The resulTs of The acuTe ToxiciTy sTudies of “dolosan forTe®” TableTs

This study aimed to evaluate the parameters of acute toxicity of the new combined tablets “Dolosan Forte®” (containing zirilon – 2,4-dichlorobenzoic acid potassium salt, pitofenone hydrochloride, fenpiverinium bromide) worked out by PJSC “Red Star”. It has been shown that LD50 for these tablets administered intragastrically equals 13231 ± 2775 mg/kg in male rats, 13216 ± 3379 mg/kg in female rats, 14393 ± 1669 mg/kg in male mice and 11963 ± 2023 mg/kg in female mice, corresponding to the V class of practically nontoxic substances (5000 mg/kg < LD50 < 15000 mg/kg). The death of the rats and mice receiving the investigated tablets can be possibly caused by the neurotoxic action of the drug with the rapid development of convulsions. The survived animals demonstrated normal state during 14 days. Macroscopic examination showed the absence of the morphological signs of the toxic effect of “Dolosan Forte®” tablets on the visceral systems of the survived rats and mice (except for the increment of the relative organ weights in the groups of animals receiving doses causing high lethality rate). Microscopic investigation did not reveal any pathological changes of the internal organs of rats after the single intragastric administration of “Dolosan Forte®” tablets at a dose of 6000 mg/kg. There were no interspecies as well as sex-specific differences in “Dolosan Forte®” toxic effects. The obtained results together with the data in the literature concerning LD50 for the components of “Dolosan Forte®” show that combined use of zirilon (2,4-dichlorobenzoic acid potassium salt), pitofenone hydrochloride, fenpiverinium bromide does not cause the increase in their acute toxicity.

inTroducTion Pain remains the common healthcare problem being one of the leading reasons for consulting a doctor. It significantly impairs quality of life and ability to work, still the effective treatment of chronic pain (which exists in the absence of tissue damage) remains suboptimal and often causes side effects and excessive healthcare costs [9,13]. So, there is a need to search for the new drugs with the high level of safety as the widely known analgesics such as sodium metamizole are able to cause blood dyscrasias and other side effects, for this reason their use is limited [10]. Zirilon (2,4-dichlorobenzoic acid potassium salt) is the promising substance in this context as it is an effective antiinflammatory agent and analgesic with the therapeutic indices significantly exceeding the values of diclofenac sodium and metamizole sodium [6].
Spasm is among the leading reasons of pain, it also contributes to the pathogenesis of the different diseases worsening the blood supply to the organs and tissues. One of the rational approaches to the pharmacological correction of pain and spasm is to combine analgesics with spasmolytics. It is expedient to use zirilon together with the spasmolytics possessing the different mechanisms of action such as pitofenone hydrochloride (myotropic spasmolytic) and fenpiverinium bromide (M-cholinolytic with additional myotropic and analgesic properties). Such combination -the tablets "Dolosan Forte ® " -was worked out by PJSC "Red Star" (Kharkiv, Ukraine). In the previous experiments [5] it has been shown that this combined drug is effective in doses lower than the reference drug "Spasmalgon ® " and has the advantage over the latter being indifferent to the normal intestinal motility.
Besides, there is evidence in the literature that pitofenone counteraction to spasm is clearly manifested in vitro in simultaneous use with antiinflammatory agents [11]. It was shown in clinical studies that diclofenac combined with pitofenone and fenpiverinium effectively reduces the pain intensity in biliary, ureteric, and intestinal colic demonstrating synergistic action [8].
Therefore, further studies of the combination of zirilon with pitofenone hydrochloride and fenpiverinium bromide is expedient. Toxicity determination represent an essential part of the preclinical drug research and is absolutely necessary for the new combined drugs. Therefore, the aim of this study was to establish the parameters of acute toxicity of the tablets "Dolosan Forte ® ".

MaTerials and MeThods
Adult random-bred mice with body weight of 22-26 g (females, n = 36) and 16-19 g (males, n = 36) as well as adult random-bred rats of both sexes (n = 36 in each group) with body weight of 170-200 g were kept in the Central Scientific-Research Laboratory of National University of Pharmacy under standard conditions. All the experimental protocols corresponded to the requirements of the "Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes." As the peroral route of administration is assumed for the tablets "Dolosan Forte ® " and is able to provide the systemic action, it was used in the experiments to reproduce the clinical signs of the acute poisoning and to determine LD 50 value. The investigated drug was administered once in the form of water suspension (the amount equalled 0.2 ml/10 g for mice and 2 ml/100 g for rats), the animals of the intact control groups received water by the similar scheme. Before the administration the animals were fasted for 12 h (rats) and 4 h (mice), after the administration -for 4 h, but they were allowed free access to water. The animals were observed for 14 days after dosing, body weight dynamics, food and water consumption were also registered. After this the animals were anesthetized and the internal organs were harvested for morphological studies and determination of relative organ weight [2]. In accordance with [1, 3] internal organs (namely the liver, kidneys, heart, adrenal glands, spleen, thymus, testicles / ovaries, as well as oesophagus, stomach, jejunum and rectum, that directly contacted with the drug) were harvested for morphological studies. The organs were fixed in 10 % solution of neutral formalin, dehydrated in ethanol solutions of the increasing concentration, embedded in celloidin-paraffin. Microtome sections were stained with hematoxylin and eosin [5]. For the analysis the microscope Granum L3000 with the appropriate camera and software were used.
Calculation of LD 50 was performed by the method of V. B. Prozorovskiy [2]. The other data were processed using the standard software "Statistica 6.0", dispersion analysis, Newman-Keuls criterion, Kruskal-Wallis method, Mann-Whitney criterion were applied. The level of significance was defined as p ≤ 0.05. resulTs and discussion 1. The results obtained in experiments in rats It was established that 10 min after the single intragastric administration of the tablets "Dolosan Forte ® " there was a decrease in the locomotor activity, then lateral position was registered, and convulsions developed in the most of the rats. Against the background of the drug at doses 10000 mg/kg and higher, extremely severe convulsions were seen, epistaxis occurred in some cases, and death was observed within 20-30 min. Such clinical symptoms are the evidence of CNS affection. The condition of the survived animals improved gradually after 30 min, still adynamia persisted. All these signs disappeared the next day, the condition of the animals did not have any differences from intact rats.
The data listed in Tab. 1 allowed calculating of LD 50 value for the tablets "Dolosan Forte ® " administered intragastrically to rats: it equals 13231 ± 2775 mg/kg in males and 13216 ± 3379 mg/kg in females.
Subsequently, the observation on the animals survived during 14 days shown that they demonstrated normal activity, appearance, and appetite, responded to auditory and visual stimuli; processes of urination and defecation were normal; respiratory disorders, and seizures were not observed. Macroscopic examination on day 14 did not show any pathological changes in animal's general state of health and well-being. The consumption of food in all the experimental animals did not differ from those in the groups of intact control, water consumption had some inter-individual differences without any consistent patterns between the groups. There was a positive dynamics in the body weight dynamics in all of the groups without any differences.
Still in some of the experimental groups higher increment in the body weight was seen, namely among the male rats receiving the investigated drug at doses of 14000 and 16000 mg/kg and among the female rats receiving it at a dose of 10000 mg/kg. This may be determined by the low number of animals in the mentioned groups due to lethality.
Macroscopic examination of the animals survived during 14 days showed no differences from intact rats in the general appearance, as well as in macroscopic structure of the thoracic and abdominal cavities, lymph nodes, thymus, heart and its valves, lungs, stomach (including mucous membrane structure), intestine and colon, liver, pancreatic gland, spleen, adrenal glands, kidneys and bladder uterus and gonads. There were no changes in the relative organs weights (namely, the liver, kidneys, heart, lungs, spleen, thymus, adrenal glands, testes) in male rats receiving the tablets "Dolosan Forte ® " at doses of 6000 and 10000 mg/kg, the increase in the spleen relative weight was seen in males receiving the drug at a dose of 14000 mg/kg. In all of the groups of female rats, the relative organs weights (mentioned above, except for testes) were within the normal physiological range. Microscopic investigation was performed for the samples of the rats receiving the tablets "Dolosan Forte ® " at a dose of 6000 mg/kg. Compared with the intact animals, no pathological changes were revealed in the histological structure of the myocardium, lungs and bronchial epithelium, liver, kidneys, adrenal glands, spleen, thymus, pancreas, testicles and ovaries (spermatogenesis and oogenesis processes were unchanged). There were no pathological shifts in the place of the primary contact with the drug -the gastrointestinal system (namely, the oesophagus mucous membrane, gastric mucosa in all parts, intestinal epithelium of the jejunum and pelvic colon).
Therefore, the results of the macroscopic examination and microscopic investigation demonstrate the absence of the morphological signs of the toxic effect of "Dolosan Forte®" tablets after the single intragastric administration at a dose of 6000 mg/kg to rats. The fast death of the rats receiving this drug at higher doses is possibly caused by the neurotoxic action and not by the visceral systems affection.

The results obtained in experiments in mice
It was registered that immediately after administration of the tablets "Dolosan Forte ® " there was an acute deterioration of the animals condition: the decrease in locomotor activity, lateral position, and loss of consciousness. After 5-10 min, severe convulsions were observed both in male and female mice. Less intensive signs of intoxication were observed in animals receiving the investigated drug at a dose of 10000 mg/kg. After 20-30 min convulsions stopped, while adynamia was present. Administration of the tablets "Dolosan Forte ® " at doses within the range of 11500-16000 mg/kg caused death in the first day of observation, and the dose of 18000 mg/kg led to the death within 15 min (Tab. 2).
The data listed in Tab. 2 allowed calculating of LD 50 value for the tablets "Dolosan Forte ® " administered in-tragastrically to mice: it equals 14393 ± 1669 mg/kg in males and 11963 ± 2023 mg/kg in females.
The further observations in mice that survived during 14 days shown that that they demonstrated normal activity, appearance, and appetite, responded to auditory and visual stimuli; processes of urination and defecation were normal; respiratory disorders, and seizures were not observed. There were no differences in the body weight increment in all of the experimental groups from intact control values.
Macroscopic examination did not reveal any pathological changes in animal's general state of health and well-being. The examination of the thoracic and abdominal cavities showed the normal physiological state of the internal organs, such as the thymus, heart, lungs, stomach (including mucous membrane structure), intestine and colon, liver, pancreatic gland, spleen, adrenal glands, kidneys and bladder, uterus and gonads. In the groups of male and female mice receiving the tablets "Dolosan Forte ® " at doses within the range of 10000-14000 mg/kg, the relative organs weights (namely, the liver, kidneys, heart, lungs, spleen, thymus, testes) did not differ from