Oral suspension with Anti-ulcerous and chemoprotective effect on colon cancer and preparation method thereof

Abstract

To provide a novel pharmaceutical composition or dietary supplement consisting of an aqueous oral suspension for preventing and treating stomach ulcers and colon cancer, and to provide a method for preparing the same.SOLUTION: A novel pharmaceutical composition or dietary supplement is characterized by containing a purified extract of a fatty alcohol from beeswax, microcrystalline cellulose, and excipients accepted by a pharmaceutical industry. A method for preparing the suspension is characterized by subjecting the fatty-alcohol extract (also known as fatty alcohol purified extract) to a process to reduce particle size to <1.5 micrometers in the presence of an emulsifying agent, at >1000 rpm. A mixture of the active ingredient and microcrystalline cellulose in a proportion and concentration in which they are formulated causes these components to act synergistically, providing the composition with strong anti-ulcerous and chemoprotective effects.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel pharmaceutical composition consisting of an oral aqueous suspension, and to a process for its preparation, which has an antiulcer effect on the gastric mucosa and a chemopreventive effect against colon cancer, characterized in that it contains as an active ingredient a refined extract containing fatty alcohols from the refined wax of the honeybee (Apis mellifera) (also simply called wax or bee wax), the particle size of which has been reduced by technical means to <1.5 μm.

[0002] In addition to fatty alcohols, refined wax extracts contain diols, paraffins and potassium salts of fatty acids as minor components.

[0003] The novel composition may also contain a range of excipients accepted by the pharmaceutical industry, including one or more emulsifiers used in the process of reducing the particle size of the fatty alcohol extract, as well as humectants, sweeteners, suspending agents, preservatives, flavoring agents, and water. [Background technology]

[0004] A gastric ulcer is an injury that extends into the mucosal layer, and sometimes into the muscular layer, of the stomach, forming a cavity with acute and chronic inflammation around it, which is one of its major complications and the main cause of gastrointestinal bleeding (Valley 2002, Sadic 2009).

[0005] Other complications include perforation, penetration, and obstruction, all of which result in significant impairment of the patient's quality of life (Sung 2010).

[0006] Ulcers are primarily caused by an imbalance between aggressive factors (acid secretion, pepsin, Helicobacter pylori, nonsteroidal anti-inflammatory drugs) and defensive factors (mucus and bicarbonate secretion, blood microcirculation, prostaglandins, growth factors), i.e. Helicobacter pylori infection and continued use of nonsteroidal anti-inflammatory drugs (Ramakrishnan 2007).

[0007] Despite current advances in H. pylori eradication therapy, mortality from peptic ulcer complications remains stable.

[0008] In clinical practice, the most commonly used drugs for the treatment of gastric ulcers are proton pump inhibitors and H2 receptor antagonists, which have various adverse effects such as nausea, constipation, gynecomastia and impotence (Forgerini 2018).

[0009] These effects limit their use and justify the search for and use of new pharmaceutical compositions that are effective as antiulcer agents and have a better safety profile.

[0010] The most widely used pharmacological models to study potential anti-ulcer substances are the induction of ulcers by alkaline substances, ethanol or by pylorus ligation, as a way to cover different mechanisms by which the substance being evaluated may exert its anti-ulcer effect.

[0011] Meanwhile, cancer is one of the current leading causes of death, with over 8 million deaths worldwide each year, and colon cancer is one of the five leading causes of death worldwide, with a growing trend, especially in countries with fewer financial resources (Torre 2015, Chatenoud 2014).

[0012] Considering the high incidence of this disease and the fact that its treatment methods are generally surgical, followed by chemotherapy, justifies the search for and use of chemopreventive pharmaceutical compositions that can be used for the prevention and treatment of patients with this type of cancer or with risk factors for it, such as ulcerative processes in the digestive tract (ulcerative colitis, Crohn's disease and others).

[0013] The most widely used pharmacological model for the study of potential chemopreventive agents against the disease is based on the use of cancer-inducing agents.

[0014] Among the latter, one of the most effective and used is 1,2-dimethylhydrazine, whose metabolism begins to produce cytotoxic substances for colon cells within hours of induction (Corpet 2003, Reddy 2004).

[0015] Due to the fact that gastric ulcers and colon cancer constitute important health problems worldwide, affecting the quality of life and potentially causing death in those who suffer from them, and due to the fact that existing conventional therapies are associated with various adverse effects that limit their clinical use and do not necessarily intend to cure or prevent the occurrence of these diseases, it is necessary to search for effective therapeutic agents for both medical conditions.

[0016] The need for new, safe and effective anti-ulcer and anti-cancer therapeutic strategies is a contemporary problem. The refined extract of refined wax used in the preparation of the composition of the present invention, as well as the process for its manufacture, has been previously patented, said extract being entitled as a natural mixture of higher primary aliphatic alcohols with anti-inflammatory activity for the treatment of gastric and duodenal ulcers, the most suitable administration form of which was in the form of tablets, pills or capsules (CU 22412 A1: reference family US6235795B1 and US6465526B1), i.e. in finished solid form.

[0017] In accordance with the above, tablets with this active ingredient standardized to a dose of 50 mg of fatty alcohol have been registered as a dietary supplement in Cuba and other countries as a functional food or alternative medicine.

[0018] However, this formulation has several drawbacks, such as: its large size (655 mg), which results in a large amount of excipients and high manufacturing costs; increased rejection in different countries due to the use of two of its excipients (croscarmellose sodium and polyvinylpyrrolidone); and the very low bioavailability (<10%) of the fatty alcohols it contains.

[0019] It should be noted that the pharmacological efficacy of said tablets as anti-ulcer agents is much lower than that achieved by the suspensions of the present invention.

[0020] When the fatty alcohol extract of the present invention is subjected to technological processes of shearing or precipitation by temperature change with stirring in the presence of a specific emulsifier to reduce its particle size, and then formulated into an aqueous suspension mixed with microcrystalline cellulose in a certain ratio and specific concentration, its pharmacological efficacy is synergistically enhanced, which was not predicted from the prior art.

[0021] The purpose of the aqueous suspension of the present invention is to significantly exceed the anti-ulcer effect of both the fatty alcohol extract and the microcrystalline cellulose in tablet form, and to have a powerful chemopreventive effect against colon cancer not previously reported for either of these two substances, which can be seen in the examples.

[0022] On the other hand, microcrystalline cellulose (derived from alpha-cellulose) is mainly used in the manufacture of creams and solid suspensions for cosmetics, detergents, and as a stabilizer in foods (Nsor-Atindana, 2017).

[0023] In the pharmaceutical industry, it is used in the preparation of tablets and capsules as a compression and filling agent, but its bile acid sequestration ability has also been reported (Paniagua 1999) and it has been used as an active ingredient in suspensions with antiulcer effects at a 12% dose (Barzaga 2004).

[0024] It should be noted that the pharmacological effect reported for the 12% suspension is much lower than that achieved with the suspension according to the invention, which contains a much lower dose of microcrystalline cellulose (≦2%), as seen in Example 3.

[0025] This is the result of a synergistic effect that occurs when microcrystalline cellulose is mixed with a purified extract of fatty alcohols from wax (beeswax) in specific ratios and concentrations, which enhances its anti-ulcer effect and allows the composition to acquire a powerful chemopreventive effect against colon cancer not seen in conventional formulations and which could not be derived from the prior art.

[0026] The use of refined waxes (refined beeswax) and microcrystalline cellulose in the pharmaceutical industry has so far been limited to two uses: in the formulation of solid medicines, where the formulation allows for a controlled release of the active ingredient (Kallai 2010; Reiner, 2018), or as an inert filler-excipient in suppositories or other formulations (Lee, 2018).

[0027] Prior to the present invention, the combined use of an extract of fatty alcohols from wax (refined beeswax) and microcrystalline cellulose in an aqueous suspension has not been reported, with the two substances acting synergistically to provide the composition with a powerful anti-ulcer and chemopreventive effect.

[0028] When using the two substances in the form of a tablet (CU 22412 A1), the effect observed with the aqueous suspension described in the present invention is not achieved, as can be seen in the examples. [Prior art documents] [Patent documents]

[0029] [Patent Document 1] CU22412A1 Publication Family US6235795B1 and US6465526B1) [Non-patent literature]

[0030] [Non-Patent Document 1] Revista Cubana De Farmacia, 2004, Vol.38, No.2 Summary of the Invention

[0031] The procedure for obtaining the pharmaceutical composition of the invention is characterized by: A) mixing a substance used as an emulsifier, polyoxyethylene hydrogenated castor oil or polysorbate (2-4%) with an extract of fatty alcohols extracted from beeswax (1-2%); B) subjecting the extract to a technological process to reduce the particle size to <1.5 micrometers, using a homogenizer capable of shearing the particles at >1000 rpm and >80°C for >1 minute, or by treating the prepared mixture at >1000 rpm and >80°C for >1 minute, followed by reducing the temperature to <60°C at the same stirring speed; C) While stirring, add: microcrystalline cellulose (1-2%) as a second active ingredient, methyl and propyl parabens (0.1-0.6%) as preservatives, glycerin (20-30%) as a humectant, sorbitol or sodium saccharin (1-6%) as a sweetener, carboxymethylcellulose (0.1-1%) as a suspending agent, essential oils (0.01-0.05%) as flavorings, and purified water (50-70%).

[0032] The result of this process is a novel pharmaceutical composition characterized by an aqueous suspension for oral use in which the active ingredients (fatty alcohol extract and microcrystalline cellulose) act synergistically to provide a powerful antiulcer and chemopreventive effect.

[0033] This novel pharmaceutical composition, taking into account its protective properties on the digestive tract and its chemoprotective effect against colon cancer, makes it possible to prevent or treat ulcerative processes and other related conditions.

[0034] The proportion in which the active ingredients are present in this pharmaceutical composition is at a specific concentration and proportion that ensures that the desired pharmacological effect is obtained as a result of the synergistic action of the active ingredients it contains, and is the result of several studies carried out to specify and standardize the content of both the active ingredients and the excipients in the final composition.

[0035] The potent pharmacological effect of the composition of the present invention is much higher than that of both active ingredients administered separately, due to the higher doses present in the composition of the present invention than when administered individually, which can be confirmed in the embodiments of the present application, demonstrating the occurrence of synergistic effects of the composition of the present invention, without prejudice to previous findings.

[0036] The advantage of this composition over other existing compositions is that its antiulcer effect is superior to that of currently registered tablets (used in medicine) containing 50 mg of wax (beeswax) fatty alcohol and also that its effect is superior to that of a previously reported 12% microcrystalline cellulose suspension; in other words, the composition has a much higher antiulcer effect than that of both previous formulations, even at lower doses of the active ingredient.

[0037] On the other hand, the composition, as shown in Example 6, was shown to have an effective chemopreventive effect against colon cancer, and was significantly more effective than a similar suspension containing both active ingredients separately.

[0038] This effect has not been previously observed with purified extracts of beeswax or microcrystalline cellulose and is the result of the synergistic action of both substances (active ingredients) in the subject by the suspension of the present invention. Furthermore, industrial production of this suspension is less expensive than producing a tablet containing 50 mg of fatty alcohol and does not contain excipients that are currently restricted for use in the pharmaceutical industry.

[0039] The present invention has industrial applications, the composition is novel, and both the suspension itself and its manufacturing process are considered to be inventive, since it is not expected from the current state of the art that a mixture of two different anti-ulcer active ingredients, in a specific ratio and concentration, showed a synergistic effect in pharmacological benefits against gastric ulcers and chemoprotective effects against colon cancer, in the state of the prior art where neither of them has been disclosed for its chemoprotective effects in carcinogenesis.

[0040] The present invention contributes to the food and pharmaceutical industries as the resulting composition can be used as a dietary supplement for its beneficial preventative effects on the digestive tract and can also be used as a medicine for the treatment of ulcers and cancer. Example 1

[0041] In a 50 L auxiliary stainless steel vessel (R1), 12 L of purified water was added and 1 kg of microcrystalline cellulose was added in small portions at intervals with stirring. It was stirred at 60 rpm for 30 minutes until a homogenous paste was obtained. 5 kg of 70% sorbitol and 24 kg of glycerin were added with stirring and stirring was continued for 10 minutes to obtain Mixture 1 (M1). Meanwhile, in a 300 L stainless steel reactor, 60 L of water was heated to 90°C, 10 L was separated into an auxiliary vessel (R2), 3 kg of polysorbate 80 was mixed in while stirring at 1200 rpm, and the mixture was stirred at 1200 rpm for 5 minutes. 1.2 kg of purified extract of fatty alcohol (also called extract of fatty alcohol) was added little by little at intervals, and the stirring was maintained for 10 minutes to obtain mixture 2 (M2), after which the temperature was lowered to 30±2°C while maintaining the stirring. In a separate vessel, 0.18 kg of methylparaben and 0.02 kg of propylparaben were dissolved in 1.5 liters of ethanol with stirring at 60 rpm. 0.2 kg of sodium carboxymethylcellulose A / V were moistened and 0.03 kg of flavoring was added and stirred for 5 minutes to obtain Mixture 3 (M3). In a separate 5 L auxiliary vessel, 1 kg of glycerin was mixed with 0.5 L of ethanol and stirred at 60 rpm for 5 minutes to obtain mixture 4 (M4). In the reactor where the mixture M2 was obtained, M1 was charged, the residue was dragged with a minimum amount of water separated in R2 and stirred at 60 rpm for 10 minutes. After this time, M3 was charged and the residue was dragged with M4. The rest of the suspension was then dragged with warm water in R2 and stirred for 10 minutes. The stirring was then stopped and the mixture was left to stand for 10 hours. After this time, the remaining alcohol was sprinkled on the surface of the suspension and 100 liters of suspension was completed to the mark with water. This was stirred for 5 minutes at 1200 rpm, the suspension was filtered and a sample was taken for quality control. The final preparation was bottled in amber glass bottles covered with plastic screw caps and the bottles were stored at room temperature. Example 2

[0042] In a 50 L auxiliary stainless steel vessel (R1), 12 L of purified water was added and 1 kg of microcrystalline cellulose was gradually charged at intervals with stirring at 40 rpm. It was stirred for 30 minutes until a homogeneous paste was obtained. 5 kg of 70% sorbitol and 24 kg of glycerin were added with stirring and stirred at 40 rpm for 10 minutes to obtain Mixture 1 (M1). Meanwhile, in a 300 L stainless steel reactor equipped with a Polytron homogenizer system, 60 L of water was heated to 90°C, 10 L was separated into an auxiliary vessel (R2) and charged under stirring. 3 kg of Cremophor RH 40 was stirred at 9000 rpm for 5 minutes. 1.2 kg of purified extract of fatty alcohols was added in small portions at intervals, stirring was maintained for 10 minutes to obtain mixture 2 (M2), and then the temperature was lowered to 30±2°C while maintaining stirring. In a separate vessel, 0.18 kg methylparaben and 0.02 kg propylparaben were dissolved in 1.5 liters of ethanol with stirring at 40 rpm. 0.3 kg sodium carboxymethylcellulose A / V were moistened and 0.03 kg flavoring was added and stirred for 5 minutes to obtain mixture 3 (M3). In a separate 5 L auxiliary vessel, 1 kg glycerin was mixed with 0.5 L ethanol and stirred for 5 minutes to obtain mixture 4 (M4). In the reactor where the mixture M2 was obtained, M1 was charged and the residue was dragged with a minimum amount of water separated in R2 and stirred for 10 minutes at 40 rpm. After this time, M3 was incorporated and the residue was dragged with M4. The rest of the suspension was then dragged with warm water in R2 and stirred for 10 minutes at 1000 rpm. The stirring was then stopped and the mixture was left to stand for 12 hours. After this time, the remaining alcohol was sprinkled on the surface of the suspension and 100 liters of suspension was completed to the mark with water. This was stirred for 5 minutes at 1000 rpm, the suspension was filtered and a sample was taken for quality control. The final preparation was bottled in amber glass bottles covered with plastic screw caps and the bottles were stored at room temperature. Example 3

[0043] The composition obtained in Example 1 (hereinafter, FAMC) was subjected to a preclinical study in animals to compare its antiulcer effect with that of a registered fatty alcohol tablet (hereinafter, RAT) and a fatty alcohol (hereinafter, FA) and microcrystalline cellulose (hereinafter, MC) suspension, the latter two prepared with FA and MC in doses equal to the doses of both substances in the FAMC suspension. The experimental model used in this study was the induction of gastric ulcers in rats by sodium hydroxide. Male Sprague-Dawley rats (250-300 g) were used, which were adapted for 7 days to normal laboratory conditions at 20-25°C, relative humidity of 60±5%, and a 12-h light / dark cycle, with free access to water and standard rod chow. After completion of the quarantine, the animals were randomly assigned to 10 experimental groups (10 animals / group): a negative control group that received vehicle only, 9 groups in which ulcers were induced with 0.2 N sodium hydroxide (positive control group), one of which did not receive any antiulcer agent, and 8 groups that received the substances to be evaluated as shown in Table 1. The suspension was administered as is, whereas the RAT was prepared in an acacia gum / water vehicle (1%). The use of a negative control group (not injured by sodium hydroxide) and a positive control group (injured and not treated with any substance) confirmed the validity of the model under experimental conditions and allowed to compare the antiulcer efficacy of the evaluated substances. All treatments and sodium hydroxide were administered orally as a single dose by gastric intubation. The doses chosen were within the range of effective doses proven in previous preliminary experience. To induce gastric ulcers, the animals were fasted for 24 hours before the experiment and had free access to water. After one hour of the single administration of the vehicle and the different treatments, each rat was administered the ulcerogenic agent (1 mL / 200 g) by gastric intubation. After one hour, the rats were sacrificed in a halothane atmosphere and the stomach was removed and opened at the greater flexure. Gastric mucosal lesions were quantified by two independent blinded observers. Lesion scores were defined as the sum of lesion sizes in mm2 (Ohara 1992). Statistical comparisons between the mean ulcer areas in the different groups were performed using the nonparametric Mann-Whitney U test. A priori, a significance level of α = 0.05 was set, using the commercially available Windows package Statistic (Release 4.2, StatSoft, Inc USA). Table 1: Effects of FAMC, MC, FA suspension, and RAT on sodium hydroxide-induced gastric ulcers in rats. [Table 1-1] [Table 1-2] As shown in Table 1, oral administration of NaOH (0.2N) produced gastric lesions in animals of the positive control group compared to healthy animals (negative control), demonstrating the validity of the model in the experimental conditions. Oral administration of FAMC, FA, and RAT reduced the gastric ulcer index, whereas CM produced only a small, non-significant reduction. FAMC suspensions (25 and 200 mg / kg) produced a significant reduction in the gastric ulcer index, not only in comparison with the positive control group, but also with the same doses of MC, FA and RAT suspensions. Thus, the highest antiulcer effect was achieved with FAMC suspensions, especially since the highest dose tested, 200 mg / kg, produced ~100% (total) inhibition of the gastric ulcer index. Furthermore, the FAMC suspension produced a higher inhibition percentage than the sum of those achieved with the MC and FA suspensions separately, indicating a synergistic effect in the FAMC, with the presence of FA extract and MC promoting greater antiulcer efficacy at the rates and concentrations shown. It should also be noted that the antiulcer effect of the FAMC suspension is much higher than that reported for a 12% CM suspension (Barzaga 2004), both of which were evaluated in an alkaline gastritis ulcer induction model. Thus, the percentage of inhibition achieved with 25 and 200 mg / kg FAMC suspensions (86.5 and 98.6%, respectively) is higher than that reported for MC (Barzaga 2004), which was 50% at a higher dose (356.38 mg / kg), which is consistent with the synergistic effect of FAMC observed in this example. It is also worth noting that in this example, the lowest dose tested, 25 mg / kg FAMC suspension, resulted in a high inhibition rate of 86.5% against gastric ulcers, indicating that in addition to high efficacy, the FAMC suspension also has high potency. Example 4

[0044] The composition obtained in Example 2 (FAMC) was subjected to a preclinical study in animals to compare its antiulcer effect with that of a registered fatty alcohol tablet (RAT) and of a fatty alcohol (FA) and microcrystalline cellulose (MC) suspension, the latter two prepared with doses of FA and MC equal to the doses of both substances in the FAMC suspension. The experimental model used in this study was ethanol-induced gastric ulcer in rats. Male Sprague-Dawley rats (250-300 g) were used and were adapted for 7 days under normal laboratory conditions at 20-25°C, 60±5% relative humidity, and a 12-h light / dark cycle with free access to water and standard rodent chow. After the end of the quarantine, the animals were randomly divided into 11 experimental groups (10 animals / group): a negative control group receiving only the vehicle, 10 groups in which ulcers were induced with 60% ethanol, a positive control group that was not treated with any of the antiulcer agents, 8 groups treated with the substances to be evaluated, and a group treated with omeprazole as a reference substance, as shown in Table 2. The suspension was administered as is, while tablets were prepared in a suspension containing 1% gum acacia. The use of a negative control group (not injured by ethanol) as well as a positive control group (injured and not treated with any substance) made it possible to validate the functioning of the model and to compare the antiulcer effect of the substances evaluated. All treatments and ethanol were administered orally as a single dose by intragastric intubation. The doses chosen were within the range of effective doses verified in previous preliminary experience, a dose of omeprazole (20 mg / kg) also effective in this model. To induce gastric ulcer, animals were fasted for 24 hours before the experiment and allowed to drink water ad libitum. After a single administration of vehicle and one hour of different treatment, each rat was administered ulcerogenic agent (1mL / 200g) by gastric intubation. After one hour, rats were sacrificed in halothane atmosphere, and stomachs were removed and opened at the greater flexure. Quantification of gastric ulcer index was performed as described in Example 3. The results for the ethanol-induced ulcer index (Table 2) show that oral administration of ethanol led to the formation of gastric lesions in the positive control animals compared to the negative control animals (healthy). At doses of 25 and 200 mg / kg, animals treated with FAMC suspension showed a lower ulcer index compared to the animals with lesions (positive control). Treatment with said suspensions markedly and significantly reduced the gastric ulcer index, reaching 93.95% and 99.85% inhibition at doses of 25 and 200 mg / kg, respectively. Statistical comparison between the effects of the different treatments used showed significant differences at similar doses, highlighting that the FAMC suspension showed the best results, with the percentage of inhibition even higher than the sum of the FA and MC suspensions, indicating a greater protection of the gastric mucosa. The standard substance used (omeprazole) also reduced the gastric ulcer index, which confirms the validity of the results obtained in our experimental conditions. It is noteworthy that the FAMC suspension was not administered at the same dose, since it was not the object of the test to compare both substances, but achieved an effect superior to that of omeprazole. Table 2: Effects of FAMC, MC, FA suspension, and RAT on ethanol-induced gastric ulcers in rats. [Table 2-1] [Table 2-2] In conclusion of the study presented in this example, it can be confirmed that oral administration with FAMC suspension markedly and significantly inhibits the formation of gastric ulcers induced by ethanol in rats, with a higher efficacy than that achieved with the registered tablets and suspensions FA and MC at the same doses. Furthermore, it was observed that the efficacy of the FAMC suspension was greater than the sum of the efficacy of the FA and MC suspensions at both individual doses evaluated, demonstrating the presence of a synergistic effect for the purposes of the composition of the present invention. Example 5

[0045] The composition obtained in Example 2 (FAMC) was subjected to a preclinical study in animals and its antiulcer effect was compared with that of registered fatty alcohol tablets (RAT) and with fatty alcohol (FA) and microcrystalline cellulose (MC) suspensions, the latter two prepared with doses of FA and MC equal to the doses of both substances in the FAMC suspension. The experimental model used in this study was a model of induction of gastric ulcer in rats by pylorus ligation, which is an acid-dependent model, different from the model shown in the previous example. Male Sprague-Dawley rats (250-300 g) were used and were adapted for 7 days under normal laboratory conditions at 20-25°C, 60±5% relative humidity, and a 12-h light / dark cycle, with free access to water and standard rodent chow. After the end of the quarantine, the animals were randomly divided into 11 experimental groups (10 animals / group): a negative control group receiving only the vehicle, 10 groups in which ulcers were induced by pylorus ligation, a positive control group that was not treated with any of the antiulcer drugs, 8 groups treated with the substances to be evaluated, and a group treated with omeprazole as a reference substance, as shown in Table 3. The suspension was administered as is, whereas a suspension containing 1% gum acacia was prepared and the tablets were administered. The use of a negative control group (not subjected to pylorus ligation) and a positive control group (injured and not treated with any substance) allowed to verify the function of the model and to compare the antiulcer efficacy of the evaluated substances. All treatments were administered orally as a single dose by gastric intubation. The doses chosen were within the range of effective doses verified in the preliminary experience, and a dose of omeprazole (20 mg / kg) was also effective in this model. To induce gastric ulcers, the animals were fasted for 24 hours before the experiment and had free access to water. After one hour of the single vehicle administration and the different treatments, the animals were anesthetized by intraperitoneal injection of sodium thiopental (35 mg / kg), a 2 cm long incision was made in the anterior third of the linear abdominal mesothelium, the stomach was located, the pyloric sphincter was ligated with silk suture No. 2, and the abdominal wall was sutured with silk. After one hour, the rats were sacrificed in a halothane atmosphere, the stomach was removed, and the gastric juice was collected in a test tube. The latter was centrifuged at 3000 rpm, and the supernatant was separated and quantified in ml. The stomach was opened through the greater flexure and the gastric ulcer index was quantified as described in Example 3. The results of ulcer index induced by pylorus ligation (Table 3) show that the formation of gastric lesions occurred in the animals of the positive control group compared to the animals of the negative control group (healthy). At doses of 25 and 200 mg / kg, the animals treated with FAMC suspension showed a lower ulcer index compared to the animals with lesions (positive control). Treatment with the suspension markedly and significantly reduced the gastric ulcer index, reaching 76.3% and 99.7% inhibition at doses of 25 and 200 mg / kg, respectively. Statistical comparison of the effects of the different treatments at similar doses showed that the FAMC suspension was the evaluated substance that showed the greatest antiulcer effect, since it was significantly superior to the RAT, and also superior to the suspensions with the respective monotherapies of FA and MC, respectively. Furthermore, the fact that the inhibition achieved by the FAMC suspension was greater than the sum of the inhibition achieved with MC and FA individually demonstrates the synergistic effect of the presence of these two components in the mixture of the FAMC suspension. On the other hand, the control substance omeprazole significantly reduced the gastric ulcer index, which confirms the validity of the results obtained in our experimental conditions. Gastric fluid volume had no effect on FAMC, MC, FA suspensions or on RAT, suggesting that the antiulcer activity of these substances is not dependent on gastric acid production. Only omeprazole reduced this variable, which is consistent with its mechanism of action as an antisecretory proton pump inhibitor. Table 3. Effects of FAMC, MC, FA suspension, and RAT on gastric ulcers induced by pylorus ligation in rats. [Table 3] FAMC: Suspension of fatty alcohol + microcrystalline cellulose MC: Microcrystalline cellulose suspension FA: Fatty alcohol suspension RAT: fatty alcohol tablets registered as a dietary supplement I inhibition, Data as Mean ± SEM (standard error of the mean) * p<0.05, ** p<0.01, *** p<0.0001 compared to positive control p<0.0001 compared with the same dose of FA. ap<0.0001 Compared with the same dose of MC bp<0.0001Compared with RAT of the same dose (Mann-Whitney U test) As a conclusion of the study presented in this example, it can be shown that oral administration with FAMC suspension significantly and significantly inhibits the formation of gastric ulcers induced by pylorus ligation in rats, with a higher efficacy than that achieved with registered tablets and FA and MC suspensions at the same doses.Furthermore, it is observed that the efficacy of FAMC suspension is greater than the sum of the efficacy of FA and MC suspensions at both doses evaluated, which demonstrates the presence of a synergistic effect in the purpose of the composition of the present invention. Example 6

[0046] The composition obtained in Example 1 (FAMC) was subjected to a preclinical study in animals to compare its chemoprotective effect with that of a registered fatty alcohol tablet (RAT) and a suspension of fatty alcohol (FA) and microcrystalline cellulose (MC) at concentrations equivalent to those present in the FAMC composition. For the evaluation, colon cancer was induced in Holtzmann rats with 1,2-dimethylhydrazine (DMH). Animals aged 3 months and 250±20 g were randomly distributed into the following groups: sodium polysorbate 2 mL / kg; DMH 20 mg / kg; and DMH+FAMC, RAT, FA; and MC suspension. DMH was prepared at 4 mg / mL in distilled water with 0.4 mg / mL EDTA as a stabilizer at pH 6.5. 20 mg / kg body weight was administered subcutaneously once a week for 18 weeks, and the suspension and polysorbate were administered orally for 18 weeks. After the scheduled time, 1 hour after the last administration, the rats were sacrificed with 100 mg / kg pentobarbital, and the colon was removed for histopathological analysis, which was gently washed with saline to remove blood and debris attached to the tissue; the tumor mass area was fixed in 10% buffered formalin solution for 7 days; then, 3-5 μm sections were selected, fixed in paraffin, and stained with hematoxylin and eosin. Evaluation was based on microscopic observation for neoplasia and dysplasia.Statistical analyses to evaluate chemoprevention efficacy were the Shapiro-Wilk normality test, the non-parametric Kruskal Wallis and Fisher tests with a 95% confidence level. Table 4 summarizes the percentage of histopathological observations of the colon of rats included in the study, induced with colon cancer and treated with the different suspensions. When applying the Kruskal Wallis test at a confidence level of 95%, p<0.05 was observed in all cases, and the results of the different groups showed significant differences between all treatment types and stages of cancer development (normal, dysplasia and neoplasia). All animals (100%) treated only with DMH developed dysplasia or neoplasia, which indicates the functionality of the model. The only group in which the appearance of neoplasms was not observed was the group treated with the FAMC composition, where only 20% of the animals developed dysplasia, meaning that 80% of the animals were not affected by the application of the carcinogen. Only 20%, 10% and 10% of the animals in the groups treated with the suspensions FA, MC and RAT, respectively, were not affected by the cancer inducer. Thus, a relevant synergistic effect between the extract of fatty alcohol and MC was demonstrated, which gives the suspension, which is the object of the present invention, a chemoprotective effect. The chemoprotective effect of the FAMC suspension was much greater than that of the suspensions of such individual substances. Table 4. Histopathological results: colons of rats treated with vehicle (PS), dimethylhydrazine (DMH) and the different suspensions (frequency percentages). [Table 4] PS: 3% sodium polysorbate. Statistical evaluation was performed using the Kruskal-Wallis test to determine the distribution of treatment categories within each cancer stage group (significance level 0.05). Normal (p=0.01), dysplasia (p=0.049), neoplasm (p=0.01). +p<0.05; ++p<0.01; +++p<0.001; (Fisher's test) compared with the group containing polysorbate vehicle. In conclusion, indications of histopathological studies revealed that the suspension containing a mixture of fatty alcohol and microcrystalline cellulose (FAMC) was the only one that showed significant antitumor activity in dimethylhydrazine-induced colon cancer in rats with 100% inhibition of the neoplasia.

Claims

[Claim 1] A pharmaceutical or dietary supplement comprising an oral suspension having antiulcer and chemopreventive effects against colon cancer, characterized in that it is an aqueous suspension containing a purified extract of fatty alcohols from honeybee (Apis mellifera bee) refined wax (hereinafter also referred to as fatty alcohol extract, which is a natural mixture of higher primary fatty alcohols described in the prior art CU22412A1, which contains, in addition to fatty alcohols, diols of fatty acids, paraffin and potassium salts as minor components) and microcrystalline cellulose as active ingredients, The ingredients are in the following proportions: 1-2% fatty alcohol extract; 1-2% microcrystalline cellulose; 2-4% emulsifier; 0.1-0.6% preservative; 20-30% moisturizer, 1-6% sweetener; 0.1-1% suspending agent; 0.01-0.05% flavoring agent 50-70% purified water, That is, The particle size of the fatty alcohol extract is less than 1.5 micrometers. Medicines or dietary supplements.

Images