PHARMACO-TOXICOLOGICAL PROPERTIES OF OXIDIZED CELLULOSE

ABSTRACT

Deaths related to uncontrolled massive blood loss caused by various injuries are now common all over the world. Hemostatic agents are used to prevent such situations. It should be noted that one of the mandatory conditions for the use of drugs and medical devices in humans is to first conduct toxicological studies in animals. The purpose of this work is to study the hemostatic efficiency and toxicity of oxidized cellulose samples obtained by exposure to ultrasound waves for the first time with the help of oxidizing agents that selectively affect cotton lint, cellulose fabric, and viscose in vivo, and compare it with the hemostatic agent "Surgicel fibrillar" currently used in medical practice. compared. The study investigated the effects of these samples on bleeding time and amount of blood loss in a parenchymal hemorrhage model. LD10, LD16, LD50 values were determined for each sample.

АННОТАЦИЯ

В настоящее время смертельные случаи, связанные с неконтролируемой массивной кровопотерей, вызванной различными травмами, распространены во всем мире. Для предотвращения подобных ситуаций применяют кровоостанавливающие средства. Следует отметить, что одним из обязательных условий применения лекарственных средств или изделий медицинского назначения является предварительное проведение токсикологических исследований на животных. Поэтому основной целью настоящей работы являлось сравнительные изучения гемостатической эффективности и токсичности образцов окисленной целлюлозы, полученных воздействием ультразвуковых волн на хлопковый ворс, целлюлозную ткань и вискозу в условиях in vivo с кровоостанавливающим средством «Surgicel фибриллярный». В проведенных исследованиях на модели паренхиматозного кровотечения было изучено влияние полученных образцов на время кровотечения и объем кровопотери. Были также найдены значения ЛД₁₀, ЛД16, ЛД50 для каждого образца.

Keywords: oxidized cellulose, hemostasis efficiency, bleeding time, LD50, Surgicel fibrillar, ultrasound, inclusion

Ключевые слова: окисленная целлюлоза, гемостатическая эффективность, время кровотечения, ЛД50, Surgicel фибриллярный, ультразвук, инклюдация.

Introduction.  One of the priority areas of chemistry and pharmaceuticals is the creation of drugs for medicine based on plant raw materials, which are not inferior to synthetic drugs in terms of effectiveness and have low toxicity. Currently, deaths related to uncontrolled massive blood loss caused by various injuries account for more than 30% of traumatic deaths worldwide [1]. As a solution to prevent and reduce such unexpected situations, the use of hemostatic agents is very effective [2].

There are many types of hemostatic agents, which are based on collagen, albumin, fibrin and polysaccharides [3]. One of the most commonly used hemostatic agents is oxidized cellulose, which is a natural polysaccharide derivative. Hemostatic agents obtained on the basis of oxidized cellulose clearly differ from their analogues with important indicators such as high efficiency, lack of side effects, biodegradability, and non-allergenicity [4]. But its synthesis is a very difficult process in practice. In particular, in obtaining oxidized cellulose, the reaction takes a long time, and various aggressive and toxic oxidants are used in oxidation.

In our previous work, we developed a method for obtaining monocarboxylic cellulose with a high degree of oxidation in a short time by applying ultrasound waves to the long-term cellulose oxidation processes [5]. Also, the effect of inclusion processes on the reaction by preliminary treatment of raw materials before oxidation reactions was studied [6].

In this work, we aimed to determine how the synthesized hemostatic agents were obtained using new synthesis conditions, how the reaction conditions affected the pharmacological properties of the obtained agent, that is, its hemostatic activity and some toxicological properties. It should be noted that one of the mandatory conditions for the use of drugs and medical devices in humans is to first conduct toxicological studies in animals. This rule is also reflected in the Declaration of Helsinki.

The more thorough the pharmacotoxicological studies in animals, the less adverse reactions are observed in clinical studies [7]. Taking this into account, it is necessary to determine a number of pharmacotoxicological properties of oxidized cellulose and conduct research on their comparative comparison with hemostatic agents currently used in medical practice.

The purpose of the study. In vivo study of the hemostatic efficiency and toxicity of oxidized cellulose samples obtained by exposure to ultrasound waves for the first time with the help of oxidizing agents that selectively act on cotton lint, medical gauze, and viscose fabric, and comparative comparison with hemostatic agents used in medical practice.

Materials and methods. The following samples were studied in the research:

1) Viscose-based oxidized viscose in powder form (oxidation level-89 mol %)

2) Oxidized cellulose obtained on the basis of cotton lint, in the form of fiber (oxidation level - 28 mol %)

3) Oxidized cellulose in the form of fabric obtained on the basis of medical gauze (oxidation level - 39 mol %)

4) Comparative agent - "Surgicel fibrillar" (fluffy oxidized viscose, oxidation level - 74 mol %)

Evaluation of hemostatic efficiency of oxidized cellulose.

Determining the hemostatic efficiency of oxidized cellulose was carried out using a model of parenchymatous bleeding in animals. Researches were conducted in purebred white rats with a body weight of 200-220±15 g. For this, 3 rats of both sexes were selected for each group (5 groups). During the study, all animals were kept in standard vivarium conditions based on World Health Organization recommendations for experimental experiments using animals and international guidelines [8,9].

Initially, rats were anesthetized by intraperitoneal injection of ethaminal sodium at a dose of 50 mg/kg, and a longitudinal incision was made along the linea alba of the abdomen. The incision was closed with a saline-moistened napkin and the intestines were removed. The front surface of the liver was also taken out and a filter paper was placed behind it. The anterior surface of the liver was resected using a surgical instrument (scalpel) using a special limiting measuring device (plastic with a small circular hole in the center). The shape of the cross-section cut in this vertical projection is circular and its dimensions are constant. The total area of the bleeding wound is about 1-1.5 cm2, the depth is about 0.3 cm. In the experiments, pre-weighed test and control samples were used in dimensions covering the wound surface. Both tests were performed simultaneously to provide a comparative evaluation of the study agent and the control. Medical gauze was used as a control. Bleeding stop was determined by observing the complete cessation of bleeding through the surface and edges of the specimen used in the evaluation (the paper felt dry when the filter paper was touched). Hemostatic agent "Surgicel fibrillar" (manufactured by ETHICON, LLC, Puerto Rico 00754, LOT MAD 0021, EXP 2020-10-31) was used as a comparator in the research.

Determination of the toxicity of oxidized cellulose.

Studies to determine the general and acute toxicity of hemostatic agents based on oxidized cellulose were carried out in male mice weighing 20±2 g. The animals were kept in standard vivarium conditions in accordance with international recommendations, following all rules of handling animals in experimental experiments [8,9]. The studies were carried out by the oral route. For fabric and fibrous samples, their extracts were used, and for powdered samples, a 3.5% solution of the powder was used. Each dose of the samples was tested in 6 animals for 14 days. Extracts of fabric and fibrous samples were prepared as follows: first, the test samples were weighed and placed in containers. Distilled water with a pH of 7.0 was measured to the nearest 1 ml using a pipette or other measuring device and poured over the studied samples. In this case, the surface of the sample and the volume ratio of the added water were calculated using the equation S:V =1.6:1.0 (cm²/ml) [10]. The extraction process was carried out at 37°C for 72 hours. Extracts from fabric and fibrous samples were administered orally to experimental animals in doses of 0.2, 0.3, 0.4 and 0.5 ml. According to methodological recommendations, the maximum physiologically acceptable volume for oral administration to mice is 0.5 ml. Therefore, it is almost impossible to study a larger volume of the extract solution. General and acute toxicity of hemostatic agents in powder form were used in doses of 250, 500, 750 and 1000 mg/kg, in the form of a 3.5% solution.

Result and discussion

During the research, experiments were carried out to determine the hemostatic efficiency of oxidized cellulose samples obtained on the basis of previously synthesized cotton lint, cellulose fabric and viscose with different degrees of oxidation.

The hemostasis efficiency of known hemostatic agents is determined in vivo using a parenchymatous bleeding model in animals. In this case, the method of calculating the bleeding time and the amount of blood loss in percentages compared to the control is used to determine the effectiveness of hemostasis. Bleeding time is determined using a stopwatch, and the amount of blood loss is determined gravimetrically. To determine the amount of blood loss in the studies, the weight of the study samples was measured before surgery (m₁, g) and after surgery (m₂, g) using an analytical balance. Their difference represents the amount of blood loss. Table 1 below lists the bleeding time and blood loss amounts of the samples determined during the experiments.

Table 1.

Effect of samples on bleeding time and amount of blood loss in a parenchymal hemorrhage model

As can be seen in Table 1 above, in the experiments conducted using cotton lint-based samples, it was found that these samples reduced parenchymatous bleeding time by 81.7%. This indicator was 75% in the drug "Surgicel fibrillar" used as a comparator. The amount of blood loss was reduced by 91.6% compared to the control group (94.4% in the drug "Surgicel fibrillar"). During the experiment, it was found that the sample obtained on the basis of cotton lint was much better in terms of bleeding time than the drug "Surgicel fibrillar", which was used as a comparator, and the amount of blood loss was almost the same.

Samples taken on the basis of medical records reduced bleeding time by 73.7% and blood loss by 43.8% compared to the control group. Taking into account that these indicators are 75 and 94.4, respectively, in the drug "Surgicel fibrillar", which was used as a comparative tool, it was found that the bleeding time is almost the same, and it is slightly behind in terms of the amount of blood loss.

Experiments conducted on samples synthesized on the basis of viscose fabric showed that the samples reduced parenchymatous bleeding time by 80.7% compared to the control and reduced the amount of blood loss by 82.5%. It was found to be superior in terms of bleeding time and slightly inferior in amount of blood loss for the sample obtained when compared to the comparator.

In general, it was found that the hemostatic efficiency of the samples synthesized by us were almost similar to each other in terms of hemostatic indicators, while some samples were slightly superior. When comparing the hemostatic efficiency of samples based on cotton lint, cellulose fabric and viscose, it was found that the efficiency of the sample based on cotton lint is higher. This can be attributed to the fiber structure and structure of this sample. In cotton cellulose microfibrils, the percentage of crystalline areas is higher than amorphous areas, which ensures a larger total surface area of the fiber. The larger the surface of the fiber, the higher its sorption properties and better sorption of the liquid part of the blood.

In toxicity studies, after the animals were given the maximum dose allowed orally, they were monitored hourly in the laboratory on the first day of the experiment. From the 2nd day of the study, every day for 2 weeks in the vivarium, the general condition and activity, behavior, heart rate, frequency and depth of breathing movements, condition of hair and skin, changes in body weight and other parameters of the animals of all groups were monitored. Survival, general condition, and mortality were used as indicators of the functional status of the animals during the experiment. In all experiments, the animals were kept under the same conditions, with ad libitum access to food and water.

The results of the study of the toxicity of the samples are presented in Table 2. It was found that when the extracts of fibrous and fabric samples were administered orally in a dose of 0.2-0.4 ml, the general condition of the animals did not change, and the frequency and depth of respiratory movements were normal.

Table 2.

Study of the toxicity of samples

After oral administration of the maximum tolerated volume of 0.5 ml to rats, slight restlessness was observed in the animals. After 20-30 minutes, the behavior of the animals did not differ from that of the control. Then the general physiological state: behavior, condition of hair and skin, condition of the tail, amount and consistency of feces, frequency of urination and other indicators did not differ from the control. No animal deaths were recorded during the entire experimental period, especially when the maximum allowed volume (0.5 ml) was introduced.

The results of the toxicological study shown in the table confirm that the average lethal dose (LD50) of samples in the form of fiber and fabric is ≥ 25000 mg/ml. It belongs to class VI of relatively non-toxic substances.

A single oral dose of 250, 500, 750, 1000 mg/kg of the powdered sample in the form of a 3.5% solution showed that the general functional status of the animals did not change during the experiment. The animals' behavior and feeding were normal. In the study, it is almost impossible to study at high doses of samples due to the high viscosity of the solution (very thick gel).

A general exposure study showed that when the powdered sample was administered at a dose of 1,000 mg/kg, animals collapsed after 10 minutes. The duration of this effect lasted 25-30 minutes. Summarizing the results obtained in the study of acute toxicity of powder samples after single oral administration, we can conclude that the LD50 is higher than 1000 mg/kg dose. This showed that according to toxicity classes, it belongs to class IV of less toxic compounds.

Conclusion

Based on the results of the experimental research, it can be concluded that in our previous studies, for the first time, the hemostatic samples synthesized using ultrasound waves were compared with the drug "Surgicel fibrillar" which is currently on sale and used in medical practice. It was found that the hemostatic indicators of the samples were almost close to the properties of the comparative drug, and some samples were slightly superior. When comparing the hemostatic efficiency of samples based on cotton lint, cellulose fabric and viscose, it was found that the efficiency of the sample based on cotton lint is higher.

From general and acute toxicity studies, it can be concluded that the median lethal dose (LD50) of cotton lint and cellulose fabric samples was ≥ 25,000 mg/ml. It was found that it corresponds to class VI of relatively harmless substances.

In the powdered sample synthesized on the basis of viscose, the LD50 value was found to be higher than 1000 mg/kg dose, which indicated that it belongs to class IV of low toxic compounds according to toxicity classes.

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