WO2016059654A1

WO2016059654A1 - Use of glucose oxidase for the control, the prevention and the cure of intestinal disorders

Info

Publication number: WO2016059654A1
Application number: PCT/IT2014/000271
Authority: WO
Inventors: Giuseppe Baricco
Original assignee: Giuseppe Baricco
Priority date: 2014-10-16
Filing date: 2014-10-16
Publication date: 2016-04-21

Abstract

A use is described of the enzyme glucose oxidase, immobilized or not, administered by oral route with the purpose of controlling, preventing or curing some common intestinal disorders of humans relatable to intestinal dysbiosis, like Irritable Bowel Syndrome and Diverticulitis,

Description

USE OF GLUCOSE OXIDASE FOR THE CONTROL, THE PREVENTION AND THE CURE OF INTESTINAL DISORDERS

The present invention refers to the use of glucose oxidase for the control, the prevention and the cure of intestinal disorders.

Many people suffer from mid-intensity, long term, high nuisance intestinal disorders. Those disorders (mainly: abdominal pain, irregular stools, intestinal gas overproduction) , when cannot be addressed from the doctor to a specific etiological diagnosis, are commonly aggregated under the name of "Irritable Bowel Syndrome" (IBS) . It is a symptomatic diagnosis, and the disease is classified as a functional gastrointestinal disorder with unknown organic cause. Together with the abdominal pain, diarrhea predominate in the most part of the cases (IBS-D), more rarely constipation (IBS-C), or an alternation of the two can be observed (IBS-A) .

Currently, stating the uncertainty about the precise etiology, no specific treatment of the disease is known, and the doctor intervention is oriented to relieving the symptoms/ acting on a classical multiple approach, working on diet adjustment, medication and/or dietary supplementation, psychological intervention. IBS normally has no or little impact on routine clinical tests, even if normally, during a visit, a higher sensibility of the large intestine to the palpation can be observed.

Between the various attempts to give an explanation of IBS, abnormalities in the normal equilibrium of the gut flora have been often theorized. This dysbiosis is claimed to be originated from a bacterial overgrowth in the small intestine (of unknown origin, with a strong linkage with a post-infective status of the patient) , to a diet poor in fermentable fibers, or finally to specific malabsorption or intolerance conditions of the patient.

Even if IBS does not have any direct effect on life expectancy, however, it is a source of chronic pain, fatigue, can easily induce depression in the patient, can deteriorate the relationship with the doctor, and, finally, has a high social cost, due to absenteeism on work, and to the continuous request of new exams, visits, solutions, that in many Countries are paid from the national welfare system.

The diagnosis of IBS, in place of the more intuitive method of the exclusion criteria (that means to establish a diagnosis of IBS once that all known and certain causes of the symptoms have been excluded by specific tests), can now be done following the flow-chart defined as "Rome III process" (2006), which appears the most modern and scientifically acceptable approach to the functional gastrointestinal disorders.

Another common intestinal disease causing similar symptoms is known as "Diverticulitis", that is a consequence of a clear anatomical variation in the patient (diverticulosis) . In this case, some pouches (diverticula) are easily seen by tomography in the bowel wall, more frequently in the colon: the causative agent of the disease is unknown, with the common theory of a dietary origin (lack of fibers in the diet) being recently discussed and questioned. As in a high percentage of the cases, a mild intestinal infection occurs (or is suspected to be potentially occurring) , the standard treatment from the doctors is based on a low- residue diet and on the prescription of prebiotics and probiotics, quite often associated with a pulsed antibiotic metaphylaxis, usually with nonabsorbable molecules administered on a regular basis to the patient at variable intervals (most frequently one week of antibiotic treatment each 4- 6 weeks) .

Therefore, for those intestinal syndromes, there is a need of a composition that full fills the following requirements:

• provide an efficient way to modulate the intestinal flora of the patients

• not to be an antibiotic, according to the growing global concerns about the use and the abuse of antibiotic molecules in the perspective of overall control of antibiotic resistance

• to be safe for the patients, allowing long- term administration

· not to be conceivable as able to produce bacterial resistance.

The above and other objects and advantages of the invention, as will appear from the following description, are obtained with the use of glucose oxidase as claimed in Claim 1. Preferred embodiments and non-trivial variations of the present invention are claimed in the dependent Claims .

It is intended that all enclosed claims are an integral part of the present disclosure.

The present invention will be better described by some preferred embodiments thereof, given as a non-limiting example, with reference to the enclosed drawings, in which:

- Figure 1 is a graphical representation of the GOD reaction;

- Figure 2 shows as Table 1 the results of GOD treatment on IBS; and

- Figure 3 shows as Table 2 the results of GOD treatment on Diverticulitis.

It will be immediately obvious that numerous variations and modifications (for example related to shape, sizes, arrangements and parts with equivalent functionality) could be made to what is described, without departing from the scope of the invention as appears from the enclosed claims.

Glucose oxidase (GOD) is a flavin enzyme with flavin adenine dinucleotide (FAD) as coenzyme. GOD catalyzes the oxidation of β-D-glucose via D-glucono-5-lactone to gluconic acid, utilizing oxygen as an electron acceptor with simultaneous production of hydrogen peroxide.

GOD is used commercially in various applications including biosensors for quantitative determination of glucose in body fluids, beverages and fermentation liquor .

GOD has been used successfully to remove residual glucose and oxygen in foods and beverages in order to prolong their shelf life, since the H₂O₂ produced by the enzyme acts as a good bactericide. GOD is used to remove glucose during the manufacture of egg powder, preventing browning during dehydration caused by the Maillard reaction, and is used also in baking industry, providing slight improvements to the crumb properties in bread and croissants. GOD can also be used to remove oxygen from the top of bottled beverages before they are sealed.

GOD system is shown to control non-enzymatic browning during fruit processing and puree storage. In addition, GOD is used to prevent color and flavor loss as well as to stabilize color and flavor in beer, fish, tinned foods and soft drinks. For example, it is used to reduce the discoloration occurring in wines and mayonnaises.

GOD enzyme system can be used to retard the lipid oxidation in mayonnaise stored at 5 °C and 25 °C. It is also used to manage dough rheology and consequently bread quality by a strengthening action on wheat dough, hence getting an improvement in bread quality.

GOD has been studied also in the wine industry, where it can lower the alcohol content of wine through the removal of some of the glucose (by converting it to δ-glucono-l, 5-lactone and gluconic acid) , which would otherwise be converted to alcohol. Tests showed that the GOD treatment of wine must could reduce the potential alcohol content of wine by about 2%. In addition, GOD is able to inhibit wine spoilage through its bactericidal effect on acetic and lactic acid bacteria.

The most common microbial sources for the industrial production of GOD enzymes are Aspergillus niger, Penlcillium notatum, P. glaucum, P. amagasakiense, even if the majority of the commercial preparations of this enzyme are obtained from Aspergillus niger . GOD producing gene from A. niger or from other sources (like P. variabile) can also be successfully inserted by genetic technology in highly productive, gene-expressing microorganisms, like yeasts (Saccharomyces cerevisiae, Pichia pastoris) , fungi, and bacteria (E.coli), thus improving the general efficiency of the industrial fermentation process.

Glucose Oxidase is classified as a food additive in the EC legislation (E 1102), and is included into the GRAS list of FDA in the United States.

The discover lies in having understood that GOD, once ingested, maintains - completely or more probably in wide part - its chemical integrity (and by consequence its activity) , along the entire intestinal tract, bypassing the acidic environment of the stomach in which is inhibited, and restarting to work in the small and large intestine, which environment, for pH, chemistry and temperature, is optimal or sub-optimal for GOD enzymatic activity.

A small amount of free glucose is always present in the intestinal tract, primarily as final step of the physiologic digestion of dietary starch and carbohydrates, but also as a result of normal processes of bacterial fermentation, therefore making available for the enzyme its major specific substrate of action.

The quantity of glucose intercepted by the GOD enzyme into the gut is quickly transformed into gluconic acid, a 6-carbon fatty acid naturally occurring in nature (fruits, honey) completely nontoxic, and in hydrogen peroxide that, immediately after having been produced from the enzyme, reacts in contact with the organic matter of the intestinal content by producing, each two molecules of H₂O₂, two molecules of water and one molecule of nascent oxygen, that explains the mild bacterial modulation and the local disinfectant action obtained from GOD at intestinal level. Small amounts of oxygen, of course, are non-toxic, if not beneficial, for higher organisms.

Therefore, the merits of the invention are two :

1. providing a new, safe, and reasonably effective tool to relief the symptoms of a variety of diffused intestinal disorders with a new use of a known enzyme 2. reducing the use of antibiotic molecules actually used for the same purposes, preserving their efficacy along time.

The invention will now be described in detail. a. Substrate

GOD is highly specific for β-D-glucose and shows only marginal activities with other sugars. β-D-glucose gets oxidized in the presence of molecular oxygen at C-l position to δ -glucono-1, 5-lactone. Luminal glucose concentration is generally assumed to be around 50-100 mM/1, therefore a quantity of the specific substrate for the enzyme can be expected to be present in the intestine. A limited oxygen tension is also normally present within the intestine, making available the second necessary co-factor for the GOD enzymatic reaction.

b. pH and stability

pH plays an important role in maintaining the proper activity of an enzyme. Most proteins are only active within a relatively narrow pH range, usually around neutrality, and GOD, with a variation between sources due to the fermentation origin, in fact has an optimal activity pH of 7.0, that is also the reference pH of the intestinal content, physiologically being around 6.5 at the small intestine, and slightly raising along the tube, reaching 7,5 at the rectum.

c. Temperature

Enzymes are known to be sensitive to changes in temperature, and is generally considered that at 70 °C most enzymes get denaturated and loose all or part of their activity. GOD optimal temperature is between 25 °C and 40 °C, that widely includes the normal range of body temperature (35-37 °C) .

d. Storage stability

GOD has an half-life of approximately 30 min at 37 °C, while in immobilized forms, as are almost all the available sources, glucose oxidase is more effective for applications at 37 °C and have a stability, if properly stored, of up to 1 year after the production date.

e. Unit definition

One GOD unit will oxidize 1.0 μπιοΐβ of β-D- glucose to D-gluconolactone and H2O2 per min at pH 5.1 at 35 °C, equivalent to an O2 uptake of 22.4 μΐ per min.

TREATMENT DESCRIPTION

GOD can be assumed from the patient once/day or better in several administrations, by oral route, taking care not to heat it at over 50 °C (for example, if it is assumed with hot drinks) . No matter if the assumption is close or far from meals .

Typically, it is a medium-term treatment, lasting from 7 to 28 days of administration.

a. In case of irritable bowel syndrome

No change in diet prescriptions or other medical recommendations is required.

The typical dosing regime is the following:

90 U/day from day 1 to day 7,

60 U/day from day 8 to day 14,

30 U/day from day 15 to day 28 (maintenance phase) .

Then, a washout period would be placed, until the re-appearance of the first symptoms, that can be faced with a complete cycle, or in some cases, just re-starting with the maintenance regimen.

b. In case of diverticulitis

Start simultaneously with the end of one of the periodical antibiotic cycles prescribed from the doctor, and follow-up with this dosing regimen: 90 U/day from day 1 to day 14

60 U/day from day 15 to day 28 Then, a 7-14 days washout period would be placed, before to repeat the treatment (without the antibiotic) .

GOD can be administered to the patient in any way (powder, tablets, capsules, candies, other) suitable to allow a precise dosing and a proper storage.

The invention will now be described through non-limiting Examples.

RESULTS OF THE PRELIMINARY TRIALS

According to current legislation about the experiments on new compounds in humans, scientifically organized, controlled trials have not been conduced, nevertheless through confidential and private relationships some cases have been treated in order to have a first response about the potential efficacy of the treatment.

GOD has been provided to the volunteers in the form of standard gelatin capsules of 450 mg, each containing 30 U of Glucose Oxidase derived from Pichia Pastoris submerged fermentation, using maltodextrin as diluting carrier, to be taken with meals .

The composition of the powder in each 450 mg gelatin capsule was: Glucose Oxidase 2000 U/g potency 15 mg (equal to 30 U GOD)

Mannan oligosaccharides 20 mg

Maltodextrin 265 mg

(carrier) a. Irritable bowel syndrome

A group of 15 volunteers, aging from 25 to 70 years, 10 males and 5 females, chronically affected from at least two of the symptoms related to IBS (abdominal pain, intermittent diarrhea, abdominal gas overproduction) have been submitted to the treatment .

As the IBS syndrome has no objective parameter to be monitored, the volunteers have been asked to self-evaluate the results, attributing a score (1 - Healthy, 2 - Intermediate, 3 - Sick) at Day 0, 7, 14 and 28^th of the treatment to each of the majors symptoms .

They have also been asked to point out any adverse (or suspected as adverse) effect along the 4 weeks of treatment.

The results are summarized in Table 1.

Overall, a significant improvement of the patient's conditions has been observed, mainly in the parameters of abdominal pain and diarrhea. In the major part of the patients, the improvement has been observed within the 7^th day of treatment, and maintained along the entire period.

No adverse effect has been pointed out, one patient (n° 9) reported no positive effect of the treatment .

b. Diverticulitis

A group of 5 volunteers, aging from 60 to 75 years, 1 male and 4 females, chronically affected from diverticulitis (with abdominal pain and intermittent diarrhea) have been observed for two subsequent cycles following the periodic antibiotic treatment prescribed by the doctor as metaphylaxis (.1 week of antibiotic metaphylaxis with Rifaximin followed from four weeks of washout), in which the first washout period was with no treatment (cycle a. ), while the second washout period included the GOD treatment according to dosing schedule (cycle b. ) .

The volunteers have been asked to self- evaluate the results, attributing a score (1 - Healthy, 2 - Intermediate, 3 - Sick) at Day 0, 7, 14 and 28^th to each of the two symptoms.

Day 0 is the last day of the antibiotic treatment. They have also been asked to point out any adverse (or suspected as adverse) effect along the 4 weeks of observation.

The results are summarized in Table 2.

Overall, a significant improvement of the patient's conditions in the washout period has been observed, ' in both the controlled parameters (AVG Day 28 score for abdominal pain = 2.2 for cycle a. and 1,2 for cycle b., while for diarrhea = 2.8 for cycle a. vs 1.2 for cycle b.). In the major part of the patients, the decline of the clinical conditions after the end of the antibiotic cycle has been significantly slowed, if not blocked, from the GOD treatment.

No adverse effect has been pointed out.

Claims

1. Use of the enzyme glucose oxidase, immobilized or not, administered by oral route with the purpose of controlling, preventing or curing some common intestinal disorders of humans relatable to intestinal dysbiosis, like Irritable Bowel Syndrome and Diverticulitis.

2. Use according to Claim 1, wherein a daily dosage of the enzyme Glucose Oxidase is from 10 to 2.000 U/head/day, preferably from 30 U to 100 U/head/day.

3. Use according to Claim 1, wherein a length of the treatment is between 2 and 90 consecutive days, preferably from 7 to 28 consecutive days.

4. Use according to Claim 1, wherein a preparation or dilution form of the pure enzyme (stabilized or not) , in any shape suitable for a precise dosing regimen, is in the form of powder, tablet, capsule, candy or cream, prepared to be ingested as a solid, or diluted in drinkable liquids .

5. Use according to Claim 4, wherein the finished product is marketed as food ingredient, food supplement, food additive, medical device or medicine .

6. Use according to Claim 1, wherein a fermentation origin of the Glucose Oxidase is from Aspergillus niger, Penicillium notatum, P. glaucum, P. amagasakiense, P. variabile, Saccharomyces cerevisiae, Pichia pastoris, E. coli, or any other suitable micro-organism.

7. Use according to Claim 1, wherein a natural origin of the Glucose Oxidase is from any natural reservoir of the same, like honey.

8. Use according to Claim 1, wherein a packaging technology and a form of preparation of the finished product according to Claim 4 or 5 is useful to extend the shelf-life of the product, like for example low temperature candies or sachets in modified atmosphere, or microencapsulation of the active principle.

9. A composition to be used according to Claim 1, in which to the main active principle (Glucose Oxidase) any of the currently known food supplements/medicines is associated, which is useful to control or modulate the intestinal flora of the patients, for example prebiotics, probiotivcs tanning, essential oils, purified fibers .