ITRM980433A1 - COMPOSITION WITH ANTIOXIDANT ACTIVITY ANTIPROLIFERATIVE ENERGETIC AND SUITABLE TO IMPROVE THE METABOLIC USE OF GLUCOSE - Google Patents

Description

Description of the invention entitled:

"Composition with antioxidant, antiproliferative, energetic activity and suitable for improving the metabolic utilization of glucose"

The present invention relates to a composition for the prevention and / or treatment of damage induced by the presence of free radicals due to environmental pollution; brain or myocardial lesions induced by free radicals after cerebral or myocardial ischemia and consequent reperfusion; atherosclerotic lesions and tissue proliferative processes; to increase the glucose utilization capacity in diabetic subjects or those with insufficient response to insulin activity; diabetic neuropathies and increased muscle energy needs.

Correspondingly, the composition can take the form and carry out the activity of a food supplement or a real medicament, depending on the support or preventive or properly therapeutic action that the composition is intended to carry out according to the particular individuals to whom is destined.

More particularly, the present invention relates to a composition that can be administered orally, parenteral, rectal or transdermal comprising in association:

(a) acetyl L-carnitine or a pharmacologically acceptable salt thereof, optionally in admixture with at least one other "carnitine"; where by "carnitine" is meant L-thymetine or an alkanoyl L-carnitine selected from the group comprising propionyl L-carnitine, valerli L-carnitine and isovaleryl L-carnitine or their pharmacologically acceptable salts; And

(b) a catechin selected from the group comprising (-) epicatechin, (-) epicatechin gallate; (-) epigallocatechin and (-) epigallocatechin gallate or mixtures thereof.

It is well known that L-carnitine and its derivatives have proved useful in the treatment of myocardial ischemia, in angina pectoris, in peripheral vasculopathies, in situations of increased muscular energy demand and in the various atherosclerotic forms.

These therapeutic activities of carnitine or its derivatives are linked to the complex biochemical activity that this substance is capable of carrying out at the cellular and tissue level.

Carnitine in addition to being essential for the β-oxidation of fatty acids and providing for the synthesis of ATP, plays an important antioxidant role as demonstrated by its protective effect on the liperoxidation of cellular phospholipid membranes and on the oxidative stress induced at the level of myocardial cells. or endothelial. In particular, carnitine was also shown to be capable of intervening on the legal metabolism and on insulin secretion. In carrying out these different activities, all the various carnitines studied prove to be effective, but with a different intensity of action so that in many cases it appears more useful to apply the different carnitines associated with each other than a single carnitine. This is probably linked to their different kinetics and their different intervention in the stages of mitochondrial functional activation which results in an amplification of the desired metabolic effect. The carnitines that are indicated as interacting with each other are above all carnitine, acetyl L-carnitine, propionyl L-carnitine and isovaleryl L-carnitine.

On the glucose metabolism and on the insulin activity as well as on the tissue proliferative reactions, as well as on the oxidative activity, also the polyphenols and in particular the catechins present in green tea have been shown to perform an important regulatory function.

Although the beneficial health effects of tea have long been known, only recently have the most important ones been isolated. soluble components of tea so as to be able to study its pharmacological properties.

Through chromatographic fractionation methods it was possible to isolate from green tea, as active components, at least five different structurally similar catechins and precisely (-) gallocatechin (GC), (-) epigallocatechin (ECG), (-) epicatechin (C ), (-) epigallocatechin gallate (ECGg) and (-) epicatechin gallate (ECg).

In the case of black tea, both of tropical origin and obtained with different extraction processes, the catechins are oxidized and preferably form theaflavins or thearubigo which also have, albeit to a lesser extent, pharmacological active. Epidemiological studies on populations subjected to the same oxidative stress such as those from environmental pollutants and cigarette smoke, in equally industrially developed countries of East Asia, have shown fewer cardiovascular accidents in those populations such as the Japanese and Chinese where, unlike in others, there is a large consumption of tea as a drink, such as to make a "Far East Paradox" appear, similarly to the "French Paradox".

The antioxidant capacity of the total catechins present in green tea or raw catechins as well as that considered individually among the different catechins, has been evaluated on numerous experimental and clinical tests. An intense antioxidant capacity has been demonstrated for all the different catechins, for example on linoleic acid, and on saturated fatty acids such as lard, a very evident ability also in comparison to other antioxidants, such as α-tocopherol.

Also significant were the experiments carried out on the oxidation of LDL ("Low Density Lipoproteins") induced by copper salts which was inhibited both by total crude catechins and by epigallocatechin gallate.

Also in an in vivo study it was possible to demonstrate the ability of tea catechins to inhibit the oxidation of LDL mediated by the presence of macrophages.

In addition to the lipid antioxidant capacity, one of the biological effects of the tea catechins mainly studied is that of the regulation of glucose metabolism.

The administration of tea catechins is in fact able to decrease the glycemic and insulin levels promoted by the administration of starch or sucrose in the rat. In animals made diabetic with streptozotocin it was also found that the administration of tea catechins reduces blood sugar and can have a preventive and curative effect on the onset of diabetes.

In addition to the antioxidant activities, the tea catechins have therefore also proved capable of intervening on glucose metabolism and insulin secretion, as well as on the regulation of tissue proliferative processes.

By administering tea, the risk of pancreatic cancer can be reduced and the expression of oncogenes decreased.

Research carried out by injecting a carcinogen such as methylcholanthrene subcutaneously into mice has shown that catechins in tea can prolong the period of appearance in these animals of subcutaneous solid tumors as well as the appearance in female mice (C3H / HeN strain) of spontaneous breast tumors.

The stimulation of glutathione peroxidase and catalases, as well as the inhibition of ornithine decarboxylase and urokinase, may be at the basis of these favorable activities of the catechins present in green tea.

Among the various catechins studied, epigallocatechin gallate proved to be the most active in inhibiting the "Tumor Necrosis Factor" (TNF) and mRNA ornithine decarboxylase gene expression.

It has surprisingly been found that a composition comprising in association as characterizing components: (a) acetyl L-carnitine or a pharmacologically acceptable salt thereof; And

(b) a catechin selected from the group comprising (-) epicatechin, (-) epicatechin gallate, (-) epigallotcatechin and (-) epigallocatechin gallate and mixtures thereof,

it is extremely effective in the prevention and / or therapeutic treatment of damage induced by the presence of free radicals due to environmental pollution; brain or myocardial lesions induced by free radicals after cerebral or myocardial ischemia and consequent reperfusion; atherosclerotic lesions and tissue proliferative processes; to increase the glucose utilization capacity in diabetic subjects or those with insufficient insulin response; of diabetic neuropathies and for the increased muscular energy requirement, due to the powerful synergistic effect exerted by its components.

It has also been found that, advantageously, component (a) may further comprise a "carnitine" selected from the group comprising L-carnitine, propionyl L-carnitine, valeryl L-carnitine, isovaleryl L-carnitine or their pharmacologically acceptable salts or their mixtures. and that component (b) may consist of a green tea extract (Camellia sinensis, Camellia thea Link, fam. Theacee).

The weight ratio (a) :( b) ranges from 1: 0.1 to 1: 1.

Toxicology

The poor toxicity and good tolerability of both tea catechins and carnitines are well known. These favorable toxicological characteristics of catechins and carnitines have been confirmed by associating these components with each other and by administering them at high doses in both rats and mice. In fact, in these animals it was possible to obtain parenteral administration of over 75 mg / kg of catechin complex and 200 mg / kg of carnitine (50 mg L-carnitine 50 mg acetyl L-carnitine 50 mg propionyl L-carnitine 50 mg isovaleryl L-carnitine) or 250 mg / kg of acetyl L-carnitine, and over 500 mg / kg of catechin complex and 400 mg / kg of carnitine (associated in an equiponderal ratio) or 400 mg / kg of oral acetyl L-carnitine, without any of the animals thus treated dying. Even the prolonged administration through the diet for thirty consecutive days, both in a group of rats and in a group of mice, of 200 mg / kg of catechin complex or of 50 mg / kg of epigallocatechin gallate together with 200 mg / kg of carnitine complex or 250 mg / kg of acetyl L-carnitine associated with each other was well tolerated and did not show any signs of toxicity. Both the weight gain and the various blood chemistry tests performed on these animals proved to be normal as well as the histopathological tests performed on the main organs after sacrificing the animals at the end of the treatment.

Evidence on neuroprotective activity in experimental cerebral ischemia

Considering that cerebral ischemic lesions are related to the production of free radicals and nitroxide (Lipton, S. A., Nature 364-625, 1993) and the favorable protective effect on free radical damage carried out by both carnitines and green tea catechins , we wanted to evaluate its activity on experimental cerebral ischemia. For this purpose, cerebral ischemia was caused by occluding the middle cerebral artery (MCA) according to the method described by Scharkey (Scharkey, Y., Nature 371-336, 1994) through the injection into the anesthetized rat of endothelin-1 ( 120 pmol in 3 ni) injected in three minutes with a microcannula placed stereotactically in the pyriform cortex at the level of the cerebral middle artery. Occlusion of the artery occurs, and the resulting ischemic area can be checked three days after this procedure with the transcardiac perfusion of a solution of paraformaldehyde (4% in PBS).

After removing the brain, it was placed for 24 hours in a fixative containing 10% sucrose, and the cryostatic sections (20 nm) fixed with resylviolet were examined under a light microscope. Either the association of the different carnitines (50 mg / kg of an association formed by L-carnitine, acetyl Lcarnitine, propionyl L-carnitine, isovaleryl L-carnitine in an equiponderal ratio between them of 1: 1) or of acetyl L-carnitine ( 50 mg / kg), either green tea catechin complex (50 mg / kg) or epigallocatechin gallate (50 mg / kg) or these associated products were administered intravenously 5 minutes after endothelin injection .

The volume of the infarcted zone was calculated according to the Park method (Park, C. K., Anns. Neurol. 20-150, 1989). The results of these tests (Table 1) show that both the carnitine complex and the acetyl L-carnitine complex, the catechin complex and the epigallocatechin gallate are able to reduce the ischemic zone, but the surprisingly greater and more significant result is it is obtained only with the association of the carnitine complex with the catechin complex or with epigallocatechin gallate and acetyl L-carnitine.

An unsuspected synergy developed by the association of these products is thus demonstrated.

Tests on the reduction of lens opacification in galactosemic rats induced by the administration of the carnitine complex. acetyl L-carnitine. complex of catechins. epigallocatechin gallate or their association

The administration of galactose with the diet causes the appearance of ocular cataracts in the rat. The opacification of the lens that occurs after about eight days of treatment is classified in ascending order with 1 ° 11 ° IIP stage according to the severity, following the method described by Sippel (Sippel, T., 0., Invest. Qphtalmol. 5 -568, 1966). Treatment for eight days, together with the diet containing galactose, with the carnitine complex (300 mg / kg) consisting of an association formed by L-carnitine, acetyl L-carnitine, propionyl L-carnitine, isovaleryl L-carnitine in an equiponderal ratio between them), with acetyl L-carnitine (300 mg / kg), with the catechinic complex (100 mg / kg) and with epigallocatechin gallate (50 mg / kg) or with the various products associated with each other, induces a decrease in severity opacification of the lens. The association of the carnitine complex with the catechin complex or better still the association of acetyl L-carnitine with epigallocatechin gallate, almost completely inhibits the appearance of opacification (Table 2). An intense and unexpected synergistic effect carried out by the various components is thus demonstrated also in these tests.

Tests on the reduction of the sorbitol content in the lens and in the sciatic nerve in rats with diabetes induced by streptozotocin Considering their ability to intervene on glucose metabolism, we wanted to verify whether the administration of the carnitine complex or of acetyl L-carnitine or of the complex catechinic or epigallocatechin gallate or these associated substances could improve insulin control over high glucose concentrations and avoid the most frequent diabetic complications such as neuropathies, retinopathies, or cataracts. The high concentrations of glucose not controlled by insulin can lead, as is known, to an intracellular accumulation of sorbitol, with a decrease in cellular integrity and osmotic capacity. In these tests, the groups of diabetic rats with streptozotocin were administered orally both the carnitine complex (300 mg / kg in the equiponderal ratio between L-carnitine, acetyl L-carnitine, propionyl L-carnitine, isovaleryl L-carnitine), both acetyl L-carnitine (300 mg / kg), both the catechinic complex (100 mg / kg), and repigallocatechin gallate (50 mg / kg) as well as these products associated with each other at the same doses. After eight days of treatment, the concentration of sorbitol present in the lens and in the sciatic nerve of the animals thus treated was measured, after appropriate chemical isolation. The decrease in the concentration of sorbitol, already found in animals treated with the carnitine complex, with acetyl L-carnitine, with the catechin complex or with epigallocatechin gallate, became very evident in animals treated with the combination of the products, confirming their powerful synergy (Table 3).

Antiproliferative tests and evaluation of ornithine decarboxylase activity

To evaluate the antiproliferative activity, the method was used which consists in injecting teleocidin subcutaneously into the mouse which, similarly to forbolmyristates, causes proliferative alterations in the skin of the animal that can lead to the formation of keratotic processes of a tumor type (Fujiki , H., Biochem. Biophvs. Res. Comm. 90, 976, 1979).

Skin alterations are accompanied by an increase in the ornithine decarboxylase enzyme and this increase is proportional to the severity of the induced lesion, so that it can be used as a marker of the tumor proliferative reaction.

Teleocidin was injected subcutaneously on the depilated back of the mouse at a dose of 5 mcg / mouse dissolved in 0.2 cc of aqueous solution. The association of carnitines (300 mg / kg of an association of L-carnitine, acetyl L-carnitine, propionyl L-camitine, isovaleryl L-carnitine present in an equiponderal ratio between them), acetyl L-carnitine alone (300 mg / kg), or associated with the catechin complex extracted from green tea (100 mg / kg) or epigallocatechin gallate (50 mg / kg) were administered orally to the animal for seven days prior to the test, or were applied to the skin area half an hour before injection with teleocidin after appropriate dispersion in lanolin, so as to reach a concentration of the association of carnitines or acetyl L-carnitine equal to 200 mg / cc and equal to 100 mg / cc of catechin complex and at 50 mg / cc of epigalocatechin gallate

Of these preparations were applied to the skin area where the 0.3 cc teleocidin was injected and the treated area was protected with occlusive bandages.

The ornithine decarboxylase assay was performed on the homogenized epidermis of the animals treated both orally and locally five hours after the teleocidin injection according to the method of O'Brien and Nakadate (O'Brien, T. G., Cancer Res. . 42, 2841, 1982).

The protein concentration of the epidermal extract was measured according to Lowry's method (Lowry, 0. H., J. Biol. Chem., 193, 265, 1951).

The results obtained from these tests (Table 4) show how the modest protection carried out on cutaneous proliferative phenomena and on the increase in ornithine decarboxylase activity both from the haemitine, both from the catechin complex and from the epigallocatecliin gallate becomes surprisingly very evident and well ascertainable using they are associated with carnitine with the catechin complex or with epigallocatechin gallate.

This test also demonstrates the unexpected and surprising synergistic effect between the different components of the compositions according to the invention.

Some examples of formulations according to the invention are given hereinafter, for illustrative and non-limiting purposes.

By pharmacologically acceptable salt of L-carnitine or alkanoyl L-carnitine is meant any salt of these with an acid which does not give rise to unwanted toxic or side effects. Such acids are well known to pharmacologists and pharmaceutical technologists.

Non-limiting examples of such salts are: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, phosphate addo; fumarate, addo fumarate; glycerophosphate; glucose phosphate; lactate; maleate, addo maleate; orotate; oxalate, oxalate addo; sulfate, acid sulfate; tridoroacetate; trifluoroacetate and methanesulfonate.

A list of FDA approved pharmacologically acceptable salts is given in Int. J, of Pharm. 33, 1986, 201-217, publication which is incorporated by reference in this decree.

The composition according to the invention can further comprise vitamins, coenzymes, mineral substances and antioxidants.

Suitable excipients to be used to prepare the compositions according to the specific route of administration will be apparent to any skilled in pharmacy and in the pharmaceutical or feeding technique.

Claims (15)

CLAIMS 1. Composition comprising in association: (a) acetyl L-carnitine or a pharmacologically acceptable salt thereof; And (b) a catechin selected from the group comprising (-) epicatechin, (-) epicatechin gallate; (-) epigallocatechin and (-) epigallocatechin gallate or mixtures thereof. Composition according to claim 1, wherein component (a) further comprises a "carnitine" selected from the group comprising L-carnitine, propionyl L-carnitine, valerli L-carnitine, isovaleryl L-carnitine or their pharmacologically acceptable salts or their blends. Composition according to claim 1 or 2, wherein the weight ratio (a) :( b) varies from 1: 0.1 to 1: 1. Composition according to any one of the preceding claims, wherein the component (b) is present in the form of an extract of plant products containing it. 5. Composition according to claim 4, wherein said extract is green tea extract. Composition according to any one of the preceding claims, wherein the pharmacologically acceptable salt of L-carnitine or alkanoyl L-carnitine is selected from the group comprising: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate, acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate, acid maleate; orotate; oxalate, acid oxalate; sulfate, sulfate addo; tridoroacetate; trifluoroacetate and methanesulfonate. 7. Composition according to any one of the preceding claims further comprising vitamins, coenzymes, mineral substances and antioxidants. 8. Composition according to any one of the preceding claims, which can be administered orally, in the form of a food supplement. 9. Composition according to any one of the preceding claims, administrable orally, parenteral, rectal or transdermal in the form of a medicament. 10. Food supplement according to claim 8 for the prevention of damage induced by the presence of free radicals due to environmental pollution; for the prevention of brain or myocardial lesions induced by free radicals after cerebral or myocardial ischemia; for the prevention of atherosclerotic lesions and tissue proliferative processes; to increase the utilization capacity of glucose in diabetic subjects or those with insufficient insulin response; for the prevention of diabetic neuropathies and for the increased muscular energy requirement. 11. Medicament according to claim 9, for the therapeutic treatment of pathologies induced by the presence of free radicals due to environmental pollution; brain or myocardial lesions induced by free radicals after cerebral or myocardial ischemia; atherosclerotic lesions and tissue proliferative processes and diabetic neuropathies. 12. Food supplement according to claim 8 or 10 packaged in solid, semi-solid or liquid form. 13. Medicament according to claim 9 or 11 packaged in solid, semi-solid or liquid form. 14. Supplement according to claim 12 in the form of tablets, tablets, pills, capsules, granulates or syrups. Medicament according to claim 13, in the form of tablets, tablets, pills, capsules, granules, syrups, ampoules or drops.