Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/27—Asclepiadaceae (Milkweed family), e.g. hoya
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/2072—Pills, tablets, discs, rods characterised by shape, structure or size; Tablets with holes, special break lines or identification marks; Partially coated tablets; Disintegrating flat shaped forms
  • A61K9/2077—Tablets comprising drug-containing microparticles in a substantial amount of supporting matrix; Multiparticulate tablets
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
  • A61K9/2806—Coating materials
  • A61K9/2833—Organic macromolecular compounds
  • A61K9/284—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone
  • A61K9/2846—Poly(meth)acrylates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/20—Pills, tablets, discs, rods
  • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
  • A61K9/2886—Dragees; Coated pills or tablets, e.g. with film or compression coating having two or more different drug-free coatings; Tablets of the type inert core-drug layer-inactive layer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism

Definitions

• GASTRO-RESISTANT MICROPARTICLES COMPRISING INOSITOL AND/OR GYMNEMA SYLVESTRE EXTRACT, PHARMACEUTICAL AND NUTRACEUTICAL COMPOSITIONS THEREOF AND USES THEREOF.
• the present invention relates to a gastro-resistant composition in the form of microparticles comprising inositol and/or extract of Gymnema sylvestre and at least one gastro-resistant agent, pharmaceutical and nutraceutical compositions comprising said gastro-resistant composition, the use of said compositions and the processes for obtaining said microparticles and compositions.
• Inositol is a cyclic polyol having nine stereoisomeric forms, of which Myo-lnositol (Ml) is the most important form, as it is the most biologically active one.
• Inositol has the same formula (ObH ⁇ Ob) but a different structure of Glucose, and it is synthesized at endogenous level from a Glucose-6-phosphate molecule.
• kidneys represent the main site of inositol synthesis and catabolism and, therefore, are the main regulators of its plasma concentration. Kidneys are estimated to synthesize 4 g/day of inositol.
• Inositol can also be introduced in the body by diet; in fact, it is present in plants in the form of phytates, that are then hydrolyzed to Inositol in the intestine thanks to enzymes called Phytases. Free Inositol is absorbed at the intestinal mucosa level by active transport and its absorption is stereospecific and pH-dependent. Hence, the inositol amounts introduced by the diet are decidedly lower (about 1 g/day) compared to the amounts endogenously produced under normal conditions.
• PCOS Polycystic Ovary Syndrome
• Hyperglycemia and insulin resistance conditions can moreover be associated with intracellular depletion of inositols; in these cases, a supplementing with these compounds proves highly useful owing to their insulin-mimetic properties.
• inositols potential use in medically assisted procreation (MAP) has received special attention.
• MAP medically assisted procreation
• the presence of high concentrations of the most active stereoisomeric form of inositol, Myo-inositol, in serum and in the follicular fluid of patients subjected to IVF (In vitro fertilization) is associated with better oocyte quality and a greater percentage of treatment success.
• Gymnema sylvestre has been used for centuries in India to reduce glycemia in the diabetic patient. Actually, it has been observed that the phyto-complex of this plant is able to lower glycemia in a statistically significant way, by acting on glucose absorption at the intestinal level.
• the gymnemic acid molecule is similar to that of glucose, yet it is larger from a dimensional standpoint. It is therefore able to bind to the receptors for glucose absorption located on the intestinal villi, thereby reducing the absorption of this sugar with a competitive inhibition mechanism at the receptor level. This blockage onsets quickly (about 60 minutes), remains for about 5-6 hours, and thanks to it sugar absorption is inhibited for about 50%. Gymnemic acids might also act by inhibiting intestinal and pancreatic amylases and, therefore, by reducing glucose availability.
• Gymnema sylvestre effect is particularly evident in subjects who follow a hypercaloric diet, imbalanced towards carbohydrates (bread, pasta, sweets, etc.); in these situations, the plant can be advantageously introduced in a body weight loss program ( Preuss HG, Bagchi D et al. Effects of a natural extract of (-)-hydroxycitric acid (HCA-SX) and a combination of HCA-SX plus niacin-bound chromium and Gymnema sylvestre extract on weight loss. Diabetes Obes Metab. 2004; 6(3) :171-80).
• Gymnema sylvestre standardized extract was successfully used, in a dosage of 400 mg/day, in the treatment of patients with non-insulin-dependent diabetes mellitus (NIDDM). 22 patients affected by type-2 diabetes took a Gymnema sylvestre extract for 18-20 months, in addition to their oral hypoglycemics-based therapy. At the end of the study, a significant decrease in glucose and HbA1c mean values was found, along with a progressive increase of pancreatic insulin release, which might mean that the extract protects pancreatic beta cells and fosters their regeneration. In the course of the study, drug dosages were reduced and as many as 5 patients were able to discontinue said drugs altogether.
• NIDDM non-insulin-dependent diabetes mellitus
• Document EP2782559B1 is an example of a patent describing the use of pharmaceutical compositions and supplements for the treatment of Polycystic Ovary Syndrome (PCOS).
• PCOS Polycystic Ovary Syndrome
• compositions for the treatment of Polycystic Ovary Syndrome or for the treatment of specific conditions in which a higher contribution of inositol and/or Gymnema sylvestre extract is required are characterized by a low absorption of said active ingredients at the intestinal level, causing a low bioavailability.
• a technical problem still unsolved is how to induce a higher and prolonged intestinal absorption of inositol and/or of Gymnema sylvestre extract.
• microparticles comprising one or both of the above-indicated active ingredients ( Gymnema sylvestre and inositol) in combination with at least one gastro-resistant agent that may be used as such or formulated in compositions for oral use in which the controlled release of the active ingredient/s, coformulated with the gastro-resistant agent, enables to significantly increase the absorption, the stability over time and the chemico-physical properties of said active ingredients.
• the Authors of the present invention have discovered that it is possible to obtain said active ingredient/gastro-resistant agent coformulations, by a process of co-solubilization thereof and production of a gastro-resistant composition (matrix) comprising them, by spray-drying.
• object of the present invention is:
• composition for oral use comprising said microparticles and at least one suitable excipient.
• a process for the preparation of gastro-resistant microparticles comprising the following steps: a) solubilizing, in an aqueous solution comprising at least one gastro- resistant agent, of inositol and/or of a Gymnema sylvestre extract, and b) producing of a micronized product by spray-drying.
• a process for the preparation of said composition for oral use in the form of tablet, bar, or lozenge comprising the steps of: compressing of the gastro-resistant microparticles as defined above and in the claims, and of suitable excipients, until forming a tablet, a bar or a lozenge, applying, onto the surface of said tablet, bar, or lozenge, of a gastro- resistant film-forming coating by nebulizing or dipping of said tablet or lozenge into a suitable solution comprising at least one gastro-resistant agent.
• a process for the preparation of said composition for oral use in the form of a powder or of a granulate comprising the following steps: dispersing, in an aqueous or alcoholic solution, the gastro-resistant microparticles as described and claimed herein with at least one gastro- resistant agent and suitable excipients in the presence of at least one granulating agent, wherein said granulating agent may be said gastro-resistant agent; subjecting said solution to a treatment with a granulator, and optionally drying the powder or the granulate so obtained.
• gastro-resistant microparticles or said pharmaceutical composition for use as a medicament.
• the present invention brings about the following advantages:
• inositol or “Myo-inositol” can be used indifferently, as interchangeable.
• nutraceutical composition in the present invention it is meant inositol in its form as claimed, supported by a specific nutraceutical form.
• methacrylate copolymer has the meaning commonly used in the literature and comprises neutral, basic, or anionic copolymers.
• Figure 1 Graph of the release of Myo-inositol + anionic methacrylic copolymer micronized product (microparticles release only 50% of inositol at pH 1).
• Figure 2. Myo-inositol + anionic methacrylic copolymer micronized product.
• Figure 3 Graph of the release of Myo-inositol + anionic methacrylic copolymer micronized product in the formulation in tablet form coated with a double film-forming layer (ethyl cellulose + anionic methacrylic copolymer).
• the double layer significantly slows down Myo-inositol release; the formulation is totally gastro-resistant; it releases only 20% of inositol at pH 6.8 in 8h).
• FIG 4. Myo-inositol + anionic methacrylic copolymer micronized product in the formulation with double film-forming layer (ethyl cellulose + anionic methacrylic copolymer).
• Figure 5. Graph of the release of Myo-inositol + anionic methacrylic copolymer micronized product in the formulation in tablet form coated with a single film-forming layer (anionic methacrylic copolymer). The formulation is totally gastro-resistant; it releases about 70% of inositol at pH 6.8 in 8h).
• FIG. 7 Graph of the release of Gymnema sylvestre extract prolonged- release micronized product (the formulation is gastro-resistant; it releases about 10% at pH 1 , 40% at pH 6.8 after 4h and 60% at pH 7.2 in 24h).
• FIG 8. Schematic depiction of a Franz-type vertical diffusion cell.
• Figure 9. Photo of Franz cells set up with the biological membrane (porcine intestinal mucosa).
• Figure 15 A) Gavage with latex flexible gastric tube for Myo-inositol administration, both pure and carried by the new release microsystem. B) Blood collection from the tail.
• Figure 16 ovarian follicles of the rats.
• Figure 17 graphical depiction of bioavailability.
• Figure 18 graphical depiction of ovarian accumulation of Myo-inositoi.
• Figure 19 absorbance/time graph of a-amylase enzyme activity.
• Figure 20 Residual glucose amount, not sensed by Caco-2 cells in the incubation time.
• Figure 21 batch 31 SEM images
• microparticles comprise both inositol and Gymnema sylvestre extract, according to a further embodiment, said particles instead comprise only one of the above-indicated two active ingredients (inositol or Gymnema sylvestre).
• the gastro-resistant agent contained in the gastro- resistant icroparticles is an anionic, basic, or neutral methacrylate copolymer. It may be, e.g., selected from Poly(ethyl acrylate-co-methyl methacrylate) or Poly(butyl methacrylate-co-(2-dimethylaminoethyl)methacrylate-comethyl methacrylate) or Poly (methyl acrylate-co-methyl methacrylate-co-methacrylic acid) or a mixture thereof.
• Gastro-resistant agents meeting the above-reported definitions are available on the market. A non-limiting example of said agents is represented by the following Evonik Health Care products:
• copolymers are also available in pharmaceutical grade, with the name Eudragit in lieu of Eudraguard; they can be used in embodiments for pharmaceutical use.
• microparticles of the invention in any one of the above-indicated embodiments, can comprise a final weight percentage of inositol from 10 to 60%, such as from 20 to 60%, 40 to 60%, 45 to 57%, 47 to 57%, e.g. about 25%, about 45%, from 47 to 51%, 52 to 57%, and all point values of the integers comprised between said ranges.
• Inositol could be selected, e.g., from Myo-inositol, d-Chiro-inositol, Scyllo-inositol, Cis- inositol, Muco-inositol, Neo-inositol, Allo-inositol, Epi-inositol, or a mixture thereof.
• said Gymnema sylvestre extract can be a leaf extract having a titer of gymnemic acids from 10% to 100%, preferably from 25% to 75%.
• said extract could have a minimum titer in gymnemic acids of 25%; in another embodiment of the invention said extract could have a minimum titer in gymnemic acids of 75%.
• the gastro-resistant microparticles as described herein could comprise gymnemic acids in a final weight percentage from 40 to 60%, preferably from 50 to 55%. Said percentage can be applied to any embodiment described herein of said microparticles.
• the gastro-resistant agent as above-defined could be present, e.g., in a final weight percentage of said microparticles from 40 to 90%.
• the microparticles according to the invention can be comprised of an amount of inositol from 10 to 60% by weight of said particles and an amount of gastro- resistant agent from 40 to 90% by weight.
• an amount of inositol from 10 to 60% by weight of said particles and an amount of gastro- resistant agent from 40 to 90% by weight.
• said microparticles are comprised of about 25-57% of inositol and 43-75% of gastro-resistant agent.
• 100g of gastro-resistant microparticles will be comprised of about 47-51 grams of inositol, preferably Myo-inositol, and about 49-53 grams of Eudraguard Biotic as above-defined.
• microparticles described in the examples are embodiments of the present invention.
• the microparticles can comprise Gymnema sylvestre extract with a final content in gymnemic acids ranging from 40 to 60%, preferably from 50 to 55% by weight.
• 100 g of microparticles can contain at least 50 g of gymnemic acids, and further excipients that are: Tween 80, soy lecithin, gum arabic, Eudraguard Biotic, Eudraguard Control, Ethyl-cellulose, NaOH.
• a further object of the present invention is a composition for oral use comprising the gastro-resistant microparticles as described and claimed herein and at least one suitable excipient.
• the technician in the field will be able to select without difficulties the suitable excipients, also based on the type of oral composition to be made.
• composition of the invention can therefore comprise pharmaceutically acceptable excipients when the object is a pharmaceutical, nutraceutical or cosmetic composition; nutraceutically acceptable excipients when the object is a nutraceutical composition, or cosmetically acceptable excipients when the object is a cosmeceutical/cosmetic composition.
• compositions for oral use in general, the same excipients could be used, excipients of which the degree of purity could vary depending on whether a pharmaceutical, nutraceutical or cosmetic composition is to be made.
• said composition could be in the form of tablet, capsule, bar, lozenge powder or granulate.
• said composition for oral use will be coated by at least one gastro-resistant film-forming agent.
• the same gastro-resistant agents used for the preparation of the microparticles described herein can be used for the preparation of the composition of the invention.
• composition could also be made in the form of preparation to be dispersed or solubilized in water or in an aqueous solution (e.g., a drink) and could therefore be packaged in suitable sachets or stick packs ready for use.
• aqueous solution e.g., a drink
• chewing gum comprising the microparticles of the invention, or chewable tablets, i.e. , tablets to be chewed and subsequently ingested.
• a further object of this invention is a process for the preparation of gastro-resistant microparticles according to any one of claims 1 to 11, comprising the following steps a. mixing inositol and/or an extract of Gymnema sylvestre titrated in gymnemic acids, in an aqueous solution comprising at least one gastro-resistant agent, and b. producing of a micronized product by spray-drying.
• the mixing step can be carried out in various stages, so as to obtain a solution, a suspension, or a dispersion, depending on the components mixed.
• said gastro-resistant agent can be anionic, basic, or neutral methacrylate, copolymer, like, e.g. (not limited thereto) Poly(ethyl acrylate-co-methyl methacrylate) or Poly(butyl methacrylate-co-(2-dimethylaminoethyl)methacrylate-comethyl methacrylate) or Poly (methyl acrylate-co-methyl methacrylate-co-methacrylic acid) or a mixture thereof.
• anionic, basic, or neutral methacrylate, copolymer like, e.g. (not limited thereto) Poly(ethyl acrylate-co-methyl methacrylate) or Poly(butyl methacrylate-co-(2-dimethylaminoethyl)methacrylate-comethyl methacrylate) or Poly (methyl acrylate-co-methyl methacrylate-co-methacrylic acid) or a mixture thereof.
• Said inositol can be selected from Myo-inositol, d-Chiro-inositol, Scyllo-inositol, Cis- inositol, Muco-inositol, Neo-inositol, Allo-inositol, Epi-inositol, or a mixture thereof, and wherein said Gymnema sylvestre extract is a leaf extract having a titer of gymnemic acids from 10% to 100%, or from 25% to 75%.
• the ratio between said inositol and/or said Gymnema sylvestre extract and said gastroprotective agent in the solubilization step is from 1:1 to 1:8.
• microparticles containing Gymnema sylvestre according to the examples provided above are prepared, in the solubilization step, additional excipients such as those exemplified above can be added, i.e. , one or more of Tween 80, soy lecithin, gum arabic, ethyl cellulose; NaOH.
• additional excipients such as those exemplified above can be added, i.e. , one or more of Tween 80, soy lecithin, gum arabic, ethyl cellulose; NaOH.
• an amount of about 2 g of Myo-lnositol (Ml) (range between 0.3 and 6 g) can be solubilized in about 40 ml of bidistilled water. Then, about 40 ml of Eudraguard (aqueous dispersion of anionic methacrylic copolymer at 30% w/v, corresponding to about 12 g of copolymer) can be added to the solution. The mixture so obtained is then micronized by spray drying.
• Ml Myo-lnositol
• Eudraguard aqueous dispersion of anionic methacrylic copolymer at 30% w/v, corresponding to about 12 g of copolymer
• the technician in the field will know how to select suitable operating conditions.
• a non-limiting example of suitable operating conditions is represented by: Inlet T: 110 °C; suction: 100%; sample collection pump speed: 15%.
• Gymnema sylvestre extract gymnemic acids titer: 75%) can be mixed in 20 ml of bidistilled water, bringing the solution so obtained to pH about 8 with NaOH, and keeping under constant stirring for 30 minutes, in order to optimize gymnemic acid dissolution. Then, the other excipients can be added. For instance, about 50 mg of Tween 80, about 50 mg of soy lecithin and about 100 mg of gum arabic can be added.
• a gastro-resistant agent can then be added, such as, e.g., about 2 ml of Eudraguard Biotic (equal to 600 mg of anionic methacrylic copolymer) and 0.667 ml of Eudraguard Control (equal to 200 mg of neutral methacrylic copolymer). Finally, a solution of ethyl cellulose in 4% ethanol (10 ml) can be added. The mixture so obtained is then micronized by spray-drying.
• suitable operating conditions is represented by: Inlet T: 110 °C; suction 100%; sample collection pump speed 20%.
• the yield obtained was of ⁇ 50% by weight (with respect to the total weight of the Gymnema extract and of all dry excipients, excluding water and ethanol that evaporate).
• Spectrometrically calculated drug loading in gymnemic acids is of 55% w/w.
• said spray-drying can be conducted by using the following parameters:
• a further object of the present invention are the processes for the preparation of a gastro-resistant oral composition in the form of tablet, bar, lozenge, capsule, powder or granulate.
• the process for the preparation of said composition for oral use in the form of tablet, bar or lozenge can comprise the steps of:
• the process for the preparation of said composition for oral use in the form of a powder or of a granulate can instead comprise the following steps:
• the gastro-resistant microparticles as described and claimed herein with at least one gastro- resistant agent and suitable excipients in the presence of at least one granulating agent, wherein said granulating agent may be said gastro-resistant agent;
• objects of the invention are the products as described and claimed, in the form of products obtainable or obtained by the processes of the invention.
• a further object of the present invention are the gastro-resistant microparticles or the pharmaceutical/nutraceutical composition according to what described above, for use as a medicament/food supplement.
• a further object of the present invention are the gastro-resistant microparticles or the pharmaceutical composition according to what described above, for use in the treatment and/or prevention of both male and female metabolic pathologies.
• a non limiting example of both male and female metabolic pathologies comprises the metabolic syndrome accompanied by insulin resistance and glucose intolerance (e.g.: diabetes, prediabetes, PCOS, menopause).
• a further object of the present invention are the gastro-resistant microparticles or the pharmaceutical composition according to what described above, for use in the treatment and/or prevention of Polycystic Ovary Syndrome.
• the gastro-resistant microparticles or the pharmaceutical or nutraceutical composition can be administered in a daily dose of inositol of 250 mg/day to 8 g/day and/or a daily dose of Gymnema sylvestre extract of 20 mg/day to 700 mg/day.
• compositions are indicated to foster carbohydrate and lipid metabolism, to contribute to normal fertility and normal reproduction, for female fertility, male fertility, menopause, cardiovascular health, thyroid function, nervous function, pulmonary function.
• a further object of the present invention are the gastro-resistant microparticles or the pharmaceutical composition or the composition for use, wherein the daily dose can be administered a single dosage unit or in multiple dosage units.
• a further object of the present invention is the use of the gastro-resistant microparticles or of the nutraceutical composition according to what described above, in the nutraceutical, cosmeceutical/cosmetic and herbalist fields.
• compositions object of the present invention could be used in a medical device, drug, plant protection product, cosmetic product, food for special medical purposes, herbal product, homeopathic product.
• compositions according to the present invention may be used for the making (production) of food supplements, medical devices, drugs, cosmetic products.
• the pharmaceutical composition or the nutraceutical composition according to the present invention can further contain folate and/or vitamins and/or further active ingredients useful to assist in the treatment of the above-described indications.
• All active ingredients and excipients used to attain the object of the present invention can be used in preparations intended for oral administration.
• the active ingredients were to be used in compositions, the active ingredients will be used jointly with excipients of a grade consistent with the type of composition to be created.
• the yield obtained was of 40-50% by weight (with respect to the total weight of inositol and anionic methacrylic copolymer; the theoretical total yield is of 14 g, excluding the water evaporating in the process) and the drug loading in myo-inositol calculated with Megazyme K-INOSL assay is of 12% w/w.
• Gymnema extract (gymnemic acids titer: 75%) was solubilized in 20 ml of bidistilled water, bringing the solution to pH about 8 with NaOH, and keeping under constant stirring for 30 minutes, in order to optimize gymnemic acid dissolution. Thereafter, 50 mg of Tween 80, 50 mg of soy lecithin and 100 mg of gum arabic were added. Once solubilization had occurred, 2 ml of Eudraguard Biotic (equal to 600 mg of anionic methacrylic copolymer) and 0.667 ml of Eudraguard Control (equal to 200 mg of neutral methacrylic copolymer) were added. Finally, an ethyl cellulose solution in 4% ethanol (10 ml) was added. The dispersion was micronized by spray-drying under the following operating conditions:
• the yield obtained was of ⁇ 50% by weight (with respect to the total weight of the Gymnema extract and of all dry excipients, excluding water and ethanol that evaporate).
• Spectrometrically calculated drug loading in gymnemic acids is of 55% w/w.
• the yield obtained is from 30 to 50% by weight of the theoretical one.
• Inositol content in the final formulation of microparticles is from 12 to 14% by weight (i.e. , 100 g of microparticles contain at least 12 g of inositol and 88 g of Eudraguard Biotic, which is the sole excipient).
• the first layer was obtained by treating the tablet surface with an ethanol solution of 5% ethyl cellulose and rapidly dried. This first layer is optional and has the purpose of slowing down an overly fast disgregation owing to water attraction by Myo-inositol, in addition to slowing down Ml release. This coating affects tablet weight for about 2%.
• the tablet was treated with an aqueous solution of Eudraguard Biotic, from 10% to 30% w/v (preferably 30%) and rapidly dried. This coating affects tablet weight for about 3-5%.
• Microparticle gastro-resistance was assayed with the pH jump procedure (2h at pH 1 + 6h at pH 6.8), both in the form of gastro-resistant micronized product as such (batch 6), on 100 mg aliquots at a time, and in the form of tablet with enteric coating.
• the Myo-inositol assay kit K-INOSL (Megazyme, US) was used. Each sample (corresponding to the individual collection at preset times of the release assays) was analyzed individually: in a first stage the sample was put to incubate in a neutral environment in the presence of ATP and hexokinase, then was brought into a basic environment and other enzymatic components of the kit were added; absorbance was read by UV spectrophotometer at 492 nm.
• an aqueous solution of from 10% to 30% w/v (preferably 30%) of Eudraguard Biotic (preferably 2 ml, equal to 600 mg of Eudraguard Biotic) and an aqueous solution of from 10% to 30% w/v (preferably 30%) of Eudraguard Control (preferably 0.667 ml, equal to 200 g of Eudraguard Control) were added.
• the mixture was homogenized under vigorous magnetic stirring for 10 minutes.
• from 1 to 10 ml (preferably 10 ml) of a solution of ethyl cellulose in from 1% to 4% ethanol (preferably 4%) Ethanol was added, stirring vigorously for other 5 minutes.
• Gymnemic acids content ranges from 50 to 55% by weight (i.e. , 100 g of microparticles contain at least 50 g of gymnemic acids, besides other excipients which are: Tween 80, soy lecithin, gum arabic, Eudraguard Biotic, Eudraguard Control, ethyl cellulose, NaOH).
• An UV spectrophotometer was used for the assessment of gymnemic acid present in each individual collection.
• the entire UV spectrum of the Gymnema extract was analyzed, and main peak profile was characterized in order to establish which peak corresponded to gymnemic acid.
• the best peak, proportional to the amount of gymnemic acid present in the sample corresponds to a 275-nm wavelength; then, solutions having a known titer of Gymnema extract were prepared in order to obtain a calibration line with which to compare sample analyses and accurately obtain the amount of gymnemic acid present in the individual releases.
• the yield obtained is from 10% to 15%.
• the yield obtained is from 35% to 40%, and the drug loading in Myo-inositol (Ml) calculated with K-INOSL assay is of 25%.
• the experiment was conducted for the whole time in an orbital incubator at a temperature of 37+/- 1°C.
• Collections were performed from the acceptor compartment every hour, for 10 (+24) hours, and the collected aliquot replaced by an equal volume of fresh buffer solution. Collected aliquots were analyzed by K-INOSL* kit (Megazyme) to determine the Ml amount permeated into the receiving compartment (P) and the residual one in the donor compartment (R).
• the studies were carried out in triplicate and the analytical data reported in concentration v time graphs, generating the bioavailability curves shown hereinafter.
• the AUC Area Under the Curve ) was calculated for each concentration/time curve, to extrapolate the bioavailability related to the absorption stage.
• the maximum concentration value measured [given by the sum of Myo-inositol permeated into the receiving compartment + that absorbed into the tissue, calculated as Tot - (P+R)] equals to about 100% of loaded inositol.
• the Inventors then planned to assess the inositol-containing controlled- release microparticles based on in vivo studies, specifically by analyzing plasma and follicle (follicular fluid) concentrations of the rat animal model, reached by inositol post administration of the inositol-containing microparticles and to compare the concentrations detected with those reached by non-micronized inositol when administered at the same dosage.
• Murine-type animal models were provided, organized as follows:
• the dosage form will be non-micronized.
• the dosage form is represented by the inositol- containing microparticles, whose amount must equal the non-micronized inositol dosage administered to Group 2.
• the dosage form is non-micronized inositol.
• Group 3 (micronized inositol): the dosage form is represented by the inositol- containing microparticles, whose amount must equal the non-micronized inositol dosage administered to Group 2.
• the selected animal models are female Wistar Rats, of 250-280 grams weight.
• the validation of the abovementioned release system consists of two stages, one for bioavailability validation (Stage 1), the other one for biodistribution validation (Stage 2).
• Stage 1 bioavailability by single administration orally and bioavailability assessment within 24 hours
• the animals (15 female rats of weight from 250 to 280 g) were subdivided into 3 groups, each consisting of five animals, as reported in Table 1. All animals were sacrificed after 24 h by cervical dislocation, with prior sedation. After the sacrificing, ovaries were collected ( Figure 16) for quantitative determination of Myo-inositol at the follicular level (extraction from ovary) by specific assay for Myo- inositol.
• Table 1 Animal model groups for bioavailabiHty validation.
• the animals (15 female rats of weight from 250 to 280 g) received a repeated administration at 24-h intervals for 7 days.
• the animals were divided into 3 groups, as reported in Table 2.
• the animals were sacrificed after 7 days by cervical dislocation with prior sedation.
• tissue collection was carried out, specifically ovaries were collected, for quantitative determination of Myo-inositol at the follicular level (extraction from ovary) by a specific assay for Myo-inositol.
• Table 2 groups of animal models for biodistribution validation.
• Plasma samples (of about 60 mI) were provided by the experimental animal room upon centrifuging of collected blood samples (100 mI) at 10.000 rpm for 5 min and immediately cryoconserved at -80°C.
• preliminary tests were carried out on blood samples of the control group.
• plasma samples (25 mI) of the control group were deproteinized by adding 1N trifluoroacetic acid (100 mI), centrifuged at 4500 rpm for 10 min, and the supernatants analyzed with the K-INOSL Megazyme kit, upon neutralization with 1N NaOH solution (100 mI).
• Ovarian follicles (Figure 16) collected after 24 hours (single administration) and after 7 days (administration once a day for 7 days) were homogenized with 800 mI of distilled water by homogenizator at 20.000 rpm for 5 minutes. The homogenates obtained were additioned with 1N trifluoroacetic acid (100 mI), put on an ice bath for 10 min. and centrifuged at 4500 rpm for 10 min at 5°C. The decanted supernatant (900 mI) was neutralized with a 1N NaOH solution (100 mI), and finally filtered with regenerated cellulose 0.2-micrometer filters, before analysis with K- INOSL Megazyme kit, on 100-mI aliquots.
• 1N trifluoroacetic acid 100 mI
• the decanted supernatant (900 mI) was neutralized with a 1N NaOH solution (100 mI), and finally filtered with regenerated cellulose 0.2-micrometer filters,
• Each Myo-inositol concentration value determined in the biological tissues represents the mean of the values of Ml tissue concentrations I in treated animals.
• the bar graph in Figure 18 reports Ml concentration in the ovaries after 24 hours (single administration) and after 7 days (administration once a day for 7 days).
• the concentration values of the graph in Figure 18 are reported in detail.
• Tables 4 Myo -inositol concentration values found in tissues, expressed in mg/ml.
• the ratio of the respective AUCs confirms what foreseen by in vitro permeation studies, i.e. that the new release system increases both overall bioavailability and Myoinositol half-life. This is due to Myo-inositol release modulation, which proves to be site-specific and prolonged, thereby dictating a slowing down and an extension of the absorption, remarkably increasing the plasma concentration peaks and lengthening Myo-inositol half-life.
• a potential total mean increase in bioavailability of a further >1.5-fold compared to the pure active ingredient can be estimated from the AUC reports obtained. Biodistribution is also remarkably improved, both with regard to the single administration and to the repeated one (Table 4), proving to be 1.25-fold higher already after 24h, and as much as 1.65-fold higher after 7 days.
• the novel Myo-inositol release system constitutes a clear technological progress of Myo-inositol dosage forms, able to increase both bioavailability and biodistribution in the follicle in the animal models used in the present study.
• the inhibitory activity of the micronized actives towards SGLT1 membrane proteins was assessed by an in vitro cell assay whose detection method will be of colorimetric type, based on the measurement of the residual ability of human intestinal epithelial CACO-2 type cells to internalize glucose when exposed to gymnemic acid-containing microparticles.
• the detection method will be of colorimetric type and based on the quantitative assessment of intracellular 2-deoxyglucose, which owing to its similarity with glucose is absorbed by cells and whose metabolism generates NADPH that, thanks to a further glutathione-recycling enzymatic amplification reaction, generates 5- thio-2-nitro-benzoic acid that is read at 412 nm.
• the inhibitory activity on a-amylases was assessed by biochemical assay based on 3,5-dinitrosalicylic acid reduction.
• Said methodology is based on the indirect measurement of a-amylase ability to hydrolyze starch, measured by colorimetric assay based on absorbance reduction at 540 nm of 3,5-dinitrosalicylic acid that is reduced to 3-amino-5-nitro-salicylic acid.
• the assay will be performed by incubating a-amylase in the presence of the gymnemic acid- containing microparticles and the subsequent assessment of residual enzymatic activity.
• microparticle batches assayed Object of the study at issue is to study the effect on a-amylase enzyme activity by the formulations of microparticles containing Gymnema sylvestre (batch 6) and Myo inositol (batch 10), even in co-administration, prepared according to the process detailed hereinafter:
• Gymnema Sylvestre microparticles (batch 6)
• Atomizer nozzle 0.7 mm Gas used: Nitrogen
• the yield obtained was of ⁇ 50% and the drug loading in Gymnema sylvestre is equal to 55%.
• the analysis was performed using the Amylase Activity Assay kit.
• the kit enables to assess a-amylase activity over time and compare it with a positive control.
• the microparticles containing Gymnema exhibit the greater inhibitory power of a-amylase enzyme activity, demonstrating even more active compared to Gymnema DE as such at the same concentration.
• a Caco-2 cell line was used: cells were grown in an appropriate culture medium in 1ml_ wells for assays, and, once adhered and almost reaching confluence, were washed with a glucose-free isotonic medium (DPBS). For each well a known amount of glucose was added, equal to 80 pg/ml.
• DPBS glucose-free isotonic medium
• Enzymatic activity assays were performed in triplicate so as to guarantee statistical significance of the data; with standard deviation (SD) ⁇ 5%.
• SD standard deviation
• the Inventors proceeded to increase the drug loading of the Myo-inositol release microsystem with excellent results.
• the data of the experiments carried out are described hereinafter: Drug loading optimization of the Myo-inositol release microsystem
• Myo-inositol was solubilized in 40 ml of bidistilled water, then Eudraguard biotic (40 ml) was added and left under stirring for 30 minutes.
• Eudraguard biotic 40 ml
• Myo-inositol was solubilized in 20 ml of bidistilled water, then Eudraguard biotic (40 ml) was added and let disperse for 30 minutes.
• Eudraguard biotic 40 ml
• a Buchi B-90 Mini Spray-Dryer was used (maximum evaporation rate 1 L/h) with the following parameters:
• Myo-inositol was solubilized in 400 ml of bidistilled water, then Eudraguard biotic (400 ml) was added and left under stirring for 60 minutes.
• Eudraguard biotic 400 ml
• a Buchi B-90 Mini Spray-Dryer maximum evaporation rate 1 L/h was used, with the following parameters:
• Myo-inositol was solubilized in 60 ml of bidistilled water, then Eudraguard biotic (40 ml) was added and left under stirring for 30 minutes.
• Eudraguard biotic 40 ml

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