RU2806636C1 - Suspension for oral administration with anti-ulcer and chemoprotective effects in colon cancer and method of its use - Google Patents

Abstract

FIELD: medicine; gastroenterology; oncology.

SUBSTANCE: invention is intended for the prevention and treatment of gastric ulcers and colon cancer. An oral suspension with antiulcer and chemoprotective effects for colon cancer is an aqueous suspension containing the following active ingredients: a purified extract of fatty alcohols from the purified wax of Apis mellifera bees and microcrystalline cellulose.

EFFECT: use of the invention provides strong antiulcer and chemoprotective effects due to the synergistic action of the components.

4 cl, 4 tbl, 6 ex

Description

The present invention relates to a new pharmaceutical composition consisting of an aqueous suspension for oral administration, as well as a method for its preparation. This suspension has antiulcer effects on the gastric mucosa and chemoprotective properties against colon cancer and is characterized by the fact that as active ingredients it contains a purified extract of fatty alcohols from purified honey bee wax, the particle size of which is reduced to <1.5 micrometers through a technological procedure, as well as microcrystalline cellulose. In addition to fatty alcohols, purified beeswax extract contains diols, paraffins and potassium salts of fatty acids as minor components. The present new composition also includes a number of excipients accepted for use in the pharmaceutical industry, including: one or more emulsifiers used in the particle size reduction step of the fatty alcohol extract, as well as humectants, sweeteners, suspending agents, preservatives, flavorings and water.

Gastric ulcer is a wound on the mucous membrane of the stomach, and sometimes on the muscular layer of the stomach, as a result of which a cavity with acute and chronic inflammation is formed around it, which is the main cause of gastrointestinal bleeding, one of its main complications (Valley 2002, Sadik 2009 ). Other complications include perforation, penetration and blockage, all of which lead to significant deterioration in the patient's quality of life (Song 2010). Peptic ulcer disease occurs due to an imbalance under the influence of aggressive (acid secretion, pepsin, H. pylori, non-steroidal anti-inflammatory drugs) and protective (secretion of mucus and bicarbonates, capillary circulation, prostaglandins, growth factors) factors, among which the main causes are H. pylori infection and long-term use of non-steroidal anti-inflammatory drugs (Ramakrishnan 2007). Despite ongoing advances in treatment to control H. pylori, mortality from complications of peptic ulcers remains stable. In clinical practice, the most widely used drugs for the treatment of gastric ulcers are proton pump inhibitors and histamine H2 receptor antagonists, although they have various side effects such as nausea, constipation, gynecomastia and impotence (Forgherini 2018). These effects limit their use and necessitate the search and use of new pharmaceutical compositions that effectively act as antiulcer agents and also have better safety indicators. The most widely used pharmacological models for testing potential substances with antiulcer activity rely on the induction of ulcers with alkaline substances, ethyl alcohol, or cross-linking of the gastric outlet as a way of capturing the various possible mechanisms by which the substance under evaluation exerts antiulcer activity.

On the other hand, cancer is one of the leading causes of death, causing more than 8 million deaths annually worldwide, and colon cancer is one of the five most common causes of death worldwide, with an increasing trend, especially in countries with a small amount of resources (Torre 2015, Chatenot 2014). Considering the high prevalence of this disease, as well as the fact that its treatment is carried out mainly by surgery followed by chemotherapy, the search and use of chemoprotective pharmaceutical compositions that can be used for the prevention and treatment of patients with this type of cancer, or who have risk factors for this disease , such as ulceration processes in the gastrointestinal tract (ulcerative colitis, Crohn's disease, etc.), are justified. The most widely used pharmacological models for studying potential chemoprotective agents to combat this disease are based on the use of carcinogens. Among the above, one of the most effective and frequently used substances is 1,2-dimethylhydrazine. Metabolism of this carcinogen results in the production of cytotoxic substances to colonocytes within a few hours of induction (Corpet 2003, Reddy 2004).

Given that both gastric ulcers and colon cancer negatively affect the quality of life and can be fatal in people suffering from these diseases, which represent an important public health concern worldwide, and given the fact that Since existing traditional treatments are associated with various negative effects that limit their clinical use, and do not always provide cure or prevention of the spread of these diseases, it is necessary to find effective drugs for both diseases. The need for new safe and effective treatment strategies for peptic ulcer disease and cancer is an ongoing challenge.

The purified beeswax extract used in the composition of the present invention, as well as the method for its preparation, have been previously patented. This extract has been claimed as a natural mixture consisting of higher primary aliphatic alcohols for the treatment of gastric ulcers and duodenal ulcers, having an anti-inflammatory effect, and the most suitable form of its administration is tablets, pills or capsules (CU 22412 A1), i.e. , ready-made solid forms. In accordance with the above, a tablet containing this active substance containing 50 mg of fatty alcohols in a standard dosage has been registered in Cuba and other countries as a dietary supplement, as a functional food or alternative medicine. However, this dosage form has several disadvantages, such as: large size (655 mg), which provokes the use of a large number of excipients and high production costs; increasing phase-out of two excipients (croscarmellose sodium and polyvinylpyrrolidone) in many countries; as well as extremely low bioavailability of the fatty alcohols included in its composition (<10%).

It should be noted that the pharmacological activity of this tablet as an antiulcer agent is much lower than that achieved by the suspension of the present invention. When the fatty alcohol extract is subjected to cutting or precipitation processes by changing temperature and stirring in the presence of specific emulsifiers to reduce its particle size, and when subsequently obtained as an aqueous suspension, mixed with microcrystalline cellulose in proportions and with a specific concentration, its pharmacological effectiveness is synergistic expands, which is unknown at this level of technology. The aqueous suspension of the present invention significantly expands the effect of both fatty alcohol extract in tablet form and microcrystalline cellulose, and also has a powerful chemoprotective effect against colon cancer, which has not previously been reported for either of these substances, as can be seen in implementation examples.

On the other hand, microcrystalline cellulose (derived from alpha cellulose) has been predominantly used to produce creams and solid suspensions in cosmetics, detergents, and as a stabilizer in foods (Nsor-Atindana 2017). In the pharmaceutical industry it is used for the preparation of tablets and capsules, as a compressing agent and excipient, although its ability to sequester bile acids has also been reported (Paniagua 1999), and it has been used as an active ingredient in suspension with antiulcer effects with a dose 12% (Barzaga 2004). It should be noted that the pharmacological effect claimed for the 12% suspension is much lower than the effect achieved with the suspension disclosed in the present invention, which contains a much lower dose of microcrystalline cellulose (≤ 2%) as shown in embodiment number 3 This is the result of the synergistic effect that occurs when microcrystalline cellulose is mixed in specific proportions and concentrations with a purified extract of fatty alcohols derived from beeswax, which improves its antiulcer effect and also gives it a powerful chemoprotective effect in the treatment of colon cancer. which is absent in previous dosage forms, which is unknown in this prior art.

The use of processed beeswax and microcrystalline cellulose in the pharmaceutical industry has so far been limited to solid dosage forms that provide controlled release of the active ingredient (Kallai 2010; Rayner 2018), or to the use of both ingredients as excipients in suppositories or other dosage forms (Lee 2018). The combined use of an extract of fatty alcohols derived from beeswax and microcrystalline cellulose in an aqueous suspension, where both substances act synergistically, imparting to the composition a powerful antiulcer and chemoprotective effect, was also not previously indicated before the advent of the present invention. When using both substances in tablet form (CU 22412 A1), the effects observed with the aqueous suspension described according to the present invention are also not achieved, as shown in the exemplary embodiments.

The procedure for obtaining the pharmaceutical composition of the present invention is characterized by the following: A) mixing a fatty alcohol extract obtained from beeswax (1-2%) with polyoxyethylene hydrogenated castor oil or polysorbate (2-4%), which are used as emulsifiers; B) subjecting the mixture to a process to reduce the particle size of the extract to <1.5 micrometers, which can be achieved using homogenization equipment capable of grinding particles at speeds >1,000 rpm and temperatures >80°C at for >1 minute, or stir the mixture at >1,000 rpm and >80°C for >1 minute, then reduce the temperature to <60°C while maintaining the same stirring speed; C) introduction of microcrystalline cellulose as the second active ingredient (1-2%) without stopping stirring, methyl and propylparabens as preservatives (0.1-0.6%), glycerin as a moisturizer (20-30%), sorbitol or sodium saccharin as sweeteners (1-6%), carboxymethylcellulose as a suspending agent (0.1-1%), essential oils as flavoring agents (0.01-0.05%) and purified water (50-70 %). As a result of this procedure, a new pharmaceutical composition is obtained, characterized by the fact that it is an aqueous suspension for oral administration, the active ingredients of which are: an extract of fatty alcohols and microcrystalline cellulose, acting synergistically, which gives it a powerful antiulcer and chemoprotective effect.

This new pharmaceutical composition is intended for the prevention or treatment of ulcer formation and other related diseases, taking into account the protective nature of its effect on the gastrointestinal tract, as well as its chemoprotective effect in the treatment of colon cancer. The proportion in which the active ingredients are present in the present pharmaceutical composition is the result of several studies conducted to standardize the elements of both the active ingredients and excipients in the finished composition in terms of specific concentrations and proportions that provide the necessary pharmacological effects as a result of the synergistic action of these active ingredients in the composition. The potent pharmacological effects of the present composition are far superior to those of separate administration of both active ingredients, even at a dosage greater than that of the present composition as demonstrated in the exemplary embodiments, and it also demonstrates the occurrence of a synergistic effect that is not present in the current state of the art.

The advantage of this composition over existing ones is that the effectiveness of its antiulcer effect is higher than that of currently registered tablets with 50 mg of fatty alcohol extract from beeswax, and is also 12% higher than that of a suspension of microcrystalline cellulose, the development of which was previously reported; in other words, the present composition has an antiulcer effect that is much superior to both previous dosage forms, even with reduced dosages of the active ingredients. On the other hand, the present composition has been demonstrated to have an effective chemoprotective effect in the treatment of colon cancer, which is significantly superior to the effect of both active ingredients separately in similar suspensions, as presented in Example 6. Previously this effect, resulting from the synergistic effect of both substances in the suspension according to the present invention, in relation to the purified beeswax extract or microcrystalline cellulose was not observed. In addition, the industrial production of this suspension is cheaper than the production of tablets with 50 mg of fatty alcohols and does not involve the use of any excipients with restrictions for use in the pharmaceutical industry at this time.

The present invention has industrial application, the composition is new, and both the suspension itself and the method of its preparation involve an inventive step, since it is not known from the previous state of the art that a mixture of two different active ingredients with antiulcer effect with specific proportions and concentration (neither of which demonstrate chemoprotection against cancer-causing processes) has shown synergistic effects with pharmacological benefits in the treatment of gastric ulcers, as well as chemoprotection in the treatment of colon cancer. The present invention relates to the food and pharmaceutical industries, since the resulting composition can be used as a dietary supplement due to its beneficial preventive effect on the gastrointestinal tract, and also as a drug for the treatment of ulcers and cancer.

Example 1.

12 L of purified water were added to a 50 L stainless steel auxiliary container (R1), and 1 kg of microcrystalline cellulose was added in small portions at intervals and stirring. Mixing was performed at 60 rpm for 30 minutes until a homogeneous paste was obtained. 5 kg of sorbitol and 24 kg of glycerol were added with stirring, after which stirring was continued for 10 minutes to obtain Mixture 1 (M1). On the other hand, in a 300 L stainless steel reactor, 60 L of water were heated to 90°C, 10 L were taken and placed in the auxiliary vessel (R2), 3 kg of polysorbate 80 were introduced with stirring at 1,200 rpm , and everything was mixed together for 5 minutes at a speed of 1,200 rpm. 1.2 kg of purified fatty alcohol extract was added in small portions at intervals, after which stirring was continued for 10 minutes to obtain Mixture 2 (M2), and then the temperature was reduced to 30 ± 2°C without stopping stirring. In another vessel, 0.18 kg of methylparaben and 0.02 kg of propylparaben were dissolved in 1.5 liters of ethyl alcohol and stirring at a speed of 60 rpm. 0.2 kg of sodium carboxymethylcellulose A/V was moistened, 0.03 kg of flavor was added and mixed for 5 minutes to obtain Mixture 3 (M3).

In another auxiliary vessel with a capacity of 5 L, 1 kg of glycerin was mixed with 0.5 L of ethyl alcohol and stirred for 5 minutes at a speed of 60 rpm to obtain Mixture 4 (M4). Mixture M1 was introduced into the reactor with mixture M2, extracting the residues with a minimum amount of water separated in R2, and stirring was carried out at a speed of 60 rpm for 10 minutes. After this time, M3 was introduced and residues were removed from M4. Subsequently, the remaining suspension was extracted using hot water in vessel R2 and stirring was carried out for 10 minutes. Then stirring was stopped, and the mixture was infused for 10 hours. After this time, the remainder of the alcohol was sprayed onto the surface of the suspension and was diluted with water to obtain 100 liters of suspension. Stirring was performed for 5 minutes at 1200 rpm, the suspension was filtered and a quality control sample was taken. The finished drug was poured into dark glass bottles, closed with plastic screw caps, and the bottles were stored at room temperature.

Example 2.

12 L of purified water were added to a 50 L stainless steel auxiliary container (R1), and 1 kg of microcrystalline cellulose was introduced little by little at intervals and stirring at 40 rpm. Mixing was carried out for 30 minutes until a homogeneous paste was obtained. 5 kg of sorbitol and 24 kg of glycerol were added with stirring at 40 rpm, after which stirring was continued for 10 minutes to obtain Mixture 1 (M1). On the other hand, in a 300 L stainless steel reactor equipped with a polytron type homogenization system, 60 L of water was heated to a temperature of 90 °C, and 10 L of water was taken and placed in the auxiliary vessel (R2) with stirring. 3 kg Cremophor RH 40 was added with stirring for 5 minutes at 9,000 rpm. 1.2 kg of purified fatty alcohol extract was added in small portions at intervals, after which stirring was continued for 10 minutes to obtain Mixture 2 (M2), and then the temperature was reduced to 30 ± 2°C without stopping stirring. In another container, 0.18 liters of methylparaben and 0.02 kg of propylparaben were dissolved in 1.5 liters of ethyl alcohol with stirring at a speed of 40 rpm. 0.3 kg of sodium carboxymethylcellulose A/V was moistened, 0.03 kg of flavor was added and mixed for 5 minutes to obtain Mixture 3 (M3).

In another auxiliary vessel with a capacity of 5 L, 1 kg of glycerin was mixed with 0.5 L of ethyl alcohol and stirred for 5 minutes to obtain mixture 4 (M4). Mixture M1 was introduced into the reactor with mixture M2, extracting the residues with a minimum amount of water separated in R2, and stirring was carried out at a speed of 40 rpm for 10 minutes. After this time, M3 was introduced and residues were removed from M4. Subsequently, the remaining slurry was recovered using hot water in vessel R2 and stirring was carried out at 1000 rpm for 10 minutes. Then stirring was stopped, and the mixture was infused for 12 hours. After this time, the remainder of the alcohol was sprayed onto the surface of the suspension and was diluted with water to obtain 100 liters of suspension. Stirring was carried out at 1000 rpm for 5 minutes, the suspension was filtered and a quality control sample was taken. The finished drug was poured into dark glass bottles, closed with plastic screw caps, and the bottles were stored at room temperature.

Example 3.

The composition obtained in Example 1 (FAMC) underwent preclinical testing in animals, during which its antiulcer effect was compared with that of the registered fatty alcohol tablet (RAT), as well as suspensions of fatty alcohols (FA) and microcrystalline cellulose (MC ) respectively, with the latter two preparations being produced with dosages of fatty alcohols and microcrystalline cellulose equal to the dosage of both substances in the FAMC suspension. The experimental model used in this trial was the induction of gastric ulcers in rats using sodium hydroxide.

Male Sprague-Dawley rats (250-300 g) were used and were acclimated to standard laboratory conditions for 7 days at a temperature of 20-25°C, relative humidity 60 ± 5%, a day/night cycle of 12 hours, and free access to water and standard rodent food. At the end of quarantine, the animals were randomly assigned to 10 experimental groups (10 rats/group): a negative control group, which was given only vehicle, and 9 groups with peptic ulcer disease induced using 0.2 N sodium hydroxide solution, and among these groups the positive control group did not receive treatment with the antiulcer agent, and 8 groups received treatment with the evaluated substances, as presented in Table 1. The suspensions were administered as is, while the RAT was prepared in a gum arabic/water vehicle. (1 %). The use of a negative control group (not exposed to sodium hydroxide) as well as a positive control group (exposed without treatment with any substance) made it possible to confirm the performance of the model under experimental conditions, as well as to compare the effectiveness of the antiulcer effect of the substances being evaluated.

All drugs and sodium hydroxide were administered orally in a single dose via intragastric intubation. The selected doses are within the range of effective doses previously confirmed experimentally. To provoke gastric ulcers, animals were not fed for 24 hours before the start of the experiment and were given free access to water. One hour after the administration of single doses of vehicle and various drugs, each rat was administered an ulcerative agent (1 ml/200 g) via intragastric intubation. One hour later, the rats were sacrificed in halothane medium, and the stomachs were removed and opened along the greater curvature.

The number of focal mucosal lesions was counted by two observers in an independent, blinded manner. The results of the focal lesion assessment were defined as the aggregate size of the focal lesions in ^mm2 (Ohara 1992). Statistical comparison of average areas of ulceration in different groups was carried out using the nonparametric Mann-Whitney U test. Initially, the significance level was set to α = 0.05, and the commercial software package Statistic for Windows (version 4.2, StatSoft, Inc, USA) was used.

Table 1. Effect of suspensions of FAMC, MC, FA and RAT on gastric ulcer in rats provoked by sodium hydroxide. Medicine Doses
(mg/kg/day) Peptic ulcer index
(mm ² ) I (%) Negative control (vehicle) 0 0 ± 0.00*** ^-- Positive control (vehicle + NaOH) 0 83.70 ± 13.8 -- MS + NaOH 25 80 ± 10.6 4.4 MS + NaOH 200 75.4 ± 12.5 9.9 FA + NaOH 25 67.2 ± 11.2 19.7 FA + NaOH 200 50.3 ± 13.5** 39.9 RAT + NaOH 25 51.4 ± 10.4 ** Δ 38.6 RAT + NaOH 200 40.5 ± 12.70*** Δ 51.6 FAMC + NaOH 25 11.3 ± 3.00*** °a b 86.5 FAMC + NaOH 200 1.2 ± 0.3*** ° a b 98.6

FAMC: fatty alcohol suspension with added microcrystalline cellulose

MC: microcrystalline cellulose suspension

FA: fatty alcohol suspension

RAT: fatty alcohol tablet, registered as a food additive

Inhibition I, averaged data ± SEM (standard mean error)

Comparison * p <0.05, ** p <0.01, *** p <0.0001 with the positive control group

Comparison of Δ p < 0.05, ° p < 0.0001 with a similar dose of FA

Comparison of a p <0.0001 with a similar dose of MC

Comparison of b p <0.0001 with a similar dose of RAT

As presented in Table 1, the oral administration of NaOH (0.2 N solution) provoked the formation of focal gastric lesions in animals in the positive control group in comparison with the group of healthy animals (negative control), which proves the performance of the model under experimental conditions.

Oral administration of FAMC, FA and RAT resulted in a reduction in gastric ulcer score, while administration of CM resulted in a small reduction that was not significant. FAMC suspension (25 and 200 mg/kg) caused a reduction in gastric ulcer scores that was significant not only compared with the positive control group, but also compared with similar doses of MC, FA and RAT suspensions. Thus, the highest antiulcer efficacy was obtained from the FAMC suspension, especially since it resulted in ~100% (complete) inhibition of the gastric ulcer score at the highest dose tested, 200 mg/kg.

In addition, the FAMC suspension demonstrated a percentage of inhibition greater than that obtained with the MC and FA suspensions alone, demonstrating synergism with FAMC, with the presence of FA and MC extract in these proportions and concentrations providing increased antiulcer efficacy.

It should also be noted that the antiulcer effect of the FAMC suspension is far superior to that of the 12% CM suspension (Barzaga 2004) when both suspensions were evaluated in ulcer induction models of alkaline gastritis. Thus, the percentage of inhibition achieved with a FAMC suspension of 25 and 200 mg/kg (86.5 and 98.6%, respectively) exceeds that of MS, the dose of which is 50% higher (356.38 mg/kg) (Barzaga 2004), which is consistent with the synergistic action of FAMC observed in the present example.

It should also be noted that the lowest dose of 25 mg/kg FAMC suspension tested in this example provided a higher percentage of gastric ulcer inhibition of 86.5%, indicating that, in addition to being highly effective, FAMC suspension is also potent. .

Example 4.

The composition obtained in Example 2 (FAMC) underwent preclinical testing in animals, during which its antiulcer effect was compared with that of the registered fatty alcohol tablet (RAT), as well as suspensions of fatty alcohols (FA) and microcrystalline cellulose (MC ), with the latter two preparations being produced with dosages of fatty alcohols and microcrystalline cellulose equal to the dosage of both substances in the FAMC suspension. The experimental model used in this trial was the induction of gastric ulcers in rats using ethyl alcohol.

Male Sprague-Dawley rats (250-300 g) were used and were acclimated to standard laboratory conditions for 7 days at a temperature of 20-25°C, relative humidity 60 ± 5%, a day/night cycle of 12 hours, and free access to water and standard rodent food. At the end of quarantine, the animals were randomly assigned to 11 experimental groups (10 rats/group): a negative control group, which was given only vehicle, and 10 groups with peptic ulcer disease induced with ethyl alcohol 60%, and among these groups a positive control group did not receive treatment with the antiulcer agent, and 8 groups received treatment with the evaluated substances and omeprazole, as presented in Table 2. The suspensions were administered as is, while the tablet was prepared in a 1% gum arabic suspension.

The use of a negative control group (not exposed to ethyl alcohol) as well as a positive control group (exposed without treatment with any substance) made it possible to confirm the performance of the model under experimental conditions, as well as to compare the effectiveness of the antiulcer effect of the substances being evaluated. All medications and ethyl alcohol were administered orally in a single dose via intragastric intubation. The selected doses are within the range of effective doses previously confirmed experimentally, and the dose of omeprazole (20 mg/kg) in this model is also effective. To provoke gastric ulcers, animals were not fed for 24 hours before the start of the experiment and were given free access to water. One hour after the administration of single doses of vehicle and various drugs, each rat was administered an ulcerative agent (1 ml/200 g) via intragastric intubation. One hour later, the rats were sacrificed in halothane medium, and the stomachs were removed and opened along the greater curvature. Calculation of the gastric ulcer index was carried out as presented in example 3.

The results of the indicator of ulceration provoked by ethyl alcohol (Table 2) showed that the introduction of ethyl alcohol orally provoked the formation of focal lesions of the stomach in animals in the positive control group in comparison with animals in the negative control group (healthy). Animals that were administered FAMC suspension at doses of 25 and 200 mg/kg showed a lower rate of ulceration compared to sick animals (positive control group). Treatment with this suspension resulted in a clear and significant reduction in gastric ulcer scores with inhibition rates of 93.95 and 99.85% at doses of 25 and 200 mg/kg, respectively. Statistical comparison of the effect when using different drugs with the same doses showed significant differences with the clear fact that the FAMC suspension showed the best results, surpassing even the total percentage of inhibition of the FA and MC suspensions, showing the best protection of the gastric mucosa. The control substance (omeprazole) also led to a decrease in the rate of gastric ulcer disease, which confirms the validity of the results obtained under these experimental conditions. It should be noted that the FAMC suspension provided an effect superior to that of omeprazole, although they were administered at different doses, since comparison was not the purpose of the study.

Table 2. Effect of suspensions of FAMC, MC, FA and RAT on gastric ulcer in rats provoked by ethyl alcohol. Medicine Doses
(mg/kg/day) Peptic ulcer index
(mm ² ) I (%) Negative control (vehicle) 0 0.15 ± 0.07*** ^-- Positive control (vehicle) 0 169.09 ± 12.8 -- MC + ethyl alcohol 25 155.5 ± 11.6 8 MC + ethyl alcohol 200 133.3 ± 13.7 21.1 FA + ethyl alcohol 25 149.13 ± 12.2 11.7 FA + ethyl alcohol 200 89.19 ± 14.5** 47.3 RAT + ethyl alcohol 25 106.60 ± 11.40** Δ 37 RAT + ethyl alcohol 200 41.74 ± 13.70*** Δ 75.4 OMP + ethyl alcohol 20 35.97 ± 14.48*** 78.8 FAMC + ethyl alcohol 25 10.38 ± 3.00*** °a b 93.95 FAMC + ethyl alcohol 200 0.40 ± 0.34*** ° a b 99.85

FAMC: fatty alcohol suspension with added microcrystalline cellulose

MC: microcrystalline cellulose suspension

FA: fatty alcohol suspension

RAT: fatty alcohol tablet, registered as a food additive

Inhibition I, averaged data ± SEM (standard mean error)

Comparison * p <0.05, ** p <0.01, *** p <0.0001 with the positive control group

Comparison of Δ p < 0.05, ° p < 0.0001 with a similar dose of FA

Comparison of a p <0.0001 with a similar dose of MC

comparison b p < 0.0001 with a similar dose of RAT

(Mann-Whitney U test)

As a conclusion regarding the test presented in this example, it can be stated that the administration of the FAMC suspension by mouth resulted in a clear and significant inhibition of the formation of gastric ulcers in rats induced by ethyl alcohol, with greater effectiveness than achieved with the registered tablet with suspensions of FA and MC at the same doses. In addition, it was observed that the effectiveness of the FAMC suspension was superior to the combined effectiveness of the FA and MC suspensions at both doses evaluated, which proves the synergistic effect of the composition of the present invention.

Example 5.

The composition obtained in Example 2 (FAMC) underwent preclinical testing in animals, during which its antiulcer effect was compared with that of the registered fatty alcohol tablet (RAT), as well as suspensions of fatty alcohols (FA) and microcrystalline cellulose (MC ), with the latter two preparations being produced with dosages of fatty alcohols and microcrystalline cellulose equal to the dosage of both substances in the FAMC suspension. The experimental model used in this test was the induction of gastric ulcers in rats by cross-linking the gastric outlet, which is an acid-dependent model in contrast to the model presented in the previous example.

Male Sprague-Dawley rats (250-300 g) were used and were acclimated to standard laboratory conditions for 7 days at a temperature of 20-25°C, relative humidity 60 ± 5%, a day/night cycle of 12 hours, and free access to water and standard rodent food. At the end of quarantine, the animals were randomly assigned to 11 experimental groups (10 rats/group): a negative control group, which was given only vehicle, and 10 groups with gastric ulcer induced by stapling of the gastric outlet, and among these groups, a positive control group did not receive treatment with an antiulcer agent, and 8 groups received treatment with the evaluated substances and omeprazole as a control substance, as presented in Table 3. The suspensions were administered as is, and a suspension with 1% gum arabic was prepared for administration in tablet form. The use of a negative control group (not subjected to crosslinking of the gastric outlet), as well as a positive control group (exposed to no treatment with any substance), made it possible to confirm the performance of the model under experimental conditions, as well as to compare the effectiveness of the antiulcer effect of the substances being evaluated.

All medications were administered orally in a single dose via intragastric intubation. The selected doses are within the range of effective doses previously confirmed experimentally, and the dose of omeprazole (20 mg/kg) in this model is also effective. To provoke gastric ulcers, animals were not fed for 24 hours before the start of the experiment and were given free access to water. One hour after the administration of a single dose of vehicle and various drugs, the animals received analgesia in the form of sodium thiopental (35 mg/kg) by intraperitoneal injection, a 2 cm incision was made in the anterior third of the middle part of the abdominal lining, opening the stomach, and suturing was performed of the pyloric sphincter using silk thread number 2, and the wall of the abdominal cavity was sutured with silk thread. One hour later, the rats were sacrificed in halothane, the stomachs were removed, and gastric juice was collected in test tubes. The latter was centrifuged at 3000 rpm, the supernatant was separated and its amount per ml was calculated. The stomachs were opened along the greater curvature, and the gastric ulcer index was calculated as described in Example 3.

The results of the indicator of ulceration provoked by cross-linking of the gastric outlet (Table 3) showed the formation of focal lesions of the stomach in animals in the positive control group in comparison with animals in the negative control group (healthy). Animals that were administered FAMC suspension at doses of 25 and 200 mg/kg showed a lower rate of ulceration compared to sick animals (positive control group). Treatment with this suspension resulted in a clear and significant reduction in gastric ulcer scores, with inhibition rates of 76.3 and 99.7% at doses of 25 and 200 mg/kg, respectively. Statistical comparison of the effects of different drugs with a similar dose showed that the evaluated substance, FAMC suspension, provided the greatest antiulcer efficacy as it was significantly superior to RAT, as well as single drug suspensions FA and MC, respectively. In addition, the fact that the inhibition provided by the FAMC suspension was superior to the combined effect achieved by using MC and FA separately provides evidence for the synergistic effect of these two components in the FAMC suspension mixture.

At the same time, omeprazole, the control substance, also led to a significant reduction in the rate of gastric ulcers, which confirms the validity of the results obtained under these experimental conditions. The volume of gastric juice did not change under the influence of suspensions of FAMC, MC, FA or RAT, which suggests that the antiulcer effect of these substances does not depend on the production of gastric juice. This variable decreased only with omeprazole, which is consistent with its mechanism of action as an antisecretory proton pump inhibitor.

Table 3. Effect of suspensions of FAMC, MC, FA and RAT on gastric ulcer in rats caused by cross-linking of the gastric outlet. Medicine Doses
(mg/kg/day) Peptic ulcer index
(mm ² ) I (%) Volume of gastric juice Negative control (vehicle) 0 0.0 ± 0.00*** ^-- 0.0 ± 0.00*** Positive control (filler + gastric outlet crosslinking) 0 23.2 ± 3.2 -- 9.9 ± 1.2 MC + stitching of the gastric outlet 25 21.1 ± 1.6 9 10 ± 1.05 MC + stitching of the gastric outlet 200 20.5 ± 1.7 11.6 9.5 ± 1.2 FA + stitching of the gastric outlet 25 18.3 ± 2.2 21.1 9.8 ± 1.3 FA + stitching of the gastric outlet 200 13.3 ± 3.1** 42.7 9.7 ± 1.1 RAT + gastric outlet stitching 25 16.1 ± 2.40** 30.6 9.8 ± 1.1 RAT + gastric outlet stitching 200 10.6 ± 3.2 *** 54.3 9.6 ± 0.8 OMP + gastric outlet stitching 20 3.9 ± 0.5 *** 83.2 3.4 ± 0.4 *** FAMC + gastric outlet stitching 25 5.5 ± 0.7 *** °a b 76.3 9.5 ± 0.7 FAMC + gastric outlet stitching 200 0.3 ± 0.3*** ° a b 98.7 9.4 ± 0.9

FAMC: fatty alcohol suspension with added microcrystalline cellulose

MC: microcrystalline cellulose suspension

FA: fatty alcohol suspension

RAT: fatty alcohol tablet, registered as a food additive

Inhibition I, averaged data ± SEM (standard mean error)

Comparison * p <0.05, ** p <0.01, *** p <0.0001 with the positive control group

Comparison of °p<0.0001 with similar dose of FA

Comparison of a p <0.0001 with a similar dose of MC

Comparison of b p <0.0001 with a similar dose of RAT

(Mann-Whitney U test)

As a conclusion to the test presented in this example, it can be stated that the administration of the FAMC suspension by mouth resulted in a clear and significant inhibition of the formation of gastric ulcers in rats caused by cross-linking of the gastric outlet, with greater effectiveness than that achieved with the registered tablet and suspensions FA and MC at the same doses. In addition, it was observed that the effectiveness of the FAMC suspension was superior to the combined effectiveness of the FA and MC suspensions at both doses evaluated, which proves the synergistic effect of the composition of the present invention.

Example 6.

The composition obtained in Example 1 (FAMC) underwent preclinical testing in animals, during which its chemoprotective effect was compared with that of the registered fatty alcohol tablet (RAT), as well as suspensions of fatty alcohols (FA) and microcrystalline cellulose (MC ), with suspensions of fatty alcohols (FA) and microcrystalline cellulose (MC) being produced at a concentration equal to the concentration of both substances in the FAMC suspension. For the evaluation, colon cancer was induced in Holtzmann mice using 1,2-dimethylhydrazine (DMH). Three-month-old animals with a body weight of 250 ± 20 g were randomly distributed into the following groups: group administered 2 ml/kg sodium polysorbate; 20 mg/kg DMH administration group; and DMH and FAMC, RAT, FA and MC suspensions administration group.

DMH was prepared in 4 mg/mL distilled water using 0.4 mg/mL EDTA as stabilizer at pH 6.5. It was administered subcutaneously at 20 mg/kg body weight once weekly for 18 weeks, while suspensions and polysorbate were administered orally during the 18-week study period. At the end of the scheduled time, mice were sacrificed with 10 mg/kg pentobarbital one hour after the last administration, and the colon was removed for histopathological analysis. She was gently washed with saline to remove blood and contents adhering to the tissue; areas of tumor formations were fixed in a solution of buffered formalin 10% for 7 days; Next, parts 3-5 µm in size were selected and fixed in paraffin, which were stained with hematoxylin and eosin. The assessment was based on microscopic observations for neoplasia and dysplasia. Statistical analysis to evaluate the chemoprotective effect was represented by the Shapiro-Wilk test for normality, the nonparametric Kruskal-Wallis test, and the Fisher test.

Table 4 provides a summary of the percentage of histopathological observations of the colon of rats included in the study with induced colon cancer and after administration of the various suspensions. Using the Kruskal-Wallis test at a 95% confidence level, p < 0.05 was found in all cases, with results across groups varying significantly across all drug types and cancer phases (normal, dysplasia, and neoplasia). All animals receiving DMH alone (100%) developed dysplasia or neoplasia, demonstrating the validity of the model. The only group that did not exhibit neoplasia was the FAMC formulation group, in which only 20% of the animals developed dysplasia, implying that 80% of these animals were not adversely affected by the carcinogen.

In the group receiving suspensions of FA, MC and RAT, only 20, 10 and 10% of animals, respectively, were not affected by the cancer-causing substance. Thus, it was demonstrated that there is a corresponding synergistic effect between the alcohol extract and MS, which gives the suspension of the present invention a chemoprotective effect. The chemoprotective effect of the FAMC suspension was significantly superior to that of suspensions with individual substances.

Table 4. Results of histopathological analysis of the colon of rats treated with vehicle (PS), dimethylhydrazine (DMH) and various suspensions (percentage frequency). Result of histopathological analysis Medicine Frequency (%) No change (norm) Filler (polysorbate solution) 100 DMH 0 +++ DMH + FAMC suspension 80 DMH + RAT suspension 10++ DMH + FA suspension 20++ DMH + MC suspension 10++ Dysplasia Filler (polysorbate solution) 0 DMH 30+ DMH + FAMC suspension 20 DMH + RAT suspension 40+ DMH + FA suspension 50++ DMH + MC suspension 60++ Neoplasia Filler (polysorbate solution) 0 DMH 70+++ DMH + FAMC suspension 0 DMH + RAT suspension 50++ DMH + FA suspension 30+ DMH + MC suspension 30+

PS: sodium polysorbate 3%. Statistical evaluation was performed using the Kruskal-Wallis test to determine the prevalence in each cancer group relative to drug categories; the significance level was 0.05. Normal (p=0.01), dysplasia (p=0.049), neoplasia (p=0.01).

Matched + p<0.05; ++p<0.01; +++ p<0.001 with the polysorbate vehicle group (Fisher's test).

In conclusion, histopathological evidence showed that a suspension containing a mixture of fatty alcohols and microcrystalline cellulose (FAMC) was the only one to demonstrate significant antitumor activity in dimethylhydrazine-induced colon cancer in rats, with inhibition of neoplasia in 100% of cases.

Claims (4)

1. An oral suspension with antiulcer and chemoprotective effects for colon cancer, characterized in that it is an aqueous suspension containing as active ingredients a purified extract of fatty alcohols from the purified wax of Apis mellifera bees and microcrystalline cellulose. 2. Suspension for oral administration according to claim 1, characterized in that the ingredients have the following proportions: 1-2% fatty alcohol extract; 1-2% microcrystalline cellulose; 2-4% emulsifiers; 0.1-0.6% preservatives; 20-30% moisturizers; 1-6% sweeteners; 0.1-1% suspending agents; 0.01-0.05% flavorings and 50-70% purified water. 3. Oral suspension according to claim 2, characterized in that the particle size of the purified extract of fatty alcohols from purified beeswax is less than 1.5 micrometers. 4. Suspension for oral administration according to claims. 2 and 3, characterized in that the emulsifier is polyoxyethylene hydrogenated castor oil or polysorbate, the preservatives are methyl and propylparabens, the moisturizer is glycerin, the sweeteners are sorbitol or sodium saccharin, the suspending agent is carboxymethylcellulose, and the flavoring is essential oil.