WO2023199241A1 - Composition comprenant de l'acide lipoïque, de la vitamine d et du glutathion, ses utilisations, et compositions pharmaceutiques et nutraceutiques associées - Google Patents

Composition comprenant de l'acide lipoïque, de la vitamine d et du glutathion, ses utilisations, et compositions pharmaceutiques et nutraceutiques associées Download PDF

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WO2023199241A1
WO2023199241A1 PCT/IB2023/053742 IB2023053742W WO2023199241A1 WO 2023199241 A1 WO2023199241 A1 WO 2023199241A1 IB 2023053742 W IB2023053742 W IB 2023053742W WO 2023199241 A1 WO2023199241 A1 WO 2023199241A1
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composition
lipoic acid
vitamin
disease
derivative
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PCT/IB2023/053742
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English (en)
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Lorenzo Secondini
Mietta CATERA
Stefano LELLO
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URIACH ITALY S.r.l.
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Priority claimed from IT102022000007232A external-priority patent/IT202200007232A1/it
Priority claimed from IT102022000007226A external-priority patent/IT202200007226A1/it
Application filed by URIACH ITALY S.r.l. filed Critical URIACH ITALY S.r.l.
Publication of WO2023199241A1 publication Critical patent/WO2023199241A1/fr

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the present invention relates to a composition comprising lipoic acid, vitamin D, and glutathione, and therapeutic and nontherapeutic uses thereof.
  • the invention also relates to pharmaceutical and nutraceutical compositions thereof.
  • composition comprising lipoic acid, or a pharmaceutically or food-acceptable salt thereof or a derivative thereof, a vitamin D, preferably vitamin D3 and/or vitamin D2, and glutathione or a derivative thereof. It is further an object of the present invention said composition for use in a, therapeutic and/or non-therapeutic, method of treatment of diseases, symptoms and/or disorders, for example associated with cerebral aging, particularly for use in a method of treatment of neuropathies, preferably peripheral neuropathies.
  • the present invention relates to said composition for use in a method of treatment of neurodegenerative diseases.
  • Aging is an extremely complex multifactorial process characterized by a gradual continuous loss of physiological functions, particularly marked in the brain, where, however, related pathologies are not necessarily related to chronological aging.
  • Cognitive frailty is emerging as one of the greatest health threats of the 21st century. Life expectancy continues to rise but at the same time the prevalence of cognitive decline is increasing. This is becoming a serious social problem causing distress and increased costs for individuals, families, and health care systems.
  • antioxidants have been known to be used in combination with symptomatic drugs to reduce oxidative stress and improve cognitive function in aging and aging-related diseases.
  • the brain is highly susceptible to oxidative damage due to high concentrations of polyunsaturated fatty acids and transition metals that are involved in hydroxyl radical generation.
  • astrocytes are responsible in maintaining neuronal and synaptic function. Oxidative stress plays a key role in astrocyte loss, mainly due to highly active mitochondria metabolism.
  • the brain has low catalytic activity and has low levels of protective antioxidant enzymes.
  • compositions for the treatment of peripheral neuropathy in particular for the treatment of pain associated with said peripheral neuropathy.
  • the composition comprises at least one endocannabinoid, at least one phytotherpene, and at least one component selected from alpha lipoic acid and vitamin D and mixtures thereof.
  • Patent US6649195 B1 describes a composition for the prevention and treatment of macular degeneration, cataracts, glaucoma, and other eye diseases.
  • the composition contains more than thirty different vitamins, minerals, phytonutrients, and amino acids, which produce a protective effect on eye well-being.
  • US2007/116779 refers to a composition suitable for counteracting major attenuating factors specific to the degenerative processes occurring in Parkinson's disease.
  • the composition comprises at least one substance that prevents 1-methyl-4-phenylpyridinium-induced toxicity through the promotion of ATP produced by anaerobic glycolysis, ANAEROBIC (+) and in addition one or more substances selected from the group consisting of pyruvic acid, succinic acid and/or oxaloacetate possibly in combination with vitamin B3, vitamin B3 derivatives, magnesium, acetyl-L-carnitine, alpha-ketoglutarate, phospho(enol)pyruvate, fructose and fructose 1,6-biphosphate.
  • W02022/016038 refers to a composition with an anti-inflammatory effect.
  • the composition comprises a nanogel containing a first fraction of hyaluronic acid and a second fraction of hyaluronic acid or a pharmacologically acceptable salt thereof, as well as lecithin liposomes encapsulating vitamin D3.
  • W02020/053754 refers to a composition comprising lipoic acid, or a salt or derivative thereof, and a vitamin D for use in a method for the treatment of neurodegenerative diseases and/or for the treatment of peripheral neuropathy.
  • Patent application US 2009/110674 describes a composition comprising glucosamine, lipoic acid, several amino acids, vitamin A, E, B12 and D.
  • the composition can be used, for example, to counteract the symptoms of Alzheimer's disease, to treat diabetes and osteoarthritis as well as to promote brain function by maintaining healthy neurons and neurotransmitters.
  • Japanese patent application No. 2010/120916 refers to the treatment and improvement of psychiatric/neurological diseases such as Alzheimer's and Parkinson's disease by an active ingredient containing at least one agent selected from pyridoxamine, pyridoxine, glutathione and pharmaceutically acceptable salts thereof.
  • Neurodegenerative diseases are a heterogeneous group of disorders characterized by progressive and selective neuronal death leading to degeneration of specific brain regions caused mainly by the natural aging process.
  • the most common neurodegenerative diseases are Alzheimer's disease (AD) characterized by progressive loss of cognitive function and Parkinson's disease (PD) characterized by motor symptoms related to dopaminergic neuronal loss in the substantia nigra.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • Astrocyte functions are altered in the aging brain, and this contributes to reduced neuronal and synaptic function.
  • the body possesses defense mechanisms based on antioxidant actions; a group of enzymatic (e.g., superoxide dismutase, catalase, and glutathione reductase) or nonenzymatic (e.g., glutathione, melatonin, vitamins A, C, and E, and flavonoids) molecules that play an important role in maintaining cellular homeostasis and vitality.
  • enzymatic e.g., superoxide dismutase, catalase, and glutathione reductase
  • nonenzymatic e.g., glutathione, melatonin, vitamins A, C, and E, and flavonoids
  • a typical mechanism of aging in a healthy subject is selective accumulation of iron that occurs in different brain regions and cell types.
  • iron accumulation in specific brain regions greater than that reported in healthy aging, occurs in many neurodegenerative diseases and is often associated with oxidative stress and cellular damage.
  • Lipoic acid is known to be a potent chelator of bivalent metal ions, and vitamin D, particularly vitamin D3 and/or vitamin D2, is known to prevent iron accumulation-induced damage.
  • the processes involved in aging-related iron accumulation and iron-induced inflammation in specific brain regions and cells are poorly understood to date.
  • compositions (drugs) or dietary supplements or nutraceuticals currently available on the market for the therapeutic and nontherapeutic treatment of diseases, symptoms, and/or disorders, for example, associated with brain aging, and in particular for the treatment of neurodegenerative diseases, such as Alzheimer's (AD) and/or Parkinson's (PD) disease, comprise lipoic acid and in some cases other compounds with co-acting function.
  • AD Alzheimer's
  • PD Parkinson's
  • the Applicant has developed a composition comprising lipoic acid and vitamin D, specifically vitamin D3, described and claimed in W02020/053754.
  • Lipoic acid has been shown to be very effective in counteracting diseases, symptoms and/or disorders associated with brain aging, for the treatment of neurodegenerative diseases and peripheral neuropathies.
  • recent data seem to indicate that the use of lipoic acid in known dosages (generally greater than or equal to 600 mg/day) may cause side effects.
  • lower dosages of lipoic acid do not appear to have the same therapeutic effect.
  • the technical problem that the present invention addresses and solves is to provide a viable solution for reducing the amount of lipoic acid in the formulation while still providing effective treatment, both therapeutic and non-therapeutic, of various diseases, symptoms and/or disorders, for example associated with cerebral aging, and/or for the treatment of neurodegenerative diseases, such as Alzheimer's (AD) and/or Parkinson's (PD) disease, and/or for the treatment of neuropathies, preferably peripheral neuropathies, in the absence or with reduced side effects.
  • the technical problem that the present invention addresses and solves is to provide a viable low-dose lipoic acid solution for the nontherapeutic treatment of subjects undergoing, or potentially undergoing, cerebral aging that slows this process effectively and in the absence of side effects.
  • the present invention provides a composition (pharmaceutical composition, nutraceutical composition, dietary supplement, or medical device composition) comprising, as active ingredients, lipoic acid or a pharmaceutically or food-grade acceptable salt thereof or a derivative thereof (e.g., lipoic acid in its racemic form or, alternatively, in its chiral form R ), a vitamin of the D group, preferably vitamin D3 (cholecalciferol) and/or vitamin D2, and glutathione or a derivative thereof.
  • a composition pharmaceutical composition, nutraceutical composition, dietary supplement, or medical device composition
  • a composition comprising, as active ingredients, lipoic acid or a pharmaceutically or food-grade acceptable salt thereof or a derivative thereof (e.g., lipoic acid in its racemic form or, alternatively, in its chiral form R ), a vitamin of the D group, preferably vitamin D3 (cholecalciferol) and/or vitamin D2, and glutathione or a derivative thereof.
  • Said composition can effectively and rapidly treat numerous diseases, symptoms, or disorders, for example, diseases, symptoms, or disorders caused by or resulting from neurodegenerative diseases and/or neuropathies, preferably peripheral neuropathies , in both pathological and healthy subjects (not yet defined as pathological).
  • diseases, symptoms, or disorders for example, diseases, symptoms, or disorders caused by or resulting from neurodegenerative diseases and/or neuropathies, preferably peripheral neuropathies , in both pathological and healthy subjects (not yet defined as pathological).
  • the present invention provides a composition free of the side effects found in treatments of the prior art that is easy to prepare and cost-effective.
  • the Applicant after intensive research and development activity, has found that the administration of the composition according to the present invention makes it possible to reduce the amount of lipoic acid present in it to as much as 5, 10 or 15 times less than the known compositions, while maintaining or even improving the efficacy of the known compositions.
  • the composition of the invention is capable of effectively and rapidly treating numerous diseases, symptoms and/or disorders, for example caused by or resulting from cerebral (biological) aging, particularly neuropathies, preferably peripheral neuropathies and/or, in a second aspect of the present invention, neurodegenerative diseases, and generally slowing down cerebral aging.
  • diseases, symptoms, and/or disorders effectively treatable by the composition of the invention include tumor, insulin resistance, metabolic disorders, obesity, thrombotic/thromboembolic diseases, ischemic episodes from surgery, transplantation bypass, endometriosis, infertility, polycystic ovary syndrome (POOS), polyabortion (i.e., the occurrence of three or more consecutive miscarriages before the 20th week of pregnancy, each with a fetus weighing less than 500 grams) and inflammation.
  • POOS polycystic ovary syndrome
  • the composition of the invention is effective in cases of comorbidity, that is, in the case of the simultaneous presence in the same subject of two or more diseases and/or disorders.
  • repeated abortion is defined as when, in a woman's obstetrical history, two consecutive episodes of abortion occur within the 20th week of pregnancy.
  • Recurrent abortion is, on the other hand, defined as the presence of three or more consecutive episodes of miscarriage.
  • polyabortion it is generically referred to as polyabortion, and screening for this is implemented from the second consecutive episode of abortion.
  • the composition of the invention exhibits marked antioxidant and anti-inflammatory activity. Therefore, the composition of the invention can be used for the treatment of diseases, symptoms and/or disorders associated with oxidative stress and/or inflammation.
  • the surprising pharmacological activity is due to the particular combination of the three active ingredients in the composition, such as lipoic acid, vitamin D group, preferably vitamin D3 and/or vitamin D2, and glutathione.
  • the three active ingredients act synergistically so that the effect of lipoic acid is amplified allowing the amount of lipoic acid in the composition to be reduced, as detailed in the experimental part.
  • Figure 1 Cell viability measured in dose-response study for glutathione in gastric cells; all p ⁇ 0.05 vs control.
  • Fig. 1 For this test in Fig. 1 an in vitro model using GTL-16 cells (gastric cells) for gastric pre-digestion, as the first physiological passage of substances that then go into the second Transwell composed of intestinal cells, classically used for the determination of plasma absorption of orally taken substances, was used.
  • GTL-16 cells gastric cells
  • This system is now recognized and accepted by the EMA and FDA to predict exactly the absorption, metabolism and bioavailability of drugs and xenobiotics after oral intake.
  • the method is versatile and it is still in the making and has enabled further development, used here, providing to join HT29MTX (mucus secreting) cells to the Caco-2 system to increase homology with the human intestinal (small intestinal) tract.
  • Figure 3(a), Figure 3(b), Figure 3(c) and Figure 3(d) Intestinal assessment: Evaluation of the proportion of lipoic acid absorbed through the intestinal epithelium.
  • FIG 4(a), Figure 4(b), Figure 4(c) and Figure 4(d) Gut assessment: permeability rate (fluorescein method).
  • Figure 5(a), Figure 5(b), Figure 5(c) and Figure 5(d) Gut assessment: basolateral level analysis, for evaluation of lipoic acid bioavailability.
  • Figure 6(a), Figure 6(b), Figure 6(c) and Figure 6(d) Hepatic assessment: Gut pre-digestion, exclusion test to evaluate efficacy after intestinal passage.
  • Figure 7(a), Figure 7(b), Figure 7(c) and Figure 7(d) Hepatic assessment: evaluation of hepatic permeability.
  • Figure 9(a) and Figure 9(b) liver assessment: analysis of ERK/MAPK and Src activity.
  • Figure 10(a) and Figure 10(b) cytokine panel assessment: analysis of TNF-alpha (Fig. 10(a)) and NF-kB (Fig. 10(b)) activity.
  • FIG. 13 Central Nervous System assessment: anti-oxidant properties evaluation.
  • FIG. 14(a) and Figure 14(b) Central Nervous System assessment: anti-inflammatory activity evaluation. Evaluation is performed on markers related to the two main mechanisms involved in inflammation prodromal to neurodegeneration, namely TNFo (Fig. 14(a)) and IL1 p (Fig. 14(b)).
  • FIG. 15(a), Figure 15(b) and Figure 15(c) Central Nervous System assessment: evaluation of intracellular markers APP (Fig. 15(a)), TRK (Fig. 15(b)) and TAU (Fig. 15(c)). Evaluation is performed on markers related to the two main mechanisms involved in neurodegeneration.
  • Figure 16 Peripheral Nervous System assessment: evaluation of mitochondrial viability and metabolism. The test can confirm that the product reaches functional the peripheral nervous system, without side effects and improves the metabolic performance of neurons.
  • Figure 17 Peripheral Nervous System assessment: antioxidant properties evaluation.
  • FIG. 18(a) and Figure 18(b) Peripheral Nervous System assessment: anti-inflammatory activity evaluation. Evaluation is performed on markers related to the two main mechanisms involved in the inflammatory response, namely TNFo (Fig. 18(a)) and IL1 p (Fig. 18(b)).
  • Figure 19 Peripheral Nervous System assessment: evaluation of the proportion of absorbed lipoic acid.
  • Figure 20 Peripheral Nervous System assessment: BDNF ("brain-derived neurotrophic factor”) precursor analysis. Involved in the phenomena of nerve fiber and synapse regeneration.
  • BDNF brain-derived neurotrophic factor
  • Figure 21 Peripheral Nervous System assessment: intracellular marker p75NTR (Fig.21 (a)) and ERbB3 (Fig. 21(b)) analysis.
  • Figure 22 Peripheral Nervous System assessment: intracellular marker NRG1 (Fig.22(a)) and MPZ (Fig. 22(b)) analysis.
  • Figure 23 Change in the amount of lipoic acid from administration to the brain/PNS when LA is administered in concentrations of 5 piM (Fig. 23(a)) and 50 piM (Fig. 23(b)).
  • Figure 24 Change in the amount of lipoic acid from brain/PNS administration when LA is administered in 5 piM concentration in combination with Vitamin D.
  • Figure 25 Change in the amount of lipoic acid from administration to the brain/PNS, when LA is administered at a concentration of 5 pi M, in combination with Vitamin D and glutathione.
  • composition of the invention comprising:
  • lipoic acid or a pharmaceutically or food grade acceptable salt or derivative thereof for example, lipoic acid in its racemic form or, alternatively, in its chiral form R );
  • composition comprises, in addition,
  • Lipoic acid in short LA -Lipoic Acid
  • LA -Lipoic Acid Lipoic acid was first isolated in 1951 from liver extracts, by American biochemists L.J. Reed and I.C. Gunsalus. Lipoic acid is produced by our bodies in small amounts, but it can be taken in, albeit in small amounts, through the diet (broccoli, brewer's yeast, and offal are its major sources).
  • Lipoic acid is a molecule having a chiral center and is produced by the human organism in the chiral R form (R-lipoic acid, IUPAC name (R)-5-(1,2-dithiololane-3-yl)pentanoic acid).
  • synthetic lipoic acid also called alpha-lipoic acid
  • R-lipoic acid is a mixture of the chiral R and S forms (racemic form).
  • lipoic acid refers interchangeably to both the racemic form (alpha-lipoic acid) and the chiral R form.
  • composition of the present invention may comprise lipoic acid in its racemic form (alpha-lipoic acid or (RS)-lipoic acid) or, alternatively, in its chiral R-form (R-lipoic acid).
  • the composition of the present invention comprises lipoic acid in the racemic form (alpha-lipoic acid) or a pharmaceutically or food grade acceptable salt thereof or derivative thereof.
  • the composition of the present invention comprises lipoic acid in its chiral R form, or pharmaceutically or food grade acceptable salt thereof or derivative thereof.
  • Lipoic acid is a low molecular weight molecule characterized by good solubility in both hydrophilic and lipophilic environments. It exists in nature in two forms: as a cyclic disulfide (oxidized form) or as an open chain, called dihydrolipoic acid, which has two sulfhydryl groups; the two forms are easily interconverted by oxidation-reduction reactions. Lipoic acid is characterized by a pronounced antioxidant capacity, due to its particular chemical structure and, mainly, the presence of the disulfide bridge that acts as an electron acceptor. As an antioxidant, lipoic acid acts not only as a scavenger of reactive oxygen species (ROS), but also has chelating properties of transition metals.
  • ROS reactive oxygen species
  • “Derivative” of lipoic acid in the context of the present invention, is understood to mean a derivative of lipoic acid, preferably alpha-lipoic acid, known to the person skilled in the art to have antioxidant properties similar to lipoic acid itself, such as the reduced form of lipoic acid such as dihydrolipoic acid (DHLA).
  • DHLA dihydrolipoic acid
  • vitamin of the D group means a compound belonging to a group of fat-soluble pro-hormones of natural origin comprising vitamin D1 (ergocalciferol and lumisterol 1 :1), vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol); vitamin D4 (dihydroergocalciferol), vitamin D5 (sitocalciferol), and mixtures thereof.
  • vitamin of the D group means a complex of at least one of the compounds that are part of the vitamins of the D group, for example, a compound selected from the protected derivatives, such as compounds that are part of the vitamins of the D group whose functional groups are protected with appropriate protecting groups, or metabolic precursors, known to the person skilled in the art.
  • Glutathione IUPAC name (2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2- yl]amino]-5-oxopentanoic acid, is a tripeptide consisting of cysteine and glycine and glutamate. Glutathione has strong antioxidant properties.
  • glutathione or a derivative thereof means glutathione in oxidized form (GSSG) or reduced form (GSH), or a mixture thereof.
  • said (II) vitamin of the D group is vitamin D3 or cholecalciferol (CAS No. 67-97-0) and/or vitamin D2 or a derivative thereof as defined above.
  • said (I) lipoic acid is the racemic form of lipoic acid or a pharmaceutically or food grade acceptable salt thereof or a derivative thereof.
  • the (i) mixture of the present invention comprises (I) lipoic acid in the racemic form (alpha-lipoic acid) or a pharmaceutically or food grade acceptable salt thereof or a derivative thereof and (II) vitamin D3 and/or vitamin D2 or a derivative thereof.
  • each component (I) and (II) may be present in the amounts defined below.
  • said (I) lipoic acid is present in said (I) mixture of the invention in an amount by weight ranging from 5% to 70%, with respect to the total weight of the mixture, preferably from 10% to 60%, more preferably from 15% to 50%, even more preferably from 20% to 40%.
  • lipoic acid is present in the composition of the invention in an amount by weight in the range between 20 mg and 800 mg, preferably between 25 mg and 600 mg, more preferably between 30 mg and 200 mg, even more preferably between 35 mg and 100 mg, and particularly 100 mg .
  • said (II) vitamin of the D group preferably vitamin D3 (or cholecalciferol) and/or vitamin D2 is present in said (i) mixture of the invention in an amount by weight from 0.0001% to 10%, with respect to the total weight of the mixture, preferably from 0.001% to 3%, preferably from 0.01% to 1%, even more preferably from 0.05% to 0.5%.
  • said (II) vitamin of the D group preferably vitamin D3 (or cholecalciferol) and/or vitamin D2 is present in the composition of the invention in an amount by weight from 2pig to 1000 pig , with respect to the total weight of the composition, preferably from 3 pig to 500 pig, more preferably from 5 pig to 100 pig, even more preferably from 5 pig to 50 pig.
  • said (III) glutathione is present in the mixture (i) of the invention in an amount by weight from 10% to 90%, with respect to the total weight of the mixture, preferably from 20% to 80%, more preferably from 30% to 70%, even more preferably from 35% to 65%, e.g. 66%.
  • said (III) glutathione is present in the composition of the invention in an amount by weight between 50 and 800 mg, preferably between 80 mg and 600 mg, more preferably between 90 mg and 400 mg, even more preferably between 100 mg and 200 mg, e.g., 75 mg, 100 mg, 125 mg or 150 mg.
  • the mixture (i) according to the present invention comprises said (I) lipoic acid in an amount by weight from 10% to 60%, said (II) vitamin of the D group, preferably vitamin D3 (or cholecalciferol) and/or vitamin D2 in an amount by weight from 0.001% to 1%, and said (III) glutathione in an amount by weight from 20% to 80%.
  • the weight ratio between lipoic acid, or a salt or derivative thereof, and glutathione, or a salt or derivative thereof is from 1 :5 to 5:1, preferably it is from 1 :3 to 3:1, more preferably it is from 1 :2 to 2:1, e.g. 1:1.
  • the weight ratio between lipoic acid, or a salt or derivative thereof, and glutathione, or a salt or derivative thereof may be:
  • the mixture (i) according to the present invention comprises said (I) lipoic acid, or a salt or derivative thereof, in an amount by weight from 10% to 60%, said (II) vitamin of the D group, preferably vitamin D3 (or cholecalciferol) and/or vitamin D2 in an amount by weight from 0.001% to 1%, and said (III) glutathione, or a salt or derivative thereof, in an amount by weight from 20% to 80%.
  • the mixture (i) according to the present invention comprises said (I) lipoic acid, or a salt or derivative thereof, and said (III) glutathione, or a salt or derivative thereof, in a weight ratio from 1:5 to 5:1, preferably from 1 :3 to 3:1, more preferably from 1:2 to 2:1, e.g., 1:1.
  • vitamin D present in mixtures and compositions containing said mixtures, which are the object of the present invention, means vitamin D2, or vitamin D3, or mixtures thereof Vitamin D2 and vitamin D3.
  • composition according to the present invention in addition to the (i) mixture of the invention and optionally (ii) additives and/or excipients, may additionally comprise other active ingredients such as, but not limited to, anti-inflammatory agents, antioxidants, probiotics, antacids, vitamins of a group other than group D (e.g., vitamins A, B, C, E and K), homotaurine, L-acetyl carnitine, plant extracts (e.g., withamnia somnifera and bacopa), nervonic acid, mineral salts (e.g., salts of Zn, Mg and others), and mixtures thereof.
  • active ingredients such as, but not limited to, anti-inflammatory agents, antioxidants, probiotics, antacids, vitamins of a group other than group D (e.g., vitamins A, B, C, E and K), homotaurine, L-acetyl carnitine, plant extracts (e.g., withamnia somnifera and bacopa
  • composition of the invention as described above, wherein said composition is for use in a method of treatment, preventive and/or curative, of diseases, symptoms and/or disorders in a subject in need thereof.
  • the composition of the invention is for use in a method of treatment, preventive and/or curative, of diseases, symptoms and/or disorders associated with (biological) aging of the brain in a subject in need thereof; and/or in a method of treatment, preventive and/or curative, of neuropathies, preferably peripheral neuropathies, and/or, in a second aspect of the present invention, in a method of treatment, preventive and/or curative, of neurodegenerative diseases and/or symptoms or disorders associated with said neurodegenerative diseases .
  • Neurodegenerative diseases are a diverse set of diseases of the central nervous system, sharing a chronic and selective process of cell death of neurons. Depending on the type of disease, neuronal deterioration can result in cognitive deficits, dementia, motor impairment, behavioral and psychological disorders.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • Huntington's disease amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • FTD progressive supranuclear palsy
  • DLB Lewy body dementia
  • CJD Creutzfeldt-Jakob disease
  • GSS Gerstmann-Straussler-Scheinker disease
  • Alzheimer's-Perusini's disease also called Alzheimer's disease, presenile dementia of the Alzheimer's type, primary degenerative dementia of the Alzheimer's type, or simply Alzheimer's, is the most common form of progressively disabling degenerative dementia with onset predominantly at presenile age (over 65 years, but can occur earlier).
  • DSM-5 it is named as a major or mild neurocognitive disorder due to Alzheimer's disease.
  • Parkinson's disease often referred to as Parkinson's, idiopathic parkinsonism, primary parkinsonism, hypokinetic rigid syndrome, or agitating paralysis, is a neurodegenerative disease.
  • the typical motor symptoms of the condition are the result of the death of cells that synthesize and release dopamine. Such cells are found in the substantia nigra, a region of the midbrain.
  • Huntington's disease is a genetic neurodegenerative disorder that affects muscle coordination and leads to cognitive decline and psychiatric problems. Its onset is typically during middle age; it is the most common genetically caused disease in clinical neurological pictures with abnormal involuntary movements.
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig's disease or Charcot's disease or motor neuron disease is a progressive neurodegenerative disease of motor neuron that selectively affects motor neurons.
  • ALS is characterized by muscle stiffness, muscle twitching and gradual weakness due to decreased muscle size. This results in difficulty with speech, swallowing, and eventually breathing.
  • the locution frontotemporal dementia identifies a heterogeneous group of non-Alzheimer neurodegenerative dementias that are characterized by the presence of alterations in a degenerative- atrophic sense of the frontal and temporal brain lobes.
  • DSM-5 it is named as major or mild frontotemporal neurocognitive disorder.
  • Progressive supranuclear palsy or Steele-Richardson-Olszewski syndrome is a neurodegenerative disease first described in 1964.
  • the neurodegeneration involves atrophy in the midbrain and other brain structures including the subthalamic nucleus, globus pallidus, the pons nuclei, and the black matter.
  • PSP falls into a group of neurological disorders classified as "Parkinson-Plus” or "atypical Parkinson's” with symptoms reminiscent of Parkinson's disease, while other features are quite different.
  • Lewy body dementia or DLB is a neurodegenerative disease, a form of dementia similar to Alzheimer's disease but with earlier onset and often related to Parkinson's disease and Parkinsonian syndromes.
  • Creutzfeldt-Jakob disease originally described in the 1920s by Hans Gerhard Creutzfeldt and Alfons Maria Jakob, is a rare neurodegenerative disease that leads to a fatal form of progressive dementia.
  • the clinical syndrome is characterized by predominantly cortical polysectorial deficits with memory loss, personality changes, hallucinations, dysarthria, myoclonus, postural rigidity, and seizures.
  • GSS Gerstmann-Straussler-Scheinker syndrome
  • TSE transmissible spongiform encephalopathy
  • the composition of the invention is for use in a method of treatment of the following peripheral neuropathies: diabetic neuropathy, carpal - tarsal - ulnar - radial tunnel syndrome, cervicobrachialgia, herpes zoster, intervertebral disc herniation, sciatic neuralgia - lumbosciatica, Guillain- Barre syndrome, toxic and deficiency neuropathies (such as, drug neuropathy, alcoholic neuropathy), infectious neuropathies (such as, HIV infection neuropathies, cytomegalovirus neuropathies).
  • peripheral neuropathies diabetic neuropathy, carpal - tarsal - ulnar - radial tunnel syndrome, cervicobrachialgia, herpes zoster, intervertebral disc herniation, sciatic neuralgia - lumbosciatica, Guillain- Barre syndrome, toxic and deficiency neuropathies (such as, drug neuropathy
  • Diabetes is the most frequent cause of diabetic neuropathy, a peripheral neuropathy that expresses itself with different clinical variants: symmetric polyneuropathy, focal neuropathy, and mixed forms.
  • diabetes mellitus DM
  • the incidence of diabetes mellitus (DM) is calculated to be around 6% in the general population; the prevalence of neuropathy is 7% at the onset of DM to rise to 25-30% after 20 years in patients with diabetes II.
  • Carpal tunnel syndrome is the most common entrapment peripheral neuropathy. In most cases it is idiopathic. Carpal tunnel syndrome is manifested by the onset of pain and paresthesias, especially at night, in the first three fingers of the hand and the lateral half of the fourth finger, as well as the lateral half of the palm. Over time, the pains and paresthesias also radiate to the forearm and sometimes to the arm.
  • Radial tunnel syndrome is compression of the radial nerve in the proximal portion of the forearm. Symptoms include forearm and elbow pain.
  • Tarsal tunnel syndrome involves pain in the ankle, foot, and sometimes the toes caused by compression or injury of the posterior tibial nerve.
  • Ulnar tunnel (or cubital tunnel) syndrome is compression or traction of the ulnar nerve at the elbow. Symptoms include elbow pain and paresthesias in the distribution area of the ulnar nerve.
  • Cervicobrachialgia is caused by compression of a cervical nerve; it is characterized by pain originating from the posterior cervical region conditioning a functional limitation of flexion-extension and rotation movements of the neck, radiating to one or both upper limbs.
  • Herpes zoster commonly called shingles, is a viral disease of the skin and nerve endings caused by the varicella-zoster virus. It is a ganglioradicular syndrome. The clinical picture of herpes zoster is characterized by pain, broader cutaneous, neurological, and infectious manifestations.
  • Intervertebral disc herniation occurs in the cervical and lumbar regions: because of the stresses to which these parts of the spine are subjected, the intervertebral discs are more likely to undergo degenerative processes. Thus, fractures of the outer fibrous ring and subsequent herniation of the disc are possible, which leaks out of the intervertebral space, compressing the nerve structures.
  • Sciatic neuralgia or sciatica
  • the symptomatology consists mainly of intense, sometimes excruciating pain in the distribution area of L5-S1 i.e., in the lower gluteal region, posterior aspect of the thigh and leg.
  • Guillain-Barre syndrome is an acute polyradiculonevritis. It is the most frequent and best known cause of ascending paralysis: the clinical presentation is that of muscle weakness with acute or subacute onset in the lower limbs and ascending evolution with reaching nadir within four weeks.
  • metal poisoning such as lead, mercury, thallium and arsenic
  • drugs such as antiblastics, chemotherapeutics, antiarrhythmics, anti-rheumatics
  • Infectious neuropathies encompass numerous clinico-pathological entities, among which are immunodeficiency virus infection neuropathies and cytomegalovirus neuropathies.
  • composition for use in a method of treatment of peripheral neuropathy resulting from kidney disease does not include the treatment of inflammation in a population with chronic kidney disease (in short CKD) at the end-stage (end-stage renal disease or stage-5, in short ESRD) on hemodialysis (in short HD).
  • the composition of the invention can effectively and rapidly treat numerous diseases, symptoms and/or disorders.
  • the composition of the invention can be used in the treatment of one or more diseases, symptoms, and/or disorders selected from tumor, insulin resistance, metabolic disorders, obesity, thrombotic/thromboembolic diseases, ischemic episodes from surgery, transplantation, bypass, endometriosis, infertility, polycystic ovary syndrome (PCOS), polyabortion, and inflammation .
  • the composition of the invention can be used for the treatment of diseases, symptoms, and/or disorders associated with oxidative stress and inflammation.
  • the particular combination of the three active ingredients in the composition such as lipoic acid, vitamin of the D group, preferably vitamin D3 and/or vitamin D2, and glutathione allows for the effective treatment of a wide variety of diseases, symptoms, and/or disorders.
  • composition of the invention is effective in cases of comorbidity, that is, in the case of the simultaneous presence in the same subject of two or more diseases and/or disorders.
  • composition of the present invention comprises such amounts of (I), (II) and (III) as to ensure the administration to individuals in need thereof of a daily dose comprising:
  • composition of the invention can be administered to subjects in need thereof once or several times a day depending on the state of the subject and the amounts of (I), (II) and (III) included in the composition.
  • the surprising efficacy of the formulation according to the invention makes it possible to reduce the amount of lipoic acid to be administered and consequently also the volume of a tablet of the formulation; thus, the same therapeutic or nontherapeutic effects can be achieved with a single daily administration.
  • the present invention describes a method of treatment, preventive and/or curative, of diseases, symptoms and/or disorders, wherein said treatment comprises administering the composition of the invention as defined above to a subject in need thereof.
  • the present invention describes a method of treatment, preventive and/or curative, of diseases, symptoms and/or disorders associated with (biological) aging.
  • the present invention describes a method of treatment, preventative and/or curative, of neuropathies, preferably peripheral neuropathies as defined above, and/or symptoms or disorders associated with said neuropathies and said peripheral neuropathies.
  • the present invention describes a method of treatment, preventive and/or curative, of neurodegenerative diseases as defined above and/or symptoms or disorders associated with said neurodegenerative diseases.
  • the present invention describes a method of treatment, preventive and/or curative, of a disease, symptom, and/or disorder select from tumor, insulin resistance, metabolic disorders, obesity, thrombotic/thromboembolic diseases, ischemic episodes from surgery, transplantation, bypass, endometriosis, infertility, polycystic ovary syndrome (PCOS), polyabortion, and inflammation .
  • PCOS polycystic ovary syndrome
  • the present invention describes a method of treatment, preventive and/or curative, of a disease, symptom and/or disorder associated with oxidative stress and inflammation.
  • the non-therapeutic use of the composition of the invention as defined above for the non-therapeutic treatment of symptoms or disorders.
  • the non- therapeutic use of the composition of the invention is directed to the non-therapeutic treatment of symptoms or disorders associated with the brain (biological) aging in a subject in need thereof.
  • composition of the invention as defined above for the non-therapeutic treatment of slowing down brain (biological) aging in a subject in need thereof.
  • compositions nutraceutical composition, nutritional supplement product or a food product or food for special medical purposes or a medical device composition comprising or, alternatively, consisting of the composition of the present invention.
  • medical device in the context of the present invention is used in the meaning according to Italian Legislative Decree Feb. 24, 1997, No. 46, i.e., it means a substance or other product, used alone or in combination, intended by the manufacturer to be used in humans for the purpose of diagnosis, prevention, control, therapy or alleviation of a disease, which product does not exert its principal action, in or on the human body, for which it is intended, by pharmacological or immunological means or by metabolic process but whose function may be assisted by such means.
  • composition of the present invention may be, as a non-limiting example, in a liquid form, such as solution, biphasic liquid system, suspension or syrup, in a semisolid form, such as gel, cream or foam, or in a solid form, such as powder, granules, flakes, aggregates, capsules, pills, bars and equivalent forms.
  • a liquid form such as solution, biphasic liquid system, suspension or syrup
  • a semisolid form such as gel, cream or foam
  • solid form such as powder, granules, flakes, aggregates, capsules, pills, bars and equivalent forms.
  • the composition of the invention is for oral use, preferably in solid or liquid form, more preferably in solid form.
  • the active ingredients of the mixture of the present invention (I), (II) and (III) can also be administered separately (preferably in a time interval from 5 minutes to 60 minutes, and preferably from 10 minutes to 30 minutes, and in any order but, preferably, (I), (II) and (III) are administered to a subject at the same time, even more preferably in a single composition to achieve a faster and more synergistic effect, and for ease of administration.
  • said single composition corresponds to the composition of the present invention.
  • Method of treatment in the context of the present invention means an intervention, comprising the administration of a substance, or mixture of substances, or combination thereof, having as its purpose the elimination, reduction/diminution, or prevention of a disease or pathology and symptoms or disorders thereof.
  • a statement that a composition "comprises" one or more components or substances means that other components or substances may be present in addition to the one, or those, specifically stated.
  • composition of the present invention is intended indifferently for human or veterinary use, that is, as a preparation to be applied to animals with the uses and methods known to the person skilled in the art.
  • compositions of the present invention are found to be suitable for the effective treatment, both therapeutic and non-therapeutic, of diseases, symptoms and/or disorders, particularly associated with brain (biological) aging, specifically for the treatment of neurodegenerative diseases as defined above and/or symptoms or disorders associated with said neurodegenerative diseases, particularly in the absence of relevant side effects.
  • a composition comprising.
  • composition according to FR1 wherein said (I) lipoic acid is the racemic form of lipoic acid or a pharmaceutically or food grade acceptable salt or derivative thereof, or wherein said (I) lipoic acid is the chiral R form of lipoic acid or a pharmaceutically or food grade acceptable salt or derivative thereof.
  • FR3 The composition according to FR1 or FR2, wherein said (II) vitamin of the D group is a vitamin D3 or cholecalciferol and/or vitamin D2 or a derivative thereof.
  • composition according to any one of FR1-3 wherein said mixture (i) comprises said (I) lipoic acid in an amount by weight ranging from 5% to 70%, said (II) vitamin of the D group in an amount by weight ranging from 0.0001% to 10%, and said (III) glutathione in an amount by weight ranging from 10% to 90%, with respect to the total weight of the mixture.
  • composition for use according to FR5 wherein said composition is for use in a method of treatment, preventive and/or curative, of a disease, symptom and/or disorder associated with brain aging in subject in need thereof.
  • FR8 The composition for use according to FR6, wherein said neurodegenerative disease is selected from Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD or presenile dementia), progressive supranuclear palsy (PSP or Steele- Richardson-Olszewski syndrome), Lewy body dementia (DLB), Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS).
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • Huntington's disease amyotrophic lateral sclerosis
  • FTD or presenile dementia frontotemporal dementia
  • PSP progressive supranuclear palsy
  • LLB Lewy body dementia
  • CJD Creutzfeldt-Jakob disease
  • GSS Gerstmann-Straussler-Scheinker disease
  • composition for use according to FR7 wherein said peripheral neuropathy is selected from: diabetic neuropathy, carpal, tarsal, ulnar, radial tunnel syndrome, cervicobrachialgia, herpes zoster, intervertebral disc herniation, sciatic neuralgia or lumbosciatica, Guillain-Barre syndrome, toxic and/or deficiency neuropathies, preferably drug neuropathy and alcoholic neuropathy, and infectious neuropathies, preferably HIV infection neuropathies and cytomegalovirus neuropathies.
  • peripheral neuropathy is selected from: diabetic neuropathy, carpal, tarsal, ulnar, radial tunnel syndrome, cervicobrachialgia, herpes zoster, intervertebral disc herniation, sciatic neuralgia or lumbosciatica, Guillain-Barre syndrome, toxic and/or deficiency neuropathies, preferably drug neuropathy and alcoholic neuropathy, and
  • FR10 The composition for use according to FR5, wherein said diesase, symptom, and/or disorder is selected from tumor, insulin resistance, metabolic disorders, obesity, thrombotic/thromboembolic diseases, ischemic episodes from surgery, transplantation, bypass, endometriosis, infertility, polycystic ovary syndrome (PCOS), polyabortion, and inflammation.
  • said diesase, symptom, and/or disorder is selected from tumor, insulin resistance, metabolic disorders, obesity, thrombotic/thromboembolic diseases, ischemic episodes from surgery, transplantation, bypass, endometriosis, infertility, polycystic ovary syndrome (PCOS), polyabortion, and inflammation.
  • PCOS polycystic ovary syndrome
  • composition for use according to FR5 wherein said disease, symptom, and/or disorder is associated with oxidative stress.
  • composition for use according to one of FR5-11 wherein said composition is administered at least once a day, e.g., two to four times, so as to ensure the administration to subjects in need thereof of a daily dose in the range:
  • Figure 1 Cell viability measured in dose-response study for glutathione in gastric cells; all p ⁇ 0.05 vs control.
  • Fig. 1 For this test in Fig. 1 an in vitro model using GTL-16 cells (gastric cells) for gastric pre-digestion, as the first physiological passage of substances that then go into the second Transwell composed of intestinal cells, classically used for the determination of plasma absorption of orally taken substances, was used.
  • GTL-16 cells gastric cells
  • This system is now recognized and accepted by the EMA and FDA to predict exactly the absorption, metabolism and bioavailability of drugs and xenobiotics after oral intake.
  • the method is versatile and it is still in the making and has enabled further development, used here, providing to join HT29MTX (mucus secreting) cells to the Caco-2 system to increase homology with the human intestinal (small intestinal) tract.
  • the GTL-16 cell line donated by the Laboratory of Histology, University of Eastern Piedmont (Italy), is a clonal line derived from a poorly differentiated gastric carcinoma cell line widely used as a model of gastric epithelial cells.
  • Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Merck Life Science, Rome, Italy) supplemented with 10% fetal bovine serum (Fetal Bovine Serum, FBS), 1% penicillinstreptomycin, in an incubator at 37 °C, 5% CO2. 1 x 1 o 4 cells were used to explore cell viability by MTT.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • penicillinstreptomycin penicillinstreptomycin
  • the Caco-2 cell line provided by the American Type Culture Collection (ATCC), was cultured in Dulbecco's modified Eagle's/ F-12 Ham Nutrient medium (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% FBS, L-glutamine 2 mM and 1% penicillin-streptomycin, in an incubator at 37 °C, 5% CO2.
  • This cell line is being used to develop a widely accepted (by EMA and FDA) experimental model to predict the absorption, metabolism, and bioavailability of drugs and xenobiotics after oral intake.
  • Cells were used at passage numbers between 26 up to 32 (Cell cultures grow in plate, confluence refers to the space occupied by the cells.
  • Passage refers to the number of times cells were moved from one plate to a new plate.
  • This cell line was co-cultured with HT29-MTX calyciform cells, which exhibit intestinal barrier properties in terms of paracellular transport and relative efflux.
  • This model makes it possible to in vitro recreate the intestinal epithelium in the most physiological way possible.
  • the co-culture was seeded at a density of 7:3 (Caco-2: HT29-MTX) on 6.5-mm Transwell® with 0.4-pm pore size polycarbonate membrane inserts (Merck Life Science, Italy) in a 24-well plate to perform uptake studies.
  • HepG2 Human epithelial cells
  • DMEM fetal bovine serum
  • L-glutamine 2 mM L-glutamine 2 mM
  • penicillin-streptomycin at 37 °C, 5% CO2.
  • the cells used in these experiments had a 90-95 passage range, and after reaching 80-90% confluence, 2 x 10 5 cells/well were grown in 96-well plates for a cytotoxicity assay using a trypan blue assay; thereafter, 1 x 10 4 cells were used for permeability detected by 0.04% fluorescein (Merck Life Science, Italy) by sinusoid transwell, and finally, 1 x 10 6 on a 6-well plate to analyze activated intracellular pathways by Western blot analysis.
  • mice astrocytes Primary cultures of mouse astrocytes were prepared from both male and female C57BL/6 mouse pups, following a standard technique described elsewhere according to the National Guidelines for the Use and Care of Laboratory Animals. Briefly, within 24 h after birth, the pups were euthanized, and the cortices were mechanically dissected and digested. The cell suspension was centrifuged at 800 rpm for 5 min. Pelleted cells were resuspended in neuronal basal medium (Sigma-Aldrich, Milan, Italy) supplemented with 5% FBS, 1% penicillin/streptomycin and L-glutamine 2 mM, plated in multiple wells and maintained in culture for 6 days before treatment.
  • neuronal basal medium Sigma-Aldrich, Milan, Italy
  • Astrocytes should be separated from microglia and oligodendrocyte precursor cells by shaking, as reported in the literature.
  • 1 x 10 4 cells were plated on a 96-well plate to study cell viability by MTT assay and ROS production by colorimetric assay; 1 x 10 6 cells were plated on a 6-well plate to analyze TNFa, IL1 b and molecular pathways by Western blot or ELISA analysis.
  • BBB Blood-Brain Barrier
  • astrocytes were co-cultured with human umbilical vein endothelial cells (HUVECs) according to methods reported in the literature.
  • HUVECs were purchased from ATCC® .
  • IFRS1 cells were seeded on 100-mm plastic discs (Greiner Bio-One GmbH, Frickenhausen, Germany, 664-160-013) at an approximate density of 2 x 10 4 /cm 2 and maintained in DMEM containing 5% FBS, recombinant human eregulin-p (EMD Millipore, Billerica, MA, USA) 20 ng/mL, forskolin (Sigma, St.
  • NSC-34 cells were seeded on 100-mm plastic discs at an approximate density of 1 x 10 4 /cm 2 and maintained in DMEM containing antibiotic antifungal solution and 5% FBS.
  • cells were detached from the discs using 0.05% trypsin/EDTA 0.53 mM solution, suspended in DMEM/F12 Ham containing 5% FBS in a 50 mL polypropylene conical tube, and reseeded on Aclar fluorocarbon coverslips coated with poly-L-lysine/laminine, 12-well culture plates coated with type I collagen, and 2-well glass chamber slides coated with type I collagen at an approximate density of 2 x 10 3 /cm 2 .
  • Cells were maintained in DMEM/F12 containing 1% FBS, 1% MEM nonessential amino acids and 10 ng/mL recombinant rat brain-derived neurotrophic factor (BDNF) for 5-7 days.
  • BDNF rat brain-derived neurotrophic factor
  • NSC-34 cells When elongation of neurites from most NSC-34 cells was observed under a phase-contrast microscope, the cells were incubated for 12- 16h with DMEM/F12 containing 5% FBS and mitomycin C 1 pig/mL. NSC-34 cells were then rinsed twice with 5% DMEM/FBS, and co-cultured with IFRS1 cells, which had been detached from the discs using trypsin/EDTA and suspended in 5% DMEM/FBS at an approximate density of 1 x 10 5 /mL. The cell density ratio of NSC-34:IFRS1 was adjusted from 1 :7 to 1 :10.
  • the co-cultured cells were then fed twice weekly in DMEM containing 5% FBS, ascorbic acid 50 pig/mL, BDNF 10 ng/mL and recombinant rat ciliary neurotrophic factor 10 ng/mL, and maintained for up to 28 days.
  • hepatic metabolism of lipoic acid (5 piM and 50 piM), alone and combined with vitamin D3 100 nM and glutathione 5mM, was examined for 24 hours (Fig. 6 - 7 - 8 - 9).
  • a HepG2 cell line was used to analyze the cytotoxic effect of the new formulation using an exclusion test and, in particular, the main intracellular pathways involved in inflammation and lipoic acid metabolism were analyzed.
  • the rate of superoxide anion release was used to examine the ROS produced by astrocytes after stimulations. After treatment, 100 pl of cytochrome C was added to all samples (treated or not), and in another sample, 100 pl of superoxide dismutase was also added for 30 min in an incubator (all substances were from Sigma-Aldrich). Absorbance was measured by a spectrometer (VICTOR X4, multiwell plate reader), at 550 nm, and O2 was expressed as mean ⁇ SD of nanomoles per reduced cytochrome C per microgram of protein versus control on percent (%).
  • TEER is used to determine that Transwells or co-cultures reach a proper maturational state that allows experimental assays to begin, as well as to eventually verify their biological integrity.
  • the data are usually used only in publications or if there is no possibility of performing specific assays. In this case we indicated it because it is part of experimental practice [Galla R, Grisenti P, Farghali M, Saccuman L, Ferraboschi P, Uberti F. Ovotransferrin Supplementation Improves the Iron Absorption: An In Vitro Gastro- Intestinal Model. Biomedicines. 2021 ;9(11): 1543; Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman J J. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 2015 Apr;20(2): 107-26. doi: 10.1177/2211068214561025],
  • transepithelial electrical resistance was measured using an epithelial voltohmeter (EVOM3; WPI, Berlin, Germany). Measurements were performed at each culture medium replacement according to the manufacturer's instructions.
  • Fluorescein was added to the apical compartment of the Transwell system to determine the rate of cell layer uptake. This was performed as previously published [Molinari C, Morsanuto V, Ruga S, et al. The Role of BDNF on Aging- Modulation Markers. Brain Sci. 2020;10(5):285. Published 2020 May 9.
  • LA concentration was measured as described in the literature [Molinari C, Morsanuto V, Ghirlanda S, et al. Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation. Oxid Med Cell Longev. 2019;2019:2843121. Published 2019 Feb 28. doi:10.1155/2019/2843121],
  • basolateral volume was analyzed by a spectrometer (VICTOR X4, multiwell plate reader) at 320 nm, and absorbance relative to the standard curve was obtained from the LA (200 ng/ml). Results were expressed as mean ⁇ SD (%) absorbance, normalized to control.
  • a single-dose cytotoxicity assay was conducted by determining the numbers of viable and dead cells using a trypan blue dye exclusion method as described by Morita et al. [Kanwal S, Abeysinghe S, Srisaisup M, Boonserm P. Cytotoxic Effects and Intracellular Localization of Bin Toxin from Lysinibacillus sphaericus in Human Liver Cancer Cell Line. Toxins (Basel). 2021; 13(4):288. Published 2021 Apr 19. doi:10.3390/toxins13040288].
  • HepG2 cells were treated with lipoic acid 5 piM and 50 piM alone and combined with vitamin D and glutathione, while 1x PBS (pH 7.4) was used as a positive control. After 24 hours of treatment, cells were harvested by trypsinization, and aliquots of the cells were mixed with an equal volume of trypan blue dye. Viable cells (the cells that excluded the dye) and dead cells (those that took up the dye) were counted using a hemocytometer, and cell viability was expressed as the percentage of total cell number.
  • TNFo concentration on basolateral environment was determined using the TNF-o ELISA kit (Sigma, Milan, Italy) according to the manufacturer's instructions.
  • the absorbance of each well was measured after addition of stop solution at 450 nm using a plate reader (VICTOR X4, multiwell plate reader).
  • Basolateral co-culture medium was collected for IL-1p quantification with the ELISA kit for IL-1p (R&D systems, MN) according to the manufacturer's instructions. Chemiluminescent absorbance was determined using a microplate reader at 450 nm with correction at 570 nm. IL-1 p was quantified by correlating sample readings to the generated standard curve. Quantification of BDNF
  • BDNF brain-derived neurotrophic factor
  • ERK/MAPK activity was measured by InstantOneTM ELISA (Thermo Fisher) on cell lysates following the manufacturer's instructions. The strips were measured by a spectrometer (VICTOR X4, multiwell plate reader) at 450 nm. Results were expressed as mean absorbance (%) compared with control.
  • the SimpleStep ELISA® in vitro SRC kit (Abeam, Cambridge, UK) is designed for quantitative measurement of SRC protein in human cell culture extracts. After 30 min of incubation, the plate was read at 450 nm using the VICTOR X4 multiwell plate reader. The results were calculated by subtracting the mean zero (blank) from the optical density of the sample.
  • APP amyloid precursor protein
  • GTL-16 cells gastric cells
  • intestinal cells classically used for the determination of plasma absorption of substances at oral intake.
  • GTL-16 cells gastric cells
  • This system is now recognized and accepted by the EMA and FDA to predict precisely the absorption, metabolism and bioavailability of drugs and xenobiotics after oral intake.
  • the method is very plastic and versatile and it is still in the making and has enabled further development, used here, providing to join HT29MTX (mucus secreting) cells to the Caco-2 system to increase homology with the human intestinal (small intestinal) tract.
  • Figure 1 shows the results of a cell viability assay, measured in dose-response studies for glutathione in cells mentioned above.
  • concentrations of glutathione were evaluated: 2 mM, 5 mM, 10 mM and 20 mM. All concentrations tested are found to be effective. The dose that seems to give the best results is 5mM. This concentration of glutathione was, therefore, maintained in all subsequent tests.
  • Figures 2(a)-(f) show the results of a cell viability assay measured in predigested gastric model at different times. Specifically, the results observed after 1 hour ( Figure 2(a)), 2 hours (Figure 2(b)), 3 hours ( Figure 2(c)), 4 hours (Figure 2(d)), 5 hours ( Figure 2(e)) and 6 hours ( Figure 2(f)) are shown. As can be seen, the presence of glutathione (G) contributes to increased cell viability. The kinetics show a physiological effect that seems to peak around 4h. Among the concentrations of lipoic acid (LA) tested, the best results were obtained using the lower concentration (5 pi M) in combination with vitamin D (VitD) and glutathione (G).
  • LA lipoic acid
  • Figures 3(a)-(d) show the results of an absorption test, conducted to assess the proportion of lipoic acid absorbed through the intestinal epithelium (intra/extra ratio), at different times. Specifically, results observed after 1 hour ( Figure 3(a)), 3 hours ( Figure 3(b)), 4 hours ( Figure 3(c)) and 6 hours ( Figure 3(d)) are shown . The test showed that the ratio of the extra to intra amount of lipoic acid confirms what has been observed on kinetics and shows that the combined with LA 5piM at 3h increases absorption by 23% and at 4h by 15% compared with LA 5pi M+vitD alone.
  • Figures 4(a)-(d) show the results of a test conducted to assess the rate of permeability through the intestinal epithelium, at different times. Specifically, results observed after 1 hour (Figure 4(a)), 3 hours ( Figure 4(b)), 4 hours (Figure 4(c)) and 6 hours ( Figure 4(d)) are shown. The test showed kinetics confirming a physiological effect that peaks around 4 h. It was also observed that glutathione (G) increases the uptake rate of LA+VitD, assuming its increased availability.
  • Figures 5 (a)-(d) show the results of a basolateral quantification test of lipoic acid, at different times . This quantification correlates with the bioavailability of the product. Specifically, results observed after 1 hour ( Figure 5(a)), 3 hours ( Figure 5(b)), 4 hours ( Figure 5(c)) and 6 hours ( Figure 5(d)) are shown. Quantification confirms what has been observed: the combined (LA+Vit.D+G) at 3h increases by 26 % and at 4h by 13 % compared to LA+vit.D alone in the presence of LA 5piM.
  • Figures 6(a)-(d) show the results of an exclusion test conducted to evaluate the efficacy of tested compounds after intestinal passage, at different times. Specifically, the results observed after 1 hour (Figure 6(a)), 3 hours ( Figure 6(b)), 4 hours ( Figure 6(c)) and 6 hours ( Figure 6(d)) are shown. The experiment observed that the compound remains active on cells, acting on mitochondrial well-being.
  • Figures 7(a)-(d) show the results of a hepatic permeability test, conducted to assess the final plasma amount of lipoic acid, at different times. Specifically, the results observed after 1 hour ( Figure 7(a)), 3 hours ( Figure 7(b)), 4 hours (Figure 7(c)) and 6 hours ( Figure 7(d)) are shown.
  • the assay showed that the combined compound (LA+VitD+G) is absorbed hepatically and seems to maintain the same kinetics as previously observed: 10.7% at 3h and 39.7% at 4h compared with LA+Vit.D alone, when the LA concentration is 5pi M .
  • Figure 8(a) and Figure 8(b) show the results of a test conducted to evaluate the effect of lipoic acid and combinations with vitamin D and glutathione on CYP1A2 (Fig. 8(a)) and CYP3A4 (Fig. 8(b)) cytochromes.
  • CYP1A2 Fig. 8(a)
  • CYP3A4 Fig. 8(b)
  • Figure 8(a) it was observed that the presence of vitamin D increases expression in support of increased uptake (it is more metabolized), and that the combination (LA+VitD+G) reduces the action on CYP1A2, thus justifying the observed increase in lipoic acid.
  • Figure 8(b) shows that the presence of vitD reduces expression in support of the increased uptake (it is less metabolized) and that the combination brings the action on CYP3A4 back to control values, thus explaining the observed increase in lipoic acid.
  • a promiscuous action between CYP1 A2 and CYP3A4 is confirmed.
  • Figure 9(a) and Figure 9(b) show an analysis of ERK/MAPK and Src activity following different stimulations. The results obtained show that both major biological and anti-oxidant activities are conserved in the liver.
  • Figures 11 (a)-(c) show the results of an experiment evaluating the viability of the blood-brain barrier following different stimulations.
  • the test makes it possible to confirm that the product used arrives functional across the blood-brain barrier without side effects.
  • the results obtained show that none of the combinations tested causes cytotoxic effects in the brain.
  • the better efficacy of lipoic acid (LA) 5 piM, compared with 50 piM, is confirmed, even when administered alone.
  • LA5 piM combined with Vit.D and with VitD+G greatly amplifies its effects.
  • the data obtained support the hypothesis that LA reaches the brain still, at least partially, active, after passing through the liver.
  • Results designed to evaluate the antioxidant properties of LA, LA+VitD, and LA+VitD+G are shown in Figure 13. Given the importance of oxygen radicals in Neurodegenerative Diseases and Peripheral Neuropathies, the tests were conducted in the presence of an oxidative state comparable to that due to aging/degeneration. The molecules tested confirmed their anti-oxidant activity. Combinations of LA with VitD and with VitD+G show an amplified effect compared with LA alone (in both 5piM and 50piM concentration of LA). The antioxidant activity does not reflect the different concentration, although the result remains better using the lowest dose of LA.
  • Figures 15(a)-(c) show the results obtained in a test evaluating the response of intracellular markers APP (Fig. 15(a)), TRK (Fig. 15(b)) and TAU (Fig. 15(c)), following different stimulations.
  • APP_ betaamyloid precursor
  • LA5 piM alone, and in combination with VitD and G, seems to better support this effect.
  • the data shown in Figure 15(b) support the hypothesis of improved trophism by driving TRK_ (BDNF receptor) activation, with direct effects on plasticity and prevention of cognitive impairment.
  • TRK_ BDNF receptor
  • LA5 pi M alone, and in combination with VitD and G, is confirmed to be more active in this regard.
  • Tau_ marker of cognitive degradation
  • the data support the hypothesis of prevention of both physiological (aging) and potentially pathological (degeneration) cognitive degradation by the products tested.
  • LA5 piM in combination with VitD and G confirms the improved biological profile.
  • Tests have, in addition, been conducted to evaluate the impact of lipoic acid, particularly in combination with vitamin D and glutathione, at the peripheral nervous system level.
  • Mitochondrial viability and metabolism were initially assessed.
  • the test is useful in order to confirm that the tested product reaches the peripheral nervous system functional, without side effects.
  • the results of this test shown in Figure 16, show that the presence of G contributes to increased mitochondrial activity.
  • the best results at the PNS level were obtained using the lower dose (5 piM), in particular, in combination with VitD and G.
  • Figure 17 shows the results aimed at evaluating the anti-oxidant properties of LA, LA+VitD and LA+VitD+G, at the peripheral nervous system (PNS) level. Similar to the same evaluation at the level of the central nervous system (CNS; see Figure 13), we tested the LA, LA+VitD and LA+VitD+G effect in the presence of oxidative stress. The antioxidant activity of the tested products is maintained, acting as scavengers on ROS, suggesting a potential anti-inflammatory effect. The results obtained confirm what has been observed previously on mitochondrial activity and that the lower concentration of LA (5 piM) has more significant effects than the higher dose (50 piM).
  • PNS peripheral nervous system
  • Figures 18(a) and 18(b) show the results obtained in a test to assess anti-inflammatory activity, at the level of the peripheral nervous system.
  • the evaluation is performed on markers related to the two main mechanisms involved in the inflammatory response, namely TNFo (Fig. 18(a)) and IL1p (Fig. 18(b)).
  • Inflammation constitutes a negative aspect of recovery after peripheral nerve injury.
  • both of the two inflammatory markers evaluated are reduced in the presence of the products tested.
  • this reduction appears to be greater in the presence of LA5piM+V+G. This finding supports the better biological activity of the lower dose (5 piM) of LA.
  • Figure 20 shows the results of BDNF ("brain-derived neurotrophic factor”) precursor analysis. These results show that there is an increase in BDNF production, in response to the products tested, suggesting a mediating action of myelination through activation of p75 (a mechanism very evident in the presence of damage).
  • BDNF brain-derived neurotrophic factor
  • Figures 21 and 22 show the results of an analysis of intracellular markers related to the peripheral nervous system, namely: p75NTR (Fig.21(a)) and ERB3 (Fig. 21 (b)); and NRG1 (Fig.22(a)) and MPZ (Fig. 22(b)).
  • p75NTR has been identified as a positive modulator for Schwann cell myelination during development and is implicated in promoting nerve regeneration after injury.
  • ERbB3 is a mediator of Schwann cell biological activity, and is implicated in the regulation of myelination, migration and axonal sorting.
  • NRG1 is a regulator of Schwann cell myelination, while MPZ is am important molecule for formation of compact myelin around neurites.
  • the results obtained, with reference to all markers evaluated, show that the highest effect is obtained using lipoic acid (LA) 5 piM in combination with VitD and glutathione (G).
  • Figures 23, 24, and 25 summarize the data on changes in the amount of lipoic acid from administration through the intestines and liver to the central and peripheral nervous system.
  • Figures 23, 24 and 25 summarize the changes in the amount of lipoic acid expressed both as a percentage amount and as an amount in mg are shown.
  • Figure 23 shows the data for LA when administered alone, in concentrations of 5 piM (Fig. 23(a)) and 50 pi M (Fig. 23(b));
  • Figure 24 shows the data for LA when administered in concentrations of 5 piM (Fig. 24(a)) and 50 piM (Fig. 24(b)), in combination with Vitamin D;
  • Figure 25 shows the data for LA when administered in concentrations of 5 piM (Fig. 25(a)) and 50 pi M (Fig. 25(b)), in combination with Vitamin D and glutathione.
  • LA a greater amount of LA reaches the central and peripheral nervous system when LA is administered in combination with VitD and G, compared with the administration of LA alone, or LA in combination with vitamin D alone.
  • higher efficacy in reaching the central nervous system is achieved by using a lower concentration of LA (5 piM).
  • the combination of lipoic acid (LA) and vitamin D3 and/or vitamin D2 (vitD) and glutathione, i.e., the composition of the invention produces numerous beneficial effects, and that these beneficial effects are markedly higher than the combination of lipoic acid with vitamin D alone.
  • the combined effect of lipoic acid with vitamin D and glutathione makes it possible to significantly reduce the amount of lipoic acid required to obtain the beneficial effects.
  • the composition of the invention allows the amount of lipoic acid to be drastically reduced, thus avoiding its possible side effects, without having a reduction in the beneficial effects of this compound.
  • the experiments discussed above made it possible to observe that the composition of the invention allows even more significant beneficial effects to be obtained by decreasing the amount of lipoic acid than with higher concentrations of lipoic acid.

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Abstract

Un objet de la présente invention est une composition comprenant de l'acide lipoïque, ou un sel de qualité pharmaceutique ou alimentaire de celui-ci ou un dérivé de celui-ci, et une vitamine D, de préférence de la vitamine D3 et/ou de la vitamine D2, et du glutathion ou un dérivé de celui-ci. La présente invention concerne en outre ladite composition destinée à être utilisée dans un procédé thérapeutique et/ou non thérapeutique de traitement de maladies, de symptômes et/ou de troubles, par exemple, associés au vieillissement cérébral, aux neuropathies, de préférence aux neuropathies périphériques et aux maladies neurodégénératives.
PCT/IB2023/053742 2022-04-12 2023-04-12 Composition comprenant de l'acide lipoïque, de la vitamine d et du glutathion, ses utilisations, et compositions pharmaceutiques et nutraceutiques associées WO2023199241A1 (fr)

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IT102022000007232A IT202200007232A1 (it) 2022-04-12 2022-04-12 Composizione comprendente acido lipoico, vitamina d e glutatione, suoi usi, e relative composizioni farmaceutiche e nutraceutiche
IT102022000007232 2022-04-12
IT102022000007226A IT202200007226A1 (it) 2022-04-12 2022-04-12 Composizione comprendente acido lipoico, vitamina d e glutatione, suoi usi, e relative composizioni farmaceutiche e nutraceutiche

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WO1994000141A1 (fr) * 1992-06-24 1994-01-06 Boehringer Mannheim Italia S.P.A. Glutathion utilise comme agent chimioprotecteur
WO1998029101A1 (fr) * 1996-12-31 1998-07-09 Antioxidant Pharmaceuticals Corporation Preparations pharmaceutiques de glutathion et modes d'administration de ces preparations
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WO2009053824A1 (fr) * 2007-10-23 2009-04-30 Laboratorio Chimico Internazionale S.P.A. Composition de pastilles d'acide lipoïque
JP2010120916A (ja) * 2008-11-22 2010-06-03 Rena Science:Kk 体内生理活性モノアミンの減少に関連して発症する精神・神経疾患の改善または治療剤
WO2019197967A1 (fr) * 2018-04-10 2019-10-17 Sochim International S.P.A. Composition pour le traitement de neuropathies périphériques
WO2020053754A1 (fr) * 2018-09-10 2020-03-19 Uriach Italia S.R.L. Composition comprenant de l'acide lipoïque et de la vitamine d pour la prévention et le traitement de maladies neurodégénératives et de neuropathies périphériques
WO2022016038A1 (fr) * 2020-07-17 2022-01-20 Dt Ip Holdings I, Llc Bases de formulation comprenant de l'acide hyaluronique et formulations de celles-ci

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000141A1 (fr) * 1992-06-24 1994-01-06 Boehringer Mannheim Italia S.P.A. Glutathion utilise comme agent chimioprotecteur
WO1998029101A1 (fr) * 1996-12-31 1998-07-09 Antioxidant Pharmaceuticals Corporation Preparations pharmaceutiques de glutathion et modes d'administration de ces preparations
US6649195B1 (en) * 2002-07-11 2003-11-18 Vitacost.Com, Inc. Eyesight enhanced maintenance composition
US20070116779A1 (en) * 2005-11-23 2007-05-24 Elizabeth Mazzio Comprehensive nutraceutical agent for treatment/ prevention of Parkinson's disease
WO2009053824A1 (fr) * 2007-10-23 2009-04-30 Laboratorio Chimico Internazionale S.P.A. Composition de pastilles d'acide lipoïque
US20090110674A1 (en) * 2007-10-24 2009-04-30 Loizou Nicos C Health supplement
JP2010120916A (ja) * 2008-11-22 2010-06-03 Rena Science:Kk 体内生理活性モノアミンの減少に関連して発症する精神・神経疾患の改善または治療剤
WO2019197967A1 (fr) * 2018-04-10 2019-10-17 Sochim International S.P.A. Composition pour le traitement de neuropathies périphériques
WO2020053754A1 (fr) * 2018-09-10 2020-03-19 Uriach Italia S.R.L. Composition comprenant de l'acide lipoïque et de la vitamine d pour la prévention et le traitement de maladies neurodégénératives et de neuropathies périphériques
WO2022016038A1 (fr) * 2020-07-17 2022-01-20 Dt Ip Holdings I, Llc Bases de formulation comprenant de l'acide hyaluronique et formulations de celles-ci

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DATABASE GNPD [online] MINTEL; 28 September 2021 (2021-09-28), ANONYMOUS: "Daily Immunity Natural Raspberry & Lemon Effervescent Tablets", XP055978781, retrieved from https://www.gnpd.com/sinatra/recordpage/9039816/ Database accession no. 9039816 *

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