RU2724883C1 - Method of correcting bacterial purulent meningitis by 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl(amino)phenyl ethanoate under experimental conditions - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to experimental pharmacology, neurology and infectious diseases, and can be used for treating bacterial purulent meningitis. That is ensured by simulating bacterial purulent meningitis in laboratory rats by administering into subarachnoid space 10 mcl of a suspension containing Streptococcus pneumoniae in concentration of 5*10CFU/ml, with subsequent correction of 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl(amino)phenyl ethanoate in dosage of 25 mg/kg, which is introduced 7 hours after meningitis is induced intramuscularly once, and subsequent correction of ceftriaxone, initiated 18 hours after meningitis induction, in dose of 100 mg/kg/day intramuscularly once day for 7 days.EFFECT: method provides pronounced correction of bacterial purulent meningitis, which is confirmed by low lethality, reduced degree of neurological deficiency, faster recovery of behavioral activity, low indices of oxidative stress.1 cl, 6 tbl, 1 ex

Description

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

According to well-known literature, bacterial purulent meningitis (hereinafter - BGM) is characterized by high mortality and disability rates. Currently, the frequency of BGM is 2-10 cases per 100,000 population. But, despite a general decrease in the incidence, mortality from bacterial meningitis has changed slightly over the past 20 years and ranges from 15% to 25% in developed countries. In countries with limited resources, mortality remains at a high level from 54 to 70%. Many surviving patients have serious residual neurological and psychoorganic consequences up to disability (5-40% of patients) [Wall, EC, Cartwright, K., Scarborough, M., Ajdukiewicz, KM, Goodson, P., Mwambene, J. , ... Lalloo, DG (2013). High Mortality amongst Adolescents and Adults with Bacterial Meningitis in Sub-Saharan Africa: An Analysis of 715 Cases from Malawi. PLoSONE, https://doi.org/10.1371/journal.pone.0069783].

The main principles of treatment for HMF are to stop the further spread of the pathological process and prevent the development of complications. The “gold” standard for the treatment of hypertension is etiotropic antibiotic therapy [Federal clinical guidelines for the diagnosis and treatment of bacterial purulent meningitis in children, 2013, Skripchenko N.V., Vilnits A.A., Ivanova M.V.]. In addition to antibiotic therapy, drugs with a neuroprotective effect (for example: Mexidol) are actively used in the treatment of HMF to reduce the severity of residual neurological deficiency. As a rule, complex therapy in the treatment of HMF includes etiotropic antibiotic therapy, pathogenetic therapy with dexamethasone, neuroprotective agents, symptomatic therapy with antipyretic, anti-inflammatory (for example: Diclofenac), anticonvulsants. Mexidol and Diclofenac preparations are used to treat bacterial purulent meningitis as part of complex therapy according to clinical recommendations.

Scientific sources highlight the results of an experimental study on animals of various drugs with neuroprotective properties. It is scientifically proven that intermediate forms of reactive oxygen species and reactive nitrogen forms in large quantities when a bacterial infection develops in the body. Antioxidants attenuate neurological damage in bacterial meningitis and are a promising strategy for treating 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].

From the prior art are known:

"A method for the treatment of purulent meningitis" (RU, patent for the invention No. 2043766, publ. 09/20/1995), including the conduct of complex pathogenetic drug therapy, with the additional introduction of intravenous piracetam at a dose of 100 mg / kg body weight twice a day 1 hour after the introduction of antibiotic therapy, treatment is continued until the normalization of the cerebrospinal fluid composition.

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 further 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 resulting to pneumococcal meningitis. Transl. Res.2014; 163 (5): 503-513.DOI: 10.1016 / j.trsl.2013.12.008].

The objective of the invention is to provide an effective method for the correction of bacterial purulent meningitis using 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate in the experiment.

The technical result of the invention is an effective method for the correction of bacterial purulent meningitis using 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate in the experiment, which is confirmed by low mortality, a decrease in the degree of neurological deficit, and a quick recovery of behavioral low rates of oxidative stress.

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

The main advantage of the proposed method is that the introduction of 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 a pronounced neuroprotective effect with the correction of bacterial purulent meningitis in the experiment, which is confirmed by low mortality, a decrease in the degree of neurological deficit, a faster recovery of behavioral activity, and low rates of oxidative stress.

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

The synthesis of the new compound was carried out by reacting equimolar amounts of 2-ethyl-6-methyl-3-hydroxypyridine with 2,6-dichlorophenyl (amino) phenylethanoic acid. 40 ml of ethyl alcohol are charged into a three-necked flask equipped with a stirrer, a thermometer and a reflux condenser. Then, 1.37 g (0.01 m) of 2-ethyl-6-methyl-3-hydroxypyridine is gradually added with stirring. After dissolution, 2.96 g (0.01 m) of 2,6-dichlorophenyl (amino) phenylethanoic acid is added. The resulting solution with constant stirring is kept for 30 minutes at 50 ° C, cooled, filtered, the solvent is distilled off. The residue was recrystallized from isopropyl alcohol. Obtain 4.01 g of a light beige crystalline powder with Tplav. = 140-141 ° C. C ₂₂ H ₂₂ Cl ₂ N ₂ O _3. m.m. 433.34 g / mol.

Found,%: C 60.93; H 5.21; Cl 16.31; N 6.52

Calculated,%: C 60.98; H 5.12; Cl 16.36; N, 6.46; About 11.08

IR, ν, cm ^-1 : 3450 (OH), 3342 (NH). 1609 (C = C _arom. ), 1565 (NHCO).

UV: 0.001% solution in 95% ethanol; absorption maximum at 283 ± 2 nm.

EXAMPLE OF SPECIFIC IMPLEMENTATION

The study was performed on 90 sexually mature Wistar female rats weighing 230-260 g. The experimental animals were divided into 5 groups: 1) intact (n = 10), 2) control group with simulated pneumococcal meningitis receiving only Ceftriaxone 100 mg / kg (n = 20), 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 = 20), 4) a group with simulated pneumococcal meningitis receiving Ceftriaxone 100 mg / kg and Mexidol 7.5 mg / kg (n = 20), 5) a group with simulated pneumococcal meningitis receiving Ceftriaxone 100 mg / kg and Diclofenac 17.5 mg / kg (n = 20). The animals were kept in standard vivarium conditions of the National Research University "BelSU" with free access to food and water. The keeping of animals and the setting up of the experiment was carried out in accordance with the requirements of orders No. 1179 of the Ministry of Health of the USSR of 10/11/1983 and No. 267 of the Russian Federation of 06/19/2003, as well as the international rules "Guide for the Careand of Laboratory Animals".

Bacterial purulent meningitis was modeled by introducing into the subarachnoid space 10 μl of a suspension containing S. Pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml. Treatment began after 18 hours. "Ceftriaxone" was administered at the rate of 100 mg / kg of 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 the CSF. The preparations - 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate, Mexidol and Diclofenac were administered intramuscularly 7 hours after the induction of meningitis in the above dosages.

50% of animals in groups 2, 3, 4, and 5 were removed from the experiment 24 hours after the induction of meningitis to assess the parameters of oxidative stress in the brain homogenate, the remaining 50% were observed for 10 days, assessing the degree of neurological deficit and behavioral status.

The severity of the cerebroprotective effect was judged by indicators of mortality, severity of neurological deficit, behavioral status, level of indicators of oxidative stress (catalase, superoxide dismutase, acetyl hydroperikisi, nitric oxide metabolites, malondialdehyde).

A clinical assessment of the health status of rats was performed as follows: every 24 hours, daily, for ten days, a clinical assessment of the health status of rats was performed. Rats were weighed and the severity of the disease was evaluated clinically using the following scale: 1 = coma; 2 = does not rotate vertically when positioning on the back; 3 = rotates vertically for 30 seconds; 4 = minimal outpatient activity, rotates vertically for <5 seconds; and 5 = normal.

Assessment of the degree of neurological deficit was carried out using the scale of assessment of neurological deficit in meningitis, meningoencephalitis (Table 1).

Table 1

The degree of neurological deficit was evaluated by the total score. Moreover: 0 points - indicates the maximum severity of violations, and 21 points - their absence.

Using the device for assessing the muscle strength of the limbs of small laboratory animals in the experiment (RU, utility model patent No. 193975, publ. 11/21/2019), the specific strength of rats was determined.

A key antioxidant defense enzyme is superoxide dismutase (SOD), which catalyzes the enzymatic dismutation of the superoxide anion radical.

SOD activity was determined spectrophotometrically using an Apel 330 PD spectrophotometer (Japan) based on determining the degree of inhibition of the quercetin autooxidation reaction [Kostyuk VA, Potapov AN, Kovaleva Zh.V. (1990) Issues. honey. Chemistry No. 2. P.88-91]. The amount of enzyme required to reduce the rate of oxidation of quercetin by 50% was taken as a standard unit of activity of SOD.

One of the enzymes in the inactivation system of reactive oxygen species is catalase, which catalyzes the decomposition of hydrogen peroxide. The biological role of the enzyme is to prevent the accumulation of hydrogen peroxide, which is formed during the dismutation of superoxide anion.

Catalase activity was determined by a method based on the ability of hydrogen peroxide to form a stable colored complex with molybdenum salts [Korolyuk MA, Ivanova LI, Mayorova IG, Tokarev V.E. (1988) Lab. Case No. 1. S.16-19]. Color intensity was measured photometrically at a wavelength of 410 nm using an Apel 330PD spectrophotometer (Japan). Catalase activity was expressed in μat / ml.

Malondialdehyde (MDA) is the final product of lipid peroxidation. The determination was made spectrophotometrically using an Apel 330 PD spectrophotometer (Japan) after extraction with butanol using the TBK-Agat kits. The results were expressed in μmol / l [Ragino Yu.I., Dushkin M.I. Oxidation resistance of heparin-resistant serum B-lipoproteins in coronary heart disease // Klin. Lab Diagnostics.-1998.-No. 11.-s.3-5.].

Acyl hydroperoxides (AGP) are an intermediate product of lipid peroxidation. The determination was made using a mixture of heptane and isopropane with the addition of hydrochloric acid. The resulting heptane layer was measured spectrophotometrically at a wavelength of 233 nm against a control sample. The measurement was carried out in arbitrary units.

The determination of stable metabolites of nitric oxide was carried out by adding Griss reagent to the bioassay, measured spectrophotometrically at a wavelength of 540 nm after 5 minutes of incubation at room temperature.

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

Laboratory animals were monitored daily and mortality was evaluated (Table 2).

table 2

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

In the group of intact rats, mortality was absent; in the control group, mortality during the observation period was 40%. The group in which 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and Ceftriaxone were used was characterized by low mortality rates, 11%. In the group receiving Diclofenac and Ceftriaxone, the highest mortality rate was 77%. In the group receiving Mexidol and Ceftriaxone, mortality was 25%. From the seventh to tenth day mortality in the studied groups was not.

On the 1st, 3rd, 5th, 7th and 8th days after modeling the pathology, a clinical assessment of the health status of rats was carried out (table 3). Rats were weighed and the severity of the disease was evaluated clinically using the following scale: 1 = coma; 2 = does not rotate vertically when positioning on the back; 3 = rotates vertically for 30 seconds; 4 = minimal outpatient activity, rotates vertically for <5 seconds; and 5 = normal.

Table 3

The dynamics of the clinical assessment of the state of health in the studied groups (according to the average score in the group) (M ± m; n = 10).

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

On the first and third days after the induction of meningitis, rats treated with 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and ceftriaxone had a higher statistically significant clinical score of 4.5 points and 5 points, compared with the control group 3.29 and 4.14 points, respectively (p <0.05). In the groups treated with Ceftriaxone and Mexidol and Diclofenac, no statistically significant differences were detected compared with the control group. The clinical activity of rats (5 points) was restored in the control group only on the 8th day, and in the group treated with 2-ethyl-6-methyl-3-hydroxypyridinium 2.6 dichlorophenyl (amino) phenylethanoate and ceftriaxone - on the 3rd day. In the group receiving Diclofenac and Ceftriaxone, there was a high mortality rate on days 5–8. For this reason, there is not enough data for statistical processing.

The neurological deficit in animals was assessed on days 1, 5, and 8 after modeling the pathology using a point scale to assess the degree of neurological deficit in meningitis, meningoencephalitis (according to the average score in the group) (Table 4).

Table 4

Dynamics of the severity of neurological damage in the studied groups on a scale for assessing the degree of neurological deficit in meningitis, meningoencephalitis (according to the average score in the group) (M ± m; n = 10).

In the group receiving 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and Ceftriaxone, on the 1st, 5th and 8th day after modeling the pathology, the degree of neurological deficit was statistically significant was less pronounced compared with the control group by 10.2%, 12% and 14.2%, respectively (p <0.05). In the groups receiving ceftriaxone therapy and Mexidol and Diclofenac preparations, no statistically significant differences were detected compared with the control group.

The severity of the condition of the animal was assessed by specific strength indicators on the 1st, 5th and 10th day after modeling of meningitis (Table 5).

Table 5

Dynamics of specific power indices in the studied groups (according to the average score in the group) (M ± m; n = 10).

According to the table, it can be seen that the specific strength indices on the 1st, 5th and 10th days after modeling meningitis are statistically significantly higher in the group receiving Ceftriaxone and 2-ethyl-6-methyl-3-hydroxypyridinium 2.6 -dichlorophenyl (amino) phenylethanoate compared with the control group by 17.4%, 12.8% and 15.6%, respectively (p <0.05). In the group treated with ceftriaxone and Mexidol, there were no statistically significant differences compared with the control group. In the group that received Ceftriaxone and the drug Diclofenac, the specific strength was statistically insignificantly lower compared to the control group on the 1st day after modeling the pathology by 8.2%.

Further, the oxidative stress indices in the rat brain homogenizate were evaluated after the induction of meningitis (Table 6).

Table 6

Oxidative stress indicators for modeling pneumococcal meningitis (M ± m; n = 10)

In the group receiving Mexidol and Ceftriaxone, there was a statistically significant decrease of 44% in the activity of malondialdehyde and nitric oxide, and a statistically significant decrease in the activity of superoxide dismutase by 11%.

In the group receiving Diclofenac and Ceftriaxone, there was a statistically significant decrease in superoxide dismutase activity by 27%. The activity of catalase, malondialdehyde, acetyl hydroperoxides and nitric oxide is comparable with similar indicators in the control group.

The group receiving 2-ethyl-6-methyl-3-hydroxypyridinium 2,6-dichlorophenyl (amino) phenylethanoate and Ceftriaxone showed a statistically significant decrease in the activity of all the estimated oxidative stress indicators compared with the control group: a 17% decrease in catalase activity superoxide dismutase by 34%, malondialdehyde and acetyl hydroperoxides by 50%, nitric oxide by 85%.

Thus, the obtained results indicate a pronounced correction of bacterial purulent meningitis in rats, simulated by introducing into the subarachnoid space 10 μl of a suspension containing Streptococcus pneumoniae at a concentration of 5 * 10 ⁹ CFU / ml, 2-ethyl-6-methyl-3-hydroxypyridinium 2 , 6-dichlorophenyl (amino) phenylethanoate at a dose of 25 mg / kg, administered once intramuscularly 7 hours after the induction of meningitis and Ceftriaxone at a dosage of 100 mg / kg / day administered intramuscularly 18 hours after the induction of meningitis, and then daily 1 time per day for 7 days.

Claims (1)

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