RU2723165C1 - Method for reducing the severity of the long-term effects of carbon monoxide poisoning - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to toxicology, and can be used to reduce severity of long-term effects of CO carbon monoxide poisoning in experiment. For this purpose, 10, 15 or 20 minutes prior to the onset of inhalation poisoning with carbon oxide, ozonised physiologic saline (OPS) is administratively orally introduced in dose of 150, 400, 600 mcg per 100 g of weight before the beginning of inhalation poisoning, concentration of dissolved ozone 200 mcg/ml. To treat the consequences of the poisoning with the therapeutic purpose, BFR is administered intraperitoneally in dose of 12.5 mcg per 100 g of the weight with the concentration of solution being 30 mg/ml, starting 1 day after the poisoning; wherein the therapeutic course is 7 injections once a day.EFFECT: invention provides reduced severity of the long-term effects of acute carbon monoxide poisoning by increasing body resistance to carbon monoxide during preventive use by increasing oxygen partial pressure, saturation of hemoglobin with oxygen, oxygen delivery and utilization by tissues; and reducing the severity of hypoxic brain injury following acute CO poisoning.1 cl, 2 dwg, 1 ex, 6 tbl

Description

The invention relates to medicine, namely to toxicology, and can be used to reduce the severity of the long-term effects of carbon monoxide poisoning.

The main mechanism of the toxic effect of carbon monoxide is hypoxia. Its development leads to cerebral insufficiency, the progression of which forms irreversible changes [Shilov, V.V. Pharmacological correction of hypoxia in patients with acute cerebral insufficiency due to acute carbon monoxide poisoning and combustion products / V.V. Shilov [et al.] // Occupational medicine and industrial ecology. - 2012. - No. 6. - S. 22-27.]. It is connected, however, with the fact that the need for neurons in energy supply is higher than that of other cells of the body. In patients who have suffered acute carbon monoxide poisoning in the long term, various persistent changes in the function of the nervous system, asthenic state and sleep disturbances may develop. As a result, the quality of life after acute carbon monoxide poisoning is sharply reduced.

The neurotoxicity of carbon monoxide is associated, in addition to hemic hypoxia, with the activation of soluble guanylate cyclase. As a result, cerebral vessels dilate and, as a result, cerebral edema [Wang, R. Resurgence of carbon monoxide: an endogenous gaseous vasorelaxing factor / R. Wang // Can. J. Phyol. Pharmacol - 1998. - Vol. 76, No. 1. - P. 1-15]. After primary hyperperfusion, hypoperfusion is noted, which plays an important role in the development of long-term neurological disorders [Mariluz, H.V. In vivo exposure to carbon monoxide causes delayed imprairment of activation of soluble guanylatecyclase by nitric oxide in rat brain cortex and cerebellum / H.P. Mariluz A.F. Castoldi, T. Cocconi // J. Neurochem. - 2004. - Vol. 9, No. 5. - P. 1157-1165]. These changes are especially pronounced in the subcortical nuclei, which is manifested mainly by their necrosis.

As an antidote, which also has a prophylactic effect, in case of carbon monoxide poisoning, the drug Atsizol (bis- (1-vinylimidazole) zinc diacetate) is used. For prophylactic purposes, the drug is administered at a dose of 1 ml intramuscularly 20-30 minutes before entering the smoke zone (gas contamination), at high risk of inhalation of carbon monoxide, during the period of work to eliminate the fire extinguishing and rescue the victims. The protective effect lasts for 1.5-2 hours. Repeated use of the drug is allowed 1 hour after the first injection. For therapeutic purposes, “Atsizol” is recommended to be used as soon as possible after poisoning, regardless of the severity of the lesion. The drug is administered at a dose of 1 ml intramuscularly immediately after removing the victim from the fire zone (gas contaminated room). Repeated administration is allowed 1 hour after the first administration:

The mechanism of action of this drug is associated with an increase in the affinity of hemoglobin for oxygen, which allows hemoglobin to be fully saturated with oxygen. At the same time, there is some difficulty in oxygen release, which may affect the blood supply to target organs. In addition, as a chemical compound, the drug can cause allergies. It has practically not been studied how the use of this drug affects the reduction in the severity of the long-term effects of carbon monoxide poisoning. The use of this drug does not replace oxygen therapy, since oxygen is a real highly effective prophylactic and therapeutic agent. The search continues for the possibility of using oxygen solutions. In this regard, the use of solutions of another form of oxygen, ozone, is promising.

Ozone therapy is a therapeutic method that has been increasingly used in clinical practice in recent years. Ozone has a wide variety of healing effects. For example, it is known that it has antibacterial, antiviral, anti-inflammatory and immunomodulating effects, enhances microhemodynamics, helps correct lipid peroxidation disorders, and increases the activity of the antioxidant defense system. All this made it possible to use this method in the treatment of a number of diseases [Chekman, I.S. Ozone and Ozonation / I.S. Chekman [et al.] - X .: “Digital drunkard No. 1”, 2013. - 144 p.]

Including the known method of detoxification of the body by intravenous drip of ozonized physiological saline (OGF) with an ozone concentration of 1.8-3 mg per 500 ml of injected fluid (application for the invention of the Russian Federation No. 94017960, A61K 33/00, publ. 05.10.1996) .

However, the authors recommend using this method for endotoxemia, and the exact doses per kg of weight are not indicated.

Ozonotherapy is not widespread for the prevention and treatment of acute poisoning. In the available literature there is a mention of the effectiveness of intravenous administration of FGF (200 ml with a concentration of dissolved ozone of 400 μg / ml) in severe poisoning with barbiturates. At the same time, there was a significant acceleration of a decrease in the concentration of toxicants in the patient’s body, improvement of the state [Gribova, N.G. Detoxification efficacy of infusion ozone therapy in acute poisoning with barbiturates / N.G. Gribova, A.A. Lavrentiev, G.N. Sukhodolova // Medicine of critical conditions. - 2010. - No. 3. - S. 24-28].

A method of using the ozone-oxygen mixture for the relief of alcohol withdrawal syndrome is described (application 2006143697/14 of 08/08/2006). With this method, the author is offered to combine the introduction of an ozone-oxygen mixture with preliminary hydrocolonotherapy. However, the rectal use of ozone mixtures is not convenient as a prophylactic, as it requires special equipment and stationary conditions. Also, the preliminary use of hydrocolonotherapy causes a violation of the microflora of the large intestine, has a number of contraindications (pathology of the cardiovascular system, diseases of the large intestine, etc.). In addition, the method is justified in case of alcohol poisoning, since the decay products of alcohol are excreted for a long time through the mucous membrane of the gastrointestinal tract, but are not suitable for the prevention and treatment of acute carbon monoxide poisoning, including to reduce the severity of long-term consequences, since acute carbon monoxide poisoning lies hemic hypoxia.

A method for the treatment of endotoxemia by performing discrete plasmapheresis with the separation of red blood cells from plasma and returning them with compensation by donor plasma is described (application 2001114865/14 from 05/30/2001). Before returning to the patient’s body, erythrocytes are processed by FGM in a volume of 200 ml and an ozone concentration of 2 mg / L. The method has disadvantages. So during plasmapheresis, not only toxins are removed from the blood, but also some of the plasma proteins. The method is effective in case of poisoning with certain substances: inorganic mercury, carbamazepine, theophylline, etc. However, plasmapheresis cannot be used in case of carbon monoxide poisoning, since in the pathogenesis of intoxication it is the formation of carboxyhemoglobin, and not the circulation of toxins in the blood plasma. In addition, the applicability of the plasmapheresis method is limited due to possible complications. Possible complications include blood coagulation disorders, infectious complications, and allergic reactions.

Thus, the possibility of using FGD in acute carbon monoxide poisoning has not been studied extensively in the available literature. In addition, the possibility of non-invasive (oral) administration of FGM has not been investigated. Highlighted close analogues do not allow us to identify specific doses, routes of administration, which are effective to reduce the severity of the long-term effects of carbon monoxide poisoning.

The basis of the invention is the task of creating a method of reducing the severity of the long-term effects of carbon monoxide poisoning by using OFs for prophylactic and therapeutic purposes.

The technical result consists in reducing the severity of the long-term effects of acute carbon monoxide poisoning by increasing the body's resistance to carbon monoxide during prophylactic use by increasing the partial pressure of oxygen in the blood, saturating hemoglobin with oxygen, delivering and utilizing oxygen to tissues; and reducing the severity of hypoxic damage to the brain after acute carbon monoxide poisoning during therapeutic use.

The invention is illustrated by drawings: FIG. 1 - dose-mortality curve in an unprotected group, FIG. 2 - curve "dose-mortality" in the protected group.

The inventive method consists in the fact that 10, 15 or 20 minutes before the start of inhalation of carbon monoxide poisoning, OGF is prophylactically orally administered at a dose of 150, 400, 600 μg / 100 g, the concentration of dissolved ozone is 200 μg / ml. For therapeutic purposes, OFs are administered intraperitoneally at a dose of 12.5 μg / 100 g at a solution concentration of 30 mg / ml 1 time per day (7 injections per course), starting 1 day after poisoning (7 injections per course).

An example of practical implementation.

The work was performed on white outbred rats - females weighing 130-140 g. Rats were kept in standard vivarium conditions on a balanced diet. The legitimacy of the study was confirmed by the decision of the local independent Ethics Committee at the Military Medical Academy. CM. Kirov (minutes No. 143 of December 24, 2013).

Static inhalation inoculation of CO was carried out in an airtight seed chamber. For intoxication, carbon monoxide obtained by the decomposition of formic acid under the action of hot concentrated sulfuric acid was used. Reaction scheme: HCOOH → Н ₂ О + СО.

The concentration of CO in the seed chamber was controlled using a gas analyzer DAX-M-03-CO-1500 "Russia". The exposure was carried out for 30 minutes. The lethal concentration of LC _{50 was} determined as a toxicometric parameter, since it represents the most accurate quantitative characteristic of the toxicity of a substance with a minimum value of 95% confidence interval and an undeniable effect (death of an animal) [Kutsenko, S.A. Fundamentals of Toxicology / S.A. Kutsenko. - St. Petersburg: LLC "Publishing House Foliant", 2004. - 720 p.].

LC _{50 was} determined by probit analysis and Finney's method. The visibility of the calculations was confirmed by the construction of dose-mortality curves.

OFs were obtained using the “Thunderstorm” ozonizer produced by Don LLC (Tomsk) using an improved methodology (certificate of rationalization proposal No. 14105/3 issued by the S. Kirov Military Medical Academy in St. Petersburg) .

The concentration of dissolved ozone in the solution was controlled by the method of iodometric titration (GOST 2332 dated 12/26/72).

The effectiveness of prophylactic use was assessed by survival during the day, the development of symptoms of poisoning, the time of restoration of neurological status after the end of the seed. Quantitatively, the effectiveness of the prophylactic administration of FGM was assessed using a protection coefficient [Kutsenko, S. Fundamentals of Toxicology / CA. Kutsenko. - St. Petersburg: LLC "Publishing House Foliant", 2004. - 720 p.], Defined as:

LC ₅₀ * / LC ₅₀ ,

where LC ₅₀ * is the lethal concentration in protected animals, LC ₅₀ is the lethal concentration in unprotected animals.

Also, the effectiveness of the prophylactic administration of OPF was evaluated by antidote power (Kutsenko, SA Fundamentals of Toxicology / SA. Kutsenko. - St. Petersburg: Foliant Publishing House LLC, 2004. - 720 p.]:

LC ₁₆ * / LC ₈₄ ,

where LC 16 * is the value determined in protected animals, LC ₈₄ is the value determined in unprotected animals. If this ratio approaches 1, the effectiveness of the antidote is considered satisfactory.

The study evaluated memory and learning ability in rats in the long-term period after acute CO poisoning. Memory was evaluated in the test “black and white camera” [Buresh, Ya. Methods and basic experiments on the study of the brain and behavior / Ya. Buresh, O. Bureshova, DP Houston; trans. from English E.N. Painting; under. ed. A.S. Batueva. - M.: Higher School, 1991. - 400 p.).

The learning ability of animals was evaluated by the method of conditioned reflex of active swimming avoidance (URAI) [Buresh, Ya. Methods and basic experiments on the study of the brain and behavior / Ya. Buresh, O. Bureshova, DP Houston; trans. from English E.N. Painting; under. ed. A.S. Batueva. - M.: Higher School, 1991. - 400 p.].

For statistical data processing, the program "Exel for Windows", "Statistica 6.0" was used with the calculation of the average value, the error of the mean and standard deviation. The differences in the mean values of the data were evaluated by a nonparametric method using the Mann-Whitney U-test. A probability of p ≤ 0.05 and higher was considered sufficient to conclude that the differences in the data were statistically significant. The study used nonparametric correlation analysis (Spearman correlation).

At the first stage of the study, the effect of FGM on the gas composition of rat blood was determined.

Rats were divided into 5 groups. The first 4 groups were rats administered orally with FGF at doses of 100 μg / 100 g, 150 μg / 100 g, 400 μg / 100 g, 600 μg / 100 g, respectively. The concentration of dissolved ozone in solutions was 200 μg / ml. This concentration was chosen so that it was possible to administer to the rat an instantly large dose of ozone and not to administer a large volume of liquid. The fifth group consisted of rats, which were injected orally with physiological saline in volume, as at the maximum of the studied doses (3 ml / 100 g). This group was conditionally called the control. Each group was further divided into 3 subgroups: in the first subgroup, blood was taken after 10 minutes. from the moment of injection of the solution, in the second subgroup - after 15 minutes, in the third - after 20 minutes.

With the administration of FGD at doses of 150 μg / 100 g, 400 μg / 100 g, 600 μg / 100 blood after 10, 15 and 20 minutes, the partial pressure of oxygen in the blood, the saturation of hemoglobin with oxygen, and the delivery and utilization of oxygen by the tissues significantly increased compared to the control group. All these effects can “resist” the effects of carbon monoxide on the body. The maximum effect with oral administration is observed after 15 minutes and is dose-dependent.

For further research, administration of FGF was used at an ozone dose of 600 μg / 100 g intragastrically 15 minutes before the onset of intoxication.

The determination of LC ₅₀ in two groups of animals unprotected and protected. The LC ₅₀ calculations were carried out using the probit analysis method and the Finney method. The protection index was determined by the formula:

LC ₅₀ * / LC ₅₀ ,

where LC ₅₀ * is the lethal concentration in protected animals, LC ₅₀ is the lethal concentration in unprotected animals

The results of the study are shown in table 1.

In addition, LC ₁₆ and LC _{84 were} determined by the probit analysis method (table 2).

The effectiveness of the antidote was determined by the formula:

LC ₁₆ * / LC ₈₄ ,

where LC 16 * is the value determined in protected animals, LC ₈₄ is the value determined in unprotected animals.

The antidote power of prophylactic administration of FGP is 0.8, which, according to the literature, is quite high.

In FIG. Figure 1 shows the dose-mortality curve in an unprotected group.

In FIG. 2 shows the dose-mortality curve in the protected group.

Thus, the prophylactic use of FGF at a dose of 600 μg / 100 g 15 minutes before carbon monoxide intoxication increased the survival of animals (protection index was 1.3; antidote power was 0.8).

Given that the main role in the development of the long-term effects of acute carbon monoxide poisoning is played by hypoxia and hypoperfusion, for therapeutic purposes we have chosen the scheme recommended in the clinic for ischemic brain damage [Methods for the use of ozone in medicine. Methodical recommendations (approved by the Ministry of Health of Ukraine, 2004). - URL: http://www.pdffaclory.com.]. Adjusted for the metabolic rate in rats, the dose of FGD was 12.5 μg / 100 g intraperitoneally once a day, the course of treatment was 7 procedures. This scheme was used from 1 day. after acute carbon monoxide poisoning.

For an experiment to study the severity of the long-term effects of carbon monoxide poisoning, all rats were divided into 4 groups. Group I - intact rats, Group II - control, Group III - therapeutic use of FGM, IV - prophylactic use of FGM. As a prophylactic agent, we used OFR with ozone concentrations of 600 μg / 100 g orally, the seed was carried out 15 minutes after administration. The effectiveness of this method was justified above.

All rats were taken in an experiment with two chambers. The purpose of this experiment was to test the memory of animals after acute carbon monoxide poisoning. Conducted stage I of the experiment - familiarization and II ethan - training. After 24 hours, we reproduced the acquired skills. The waiting time for the transition from camera to camera amounted to a maximum of 180 seconds. At the end of this time, if the animal itself did not go from camera to camera, it was removed and returned to the living cage. Moreover, the latent period of transition from the camera to the camera did not significantly differ in all groups. Data on the experiment are given below (table. 3).

The day of testing the skills developed was considered a zero day. Following the two-chamber test, animals were seeded with carbon monoxide at a dosage of 3200 ppm. This dosage is less than LC50. It was experimentally established that this particular concentration causes severe poisoning in animals of the control group with signs of damage to the central nervous system and does not cause the death of animals. Animals of each group were monitored and a lesion clinic was determined. At the same time, the time of development in animals of successively muscle weakness, lateral position, generalized muscle cramps, and the onset of a fatal outcome was recorded. The total data are given in table 3.

As can be seen from table 3, in animals of group II and III developed all stages of severe acute carbon monoxide poisoning. Moreover, the time of onset of each stage in one of the groups did not differ from the corresponding indicator in the other group. In addition, after 10 hours, 1 animal died in each group. These facts suggest that in the control and in the treatment group poisoning of the same severity developed and further comparison of animals of both groups is correct.

As for group IV, in which rats were given prophylactically FGM, in 5 animals signs of poisoning did not develop at all, all these animals were active within the seed chamber. 5 animals developed muscle weakness. The time of its development averaged 10.1 ± 0.9 min, which was 6 minutes later than in the groups without FGP (p <0.05).

When comparing the frequency of respiratory movements of animals (NPV) by the end of the seed, on average in group II and III it was 96.1 ± 14.4 per minute, and in group IV 72.5 ± 7.9 per minute, which is significantly less than in other groups by 1.3 times (p <0.05).

That is, the introduction of FGM causes, at the same concentration of carbon monoxide, less severe poisoning, or does not cause it at all.

After 30 minutes of seeding, the animals were taken out of the chamber and the temporal indicators of the restoration of various functions (motor and sensory) were evaluated. The total data are given in table 4.

As can be seen from the table, in animals of group II and III, the recovery time of motor and sensory functions did not differ significantly: on average, after 5.3 minutes, the animals were in a position on their stomach, on average, after 6.6 minutes, the animals raised to their paws. A partial restoration of pain sensitivity was observed on average after 2 minutes, on average after 7.5 minutes a complete restoration of pain sensitivity was observed. This fact once again emphasizes that animals of groups II and III developed carbon monoxide poisoning of the same severity. At the same time, in animals of group IV, the restoration of functions was faster. So, reliably 2.5 times faster (p <0.05) the animal was on its stomach. Significantly 2.3 times faster animals stood on their paws. The time for partial restoration of pain sensitivity did not significantly differ. At the same time, complete restoration of pain sensitivity occurred 1.8 times faster (p <0.05). It should be noted that 5 animals from group IV did not develop motor and sensory disturbances at all.

After 10 minutes of being out of the seed chamber, the NPV in the II and III groups did not significantly differ and averaged 86.7. In group IV, the NPV after 10 min was significantly 1.4 times less than in II and III (p <0.05).

Thus, in animals of groups II and III, severe carbon monoxide poisoning with generalized seizures developed. The severity of poisoning and the rate of restoration of body functions were the same in these groups. In group IV, due to the prophylactic administration of FGD, the severity of poisoning, ceteris paribus, was less and the recovery of nervous system functions was faster.

Table 5 presents the data of testing animals in a black and white camera.

As can be seen from table 5, at day 0, before the seed, the latent period in all groups did not differ significantly. Subsequently, in group I, the latency period in intact rats did not change, which indicates the absence of memory impairment, the preservation of the acquired skill. In the control (group II), starting from the 1st day, the latent period of the Transition from the light chamber to the dark one significantly decreased by 1.5 times (p <0.05). This indicates the development of memory impairment already from 1 day after seeding. Subsequently, memory impairments increased on the 7th, 14th and 21st days, latent decreased by 2; 3.2; 5.1 times, respectively, but compared with the original (p <0.05). In group III, memory impairment did not develop on day 1, starting from the 7th knot the latent period decreased by 1.2 times (p <0.05), on day 14 it decreased by 1.4 times compared to the initial (p <0, 05), on day 21 it did not significantly change compared to day 14. In group IV, on days 1 and 7, the latent period did not significantly change compared to day 0. On the 14th day, the latent period decreased by 1.2 times compared to the initial (p <0.05), on the 21st day the latent period remained at the level of 7 days.

Thus, in the control group of animals after severe acute carbon monoxide poisoning, from 1 day, memory impairments developed, which increased by 21 days. In group III of animals, in which acute carbon monoxide poisoning of the same severity was simulated as in the control and then FGM was administered according to the treatment regimen, memory impairments began to develop, starting from 7 days, increasing by 14 days, and by 21 day did not change compared to 14. Moreover, the severity of violations was less than in the control group. In group IV, the degree of poisoning was less and memory impairments in the distant period developed less pronounced.

The learning ability of animals was studied by the method of rescue from water. The experiment was carried out from 7 to 11 days after poisoning. Temporary latency indicators are shown in table 6.

In the group of intact animals (I), the latent period, starting from 8 days. significantly decreased, namely 2.1 times for 8 days. in comparison, 5 times for 9 days., 9 times for 10 and 11 days. (p <0.05) (all comparisons with data on day 7) That is, the animals were gradually trained to find a way out of the water (Table 6). At the same time, learning disabilities were observed in the control group, which is expressed in a longer latent period, compared with similar indicators on the same day. So, the latent period for 7 days is 1.6 times greater in group II (p <0.05), for 8 days. 1.5 times more in group II (p <0.05), for 9 days 2.8 times more in group II (p <0.05), for 10 days. 2.7 times more in group II (p <0.05), by 11 days. 1.5 times more in group II (p <0.05). Thus, in severe acute carbon monoxide poisoning, learning disabilities occur most pronounced on days 9 and 10. In group IV, in which animals developed less severe poisoning, the latent period was 7 and 8 days. not significantly different from group I. On the 9th and 10th day. the latent period is greater than in group I by 1.2 and 1.4 times, respectively (p <0.05). For 11 days. the latent period does not significantly differ from group I. When comparing the latent period on different days of training, a significant excess of similar indicators in group II was revealed compared with group IV. In group III, the temporary indicators of saving animals from water significantly exceed the same indicators for the corresponding day in animals of groups I and IV. At the same time, when compared with the control group, animals of group III learn faster, only on day II. the latent period in groups III and II is not significantly different.

Thus, in severe acute carbon monoxide poisoning for 7-11 days. learning disabilities develop. The use of OFR for both prophylactic and therapeutic purposes reliably levels these disorders.

Claims (1)

A way to reduce the severity of the long-term effects of carbon monoxide poisoning in an experiment, which is that 10, 15 or 20 minutes before the start of carbon monoxide poisoning, ozone physiological saline (OGF) is prophylactically administered orally at a dose of 150, 400, 600 μg per 100 g weight before the onset of inhalation poisoning, the concentration of dissolved ozone is 200 μg / ml; for the treatment of the consequences of poisoning for therapeutic purposes, FGM is administered intraperitoneally at a dose of 12.5 μg per 100 g of weight at a solution concentration of 30 μg / ml, starting from 1 day after poisoning; while the course of treatment is 7 injections 1 time per day.

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