Formulation comprising vanilloid polyphenols supported on lamellar solids to reduce plasma cgrp levels and its applications

Abstract

A class of new formulations based on vanilloid polyphenols supported on lamellar solids for the reduction of Calcitonin gene-related peptide (CORP) levels in plasma is described. The preparation processes of the formulations are described and their better dissolution properties in a hydroalcoholic environment compared to vanilloid polyphenols itself.

Description

[0001] Formulation comprising vanilloid polyphenols supported on lamellar solids to reduce plasma CGRP levels and its applications

[0002] DESCRIPTION

[0003] Field of application

[0004] The invention concerns new formulations based on vanilloid polyphenols supported on lamellar solids for the reduction of Calcitonin gene-related peptide (CGRP) levels in plasma. The invention also concerns formulation preparation processes and their better dissolution properties in a hydroalcoholic environment compared to vanilloid polyphenols itself. For these formulations administered in vivo to rabbits, a different first pass metabolism at plasma level was also observed by LC-MS (liquid chromatographymass spectrometry) chromatographic analysis. Vanilloid polyphenols supported on lamellar solids are suitable for use in various pharmaceutical, nutraceutical, food, dermatological, cosmeceutical applications and in medical devices as CGRP is involved in neuroinflammation processes underlying the transmission of pain and in pathologies such as polycystic ovarian disease (PCOS).

[0005] The present invention therefore concerns strategies to improve the activities of polyphenols such as antioxidant, anti-inflammatory, antiviral, antidiabetic, cardioprotective, neuroprotective, chemoprotective, antibiotic, antiischemic, antiobesity, antihypertensive, anticancer, antitumor, anti angiogenic and antiaging and pathologies such as pain and PCOS.

[0006] Prior art

[0007] Neuropeptides are chemical messengers made up of small chains of amino acids that are synthesized and released by peptidergic neurons. There are over 100 known neuropeptides, representing the largest and most diverse class of signaling molecules in the nervous system. Neuropeptides are synthesized from large precursor proteins that are cleaved and processed post-translationally, then packaged into dense central vesicles. Neuropeptides are often released together with other neuropeptides and neurotransmitters in a single neuron, producing a multitude of effects. Once released, neuropeptides can spread widely to affect a wide range of targets. Among the neuropeptides of particular importance in physiological and pathological processes are: CGRP, substance P, VIP, NK, CCK, somatostatin, gastrin, secretin, ghrelin, enkephalins, bombesin, galamin.

[0008] Calcitonin alpha gene-related peptide (CGRP) is a neuropeptide consisting of 37 amino acids, produced by alternative splicing of the calcitonin gene and synthesized mainly in sensory neurons, for example those whose cell bodies are located in the dorsal root ganglia. There is a beta-type CGRO isoform which is encoded by a different gene whose biological action is comparable. CGRP mediates its effects through a receptor composed of a G protein-coupled receptor called calcitonin receptor-like receptor (CRLR) co-assembled with receptor activity modifying protein 1 (RAMP1). CGRP has been well documented to exert complex beneficial cardiovascular effects including a potent vaso-relaxant effect and protective effects on cardiomyocytes and endothelial cells. CGRP is involved in pain transmission and neuroinflammation processes that are involved in the pathogenesis of migraine and skin pain. CGRP has also been linked to metabolic disorders including insulin resistance and obesity. See in this regard:

[0009] Kumar A, Potts JD, DiPette DJ. Protective Role of a-Cal citonin Gene-Related Peptide in Cardiovascular Diseases. Front Physiol. 2019 Jul 2;10:821. doi: 10.3389 / fphys.2019.00821. PMID: 31312143; PMCID: PMC6614340;

[0010] Karsan N, Goadsby PJ. Calcitonin gene-related peptide and migraine. Curr Opin Neurol. 2015 Jun;28(3):250-4. doi: 10.1097 / WC0.0000000000000191. PMID: 25887765;

[0011] Gram DX, Hansen AJ, Wilken M, Elm T, Svendsen O, Carr RD, Ahren B, Brand CL. Plasma calcitonin gene-related peptide is increased prior to obesity, and sensory nerve desensitization by capsaicin improves oral glucose tolerance in obese Zucker rats. Eur J Endocrinol. 2005 Dec;153(6):963-9. doi: 10.1530 / eje.1.02046. PMID: 16322403;

[0012] Kreutter DK, Orena SJ, Torchia AJ, Contillo LG, Andrews GC, Stevenson RW. Amylin and CGRP induce insulin resistance via a receptor distinct from cAMP- coupled CGRP receptor. Am J Physiol. 1993 Apr;264(4 Pt l):E606-13. doi: 10.1152 / ajpendo.l993.264.4.E606. PMID: 8386456.

[0013] Peptidergic neurons (CGRP positive have been) described to also innervate the vascular districts, the medulla and the ovarian cortex. CGRP is capable of altering the production of estrogenic and androgenic hormones and the altered levels along with metabolic symptoms have been seen to correlate with high plasma CGRP levels in patients with PCOS. See in this regard:

[0014] Inyama CO, Wharton J, Su HC, Polak JM. CGRP-immunoreactive nerves in the genitalia of the female rat originate from dorsal root ganglia T11-L3 and L6-S1 : a combined immunocytochemical and retrograde tracing study. Neurosci Lett. 1986 Aug 15;69(1): 13-8. doi: 10.1016 / 0304-3940(86)90406-4. PMID: 3528934;

[0015] Uddman R, Edvinsson L, Ekblad E, Hakanson R, Sundler F. Calcitonin gene- related peptide (CGRP): perivascular distribution and vasodilatory effects. Regul Pept. 1986 Aug;15(l): l-23. doi: 10.1016 / 0167-0115(86)90071-6. PMID: 3532219;

[0016] Calka J, McDonald JK, Ojeda SR. The innervation of the immature rat ovary by calcitonin gene-related peptide. Biol Reprod. 1988 Dec;39(5): 1215-23. doi: 10.1095 / biolreprod39.5.1215. PMID: 3265338.

[0017] Capsaicin treatment depletes the presence of CGRP in the ovary indicating the presence of the neuropeptide within unmyelinated sensory fibers (Ghatei MA, Gu J, Mulderry PK, Blank MA, Allen JM, Morrison JF, Polak JM, Bloom SR. Calcitonin gene- related peptide (CGRP) in the female rat urogenital tract. Peptides. 1985 Sep- Oct;6(5):809-15. doi: 10.1016 / 0196-9781(85)90306-7. PMID: 2417202).

[0018] The release of CGRP and SP (substance P) from nerve terminals by polymodal neurons promotes neuro-inflammation. Substance P can induce extravasation of the plasma to allow other substances (bradykinin, ATP, histamine) to access the site of injury and therefore the sensory nerve terminals. This process has been considered important in neurogenic inflammation due to the release of inflammatory mediators such as histamine and serotonin and the release of proteolytic enzymes that catalyze the production of bradykinin. CGRP apparently does not produce plasma extravasation, but is a potent vasodilator and also acts synergistically with SP and other inflammatory mediators to enhance plasma extravasation. The release of neuropeptides, in addition to coordinating local vascular reactions, promotes the excitation of sensory fibers with induction of inflammatory pain. Inflammatory pain is generated, for example, when tissue is damaged, as may result from surgery or due to an adverse physical, chemical or thermal event or an infection by a biological agent or insect bite. Neuroinflammation coordinated by neuropeptides released from nerve terminals is involved in the skin aging process. See in this regard:

[0019] Elewa R, Makrantonaki E, Zouboulis CC. Neuropeptides and skin aging. Horm Mol Biol Clin Investig. 2013 Dec;16(l):29-33. doi: 10.1515 / hmbci-2013-0062. PMID: 25436744;

[0020] Siiskonen H, Harvima I. Mast Cells and Sensory Nerves Contribute to Neurogenic Inflammation and Pruritus in Chronic Skin Inflammation. Front Cell Neurosci. 2019 Sep 18;13:422. doi: 10.3389 / fncel.2019.00422. PMID: 31619965; PMCID: PMC6759746.

[0021] Polyphenols constitute a structurally heterogeneous group of compounds produced by the secondary metabolism of plants characterized by the presence of phenolic groups. From a health and pharmacological point of view, the activity of polyphenols can be summarized in the following actions: antioxidant, anticarcinogenic, antiatherogenic, anti-inflammatory, antibacterial and antiviral (Visioli et al., 2011, Polyphenols and human health: a prospectus. Crit Rev Food Sci Nutr.;5:524-46).

[0022] A group of polyphenols that has attracted considerable interest for its healthpromoting properties is that of vanilloid polyphenols. A vanilloid is a compound that has a vanillic group. In organic chemistry, a vanyl group is a functional group consisting of a benzyl ring replaced by a hydroxyl group (phenol) in the para position and a methoxy group in the meta position.

[0023] Among the vanilloid polyphenols we can find vanillic alcohol (CAS 498-00- 0), vanillin CAS 121-33-5), vanillic acid (CAS 121-34-6), acetovanillone (CAS 498-02 - 2), vanillylmandelic acid (CAS 55-10-7), homovanillic acid (CAS 306-08-1), capsaicin (CAS 404-86-4), curcumin (CAS 458-37-7 ), demethoxy curcumin and bis demethoxy curcumin.

[0024] Curcumin is a vanillic polyphenolic substance that constitutes 2-5% of the powder of the Turmeric root (Curcuma longa) and represents the most abundant of the curcuminoids. The other relevant curcuminoids are demethoxy curcumin and bis demethoxy curcumin.

[0025] Curcumin has two tautomeric forms and is practically insoluble at room temperature in aqueous solutions at neutral and acidic pH, conversely it is soluble in organic solvents such as methanol, ethanol, acetone and dimethyl sulfoxide.

[0026] The therapeutic potential of curcumin is limited by its low bioavailability and poor pharmacokinetic profile (ADME; absorption, distribution, metabolism and excretion) and short half-life in the gastrointestinal tract. See in this regard:

[0027] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm. 2007 Nov-Dec;4(6):807-18. doi: 10.1021 / mp700113r. Epub 2007 Nov 14. PMID: 17999464.

[0028] Ghosh S, Banerjee S, Sil PC. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food Chem Toxicol. 2015 Sep;83: l l l-24. doi: 10.1016 / j.fct.2015.05.022. Epub 2015 Jun 9. PMID: 26066364.

[0029] Curcumin is unstable in neutral and acidic environments due to the reactivity of the a,P-unsaturated ketone group (Michael addition). Curcumin is readily subject to autoxidation through a radical chain reaction leading to the incorporation of oxygen to produce a bicyclopentadione product. Curcumin, both solid and in solution, is also photounstable and the photodegradation products generate ferulic acid, ferulic aldehyde, vanillin and vanillic acid (Nelson KM, Dahlin JL, Bisson J, Graham J, Pauli GF, Walters MA. The Essential Medicinal Chemistry of Curcumin. J Med Chem. 2017 Mar 9;60(5): 1620-1637. doi: 10.1021 / acs.jmedchem.6b00975. Epub 2017 Jan 11. PMID: 28074653; PMCID: PMC5346970).

[0030] Curcumin is therefore an active ingredient of great interest, however its use is limited due to problems linked to its reduced bioavailability. Different types of chemical modifications have been developed to increase the bioavailability of curcumin such as the use of liposomes, phytosomes, nanoparticles, micelles, phospholipid complexes, polymers, adjuvants. See in this regard:

[0031] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm. 2007 Nov-Dec;4(6):807-18. doi: 10.1021 / mp700113r. Epub 2007 Nov 14. PMID: 17999464;

[0032] Hussain Z, Thu HE, Ng SF, Khan S, Katas H. Nanoencapsulation, an efficient and promising approach to maximize wound healing efficacy of curcumin: A review of new trends and state-of-the-art. Colloids Surf B Biointerfaces. 2017 Feb l;150:223-241. doi: 10.1016 / j.colsurfb.2016.11.036. Epub 2016 Nov 30. PMID: 27918967;

[0033] Mirzaei H, Shakeri A, Rashidi B, Jalili A, Banikazemi Z, Sahebkar A. Phytosomal curcumin: A review of pharmacokinetic, experimental and clinical studies. Biomed Pharmacother. 2017 Jan;85: 102-112. doi: 10.1016 / j.biopha.2016.11.098. Epub 2016 Dec 5. PMID: 27930973;

[0034] Jager R, Lowery RP, Calvanese AV, Joy JM, Purpura M, Wilson JM. Comparative absorption of curcumin formulations. Nutr J. 2014 Jan 24;13: 11. doi: 10.1186 / 1475-2891-13-11. PMID: 24461029; PMCID: PMC3918227;

[0035] Naksuriya O, Okonogi S, Schiffelers RM, Hennink WE. Curcumin nanoformulations: a review of pharmaceutical properties and preclinical studies and clinical data related to cancer treatment. Biomaterials. 2014 Mar;35(10):3365-83. doi: 10.1016 / j .biomaterials.2013.12.090. Epub 2014 Jan 15. PMID: 24439402;

[0036] Purpura M, Lowery RP, Wilson JM, Mannan H, Munch G, Razmovski- Naumovski V. Analysis of different innovative formulations of curcumin for improved relative oral bioavailability in human subjects. Eur J Nutr. 2018 Apr;57(3):929-938. doi: 10.1007 / s00394-016-1376-9. Epub 2017 Feb 16. PMID: 28204880; PMCID: PMC5861163;

[0037] Rahimi HR, Nedaeinia R, Sepehri Shamloo A, Nikdoust S, Kazemi Oskuee R. Novel delivery system for natural products: Nano-curcumin formulations. Avicenna J Phytomed. 2016 Jul-Aug;6(4):383-98. PMID: 27516979; PMCID: PMC4967834;

[0038] Szymusiak M, Hu X, Leon Plata PA, Ciupinski P, Wang ZJ, Liu Y. Bioavailability of curcumin and curcumin glucuronide in the central nervous system of mice after oral delivery of nano-curcumin. Int J Pharm. 2016 Sep 10;511 (1):415-423. doi: 10.1016 / j .ijpharm.2016.07.027. Epub 2016 Jul 15. PMID: 27426105; Lu PS, Inbaraj BS, Chen BH. Determination of oral bioavailability of curcuminoid dispersions and nanoemulsions prepared from Curcuma longa Linnaeus. J Sci Food Agric. 2018 Jan;98(l):51-63. doi: 10.1002 / jsfa.8437. Epub 2017 Jul 10. PMID: 28516478;

[0039] Steigerwalt R, Nebbioso M, Appendino G, Belcaro G, Ciammaichella G, Comelli U, Luzzi R, Togni S, Dugall M, Cesarone MR, Ippolito E, Errichi BM, Ledda A, Hosoi M, Corsi M. Meriva®, a lecithinized curcumin delivery system, in diabetic microangiopathy and retinopathy. Panminerva Med. 2012 Dec;54(l Suppl 4): 11-6. PMID: 23241930.

[0040] To try to overcome the bioavailability problems of curcumin, special formulations have been created that make it more bioavailable. Such formulations are described for example in patents EP1837030A1, EP2627195B1, US8785380B2, US9259401B2, CN102274163B, ES2739194T3, CN111407752A, EP2964243A1 and JP2017533970A

[0041] Curcumin has multiple health and pharmacological properties such as antiinflammatory, antioxidant, antiviral, proapoptotic, chemopreventive, chemotherapeutic, antinociceptive, antiproliferative, antiparasitic and antimalarial and is used as a wound healing agent. (Urosevic M, Nikolic L, Gajic I, Nikolic V, Dinic A, Miljkovic V. Curcumin: Biological Activities and Modem Pharmaceutical Forms. Antibiotics (Basel). 2022 Jan 20; 11(2): 135. doi: 10.3390 / antibioticsl 1020135. PMID: 35203738; PMCID: PMC8868220).

[0042] Curcumin shows an important interest in the field of scientific and clinical research as demonstrated by the increase in scientific articles and clinical studies concerning it (for example Kotha RR, Luthria DL. Curcumin: Biological, Pharmaceutical, Nutraceutical, and Analytical Aspects. Molecules. 2019 Aug 13;24(16):2930. doi: 10.3390 / molecules24162930. PMID: 31412624; PMCID: PMC6720683).

[0043] Particularly interesting for the purposes of the present invention is its potential use in the treatment of PCOS. Polycystic ovary syndrome (PCOS), the most common endocrine disorder, is characterized by ovulatory dysfunction, hyperandrogenism, and polycystic ovaries. PCOS has been linked to a number of increased risks of metabolic disorders, including insulin resistance (IR), glucose intolerance, type 2 diabetes, obesity, dyslipidemia, and cardiovascular disease. See in this regard:

[0044] Wekker V, van Dammen L, Koning A, Heida KY, Painter RC, Limpens J, Laven JSE, Roeters van Lennep JE, Roseboom TJ, Hoek A. Long-term cardiometabolic disease risk in women with PCOS: a systematic review and meta-analysis. Hum Reprod Update. 2020 Nov l;26(6):942-960. doi: 10.1093 / humupd / dmaa029. PMID: 32995872; PMCID: PMC7600286%.

[0045] Shen W, Qu Y, Jiang H, Wang H, Pan Y, Zhang Y, Wu X, Han Y, Zhang Y. Therapeutic effect and safety of curcumin in women with PCOS: A systematic review and meta-analysis. Front Endocrinol (Lausanne). 2022 Oct 27; 13: 1051111. doi: 10.3389 / fendo.2022.1051111. PMID: 36387924; PMCID: PMC9646792.

[0046] Shojaei-Zarghani S, Molani-Gol R, Rafiraf M. Curcumin and Polycystic Ovary Syndrome: a Systematic Review. Reprod Sci. 2022 Aug;29(8):2105-2118. doi: 10.1007 / s43032-021-00826-6. Epub 2022 Feb 14. PMID: 35157259.

[0047] The plasma CGRP level in women with PCOS has been found to be higher than that of normal subjects and to correlate with hormonal and metabolic parameters. CGRP is capable of stimulating the production of estrogens and androgens processed by the granulosa cell (Zhang Z, Gong F, Lu GX. Plasma level of calcitonin gene-related peptide in patients with polycystic ovary syndrome and its relationship to hormonal and metabolic parameters. Peptides. 2012 Apr;34(2):343-8. doi:

[0048] 10.1016 / j. peptides.2012.01.018. Epub 2012 Jan 31. PMID: 22314079).

[0049] Summary of invention

[0050] The Applicant has now found that compositions or formulations comprising one or more vanillic polyphenols supported on one more lamellar solids through a weak intramolecular interaction, for example by hydrogen or Van der Waals bonds, lamellar solids are useful for the treatment of pathologies related to dysregulation of neuropeptide levels, where neuropeptides are CGRP, substance P, VIP, NK, CCK, somatostatin, gastrin, secretin, ghrelin, especially CGRP.

[0051] In particular, the Applicant has found that a solid ingredient consisting of an extract of Curcuma longa having a content of curcuminoids equal to 95%, of which 70% curcumin (defined as Curc95), supported on magnesium hydroxide is capable of acutely reducing plasma levels of CGRP and improve the clinical picture of patients suffering from polycystic ovaries. This effect is observed only when Curc95 is administered under appropriate conditions, i.e. in a formulation in which Curc95 is supported on a matrix comprising or made up of lamellar solids.

[0052] In the context of the present invention when vanilloid polyphenols supported on lamellar solids are indicated, it means an ingredient consisting of venilloid polyphenols, in particular the curcuminoids contained in curcumin supported on the particles of a lamellar solid.

[0053] In the context of the present invention, by lamellar solid we mean an inorganic compound not containing organic carbon, which, due to its chemical structure, forms a solid which is spatially arranged as a succession of lamellae, i.e. planar macrocrystals which have a very large size, smaller (thickness) than the other two (see chapter 1 of Volume VII of Comprehensive Supramolecular Chemistry, Pergamon Press, Oxford, 1996). If the lamellae have an electric charge (positive or negative), ions of opposite charge are positioned inside the tunnels, or between the lamellae, to preserve the electrical neutrality of the solid. These ions can be replaced through ion exchange reactions by other ions due to the concentration effect or as a consequence of the greater affinity of some ions compared to others towards the lamellar matrix.

[0054] Among the lamellar solids, magnesium hydroxide, anionic hydrotalcites and double hydroxide salts are of particular importance in the context of the present invention. These lamellar solids have attracted attention for their applications in the pharmaceutical, health, cosmeceutical and food fields.

[0055] Magnesium hydroxide is an inorganic compound with the formula Mg(0H)2, present in nature in the form of a mineral known as Brucite. It has a crystalline structure made up of layers with (OH-) groups organized in a compact hexagonal packing. Each Mg atom is octahedrally coordinated with six (OH-) groups and these octahedra share sides to form the various layers. It is an odorless white solid and has considerable importance from an industrial application point of view in various sectors including nutraceuticals and pharmaceuticals.

[0056] Hydrotalcites have a structure referable to that of brucite or Mg(0H)2, which, due to the isomorphic substitution of Mg (M(II)) with another trivalent metal (M(III)), acquire an excess of positive charge, compensated by anions present in the interlayer region (e.g. Cl- or NO3-). The general formula of synthetic hydrotalcites can be written as [M(II)l-xM(III)x(0H)2]x+[An-]x / n x mS, where M(II) is a valence (II) metal preferably chosen among Mg, Zn, Co, Ni, Mn, Cu; M(III) is a valence (III) metal preferably selected among Al, Cr, Fe, V, Co; An- is an anion with a negative charge n, which compensates for the positive charge of the lamellae; m is the number of cointercalated solvent molecules, usually water (S), per formula weight of the compound. Double hydroxide salts are anionic lamellar solids formed by a single cationic species with the same valence, usually divalent such as Mg, Cu, Ni and Zn, where the interlamellar anion exchange site is created by differences in the coordination geometry (octahedral and tetrahedral) of the divalent cation (Biswick et al., 2006, Journal of Solid state Chemistry 179: 49-55).

[0057] From a structural point of view, lamellar solids are anisotropic systems and, when analyzed by X-ray diffractometry, reveal reflections relating to the crystalline structure, from which it is possible to obtain various structural information, including the interlayer distance (distance between two adjacent lamellae).

[0058] In the context of the present invention, the solid dispersion consisting of Curc95 supported on magnesium hydroxide and called Curc@MDH is of particular importance.

[0059] Various preparation methods of Curc@MDH are described herein, some of which are based on methods described in patents EP2679243 and EP3338802B1 whose descriptions are taken up in full in this regard.

[0060] One aspect of the invention is the preparation of products containing vanilloid polyphenols including curcumin supported on lamellar solids. Curc@MDH, an example of application of the invention, shows a greater dissolution speed of curcuminoids in an acidic environment compared to Curc95. The pharmacokinetic profile of Curc@MDH shows an unexpectedly different metabolite profile compared to Curc95. The product is capable of reducing plasma levels of CGRP in vivo and reduces the effects of conditions typical of neuroinflammation linked to the release of neuropeptides such as PCOS, migraine, psoriasis.

[0061] Brief description of the figures

[0062] FIGURE 1. Characterization of the product obtained from the curcumin support process in the lamellar solid magnesium hydroxide. A) X-ray diffractograms of Curc95 powder (red line), MDH (black line) and Curc@MDH (blue line). B) Particle size analysis and particle size distribution of the Curc@MDH product obtained according to example 3. The Curc@MDH product has dimensions between 0.7-25 pm with D90 = 12.1 pm, D50 = 5.5 pm and DIO = 1.9 pm.

[0063] FIGURE 2. Curcuminoids dispersed in magnesium hydroxide show a better dissolution rate than non-dispersed curcuminoids. Comparative dissolution process in a hydroalcoholic environment (30% ethanol) of a mixture of curcuminoids (curcumin demethoxy curcumin and didemethoxy curcumin) between the mixture as it is and the same mixture of curcuminoids dispersed in magnesium dihydroxide (Curc@MDH) obtained according to example 3. The symbols represent the dissolution of the curcuminoids at the indicated time while the continues line represents the migration of the data using the Wibul equation. Note the different dissolution kinetics which is monoexponential for the product as such and biexponential for Curc@MDH. For the theory, see lannitti RG, Floridi A, Lazzarini A, Tantucci A, Russo R, Ragonese F, Monarca L, Caglioti C, Spogli R, Leonard! L, De Angelis M, Palazzetti F, Fioretti B. Resveratrol Supported on Magnesium DiHydroxide (Resv@MDH) Represents an Oral Formulation of Resveratrol With Better Gastric Absorption and Bioavailability Respect to Pure Resveratrol. Front Nutr. 2020 Nov 13;7:570047. doi: 10.3389 / fnut.2020.570047. PMID: 34422874; PMCID: PMC8377765.

[0064] FIGURE 3. Comparative pharmacokinetic profile of the glucuronide metabolite of curcumin (Curcumin O glucuronide) after oral administration of the mixture of curcuminoids as such and Curc@MDH used for the comparison of the dissolution rate described in Figure 2. Single oral administration at time 0 is of 50 mg / kg. Note how Curcumin OR glucuronide are observed only after administration of Curc@MDH. No evidence of free curcumin is observed with the two formulations (data not shown) after dipping in magnesium hydroxide compared to unsupported curcuminoids. The dosage of curcumin and its metabolite was performed by mass LC according to the analytical methodology as described in lannitti RG, Fiori di A, Lazzarini A, Tantucci A, Russo R, Ragonese F, Monarca L, Caglioti C, Spogli R, Leonard! L , De Angelis M, Palazzetti F, Fioretti B. Resveratrol Supported on Magnesium DiHydroxide (Resv@MDH) Represents an Oral Formulation of Resveratrol With Better Gastric Absorption and Bioavailability Respect to Pure Resveratrol. Front Nutr. 2020 Nov 13;7:570047. doi: 10.3389 / fnut.2020.570047. PMID: 34422874; PMCID: PMC8377765.

[0065] FIGURE 4. Curcumoids dispersed in magnesium hydroxide acutely reduce plasma CGRP concentration. Measurements using a commercial ELISA kit of plasma CGRP obtained from blood samples from rabbits before and after 180 minutes from oral administration of the 50 mg / kg dose of Curc@MDH as described in Figure 3.

[0066] Detailed description of the invention

[0067] The technique of supporting vannilloid polyphenols on lamellar solids, in particular magnesium hydroxide, involves mixing magnesium hydroxide and curcumin extract in precise ratios, subjecting the different particles to appropriate collision conditions during mixing. In particular, for obtaining the Curc@MDH product, the following are of particular importance: 1) the type of technique used to collide the particles, 2) the collision energies of the particles, 3) the treatment time, 4) the initial granulometry of the particles used and 5) the mass ratio of the two solids.

[0068] In the context of the present invention, the process of supporting one solid on another means the product obtained from the particle collision process of a mixture of two solids and created using a particular technique which leads to the modification of a chemical-physical property of at least one of the two initial solids. Of the two solids, the one whose process induces a modification of at least one chemi cal -physical property is defined as a supported solid while the other solid is defined as a carrier solid.

[0069] In the context of the present invention, curcumin supported on a solid means a product consisting of a physical mixture of curcumin and a solid carrier (in particular magnesium hydroxide), in which the support process leads to the formation of weak intermolecular interactions, potentially hydrogen bonds and van der Waals forces, between the solid carrier and the curcumin. The dispersion of curcuminoids in magnesium hydroxide can be obtained by: a) grinding of the solids through ball mill,jet mill, hammer mills or rotary blade mills; b) impregnation, through the solubilization of curcumin in a solvent, typically hydroalcoholic solution, in which the support solid is suspended and subsequent evaporation of the solvent; c) co-co-precipitation processes as described in the Italian patent application for polyphenols with a stilbenic structure EP2679243.

[0070] The characteristic of the solid carrier is a high specific interaction area. The curcumin-supported product on magnesium hydroxide is distinguished from curcumin as such by some chemi cal -physical properties such as: a) the dissolution speed of curcumin in a hydroalcoholic environment 30% v / v (Figure 2). In the context of the present invention, the form of "curcumin supported on magnesium hydroxide" (Curc@MDH) is of particular relevance, obtained through a particular interaction technique that involves collisions of curcumin particles with a type of magnesium hydroxide particles having precise and certain dimensional and morphological characteristics.

[0071] The techniques used to produce the appropriate collision energies between the solid carrier and the curcumin in order to create interactions were: High Energy Ball Milling" (HEBM), jet milling and high shear granulator techniques (HSG).

[0072] The HEBM technique allows the transfer of high mechanical energy to powder mixtures to induce structural transformations and it has also been used to induce the exfoliation of lamellar materials including hydrotalcite, which has a brucitic-type crystallographic structure like magnesium hydroxide, in organic polymers (Vittoria. V., Costantino U., Polymer. 2005, 46(5), 1601-1608). Jet milling is a technique that enables the collision of solid particles using a high-pressure gas flow. The high shear granulator technique requires the particles to be inserted into a chamber with blades rotating at high speed and capable of generating high particle collision forces; the aforementioned chamber is equipped with a wall with selector holes from where the particles escape once they reach the right size.

[0073] The preparation method of the formulation according to the invention involves the following steps which for simplicity are described with reference to the preparation of a formulation containing curcumin supported on magnesium hydroxide:

[0074] - mixing the curcumin and magnesium hydroxide in a mixer for a time between 10-60 minutes, preferably between 10-40 minutes, more preferably between 20- 30 minutes;

[0075] - turbomixing or other technology described above capable of exerting collision forces sufficiently adequate for the production of the product, in which the collision speed of the particles is between 10-50 m / s, preferably between 15-30 m / s, more preferably between 15-20 m / s for a time between 10-30 minutes, preferably between 15- 25 minutes, more preferably 20 minutes;

[0076] - stabilization of the mixture for a time interval between 1-25, preferably between 3-20 minutes, more preferably between 5-15 minutes;

[0077] - final micronization of the magnesium hydroxide-curcumin mixture with a rotation speed between 5-60 rpm, preferably between 10-40 rpm, more preferably between 15-25 rpm for a time between 5-40 minutes, preferably between 10- 30 minutes, more preferably 15-25 minutes; - sieving of the mixture with a 90-110 micron selector filter.

[0078] The average values of the collision energies of the magnesium hydroxide particles are between 1-50 nJ, preferably between 3-40 nJ, more preferably between 5-30 nJ; while those of the curcumin particles are between 5-60 nJ, preferably between 10-45 nJ, more preferably between 15-35 nJ.

[0079] The initial size distribution of the magnesium hydroxide particles is between 1-250 pm, preferably between 1-150 pm, more preferably between 1-50 pm. The initial size distribution of the curcumin particles is between 1-400 pm, preferably between 15- 250 pm, more preferably between 20-100 pm.

[0080] The mass ratio between the vinyl polyphenol component and the solid lamellar component is the following (for simplicity it is indicated with reference to a formulation containing curcumin supported on magnesium hydroxide):

[0081] - magnesium hydroxide 50-80% - curcumin 20-50%;

[0082] - preferably magnesium hydroxide 60-75% - curcumin 25-40%;

[0083] - more preferably magnesium hydroxide 70% - curcumin 30%.

[0084] The Curcuma longa extracts that can be used in the present invention have a curcuminoid content of between 10% and 95%, preferably between 50% and 95%, more preferably between 90 and 95%. The curcumin content is instead between 10% and 80%, preferably between 20% and 75%, more preferably between 70% and 75%.

[0085] Esempi

[0086] Materials:

[0087] Curc95: curcumin extract CoA CPE-095G2108B-23 - Turmeric dry extract 95%.

[0088] Magnesium hydroxide Mg(OH)2: Magnesia GMBH CoA 1725003 - Magnesia 725, 95-100%. Magnesium nitrate hexahydrate Mg(NO3)2 * 6H2O: Brenntag S.p.A., CoA T402229 - Magnesium nitrate > 98%.

[0089] Aluminum nitrate nonahydrate A1(NO3)3 * 9H2O: Poletto Aldo S.r.l., CoA 20AL0022 - Aluminum nitrate CL ST / 044 > 98%.

[0090] Magnesium chloride hexahydrate MgC12 * 6H2O: Brenntag S.p.A., CoA C309022 - Magnesium chloride > 98%.

[0091] NaOH 1 M: prepare by dilution from Soda 50% by weight - Brenntag S.p.A., CoA SODO 1800007 - Caustic Soda 48-50%.

[0092] EXAMPLE 1 : one method for preparing Curc@MDH involves the use of a high shear granulator and the collision of the particles for a period of approximately 20 minutes with a speed of the rotating blade between 18-22 m / s. Using these parameters the collision energy generated corresponds to the following average values of kinetic energy: magnesium hydroxide particles 5-30 nJ; curcuminoids 18-32 nJ. Magnesium hydroxide has a particle distribution between 1-50 microns and a D90 < 20 microns, furthermore the powder shows an ability to flow (measured according to F.U. XII Ed.) classified as "Compliant". The Curcumin Curc95 used is characterized by a grain size between 25-80 pm.

[0093] EXAMPLE 2: a method for preparing Curc@MDH involves the collision of magnesium hydroxide and Curc95 in weight ratios of 70 / 30 respectively in an HSG chamber, with a rotating blade at a speed of 19.5 m / s for 15-20 minutes. Then the product obtained from the first operation, after a rest period of 10 minutes, is placed again in the same mixing chamber with a rotation speed of the blades of 20 m / s for 20 minutes. The final product is obtained at the exit from the selector with a 100 micron selector filter.

[0094] EXAMPLE 3: a method for preparing Curc@MDH according to the invention includes the following steps: 1) mixing the two components in a mixer for 20- 40 minutes in order to obtain a homogeneous mixture of magnesium hydroxide and curcumin; 2) micronization of the magnesium hydroxide-curcumin mixture in a centrifugal micronizer with ventilated grinding at a rotation speed of 19-20 m / s and for a time of 15-25 minutes; 3) stabilization of the micronized mixture for a time interval between 3 and 15 minutes in order to obtain a stabilized mixture; 4) final micronization of the magnesium hydroxide-curcumin mixture in a centrifugal micronizer with ventilated grinding at a rotation speed of 15-25 rpm for 15-25 minutes; 5 sieving of the mixture with a 90-110 micron selector filter. The product obtained has the following chemi cal -physical and functional characteristics:

[0095] 1) the powder X-ray diffraction spectrum shows that both solids have maintained their crystalline structure (Figures 1.1, 1.2, 1.3 and 1.4),

[0096] 2) the final particle size of the product is between 0.7-25 pm with D90 = 12.1 pm, D50 = 5.5 pm and D10 = 1.9 pm (Figure 1.5),

[0097] 3) it has an increased dissolution speed in a hydroalcoholic environment (Figure 2).

[0098] 4) the oral administration of the Curc@MDH product at a concentration of 50 mg / kg in rats determines the appearance in the plasma of the metabolite Curcumin O glucuronide, an event that is not observed with the unsupported mixture of curcuminoids (Figure 3).

[0099] 5) the same dose of Curc@MDH determines a plasma reduction of approximately 30% in the concentration of the neuropeptide CGRP 180 minutes after administration (Figure 4).

[0100] EXAMPLE 4: a method for preparing Curc@LDH according to the invention involves the following recipe. Solution 1 is prepared by dissolving 641 mg of Mg(NO3)2 * 6H2O, 401.4 mg of A1(NO3)3 * 9H2O in 18 mL of de-ionized water. Solution 2 is prepared by dissolving 300 mg of curcumin (100% pure of synthetic origin) in 18 mL of hydroalcoholic solution. 3 mL of a IM NaOH solution (up to a pH between 10-11) is added to solution 2, kept away from light, under stirring and under a nitrogen flow. Subsequently, 16 mL of solution 1 are added together with 6 mL of 1 M NaOH in such a way as to maintain the pH between 10-11 during the additions. The precipitated solid is left to digest for 15 minutes, then separated by centrifugation. The recovered solid is washed twice with 30 mL of de-ionized water and finally once with 20 mL of ethanol. The product is then dried at 50°C.

[0101] EXAMPLE 5: a method for preparing Curc@MDH according to the invention involves the following recipe. Solution 1 is prepared by dissolving 525 mg of MgC12 * 6H2O in 18 mL of de-ionized water. Solution 2 is prepared by dissolving 300 mg of curcumin (100% pure of synthetic origin) in 18 mL of hydroalcoholic solution. 3 mL of a IM NaOH solution (up to a pH between 10-11) is added to solution 2, kept away from light, under stirring and under a nitrogen flow. Subsequently, 16 mL of solution 1 are added together with 6 mL of 1 M NaOH in such a way as to maintain the pH between 10-11 during the additions. The precipitated solid is left to digest for 15 minutes, then separated by centrifugation. The recovered solid is washed twice with 30 mL of deionized water and finally once with 20 mL of ethanol. The product is then dried at 50°C.

[0102] EXAMPLE 6. An exemplary formulation of the composition according to the invention is described below:

[0103] EXAMPLE 7. An exemplary formulation of the composition according to the invention is described below:

[0104] The composition described in examples 6 and 7 can be contained in a variety of formulations such as: capsules, tablets, syrups, suspensions and emulsions. The formulation of example 2 can be integrated with nutrients with female antioxidant properties such as N-acetyl-cysteine; melatonin; vitamins A, C, D, E, B2, B3, B6, B12, folic acid, inositols, zinc, selenium, Coenzyme Q10, a-lipoic acid, betaine and Ajuga reptans extracts

[0105] EXAMPLE 8. Another exemplary formulation of the composition according to the invention is described below:

[0106] The formulations of examples 6 and 8 also include eventual excipients, technological additives, co-formulations, polar and semi-polar polymer 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, acidifiers such as citric acid and flow agents such as stearic acid. The compositions described in examples 6-8 can be contained in a variety of formulations such as: capsule, compress, syrup, suspension and emulsion. Formulations of which examples 2 and 3 can be administered in divided doses multiple times a day, preferably twice a day.

[0107] EXAMPLE 9. 32-year-old GC presents a 7-month history of oligoamenorrhea and no vasomotor symptoms or night sweats. The laboratory evaluation has revealed an altered concentration of testosterone and an ultrasound picture with the presence of ovarian cysts arranged in a "rosary crown". The clinical picture is in accordance with the diagnosis of polycystic ovary syndrome based on the Rotterdam criteria. She was subjected to treatment with food supplementation with inositol without a real improvement in the ovulatory picture. So in his dietary integration scheme he has taken separately for 1 month an integrator based on Curc@MDH (500 mg per day, according to example 7) divided into two daily administrations (250 mg per tablet, one in the morning and one in the evening). A return of the ovarian cycle is observed.

[0108] EXAMPLE 10. FB of 42 shows a regular ovarian cycle with the presentation after menstruation of a migraine attack. During the acute phase, he is forced to isolate himself due to the discomfort caused by light (photophobia) and to remain in the supine position for a variable period until the pain symptoms disappear. This situation is resolved by the use of anti-migraine agents such as sumatriptan administered prior to chronic pain. Surprisingly, the patient observes that if a tablet containing Curc@MDH (500 mg, according to example 6) is ingested, the acute phase of the pain attack is more bearable and in some cases aborts with the resolution of the migraine.

Claims

CLAIMS1. Formulation comprising one or more vanillyl polyphenols supported on one or more lamellar solids.

2. Formulation according to claim 1, wherein the vanillyl polyphenols are selected from the group consisting of vanillyl alcohol (CAS 498-00-0), vanillin CAS 121- 33-5), vanillyl acid (CAS 121-34-6), acetovanilone (CAS 498-02-2), vanillylmandelic acid (CAS 55-10-7), homovanillyl acid (CAS 306-08-1), capsaicin (CAS 404-86-4), curcumin (CAS 458-37-7), demethoxy curcumin and bis demethoxy curcumin.

3. Formulation according to claim 1 or 2, wherein the lamellar solids are selected from the group consisting of magnesium hydroxide, hydrotalcites and double hydroxide salts.

4. Formulation according to one of the preceding claims, comprising a curcuminoid-based product containing curcumin supported on magnesium hydroxide.

5. Method for preparing a formulation according to one of claims 1 to 4 comprising mixing one or more vanillyl polyphenols and one or more lamellar solids by dry turbomixing.

6. Method for preparing a formulation according to one of claims 1 to 4 comprising mixing one or more vanillyl polyphenols and one or more lamellar solids by co-precipitation followed by drying.

7. Formulation according to one of claims 1 to 4 for use in the treatment of a pathology linked to the dysregulation of neuropeptide levels.

8. Formulation for use according to claim 7, wherein the neuropeptides are selected from CGRP, substance P, VIP, NK, CCK, somatostatin, gastrin, secretin and ghrelin.

9. Formulation for use according to claim 8, for use in the treatment forpathologies related to CGRP dysfunction.

10. Formulation for use according to claims 1-9, for use in the treatment of migraine.

11. Formulation for use according to claims 1-9, for use in the treatment of polycystic ovary syndrome (PCOS).

12. Formulation for use according to claims 1-9, for use in the treatment of psoriasis.

13. Formulation for use according to claims 1-9, for use in the treatment of neurogenic inflammation.

14. Formulation for use according to claims 1-9, for use in the treatment of itching.

15. Formulation for use according to claims 1-9, for use in the treatment of ovulation disorders.

16. Formulation for use according to claims 1-9, for use in the treatment of premature ejaculation.

17. Formulation for use according to claims 1-9, for use in the treatment of prostatic hypertrophy.

18. Formulation for use according to claims 1-9, for use in the treatment of prostatitis.

19. Formulation for use according to claims 1-9, for use in the treatment of dysregulation of female ovarian cycle.

20. Formulation for use according to claims 1-9, for use in the treatment of premature ejaculation.

21. Formulation for use according to claims 1-20 in the form of capsules, tablets, syrups and preparations for sublingual administration.

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