WO2003037343A1

WO2003037343A1 - Composition comprising folic acid and carnitine useful to enhance male fertility

Info

Publication number: WO2003037343A1
Application number: PCT/EP2002/011201
Authority: WO
Inventors: Paula Gaynor
Original assignee: Lonza Ag
Priority date: 2001-10-30
Filing date: 2002-10-07
Publication date: 2003-05-08

Abstract

Composition and use thereof for enhancing male fertility.

Description

COMPOSITION COMPRISING FOLIC ACID AND CARNITINE USEFUL TO ENHANCE MALE FERTILITY

The present invention relates to compositions comprising folic acid and carnitine and to uses thereof.

In the industrialized countries, increasingly, the deterioration of male fertility is observed. Changed nutritional habits, stress, smoking, traces of man-made polluting chemical with ostrogen-like physiological effects and the like have been suspected being a likely cause of that.

Ever since, the influence of nutritional factors amongst others has been gained ever increasing attention to counteract this development. EP-B1-1039 894 devises a mixture of L-carnitine and Acetyl-L-carnitine for increasing male sperm quality. However, the amount of that composition intended for human administration is in the range of several grams of substance; apart from cost considerations for human consumers, such amounts may cause symptoms of diarrohea if taken in amounts of >3 g a day.

It is an object of the present invention to devise another nutritional composition improving the fertility of a human male. Another object is the use of such composition for improving male fertility.

This object is solved by a composition according to claim 1 and a use therof according to claim XXX. The composition comprises Carnitine or an Acyl-Carnitine or a salt thereof and folic acid or a salt thereof. Folic acid, or folate, is known to be a carrier of CI -units in the organism. Folic acid is also known under the non-systematic name of pteroylglutamic acid (PGA). Under folic acid in the context of the present invention, any known active metabolic equivalent of folic acid is to be understood, e.g. folate, tetrahydrofolate, 5- methyl-tetrahydrofolate, 5,10-methylentetrahydrofolate, 10-formyltetrahydrofolate. More preferably, the folic acid is 5-methyl-tetrahydrofolate, 5,10-methylentetrahydrofolate or salts thereof. Most preferably, the folic acid is 5-methyl-tetrahydrofolate. The methylated form of folic acid (including its salts which dissolve in the gastrointestinal tract) is the form actually absorbed most optimally in the intestine amongst all CI -modified forms of folic acid; non-methylated folic acid is converted to the methyl form for transport across the intestinal barrier by specialized enzyme systems.

Preferably, such folic acid is man-made and is not comprised as a natural compound of food stuff. Due to this, the folic acid content of the present composition is most efficiently absorbed in the guts. - Foods deliver folate mostly in the 'bound' form, that is, combined with a string of amino acids, predominantly glutamate, therefore termed 'polyglutamate'. The intestine prefers to absorb the free folate form, prejudice to the above said on specific methylation systems for efficient transport. Due to this, nutritionists calculate the folic acid contents of diets in dietary folate equivalents (DFE): Synthetic folate is considered to be 1.7 more potent (1.7 DFE) upon ingestion than naturally comprised dietary 'folate'.

Carnitine according to the present invention may be (DL)-Carnitine or, preferably, essentially pure L-Carnitine. Such Carnitine may as well be Acyl-Carnitine, in particular 2- Acetyl-Carnitine. Such Carnitine may be employed either as an inner salt or as a simple or complex salt together with other ionic substances such as, but not limited to, chloride, fumarate, tartrate, citrate, isocitrate, (-)-hydroxycitrate, magnesium, calcium, cholin, either alone or in suitable combinations, particularly as non-hygroscopic complex salts, e.g. L- Carnitine-magnesium-citrate, L-Carnitine-magnesium-hydroxycitrate or L-Carnitine- cholin-tartrate. In even more preferred embodiments, Carnitine according to the present invention is either L-Carnitine-tartrate, L-Carnitine-magnesium-citrate or L-Carnitine- magnesium-(-)-hydroxycitrate. Carnitine is, in its L-form, a naturally occurring substance involved in energy metabolism in mitochondria that is widely used as a nutritional supplement, e.g. for slimming and is a well-known substance without adverse effects.

Surprisingly, upon oral adminstration to a human, the composition of the present invention displays a synergistic, male fertility enhancing effect. Sperm count and density of sperm cells with normal, non-pathologic morphology are synergistically enhanced. In addition, L- carnitine contributes to sperm motility. Sperm count and density can be determined using standard microscopic techniques (Adelmann, M. eta 1., Atlas of sperm cell morphology, Chicago, ASCP Press, 1989; Schover, L. et al., Overcoming Male Infertility, New York, Wiley, 2000). Preferably, sperm count is determined according to the method of Overstreet, J. et al. (Overstreet et al, 1997, In: Hellstrom, W. (ed.), Male infertility and sexual dysfunction, New York, Springer- Verlag, p.39 ff). Whereas sperm concentration relates to the number of sperm that is found in each milliliter of semen, the sperm count - total sperm count - is the total number of sperm opresent in the semen (semen volme multiplied by sperm concentration).This value accurately describes the sperm production by the testicles. Briefly, appropriate dilutions of semen (1:10 or 1:20 using w3ater were applied to a Neubauer hemacytometer (Fisher, Pittsburgh, PA), allowed to settle and counted. From this count, density (10⁶ sperm/mL) was determined, and total count was calculated by multiplying the density by the measured ejaculate volume.

For male individuals considered healthy, the normal value for sperm concentration is approx. > 20 million/ml. The normal total sperm count is > 40 million. The composition and its use, respectively, according to the present invention, is most effective in individuals having abnormally lowered values of sperm count and density as compared to these standard values.

Preferably, the male person to whom the composition according to the present invention is administered has a daily intake of folic acid, calculated as DFE, that is below the recommended 400 μg/day of folic acid according the RDA guidelines (RDA: Recommended daily allowance; US health services). The blood plasma total folate concentration should thus be below a threshold of about 7nmol/L as is easily determined by commercial radio immunoassy (Quantaphase B-12 folate Radioassay; BioRad, Hercules, CA). It should be noted that folate is comprised mostly in fresh vegetables or certain fruits, but is very poorly present e.g. in milk products. Furthermore, storage/processing of foods, and in particular cooking of food, very efficiently destroys folic acid in foodstuffs. Only raw, fresh vegetables or freshly prepared orange juice do provide sufficient amounts of folic acid if taken in effective amounts.

Preferably, the male person to to whom the composition according to the present invention is administered is deficient in folic acid for other reasons than insufficient nutritional intake. Deficiency in this context does not relate to lowered total levels of folate e.g. in blood plasma. Quite in contrast, the blood plasma levels in such individuals are not significantly lowered as compared to healthy, balancedly fed individuals. Rather, the metabolic balance of biochemically active methyl- and non-methyl derivatives of tetrahydrofolic acid is shifted. The deficiency consists in lowered amount of non-methyl tetrahydrofolic acid. For instance, it has been observed that medicaments, e.g. such often taken substances such as gastric antiacidants or aspirin (acetyl-salicylic acid) and common acitivities such as smoking or consumption of alcoholic beverages interfered with normal folic acid metabolism, lowering the effective amounts of non-methyl tetrahydrofolic acid vs. methylated tetrahydrofolic acid (5-methyl tetrahydrofolic acid) in vivo. Low seminal plasma concentration of non-methylated- folate has been found to correlate with inferior sperm count and density (Wallock, L. et al, 2001, Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and non-smokers, Fertility and sterility, 75, 2:252-259). Also, fairly common congential reasons for such subtle in vivo folate deficiencies exist: A common genetic polymorphism (C677T) of 5,10- methylentetrahydrofolate reductase (EC 1.5.1.20) exists, homozygous individuals having lowered levels of methionine when folate status is compromised (Ma, J. et al., 1996,

Methylentetrahydrofolate reductase polymorphisms, plasma folate, homocysteine, and risk of mycardial infarction in US physicians, Circulation 1996, 94:2410-2416 and references cited therein). According to the present invention, a male-fertility affecting deficiency in non-methyl tetrahydrofolic acid or its corresponding salt is determined by measuring the concentration of folates in seminal plasma (according to the method of Tamura, T.,

Microbiological assays of folates. In: Picianno, M.F. et al., Folic acid metabolism in health and disease, New York, Wiley-Liss, 1990: 121-137). Employing two different strains of microorganims, the value for total and non-methyl tetrahydrofolic acid can be determined. A proportion of less than 40%, more preferably of less than 35%, and most preferably of less than 30 % of the non-methyl tetrahydrofolic acid in total seminal plasma folat (which may be in the range of approx. 5-25 nmol/L folat, more commonly in the range of 10-20 nmol/L folat) is indicative of a deficiency in non-methyl tetrahydrofolic acid according to the present invention.

Whereas lowered folate intake or disbalanced folate metabolism is crucial for maximum effectiveness of using the composition of the present invention for enhancing male fertility, the person administered the present composition does not need to be abnormally deficient in carnitine. L-carnitine can be taken over a wide range of dosage with ever improving effect up to the range of several grams.

A further beneficial side effect of carnitine administration is the reduction of serum cholesterol levels based on metabolic effects (Cacciatore, L. et al., (1991), Drugs Exp. Clin. Res. 18, 355 ff.). This further promotes a patient's health. Folic acid itself is an essential vitamin needed for anabolic acitivities (nucleotide synthesis, aminoacid synthesis) in the body.

Preferably, the composition in accordance with the present invention comprises at least 5 mg up to 8000 mg carnitine per 100 μg of folic acid inhibitor, more preferably 100 to 2000 mg carnitine per 100 μg of folic acid, most preferably 250 to 850 mg carnitine per 100 μg of folic acid.

According to the present invention, the folic acid may amount to between 10 to 1000 μg, preferably between 40 to 400μg in suitable oral dosage forms comprising the composition of the present invention. For example, a daily dose for folic acid supplementing the diet can be 50-300 μg for an average adult and is achieved by multiple oral intake of a capsule or tablet. Carnitine may amount to between 50 to 8000 mg in suitable oral dosage forms of the composition. Preferably, it amounts to in between 250 to 1000 mg for the purpose of enhancing the male fertility.

Preferably, the composition of the present invention should be administered for at least 2-4 weeks prior to detecting the effect on sperm quality as described above. The present invention should essentially work in mammalian males of all species, preferably it works in humans. More preferably, said human males are older than 30 years of age. At this age, folic acid metabolism is more easily disturbed and a deficiency in non-methyl tetrafolic acid provoked than in younger human males.

The composition according to the present invention may be added to foodstuff, i.e. the diet, or swallowed as a freshly prepared suspension. For example, the composition may be added to low-calorific cereal or chocolate bars or similiar snacks comprising a certain amount of fat. Dispersible powders and granules comprising the composition according to the present invention are a prefered embodiment. Such powders or granules may be suitable for preparation of an aqueous suspension by the addition of water and may provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Preferably, the composition is prepared as an oral dosage form and may comprises further, pharmaceutically acceptable exciepients.

Such excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch and alginic acid; binding agents such as starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated with known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or with a wax may be employed.

In another preferred embodiment, oral dosage forms such as tablets, capsules or microbeads are coated with an enteric coating which prevents dissolution in the acidic environment of the stomach. Instead, this coating dissolves in the small intestine at a more neutral pH. Complex organic molecules such as lipase inhibitors, in particular lipstatin, are susceptible in varying degrees to acid hydrolysis. Such enteric coated compositions are described by Bauer et al. (Coated Pharmaceutical Dosage Forms: Fundamentals, Manufacturing Techniques, Biopharmaceutical Aspects, Test Methods and Raw Materials, CRC Press, Washington, D.C., 1998) .

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Oil suspensions may be formulated by suspending the active ingredients in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspension may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agent, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by an added antioxidant such as ascorbic acid.

Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. It is also possible to add further slimming agents such as glycosidase inhibitors, e.g. acarbose, whose funciton is to hinder breakdown of carbohydrates such as starch in the intestine. This further reduces the calorific value of the diet.

In another preferred embodiment, the composition of the present invention further comprises one or several vitamins such as e.gNitamine A, Vitamine D or Nitamine E. Such composition may further comprise essential fatty acids such as linolic acid, linolenic acid or omega-3 polyunsatured fatty acids.

It is a further object of the present invention to provide a method to using the composition of the present invention to enhance male fertility, i.e. to increase sperm count and density. All of the above said applies to this further object of the present invention alike, alone or in suitable combination.

It is possible to achieve said fertility enhancing effect not only by ingesting compositions combining carnitine and folic acid as described above, but as well by injecting pharmaceutical preparations of folic acid concomitant with oral ingestion of carnitine or by ingesting stepwise separately prepared formulations of carnitine and folic acid, respectively. It is also possible to saturate the body's need for carnitine once a day by a certain dose of carnitine, effective amounts being described above, whilst ingesting folic acid repeatedly concomitant with the ingestion of diet, or vice versa. However, it is equally possible to employ retard-capsules or -tablets which continously release the carnitine over a prolonged period of time.

It is also possible to prepare dosage forms combining , according to the present invention, folic acid and Carnitine whilst not bringing them physically in admixture. Such pharmaceutical dosage forms might be e.g. gelatine capsules having a partitioning wall or double walled capsules consisting essentially of two separate capsules one being inserted into the other.

Dosage forms combining carnitine and folic acid, separately as said before or in physical admixture, are a further object of the present invention. Even more generally, a produce comprising folic acid and carnitine or an Acyl-carnitine or a salt thereof as a combinatorial medicine kit for simultaneous, separate or temporally staggered application is an object of the present invention. Such items a generally termed kits-of-parts and may e.g. consist of separate blister bags comprised in a single package.

Example 1

Composition and pharmaceutical dosage form comprising Carnitine (commercial L- Carnitine, Lonza Ltd.) and folic acid.

A hard gelatine capsule is filled with approx. 447 mg of a powder mixture. The particle size is <0.8 μm. The powder has been mixed by addition of the fine-milled, solid compounds in a conventional knedding machine. The composition of the powder mixture is given below:

L-Carnitine (Carnitine-Mg-Citrate, Lonza Ltd.) 300 mg folic acid 400 μg of folic acid

Sodium-stearate 1.5 mg microcrystalline cellulose 60

Vitamine E 2 mg

Lactose 37 mg

Talcum 4.5 mg

Sodium-Carboxymethyl-starch 8.5 mg Polyvinylpolypyrrolidon 8.5 mg

Acarbose 25.0 mg

Claims

1. Composition comprising folic acid or a salt thereof and carnitine or an acyl-camitine or a salt thereof.

2. Composition according to claim 1, characterised in that the composition further comprises a pharmaceutically acceptable excipient .

3. Composition according to one of the preceding claims, characterised in that the ratio of Carnitine to folic acid is 10 to 8000 mg L-Carnitine per 100 μg of folic acid.

4. Produce comprising folic acid and carnitine or an Acyl-camitine or a salt thereof as a combinatorial medicine for simultaneous, separate or temporally staggered application.

5. Oral dosage form comprising a composition according to claim 1.

6. Dosage form according to claim 5, characterised in that it is a tablet or a capsule.

7. Use of a composition according to claim 1 or a produce according to claim 4 for enhancing male fertility.

8. Use of a composition according to claim 1 or a produce according to claim 4 for enhancing sperm count or density in a mammalian, preferably human, male.

9. Use of a composition according to claim 1 or a produce according to claim 4 for enhancing sperm count and density in a mammalian, preferably human, male.

10. Method for enhancing the fertility of a human male wherein an effective, sperm count and density enhancing amount of a composition comprising folic acid and a carnitine or acyl-camitine, or salts thereof, is administered to said male.