WO2002040013A1

WO2002040013A1 - Composition for stimulating the immune defence and iron metabolism, containing propionyl l-carnitine and lactoferrin

Info

Publication number: WO2002040013A1
Application number: PCT/IT2001/000395
Authority: WO
Inventors: Pietro Pola
Original assignee: Sigma Tau HealthScience SpA
Current assignee: Sigma Tau HealthScience SpA
Priority date: 2000-11-17
Filing date: 2001-07-24
Publication date: 2002-05-23
Anticipated expiration: 2003-05-17
Also published as: AU2001277688A1; ITRM20000601A1; ITRM20000601A0; IT1317937B1

Abstract

A composition is disclosed which can be used as a health food/dietary supplement or as a drug for the prevention and/or treatment of peroxidative lipid disorders, of myocardial and cerebral disorders and of abnormalities of iron metabolism and for regulating and stimulating the immune defences, containing as its characterising components propionyl L-carnitine and lactoferrin or a natural product containing lactoferrin.

Description

COMPOSITION FOR STIMULATING THE IMMUNE DEFENCE AND IRON METABOLISM, CONTAINING PROPIONYL L-CARNITINE AND LACTOFERRIN

The present invention relates to a composition suitable for the prevention and/or treatment of peroxidative lipid disorders such as atherosclerosis, of myocardial and cerebral distress, of abnormalities related to iron metabolism and growth factor disorders, of bacterial and viral infections and of forms of cell degeneration. The composition is also suitable for regulating and stimulating the immune defences.

Accordingly, the composition may take the form and exert the activity of an actual medicine, depending upon the support or preventive action or the strictly therapeutic action that the composition is intended to exert according to the particular individuals in whom it is to be used.

More particularly, the composition according to the invention contains as its characterising ingredients:

(a) propionyl L-carnitine or one of its pharmacologically acceptable salts; and

(b) lactoferrin or a natural product containing lactoferrin such as whey or colostrum, or mixture thereof.

The metabolic action both of L-carnitine and of the alkanoyl L-carnitines such as acetyl L-carnitine, propionyl L-carnitine, isovaleryl L-carnitine and butyryl L-carnitine is well known. Their function, in fact, is very important for the production of energy, which takes place mainly through the intramitochondrial β-oxidation of fatty acids, the oxidation of branched-chain amino acids and the regulation of insulin activity. Also important is their protective activity against peroxidative phenomena which has been observed both at cerebral level and at myocardial and renal endothelial level. Another important property is their ability to stabilise cellular phospholipid membranes and the deformability of erythrocytes as well as the stimulation of erythropoietic activity.

All these biological activities account for the favourable effects obtained by the administration of "carnitines" (a term used to denote collectively both L-carnitine and the above-mentioned alkanoyl L-carnitines) in many pathological conditions depending above all on an energy production defect or on lipid peroxidation phenomena such as are encountered in myocardial insufficiency, vascular and cerebral disorders and in prolonged physical exercise.

Lactoferrin, too, like the "carnitines", has a particular physiological role in the body, not only in the regulation of iron metabolism, but also in many mechanisms underlying the immune responses and operating against bacterial and viral infections.

Lactoferrin, which is regarded as belonging to the transferrin group, is a glycoprotein formed by 703 amino acids which is isolated both from cow's milk and human milk, and is above all present in high percentages in colostrum. As well as being present in milk, lactoferrin is also present in plasma, derived from neutrophils, in seminal fluid, in the mucous membranes, in tears and in sweat.

Colostrum, i.e. the mother's milk secreted immediately after childbirth, is particularly rich in lactoferrin, which constitutes 20% of the total proteins present in colostrum.

The high lactoferrin content in colostrum is due to the fact that the immune defences in the newborn are weak and it is precisely lactoferrin that provides the organic defences. To this end it is important that it should not be degradable by tryptic enzymes and that it is well absorbed by the stomach.

In organic fluids lactoferrin is present as a monoferric, diferric or iron-free structure.

Three isoferrins of lactoferrin have been isolated, two of which, i.e. lactoferrin-beta and lactoferrin- amma, with RNase activity, and one, lactoferrin-alpha, without RNase activity.

Specific receptors for lactoferrin have been identified in intestinal tissue, in macrophages and monocytes, in neutrophils, in platelets and in certain bacteria.

The antibacterial activity of lactoferrin is related above all to its ability to bind to iron and therefore to prevent the growth of those bacteria which need iron for their proliferation. In addition to its effect on bacterial growth, a further antibacterial action of lactoferrin has been demonstrated, caused by incorporation of lactoferrin in the bacterial membrane and consequent dispersion of the lipopolysaccharides of the bacterial wall with permanent alterations of its permeability. Lactoferrin, moreover, is capable of exerting an antifungal and antiviral effect, as well as of exerting an inhibiting effect on the growth of many forms of tumours and of regulating and enhancing the immune defences, especially when these are depressed or insufficient.

Other known properties of lactoferrin are its antioxidant ability, its inhibition of platelet aggregation and of the formation of thrombi, and its anticholesterolaemic activity.

It has now surprisingly been found that a composition containing as its characterising components a combination of: (a) propionyl L-carnitine or one of its pharmacologically acceptable salts; and

(b) lactoferrin or a natural product containing lactoferrin, selected from the group consisting of whey, colostrom or mixture thereof, is extremely effective in the prevention and treatment of peroxidative lipid disorders such as atherosclerosis, of myocardial and cerebral distress, of abnormalities related to disorders of iron metabolism and growth factors, of bacterial and viral infections and forms of cell degeneration, and is also suitable for regulating and stimulating the immune defences owing to the potent synergistic effect exerted by its components.

The composition may also contain an additional "carnitine" selected from the group consisting of L-carnitine, acetyl L-carnitine, butyryl L- carnitine, valeryl L-carnitine and isovaleryl L-carnitine or their pharmacologically acceptable salts or mixtures thereof.

The weight-to-weight ratio of component (a) to component (b) ranges from 1:0.1 to 1:10, and preferably from 1:0.5 to 1:2.

Here below are described a number of the most significant tests confirming the unexpected, potent synergistic effect exerted by the combination according to the invention described herein.

Tests on oxidant effects induced by HsOa

For these tests a culture of pheochromocytoma cells (PC-12) containing 3 x 10⁵ M cells/mL was used, which, according to the procedure described by Nordman (Nordman R., Free Rad. Biol. Med.. 1227, 1996), was subjected for 30 minutes to a solution of H2O2 0.1 mM. At the start of the test, to the cell culture were added 100 μg/cc of lactoferrin or the same amount of propionyl L-carnitine or the two compounds in combination. After 24 hours, survival of the cells incubated with H2O2 alone was observed or survival of cells which, in addition to H2O2, were incubated with lactoferrin alone or propionyl L-carnitine alone or with the two compounds in combination.

It could be noted that, as a result of the peroxidation induced by H2O2, the cell survival was 30%, while the survival rates were 42% and 34% when the cells were incubated with H2O2 and lactoferrin and with H2O2 and propionyl L-carnitine, respectively.

Surprisingly, the cell survival rate increased to 90% when lactoferrin was given in combination with propionyl L-carnitine, thus demonstrating the potent synergistic effect of the above-mentioned compounds in protecting the cells against peroxidative damage induced by H2O2.

Tests on the immunosuppressive and toxic effects induced by mitomycin C

Intraperitoneal injections of mitomycin C in mice (50 μg/mouse/day) for five consecutive days caused substantial leukopenia, which in approximately 12 days led to the deaths of all the animals thus treated. The results of this test also revealed the potent synergistic effect exerted by lactoferrin and propionyl L-carnitine in protecting animals treated with mitomycin C against the lowering of the number of leukocytes and the mortality induced by mitomycin.

In fact, the oral administration of the combination of lactoferrin (20 mg/mouse) and propionyl L-carnitine (30 mg/mouse) from the first day of administration of mitomycin for five consecutive days, led to an above 70% survival rate in the animals thus treated compared to controls, as well as to a much more limited reduction in the number of leukocytes. By contrast, the protective effect exerted by lactoferrin alone was only modest (survival: approximately 30%) as was that exerted by propionyl L- carnitine alone (survival: 15%).

The results of these tests are given in Tables 1 and 2 here below.

Table 1

Leukopenia in mice treated with mitomycin C after administration of lactoferrin and propionyl L- carnitine

Treatment No. leukocytes after

5 days 10 days 12 days

Mitomycin C 5.600±360 3,100±290 l-300±260

Lactoferrin 5,900±280 5,000±380 4,600±390

Propionyl L-carnitine 6,100±320 4,200±405 4,300±425

Lactoferrin + propionyl L-carnitine 7,000±420 6,200±410 6,000±425

Table 2

Survival in mice treated with mitomycin C after administration of lactoferrin and propionyl L- carnitine

Treatment % animals surviving after

5 days 10 days 12 days

Mitomycin C 60 30 10

Lactoferrin 75 45 30

Propionyl L-carnitine 70 30 20

Lactoferrin + propionyl L-carnitine 90 80 75

Some non-limiting examples of combination compositions according to the present invention are given hereinbelow:

1) Propionyl L-carnitine 300 mg

Lactoferrin 300 mg 2) Propionyl L-carnitine 100 mg Acetyl L-carnitine 100 mg L-carnitine 100 mg Lactoferrin 300 mg

3) Propionyl L-carnitine 300 mg Bovine colostrum 300 mg

4) Propionyl L-carnitine 100 mg Acetyl L-carnitine 100 mg L-carnitine 100 mg Bovine colostrum 300 mg

5) Propionyl L-carnitine 300 mg Lactoferrin 200 mg

Vit. C 50 mg β-carotene 2 mg Pyridoxine 5 mg Folic acid 50 μg

Vit. PP 25 μg

6) Propionyl L-carnitine 75 mg Acetyl L-carnitine 75 mg L-carnitine 75 mg Butyryl L-carnitine 75 mg Lactoferrin 200 mg Colostrum 200 mg

Lysine 100 mg Vit. C 50 mg

Coenzyme Qio 25 mg Pyridoxine 5 mg

What is meant by a pharmacologically acceptable salt of the various aforesaid carnitines mentioned in the present specification is, in addition to the respective "inner salts", any salt of these with an acid which does not give rise to unwanted toxic or side effects. These acids are well known to pharmacologists and to experts in pharmaceutical technology.

Non-limiting examples of such salts are the following: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate; acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate; acid maleate; mucate; orotate; oxalate, acid oxalate; sulphate, acid sulphate; trichloroacetate; trifluoroacetate and methane sulphonate.

Among these salts, L-carnitine acid fumarate (US 4,602,039), acetyl L- carnitine mucate, propionyl L-carnitine mucate (US 5,952,379) and isovaleryl L-carnitine acid fumarate (US 5,227,518) are particularly preferred.

A list of FDA-approved pharmacologically acceptable acids is given in Int. J. Pharm.. 33, 1986, 201-217, the latter publication being incorporated in the present specification by reference.

The supplement of the invention may further comprise vitamins, coenzymes, mineral substances, aminoacids and antioxidants. The supplement may be manufactured in the form of tablets, lozenges, capsules, pills, granulates, syrups, herb teas, vials or drops.

Claims

1. A combination composition comprising:

(a) propionyl L-carnitine or a pharmacologically acceptable salt thereof; and

(b) lactoferrin or natural product comprising lactoferrin selected from the group consisting of whey, colostrum or mixture thereof.

2. The composition of claims 1 wherein component (a), further comprises a "carnitine" selected from the group comprising L-carnitine, acetyl L-carnitine, butyryl L-carnitine, valeryl L-carnitine and isovaleryl L-carnitine or the pharmacologically acceptable salts or mixtures thereof.

3. The composition of claims 1 and 2 wherein the weight ratio (a):(b) ranges from 1:0.1 to 1:10, preferably from 1:0.5 to 1:2

4. The composition of anyone of the preceding claims which further comprises vitamins, sugars, coenzymes, mineral substances, aminoacids, peptides and antioxidants.

5. The composition of anyone of the preceding claims wherein the pharmacologically acceptable salt is selected from the group comprising: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate; acid fumarate; glycero- phosphate; glucose phosphate; lactate; maleate; acid maleate; mucate; orotate; oxalate; acid oxalate; sulphate, acid sulphate; trichloroacetate; trifluoroacetate and methane sulphonate.

6. The composition of any of the preceding claims, orally administrable, in the form of a health food or dietary supplement.

7. The composition of anyone of claims 1-5, orally, parenterally, rectally, sublingually or transdermally administrable, in the form of a medicament.

8. The health food or dietary supplement of claim 6 manufactured as a solid, semisolid or liquid preparation.

9. The health food or dietary supplement of claim 8 manufactured as tablets, capsules, lozenges, pills, granulates, syrups or drops.

10. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 300 mg

Lactoferrin 300 mg

11. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 100 mg

Acetyl L-carnitine 100 mg

L-carnitine 100 mg

Lactoferrin 300 mg

12. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 300 mg

Bovine colostrum 300 mg

13. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 100 mg Acetyl L-carnitine 100 mg L-carnitine 100 mg Bovine colostrum 300 mg

14. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 300 mg

Lactoferrin 200 mg it. C 50 mg β-carotene 2 mg

Pyridoxine 5 mg

Folic acid 50 μg

Vit. PP 25 μg

15. The health food of claim 9 in unit dosage form, which comprises:

Propionyl L-carnitine 75 mg

Acetyl L-carnitine 75 mg

L-carnitine 75 mg

Butyryl L-carnitine 75 mg

Lactoferrin 200 mg

Colostrum 200 mg

Lysine 100 mg

Vit. C 50 mg

Coenzyme Qio 25 mg

Pyridoxine 5 mg

16. A therapeutic method for the prevention and/or treatment of peroxidative lipid disorders, such as atherosclerosis, myocardial and cerebral disorders, abnormalities related to iron metabolism and growth factor disorders, bacterial and viral infections and cell degeneration and for regulating and stimulating the immune difenses which comprises administering to an individual in need thereof a combination composition comprising the following ingredients:

(a) propionyl L-carnitine or a pharmacologically acceptable salt thereof; and (b) lactoferrin or natural product comprising lactoferrin selected from the group consisting of whey, colostrum or mixture thereof.

17. The therapeutical method according to claim 16, wherein the component (a) further comprises a "carnitine" selected from the group comprising L-carnitine, acetyl L-carnitine, butyryl L-carnitine, valeryl L- carnitine and isovaleryl L-carnitine or the pharmacologically acceptable salts or mixtures thereof.

18. The therapeutical method according to claims 16 or 17, wherein, in said composition, the weight ratio (a):(b) ranges from 1:0.1 to 1:10, preferably from 1:0.5 to 1:2