RU2750964C1 - Method for correction of bacterial purulent meningitis with bis (2-ethyl-6-methyl-3-hydroxypyridinium)2,6-dichlorophenyl (amino)phenylethanoate under experimental conditions - Google Patents

Abstract

FIELD: medicine, experimental pharmacology in particular.

SUBSTANCE: invention relates to medicine, in particular to experimental pharmacology, neurology and infectious diseases, and can be used for the treatment of bacterial purulent meningitis. The method for correcting bacterial purulent meningitis in the experiment consists in simulating bacterial purulent meningitis in rats by injecting 10 µl of a suspension containing Streptococcus pneumoniae at a concentration of 5*10⁹ CFU/ml into the subarachnoid space. Subsequent correction is performed by bis (2-ethyl-6-methyl-3-hydroxypyridinium)2,6-dichlorophenyl (amino)phenylethanoate at a dosage of 25 mg/kg, which is administered 7 hours after the induction of meningitis intramuscularly once, and followed by correction with ceftriaxone, initiated 18 hours after the induction of meningitis, at a dosage of 100 mg/kg/day intramuscularly once a day for 7 days.

EFFECT: invention provides a pronounced correction of bacterial purulent meningitis, which is confirmed by high recovery rates of neurological and cognitive functions, as well as rapid recovery of behavioral activity and low mortality.

1 cl, 5 tbl, 1 ex

Description

The invention relates to medicine, in particular to experimental pharmacology, neurology and infectious diseases.

According to well-known literary sources, bacterial purulent meningitis is an infectious and inflammatory disease of the central nervous system, characterized by the development of purulent inflammation of the pia mater in response to the penetration of various bacterial agents through the blood-brain barrier. The disease has a severe course, is characterized by high mortality and the formation of residual neurological deficit after the previous disease.

If untreated, death in bacterial purulent meningitis occurs in 100% of cases. In addition, death and the development of residual neurological complications are possible despite adequate therapy.

Scientific sources highlight the results of experimental studies on animals of various drugs with neuroprotective properties. Antioxidants attenuate the degree of neurological damage in bacterial meningitis and are a promising strategy in the treatment of bacterial meningitis [Mook-Kanamori BB, Geldhoff M, vander Poll T, van de Beek D; Pathogenesis and pathophysiology of pneumococcal meningitis. ClinMicrobiolRev 2011; 24: 557-91].

The prior art known "Method for the treatment of purulent meningitis" (patent RU No. 2043766, publ. 20.09.1995), including the management of complex pathogenetic drug therapy, with the additional introduction of intravenous piracetam at a dose of 100 mg / kg body weight twice a day through 1 hour after the introduction of antibiotic therapy, treatment is continued until the cellular composition of the cerebrospinal fluid normalizes.

A known method of modeling bacterial purulent meningitis in rats by introducing into the subarachnoid space 10 μl of a suspension containing S. Pneumoniae at a concentration of 5 * 10 ⁹ CFU \ ml, followed by treatment with ceftriaxone at a dose of 100 mg / kg / day, administered intramuscularly 18 hours after induction and then daily 1 time per day for 7 days [Barichello T., Simões LR, Generoso JS, Sangiogo G., Danielski LG, Florentino D., Dominguini D., Comim C., Petronilho F., Quevedo J. Erythropoietin prevents cognitive impairment and oxidative parameters in Wistar rats subjected to pneumococcal meningitis. Transl. Res. 2014; 163 (5): 503-513. DOI: 10.1016 / j.trsl.2013.12.008].

Also known is a method for correcting bacterial purulent meningitis using 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate in the experiment (patent RU No. 2724883, publ. 06/26/2020), which is carried out as follows : modeling bacterial purulent meningitis in rats by introducing 10 μl of a suspension containing Streptococcus pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml into the subarachnoid space, followed by correction of 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino ) phenylethanoate at a dosage of 25 mg / kg, which is administered 7 hours after the induction of meningitis intramuscularly once, and subsequent correction with ceftriaxone, initiated 18 hours after the induction of meningitis, at a dosage of 100 mg / kg / day intramuscularly 1 time per day for 7 days ...

The disadvantages of this method are incomplete recovery of neurological and cognitive functions, as well as incomplete recovery of behavioral activity after completion of treatment.

The objective of the present invention is to create a more effective method for the correction of bacterial purulent meningitis using bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate in the experiment for a more complete restoration of neurological and cognitive functions, as well as full recovery of behavioral activity after completion of treatment and a decrease in the mortality rate.

The technical result of the proposed invention is an effective method for correcting bacterial purulent meningitis using bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate in the experiment, confirmed by high recovery rates of neurological and cognitive functions, as well as rapid recovery of behavioral activity and low mortality.

The task is achieved by the fact that the proposed method for the correction of bacterial purulent meningitis using bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate in the experiment. The method is carried out as follows: modeling of bacterial purulent meningitis in rats is carried out by introducing 10 μl of a suspension containing Streptococcus pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml into the subarachnoid space, and subsequent correction with ceftriaxone, initiated 18 hours after the induction of meningitis, at a dosage of 100 mg / kg / day intramuscularly 1 time per day for 7 days. Moreover, the subsequent correction after the introduction of a suspension containing Streptococcus pneumoniae is carried out by bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate at a dosage of 25 mg / kg, which is administered 7 hours after induction meningitis intramuscularly once.

The main advantage of the proposed method is that the introduction of bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate at a dose of 25 mg / kg / day intramuscularly once 7 hours after the induction of meningitis leads to pronounced neuroprotective effect in the correction of bacterial purulent meningitis in the experiment, which is confirmed by the high rates of recovery of neurological and cognitive functions, as well as the rapid recovery of behavioral activity and low mortality, which is confirmed by a specific example of research.

METHOD FOR OBTAINING BIS-2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate

The synthesis of the new compound was carried out by the interaction of two molecules of 2-ethyl-6-methyl-3-hydroxypyridine with 2,6-dichlorophenyl (amino) phenylethanoic acid. In a three-necked flask equipped with a stirrer, thermometer and reflux condenser, load 40 ml of ethyl alcohol. Then, with stirring, 2.74 g (0.02 m) of 2-ethyl-6-methyl-3-hydroxypyridine are gradually added. After dissolution, 2.96 g (0.01 ml) of 2,6-dichlorophenyl (amino) phenylethanoic acid are added. The resulting solution is kept with constant stirring for 30 minutes at 50 C, cooled, filtered, the solvent is distilled off. The residue is recrystallized from isopropyl alcohol. Get 5.38 g of a light beige crystalline powder with T melt. = 156 - 158 ° C. C ₃₀ H ₃₃ Cl ₂ N ₃ O _4. M.W. 570.56 g / mol.

Found,%: C 63.06; H 5.91; Cl 13.39; N 7.42

Calculated,%: C 63.15; H 5.84; Cl 12.43; N 7.37; About 11.22

IR, ν, cm ^-1 : 3450 (OH), 3342 (NH). 1610 (C = C _arom .), 1565 (NHCO).

UV: 0.001% solution in 95% ethyl alcohol maximum absorption at 283 ± 2 nm.

EXAMPLE OF A SPECIFIC PERFORMANCE

The study was carried out on 40 sexually mature female Wistar rats weighing 230-260 g. The experimental animals were divided into 4 groups: 1) intact (n = 10); 2) a control group with simulated pneumococcal meningitis who received only ceftriaxone 100 mg / kg (n = 10); 3) a group with simulated pneumococcal meningitis receiving ceftriaxone 100 mg / kg and 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate 25 mg / kg (n = 10); 4) a group with simulated pneumococcal meningitis who received ceftriaxone 100 mg / kg and bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate 25 mg / kg (n = 10). The animals were kept in standard conditions of the NRU "BelSU" vivarium with free access to food and water. Keeping animals and setting up the experiment was carried out in accordance with the requirements of orders No. 1179 of the Ministry of Health of the USSR dated 11.10.1983 and No. 267 of the Russian Federation dated 19.06.2003, as well as the international rules "Guide for the Careand of Laboratory Animals".

Bacterial purulent meningitis was modeled by injecting 10 μl of a suspension containing S. Pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml into the subarachnoid space. Treatment was started 18 hours later. The animals received ceftriaxone (100 mg / kg body weight) intramuscularly for 7 days. After 10 days, the animals were free of infection. The absence of infection was confirmed by puncture of the subarachnoid space on the 10th day and the subsequent negative result of bacteriological culture of CSF. The drugs - 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and bis-2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate were injected intramuscularly after 7 hours after the induction of meningitis in the above dosages.

The pronounced effect of exposure to bis-2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate was judged by the high rates of recovery of neurological and cognitive functions, as well as the rapid recovery of behavioral activity and low mortality.

To assess the speed and degree of recovery of neurological functions and behavioral activity, a clinical assessment of the health status of rats was carried out, the degree of neurological deficit was determined using a special rating scale for meningitis and meningoencephalitis.

The clinical assessment of the health status of the rats was carried out as follows: every 24 hours, daily, for ten days, a clinical assessment of the health status of the rats was carried out. The rats were weighed and the severity of the disease was assessed clinically using the following scale: 1 = coma; 2 = does not pivot vertically when positioned on the back; 3 = rotates vertically within 30 s; 4 = minimal ambulatory activity, turns vertically within <5 s; and 5 = normal.

The degree of neurological deficit was assessed using a scale for assessing neurological deficit in meningitis, meningoencephalitis (Table 1).

Table 1. Scale for assessing neurological deficit in meningitis, meningoencephalitis.

The degree of neurological deficit was assessed by the total score. 21 points indicate the absence of neurological disorders, 0 points indicate their maximum severity.

The assessment of the cognitive functions of rats was carried out on the 10th day after modeling of meningitis using the test "Object recognition problem". This test evaluates spatial memory. The experiment was carried out in an open field made of wood (field size 40 by 50 cm, wall height 50 cm). First, an addiction session was conducted for 5 minutes, during which the animals freely explored the open field. At this time, there were no objects in the open field. After the habituation session, a training session was carried out: the rats, one at a time, examined an open field for 5 minutes, in which there were 2 identical objects (objects A1 and A2, both cubes). The objects were located at a distance of 10 cm from the walls in 2 adjacent corners.

The analysis of short-term memory (CP) of object recognition was carried out 90 minutes after the training session. The animals explored for 5 minutes an open field in which there was one familiar object (A) and one new object (B, a pyramid with a square base). The recognition index was calculated using the formula TB / (TA + TB); where TA is the time spent learning a familiar subject A and TB is the time spent learning a new subject B.

Testing of rats for long-term memory (LR) analysis of object recognition was performed 24 hours after the training session. The animals explored the open field for 5 minutes in the presence of one familiar object A and one new object C (a ball with a square base). Recognition memory was assessed in the same way as in short-term memory analysis. Investigation of an object was considered to be sniffing (examining an object from a distance of 3-5 cm) or touching an object with the nose and / or forepaws. All objects used in the task had similar texture (smooth), color (blue), and dimensions (weight, 150-200 g), but differed in shape.

The reliability of changes in the absolute parameters was determined by the difference method of variation statistics with finding the mean values of the shifts, the arithmetic mean and the probability of a possible error (p) according to the Student's tables. Differences were assessed as significant at p <0.05. For the calculations, the statistical analysis program Microsoft Excel 2016 was used.

Laboratory animals were monitored daily and mortality was assessed during 10 days of the experiment (Table 2).

table 2

Mortality rate expressed as a percentage (n = 10).

In the group of intact rats, there was no lethality; in the control group, the lethality during the observation period was 40%. The group that used 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone had a low mortality rate, 11%. The group receiving bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone had the lowest mortality rate of 10%.

On the 1st and 3rd day after modeling the pathology, a clinical assessment of the health status of the rats was carried out (Table 3).

Table 3

Dynamics of the clinical assessment of the state of health in the studied groups (by the mean value of the points in the group) (М ± m; n = 10).

Note: here and everywhere below * - p <0.05, # - p> 0.05 in relation to the control group of rats.

Rats receiving 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone had a clinical score 36% higher compared to the control group (p <0.05). In the group receiving bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone, the animals had the highest clinical assessment of the health status of rats on the 1st day of the disease, 46% higher relative to the control group (p <0.05).

The assessment of neurological deficit in animals was carried out on the 1st, 5th and 8th days after modeling the pathology, using a point scale to assess the degree of neurological deficit in meningitis, meningoencephalitis (according to the average value of the points in the group) (Table 4).

Table 4

Dynamics of the severity of neurological damage in the study groups according to the scale for assessing the degree of neurological deficit in meningitis, meningoencephalitis (according to the mean value of the points in the group) (М ± m; n = 10).

In the group that received 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone, on the 1st, 5th and 8th days after modeling the pathology, the degree of neurological deficit was statistically significantly less expressed in comparison with the control group by 15.4%, 16.6% and 17.6%, respectively (p <0.05). Animals receiving bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone on days 1, 5 and 8 after modeling of meningitis had a statistically significant lower degree neurological deficit by 20.4%, 21.3% and 20%, respectively (p <0.05).

Further, the assessment of the cognitive abilities of the rats was carried out on the 10th day after the modeling of meningitis (Table 5).

Table 5

Recognition indices of short-term and long-term memory in rats on the 10th day after modeling pneumococcal meningitis (in a.u.) (М ± m; n = 10)

In the group receiving ceftriaxone and 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate at a dose of 25 mg / kg, the recognition index of short-term memory is 37.6% lower than in the control group, and the recognition index long-term memory is 28.4% less than in the control group (p <0.05). Animals treated with ceftriaxone and bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate at a dose of 25 mg / kg had a short-term memory recognition index by 44% less than in the control group, and the index recognition of long-term memory by 33.6% less than in the control group (p <0.05).

Thus, the results obtained indicate a pronounced correction of bacterial purulent meningitis in rats using bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate in an experiment simulated by introducing 10 μl into the subarachnoid space a suspension containing Streptococcus pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml, and subsequent correction with ceftriaxone, initiated 18 hours after the induction of meningitis, at a dosage of 100 mg / kg / day intramuscularly 1 time per day for 7 days. Moreover, the subsequent correction after the introduction of a suspension containing Streptococcus pneumoniae is carried out by bis (2-ethyl-6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate at a dosage of 25 mg / kg, which is administered 7 hours after induction of meningitis intramuscularly once.

Claims (1)

A method for correcting bacterial purulent meningitis in an experiment, including modeling bacterial purulent meningitis in rats by introducing 10 μl of a suspension containing Streptococcus pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml into the subarachnoid space, characterized in that the subsequent correction is carried out with bis (2-ethyl 6-methyl-3-hydroxypyridinium) 2,6-dichlorophenyl (amino) phenylethanoate at a dosage of 25 mg / kg, which is administered 7 hours after the induction of meningitis intramuscularly once, followed by correction with ceftriaxone, initiated 18 hours after the induction of meningitis, in a dosage of 100 mg / kg / day intramuscularly 1 time per day for 7 days.