SOME ASPECTS OF ENISAMIUM IODIDE NASAL SPRAY SAFETY: PRE-CLINICAL STUDY RESULTS

The aim. To substantiate the safety using of the new nasal spray with Enisamium Iodide via study results of acute local drug-induced irritant action of the test object single-dose to eyes and nasal cavity mucosa. 
 
Material and methods. Enisamium Iodide 10 mg/mL (nasal spray) was the test object. The reference drug was 0.9 % saline. Flemish Giant rabbits were used to induce the experiment (2 groups, 9 rabbits in each group). All study objects were administered in single-dose into the eye conjunctival sacs (0.01 mL) and nasal passages (0.1 mL) by instillation. The eye examination we performed in different time observation point (through 1, 24, 48 and 72 h after drug instillation). Nasal endoscopy was used for control of nasal cavity in all stages of study (15 minutes before, 1-hour and 24 hours after drug instillation) under general anesthesia. The scales of the assessment were used to the result objectivity. 
 
Results. The total score was 0 points in all groups at all-time points according to the relevant scale and the scale of the assessment of rabbit nasal cavity mucosa by nasal endoscopy results. This corresponds to the condition of a healthy eye and healthy nasal mucosa. 
 
Conclusions. Enisamium Iodide 10 mg/mL (nasal spray) in the single-dose instillation into the rabbit eye conjunctival sacs and rabbit nasal passages did not show local drug-induced irritant action on the eye conjunctiva and nasal cavity mucosa in the experimental animals. Nasal endoscopy could be used as an informative visual method in preclinical studies.

of corticosteroids, despite the high-level evidence in the treatment of individual forms of ARS, is not rational in the treatment of viral ARS. The use of irrigation therapy with saline solutions of various concentrations based on sea or mineralized water, despite the presence of topical forms, will not always lead to a rapid decrease in all symptoms of ARS, for example, such as facial pain.
However, despite the presence of a large number of drugs for treatment of various ARS forms, the use of an intranasal dosage form ensures high compliance due to ease of use and high safety. The presence of a drug with various pharmacodynamic effects (anti-inflammatory and analgesic effects, antiviral and interferonogenic activity) in an intranasal dosage form can be effective.
A new nasal spray with Enisamium Iodide aqueous solution (N-methyl-4-benzylcarbamidopyridinium Iodide) original dosage form of a well-known pharmaceutical substance -has been developed by Farmak JSC (Ukraine). The anti-inflammatory action of Enisamium Iodide (EI) has been proved in the pre-clinical studies [8][9][10], potentially justifies high drug effectiveness in the nasal spray dosage form for ARS treatment. It causes a direct positive effect on the inflammation and swelling into the nasal cavity, paranasal sinuses, and sinus ostium. However, studying the safety of a medicinal product with a new route of administration is an integral component of creating a new drug or the scientific rationale for using an existing drug according to new indications.
The aim of the study to substantiate the safety using of the new nasal spray with anti-inflammatory action, which contains EI at a concentration of 10 mg/mL administered intranasally via study results of acute local drug-induced irritant action of the single-dose of the test object to eyes and nasal cavity mucosa. We developed the study design, which is shown in Fig. 1.   Fig. 1. Design of the pre-clinical study «Acute local drug-induced irritant action of the test objects in the single-dose to eyes and nasal cavity mucosa»: -the studied animal group where 0.9 % saline were instilled; -the studied animal group where Enisamium Iodide 10 mg/mL (nasal spray) were instilled.

Material and Methods
EI nasal spray (JSC Farmak, Ukraine) with an active substance concentration of 10 mg/mL was the test object. This concentration has been chosen considering the previous stage results of the of EI (nasal spray) pre-clinical studies [8][9][10][11]. The reference drug was 0.9 % saline.
Eighteen Flemish Giant rabbits of both sexes aged around 90-110 days weighing 2.5-3 kg were used to induce the experiment. Study of local drug-induced irritant action was conducted according to the methodological recommendations «Pre-clinical study of the local drug-induced irritant action» [12]. The baseline data related to all study groups are illustrated in Table 1.
The test animals were kept in the Central Scientific-Research Laboratory of the National University of Pharmacy following the sanitary standards and the required diet. All studies were conducted in compliance with the general ethical principles of animal experiments established by Directive 2010/63/EU concerning the protection of animals used for experimental and other scientific purposes [13]. The Bioethics Commission of the National University of Pharmacy (Kharkiv, Ukraine) approved study design (Protocol No 3 of 15 March 2017).
Intact controls were contralateral anatomical structures. These were right rabbit eyes and right rabbit nasal passages, respectively. All study objects were administered in an unchanged state into the eye conjunctival sacs and nasal passages using Biohit Proline fixed volume pipettor 200 µl with Biohite pipettor tips [14]. Before drug administration, rabbits were fixed in special boxes. Prior to the study object instillation, we examined both eyes and both nasal passages in all experimental groups.
The eye examination we performed 1 hour before instillation of the study objects for initial control of eye condition, as described in the methodological recommendations [12]. Observations were made through 1, 24, 48 and 72 h after drug administration (instillation of the study objects in single-dose into the conjunctival sac of the eyes). Local drug-induced irritant action on the eye mucous membranes was studied on the relevant scale of evaluation of the indicators: the state of the tissues around the eyes, including eyelids, the condition of the conjunctiva, blinking membrane, cornea and iris in each animal.
Nasal endoscopy was used for control of nasal cavity mucosa in all stages of study (15 minutes before instillation of the study objects into the nasal passage, 1-hour and 24 hours after drug instillation with digitally capturing). The procedure was performed under general anesthesia with Ketamine/Xylazine (35/5 mg/kg, intramuscularly [15]) using Karl Storz rhinoscopes (Germany): the one was diameter 4 mm, length 150 mm, 30° angle and the other was diameter 2.7 mm, length 110 mm, 0° angle. The pictures were digitally captured using the rhinoscope and then examined to reveal the pathological changes. The condition of the nasal cavity mucosa was evaluated according to the following nasal endoscopy parameters: swelling and hyperemia of the middle turbinate, amount and character of discharge into the nasal passage ( Statistical analysis of the results was performed using Kruskal-Wallis one-way analysis of variance and Mann-Whitney U test for a posteriori pairwise comparisons [16,17]. Utilized computer software included IBM SPSS Statistics v. 22 (IBM Сorp., USA) and MS Excel 2016 (Microsoft Corp., USA). The level of statistical significance was considered as p<0.05.

Results
In the observation of animals after instillation of the studied objects, no changes in the animal behavior were observed, which could serve as signs of pain or discomfort. Signs of short-term blepharospasm (from 10 to 90 seconds) were observed in groups I a, I b and I c, which is most likely associated with the procedure of instilling the studied objects in the eye than with the properties of these drugs. These phenomena stopped on their own and did not require any additional treatment. In general, during of the eye examination at all-time points of observation, there were no signs of irritation. The eyes of all animals in I b and I c studied groups remained unchanged compared to the control eye (I a group) (Fig. 2). The cornea remained transparent without signs of turbidity; its surface was smooth and shiny. The iris of the left eye of the studied animals looked intact; a positive reaction to light was quick and full. None of the animals had erythema, edema and secretions (Fig. 2).
The total score on the relevant scale [12] was 0 points in all groups at all-time points, which corresponds to the condition of a healthy eye.
In studying of the acute local drug-induced irritant action of the test object single-dose on the nasal mucosa, the application of the studied drugs did not attract significant changes in the general condition and behavior of animals. The combination of drugs given by materials and methods (general anesthesia with Ketamine/Xylazine) led to the formation of sufficient depth and duration of drug sleep; however, all animals were in satisfactory condition, alert and active after its completion. General anesthesia created satisfactory conditions for nasal endoscopy conducting before the instillation of the studied objects and 1-hour after this instillation (this was the first and the second episode of general anesthesia). Repeated general anesthesia (24 hours after the studied object instillation) also did not cause any negative changes either in the general condition or in the behavior of the animals. Nasal endoscopy allowed an intravital objective assessment of the condition of the nasal mucosa of experimental animals, significantly increased the possibility of an objective assessment, and allowed the material to be digitally captured for further analysis that is more thorough. In addition, a significant fact in favor of intravital nasal endoscopy is that this technique allows you to more closely follow modern bioethical standards for conducting experiments on animals, causing them the least possible harm. Therefore, the results of nasal endoscopy of the right (intact) nasal passage in II a group (before, 1-hour and 24 hours after the studied object instillation) were as follows (Fig. 3): − the nasal mucosa was pale pink; − on the surface of the mucosa was a small amount of transparent mucous discharge; − sometimes a vascular pattern was visualized, consisting of unexpanded and unconvoluted vessels that were not overfilled with blood; − no areas of hyperemia or swelling were observed. c -24 hours after instillation The same rhinoscopic pattern was observed with nasal endoscopy of the left nasal passage in animals of group II b (Fig. 4): − the nasal mucosa was pale pink with a small amount of transparent mucous discharge; − the vascular pattern was unchanged; − without signs of hyperemia or swelling. The total score was 0 points in both groups (II a and II b) at all-time points according to the scale of the assessment of rabbit nasal cavity mucosa by nasal endoscopy results (Table 2), which corresponds to the condition of a healthy nasal cavity mucosa.

Discussion
After statistical analysis of the total score on the relevant scale [12] of the comparative study results obtained we have not observed acute local drug-induced irritant action of the test objects in the single-dose instillation into the rabbit eye conjunctival sac. The total score was 0 in all groups at all-time points, which corresponds to the condition of a healthy eye. The absence of negative changes in the cornea, iris, conjunctiva and blinking membrane for 72 hours in a single-dose installation of the study objects in a conjunctival sac at a dose of 0.1 ml indicates the absence of the local drug-induced irritant action of the Enisamium Iodide 10 mg/mL (nasal spray) on the test animal eyes.
After statistical analysis of the total score on the relevant scale (Table 2) of the comparative study results obtained we have not observed acute local drug-induced irritant action of the test objects in the single-dose instillation into the rabbit nasal passages. The total score was 0 points in both groups (II a and II b) at all-time points according to the scale of the assessment of rabbit nasal cavity mucosa by nasal endoscopy results (Table 2). Thus, it is possible to assume that the test object Enisamium Iodide 10 mg/mL (nasal spray) at the single-dose administration did not show local drug-induced irritant action on the rabbit nasal mucosa in our experiment.
However, it is worth noting that in the nasal rhinoscopy evaluation, we observed a single line hemorrhages of up to 3 mm in length on nasal mucosa into right and left nasal passages in some animals (four animals in II a group and three animals in II b group) (Fig. 3, 4). Probably, these phenomena are associated with the procedure of nasal endoscopy, during which it is impossible to completely avoid minor trauma to the nasal mucosa, but this does not affect the condition of the mucosa and animals in general.
Therefore, we can argue that nasal endoscopy is one of the most effective and safe methods of visual assessment in experimental rhinology and could be used in preclinical studies of new drugs.
Study limitations. However, studying of drug safety with a new route of administration is not limited to studying an acute local drug-induced irritant action. We have got good results of the pre-clinical studies [8][9][10][11] and clinical trial (phase I has been conducted as a single-centre, randomized, double-blinded, placebo-controlled clinical trial with dose-escalation -the study of local tolerance and safety of different doses with healthy volunteers). Despite this, we do not have enough data to make conclusion about the complete pharmacological profile of the Enisamium Iodide 10 mg/mL (nasal spray).
Prospects for further researches. We are planning to continue studying Enisamium Iodide 10 mg/mL (nasal spray) through preclinical studies and clinical trials. The preclinical study aims will be an in-depth study of Enisamium Iodide effectiveness in experimental RS. Clinical trials will aim to study the effectiveness and safety of EI in topical use in patients with acute rhinosinusitis. At all stages of research, we will plan to use nasal endoscopy, as the most informative visual method in experimental rhinology.

Conclusions
Test object Enisamium Iodide 10 mg/mL (nasal spray) has no local irritant effect on rabbit eye conjunctiva.
Test object Enisamium Iodide 10 mg/mL (nasal spray) has no local irritant effect on the rabbit nasal cavity mucosa.
It is possible to assume that Enisamium Iodide 10 mg/mL (nasal spray) does not have local irritant properties. № 2(35)2020 Enisamium Iodide 10 mg/mL (nasal spray) is a perspective object for further pre-clinical studies and clinical trials aiming to substantiate of its safety and effectiveness for ARS treatment as well as its implementation to the clinical practice.
Nasal endoscopy could be used in preclinical studies of new drugs as an informative visual method for assessment in experimental rhinology.