HK1225295B - Composition useful for promoting female fertility - Google Patents

Description

Composition for promoting female fertility

Technical Field

The field of the invention relates to female fertility during hormonal or pharmacological stimulation.

Background Art

Female infertility affects approximately 48 million women worldwide.

In the United States, approximately 10% of couples of childbearing age have difficulty conceiving.

Ovulation stimulation with fertility hormones or medications is the mainstay of treatment for women with infertility due to ovulatory disorders. These medications regulate or induce ovulation.

Medications used to stimulate ovulation are:

Clomiphene citrate: This medication works by acting on the pituitary gland to induce ovulation. It is often used for women with polycystic ovary syndrome (PCOS) or other ovulation problems. Clomiphene citrate is used as a fertility drug in women by inducing superovulation, the release of multiple eggs in a given menstrual cycle. This medication is taken orally.

Bromocriptine and cabergoline: These drugs are used to reduce the amount of prolactin released by the pituitary gland in women who have problems ovulating due to high prolactin levels. Potential side effects include nausea, vomiting, nasal congestion, headache, dizziness, fainting, and low blood pressure.

Medrol: A steroid given daily for four days during the cycle to assist with pre-implantation embryos.

Injectable hormones used to stimulate ovulation are:

-Human chorionic gonadotropin (hCG), often combined with other fertility medications and nutritional supplements to trigger the ovaries to release one or more mature eggs;

-Follicle-stimulating hormone (FSH);

- Human menopausal gonadotropin (hMG). This drug combines both FSH and LH (luteinizing hormone).

- Gonadotropin-releasing hormone (GnRH). This hormone stimulates the release of FSH and LH from the pituitary gland.

These hormones are rarely prescribed in the United States.

- Gonadotropin-releasing hormone agonists (GnRH agonists);

-Gonadotropin-releasing hormone antagonists (GnRH antagonists).

All of these drugs/hormones can be used to increase oocyte fertilization in vivo or in vitro.

In vitro fertilization (IVF) is a method in which an oocyte is fertilized by sperm outside the body (external). When other methods of assisted reproductive technology have failed, IVF is the primary therapy for infertility. The method involves monitoring and stimulating the female ovulation process, removing one or more oocytes from the female ovary, and fertilizing them by sperm in a liquid culture medium in the laboratory. The fertilized oocyte (zygote) cultivated for 2-6 days in a growth medium is subsequently transferred to the patient's uterus for the purpose of establishing a successful pregnancy.

Fertility hormones and medications used in the initial management of women with ovulatory disorders have been reported to have side effects at www.webmd.com/infertility-and.../fertility-drugs .

It is reported in www.ncbi.nlm.nih.gov/pmc/articles/PMC2749720/ that female fertility is affected by oxidative stress caused by exposure to alcohol, tobacco smoke, and/or environmental pollutants.

It was reported in Curr Opin Obstet Gynecol. 2009 Jun;21(3):219–222 that the role of oxidative stress in female fertility is an area of ongoing research and urgent investigation; and identifying modifiable factors that reduce oxidative stress in the gynecological setting may be an inexpensive and non-invasive therapy to improve fertility. In order to improve the reproductive health of women (humans or animals), it has been recommended to provide a good multivitamin preparation and include adequate amounts of antioxidants and supplements in the diet.

Vitamin E is crucial for proper reproductive function in women. In fact, its chemical name, "tocopherol," comes from the Greek words tokos, meaning "offspring," and phero, meaning "to reproduce." Vitamin E protects hormones from oxidation. While vitamin E becomes less available in selenium-rich processed foods and through exposure to harmful oxidants, daily supplementation with 400-800 IU of vitamin E may improve fertility.

It was reported in Fertil Steril. 2008 Mar;89(3):668-76. Epub 2007 Jul 10 that folic acid supplementation can improve female fertility.

In Hum Reprod. 1999 Jul;14(7):1690-7 it was reported that oral supplementation of L-arginine improved ovarian response, endometrial receptivity and pregnancy rates in poor responders.

Anim. Reprod. Sci. 2012 Sep; 134(1-2): 69-75 reported that carnitine has beneficial effects on oocyte and embryo development in vitro or in vivo.

It was reported in Acta Obstet Gynecol Scand. 2007; 86(2): 218-22 that N-acetylcysteine was shown to be effective in inducing or enhancing ovulation in patients with polycystic ovary.

Vitamin D was reported to be associated with female reproduction in Eur J Endocrinol. 2012 May; 166(5): 765-78.

US Patent 6,569,857 teaches a method for increasing the likelihood of pregnancy by administering specific doses of vitamin B6 and folic acid to an animal over a period beginning at least two weeks prior to conception, while simultaneously increasing nutrient stores for embryonic or fetal development before and during pregnancy.

U.S. Patent 6,861,079 teaches a fertility kit for enhancing natural fertility, comprising specific dosage ranges of L-arginine, L-cysteine, selenium, vitamin C, vitamin E, zinc, vitamin B-6, para-aminobenzoic acid (PABA), vitamin A, folic acid, at least one phytoestrogen, and several devices for promoting ovulation.

As reported above, the use of fertility hormones/drugs has side effects, which may be mild or moderate.

Therefore, there is a need for new pharmaceutical/nutraceutical compounds/compositions for improving female fertility while reducing the side effects caused by the use of fertility hormones/drugs.

Summary of the Invention

The present invention provides a synergistic combination of biologically effective compounds for promoting female fertility. All components have also been studied separately to determine their individual efficacy.

Specifically, the present invention relates to a synergistic composition for promoting female fertility, which comprises L-carnitine, acetyl L-carnitine, N-acetyl cysteine as active ingredients, as well as several specific vitamins, amino acids, antioxidants and trace elements.

DETAILED DESCRIPTION

It has now been discovered that a combination composition comprising, as active ingredients, L-carnitine or a salt thereof, acetyl L-carnitine or a salt thereof; N-acetyl cysteine; and several specific vitamins, amino acids, antioxidants, and trace elements has an unexpected synergistic effect in promoting female fertility during pharmacological and/or hormonal stimulation.

Therefore, one object of the present invention is a combination composition comprising as active ingredients L-carnitine or a salt thereof, acetyl-L-carnitine or a salt thereof, vitamin B9 (folic acid), vitamin A, vitamin B12, vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), arginine, vitamin C, vitamin E (α-tocopherol), selenium, zinc, copper, iron, vitamin D and N-acetylcysteine.

The above-mentioned composition may further comprise a diluent and/or an excipient, and/or other active ingredients for promoting female fertility.

Another object of the present invention is a composition comprising:

(a) L-carnitine fumarate in a dosage of 2589 to 287.6 mg (corresponding to 1505.2 to 167.2 mg L-carnitine inner salt), preferably in a dosage of 1726 to 431.5 mg (corresponding to 1000 to 250 mg L-carnitine inner salt), and most preferably in a dosage of 863 mg (corresponding to 501.73 mg L-carnitine inner salt);

(b) acetyl-L-carnitine in a dosage of 750 to 25.0 mg, preferably in a dosage of 500 to 125 mg, and most preferably in a dosage of 250 mg;

(c) folic acid (vitamin B9) in a dose of 600 to 66.6 mcg, preferably in a dose of 400 to 100 mcg, and most preferably in a dose of 200 mcg;

(d) Vitamin A from beta carotene in a dosage of 2400 to 266.6 mcg, preferably in a dosage of 1600 to 400 mcg, and most preferably in a dosage of 800 mcg;

(e) Vitamin B12 in a dosage of 7.5 to 0.833 mcg, preferably in a dosage of 5.0 to 1.250 mcg, and most preferably in a dosage of 2.5 mcg;

(f) Vitamin B5 (pantothenic acid) in a dosage of 18.0 to 2.0 mg, preferably in a dosage of 12.0 to 3.0 mg, and most preferably in a dosage of 6 mg;

(g) Vitamin B6 (pyridoxine) in a dosage of 6.0 to 0.666 mg, preferably in a dosage of 4.0 to 1.0 mg, and most preferably in a dosage of 2 mg;

(h) arginine in a dosage of 1500 to 166.6 mg, preferably in a dosage of 1000.0 to 250.0 mg, and most preferably in a dosage of 500 mg;

(i) Vitamin C in a dosage of 270.0 to 30.0 mg, preferably in a dosage of 180.0 to 45.0 mg, and most preferably in a dosage of 90 mg;

(j) Vitamin E (α-tocopherol) in a dosage of 90.0 to 10.0 mg, preferably in a dosage of 60.0 to 15.0 mg, and most preferably in a dosage of 30 mg;

(k) Selenium in a dose of 150 to 16.6 mcg, preferably in a dose of 100.0 to 25.0 mcg, and most preferably in a dose of 50 mcg;

(1) zinc, in a dosage of 30.0 to 3.33 mg, preferably in a dosage of 20.0 to 5.0 mg, and most preferably in a dosage of 10 mg;

(m) copper in a dose of 4.95 to 0.55 mg, preferably in a dose of 3.3 to 0.825 mg, and most preferably in a dose of 1.65 mg;

(n) iron in a dose of 42.0 to 4.66 mg, preferably in a dose of 28.0 to 7.0 mg, and most preferably in a dose of 14 mg;

(o) Vitamin D in a dosage of 15.0 to 1.66 mcg, preferably in a dosage of 10.00 to 2.50 mcg, and most preferably in a dosage of 5 mcg;

(p) N-acetylcysteine, in a dosage of 150 to 16.6 mg, preferably in a dosage of 100.0 to 25.0 mg, and most preferably in a dosage of 50 mg.

Another subject of the present invention is the composition mentioned above for promoting female fertility.

Another object of the present invention is the above-mentioned composition for promoting female fertility in combination with drugs and/or hormonal stimulation.

Another object of the present invention is the above-mentioned composition for promoting female fertility in combination with drugs for supporting ovulation and/or hormonal stimulation.

Another subject of the present invention is the composition mentioned above for use in promoting female fertility during pharmaceutical and/or hormonal stimulation to support the fertilization of the oocyte.

Another subject of the present invention is the above-mentioned composition for use in promoting female fertility during pharmaceutical and/or hormonal stimulation for promoting in vivo fertilization of oocytes.

Another subject of the present invention is the composition mentioned above for use in promoting female fertility during pharmaceutical and/or hormonal stimulation for promoting in vitro fertilization of oocytes.

Another object of the present invention is the above-mentioned composition for reducing the side effects caused by the use of hormones and/or drugs for promoting female fertility.

The composition of the present invention can be administered to a subject in need thereof at least one month before starting drug and/or hormone stimulation; the administration must be started at least one week before starting drug and/or hormone stimulation; the administration can continue after oocyte fertilization under the control of a physician.

Another object of the present invention is the use of the composition mentioned above as a dietary supplement.

The composition of the present invention may further comprise coenzymes, other minerals, antioxidants, vitamins, and drugs for promoting female fertility or assisting in the treatment of infertility.

The salt of L-carnitine or acetyl L-carnitine refers to any salt of L-carnitine or acetyl L-carnitine with an acid that does not produce toxicity or side effects.

Non-limiting examples of such salts are chloride, bromide, orotate, aspartate, acid aspartate, acid citrate, magnesium citrate, phosphate, acid phosphate, fumarate and acid fumarate, magnesium fumarate, lactate, maleate and acid maleate, oxalate, acid oxalate, pamoate, acid pamoate, sulfate, acid sulfate, glucose phosphate, tartrate and acid tartrate, glycerophosphate, mucate, magnesium tartrate, 2-amino-ethanesulfonate, magnesium 2-amino-ethanesulfonate, methanesulfonate, choline bitartrate, trichloroacetate, and trifluoroacetate.

A list of pharmaceutically acceptable salts approved by the FDA is given in the publication Int. J. of Pharm. 33 (1986), 201-217.

According to the present invention, L-carnitine, acetyl-L-carnitine, vitamin B9, vitamin A, vitamin B12, vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), arginine, vitamin C, vitamin E (α-tocopherol), selenium, zinc, copper, iron, vitamin D, and N-acetylcysteine can be administered in a "co-ordinated manner." "Co-ordinated manner" of the aforementioned compounds refers to co-administration (i.e., substantially concurrent administration) or sequential supplementation of L-carnitine and at least one acetyl-L-carnitine, vitamin B9, vitamin A, vitamin B12, vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), arginine, vitamin C, vitamin E (α-tocopherol), selenium, zinc, copper, iron, vitamin D, and N-acetylcysteine, or administration of a composition comprising a combination of the aforementioned active ingredients and optionally further comprising a mixture of one or more pharmaceutically acceptable excipients or diluents.

The following non-limiting examples further illustrate the present invention.

Example 1

Materials and methods

animal

The study used CD1 mice (60 females and 60 males; 6 weeks of age; weighing ≥16.2 g; 5 mice/group). Mice were acclimated for one week before weighing. Female mice began superovulation 2 to 3 days after reaching the appropriate weight. All mice were maintained under SPF conditions at a temperature of 21 to 24°C with a 12:12-h light:dark cycle (lights, 0700 to 1900). Mice were housed in static microisolator cages with ad libitum access to food and water.

deal with

All female mice, except the "control" group, were orally treated (0.5 ml, gastric gavage, twice a day) with a composition of the invention (DNC064) having the following composition for 7 days (including days when the mice were treated with hormones for superovulation):

L-Carnitine Fumarate 0.4mg+

Acetyl-L-carnitine HCl. 0.12mg+

Vitamin B12 0.0012mcg+

Vitamin B9 0.09mcg+

Vitamin B6 0.0009mg+

Vitamin B5 0.003mg+

Vitamin A 0.4mcg+

Vitamin D 0.0022mcg+

Selenium 0.022mcg+

Vitamin C 0.04mg+

Vitamin E 0.014mg+

Zinc 0.004mg+

Copper 0.0008mg+

Iron 0.007mg+

L-Arginine 0.24mg+

N-acetylcysteine 0.24mg,

Their combinations are reported in Table 1.

Superovulation and pairing

The superovulation technique used is described in J.Am.Assoc.Lab.Anim.Sci. 2011 Jul 50(4); 471-478. Briefly, female mice were treated with 5 IU (0.1 mL, intraperitoneal) pregnant mare serum gonadotropin (PMSG). These mice received 5 IU (0.1 mL IP) of human chorionic gonadotropin (HCG) 47 to 49 hours after the last injection of PMSG. Immediately after the HCG injection, the female mice were paired with male mice in a 1:1 ratio. All female mice underwent this superovulation treatment.

Oocyte collection and analysis

On the second day after pairing, all female donors were euthanized by cervical dislocation and oviducts were collected from all female mice and placed in 2 mL M2 medium (Sigma-Aldrich) in a 35-mm culture dish (Fisher Scientific). Each oviduct was then moved to a culture dish containing 2 mL M2 medium (Sigma-Aldrich) and 75 μL hyaluronidase (10 mg/mL; Sigma-Aldrich), where the ampulla was torn open to release the oocytes.

After all oviducts in the group were processed, all oocytes were collected and placed in 100-μL KSOM drops (Millipore, Billerica, MA) in embryo-test mineral oil (Sigma-Aldrich) equilibrated to 37° C. in 5% CO _2. The oocytes were incubated for 24 h, after which the drops were scored for the number of 2-cell fertilized oocytes.

The results obtained are reported in Table 1 below.

Table 1

Statistical analysis

Significance was calculated using the Student's t-test, and a value of p < 0.05 was considered to represent a significant difference.

The results reported in Table 1 show that the treatment according to the invention with either the single components or different combinations thereof statistically increased the number of fertilized oocytes.

The compositions of the present invention may be administered in any suitable form for oral administration.

Examples of administration forms are liquid, semi-liquid or solid forms in sachets, pills, vials, gels or liposomes.

L-Carnitine and acetyl L-carnitine are known compounds and their preparation is described in US Pat. No. 4,254,053.

Vitamin A, vitamin B9, vitamin B12, vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), arginine, vitamin C, vitamin E (alpha-tocopherol), selenium, zinc, copper, iron, vitamin D, and N-acetylcysteine have long been widely commercially available for human use.

The compositions of the present invention are composed of active ingredients that are familiar and already in use by practitioners in the medical field.

Therefore, it is very easy to purchase because these ingredients are products that have been available on the market for a long time and are of a grade suitable for human administration.

For any compound, the therapeutically effective dose can be estimated initially in cell culture assays or in animal models, usually mice or rats.

Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine suitable doses and routes for administration in humans.

The precise effective dose for a human subject will depend on the severity of the disease state, the subject's general health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combination, reaction sensitivity, and tolerance/response to treatment. Such amounts can be determined by routine experimentation and are within the judgment of the clinician.

In the following, non-limiting examples of compositions according to the invention are reported.

Composition 1

Composition 2

Composition 3

Composition 4

Claims (20)

1. A combination composition for promoting female fertility, comprising: -L-carnitine fumarate, at a dose of 2589 to 287.6 mg (corresponding to 1505.2-167.2 mg L-carnitine internal salt); -Acetyl-L-carnitine, dosage from 750 to 25.0 mg; - Folic acid (vitamin B9), dosage 600 to 66.6 mcg; - Vitamin A from beta-carotene, at a dose of 2400 to 266.6 mcg; - Vitamin B12, dosage 7.5 to 0.833 mcg; - Vitamin B5 (pantothenic acid), dosage 18.0 to 2.0 mg; - Vitamin B6 (pyridoxine), dosage 6.0 to 0.666 mg; -Arginine, dosage 1500 to 166.6 mg; - Vitamin C, dosage 270.0 to 30.0 mg; - Vitamin E (α-tocopherol), dosage 90.0 to 10.0 mg; -Selenium, at a dose of 150 to 16.6 mcg; - Zinc, dosage 30.0 to 3.33 mg; - Copper, dosage from 4.95 to 0.55 mg; - Iron, at a dose of 42.0 to 4.66 mg; - Vitamin D, dosage 15.0 to 1.66 mcg; -N-acetylcysteine, dosage 150 to 16.6 mg. 2. The combination composition of claim 1, comprising: -L-carnitine fumarate, dosage from 1726 to 431.5 mg (corresponding to 1000-250 mg L-carnitine internal salt); -Acetyl-L-carnitine, dosage is 500 to 125 mg; - Folic acid (vitamin B9), dosage 400 to 100 mcg; - Vitamin A derived from beta-carotene, at a dose of 1600 to 400 mcg; - Vitamin B12, dosage 5.0 to 1.250 mcg; - Vitamin B5 (pantothenic acid), dosage 12.0 to 3.0 mg; - Vitamin B6 (pyridoxine), dosage 4.0 to 1.0 mg; -Arginine, dosage from 1000.0 to 250.0 mg; - Vitamin C, dosage 180.0 to 45.0 mg; - Vitamin E (α-tocopherol), dosage 60.0 to 15.0 mg; -Selenium, at a dose of 100.0 to 25.0 mcg; - Zinc, dosage 20.0 to 5.0 mg; - Copper, dosage from 3.3 to 0.825 mg; - Iron, dosage is 28.0 to 7.0 mg; - Vitamin D, dosage 10.00 to 2.50 mcg; -N-acetylcysteine, dosage from 100.0 to 25.0 mg. 3. The combination composition of claim 1, comprising: -L-carnitine fumarate, the selected dose is 863mg (corresponding to 501.73mg L-carnitine inner salt); -Acetyl-L-carnitine, dosage 250mg; - Folic acid (vitamin B9), dosage 200mcg; - Vitamin A derived from beta-carotene, at a dose of 800 mcg; - Vitamin B12, dosage 2.5 mcg; - Vitamin B5 (pantothenic acid), dosage 6mg; - Vitamin B6 (pyridoxine), dosage 2mg; -Arginine, dosage 500mg; - Vitamin C, dosage 90mg; - Vitamin E (α-tocopherol), dosage 30mg; -Selenium, dosage 50mcg; - Zinc, dosage 10mg; - Copper, dosage 1.65 mg; - Iron, dosage 14mg; - Vitamin D, dosage 5 mcg; -N-acetylcysteine, dosage 50mg. 4. The combination composition of claim 1, further comprising coenzymes, minerals, antioxidants, vitamins, and medicaments for promoting female fertility. 5. The combination composition of claim 1, as a dietary supplement. 6. The combination composition of claim 1, used to promote female fertility under pharmaceutical and/or hormonal stimulation. 7. The combination composition of claim 1, used to promote female fertility during medicament and/or hormonal stimulation for supporting ovulation. 8. The combination composition of claim 1, used to promote female fertility during drug and/or hormone stimulation for promoting oocyte fertilization. 9. The combination composition of claim 1, used to promote female fertility during drug and/or hormone stimulation for promoting in vivo fertilization of oocytes. 10. The combination composition of claim 1, used to promote female fertility during pharmaceutical and/or hormonal stimulation for promoting in vitro fertilization of oocytes. 11. The combination composition of claim 1, wherein the salt of said L-carnitine or acetyl L-carnitine is selected from: chloride, bromide, orotate, aspartate, magnesium citrate, phosphate, fumarate, lactate, maleate, oxalate, dihydroxynaphthyl salt, sulfate, glucose phosphate, tartrate, glycerol phosphate, mucilage, 2-aminoethanesulfonate, methanesulfonate, choline tartrate, trichloroacetate, or trifluoroacetate. 12. The combination composition of claim 1, for oral administration. 13. The combination composition of claim 12, wherein the administration is initiated at least one month prior to the start of drug and/or hormone stimulation. 14. The combination composition of claim 12, wherein the administration is initiated at least one week prior to the start of drug and/or hormone stimulation. 15. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility. 16. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility under pharmaceutical and/or hormonal stimulation. 17. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility during medicament and/or hormonal stimulation for supporting ovulation. 18. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility during medicaments for promoting oocyte fertilization and/or hormone stimulation. 19. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility during medicament and/or hormone stimulation for promoting in vivo fertilization of oocytes. 20. Use of the combination composition of any one of claims 1-4 in the preparation of a medicament for promoting female fertility during medicament and/or hormone stimulation for promoting in vitro fertilization of oocytes.