WO2008155651A2

WO2008155651A2 - Inositol for induction of ovulation

Info

Publication number: WO2008155651A2
Application number: PCT/IB2008/001639
Authority: WO
Inventors: Vittorio Unfer
Original assignee: Lo.Li. Pharma S.R.L.; Marelli, Mariapina
Priority date: 2007-06-20
Filing date: 2008-06-18
Publication date: 2008-12-24
Also published as: ITRM20070341A1; WO2008155651A3

Abstract

A composition comprising inositol for induction of ovulation in a subject by administering to the subject the composition in such a quantity so as to provide at least 2 g/day of inositol.

Description

SUPPLEMENTARY COMPOSITION COMPRISING INOSITOL FOR CONTROLLING INDUCTION OF OVULATION Technical Field

The present invention relates to a composition comprising inositol for controlling induction of ovulation in a subject, particularly but not exclusively for treating patients affected by hyperinsulinemia and/or polycystic ovary syndrome and/or patients subjected to medically assisted procreation procedures. Background art Polycystic ovary syndrome (PCOS) is a medical condition which causes irregular menstrual cycles, absence of ovulation and excess of male traits in affected women. Moreover, this condition is often associated with hyperinsulinemia. As a whole, these conditions are responsible for infertility in a large number of women. In order to obviate infertility, caused by PCOS or by other causes, many women undergo in vitro fertilization procedures.

The therapeutic efficiency of in vitro fertilization (IVF-ET) procedures is closely linked to the resulting number of zygotes, which in turn depends on the number of picked-up ova. Accordingly, ovarian hyperstimulation is indispensable in order to increase the number of ova, which is one in the natural menstrual cycle. Several protocols and active ingredients have been used up to now (domitene citrate, FDVIG, purified FsH, recombinant FsH, GnRH agonists or antagonists) with the goal of obtaining an optimum number of ova.

In the vast majority of cases, a "long" protocol is selected. This comprises a hypophyseal blockage with a GnRH analog, followed by hyperstimulation with gonadotropins FsH (Gonal-F, Puregon, Fostimon) or hMG (Merional or Menopor). Other protocols are reserved to particular cases, such as hyper- or hyporesponsive patients. The specific treatment selected is presented by the physician prior to any hyperstimulation. In general, the contraceptive pill (Microgynon 30) is used briefly. This approach allows to synchronize all the small follicles that are present in the ovaries. This leads to a more harmonious stimulation and probably to better results. Currently, this approach allows better control over the precise moment (day of the week) in which ovum pick-up occurs. This approach, which uses the pill briefly (7-30 days) ("double suppression"), is used by most IVF programs in the USA.

IVF-ET comprises several steps. The first step consists in ovarian hyperstimulation by using two drugs capable of achieving stimulation of the ovaries and the production of many ova. The first drug is a GnRH analog (Decapeptyl®), which blocks the synthesis of gonadotrope hypophyseal hormones (FSH, LH) and induces a block of the natural cycle. Daily subcutaneous administration begins generally 4 days before suspending the taking of the pill and continues to induction of ovulation (approximately 20- 25 days in total). The second drug is a follicle stimulating hormone (FsH; Gonal-F®, Puregon®, Fostimon®, Merional® HMG, Menopor®), which is administered every day in the form of a subcutaneous injection and induces the growth of ovarian follicles (ovary stimulation). The growth of these follicles is followed by echographies (see photograph) and by blood hormone dosages. The second step consists of induction of ovulation. The ovum can be fertilized with the sperm only if it has resumed its meiotic division and has expelled the first polar globule. It is then considered to be mature. In a natural cycle, maturation of the ovum and ovulation are induced by the peak of LH, the luteinizing hormone. In a stimulated cycle, the administration of hCG (pregnancy hormone) allows to mimic the action of the LH. One then speaks of induction of ovulation. Several natural or synthetic hormones, such as urinary hCG, recombinant hCG or recombinant LH, can be used for this purpose. Induction of ovulation is generally programmed 35 hours before the hour intended for follicular puncture. The third step consists in ovum pick-up (OPU). Most ovum pick-ups are performed under general anesthesia in an operating room. Exceptionally, a local or local-regional anesthesia can be proposed. This fact is discussed with the physician and with the anesthesiologist before the operation.

The follicles are perforated transvaginally by means of a needle which is introduced in the follicle by means of an echography probe. The resulting liquid is collected in tubes which are labeled with the name of the patient and are taken to the laboratory. The cumulus-oocyte (COC) complexes are searched (Figure 3) by means of a binocular magnifying lens and are then cultured in an incubator. Step 4 consists in collecting and preparing the sperm. Sperm collection is performed in the immediate proximity of the laboratory on the day of ovum pick-up. An abstinence of 2-3 days is recommended, since this duration allows to achieve a maximum of motile sperm cells. In cases in which ejaculation by masturbation is not possible, sperm collection during coitus is possible but must be performed with a special condom provided by the laboratory. This possibility must be discussed in advance with the physician and the laboratory. In very rare cases, when the first sample contains too few sperm cells or none, collection of a second sample may be necessary. To avoid the risk of functional problems on the day of ovum pick-up, the partner is recommended to freeze a "backup" sperm sample which might be used if it is impossible to obtain fresh sperm. Once informed, it is the partner who decides whether this precaution is necessary. To freeze a sperm sample, the husband must contact the laboratory and take and reserve an appointment. The same precaution of 2-3 days of abstinence is applied.

The length of the head of the sperm cells is 4-5 pm, the width is 1.50- 1.75 pm, flagella length is 45 pm and is variable from person to person.

Step 5 consists in in vitro fertilization proper. In classic IVF, a few hours after OPU, 50,000 sperm cells are placed in the presence of each cumulus-oocyte complex. This fertilization is performed in a four-cup culture box (Figure 7). Each box is identified with the name of the patients and placed to incubate at 37°C in the presence of 5% CO₂, 5% O₂ and 90% N₂ in a special incubator.

When the low number of sperm cells makes classical fertilization impossible, ICSI (intracytoplasmic sperm injection) is used.

The sixth step consists in identifying the embryo zygotes and in freezing the supernumerary zygotes. Once the sperm cell has penetrated the ovum, the genetic material of paternal and maternal origin appear in the form of structures known as pronuclei. Thus, 16-20 hours after placement for fertilization, the zygotes (fertilized ova) are identified by the presence of two pronuclei (the maternal and paternal centers) and of polar globules.

A maximum of three zygotes is cultured for transfer of fresh embryos, but generally the number is limited to 2. All the supernumerary zygotes are frozen immediately (this must be done before the 20^th hour, which corresponds to the moment when the pronuclei disappear and fuse their content, in accordance with LPMA).

Step 7 consists of the so-called "embryo transfer". Embryo transfer is performed 48-72 hours after ova pick-up. The transfer is performed transvaginally by means of a fine catheter and is not painful. Transfer is performed by means of an echography with half-full bladder. Sometimes it is necessary to grip the neck of the uterus with a clamp in order to straighten the uterus and allow catheter insertion. Once the embryos have been inserted in the uterus, the patient remains lying down for approximately 30 minutes. Progesterone, administered vaginally, is prescribed from the evening of the day of ovum pick-up.

Transfers of thawed embryos are generally performed during cycles replaced by taking estrogens (Progynova® or transdermal patches of Estradot®) and progesterone (Utrogestan®, Crinone®). A maximum of 2 embryos is transferred. There is, therefore, the need to provide a composition which is capable of controlling the induction of ovulation in a subject, particularly if the subject is affected by PCOS, hyperinsulinemia or both diseases. Disclosure of the invention The aim of the present invention is to provide a composition for controlling induction of ovulation.

An object of the invention is to provide a composition which is capable of controlling ovulation in subjects affected by hyperinsulinemia, polycystic ovary syndrome, secondary amenorrhea or undergoing in vitro fertilization procedures.

Another object of the invention is to provide a composition for ovulation control which is highly reliable, relatively easy to provide and at competitive costs.

This aim and these and other objects, which will become better apparent hereinafter, are achieved by a composition comprising inositol for controlling the induction of ovulation in a subject by administering to said subject said composition in such a quantity so as to provide at least 2 g/day of inositol.

Brief Description of the Drawings Further characteristics and advantages of the invention will become better apparent from the following detailed description and from the accompanying drawings, wherein:

Figure 1 is an echography of an ovary in which the rounded black regions correspond to the follicles, the largest of which measure approximately 2 cm;

Figure 2 is a photograph of a mature ovum, in which it is possible to distinguish the polar globule at 3 o'clock, which indicates resumption of meiosis;

Figure 3 is a photograph of three cumulus-oocyte complexes observed by means of a binocular magnifying lens; Figure 4 is a photograph of a sperm sample;

Figures 5 a and 5 b are photographs of an incubator with control of the levels of oxygen and nitrogen which can be used during the in vitro fertilization step; Figure 6 is a photograph of cumulus-oocyte complexes (2 mm diameter), in which the ovum is contained in the mass of cells at the center (0.2-0.3 mm diameter);

Figure 7 is a photograph of a culture box in which fertilization is performed; Figure 8 is a photograph of a zygote in the step with two pronuclei, which are visible next to each other at the center of the ovum. The two polar globules are visible at 12 o'clock;

Figure 9 is a photograph of a catheter which can be used to transfer embryos in an in vitro fertilization procedure; Figure 10a is a photograph of an embryo with four blastomeres or cells respectively 45 hours after insemination;

Figure 1 Ob is a photograph of an embryo with eight blastomeres or cells respectively 72 hours after insemination;

Figure 1 Oc is a photograph of an embryo in the morula stage 96 hours after insemination;

Figure 1Od is a photograph of an embryo in the blastula stage 120 hours after insemination; and

Figure 1 Oe is a photograph of an embryo in the complete blastocyst stage 144 hours after insemination. Ways of carrying out the Invention

Other objects, characteristics and advantages of the invention will become apparent in the following detailed description.

The composition of the present invention comprises as its active ingredient inositol, a substance which has a controlling activity on the induction of ovulation and on oocyte quality. The inventor of the present invention has in fact discovered that the administration of inositol allows to control the induction of ovulation in a subject, particularly by administering a quantity of composition capable of providing at least 2 g/day of inositol. Preferably, in the context of the present invention, the term "inositol" is used to reference the isoform known as myo-inositol.

In one embodiment, the composition described here may be administered to the subject in such a quantity so as to provide at least 2 g/day of inositol, preferably 2 to 4 g/day, more preferably 2 g/day.

Administration of the composition to the subject may be single-dose or may be spread over two or more daily doses, for example two doses of at least 1 g of inositol each or four doses of at least 500 mg of inositol each.

In another embodiment, the composition according to the invention may be characterized in that it further comprises folic acid or may be administered simultaneously, sequentially or separately with respect to the folic acid.

Preferably, the composition comprises folic acid, so as to allow the administration in a single formulation of both active ingredients. During some studies it has in fact been demonstrated that the use of folic acid as a supplement controls the tendency to hypercoagulation that is present in patients undergoing ovulation induction. Preferably, the composition is administered in such a quantity so as to provide at least 200 μg/day of folic acid, more preferably 200 to 400 μg/day of folic acid.

The inventor of the present invention has in fact discovered that the combination of inositol and folic acid, due to a synergistic action, helps to control ovulation induction, particularly in patients affected by PCOS and in patients subjected to controlled ovarian hyperstimulation in medically assisted procreation protocols. The simultaneous presence, in a single formulation, of inositol and folic acid enhances the individual properties of the two substances, determining a synergistic action whose benefits become apparent in better control of ovarian stimulation and in a reduction in the incidence of uncontrolled ovarian hyperstimulation (OHSS).

The composition described here may be used in particular to control ovulation in subjects affected by hyperinsulinemia in which the ovulation cycle is compromised. Moreover, the present composition may be used to control ovulation in subjects affected by polycystic ovary syndrome (PCOS). This disorder is a known cause of female infertility and its correlation with alterations in the insulin level is known. PCOS is observed in at least 10% of women and causes irregular menstrual cycles, chronic absence of ovulation and excessive secretion of male hormones, with consequent development of male traits in the subject.

Moreover, the composition according to the invention may be used also to control ovulation in subjects affected by secondary amenorrhea.

In another embodiment, the composition described here may be used to control ovulation in subjects subjected to a medically assisted procreation procedure, such as in vitro fertilization/embryo transfer (IVF-ET).

The inventor of the present invention has in fact observed that the administration of inositol in patients affected by PCOS, hyperinsulinemia or amenorrhea is capable of regularizing the ovulation cycle with a positive effect on oocyte maturation. Without intending to be bound to any theory in particular, it is believed that the therapeutic action of the composition according to the invention is due to the effect of inositol on insulin secretion, thanks to whose reduction it is possible to restore spontaneous ovulation. Inositol

This substance is part of the group of so-called non- vitamin Bs and is tightly linked to choline and biotin. Studies conducted on animals have shown that vitamin B6, folic acid, pantothenic acid and p-aminobenzoic acid (PABA) have a very close activity association with inositol. Myo- inositol, the main isoform of inositol, is the only active constituent, from the nutritional standpoint, of phosphatidylinositol. Inositol is active in cell membranes and in sending messages which control cellular functions in the nervous system. Like choline, inositol is also present in lecithin.

Animal tissues and some plants contain inositol. In animal tissues, it is present as a component of phospholipids, substances which contain phosphor, fatty acids and nitrogenous bases. In plant cells it appears in the form of phytic acid, an organic acid which bonds calcium and iron in an insoluble complex and interferes with their assimilation.

Inositol is found to be effective in stimulating the production of lecithin in the body. Fats are transferred from the liver to the cells with the aid of lecithin and inositol thus contributes to fat metabolism and helps to reduce the rate of cholesterol in blood. Combined with choline, it prevents the hardening of fats in the arteries and protects the liver, kidneys and heart. Moreover, the usefulness of inositol has been observed also in the nutrition of brain cells. Large quantities of inositol are present in the nerves of the spinal cord, in the brain and in cerebrospinal fluid. Inositol is necessary for the growth and survival of cells of bone marrow, for ocular membranes and for the intestines. It is vital for hair growth and can also prevent hair fraying and loss. Inositol is present in untreated wholemeal cereals, in citrus fruits, in beer yeast, in unrefined raw molasses and in liver. Approximately 7% of ingested inositol is converted into glucose. Its effectiveness in alleviating ketosis, i.e., the incomplete metabolism of fatty acids, is only one third that of glucose. Inositol-rich foods are fresh fruit and dry fruit, legumes and cereals. Fresh fruit and vegetables contain more myo-inositol than frozen products.

The recommended dose of inositol has not yet been set, but most physicians recommend consuming the same quantity of inositol and choline. The daily consumption of inositol in food is approximately 1 g, which originates from animal sources such as phospholipids, or from vegetable sources such as phytic acid. The human body contains a larger quantity of inositol than other vitamins, except for niacin. A spoonful of yeast yields approximately 40 mg of choline and inositol. Therapeutic doses vary from

500 to 1000 mg per day. Patients affected by peripheral neuropathies caused by diabetes have been administered 500 mg twice per day for two weeks with considerable results. 3 g of myo-inositol orally and 1 g intravenously have been administered without side effects. No toxicity has been observed.

Research conducted on yeast cells has shown that when deprived of inositol, the metabolic processes are hindered in their function and consequently most cells die. In other studies, the lack of inositol in yeast cells leads to malformations of the walls of the cells and to the inability of daughter cells to separate from the mother cell. Inhibition of fermentation and of oxidation activity, and a reduction in the level of coenzyme nucleotides, have also been observed. Caffeine can lead to a deficit of inositol in the body. The use of sulfa drugs increases the demand for inositol. Diuresis, as in the case of diabetes insipidus and in excessive ingestion of water, can cause loss of inositol.

Deficit of inositol can cause constipation, eczema and eye anomalies. It contributes to hair loss and to a high rate of cholesterol in blood, which can become manifest with cardiac and arterial diseases. An inositol deficit can have negative effects on the nervous system of diabetics who are affected by peripheral neuropathies.

Inositol is beneficial in treating constipation because it has a stimulating effect on the muscular action of the food canal. It is also recommended to people affected by baldness and is essential in contributing to lower the level of cholesterol in blood and therefore is a protective factor with respect to cardiovascular diseases. It helps to eliminate fats from the liver in patients who need to be operated for stomach cancer.

The use of inositol combined with choline has a positive effect on peripheral diabetic neuropathies. The same combination is recommended in female nutrition. A dose of 500 mg of inositol and 1000 mg of choline helps in cases of hypoglycemia. Inositol, vitamin E and octacosanol are known for their positive effects on cerebral palsy when there are no allergies. Nervous transmission is improved by taking B-complex vitamins and inositol. Dr Cari Pfeiffer, in his Brain Bio Center, has studied the effects of inositol on brain waves. The results have shown that this vitamin has an anxiolytic power which is similar to that of Librium. Dr Pfeiffer believes that people who take Librium or Valium could interrupt the treatment, taking instead an adequate dose of inositol. Since inositol has an effect which is similar to that of sedatives, it not only acts as a tranquilizer without any side effect, but it can be effective against insomnia. It solves cases of slight hypertension by gradually reducing blood pressure; one gram in the morning and one at night are recommended. Inositol is useful for treating schizophrenia, hypoglycemia and for people with a high rate of copper and a low rate of zinc in serum. It has been discovered that inositol prevents liver enlargement.

Folic acid

Folic acid (folate) is vitamin B9, a substance which is not produced by the body and must therefore be taken with food or by way of the action of the intestinal bacterial flora. The daily requirement for folic acid in normal conditions is approximately 0.2 mg. In recent decades, folic acid has been recognized as essential in preventing neonatal malformations, particularly those affecting the neural tube, which can occur in the first stages of embryo development. During pregnancy, therefore, the demand for folate doubles to 0.4 mg because the fetus uses maternal reserves. Although its role is not known in detail, folic acid is in fact essential for synthesis of DNA and proteins and for forming hemoglobin, and is particularly important for tissues which undergo proliferation and differentiation processes, such as indeed embryo tissues. However, folic acid also helps to prevent other situations of health risk. The presence of folic acid in fact lowers the levels of the amino acid homocysteine, which is associated with the risk of cardiovascular diseases and infarctions, although at present it is not possible to establish a direct association between folate taking and reduction of cardiac risk. Moreover, folic acid seems to have a role, which has not yet been clarified, in preventing other congenital defects and malformations such as cleft lip and palate and some congenital heart defects.

A deficit of folic acid in the first stages of pregnancy increases greatly the risk of fetal malformation, in particular of neural tube defects (NTD) associated with spina bifida or anencephaly. In general, a folate deficit can give rise more easily to adverse outcomes, such as for example delayed intrauterine growth, premature birth or placental lesions.

In adults, folic acid deficit can become manifest with megaloblastic anemia. Moreover, the absence of folic acid is often associated with deficits of other oligonutrients (zinc, vitamin B 12), which in turn are further factors of teratogenic risk (e.g., neural tube defects).

A reduction in the absorption of folic acid and/or a consequent increase in its demand may also derive from taking certain drugs (barbiturates, estroprogestins), from high consumption of alcohol, from insulin-dependent diabetes mellitus, coeliac disease, or some specific variations of genes involved in folate metabolism (methylene- tetrahydrofolate-reductase, folate receptor). If women of fertile age have one of these risk factors, therefore, they must take folic acid with particular attention in the periconception period. Women in high-risk groups (who have a certain family history with neural tube diseases or have had a previous pregnancy with a NTD or are affected by diabetes mellitus, obesity or epilepsy) should be monitored with particular care by health workers, since they might require larger quantities of folic acid. In practice it has been found that the composition according to the invention fully achieves the intended aim, since the composition comprising inositol in such a quantity so as to provide at least 2 g/day of inositol to a subject to which it is administered allows to control ovulation induction.

The disclosures in Italian Patent Application no. RM2007A000341, from which this application claims priority, are incorporated herein by reference.

Claims

I . A composition comprising inositol for controlling induction of ovulation in a subject by administering to said subject said composition in such a quantity so as to provide at least 2 g/day of inositol.

2. The composition according to claim 1, wherein said inositol is myo-inositol.

3. The composition according to one or more of the preceding claims, wherein said subject is affected by hyperinsulinemia.

4. The composition according to one or more of the preceding claims, wherein said subject is affected by polycystic ovary syndrome.

5. The composition according to one or more of the preceding claims, wherein said subject is affected by secondary amenorrhea.

6. The composition according to one or more of the preceding claims, wherein said subject is subjected to an in vitro fertilization and embryo transfer (IVF-ET) procedure.

7. The composition according to one or more of the preceding claims, wherein said composition is administered in such a quantity so as to provide 2 to 4 g/day of inositol.

8. The composition according to claim 7, wherein said composition is administered in such a quantity so as to provide 2 g/day of inositol.

9. The composition according to one or more of the preceding claims, characterized in that it further comprises folic acid or is administered simultaneously, sequentially or separately with respect to said folic acid.

10. The composition according to claim 9, characterized in that it further comprises folic acid.

I I . The composition according to claim 9 or 10, wherein said composition is administered in such a quantity so as to provide at least 200 μg/day of folic acid.

12. The composition according to claim 1 1, wherein said composition is administered in such a quantity so as to provide 200 to 400 μg/day of folic acid.