https://orcid.org/0000-0002-6580-2886
Abstract
To analyse the antinociceptive interaction between quercetin (QUER) and diclofenac (DIC) in experimental arthritic gout-pain.
The antinociceptive effect of DIC and QUER alone and in combination were evaluated using an arthritic gout-pain model. Pain was induced through intra-articular administration of uric acid in the rats and the treatments were administered 2 h later. Additionally, the cyclooxygenase (COX) activity was determined in rats treated with DIC, QUER and their combination.
DIC induced a maximal effect of 69.7 ± 2.7% with 3.1 mg/kg; whereas QUER only produced 17.6 ± 2.6% with the maximal dose (316 mg/kg). Ten of twelve DIC + QUER combinations showed a lesser antinociceptive effect than DIC alone did (P < 0.05). Moreover, DIC reduced total-COX (70.4 ± 1.3 versus 52.4 ± 1.8 and 77.4 ± 9.0 versus 56.1 ± 1.3, P < 0.05) and COX-2 (60.1 ± 1.0 versus 42.4 ± 1.8 and 58.1 ± 2.4 versus 48.7 ± 1.3, P < 0.05) activity after 1 and 3 h, respectively. Nevertheless, only the COX-2 activity induced by DIC was prevented in the presence of QUER (63.2 ± 3.0 versus 60.1 ± 1.0 and 56.6 ± 1.3 versus 58.1 ± 2.4 at 1 and 3 h, respectively).
All these data demonstrated that the simultaneous administration of QUER + DIC produces an unfavorable interaction on the antinociceptive effect of DIC. Therefore, this combination might not be recommendable to relieve arthritic gout-pain.
Introduction
Pain is considered one of the leading causes of medical attention at the first level of attention. It is defined by the International Association for the Study of Pain (IASP) as ‘an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage’ that decreases the quality of life of patients.[1] It can be associated with tissue damage mediated by activation of nociceptors through the release of a variety of chemical mediators, such as prostaglandins, bradykinin, excitatory amino acids and cytokines, among others, which in turn act on specific receptors and ion channels contributing to the induction of inflammatory pain.[2, 3] This kind of pain is caused by several arthritic diseases, which have a high prevalence worldwide. It is estimated that ~1.49% population suffer from rheumatoid arthritis and 0.4% from gouty arthritis.[4] Several types of medications, such as non-steroidal anti-inflammatory drugs (NSAIDs) and other treatments (herbs and pure natural products, such as flavonoids) can bring a relief. Diclofenac (DIC) is one of the most prescribed NSAIDs for the treatment of acute and chronic pain, especially in inflammatory pain induced by diseases of rheumatic origin. It is considered to have an adequate clinical efficacy and lower incidence of adverse effects than other NSAIDs do.[5] However, its long-term use is not recommended due to the risk of gastrointestinal, cardiovascular and kidney complications.[6]
Currently, the use of medicinal plants, food supplements or some of their metabolites in an individual manner has increased considerably, whether under medical recommendation or not.[7] It is believed that an alternative to improve the effects of DIC and others NSAIDs, especially in its chronic administration, is to use them in combination with natural products to produce less or no side effects. In this sense, preliminary preclinical studies have shown that the combination of metabolites of medicinal plants with some NSAIDs, as hesperidin with ketorolac[8] or metamizole[9] as well as curcumin or ursolic acid with diclofenac,[10, 11] increases the efficacy of these analgesics.
Quercetin (QUER) is one of the most abundant flavonoids in fruits and vegetables, such as berries, onions, apples and beverages as white tea.[12] It has also been isolated from several medicinal plants as Tilia americana var. mexicana, Ginkgo biloba L. and Hypericum perforatum L., among others[13, 14]; mainly in those used in traditional medicine pain relief such as Bridelia ferruginea Benth and Polygonum hydropiper L.[15] In addition, pharmacological assays QUER have demonstrated its antioxidant,[16] anti-inflammatory,[17] cytotoxicity,[18] antihypertensive[19] and antinociceptive properties.[20] In a preliminary study, QUER was the principal metabolite responsible in part of the antinociceptive activity of T. americana evaluated in an arthritic gout-pain model.[21]
Since the combination of metabolites of medicinal plants with NSAIDs has shown antinociceptive potentiation and that QUER, a product abundant in medicinal plants, has shown antinociceptive effects in the experimental arthritic pain. The aim of this study was to analyse its pharmacological interaction with DIC, one of the most used NSAID analgesics for arthritic pain relief, to know the kind of antinociceptive interaction of their combination in the model of inflammatory pain type gouty arthritis. Additionally, we analysed if the effect of this combination involves the inhibition of the COX, as the main mechanism of action of NSAIDs.
Method
Animals
Male Wistar rats (180–200 g) obtained from the Central Bioterium of the Faculty of Medicine of Universidad Nacional Autónoma de México (UNAM) were used in this study. Animals were kept in a room with controlled temperature and humidity (25°C and 45–65%, respectively), under a 12 h light/dark cycle, with water and food ad libitum. The experiments were carried out following Guidelines on Ethical Standards for Investigations of Experimental Pain in Animals,[22] and the Official Mexican Standard (NOM-062-ZOO 1999). The protocol was approved by the local Ethics and Research Committees of the Faculty of Medicine (FM/DI/054/2018) on 2 October 2018. All experiments were carried out between 7:00 am and 2:00 pm. Each animal was used one time and was sacrificed after experiment by cervical dislocation.
Compounds
Diclofenac sodium salt (DIC), quercetin (QUER), uric acid and mineral oil were purchased from Sigma–Aldrich (St. Louis, MO, USA). Suspension of uric acid (30%) was prepared with mineral oil and used to induce nociception. DIC was prepared in a 0.9% saline solution (SS) and QUER was suspended using two drops of Tween 80 in SS. DIC and QUER solutions were freshly prepared on the day of the experiments and administered in a volume of 0.1 ml/100 g body weight rats. Control animals received the same volume of either vehicle or SS.
Pain-induced functional impairment in the rat model
For the induction of pain, we used the ‘pain-induced functional impairment in the rat’ model (‘PIFIR’),[23] which consists of intra-articular administration of 50 µL of uric acid (30%) into the knee joint of the right hind limb of the rats and the dysfunction that it produces. After uric acid administration, electrodes were attached to the plantar surface of each hand paw of the animal and they were placed in a steel cylinder of 30 cm of diameter, which was rotated at a speed of 4 turns per minute for 2 min, forcing the animals to walk. Electrodes were connected to an interface (OmniAlva's OASDA System) to record the contact time of each hind paw with the cylinder and calculate the functionality index (FI) expressed as a percentage. It was calculated with the contact time of the right paw between the contact time of the left paw multiplied by 100. Two hours after the administration of uric acid, a painful and inflammatory process was developed in the injected paw, which stopped the animal using that limb for walking, registering a %FI below 10%.
Experimental protocol
The experimental protocol consisted of two sets of experiments. In the first set, a dose–response curve for each individual drug was built using a logarithmic increase among doses to evaluate the antinociceptive effect. For this, DIC was tested at doses of 0.1, 0.31, 1.0, 3.1 or 10 mg/kg, the same doses previously reported in the PIFIR model.[23] Whereas for QUER, it was required to establish the range of doses to produce its maximal effect, a screening was performed based on the antinociceptive effects showed in a preliminary study.[24] Thereby, four doses of QUER (31, 100, 316 or 562 mg/kg) were tested by intraperitoneal administration (ip) and three doses (316, 562 or 1000 mg/kg) were assayed by oral administration (po) using different groups of rats. In the second set of experiments and according to the results of the antinociceptive effects of the individual drug administration, four doses of DIC (0.1 to 3.1 mg/kg, po) and three doses of QUER (31 to 316 mg/kg, ip) were chosen to be simultaneously administered to evaluate the antinociceptive effect of a total of 12 combinations of DIC + QUER: 0.1 + 31.6, 0.1 + 100, 0.1 + 316.2, 0.31 + 31.6, 0.31 + 100, 0.31 + 316.2, 1.0 + 31.6, 1.0 + 100, 1.0 + 316.2, 31.6 + 31.6, 31.6 + 100 and 31.6 + 316.2 mg/kg. Each dose of the individual drug or combination was given to six animals 2 h after uric acid administration, when the FI was below 10%. The antinociceptive effects were recorded every 15 min for the first hour, and every 30 min for 3 h more. The recovery of FI after administration of each treatment was considered as antinociceptive effect. To know the kind of interaction induced by DIC + QUER, the antinociceptive effect obtained with each combination was compared with the antinociceptive effect expected, which was obtained considering the sum of the effects of DIC and QUER administered individually in the respective combination. To determine the antinociceptive effect of the different treatments 144 rats were used.
Determination of cyclooxygenase activity after treatments
A total of 24 rats were administered with 50 µL of uric acid (30%) in the right paw and randomly divided into four groups to receive the following treatment 2 h later: (1) group administered with SS (control), (2) group DIC (3.16 mg/), (3) group QUER (316 mg/kg) and (4) group DIC + QUER. Animals were anesthetized with sodium pentobarbital (40 mg/kg, ip) and their caudal veins were cannulated. Blood samples of 0.5 ml were obtained from the 12 animals at 1 and 3 h after treatments. The serum of each blood sample was separated by centrifugation at 14 706 g for 5 min and stored at −20°C. COX activity was measured using an Elisa assay kit (Cayman Chemical, Ann Arbor, Michigan, USA, No. 760151). The peroxidase COX activity was obtained by a colorimetric assay monitoring the appearance of oxidized N,N,N′,N′, tetramethyl-p-phenylenediamine (TMPD) at 590 nm. The kit included isozyme-specific inhibitors for distinguishing COX-2 from COX-1 activity, DuP 697 (60 µ□, inhibitor of COX-2) and SC560 (66 µm, inhibitor of COX-1). Each sample was assayed by triplicate according to the instructions of the maker.
Statistical analysis
The percentage of the antinociceptive effect of each treatment was obtained from the temporal course curves (TCC) of a 4 hours-period by recording data every 15 min for the first hour and every 30 min for 3 h more. To build the dose–response plot, the area under the curve (AUC) of each treatment was obtained from TCC using the trapezoid addition method.[25]
To analyse the antinociceptive interaction of DIC plus QUER, we compared the AUC of the combination to the AUC of the expected effect. The AUC expected was calculated with the theoretical sum of AUC of the individual effects of DIC and QUER. A supra-additive or synergistic response was considered when the AUC value of the combination was higher than the one expected. If the AUC value of the combination was similar to the theoretical sum, the interaction was additive. Furthermore, an AUC value of the combination less than the expected indicated an infra-additive or antagonic interaction.[26] The effects of each treatment were analysed using six animals in each group and the results are expressed as the average ± standard error of the mean (SEM).
The sample size for each group was calculated considering the average values of the different experimental groups, which refers to the estimate of the value that could represent a minimal significant relief with clinical relevance (20% antinociceptive effect) in comparison with a lack of relief of 5% (μ2−μ1 = 15); the standard deviation (SD = 9) is a representation of the variability that we expect to find between subjects with the same treatment; a significant level of 95% corresponds to an α = 0.05 (Zα/2 = 1.96) and, a statistical power of 80% (Zβ = 0.84). The P < 0.05 represents 5% of the probability that the observed difference is due to chance; and, the power of the test (80%) represents the probability of observing a statistically significant difference between the treatments. With these parameters and the following formula,[27] an n = 5.6 was calculated; for which six animals were used per experimental group.
Considering that 29 groups of animals (6 animals per group) were used, the total number of animals used in this study was one hundred and seventy-four.
Statistical differences between treatments in the TCC were determined using a two-way analysis of variance (ANOVA) followed by Dunnett's test in comparison to the effect of DIC in individual administration at each time. Statistical differences between treatments in the dose–response curves were determined by a one-way analysis of variance (ANOVA) followed by Dunnett's test in comparison to the effect of vehicle (VEH). To compare the AUC values of combination versus expected, we used the Student's t-test. In all cases, the significance level was P < 0.05.
Results
Antinociceptive effects of diclofenac or quercetin in individual administration
Intra-articular administration of uric acid-induced a progressive diminution of the functionality of animals. When the %FI reached the zero value (approximately 2 h after uric acid administration), oral administration of DIC produced a dose-dependent antinociceptive effect that reached a maximum effect (Emax) of 69.7 ± 2.7% with a dosage of 3.1 mg/kg and an adequate-effective dose 50 (ED50) calculated as 1.7 ± 0.45 mg/kg. On the other hand, QUER orally administered did not produce any antinociceptive effect. In contrast, QUER intraperitoneally administered produced a lower but significant antinociceptive effect to the doses of 100 and 316 mg/kg (9.3 ± 3.2 and 17.6 ± 2.6%, respectively). Although, it was not possible to calculate their ED50 (Figure 1). To analyse the antinociceptive response of combinations, we administered QUER by intraperitoneal way, since oral administration did not produce antinociceptive response.
Dose–response curves of the antinociceptive effect of DIC (po) and QUER (po and ip). Each point represents the average of six rats ± SEM. * P < 0.05 versus VEH, one-way ANOVA followed by a Dunnett's test.
Antinociceptive effects of diclofenac + quercetin combination
Figure 2 shows temporal courses of the antinociceptive effect of DIC individually administered or in combination with several doses of QUER. A dosage of 0.1 mg/kg of DIC in individual administration reached an Emax of 28.3 ± 5.0% at 30 min, which did not change in the course of the entire experiment. This effect remained in the presence of 31.6 mg/kg of QUER (28.3 ± 5.0% versus 26.8 ± 6.8), but it was abolished in the presence of doses of 100 and 316 mg/kg of QUER (28.3 ± 5.0% versus 3.9 ± 1.0 and 3.3 ± 1.8%, P < 0.05) (Figure 2a). Also, the antinociceptive effect of 0.31 mg/kg of DIC in individual administration at 30 min was significantly reduced in combination with the three doses of QUER (31.6, 100 and 316 mg/kg) (from 23.6 ± 4.8 to 2.7 ± 1.7, 6.1 ± 2.6 and 1.9 ± 0.6%, respectively; P < 0.001) at 30 min and almost at all times, except between 45 and 180 min, where the effect of DIC alone did not show a significant difference in the presence of 316 mg/kg of QUER (23.5 ± 5.9 versus 22.9 ± 6.9 at 45 min and 11.1 ± 1.8 versus 3.4 ± 2.7% at 180 min) (Figure 2b). Regarding the effect of 1.0 mg/kg of DIC, it reached an Emax of 41.0 ± 5.9% at 45 min after its individual administration, which was maintained during the 3 h following its evaluation. In this case, only doses of 31.6 and 316 mg/kg of QUER significantly decreased the antinociceptive effect of DIC in almost all times (from 41.0 ± 5.9 to 22.0 ± 7.3 and 17.8 ± 4.6 at 45 min, P < 0.05; and from 46.7 ± 1.6 to 13.8 ± 5.0 and 11.9 ± 2.4 at 4 h, respectively, P < 0.001) (Figure 2c). Finally, 3.1 mg/kg of DIC reached an Emax of 72.2 ± 4.0 at 45 min after its individual administration, which remained throughout the evaluation period (74.2 ± 1.5 at 4 h) and; when it was administered at 100 or 316 mg/kg of QUER, its antinociceptive effect significantly diminished from 30 min of evaluation (from 60.9 ± 10.4 to 27.5 ± 5.1 and 30.9 ± 8.4%, P < 0.01 and P < 0.05; respectively); while, the combination of this DIC dose with 31.6 QUER only diminished its effect after 2.5 h of its administration (from 73.2 ± 5.0 to 39.1 ± 11.7%, P < 0.01) (Figure 2d).
Temporal courses of the antinociceptive effect of 0.1 (a), 0.31 (b), 1.0 (c) and 3.1 mg/kg (d) of DIC in individual administration and in the presence of 31.6, 100 or 316 mg/kg of QUER. The data are expressed as an average of six rats ± SEM. * P < 0.05 versus DIC in individual administration at each time, two-way ANOVA followed by a Dunnett's test.
Figure 3 shows the analysis of the antinociceptive effect during 4 h of DIC (0.1, 0.31, 1.0 and 3.1 mg/kg) and QUER (31.6, 100 and 316 mg/kg), and their combinations. The first bars show the antinociceptive effect of DIC and QUER in individual administration and represent the sum of the antinociceptive effect of both drugs that was the expected effect. Whereas, the second bars show the antinociceptive effect obtained with the co-administration of both drugs in their respective doses. The co-administration of 31.6 mg/kg of QUER with DIC (0.31, 1 and 3.1 mg/kg) showed less antinociceptive effect than the expected (2.2 ± 0.9 versus 20.0 ± 2.5%, 13.9 ± 3.3 versus 41.1 ± 3.5%, 43.0 ± 7.2 versus 73.7 ± 4.0%, respectively; P < 0.05) (Figure 3a). The co-administration of 100 mg/kg of QUER with DIC (0.1, 0.31 and 3.1 mg/kg) also showed less antinociceptive effect than the sum of the individual effects of each drug (1.9 ± 1.6 versus 26.5 ± 8.0%, 5.1 ± 2.6 versus 25.3 ± 4.5%, 39.9 ± 8.5 versus 79.1 ± 5.9%, respectively; P < 0.05) (Figure 3b). Finally, the co-administration of 316 mg/kg of QUER with DIC (0.1, 0.31, 1 and 3.1 mg/kg) also induced less antinociceptive effect than the expected (0.7 ± 0.4 versus 35.3 ± 7.4%, 2.0 ± 1.0 versus 33.6 ± 3.9%, 10.3 ± 3.6 versus 54.7 ± 4.8%, 20.3 ± 7.2 versus 87.3 ± 5.3%, respectively; P < 0.05) (Figure 3c). In general, ten of the twelve DIC–QUER combinations (0.31 + 31.6, 1.0 + 31.6, 3.1 + 31.6, 0.1 + 100, 0.31 + 100, 3.1 + 100, 0.1 + 316, 0.31 + 316, 1.0 + 316 and 3.1 + 316 mg/kg, respectively) showed less antinociceptive effect than the expected. Only in two combinations of DIC + QUER (0.1 + 31.6 and 1.0 + 100 mg/kg, respectively), the antinociceptive effects obtained did not show significant difference to the antinociceptive effect expected with the corresponding combinations (18.1 ± 3.8 versus 21.7 ± 6.1% and 36.8 ± 5.8 versus 46.4 ± 5.4%, respectively) (Figure 3a and b). Combinations of DIC with QUER that showed more than a 70% diminution in comparison with the effect of DIC alone were 0.31 + 31.6 (87.5%), 0.1 + 100 (90%), 0.1 + 316 (96%), 0.31 + 316 (87%), 1 + 316 (73%) and 3.1 + 316 mg/kg (71%).
Antinociceptive effect of the administration of different doses of DIC and QUER 31.6 mg/kg (a), 100 mg/kg (b) or 316 mg/kg (c) in individual administration and their combinations. In the first bars, the effects of DIC or QUER in individual administration are showed together and represent the expected effect; while in the other bars, the effect of each combination is showed and it represents the obtained effect. Each bar represents the average of six rats ± SEM of the AUC obtained of the temporal course the antinociceptive effect of each treatment during 4 h of evaluation. * P < 0.05, Student's t-test.
Determination of cyclooxygenase activity with one combination of diclofenac + quercetin
The recovery in the FI% (antinociceptive effect) of animals treated with 3.1 mg/kg of DIC alone was related to a diminution of total COX and COX-2 activity on the serum samples at 1 and 3 h after administration of treatments in comparison with that of the control group (Figure 4). QUER alone (316 mg/kg) induced a recovery in the FI% only at 3 h (26.8 ± 5.0%) (Figure 4a), this effect was related to a diminution of activity of total COX (from 77.9 ± 1.1 to 63.6 ± 1.6, P < 0.05) (Figure 4b) at the same time, but not with activity of COX-2 (58.1 ± 2.4 versus 60.9 ± 1.3) (Figure 4c), which did not diminish at any time. Finally, the combination of DIC + QUER showed recovery in the FI% but much less than the effect induced by DIC alone (26.7 ± 3.7 versus 71.4 ± 3.2% and 37.4 ± 7.9 versus 76.4 ± 3.1, P < 0.05) (Figure 4a) at 1 and 3 h, respectively. This effect of the combination was related to a significant diminution of activity of total COX (Figure 4b) only at 3 h (from 77.9 ± 1.1 to 68.3 ± 1.1, P < 0.05), but not with the activity of COX-2, which did not change at any time (60.1 ± 1.0 versus 63.2 ± 3.0 at 1 h and 58.1 ± 2.4 versus 56.6 ± 1.3 at 3 h) (Figure 4c).
(a) Functionality index (%), (b) activity of total-COX and (c) activity of COX-2 of control, DIC (3.1 mg/kg), QUER (316.2 mg/kg) and DIC + QUER groups at 1 and 3 h after administrations of each treatment. Each bar represents the average of six measurements ± SEM for treatment. * P < 0.05 versus their respective control group at each time, two-way ANOVA followed by a Dunnett's test.
Discussion
In this study, the pharmacological interaction produced by the co-administration of DIC + QUER was determined and compared with the antinociceptive effect of DIC alone and the sum of each drug's effects in the PIFIR model. In this model, an acute inflammatory pain accompanied by dysfunction of the limb is induced when a suspension of uric acid is administered in rats, similar to that reported by patients suffering of gouty arthritis.[23]
Under our experimental conditions, DIC produced antinociceptive effects resembling those previously reported in this model,[24] and observed in the formalin and writhing tests as nociceptive and abdominal pain models, respectively.[10, 28] It is well-known that the main mechanisms of action of DIC is by the inhibition of cyclooxygenase (COX), the enzyme responsible for synthesizing prostaglandins (PGs) from arachidonic acid, with the consequent diminution in the synthesis of these prostanoids that participate as mediators of pain and inflammation. In general, DIC is well-tolerated and produces adequate clinical efficacy mainly due to its higher selectivity for COX-2 than for COX-1.[5, 29] Since COX-2 is the isoform responsible for synthesizing the PGs that participate in pain after tissue damage, while COX-1 synthesizes the PGs involved in the homeostasis of the organism.[30] However, in chronic administration, DIC can also induce adverse effects such as gastrointestinal lesions or cardiovascular complications.[5, 6]
In the case of QUER, it has been reported its significant antinociceptive effects assayed in acute pain models, such as the formalin, writhing[20] and tail-flick[31] tests. In contrast, QUER did not produce antinociceptive effects after enteral administration at doses tested in the PIFIR model. Whereas it produced a significant but poor antinociceptive response using parenteral administration. Efficacy of QUER appears to depend on the route of administration, but also on the experimental model used to induce pain-like behaviour. In acute pain, treatment of QUER was given previous to the algogenic stimulus, whereas, in our study, treatment was given after the painful and inflammatory process was already installed (2 h after uric acid administration). In a similar manner, the intensity of pain might play a role in the antinociceptive efficacy of QUER like in the hot plate test, where this flavonoid was not effective to reduce nociception.[32] The lack of effect obtained in the presence of QUER after enteral administration suggests its low oral bioavailability because of its physicochemical characteristics and the first-step effect.[33] Therefore, a parenteral administration was decided to improve its bioavailability and reduce its possible interaction with DIC.
Anti-inflammatory efficacy of QUER was preliminarily demonstrated in a model of gouty arthritis by reducing inflammatory mediators (IL-1β, TNF-α, COX-2 and PGE2) in serum, liver and synovial tissue.[17] Nevertheless, it protected cartilage and bone from collagen-induced damage in a mouse arthritis model.[15] Thus, despite its low bioavailability, the anti-inflammatory efficacy of this flavonoid has been compared with that of NSAIDs[34] suggested as a good candidate to study its interaction with DIC in a model of arthritic pain.
The DIC + QUER combination has been preliminarily studied showing advantages in comparison to the efficacy of DIC alone in an inflammatory condition, since it decreased joint damage induced in the collagen arthritis model.[35] Also, QUER decreased DIC-induced gastric ulcers more effectively than famotidine, an H2 antagonist used as a gastroprotector.[36] However, the antinociceptive interaction of this combination had not been studied until now. Our results showed that QUER in acute administration decreased the antinociceptive effect of DIC suggesting an antagonic interaction for this effect. These results surprised us since we expected the combination of DIC + QUER to produce a supra-additive antinociceptive interaction, as in other experimental pain models or in combination with other metabolites of medicinal plants such as curcumin or xylopic acid with DIC,[10, 28] as well as combinations of flavonoids (hesperidin and rutin) with other NSAIDs (ketorolac, metamizole and naproxen, respectively),[8, 9, 37] which have shown supra-additive effects. However, QUER + DIC interaction resembled the results of ursolic acid combined with tramadol, another combination that produced an infra-additive or antagonic effect.[11] It is clear that there is no certainty that combinations of natural products derived from medicinal plants and clinical drugs used together will always produce beneficial effects since these can alter the effect of the drug through a pharmacodynamic or pharmacokinetic mechanism,[38] so it is necessary to analyse the pharmacological interaction of each combination.
The possible explanation of an antagonic effect induced by the combination of two drugs could be by pharmacodynamic or pharmacokinetic mechanisms. Regarding the pharmacodynamic mechanism, it is known that two drugs can show a pharmacological effect of antagonism if both act on the same mechanism of action, since they saturate the same pathway.[39] In the case of DIC + QUER combination, this hypothesis is difficult to demonstrate because the analgesic efficacy of both drugs involves different mechanisms. For example, in the analgesic effect of DIC, the participation of endogenous opioids[40] and the activation at the peripheral level of the l-arginine-NO-cGMP-K+ [41] pathway have been reported. While in the antinociceptive effect of QUER, the participation of these pathways might not be involved.[20] The antinociceptive effect of QUER seems to be by its modulation on NMDA receptors, as well as by inhibition of pro-nociceptive cytokine production in inflammatory pain.[42]
Such the main mechanism of DIC is the inhibition of COX-1 and COX-2,[5] and QUER has shown the participation of COX-2,[17] we explored the effect of DIC, QUER and their combination on the total COX and COX-2 activity. The results of this test showed that DIC diminished the COX-total and COX-2 activity in serum of rats, suggesting that in the antinociceptive effect of DIC both COX-1 and COX-2 are involved; while QUER diminished only the total-COX activity at 3 h but not the COX-2 activity, suggesting that the poor antinociceptive effect showed by QUER at that time could be have been due only to a partial inhibition of COX-1 activity. This is possible considering that in the PIFIR model used in this study, peripheral administration of both selective COX-2 and COX-1 inhibitors reduced uric acid-induced dysfunction, suggesting that both COX isoforms contribute to the development and maintenance of local inflammatory nociception induced in this model.[43] On the other hand, the combination of DIC + QUER diminished only the total-COX activity but not the COX-2 activity, suggesting that acute administration of QUER avoids the inhibitory effect of DIC on the COX-2 activity maybe by a stimulating effect on this isoenzyme. This could explain the reduction in the antinociceptive effect of DIC in the presence of QUER, and the antagonist effect showed by the combination in comparison with the antinociceptive activity of DIC alone. This agrees with an in vitro study where QUER shows a direct stimulatory effect on the catalytic activity of COX-2, increasing the formation of prostaglandins as PGE2.[44] The molecular mechanism by which flavonoids stimulate the catalytic activity of COX-2 is not clear; however, Bai and Zhu[44] suggest that the COX rapidly undergo suicidal inactivation, which is likely mediated by reactive radical intermediates formed during enzymatic catalysis of cyclooxidation and peroxidation; flavonoids mainly act to slow down the suicidal inactivation of the COX enzymes, but they cannot reactivate the inactive enzymes.
Also, a pharmacokinetic interaction between QUER and DIC could be considered. It is known that QUER inhibits several enzymes of the cytochrome family, including the CYP2C9 isoform,[45] which is responsible for metabolizing DIC in 4-hydroxy-diclofenac.[46] This metabolite of DIC comprises between 30% and 40% of the biotransformation products of DIC and is responsible for about 30% of the analgesic, anti-inflammatory and antipyretic activity of the original compound.[47] In one study, the simultaneous administration of QUER + DIC produced a significant diminution in Cpmax and AUC0-∞ of 4-hydroxy-diclofenac,[48] suggesting that QUER decreased the formation of the active metabolite of DIC. Therefore, the decrease in the antinociceptive effect of DIC when administered in combination with QUER, as observed in this study, could be due to the inhibition of CYP2C9 induced by QUER that prevents the hydroxylation of DIC and the subsequent formation of 4-hydroxy-diclofenac. Overall, an alteration in the pharmacokinetic parameters of DIC after the administration of natural compounds such as diosmin,[49] resveratrol[50] and piperine[51] have been attributed to the inhibitory effect that these natural compounds induce in the CYP2C9 enzyme.
The main strength of this study is that antinociceptive effect of DIC alone and in combination with QUER was tested in a pain model with characteristics of predictability, construction and appearance, since it is installed before treatment as in the disease and respond to the presence of anti-inflammatory drugs just like that observed in the clinic, unlike other models in which treatment is administered before the algogenic agent or nociceptive stimulus. Moreover, in this study, ten of 12 combinations of several doses of DIC + QUER produced lesser effects than those with DIC alone suggesting an antagonic effect between the interaction of these two molecules. It is important to mention that this study might have limitations, since our experimental design did not allow to explain if interactions involve pharmacodynamic or pharmacokinetic mechanisms. Thus, other studies are required to determine the precise mechanisms involved in the unbeneficial interaction of QUER on the antinociceptive effect of DIC. Also, it is necessary to study the antinociceptive effect of the DIC + QUER combination in other experimental models to ensure that the antagonic effect will be reproduced.
Conclusion
In conclusion, this study gives preclinical evidence that a combination of DIC + QUER produces unfavorable interaction on the antinociceptive effect of DIC alone in gout arthritis. The antagonistic interaction might be in part mediated by avoiding its mechanism of action on the COX-2 activity. These results suggest that the combination of this flavonoid (QUER) and DIC is not a functional alternative in the arthritic pain therapy suggesting that patients who use DIC effectively for the inflammatory pain relief should not use dietary supplements containing QUER.
Acknowledgements
We thank M. in Cs. Mariana Y. Hernández and Miguel A. Zumaya for their Technical assistance and to Mrs. Josefina Bolado (Head of the Scientific Paper Translation Department, from División de Investigación at Facultad de Medicina UNAM) for reviewing the English-language version of this manuscript. A.B.M. thanks the fellowship (No. 78097) granted by Council for Science and Technology (CONACYT).
Funding
This work was supported by the Program for Technological Research and Innovation Projects UNAM-DGAPA-PAPIIT (grant number IN201820).
Author Contributions
R.V.M. designed the study, interpreted the data and wrote the manuscript. M.D.C. and G.E.A.L. supervised the study. A.B.M. and J.A.H performed the experiments, collected and evaluated the data. M.E.G.T. and G.N.V. participated in draft the article. All authors critically revised the manuscript for important intellectual content and approved the final draft.
Ethical Statement
The study was approved by the local Ethic and Research Committees of the Faculty of Medicine of the National Autonomous University of Mexico (FM/DI/054/2018) on October 2, 2018. The experiments were carried out following Guidelines on Ethical Standards for Investigations of Experimental Pain in Animals (Zimmermann, 1983), and the Official Mexican Standard for the production, care and use of laboratory animals (NOM-062-ZOO 1999).
Conflict of interest
The authors declared that they have no conflicts of interest to disclose.
References
