Study on the anti-inflammatory effects of clavulanic acid on carrageenan induced paw edema in rats

Abstract

Clavulanic acid is a β-lactamase inhibitor isolated from Streptomyces clavuligerus. The resemblance of chemical structure in clavulanic acid and beta-lactam antibiotics allows the interaction of clavulanic acid with the enzyme β-lactamase that is secreted by certain bacteria. Previous studies confirm that there are tertiary structural similarities between β-lactamase and D-alanyl-D-alanin carboxypeptidase-transpeptidase. Because of such structural similarity, β-lactamase inhibitors can inhibit carboxypeptidase in that manner in mammals. As previous studies confirmed contribution of glutamate in inflammatory responses, the present investigation was based on the assumption that GCP-II could be inhibited by β-lactamase inhibitors to exhibit anti-inflammatory effects by reducing the level of glutamate in peripheral and central nervous systems. We suggested that clavulanic acid known for its activity as a β-lactamase inhibitor was also an inhibitor of mammalian GCP-II (NAALADase) activity and reduced the level of glutamate in peripheral and central nervous systems. Since glutamate contributes to the inflammatory processes and NAAG reduces the level of glutamate in the peripheral and central nervous systems by acting at mGluR3, we aimed at evaluating the protective effects of clavulanic acid in a rat model of acute local inflammation (carrageenan-induced paw edema). NAAG was used as a reference drug and the effect of clavulanic acid on the inflammatory and biochemichal factors in the carrageenan induced rat paw edema model was compared to that of NAAG in the paw tissue of rats. The present study was designed to evaluate the anti-inflammatory effects of clavulanic acid, as a GCP-II inhibitor on carrageenan-induced paw edema in rats.

Methods:

Clavulanic acid was administered intraplantary (25, 50, 100, 150 and 200 µg/kg) and intraperitoneally (100, 200 or 300 mg/kg) and NAAG (10 mg/kg) [as reference drug], was administered intraperitoneally 20 min before the intraplantar injection of the carrageenan 1% w/v. The volume of the paw was measured immediately prior to the carrageenan injection and then at hourly intervals from 1 to 4 hours afterward. Data were expressed as a percentage of increase in the paw volume and compared with those of pre-injection values. Neutrophil infiltration (MPO activity) was assessed in the inflamed paw tissue of intraplantar or intraperitoneal clavulanic acid treated rats and intraperitoneal NAAG treated rats and compared with control group. Lipid peroxidation (MDA assay), free radical scavenging activity (SOD and GPx assay), IL1β, TNF-α and PGE2 levels were assessed in the paw tissue of intraperitoneal clavulanic acid treated rats or intraperitoneal NAAG treated rats and compared with that of control rats. Histological studies were carried out to compare the microscopic structure of inflamed paw tissue of intraperitoneal clavulanic acid treated rats with that of control rats.

Results and Discussion: Intraperitoneal clavulanic acid caused a potent and dose-related inhibition of the carrageenan-induced inflammatory response. Results showed that intraplantar clavulanic acid (100 or 150 µg/kg), intraperitoneal clavulanic acid (200 or 300 mg/kg) or intraperitoneal NAAG (10 mg/kg) significantly reduced maximum paw volume at the 4th hour respectively by 46.87%, 48.65%, 54.51%, 59.81% and 52.57% (p<0.05), (p<0.01), (p<0.001) and total inflammatory response (AUC) during 4 hours respectively by 41.3%, 35.67%, 53.17%, 59.13% and 56.9% in comparison with that of control group (p<0.05), (p<0.01), (p<0.001). Intraplantar clavulanic acid (100, 150 µg/kg), intraperitoneal clavulanic acid (100 or 200 mg/kg) or intraperitoneal NAAG (10 mg/kg) treated rats resulted in a marked reduction of MPO activity in the inflamed paw respectively by 51.15% , 43.9%, 79%, 82% and 80.19% in comparison with that in the control group (p<0.05), (p<0.01), (p<0.001). Moreover, intraperitoneal clavulanic acid (200 mg/kg) or NAAG (10 mg/kg), reduced the MDA levels respectively by 53.22% and 54.3%, in comparison with that in the control group (p<0.01), (p<0.001). SOD activity in NAAG (10 mg/kg), treated group was increased up to 25% in comparison with that in the control group (p<0.05). Treatment by intraperitoneal clavulanic acid (200 mg/kg) increased SOD activity slightly but not significantly. Intraperitoneal NAAG (10 mg/kg) or clavulanic acid (200 mg/kg), didn’t have any significant effect on GPx activity. The levels of IL-1β in the intraperitoneal clavulanic acid (300 mg/kg) or NAAG (10 mg/kg) treated rats, were reduced by 61.2% and 72.4%, respectively in comparison with the control group (p<0.05), (p<0.01). TNF-α level in clavulanic acid (300 mg/kg) treated rats was reduced by 35.15% in comparison with that in the control group (p<0.05), but NAAG (10 mg/kg) didn’t have any significant effect on TNF-α level. PGE2 level in clavulanic acid (200 or 300 mg/kg) treated rats was reduced by 12.77% and 13.28% respectively (p<0.05) and in NAAG (10 mg/kg) treated rats was reduced by 13.9% in comparison with that in the control group (p<0.05). Data showed that administration of clavulanic acid (200 or 300 mg/kg) or NAAG (10 mg/kg) significantly reduced PGE2 level in comparison with that of control group.

It has been previously mentioned that clavulanic acid, a recognized β-lactamase inhibitor, is also a potent inhibitor of GCP-II. In accordance with the hypothesis of the present study, clavulanic acid, a β-lactam containing compound which is an effective β-lactamase and peptidase inhibitor, can function in neurochemical mediation of glutamatergic pathways and has the ability of reducing inflammatory responses. In the present study, the authors clearly demonstrated that the intraperitoneal injection of clavulanic acid (200 or 300 mg/kg) attenuated the peripheral inflammatory response induced by subplantar carrageenan injection. Inhibition of the NAAG-peptidase (GCP-II) reduces inactivation of NAAG, increases the levels of NAAG and decreases the levels of glutamate. We suggest that the anti-inflammatory response seen by clavulanic acid is related to the action of clavulanic acid on GCP-II inhibition resulting in elevation of NAAG level and reduction of glutamate level increase in the peripheral afferent nerve terminals. Clavulanic acid was injected 20 minutes prior to the induction of inflammation by carrageenan and its significant anti-inflammatory effect was detectable at both phases of the carrageenan induced inflammation model. In accordance with our data, we showed for the first time that clavulanic acid was an effective in vivo inhibitor of the neutrophil infiltration; as determined by the MPO levels in the inflammatory paw tissue. We demonstrated that paw tissue concentration of MDA, an end product of lipid peroxidation and free radical formation, was significantly decreased by intraperitoneal administration of clavulanic acid (200 mg/kg) and the result was similar to that of NAAG as reference drug. Reduction of MDA level observed in inflamed paw tissues indicated the ability of clavulanic acid to attenuate the oxidative stress. Prostaglandins and interleukins are produced during inflammation and interact with their respective receptors located on the peripheral terminals of primary afferents. Prostaglandin E2 (PGE2) is one of the most important metabolites of arachidonic acid generated through an enzymatic cascade controlled by cyclooxgenase (COX) enzymes. PGE2 elicits a wide range of inflammatory responses including increase of vascular permeability. The significance of this role is emphasized by the broad clinical administration of cyclooxgenase inhibitors to reduce inflammation in a variety of inflammatory disorders. We found that intraperitoneal clavulanic acid (200 or 300 mg/kg) significantly reduced the levels of PGE2 in inflamed paw tissue and the results were similar to that of NAAG as reference drug. Agents derived from the inflamed tissue, such as bradykinin, stimulate the release of TNF-α which in turns stimulates the release of IL-1β. IL-1β promotes the release of COX enzymes, which converts arachidonic acid to prostaglandins. Herein, we report that injection of the clavulanic acid reduces TNF-α and IL-1β levels in the paw tissue. Four hours after the challenge, the levels of TNF-α were reduced coincident with the significant reduction of the IL-1β and PGE2 levels. At the 4th hour time point, when TNF-α, IL-1β and PGE2 were inhibited, MPO activity was also reduced significantly in comparison with that in the control group.

Conclusion: This experiment showed that the carrageenan successfully induced edema in the paw. Clavulanic acid reduced the paw volume; the responses were great at both phases of the inflammation. MDA level was reduced by clavulanic acid. We found that the pattern of proinflammatory mediator concentration in the paw tissue changed by clavulanic acid was correlated with the number of infiltrating cells. Overall we suggest that the anti-inflammatory properties of clavulanic acid could be related to its inhibitory effect on GCP-II activity results in elevating NAAG level and reducing glutamate level increase.