AU2015263221A1

AU2015263221A1 - Food compositions comprising tungsten (VI) salts

Info

Publication number: AU2015263221A1
Application number: AU2015263221A
Authority: AU
Inventors: Agnes Arbat Bugie; Ignacio Canals Almazan
Original assignee: Oxolife SL
Current assignee: Oxolife SL
Priority date: 2014-05-21
Filing date: 2015-05-21
Publication date: 2016-12-22
Anticipated expiration: 2035-05-21
Also published as: AU2015263221B2; TW201600022A; UY36133A; ES2551828B1; NZ726934A; WO2015177393A1; ES2551828A1; AR100545A1

Abstract

The invention relates to food compositions comprising tungsten (VI) salts and to the production thereof, for stimulating and normal reproduction and fertility in a non-diabetic female mammal.

Description

1

DESCRIPTION “Food compositions comprising tungsten (VI) salts”

The present invention relates to the use of tungsten (VI) salts for the preparation of food compositions, as well as to said food compositions, particularly for favoring normal reproduction and fertility in a non-diabetic female mammal.

PRIOR ART

Fertility in mammals is a multistage process that is not totally efficient naturally. Factors such as age, eating habits or life style, among others, modulate the success of this process. In this sense, it is known for example that pregnancy rate in women 20 to 24 years of age is about 86%, while it diminishes to 50% in women 35 to 32 years of age [Management of the Infertile Woman by Helen A. Carcio; The Fertility Sourcebook by M. Sara Rosenthal, ASAS Summary of FAIR 2012], Other factors such as overweight or underweight are also responsible for delays in achieving pregnancy naturally or even for the impossibility in achieving same [Fertil Steril 2013;100:631-7\. It is estimated that about 66% of couples do not conceive naturally during the first 3 months of unprotected sexual intercourse. This figure decreases to 15% of couples in the course of the first year of unprotected sexual intercourse. Infertility is defined (WHO, ASRM, NICE) as the inability to conceive after 12 months of regular, unprotected sexual intercourse. The term “subfertility” has also been coined, particularly in Europe, to define those couples with any form or degree of reduced fertility leading to delay in conception [Gnoth C et al Definition and prevalence of subfertility and infertility, Human reproduction 2005;20(5):1144-1147\.

The main causes of infertility among women include ovulatory dysfunctions, pathologies of the reproductive tract, reduced oocyte quality and follicular depletion inherent to aging. However, there is also a considerable percentage of women with unexplained infertility, also referred to as idiopathic infertility, one of the possible causes of which relates to deficiencies in the implantation process. 2

Follicular development, ovulation, migration of immature egg (oocyte), as well as subsequent conception and zygote implantation on the uterine wall is regulated by hormonal secretions of certain endocrine organs such as the pituitary gland, the hypothalamus and the thyroid gland, for example. Among the different biological causes of infertility in women, causes linked to disorders in the hormonal stimuli regulating this whole process can also be pointed out.

Different treatments for female infertility including, among others, the administration of medicinal products for treating hormonal problems involving ovulation disruption, are known. Likewise, some beneficial effects have been postulated with various treatments based on taking vitamin supplements, particularly vitamin B, vitamin C, vitamin E and folic acid, mineral supplements such as selenium, zinc or iron complexes or salts, essential fatty acids (omega-3), as well as extracts from plants such as chaste tree {Vitex agnus-castus), damiana, licorice, red clover flower, chasteberry, black cohosh, dong quai (Angelica sinensis), wild yam or sweet potato {Dioscorea villosa), false unicorn root, green tea, nettles (Urtica dioica), wild oats (Avena sativa), dandelion (Taraxacum officinale), etc., although the efficacy has not been clearly demonstrated in any of these treatments.

The percentage of pregnancies achieved by means of the aforementioned treatments has limitations. In this sense, for example, it has been observed that treatment of women with irregular or no ovulation by means of administering clomiphene citrate, a drug of the stilbene family, allows restoring ovulation in a high percentage, but the pregnancy rate remains low, equal to or less than about 50%.

In vitro fertilization treatments are very effective in the oocyte fertilization step. However, the embryo implantation rate on the uterine wall is low. This promotes multiple embryo transfers per each in vitro fertilization cycle, with the consequence of a higher percentage of risky multiple pregnancies.

Finally, it is known that metabolic disorders such as diabetes or obesity entail a limitation of fertility. It is known that partial or complete recovery of glycemia, insulinemia and/or body weight in diabetic or obese female mice with damaged reproductive function, involves an improvement in fertility. 3

Different pharmacological treatments, such as metformin or tungsten (VI) salts, or even life style changes, have been shown to improve diabetes or insulin disorders, such as insulin deficiency or insulin resistance, for example, completely or partially recovering the reproductive function, when the infertility is due to diabetes or insulin disorders.

In the specific case of sodium tungstate, it is known that rats with diabetes and insulinopenia induced by the injection of streptozotocin partially recover circulating insulin levels after a prolonged treatment (10 weeks) with sodium tungstate as a result of partially reversing diabetes. Subsequently, when the female rats partially recovered from diabetes mate with healthy male rats, it is observed that, parallel to the partial recovery from diabetes, they have partially recovered their reproductive capacity. Particularly, it is observed that the percentage of births with respect to the number of positive curettages in female mice which had partially recovered from diabetes after treatment with sodium tungstate increases to 66%, a percentage which is lower than non-diabetic female mice where the percentage is 100%. (cf. J. Ballester et. al., “Tungstate administration improves the sexual and reproductive function in female rats with streptozotocin-induced diabetes”, Human Reproduction, 2007, vol. 22, pp. 2128-2135). Nevertheless, the tungsten (VI) salts were neither observed nor postulated to have effect on non-diabetic female animals.

Tungsten is found in trace amounts in animals and plants. For example, tungsten content of up to a maximum of 100 mg/kg has been described in plants [Gbaruko B.C. & Igwe J.C. Global Tungsten: Occurrence, Chemistry, Environmental and Health Exposure Issues. Journal of Environmental Research 2007;1(1):27-32].

In view of the foregoing, despite the extensive research and progress in understanding and handling the reproductive process in mammals and particularly in humans, there are still many couples who do not benefit from the different approaches available today for increasing fertility as they are ineffective. Therefore, there is still a need to find new alternatives for improving natural reproductive efficiency and/or treating female infertility that provide greater efficacy. 4

DISCLOSURE OF THE INVENTION

It has now been found that the administration of a tungsten (VI) salt or a solvate of said salt is effective for favoring normal reproduction and fertility in a non-diabetic female mammal. Despite the fact that sodium tungstate was known to be able to partially reverse impairments in the reproductive function of diabetic female mice because it normalizes blood glucose levels, the possibility of sodium tungstate having an effect on the function of the female reproductive system in non-diabetic female mammals, favoring normal reproduction and fertility, has not been described nor suggested. A first aspect of the present invention therefore relates to the use of a tungsten (VI) salt or a solvate thereof for the preparation of a food composition for favoring normal reproduction and fertility in a non-diabetic female mammal. A second aspect of the present invention relates to a food composition which comprises a tungsten (VI) salt or a solvate thereof at a concentration greater than 100 mg/kg.

DETAILED DISCLOSURE OF THE INVENTION

As indicated above, according to a first aspect the present invention relates to the use of a tungsten (VI) salt or a solvate thereof for the preparation of a food composition for favoring and/or contributing to normal reproduction and fertility in a non-diabetic female mammal.

In the context of the present invention, the term “food composition” would encompass any solid or liquid tungsten (VI) salt-enriched food, as well as any nutritional supplement or complement containing at least one tungsten (VI) salt.

The term “favoring normal reproduction and fertility” or “contributing to normal reproduction and fertility” refers to contributing to normal reproduction and fertility or improving normal reproduction and fertility (reducing the times for achieving pregnancy), “normal reproduction and fertility” being understood as a state of fertility 5 in which pregnancy is achieved following the definitions provided above: * in a subfertility situation: pregnancy is achieved in a period of 12 months of regular, unprotected sexual intercourse; and * in an infertility situation: pregnancy is achieved after a period 12 months of regular, unprotected sexual intercourse. The need to restore and/or promote ovulation, improve oocyte and embryo quality, increase zygote implantation on the uterine wall, regulate hypothalamic-pituitary-ovarian axis impairment, including polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic kidney failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia are included herein.

According to a particular embodiment, the food composition of the invention contains a total concentration (i.e., the sum of all tungsten (VI) salts contained in the composition) of tungsten salts of at least 100 mg/kg; according to other particular embodiments, said total concentration is preferably of at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 300 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least 850 mg/kg, at least 900 mg/kg or at least 850 mg/kg.

According to another particular embodiment, the total concentration of tungsten (VI) salts is equal to or less than 1000 mg/kg.

The female mammal in the context of the present invention can be any mammal, including, but not limited to, humans, mice, rats, rabbits, dogs, cats, guinea pigs, hamsters, cows, horses, pigs, sheep, goats, etc.

According to a particular embodiment, the female mammal is a human. According to another particular embodiment, the food composition will be suitable for the administration of tungsten salts in a daily dose between 0.001 mg per kg of body weight of the female mammal (hereinafter, mg/kg/day) and the maximum tolerated dose for the corresponding female mammal species.

According to a further particular embodiment, the maximum daily dose will be 1000 6 mg/kg/day; according to additional particular embodiments, the administration would be carried out in doses of at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 0.5 mg/kg/day, at least 1 mg/kg/day, at least 10 mg/kg/day, at least 25 mg/kg/day, at least 50 mg/kg/day, at least 100 mg/kg/day, at least 200 mg/kg/day, at least 300 mg/kg/day, at least 400 mg/kg/day, at least 500 mg/kg/day, at least 600 mg/kg/day, at least 700 mg/kg/day, at least 800 mg/kg/day or at least 900 mg/kg/day.

The tungsten salt generally comprises a tungsten (VI) anion and a dietetically or pharmaceutically acceptable cation. The cation is preferably an alkaline or alkaline earth cation. The cation is more preferably selected from the group consisting of sodium, potassium, magnesium, calcium and zinc. According to a particular embodiment, the tungsten (VI) salt is the tungsten (VI) sodium salt.

The tungsten (VI) anion is in turn preferably selected from the list consisting of W042", HW04", W2072- and HW207". The anion is preferably W042'.

According to a particular embodiment, the salt is in the form of a solvate; the solvate is preferably a hydrate, more specifically a dihydrate.

All the features listed individually for different elements of the invention can be combined with one another, all the possible combinations being included within the scope of the present invention. For example, according to a preferred embodiment the solvate of the tungsten (VI) salt is a dihydrate and the cation is a sodium cation. Similarly, the rest of the possible combinations is also intended to be included within the scope of the present invention.

The food composition can be a liquid composition, i.e., a beverage, according to terms most generally used in the society. In the context of the present invention, such liquid composition includes, but is not limited to, any beverage selected from the group consisting of animal or plant milk, as well as any derivative thereof, such as for example, milk shakes, yogurt, kefir, etc.; fruit and/or vegetable juices; still water or sparkling water, or flavored or sweetened water or beverages (by means of nutritive sweeteners (sucrose, fructose...) or artificial sweeteners); seasonings, such as for example, any salsa, dressing, ketchup, oil, vinegar or vinegar preparations; 7 alcoholic beverages of any type; tea, coffee; as well as all types of refreshing beverages or soft drinks, or energizing beverages.

The food composition can also be a solid composition. Such solid composition can, for example, be selected from, but is not limited to, the group consisting of animal or plant milk derivatives, such as cheese, butter, margarine and tofu; any type of bread, including fresh, packaged or frozen bread, sliced bread, wholemeal bread, spiced bread, sweet bread, salty bread, etc; pasta prepared from any cereal flour, such as wheat or semolina flour (macaroni, spaghetti, noodles, etc); baked goods, including cakes, cookies, muffins, doughnuts, etc.; infusions, tea or coffee, in bulk or in sachets, for preparing beverages; jellies, candies, including gummy candies, better known as “soft fruit candies”; as well as any type of solid seasoning, for example, oregano, salt, coriander, parsley, basil, etc, or mixtures thereof.

Finally, the food composition can also be a nutritional, dietary or food supplement or complement, any of these terms being used in the context of the present invention in an equivalent manner. These terms are normally used for compositions consumed orally, which contain an ingredient intended for complementing the diet, in the case of the present invention, the tungsten (VI) salt/salts. They shall never replace a conventional food, or be the only component of a meal or of the diet. They can be found in different presentations, such as pastilles, pills, tablets, capsules, soft gelatin capsules, gelatin capsules, wafers, effervescent tablets, liquids (solution, suspension, syrup), granules and powders, all of which are included as particular embodiments within the scope of the present invention. Dietetically or pharmaceutically acceptable excipients are obvious for the skilled person for obtaining any of the preceding presentations, and they are included within the scope of the present invention.

According to a particular embodiment, any of the preceding food compositions (solid composition, liquid composition or nutritional supplement/complement) comprises in their composition at least one nutritive sweetener, such as sucrose or fructose. Ingestion of these nutritive sweeteners must be controlled and limited in diabetic patients, therefore a food composition including such nutritive sweetener would be contraindicated in diabetic mammals or would at least be taken into account in controlling their diet. 8

According to a particular embodiment, the non-diabetic female mammal is selected from the group consisting of female mammals that require restoring and/or promoting ovulation, improving oocyte and embryo quality, increasing zygote implantation on the uterine wall, regulating hypothalamic-pituitary-ovarian axis impairment, including polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic kidney failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia.

According to another particular embodiment, the non-diabetic female mammal is selected from the group consisting of female mammals having insulin resistance (also known as resistance to insulin or insulinic resistance); when insulin resistance occurs together with hyperglycemia, it can lead to the development of diabetes, nevertheless insulin resistance alone does not lead to a diabetic condition. The female mammals having insulin resistance are preferably selected from the group consisting of female mammals having one or more of the following conditions: obesity or overweight, metabolic syndrome, prediabetes, polycystic ovarian syndrome, hypertension, heart disease, hyperlipidemias or dyslipidemia, hyperthyroidism, hyperparathyroidism, hyperleptinemia or leptin resistance, sedentary life style, eating disorders, obstructive sleep apnea syndrome, fetal malnutrition, Prader-Willi syndrome, Rabson-Mendenhall syndrome, fatty liver, leprechaunism, pathologies associated with excessive glucocorticoid secretion (e.g., acromegaly), insulin resistance caused by pharmacological treatments (e.g., glucocorticoids, thiazide diuretics, beta-blockers), stress or early menarche [Egas Be jar, Daniela et.al Insulinorresistencia/lnsulin resistance. Medicina (Guayaquil);10(2):159-166, April 2005; Graves, Thomas K. WHY ISN’T THIS INSULIN WORKING? Western Veterinary Conference 2013 (SA113); Ximena Gaete V. Adelanto de la pubertad en Chile y el mundo. Rev Chil Pediatr 77 (5); 456-465, 2006; Leszek Szablewski Glucose Homeostasis and Insulin Resistance (elSBN: 978-1-60805-) Bentham e-books]. Many of these indications have been described to be unrelated to infertility, including prediabetes [Acta Diab. Latina 4, 507, 1967\.

Another aspect of the present invention relates to a food composition which comprises tungsten (VI) salt or salts or a solvate thereof at a concentration of at least 9 100 mg/kg. According to particular embodiments, the tungsten (VI) salt or salts are comprised in the composition at concentrations of at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 300 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least 850 mg/kg, at least 900 mg/kg or at least 850 mg/kg.

All the definitions and preferred meanings provided above in relation to the first aspect of the invention are applicable to this second aspect of the invention. Therefore, for example, the tungsten (VI) salt will comprise a tungsten (VI) anion and a dietetically or pharmaceutically acceptable cation, which will preferably be an alkaline or alkaline earth cation, even more preferably a sodium, potassium, magnesium and calcium and zinc cation. Particularly, the salt will be tungsten (VI) sodium salt. The tungsten (VI) anion is once again selected from W042", HW04“, W2072- and HW207" ions, preferably W042-, and in the case of being presented in the form of a solvate, the solvate will preferably be a hydrate, more specifically a dihydrate. The rest of the particular embodiments provided above for the first aspect also relate to this second aspect.

The food composition of the second aspect of the invention can be a liquid composition or beverage, a solid composition or a nutritional supplement or complement (also referred to as dietary or food supplement or complement).

The liquid composition has already been defined above in relation to the first aspect of the invention, and all the preferred embodiments are also applicable to this second aspect of the invention.

Likewise, the solid composition has been defined above in relation to the first aspect of the invention, and all the preferred embodiments are also applicable to this second aspect of the invention.

Similarly, the nutritional supplement or complement (or dietary or food supplement or complement) has been defined above in relation to the first aspect of the invention, and all the preferred embodiments are also applicable to this second aspect of the invention. 10

The composition preferably comprises a nutritive sweetener, such as sucrose or fructose. A series of non-limiting, illustrative examples of the present invention are included below.

EXAMPLES

Example 1. Description of the animal model (IRS2 -/- female mice)

The mouse model used for determining the activity of tungsten (VI) salts for favoring normal reproduction and fertility is an Irs2 gene knock-out mouse, IRS27 [Burks et. al., “IRS-2 pathways integrate female reproduction and energy homeostasis”, Nature, 2000, vol. 407, pp. 377-382\. Irs2 gene deletion translates into a clear sexual dimorphism in relation to fertility and carbohydrate metabolism.

The male mice of this model have insulin resistance and severe hyperglycemia that started at an early age. In contrast, the female mice remain relatively euglycemic at an early age and develop slight insulin resistance which remains until a later age (4-5 months). IRS2_/" female mice at an early age, about 10 weeks old, show a low follicular development and persistent anovulation, accompanied by the absence of estrous cycle in most mice. The pregnancy rate in IRS2_/" female mice is 9% compared to the rate of 100% in IRSwt (IRS-2+/+ wild type) female mice. Given that female mice at these ages remain euglycemic and that they only develop slight insulin resistance, the profound impairment in fertility is not a direct result of anomalies in glucose metabolism.

Example 2. Ovulation, implantation and pregnancy study A. Animals

Ten IRS2_/" female mice of ages comprised between 6 and 8 weeks.

Six “wild-type” (^82^) male mice of ages comprised between 6 and 8 weeks. 11

The female and male mice were housed separately in normal conditions, i.e., a 12h light/darkness cycle and controlled temperature and humidity. The animals were fed at will (also referred to as ad libitum) with a standard feed diet. B. Method

Pre-treatment phase

After an acclimatization period, the IRS27 female mice were housed in groups of 4-6 mice/cage. Tungstate salt-free drinking water was administered to the animals during the pre-treatment phase (2 weeks).

Treatment phase

Sodium tungstate was administered in the drinking water (ad libitum) by means of a solution of 2 mg/ml of sodium tungstate dihydrate (marketed by Carlo Erba) in distilled water after the pre-treatment phase (day 0 of treatment) and up to 4 weeks before sacrificing the animals. The daily dose of sodium tungstate ingested by the mice was about 180 mg/kg of body weight.

Mating

After the first three weeks of treatment, the IRS2"7" female mice were housed in cages in pairs together with a ^82^ male mouse continuously.

The mice were observed daily to look for signs of pregnancy or birth.

After 4 weeks, the male mice were exchanged between the cages and they were kept therein for another 4 weeks.

The administration of tungstate was maintained for 8 weeks in which male and female mice were housed together. After these 8 weeks, treatment was withdrawn and the male and female mice were housed together for 4 additional weeks.

Sacrifice 12

After this period, the female mice were sacrificed and biopsy was carried out to look for indications of pregnancy. C. Results

Results of the ovulation study

During the pre-treatment period and during the first 3 weeks of treatment, vaginal smears were performed in 6 randomly chosen female mice on days -8; -5; -2; -1; 7; 8; 14; 15 and 22, to determine the phase of the estrous cycle they were in.

Between 1 and 2 ml of saline solution was introduced into the vagina of the mice with a Pasteur pipette. Vaginal exudate was collected with the same pipette and spread on a slide. Once air-dried, it was fixed and stained with the Papanicolau technique.

The Papanicolau technique comprises staining the vaginal smears fixed on the slide in the following manner: -10 immersions in 50% v/v alcohol; - Immersing in Harris hematoxylin solution for 3 minutes; - Rinsing with running water; -10 immersions in acid alcohol (1% hydrochloric acid); - Rinsing with running water; -10 immersions in 95% v/v alcohol; - Immersing in OG-6 solution for 30 seconds; -10 immersions in 96% v/v alcohol; - Immersing in eosin solution for 1 minute; -10 immersions in 96% v/v alcohol; -10 immersions in 86% v/v alcohol; and -10 immersions in xylol

The preparations were analyzed by trained staff using a coding in a single-blind manner for eliminating observer bias.

The samples were identified in the following phases: diestrus, proestrus, estrus, metestrus, anestrus or non-evaluable. The cyclical nature of the four phases in 13 periods of 4 to 6 days are indicative of a normal estrous cycle, whereas the absence of this cyclical nature and the persistence in anestrus, diestrus or proestrus phases are indicative of the absence of estrous cycle. 5 Of the 10 female mice subjected to study, Table 1 summarizes the phases of the estrous cycle in the vaginal smears performed in 6 of these IRS2_/" female mice.

Table 1

Mouse Time (davs) No. -8 -5 -2 -1 7 8 14 15 22 72 A A P D E E-M E E A 898 D D P P E E E-M M M 926 D D P P E-M E-M D E P 928 D-P D P P E E E-M M A 942 A D P P E E-M E-M A D 972 D D P M M D D 10 The phases of the estrous cycle are the following: A: Anestrus; D: Diestrus; P: Proestrus; E: Estrus; and M: Metestrus.

The phases of the estrous cycle found in the vaginal smears of the IRS2_/" female mice of Table 1 show that during the pre-treatment period all the mice were in the 15 proestrus (P) or diestrus (D) phase, i.e., with the absence of estrous cycle.

However, after starting the administration of tungstate, it is observed that the IRS2_/" female mice are in the late phases of estrous cycle, i.e., estrus (E) and metestrus (M) phases, which is indicative of the recovery of a normal estrous cycle. 20

These results indicate that the administration of a tungsten (VI) salt allows quick recovery (on the seventh day of treatment) of the estrous cycle in infertile, nondiabetic IRS2_/" female mice in 100% of analyzed mice. 14

Results of the implantation and pregnancy study

After the cross-breeding period of the method of section B, the female mice were sacrificed and biopsies were carried out to look for indications of pregnancy.

Table 2 summarizes the age at the time of treatment, whether or not there was pregnancy and the number of embryos per female mouse.

Table 2

Mouse No. Age (months) at the start of Pregnancy Number of embryos implanted or young born treatment cross-breeding 72 10 12 YES 1 young 942 10 12 YES 8 young 928 10 12 YES 7 embryos 931 10 12 YES 3 young 898 10 12 YES 7 embryos 900 10 12 NO - 921 10 12 YES 6 embryos 926 10 12 YES 8 young 972 8 10 NO - 973 8 10 YES 7 embryos

The results of Table 2 show that while the pregnancy rate in untreated IRS2_/' female mice is 9%, the pregnancy rate in IRS2_/" female mice treated with tungstate increases to 80%. 15 Furthermore, these results also show that the mean of young/implanted embryos per pregnant female mouse is about 5, a number which can be considered comparable 15 to the number of young of a female mouse.

Therefore, the results of Tables 1 and 2 demonstrate that a tungsten (VI) salt is an effective treatment for recovering ovulation and/or increasing oocyte implantation. Therefore, the administration of a tungsten (VI) salt as defined in the present invention is effective for the treatment of infertility in non-diabetic female mammals.

Example 3. Glvcemia and body weight study A. Animals

Six IRS2 Λ female mice of ages comprised between 6 and 8 weeks.

The female mice were housed in normal conditions, i.e., a 12h light/darkness cycle and controlled temperature and humidity. The animals were fed at will (also referred to as ad libitum) with a standard feed diet. B. Method

Treatment phase

After an acclimatization period, sodium tungstate was administered in the drinking water (ad libitum) by means of a solution of 2 mg/ml of sodium tungstate dihydrate (marketed by Carlo Erba) in distilled water after the pre-treatment phase (day 0 of treatment) and for 12 days. C. Results

The body weight was monitored on days 0, 2, 5, 7, 9 and 12 of the treatment period, and the blood glucose was determined after 6 hours of fasting on each of the aforementioned days by means of drawing blood from the tail vein and with a glucose sensor (AccuTrend glucose sensor, Roche, Mannheim, Germany).

Table 3 summarizes the glycemic levels expressed in mg/dl, and Table 4 summarizes the body weight of the mice under study expressed in grams. 16

Table 3

Glycemia (mg/dl) R1327 R1345 R1347 R1354 R1376 R1381 Average Day 0 146 129 115 126 141 112 128.17 Day 2 136 128 118 110 105 107 117.33 Day 5 125 142 114 111 137 105 122.33 Day 7 140 110 106 89 133 135 118.83 Day 9 125 153 109 108 131 125 125.17 Day 12 136 116 111 86 123 141 118.83

Table 4

Body weight (g) 1327 1345 1347 1354 1376 1381 Average Day 0 22.7 15.33 18.21 16.5 13.99 15.45 17.03 Day 2 22.9 14.84 18.98 17.01 14.18 16.04 17.33 Day 5 22.61 14.89 18.2 16.61 13.79 15.72 16.97 Day 7 22.42 15.07 17.81 16.35 13.74 15.66 16.84 Day 9 22.85 15.56 17.9 16.89 14.5 16.39 17.35 Day 12 23.15 15.91 18.24 17.19 14.92 16.89 17.72 5

The results of Tables 3 and 4 show that during the administration of sodium tungstate no variations were observed in the body weight or in the glycemia in the first 12 days of treatment, the same period in which ovulation is successfully reestablished in the tested female mice from day 7 of treatment (cf. Table 1). 10

Therefore, the results of Tables 1 to 4 demonstrate that a tungsten (VI) salt is a treatment effective for recovering ovulation and/or increasing oocyte implantation, regardless of the changes in body weight and in carbohydrate metabolism. It is therefore demonstrated that the administration of a tungsten (VI) salt as defined in 15 the present invention or a composition containing such tungsten (VI) salt has a direct 17 effect on the female reproductive system and is therefore effective for favoring normal reproduction and fertility in non-diabetic female mammals.

Example 4. Endometrial embryonic adhesion study A. Model

An “in vitro" human-human embryonic adhesion model made up the HEC1-A endometrial cell line and the JEG-3 trophectoderm cell line was used to determine the effect of sodium tungstate on endometrial receptivity.

The JEG-3 cell line which simulates the trophectoderm cells of a human embryo are cells that grow in monolayers in the laboratory using low adherence plates and are capable of forming spheroids simulating the human embryo; this is one of the most widely used cell lines for conducting in vitro embryonic adhesion assay. B. Method

The cell lines were commercially purchased (American Type Culture Collection (ATCC); Rockville, MD, USA) for conducting the endometrial embryonic adhesion experiments. They were thawed and expanded in 4 passes for obtaining sufficient cells to perform all the assays.

The HEC1-A cells were plated in 24-well plates and cultured with McCoy 5A culture medium supplemented with 10% fetal bovine serum and 0.1% antibiotics (fungizone and penicillin), until reaching 90% confluence. After reaching the confluence, there was added to the culture medium sodium tungstate at a final concentration of 10 μΜ, as well as Withaferin A (as a control, a compound that prevents embryonic adhesion) or culture medium (baseline adhesion data), without any additional component, for 24 hours.

The JEG-3 cells were plated in low adherent plates with Eagle’s minimal essential medium (EMEM) culture medium supplemented with 10% fetal bovine serum and 0.1% antibiotics (fungizone and penicillin). Twenty-four hours before the adhesion assay, the JEG-3 trophoblast spheroids were formed from this culture. To that end, 18 JEG-3 cells in the medium described above were suspended in an Erlenmeyer flask under stirring at a concentration of 6x105 cells/6 ml. The resulting spheroids were collected for the adhesion assay. 5 For the adhesion assay, 24 hours after culturing the HEC-1A cells with culture medium adding sodium tungstate, Withaferin A or without adding any treatment, the culture medium was changed with fresh culture medium without treatments under study. The trophectoderm spheroids were added onto the monolayer of HEC-1A cells, placing 6 to 10 spheroids per well. The adhesion of the spheroids was 10 measured after 60 minutes, counting spheroid that was floating in the culture medium as non-adhered spheroid and spheroid that was not floating as adhered spheroid. The examination was conducted with an inverted microscope (Nikon Diaphot 300; Nikon Corp., Tokyo, Japan). The assay was conducted in triplicate. 15 C. Results

The results obtained are listed in Table 5. A higher percentage of embryonic adhesion was observed in the conditions in which the HEC-1A lines were treated with sodium tungstate with respect to those in which they were not treated. No 20 adhesion whatsoever is observed in cells treated with the negative control (C-), Withaferin A.

Table 5

Absolute adhesion rates Exp 1 Exp 2 Exp 3 Mean Baseline adhesion 12% 48% 31% 30% 10 μΜ sodium tungstate 31% 65% 35% 44% Withaferin A (C-) 0% 0% 0% 0% 25

PARTICULAR COMPOSITIONS OF THE INVENTION

Food compositions according to the invention are prepared below in an illustrative manner. They must in no way be interpreted as limiting the scope of the invention. 19

Example 5 A tungsten salt-enriched salad dressing is prepared. An amount of 25 mg of Na2W04 (100 mg/kg) is added to 250 grams of a commercially available dressing and it is mechanically stirred for 30 minutes.

Example 6 A tungsten salt-enriched ketchup-type salsa is prepared. To that end, 170 mg of CaW04 are added to 200 grams of commercially available ketchup and it is mechanically stirred for 1 hour.

Example 7 A tungsten salt-enriched milk is prepared. To that end, 1 kg of whole cow’s milk is provided, 150 mg of ZnW04 are added and it is mechanically stirred for 20 minutes. It is recommended to shake the packaging immediately before consumption.

Example 8 A tungsten salt-enriched fruit juice is prepared. Five-hundred mg of MgW04 are added to 1 kg of commercial juice and it is mechanically stirred for 10 minutes. It is recommended to shake the packaging immediately before consumption.

Example 9

Coated tablets with the following composition are prepared: PH200 Microcrystalline cellulose (diluent/gliding agent) 250.00 mg Colloidal anhydrous silica (gliding agent/adsorbent) 3.00 mg Magnesium stearate (lubricant) 5.00 mg Talc (lubricant) 7.00 mg

Opadry® white (Opadry® and-1-7000 White)(*) (coating film) 8.00 mg Sodium tungstate dihydrate 200.00 mg (*) mixture of hydroxypropylmethylcellulose, polyethylene glycol 6000 and 20 titanium dioxide (E-171)

Example 10 5 Effervescent tablets with the following composition are prepared:

Sorbitol, aspartame, sucralose and xylitol (sweeteners) 0.025mg per 10 mg Calcium carbonate 0.350 mg per 10 mg Citric acid (acidulant) 0.650 mg per 10 mg Sodium acid carbonate (acidity regulator) 0.350 mg per 10 mg 10 Orange flavoring 0.25 mg per 10 mg Tungsten salt

Example 11 15 Gelatin capsules with the following composition are prepared:

Nifedipine 6 mg Xanthan gum 5 mg Orange flavoring 0.3 mg Citric acid 0.4 mg 20 Gelucire 44/14 90 mg PH10 microcrystalline cellulose (diluent) 92.00 mg/capsule Sodium tungstate dihydrate 100.00 mg/capsule

Claims

1. Use of a tungsten (VI) salt or a solvate thereof for the preparation of a food composition for favoring normal reproduction and fertility in a non-diabetic female mammal.

2. Use according to claim 1, characterized in that the total concentration of tungsten salts in the composition is equal to or greater than 100 mg/kg.

3. Use according to any one of claims 1 and 2, characterized in that the mammal is a human.

4. Use according to claim 3, characterized in that the composition is administered in a daily dose between 0.001 mg and 1000 mg of tungsten (VI) salt per kg of body weight of the female mammal.

5. Use according to any one of claims 1 to 4, characterized in that the salt comprises a tungsten (VI) anion and a dietetically or pharmaceutically acceptable cation.

6. Use according to claim 5, characterized in that the cation is an alkaline or alkaline earth cation.

7. Use according to any one of claims 5 or 6, characterized in that the cation is selected from the group consisting of sodium, potassium, magnesium, calcium and zinc.

8. Use according to any one of the preceding claims, characterized in that the tungsten (VI) salt is the tungsten (VI) sodium salt.

9. Use according to any one of claims 5 to 8, characterized in that the tungsten (VI) anion is selected from W042', HW04~, W2072” and HW207_ ions.

10. Use according to claim 9, characterized in that the anion is W042”.

11. Use according to any one of the preceding claims, characterized in that the solvate is a dihydrate.

12. Use according to claims 6 to 11, characterized in that the solvate of tungsten (VI) salt is a dihydrate and the cation is a sodium cation.

13. Use according to any one of the preceding claims, characterized in that the composition is a liquid composition or a beverage.

14. Use according to claim 13, characterized in that the liquid composition or beverage is selected from the group consisting of animal or plant milk as well as derivatives thereof, such as milk shakes, yogurt and kefir; fruit and/or vegetable juices; still water, sparkling water and flavored or sweetened water; seasonings; alcoholic beverages; tea; coffee; and refreshing beverages or soft drinks.

15. Use according to any one of claims 1 to 12, characterized in that the composition is a solid composition.

16. Use according to claim 15, characterized in that the solid composition is selected from animal or plant milk derivatives, such as cheese, butter, margarine and tofu; flours; breads; pasta; baked goods, including cakes and cookies; jellies, candies, soft fruit candies; infusions, tea or coffee; seasonings.

17. Use according to any one of claims 1 to 12, characterized in that the food composition is a nutritional supplement or complement.

18. Use according to claim 17, characterized in that it comprises at least one pharmaceutically acceptable excipient or carrier presented in the form of a pill, tablet, pastille, capsule, powder, wafer, effervescent powder or tablet, solution, suspension, syrup or granules.

19. Use according to any one of the preceding claims, characterized in that the composition comprises sucrose.

20. Use according to any one of the preceding claims, characterized in that the non-diabetic female mammal is selected from the group of mammals that require restoring and/or promoting ovulation, improving oocyte or embryo quality, increasing zygote implantation on the uterine wall, regulating hypothalamic-pituitary axis impairment, including polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic kidney failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia.

21. A food composition, characterized in that it comprises a tungsten (VI) salt or a solvate thereof at a concentration equal to or greater than 100 mg/kg.

22. The composition according to claim 21, characterized in that the salt comprises a tungsten (VI) anion and a dietetically or pharmaceutically acceptable cation.

23. The composition according to claim 22, characterized in that the cation is an alkaline or alkaline earth cation.

24. The composition according to any one of claims 22 and 23, characterized in that the cation is selected from the group consisting of sodium, potassium, magnesium and calcium and zinc.

25. The composition according to any one of claims 21 to 24, characterized in that the tungsten (VI) salt is the tungsten (VI) sodium salt.

26. The composition according to any one of claims 22 to 25, characterized in that the tungsten (VI) anion is selected from W042", HW04~, W2072" and HW207~ ions.

27. The composition according to claim 26, characterized in that the anion is W042-.

28. The composition according to any one of claims 21 to 27, characterized in that the solvate is a dihydrate.

29. The composition according to any one of claims 21 to 28, characterized in that the solvate of tungsten (VI) salt is a dihydrate and the cation is a sodium cation.

30. The composition according to any one of claims 21 to 29, characterized in that it is a liquid composition or a beverage, a solid composition or a nutritional supplement or complement.

31. The composition according to claim 30, characterized in that it is a liquid composition or a beverage as defined in claim 14.

32. The composition according to claim 30, characterized in that it is a solid composition as defined in claim 16.

33. The composition according to claim 30, characterized in that the food composition is a nutritional supplement or complement as defined in claim 18.

34. The composition according to any one of claims 21 to 33, characterized in that it comprises sucrose.