RETRACTED ARTICLE: Release-Active Dilutions of Diclofenac Enhance Anti-inflammatory Effect of Diclofenac in Carrageenan-Induced Rat Paw Edema Model

The study was aimed to investigate the effect of technologically treated diclofenac (release-active dilutions of diclofenac (RAD of diclofenac)) on anti-inflammatory activity of diclofenac in carrageenan-induced rat paw edema model. Ninety male Wistar albino rats (6–8 weeks) divided into nine groups (n = 10) were used. Anti-inflammatory activity was assessed at 1, 2, 3, 4, and 6 h after subplantar injection of carrageenan (0.1 ml of a 1 % solution in normal saline). Diclofenac alone was studied at 5 and 20 mg/kg, RAD of diclofenac alone at 7.5 ml/kg and their combination at 5 and 7.5 ml/kg, respectively. Diclofenac reduced (p < 0.05 at least) paw edema at all time points. RAD of diclofenac enhanced (p < 0.05) anti-inflammatory effect of diclofenac (5 mg/kg) at 2, 4, and 6 h on concurrent and at 2 and 4 h on sequential administration. Moreover at 2 h, anti-inflammatory effect of combination treatment reached values comparable to those of diclofenac (20 mg/kg). In conclusion, RAD of diclofenac enhanced anti-inflammatory effect of diclofenac.


INTRODUCTION
Diclofenac belongs to nonsteroidal anti-inflammatory class of drugs (NSAIDS) and it is the most well-known NSAID globally [1]. Diclofenac produces analgesic, antipyretic, and anti-inflammatory effects and is widely used for the treatment of moderate pain and inflammation, which are common symptoms of various diseases [1]. Diclofenac effect is mediated by inhibition of cyclooxygenase (COX), an enzyme converting arachidonic acid into prostaglandins (PGs), thromboxanes, and prostacyclins. Due to adverse effects, diclofenac should be used in at the lowest dose and for the shortest term possible [1]. Thus, many attempts were made to develop an approach of combining diclofenac with different agents which could increase sensitivity to diclofenac and decrease its dose [2][3][4][5][6][7][8][9][10][11].
One such approach might consist in using releaseactive therapeutics with their effect mediated by specifically processed ultradilutions of the starting substance [12]. The efficacy of such technologically treated compounds, releaseactive dilutions (RAD), was shown for both antibodies and chemical substances [12][13][14]. RAD corresponds to a new class of therapeutics which demonstrated specific modulating activity [12]. Modifying effects of RAD were discovered in 1996 when it was established that combined application of prednisolone in therapeutic doses and its RAD to experimental animals alleviated toxic side effects and tended to enhance its anti-inflammatory activity [15]. Further studies confirmed this phenomenon. It was shown that modulating action of RAD of phenazepam significantly potentiated anxiolytic and anticonvulsant effects of therapeutic doses of phenazepam in Vogel conflict test and in rats with corazolinduced seizures, both after prior or simultaneous administration [16]. Combination of RAD of haloperidol and therapeutic doses of haloperidol significantly increased psychotropic neuroleptic effect of conventional doses of the drug administered preliminarily or simultaneously [17]. In addition, RAD of haloperidol served to reduce significantly cataleptogenic side effect of haloperidol.
Based upon the beneficial effects of RAD technology, the present study in carrageenan-induced paw edema test was designed to develop and investigate potential novel approach in altering anti-inflammatory activity of diclofenac by combining it with its release-active form.

Experimental Animals
The experimental design and use of animals were approved by Institutional Animal Ethics Committee (IAEC proposal no. IAEC/20/181) of Dabur Research Foundation, Ghaziabad, India. Animals (90 male Wistar albino rats (Rattus norvegicus) weighing 178-295 g (6-8 weeks)) were supplied by Animal Facility, Dabur Research Foundation and housed five animals per cage at 21.4-23°C and relative humidity between 52 and 58 %. They had access to food and water ad libitum and were exposed to alternate 12 h light and dark cycles.

Preparation of diclofenac release-active dilutions
RAD of diclofenac is a clear solution for oral use manufactured by ООО "NPF "MATERIA MEDICA HOLDING", Russia, using diclofenac sodium salt (Sigma-Aldrich, Cat No. D6899) as a starting substance. Release-active dilutions were manufactured using routine methods described in the European Pharmacopoeia (6th Edition, 2007) as described previously [12]. All ultrahigh dilutions were prepared in glass vials. Starting substance (diclofenac sodium salt) was dissolved in a solvent (ethanol-water solution) and shaken for 1 min to produce the first centesimal (C1) dilution. All subsequent dilutions consisted of one part of the previous dilution and 99 parts of solvent (ethanol-water solution for intermediate dilutions and distilled water for preparation of the final dilution) with succession between each dilution. Thus, RAD of diclofenac contain release-active dilutions of diclofenac, which was diluted up to receiving the mixture of final dilutions C12+ C30+C200. Solutions were prepared in sterile conditions avoiding direct intense light and stored at room temperature. Quality of RAD of diclofenac was confirmed by quality control of diclofenac substance fulfilled in accordance with the requirements of the European Pharmacopoeia monograph 'Diclofenac sodium' (01/2008:1002), quality control of incoming materials and excipients, as well as usage of validated manufacturing process.
In case of placebo, solvent (ethanol-water solution) was used to prepare ultrahigh dilutions instead of diclofenac powder using the same method described above. RAD of diclofenac and placebo were coded by manufacturer and used blinded in the study.

Experimental Design
In the present study, we investigated the antiinflammatory activity of RAD of diclofenac alone or in combination with diclofenac using carrageenan-induced paw edema test according to the modified method of Winter et al. [18]. Male Wistar rats were selected for the study based on available literature [19][20][21][22]. The animals were randomized based on their body weight and divided into nine different groups consisting of 10 animals each. Rats were given a subcutaneous (s.c.) injection of carrageenan (0.1 ml of 1 % solution in normal saline) into the plantar side of the left hind paw. The paw was marked with ink at the level of lateral malleolus. The paw volume was measured up to the mark using digital plethysmometer (LE 7500, Panlab, Harvard Apparatus) before (−1 h) and at 1, 2, 3, 4, and 6 h after injection of carrageenan for all the animals (Fig. 1). Paw edema volume was calculated by subtracting −1 h paw volume from the respective paw volumes at 1, 2, 3, 4, and 6 h. Similarly, percentage of anti-inflammatory activity was calculated for each animal using the following formula: paw edema of control (in milliliter) −paw edema of test (in milliliter)/paw edema of control (in milliliter) ×100 %.

Test Drug Treatment Schedule
For groups 1 and 2 (concurrent administration), diclofenac powder was dissolved in RAD of diclofenac (water solution) or placebo (water solution), respectively, in order to achieve the final concentration of diclofenac in the solutions equal to 5 mg/kg. For groups 3 and 4 (sequential administration), rats received 7.5 ml/kg of RAD of diclofenac or placebo, respectively, via gavage. After 10 min dosing, diclofenac was administered at the dose of 5 mg/kg. For groups 5 and 6, rats received 7.5 ml/kg of RAD of diclofenac or placebo, respectively, via gavage. For groups 7 and 8, rats received diclofenac at the dose levels of 5 and 20 mg/kg, respectively. Group 9 rats received 7.5 ml/ kg of 0.25 % Na-CMC solution via gavage. All diclofenac formulations were prepared in 0.25 % Na-CMC. Similarly, all the formulations were administered to the respective group of animals at the dose volume of 7.5 ml/kg. The groups, the drug treatment and carrageenan administration are summarized in Table 1.

Statistical Analysis
The data are expressed as mean±standard error of mean (SEM) for each group. Data of control group and the groups receiving test drugs were analyzed by one-way analyses of variance (ANOVA). In post hoc analysis, mean response of two groups was compared using Dunnett's t test. Data of the groups receiving test drugs were analyzed by generalized estimating equations. Calculation was carried out by GENMOD. A value of p<0.05 were considered statistically significant.

RESULTS
Subcutaneous injection of carrageenan in rats led to a time-dependent gradual enhancement of paw volume with A Combination of RAD of diclofenac (7.5 ml/kg) and diclofenac (5 mg/kg; diclofenac was administered concurrently with RAD of diclofenac), G2 combination of placebo (7.5 ml/kg) and diclofenac (5 mg/kg; diclofenac was administered concurrently with placebo), G5 RAD of diclofenac (7.5 ml/kg), G6 placebo (7.5 ml/kg), G7 diclofenac (5 mg/kg), G8 diclofenac (20 mg/kg), G9 control (0.25 % Na-CMC solution; 7.5 ml/kg). B G3 Combination of RAD of diclofenac (7.5 ml/kg) and diclofenac (5 mg/kg) (diclofenac was administered after 10 min of RAD of diclofenac), G4 combination of placebo (7.5 ml/kg) and diclofenac (5 mg/kg; diclofenac was administered after 10 min of placebo).  Fig. 2). Administration of RAD of diclofenac (7.5 ml/kg) for 1 h prior to carrageenan injection significantly (p<0.01) inhibited carrageenan-induced edema in rat paw at the 2 and 6 h time points as compared to control. There was a reduction in paw volume at the 3 and 4 h time points as well but the difference was insignificant (Table 2, Fig. 3). In contrast, administration of placebo did not prevent enhancement of paw volume at any time points (Table 3, Fig. 4).
Co-administration of RAD of diclofenac with low dose of diclofenac (5 mg/kg) enhanced inhibitory effect of diclofenac (Table 2, Fig. 3). Anti-inflammatory effect of a combination exceeded (p<0.05) that of diclofenac alone (5 mg/kg) at 2, 4, and 6 h in concurrent administration and at 2 and 4 h in sequential administration. More importantly, at 2-h time point, anti-inflammatory effect of the combinations reached values comparable to group of diclofenac alone at high dose (20 mg/kg). In addition, the effect of individual treatment with diclofenac (5 mg/kg) was significantly lower (p< 0.05 at least) than the one of diclofenac at high dose (20 mg/kg) at all time points. However, combined treatment with diclofenac (5 mg/kg) and RAD of diclofenac produced greater inhibition in paw edema, which was not significantly different from the effect of diclofenac at high dose (20 mg/kg) at 2 and 4 h after carrageenan injection for concurrent administration and at 2, 4, and 6 h after sequential administration. Coadministration of placebo did not have any effect on inhibitory activity of diclofenac (5 mg/kg) despite the fact of no significant difference in anti-inflammatory activity at 1 and 2 h after carrageenan injection as compared to diclofenac 20 mg/kg for concurrent and sequential administration, respectively (Table 3, Fig. 4).

DISCUSSION
Our primary aim was to assess whether antiinflammatory activity of diclofenac could be enhanced by RAD of diclofenac and, therefore, allow to decrease the dose of diclofenac or duration of its administration.
Among many methods aimed at screening of antiinflammatory drugs, one of the most useful techniques is based upon ability of such agents to inhibit edema produced in rat hind paw after injection of 1 % carrageenan. It has been widely used as a simple and reliable model to assess anti-inflammatory activity of various agents [19][20][21][22][23][24][25]. Also, inhibition of carrageenan-induced inflammation has been shown to be highly predictive of anti-inflammatory drug activity in human inflammatory disease and doses of NSAIDs in this model correlate well with effective dose in patients [26]. In addition, this model allows to detect orally active anti-inflammatory agent particularly in acute phase of inflammation [27]. The paw volume is measured before and after injection of carrageenan and the paw volume of treated animals is compared to the controls. Any substance having ability to reduce paw volume in this model potentially acts as an anti-inflammatory agent by inhibiting synthesis of release of inflammatory mediators. Suitability of this model in terms of assessment of diclofenac anti-inflammatory activity as well as possibility to increase diclofenac-induced anti-inflammatory response or maintain its efficacy but with reduction of adverts effects has been the subject of several experiments [2][3][4][5][6][7][8][9][10][11]. Also, diclofenac and/or its metabolites were shown to have a tendency for reaching higher concentration in inflamed footpads in the carrageenan-injected animals [28] inhibiting COX activity. COX-2 isoform, selective target for diclofenac [29], is the rate limiting enzyme for the synthesis of PGs, important mediators of inflammation [30,31]. Also, COX-2 is known to play a role in PGs production under inflammatory conditions as well [32,33]. Development of edema after carrageenan injection is believed to be biphasic and mediators operate in sequence to produce inflammatory response [30,34]. The early phase of inflammation (0-2 h after injection of carrageenan) is acknowledged by the role of mediators like histamine, 5-hydroxytryptamine and bradykinin, while the late accelerating phase (2-6 h postcarrageenan injection) has been attributed to elevated production of PGs, oxygen-derived free radicals, and production of COX-2 [20,21,27].
In this study, anti-inflammatory effect of diclofenac was confirmed as it reduced carrageenan-induced edema in rat paw in a dose-dependent manner. Diclofenac showed maximum anti-inflammatory activity at 2 and 3 h for the low (5 mg/kg) and high doses (20 mg/kg), respectively, and maintained the activity until the last time point analyzed. These findings are in line with the available literature suggesting the effects of diclofenac in late phase (2-6 h) of inflammatory response [6][7][8][9][10][11].
Co-administration of RAD of diclofenac (7.5 ml/kg) and diclofenac (5 mg/kg) produced comparable antiinflammatory activity to that of high dose of diclofenac (20 mg/kg) at 2-h time point. Among the methods studied, sequential administration revealed marginal advantage over concurrent administration. Sequential administration of RAD of diclofenac and diclofenac resulted in >50 % anti-inflammatory activity at 2 h and at subsequent time points studied. This clearly indicates that RAD of diclofenac effectively enhances anti-inflammatory effects of diclofenac (5 mg/kg) at all time points at and after 2 h.
Ability of RAD of diclofenac to enhance efficacy of diclofenac and improve its safety has been already confirmed in previous studies [12,35]. Thus, antinociceptive effect of diclofenac, as shown in behavioral test of acetic acid seizures, was significantly improved, when it was administrated in combination with RAD of diclofenac [12]. According to Petrov VI et al., RAD of diclofenac decrease toxicity of diclofenac by increasing LD 50 value and reducing duration of intoxication symp- Fig. 2. Anti-inflammatory activity (%, mean±SEM) of diclofenac (5 and 20 mg/kg) given orally 60 min before carrageenan injection. Fig. 3. Anti-inflammatory activity (%, mean±SEM) of diclofenac (5 and 20 mg/kg), RAD of diclofenac (7.5 ml/kg) and their combination in carrageenan-induced rat paw edema model. Combination of RAD of diclofenac (7.5 ml/kg) and diclofenac (5 mg/kg) were given orally 60 min before carrageenan injection (CA concurrent administration) or sequentially starting with RAD of diclofenac administration (7.5 ml/kg) followed by diclofenac administration (5 mg/kg) in 10 min time interval (SA sequential administration). toms and the period of rehabilitation in a model of acute toxicity [35]. Therefore, the results of the current study are in line with the previous findings. The distinct mechanism mediating enhanced effect of diclofenac by RAD of diclofenac is still under investigation. However, long-term experience of development target-based therapeutics containing release-active dilutions [12,36] and also investigation of two regimes of RAD of diclofenac and diclofenac administration (coadministration and sequential administration) provided us with the basis to make a conclusion on the initial target for RAD of diclofenac. For therapeutics containing releaseactive dilution of different substances that are currently on market and under development, the ability to modify substance-associated targets was shown [12,36]; it means that in case of RAD of diclofenac, COX is one of the targets. This assumption found confirmation in the results of this study. RAD of diclofenac possessed anti-inflammatory activity itself which means that this agent could inhibit inflammation-related processes. Sequential administration showed more pronounced results vs. concurrent administration showing that RAD of diclofenac-increased sensitivity of diclofenac targets, i.e., COX. Another support for potential involvement of COX as a target of RAD of diclofenac is that enhanced diclofenac activity was shown on the late phase of inflammation with COX dominating. Obviously, suggestion on mechanism of RAD of diclofenac effect resulting from this study should be the subject of further research work. Fig. 4. Anti-inflammatory activity (%, mean±SEM) of diclofenac (5 and 20 mg/kg), placebo (7.5 ml/kg), and their combination in carrageenan-induced rat paw edema model. Combination of placebo (7.5 ml/kg) and diclofenac (5 mg/kg) were given orally 60 min before carrageenan injection (CA concurrent administration) or sequentially starting with placebo administration (7.5 ml/kg) followed by diclofenac administration (5 mg/kg) in 10-min time interval (SA sequential administration).
Previous findings and the results of the current study provide encouraging basis for simultaneous use of RAD of diclofenac and diclofenac. Particularly, based on additive anti-inflammatory activity achieved by this coadministration, one can suggest possible reduction in the dose of diclofenac in the future practice which may be useful to achieve the desired efficacy and yet also avoid its well-known gastric toxicities such as ulceration and subsequent hemorrhage [37][38][39][40].

ACKNOWLEDGMENTS
This work was supported by a grant from OOO "NPF "MATERIA MEDICA HOLDING", 3rd Samotyochny per., 9, 127473, Moscow, Russian Federation. The authors have no competing interests or potential conflicts of interest to report.
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