HK1056549B - Double salts offumaric acid with a carnitine and an amino acid and food supplements, dietary supplements and drugs containing same - Google Patents

Description

Fumaric acid double salt of carnitine and amino acid, and food additive, dietary supplement and medicine containing the same

The present invention relates to a stable, non-hygroscopic, complex salt of fumaric acid (hereinafter referred to as "complex fumarate") with "carnitine" which refers to either L-carnitine or isovaleryl L-carnitine. The invention also relates to food additives, dietary supplements, nutraceuticals and pharmaceuticals comprising the fumaric acid double salt.

Fumaric acid [ (E) -2-butenedioic acid ] shows interesting applications in both nutritional and medical terms.

Fumaric acid can be used as a substitute for tartaric acid (which can cause unpleasant gastrointestinal side effects due to its laxative effect) in the preparation of beverages and baking powder, and also as a substitute for citric acid in fruit juice beverages.

Heart protective Effect of fumaric acid the heart protective effect of fumaric acid has been shown in perfused rat hearts (La Plante et al, "role and metabolism of fumarate in perfused rat hearts: -an item¹³C isotope research (Effects and Metabolism of fumarate in the saturated rat heart: A)¹³C mass isoboromer study) ", am.j. physiol.272: E74-E82, 1997) and in immature myocardium (Pearl et al, "Fumarate-rich hematological cardioplegia leads to complete restoration of immature myocardial function (Fumarate-enriched cardiac therapies in complete functional recovery of myocarpium)", ann. 1636-41, 1993).

Furthermore, fumaric acid is a "pharmacologically" acceptable acid ": in fact, its salts are in the list of "FDA approved commercially available salts", which is published e.g.in the Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences, Vol.66, No.1, (1977) pages 1-19).

The conversion of a drug into the respective pharmacologically acceptable salt form is a widely adopted means to optimize the administration form or certain properties of the drug, such as stability, water absorption, flowability and the like.

The fumaric acid salts of L-carnitine and isovaleryl L-carnitine are both known compounds. (fumaric acid is a dicarboxylic acid: only one of the carboxyl groups in the acid fumaric acid salts mentioned above participates in the salt formation.)

The preparation and physicochemical properties of acidic salt of L-carnitine fumarate have been disclosed in U.S. Pat. No. 4,602,039, which was developed to solve the complicated problems of storage and processing due to the moisture absorption of the inner salt of L-carnitine. In fact, the fumaric acid salt of L-carnitine is very stable, does not cause gastrointestinal side effects, and shows a moisture resistance even exceeding that of L-carnitine tartrate, which is also a less deliquescent salt developed to overcome the water absorption of L-carnitine.

However, tartrate has the advantage that both of its carboxyl groups form salts with L-carnitine and therefore contains a high percentage of L-carnitine (68% versus 58%).

Isovaleryl L-carnitine fumarate is also a stable compound with considerable resistance to moisture, and its preparation is described in U.S. Pat. No. 5,227,518.

Each attempt to form the free carboxyl group of the acid fumarate salt, in which the other carboxyl group of the acid fumarate salt has been salted with L-carnitine or isovaleryl L-carnitine, has failed so far. For example, attempts to prepare the fumarate salt of L-carnitine (i.e. a neutral salt with a high percentage of L-carnitine favouring conditions, about 73.5% versus 68% compared to the tartrate salt and about 73.5% versus 58% compared to the fumarate salt) resulted in a highly hygroscopic substance, likely consisting of a mixture of the acid fumarate salt of L-carnitine and the inner salt of L-carnitine. The latter salt gives the final product overall a high water absorption.

Attempts to form salts of the free carboxyl group of the acid fumarate with alkanoyl L-carnitines, such as acetyl L-carnitine and propionyl L-carnitine, have also failed.

It is an object of the present invention to provide a stable and non-deliquescent complex salt of fumaric acid in which one carboxyl group of the fumaric acid forms a salt with L-carnitine or isovaleryl L-carnitine and the other carboxyl group also forms a salt with a compound which has useful nutritional, dietetic and medical properties and which can also be used as a veterinary feed additive.

Thus, it is clear that the utility of the salt of the invention is not only in its non-deliquescence and high stability, but also in that its two cationic moieties together contribute to the nutritional, energetic, and/or medical efficacy of the salt. Thus, the aforementioned efficacy of these new salts is not due to the "carnitine" moiety of the salt.

The aforementioned objects are achieved by a fumaric acid double salt formed by L-carnitine or isovaleryl L-carnitine and an amino acid, the double salt having the formula (I):

wherein: r is hydrogen or isovaleryl; [ A ]⁺]Is a positively charged amino acid selected from creatine, ornithine, lysine, arginine and histidine.

The present invention should be considered to specifically include the following compounds having the formula (I):

-fumarate salts of L-carnitine and creatine;

isovaleryl L-carnitine and creatine fumarate;

-fumarate salts of L-carnitine and ornithine;

isovaleryl L-carnitine and ornithine fumarate;

-fumarate of L-carnitine and lysine;

isovaleryl L-carnitine and lysine fumarate;

-fumarate salts of L-carnitine and arginine;

isovaleryl L-carnitine and arginine fumarate;

-fumarate salt of L-carnitine and histidine;

isovaleryl L-carnitine and histidine fumarate;

lysine, arginine and histidine are amino acids occurring in proteins, i.e., they are three of the twenty amino acids that can be obtained by controlled hydrolysis of natural proteins (see, e.g., j.dayid rain, incBiochemistry(Biochemistry), Chapter 3, "Primary Structure of Amino acids and proteins" (Amino acids and the primary structures of proteins); McGraw-Hill, 1990).

Although reference is made to the very large number of documents published in this respect so far (see, for example, f.fi danza and g.liguori,Nutrizione umanachapter 3: "Le protein", Casa Editorliania Idelson, 1995; and I.Goldberg (Ed.), (functional foods)Functional Foods)Chapman, "Amino acids, peptides and proteins (peptides and proteins)" Chapman&Hall inc.1994), considering the specific physiological functions of creatine and ornithine, it is believed that it is useful to briefly discuss both.

Creatine is an amino acid present in considerable amounts in skeletal muscle tissue of vertebrates, of which about 2/3 is present in the form of phosphocreatine.

Creatine is biosynthesized from three amino acids mainly in the liver and kidney: that is glycine provides the carbon backbone, arginine releases the amidino group, and methionine releases the methyl group. Creatine is secreted with urine in the form of creatinine. Creatine is obtained from food as it is mainly present in meat. However, in order to obtain 10 g of creatine per day, 2.5 kg of meat should be consumed. Exogenous supply and endogenous biosynthesis should compensate for the daily loss of conversion of creatine to creatinine, an amount estimated to be about 2 grams for a 70 kilogram male.

The physiological function of creatine is very important: it acts as a reservoir of high-energy phosphate groups by reversibly taking up phosphate groups of ATP, mainly in skeletal muscle, but also in brain, liver and kidney. This response is extremely important because ATP cannot be stored in the tissue after a very limited threshold has been exceeded. That is, phosphocreatine provides a phosphate group that is 5 times the amount of ATP in the tissue. Creatine phosphate decreased in skeletal muscle by a correspondingly greater amount than ATP after a moderate tired physical exercise, indicating that creatine phosphate re-phosphorylates ADP when ATP was dephosphorylated.

When the rate of ATP metabolic synthesis exceeds the rate of ATP usage, phosphocreatine formation will result. Therefore, phosphocreatine is a reserve of immediate available energy suitable for balancing the energy requirements over the rate of ATP synthesis during metabolic phosphorylation.

Since creatine can increase the skeletal muscular system only when its intake is accompanied by prolonged physical exercise, it is mainly athletes and sportsmen who obtain creatine. Creatine uptake results in a reduction of fat, but it can strengthen skeletal muscle. Recent studies have shown that the simultaneous intake of creatine and saccharides can promote the effect of creatine, because monosaccharide-stimulated insulin production plays a role in transporting creatine to muscle cells.

Ornithine is a non-proteinogenic amino acid, a lower homolog of lysine, an important intermediate in the urea biosynthetic cycle, where it is produced by guanidyl conversion of arginine. Ornithine may also be converted to glutamic acid.

The fumarates of formula (I) fully achieve the objects of the present invention in that they are not only stable and non-deliquescent compounds, which make them easy to prepare in the solid forms preferred in nutraceuticals, nutritional and dietary supplements, but also in that they combine the complementary physiological activities of "carnitine" and the aforementioned amino acids in a single salt molecule.

For example, those fumarates which combine creatine and "carnitine" synergistically in a single compound stimulate on the one hand the production of energy in muscle cells, especially in type I muscle fibers, thus allowing the entry of important energy carriers (fatty acids) into the mitochondria; on the other hand, it can also stimulate ATP formed by cellular respiration (oxidative phosphorylation) to leave the same organelles. This ATP provides the mechanical energy required for muscle contraction.

The following non-limiting examples illustrate the preparation and physicochemical properties of some of the compounds of the present invention.

Example 1

Fumarate of L-carnitine and creatine (BS/231)

14.9g (0.1 mole) of creatine monohydrate with 16.1g (0.1 mole) of L-carnitine inner salt were dissolved in 500mL of water.

To the resulting solution was added 11.6g (0.1 mol) of fumaric acid with constant stirring. After complete dissolution, isobutanol was added and the mixture was distilled under vacuum at 40 ℃. The residue was taken up in acetone and the mixture was stirred for several hours.

The mixture was then filtered in vacuo and the resulting solid was dried overnight in a constant temperature oven at 30 ℃. 40.5g of fumarate salt of L-carnitine and creatine are obtained, which has proved to be a non-deliquescent and pleasant tasting white crystalline solid.

The yield was 96%.

m.p. ＝134℃(dec.)

K.F. ＝0.7％

[α]²⁰ _D ＝-10.7(c＝1％H₂O)

pH ＝5.5(c＝1％H₂O)

The proportion is as follows:

l-carnitine 40%

Creatine content 32%

Fumaric acid 28%

Elemental analysis C% H% N%

Calculated value 44.226.6713.75

Measured value 44.016.5913.68

NMR：D₂O δ＝6.6(2H，s， )；4.6-4.4(1H，m， )；

3.9(2H，s，N- CH ₂-COOH)；3.4-3.3(2H，d，N- CH ₂-CH)；

3.2(9H，s，( CH ₃)₃-N)；2.9(3H，s，N- CH ₃)；2.5-2.4(2H，d， CH ₂-COOH)

HPLC：

Column: hypersil APS-2(5 μm) 200X 4.6

Temperature: 30 deg.C

Mobile phase: CH (CH)₃CN/H₂O+0.05M KH₂PO₄/CH₃CN(65-35v/v)

pH: 4.7 with phosphoric acid

Flow rate: 0.7mL/min

Rt ═ fumaric acid 12.5; creatine 7.4; l-carnitine 10.8.

Example 2

Isovaleryl L-carnitine and creatine fumarate (BS/232)

14.9g (0.1 mole) of creatine monohydrate with 24.5g (0.1 mole) of isovaleryl L-carnitine inner salt were dissolved in 500mL of water.

To the resulting solution was added 11.6g (0.1 mol) of fumaric acid with constant stirring. After complete dissolution, isobutanol was added and the mixture was distilled under vacuum at 40 ℃. The residue was taken up in acetone and the mixture was stirred for several hours.

The mixture was then filtered in vacuo and the resulting solid was dried overnight in a constant temperature oven at 30 ℃. 47.2g of isovaleryl L-carnitine and creatine fumarate were obtained as a white crystalline solid which proved not to deliquesce and could be crystallized in 95% ethanol.

Yield: 97 percent

m.p. ＝125-127℃(dec.)

K.F. ＝0.5％

[α]²⁰ _D ＝-8(c＝1％H₂O)

pH ＝5.3(c＝1％H₂O)

Elemental analysis C% H% N%

Calculated value 48.97.1311.4

Measured value 48.77.1110.98

NMR：D₂Oδ＝6.6(2H，s， )；4.6-4.4(1H，m， )；

3.9(2H，s，N- CH ₂-COOH)；3.4-3.3(2H，d，N- CH ₂-CH)；

3.2(9H，s，( CH ₃)₃-N)；2.9(3H，s，N- CH ₃)；

2.5-2.4(2H，d， CH ₂-COOH)；2.4-2.2(2H，d， CH ₂- )；

2.2-1.9(1H，m， )；1-0.8(6H，d， )

HPLC：

Column: hypersil APS-2(5 μm) 200X 4.6

Temperature: 30 deg.C

Mobile phase: CH (CH)₃CN/H₂O+0.05M KH₂PO₄/CH₃CN(65-35 v/v)

pH: 4.7 with phosphoric acid

Flow rate: 0.7mL/min

λ： 205nm

Rt ═ fumaric acid 12.5; creatine 7.4; isovaleryl L-carnitine 6.3.

The proportion is as follows:

isovaleryl L-carnitine 50%

Creatine 27%

23 percent of fumaric acid.

Example 3

Fumarate of L-carnitine and L-ornithine (BS/238)

8g (0.05 mol) of L-carnitine inner salt, 5.8g (0.05 mol) of fumaric acid and 6.6gL-ornithineDissolve in 7.5mL of water at 60 deg.C, and slowly pour the resulting clear viscous mass into an acetone solution (800mL) with vigorous mechanical stirring. The precipitated solid was filtered off and dried. 17g of the title compound as a white solid was obtained without deliquescence.

Yield: 92 percent of

m.p. ＝185-187℃(dec.)

K.F. ＝0.9％

[α]²⁰ _D ＝-7.5(c＝1％H₂O)

pH ＝4.7(c＝1％H₂O)

NMR：D₂O＝6.6(2H，s， CH＝CH)；4.6-4.4(1H，m，- CH-)；

3.8-3.6(1H，t，- CH-NH₂)；3.4-3.3(2H，d，N- CH ₂)；3.2(9H，s，( CH ₃)₃)；

3-2.9(2H，t， CH ₂-NH₂)；2.6-2.5(2H，d，- CH ₂-COOH)；

2-1.8(2H，m， CH ₂-CH₂-NH₂)；1.8-1.6(2H，q，CH₂- CH ₂-CH).

HPLC：

Column: hypersil APS-2(5nm) 200X 4.6

Temperature: 30 deg.C

Mobile phase: CH (CH)₃CN/H₂O+0.05M KH₂PO₄/CH₃CN(65-35 v/v)

pH: 4.7 with phosphoric acid

Flow rate: 0.7mL/min

Rt ═ fumaric acid 12.5; 10.8 parts of L-carnitine; l-ornithine 9.

The proportion is as follows:

fumaric acid 28.3%

L-carnitine 39.4%

L-ornithine 32.3%

Example 4

Fumarate of lysine and L-carnitine/fumarate of lysine and L-carnitineFumarate of valeryl L-carnitine (BS/239，BS/240)

Following the procedures of example 1 and example 2, substituting 0.1 mole of lysine for 0.1 mole of creatine monohydrate yielded the white non-deliquescent compounds L-carnitine and lysine fumarate and isovaleryl L-carnitine and lysine fumarate, respectively.

In table 1 below, some of the compounds of the present invention are shown to increase (%) in weight and appearance by exposure to an environment having a relative humidity of 60.5 ± 5% at 25% for 24 hours, as compared to L-carnitine inner salt and isovaleryl L-carnitine inner salt and anhydrous creatine.

Reference documents: pharmaeuropa, 11 months 1996.

TABLE 1

Compound (I)	Weight gain (%)	Appearance of the product
			L-Carnitine inner salt Isovaleryl L-Carnitine inner salt Anhydrous creatine example 1 (BS/231) Compound of example 2 (BS/232) Compound of example 3 (BS/238)	192030.180.190.16	No change in deliquescence and deliquescence, free-flowingChange without change

The preparation of a composition comprising at least one fumaric acid double salt of the formula (I) is obvious to any expert in pharmaceutical technology or pharmacy.

The composition may further comprise other ingredients such as antioxidants, coenzymes and minerals, and may be in the form of tablets, chewable tablets, pills, troches, lozenges, capsules, granules or powders.

In unit doses, these compositions may contain an amount of fumarate of formula (I) which provides 50-2000mg, preferably 100-1000mg of L-carnitine inner salt or isovaleryl L-carnitine inner salt.

Claims (10)

1. A fumarate salt of the formula (I):

wherein: r is hydrogen or isovaleryl; [ A ]⁺]Is a positively charged amino acid selected from creatine, ornithine, lysine, arginine and histidine.

2. The fumarate salt of claim 1, selected from the group consisting of:

-fumarate salts of L-carnitine and creatine;

isovaleryl L-carnitine and creatine fumarate;

-fumarate salts of L-carnitine and ornithine;

-fumarate of L-carnitine and lysine;

isovaleryl L-carnitine and lysine fumarate.

3. A composition comprising as active ingredient the fumarate salt of formula (I) according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. The composition of claim 3, further comprising one or more additional ingredients selected from the group consisting of antioxidants, coenzymes, and minerals.

5. The composition of claim 3 or 4 in the form of a tablet, pill, troche, lozenge, capsule, granule or powder.

6. The composition of claim 5, wherein the tablet is a chewable tablet.

7. A composition according to claim 3 or 4 in unit dosage form comprising as active ingredient a fumarate salt of formula (I) comprising 50-2000mg L-carnitine inner salt or isovaleryl L-carnitine inner salt.

8. The composition of claim 7, comprising 100-1000mg of L-carnitine inner salt or isovaleryl L-carnitine inner salt.

9. Use of a composition according to any one of claims 3 to 4 for the preparation of a food additive, dietary supplement or medicament for human consumption.

10. Use of a composition according to any one of claims 3 to 4 as a veterinary feed additive.