MXPA97002280A - Use of l-carnitine or derivatives of the same yantioxidants for the prevention and treatment of diseases produced by oxidative tension alos systems nerviosso and cardiovascu - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of an alteration or disease caused by oxidative stress caused by oxygen free radicals consisting of an L-carnitine or an alkanoyl L-carnitine or the pharmacologically acceptable salts thereof , characterized in that the linear or branched alkanoyl group has 2-6 carbon atoms in combination and is mixed with, independently a lipophilic or hydrophilic antioxidant.

Description

USE OF L-CARNITINE OR DERIVATIVES OF THE SAME AND ANTIOXIDANTS FOR THE PREVENTION AND TREATMENT OF DISEASES PRODUCED BY OXIDATIVE TENSION TO THE NERVOUS SYSTEMS AND CARDIOVASCULAR DESCRIPTION OF THE INVENTION The present invention relates to a novel therapeutic use of the L-carnitine, some alkanoyl L-carnitines and pharmacologically acceptable salts thereof, for the prevention and treatment of alterations and diseases caused by oxidative stress caused by free oxygen radicals. More specifically, the present invention relates to the corked use of L-carnitine or an alkanoyl L-carnitine, or the pharmacologically acceptable salts thereof, with natural lipophilic antioxidants such as vitamin E or vitamin A and / or natural hydrophilic antioxidants such as vitamin C, glutathione (GSH) or setenium. Various therapeutic uses of both, L-carnitine and alkanoyl L-carnitine are already known, none of which is related to the use described herein. According to the present invention, "co-ordinated use" of the aforementioned compounds is understood to mean either co-administration, that is, the substantially concomitant supplementation of L-carnitine, alkanoyl L-carnitine or a salt of them pharmacologically acceptable, and a natural lipophilic antioxidant such as vitamin E or vitamin A, or a natural hydrophilic antioxidant such as REF: 24322 vitamin C, GSH or selenium, or the administration of a preparation in combination or mixture of the above-mentioned active ingredients, in addition to suitable excipients, if any. Therefore, the present invention also relates to orally, parenterally, rectally or transdermally administrable pharmaceutical compositions suitable for treating disorders and pathologies related to oxidative stress to body proteins and fatty acids comprising, as active ingredients, L -carnitine, an alkanoyl L-carnitine, or a pharmacologically acceptable salt thereof, and a natural lipophilic antioxidant such as vitamin E or vitamin A and / or a natural hydrophilic antioxidant such as vitamin C, GSH or selenium. Alterations and diseases associated with protein oxidation include diseases of the central nervous system such as parkinsonism, trauma, cerebral palsy, diabetic neuropathy and aging; diseases of the peripheral nervous system such as diabetic peripheral neuropathy and traumatic nerve damage; diseases of the cardiovascular system such as intermittent claudication, ischemic reperfusion injury and stroke; and abnormalities of the immune system under conditions of low oxygen pressure. Although it has long been postulated that the formation of free radicals is the probable cause of ischemic damage, it has proven difficult to directly demonstrate that such formation occurred, and / or that it was pronounced enough to overcome the antioxidant defenses of tissues, such as it is reported by Curran et al., Mol. Cell. Biol. 5, 167-172, 1985. In many cases, oxygenated tissues suffer damage, which may even be permanent, if they become ischemic and are reperfused. It is considered that the products of molecular oxygen reduction (PROM) are responsible for cell damage in the course of ischemia and postischemic reperfusion of organs such as the brain, heart, intestines and kidneys (Braughler, JM and Hall, ED (1989) Free Rad. Biol. Med. 6, 289 -301). On the other hand, in inflammation, aging and other important biological processes, it is known that PROMs are mediators and / or modulators of several cellular responses (Turner, E. et al., (1988) Science 242, 939-941) . Several therapeutic protocols have been developed, mainly aimed at eliminating the toxic effect of PROMs on objective biological structures such as the cell membrane. The most commonly adopatada approach has been to use molecules that mimic the well-known antioxidant action of natural compounds, which are part of the so-called primary antioxidant front (Bolli, R. (1989)., J. Am. Coll. Cardioi., 12, 239-249). The most commonly used substances are, for example, superoxide dismutase, an enzyme capable of specifically eliminating superoxide anion, and vitamin E, a molecule that belongs to the tocopherol family, capable of interrupting dangerous oxidant reactions that affect acids. polyunsaturated fatty acids Recent years have witnessed the sale of FVL products, in which several combinations of non-enzymatic antioxidants are present (vitamin E, vitamin A, vitamin C, selenium, glutathione, etc.). Clearly, this type of approach tends exclusively towards the elimination of PROMs or other reactive molecules that are produced by the interaction of PROMs with macromolecules of biological interest. This strategy does not take into account those biological abnormalities that occur even when it is possible to reduce the original toxic potential of PROMs. Additionally, pathological events of unpredictable onset, such as organ ischemia, often do not allow effective therapeutic intervention designed to get ahead with the antioxidant action of PROMs. Thus, it is necessary to sensitize and / or enhance the mechanisms of cellular repair, to minimize the toxic effects due to the harmful action of the PROM. The exclusion of a direct antioxidant action, that is to say, of the primary type, of L-carnitine and its esters against the PROM has already been abundantly demonstrated (Arduini, A., et al. (1990), Free Rad. Res. Commun. 10, 325-332). The typical objective of PROMs in the course of oxidative stress is the plasma membrane, whose integrity is essential for cell survival. The polyunsaturated fatty acids of the membrane phospholipids are particularly sensitive to the oxidizing action of PROMs. These fatty acids are capable of propagating the peroxidative reactions triggered by the PROM, thus stabilizing the intermediary radical species (Mead, JF (1976) in "Free Radicáis in Biology" (Pryor WA ed.) Vol. 1, pages 51-80 , Academic Press, New York). The peroxidation of the biological membranes can generate several oxidation products, which cause significant alterations of the structure and function of the membrane (Arduini, A. et al., (1989) Arch. Biochem. Biophys., 273, 112-119 ). The invention described herein is based on the surprising synergistic effect that occurs between L-carnitine, or an alkanoyl L-carnitine (as will be specified below) or a pharmacologically acceptable salt thereof, and a natural lipophilic antioxidant such as vitamin E or vitamin A and / or a natural hydrophilic antioxidant such as vitamin C, GSH or selenium. This synergistic effect is particularly surprising, because of the lack of direct antioxidant action mentioned above of L-carnitine and its referred derivatives. Now it has been found that the coordinated use of L-carnitine, an alkanoyl L-carnitine or the pharmacologically acceptable salts thereof and the above antioxidant agents, while eliminating the toxic effect produced by PROM's against the biological structures of cells taken as objective, not only prevents cell damage, but also improves the process of cell repair, thus allowing remarkable therapeutic results.

The alkanoyl L-carnitines useful for the novel therapeutic use of the present invention are those wherein the alkanoyl group is a straight or branched chain group having from 2 to 8, preferably from 2 to 6, carbon atoms. Particularly preferred are acetyl, propionyl, butyryl, valeryl and isovaleryl L-carnitine. The pharmaceutically acceptable salts of carnitine or alkanoyl L-carnitine include, in addition to the switterions, all pharmaceutically acceptable salts which are prepared by the addition of an acid to L-carnitine, respectively, and which do not give rise to toxic effects and undesirable side effects The formation of pharmaceutically acceptable acid addition salts is well known to those skilled in pharmacy and pharmaceutical technology. Non-limiting examples of suitable salts include the chloride, bromide, orotate, aspartate acid, acid citrate, acid phosphate, fumarate, acid fumarate, lactate, maleate, acid maleate, oxalate acid, acid sulfate, glucose phosphate, tartrate and acid tartrate. For convenience of simplicity and clarity, reference will be made below to L-carnitine only, it being understood, however, that anything described in connection with L-camitine applies equally to the alkanoyl L-carnitines identified above, and the pharmacologically acceptable salts thereof.

The compositions of the present invention prove to be particularly effective in inhibiting the toxic effect of PROMs, acting in both ways, as primary and secondary antioxidants, with the result that they can be used in the pharmaceutical field for the prevention or treatment of diseases of the central nervous system such as parkinsonism, cerebral palsy, and diabetic neuropathy; diseases of the peripheral nervous system such as diabetic peripheral neuropathy and traumatic nerve damage; diseases of the cardiovascular system such as stroke, ischemia-reperfusion injury and intermittent claudication; and damage to the immune system in conditions of low oxygen pressure. The efficacy of co-ordinated use according to the invention has been confirmed by several pharmacological tests, some of which are reported here below. PHARMACOLOGICAL TESTS I) index of oxidative damage by evaluation of the reaction products of thiobarbituric acid f PRATB) produced by erythrocytes Animal treatment For this study, 20 male Wistar rats were used with an age of 3 months. All animals were treated with a mixture of primary antioxidants (200 IU / kg of vitamin E and 30 mg / kg of ascorbic acid) administered orally for 30 consecutive days. In a subgroup of 10 rats, oral L-carnitine was added to the basic treatment (50 mg / kg). The study was conducted according to the "open" experimental design, and the selection of the subgroup of animals receiving the treatment of the combined antioxidants plus L-carnitine was done using a randomized method. Preparation of red blood cells At the end of the treatment, venous blood samples were taken in test tubes containing heparin. The leukocytes and platelets were removed via a chromatographic column containing cellulose and alpha-cellulose (1: 1 w / w). The red blood cells were then washed three times with saline. Incubation conditions All incubations were done in an oscillating bath at 37 ° C. For experiments with intact cells, the red blood cells were washed one last time with Krebs incubation buffer (120 mM NaCl, 5 mM KCI, MgSO4). 1 mM, 1 mM NaH2P04, 40 mM sucrose, 5 mM glucose, 10 mM Tris-HCl, pH 7.4), and were resuspended in the same buffer, at a hematocrit of 5%. The erythrocytes were treated with tert-butyl hydroperoxide (t-BuOOH), a chemical agent capable of generating lipoperoxidative phenomena in the erythrocyte membrane, at a concentration of 2 mM. At the end of treatment with t-BuOOH, the erythrocytes were washed three times with incubation buffer. All the washes were done at 4o C. Determination of the oxidative damage index The index of oxidative damage selected was the index of the reaction product of thiobarbituric acid (PRATB). For the determination and quantification of the PRATB produced by erythrocytes, the method described by Tsun-Yee Chiu and Claster (Methods in Haematology, Vol. 19, pages 203-236, Churchill Livingstone, New York) was used. The results are presented in Figure 1. II) Oxidative stress index by evaluation of the phosphatidyl ethanolamine / sphingomyelin ratio The animals were treated and the red blood cells were prepared as in the previous test. The incubation conditions were also identical to those used in the previous test. Extraction and separation of phospholipids The lipid components of the erythrocyte membrane were extracted according to the method described by Fióse and Okiander (Rose, H. G. and Okiander, M., J. Lipid Res. (1965) 6: 428-431). To prevent the oxidative phenomenon, butylhydroxytolene (BHT, 0.1%) was added to the extraction solvents. The lipid extract was dried under a flow of nitrogen, and re-suspended with toluene. For separation of the individual classes of phospholipids, bi-dimensional thin layer chromatography (Rouser, G. et al., Lipids (1970) 5: 494-496) was used. The phospholipid classes were highlighted with iodine vapors. Individual classes of phospholipids were identified through the use of standards. The determination of the phosphorus present in the individual classes of phospholipids was made according to Bottcher (Bottcher, C.J. F. et al., Anal. Chim. Acta (1961) 24: 203-208). The results are presented in Figure 2.

III) Hemolysis in the course of oxidative stress The animals were treated and the red blood cells were prepared as in the previous test. The incubation conditions were also identical to those used in the previous test. Determination of hemolysis Hemolysis of erythrocytes was evaluated by measuring the amount of hemoglobin in the course of incubation with the oxidizing agent. At the end of the incubation, the erythrocytes were centrifuged, and the buffy coat was used for the determination of the hemoglobin released in the course of the oxidative stress. The determination of hemoglobin was made spectrophotometrically, according to the method described by Winterbourn C, in Handbook of Methods for Oxygen Radical Research (ed R. Greenwad), pages 13-41, CRC Press, Boca Raton, 1985. The results were presented in Figure III. The addition of L-carnitine to the antioxidant mixture significantly reduces oxidative damage to the cell membrane. The amount of PRATBs was significantly reduced in the erythrocytes obtained from the rats treated with L-carnitine and antioxidants, compared to those treated with the antioxidants alone (Figure 1). Following the oxidative stress, the phosphatidylethanolamine / sphingomyelin ratio of the erythrocytes of the animals treated with antioxidants was only reduced to a greater degree than that of the erythrocytes treated with antioxidant plus L-carnitine (Figure 2). Finally, hemolysis measured in the course of oxidative stress was significantly increased in erythrocytes of animals treated with antioxidants only, compared to those treated with antioxidants plus L-carnitine (Figure 3). An appropriate pharmaceutical composition in unit dosage form comprises from about 0.3 to about 0.5 grams of L-carnitine, or an equivalent amount of alkanoyl L-carnitine or its pharmacologically acceptable salts, and from about 50 to about 2000 U / L, preferably, from about 300 to about 1000 U / l of vitamin E, and / or from about 50 to about 500 mg or, preferably, from about 100 to about 300 mg of vitamin C. The following non-limiting examples show some compositions according to present invention. Examples 1) L-carnitine 500 mg; Vit. E 1000 U / l; Vít. C 300 mg 2) acetyl L-carnitine 500 mg; Vit. E 1000 U l; Vit. C 300 mg 3) propionyl L-carnitine 500 mg; Vit. E 1000 U / l; Vit. C 300 mg 4) isovaleryl L-carnitine 500 mg; Vit. E 1000 U / l; Vit. C 300 mg 5) valeryl L-carnitine 500 mg; Vít. E 1000 U / l; Vit. C 300 mg 6) butyryl L-carnitine 500 mg; Vit. E 1000 U / l; Vit. C 300 mg 7) L-carnitine 500 mg; Vit. At 1000 mg; Vit. C 300 mg 8) acetyl L-carnitine 500 mg; Vit. At 1000 mg; Vrt. C 300 mg 9) propionyl L-carnitine 500 mg; Vít. At 1000 mg; Vit. C 300 mg 10) isovaleryl L-carnitine 500 mg; Vit. At 1000 mg; Vit. C 300 mg 11) valeryl L-carnitine 500 mg; Vit. At 1000 mg; Vit. C 300 mg 12) butyryl L-carnitine 500 mg; Vit. At 1000 mg; Vit. C 300 mg 13) L-carnitine 500 mg; Vit. E 1000 U / l; GSH 500 mg 14) L-carnitine 500 mg; Vit. E 1000 U / l; Vit. C 300 mg; GSH 500 mg 15) L-carnitine 500 mg; Vit. At 100 mg; Vit. C 300 mg; selenium 40 mg It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (13)

1. CLAIMS 1. The use of L-carnitine, an alkanoyl L-carnitine wherein the alkanoyl group, straight or branched chain, has 2-6 carbon atoms, or the pharmacologically acceptable salts thereof in combination and mixture with a lipophilic antioxidant and / or a hydrophilic antioxidant, the use is characterized in that it is for preparing a medicament for the prevention and treatment of alterations and diseases produced by oxidative stress originated by oxygen free radicals.
2. 2. The use according to claim 1, characterized in that the aforementioned disorders and diseases comprise the diseases of the central nervous system such as parkinsonism, trauma, cerebral palsy, diabetic neuropathy and aging; diseases of the peripheral nervous system such as diabetic peripheral neuropathy and traumatic nerve damage; diseases of the cardiovascular system such as damage by ischemic-reperfusion stroke and intermittent claudication; and abnormalities of the immune system under conditions of low oxygen pressure.
3. 3. The use according to any of claims 1 or 2, characterized in that the alkanoyl L-carnitine is selected from acetyl-, propionyl-, butyryl-, valeryl- and isovaleryl-L-carnitine.
4. 4. The use according to claims 1-3, characterized in that the lipophilic antioxidant is selected from vitamin E and vitamin A.
5. 5. The use according to claims 1-3, characterized in that the hydrophilic antioxidant is selected from vitamin C, glutathione (GSH) and selenium.
6. 6. A pharmaceutical composition orally administrable, parenterally, rectally or transdermally, for the prevention and treatment of alterations and diseases produced by oxidative stress originated by oxygen free radicals, characterized in that it comprises L-carnitine, an alkanoyl L-carnitine wherein the alkanoyl group, straight or branched chain, has 2-6 carbon atoms, or the pharmacologically acceptable salts thereof, in combination and mixture with a lipophilic antioxidant and / or a hydrophilic antioxidant, and a pharmacologically acceptable excipient thereof .
7. The composition according to claim 6, characterized in that it is for the prevention and treatment of disorders and diseases of the central nervous system such as parkinsonism, trauma, cerebral palsy, diabetic neuropathy and aging; diseases of the peripheral nervous system such as diabetic peripheral neuropathy and traumatic nerve damage; diseases of the cardiovascular system such as damage by ischemic-reperfusion stroke and intermittent claudication; and abnormalities of the immune system under conditions of low oxygen pressure.
8. 8. The composition according to claim 7, characterized in that the alkanoyl L-carnitine is selected from acetyl-, propionyl-, butyryl-, valeryl- and isovaleryl-L-camitine.
9. 9. The composition according to claims 7 and 8, characterized in that the lipophilic antioxidant is selected from vitamin E and vitamin A.
10. 10. The composition according to claims 7 and 8, characterized in that the hydrophilic antioxidant is selected from the vitamin C, glutathione (GSH) and selenium.
11. The composition according to claim 9, characterized in that it is in unit dosage form comprising 0.3-0.5 g of L-carnitine or an alkanoyl L-carnitine and 50-2,000, preferably 300-1,000 U / l of vitamin E.
12. 12. The composition according to claim 10, characterized in that it is in unit dosage form comprising 0.3-0.5 g of L-carnitine or an alkanoyl L-carnitine and of 300-500 mg of vitamin. C. The composition according to any of the preceding claims, characterized in that it additionally comprises polyphenols, anthocyanins, anthocyanosides, mineral salts and vegetable fibers.