [17].

Under these circumstances, as limited data are available on the effectiveness of the pharmacological antinociceptive interaction of vitamin C plus NSAID, the aim of this study was to assess whether the combination of ketorolac and ascorbic acid could enhance the antinociceptive effect in a formalin-induced pain model, considering the potential synergy between both compounds and their ability to provide a more effective treatment.

2. Materials and Methods

2.1. Animals

Seventy-eight male CD1 mice, weighing between 25 and 30 g, were used. The animals were obtained from the School of Chemical Sciences at the Autonomous University of Chiapas. They were housed in polycarbonate cages at room temperature (24 ± 2 °C) with a 12 h light/dark cycle and had ad libitum access to food before treatment. Six mice were used in each experimental group. The duration of the experiment was kept as short as possible, always ensuring that the number of animals used was the minimum necessary. Each animal was used for a single experiment and was euthanized immediately by cervical dislocation, afterward, following ethical guidelines for pain research as outlined in the Official Mexican Standard NOM-062-ZOO-1999 (Technical Specifications for the Production, Care, and Use of Laboratory Animals) [18] and adhering to ethical standards for animal experimentation [19]. The experimental procedures were approved by the research committee of the Autonomous University of Chiapas (Approval date: 19 April 2021; protocol number: 03/ECQ/RPR/247/20)

2.2. Drugs

Formaldehyde (37%) was purchased from J.T. Baker (Easton, Pennsylvania, USA). Ketorolac (AMSA Laboratories, Mexico City, Mexico) and ascorbic acid (Cayman Chemical, Ann Arbor, Michigan, USA) were used. The drugs were dissolved in an isotonic saline solution (0.9% NaCl, PiSA Laboratories, Mexico City, Mexico). All substances were freshly prepared for each use and administered subcutaneously (s.c.) into the right hind paw of the mouse with a 30-gauge needle in a 20 µL volume.

2.3. Pain Model

The formalin-induced pain model was used [20], which involves the subdermal administration of 20 µL of 2% formaldehyde into the dorsal area of the right hind limb using 1 mL syringes with a 30 G × 13 mm needle. After administration, the behavior was observed by counting the number of flinches of the affected limb for 1 min, every 5 min, over a period of 45 min. For this, the animals were placed inside polycarbonate cylinders, and 15 × 15 cm mirrors were used, positioned perpendicularly, to ensure complete visibility during the experiment. The observations were divided into two phases: the first phase covered the behavior evaluated from 0 to 10 min, and the second phase from 10 to 45 min.

The time course curve for each drug concentration was constructed by plotting the number of paw flinches induced by formalin as a function of time. The area under the curve (AUC) for the formalin phases was calculated using the trapezoidal method [21].

2.4. Experimental Groups

Two sets comprising 12 experimental groups (6 mice per group) were used (ketorolac, ascorbic acid, and ketorolac + ascorbic acid), to characterize the concentration–response curve of the individual drugs and the combination, as well as a control group that received a volume of vehicle (saline solution).

The first set (8 groups) received ketorolac. The drug was administered subcutaneously 15 min before the formalin injection at the following logarithmic concentrations: 10, 31.6, 56.2, and 100 µg/paw. Ascorbic acid was administered at concentrations of 10, 31.6, and 100 µg/paw, also 15 min prior to the formalin and vehicle application.

Once the concentration–effect curves for each individual drug were obtained, the effective concentration was found to be 30 (EC₃₀), according to the lineal logarithmic analysis. Based on these results, the EC₃₀ for the ketorolac–ascorbic acid combination were evaluated in the second set (4 groups). Four different doses of KET and ascorbic acid, AA, were administered in the combination (

Table 1. Doses of ketorolac and ascorbic acid evaluated in combination, at a 1:1 ratio.

Combination	Dose (µg/paw)
	Ketorolac	Ascorbic Acid	Total
EC30 + EC30	6.26	3.21	9.48
(EC30 + EC30)/2	12.53	6.42	18.96
(EC30 + EC30)/4	25.06	12.85	37.91
(EC30 + EC30)/8	50.12	25.70	75.82

).

Subsequently, the EC₃₀ experimental for the combination was calculated and compared with the theoretically obtained EC₃₀ to determine the interaction type of the combination, using the isobologram as described by Talladira [22]. All concentrations of the individual drugs and the combination were administered in a 20 µL volume.

2.5. Statistical Analysis

Data on antinociceptive effects are presented as the mean ± standard error of the mean for each group. To analyze the antinociceptive effects at the different treatment time points, a two-way analysis of variance (ANOVA) was performed, followed by a Tukey post hoc test. Results from the isobolographic analysis were compared using a Student’s t-test. All analyses were conducted using GraphPad Prism 6.0 software (SPSS Inc., Chicago, IL, USA).

3. Results

The formalin-induced nociception model (2%) resulted in a biphasic paw flinching behavior. The first phase began immediately after the formalin injection and gradually decreased over the first 10 min. The second phase started around 15 min and lasted until approximately 45 min. The maximum average number of paw flinches observed during phase 2 was 10.38 ± 3.13. The area under the curve (AUC) for the vehicle group revealed that the highest nociceptive activity occurred during phase 2 (Figure 1a), with an average of 271.87 ± 34.91 Area Units (AU), while phase 1 showed an average of 110.31 ± 20.37 AU.

3.1. Effect of Ketorolac on Nociceptive Behavior in the Formalin-Induced Pain Model

KET was administered in doses of 10.0, 31.6, 56.2, and 100.0 µg/paw to the dorsal region of the right hind limb 15 min prior to formalin injection. The temporal course of paw flinches showed an increase in flinches starting at 15 min, which decreased at 30 min and ceased at 45 min. As observed in the temporal course of KET (Figure 1a), doses of 31.6, 56.2, and 100.0 µg/paw resulted in a decrease in the number of flinches, although these reductions were not statistically significant compared to the vehicle group.

A global analysis of the effects, based on the area under the curve (AUC), revealed a slight antinociceptive effect during phase 1, with the higher doses showing AUC values below those of the vehicle group (Figure 1b). However, no statistically significant differences were observed in this phase. In contrast, a significant difference was found during phase 2 when administering 56.2 and 100.0 µg/paw of KET (200 ± 57.00 AU and 201.25 ± 21.35 AU, respectively), compared to the vehicle.

3.2. Effect of Ascorbic Acid on Nociceptive Behavior in the Formalin-Induced Pain Model

The AA was administered in doses of 10.0, 31.6, and 100.0 µg/paw to the dorsal region of the right hind limb 15 min prior to formalin injection. In phase 1, nociceptive activity was highest during the first minute and persisted until the 5 and 10 min marks, after which there was a drastic decrease. Following this, the number of paw flinches began to increase again starting at 15 min, peaking at 25 min, and gradually decreasing until ceasing at 45 min (Figure 1c). The temporal course of AA showed that all doses (10.0, 31.6, and 100.0 µg/paw) caused a considerable decrease in the number of flinches compared to the vehicle group, indicating an antinociceptive effect.

A global analysis of the results revealed that the AUC for all doses of AA (10.0, 31.6, and 100.0 µg/paw) showed a significant antinociceptive effect during phase 1, with the AUC values for the doses falling below that of the vehicle, with averages of 61.66 ± 17.44, 56.25 ± 9.18, and 56.66 ± 16.02 AU, respectively, and statistically significant differences. Additionally, during phase 2, statistically significant differences were also found compared to the vehicle, with average AUC values of 186.25 ± 46.19, 149.58 ± 67.70, and 148.33 ± 52.95 AU for each dose, respectively (Figure 1d). This indicates that the antinociceptive effect was observed in both phases of the experiment.

3.3. Dose–Response of Ketorolac and Ascorbic Acid Combinations

As with the individual treatments, these combinations were applied to the dorsal area of the right hind paw 15 min before the formalin test. The temporal course showed a gradual decrease in the number of paws shakes from minute 5, which persisted until the end of the evaluation (Figure 2a).

The AUC values for the combinations KET 6.26/AA 3.21, KET 12.53/AA 6.26, KET 25.06/AA 12.85, and KET 50.12/AA 25.7 in phase 1 were 72.9 ± 15.11, 71.25 ± 12.62, 64.16 ± 7.35, and 54.58 ± 7.48 UA, respectively. In phase 2, the AUC values were 72.5 ± 23.87, 76.25 ± 32.20, 80.41 ± 31.67, and 60.41 ± 23.09 UA, respectively (Figure 2b). Except for the combination KET 6.26/AA 3.21, which did not show a significant effect in phase 1, all other combinations exhibited statistically significant differences in both phases. In comparison to the vehicle group, the administration of KET and AA combinations produced a noticeable antinociceptive effect in both phases. The effect was more prominent during phase 2, when all combinations showed statistically significant differences.

We contrasted the effects of the combination that used the least amounts of the active principles with the individual doses that had the strongest antinociceptive effects in order to better understand these findings. The temporal analysis revealed that the KET 6.26/AA 3.21 combination resulted in the lowest number of paw flinches when compared to the vehicle (Figure 3a).

The AUC analysis indicated that the KET 6.26/AA 3.21 combination exhibited a greater antinociceptive effect than the 100 µg/paw doses of KET and AA (Figure 3b), as well as the vehicle, in both phases of the formalin test. Furthermore, all treatments showed statistically significant differences during phase 2 compared to the vehicle. In phase 1, the average AUC values were 79.16 ± 17.16 UA for KET 100 µg/paw, 72.91 ± 15.11 UA for AA 100 µg/paw, and 56.66 ± 16.66 UA for the KET 6.26/AA 3.21 combination. In phase 2, the average AUC values were 201.25 ± 51.35 UA for KET 100 µg/paw, 148.33 ± 52.95 UA for AA 100 µg/paw, and 72.50 ± 23.87 UA for the KET 6.26/AA 3.21 combination.

The combination KET 6.26/AA 3.21 exhibited the most pronounced antinociceptive effect in both phase 1 and phase 2 compared to the vehicle and the highest individual doses of KET and AA.

3.4. Isobolographic Analysis of the Antinociceptive Interaction Between the Combination of Ketorolac and Ascorbic Acid

An isobologram was constructed based on the EC₃₀ values of KET and AA on fixed proportions (1:1), graphically representing the pharmacological interaction between these two compounds (Figure 4). The X-axis represents the EC₃₀ of AA (25.7 ± 1.34 µg/paw), while the Y-axis represents the EC₃₀ of KET (52.12 ± 1.30 µg/paw). The midpoint of the isobole indicates the theoretical additive dose calculated from the EC₃₀ values of the individual treatments. The experimental EC₃₀ for the combination fell well below the isobole, indicating a synergistic interaction. The resulting experimental EC₃₀ was 2.6 ± 2.1 µg/paw, suggesting that the combination of these treatments potentiates their individual effects, thereby reducing the required dose to achieve antinociceptive efficacy.

4. Discussion

This study evaluated the combination of an antioxidant molecule, AA, and a NSAIDs, KET, in a formalin-induced pain model. The results confirmed the biphasic nature of the nociceptive response, with a neurogenic phase (0–10 min) followed by an inflammatory phase (15–45 min), consistent with the previous studies [23,24].

The individual administration of ketorolac produced dose-dependent antinociceptive effects, in agreement with the previous reports demonstrating the analgesic effect of this drug in the same model using systemic ketorolac and local tramadol [25,26]. Its primary mechanism of action involves COX-2 inhibition, leading to reduced synthesis of inflammatory prostanoids [27,28,29]. Evidence suggests that ketorolac exhibits synergistic effects in nociceptive pain models in rats when combined with tizanidine [30], methyleugenol [31], and B-complex vitamins [8,32]. Furthermore, ketorolac has shown enhanced analgesic effects when combined with other substances, such as tapentadol in a trigeminal pain model in mice [33]. Additional studies have reported a synergistic interaction when ketorolac is co-administered with drugs as tramadol [34].

Ascorbic acid, widely recognized for its antioxidant properties, also exhibited antinociceptive effects. Its action is attributed to its anti-inflammatory and antinociceptive properties, resulting from its ability to scavenge reactive oxygen species (ROS), reduce pro-inflammatory cytokine production, and modulate glutamatergic ionotropic receptors involved in pain signaling; additionally, ascorbic acid plays a key role as a cofactor in the synthesis of neurotransmitters implicated in pain processing [35,36,37,38]. Previous research has shown that its co-administration with other antioxidants or analgesics enhances the analgesic response [12,36,39,40]. Furthermore, studies have reported decreased ascorbic acid or vitamin C levels in hospitalized patients, elderly individuals, and patients with fractures or cancer [41,42], suggesting a correlation between vitamin C deficiency and increased pain perception.

This study provides the first evidence of the combined use of ketorolac and ascorbic acid in a formalin-induced pain model. The tested combinations demonstrated superior antinociceptive effects compared to individual treatments, suggesting an enhancement of the analgesic effect. Isobolographic analysis confirmed a synergistic interaction, indicating that the combination reduces the required dose for achieving significant analgesia. This finding aligns with previous research on pharmacological synergy between NSAIDs and antioxidants or vitamins [12,26,39].

From a methodological perspective, the evaluation of pharmacological interactions relies on dose–response strategies, with isobolographic analysis being the reference method due to its ease of interpretation and robustness. The observed synergy between ketorolac and ascorbic acid suggests that their complementary mechanisms contribute to pain relief. While ketorolac inhibits COX-2 and prostanoid synthesis, ascorbic acid reduces oxidative stress and inflammation by modulating cytokines and ROS, potentially acting together to enhance pain relief [30,32,36].

Future research could explore its application in other pain models, mechanisms of action, adverse effects and clinical studies to validate its efficacy in humans.

5. Conclusions

The combination of KET with AA represents a promising strategy to enhance the analgesic effects of NSAIDs, potentially reducing the required doses, supporting the potential use of this interaction in the treatment of inflammatory pain.