COMPOSITION FOR USE IN PREVENTION OR TREATMENT OF OESOPHAGEAL DISEASES LINKED TO EPITHELIAL BARRIER DEFECTS

Abstract

A composition comprising quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) and/or myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one) is used in the prevention or treatment of heartburn, gastroesophageal reflux disease (GERD) or Eosinophilic Esophagitis (EoE). The composition reinforces tight junction strength in the oesophagus wall at low concentrations, restoring healthy barrier function to the oesophagus and resistance to acid. The composition prevents acid from penetrating the oesophagus, irritating underlying nerves and causing pain.

Claims

1. A method for prevention or treatment of gastroesophageal reflux disease in a patient in need thereof comprising administering to the patient a composition comprising quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) and/or myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one), wherein the quercetin and/or myricetin is comprised in the composition in an amount of 0.01-100 mg, and wherein the composition does not comprise a hydrolyzed protein source comprising whey and casein; a lipid; at least one pre-gelatinized starch; a low-methylated pectin; and/or at least one additional carbohydrate.

2. A method for prevention or treatment of Eosinophilic Esophagitis in a patient in need thereof comprising administering to the patient a composition comprising quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) and/or myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one).

3. The method according to claim 1, wherein the method reinforces oesophagus wall barrier function in the patient.

4. The method according to claim 1, wherein the patient is a pregnant woman or a paediatric patient.

5. The method for use according to claim 1, the composition comprising quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) and myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H benzopyran-4-one).

6. The method for use according to claim 1, wherein the composition comprises genistein (5,7-dihydro-3-(4-hydroxyphenyl)chromen-4-one) and/or berberine (5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a] quinolizinium).

7. The method for use according to claim 1, wherein an amount of quercetin ranges between 25 and 100 mg.

8. The method for use according to claim 1, wherein an amount of myricetin ranges between 25 and 50 mg.

9. The method for use according to claim 6, wherein an amount of genistein ranges between 0.01 and 600 mg.

10. The method for use according to claim 6, wherein an amount of berberine ranges between 0.01 and 1500 mg.

11. The method for use according to claim 1, wherein the composition is administered in a solid dosage form.

12. The method for use according to claim 1, wherein the composition is administered present in a liquid dosage form.

13. Composition for use according to claim 12, wherein the composition comprises a viscosity controlling agent selected from acacia gum, tragacanth, alginic acid, carrageenan, locust bean gum, guar gum, gelatine, polyacrylate, methylcellulose, carboxymethylcellulose, xanthan gum, gellan gum, curdlan, dextran, pullulan, scleroglucan, or a combination thereof

14. The method according to claim 13, wherein the viscosity controlling agent is alginic acid.

15. The method according to claim 13, wherein the viscosity controlling agent is present in an amount ranging from 0.05 — 10 wt. %, based on the total weight of the composition.

16. The method according to claim 14, wherein the viscosity controlling agent is present in an amount ranging from 0.05-10 wt. %, based on the total weight of the composition.

17. The method according to claim 2 wherein the method reinforces oesophagus wall barrier function in the patient.

18. The method according to claim 2, wherein the patient is a pregnant woman or a paediatric patient.

19. The method according to claim 2, the composition comprising quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) and myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one).

20. The method according to claim 2, wherein the composition comprises genistein (5,7-dihydro-3-(4-hydroxyphenyl)chromen-4-one) and/or berberine (5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0051] FIG. 1 is a schematic drawing of the functioning of the oesophagus and the effect of the composition according to the invention.

[0052] FIG. 2 is a graph of activity data for compounds Myricetin and Quercetin.

EXAMPLES

[0053] Referring now to FIG. 1. When food is swallowed and travels down the oesophagus (1), the lower esophageal sphincter (LES) opens, allowing food to enter the stomach and a little bit of acid travels upwards into the oesophagus (2). Subsequently, the LES closes to prevent food and more acid from flowing back (3). In a healthy oesophagus, acid is contained because cells are tightened together (4). Patients with heartburn and GERD have an acid-sensitive oesophagus. Cells are spaced apart and stomach acid can reach the underlying nerves, causing pain (5). The composition according to the invention effectively reinforces the oesophagus wall, restoring oesophagus health (6), and reducing or eliminating heartburn and GERD symptoms.

[0054] The biological barriers of concern in heartburn, GERD and EoE are epithelia, which consist of epithelial cells. Epithelia are oesophagus epithelium and connections between the epithelial cells that seal the space between cells, tight junctions (TJs), see FIG. 1.

Active Compounds

[0055] The present inventors used a unique approach in order to identify compounds which reinforce the TJ system via the control of TJ initiation. Par6 was selected as the biological target; it acts as a regulator of tight junction initiation. Par6 has a polar distribution, with a defined zone of high abundance at one end of the cell. This Par6 distribution is key to the biological function, not just the absolute Par6 protein level.

[0056] Standard tools for RNA and protein level measurement are not adequate to discover compounds which target Par6 and the present inventors devised and implemented an assay where the distribution of Par6 in living tissues can be monitored: Thelial Live Targeted Epithelia (theLiTE™; U.S. Pat. No. 9,416,391).

[0057] The technology of theLITE™ allows monitoring the distribution of Par6—an intracellular protein that controls tight junction assembly. The assay was performed in live follicular epithelia of the egg chamber of the fruit fly Drosophila. Par6 was tagged with a green fluorescence protein (GFP) and its distribution within the cell was monitored by the GFP signal. In barrier cells Par6 has a polar distribution, with a defined zone of high abundance for example at one end of the cell. This protein distribution is key to the biological function, not just the absolute protein level. Standard tools for RNA and protein level measurement are not adequate because these techniques do not inform about the distribution of Par6 within the cell.

[0058] In theLiTE, the present inventors tested whether compounds modulate the distribution of Par6 protein in live follicular epithelia. In theLiTE more than 20 live egg chambers were exposed to test each compound and monitored for Par6 distribution after a set incubation time; any positive compounds were tested in three separate runs of experiments and over a broad range of concentrations. The full method is disclosed in the US patent document. In total over 2000 individual compounds were screened; four candidates with potent Par6 modulation and low toxicity were short-listed. Two, Myricetin and Quercetin, are natural flavonoid compounds either or both of which can be formulated in the composition according to the present disclosure. The activity data for compounds Myricetin and Quercetin are shown in FIG. 2. The positive control in FIG. 2, indicated with a “+” sign, is Antimycin A, at a concentration of 40 micromolar. The negative control, indicated with a “−” sign is the solvent medium (Insect cell culture medium plus 10% dimethyl sulfoxide). Antimycin A (CAS number 1397-94-0) is toxic, and therefore not useful in therapy. Nevertheless, it was identified as the most potent compound in the test and as such included as a positive control.

[0059] Myricetin and Quercetin are widely known and well characterized flavonoid compounds. Name and industry standard identifications are listed in Table 1.

TABLE-US-00001 TABLE 1 Quercetin and myricetin data Name: Quercetin Myricetin CAS 117-39-5 529-44-2 Number: IUPAC 2-(3,4-dihydroxyphenyl)-3,5,7- 3,5,7-Trihydroxy-2-(3,4,5- Name: trihydroxychromen-4-one trihydroxyphenyl)-4H-1- benzopyran-4-one

Preclinical Data—Cell Culture

[0060] The industry standard assays for measuring barrier strength and reinforcement in vitro are based on cell culture. Briefly, cells are cultured on a permeable substrate in a two-chamber device until they form a complete barrier layer, separating the two chambers. Fluorescent dye Lucifer yellow is added to one chamber and passage through the cell monolayer to the second chamber monitored. The lower the dye efflux, the stronger the barrier.

[0061] Quercetin is reported to have similar barrier reinforcing effects in Caco-2 and oesophagus derived cell line HET2A at a concentration of 20 to 50 micromolar (e.g. NIH project 5R21DK097529-02 accessed 19.02.2020 via http://grantome.com/grant/NI H/R21-DK097529-02).

Preclinical Data—Animal Models

[0062] A variety of animal models which recapitulate barrier defects in oesophagus are available. However they are considered poor models for the clinical situation because of structural differences and the fact rodent oesophagus' tissue is keratinized, whilst the human oesophagus is not.

Clinical Data

[0063] Clinical Trial—Patients with Non-Erosive GERD

[0064] A clinical trial of Quercetin in patients with non-erosive Gastro esophageal Reflux Disease (GERD) has been approved by the relevant ethical committees and is completed by team directed by Professor Roy C Orlando at the University of North Carolina at Chapel Hill. The trial is funded by the U.S. National Institutes of Health (NIH), Grant #R21-DK097529, and study description available at NIH's ClinicalTrials.gov website, identifier #NCT02226484. Summary: In this open-label trial subjects are all given 500 mg of Quercetin orally twice daily (tablet form) 1 hour before the morning and the evening meal for 6 weeks. Endoscopic esophageal biopsies are scheduled before (day 0) and after (day 56) Quercetin therapy for evaluation of esophageal epithelial barrier function. Quercetin increases esophageal epithelial barrier function, and specifically its resistance to injury upon contact with hydrochloric (gastric) acid (Clinicaltrials.gov NCT02226484, unpublished).

Efficacy in a Commercially Relevant Population

[0065] 27 pregnant women self-identifying as suffering from heartburn symptoms volunteered and were provided with a monthly supply of a lozenge format of the invention disclosed here (a 2,5 g isomalt lozenge comprising 38 mg Quercetin as active ingredient plus selected B vitamins), to be consumed daily through dispersion by sucking in the mouth for 30 consecutive days. Participants self-report symptom improvement upon completion in 70 percent of cases. Of the participants who experienced relief, 62% reported near immediate relief (15 to 30 mins after usage) strongly consistent with a localized effect of the invention disclosed here. By comparison, 14 GERD sufferers in clinical trial NCT02226484 who were administered twice 500 mg quercetin daily for a 6-week period were monitored for heartburn symptom improvement. There is no report of consistent effects.

[0066] 10 persons diagnosed with EoE are provided with a monthly supply of a lozenge format of the invention disclosed here (a 2,5 g isomalt lozenge comprising 38 mg Quercetin as active ingredient plus selected B vitamins), to be consumed daily after breakfast through dispersion by sucking in the mouth for 30 consecutive days. Participants self-report symptom improvement including reduced food sensitivities upon completion in 70 percent of cases.

Synergy Between Active Compounds

[0067] In a 6-weeks treatment, 50 subjects suffering from GERD receive a twice daily oral dose (1 hour before the morning and the evening meal) with either [0068] Quercetin (500 mg); [0069] Myricetin (500 mg); [0070] Quercetin (500 mg) and Myricetin (500 mg); [0071] Quercetin (500 mg) and Genistein (300 mg); [0072] Myricetin (500 mg) and Berberine (750 mg).

TABLE-US-00002 No. of GERD patients Treatment Observed Effect 14 Quercetin 12 subjects report reduction in severity of GERD, as well as reduced usage of PPIs 14 Myricetin 10 subjects report reduction in severity of GERD, as well as reduced usage of PPIs 8 Quercetin + 8 subjects report suppression of GERD symptoms Myricetin and a near complete elimination of the need to use PPIs and antacids, for limited period of time (~4 hours) 7 Quercetin + 6 subjects report that GERD is resolved for Genistein limited period of time (~4 hours) 7 Myricetin + 5 subjects report that GERD is resolved for Berberine limited period of time (~4 hours)

[0073] The use of Quercetin alone or Myricetin alone leads to a reduction of GERD severity and a reduction in the need to use PPIs to resolve remaining symptoms. The use of the combination of Quercetin and Myricetin leads to suppression of GERD symptoms and a near complete elimination of the need to use PPIs and antacids.

[0074] The use of the combination of Quercetin and Genistein, as well as the combination of Myricetin and Berberine show further synergistic effects for completely resolving GERD. The present inventors consider that this may be due to their complementing mechanisms of action. Myricetin and Quercetin both act through modulating Par6, while Genistein and

[0075] Berberine are examples of flavonoids which reinforces tight junctions but not through modulating Par6 (data not shown). Having two molecules which both reinforce the epithelial barrier but acting via different routes leads to considerable synergy.

Formulation

[0076] The prototype formulation used in the first clinical trial was a tablet. As with all solid foods, it typically passes through the oesophagus in under 10 seconds, too rapidly for the active compounds to have their full effect.

[0077] Usage of a dissolve in the mouth tablet, such as 2,5 g compressed lozenge comprised of isomalt plus active ingredients allows a prolonged, controlled and localized exposure of the oesophagus tissue to the active ingredient; consumption of such a lozenge by probands revealed times to complete dissolution in excess of five minutes.