Patent Publication Number: US-2021169825-A1

Title: Mixture of resveratrol supported on metal hydroxide and non-supported pure resveratrol for treating female fertility

Description:
FIELD OF APPLICATION 
     This invention concerns the pharmaceutical, nutraceutical or in the form of a dietary supplement based on mixture of resveratrol with different kinetics of dissolution for use in the treatment of female infertility. A couple who tried to conceive for over a year without success is considered infertile or less fertile than a typical couple (Evers, 2002). The factors influencing the state of infertility are often related to women and include ovulatory insufficiency, tubal damage, endometriosis, poor egg quality and unexplained sub fertility. (Evers J L. Female subfertility. Lancet. 2002 Jul. 13; 360(9327):151-9). 
     To overcome these fertility problems, many couples undergo assisted Reproductive Techniques (ART). These include stimulation of ovulation, intrauterine insemination (IntraUterine inseminations, IUI), in vitro fertilization (In Vitro Fertilization, IVF) and intracytoplasmic sperm injection (ICSI). Some women use supplements with antioxidants in preparation or at the same time as treatment ART, and some women use supplements with antioxidants without practicing ART in an attempt to improve their fertility outcome. 
     Nutrients are defined as those organic or inorganic substances that are absorbed and assimilated by the organism through the process of digestion of food or taken through dietary supplements. Nutrients maintain homeostasis of the body composition and the state of health of an individual. Some nutrients are readily available for absorption from the food (e.g. vitamins, trace elements, antioxidants and active ingredients), others are produced during the digestive process (e.g. monosaccharides from polissaccharides or amino acids by proteins). 
     Many nutrients have demonstrated the ability to increase the probability of a conception, both in a natural way and through ART. Among these substances included are vitamins, provitamins, minerals and phytocomplexes, many of which have antioxidant properties. Antioxidants, in particular, are biological and chemical compounds that reduce oxidative damage (imbalance between the creation of reactive oxygen species and the ability of the body to eliminate them). Among the nutrients with anti-oxidant and non-antioxidant properties, used in female subfertility, they cite, but are not limited to: N-acetil-cysteine; Melatonin Vitamins A, C, D, E, B2, B3, B6, B12, folic acid, inositol, zinc, selenium, Coenzyme Q10, arginine, a-lipoic acid and resveratology. Showell M G, Mackenzie-Proctor R, Jordan V, Hart R J. Antioxidants for female subfertility. Cochrane Database Syst Rev. 2017 Jul. 28; 7:CD007807. In the Italian application 102016000098988, extracts of  Lycium barbarum  berries were claimed to improve female fertility. 
     Vitamin D is well known for its role in the maintenance of calcium homeostasis and in the promotion of bone mineralization. Increasing evidence indicates a correlation between vitamin D deficiency and decreased female fertility. Lerchbaum E, Obermayer-Pietsch B. Vitamin D and Fertility: a systematic review. Eur J Endocrinol. 2012 May; 166(5):765-78. 
     Vitamin E is a well-known endogenous antioxidant and the benefits of fertility include the improvement of the growth epithelium in the blood vessels and in the endometrium. Lédée-Bataille N, Olivennes F, Lefaix J L, Chaouat G, Frydman R, Delanian S. Combined treatment by pentoxifylline and tocopherol for recipient women with a thin endometrium enrolled in an oocyte donation programme. Hum Reprod. 2002 May; 17(5):1249-53. 
     Vitamin B6 (pyridoxine) is involved in various biochemical reactions affecting both the amino acid metabolism and that of various other compounds. Higher levels of vitamin B6 were found in fertile women than infertile women. Grajecki D, Zyriax B C, Buhling K J. The effect of micronutrient supplements on female fertility: a systematic review. Arch Gynecol Obstet. 2012 May; 285(5):1463-71. doi: 10.1007/s00404-012-2237-2. 
     Vitamin B12 (Cyanocoballamine) is necessary for the metabolism of fatty acids and therefore necessary for energy metabolism and cellular proliferation. Supplementation with folic acid and vitamin B-12 have been associated with positive reproductive outcomes in both natural pregnancies and those after treatment with assisted reproductive technology (ART). Gaskins A J, Chiu Y H, Williams P L, Ford J B, Toth T L, Hauser R, Chavarro J E; EARTH Study Team. Association between serum flurries and vitamin B-12 and outcomes of assisted reproductive technologies. Am J Clin Nutr. 2015 October; 102(4):943-50. Moreover, folic acid is necessary for all DNA synthesis, repair and methylation reactions, for homocysteine metabolism and in biochemical reactions involved in cell division. Dattilo M, Giuseppe D, Ettore C, Ménézo Y. Improvement of gamete quality by stimulating and feeding the endogenous antioxidant system: mechanisms, clinical results, insights on gene-environment interactions and the role of diet. J Assist Reprod Genet. 2016 December; 33(12):1633-1648. 
     Inositols such as my-inositol and D-chiro-inositol help ovarian function and decrease hyperandrogenism and insulin resistance. Nestler J E, D J Jakubowicz, de Vargas A F, Brik C, Quintero N, Medina F. Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system. J Clin Endocrinol Metab. 1998 June; 83(6):2001-5. 
     Coenzyme Q10 is an important cofactor in mitochondrial physiology and its supplementation has been proposed to protect the ovarian reserve by combating mitochondrial ovarian ageing. Özcan P, Fiçicioğlu C, Kizilkale O, Yesiladali M, Tok O E, Ozkan F, Esrefoglu M. Can Coenzyme Q10 supplementation protect the ovarian reserve against oxidative damage? J Assist Reprod Genet. 2016 September; 33(9):1223-30. 
     N-Acetil cysteine is an important endogenous antioxidant molecule that has been shown to be necessary for the secretion of cervical mucus and ovulation. Badawy A, State O, Abdelgawad S. N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: a cross-over trial. Acta Obstet Gynecol Scand. 2007; 86(2):218-22. 
     Resveratol is a polyphenol with a stilbenic structure contained in micromolar concentration in red wine and grape husk as well as in some plant species such as  Polygonum cuspidatum . After absorption, resveratrol is subjected to reactions in metabolism: in particular, it is conjugated with sulphuric acid and glucoric acid, which therefore represent the metabolites. These metabolites can release resveratrol by acting as precursors (see for example during Enterohepatic recirculation). For this reason, when we refer to the plasmatic resveratrol we mean total resveratrol, which is the sum of free resveratrol and its metabolites. Resveratrol has been shown to protect cells from oxidative stress by behaving as an antioxidant and by improving mitochondrial function. Resveratrol is an activator of SIRT-1, a deacetylase NAD+employee that is involved in the post-translational modification of certain proteins. 
     SIRT-1&#39;s are involved in epigenetic regulation, as there are histone proteins among their target proteins. Through the activation of SIRT-1 resveratrol has been suggested to prevent ovarian ageing, as demonstrated by the decrease in the frequency of mitotic errors and by the mitochondrial distribution, thus allowing the maintenance of Ovarian reserve (Li et al., 2013). During the in vitro maturation of resveratrol supplementation improves oocyte quality and embryonic development (Tatone et al., 2018). Liu M, Yin Y, Ye X, Zeng M, Zhao Q, Keefe D L, Liu L. Resveratrol protects against age-associated infertility in mice. Hum Reprod. 2013 March; 28(3):707-17. Tatone C, Di Emidio G, Barbonetti A, Carta G, Luciano A M, Falone S, Amicarelli F. Sirtuins in gamete biology and reproductive physiology: emerging roles and therapeutic potential in female and male infertility. Hum Reprod Update. 2018. 
     Resveratrol possesses an optimal breakdown coefficient to cross the biological membranes, but the absorption and therefore its bioavailability is limited by the low solubility in water. Molecules such as resveratrol (membrane permeable, but poorly soluble in water) are classified according to the biopharmaceutical system in class II and their absorption can be increased by increasing the dissolution rate. Resveratrol formulations co-precipitated or supported with bivalent or trivalent metal salts, known in the art as described in the patent application EP 2679243 whose contents are here completely incorporated by reference, present a better dissolving speed versus a pure resveratrol not supported. 
     According to a particularly preferred form of accomplishment, a formulation containing trans-Resveratrol supported on magnesium hydroxide is used in the composition according to the invention. The content of Trans-resveratrol in such formulation can be between 10% and 50% by weight on the weight of the formulation, preferably it is 30% by weight on the weight of the formulation and sold commercially under the trade name REVIFAST®. REVIFAST® presents an increased dissolution rate at the pH of the gastric environment compared to unsupported pure resveratrol. Non-supported pure resveratrol means a crystalline product with a purity exceeding 98% of both natural origin (e.g.  Polygonum cuspidatum ) and synthesis or mixtures of the two. 
     In the patent application US2017/0312251 is reported a formulation of dietary supplements to increase female fertility. The Formulation has a composition that includes L-carnitine tartrate, coenzyme Q10, vitamin B12, N-acetyl cysteine and plant extracts containing various antioxidants including resveratrol. In Addition, a dosage plan is claimed which includes a fractional daily dosage (2 cps/day) of the formulation described above for a period of at least three months. 
     In the PCT/IB2017/052151 patent application, a formulation is claimed including a mixture of inositols in precise stoichiometric ratios and resveratrol co-precipitated or supported with bivalent or trivalent Salts in the form of hydroxides able, among the various applications, to reduce the symptomatology associated with polycystic ovary syndrome, including infertility. 
     After ingestion and absorption of a single dose of the nutrient, the plasma concentration increases until it reaches a peak of maximum concentration, after which it decreases until it disappears over time following processes related to metabolism, distribution and elimination of nutrients. 
     The physiological action of the nutrient is partly mediated by the chemical-physical interaction of the same with specific receptors present in our organism. Receptor means a protein, a nucleic acid or a lipid that has the ability, on the one hand, to interact with the nutrients and on the other to modify the biochemical reactions within the cells expressing the above-mentioned receptors. The nutrient interaction with the receptor leads to the formation of a nutrient/receptor complex hereinafter referred to as the complex. The formation of the complex is through the formation of chemical bonds between the nutrient and the receptor and can be described by the chemical reaction shown in diagram 1: 
       Nutrient+Receptor&lt;-&gt;Complex  [scheme 1]
 
     The affinity of a nutrient for its receptor means the energy associated with the formation of the complex and expresses the tendency of a nutrient to associate with a receptor or to remain free. At the same affinity of a nutrient for its receptor the concentration of the complex is solely dependent on the nutrient concentration according to the scheme 1. 
     The efficacy of a complex means the ability of the complex to activate, inhibit or modify biochemical reactions within the cell and between cells. The efficacy of a complex determines the biological effect of the nutrient itself on the organism according to the pattern 2. 
       Nutrient+Receptor&lt;-&gt;Complex-&gt;biological effect  [scheme 2]
 
     From the above, a nutrient evokes a biological effect in precise concentration ranges that are closely related to the affinity for the receptor (s) that mean/no the biological effect. The therapeutic range of a substance is the range of concentrations that can actually evoke a therapeutically effective biological effect. This range is subjective and can vary significantly from patient to patient, and obviously from substance to substance. When a substance such as a drug or nutrient is administered in a preventive manner, as in the case of the object of this invention, it is reasonable to use the target level strategy. In particular, a concentration of the desired stationary state (target concentration, CB) of the drug or nutrient (usually in the plasma) is chosen and a dosage is calculated to allow it to reach this value. Except when you do not have specific information about the drug and nutrient, the target concentration represents the center of the Therapeutic range. It Is generally accepted that side effects or loss of efficacy of a nutrient are related to the difficulty of quickly reaching and maintaining the target concentration of the drug or nutrient in a specific therapeutic window. (Goodman &amp; Gilman, The pharmacological bases of the therapy, Zanichelli 8 edition, 1992). Many biological effects of nutrients are observed in vitro, a condition where the concentration of the same can be defined by the experimenter and is stable over time. Unfortunately, the concentration reached by a nutrient in vivo is not constant but dynamic and takes some time, from the time of administration, to reach and maintain the target concentration. Due to this dynamic concentration many biological effects observed in vitro cannot be reproduced in vivo. The rapid attainment of the target concentration of the nutrient and its maintenance would theoretically allow to overcome the limits posed by the complexity of the biological systems in vivo. 
     Surprisingly, the applicant found that the administration of a composition of resveratrol supported on hydroxides of bivalent or trivalent metals, particularly on magnesium hydroxide together with pure non-supported resveratrol, allows the concentration of the molecule to reach and maintain a target concentration useful for improving female fertility. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Plasma profile of total resveratrol (free resveratrol+its metabolites) following the administration to voluntary subjects of a single dose of resveratrol of 180 mg REVIFAST® (circles), from pure resveratrol not supported (squares) and a simulation of the combination of resveratrol from REVIFAST® and pure resveratrol not supported in the ratio 1:2 (triangles). A dotted line shows the value of the target concentration (CB). 
     
    
    
     SUMMARY OF THE INVENTION 
     The Applicant is designed with a composition or formulation comprising resveratrol supported on bivalent or trivalent metal hydroxide and unsupported pure resveratrol, which are present in specific stoichiometric ratios and have different speeds of dissolution when administered, is able to:
         a) Rapidly reach the target concentration of total resveratrol;   b) Increase the duration that the plasma concentration of total resveratrol maintains at the target concentration (CB);   c) To increase fertility outcome       

     In fact, as demonstrated in the experimental part given below, the applicant found, surprisingly, by means of food supplements of resveratrol having different dissolution rates in reports defined stoichiometry increases the rate of pregnancy in women. 
     In the composition according to the invention, the term “formulation” means a mixture comprising: Resveratrol with different dissolution rates as defined above and nutrients with antioxidant properties used in female subfertility as N-Acetil-cysteine; Melatonin Vitamins A, C, D, E, B2, B3, B6, B12, folic acid, inositol, zinc, selenium, Coenzyme Q10, A-lipoic acid, arginine and extracts from the berries of the  Lycium barbarum.    
     Preferably, in the composition according to the invention, the total amount of resveratrol administered daily is between 20 mg and 300 mg, preferably between 150 and 180 mg even more precisely 150 mg. 
     Preferably, in the composition according to the invention, the relationship between resveratrol supported on hydroxides of bivalent or trivalent metals (in particular resveratrol arising from REVIFAST®) and that resulting from a pure resveratrol unsupported is Between 0.1 and 10, preferably between 0.3 and 3 even more preferably 0.5. 
     Preferably, in the composition according to the invention, the unsupported pure resveratrol is a crystalline product also in the form of granulated with a purity greater than 98% both of natural origin (for example from  Polygonum cuspidatum ) that of synthesis or Mixtures of the two. 
     Preferably, in the composition according to the invention, the quantity of N-acetil-cysteine administered daily is between 100 mg and 1000 mg, preferably between 200 and 800 mg even more preferably 600 mg. 
     Preferably, in the composition according To the invention, the quantity of vitamins A daily administered is between 200 IU and 2000 micrograms, preferably between 500 and 1500 micrograms even more preferably 1200 micrograms. 
     Preferably, in the composition according to the invention, the quantity of vitamins C administered daily is between 100 mg and 1500 mg, preferably between 300 and 1200 mg even more preferably 1000 mg. 
     Preferably, in the composition according to the invention, the quantity of vitamins administered daily is between 10 200 mg, preferably between 20 and 100 mg IU even more preferably 60 mg. 
     Preferably, in the composition according to the invention, the quantity of vitamins B2, administered daily is between 0.5 mg and 50 mg, preferably between 1 and 30 mg even more preferably 15 mg. 
     Preferably, in the composition according to the invention, the quantity of vitamins B3, administered daily is between 10 mg and 100 mg, preferably between 20 and 70 mg even more preferably 50 mg. 
     Preferably, in the composition according to the invention, the quantity of betaine, administered daily is between 20 mg and 1000 mg, preferably between 100 and 500 mg even more preferably 200 mg. 
     Preferably, in the composition according to the invention, the amount of vitamins B6, administered daily is between 0.5 mg and 10 mg, preferably between 1 and 8 mg even more preferably 3 mg. 
     Preferably, in the composition according to the invention, the quantity of vitamin B12, administered daily is between 0.5 micrograms and 2000 micrograms, preferably between 2 micrograms and 500 micrograms even more preferably 10 micrograms. 
     Preferably, in the composition according to the invention, the amount of folic acid or reduced and methylated forms such as the 5-Methyl-Tedra-Hydro-folate administered daily is between 50 mcg and 800 mcg, preferably between 200 mcg and 500 mcg even more Preferably 400 mcg. 
     Preferably, in the composition according to the invention, the quantity of Inositols with particular attention to my-inositol and the Chiro-inositol, which administered daily are between 10 mg and 1000 mg, preferably between 100 and 800 mg even more Preferably 700 mg. 
     Preferably, in the composition according to the invention, the quantity of zinc administered daily is between 1 mg and 100 mg, preferably between 5 and 50 mg even more preferably 15 mg. 
     Preferably, in the composition according to the invention, the amount of selenium, administered daily is between 30 mcg and 200 mcg, preferably between 50 and 100 mcg even more preferably 70 mcg. 
     Preferably, in the composition according to the invention, the quantity Coenzyme Q10, administered daily is between 100 mg and 500 mg, preferably between 150 and 400 mg even more preferably 200 mg. 
     Preferably, in the composition according to the invention, the amount of a-lipoic acid, administered daily is between 10 mg and 300 mg, preferably between 20 and 200 mg even more preferably 150 mg. 
     Preferably, in the composition according to the invention, the amount of arginine, administered daily is between 100 mg and 1000 mg, preferably between 200 and 500 mg even more preferably 280 mg. 
     Example 1 
     The pharmacokinetics in humans of different types of resveratrol formulations. The administration of 180 mg Resveratrol supported on magnesium hydroxide (REVIFAST®) in voluntary subjects leads to a transient increase in total concentration (free resveratrol+resveratrol metabolites) plasmatic resveratrol with Time at the peak concentration of 30 minutes from administration, followed by a decay of about 40% in the period up to 180 minutes from administration. On the contrary, the total plasma concentration of resveratrol achieved with 180 mg of pure resveratrol results in absolute lesser value and with a time at a peak of 90 minutes from administration. The simulation of the combination of resveratrol with different dissolving kinetics REVIFAST®+pure resveratrol not supported in report 1:2) shows a spike in plasma concentration of total resveratrol (Target Concentration, CB) with a peak time of 30 minutes that is kept stable throughout the period examined (180 minutes). 
     Example 2. An Illustrative Table with the Formulation of the Composition According to the Invention is Described Below 
       
     
       
         
           
               
               
             
               
                   
               
               
                 Component 
                 Dose 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 REVIFAST ® 
                 160 
                 mg (of which 48 mg di resveratrol) 
               
               
                 30% resveratrol 
               
               
                 Sintetic resveratrol 
                 104 
                 mg (of which 102 mg di resveratrol) 
               
               
                 98% resveratrol 
               
               
                 Total resveratrol 
                 150 
                 mg 
               
               
                   
               
            
           
         
       
     
     The composition described in Example 1 can be contained in a variety of formulations such as: capsules, tablets, syrups, suspensions and emulsions. The formulation of Example 2 can be supplemented with nutrients with antioxidant properties and not used in female subfertility such as N-acetil-cysteine; Melatonin Vitamins A, C, D, E, B2, B3, B6, B12, folic acid, inositol, zinc, selenium, Coenzyme Q10, A-lipoic acid, betaine and berry extracts of  Lycium barbarum.    
     Example 3 
     Another illustrative table with the formulation of the composition according to the invention is described below: 
     
       
         
           
               
               
             
               
                   
               
               
                 Componente 
                 Dose 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 REVIFAST ® 
                 160 
                 mg (di cui 48 mg di resveratrolo) 
               
               
                 30% resveratrolo ponderale 
               
               
                 Resveratrolo sintetico 
                 104 
                 mg (di cui 102 mg di resveratrolo) 
               
               
                 98% resveratrolo ponderale 
               
               
                 Acido folico (5-Metil-Tedra- 
                 400 
                 microgrammi 
               
               
                 Idro-folato) 
               
               
                 Vitamina D3 
                 25 
                 microgrammi 
               
               
                 Vitamina B6 
                 1.4 
                 mg 
               
               
                 Vitamina B12 
                 2.5 
                 microgrammi 
               
               
                   
               
            
           
         
       
     
     The formulations of examples 2 and 3 also include possible excipients, technological additives, co-formularies, polar and semipolar polymeric matrices, carriers and stabilizers for both pharmaceutical and nutraceutical use. Examples of excipients are xanthan gum and guar gum, sweeteners such as glucose and sucrose, acidic substances such as citric acid agents such as stearic acid. The composition described in Example 2 can be contained in a variety of formulations such as: capsules, tablets, syrups, suspensions and emulsions. The formulations referred to in examples 2 and 3 may be administered by splitting them several times a day, preferably twice daily. 
     Example 4. Case Report n1 of Fertility Enhancement 
     Ms. MM is 37 years old and is classified as infertile since has tried to conceive for more than 12 months without the initiation of a state of pregnancy. She is not affected by polycystic ovary syndrome according to the Rotterdam criteria. She was supported with a food supplement according to the scheme for 3 months based on pure resveratrol (100 mg per day) without having a pregnancy and subsequently doubling the dosage (200 mg) has not changed its situation. Ms. MM then added a REVIFAST®-based supplement (48 mg/day) together with a non-supported pure Resveratrol-based supplement (100 mg/day) for a period of three months after which she successfully began a pregnancy As, demonstrated by the sonographic framework. The quantities taken by Ms MM are such that the dosage used is that shown in examples 2 and 3. 
     Example 5. Case Report n2 of Fertility Enhancement 
     Miss. CL, a 32 year old women classified as being sub-fertile because she has been trying to conceive for more than 12 months unsuccessfully. She is not affected by metabolic disorders such as polycystic ovary syndrome according to the Rotterdam criteria. In her dietary supplement scheme, she took a resveratrol supplement supported on magnesium hydroxide (48 mg daily) for 2 months without having a pregnancy. Subsequently, on her own initiative, Ms CL changed her integration scheme by adding a supplement of unsupported pure resveratrol (100 mg per day). After a period of 53 days she resulted pregnant as shown by the ultrasound picture. The quantities assumed by Ms CL are such that the dosage used is that shown in examples 2 and 3. 
     Example 6. Case Report n3 of Fertility Enhancement 
     Ms. TB is 39 years old and is classified as a sub-fertile because she has been trying to conceive for more than 12 months without starting a state of pregnancy. It is not affected by metabolic disorders such as polycystic ovary syndrome according to the Rotterdam criteria. In his dietary supplement scheme he took a resveratrol supplement supported on magnesium hydroxide (48 mg per day)+inositol (1400 mg) for 2 months daily without becoming pregnant. Subsequently, on her own initiative, Ms TB changed her integration scheme by adding a supplement of unsupported pure resveratrol (100 mg per day). After a period of 68 days she had a pregnancy as shown by the ultrasound picture. The quantities assumed by Ms TB are such that the dosage used is that shown in examples 2 and 3