Pharmaceutical composition of pediphene for treating damage by non-lethal irritants

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is a pharmaceutical composition in the form of a spray for treating a damage by non-lethal irritants (e.g. pelargonic acid morpholide), containing pediphene in the following proportions, wt/volume %: pediphene hydrochloride 0.01-10.0; sodium chloride 0.1-10.0; water for injections up to 100 ml. What is shown is the efficacy of pediphene in compliance with the declared application ensured by the local anaesthetic effect of the drugs.

EFFECT: drug preparation has no allergenic and immunotoxic properties.

3 dwg, 40 tbl

 

The alleged invention relates to the field of medicine, namely, to the chemical-pharmaceutical industry, specifically, to the production of pharmaceuticals for the treatment of lesions of non-lethal means annoying. The main active drug compound of the proposed pharmaceutical composition is pedifen.

Pedifen is a hydrochloride of N,N-diethyl-5,5-diphenyl-2-pentylamine.

(C6H5)2CN-CH2-C≡C-CH2N(C2H5)2·HCl

Pedifen relates to derivatives of acetylenic amines with n-cholinolytic action.

The term "pedifen" in the medical literature refers to both salts of Pedavena and its Foundation. In practical medicine, this term is used to denote a solution of Pedavena, and pharmaceutical compositions containing pedifen.

The substance of pedifen produced FSUE RPC "parsedata" FMBA of Russia № R N002888/01 dated 06.06.2008.

Pharmaco-Toxicological assessment of pedifen given in thesis Onufrienko ME (Onufrienko ME, Pharmaco-Toxicological assessment of Pedavena, St. Petersburg, 1995). Installed anti-stress, immunostimulirutuyu and growth-stimulating activity of Pedavena. Other types of pharmacological activity not described in literature.

Russia, along with other with the wounds signed the Convention on the prohibition of Chemical weapons, which includes all toxic substances lethal actions. A number of substances irritants (irritant) now, as in other countries, are seen as the "police" of gases and chemical basis for civil gas weapons. Although irritants in the gas weapons are used in irritating concentrations, they can cause chemical burns to the eyes, which are characterized by the duration of the pathologic process and the development of complications (post-traumatic keratitis, keratouveitis, traumatic cataract, and others),characterized by the duration of the pathologic process and in some cases leading to decreased visual acuity and disability of the victims. This necessitates the development of effective and safe prophylaxis and therapy of lesions substances irritants.

Known treatment unithiol chemical eye injuries caused by the irritant chloracetophenone (Mukovskyy L.A., Panchasara V.M., Brzeski CENTURIES and others, Toxicol. Gazette, 2009, No. 6 page 7-10). This issue is devoted to thesis Anesini V.M. "Features eye lesions aerosol gas weapons and their treatment" SPb, 2007, Where it is concluded that at present, the most effective way of providing medical aid in case of eye injury to lacrimators who are washing the conjunctival cavity with a solution of unithiol or frequent instillation of sodium isopentane polysulphonate. However, the necessary conditions for this hospital.

The closest to this technical solution is the use of 4% spray lidocaine for the treatment of lesions of non-lethal irritant agents (patent RF №2011125817/15 from 24.06.2012,). A disadvantage of the known treatments are relatively common side effects of lidocaine, causing allergic reactions, including anaphylactic shock, and restrictions on the doses of this preupdate.

The present invention is an improved method of treatment of lesions of non-lethal means annoying.

The problem is solved in that in the proposed pharmaceutical composition in the form of a spray contains as active substance - pedifen. In the proposed pharmaceutical composition and the experimental part, the term of pedifen describes hydrochloride N,N-diethyl-5,5-diphenyl-2-pentylamine.

As established in the course of this study, Pedifen has a strong local anesthetic action. When studying the terminal anesthesia is established that the activity he is not inferior to the dikaina, and when wiring exceeds the novocaine.

Pedifen have a stimulating effect on the Central nervous system.

Pedifen is an antioxidant. In vitro experiments have shown that Pedifen, about 10 times Prevost who goes ascorbic acid, yielding to the effect of tocopherol. The introduction of Pedifen "intact animal is accompanied by a decrease in free-radical oxidation of the tissues and increase their antioxidant activity. Prevents activation of lipid peroxidation (LPO). Pedifen detected and membrane-stabilizing properties, which may also be associated with its antioxidant activity.

Pedifen has a pronounced spasmolytic activity, exceeding papaverine this figure 6.5 times.

It is shown that pedifen has no allergenic properties.

Pedifen possesses antimicrobial activity against gram-positive (staphylococci) and gram-negative (Pseudomonas aeruginosa and Escherichia coli) microflora, therefore, the need for preservatives in the creation of dosage forms is missing.

According to the present invention for the treatment of lesions of non-lethal means annoying the pharmaceutical composition:

Pedifen0.01 to 5.0 g
Sodium chloride0.1 to 10.0 g
Water for injectionto 100 ml

The predominant content of pedifen in a solution of 0.1 to 1.0 g per 100 m is solution.

Distinctive features of the proposed pharmaceutical compositions are using the original ingredients (in the above ratio), and sodium chloride to create the optimum osmotic pressure of the composition.

Given the characteristics of the mucous membranes of the eyes, and subjected to the action of irritating the tools you will need the exact observance of the ratios of the original ingredients. The use of pedifen below the lower limit does not guarantee the occurrence of the desired effect. When the concentration of pedifen more than 10% limited the permitted use of the drug in this naznacheniju.

The final selection of concentrations was based on data from biological research.

Possible use in the proposed composition of the softening component, for example, hypromellose, hydrocapillary or hyaluronic acid, however, there is a special need.

Advantages of pedifen before lidocaine in that the latter often has allergies and even anaphylactic action.

Subacute (14 days) daily intracrystalline application pharmaceutical form of pedifen experimental animals at doses exceeding therapeutic for a person in the tens to hundreds of times, had no harmful effects on the basic adaptive system the volumes (nervous, cardiovascular, hematopoietic, excretory, antitoxic), metabolism, overall health and development, basic homeostatic parameters of the organism.

Note also the absence of the drug dosage form of Pedifen 0.25% irritating to tissues at the site of application (intracrystalline). All this applies to rodents and nigritana, i.e. testifies to the universal nature of harmlessness of a new drug.

A study of the possible allergic effects of drug Pedifen 0.25% spray on Guinea pigs of both sexes has been shown that it does not cause the total reaction anaphylaxis using the drug even at doses 10 times more therapeutic. In addition, conjunctival samples, reactions degranulation of mast cells and reactions of the immune complexes were negative. These data allow us to conclude that the drug has no allerheiligen action.

The experiments showed that the introduction of drug Pedifen 0.25% spray does not exert immunotoxic action

The following examples illustrate but do not limit the claims of the applicants.

EXPERIMENTAL EXAMPLES

For the preparation of pharmaceutical compositions in the form of spray take about 70 ml of water for injection (FS 42-2620-97), in which is dissolved not the required number of pedifen hydrochloride (ND 42-14192-06) (based on anhydrous substance), sodium chloride (mm 42-0474-4119-03 or Heb. Pharm.) and stirred for 15 minutes, bring up to the required volume with water for injection (FS 42-2620-97), filtered through a sterile filter 0,22 µm, poured into containers, usually equipped with a spray bottle, Packed in a plastic bag and sterilized by radiation.

On the basis of preliminary biological studies we have proposed in this patent pharmaceutical composition called "pedifen 0.25% in the form of spray" of the following composition:

Pedifen hydrochloride (ND 42-14192-06) (based on anhydrous substance)0.25 g
Sodium chloride (mm 42-0474-4119-03, Heb. Pharm.)0.6 g
Water for injection (FS 42-2620-97)to 100 ml

EVALUATION of ACUTE toxicity of the DRUG DOSAGE FORM of PEDIFEN 0.25%

To measure acute toxicity of the drug was administered per os to white mice and rats of both sexes intragastrically (W/W) through noninvasive metal probe in increasing doses according to the Litchfield-Wilcoxon signed. Calculations of average lethal doses was performed by V.B. have been the Prozorovsky (Pharmacology and toxicology, 1978, No. 4, s-502). To achieve large doses of the Reparata introduction was carried out by repeated at intervals of 20-30 minutes for 4 hours. Control animals received a similar volume of introduction of the solvent - water for injection.

Dosing was performed on the total weight of the drug (the current start and auxiliary substances)conditionally assuming that 1 ml weighs approximately (including specific gravity) 1,

To study the effects of the drug when backfilling in his eyes buried in the conjunctival bags and on the cornea in maximum doses. The control animals were administered the same way a similar dose of water for injection. After that, animals were placed for 14 days in the cells for observation.

Dependent doses effects of drug Pedifen 0.25% presented in tables 1-4.

Table 1
Lethal effects (Palo/all) dosage forms of the drug Pedifen 0.25% when the/W introduction mice-males
Dose, g/kg357.51015
Pedifen 0.25%0/60/60/60/60/6
LD 50>15.0 g/kg

Table 2
Lethal effects (Palo/all) dosage forms of the drug Pedifen 0.25% when the/W introduction mice-females
Dose, g/kg357.51015
Pedifen 0.25%0/60/60/60/60/6
LD50>15.0 g/kg

td align="center"> 0/6
Table 3
Lethal effects (Palo/all) dosage forms of the drug Pedifen 0.25% when the/W rats-males
Dose, g/kg68101215
Pedifen 0.25%0/60/60/60/6
LD50>15.0 g/kg

Table 4
Lethal effects (Palo/all) dosage forms of the drug Pedifen 0.25% when the/W rats-females
Dose, g/kg68101215
Pedifen 0.25%0/60/60/60/60/6
LD50>15.0 g/kg

The data obtained show that sex differences in the rates of acute toxicity dosage form of the drug is not shown. This allows you to combine data on mortality in males and females and to calculate the lethal dose without regard to gender. As you can see, the difference in values of LD50the dosage form of the drug Pedifen 0.25% for two species of laboratory animals are available.

Intragastric administration

The death of rats and mice at the dose of the dosage form to a maximum of 15 g/kg) were observed. P the pout and the only external manifestation of toxic action was intermittent numbness animals which had survived the first few minutes after injection. In the future, the condition of the animals was not different from the control group. Mortality in the control groups was not observed. In the first and second days was observed a slight decrease of potreblenia feed. In the remaining days of their condition did not differ from normal.

Sex differences in the course of intoxication were noted.

The results of measuring the body weight of the animals survived intoxication at the maximum dose are presented in tables 5 and 6.

Table 5
Effect of acute intragastric drug dosage form
Pedifen 0.25% on body weight of mice, g
Time studiesThe intactControl"Pedifen"
1234
Males 14 days
Background19.9±0.320.1±0.419.6±0.1
7 days20.7±0.219.9±0.3 21.0±0.1
14 days21.9±0.421.8±0.522.0±0.3
Females 14 days
Background18.7±0.119.2±0.419.7±0.1
7 days20.4±0.121.0±0.320.1±0.4
14 days21.0±0.421.4±0.420.4±0.1

Table 6
Effect of acute intragastric drug dosage form
Pedifen 0.25% on body weight of rats, g
Time studiesThe intactControl"Pedifen"
1234
Males 14 days
Background180.2±1.4180.6±1.1 180.6±1.4
7 days182.8±1.4194.7±2.1180.2±1.3
14 days200.9±1.7201.8±1.2198.3±1.7
Females 14 days
Background174.6±1.9181.3±1.0177.2±0.9
7 days186.4±1.9187.6±1.6188.6±1.7
14 days188.6±2.4189.8±1.9190.0±1.8

Analysis of the data shows a decrease in average body weight in all animals receiving the drug, on the second day after injection. This same effect was observed in control (although to a lesser extent), so that it can be caused by stress.

Pedifen has a relatively low toxicity. Not cumulated.

The study of toxicity when used in the eye

When backfilling of the drug in the eye, mortality was not observed. The volume of injected drug in defining the parameters of acute toxicity when intracrystalline apt the paths corresponded to the maximum technically possible to introduce in the course of the day. Respectively, for rats and rabbits these amounts equal to 1.0 and 8.0 ml. No external pathological manifestations, which could be mistaken for signs of intoxication were observed. However, in animals treated with the model sample and water for injection into the eye, marked hyperemia of the mucous membranes of the eyes, watering of the eyes. Significant changes in the pupil diameter and reaction to light (pupillary reflex) is not registered. When examining animals in 24 hours in rabbits was observed symptoms of conjunctivitis in mild (redness at 1 point, serous discharge from the conjunctival cavity). Animals of the control group these symptoms were less pronounced. The test results with fluorescein in rabbits all experimental groups is negative (the absence of organic lesions of the cornea).

The state and behavior of experimental animals in the future, the follow-up period did not differ from the behavior of control and all groups corresponded to the normal state and the behavior of intact rats and rabbits. Dynamics of body weight of animals in all experimental groups also did not differ from the control.

These autopsy (necropsy)

At the necropsy of rats and mice at the end of the experiment (14 days) differences between animals treated with the drug has not been established.

Wool experimental animals had a neat appearance, b is La shiny, without foci of alopecia. Food animals was satisfactory.

Upon examination of the thoracic and abdominal cavities irregularities in the arrangement of the internal organs were noted.

The submandibular lymph nodes and salivary glands had an oval or rounded shape, uniform pinkish or yellowish color and a moderate density.

The thyroid gland is tightly belonged to the larynx, had normal size and density, pinkish-reddish color. Thymus had a triangular shape, whitish color and moderately firm consistency.

The size and shape of the heart changes were introduced. The heart muscle was brownish, thick.

The surface of the lungs were pale pink color; the light was spadolini at the opening of the chest. The fabric on the section also had a uniform pale pink color. The mucous membrane of extrapulmonary bronchi was smooth, shiny, pale pink.

The spleen had a dark cherry color, smooth surface and plotnovato consistency.

The pancreas was pale pink, lobed.

The size and shape of the liver changes were introduced. The liver capsule was thin, transparent. The liver tissue had a brownish color and moderately firm consistency.

The size and shape of the kidneys did not differ from the control, the capsule can be easily removed. The surface of the body was smooth, uniform brownish-with rovati color. In the context of kidney clearly differed cortex and the medulla.

The shape, size and density of the adrenal glands, ovaries or testes did not differ from the control.

The lining of the brain were thin, transparent. The brain substance had moderate density. Enlargement of the ventricles of the brain were observed.

In animals that received the drugs and water for injection intragastrically, apparent violations on the part of organs of the gastrointestinal tract were observed. Mucosa of the esophagus was shiny, smooth, pale color. The size and shape of the stomach changes were introduced. The lumen was filled with food content. Hyperemia, erosions, hemorrhages, indicating annoying action of the drug, was not observed. The lumen of the duodenum 12 changes were introduced, the mucosa of the intestine was shiny, smooth, pale pink. The mucous membrane of the small intestine was as pale pink, shiny, smooth. The mucous membrane of the colon had a slightly grayish hue, was smooth and shiny.

Animals treated model sample and water for injection into the eye, marked hyperemia of the mucous membranes of the eyes, watering of the eyes. Significant changes in the pupil diameter and reaction to light (pupillary reflex) is not registered. When examining animals in 24 hours in rabbits OBS who had the symptoms of conjunctivitis in mild (redness at 1 point, serous discharge from the conjunctival cavity). Animals of the control group these symptoms were less pronounced. The test results with fluorescein in rabbits all experimental groups is negative (the absence of organic lesions of the cornea).

In tables 7 and 8 shows the mass ratios of the internal organs of mice and rats, averaged over the group.

6.7±0.2
Table 7
Mass (µ) bodies in white mice in acute intragastric administration of the dosage form of the drug Pedifen 0.25% (g/kg body weight)
BodyThe intactControl"Pedifen"
1234
Males 14 days
Heart3.3±0.13.2±0.23.6±0.09
Lungs with trachea6.4±0.26.4±0.26.2±0.09
The thymus 0.88±0.020.90±0.020.87±0.02
Liver38.6±1.740.0±1.339.8±2.6
Spleen2.9±0.082.9±0.13.3±0.09
Kidney (left)4.2±0.24.2±0.14.3±0.2
1234
The adrenal glands0.18±0.010.18±0.010.17±0.01
The brain15.1±0.515.7±0.415.6±0.4
The testes or ovaries4.0±0.094.0±0.13.9±0.2
Females 14 days
Heart3.4±0.33.4±0.23.2±0.3
Lungs with trachea6.5±0.16.7±0.2
The thymus0.85±0.060.87±0.060.88±0.06
Liver35.6±1.437.0±1.436.9±2.3
Spleen3.2±0.13.0±0.093.0±0.2
Kidney (left)4.3±0.24.3±0.34.6±0.09
The adrenal glands0.17±0.0090.15±0.010.17±0.01
The brain16.5±0.415.9±0.316.2±0.4
The testes or ovaries0.20±0.010.24±0.020.19±0.01

Table 8
Mass (µ) bodies in white rats with acute intragastric administration of the dosage form of the drug Pedifen 0.25% (g/kg weight of the body)
BodyThe intactControl"Pedifen"
1234
Males 14 days
Heart3.8±0.023.8±0.13.7±0.03
Lungs with trachea7.4±0.097.2±0.27.6±0.07
The thymus1.36±0.041.34±0.111.41±0.06
Liver42.6±0.936.8±1.543.7±0.9
Spleen4.7±0.074.2±0.14.4±0.2
Kidney (left)4.0±0.014.4±0.094.0±0.009
The adrenal glands0.12±0.0020.10±0.020.10±0.001
The brain6.9±0.018.7±0.47.0±0.05
The testes or ovaries12.5±0.0912.7±0.0911.7±0.09
Females 14 days
Heart3.4±0.033.6±0.023.6±0.02
Lungs with trachea8.2±0.078.4±0.078.3±0.1
The thymus1.46±0.051.52±0.041.50±0.05
Liver40.5±0.940.8±0.741.0±0.9
Spleen4.8±0.054.8±0.074.9±0.09
Kidney (left)4.1±0.024.3±0.014.2±0.02
The adrenal glands0.12±0.0010.11±0.0010.10±0.001
The brain7.31±0.097.03±0.087.13±0.06
The testes or ovaries0.61±0.030.61±0.040.60±0.05

The analysis values of the mass ratios did not reveal any significant differences between groups of animals treated with a model sample of the drug compared to the control group.

Conclusion according to the results of studies of acute toxicity of the investigational drug dosage form:

Study of acute toxicity in rodents nonlinear white mice and rats showed that the drug Pedifen 0.25% parameters of acute toxicity refers to practically non-toxic substances. This is evidenced by the following experimental data.

Values LD50drug Pedifen 0.25% for outbred white mice and rats of both sexes in the/W introduction knowingly lie above 15 g/kg, which is approximately seven times greater than the maximum daily dose for humans is 20 mg/kg

Preparation of Pedifen 0.25% at its acute/W administration in doses up to 15 g/kg mongrel Belm mice and rats of both sexes were not led to death, did not cause macroscopic changes in the brain, internal organs and endocrine, did not cause di the trophic status of internal organs, as evidenced by the values of their mass ratios, and was not observed its irritating effect on the mucous membranes of the digestive tract.

Intracrystalline the introduction of the drug in the highest possible dose of more than 5 g/kg has expressed no irritating effect on the mucous membranes of the eyes of laboratory animals.

In acute experiments on the use of drug, Pedifen 0.25% not observed evidence of the annoying effects of the drug on the mucous membranes of the digestive tract and the eyes of laboratory animals.

Thus, the results of toxicomanie, observations of experimental animals in the 14-day period after acute application and data pathomorphological studies allow us to classify drug Pedifen 0.25% to class VI relatively harmless drugs (.Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed.IV, Baltimore, 1975, 427 p.; Sidorov, K.K., 1973) (table 9). The status of survivors of acute animal experiments shows good tolerability and safety of the drug in doses exceeding the maximum therapeutic hundreds of times.

Studies indicate the absence of contraindications for indicators of acute toxicity for clinical trials or medical use of the drug Pedifen 0.25%.

Table 9
The degree of toxicity of drugs (Hodge and Sterner)
ToxicityThe termLD50once per os, rats (mg/kg)LD50once the i/v*, rats (mg/kg)
1Extremely toxic<1<0.1
2Highly toxic1-500.1-50
3Moderately toxic50-5005-50
4Allatoxin500-500050-500
5Practically non-toxic5000-15000500-1500
6Relatively harmless>15000>1500
* - gradation of degrees of toxicity when the intravenous route that call is to be placed is determined by multiplying the values of standard doses for assessing toxicity oral route of administration by a factor of 0.1

The STUDY of SUBACUTE TOXICITY of the DRUG DOSAGE FORM of PEDIFEN 0.25% AT INTRACRYSTALLINE INTRODUCTION

Daily therapeutic dose for humans is 2 drops in each eye once, with the possibility of re-introduction after 30 minutes, but not more than 0.8 ml once, with the possibility of re-introduction after 30 minutes, but not more than 0.8 ml for an adult. The maximum therapeutic dose for the average human weighing 60 kg will be of 0.013 ml/kg (approximately 0.01 ml/kg). According to the rules of extrapolation therapeutic dose for rodents 5.7 times more than for a person who is 0,057 ml/kg (0,006 ml/100 g). This will be the first therapeutic dose. It was dosaged drops using an automatic micropipette. Therapeutic dose for rabbits weighing between 2 kg according to calculations using the conversion factor of doses 3 times more than for a man - 0,039 ml/kg (approximately 0.04 ml/kg). For the convenience of dosing and calculations as the maximum therapeutic dose, it was decided to adopt 0.05 ml/kg

The studied doses

1. For rats: 1 dose 0,006 ml in one eye. Accordingly 0,012 ml per day is the maximum therapeutic dose (0.06 ml/kg). 2nd dose was on 0,018 ml in both eyes. Accordingly 0.036 ml per day - three times the maximum therapeutic dose (of 0.18 ml/kg). 3rd dose with the Tawil 0.6 ml in both eyes, what is 0.12 ml / day (0.6 ml/kg), a dose of 60 times the maximum therapeutic dose for humans, or 12 drops per animal; burial was made in several stages in both eyes.

2. For rabbits: 1 dose of 0.05 ml in one eye. Accordingly 0.1 ml per day is the maximum therapeutic dose (0.05 ml/kg). 2nd dose was 0.15 ml in both eyes. Accordingly 0.3 ml per day - three times the maximum therapeutic dose (0.15 ml/kg). 3rd dose was 0.5 ml in both eyes, which is 1.0 ml per day (0.5 ml/kg), the dose to 50 times the maximum therapeutic dose, or 10 drops per animal; burial was made in several stages in both eyes.

The frequency of application of 1 times a day for 14 days.

The total duration of follow - up 21 days.

Physiological parameters were recorded before the start of the experiment (background), after 14 and 21 days.

Hematological and biochemical parameters were assessed at 14 and 21 day study.

Macroscopic and histological examination of internal organs was performed posthumously after the introduction and at the end of the observation period.

In the ophthalmic research in animals has been evaluated condition of the mucous membranes, the presence of corneal reflexes, measured the size of the pupil (p) and the width of the palpebral fissure (f) left (s) and right (d) eyes. In t the value of the whole observation period the animals from the control and experimental groups edema or hyperemia of the mucous was not recorded.

Table 10 presents the results of measuring the size of the pupil and the width of the palpebral fissure. When analyzing the data presented significant differences of performance in all the experimental groups were not found.

1.05±0.13
Table 10
The influence of the drug dosage form of Pedifen 0.25% on the value of the pupil and the width of the palpebral fissure in white rats (mm, M±m)
IndexThe intactControlDosage
0.06 ml/kg0.18 ml/kg0.6 ml/kg
MFMFMFMFMF
Background
pd 1.08±0.091.08±0.091.02±0.041.08±0.091.06±0.011.08±0.091.02±0.111.03±0.041.01±0.051.08±0.1
ps1.06±0.11.05±0.051.06±0.11.06±0.011.03±0.041.05±0.051.06±0.091.08±0.031.08±0.011.06±0.09
Fd2.8±0.12.7±0.093.1±0.132.8±0.12.7±0.092.87±0.113.1±0.132.8±0.113.08±0.142.74±0.1
Fs2.6±0.12.8±0.11 3.8±0.112.84±0.072.74±0.152.91±0.093.08±0.142.84±0.073.03±0.12.9±0.12
Duration of anesthesia000024.1±0.823.2±0.924.6±0.924.1±0.826.5±0.925.8±0.9
14 days
pd1.08±0.091.08±0.091.02±0.051.1±0.11.02±0.111.02±0.041.08±0.051.10±0.091.03±0.051.1±0.09
ps1.03±0.041.05±0.051.03±0.041.08±0.071.05±0.091.06±0.11.05±0.051.05±0.051.06±0.1
fd3.0±0.013.0±0.013.03±0.112.84±0.072.83±0.112.95±0.163.03±0.12.76±0.153.1±0.132.72±0.09
fs2.84±0.073.03±0.113.0±0.012.76±0.152.87±0.092.95±0.173.00±0.12.83±0.093.0±0.12.8±0.12
Duration of anesthesia000024.6±0.924.1±0.823.2±0.925.8±0.926.5±0.9

The EFFECT ON BODY WEIGHT

Dynamics of changes of body weight of the animals of the experimental groups are presented in table 11.

Growth dynamics of body weight in all experimental groups was the same.

The data analysis confirms the absence of any differences between groups: the value of F-test for the significance test is equal to 0.73 at the critical value of F 0.95; 10,90=1.99.

Table 11
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% by weight of white rats
Time studiesThe intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
(days)MFM FMFMFMF
Background184±2.0179±2.7186±2.6185±1.8188±2.9183±2.8185±1.9187±2.9187±1.9180±2.8
day 7199±1.8187±2.0193±2.5189±2.2193±2.4184±2.0200±1.8201±2.9200±1.8188±2.0
day 14211±2.5207±2.3215±1.9206±2.4210±2.0204±2.4211±2.5201±2.3211±2.5204±2.3
21 days216±2.1 206±2.3218±1.7216±2.5220±1.8206±2.2217±2.1209±3.0217±2.1209±2.4

EFFECT ON FEED AND WATER intake

To determine the amount of feed consumed and the water rat was placed on the day of the exchange of the cell.

Data for all experimental groups are presented in tables 12 and 13.

Table 12
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% on feed intake of white rats (g, M±m)
Time studiesGroups of animals
The intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
(days)M FMFMFMFMF
Background20.2±0.919.2±0.419.4±0.819.1±1.019.6±0.719.3±1.019.3±0.919.7±0.919.4±1.019.3±0.9
day 720.4±0.620.1±0.620.1±0.920.4±0.919.9±0.920.4±0.920.8±0.920.0±1.020.9±0.920.8±1.0
day 1421.5±0.621.3±0.921.3±0.920.3±0.921.2±1.020.5±1.021.3±0.920.2±0.821.4±0.921.2±0.9
21 days20.9±0.921.3±0.719.7±1.019.7±0.819.6±1.019.9±0.920.8±0.820.9±0.920.9±0.820.7±0.9

Table 13
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% on water consumption white rats (g, M±m)
Time
research
Groups of animals
The intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
(days)MFMFMF MFMF
Background15.5±0.516.0±0.715.3±0.715.6±0.515.3±0.816.1±0.616.3±1.515.3±1.016.4±1.515.5±0.5
day 715.6±0.816.6±0.915.5±0.916.4±0.816.4±0.616.4±1.016.8±1.116.4±0.716.9±1.116.4±0.8
day 1416.7±0.717.0±1.117.0±1.015.2±0.815.5±0.617.2±1.117.7±0.617.4±0.917.6±0.515.4±0.9
21 days16.6±0.916.5±0.916.4±0.6 16.4±0.716.4±0.816.2±0.917.1±0.916.6±0.917.3±0.816.4±0.7

EFFECT ON LOCOMOTOR AND EXPLORATORY ACTIVITY

Table 14 presents data on the influence of the drug dosage form of Pedifen 0.25% on the spontaneous locomotor activity of rats.

Table 14
The influence of the drug dosage form of Pedifen 0.25% on the structure of the behavior of white rats (M±m)
The studied parametersThe intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
123456
Males, the background
The latent period96±1286±10 99±2292±1586±12
Horizontal activity1.7±0.12.1±0.52.1±1.22.2±0.21.4±0.1
Vertical activity2.0±0.31.8±0.42.0±0.61.3±0.21.9±0.1
Peeking1.5±0.24.4±0.51.8±0.32.1±0.41.9±0.1
Grooming0.8±0.21.3±0.091.7±0.30.9±0.31.5±0.1
Boles1.6±0.091.8±0.41.7±0.21.2±0.21.1±0.2
Females, the background
The latent period88±1889±1486±10 93±2086±9
Horizontal activity2.2±0.32.2±0.22.0±0.41.6±0.42.0±0.4
Vertical activity2.6±0.22.1±0.21.5±0.31.8±0.41.7±0.3
Peeking3.0±0.72.5±0.22.1±0.23.0±0.62.4±0.5
Grooming1.8±0.090.7±0.20.4±0.21.6±0.10.6±0.1
Boles1.4±0.21.7±0.21.6±0.41.4±0.11.5±0.08
Males, 14 days
The latent period90±1391±1391±1990±11 89±11
Horizontal activity1.6±0.52.3±0.62.3±1.12.1±0.12.0±0.5

Vertical activity1.9±0.31.6±0.41.9±0.62.3±0.21.7±0.4
Peeking3.5±0.23.4±0.31.8±0.32.5±0.53.4±0.5
Grooming1.1±0.091.9±0.41.6±0.30.6±0.31.3±0.09
Boles1.3±0.11.8±0.31.7±0.21.5±0.091.7±0.3
Females, 14 days
The latent period88±1684±1488±18 92±2193±17
Horizontal activity1.4±0.42.3±0.42.1±0.31.5±0.41.2±0.09
Vertical activity1.4±0.72.1±0.62.5±0.11.7±0.31.8±0.6
Peeking4.1±0.42.1±0.22.7±0.32.9±0.63.4±0.7
Grooming1.4±0.10.5±0.10.7±0.21.6±0.21.6±0.2
Boles1.5±0.11.6±0.31.8±0.41.3±0.31.5±0.3
Males 21 days
The latent period91±1185±1290±1394±21 88±16
Horizontal activity1.7±0.21.4±0.092.1±0.21.4±0.41.2±0.4
Vertical activity2.0±0.31.9±0.091.3±0.31.8±0.31.6±0.7
Peeking1.4±0.11.9±0.092.0±0.32.9±0.84.3±0.5
Grooming0.8±0.11.5±0.20.7±0.21.6±0.11.3±0.1
Boles1.5±0.081.0±0.21.3±0.21.3±0.21.5±0.1
Female 21 day
The latent period95±1095±1894±2194±1796±18
Horizontal activity1.5±0.11.8±0.42.3±1.12.2±0.31.7±0.4
Vertical activity2.0±0.12.1±0.32.0±0.62.5±0.21.9±0.5
Peeking1.5±0.23.8±0.31.8±0.32.5±0.23.8±0.3
Grooming0.8±0.11.4±0.21.6±0.40.8±0.21.3±0.1
Boles1.7±0.091.1±0.31.2±0.31.7±0.11.1±0.2

Any differences between the groups were not detected: the value of F0 - 0.11, what makes exclude the effect of the drug on the studied parameter.

IMPACT ON DURATION GEKSENALOVY SLEEP

Detoxifying the liver function was assessed by the duration geksenalovy sleep. D. the nnye presented in table 15.

The obtained data indicate the absence of significant changes in the duration geksenalovy sleep in all experimental groups after 14 days of application pharmaceutical form of the drug and at the end of the observation period (21 days).

Table 15
The influence of the drug dosage form of Pedifen 0.25% duration geksenalovy sleep albino rats (M±m)
Time study-ingGroups of animals
The intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
(days)MFMFMFMFMF
day 14 28.5±1.329.9±2.026.8±2.325.9±2.032.6±2.727.9±2.230.4±2.127.7±2.227.6±2.332.1±2.7
21 days33.5±1.531.1±.926.2±2.032.2±2.830.8±2.328.8±2.432.2±1.532.7±2.927.9±1.326.7±2.4

The EFFECT ON the FUNCTIONAL STATE of the KIDNEYS

Urine tests conducted prior to the beginning of the experiment, on day 14, and at the end of the observation period. The results are shown in tables 16

0
Table 16
The results of the analysis of urine in white rats in the study of subacute toxicity of the drug dosage form of Pedifen 0.25% (M±m)
The studied parametersThe intactControl Dose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
123456
Males, the background
Number of allocated urine, ml15.6±1.715.4±1.514.57±1.616.1±2.314.7±1.6
pH5.9±0.066.0±0.095.9±0.086.0±0.086.0±0.09
Specific gravity, g/ml1.03±0.00081.01±0.00081.00±0.0011.00±0.00091.01±0.0009
Glucose, g/ml00000
Bilirubin, feature (1), no (0)00 00
Ketone bodies, there is (1), no (0)00000
Protein, g/l3.1±0.082.8±0.082.9±0.082.9±0.083.1±0.08
The leukocyte count in a field of view0-10-10-10-10-0
The erythrocyte count in a field of view0-10-10-10-10-1
ColorFrom light yellow to dark yellow
TransparencyBasically transparent
Females, the background
Number of allocated urine, ml15.5±0.716.4±2.615.3±1.6 19.2±0.716.7±2.7
pH5.7±0.085.9±0.076.2±0.086.0±0.065.7±0.08
Specific gravity, g/ml1.0±0.0020.98±0.00090.96±0.0021.0±0.0041.0±0.0008
Glucose, g/ml00000
Bilirubin, feature (1), no (0)00000
Ketone bodies, there is (1), no (0)00000
Protein, g/l3.2±0.083.0±0.093.0±0.12.9±0.093.0±0.06
The leukocyte count in a field of view 0-10-11-21-20-0
The erythrocyte count in a field of view0-01-20-10-10-0
ColorFrom light yellow to dark yellow
TransparencyBasically transparent
Males, 14 day
Number of allocated urine, ml14.6±1.714.5±1.615.2±1.919.0±0.815.48±1.8
pH6.0±0.066.2±0.086.0±0.096.0±0.076.0±0.08
Specific gravity, g/ml0.98±0.0011.01±0.0011.00±0.0011.01±0.0051.02±0.001
Glucose, g/ml0000
Bilirubin, feature (1), no (0)00000

0
Continuation of table 16
123456
Ketone bodies, there is (1), no (0)00000
Protein, g/l2.9±0.072.9±0.092.9±0.073.1±0.083.1±0.09
The leukocyte count in a field of view0-10-10-10-00-1
The erythrocyte count in a field of view0-20-1 0-10-10-1
ColorFrom light yellow to dark yellow
TransparencyBasically transparent
Females, 14 days
Number of allocated urine, ml17.1±2.815.7±2.616.0±1.715.6±4.913.9±3.1
pH5.8±0.076.1±0.096.2±0.085.7±0.086.0±0.09
Specific gravity, g/ml1.04±0.0011.04±0.0010.96±0.0021.04±0.0011.04±0.002
Glucose, g/ml00000
Bilirubin, feature (1), no (0)000 00
Ketone bodies, there is (1), no (0)00000
Protein, g/l3.3±0.092.9±0.093.1±0.093.1±0.13.1±0.08
The leukocyte count in a field of view0-10-10-01-20-1
The erythrocyte count in a field of view0-01-20-10-11-2
ColorFrom light yellow to dark yellow
TransparencyBasically transparent
Males 21 days
Number of allocated urine, ml17.3±2.615.4±1.816.3±1.814.9 the 1.9 15.9±2.6
pH6.0±0.065.9±0.075.8±0.095.9±0.096.0±0.08
Specific gravity, g/ml1.00±0.00081.01±0.00091.00±0.00091.00±0.0011.00±0.0009
Glucose, g/ml00000
Bilirubin, feature (1), no (0)00000
Ketone bodies, there is (1), no (0)00000
Protein, g/l3.0±0.072.9±0.093.1±0.13.2±0.083.2±0.1
The leukocyte count in a field of view 0-00-10-00-00-1
The erythrocyte count in a field of view0-00-10-10-10-0
ColorFrom light yellow to dark yellow
TransparencyBasically transparent
Female 21 day
Number of allocated urine, ml16.2±0.915.3±0.915.9±1.816.9±2.584±14
pH6.2±0.075.7±0.095.9±0.086.0±0.076.0±0.06
Specific gravity, g/ml1.02±0.0051.02±0.0041.00±0.0020.99±0.0010.99±0.001
Glucose, g/ml0000
Bilirubin, feature (1), no (0)00000
Ketone bodies, there is (1), no (0)00000
Protein, g/l3.1±0.083.1±0.093.2±0.093.0±0.073.1±0.09

Continuation of table 16
123456
The leukocyte count in a field of view0-00-10-00-00-1
The erythrocyte count in a field of view0-10-2 0-10-00-1
ColorFrom light yellow to dark yellow
TransparencyBasically transparent

As you can see, the urine parameters in all experimental groups close in meaning to the significant differences between any groups not detected.

Impact ON the CHARACTER of ELECTROCARDIOGRAPHY

Data on the impact of the introduction subacute 14-day drug on electrocardiography parameters are presented in table 17.

Table 17
The influence of the drug dosage form of Pedifen 0.25% on the variation and the nature of the ECG albino rats (M±m)
The studied parametersThe intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
1234 56
Males, the background
HR/min453±18465±21445±36459±21439±28
R mV0.09±0.010.08±0.010.08±0.010.09±0.020.10±0.02
R mV0.60±0.050.61±0.100.63±0.090.60±0.060.64±0.10
S MB0.02±0.030.04±0.040.04±0.030.03±0.040.04±0.04
T, mV0.15±0.020.13±0.0090.13±0.030.16±0.010.14±0.04
PQ, MS46.3±1.046.4±2.146.1±1.145.3±1.144.1±1.0
QT, MS53.8±2.154.5±3.258.0±2.257.4±2.256.2±3.1
Females, the background
HR/min459±27442±20470±23467±33425±12
R mV0.09±0.010.10±00.10±00.09±0.020.10±0.009
R mV0.62±0.060.66±0.100.63±0.110.60±0.100.69±0.09
S MB0.04±0.040.01±0.010.02±0.020.03±0.030.01±0.02
T, mV0.14±0.010.16±0.020.15±0.030.16±0.010.14±0.03
PQ, MS46.6±2.145.±1.0 47,6±2.146.4±2.146.0±1.0
QT, MS61.3±3.155.9±3.258.3±1.156.1±3.255.9±3.2
Males, 14 day
HR/min472±23454±22453±21426±21455±20
R mV0.09±00.09±0.020.09±00.09±00.10±0
R mV0.61±0.090.59±0.100.60±0.100.66±0.090.62±0.10
S MB0.02±0.010.03±0.030.02±0.020.009±0.010.02±0.02

Continuation of table 17
1 23456
T, mV0.17±0.0090.16±0.020.16±0.020.16±0.020.16±0.03
PQ, MS46.4±1.145.6±2.247.0±2.245.3±1.147.7±2.0
QT, MS57.8±3.256.1±3.156.5±1.056.1±3.159.2±1.1
Females, 14 day
HR/min462±23440±13429±19474±20450±36
R mV0.09±00.11±0.010.09±00.10±00.08±0.01
R mV0.62±0.080.68±0.100.64±0.09 0.61±0.110.61±0.09
S MB0.01±0.010.02±0.020.01±0.010.03±0.020.03±0.03
T, mV0.16±0.010.15±0.030.15±0.030.16±0.020.13±0.02
PQ, MS46.7±1.046.0±1.145.5±1.146.6±2.147.0±1.1
QT, MS57.2±3.156.0±3.257.0±3.157.5±1.158.3±2.1

Analysis of the data shows no differences in the magnitude of heart rate and nature of the ECG in all experimental groups. On day 14, the value of intergroup differences - the value of F0 was equal to 0.71 at the critical value F0.95; 10,90=1.99.

Figure 1 shows the ECG of white rats after 14 days of study at the maximum dose.

The EFFECT ON the PERIPHERAL BLOOD

Hematological and biochemical blood tests were carried out after 14 d the her and in the end of the study. Blood was obtained after simultaneous giltinane.

The data obtained on the 14th day of the experiment are presented in table 18.

Table 18
The influence of the drug dosage form of Pedifen 0.25% basic hematological parameters of peripheral blood white rats (M±m, n=10)
The studied parametersThe intactControlDose
0.06 ml/kg0.18 ml/kg0.6 ml/kg
123456
Males, 14 days
Hemoglobin, g/DL11.9±0.211.9±0.212.1±0.112.1±0.112.0±0.1
Hematocrit, %49.8±1.149.9±1.250.5±1.1 51.4±1.250.1±1.2
Erythrocytes, 1012/l6.5±0.26.5±0.36.4±0.16.3±0.36.3±0.2
Color index (sit), PG24.8±0.823.9±0.822.8±0.722.7±0.523.7±1.0
The average volume of red blood cells MCV, μm377.6±0.776.5±0.777.8±0.576.8±0.677.6±0.6
Average conc. hemoglobin in the erythrocyte ICSU, %24.5±0.323.5±0.223.5±0.124.4±0.223.4±0.1
ESR, mm/h4.2±0.44.8±0.45.3±0.34.6±0.44.9±0.3
Platelets, 109/l580±12.1582±10.9630±10.9 614±10.3600±11.0
Leukocytes, 109/l6.7±0.46.6±0.27.0±0.096.4±0.26.5±0.2
Stab neutrophils, %0.3±0.0090.2±0.0090.2±0.010.3±0.010.2±0.01
Segmented neutrophils, %12.4±1.012.7±1.112.5±1.112.7±1.212.6±1.1
Basophils, %00000
Eosinophils, %3.4±0.23.6±0.23.4±0.23.3±0.23.5±0.2
Ionicity, %3.2±0.22.8±0.33.3±0.43.0±0.22.9±0.3
Lymphocytes, %77.4±0.977.0±1.077.9±1.278.9±1.276.9±1.0
Plasma cells, %0.2±0.020.1±0.010.1±0.020.1±0.010.1±0.01
Activated. partials. thromboplastin. time42.7±1.842.1±2.041.2±2.041.3±1.8141.7±1.9
Prothrombin time15.4±1.115.0±1.114.2±1.213.7±0.914.9±1.2
Females, 14 day
Hemoglobin, g/DL12.2±0.312.3±0.212.4±0.112.4±0.212.1±0.1
Hematocrit, %49.75±1.050.4±0.949.2±1.149.4±1.0 50.2±0.9
Erythrocytes, 1012/l6.3±0.16.5±0.36.2±0.16.3±0.26.5±0.2
Color index (sit), PG23.2±0.722.6±0.623.7±0.923.6±1.025.1±0.8
The average volume of red blood cells MCV, μm377.8±0.777.6±0.677.4±0.577.3±0.578.1±0.6
Average conc. hemoglobin in the erythrocyte ICSU, %24.0±0.223.5±0.224.4±0.224.4±0.224.4±0.3
ESR, mm/h4.7±0.33.8±0.33.3±0.23.4±0.24.5±0.3

Continuation of table 18
12 3456
Robocity, 109/l623±10.9648±9.9619±10.0578±11.0580±10.4
Leukocytes, 109/l6.8±0.26.2±0.26.5±0.25.9±0.25.6±0.2
Stab neutrophils, %0.1±0.010.2±0.010.2±0.010.1±0.010.3±0.01
Segmented neutrophils, %12.0±0.812.5±0.912.1±1.111.9±1.212.0±1.1
Basophils, %00000
Eosinophils, %3.3±0.23.5±0.33.1±0.32.9±0.1 3.0±0.2
Monocytes, %3.2±0.33.7±0.33.7±0.13.8±0.23.6±0.1
Lymphocytes, %78.5±0.878.8±1.078.5±1.176.5±1.278.7±1.0
Plasma cells, %0.2±0.010.2±0.0080.3±0.0080.1±0.0080.1±0.02
Activated. partials. thromboplastin. time39.2±1.840.3±1.940.8±2.042.3±1.943.8±2.0
Prothrombin time13.8±1.114.0±1.214.7±1.015.7±1.016.4±1.1
Males, 21 days
Hemoglobin, g/DL12.3±0.212.5±0.2121±0.2 12.0±0.212.2±0.2
Hematocrit, %49.6±1.049.7±1.150.2±1.250.3±1.349.9±1.0
Erythrocytes, 1012/l6.3±0.26.2±0.26.4±0.36.5±0.36.3±0.2
Color index (sit), PG23.9±1.023.4±1.023.7±1.023.9±0.923.1±0.6
The average volume of red blood cells MCV, μm377.5±0.677.4±0.676.5±0.776.4±0.877.4±0.7
Average conc. hemoglobin in the erythrocyte ICSU, %24.6±0324.4±0.223.4±0.123.5±0.223.6±0.3
ESR, mm/h3.4±0.33.5±0.34.9±0.4 4.8±0.44.8±0.3
Platelets, 109/l578±12.0589±11.0594±11.0584±11.0627±11.0
Leukocytes, 109/l6.3±0.46.6±0.56.6±0.26.7±0.26.8±0.4
Stab neutrophils, %0.2±0.010.2±0.010.2±0.010.2±0.010.3±0.01
Segmented neutrophils, %12.0±1.212.1±1.312.6±1.112.8±1.112.2±1.0
Basophils, %00000
Eosinophils, %3.2±0.33.1±0.33.6±0.23.7±0.2 3.2±0.3
Monocytes, %3.8±0.33.6±0.32.8±0.32.9±0.33.4±0.4
Lymphocytes, %77.2±1.177.0±1.276.9±1.077.4±1.078.2±1.0
Plasma cells, %0.2±0.010.3±0.010.1±0.010.1±0.010.4±0.01

Continuation of table 18
123456
Activated. partials. thromboplastin. time42.5±2.041.5±1.941.7±1.942.3±2.040.0±1.83
Prothrombin time14.9±1.214.7±1.114.9 the 1.2 15.0±1.113.9±1.0
Female, 21 days
Hemoglobin, g/DL12.1±0.212.1±0.212.1±0.112.1±0.212.2±0.3
Hematocrit, %50.1±1.251.3±1.051.0±1.250.7±1.352.0±1.1
Erythrocytes, 1012/l6.3±0.36.5±0.16.5±0.16.3±0.096.1±0.2
Color index (sit), PG22.2±0.622.9±0.723.1±0.923.8±1.024.4±0.8
The average volume of red blood cells MCV, μm376.6±0.777.5±0.777.5±0.776.7±0.777.6±0.8
Average conc. hemoglobin in the erythrocyte ICSU, %3.9±0.1 23.9±0.323.3±0.123.5±0.124.3±0.2
ESR, mm/h4.7±0.35.2±0.34.8±0.45.8±0.37.3±0.5
Platelets, 109/l615±10.4635±9.9563±11.1599±9.9586±12.0
Leukocytes, 109/l7.3±0.17.4±0.16.3±0.16.1±0.27.0±0.1
Stab neutrophils, %0.3±0.010.2±0.010.1±0.010.1±0.010.4±0.02
Segmented neutrophils, %12.3±1.312.7±1.212.4±1.012.7±1.013.0±1.0
Basophils, %00 000
Eosinophils, %3.5±0.33.4±0.23.6±0.13.7±0.13.8±0.3
Monocytes, %3.1±0.34.3±0.53.8±0.33.4±0.34.1±0.3
Lymphocytes, %77.7±1.378.6±1.276.7±1.077.1±0.977.8±0.9
Plasma cells, %0.09±0.010.09±0.010.1±0.010.09±0.010.1±0.01
Activated. partials. thromboplastin. time43.8±1.544.2±1.743.6±2.044.2±1.943.4±1.9
Prothrombin time15.4±0.915.8±1.114.9±1.215.8±1.3 15.4±1.2

Statistical analysis of the obtained data revealed no significant differences between all experimental groups on any of the indicators (all indicators criterion of heterogeneity of the groups was below the critical value).

Microscopic examination of blood smears showed the following. Lymphocytes are strongly picotesla the core is sometimes found significant Ashrafinia grain. The bulk of segmented cells has a gentle, neutrophilic, not quite clear grain in smears. Kernel eosinophilic cells formed from loose chromatin substance and have an almost circular shape. Kernel eosinophils and neutrophils are formed on ringed type, so often found the annular core has a rod-shaped forms. Monocytes are very different from lymphocytes. The latter is equal to the value of two cells, have a great bobbygee core and wide protoplasmatic the border that is painted in blue or purple color with a delicate granulation. Blood platelets are large piles. Significant differences between the parameters of the control and test groups were not observed. Sex differences were not found. Thus, the peripheral blood of rats of all experimental groups after the 14-day studies on its share of the governmental and qualitative composition corresponded to specific physiological norm.

IMPACT ON BLOOD BIOCHEMICAL parameters

Table 19 shows the values of the main biochemical parameters of blood of rats of all experimental groups obtained on day 14 of the study.

Analysis of the data in table 19 did not reveal any shift of biochemical parameters of blood in experimental groups as compared to control, and in relation to each other. Thus, to the 14th day of the study the result of applying the drug dosage form of Pedifen 0.25% did not lead to any significant effects on the biochemical parameters of blood.

Significant differences between the levels of the studied parameters at the end of the observation period (21 days) is also not detected.

DATA PATHOMORPHOLOGICAL STUDIES

This section provides summary data of necropsy rats all studied groups, as the results of macroscopic studies of the studied organs of differences between groups is not installed.

Rats right physique, satisfactory power. At external examination of discharges from natural orifices is not detected. Most animals coat is glossy, neat appearance, lesions of alopecia is not defined.

Eyes saved. The eye slit of the usual dimensions. The mucous membranes of the conjunctiva normal OCD the ski. Signs of irritation not present.

Teeth saved. Visible mucous membranes pale coloring, brilliant. The mammary glands of females without seals to the touch, nipple discharge was absent. Male reproductive organs are developed correctly, deformation or swelling of the limbs not.

Thoracic and abdominal effusion does not contain. The position of the internal organs of the thoracic and abdominal cavities violations is not. Parietal and visceral pleura and peritoneum thin, shiny, smooth.

The submandibular lymph nodes and salivary glands are oval, pale yellow or pinkish color, with a smooth surface, a thin capsule, not soldered between themselves and the underlying tissues. The surface of the incision uniform color.

The thyroid gland is a reddish color, normal size and shape, moderately thick consistency. The thymus gland is triangular in shape, whitish, moderately thick consistency, normal size.

The intima of the aorta smooth, shiny, whitish color. The diameter of the aorta is not modified. The leaves of the pericardium is a thin, transparent, smooth. The size and shape of the heart changes are not present. The left ventricle is reduced, in the right contains a small amount of dark liquid blood. Heart valves are thin, shiny, smooth. The heart muscle in the context of a uniform brownish color, moderately dense.

Illuminate the trachea and large bronchi is not modified, the mucous membrane is shiny, smooth, pale color. Light aircraft, without seals to the touch, pale pink color.

Mucosa of the esophagus shiny, smooth, pale color. Stomach normal size and shape, filled with food content. The mucous membrane is iron free part of the stomach is folded, pink, brilliant. The mucosa of the gastric body folded, pink, brilliant.

The mucous membrane of the small intestine pale pink, shiny, smooth. The mucous membrane of the colon grayish color, shiny, smooth.

The size and shape of the liver changes are not present. The surface of the liver is smooth, uniform dark red color, the capsule is a thin, transparent. The liver tissue in the context of a full, moderately dense.

The pancreas is a flat-shaped, pale pink, lobed, moderately thick consistency.

The spleen is of normal shape, dark cherry, moderately thick consistency. Surface of body smooth, thin capsule. In the section on dark red background spleen visible small grayish color follicles.

The size and shape of the kidneys is not changed. The surface of the kidney brownish, smooth, capsule thin, transparent, easy to remove. In the context of organ distinct cortical and brain substance.

The adrenal glands rounded, pale-zelcogetvica, smooth, moderately dense. The incision is in dark-colored brain substance.

The bladder is filled with a transparent urine. The mucous membrane of the bladder smooth, shiny, pale color.

The body of the uterus in females normal density, size and shape. The horns of the uterus thin, slimy shiny, pale. Ovaries dark red color, with a rough surface, moderately dense. The testes of males whitish color, normal size and density.

Shell brain thin, transparent. The substance of the brain normal density, the surface of the brain is smooth. On frontal sections of the brain are clearly distinguished gray and white matter. The ventricles of the brain normal values, there is no such extension.

Table 20 presents data on the mass ratios of the organs of white rats of all experimental groups on day 14 of the experiment.

From the data given in the table it follows that the significant differences of the mass coefficients of internal organs of rats in any of the experimental groups from the reference no. Pathological changes are not marked and 21 days.

Thus, the 14-day registration in the eyes of the dosage form of the drug Pedifen 0.25% in three ranges of doses, does not cause significant morphological changes in the internal organs, including brain, who has no local irritating effect on the mucous membranes of the eye.

The RESULTS of HISTOLOGICAL RESEARCH

The objects were fixed in 10% formalin or fluid Carnoy and embedded in paraffin. The brain was fixed in 96% alcohol and placed in celloidin or paraffin. The sections of the internal organs were stained with hematoxylin-eosin and van gieson, brain slices were stained by Nissly.

When viewing a histological study of the organs of the differences between groups were found.

Cytoarchitectonics of the cerebral cortex of the brain is not broken, foci of no loss. The vessels of the meninges moderately full; cortical vessels of uniform diameter. Contain a single erythrocytes. The signs of acute or chronic disease of the neurons are absent. Nucleus neurons bright, the nuclear membrane is a thin, clear nucleoli. In the cytoplasm is determined enough chromatopelma the grit Nissle: dust in the cytoplasm of neurons 2-3-th layer and larger in the cytoplasm of neurons of the 5th layer of the cerebral cortex. Neurons of different nuclear formations of the middle and medulla oblongata contain large glybki of tigroid. The nucleus of nerve cells and glial cells is not changed - the nuclear membrane is thin, the content of chromatin normal nucleoli clear.

Endothelium cells of the inner lining of the aorta with clear nuclei. Destruction of elastic fibers among which it shell not. Cross-striated myofibrils in all parts of the heart are distinct, core cardiomycytes contain a sufficient amount of chromatin, nuclear membrane thin. Foci violations tinctorially properties of cytoplasm no. Excessive proliferation of stroma (cardiovirus) no.

The epithelium of the larynx, trachea, large bronchi is not changed, the engine clear. The alveoli of all lobes of the lungs contain air. Kernel alveolar epithelium clear, cytoplasm oxytelinae. The internal-lung epithelium of the bronchi unchanged. Acute inflammatory changes in the lung tissue was absent.

Trabecular structure of liver sections obtained from different fractions of liver disorders is not. Border hepatocytes distinct, granular cytoplasm, slightly oxytelinae. Focal violations tinctorially properties of cytoplasm no. Nuclei contain a clear nucleoli and a sufficient amount of chromatin. Nuclear membrane is thin. The sinusoids of the liver worthwhile.

The capillaries of the glomeruli and interstitial tissue of the kidney worthwhile, the cytoplasm of the epithelium of the proximal tubules of the kidneys oxytelinae, cell borders are visible, kernel neuroepithelial bright, clear.

The vessels of the cortex and the medulla of the adrenal glands worthwhile. All zones of the adrenal cortex is clearly expressed, the cell nucleus contains a sufficient amount of chromatin. C is toplasma cell beam zone vacuolation due to the content of a large number of lipids. The cells of the medulla of large, oval, United in clusters and cords.

Lymphoreticular elements of lymph nodes and spleen with clear nuclei, destruction or atrophy of the follicles no. In the red pulp of the spleen visible foci of hematopoiesis with isolated megakaryocytes.

The mucous membrane is iron free part of the stomach is lined by stratified squamous epithelium, the cells of which changes are not present. The covering epithelium of the mucosa of the gastric body formed mucous cylindrical cells, defects in the epithelial lining no. Main and tuck the glands of the body of the stomach are not changed. Defects stratified squamous non-squamous epithelial lining of the esophagus is not.

Lobed structure of the pancreas saved. Cells of the islets of Langerhans contain bright, clear nucleus, cytoplasm weakly oxytelinae. The epithelial cells of the exocrine part of the gland basophilic, kernel, located in the middle part, clear, with a sufficient quantity of chromatin Vessels stroma pancreas moderately full.

Thyroid follicles filled with a small amount of oxytelinae, slabomineralizovannogo colloid. The epithelium of the follicle regular height, kernel clear. The vascular stroma moderately full. The structure of the submandibular glands of violations does not belong to yet. Epithelial cells of the leaf departments and excretory ducts with clear nuclei, destruction of cells no.

Thymus retains expressed lobed structure. The cortical substance of the lobules filled with lymphocyte/thymocyte. The medulla of the thymus contains fewer lymphocytes and bright epithelial cells, sometimes forming a concentric shape, and the bullock of Kassala. The stroma of the thymus moderately full.

In the cortical substance of the ovaries of females visible follicles of different size and degree of ripening. The follicular epithelium is not changed, the engine light, clear, the medulla of the ovary worthwhile. Cells of the seminiferous tubules of the testes of males are at different stages of spermatogenesis. The epithelium of the seminiferous tubules and interstitial cells are not changed. Kernel-clear.

Thus, the results of the necropsy and histological examination daily use dosage forms drug Pedifen 0.25% in three ranges of doses for 14 days in rats of both sexes does not cause irritation, inflammation or destruction the eye tissues, and is not accompanied by the development of dystrophic and destructive, focal sclerotic changes in parenchymatous cells and stroma of the internal organs of rats.

The STUDY of SUBACUTE TOXICITY IN INTRACRYSTALLINE the INTRODUCTION of RABBITS

The INFLUENCE OF ETIMOLOGICALLY INDICATORS

During the experiment we recorded the following parameters: the intensity of blepharospasm, tearing, redness of the conjunctiva of the eyelids and sclera on its intensity and duration, the severity of pupillary reflex, sensitivity of the cornea (corneal reflex), damage to the cornea (fluoresceine test).

Evaluation of blepharospasm conducted visually on the duration of contraction of the circular muscles of the eye (s).

The severity of watery eyes were estimated by a method Schirmer (modification for rabbits) according to the intensity of wetting of the paper tape (mm).

The intensity of hyperemia of the conjunctiva of the eyelids and the sclera of the eyeball was evaluated visually: 1 point - vessels injected, 2 points - individual veins indistinct, 3 credits diffuse deep redness.

The severity of swelling of the eyelids were also evaluated visually: mild swelling of the eyelids - 1 point, marked swelling with partial eversion of the eyelids - 2 points, resulting in swelling of the eyes closed half - score of 3, the result of swelling of the eyelids closed for more than half a score of 4.

Evaluation of the pupillary reflex indoors with low light. Pupil diameter is determined by the linear pupillometer. As a light stimulus using a narrow beam directional light (medical flashlight).

The condition of the corneal epithelium was estimated by the method of the m fluorescein samples In the conjunctival SAC of the eyes injected with 1-2 drops of 0.5% solution of fluorescein sodium. A positive test (+) when the green staining of the corneal epithelium indicates damage of the corneal epithelium. All animals of the three experimental groups were absent blepharospasm and swelling of the eyelids, fluoresceine test was negative. The results are presented in table 21

Table 21
The severity of response from the eyes of rabbits after 14 days of installation of the dosage form of the drug Pedifen 0.25% (M±M)
The studied parametersThe intactControlPedifen 0.25%
0.05 ml/kg0.15 ml/kg0.5 ml/kg
123456
Males, 14 days
Blepharospasm (C)0000 0
Tearing, mm9.8±0.99.5±0.911±1.812.5±1.811.9±2.3
Conjunctival hyperemia (point)00-10-11.72.0±0.0
Swelling of the conjunctiva of the eyelids (point)00000
Fluoresc. test (+)(-)-----
The duration of anesthesia (min)0026,3±1.826,3±1.628,0±1.8
Pupil diameter, mmdispelled. St.8.2±0.358.0±0.185.7±0.357.7±0.18 7.5±0.16
healthy lifestyles. St.6.2±0.56.1±0.56.1±0.56.3±0.55.6±0.18
Females, 14 days
Blepharospasm (C)00000
Tearing, mm9.5±0.110.5±2.310.8±2.310.8±2.312.8±1.4
Conjunctival hyperemia (point)01.71.8±0.412.4±1.21.7±0.45
Swelling of the conjunctiva of the eyelids (point)01000
Fluoresc. test (+)(-)--- --
The duration of anesthesia (min)0024.8±1.428,3±2.728,0±1.8
Pupil diameter, mmdispelled. St.8.3±0.58.2±0.38.5±0.58.0±0.357.8±0.35
healthy lifestyles. St.6.3±0.56.1±0.56.1±0.76.2±0.355.8±0.35

In rabbits the first group at 7-14 day of the experiment demonstrated a slight increase watery eyes, the appearance of hyperemia of the conjunctiva of the eyelids, is recorded in 2/3 of experimental animals with intensity 1 point, the duration of anesthesia (absence of corneal reflex) accounted for up to 25 minutes All symptoms irritation eyes after instillation of the drug at a dose of 0.05 mg/kg remained no more than 30-60 minutes On the next day after the termination of the 14-day exposure of Pedifen 0.25% in the dose, the eyes of experimental animals were without pathology and did not differ from the control.

In the first the following days after instillation of the drug Pedifen 0.25% at a dose of 0.15 mg/kg, there was an increase in the intensity of watery eyes. On the first day appeared hyperemia of the conjunctiva of the eyelids to 1 point, the intensity of which gradually increased to a 14-day introduction to 1.7 points. However, the duration of hyperemia was not more than 2 hours after drug administration. The duration of anesthesia after each installation was 26-27 minutes On the following day after 14 injections all the symptoms of irritation on the part of the membranes of the eyes had disappeared.

In all animals of group III after daily instillation of the drug dosage form of Pedifen 0.25% noted a small increased watery eyes. Already after 1 day of injection in all animals (100%) appeared hyperemia (not less than 1 point), starting from the 7th day of the introduction, the intensity of hyperemia of the conjunctiva of the eye increased to 2 points and remained on the following day, decreasing to 1 point. 2 days after the last instillation of the drug conjunctival hyperemia was gone. The intensity watery eyes returned to normal (8,5±0.5 mm). The duration of local anesthesia after each instillation of drug Pedifen 0.25% registered for 28-30 minutes 48 hours after the last injection in the conjunctival SAC of the aqueous solution of the drug all signs of irritation were practically absent (figure 2).

Animals of the control group were daily introduced into the conjunctival mesh is to the left eye of the maximum volumes of distilled water, 0.5 ml for 14 days.

The results show that the used in the present work, the solvent of the model drug (distilled water), if taken daily for 14 days does not cause a response from the conjunctival membranes of the eye (blepharospasm, lacrimation, hyperemia of the conjunctiva, conjunctival edema, inhibition of corneal reflex, organic lesions of the conjunctiva of the eye). Minor redness, marked with 7 days, most likely related to daily manipulations (assessment watery eyes, repeated instillation of large volumes) and had a short duration (approximately 2 hours).

Thus, the daily intracrystalline introduction of the dosage form in three doses (maximum therapeutic, 3-and 10-fold the maximum therapeutic) has expressed no irritating effect on the mucous membranes of the eyes. Marked irritation symptoms are intermittent and can be provoked daily manipulations associated with ophthalmologic examination.

The EFFECT ON BODY WEIGHT

The results of the weighing of rabbits are presented in table 22

As can be seen from the data presented in the table, the growth dynamics of the body weight in all experimental groups was the same. Analysis of the data which confirms the absence of any differences between groups: the value of F-test for the significance test is equal to 0.73 at the critical value F0.95; 10,90=1.99.

Table 22
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% on body weight of rabbits (g, M±m)
Survey duration (days)Groups of animals
InjectieControl0.05 ml/kg0.15 ml/kg0.5 ml/kg
MFMFMFMFMF
Background2529±302478±262471±302437±232499±312500±242548±302505±222521±322438±25
day 72569±322481±262564±312550±252560±332561±242663±332485±252545±322486±26
day 142736±342560±252737±312558±262696±322531±262660±312625±262712±322593±28
21 days2791±332687±272803±322682±282820±312696±292758±312657±282764±322697±29

Impact ON the CONSUMPTION of FOOD AND WATER

The definition of consumption of water and feed were determined in all groups of animals once a week and 7 days after instillation. The results are presented in table 23 and 24

Table 23
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% on feed intake of rabbits (g, M±m)
Survey duration (days)Groups of animals
The intactControl0.05 ml/kg0.15 ml/kg0.5 ml/kg
MFMFMFMFMF
Background194±1189±2191±2193±1191±3192±4195±2200±1196±1188±3
day 7195±1193±1 201±2199±2194±3192±1190±1200±2193±3195±2
day 14196±2199±3194±3197±1187±3196±3194±3196±1202±3197±2
21 days206±1205±2199±2197±3192±1195±3204±1205±2198±2210±2

Table 24
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% on water consumption rabbits (ml, M±m)
Time studiesThe group's belly is s
The intactControl0.05 ml/kg0.15 ml/kg0.5 ml/kg
(days)MFMFMFMFMF
Background76±273±171±372±175±375±173±275±378±178±2
day 770±176±180±170±377±272±278±379±176±277±3
day 1477±2 72±279±379±174±377±280±172±280±272±2
21 days81±277±281±279±178±280±281±277±378±377±2

Significant differences in feed intake and water in rabbits of all experimental groups were not found.

EFFECT ON CARDIOVASCULAR ACTIVITY

Data on the impact of subacute 14-day drug on heart rate are presented in table 25.

Table 25
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% heart rate (HR) rabbits (BPM), M±m)
Survey duration (days)Group alive is data
The intactControl0.05 ml/kg0.15 ml/kg0.5 ml/kg
MFMFMFMFMF
Background301±9309±11304±8324±10298±12309±10321±11335±9334±9317±8
day 14324±11296±12324±9338±11311±10322±11304±11270±12289±12321±10

Significant differences in the indices of heart rate in rabbits experiment the selected groups compared with the control was not detected (figure 3).

The EFFECT ON the PERIPHERAL BLOOD

Hematological analysis of blood was carried out at the end of the study. Blood was obtained after simultaneous giltinane.

The obtained data are presented in table 26.

3
Table 26
Effect of subacute intracrystalline the introduction of the drug dosage form of Pedifen 0.25% basic hematological parameters of peripheral blood of rabbits (M±m)
The studied parametersThe intactControlDose
0.05 ml/kg0.15 ml/kg0.5 ml/kg
123456
Males, 14 days
Hemoglobin, g/DL16.7±0.0816.1±0.117.1±0.0917.0±0.0916.4±0.09
Hematocrit, %41.8±1.745.1±1.543.4±1.545.5±1.742.6±1.9
Erythrocytes, 1012/l6.9±0.095.8±0.096.8±0.096.7±0.096.4±0.1
Color index (sit), PG23.8±0.923.7±0.822.9±0.723.1±0.722.7±0.6
The average volume of red blood cells MCV, μm363.7±1.665.0±1.163.0±1.662.8±0.862.4±0.9
Average conc. hemoglobin in the erythrocyte ICSU, %38.5±0.437.5±0.436.8±0.437.3±0.638.2±0.5
ESR, mm/h5.2±0.54.7±0.45.6±0.45.4±0.45.0±0.4
Platelets, 109/l247±9238±9269±7263±8268±10
Leukocytes, 109/l10.3±0.412.6±0.510.2±0.610.1±0.510.2±0.5
Stab neutrophils, %1.5±0.121.1±0.111.5±0.101.4±0.101.2±0.13
Segmented neutrophils, %70.7±0.871.2±0.973.0±0.472.9±0.770.4±0.9
Basophils, %00000
Eosinophils, %2.1±0.092.1±0.12.0±0.091.8±0.11.9±0.1
12456
Monocytes, %5.7±0.45.7±0.55.9±0.36.3±0.35.8±0.4
Lymphocytes, %18.7±0.518.0±0.918.9±0.618.0±0.918.1±1.0
Plasma cells, %0.4±0.010.4±0.020.3±0.020.3±0.030.5±0.04
Activated. partials. thromboplastin. time49.9±1.450.8±1.248.5±1.349.1±1.448.1±1.5
Prothrombin time19.5±1.419.8±1.318.7±1.118.7±1.218.8±1.0
Females, 14 days
Hemoglobin, g/DL17.2±0.1 16.8±0.515.8±0.215.9±0.116.4±0.09
Hematocrit, %45.9±1.345.9±1.646.1±1.345.6±1.644.4±1.7
Erythrocytes, 1012/l6.4±0.096.1±0.086.2±0.096.1±0.16.1±0.09
Color index (sit), PG22.7±0.723.0±0.624.4±0.622.7±0.622.5±0.6
The average volume of red blood cells MCV, μm361.9±0.961.1±1.063.5±0.960.9±0.660.0±0.8
Average conc. hemoglobin in the erythrocyte ICSU, %32.6±0.236.0±0.633.8±0.0935.5±0.435.0±0.3
ESR, mm/h4.6±0.9 4.0±0.94.2±1.05.2±0.55.0±0.4

Continuation of table 26
123456
Platelets, 109/l243±10235±10269±8269±9273±9
Leukocytes, 109/l12.6±0.311.6±0.711.9±0.311.3±0.611.4±0.6
Stab neutrophils, %1.1±0.120.9±0.151.3±0.121.1±0.101.2±0.11
Segmented neutrophils, %67.9±0.868.7±0.567.8±0.769.3±1.0 68.7±0.8
Basophils, %00000
Eosinophils, %3.4±0.23.7±0.23.1±0.33.5±0.13.3±0.1
Monocytes, %6.6±0.56.2±0.36.5±0.47.0±0.37.0±0.3
Lymphocytes, %19.6±0.519.8±0.519.8±0.620.3±1.420.2±1.5
Plasma cells, %0.3±0.030.2±0.020.3±0.010.4±0.010.3±0.02
Activated. partials. thromboplastin. time49.9±1.950.6±1.250.7±1.747.7±1.6 48.7±2.0
Prothrombin time19.8±1.018.8±1.118.9±1.018.4±1.118.7±1.0
Males 21 days
Hemoglobin, g/DL16.6±0.0916.7±0.116.7±0.0916.6±0.0816.3±0.1
Hematocrit, %43.3±1.846.8±1.444.1±1.843.7±1.742.9±1.5
Erythrocytes, 1012/l6.7±0.095.8±0.086.8±0.087.0±0.15.9±0.09
Color index (sit), PG22.8±0.623.9±0.823.9±0.523.5±0.923.7±0.6
The average volume of red blood cells MCV, μm3 63.3±0.759.1±0.760.8±0.864.1±.763.7±1.1
Average conc. hemoglobin in the erythrocyte ICSU, %37.7±0.333.4±0.239.0±0.433.5±0.0936.1±0.4
ESR, mm/h4.9±0.24.5±0.95.3±0.54.1±1.03.9±0.9
Platelets, 109/l233±9236±9253±9267±8239±10
Leukocytes, 109/l12.7±0.412.4±0.310.5±0.411.8±0.311.6±0.7
Stab neutrophils, %1.0±0.091.0±0.121.4±0.111.1±0.120.9±0.15
Segmented neutrophils, %71.1±0.773.0±0.371.9±0.768.4±0.470.5±0.4
Basophils, %0000
Eosinophils, %2.0±0.091.8±0.082.0±0.083.8±0.13.7±0.2
Monocytes, %5.6±0.56.0±0.35.4±0.36.3±0.46.3±0.3
Lymphocytes, %18.5±0.818.6±0.619.0±0.43.7±0.119.3±0.5
Plasma cells, %0.2±0.020.2±0.020.3±0.0096.2±0.40.3±0.01
Activated. partials. romiplostim. time51.8±1.350.3±1.349.7±1.450.8±1.349.2±1.3
Prothrombin time19.4±1.319.5±0.919.9±1.319.4±0.917.7±1.1
Female 21 day
Hemoglobin, g/DL17.2±0.0816.3±0.0916.0±0.0916.0±0.116.5±0.09
Hematocrit, %43.9±1.644.0±1.745.6±1.345.3±1.744.3±1.3
Erythrocytes, 1012/l6.8±0.16.5±0.096.0±0.15.9±0.16.2±0.09
Color index (sit), PG23.5±0.8230±0.6 23.3±0.823.1±0.723.5±0.7

td align="center"> 20.4±0.3
Continuation of table 26
123456
The average volume of red blood cells MCV, μm363.1±1.662.7±0.862.3±0.861.9±0.662.2±0.7
Average conc. hemoglobin in the erythrocyte ICSU, %35.6±0.637.4±0.633.3±0.0934.2±0.232.6±0.3
ESR, mm/h3.9±0.94.1±0.94.1±0.95.1±0.44.6±0.8
Platelets, 109/l233±9268±9268±9265±8239±8
Leukocytes, 10 9/l12.2±0.711.6±0.211.7±0.311.0±0.412.0±0.4
Stab neutrophils, %0.9±0.141.2±0.141.2±0.121.0±0.131.0±0.12
Segmented neutrophils, %66.7±0.369.2±0.670.8±0.266.8±0.466.3±0.6
Basophils, %00000
Eosinophils, %3.6±0.093.1±0.13.6±0.093.7±0.093.2±0.2
Monocytes, %5.9±0.26.2±0.46.3±0.26.5±0.36.6±0.1
Lymphocytes, %18.9±0.420.3±0.519.4±0.519.2±0.3
Plasma cells, %0.2±0.0090.2±0.0080.2±0.020.3±0.0090.3±0.02
Activated. partials. thromboplastin. time51.6±1.250.9±1.650.6±1.149.6±1.347.6±1.7
Prothrombin time18.1±1.119.4±1.018.1±1.118.7±1.118.0±1.0

Statistical analysis of the obtained data does not reveal significant differences between all experimental groups on any of the indicators (all indicators criterion of heterogeneity of the groups was below the critical value).

IMPACT ON BLOOD BIOCHEMICAL parameters

In table 27 presents the core values of blood biochemical parameters of rabbits all experimental groups obtained at the end of the study.

Analysis of the data presented in table 27, are not being identified is l any shift of biochemical parameters of blood in experimental groups as compared to control, and in relation to each other. Thus, by the end of the 14-day study, the result of the application of both drugs did not lead to any significant effects on the biochemical parameters of blood.

Data pathomorphological studies

At external examination found that rabbits correct physique, satisfactory power. Discharge from natural holes were not found. Coat is glossy, neat appearance, lesions of alopecia is not defined. Teeth saved. Visible mucous membranes pale coloring, brilliant.

Eyes without inflammation. The subordinate apparatus is not changed. Conjunctiva pink color. Abnormal discharge from the lacrimal canals and conjunctival cavity no. Iris without pathological inclusions. The cornea has a smooth, transparent surface.

The mammary glands of females without seals to the touch, nipple discharge was absent. Male reproductive organs are developed correctly.

Deformation or swelling of the limbs not.

Thoracic and abdominal effusion does not contain. The position of the internal organs of the thoracic and abdominal cavities violations is not. Parietal and visceral pleura and peritoneum thin, shiny, smooth. Lymph nodes grayish color, round shape, with a smooth surface and dense whitish capsule. M is waiting for him and subjacent tissues nodes are not soldered. The thyroid gland is pinkish-red, normal size and shape, moderately thick consistency. The thymus is a flat formation, pale, slightly pinkish, lying among adipose tissue anterior mediastinum at the arm of the sternum. The dimensions and density are not changed.

The intima of the aorta smooth, shiny, whitish color. The diameter of the aorta is not modified. The leaves of the pericardium is a thin, shiny, smooth. The size and shape of the heart changes are not present. The left ventricle is reduced, in the right are dark red clots and liquid blood. Heart valves are thin, shiny, smooth. The heart muscle in the context of a uniform brownish color, moderately dense.

The lumen of the trachea and large bronchi is not changed, the mucous membrane is shiny, smooth, pale color. Light aircraft, without seals to the touch, pale pink color.

Salivary glands are oval, yellowish, moderately dense, in the context of granular form. Mucosa of the esophagus shiny, smooth, pale color. Stomach normal size and shape, filled with food content. The mucous membrane of the stomach is folded, glossy, pale color. The mucous membrane of duodenum 12 pale pink, smooth, shiny. The mucous membrane of the small intestine pale pink, shiny, smooth. The mucous membrane of the large Ki is Ki grayish color, shiny, smooth. The size and shape of the liver changes are not present.

The surface of the liver is smooth, uniform dark red color, the capsule is a thin, transparent. The liver tissue in the context of a full, moderately dense. Pancreas normal shape and size, moderate density, whitish, slightly pink, lobed. The spleen is of normal shape, dark cherry, moderately thick consistency, with wrinkled capsule. In the section on dark red background spleen visible small grayish color follicles.

The size and shape of the kidneys is not changed. The surface of the kidney brownish, smooth, capsule dense, whitish, easy to remove. In the context of organ distinct cortical and brain substance. The adrenal glands are oval, whitish, normal size and density. The bladder filled with a small amount of clear urine. The mucous membrane of the bladder folded, glossy, pale color. The body of the uterus in females moderately thick consistency, shape and dimensions are not changed, the mucous membrane tubes pale, shiny, smooth. The ovaries are of normal size and density, with a rough surface, grayish color. The testes of males whitish color, normal size and density, the cut surface granular, homogeneous pale color. Capsule dense, opaque.

The solid is th mater brain dense, whitish removed easily, binding with the substance of the brain is not. The substance of the brain normal density. Sulcus and gyrus brain well-defined. On frontal sections of the brain are clearly distinguished gray and white matter. The ventricles of the brain normal values, there is no such extension.

In table 28 presents data on the mass ratios of the organs of rabbits from all experimental groups.

The RESULTS of HISTOLOGICAL RESEARCH

Cytoarchitectonics of the cerebral cortex of the brain is not broken. Neurons of all layers in the motor region of the cortex contain the usual amount chromatopelma the grit Nissle, having the appearance of a large deep pyramidal cells of the 5th layer. Engine light, the nuclear membrane is a thin, clear nucleoli. Neurons of different nuclear formations of the mid-brain (the oculomotor nerve, the red nucleus, the black substance) contain large glybki of tigroid and bright core with a thin membrane. Neurons of different nuclear systems oblong brain (the reticular formation nuclei of the cranial nerves) contain large glybki of tigroid and light nuclei with distinct nucleoli. The signs of acute or chronic disease of the neurons are absent. Glial cells (astrocytes and oligodendroglia) are not changed. Kernel glial the notches contain a sufficient amount of chromatin. Regressive or progressive changes glia are missing.

Endothelium cells of the inner lining of the aorta with clear nuclei. Changes of elastic frame of the aortic wall no. Cross-striated myofibrils left and right ventricles and interventricular septum distinct, core cardiomycytes bright, cytoplasm oxytelinae. Foci violations tinctorially properties of cytoplasm no. Changes in the connective tissue stroma of the myocardium is not defined. The epithelium of the mucous membrane of the larynx, trachea, large bronchi saved, the nucleus of epithelial cells of the light. Vessels beneath the mucous layer of conventional diameter. Acute inflammatory changes are missing.

The alveoli of all lobes of the lungs contain air. Kernel alveolar epithelium clear. The epithelium of the alveoli and intra-lungs bronchi unchanged. The bronchial lumen wide. Acute inflammatory changes are missing.

The liver has expressed lobed structure. Trabecular structure cloves of violations is not. Border hepatocytes distinct, granular cytoplasm, slightly oxytelinae. Focal violations tinctorially properties of cytoplasm no. Nuclei contain a clear nucleoli and a sufficient amount of chromatin. The sinusoids of the liver worthwhile.

The epithelium of convoluted tubules of the kidneys is not changed. The cytoplasm oxytelinae, kernel clear, the thin membrane and a sufficient number of chromatin, cell borders are visible. The capillaries of the glomeruli and vessels located between the tubules, full-blooded. All zones of the adrenal cortex is clearly expressed. Cell boundaries are distinct, engine light. In the cytoplasm of the cell to the beam area visible vacuoles, representing a drop of oil on preparations stained with Sudan. The vessels of the brain substance worthwhile.

Lymphoid elements of the spleen with clear nuclei, destruction or atrophy of the follicles no. Reticular cells with a bright, large nuclei. The endothelial sinuses are not changed.

Defects in the epithelial lining of the stomach is not observed. Main and tuck the glands of the body of the stomach unchanged. Flat non-squamous epithelium of the esophageal mucosa pronounced layered structure, with distinct basal membrane and light nuclei. Lobed structure of the pancreas saved. Cells of the islets of Langerhans contain bright, clear nucleus, cytoplasm is weakly oxytelinae. The epithelial cells of the exocrine part of the gland basophilic, kernel, located in the middle part, clear. The vascular stroma of the pancreas moderately full.

Thyroid follicles are different sizes and filled oxyphyllum colloid. The epithelium of the follicle cubic, regular height, kernel clear. The vascular stroma moderately full. The structure porcellus the different salivary glands disorders is not. Bright epithelial cells of mucous Department glands surrounded by basophilic protein paloniemi. The epithelium of the excretory ducts has a clear cell borders and bright core. Lymphoreticular elements submandibular lymph nodes are not changed. Destruction, inflammation or atrophy of nodes not.

Thymus retains expressed lobed structure. Lymphoid cells (thymocytes), contain a clear core with a sufficient quantity of chromatin and thin nuclear membrane. The medulla of the thymus contains a small amount of lymphoid elements, as well as bright epithelial cells with large, pale colored kernels. Epithelial cells form a concentric shape, resembling pearls - calf, Kassala. The stroma of the thymus moderately full.

In the cortical substance of the ovaries of females visible follicles of different size and degree of ripening. The follicular epithelium is not modified, the kernel clear, the medulla of the ovary worthwhile. The testes of males - the epithelium of the seminiferous tubules and interstitial cells are not changed. Core clear cytoplasm oxytelinae.

Eyes. Histological examination of sections of the bulbar conjunctiva was characterized by the absence of necrosis of the epithelium. The epithelial layer was retained throughout. The top layers of the epithelium intact. Bezepitelialnye gap report is willow. Blood vessels and inflammatory infiltration was not determined. In the sclera under the conjunctiva changes of an inflammatory nature not mentioned. Microscopically in the lens without visible changes.

Thus, the daily intracrystalline introduction of the dosage form of the drug Pedifen 0.25% in different doses for 14 days to rabbits of both sexes does not cause dystrophic and destructive, focal sclerotic changes in parenchymatous cells and stroma of the studied organs, and is not accompanied by a local irritant effect on the eye.

Assessment of the effectiveness of the DRUG DOSAGE FORM of PEDIFEN 0.25% WITH the DEFEAT of EYE IRRITANTS

When conducting an experimental evaluation of the effectiveness of the drug dosage form of Pedifen 0.25% as the comparison drug was used 2% solution Dikaina (in the form of eye drops).

The effectiveness of the TOOLS of THERAPY WITH the DEFEAT of the EYE MORPHOLINO PELARGONII ACID

Experiments to test regimens eye lesions caused by exposure of morpholide pelargonii acid (IPC), was conducted on rabbits breed "Chinchilla". Experimental animals were divided into three groups: 1 - as a means of therapy was used the drug Pedifen 0.25%; 2 - solution Dikaina 2% (eye drops); 3 - control without treatment. Schema therapy use is alas on previously well-established technique, namely twice intracrystalline the introduction of the drug and every 30 minutes after installation of the IPC solution (3% solution in a volume of 0.02 ml). Observation of the animals was carried out within 14 days. During the experiment it was established that in the first day after exposure IPC status of the mucous membranes of the eyes of rabbits did not differ. It should be noted a more rapid decrease in severity of watery eyes in group No. 1. Further, since the 3 day marked subsidence of the inflammatory phenomena in group No. 1. Test with fluorescein was positive in all animals within 3-4 days. Dynamics of remitting inflammatory and destructive changes are shown in tab.

Table 29
The effectiveness of medicines use (terms of normalization of acute damage to the eyes of rabbits substance IPC).
The studied parametersThe timing of the disappearance of symptoms (days)
group 1group 2group 3
Conjunctival hyperemia3-4 6-812
Conjunctival edema1-248
Detachable122-3
Fluoresceine sample48-910-14

Thus, despite the fact that when exposed to IPC revealed no organic damage to the cornea and recover on their own, the use of the drug, Pedifen 0.25% in comparison with dikainom early after exposure to PB significantly accelerates the dynamics of the healing process.

The effectiveness of the MEANS of TREATMENT WHEN the LESION is in the EYE of CHLOROBENZYLIDENEMALONONITRILE

The effect of ortho-chlorobenzylidenemalononitrile (CS) was accompanied by the profuse lacrimation (40-60 mm) for 15-30 minutes, blepharospasm, conjunctival hyperemia intensity 2-3 points, swelling of the conjunctiva (1-2 points), decreased corneal sensitivity. Ophthalmoscopy using a slit lamp through the day revealed local and focal opacities in the corneal epithelium (1-2 points), micro - and patchy opacities in the surface and anterior stromal layers in the form of Christ is lychesky colorless and brown inclusions. In one case observed extensive infiltration of corneal vascularization and Robovie changes. Thus, in animals without treatment in the first days after exposure to CS was developed typical picture of the burn process of the second degree. The reverse process of the development of symptoms of the eyes of animals without treatment (group 3) was 7-9 days. The results are presented in table 31.

Animals of group 1 after instillation of the substance CS in the eye after 10 min was performed instillation of drug Pedifen 0.25% according to the above diagram. Observation of the animals ' eyes started immediately after exposure to CS and was conducted within 14 days. In rabbits, which we carried out the treatment of the aforementioned drug, these symptoms of chemical burn, as swelling of the eyelids, redness of the mucous membranes of the eyes were already after 3-5 days. The restoration of damaged corneal epithelium (negative fluoresceine sample) occurred after 4 days. After 3-4 days disappeared conjunctival hyperemia, 5 days completely disappeared swelling of the conjunctiva. The apparent complete disappearance of symptoms of inflammation occurred on the 6th day.

Animals 2 groups as a means of first aid intracrystalline solution was used dikaina 2%. In the first day of the clinical lesions of the mucous membranes of the eyes was not significantly different from group 1. Purchased, installed, the disappearance of the inflammatory phenomena in animals of this group is slower, but compared with the control group, with positive dynamics.

The results of the effectiveness of treatment is presented in table 30.

With the aim of studying the peculiarities of the development of the remote consequences of eye lesions substance CS in terms of drug therapy the eyes of all animals were subjected to 3 months biomicroscopy of the cornea and conjunctiva using a slit lamp. In the survey it was established that in some cases, even with a shiny smooth (healthy) of the cornea on its surface was detected as a light, superficial and deep foci of stromal opacities of various shapes, sprouting through the limb superficial and deep vessels. Results the influence of drugs on the prevention of late effects (observations 3 months) after the defeat of the eye CS are presented in table 31

Table 30
The effectiveness of medicines use (terms of normalization of acute damage to the eyes of rabbits substance CS)
The studied parametersThe timing of the disappearance of symptoms (days)
group 1group 2group 3
Conjunctival hyperemia3-567-8
Conjunctival edema3-569
Detachable124
Fluoresceine sample46-77-9
Table 31
The influence of drugs on the prevention of late effects (observations 3 months) after the defeat of the eye CS.
Integral resultThe observed group of animals
group 1group 2group 3
The number of rabbits b is C pathology of the conjunctiva of the eye 441
The number of rabbits with corneal pathology124

Thus, the use of the drug, Pedifen 0.25% can reduce recovery time and prevent the development of further complications from the side of the eyes caused by substance after the defeat of the substance CS.

The EFFECTIVENESS of THERAPY WITH the DEFEAT of the EYE DIBENZ[b,f]-1,4-OXAZEPINE (CR)

Exposures CR was accompanied by a growing blepharospasm, increasing lacrimation, hyperemia of the conjunctiva, swelling of the conjunctiva, decreased sensitivity of the cornea. Through 20-120 minutes after installation of the solution of the substance CR all animals were monitored sufficiently intense lacrimation, mild blepharospasm, intense hyperemia of the conjunctiva, multiple subconjunctival hemorrhage, chemosis, swelling of the eyelids, swelling of the third century. When conducting fluorescein samples were observed intense staining in the lower quadrants of all eyes. After 24 and 48 hours was observed pronounced redness of the cornea; fluoresceine sample was accompanied by a large (sometimes total colouring of the anterior cornea, which indicates that erosion Horny oblock the eyes. The staining with fluorescein anterior segment of the eye in some animals of the control group, expressed to a lesser extent, was observed up to 8 days after instillation of the substance CR.

A significant reduction in inflammation of the conjunctiva and eyelids, no swelling of the eyelids and of the third century. In some animals positive fluoresceine sample in the lower segments of the pupil. Within 14 days after instillation some animals have registered slight hyperemia (1 point), spot painting the lower part of the cornea when conducting fluorescein samples.

Thus, the full restoration of the anterior eye after injury caused by substance CR in the amount of 0,05 mg, occurs within 2 weeks.

After 10 min of exposure irritant against the background of significant watery eyes, redness of the conjunctiva, eyelids and third century, progressive swelling of the eyelids held twice instillation of drug Pedifen 0.25% with an interval of 10 seconds and re-instillation of the drug Pedifen 0.25% after 30 min after 10 min of re-instillation of the drug Pedifen 0.25% within 20 min recorded the absence of corneal reflex, followed by a decrease in the sensitivity of the cornea. After 60 min was observed moderate lacrimation, hyperemia of mucous century up to 3 points, chemosis, conjunctivitis with serous secretions after 10 min, the surface is ornago instillation of drug Pedifen 0.25%. Within 20-30 minutes after each instillation of the drug was noted the absence or significant reduction in corneal reflex, positive fluoresceine the sample in the lower segments to grip the optic zone. During the first days of acute inflammation was not.

Two days after treatment intensity watery eyes closer to the source data, hyperemia of the conjunctiva of the eyelids, of the third century was rapidly decreased, swelling of the eyelids was 1 point.

After 3 days occurs practically normalization watery eyes, remains moderate redness of the eyelids (2 points), swelling of the eyelids to 1 point, positive fluoresceine the sample with the capture of the lower parts of the iris. Procrasinate petechial haemorrhages.

5 days after exposure to a substance CR and subsequent treatment of Pedifen 0.25% noted the absence of most of the manifestations of intoxication: increased watery eyes, redness of the mucous membranes of the eyelids, swelling of the conjunctiva, eyelids, complete subsidence of the inflammatory manifestations of eye tissues. However, continued violation of the integrity of the corneal epithelium and in some animals, which was confirmed partial colouring when conducting fluorescein samples. Full recovery was recorded on a 7-8 day, which is almost 2 times greater than the time normalization condition of the mucous membranes of the eyes La the oratorial animals in respect of which has not been conducted remedial measures.

When used as the comparison drug solution Dikaina, recovery time was slightly reduced, which amounted to 10-12 days.

Thus, in the experimental evaluation of the effectiveness of the drug dosage form of Pedifen 0.25% shows that the use of this drug in the early stages after the defeat of eye irritants significantly reduces symptoms of irritation, promotes more rapid recovery of damaged mucous membranes from exposure to a chemical factor gas weapons and prevents the development of remote consequences. The drug in experimental studies demonstrated the best results in comparison with the reference product, because along with local antiserum action has other pharmacological properties. Pedifen 0.25% antioxidant, which significantly reduces the time course of the inflammatory process and promotes regenerative processes.

The STUDY of ALLERGENICITY AND IMMUNOTOXICITY assessment of the ALLERGENIC PROPERTIES

The total reaction anaphylaxis (anaphylactic shock)

Guinea pigs administered intracrystalline within 5 days. After 21 days after sensitization the animals intracardiac have introduced R is treausry dose, is equal to the total sensitizing. The same dose was administered to Guinea pigs of the control group.

A record of the intensity of anaphylactic shock was carried out in the indices Weigle:

++++ - shock fatalities;

+++ - shock severe (General convulsions, asphyxia, the animal loses the ability to stand on feet, falls on its side, does not die);

++ moderate shock (small convulsions, severe symptoms of bronchospasm);

+ - shock weak (some anxiety, shortness of breath, scratching faces, involuntary urination, defecation, hair disheveled);

0 - shock did not develop signs are missing.

The reaction of immune complexes

For the given reaction used Guinea pigs which were injected drug Pedifen 0.25% intracrystalline. In the control groups used water in the same volume.

Within 10 days after the last injection of the drug was carried out by the introduction of the resolving dose intradermally.

The resolving dose of the drug was determined on intact animals. For the resolving dose took the highest dilution of antigen does not cause visible changes in the skin within 1 hour after injection. In the control groups in the same way was injected solvent.

Accounting reactions were performed visually (in points on Savesave). In the positive case, after 30 minutes on the site of injection the AI can develop edema and hyperemia. After 2-3 hours of inflammatory focus can be compacted, and the skin is brown-red. Histological examination in the case of a positive reaction can be detected acute hemorrhagic inflammation, infiltrated the plot can be seen polymorphonuclear leukocytes.

Indirect reaction degranulation of mast cells.

To obtain the fat cells used outbred rats. For this bloodless rats in the abdominal cavity was injected 6 ml of sterile physiological solution (pH=7.4), heated to a temperature of 37°C. After the massage for 1 min abdominal wall in the midline of the abdomen was made the cut length of 1.5 see Turned the carcass is cut down and collected exudate flowing down the intestinal loops in a sterile tube.

The drugs were prepared on slides, pre-painted 0.3% alcoholic solution of neutral red and dried at room temperature.

To 30 μl of a suspension of fat cells was added 30 μl of serum experimental animal and 30 µl sample of the drug in a pre-selected dose so that degranulation of mast cells in the control did not exceed 5%. In the case of Pedifen 0.25% such optimal breeding has been a dilution of 1 mg/ml

Then the drugs cover cover glass, the edges of which are smeared with vaseline, and incubated in a thermostat at 37°C in ECENA 15 minutes.

After this drug mikroskopiruyut magnification ×20. The evaluation results are differential counting method, counting indicator degranulation of mast cells (PTC) according to the formula:

PTC=(1·a+2·b+3·e+3·d)/100,

where a, b, C, d - number (the average of three repetitions) degranulating cells, respectively, the degree of degranulation (mild, moderate, severe and complete degranulation of cells). Each drug was calculated 100 cells. The reaction was considered positive if PDTK exceeded 0.2.

Conjunctival test

For staging sample of 50 mg (ml) of the drug was administered under the upper eyelid Guinea pigs of the control and experimental groups. Under the upper eyelid of the second eye was injected with the same amount of water (control).

The reaction was taken into account in 15 minutes and after 24 to 48 hours in points.

Assessment of the ALLERGENIC PROPERTIES

The total reaction anaphylaxis (anaphylactic shock)

After 21 days after injection of the drug intracardiac all experimental and control groups was introduced resolving dose of Pedifen 0.25%.

Evaluation of response was performed in the indices Weigle. The results obtained are presented in table 32.

Table 32
Estimation of the total reaction anaphylaxis (indexes Weigle) mo the ski pigs after application of the drug Pedifen 0.25%
The experimental groupFloorNumbers of animals
123456
ControlM000000
F000000
Pedifen 0.25%M000000
F000000

Thus, the signs of even moderate shock were not identified nor real the animal from a group of females and males, receiving Pedifen 0.25% in the investigated dose range.

The reaction of indirect degranulation of mast cells

The experiments were performed on Guinea pigs, which on day 10 after intracrystalline introduction of Pedifen 0.25% was taken by the blood to obtain serum. The figures obtained degranulation of mast cells of rats in the presence of the test and control sera, drug Pedifen 0.25% or saline in the control is presented as table 33.

Table 33
The reaction degranulation of mast cells in rats (PTC) in the presence of sera of Guinea pigs treated with PegIFN 0.25% per month
The experimental groupFloorNumbers of animals
123456
ControlM0.090.100.120.160.140.11
F0.150.160.140.120.120.13
Pedifen 0.25%M0.160.100.140.140.120.15
F0.180.140.130.170.160.15

Among the resulting figures degranulation of mast cells in none of the cases did not exceed the value of 0.2, after which the reaction is considered positive. In all other cases it was below this value and, therefore, negative. The obtained data are processed statistically, are presented in table 34.

Table 34
A comparison of degranulation of mast cells in experimental and control groups of Guinea pigs
The experimental group FloorPDTK (M±m)t-student testThe probability (p)
ControlM0.120±0.011--
F0.137±0.007--
Pedifen 0.25%M0.135±0.0091.08>0.05
F0.155±0.0081.81>0.05

Thus, significant differences in the mean values PDTK in the experimental and control groups of female and male Guinea pigs treated with the drug Pedifen 0.25% in one month, is not revealed. These data allow us to conclude that when intracrystalline the introduction of the drug Pedifen 0.25% has no allergenic properties, which are identified in the indirect reaction degranulation of mast cells.

The reaction of immune complexes

Sensitization of Guinea pigs was carried out by intracrystalline injection drug Pedifen 0.25% within 5 days. Within 10 days after the last introduction is of intradermally injected resolving dose of the drug. This dose was determined on intact animals and the highest dilution of the drug, not causing visible changes in the skin through the hours after intradermal injection. In the control was injected with the same dilution solvent is physiological saline. The results obtained are presented in table 35.

Accounting reactions were performed visually, starting 30 min after the introduction of the resolving dose. At this time no manifestations on the skin were found. No signs of reaction was not detected after 1, 4 and 24 hours after its production.

td align="center"> 0/0
Table 35
The reaction of immune complexes in Guinea pigs after administration of the resolving dose of Pedifen 0.25%
The experimental groupFloorNumbers of animals
123456
After 1 hour
ControlM0/00/00/00/00/0
F0/00/00/00/00/00/0
Pedifen 0.25%M0/00/00/00/00/00/0
F0/00/00/00/00/00/0
After 4 h
ControlM0/00/00/00/00/00/0
F0/00/00/00/00/00/0
Pedifen 0.25% M0/00/00/00/00/00/0
F0/00/00/00/00/00/0
After 24 h
ControlM0/00/00/00/00/00/0
F0/00/00/00/00/00/0
Pedifen 0.25%M0/00/00/00/00/00/0
F0/00/00/00/0 0/00/0
Note: the numerator is the result of reactions to the experience, the denominator of the result in the control.

Given the lack of response indicators in the experience and control in all periods of observation, we can conclude that when intracrystalline the use of the drug Pedifen 0.25% has no allergenic properties identified in the reaction of immune complexes.

Conjunctival test

Conjunctival samples was performed on day 10 after intracrystalline introduction of Pedifen 0.25% (experimental group) or an equal volume of water for 5 days.

None of the animal experimental and control groups was not a reaction to a drug, as in the control with distilled water.

The obtained data testify to the lack of Pedifen 0.25% allergenic properties identified in conjunctival sample.

The STUDY IMMUNOTOXIC PROPERTIES

Determination of phagocytic activity of macrophages

Assessing the impact of the drug on the natural resistance of the body was performed, determining the phagocytic activity of macrophages in experimental (treated with the drug for 14 days) and control (treated with water in an appropriate amount) groups of CBA mice of both sexes (6 animals in each group).

Mice experienced and the completed groups scored by dislocation of the cervical vertebrae. Filled liberated peritoneum and intraperitoneally injected with 5 ml of medium 199 containing 20% fetal calf serum. After the massage the abdominal cavity medium was aspirated with a syringe. Portions of the cell suspension obtained from all groups of animals, were United in a common pool. Counted the number of cells per ml of medium and drove concentration up to 1.5 106 cells/ml Suspension was poured into 200 μl into the wells of the microplate for cultivation and washed with medium 199 special nozzle for centrifuges CLR-1. Incubated 2 hours at 37°C in a CO2 incubator in an atmosphere of absolute humidity with 5% CO2. To the precipitate cells were added to 200 μl of 0.1% solution of neutral red with medium 199. Then the microplate was incubated at 37°C for 1 hour.

The microplate was centrifuged, the precipitate was washed 2 times with saline and added to 200 ál lyse solution. Thereafter, the microplate was photometrically on spectrophotometer "MultiScan" at 450 nm.

A similar method described in working Uede T. et al. (1986)on the colorimetric determination of tumor necrosis factor using the microplate for cultivation and the same dye. The advantage of micromodification before macromethod is less consumption of cells, the smaller volume of reagents and, therefore, the ability to perform more repetitions of the measurements in one is the same group: in our micromethod was used at least 8 measurements in each group.

Determination of hemagglutinin titer in serum

To determine the effect of the drug on the humoral immune response used the definition of the titer of antibodies to sheep erythrocytes (EB) in the experimental and control groups of mice CBA of both sexes who had injected the drug.

Then mice were immunized intraperitoneally 0.2 ml of 10% EB in physiological solution. On day 7 got the serum, its rise at 56°C to inactivate complement within 30 minutes.

To determine the titer of hemagglutinin used micromodification reaction of haemagglutination with microtitration mostly saddle manufactured by MOM (Hungary). Dispenser microtitration contributed 25 ál serum dilution of 1:5 in 1 and 2 well, where, as in all subsequent holes (2 to 12) of each row of the microplate, in advance were made 25 µl of 1% normal rabbit serum in 0.15 M NaCl. Then use the device for cultivation in 25 μl of microtitration serum was mixed and transferred to 25 μl of the second hole of this series and so on up to 11 wells, inclusive. Hole 12 served as a control, which was only 1% normal rabbit serum. After dilution of the serum received a number of two-fold dilution ranging from 1:5, 1:10, etc.

Then all wells were made of 25 μl of 0.5% suspension of DL 1% normal rabbit serum. The tablets were shaken and left p and room temperature for 1 hour. During this time, the reaction was ended and in control with 1% rabbit serum precipitate was formed, which indicated a negative reaction in the hole. In the pilot holes determined the titer of the antibody, i.e. the highest serum dilution at which there is a clear agglutination of erythrocytes. The titer of the serum was expressed as the reciprocal of titer or in log base 2.

To determine the isotype of antibodies to 25 μl of the diluted 5 times the immune serum was added 25 μl of a 0.2 M solution of 2-mercaptoethanol. The mixture is incubated at 37°C for 30 minutes. The remaining stages of the formulation of the reaction (serum dilution, making red blood cells) was carried out as described above. Assessment class immunodeficiency-noglobulins antibodies to EB made as follows: total antibody titer (without 2-me) is the sum of the interactions of IgM - and IgG-antibodies to EB, and the titer of IgG antibodies is the title reaction in the presence of 2-me, because IgM antibodies are sensitive to 2-me.

Determining the severity of the reaction of hypersensitivity delayed-type Sensitization of mice CBA was carried out 1-107 DL in a volume of 0.1 ml subcutaneously. On the 5th day as the resolution of the dose was administered 2-108 DL in a volume of 20 µl under aponeurotic plate of one of the limbs. In the contralateral paw was injected in the same volume of saline. After 24 hours were evaluated viragen is here local reactions on the ratio of the amount of swelling stop the test and control paws. The volume of the foot was measured using Vernier calipers. Index response (IR) was calculated by the formula

IL=[(ho-hk)/hk]·100%,

where ho and hk - volume foot in the experiment and the control, respectively.

To assess the impact of the drug on the severity of hypersensitivity reactions of the delayed type used mice of CBA of both sexes, who have introduced the drug Pedifen 0.25% within 5 days. At least 6 animals were in each group. In the control were used animals of both sexes of the same batch of CBA mice, which was also administered intracrystalline water in an appropriate amount.

The experimental results were processed by methods of variation statistics using t-student test.

The DEFINITION of IMMUNOTOXIC PROPERTIES

Determination of the mass of lymphoid organs

Body mass and mass ratios of the internal organs was determined in rats of all experimental groups. The obtained experimental data with the drug at a dose of 14 mg/kg are presented in table 36. Practically there was no difference in the mass ratios of the thymus and spleen in the control and experimental groups.

If microscopii the thymus of control and test groups had a triangular shape, whitish color and a little plotnovato consistency. The size and shape of the spleen in the experience changes were not compared with controlment spleen had a uniform dark cherry color was smooth. The consistency of the spleen was moderately dense. In the context of organ stood out greyish small-cell follicles.

Table 36
Mass (µ) organs of white rats with the introduction of Pedifen 0.25% (g/kg body weight)
BodyControlPedifen 0.25%
MFMF
Heart3.7±0.13.5±0.13.6±0.13.7±0.1
Lungs with trachea8.9±0.29.0±0.29.1±0.38.8±0.4
The thymus1.45±0.061.46±0.071.38±0.041.36±0.06
Liver41.1±1.540.7±2.341.3±1.440.9±2.0
Spleen5.7±0.26.1±0.16.0±0.35.8±0.2
Kidney (left)4.2±0.14.1±0.14.1±0.14.2±.2
The adrenal gland (left)0.14±0.010.16±0.020.15±0.030.14±0.02
The brain7.18±0.157.20±0.167.19±0.127.17±0.14
The testes or ovaries13.2±0.200.68±0.0413.5±0.30.74±0.05

The data obtained allow to conclude that according to the autopsy and microscopic studies of the drug, Pedifen 0.25% in subacute and chronic experiment does not change the mass of the Central organs of the immune system. In this regard, the definition of cellularity of these bodies did not.

Determination of phagocytic activity of macrophages

Assessing the impact of the drug on the natural resistance of the body was performed, compare the th phagocytic activity of peritoneal macrophages of mice CBA experienced (daily administration of the drug for 5 days) and control (water or in an appropriate volume) groups. The figures obtained optical density in different experimental groups are presented in table 37 and in aggregated form in table 38.

Table 37
The results of the measurement of optical density in different experimental groups
The experimental groupFloorThe measured values of optical density
ControlM0.5430.6180.5310.5290.6020.5630.5860.625
F0.6290.6210.5700.5990.6440.6230.6190.545
Pedifen 0.25%M0.6370.606 0.5770.5750.5720.5730.5360.597
F0.6020.5730.5290.6220.5800.6110.5880.546

Table 38
Indicators phagocytic activity of peritoneal macrophages of mice CBA after applying the drug Pedifen 0.25%
The experimental groupFloorThe number of dimensionsThe optical densityt-student testThe probability (p)
ControlM80.575±0.014--
F80.606±0.012- -
Pedifen 0.25%M80.584±0.0110.54>0.05
F80.581±0.0111.53>0.05

The data obtained show that the use of the drug Pedifen 0.25% does not cause changes in phagocytic activity in experimental groups compared with control groups of males and females.

Thus, the drug Pedifen 0.25% does not have immunotoxic properties identified on the evaluation of phagocytic activity of macrophages.

Determination of the effect of drug Pedifen 0.25% on the humoral immune response to sheep erythrocytes

To determine the effect of the drug on specific humoral immune response used the definition of hemagglutinin titer in experimental and control groups of CBA mice immunized with sheep erythrocytes. On day 7 after immunization, sera were determined by the titer of antibodies to EB and the titer of IgG antibodies to this antigen. The results obtained are presented in table 390.

Table 39
the engineers of hemagglutinin in the blood serum of the experimental and control groups of CBA mice after application of Pedifen 0.25%
No. of animalsControlPedifen 0.25%
MFMF
General. titleThe IgG titerGeneral. TitleThe IgG titerGeneral. titleThe IgG titerGeneral. TitleThe IgG titer
15.315.315.315.318.317.318.317.31
26.315.315.315.316.316.318.317.31
39.318.317.316.31 9.319.318.318.31
47.317.317.317.318.318.315.315.31
58.318.318.318.316.316.317.317.31
68.318.316.316.318.318.318.317.31
77.317.317.317.319.318.317.316.31
88.318.31- -9.319.318.318.31
M±m7.56±0.457.31±0.466.74±0.436.60±0.428.19±0.447.94±0.427.69±0.387.19±0.35
Note: the Titer of antibodies are presented in units of log2. In two groups the number of animals (females) is 7.

Although according to the table in the experimental groups, there can be observed a tendency to increase as the total antibody titer and IgG antibodies, these increases were not statistically significant character - the maximum value for t-test, when comparing the groups, and which is achieved for a group of males (average is 8.19±0.44 vs. 7.56±0.45 in the control), is equal to 1.66, which is significantly below the critical value.

Determination of the effect of drug Pedifen 0.25% on the severity of hypersensitivity reactions of the delayed type.

To study the effect of the drug on specific cellular immunity investigated the severity of hypersensitivity reactions of the delayed type experienced in receiving study drug for 5 days, and counter the selected groups of mice.

For the induction of delayed-type hypersensitivity CBA mice were senzibilizirani with sheep erythrocytes and on the 5th day they were introduced allowing the dose of the antigen under aponeurotic plate. In the contralateral paw was injected with the same volume of saline solution and after 24 hours was assessed by the severity of local reactions at the ratio of the amount of swelling stop the test and control paws.

Table 40 presents the indices of the response of individual animals in the experimental and control groups:

Table 40
The influence of the drug, Pedifen 0.25% on the severity of hypersensitivity reactions of the delayed type mice CBA
The experimental groupFloorIndexes reactions, %M±Mt-student test
ControlM10067806710010085.7±6.7
F 6792100676710082.2±6.9
Pedifen 0.25%M10010093671007789.5±5.80.43 (p>0.05)
F938967679210084.7±5.80.28 (p>0.05)

The comparison of the obtained data shows that the drug Pedifen 0.25% does not have a stimulating or inhibiting effect on the reaction of hypersensitivity delayed-type female mice CBA.

Thus, a study of the possible allergic effects of drug Pedifen 0.25% Guinea pigs of both sexes were shown that he has no allergenic properties, judging by the reaction of General anaphylaxis using the drug even at doses 10 times more therapeutic. Cu is IU, conjunctival samples, reactions degranulation of mast cells and reactions of the immune complexes were negative.

In the study likely immunotoxic properties of the drug on mice CBA both sexes, it was shown that the drug Pedifen 0.25% with long-term use does not affect the weight of the Central lymphoid organs and phagocytic activity of macrophages.

The data obtained allow to conclude that the drug Pedifen 0.25% has no allergenic and immunotoxicity properties.

Experimental studies of General toxicity (acute, subacute, local irritant) dosage forms of the new drug Pedifen 0.25% spray showed that the drug in conditions as introduction acute and subacute application within 14 days of virtually no toxic effect on an organism of warm-blooded laboratory animals (rodents and negrisolo - rabbits).

When intracrystalline route of administration to rats and rabbits, the LD50 value for the preparation of Pedifen 0.25% is higher than the dose of 5 g/kg, which is the maximum possible volume.

Subacute (14 days) daily intracrystalline the use of the drug dosage form of experimental animals at doses exceeding therapeutic for a person in the tens to hundreds of times, had no harmful environmenta what I am on main adaptive systems (nervous, cardiovascular, hematopoietic, excretory, antitoxic), metabolism, overall health and development, basic homeostatic parameters of the organism.

Note also the absence of the drug dosage form of Pedifen 0.25% irritating to tissues at the site of application (intracrystalline). All this applies to rodents and nigritana, i.e. testifies to the universal nature of harmlessness of a new drug.

A study of the possible allergic effects of drug Pedifen 0.25% spray on Guinea pigs of both sexes has been shown that it does not cause the total reaction anaphylaxis using the drug even at doses 10 times more therapeutic. In addition, conjunctival samples, reactions degranulation of mast cells and reactions of the immune complexes were negative. These data allow us to conclude that the drug has no allerheiligen action.

The experiments showed that the introduction of drug Pedifen 0.25% spray does not exert immunotoxic action.

Application:

SYMBOLS AND ABBREVIATIONS

F - female - female

fd(s) - fissura dextra (sinistra) - the eye slit is left (right)

fd(s) - fissura dextra (sinistra) - palpebral left (right

GLP - Good Laboratory Practice" - "Good laboratory practice"

IPC - morpholin Pelago the OIC acid

CR - dibenz(b,f)-1.4-oxazepam

CS - ortho-chlorobenzylidenemalononitrile

The sit is the average amount of hemoglobin in red blood cells

ICSU - average concentration of hemoglobin in red blood cells

MCV is the average volume of red blood cells

pd(s) - pupilla dexter (sinister) - pupil right (left)

pd(s) - pupilla dexter (sinister) - pupil right (left)

HELL - blood pressure systolic

ALT - alanine aminotransferase

ACT - aspartate aminotransferase

in/W - vnutrijeludocna

M - male - male

MK - mass ratio

ESR - erythrocyte sedimentation rate

BH - respiratory rate

HR - heart rate

Alkaline phosphatase alkaline phosphatase

ECG - electrocardiogram

1. Pharmaceutical composition in the form of a spray for the treatment of lesions of non-lethal irritant means, characterized in that it contains the active ingredient, pedifen and additionally sodium chloride, the ratio of ingredients is, g:

Pedifen hydrochlorideof 0.01 to 10.0
Sodium chloride0,1-10,0
Water for injection, ml100

2. The pharmaceutical composition according to claim 1, characterized in that pedifen used in an amount of 0.1 to 1.0 is.



 

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