ES2551828A1 - Food compositions comprising tungsten salts (VI) (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Food compositions comprising tungsten (VI) salts as well as their preparation, to promote fertility and normal reproduction in a non-diabetic female mammal.

Description

DESCRIPTION

Food compositions comprising tungsten salts (VI)

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

STATE OF THE TECHNIQUE

Fertility in mammals is a multi-stage process that naturally is not totally efficient. Factors such as age, eating habits or lifestyle, among others, 10 modulate the success of this process. Thus, it is known, for example, that the pregnancy rate in women aged 20 to 24 is approximately 86%, while in women aged 35 to 32, up to 50% decline [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 being overweight or underweight, are also responsible for delays in achieving a pregnancy 15 naturally or even for the impossibility of achieving it [Fertil Steril 2013; 100: 631–7]. It is estimated that approximately 66% of couples do not conceive naturally during the first 3 months of unprotected sex. This figure decreases to 15% of couples during the first year of unprotected sex. Infertility is defined (WHO, ASRM, NICE) as the inability of 20 to conceive after 12 months of regular unprotected sexual intercourse The term "subfertility" has also been coined, especially in Europe, to define those couples with some form or degree of reduced fertility, which entails a delay in conception [Gnoth C et al Definition and prevalence of subfertility and infertility Human reproduction 2005; 20 (5): 1144-1147]. 25

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

Follicular development, ovulation, migration of the immature ovum (oocyte), as well as the subsequent conception and implantation of the zygote in the uterine wall is regulated by 35 hormonal secretions of certain endocrine organs such as the pituitary gland, the

Hypothalamus and thyroid gland. Among the multiple biological causes of infertility in women, we can also highlight the causes linked to disorders of hormonal stimuli that regulate this whole process

Different treatments for female infertility are known, among which are the administration of medications to treat hormonal problems that lead to ovulation disorders. Some beneficial effects have also been postulated with numerous treatments based on the intake of vitamin supplements, especially vitamin B, vitamin C, vitamin E and folic acid, mineral supplements such as salts or complexes of iron, zinc, or selenium, essential fatty acids ( omega 3), as well as 10 extracts of plants such as Chasteberry (Vitex agnus chastus), Damiana, licorice, Red Clover flower, Agnocasto Berry, Black Cohosh, Dong Quai (Angelica sinensis), Yam or Sweet Potato (Dioscorea villosa) , False Unicorn Root, green tea, Nettles (Urtica dioica), Wild oats (Avena sativa), Dandelion (Taraxacum officinale), etc. although for none of these treatments its effectiveness has been clearly demonstrated. fifteen

The percentage of pregnancies achieved through the treatments mentioned above have limitations. Thus, for example, it has been observed that the treatment of women whose ovulation is irregular or absent through the administration of clomiphene citrate, a drug of the stilbene family, allows the restoration of ovulation 20 in a high percentage, but the pregnancy rate remains low, approximately equal to or less than 50%.

In vitro fertilization treatments are very effective at the stage of oocyte fertilization. However, the implantation rate of embryos in the uterine wall is low. 25 This promotes multiple embryo transfers for each in vitro fertilization cycle, with the consequence of a higher percentage of multiple pregnancies considered at risk.

Finally, it is known that metabolic disorders such as diabetes or obesity 30 lead to a limitation of fertility. It is known that the partial or total recovery of blood glucose, insulinemia and / or body weight of diabetic or obese female mice with impaired reproductive function, leads to an improvement in fertility.

Different pharmacological treatments, such as metformin or tungsten (VI) salts, or even changes in lifestyle, have been shown to improve diabetes or alterations in the

Insulin, such as insulin deficiency or resistance, recovering the reproductive function totally or partially, when infertility is due to diabetes or changes in insulin.

In the specific case of sodium tungstate, it is known that rats with diabetes and insulinopenia 5 induced by streptozotocin injection partially recover circulating insulin levels after prolonged treatment (10 weeks) with sodium tungstate as a result of partially reversing diabetes. Subsequently, when female rats partially recovered from diabetes mate with healthy males, it is observed that, parallel to the partial recovery of diabetes, they have partially recovered their reproductive capacity. In particular, it is observed that the percentage of deliveries with respect to the number of positive scrapes in female mice that had been partially recovered from diabetes after treatment with sodium tungstate increases up to 66%, lower percentage than females that did not suffer from diabetes where the percentage is 100%. (cf. J. Ballester et al., "Tungstate administration 15 improves the sexual and reproductive function in female rats with streptozotocin-induced diabetes", Human Reproduction, 2007, vol. 22, pp. 2128-2135). However, no effect of tungsten (VI) salts was observed or postulated in non-diabetic female animals.

Tungsten is found in trace amounts in animals and plants. For example, contents of up to 100 mg / kg have been described in 20 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 all of the above, despite intensive research and advances in the understanding and management of the reproductive process of mammals and humans in particular, many couples still do not benefit from the different approaches currently available to increase Fertility for being ineffective. Therefore, there is still a need to find new alternatives for the improvement of natural reproductive efficiency and / or the treatment of female infertility that give greater efficacy. 30

SUMMARY OF THE INVENTION

It has now been found that the administration of a tungsten salt (VI), or a solvate of said salt is effective in promoting fertility and normal reproduction in a non-diabetic female mammal. Although it was known that sodium tungstate can partially reverse the alterations in reproductive function in diabetic female mice

Because it produces a normalization of blood glucose levels, it has not been described or suggested that sodium tungstate can have an effect on the function of the female reproductive system in non-diabetic female mammals, favoring fertility and normal reproduction .

 5

Therefore, a first aspect of the present invention relates to the use of a tungsten salt (VI) or a solvate thereof, for the preparation of a food composition to promote fertility and normal reproduction in a female mammal. diabetic.

A second aspect of the present invention relates to a food composition, which comprises a tungsten salt (VI) or a solvate thereof in a concentration greater than 100 mg / kg.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, according to a first aspect the present invention relates to the use of a tungsten salt (VI) or a solvate thereof, for the preparation 15 of a food composition to favor and / or contribute to the Normal fertility and reproduction in a non-diabetic female mammal.

Within the framework of the present invention, the term "food composition" would encompass any food, solid or liquid, enriched with tungsten salt (VI), as well as any nutritional supplement or supplement containing at least one tungsten salt (VI) .

The term "promote fertility and normal reproduction" or "contribute to fertility and normal reproduction" refers to a contribution to normal fertility and reproduction, or improve fertility and normal reproduction (reducing the time to achieve pregnancy), meaning "normal fertility and reproduction" a state of fertility in which it is achieved, following the definitions provided previously:

* in a situation of subfertility: a pregnancy in a period of 12 months of regular and unprotected sex; and 30

* In an infertility situation: a pregnancy after a period of 12 months of regular and unprotected sex. This includes the need to restore and / or enhance ovulation, improve oocyte and embryo quality, increase the implantation of the zygote in the uterine wall, regulate an alteration of the hypothalamic-pituitary-ovarian axis, including polycystic ovary syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus,

cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia.

According to a particular embodiment, the food composition of the invention contains a total concentration (ie, the sum of all tungsten salts (VI) 5 contained in the composition) of tungsten salts of at least 100 mg / kg; according to other particular embodiments, said total concentration 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 10 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 salts (VI) is equal to or less than 1000 mg / kg. fifteen

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

 25

According to one more particular embodiment, the maximum daily dose will be 1000 mg / kg / day; according to additional particular embodiments, the administration would be carried out in doses of at least 0.001 mg / kg / day, of at least 0.01 mg / kg / day, of at least 0.1 mg / kg / day, of 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, of at least 100 30 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.

In general, the tungsten salt comprises a tungsten anion (VI) and a dietary or pharmaceutically acceptable cation. Preferably, the cation is a cation

alkaline or alkaline earth. More preferably, the cation is selected from the group consisting of sodium, potassium, magnesium, calcium and zinc. According to a particular embodiment, the tungsten salt (VI) is the tungsten sodium salt (VI).

In turn, the tungsten anion (VI) is preferably selected from the list consisting of WO42−, HWO4−, W2O72− and HW2O7−. Preferably, the anion is WO42−.

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

All features listed individually for different elements of the invention can be combined with each other, leaving all possible combinations included within the scope of the present invention. Thus, for example, according to a preferred embodiment, the solvate of the tungsten salt (VI) is dihydrate and the cation is a sodium cation. Likewise, the remaining possible combinations are also intended to be included within the scope of the present invention. fifteen

The food composition can be a liquid composition, that is, a beverage according to terms more generally used in society. Such liquid composition, within the framework of the present invention, includes, but is not limited to, any beverage selected from the group consisting of milk of animal or vegetable origin, as well as any derivative thereof, such as, for example, shakes, yogurt , kefir, etc .; fruit and / or vegetable juices; 20 water without gas or with gas, or water or flavored or sweetened beverages (by means of nutritive (sucrose, fructose…) or artificial sweeteners); seasonings, such as any sauce, dressing, ketchup, oil, vinegar or vinegar preparations; alcoholic beverages of any kind; Tea coffee; as well as all kinds of soft drinks or soft drinks, or energy drinks. 25

The food composition can also be a solid composition. For example, such a solid composition may be selected, but not limited to, from the group consisting of milk derivatives of animal or vegetable origin, such as cheese, butter, margarine and tofu; any type of bread, including fresh, packaged or frozen, of mold, whole wheat, spiced, sweet, salty, etc; pasta prepared from any cereal flour, such as wheat or semolina (macaroni, spaghetti, noodles, etc); bakery products, including biscuits, cookies, muffins, donuts, etc .; infusions, tea or coffee, in bulk or in sachets, to prepare drinks; jellies, candies, including rubber, better known as "jelly beans"; 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 nutritional supplement or supplement, any of these terms being used in the context of the present invention. These terms are commonly used for compositions consumed orally, which contain an ingredient intended to supplement the diet, in the case of the present invention the tungsten salt (s) (VI). They will never be a substitute for a conventional food or a single component of a meal or diet. They can be given in different presentations, such as pills, 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 in the scope of the present invention. Dietary- or pharmaceutically acceptable excipients are apparent to the expert for obtaining any of the above presentations, and are intended to be included within the scope of the present invention.

According to a particular embodiment, any of the above food compositions (solid composition, liquid composition or nutritional supplement / supplement) comprises in its composition at least one nutritive sweetener, such as sucrose or fructose. The intake of these nutritive sweeteners should be controlled and limited in diabetic patients, therefore a food composition that includes such a nutritive sweetener would be contraindicated in diabetic mammals or at least should be taken into account during the control of your diet.

According to a particular embodiment, the non-diabetic female mammal is selected from the group consisting of female mammals that need to restore and / or enhance ovulation, improve oocyte and embryo quality, increase zygote implantation in the uterine wall. , regulate an alteration of the hypothalamic-pituitary-ovarian axis, including polycystic ovary syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, idiopathic causes eating disorders, such as anorexia nervosa and bulimia. 30

According to another particular embodiment, the non-diabetic female mammal is selected from the group consisting of female mammals that have insulin resistance (also known as insulin resistance or insulin resistance); Insulin resistance, when presented together with hyperglycemia, can lead to the development of diabetes, however insulin resistance alone does not lead to a diabetic state. Preferably, the

Female mammals that have insulin resistance are selected from the group consisting of female mammals that have one or more of the following conditions: Obesity or overweight, Metabolic syndrome, Prediabetes, Polycystic ovary syndrome, Hypertension, Coronary heart disease, Hyperlipidemias or dyslipidemia, Hyperthyroidism, Hyperparathyroidism , Hyperleptinemia or leptin resistance, Sedentarism, Eating disorders 5, Obstructive sleep apnea syndrome, Fetal malnutrition, Prader Willi syndrome, Rabson-Mendenhall syndrome, Liver steatosis, Leprechaunism, Pathologies associated with excess glucocorticoid secretion ( eg acromegaly), insulin resistance caused by pharmacological treatments (eg glucocorticoids, thiazide diuretics, beta blockers), Stress or Early Menarche [Egas Béjar, Daniela et.al 10 Insulinorresistencia / Insulin resistance. Medicine (Guayaquil); 10 (2): 159-166, Apr. 2005; Serious, Thomas K. WHY ISN’T THIS INSULIN WORKING? Western Veterinary Conference 2013 (SA113); Ximena Gaete V. Advancement of puberty in Chile and the world. Rev Chil Pediatr 77 (5); 456-465, 2006; Leszek Szablewski Glucose Homeostasis and Insulin Resistance (eISBN: 978-1-60805-) Bentham e-books]. Many of these indications have been described that are not related to infertility, including prediabetes [Acta Diab. Latin 4, 507, 1967].

Another aspect of the present invention relates to a food composition, which comprises the salt or salts of tungsten (VI) or a solvate thereof in a concentration of at least 100 mg / kg. According to particular embodiments, the tungsten (VI) salt or salts are comprised in the composition in 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 25 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 preferred definitions and meanings provided above in relation to the first aspect of the invention are applicable to this second aspect of the invention. Thus, for example, the tungsten salt (VI) will comprise a tungsten anion (VI) and a dietary or pharmaceutically acceptable cation, which preferably will be an alkaline or alkaline earth cation, even more preferably a sodium, potassium, magnesium cation and calcium and zinc Particularly, the salt will be tungsten sodium (VI). Again, the tungsten anion (VI) is selected from the ions WO42−, HWO4−, W2O72− and HW2O7−, preferably WO42−, and if presented in the form of a solvate, the same

preferably it will be the hydrate, more specifically the dihydrate. The other particular embodiments provided above for the first aspect also refer to this second aspect.

The food composition of the second aspect of the invention may be a liquid or beverage composition, a solid composition or a nutritional supplement or supplement (also called a dietary or nutritional supplement).

The liquid composition has already been defined above in relation to the first aspect of the invention, and all preferred embodiments are also applicable to this second aspect of the invention. 10

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

 fifteen

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

Preferably, the composition comprises a nutritive sweetener, such as sucrose or fructose.

A series of illustrative, non-limiting examples of the present invention are included below.

 25

EXAMPLES

EXAMPLE 1. DESCRIPTION OF THE ANIMAL MODEL (IRS2 FEMALE MOUSES - / -)

The mouse model used to determine the activity of tungsten salts (VI) to promote normal fertility and reproduction is the IRS2 - / - mouse, a knock-out of the Irs2 gene [Burks et al., "IRS-2 pathways integrate female reproduction and energy 30 homeostasis ", Nature, 2000, vol. 407, pp. 377-382]. The deletion of the Irs2 gene translates into a clear sexual dimorphism in relation to fertility and carbohydrate metabolism.

Males of this model have insulin resistance and severe hyperglycemia started at an early age. In contrast, at an early age, females remain relatively euglycemic and develop a slight insulin resistance, which is maintained until advanced ages (4-5 months). Female IRS2 - / - at an early age,

around 10 weeks of age, they show low follicular development and persistent anovulation, accompanied by the absence of the estrous cycle in most mice. The pregnancy rate of female IRS2 - / - mice is 9% compared to a 100% rate in female IRSwt mice (IRS-2 + / + wild type). Since in these ages the females remain euglycemic and only develop a slight insulin resistance, the profound alteration in fertility is not a direct consequence of abnormalities in glucose metabolism.

EXAMPLE 2. OVULATION, IMPLEMENTATION AND PREGNANCY STUDY

A. Animals 10

10 female IRS2 - / - mice aged between 6 and 8 weeks.

6 male wild-type mice (IRS2wt) aged between 6 and 8 weeks.

Female and male mice were housed separately under normal conditions, ie 12h light / dark cycle and controlled temperature and humidity. The animals were fed at will (also called ad libitum) with a standard feed diet. fifteen

B. Method

Pre-treatment phase

After an acclimatization period, the female IRS2 - / - mice were housed in groups of 4-6 mice / cage. During the pre-treatment phase (2 weeks), animals were given drinking water without tungstate salt.

Treatment phase

Sodium tungstate was administered in drinking water (ad libitum) using 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 the animals are slaughtered. The daily dose of sodium tungstate ingested by the mice was approximately 180 mg / kg body weight.

Cross 30

After the first three weeks of treatment, the female IRS2 - / - mice were housed in cages in pairs next to a male IRS2wt mouse continuously.

Mice were observed daily for signs of pregnancy or childbirth.

After 4 weeks the males were exchanged between the cages and kept for another 4 weeks. 35

The administration of tungstate was maintained during the 8 weeks in which males and females settled together. After these 8 weeks the treatment was withdrawn and the joint establishment of males and females was maintained for an additional 4 weeks.

Sacrifice 5

After this period, the females were sacrificed and biopsied for signs of pregnancy.

C. Results

Results of the Ovulation Study 10

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

Between 1 and 2 ml of saline was introduced into the mice's vagina with a 15 pasteur pipette. The vaginal exudate was collected with the same pipette and spread on a slide. Once air dried, it was fixed and stained with the Pap smear.

The Pap smear technique involves staining the vaginal smears fixed on the slide as follows:

- 10 dives in 50% v / v alcohol;

- Immerse in Harris hematoxylin solution for 3 minutes;

- Rinse with running water;

- 10 dives in Acid Alcohol (1% Ac. Hydrochloric);

- Rinse with running water; 25

- 10 dives in Alcohol 95% v / v;

- Immerse in OG-6 solution for 30 seconds;

- 10 dives in alcohol 96% v / v;

- Immerse in eosin solution for 1 minute;

- 10 dives in alcohol 96% v / v; 30

- 10 dives in alcohol 86% v / v; Y

- 10 dives in xilol

Preparations were analyzed by trained personnel and using a simple blind and coding to eliminate observer bias.

 35

The samples were identified in the following phases: right, proestrus, estrus, metaestrus, anestrus or not evaluable. The cyclicity of the four phases in periods of 4 to 6 days, are indicators of a normal estrous cycle, while the absence of this cyclicity and persistence in anestrus, right or proestro phases are indicators of the absence of the estrous cycle. 5

  Of the 10 female mice under study, Table 1 summarizes the phases of the estrous cycle of vaginal smears performed on 6 of these female mice IRS2 - / -.

Table 1 10

 Mouse No.

 Time (days)

 -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

The phases of the estrous cycle are the following: A: Anestro; D: right handed; P: Proestro; E: Estro; and M: metaestro.

The phases of the estrous cycle found in the vaginal smears of the female mice IRS2 - / - 15 of Table 1 show that during the pre-treatment period all the mice were in the proestro (P) or right-handed (D) phase, it is say in the absence of estrous cycle.

While after the start of tungstate administration, it is observed that the female mice IRS2 - / - are in late stages of the estrous, estro (E) and metaestro (M) cycle, which is an indicator of the recovery of the cycle normal estral.

These results indicate that the administration of a tungsten salt (VI) allows rapid recovery (on the seventh day of treatment) of the estrous cycle in infertile female IRS2 - / - non-diabetic mice in 100% of mice analyzed.

Results of the Implantation and Pregnancy Study 5

After the crossing period of the method of section B, the females were sacrificed and biopsies were performed in search of signs of pregnancy.

Table 2 summarizes the age at the time of treatment, if there was pregnancy and the number of embryos per female. 10

Table 2

 Mouse No.

 Age (months) at the beginning of pregnancy Number of implanted embryos or babies born

 treatment

 crossing

 72

 10 12 YES 1 breeding

 942

 10 12 YES 8 offspring

 928

 10 12 YES 7 embryos

 931

 10 12 YES 3 offspring

 898

 10 12 YES 7 embryos

 900

 10 12 NO -

 921

 10 12 YES 6 embryos

 926

 10 12 YES 8 offspring

 972

 8 10 NO -

 973

 8 10 YES 7 embryos

The results in Table 2 show that while the pregnancy rate of female IRS2 - / - untreated mice is 9%, the pregnancy rate of female IRS2 - / - 15 mice treated with tungstate is increased to 80 %.

In addition, these results also show that the average number of offspring / embryos implanted by a female pregnancy mouse is approximately 5, a number that can be considered comparable to the number of offspring of a female mouse.

Thus, the results of Tables 1 and 2 demonstrate that a tungsten salt (VI) is an effective treatment for ovulation recovery and / or increased oocyte implantation. Thus, 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.

 10

EXAMPLE 3. STUDY OF GLUCEMIA AND BODY WEIGHT

A. Animals

6 female IRS2 - / - mice aged between 6 and 8 weeks.

Female mice were housed in normal conditions, ie 12h light / dark cycle and controlled temperature and humidity. The animals were fed at will (also called ad libitum) with a standard feed diet.

B. Method

Treatment phase

After an acclimatization period, the sodium tungstate was administered in the drinking water 20 (ad libitum) by means of a 2 mg / ml solution of sodium tungstate dihydrate (commercialized by Carlo Erba) in distilled water after the pre-treatment phase ( day 0 of treatment) and for 12 days.

C. Results 25

On days 0, 2, 5, 7, 9, and 12 of the treatment period, body weight was monitored, and after 6 hours of fasting on each of the days mentioned above, blood glucose was determined by extraction of blood from the caudal vein and with a glucose sensor (AccuTrend glucose sensor Roche. Mannheim, Germany).

 30

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

Table 3

 Glycemias (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

The results of Table 3 and 4 show that during the administration of sodium tungstate 5 there are no variations in body weight or blood glucose in the first 12 days of treatment, the same period during which from the 7 day of treatment the reestablishment of ovulation is already achieved in the female mice tested (cf. Table 1).

Thus, the results of Tables 1 to 4 demonstrate that a tungsten salt (VI) is an effective treatment for ovulation recovery and / or increased oocyte implantation, independently of changes in body weight. and changes in carbohydrate metabolism. Thus, it is demonstrated that the administration of a tungsten salt (VI) as defined in the present invention or a composition containing such a tungsten salt (VI) has a direct effect on the female reproductive system and by

Therefore, it is effective in promoting fertility and normal reproduction in non-diabetic female mammals.

EXAMPLE 4. STUDY OF EMBRYO ADHERENCE IN THE ENDOMETRY

A. Model 5

To determine the effect of sodium tungstate on endometrial receptivity, an “in vitro” human-human embryonic adhesion model was used, composed of the HEC1-A endometrial cell line and the JEG-3 human trophoectoderm cell line.

The JEG-3 line, a cell line that simulates the trophoectoderm cells of a human embryo 10, are cells that grow in monolayer in the laboratory, using low adhesion plaques, are capable of forming spheroids that simulate the human embryo; This is one of the most used cell lines for in vitro embryonic adhesion assays.

B. Method 15

Cell lines were purchased commercially (American Type Culture Collection (ATCC); Rockville, MD, USA) to perform embryonic adhesion experiments on the endometrium. They were thawed and expanded for 4 passes to have enough cells to perform all the tests.

 twenty

HEC1-A cells were planted 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 a confluence of 90%. After reaching the confluence, sodium tungstate was added to the culture medium at a final concentration of 10, as well as Withaferrin A (as a control, compound that prevents embryonic adhesion) or culture medium (baseline adhesion data), without any component additional, for 24 hours.

JEG-3 cells were planted on low adhesive plates with ‘Eagle’s minimal essential medium’ (EMEM) culture medium, supplemented with 10% fetal bovine serum and 0.1% antibiotics (fungizone and penicillin). 24 hours before the adhesion test, the formation of JEG-3 trophoblastic spheroids was carried out from this culture. To do this, JEG-3 cells were suspended in an Erlenmeyer under stirring in the medium previously described at a concentration of 6x105 cells / 6ml. The resulting spheroids were collected for the adhesion test.

 35

For the adhesion test, after 24h of culture of the HEC-1A cells with culture medium adding sodium tungstate, Withaferrin A or without adding any treatment, the culture medium was changed by means of fresh culture without treatments under study. Trophoectoderm spheroids were added on the monolayer of HEC-1A cells, placing 6 to 10 spheroids per well. The adhesion of the spheroids was measured after 60 minutes, counting as the non-adhering spheroid that was floating in the culture medium and adhering spheroid that was not floating. The examination was performed with an inverted microscope (Nikon Diaphot 300; Nikon Corp., Tokyo, Japan). The test was performed in triplicate.

 10

C. Results

The results obtained are shown in table 5. A higher percentage of embryonic adhesion was observed under the conditions in which the HEC-1A lines were treated with sodium tungstate versus those in which it was not. No adhesion is observed in cells treated with the negative control (C-) Withaferrin A. 15

Table 5

 Absolute Adhesion Rates

 Exp1 Exp2 Exp3 Media

 Basal Adhesion

 12% 48% 31% 30%

 10 μM sodium tungstate

 31% 65% 35% 44%

 Withaferrin A (C-)

 0% 0% 0% 0%

SPECIFIC COMPOSITIONS OF THE INVENTION

Food compositions according to the invention are prepared below, by way of illustration. In no case should they be construed as limiting the scope of the invention.

EXAMPLE 5

A salad dressing enriched with a tungsten salt is prepared. A quantity of 25 mg of Na2WO4 (100 mg / kg) is added to 250 g of a commercially available dressing and stirred mechanically for 30 minutes.

EXAMPLE 6

A ketchup sauce is prepared, enriched with a tungsten salt. For this, 170 mg of Ca WO4 is added to 200 g of commercially available ketchup and 30 is mechanically stirred for 1 h.

EXAMPLE 7

Milk enriched with a tungsten salt is prepared. For this, 1kg of whole cow's milk is available, 150 mg of ZnWO4 is added and mechanically stirred for 20 minutes. Immediately before consumption it is recommended to shake the container.

 5

EXAMPLE 8

A fruit juice enriched with a tungsten salt is prepared. 500 mg of MgWO4 is added to 1 kg of commercial juice and stirred mechanically for 10 minutes. Immediately before consumption it is recommended to shake the container.

 10

EXAMPLE 9

Coated tablets with the following composition are prepared:

Microcrystalline cellulose PH200 (Diluent / slider) 250.00 mg

Colloidal anhydrous silica (Sliding / adsorbent) 3.00 mg

Magnesium stearate (Lubricant) 5.00 mg 15

Talc (Lubricant) 7.00 mg

Opadry® white (Opadry® y-1-7000 White) (*) (Coating film) 8.00 mg

Sodium tungstate dihydrate 200.00 mg

(*) Mixture of hydroxypropyl methylcellulose, polyethylene glycol 6000 and titanium dioxide (E-171)

  twenty

EXAMPLE 10

Effervescent tablets with the following composition are prepared:

Sorbitol, aspartame, sucralose and xylitol (sweeteners) 0.025mg per 10 mg

0.350mg calcium carbonate per 10 mg

Citric acid (acidulant) 0.650mg per 10 mg 25

Sodium acid carbonate (acidity corrector) 0.350mg per 10 mg

Orange aroma 0.25mg per 10 mg

Tungsten salt

Example 11 30

Gelatin capsules are prepared with the following composition:

Nifedipine 6 mg

Xanthan gum 5 mg

0.3 mg orange flavor

Citric acid 0.4 mg 35

Gelucire 44/14 90 mg

Microcrystalline cellulose PH10 (Diluent) 92.00 mg / capsule

Sodium tungstate dihydrate 100.00 mg / capsule

Claims (34)

1. Use of a tungsten salt (VI) or a solvate thereof, for the preparation of a food composition to promote fertility and normal reproduction in a non-diabetic female mammal. 5 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 human. 4. Use according to claim 3, characterized in that the administration of the composition is carried out in a daily dose between 0.001 mg and 1000 mg of tungsten salt (VI) per kg of body weight of the female mammal. fifteen 5. Use according to any one of claims 1 to 4, characterized in that the salt comprises a tungsten anion (VI) and a dietary or pharmaceutically acceptable cation.  twenty 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 25 and zinc. 8. Use according to any one of the preceding claims, characterized in that the tungsten salt (VI) is the tungsten sodium salt (VI).  30 9. Use according to any one of claims 5 to 8, characterized in that the tungsten anion (VI) is selected from the ions WO42−, HWO4−, W2O72− and HW2O7−. 10. Use according to claim 9, characterized in that the anion is WO42−.  35 11. Use according to any one of the preceding claims, characterized in that the solvate is dihydrate. 12. Use according to claims 6 to 11, characterized in that the tungsten salt solvate (VI) is dihydrate and the cation is a sodium cation. 5 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 10 is selected from the group consisting of milk of animal or vegetable origin as well as its derivatives, such as shakes, yogurt and kefir; fruit and / or vegetable juices; sparkling water, sparkling water and flavored or sweetened water; seasonings; alcoholic drinks; tea; coffee; and refreshing drinks or sodas.  fifteen 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 milk derivatives of animal or vegetable origin, such as cheese, butter, margarine and tofu; flours; breads; pasta; bakery products, including biscuits and cookies; jellies, candies, jelly beans; 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 supplement. 18. Use according to claim 17, characterized in that it comprises at least one pharmaceutically acceptable excipient or vehicle and is in the form of a pill, tablet, tablet, 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.  35 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 need to restore and / or enhance ovulation, improve oocyte or embryo quality, increase zygote implantation in the uterine wall, regulate an alteration of the hypothalamic-pituitary axis, including polycystic ovary syndrome 5, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, idiopathic causes and eating disorders, such as anorexia nervosa and bulimia.  10 21. A food composition, characterized in that it comprises a tungsten salt (VI) or a solvate thereof in a concentration equal to or greater than 100 mg / kg. 22. Composition according to claim 21, characterized in that the salt comprises a tungsten anion (VI) and a dietary or pharmaceutically acceptable cation. fifteen 23. Composition according to claim 22, characterized in that the cation is an alkaline or alkaline earth cation. 24. 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. Composition according to any one of claims 21 to 24, characterized in that the tungsten salt (VI) is the tungsten sodium salt (VI). 25 Composition according to any one of claims 22 to 25, characterized in that the tungsten anion (VI) is selected from ions WO42−, HWO4−, W2O72− and HW2O7−.  30 27. Composition according to claim 26, characterized in that the anion is WO42−. 28. Composition according to any one of claims 21 to 27, characterized in that the solvate is dihydrate.  35 29. Composition according to any one of claims 21 to 28, characterized in that the tungsten salt solvate (VI) is dihydrate and the cation is a sodium cation. 30. 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 supplement. 31. Composition according to claim 30, characterized in that it is a liquid composition or a beverage as defined in claim 14. 10 32. Composition according to claim 30, characterized in that it is a solid composition as defined in claim 16. 33. Composition according to claim 30, characterized in that the food composition is a nutritional supplement or supplement as defined in claim 18. 34. Composition according to any one of claims 21 to 33, characterized in that it comprises sucrose. twenty