Compositions for the treatment of gastro-esophageal reflux disease (GERD)

Abstract

Oral pharmaceutical, nutraceutical, health foods and medical device compositions based on polysaccharides from Opuntia Ficus Indica cladodes in combination with other active substances are useful for the prevention or treatment of GERD (Gastro-Esophageal Reflux Disease), gastric esophagitis and related diseases (e.g. dyspepsia, esophagitis, esofageal tumour, gastro-intestinal symptoms, chronic pharyngitis, Barrett's esophagus, esophageal adenocarcinoma, GERD-related pulmonary symptoms).

Claims

1. An oral solid composition for treating diseases of the upper gastrointestinal tract, comprising a synergistic combination of: 1-20 g Opuntia ficus indica cladodes polysaccharides; 5-20 g Olea europeae leaf extract; 5-10 g sodium bicarbonate; and 10-20 g sodium alginate, wherein said diseases are selected from the group consisting of gastroesophageal reflux, GERD, dyspepsia, gastrointestinal disturbs, pharyngitis, Barrett's esophagus, esophageal adenocarcinoma, and GERD-related pulmonary symptoms.

2. The composition according to claim 1, wherein said cladodes polysaccharides are provided as mucilages, cladode juice, or a filtered cladode juice aqueous solution that is optionally concentrated and dried.

3. The composition according to claim 1, which is in the form of a pharmaceutical or nutraceutical preparation, health food or medical device composition.

4. The composition according to claim 3, which is in the form of a tablet, capsule, or powder.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Further characteristics and advantages of the present invention will be more evident from the following description and non-limiting examples with reference to the attached figures, wherein:

(2) FIG. 1: photograph of cladodes and fruits of Opuntia Ficus Indica;

(3) FIG. 2: in vitro model of mucoadhesion;

(4) FIG. 3: comparison of the synergistic effect of polysaccharides extracted from Opuntia Ficus Indica cladodes with sodium alginate with respect to the individual components in an in vitro model on epithelial buccal mucosa cells;

(5) FIG. 4: mucoadhesion (inhibition percentage of lectin binding after treatment) for each tested substances compared in FIG. 3.

(6) FIG. 5: gastric anti-ulcerogenic effect of different substances or substances combination tested in different experimental models of chemical gastric ulcer in rats. Data are reported as meanSD of 6 animals; in parenthesis the percent inhibition compared to the control group.

(7) FIG. 6: esophageal anti-ulcerogenic effect of different substances or substances combination tested in a model of gastroesophageal reflux surgically induced. Data are reported as meanSD of 6 animals; in parenthesis the percent inhibition compared to the control group.

(8) Non limiting examples of compositions according to the invention are reported in the following tables.

(9) TABLE-US-00001 TABLE 1 Oral suspension 1 Components % Polysaccharides from Opuntia 2 g Ficus Indica cladodes Water up to 100 ml

(10) TABLE-US-00002 TABLE 2 Oral suspension 2 Components % Sodium Alginate 5 g Sodium Bicarbonate 2.67 g Polysaccharides from Opuntia Ficus 2.0 g Indica cladodes Water up to 100 ml

(11) TABLE-US-00003 TABLE 3 Oral powder (bags) 1 Components Amount Sodium Alginate 10-20 g Sodium Bicarbonate 5-10 g Polysaccharides from Opuntia Ficus 1-20 g Indica cladodes

(12) TABLE-US-00004 TABLE 4 Oral powder (bags) 2 Components Amount Sodium Alginate 10-20 g Sodium Bicarbonate 5-10 g Olive leaves extract 5-20 Polysaccharides from Opuntia Ficus 1-20 g Indica cladodes

(13) TABLE-US-00005 TABLE 5 Capsule 1 Components mg/capsule Cimetidine 200 mg Polysaccharides from Opuntia Ficus 100-200 mg Indica cladodes Olive leaves extract 100-200 mg Lactose up to capsule filling

(14) TABLE-US-00006 TABLE 6 Capsule 2 Components mg/capsula Ranitidine chloridrate 84 mg Alginic acid 500.0 mg Polysaccharides from Opuntia Ficus 200 mg Indica cladodes Olive leaves extract 200 mg Sodium bicarbonate 165.25 mg Magnesium stearate 0.75 mg Filler 350 mg

(15) In Vitro Determination of Mucoadhesion of Polysaccharides from Opuntia Ficus Indica Cladodes Alone and in Combination with Alginic Acid on Buccal Mucosae Epithelial Cells

(16) In order to assess the mucoadhesive capability of polysaccharides from Opuntia Ficus Indica cladodes, the in vitro mucoadhesion assay described by Patel (Patel D. and al., An in vitro mucosal model predictive of bioadhesive agents in the oral cavity. J Control Release, 1999, 61(1-2): 175-183) was applied to suspended epithelial cells from buccal mucosa. In this test, mucoadhesion (adhesive capability of the tested substances to the mucosal cells) is determined as a function of lectin-membrane glycoprotein binding inhibition induced by the cellular surface-masking due to the mucoadhesive substance.

(17) Materials and Methods

(18) The treatment of mucosal cells with biotinylated lectin and streptavidin peroxidase confer a yellow colour to the sample; pre-treatment of mucosal cells with a mucoadhesive substance (30 C., for 15 minutes before the treatment with lectin) hides the lectin binding site, reducing the final yellow colouring intensity.

(19) Lectin is a protein contained in some plants of Leguminosae (Canavalia ensiformis), with high tendency to bind glucosidic and mannosidic residues of membrane glycoproteins; biotin (vitamin H) is bonded to lectin through streptavidin peroxidase that is added to cellular suspension to obtain the complex protein-glucose-lectin-biotin-streptavidin peroxidase.

(20) By addition of o-phenylendiamine dihydrochloride (o-pd) and hydrogen peroxide to the cellular suspension, o-pd is oxidized by the complex protein-glucose-lectin-biotin-streptavidin peroxidase to 2,3-diaminophenazine, and the sample becomes yellow (FIG. 2).

(21) The reaction is then blocked after 1 min adding H.sub.2SO.sub.4 1M. The colour intensity is read at the spectrofluorimeter and it is proportionally related to the binding between lectin and glycosidic residues on cellular membrane.

(22) Buccal mucosa cells were obtained from male and female healthy volunteers, by gently scraping the inner cheek with a wooden spatula. The cells were then pulled and suspended in 10 ml isotonic Tris 0.05M (TBS) pH 7.6 (Patel D. et al., An in vitro mucosal model predictive of bio-adhesive agents in the oral cavity. J Control Release, 1999, 61(1-2): 175-183). A suspension sample was then added (0.1 ml trypan blue 0.5% (w/v)) and the total number of cells was determined using an haemocytometer: each sample contained 4810.sup.4 buccal mucosa cells. Polysaccharides from Opuntia Ficus Indica cladodes and sodium alginate were solubilised in NaCl 0.9% isotonic solution at 0.5% w\v and these solutions were then used for the pre-treatment of buccal mucosa cells. Results are expressed as percentage of tested substance and mucosa cells binding, which is inversely proportional to percent reduction of sample colouring compared to the control.

(23) The whole experiment was repeated 5 times for the control and for each tested substance.

(24) Statistical analysis of results was performed by means of one-way ANOVA followed by Bonferroni post hoc test.

(25) Results

(26) As shown in FIGS. 3 and 4, the assay shows a 55.81% Opuntia Ficus Indica polysaccharides mucoadhesion to buccal mucosa suspended cells, significantly higher than that observed with sodium alginate (mucoadhesion 5.36%; p<0.05). It was moreover observed that by adding sodium alginate to Opuntia ficus Indica cladodes polysaccharides, mucoadhesion reaches 74.83%.

(27) Conclusions

(28) The results obtained with the mucoadhesion in vitro assay showed that Opuntia Ficus Indica cladodes polysaccharides exhibit higher mucoadhesion than that observed with sodium alginate. It was moreover unexpectedly found that the combination of these substances determines a synergistic effect that guarantees a strengthened mucoadhesive activity.

(29) In Vivo Evaluation of Antiulcer Activity of a Composition Containing Sodium Alginate/Antacid/Opuntia Ficus Indica Cladodes Polysaccharides/Olea Europeae Leaves Extract

(30) Aim of this study was to assess the antiulcer activity of the combination of sodium alginate/antacid, Opuntia Ficus Indica cladodes polysaccharides and Olea Europeae leaves extract in an animal model of ethanol- and Indomethacin-induced ulcer (rats).

(31) Materials and Methods

(32) Experimental protocol for the evaluation of 5 test compositions compared to the vehicle: alginate/antacid combination (A), Opuntia Ficus Indica cladodes polysaccharides (B), Olea Europeae leaves extract (C), alginate\antacid\Opuntia Ficus Indica cladodes polysaccharides combination (D), alginate\antacid\Opuntia Ficus Indica cladodes polysaccharides\ Olea Europeae leaves extract combination (E) and Carbopol 919 0.3% w/v suspension in water as vehicle (F).

(33) Each sample was obtained by water suspension (concentrations reported in Table 7) and administered with an oral pump (1 ml 100 g weight).

(34) TABLE-US-00007 TABLE 7 test compositions Compositions (% w/v) Components A B C D E F Sodium Alginate 5 5 5 Sodium Bicarbonate 2.67 2.67 2.67 Opuntia Ficus Indica 2 2 2 cladodes polysaccharides Olea Europeae leaves 1 1 extract Carbopol 919 0.3

(35) The experiment was performed on male Wistar (Rattus norvegicus) adult rats, weighing from 180 to 200 g. The animals were exposed to natural light (12:12 h light and night cycles) in controlled temperature (241 C.) and humidity (60%4) conditions, and supplied standard food (Morini Mill rat GLP) and water ad libitum for orie week. The animals were then deprived of food 24 hours, and of water 2 hours before the start of the experiment (F. Vitali, Antiulcer potential of a standardized extract of red orange juice in the rat, Int J Food Properties, 10, 331-344, 2007).

(36) Experimental Model of Ethanol Induced Ulcer

(37) The animals were randomly divided in 6 groups (1-6) of 6 animals each. Group 1 was used as control and was treated with the vehicle (sample F, Carbopol 919 0.3% w/v and calcium carbonate 1% w/v in distilled water neutralized with NaOH, 1 ml/100 g weight) and, after 1 hour, with the ulcerogenic agent: absolute ethanol 1 ml/rat; groups 2-6 were orally administered samples A (767 mg/kg weight, Group 2), B (200 mg/kg weightGroup 3), C (100 mg/kg weightGroup 4), D (967 mg/kg weightGroup 5) and E (1067 mg/kg weightGroup 6). After 1 hour from administration of samples A-E, the animals of groups 2-6 received 1 ml/animal of absolute ethanol, to induce the ulcer. 1 hour after treatment with ethanol, all animals were anesthetised with diethyl ether and sacrificed. The stomach of each animal was then removed, cutted at the big curve level and gently rinsed with NaCl 0.9% solution, without removing the superficial mucosa.

(38) Experimental Model of Indomethacin-Induced Ulcer

(39) The animals were randomly divided in 6 groups (1-6) of 6 animals each. The animals of Group 1 (control) were orally administered the vehicle (sample F, Carbopol 919 0.3% w/v and calcium carbonate 1% w/v in distilled water neutralized with NaOH, 1 ml/100 g weight) and, after 1 hour, the ulgerogenic agent Indomethacin (suspension in distilled water containing carboxymethylcellulose 1%) at 0.03 g/Kg dose, (1 ml/Kg weight) s.c. Groups 2-6 were treated as described above for ethanol-induced ulcer, with samples A-E and, after 1 hour, with Indomethacin suspension. After 5 hours from the treatment with Indomethacin, the animals were anesthetised with diethyl ether and sacrificed. The stomach of each animal was then removed, and analysed as described in the experiment with ethanol.

(40) Evaluation of the Efficacy of the Tested Compositions

(41) Gastric mucosal surface was visually analysed. The length (mm) of each lesion on the mucosa was determined using an arbitrary scale (Table 8).

(42) In both the experimental models, the ulceration index (U.I) was then calculated for each stomach, adding the scores assigned to the ulcer and the Ulceration Inhibition Percentage (P.I.U.): [(U.I..sub.ControlU.I..sub.Sample)\U.I..sub.Control]*100. Data are expressed as arithmetic mediaSEM for each group; data significance was analysed by means of Kruskal-Wallis followed by Dunn's test. A p<0.05 value was considered significant.

(43) The ulcer evaluation score for the 2 models is reported in Table 8.

(44) Results

(45) The results of these experiments are illustrated in FIG. 5. In both the experimental models the Ulceration Inhibition Percentage (P.I.U.) obtained with group D (treated with the sodium alginate/antacid/Opuntia Ficus Indica cladodes polysaccharides combination) is higher than that obtained with the individual components of the same combination and higher than the sum of their P.I.U.s, indicating a synergistic effect of the combination of individual substances. Unexpectedly, the addition of Olea Europeae leaves extract (group E) increases the P.I.U. by more than the sum of P.I.U.s of Olea Europeae leaves extract individual component and sodium alginate/antacid/Opuntia Ficus Indica cladodes polysaccharides combination, revealing their synergistic.

(46) TABLE-US-00008 TABLE 8 Ulcer evaluation in the chemically induced ulcer models Score Lesion 0 No ulcer 0.5 Hyperemia 1 Hemorrhagic spots 2 1-5 small ulcers 3 Many small ulcers 4 1-5 small ulcers and 1-3 large ulcers 5 Many small and large ulcers 6 Stomach full of ulcers along with perforation

(47) Conclusions

(48) This in vivo test revealed an unexpected synergistic potentiation of the sodium alginate/antacid effect determined by Opuntia Ficus Indica cladodes polysaccharides. The synergistic effect is further potentiated by the Olea Europeae leaves extract. The ulcer lesions observed in animals receiving conventional treatments (sodium alginate plus sodium bicarbonate) scored very high compared to the animals treated with the combination of the invention, demonstrating a synergistic effect of its individual components.

(49) In Vivo Evaluation of Sodium Alginate/Antacid/Polysaccharides Cladodes Opuntia Ficus Indica/Olea Europeae Leaves Extract Combination in Reducing Surgically-Induced Gastroesophageal Reflux (Pyloric Ligation)

(50) Aim of this study was to evaluate the efficacy of the sodium alginate/antacid/polysaccharides cladodes Opuntia Ficus Indica combination added with Olea Europeae leaves extract in reducing the gastroesophageal reflux surgically induced by pyloric ligation in rats.

(51) The reduction of gastroesophageal reflux was measured from the esophageal ulcers in animals treated with the active combinations after surgical induction.

(52) Adult Wistar male rats (Rattus norvegicus) weighing 180-200 grams were randomly divided in 6 groups (1-6), each of 6 animals. According to the experimental protocol described by Min (Min et al.: The effects of apigenin-7-O--D-glucurono-pyranoside on reflux oesophagitis and gastritis in rats, Autonomic & autacoid Pharmacology 2005 July; 25(3):85-91; Hayakawa T. et al.: Roles of cyclooxygenase 2 and microsomal prostaglandin E synthase 1 in rat acid reflux oesophagitis, Gut 2006, 55: 450-456), the animals were anesthetised with pentobarbital and acid reflux esophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Group 1 (not treated) was the positive control. Groups 2-6 were intraduodenally treated with compositions A-E immediately after the ligation of pylorus and limiting ridge, using the same dosages as in the chemically-induced ulcer models described above. Afterwards, a longitudinal cardiomyotomy of about 1 cm across the gastro-oesophageal junction was performed to enhance reflux from the stomach into the esophageal body. After 6 h, the animals were sacrificed under diethyl ether anaesthesia and the esophagus was harvested and the lesions total area scored as reported in Tab. 9.

(53) The final ulceration index (U.I.) and the P.I.U. for each group were calculated as described above.

(54) TABLE-US-00009 TABLE 9 Esophageal mucosa lesions evaluation Score Total area (mm.sup.2) 0 No visibile lesions 1 Rare lesions 2 Total lesions area 30 mm.sup.2 3 Total lesions area 30 mm.sup.2 4 Perforation

(55) Results

(56) The results of this experiment are reported in FIG. 6. This experimental model confirmed the higher Ulceration Inhibition Percentage (P.I.U.) obtained with group D (treated with the sodium alginate/antacid/Opuntia Ficus Indica cladodes polysaccharides) than that observed in the experimental groups treated with the individual components and even higher than the sum of their P.I.U.s, revealing a marked synergistic effect of the sodium alginate/antacid/Opuntia Ficus Indica cladodes polysaccharides combination in reducing the gastroesophageal reflux. The further addition of Olea Europeae leaves extract to this combination (group E) increased the P.I.U. to a level higher than the sum of Olea Europeae leaves extract individual component and combination D P.I.U.s., confirming the results obtained in the chemically-induced gastric ulcer models.

CONCLUSIONS

(57) This in vivo test shows that Opuntia Ficus Indica cladodes extract has a potentiating effect in reducing the gastroesophageal reflux. This effect is further increased by the addition of Olea Europeae leaves extract.