MXPA98000916A - Use of basic amino acids and derivatives to decrease ceram levels - Google Patents

Abstract

The present invention relates to the therapeutic use of basic amino acids, acylated basic amino acids and their pharmacologically acceptable salts, for the preparation of a medicament for the prophylaxis of diseases or the therapeutic treatment of cellular disorders accompanied by high levels of ceramics.

Description

USE OF BASIC AMINO ACIDS AND DERIVATIVES TO DECREASE CERAMIDE LEVELS DESCRIPTION OF THE INVENTION c The present invention relates to a new therapeutic use of the basic amino acids, the basic amino acids, acylates, and their pharmacologically acceptable salts, for the prophylaxis of diseases or the therapeutic treatment of cellular disorders accompanied by high ceramide levels. In particular, the present invention relates to the use of L-carnitine, acyl-L-carnitine derivatives and pharmacologically acceptable salts thereof, for the prophylaxis of diseases or the therapeutic treatment of cellular disorders accompanied for high levels of ceramide. Ceramide is the basic molecule for the structure of sphingolipids and the metabolism of the same. All sphingolipids contain ceramide as the main hydrophilic component, and originate from ceramide through biosynthetic pathways that mainly modify the 1-hydroxyl position thereof. In turn, sphingolipids play a role REF: 26799 important in the transduction of the signal through the cell membrane. Ceramide plays an important role in the transduction of the signal through the cell membrane. Molecules capable of acting on intracellular receptors (eg calcitriol) or transmembrane receptors [eg, gamma-interferon, (IFN-γ), interleukin-1 (IL-1) and nerve growth factor (NGF) J hydrolyze sphingomyelin to ceramide. Ceramide activates phosphatases and protein kinases and, from a biological point of view, induces cellular apoptosis, cell growth and differentiation, modulates the expression of cyclooxygenases and phospholipases and the activation of nuclear factors kB (NFkB ) [Kuno, K. and collaborators, J. Leukoc. Biol., 56 (5): 542-7; Cifone, M. G. et al., J. Exp. Med., 180 (4): 1547-52; Kolesnick R., Mol. Chem. Neuropathol. , 21 (2-3): 287-97; Jarvis, W.D. and collaborators, Proc. Nati Acad. Sci. U.S.A., 91 (1): 73-7; Obeid, L.M. et al., Science, 259 (5102): 1769-71]. It has now been found that variations in the concentration or metabolism of ceramide contribute to the pathogenesis of numerous diseases, or contribute to the induction of metabolic disorders cell phones. Unfortunately, to date, there are no methods to reduce the levels of ceramide in vi vo. Accordingly, an object of this invention is to provide a novel use of the basic amino acids, the basic acylated amino acids and their pharmacologically acceptable salts to reduce the ceramide levels in vi vo. A second objective of the present invention is to provide a new use of the basic amino acids, the acylated basic amino acids and their pharmacologically acceptable salts, for the prophylaxis of diseases or the therapeutic treatment of cellular disorders accompanied by high levels of ceramide. It has in fact been found that the administration of high doses of basic amino acids, low molecular weight basic compounds or acylated derivatives thereof and pharmacologically acceptable salts thereof, reduces ceramide levels, and such compounds can thus be used for the treatment of diseases characterized by high levels of ceramide. In particular, it has been found that basic amino acids such as arginine, usin, histidine, ornithine, and carnitine or acylated derivatives thereof and pharmacologically acceptable salts of they can be used for the treatment of diseases characterized by high levels of ceramide. According to the invention, there is provided a new use of the basic amino acids, acylated derivatives of basic amino acids and pharmacologically acceptable salts thereof, to decrease the ceramide levels in vi vo. Suitable amino acids include any amino acid with a basic charge, such as arginine, plant, histidine, ornithine and carnitine. These compounds are commercially available. Preferably, the L-amino acids are used. More preferably, carnitine is used. These compounds can be used as free amino acids or as pharmaceutically acceptable salts. Acylated derivatives of the basic amino acids can also be used in the present invention. Acyl amino acids of 2 to 6 carbon atoms that are linear or branched can also be used. These amino acids are well known to pharmacologists and to those skilled in the pharmaceutical art. Particularly preferred acyl groups are acetyl, propionyl, butyryl, valeryl and isovaleryl.

Suitable pharmaceutical salts can be formed between the above basic amino acids and any conventional anions such as chlorine, bromine, iodine or an aspartate acid such as aspartate, an acid citrate such as citrate, an acid tartrate such as tartrate, an acid phosphate such as phosphate, an acid fumarate, a glycophosphate such as glycophosphate, acid lactate, acid maleate, orotate; oxalate acid, particularly oxalic acid; a sulfate, particularly and preferably sulfate, trichloroacetate, trifluoroacetate and methanesulfonate. Examples of diseases or disorders characterized by high levels of ceramide include inflammatory bowel diseases, diffuse intravascular coagulation, fever, protein catabolism and / or lipid depletion, hepatosplenomegaly associated with inflammatory or metabolic liver diseases, endomyocarditis, endothelial cell activation and of leukocytes, capillary thrombosis, meningoencephalitis due to infectious agents, organ transplantation, rheumatoid arthritis and connective tissue diseases, and autoimmune diseases, hyperthyroidism, radiation damage and / or chemotherapeutic agents and chronic fatigue syndrome.

Since the use of some drugs may also induce high levels of ceramide, the present invention also contemplates the decrease of ceramide levels in patients treated with such a drug. For example, a basic amino acid according to the present invention can be co-administered with corticosteroids (such as dexamethasone), anti-inflammatory agents (such as indomethacin), antivirals (such as interferon), immunosuppressants (such as cyclosporin), chemotherapeutics (such as adriamycin). ), immunopotentiators (such as immunoglobulins and vaccines) and endocrinological agents (such as methimazole) to prevent increased levels of ceramide. Normal levels of ceramides in healthy patients depend on the age, size and weight of the individual, but are generally in the range of 5 to 50 picomoles / 106 cells (preferably, peripheral blood lymphocytes). Higher levels of 50 picomoles / 10th cells are considered high levels. The use of basic amino acids, acylated basic amino acids and the pharmacologically acceptable salts thereof of the present invention, reduce such high levels by at least 25%.

In general, the basic amino acids are administered according to the present invention in concentrations that reduce the ceramide levels by at least 25%. Suitably, this result is achieved by the administration of 50 mg to about 15 g / day of basic amino acids by the oral or parenteral route. Preferably, the high levels of these basic amino acids should be administered, for example, to more than 1 g per day, more than 2 g per day; particularly and preferably, 4-10 g per day. Periodic verification of ceramide levels can be conducted either by directly verifying the levels of ceramide in a cell (such as a lymphocyte) or by indirectly checking the concentrations of a ceramide metabolite in a cell. Preferably, the patient's ceramide levels are checked periodically before and after the administration of the basic amino acid, in order to evaluate the amount of the reduction. Periodic verification can begin at any time after administration, but is properly started after 3 hours to ensure accurate results. The periodic verification can be continued indefinitely.

Ceramide levels can be directly measured by isolating peripheral blood lymphocytes from the patient. After this, the cells are centrifuged to remove the supernatant, and the lipids are removed from the cell button. The organic phase containing the ceramide can be evaluated using the "DAG-kinase assay" for the phosphorylation of ceramide, which is evidenced by autoradiography [Cifone, MG et al., J. Exp. Med., 180 ( 4): 1547-52]. Having generally described this invention, further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

Example 1 The peripheral blood lymphocytes were isolated according to the classical methodologies. The cells were incubated with L-carnitine (200 mcg / ml) or with isovaleryl-L-carnitine for 30 minutes at 37 ° C, and thereafter with a monoclonal anti-Fas antibody for another 30 minutes. The cells were then centrifuged, removing the supernatant, and the cell button was delipidated. The organic phase (containing the ceramide) was evaluated in the "DAG-kinase assay" for the phosphorylation of ceramide, which was subsequently evidenced by autoradiography. The results are indicated below in Table 1.

Table 1 Ceramide (picomoles per 106 cells) Control 20 Control + anti-Fas antibody 81.6 Control + anti-Fas + antibody 7.3 L-carnitine (100 mcg / ml) Control + anti-Fas + antibody 8.6 isovaleryl-L-carnitine (50 mcg / ml) Control + anti-Fas + 7.3 isovaleryl-L-carnitine antibody (100 mcg / ml) It is known that cells suitably stimulated (for example with Fas-L, interleukin-1, etc.) generate ceramide. An anti-Fas antibody was used to increase the production of ceramide from a baseline value (20 picomoles per 106 cells) to 81.6 picomoles per 106 cells. It was thus shown that L-carnitine and isovaleryl-L-carnitine inhibit the synthesis of ceramide in vi tro.

Example 2 Two patients affected by symptomatic neuromyopathy (chronic fatigue syndrome) were treated with 3 g per day of L-carnitine by the oral route in two months. Ceramide was measured in the muscles before and after administration. The results are indicated below in Table 2.

Table 2 Pre-treatment with ceramide Post-treatment with ceramide (picomoles per mg of (picomoles per mg protein) proteins) Patient 1 76 28 Patient 2 142 46 Example 3 Four patients affected by hyperthyroidism who had been treated with methimazole (15 mg oral route, daily) for more than eight months, were treated for 4 weeks with 8 g per day of L-carnitine by the oral route. Ceramide associated with lymphocytes was determined before and after treatment. The results are shown in Table 3 below.

Table 3 Pre-treatment with ceramide Post-treatment with ceramide (picomoles per mg of (picomoles per mg of protein) proteins) Patient 1 73 26 Patient 2 45 27 Patient 3 111 36 Patient 4 69 18 Example 4 Three patients with hepatosplenomegaly due to viral hepatitis C were treated with 4 g of bolus of L-carnitine via the intravenous route. The ceramide associated with the lymphocytes was determined before and after 3 and 48 hours after the infusion. The results are shown in Table 4 below.

Table 4 Ceramide Ceramide Ceramide Patient 1 Patient 2 Patient 3 (picomoles / 106 (picomoles / 106 (picomoles / 106 cells) cells) cells) Pre-treatment 65 77 79 After 3 12 32 24 hours After 48 31 21 23 hours It is apparent that the administration of a bolus of L-carnitine inhibited the increase in levels of Ceramide already after 3 hours from the infusion, The effect remains for at least two days.

Example 5 Four patients affected by protein catabolism and lipid reduction as a consequence of tubercular infection were treated for two weeks with 8 g per day of L-carnitine by the parenteral route. The ceramide associated with peripheral blood lymphocytes was determined before and after treatment. The results are shown in Table 5 below.

Table 5 Pre-treatment with ceramide Post-treatment with ceramide (picomoles / 106 cells) (picomoles / 106 cells) Patient 1 127 59 Patient 2 265 77 Patient 3 301 152 Patient 4 78 54 Having now fully described the invention, it will be apparent to someone of experience ordinary in the art, many changes or modifications may be made to this without departing from the spirit or scope of the invention, as claimed herein.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

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

Claims (15)

1. The use of a basic amino acid or an acylated basic amino acid, characterized in that the basic amino acid is selected from the group comprising arginine, usin, histidine and ornithine or a pharmacologically acceptable salt thereof, L-carnitine or an acyl-L-carnitine wherein the acyl group is selected from the group comprising acetyl, propionyl, butyryl, valeryl and isovaleryl, or a pharmacologically acceptable salt thereof, to produce a medicament for the prophylaxis or therapeutic treatment of cellular diseases or disorders caused by high levels of ceramide.

2. The use according to claim 1, characterized in that the basic amino acid, the basic acylated amino acid, the L-carnitine, the acyl-L-carnitine or the pharmacologically acceptable salt thereof, are co-administered with corticosteroids, anti-inflammatory agents, antiviral agents , immunosuppressive agents, cytostatic agents and immunostimulatory agents.

3. The use of isovaleryl L-carnitine for the prophylaxis or therapeutic treatment of diseases or cellular disorders caused by high levels of ceramide, said diseases or disorders are characterized because they include inflammatory diseases of the intestine, diffuse intravascular coagulation, fever, disorders induced by catabolism of proteins and / or lipid depletion, hepatosplenomegaly associated with inflammatory or metabolic liver diseases, endomyocarditis, diseases induced by activation of endothelial cells and leukocytes, capillary thrombosis, meningoencephalitis due to infectious agents, disorders induced by organ transplantation, rheumatoid arthritis and of connective tissue diseases, and autoimmune diseases, hyperthyroidism, radiation damage and / or chemotherapeutic agents and chronic fatigue syndrome.

4. An oral or parenterally administrable pharmaceutical composition for the prophylaxis or therapeutic treatment of cellular diseases or disorders caused by high levels of ceramide, said diseases or disorders comprising inflammatory bowel diseases, coagulation diffuse intravascular disease, endothelial cell-induced disorders and activation of leukocytes, capillary thrombosis, meningoencephalitis due to infectious agents, connective tissue diseases and radiation damage, the composition characterized in that it comprises as an active ingredient an amount of a basic amino acid or an acylated basic amino acid , wherein the basic amino acid is selected from the group comprising arginine, Usin, histidine and ornithine or a pharmacologically acceptable salt thereof, L-carnitine or an acyl-L-carnitine, wherein the acyl group is selected from the group comprising acetyl, propionyl, butyryl, valeryl and isovaleryl or a pharmacologically acceptable salt thereof, effective to reduce ceramide levels, and at least one pharmacologically acceptable excipient therefor.

5. An oral or parenterally administrable pharmaceutical composition for the prophylaxis or therapeutic treatment of cellular diseases or disorders caused by high levels of ceramide, comprising inflammatory bowel diseases, diffuse intravascular coagulation, fever, disorders induced by the catabolism of proteins and / or lipid depletion, hepatosplenomegaly associated with inflammatory or metabolic liver diseases, endomyocarditis, disorders induced by activation of endothelial cells and leukocytes, capillary thrombosis, meningoencephalitis due to infectious agents, disorders induced by organ transplantation, rheumatoid arthritis and connective tissue diseases, and autoimmune diseases, hyperthyroidism, damage by radiation and / or chemotherapeutic agents and chronic fatigue syndrome, the composition characterized in that it comprises as an active ingredient an amount of isovaleryl-L-carnitine or a pharmacologically acceptable salt thereof, effective to reduce the levels of ceramide, and at least one excipient pharmacologically acceptable for it.

6. The use according to any of claims 1 to 3, characterized in that the acyl group of the basic amino acid is selected from acetyl, propionyl, butyryl, valeryl and isovaleryl.

7. The use according to claim 6, characterized in that the acylated basic amino acid is isovaleryl-L-carnitine.

8. The use according to any of claims 1 to 3, characterized in that the anion of the pharmacologically acceptable salt is selected from the group consisting of chloride, bromide, iodide, aspartate, acid, acid citrate, acid tartrate, acid phosphate, acid fumarate , glycophosphate, acid lactate, acid maleate, orotate, oxalate acid and sulfate.

9. The use according to claim 8, characterized in that the anion is sulfate, trichloroacetate, trifluoroacetate or methanesulfonate.

10. The use according to claim 1, characterized in that the daily dose administered orally or parenterally is 50 mg to 15 g.

11. The use according to claim 1, characterized in that the daily dose administered orally or parenterally is from 4 to 10 g.

12. An orally or parenterally administrable pharmaceutical composition, for the prophylaxis of diseases or the therapeutic treatment of cellular disorders accompanied by high levels of ceramide, The composition is characterized in that it comprises as an active ingredient an amount of basic amino acids, basic acylated amino acids or pharmacologically acceptable salts thereof, effective to reduce ceramide levels, and at least one pharmacologically acceptable excipient therefor.

13. The orally or parenterally administrable pharmaceutical composition according to claim 12, wherein the disease or disorders characterized by high levels of ceramide comprise inflammatory bowel diseases, diffuse intravascular coagulation, fever, protein catabolism and / or lipid depletion, hepatosplenomegaly associated with inflammatory or metabolic liver diseases, endomyocarditis, activation of endothelial and leukocyte cells, capillary thrombosis, meningoencephalitis due to infectious agents, organ transplantation, rheumatoid arthritis and connective tissue diseases, and autoimmune diseases, hyperthyroidism, radiation damage and / or chemotherapeutic agents and chronic fatigue syndrome.

14. The composition according to claim 12 or 13, characterized in that it is suitable for oral or parenteral administration from 50 mg to 15 g / day of basic amino acid.

15. The composition according to claim 12 or 13, characterized in that it is suitable for oral or parenteral administration from 4 g to 10 g / day of basic amino acid.