INTRODUCTION:

Probiotics are living non-pathogenic microorganisms that, when consumed in quantities greater than n x 106 colony-forming units (CFU)/g, have a positive effect on the state of the intestinal microbiota of the human body¹. Probiotic microorganisms have special properties, such as resistance to environments with high acidity, tolerance to bile and pancreatic secretions, and the ability to adhere to and invade intestinal epithelial cells². The above properties allow them to remain viable in the conditions of the gastrointestinal tract and influence the composition of the intestinal microbiota.

Among the main mechanisms of action of probiotic microorganisms, one can name the ability to colonize and normalize disturbed microbiocenosis; competitive exclusion of pathogens and production of bacteriocins; production of short-chain and branched-chain fatty acids, which can modulate tissue sensitivity to insulin and other features^{3, 4}. The presence of such a wide range of activity allows probiotics to be used as an effective means for the prevention and treatment of infectious^{5, 6}, allergic, and autoimmune diseases⁷. In addition, probiotic-based medications are increasingly being offered as an alternative to antibiotics ⁸.

The use of probiotic microorganisms as agents for the treatment and prevention of a wide range of diseases of the gastrointestinal tract has gained the greatest popularity⁹. Many studies have proven the effectiveness of probiotics in infectious diarrhea^10-127-9, antibiotic-associated diarrhea¹³, Clostridioides difficile-associated diarrhea¹⁴, traveler's diarrhea¹⁵, Helicobacter pylori-associated gastritis¹⁶, Crohn's disease, and necrotic enterocolitis^17-19.

Despite the high effectiveness of probiotic preparations²⁰, there are currently several problems associated with their use^{21, 22}. One such problem is the probability of horizontal gene transfer between probiotic and pathogenic microorganisms by transformation, transduction, or conjugation, which can lead to the development of antibiotic resistance of strains and the acquisition of virulence factors^23,
24. In addition, probiotic bacteriophages can cause mutations in intestinal epithelial cells^{25, 26}. There is also evidence that indicates the occurrence of bloating due to the accumulation of D-lactate produced by lactic acid bacteria²⁷.

Uncontrolled intake of probiotic preparations can serve as the main factor contributing to the development of the above-mentioned adverse events^{28, 29}. Thus, there is a need for the production and sale of preparations based on probiotic microorganisms following established international standards^30-32.

The main stage necessary to initiate the production process of a medicinal product that meets the requirements of safety and efficacy is the preclinical trial^{33, 34}.

Preclinical trials are studies that are conducted on in vitro, in vivo, ex vivo, and in silico models to obtain basic information about the safety and biological performance of a potential medication before testing it on humans^{35, 36} Preclinical trials are mainly conducted following the principles of GLP/GSP (Good Laboratory Practice and Good Scientific Practice) to ensure the reliability and reproducibility of results³⁷.

Currently, many products in the pharmaceutical market of Kazakhstan contain live probiotic microorganisms. However, so far, no Kazakhstan-made probiotic medication with clinically proven safety and performance has been presented.

The developed AS-Probionorm probiotic medication (Industrial Microbiology LLP, Kazakhstan) based on the association of specific action lactic acid bacteria Lactobacillus fermentum 30 and Lactobacillus cellobiosus 36 has high antimicrobial activity against pathogens of intestinal diseases.

The purpose of this paper is to describe the protocol and results of a preclinical trial of the AS-Probionorm medication intended for the treatment of human intestinal infections.

MATERIALS AND METHODS:

Preparation of AS-Probionorm:

To obtain a stock culture, pure cultures of L. fermentum 30 and L. cellobiosus 36 were covered with purified water in a volume of 6-8 ml on slant agar. The culture was scraped off the slant agar with an inoculating loop, shaken until a uniform suspension was formed, and transferred from a test tube to a flask with a liquid nutrient medium at the rate of 1 tube per 250 ml of nutrient medium. Cultivation was carried out in a thermostat at 37^оC for 24 hours.

The inoculant material was obtained as follows: stock culture of lactic acid bacteria was introduced into flasks with 250 ml of nutrient medium at the rate of 3-5% of each culture. After inoculation, the flasks were cultivated in a thermostat at a temperature of 37^оC for 24 hours. The presence of an association of lactic acid bacteria was determined by the presence of sediment and turbidity of the nutrient medium.

To obtain the active pharmaceutical substance (APS), the association of L. fermentum 30 and L. cellobiosus 36 was cultured under fermenter conditions. The nutrient liquid medium was sterilized in a fermenter for 30 minutes at a temperature of 121^оC. Inoculation of the fermenter with a sterile nutrient medium cooled to a temperature of (35± 1)^оC was carried out by transferring the inoculation culture from flasks in an amount of 5-6% of the volume of the nutrient medium. The following mode was chosen for cultivation: pressure: 0.3-0.5 atm., temperature: 37^оC, duration of cultivation: 24 hours without aeration.

At the end of the cultivation process, a sample was taken to control the microbiological purity and determine the titer of the grown culture on a cell viability analyzer.

After receiving the APS corresponding to the stated requirements, the centrifugation stage was started. Centrifugation was carried out by continuously feeding culture liquid into a flow centrifuge to produce biomass, followed by the introduction of sterile components of a stabilizing drying medium. The following centrifugation mode was selected: the feed rate of the culture liquid into the rotor was 0.25 l/min, the centrifugation speed was 3,200 rpm, and the cooling temperature of the centrifuge rotor was not higher than 8^оC. The total time equaled 6.5 hours.

The resulting biomass was transferred to obtain the finished form of the preparation. Then, a sterile stabilizing medium was introduced into the biomass, containing 1.5% food gelatin, 5% sucrose, and skimmed milk powder based on the final concentration. Then, in the lyophilization box, it was poured into trays made of polished stainless steel and placed in a freeze dryer to obtain a dry preparation.

Drying of biomass with added protectors was carried out in a Liobeta-35 freeze dryer. The following drying mode was selected: freezing (-30^оC): 10 h, freezing (-60 ^оC): 5 h, vacuum: 0.9 MPa, drying 1 (-26^оC): 6 h, vacuum: 1.0 MPa, drying 2 (+20^оC): 18 h, drying 3 (+30^оC): 2 hours.

The duration of the entire drying process was at least 26 hours and the final temperature of the product was + (25-27)^оC.

At the end of the drying process, the vacuum pumps were turned off and air was supplied to the chamber through a valve. After equalizing the pressure, the hatch of the apparatus was opened and the dried material was discharged. The dried preparation was transferred to the grinding and packaging stage.

The grinding was carried out in a device for grinding dry products (mill) to obtain a homogeneous dry powder. The ground preparation was transferred to the packing and packaging stage.

The resulting dry preparation was packed in doses of 1 g in a sachet of laminated paper. 14 sachets were packed in a cardboard box together with instructions for medical use in Kazakh and Russian.

Plan of the study:

Conducting preclinical trials of AS-Probionorm included the following main stages:

1) Determination of antibacterial activity on strains of Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis by diffusion into agar;

2) Determination of subacute toxicity in experimental animals in comparison with Fertal (ESI srl, Italy);

3) Determination of therapeutic and preventive performance on the enteritis (colibacteriosis) model in an experiment on 50 white laboratory mice in comparison with the commercial medication Lacidofil-WM (Canada);

4) Determination of the acute toxicity class and the mean lethal dose (LD₅₀) in vivo;

5) Determination of chronic toxicity in vivo;

6) Determination of the presence of phages and resistance to the action of gastric juice and bile.

The study of the antibacterial activity of the medication AS-Probionorm was carried out on strains of E. coli, S. aureus, and S. enteritidis by diffusion into agar. As a result of the conducted studies, we found that AS-Probionorm had antibacterial activity in the declared spectrum of action (growth inhibition zones of test cultures in the range from 17 to 28 mm).

The safety assessment of AS-Probionorm in terms of subacute toxicity was carried out on experimental animals in comparison with Fertal.

The main objective of this study was to identify the most sensitive organs and organ systems, as well as the nature and degree of pathological changes after the use of the medications under study.

The experiment was conducted on young, healthy, adult rats of both sexes. The determination of subacute toxicity was carried out following the requirements of the GLP Standard of the Republic of Kazakhstan and based on the Guidelines for the experimental (preclinical) trials of new pharmacological substances.

With repeated intragastric administration of the studied medications AS-Probionorm and Fertal, no changes were found in the general condition, motor activity, and emotional status of the animals, which was confirmed by the positive dynamics of body weight of the control and experimental groups throughout the experiment. Mortality was not observed among experimental animals. The necropsy data demonstrated that the medication under study did not cause pathological changes in animals' internal organs. Based on the obtained experimental data on the comparative study of the subacute toxicity of AS-Probionorm compared to Fertal, their comparable safety was established.

As part of the trial of the therapeutic and preventive performance of AS-Probionorm, a study was conducted in comparison with the commercially available medication Lacidofil-WM. During the study, the medications were administered and the physiological state of laboratory animals was monitored. Furthermore, bacteriological studies and the study of the persistence and elimination of various microorganisms in the experiment on healthy mice and mice infected with the E. coli strain O15 were carried out. Before the experiment, experimental groups of mongrel white mice of the same age (3 months) and sex (male), weighing 30-32 g, were formed, which were kept in the same quarantine regime for 10 days. Each mouse was weighed and thermometrized, and a veterinary clinical examination was performed. In total, 50 clinically healthy mongrel white mice were taken into the experiment, not emaciated and without symptoms of any disease. The white mice were quarantined with 10 heads per cage equipped with an individual drinking bowl and feeder, in a specially designated vivarium room in a clean area. Daily single administration of the AS-Probionorm to laboratory animals for 10 days at a dose of 5% of body weight 15 minutes before feeding increased the growth of lactobacilli by 247 times and reduced the number of opportunistic intestinal microflora by 16 times. The commercially available medication Laciodofil-WM also increased the growth of lactobacilli by 32 times and reduced the content of E. coli by 6 times. The obtained data indicate more pronounced restorative properties of the AS-Probionorm compared to Laciodofil-WM. The use of AS-Probionorm for therapeutic purposes at a dose of 5% of body weight 15 minutes before feeding 3 times a day protected experimental mice from the development of septic infection by 100%. On the 4th or 5th day, the animals showed an improvement in general well-being, and their diarrhea stopped. The death of laboratory animals has not been noted.

The use of Laciodofil-WM for therapeutic purposes, at a dose of 5% of body weight 15 minutes before feeding 3 times a day, protected animals from severe disease by 90%. In this group, one mouse died on the 4th day. During the bacteriological analysis of the pathological material obtained from the dead animal, a culture of E. coli O15 was isolated. The rest of the animals (9 heads) showed an improvement in their general condition on the 6th day, and diarrhea stopped. Experimentally infected white mice of the experimental group, who received AS-Probionorm for preventive purposes 10 days before infection at a dose of 5% of body weight 15 minutes before feeding 1 time a day and after infection without interruption until the end of the experiment before feeding 3 times a day, fell ill with colibacteriosis in a milder form. Only during the first 2 days after infection, slight depression and palpitations were noted. On the 3rd day, there was an improvement in the general condition. Diarrhea in all experimental mice stopped on the 3rd or 4th day after infection. Experimentally infected white mice of the experimental group, who received Laciodofil-WM for prophylactic purposes 10 days before infection at a dose of 5% of body weight 15 minutes before feeding 1 time a day and after infection without interruption until the end of the experiment before feeding 3 times a day, also became ill with colibacteriosis in a milder form. The animals had a local reaction in the form of fever, edema, hyperemia at the site of injection of the infecting material, diarrhea, and increased heart rate for two days. The cessation of symptoms of the disease was observed on the 6th day. Weight gain was established in 2 experimental groups of laboratory mice treated with AS-Probionorm and Laciodofil-WM during 24 days of the experiment. On average, this value equaled 4 g for the group of animals receiving AS-Probionorm and 2 g for animals receiving Laciodofil-WM.

RESULTS:

As a result of the experiment conducted on white mice, it was found that the greatest therapeutic and preventive performance of AS-Probionorm was observed in the 4th experimental group in animals who had received the medication for a long time (10 days before infection and for 14 days during the experiment, in total 24 days). After 10 days of use of AS-Probionorm, positive dynamics in the composition of the intestinal microbiota of laboratory animals were established. There was an increase in the total number of microorganisms, in particular lactobacilli, by 247 times, and a decrease in the conditionally pathogenic microflora by 16 times compared to the state before the use of the preparation. The use of AS-Probionorm protected experimental mice from the development of septic infections. The animals receiving the preparations became only slightly ill, and the pathogen did not enter the bloodstream. In control mice infected with E. coli O15, intestinal damage with the formation of hemorrhages was noted. Experimentally infected white mice of experimental groups receiving AS-Probionorm became ill with colibacteriosis in a milder form only within three to four days after infection. The experimental white mice showed slight depression, local temperature reaction, and edema at the site of injection of the infecting material. After three to four days, there was a rapid improvement in the condition of the experimental white mice. The use of AS-Probionorm for the treatment of white mice from experimental infection significantly reduced the time of elimination of pathogens (on average up to 4 days). Administration of the medication to the animals of the experimental groups protected them from adhesion and penetration of microorganisms into the cells, thus preventing the penetration of pathogens into the bloodstream and, as a result, the septic development of the infectious process. A significant increase in the live weight of white mice who regularly received the preparation (on average by 3-5 g) indicates a high immunostimulating effect of the probiotic. The use of the preparation allowed laboratory animals to easily live through the infection and recover in a short time. Thus, as a result of the experiment conducted on white mice, the high therapeutic and preventive performance of AS-Probionorm was demonstrated.

In the course of this study, the toxicity class and the LD₅₀ of the studied compound in vivo were also determined.

Healthy sexually mature outbred mice in the amount of 30 heads, weighing 22 g ± 10%, were used to study acute toxicity. The preparation under study was administered once orally in the following dosages: 50.0 mg/kg; 300.0 mg/kg; 1,000.0 mg/kg; 2,000.0 mg/kg; 4,000.0 mg/kg; and 5,001.0 mg/kg of weight. The follow-up period was 14 days, during which daily weighing, temperature measurement, and veterinary clinical examination of laboratory mice were carried out. After the observation period, the animals were euthanized, followed by an assessment of the condition of the internal organs.

During the observation period, no deaths of animals were observed after administration of the studied solutions in different doses, as well as no changes in somatic parameters, were registered. When studying the dynamics of the body weight of mice, no significant changes were found between the experimental groups. There was no significant difference between the absolute weight values and the relative weight coefficients of the internal organs of the animals of the experimental groups. The location and appearance of internal organs and other parameters without deviations correspond to the species characteristics of the animals. The macroscopic picture did not show pathological changes in the internal organs of the animals of the experimental groups. We could not calculate the LD₅₀ due to the absence of animal deaths at all doses used. Thus, the toxicity class can be defined as a non-toxic substance.

To determine the chronic toxicity of AS-Probionorm, a study was conducted on white outbred mature laboratory mice in the amount of 100 individuals (10 heads of each sex for each dose, for the negative control group and intact control group), weighing 20 g ± 10%.

The preparation under study was administered once orally in the following dosages: 5.0х10⁶ CFU; 10.0х10⁶ CFU; and 15.0х10⁶ CFU/g. The follow-up period was 60 days, during which daily weighing, temperature measurement, and veterinary clinical examination of laboratory mice were carried out. After the observation period, the animals were euthanized, followed by a histological examination of the internal organs. It was found that AS-Probionorm in the studied doses did not have a toxic effect on the body with prolonged use. In the course of this study, no mice deaths were registered in the experimental groups, and no deviations in the appearance of animals and the level of feed consumption were observed. According to the results of physiological studies of spontaneous motor activity in the open field tests and the research reflex of deviations/changes were not observed, and animals of all experimental groups showed the same type of behavior. The initial weight and body weight gain corresponded to the physiological norm. Assessment of secretory renal function showed no changes in the reabsorption activity of epithelial cells of the proximal tubules. The values of rectal temperature, and hematological and biochemical parameters of blood and urine corresponded to this animal species. Macroscopic examination showed that the probiotic preparation under study did not cause visual pathological changes in the internal organs of mice or significant changes in the relative weight coefficients of the internal organs of animals of all experimental groups. Histological examination of the internal organs of mice of the negative control group and the group of mice receiving AS-Probionorm at a maximum dose of 15.0×10⁶ CFU did not show structural changes in organs or differences between the groups.

Besides, the presence of phages in the composition of AS-Probionorm and its resistance to the action of gastric juice and bile were determined. As a result of the conducted studies, the absence of phagolysis zones was established, as well as the preservation of the number of viable cells at a sufficient level was confirmed.

A study was conducted to determine the resistance to the action of gastric juice and bile. After exposure of lactic acid bacteria to acidin-pepsin, the cell titer was reduced by only one order of magnitude.

DISCUSSION:

As a result of the conducted studies, we found that AS-Probionorm had antibacterial activity in the declared spectrum of action (growth inhibition zones of test cultures in the range from 17 to 28 mm).

Based on the obtained experimental data on the comparative study of subacute toxicity of AS-Probionorm compared to Fertal in white mice, their comparable safety was established. The use of this medication for the treatment of white mice from experimental infection significantly reduced the time of elimination of pathogens (on average up to 4 days). The administration of the preparation to the animals of the experimental groups protected them from adhesion and penetration of microorganisms into the cells, thus preventing the penetration of pathogens into the bloodstream and, as a result, septic development of the infectious process. A significant increase in the live weight of white mice who regularly received the preparation (on average by 3-5 g) indicates a high immunostimulating effect of the probiotic. The use of the preparation allowed laboratory animals to easily live through the infection and recover in a short time.

In the course of this study, the toxicity class and the average lethal dose of LD₅₀ of the studied compound in vivo were also determined. It was not possible to calculate the LD₅₀ due to the absence of animal deaths at all doses used. Thus, the toxicity class can be defined as a non-toxic substance.

When determining the chronic toxicity of this probiotic medication on white outbred mature laboratory animals, no structural changes of internal organs were detected during the observation period of 60 days.

The presence of phages in the composition of AS-Probionorm and its resistance to the action of gastric juice and bile were determined.

As a result of the conducted studies, the absence of phagolysis zones was established, as well as the preservation of the number of viable cells at a sufficient level (at least 2 billion CFU).

The results of preclinical trials of AS-Probionorm confirming its safety and medical and preventive performance make it possible to conduct clinical trials.

CONCLUSION:

Functionalization of MWCNT was performed successfully to assess toxicity taking into consideration various parameters known to quantify functionalization and toxicity. It was observed that the functionalization of MWCNT reduced material toxicity. Additionally, it can be concluded that MWCNT after functionalization is safe, potential drug carrier in inhalation drug delivery and enhance regional lung deposition with better flow properties. The formulation of cefdinir loaded FMWCNTs DPI for drug delivery is useful to reduce the dose of the drug and synergistically enhance the activity against gram-positive and gram-negative bacteria due to site-specific delivery with increased bioavailability.

CONFLICT OF INTEREST:

During the preclinical trial of AS-Probionorm, the safety and biological performance of the preparation were confirmed. AS-Probionorm demonstrated significant antagonistic activity against the most common pathogens of the human gastrointestinal tract, resistance to gastric juice and bile, and the absence of extraneous microflora. In addition, the safety of AS-Probionorm was proven in comparison with the commercially available Fertal. AS-Probionorm also demonstrated a more pronounced therapeutic and preventive effect than Lacidofil-WM available on the international pharmaceutical market.

Since the safety and performance of AS-Probionorm were proven in experimental animal models in vivo, the next step is to evaluate the therapeutic and preventive effects on the human body. Thus, the positive results obtained during the preclinical study of AS-Probionorm indicate the possibility of conducting the first phase of clinical trials of this medication.

ACKNOWLEDGMENTS:

The publication of this paper was for from the budget funds under the grant financing of project AR14870162 "Clinical trials of the made-in-Kazakhstan probiotic medication AS-Probionorm with a wide spectrum of action against human intestinal infections".

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Received on 22.04.2024 Revised on 08.07.2024

Accepted on 14.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5960-5966.

DOI: 10.52711/0974-360X.2024.00904