WO2002049636A1

WO2002049636A1 - An antidiabetic composition of amino acids

Info

Publication number: WO2002049636A1
Application number: PCT/IN2000/000128
Authority: WO
Inventors: Rajagopal Thiruvengadam; Malladi Surya Prakasa Sastry
Original assignee: Rajagopal Thiruvengadam; Malladi Surya Prakasa Sastry
Priority date: 2000-12-19
Filing date: 2000-12-19
Publication date: 2002-06-27
Also published as: AU2001235976A1

Abstract

Present invention provides a selected composition of L-Lysine Hydrochloride, L-Leucine, L-Glycine, L-Cysteine hydrochloride and L-Glutamic acid, with very effective and safe antidiabetic properties and which in addition to reducing blood glucose levels in diabetes, has the potential to alleviate the secondary complications of the diabetes. The invention describes the composition of the above amino acids, process for the preparation of different formulations, including the preferred ranges and their use as anti diabetic agents.

Description

AN ANTIDIABETIC COMPOSITION OF AMINO ACIDS

Technical Field

This invention relates to a novel composition consisting of bio-active and bio-acceptable amino acids for treatment and control of various forms of diabetes.

Diabetes Mellitus is an insidious disease, which has no cure at present and is considered to be a major global health problem. About 160 million people all over the world are currently estimated to be suffering from diabetes which according to WHO predictions is likely to cross the 300 million mark by the end of 2025. Incidence of diabetes has increased by 40% among the people in the age group of 40 and by 70% for people in the age group of 30 during 1990-1998. In India alone over 20 million diabetes patients need treatment. Diabetes is estimated to affect a significantly higher percentage of the population in India when compared with other countries. It is noticed that the Indian racial groups settled in other countries also show a higher rate of incidence when compared to other nationalities. These facts clearly indicated that Indians are more prone to diabetes.

There are two major types of diabetes (1) Insulin Dependant Diabetes Mellitus (IDDM) and (2) Non Insulin Dependant Diabetes Mellitus (NIDDM). It is shown that incidence of insulin dependent diabetes is particularly high in Indian children below the age of 15 when compared to statistics from other countries. Insulin dependent diabetes is shown to have a strong genetic aetiology. Conventional treatment for this type of diabetes is administration of insulin extraneously in a rigorous and disciplined manner.

Non insulin dependent diabetes can be controlled by proper and regulated diet and exercise. Blood sugar levels may thus be controlled and medication is avoided at least in the early stages of detection and in borderline cases. Howeyer, chronic cases require treatment both insulin and other anti-diabetic drugs. Incidence of this type of diabetes is also found to be on the increase and various anti-diabetic drugs and formulations are available in the market. These drugs act by stimulating insulin secretion, improving absorption of glucose and by increasing insulin sensitivity. In non-insulin dependent diabetes, insulin production may be normal but impaired glucose absorption and consequent increase in blood sugar level may be due to insulin resistance. Major problems facing chronic diabetic patients are diabetic retinopathy, nephropathy, diabetic neuropathy, non- healing diabetic ulcers, neuritis, cataract retinopathy and heart ailments.

Proteins are known to stimulate insulin secretion and they do play an important role in the absorption and utilization of dietary carbohydrates. While there have been suggestions on the utility of amino acids for the control of blood sugar in hyperglycemic patients based on animal experiments there has been no systematic evaluation of their utility. For example, it has been reported that cataractogenis could be prevented by preventing glycation of lens proteins mediated by chronic diabetes. This, in turn, has lead to the suggestion lysine or a mixture of amino acids could be useful in the treatment of diabetes. However, it is reported that lowering of blood sugar is not immediate and sustained which lead to the hypothesis that the mechanism may be mostly through the scavenging of glucose by the amino acids administered (Sulochana et. al. Exp. Eye Research (1998) 67-, 597). Similarly while it is reported that amino acids can stimulate insulin release (Fajans et. al.), the pancreatic beta cells do not respond equally well to all amino acids. It was not clear as to what extent, the different capacities of individual amino acids to stimulate insulin release are related to differences in their metabolism (Hellmann et al., Biochem J (1971) 123, 513). For example, studies on rabbit and rat pancreas have indicated that leucine is able to elicit insulin release in a glucose free medium. Lysine has also been reported to ameliorate cataractogenesis in rats (Sulochana et al. Insight Vol. XV(3) 1997.

In addition to the free individual amino acids, such as leucine and lysine, glutathione, the tripeptide containing glycine, glutamic acid and cysteine residues has been studied with respect to its effect on diabetic animals and patients. It has been surmised that diabetes condition lowers the glutathione content in the liver and reduces the activity of Super oxide dismutase, an enzyme responsible for the elimination of active oxygen radicals, administration of glutathione to diabetic rats led to the recovery of liver glutathione concentration due to increased Super oxide dismutase activity. The impairment of glutathione metabolism weakens the defence mechanism against oxidative stress that the cells experience during the absorption phase of glucose. It has been reported that presence of diabetic complications correlated negatively with the concentration of reduce glutathione. Experimental work with animals has shown the protective effect of glutathione on beta cell toxicity in tiials aimed at reducing the beta cell damage in insulin dependent diabetes.

Earlier experimental data has suggested that diabetes bring about considerable oxidative stress which leads to organic damage in the long run. There is also evidence that glutathione, the master physiological anti-oxidant confers protection against the degenerative changes resulting from oxidative stress.

Administration of glutathione orally, however, may not be the right answer for solving the problems associated with control of diabetes, mediated through the use of use of glutathione, since there are doubts that administration of oral glutathione may not provide the blood levels required for pharmacological activity.

In summary, therefore, it is obvious that present day knowledge on the potential utility of amino acids for the control of hyperglycemia suffer from lack of information and evidence on the appropriate composition of amino acids required to meet the challenges of developing a product which has the ability to control blood sugar levels, reduce to the extent possible the long- term complications of chronic diabetes, tackle the problems of Insulin resistance and provide a physiologically acceptable preparation with little or no side effects. Disclosure of fhe invention

The object of the present invention is to address the basic drawbacks associated with the current management of diabetes, particularly, Non- insulin Dependent Diabetes Mellitus by developing an anti-diabetic preparation which combines many of the desirable features required for an ideal Anti-Diabetic Drug. The essence of the present invention is related to the development of a novel concept for a novel and innovative method of treatment of acute and chronic diabetes and their attendant long-term complications, which affect the various tissues and organs in the human body. The new approach is to utilize the most desirable combination of amino acids, based on their effect on insulin secretion, glucose absorption, insulin resistance, oxidative stress, diabetic cataractogenesis and retinopathy, and vascular complications.

Surprisingly, it has now been found that a composition of amino acids essentially containing lysine, leucine, cysteine, glycine and glutamic acid, the last three being the building blocks for the anti-oxidant glutathione exhibit excellent antidiabetes properties. The influence of glutathione as an effective anti-oxidant for reducing the oxidative stress in tissues and cells is well documented. There is also evidence that diabetes induces increased oxidative stress in cells and tissues, which may fiirther aggravate problems of glucose absorption. Decreased levels of glutathione and increase in the levels of glutathione oxidation products are noticed in diabetic patients. It is observed that glutathione is minimally absorbed and has low bioavailability. We have noticed that administration of component amino acids for the biosynthesis of glutathone namely glycine, glutamic acid and cysteine lead to increase in bioavailability of glutathione in vivo.

The present invention resides in the remarkable finding that a composition of amino acids essentially containing lysine, glycine, leucine, glutamic acid an cysteine which may contain other biologically active and biologically acceptable amino acids has powerful anti diabetic properties. Such compositions are found to exhibit blood sugar reducing properties and also inhibit insulin resistance. Several long-term secondary complications of diabetes such as cataractogenesis, retinopathy, diabetic neuropathy and heart diseases are minimized on treatment with compositions of this invention.

A unit dosage of the composition of this invention may have the following composition;

L- Lysine hydrochloride -125.00 to 1000 mg

L- Leucine - 25.85 to 206.80 mg

L- Cysteine hydrochloride - 16.58 to 132.64 mg

L- Glycine - 10.275 to 82.20 mg

L- Glutamic acid - 20.01 to 160.08 mg

In addition to the above referenced amino acids, any known biologically acceptable amino acids may also be added to the composition.

The composition of amino acids formulated, according to this invention, were tested in Streptozotocin induced diabetic rats for its antihyperglycemic activity, in accordance with the defined protocol, with the positive (Phenformin) control and solvent control comparisons.

Albino rats of wistar strain of either sex weighing between the narrow range of weight of 150 to 200 gms were selected and were provided with water and standard commercial rat feed ad libitum. Animals were housed at temperature of 18 to 25 degrees C. Diabetes were induced in the rats by the administration of Streptozotocin at a single dose 50mg kg, intraperitonially to overnight fasted animals. Blood glucose levels were estimated by obtaining drop of blood from the tip of the rats toil and placing on a strip of lifescan Inc., USA and the values were measured using the glucometer. Blood glucose is estimated at intervals of 48 and 72 hours after Streptozotocin administration, to the overnight fasted animals which was considered as zero blood glucose value. Animals with blood glucose levels 180 to 300 mg dl were selected for study. In ail, there were 3 groups one for amino acid composition, one for Phenformin and one for solvent control. The effect of the preparation of amino acids on blood glucose levels, was measured and evaluated in comparison to solvent and positive control groups. Amino acid composition and Phenformin were administered orally as suspension in 0.3% CMC twice a day for 6 days and blood glucose levels were measured on the 6^th day. The statistical significance of the antihyperglycemic activity vis-a-vis solvent and positive control was evaluated using ANOVA. Results

The blood glucose levels after administration of amino acids were 45% lower than that of the control group and 29% lower than that of the Phenformin group (Positive control) indicating significant anti-diabetic activity for the amino acid composition.

It is to be understood that the dosage levels required for anti-diabetic activity are to be evaluated through pharmacokinetic studies and human trials. Since the composition of the present invention contains nutritional principles, safety is guaranteed and toxic effects are bound to be minimum.

The composition can be administered in a solid dosage form such as plain and coated tablets/hard gelatine capsules/soft gelatine capsules/powder in sachet/liquid dosage form like solution suspension and parental preparations.

Different dosage forms are prepared using the conventional excipients and the composition of the excipients may change depending upon the dosage form formulated.

Tablets or capsules are formulated by using suitable excipients like microcrystalline cellulose, potato starch, methyl cellulose, hydroxy propyl cellulose, dicalcium phosphate, poly ethylene glycol, hydroxy propyl methyl cellulose, talc, magnesium stearate etc. are used and the process for tabletting normally, not necessarily the same steps.

1. Dispensing 2. Sieving of raw material

3. Dry mixing

4. Wet granulation

5. Drying

6. Dry granulation

7. Blending

8. Compression

The capsules are formulated in two ways. They are

1. Direct filling of the amino acids with or without excipients in capsules.

2. Filling of extruded and spheronized granules into hard gelatine capsules. These granules are filled in 1/2/0/00 size capsules.

The same composition with or without the excipients can be used as a powder in a suitable packing like sachet etc..

This composition can be formulated into a liquid using pharmaceutical vehicles like sorbitol, water, sugar syrup, propylene glycol, glycerine in addition to flavours, buffers, antioxidants and preservatives.

EXAMPLE 1

The following non-limiting example shall serve to describe the invention.

The composition and process for extrusion and spheronisation technique is as follows.

Granules I mg cap

L-Lysine hydrochloride 250.00

L-Leucine 51.70

L-Cysteine hydrochloride 33.16

L-Glycine 20.55

Microcrystalline cellulose 45.00

Potato starch 5.99

Methyl cellulose 4.00

Hydroxy propyl cellulose 2.00

Tartrazine 0.60

P.E.G, 6000 2.00

Granules II mg cap

L-Glutamic acid 40.02

Microcrystalline cellulose 14.60

Potato starch 5.98

Methyl cellulose 2.00

Hydroxy propyl cellulose 2.00

Indigo carmine 0

The above ingredients are sieved through 40# and mixed in a suitable mixer for 30 mnts. And water is added slowly to obtain a soft mass. This is passed through an extruder and then spheronised. The spheronised granules are dried at 45 deg C for about 3 hours or till moisture content is less than

2.0% w/w. The above 2 granules are mixed in a suitable mixer for 1 hour. These granules are filled in 'O' size capsules.

The description given as above with respect to the material (excipients etc.) and process of the extrusion and spheronosation is an example. The same granules may be produced with different excipients and by modifying the process steps in the same technique.

These two granules can also be taken as single granules, by taking all the materials together.

EXAMPLE 2

The following non-limiting example shall serve to described the invention.

All the 5 amino acids namely, L-lysine hydrochloride, L-leucine, L- glycine, L-cysteine hydrochloride, L-glutamic acid were blended geometrically and the blended mix was used to test the hypoglycaemic effect in rats. Thirty six diabetic rats of either sex of Albino strain were used in the study. Animals were given streptozotocin (50mg/kg) to induce diabetes. To one group, the amino acid mix in 0.3% sodium carboxy methyl cellulose dispersion was administered twice a day, at a dose of 131.796 mg per dose for six days and blood glucose levels were checked at initial and at sixth day. However, the drug mix is administered for six days, twice a day. The other group was administered only 0.3% sodium carboxy methyl cellulose dispersion (solvent control group) at the same time intervals as that of the test group. The results are as follows :

Group Blood Glucose levels Initial 6^th day

Control 260 510

Test 284 281

Positive control 275 398

(Phenformin)

As can be seen from the above data, the blood glucose levels of the test group were 45% lower than that of the control group and 29% lower than that of positive control group. The amino acid mixture could contain the raise in blood glucose level, in test group from the initial level, where as solvent treated control group continued to show significantly increasing blood glucose levels.

Claims

CLAIMS :

1. An anti-diabetic amino acid composition which comprises a combination essentially of L-Lysine, L-Leueine, L-Glutamic acid, L- Glycine and L-Cysteine and/or pharmaceutically acceptable salts thereof.

2. The composition as claimed in claim 1, a unit dose of which comprises of:

L- Lysine hydrochioride - 125.00 to 1000 mg L- Leucine - 25.85 to 206.80 mg

L- Cysteine hydrochloride - 16.58 to 132.64 mg L- Glycine - 10.275 to 82.20 mg

L- Glutamic acid - 20.01 to 160.08 mg, the balance being other amino acids and excipients.

3. The composition as claimed in claims 1 and 2, wherein L-Lysine is one part by weight, L-Leucine is 0.2068 part by weight, L-cysteine hydrochloride is 0.1326 part by weight, L-glycine is 0.0822 part by weight and L-glutamic acid is 0.1600 part by weight, the balance being diluents, excipients and other amino acids.

4. The composition as claimed in claim 1, which consists of 250 mg of L-Lysine hydrochloride, 51.7 mg of L-Leucine, 33.16 mg of L- cysteine hydrochloride, 20.55 mg of L-glycine and 40.02 mg of L-

5. glutamic acid in addition to excipients, colourants and other amino acids.

6. The composition as claimed in claims 1 to 4 in the form of powder, tablets, soft and hard gelatin capsules, suspensions, syrups, parenteral preparations.

7. The composition as claimed in claim 5, wherein the composition is in the form of pellets or spheronised granules.

8. Use of a composition essentially consisting of lysine, leucine, glycine, glutamic acid, and cysteine in treating Diabetes Mellitus.

9. Use of the composition as claimed in claim 7, either alone or in a mixture with known hypoglycemic agents and or insulin for treating diabetes.

AMENDED CLAIMS

[received by the International Bureau on 20 March 2002 (20.03.02); original claims 1-9 replaced by new claims 1-8 (2 pages)]

1. An anti-diabetic amino acid composition which comprises a combination of L-Lysine, L-Leucine, L-Glutamic acid, L-Glycine and L-Cysteine and/or pharmaceutically acceptable salts thereof.

2. The composition as claimed in claim 1, a unit dose of which comprises of:

L- Lysine hydrochloride - 125.00 to 1000 mg L- Leucine - 25.85 to 206.80 mg

L- Cysteine hydrochloride - 16.58 to 132.64 mg L- Glycine - 10.275 to 82.20 mg

L- Glutamic acid - 20.01 to 160.08 mg, the balance being excipients.

3. The composition as claimed in claims 1 and 2, wherein L-Lysine is one part by weight, L-Leucine is 0.2068 part by weight, L-Cysteine hydrochloride is 0.1326 part by weight, L-Glycine is 0.0822 part by weight and L-Glutamic acid is 0.1600 part by weight, the balance being diluents and excipients .

4. The composition as claimed in claim 1, which consists 250mg of L- Lysine hydrochloride, 51.7mg of L-Leucine, 33.16mg of L-Cysteine hydrochloride, 20.55mg of L-Glycine and40.02mg of L-Glutamic acid in addition to excipients and colourants.

5. The composition as claimed in claims 1 to 4 in the form of powder, tablets, hard gelatin and soft gelatin capsules, suspensions and syrups.

6. The composition as claimed in claim 5, wherein the composition is in the form of pellets or spheronised granules.

7. Use of a composition consisting of Lysine, Leucine, Glycine, Glutamic acid and Cysteine in treating diabetes mellitus.

8. Use of the composition a consisting of Lysine, Leucine, Glycine, Glutamic acid and Cysteine either alone or in a mixture with known hypoglycemic agents and/or insulin for treating diabetes.