TECHNICAL FIELD

The subject matter of the invention is a composition containing myo-inositol and D-chiro-inositol for use in method of weight reduction, improvement of metabolic and hormonal parameters and improvement of condition of skin in women with polycystic ovary syndrome (PCOS).

BACKGROUND OF THE INVENTION

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in women of reproductive age, with a prevalence of 4-18% (Knochenhauer ES et al. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 83:3078-3082). According to the Rotterdam criteria from 2003, PCOS is diagnosed when 2 of 3 conditions are met: oligo- and/or anovulation, clinical and/or biological signs of hyperandrogenism and an ultrasound image of polycystic ovaries, excluding other etiologies of hyperandrogenism. PCOS is usually manifested by irregular menstrual cycles, infertility, and clinical signs of hyperandrogenism, including hirsutism, acne and androgenetic alopecia (Helvaci N. Polycystic ovary syndrome and the risk of obstructive sleep apnea: a meta-analysis and review of the literature. Endocr Connect. 2017, 6(7):437-445, Jakimiuk A, Szamatowicz J. Rola niedoboru inozytolu w patofizjologii zaburzeh wyst^pujqcych w zespole policystycznych jajnikow. Ginekol Pol, 2014, 85, 54-57).

Although pathogenesis of PCOS still remains unclear, more and more evidence supports the thesis about the important role of insulin resistance and compensatory hyperinsulinemia, often observed in these patients, both overweight and lean (Abdelhamid EMS et al. Inositol versus metformin administration in polycystic ovarian disease patients: a case-control study. Evidence Based Women's Health Journal: May 2015 - vol. 5/2, pp. 61- 66; Bevilacqua A. Results from the International Consensus Conference on myo-inositol and D-chiro-inositol in Obstetrics and Gynecology-assisted reproduction technology. Gynecol Endocrinol. 2015, 31 (6):441 -6). It has been hypothesized that in patients with PCOS, insulin resistance and compensatory hyperinsulinemia stimulate the secretion of androgens in both ovaries and adrenal glands and inhibit the synthesis of sex hormone-binding globulin (SHBG) in the liver. This leads to hyperandrogenism and further, premature follicular atresia and anovulation, and gonadotropin imbalance (increase in LH and decrease in FSH), which are characteristic of PCOS (Artini PG et al. Endocrine and clinical effects of myo-inositol administration in polycystic ovary syndrome. A randomized study. Gynecol Endocrinol. 2013, 29(4): 375-9; Carlomagno G. Contribution of myo-inositol and melatonin to human reproduction Eur J Obstet Gynecol Reprod Biol. 201 1 Dec; 159(2): 267-72). Insulin resistance determines also an increased exposure to cardiovascular and metabolic disorders, such as obesity, resistance to insulin, impaired glucose metabolism, type 2 diabetes, dyslipidemia, hypertension, metabolic syndrome, and cardiovascular diseases (Dunaif A et al. Insulin Resistance and the Polycystic Ovary Syndrome Revisited: An Update on Mechanisms and Implications. Endocr Rev., 2012; 33(6): 981 -1030; Dunaif A et al. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 1989; 38(9):1 165-1 174).

The relationship between the insulin resistance and hyperinsulinemia in the pathogenesis of PCOS is confirmed by the beneficial effect of insulin sensitizers in patients with PCOS who have shown ovulation improvement and androgen level decrease in the bloodstream (Bevilacqua A., supra).

Insulin resistance is thought to be associated with an altered insulin signaling, possibly as a result of disruption of the inositol phosphoglycan (IPG) pathway, which are mediators of intracellular insulin action and cell signal transducers (Baillargeon JP et al. Altered D-chiro-inositol urinary clearance in women with polycystic ovary syndrome. Diabetes Care. 2006; 29(2):300-5; Lamer J. D-chiro-inositol— its functional role in insulin action and its deficit in insulin resistance. Int J Exp Diabetes Res. 2002;3(1 ):47-60).

Inositol is a glucose isomer, also called a sugar alcohol, a hexahydric alcohol, and vitamin B8. Inositol is involved in a number of functions, including cell membrane formation, cell growth, morphogenesis, cytoskeleton rearrangements, proliferation regulation, intracellular signaling, peripheral nerve development and function, osteogenesis, and reproduction (Unfer V et al. Updates on the myo-inositol plus D-chiro-inositol combined therapy in polycystic ovary syndrome. Expert Rev Clin Pharmacol. 2014;7(5): 623-31 ; 31 Carlomagno G., Unfer V. Inositol safety: clinical evidences. Eur Rev Med Pharmacol Sci. 201 1 ; 15(8):931 -6). In cells, inositol is present both in free form and as a structural element of membrane phosphatidylinositols. The epimerization of six hydroxyl groups causes that the inositol has nine isomeric forms, with myo-inositol (Ml) and D-(+)-chiro-inositol (DCI) being the most important.

Myo-inositol is a phosphatidylinositol cycle precursor and a component of phospholipids and cell membranes. In addition, Ml is a precursor in the synthesis of secondary hormonal transmitters such as gonadoliberin (GnRH), TSH, and insulin.

In the ovaries, DCI is involved in the insulin-mediated synthesis of androgens, while Ml mediates the glucose uptake and FSH signaling (Unfer V et al. Effects of Inositol(s) in Women with PCOS: A Systematic Review of Randomized Controlled Trials. J Endocrinol 2016: 1849162). The most common form in the ovaries is the myo-inositol (> 99%), followed by D-chiro-inositol (Carlomagno G., Unfer V. Inositol safety: clinical evidences. Eur Rev Med Pharmacol Sci. 201 1 ; 15 (8):931 -6).

Insulin resistance has been shown to be associated with an MI/DCI imbalance in the cell (Lamer J. D-chiro-inositol in insulin action and insulin resistance - old-fashioned biochemistry still at work. IUBMB Life. 2001 ;51 (3):139-48; Sun TH et al. Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls. Biochem Biophys Res Commun. 2002; 293(3): 1092-8).

At the moment, the inositol therapy is recommended primarily for chronic treatment of PCOS (Jakimiuk et al., supra). The prior art describes the effect of using in patients with PCOS both myo-inositol alone (see, i.a., L. Ciotta, M. Stracquadanio, I. Pagano, A. Carbonaro, M. Palumbo, and F. Gulino, "Effects of Myo-lnositol supplementation on oocyte's quality in PCOS patients: a double blind trial," European Review for Medical and Pharmacological Sciences, vol. 15, no. 5, pp. 509-514, 201 1 ; A. D. Genazzani, C. Lanzoni, F. Ricchieri, and V. M. Jasonni, "Myo-inositol administration positively affects hyperinsulinemia and hormonal parameters in overweight patients with polycystic ovary syndrome," Gynecological Endocrinology, vol. 24, no. 3, pp. 139-144, 2008), and the D-chiro-inositol alone (Nestler JE et al., Ovulatory and metabolic effects of D-chiro-inositol in the polycystic ovary syndrome. N Engl J Med. 1999 29;340(17): 1314-20R; Isabella and E. Raffone, "Does ovary need D-chiro-inositol?" Journal of Ovarian Research, vol. 5, no. 1 , art. 14, 2012). A potentially beneficial effect of using a combination of these two isomers has also been described (see, i.a., E. Benelli, S. Del Ghianda, C. Di Cosmo, and M. Tonacchera, "A combined therapy with myo-inositol and D-chiro-inositol improves endocrine parameters and insulin resistance in PCOS young overweight women," International Journal of Endocrinology, vol. 2016, art. ID 3204083, 2016).

EP1052997 describes compositions containing DCI for use in the treatment of e.g., PCOS.

Japanese patent application JP2006213684 describes the use of a combination of myo-inositol and D-chiro-inositol in mice, in different weight ratios of these two components, and the effect of such a combination on lowering glucose and triglyceride level in blood and reducing insulin resistance. In experimental results it has been shown that it is preferable to increase the ratio of myo-inositol to D-chiro-inositol.

European patent application EP2782559 discloses the use of myo-inositol and D-chiro-inositol compositions in PCOS. This application was related to compositions containing myo-inositol and D-chiro-inositol in a ratio in the range above 10:1 and below 100:1 (w/w), with the application description showing only that for myo-inositol and D-chiro-inositol in a ratio of 40:1 w/w (so in the normal ratio observed in blood plasma, see Dinicola S, Chiu TT, Unfer V, Carlomagno G, Bizzarri M. The rationale of the myo-inositol and D-chiro-inositol combined treatment for polycystic ovary syndrome. J Clin Pharmacol. 2014;54(10):1079-92), after two weeks of use, restoration of normal ovulation cycle and normalization of hormone levels was observed.

The publication of Nordio M, Proietti E.“The combination therapy with myo-inositol and D-chiro-inositol reduces the risk of metabolic disease in PCOS overweight patients compared to myo-inositol supplementation alone”. Eur Rev Med Pharmacol Sci. 2012;16(5):575-81 , describes the effect of treating overweight patients (BMI > 27 kg/m ² ) with a composition containing Ml and DCI in a physiological weight ratio of 40:1 . This composition allowed to improve metabolic and hormonal parameters and was effective faster than the composition of myo-inositol alone. There were no effects of both compared therapies on body weight, including the BMI value.

Authors of the present invention have unexpectedly found that the use of a combination of myo-inositol and D-chiro-inositol, at a lower ratio of myo-inositol to D-chiro-inositol, than indicated as optimal in state of the art, i.e. at a ratio of 10:1 or lower, for example, at a ratio ranging from 10:1 to 1 :10, particularly preferably 10:1 , in patients with PCOS, allows, after 3 months, not only lowering blood glucose and normalizing hormone levels, but also causes significant and persistent weight loss and improvement of skin condition, and suppression of skin symptoms associated with PCOS, including acne. So, the use of a composition containing myo-inositol and D-chiro-inositol in a weight ratio of 10:1 or lower in patients with PCOS allows not only to obtain known therapeutic effects of the composition of these two forms of inositol (this is, i.a., hormonal parameter improvement, blood glucose reduction and insulin resistance decrease), but also provides additional beneficial effects: weight loss and skin condition improvement, not observed with the state of the art recommended ratio of myo-inositol to D-chiro-inositol of 40:1 .

The subject matter of the present invention is therefore a composition containing myo-inositol and D-chiro-inositol for use in method of weight reduction, improvement of metabolic and hormonal parameters and improvement of condition of skin in women with polycystic ovary syndrome (PCOS), wherein myo-inositol and D-chiro-inositol are in a weight ratio of 10:1 or lower, respectively.

In a preferred embodiment, the weight loss in a woman is observed no later than 3 months after the beginning of the composition intake.

In a preferred embodiment, the composition is administered twice a day for at least 3 months, preferably at least 6 months.

In a preferred embodiment, myo-inositol and D-chiro-inositol are in a weight ratio of 10:1 , respectively.

In another preferred embodiment, myo-inositol and D-chiro-inositol are in a weight ratio of 9:1 , respectively.

In a preferred embodiment, the daily dose of myo-inositol and D-chiro-inositol in combination is 1 100 mg.

In a preferred embodiment, the composition further comprises additional active ingredients, preferably selected from the group consisting of folic acid, vitamin B6, pantothenic acid, vitamin B12, and vitamin D.

In a preferred embodiment, the improvement of the condition of the patient’s skin is observed no later than 6 months after the beginning of composition intake, preferably no later than 3 months after the beginning of the composition intake.

By improving the condition of skin, as used herein, it is meant to reduce or attenuate or suppress skin symptoms associated with PCOS, e.g., acne symptoms. The improvement of the condition of skin can be evaluated in any manner known in the art, for example by self-assessment by the patient receiving the composition (e.g., in a survey), doctor's assessment, scoring on a point scale (e.g., Acne Global Severity Scale).

Description of the figures

Fig. 1 shows the results for body weight analysis.

Fig. 2 shows the results for fT (free testosterone) analysis.

Fig. 3 shows the results for SHBG analysis.

Fig. 4 shows the results for FSH analysis.

Fig. 5 shows the results for LH analysis.

Fig. 6 shows the results for LDL analysis.

Fig. 7 shows the results for HDL analysis. Fig. 8 shows the results for triglyceride analysis.

Fig. 9 shows the results for an OGTT analysis after administration of 75 g of glucose.

Fig. 10 shows the results for OGTT analysis after administration of 75 g of glucose (after 1 hour).

Fig. 1 1 shows the results for OGTT analysis after administration of 75 g of glucose (after 2 hours).

Fig. 12 shows the results for fasting insulin analysis.

EXAMPLES

Example 1 - Composition A containing myo-inositol and D-chiro-inositol

The composition was prepared in tablets, with the active ingredient content listed in Table 1 below. The technology of product manufacture is based on the combination of the granule production process (MIO + DCI) with the powder mixture of other substances (actives and excipients). The raw materials used for production are suitably sieved and homogenized so that the mixture is as homogeneous as possible. For this purpose, parameters such as humidity, bulk volume, grain size distribution of both powder mixture and granulate are tested.

The tablets are then manufactured by pressing the mixture of powder and granules. The substances are poured into a mould, where they are crushed by the lower and upper punch. The mould volume determines the weight of the tablet and is adjusted by the height of the initial setting of the lower punch. The compression pressure, regulated by the distance between the punches, determines the tablet parameters: hardness, thickness, mechanical resistance, as well as their disintegration time and release rate of active substances.

Table 1 - Active ingredients of composition A (values for one tablet)

Example 2 - Composition B containing myo-inositol and D-chiro-inositol

The composition was prepared in an analogous manner to Example 1 , with the active ingredient content listed in Table 2 below.

Table 2 - Active ingredients of composition B (values for one tablet)

Example 3 - Study of composition of the invention effect on the metabolic and hormonal parameters of patients with PCOS

Study design

In the study participated 100 women diagnosed with PCOS according to Rotterdam criteria, with an average age of 28.4 ± 5.1 years, a median age of 29 years, age range of 17 to 40 years, with an average height of 165.2 ± 12.2 cm, median height of 166 cm, range of 160 cm to 188 cm. Informed consent was obtained from all patients participating in the study. During the interview a gynecological history for each patient was collected. During the first visit a gynecological interview was conducted during which it was examined:

1 . Is the patient seeking a child?

2. Is the patient diagnosed with an oligoovulation/lack of ovulation?

3. Is the patient diagnosed with polycystic ovaries in an ultrasound scan? 4. Is the patient diagnosed with acne?

5. Is the patient diagnosed with hirsutism?

The patients who participated in the study took twice a day one tablet containing composition A, prepared as in Example 1.

At each follow-up visit (V1 - before the beginning of administration, V2 - 3 months after the beginning of administration, V3 - 6 months after the beginning of administration) weight, height and BMI measurements were taken, and free testosterone (fT), SHBG, FSH, LH, LDL, HDL, triglyceride, and fasting insulin levels were determined. An oral glucose tolerance test (OGTT) was also performed, including oral administration of 75 g of glucose and plasma glucose determination prior to administration, 1 hour after administration and 2 hours after administration. In addition, an ultrasound scan was performed at each visit. The condition of the skin, according to the Acne Global Severity Scale scoring scale was also assessed. During the visits V2 and V3, patients’ compliance and their assessment of improvement of condition of skin was also checked with the help of a survey.

Statistical analysis

To characterize the tested parameters, in case of quantitative variables, the basic measures of location and dispersion were used: mean, median, standard deviation, minimum and maximum value of the analyzed variables. In case of quantitative variables, the Wilcoxon signed-rank test was used to determine whether there were statistically significant differences between the first and the second and the first and the third visit. Due to multiple comparisons Bonferroni corrections were applied. The level of significance was assumed to be 0.05/2 = 0.025. The obtained results of the probability value p for each parameter between the first and the second, and the first and the third visit were smaller than the assumed level of significance, which indicates that there are statistically significant differences for the analyzed parameters between the first and the second and the first and the third visit.

For each of the questions asked in the gynecological interview of the patient, the percentage distribution of the results obtained is presented.

In addition, in case of qualitative variables: ultrasound scan at each visit, condition of the skin assessment, the percentage distribution of the results obtained (during visits V2 and V3) is presented and compared.

Quantitative data are presented as mean ± standard deviation (SD).

In order to determine if differences in data distribution are statistically significant, the chi-squared test is used, and when the predicted frequency in any cell is below 5, Fisher's exact test is used. The significance level is set at a = 0.05.

Results

The results for the analyzed parameters from individual visits are presented in Tables 3-5 below.

Due to multiple comparisons, Bonferroni corrections were applied, the significance level is assumed as a = 0.05/2 = 0.025 (a = significance level/number of comparisons). For each of the considered variables, the differences between the first and the second, and the first and third visit are statistically significant. The probability values for each case are given in Table 6. In addition, the results are illustrated graphically (see Figs. 1 -12).

For the SHBG and HDL variables, the median value is statistically significantly higher at the second and the third visit in relation to the first visit. For the other variables, the median values are statistically significantly lower at the second and third visit compared to the first visit.

Table 6. Wilcoxon signed-rank test. Results are statistically significant at p <0.025

In addition, the percentage distribution of qualitative results obtained during the second and third visit is analyzed. In order to check if there are statistically significant differences in the percentage distribution of results for visits V2 and V3 a chi2 test is used, if the frequency in cells is less than 5, the Fisher's exact test is used. The significance level is assumed as 0.05.

Statistically significant differences between visits V2 and V3 were found in case of the ultrasound image assessed by the researcher, and condition of the skin, in the survey (Table 7). Here, the obtained p value was lower than the assumed significance level and was respectively p<0.001 in the ultrasound image and p=0.004 in case of condition of the skin. Table 7. Assessment of qualitative parameter improvement occurrence at visits V2 and V3, vs. V1 .