The preclinical study of a new nasal spray with the anti-inflammatory properties : the effect on the leukotriene-induced inflammation

The use of medicines of the local action with complex effects on the pathogenic cascade of acute rhinosinusitis (ARS) is expedient. Farmak Joint-Stock Company (Ukraine) has developed a new original dosage form of a nasal spray, which contains an aqueous solution of the known pharmaceutical substance Enisamium Iodide (EI). The presence of the effect on the leukotriene link inflammation in EI can determine the high rate of the antiexudative action development in the topical (intranasal) application. This may be useful for pathogenetic therapy of ARS on the background of acute respiratory viral infections taking into account the potential effectiveness for nasal congestion relief. Aim. To substantiate experimentally the expediency of application of a new nasal spray with EI under conditions of leukotriene-induced exudative inflammation. Materials and methods. A new nasal spray EI (JSC Farmak (Ukraine)) – “Amizon” – with the active substance concentration of 10 mg/ml was chosen as the study object. Ibuprofen – “Nurofen” (Reckitt Benckiser Helchere International Limited (UK)), film-coated tablets, 200 mg – was used as the reference drug and was administered once daily in the dose of 48 mg/kg intragastrically. The study of anti-inflammatory properties of EI (nasal spray, 10 mg/ml) in the external (epicutaneous) application was performed on white nonlinear rats of both genders in the experimental zymosaninduced inflammation development. The anti-inflammatory activity (AIA) of EI (nasal spray, 10 mg/ml) compared to Ibuprofen was assessed in percentage in dynamics in 0.5, 1 and 2 hours of observation. Results. According to the results of the studies conducted EI in the dosage form of a nasal spray in the active substance concentration of 10 mg/ml on the model of zymosan-induced inflammation of the rat foot showed the anti-inflammatory properties. According to the degree of its anti-inflammatory properties EI was not inferior to the reference drug Ibuprofen, but even exceeded its activity as of the 30th minute of observation after zymosan injection. The result obtained allows suggesting the presence of high rate of the antiexudative action in the experimental object in the topical (intranasal) application in the treatment of patients with ARS due to the ability to inhibit the lipoxygenase route of the arachidonic acid metabolism. Conclusions. The presence of the anti-inflammatory activity of Enisamium Iodide (nasal spray) has been proven under conditions of leukotriene-induced inflammation reproduction on the model of the zymosan edema. By expression of the anti-inflammatory action during all periods of observation EI is not inferior to the reference drug Ibuprofen, exceeding the activity of the classical NSAID by 1.5 times as of the 30th minute after the pathology onset. The anti-inflammatory activity of Enisamium Iodide in the dosage form of nasal spray experimentally substantiates the expediency and perspectivity of its further preclinical studies.

It is known that viral adhesion and the subsequent viral replication in the nasal mucosa cells cause viral destruction and excessive production of proinflammatory mediators and cytokines.The lipoxygenase pathway activation of the arachidonic acid degradation, preferably of leukocyte origin, with the leukotriene formation is the cause of the conjugate vascular stage of the infectious inflammatory process.Leukotrienes play a key role in the cell-mediated immune response to viruses.LTC 4 , LTV 4 , LTE 4 are excreted by labrocytes and basophils in large quantities.They can be the cause of vasoconstriction and the permeability increase of mainly venules by direct contraction of endothelial cells of blood vessels; they can promote hypersecretion of bronchial mucous membranes and play an important role in the pathogenesis of anaphylaxis.LTV 4 is a chemoattractant, stimulates adhesion and chemotaxis of polymorphonuclear blood cells.LTV 4 enhances exocytosis of proteolytic enzymes, synthesizes and releases of oxygen free radicals [7].The effect on the prostaglandin-induced and leukotriene-induced exudative inflammation presents in the anti-inflammatory action of EI.This effect is due to stabilization of the cellular and lysosomal membranes, slowing down of basophil degranulation, the antioxidant action, and normalization of the level of prostaglandins, cyclic nucleotides and energy metabolism in the inflammatory focus.Therefore, the presence of the effect on the leukotriene link inflammation in EI can determine the high rate of the antiexudative action development in the topical (intranasal) application, and it is very useful for the pathogenetic therapy of acute rhinosinusitis (ARS) on the background of ARI taking into account the potential effectiveness for nasal congestion relief.
The aim of the work was to substantiate experimentally the expediency of application of a new na-sal spray with EI under conditions of leukotrieneinduced exudative inflammation.

Materials and methods
A new nasal spray EI (JSC Farmak (Ukraine)) -"Amizon" -with the active substance concentration of 10 mg/ml was chosen as the study object.This concentration was chosen taking into account the results of the previous stage of the EI (nasal spray) experimental study [8].
The study of anti-inflammatory properties of EI (nasal spray, 10 mg/ml) in the external (epicutaneous) application was performed on white nonlinear rats of both genders weighing 150-180 g in the experimental zymosan-induced inflammation development [7,10].Distribution of experimental animals to the groups is shown in Tab. 1.
The test animals were kept in the Central Research Laboratory of the National Pharmaceutical University (CRL of NPhU) in accordance with the standard sanitary norms on the required diet [11][12][13].All studies were conducted in accordance with the general ethical principles of experiments in animals.These principles are regulated by the Directive 2010/63/EU meeting laws, regulations and administrative provisions of the EU states concerning protection of animals used for experimental and other scientific purposes [12] and the Law of Ukraine No. 3447-IV (21.02.2006, with amendments) [13].
At beginning of the experiment the solution of EI (10 mg/ml) was administered to the second animal group to the foot region of the right hind paw below the hair follicle zone, so that the fluid evenly covered the entire animal foot, including the ankle joint.To do this, EI solution (10 mg/ml) was removed from the nasal spray bottle and the tampon, with which the solution was applied to the foot, was im- pregnated.Previously, the rat's paw was cleaned of various contaminants.Next, we carefully observed for the animals to exclude the possibility of licking the test solution from the skin surface.
A single intragastric administration of Ibuprofen in the dose of 48 mg/kg in suspension was performed in the third animal group.The suspension was previously prepared on a saline using the stabilizer Tween-80.The first (control) animal group received a saline in an equivalent volume.
The pathology was reproduced in all animals an hour after the application of the test objects.Aseptic exudative inflammation was induced by a subplantar injection of 2 % zymosan suspension (Fluka, Switzerland) in the dose of 0.1 ml [7] to the right hind paw.
The edema volume was measured on the right hind paw 30 min before the flogogen injection and in dynamics in 0.5, 1 and 2 hours using a digital plethysmometer (IITC Life Science, USA).The edema volume was displayed in cm 3 .
The anti-inflammatory activity (AIA) was assessed in a percentage by the rate of edema reduction in experimental animals received the drug under study compared to the control group of animals.The AIA was calculated by the formula: where *AIA -is the anti-inflammatory activity, %; 1 ∆V control -is the average percentage of the edema volume in the control group; 2 ∆V test -is the average percentage of the edema volume in the test group.
The statistical processing of the results obtained was carried out by the methods of variation statistics using the Student's t-test and the non-parametric methods of analysis (Mann-Whitney U-test) and STATISTICA 7.0, StatPlus 2009 and MS Excel 2007 software [14][15][16].The results obtained were presented in the form of comparative tables with the results of different groups.

Results and discussion
Indicators of the antiexudative action of EI (10 mg/ml) in the epicutaneous application compared to Ibuprofen in the intragastric administration on the model of zymosan-induced inflammation in Notes: 1) *n -number of the experimental animals; 2) 1 -differences are reliable with animals of the control group (p<0.05); 3) 2 -differences are reliable with the animals received the reference drug Ibuprofen (p<0.05); 4) **AIA − the anti-inflammatory activity.
ISSN 1562-725X (Print) ISSN 2518-1572 (Online) rats by their ability to prevent the exudative edema development compared to the control group of animals were determined during the study.The results are shown in Tab. 2. The experimental data show that the active formation of the corresponding exudative inflammatory reaction on flogogen in the animal paws occurred within two hours in the control group after reproduction of the inflammatory reaction induced by the zymosan injection.Thus, in comparison with the initial values the foot volume increased by 34.8 % in 30 min, it caused the appearance of edema in the volume of 0.48 cm 3 .Then after one hour from the start of the experiment the edema increased by 1.3 times to 43.3 % of the initial volume, which was 0.60 cm 3 .Then in two hours of the experiment the edema decreased to 33.0 %, which was 0.45 cm 3 (Tab.2).
According literature data the mechanism of the pro-inflammatory action of flogogen of the polysaccharide structure (including zymosan) is due to the increased vascular permeability and a significant increase of the leukocyte response -"respiratory (oxidative) burst", and, consequently, lipoxygenase branch activation of the eicosanoid metabolism, especially in blood cells [17].The dynamics of changes in reproduction of the zymosan edema in our experiment was in strict accordance to the above data.The vascular response intensification plays a leading role in forming the exudative edema, which was reflected by an increase in the rat foot volume after the zymosan injection.
According to the data presented in Tab. 2 the study object showed a moderate antiexudative effect.Thus, under its effect in 30 min of observation the edema volume decreased statistically significantly compared to the control group and reached 24.8 % of the initial values, which was 0.37 cm 3 on average.Subsequently, this value was also significantly lower than in the control group.After the first hour of observation it was 33.1 % (0.43 cm 3 ), and for the second hour it was 26.4 % (0.34 cm 3 ).
The experimental data resulted in the following calculated parameters of the study object by AIA: AIA was 18.6 % in 30 min after the zymosan injection, 23.6 % for the first hour and 19.9 % for the second hour.
In the course of the study the reference drug Ibuprofen showed moderate antiexudative properties on the model of the experimental zymosan-induced inflammation.Thus, compared to the control group it reliably reduced edema of the animal paws in 30 min after the zymosan injection, the edema volume was 30.5 % (0.41 cm 3 ), then in one hour of the experiment it was 34.1 % (0.46 cm 3 ), and in two hours it was 25.0 % (0.34 cm 3 ) of the initial values, respectively (Tab.2).In this connection, AIA of Ibuprofen was 12.6 % in 30 min, 21.1 % -in one hour, and 24.3 % -in two hours of the experiment.
The statistical analysis of the results indicates that the calculated AIA level of EI was statistically significant higher than the activity of Ibuprofen in 30 min of observation, and then it was statistically insignificant higher than the activity of Ibuprofen in the first hour of observation, further the results were at the same level without any statistically significant differences (Tab.2).
Thus, EI (nasal spray, 10 mg/ml) at this experimental stage showed a higher antiexudative action than the reference drug Ibuprofen, it was manifested in a statistically higher level of activity in 30 min of the pathology development.The ability of EI to suppress the zymosan-induced inflammation may be due to its specific antileukotriene properties, which needs to be clarified in the course of additional experimental studies.
Therefore, according to the results of the studies conducted EI in the dosage form of a nasal spray in the active substance concentration of 10 mg/ml on the model of zymosan-induced inflammation of the rat foot showed the anti-inflammatory properties.According to the degree of its anti-inflammatory properties EI was not inferior to the reference drug Ibuprofen, but even exceeded its activity as of the 30 th minute of observation after zymosan injection.The result obtained allows suggesting the presence of high rate of the antiexudative action in the experimental object in the topical (intranasal) application in the treatment of patients with ARS due to the ability to inhibit the lipoxygenase route of the arachidonic acid metabolism.CONCLUSIONS 1.The presence of the anti-inflammatory activity of Enisamium Iodide (nasal spray) has been proven under conditions of leukotriene-induced inflammation reproduction on the model of the zymosan edema.The drug in the dose of 10 mg/ml significantly inhibits development of foot edema in rats in the early phase of exudation (in 30 min).This indicates its depressant effect on the lipoxygenase link of inflammation.
2. By expression of the anti-inflammatory action during all periods of observation Enisamium Iodide is not inferior to the reference drug Ibuprofen, exceeding the activity of the classical NSAID by 1.5 times as of the 30 th minute after the pathology onset.
3. The anti-inflammatory activity of Enisamium Iodide in the dosage form of nasal spray (10 mg/ml) experimentally substantiates the expediency and perspectivity of its further preclinical studies.
Conflict of interests: authors have no conflict of interests to declare.