Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
• • 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
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
• • 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
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
  • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
  • C07C57/03—Monocarboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/40—Unsaturated compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/40—Unsaturated compounds
  • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
  • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

• the present invention relates to fatty acids with one or more unsaturations, of odd hydrocarbon chain, where the chemical structure of said fatty acids corresponds to that of the metabolites of alpha-hydroxylated mono or polyunsaturated fatty acids.
• the present invention also relates to the compositions comprising said odd-chain fatty acids, to their medical uses, as well as to their use as indicators of efficacy when treating a patient with the mono- or polyunsaturated alpha-hydroxylated fatty acids of the which are metabolites.
• fatty acids whose chemical structure has an odd number of carbon atoms have not been considered of therapeutic relevance, given that, in humans and, in general, in mammals, the vast majority of fatty acids present they are even chain, usually between 14 and 24 carbon atoms, being the presence of fatty acids of odd chain very rare, being limited to traces.
• Prodrugs or prodrugs are compounds that, when ingested, undergo metabolic reactions and give rise to a drug or drug, its metabolite, which provides an effect on the health of a patient or subject.
• the administration of a therapeutically active compound in the form of a prodrug allows modulating the distribution and absorption of said compound over time, since its metabolism allows the generation of the drug, that is, the metabolite, only in those cells or tissues in which metabolic reactions occur that transform said prodrug into its active metabolite.
• said prodrugs have other advantages, such as allowing a delayed or controlled administration of the active metabolite, avoiding its accumulations, which could produce harmful effects on the body.
• the identification and synthesis of said therapeutically active metabolites make it possible to act more intensively, making it possible to administer higher therapeutically active doses and, for controlled periods of time, than those that would occur during the spontaneous metabolism of its prodrug. correspondent.
• the present invention relates to a compound selected from the group consisting of: a compound of formula (II), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
• the present invention also relates to a compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), as described in the present invention, for use as a medicine and, in particular, for use in the induction of neuroregeneration and in the prevention and / or treatment (including maintenance treatment) of a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology;
• the present invention also relates to a pharmaceutical or nutraceutical composition
• a pharmaceutical or nutraceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III) and, optionally, a compound of formula ( I), as described in the present invention.
• the present invention relates to an in vitro method to determine the efficacy of a therapeutic or preventive treatment of a disease or pathology with a compound of formula (I), or with a pharmaceutically acceptable salt or ester thereof, in a subject.
• said method comprises determining in vitro in a biological sample from said subject, the amount of a compound of formula (II) or of formula (III), as described in the present invention, or of its carboxylate anion, or of a derivative formed therefrom in vivo or in vitro, wherein said amount is related to the efficacy in treating said disease or pathology
• the present invention relates to a compound selected from the group consisting of: a compound of formula (II), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
• All values of a, b, c and m of the present invention are integers greater than or equal to zero.
• the value of b is defined in a formula
• the value of m is defined as an integer between 0 and where said value of b is the one that has already been defined between 0 and Jb-1.
• the values of a, b, c and m are applicable to all formulas and compositions described herein.
• a is an integer between 1 and 7; b is an integer between 2 and 7; c is 0, 3, or 6, m is 0, and a + 3b + c + 3 is an even integer.
• a is an integer between 1 and 14; b is 1; c is 0, 3 or 6, m is 0; and where a + 3b + c + 3 is an even integer.
• m 0 and therefore the present invention refers to a compound selected from the group consisting of: a compound of formula (II), or a salt or a pharmaceutically or nutraceutically acceptable ester thereof:
• a preferred embodiment of the invention relates to a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II) or formula (III). More preferably, said salt is a sodium salt, and said ester is a methyl or ethyl ester.
• the compounds of formula (II) or of formula (III) of the present invention correspond to the formulas of the metabolites of a compound of formula (I), or of a pharmaceutically or nutraceutically acceptable salt or ester thereof :
• the compounds of formula (I) are therefore alpha-hydroxylated mono or polyunsaturated fatty acids with an even number of carbon atoms (a + 3b + c + 3 is an even integer).
• the even-chain 2-hydroxymonounsaturated fatty acids of formula (I) are prodrugs of other odd-chain monounsaturated fatty acids of formula (II), as said prodrugs undergo a decarboxylation process.
• the 2-hydroxypolyunsaturated fatty acids of formula (I), of the even chain are prodrugs of other fatty acids, mono or polyunsaturated, of the odd chain, which, in the case in which decarboxylation occurs, but not occurs the hydrogenation of one of the double bonds of the compound of formula (I), the compound derived from the prodrug of formula (I) will be a compound of formula (II), while, in the case where the hydrogenation of one of the double bonds and a decarboxylation of the compound of formula (I), the compound derived from the prodrug of formula (I) will be a compound of formula (III), in which the hydrogenated double bond can be in a different position depending on the value of m.
• figure 1A and figure 8F show a scheme of the metabolism by a-oxidation of 2-hydroxydocosahexaenoic acid (DHA-H), a compound of formula (I), at the cellular level giving rise to the acid (6Z, 9Z, 12Z, 15Z, 18Z) -heneicosse- 6,9,12,15,18-pentaenoic (HPA), its metabolite of formula (III).
• DHA-H 2-hydroxydocosahexaenoic acid
• HPA -heneicosse- 6,9,12,15,18-pentaenoic
• the DHA-H prodrug is a 2-hydroxylated polyunsaturated fatty acid, which is converted to HPA by a sequence of metabolic steps: (1) activation of DHA-H by conjugation with coenzyme A; (2) cleavage mediated by 2-hydroxyacyl-CoA Nase, giving rise to an aldehyde with an odd number of carbon atoms; (3) action of the aldehyde dehydrogenase on the aldehyde, hydrogenating one of the double bonds, to transform it into an acid (HPA).
• HPA acid
• Figure 8 shows the metabolization schemes for other fatty acids of formula (I).
• ARA-H 2-hydroxy-arachidonic acid
• EPA-H 2-hydroxy-eicosapentaenoic acid
• the compounds of formula (I) can also be monounsaturated alpha-hydroxylated fatty acids with an even number of carbon atoms.
• the prodrug sodium salt of 2-hydroxyoleic acid (20HOA) is a 2-hydroxylated monounsaturated fatty acid, which is converted into 8Z-heptadecenoic acid (8Z-heptadecenoic or C17: 1n-9), through a sequence of metabolic steps: (1) activation by an Acyl-CoA ligase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg 2+ ); (2) 20HOA-CoA would be subject to the activity of 2-hydroxyphytanoyl-CoA Nase (2-hydroxyacyl-CoA Nase 1, HACL1), forming an intermediate monounsaturated aldehyde; (3) the enzyme aldehyde dehydrogenase would be responsible for the conversion of said intermediate aldehyde
• formula (II) the medical uses of compounds of formula (I) are known
• the present invention describes the formula of its metabolites, compounds of formula (II) or formula (III) that provide a specific and differentiated therapeutic action in the body. , after the metabolism of said compounds of formula (I), which therefore act as prodrugs of these.
• the present invention provides a way of tailoring a therapeutic treatment depending on the nature of the disease and prognosis of the patient to be treated.
• the present invention discloses specific formulas of alpha-hydroxylated, mono, or polyunsaturated fatty acid metabolites that are therapeutically effective.
• the present invention also describes the uses as a medicine of said metabolites of formula (II) or formula (III), by themselves, allowing the amount administered to be controlled; or its use as a medicine in combination with its prodrug of formula (I); or its use as a medicine by administering said prodrug of formula (I), allowing the intensity and dose administered to be regulated over time during a treatment.
• the administration of the compounds of formula (II) or formula (III), through the administration of their prodrug of formula (I) thus allows modulating the distribution and absorption of a drug, since its metabolism allows the generation of the drug.
• the present specification refers to both the compounds of formula (II) and the compounds of formula (III), either obtained by chemical synthesis (see example 1), or obtained during the metabolism of the compounds of formula (I).
• the term "metabolites” is used to designate said compounds of formula (II) or of formula (III), whether their origin is the metabolism of a compound of formula (I) in the body of a subject, as if said compounds of formula (II) or formula (III) are synthetically obtained products.
• the term compound of formula (I) is used interchangeably with the term “prodrug of formula (I)” and, likewise, the terms “compound of formula (II)” and “compound of formula (III) “are used interchangeably, respectively, to the terms” metabolite of formula (II) "and” metabolite of formula (III) ", because said compounds of formula (II) and formula (III) , both if they are obtained by chemical synthesis, as if they result from the natural metabolism of a compound of formula (I), they have the chemical structure or chemical formula of a metabolite of the compound of formula (I) that acts, consequently, as a prodrug of these .
• the examples of the present invention show how the therapeutic effect exerted by the prodrugs of formula (I) is directly related to the therapeutic effect exerted by the metabolite of formula (II) or formula (III) in the body. , Y they also show the therapeutic effect that, per se, the compounds of formula (II) and formula (III) exert.
• the administration of the sodium salt of 2-hydroxyoleic acid (OHOA), a compound of formula (I) produced a greater reduction in the size of xenographic tumors in mice the greater the cellular accumulation of the C17: 1n-9 metabolite of formula (II).
• Example 6.2 of the present invention shows that the formation of the metabolite C17: 1n-9 (8Z-heptadecenoic acid), from the incorporation of 20HOA, differs between tumor and non-tumor cells.
• the glioma cells showed a significant increase in their levels of C17: 1n-9 compared to 20HOA ( Figure 13B and D), while, in non-tumor cells, the detected levels of 20HOA were significantly higher than those of its metabolite C17. : 1n-9.
• the examples and figures show that the anti-proliferative effect mediated by the compounds of formula (I), exemplified by DHA-H, is mediated, at least in part, by its metabolite HPA (compound of formula (III)), since the inhibition of the formation of this compound from DHA-H, translates into a lower antiproliferative effect of DHA-H (figure 4B).
• the therapeutic value of the compounds of formula (I) is linked in part to the biological activity of their metabolite of formula (II) or of formula (III), said compound of formula (I) acting as a true prodrug of the compound of formula (II).
• the treatment with the sodium salt of HPA induces a degree of mortality on the culture much more evident than the which induces treatment with the sodium salt of DHA-H (prodrug) or the sodium salt of DHA (natural analog), under the same conditions. Therefore, the administration of the metabolite makes it possible to provide a more pronounced therapeutic action than that obtained by the administration of the prodrug.
• C17: 1n-9 had an antiproliferative effect when administered directly instead of its prodrug, both in tumor cells and in non-tumor cells, so the administration of said metabolite of formula (II), C17: 1 n-9, through its prodrug of formula (I), 20H0A, provides a selective way to produce a therapeutic effect, allowing a more prolonged administration of said therapy without producing undesirable adverse effects and being equally useful in maintenance therapy.
• odd-chain fatty acids are metabolized by b-oxidation, giving rise to propionyl-CoA.
• Unlike the pair chain fatty acids whose metabolism ends in the production of acetyl-CoA which, in turn, is metabolized via the Krebs cycle.
• Propionyl-CoA can be transformed into propionic acid, which causes, as an adverse effect, metabolic acidosis if it accumulates.
• Propionyl-CoA can be metabolically transformed into succinyl-CoA (which is metabolized via the Krebs cycle), in a process dependent on biotin and vitamin B12.
• This process is not a common metabolic pathway for fatty acids, because in the mammalian body, the vast majority of fatty acids are pair-chain. Consequently, this metabolic pathway selectively affects odd-chain polyunsaturated fatty acids, such as the metabolites of formula (II) and formula (III), and can become saturated in the event of excessively high intracellular concentrations of said odd-chain metabolites. or specific pathological situations that present with a deficiency of biotin or vitamin B12, leading to the referred adverse effect of propionic acidosis.
• the metabolite administered directly to the cells has an undesirable toxic in some cases.
• This toxicity is achieved modulating when the prodrug or compound of formula (I) is administered, it manages to regulate the effect and toxicity of the metabolite of formula (II) or (III).
• the slower uptake of the prodrug compared to non-hydroxylated fatty acids could be useful in avoiding excessively high intracellular metabolite concentrations and, consequently, a possible accumulation of propionic acid.
• a metabolite of formula (II) or of formula (III), or of a salt or an ester pharmaceutically acceptable thereof may be convenient, through the use of the prodrug of formula (I), or of a pharmaceutically acceptable salt or ester. thereof, such as the sodium salt of DHA-H.
• the administration of the metabolites of formula (II) or of formula (III) by using the even-chain compounds of formula (I) as prodrugs allows a time-regulated administration of their metabolites of formula ( II) or formula (III), which have an odd chain.
• the present invention shows how the administration of the compounds of formula (II) or of formula (III) by means of the administration of their prodrugs of formula (I), allows a time-regulated administration of their odd-chain metabolites .
• the present invention makes it possible to adapt the therapy, depending on the nature of the disease and prognosis of the patient to be treated, to use the metabolite, or the compound of formula (I), that is, the prodrug, as a medicine.
• the use of the metabolite would be more appropriate or prioritized, to obtain a rapid and significant effect.
• the use of the prodrug, or of compositions that combine prodrug and metabolite in different ratios may be recommended or prioritized. , depending on the moment and situation / severity of the disease.
• the prodrugs having formula (I) provide a way of administering the metabolites of formula (II) or formula (III) with less risk of occurrence of adverse side effects and providing a therapeutically effective amount of said metabolites in a manner sustained over time, as the prodrug having formula (I), once administered, is metabolized.
• one aspect of the invention relates to a compound selected from the group consisting of a compound of formula (II), or a pharmaceutically acceptable salt or ester thereof:
• a + 3b + c + 3 is an even integer, for use as a medicine, and in particular for use in the induction of neuroregeneration and in the prevention and / or treatment (including maintenance treatment) of a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective
• maintenance treatment or “maintenance therapy” is defined as a therapeutic treatment administered as an adjunct to a primary or main treatment or therapy, with the purpose of either preventing or delaying recidivism. of the disease, which has remitted completely or partially after treatment with a primary treatment or therapy, or to slow the development of a disease after finishing treatment with a primary therapy.
• the present invention relates to a compound of formula (II) or a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, for use in a treatment or maintenance therapy of a disease or pathology, and more preferably in a cancer maintenance treatment or therapy.
• An embodiment of the invention therefore relates to the use of a compound selected from the group consisting of a compound of formula (II), or a pharmaceutically acceptable salt or ester thereof, and a compound of formula (III ), or a pharmaceutically acceptable salt or ester thereof, as described in the present invention, in the manufacture of a medicament for the induction of neuroregeneration or for the prevention and / or treatment (including maintenance treatment) of a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease
• Another embodiment of the present invention relates to a method of prevention and / or treatment of a disease or pathology, or to a method of inducing neuroregeneration; in a patient, wherein said method comprises administering to said patient an effective amount of a compound selected from the group consisting of a compound of formula (II), or a pharmaceutically acceptable salt or ester thereof:
• an effective amount, or a therapeutically effective amount, for the purposes of the present invention is understood as that which provides a therapeutic effect without providing unacceptable toxic effects in the patient.
• the effective amount or dose of the drug depends on the compound and on the pathology or disease treated and on, for example, the age, weight and clinical condition of the treated patient, the method of administration, the clinical history of the patient, the severity of the disease. and the potency of the administered compound.
• an embodiment of the invention relates to a compound selected from the group consisting of a compound of formula (II):
• said disease is selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• another aspect of the present invention refers to a method for administering an effective amount of a compound of formula (II), or of a compound of formula (III), as described in the present invention, for the prevention and / or the treatment of a disease or pathology, or for the induction of neuroregeneration and / or the prevention of neurodegeneration, wherein said compound of formula (II) or said compound of formula (III), is administered as a prodrug of formula (I), or as a pharmaceutically acceptable salt or ester thereof, as described in the present invention.
• the invention further relates to a method for the prevention and / or treatment of a disease or pathology; wherein said method comprises administering an effective amount of a prodrug of a compound of formula (II), or of a pharmaceutically acceptable salt or ester thereof; or of a prodrug of a compound of formula (III), or of a pharmaceutically acceptable salt or ester thereof, as described in the present invention; wherein said prodrug of the compounds of formula (II) or formula (III) has formula (I), or a pharmaceutically acceptable salt or ester thereof, as described in the present invention.
• the present invention relates to a method for the prevention and / or treatment of a disease or pathology, or for the induction of neuroregeneration and / or the prevention of neurodegeneration, wherein said method comprises administering, to a patient in need, an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:
• said compound of formula (I) is metabolized in more than 1%, 10%, in more than 40%, more than 50%, and up to 99% in a metabolite of formula (II) or of formula (III) when administered.
• Another embodiment relates to a method for the prevention and / or treatment of a disease or pathology selected from the group consisting of: neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and one metabolic disease, and for the induction of neuroregeneration and / or the prevention of neurodegeneration; wherein said method comprises administering to a patient an effective amount of a prodrug with the structure of formula (I), or a pharmaceutically acceptable salt or ester thereof:
• said conversion is a chemical or physiological process.
• the term “chemical process” refers to the conversion of the prodrug in vivo to release the active compound through a chemical reaction, wherein the prodrug is a reagent or substrate of the chemical reaction, and the active compound it is a reaction product.
• physiological process refers to a conversion due to an event or process that occurs naturally in an organism, for example due to the activity of enzymes.
• another embodiment of the invention relates to a compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, as described describe in the present invention, for use as a medicine and, in particular, for use in the induction of neuroregeneration and / or the prevention of neurodegeneration and / or for use in the prevention and / or treatment of a disease or pathology, according to the present invention, characterized in that said compound is administered before, after or together with a compound of formula (I), or with a pharmaceutically acceptable salt or ester thereof :
• the present invention relates to a method of inducing neuroregeneration and / or preventing neurodegeneration, or to a method of preventing and / or treating a disease or pathology, which comprises administering an effective amount of a compound of formula ( II), or of a compound of formula (III), or of a pharmaceutically acceptable salt or ester thereof, to a patient and, wherein said method is characterized in that it comprises the additional administration of a compound of formula (I), or of a pharmaceutically acceptable salt or ester thereof, as described in the present invention; and wherein said compound of formula (I) is administered before, after or together with said compound of formula (II) or of formula (III) ,.
• prodrug refers to a compound that, when administered to a subject, is transformed, through a metabolic process, into a second therapeutically active compound.
• the term “subject” refers, for the purposes of the present invention, to a human or an animal.
• pharmaceutically acceptable refers, for the purposes of the present invention, to that compound or substance authorized or authorized by a regulatory agency of the federal government or a state government or listed in the European, United States, or other generally recognized pharmacopoeia for its use in animals or humans. Throughout the present specification, said term applies, above all, to the salts and esters of the compounds of formulas (I), (II) and (III), which are defined in accordance with the present description.
• the term "pharmaceutically acceptable salt” refers to a salt of a compound that also possesses the desired pharmacological activity of the parent compound from which it is derived.
• the pharmaceutically acceptable salt is the sodium salt.
• ester refers to any compound in the that the hydroxyl group belonging to a carboxylic acid residue has been replaced by an alkoxide group.
• the ester is a methyl or ethyl ester. Most preferably the ester is an ethyl ester.
• bitterraceutical acceptable refers, for the purposes of the present invention, to everything that is of use in nutraceutical products.
• the term “nutraceutical” or “nutraceutical composition” refers to a dietary supplement, to be taken alone, or in combination with other foods and that produces a beneficial effect for the health of the subject that ingests it, especially in disease prevention.
• said term applies, above all, to the salts and esters of the compounds of formulas (I), (II) and (III), which are defined in accordance with the present description.
• stereoisomer refers to those compounds that have the same chemical formula and the same sequence of atoms, but have a different three-dimensional orientation in space, and includes the R and S stereoisomers (which They also use the nomenclature (+) and (-)) resulting from the presence of a chiral carbon, as well as the E and Z stereoisomers (which also use the cis / trans nomenclature) resulting from the arrangement of the carbon substituents that constitute a double bond.
• the invention also includes the two stereoisomers R and S, as well as any mixture of both, with respect to the configuration of said chiral carbon.
• both the prodrugs of formula (I) and their metabolites of formula (II), or (III), comprise C C double bonds
• the invention also includes all the E and Z stereoisomers for each of its double bonds.
• all the double bonds of the prodrug of formula (I), of the compound of formula (II) and of the compound of formula (III) have an all-cis configuration.
• the prodrug of formula (I) has a certain cis / trans (or E / Z) stereochemical configuration of its double bonds
• the metabolite of formula (II) or formula (III) will also have said configuration for the double bonds. links it contains.
• the term “comprises” indicates that it includes a group of certain characteristics (for example, a group of characteristics A, B and C) is interpreted to mean that it includes those characteristics (A, B and C), but it does not exclude the presence of other characteristics (for example, characteristics D or E), provided that they do not make the claim impracticable. Additionally, the terms “contains”, “includes”, “has” or “encompasses”, and the plural forms thereof, should be taken synonymously with the term “comprises” for the purposes of the present invention. On the other hand, if the expression "consists of” is used, then no additional characteristics are present in the apparatus / method / product, apart from those that follow that expression.
• the term “comprises” may be replaced by any of the terms “consists of”, or “consists essentially of”. Accordingly, “comprises” may refer to a group of characteristics A, B and C, which may additionally include other characteristics, such as E and D, provided that said characteristics do not make the claim impracticable, but said term “comprises ”Also includes the situation where the feature group“ consists of ”or“ consists essentially ”of A, B, and C.
• the present invention makes it possible to support an administration of a compound of formula (I), in particular of 20H0A, or a pharmaceutically acceptable salt or ester thereof, more preferably the sodium salt of 20H0A, in a maintenance treatment (maintenance therapy ), wherein said compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, is administered at different intervals over a period of time, the cumulative concentration of its metabolite of formula (II), or of formula (III), a measure of the effectiveness of the treatment.
• a maintenance treatment maintenance therapy
• said in vitro method for determining the efficacy of a treatment with a compound of formula (I), or with a pharmaceutically acceptable salt or ester thereof comprises determining the amount of a compound of formula (II), or of formula (III), or of its carboxylate anion, or of a derivative formed therefrom.
• biomarker refers to a first compound or substance, or a derivative of said first compound or substance, which can be used to determine the response and / or efficacy of a treatment. with a second compound or substance.
• the metabolites of formula (II), or of formula (III) can be used as biomarkers for determining the response and / or efficacy of a treatment with a compound of formula (I) .
• another aspect of the invention relates to an in vitro method to determine the efficacy of a therapeutic or preventive treatment of a disease or pathology, or of a treatment to induce neuroregeneration, with a compound of formula (I), or with a pharmaceutically acceptable salt or ester thereof:
• said method comprises determining the amount of a compound of formula (II), or of formula (III), of its respective carboxylate anions, or of a derivative formed therefrom.
• Said derivative of the compound of formula (II), or (III) can be formed in vitro, by reacting said compound of formula (II) or (III), comprised in the in vitro sample, with a substance to obtain a derivative thereof .
• the method of the invention comprises determining the amount of said derivative of formula (II), or of formula (III), formed in vitro.
• some techniques for the detection of fatty acids require their prior chemical modification and thus, it is usual that detection by gas chromatography requires that the fatty acid sample (in this case a compound of formula (II), or of formula (III)) is transformed into its respective methyl ester for detection and quantification.
• said derivative of the compound of formula (II), or of formula (III) can be a metabolic derivative or a derivative formed in vivo (the result of a reaction that occurred in vivo), formed as a result of the reaction of said compound of formula (II), or of formula (III), with another lipid, protein, enzyme, nucleotide, carbohydrate, etc.
• said derivative can be an ester of said compound of formula (II), or of formula (III), such as, for example, a glycerophospholipid (such as phosphatidylcholine, phosphatidylethanolamine, fostaidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid or any of its forms smooth, such as lysophosphatidylcholine, lysophosphatidylethanolamine, etc.), a plasmalogen (alkyl or alkenyl), a cholesterol ester, a glycerolipid such as triacylglycerol (triglyceride) or diacylglycerol, a cardiolipin, a sphingolipter (a coenzyme ester, a coenzyme ester, acyl-CoA), or an acylcarnitine, among others.
• a glycerophospholipid such as phosphatidyl
• the method of the invention comprises determining in vitro the amount of said metabolic derivative (or derivative formed in vivo) in the biological sample.
• the amount of said compound of formula (II), or of formula (III), or of their respective carboxylate anions, or of a derivative formed from them in vivo or in vitro is related to the efficacy of the treatment.
• Another aspect of the invention relates to the use of a compound: of formula (II):
• the disease or pathology is selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• the disease or pathology is selected from a neurological or neurodegenerative disease; a cancer; an inflammatory disease; and a metabolic disease.
• the method determines the efficacy upon treatment with a pharmaceutically acceptable salt of the compound of formula (I) and even more preferably with the sodium salt of the compound of formula (I).
• the biological sample is a blood sample (including plasma or serum), a urine sample, a saliva sample, a tissue biopsy, cerebrospinal fluid, or a sweat sample.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least a first compound selected from the group consisting of: a compound of formula (II), or a pharmaceutically acceptable salt or ester thereof:
• composition optionally comprises a second compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least a first compound selected from the group consisting of: a pharmaceutically acceptable salt or ester of a compound of formula (II):
• composition optionally comprises a pharmaceutically acceptable salt or ester of a compound of formula (I):
• compositions comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), wherein said composition optionally comprises a salt or a pharmaceutically acceptable ester of a compound of formula (I), as described above; and at least one pharmaceutically acceptable excipient; for use as a medicine; and in particular, for use in the induction of neuroregeneration and / or the prevention of neurodegeneration, and for use in the prevention and / or treatment of a disease or pathology selected from the group consisting of: a neurological disease or neurodegenerative; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• a neurological disease or neurodegenerative selected from the group consisting of: a neurological disease
• said at least first compound is a pharmaceutically acceptable salt or ester of a compound of formula (II), or of a compound of formula (III), and / or said second compound is a salt or a pharmaceutically acceptable ester of a compound of formula (I).
• One embodiment of the invention relates to the use of a pharmaceutical composition
• a pharmaceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, as described above; and at least one pharmaceutically acceptable excipient; in the manufacture of a medicine for the induction of neuroregeneration and / or the prevention of neurodegeneration, and / or for the prevention and / or treatment of a disease or pathology.
• Another embodiment of the invention relates to a method for the prevention and / or treatment of a disease or pathology, or for the induction of neuroregeneration and / or the prevention of neurodegeneration; wherein said method comprises administering, to a patient in need, an effective amount of a pharmaceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, as described above; and at least one pharmaceutically acceptable excipient.
• said disease or pathology is selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• compositions are suitable to be administered to both a human subject and an animal.
• said excipient is albumin, for example: ovalbumin, lactalbumin, native or recombinant albumin of human, bovine, murine or rabbit origin, more preferably, human serum albumin or bovine serum albumin.
• compositions disclosed in the present invention may also be co-administered, prior to, or subsequent to, additional therapy.
• additional therapy is radiotherapy, electric fields for the treatment of tumors (Tumor Treatment Fields), immunotherapy or chemotherapy.
• pharmaceutical compositions disclosed in the present invention may also be administered in conjunction with, prior to, or subsequent to, a therapy comprising the administration of temozolomide.
• Said administration can be within a treatment of an adult or a pediatric patient.
• said pharmaceutical composition is administered together, before, or after a radiotherapeutic treatment, a chemotherapeutic treatment, a treatment with electric fields for the treatment of tumors (Tumor Treatment Fields), or an immunotherapeutic treatment.
• the pharmaceutical compositions disclosed herein comprise at least one additional therapeutic component or active compound.
• Said additional therapeutic component or active compound provides additive or synergistic biological activities.
• the terms "active compound” or “therapeutic component” should be taken synonymously and mean a chemical or biological entity that exerts therapeutic effects when administered to humans or animals.
• Said active compound or additional therapeutic component can be a cell therapy, a small molecule therapy, an immunotherapy, radiotherapy, among others.
• therapeutic components or additional active compounds are compounds for the treatment of neurodegenerative diseases, anticancer agents, metabolism regulatory compounds, cardiovascular agents, and regulatory agents for obesity and overweight.
• therapeutic components or additional active compounds are compounds for the treatment of neurodegenerative diseases, chemotherapeutic agents, metabolism regulatory compounds, cardiovascular agents, and obesity and overweight regulatory agents.
• said active compound or said therapy is a chemotherapeutic agent, a cell therapy agent or an immunotherapeutic agent.
• said pharmaceutical composition further comprises a chemotherapeutic agent that is selected from the group consisting of: platinum-based antineoplastic agents; antimitotic chemotherapeutic agents; a poly adenosine diphosphate ribose polymerase (PARP) inhibitor; type I topoisomerase inhibitors; type II topoisomerase inhibitors; epothilones; cyclo-skeletal disruptors; alkylating agents; histone deacetylase inhibitors; kinase inhibitors; antifolates; peptide antibiotics; retinoids; vinca alkaloids and thymidylate synthase inhibitors.
• a chemotherapeutic agent that is selected from the group consisting of: platinum-based antineoplastic agents; antimitotic chemotherapeutic agents; a poly adenosine diphosphate ribose polymerase (PARP) inhibitor; type I topoisomerase inhibitors; type II topoisomerase inhibitors; epoth
• the chemotherapeutic agent is selected from the group consisting of: bevacizumab, carmustine, cyclophosphamide, melphalan, ifosfamide, busulfan, temozolomide, mechlorethamine, chlorambucil, melphalan, dacarbazine, daunorubicin, doxorubicin, epithelialubicin, valubicin, eporubicin docetaxel, abraxane, taxotere, epothilone, vorinostat, romidepsin, irinotecan, topotecan, camptothecin, exatecan, lurtotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemuragadibine, azpeatibrine, citratene, ap
• the additional chemotherapeutic agent is temozolomide.
• lipids when they are part of the cell membrane, can control cell signaling, means that they can also regulate the physiological state of cells and, therefore, the general state of health.
• the compounds described in the present document are useful in the induction of neuroregeneration and in the prevention and / or treatment of different diseases and pathologies, in particular selected from the group consisting of a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• diseases of the nervous system are all those diseases that affect the nervous system (both central and peripheral).
• neurodegenerative diseases which, for the purposes of the present invention, are a heterogeneous group of disorders characterized by progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.
• the neurodegenerative disease is selected from the group consisting of spinal cord injury and pain of neurological origin.
• the term “induction of neuroregeneration” refers to the regeneration of neurological functions.
• prevention of neurodegeneration indicates that the treatment causes the arrest of a neurodegenerative process already underway, or that the treatment prevents the appearance or progression of neurodegeneration.
• Neurodegenerative processes involve a significant decrease in the cognitive capacity of patients or motor alterations.
• Neurodegenerative processes, neurological and neuropsychiatric disorders have a common basis of neuronal degeneration or alterations of their components, such as lipids (for example, myelin) or membrane proteins (for example, adrenergic receptors, serotonergic receptors, etc.).
• lipids for example, myelin
• membrane proteins for example, adrenergic receptors, serotonergic receptors, etc.
• neurodegenerative diseases are selected from the group consisting of: (i) inflammatory diseases of the central nervous system such as bacterial meningitis, non-bacterial meningitis, acute hemorrhagic necrotizing encephalopathy, other encephalitis, myelitis and encephalomyelitis, cerebral ventriculitis not otherwise specified NOS, intracranial and intra-spinal abscess and granuloma, extradural and subdural abscess, phlebitis, intracranial thrombophlebitis in intra-spinal cord and sequelae of inflammatory diseases of the Central Nervous System; (ii) systemic atrophies mainly affecting the central nervous system such as Guillan-Barré, diabetic neuropathy, Wallerian degeneration, Levy body dementia, fronto-temporal dementia, Huntington's chorea, Huntington's dementia, hereditary ataxia; spinal muscular atrophy and related syndromes such as Werdnig-Hoffman; systemic atrophic
• neurodegeneration pain is defined as pain caused by an injury or disease of the somatosensory nervous system, in accordance with the definition of the International Association for the Study of Pain (IASP).
• the somatosensory nervous system comprises sensory neurons and neural pathways that respond to changes on the surface or within the body.
• paralysis refers, for the purposes of the present invention, to the partial or total loss of mobility in some part of the body, caused by an injury or disease of the central or peripheral nervous system.
• sleep disorders refers to those disorders that include problems in the initiation and maintenance of sleep caused by a problem or pathology of the central or peripheral nervous system. Non-limiting examples of such sleep disorders include insomnia, hypersomnias such as narcolepsy, sleep apnea, restless leg syndrome, circadian rhythm disorders, and parasomnia, among others.
• Certain neurodegenerative diseases can lead to processes in which blindness, hearing problems, disorientation, alterations in mood, etc. develop.
• An example of a well-characterized neurodegenerative disorder is Alzheimer's disease, in which the formation of plaques has been observed, formed mainly by the b-amyloid peptide that comes from altered protein processing. followed by an accumulation on the outside of the cells.
• tangles of hyperphosphorylated tau protein neuro-filaments appear inside the cell. This process has been associated with alterations in cholesterol metabolism and the consequent alteration in the levels of certain membrane lipids, such as docosahexaenoic acid.
• AD Alzheimer's disease
• sclerosis and other neurodegenerative processes are related to "demyelination", the net result of which is the loss of lipids in the covering of neuronal axons, with the consequent alterations in the process of propagation of electrical signals that this supposed.
• Myelin is a lipid layer that surrounds the axons of many neurons and is formed by a succession of spiral folds in the plasma membrane of glial cells (Schwann cells and oligodendrocytes, at the peripheral and central levels, respectively). Therefore, it has been shown that lipids play an important role in the development of neurodegenerative diseases. Furthermore, it has been proven that natural polyunsaturated fatty acids have a moderate preventive effect on the development of neurodegenerative processes.
• DHA docosahexaenoic acid
• the metabolic disease is preferably selected from the group consisting of obesity, overweight, hypercholesterolemia, hypertriglyceridemia, diabetes, and insulin resistance.
• Metabolic diseases form a set of pathologies characterized by the accumulation or deficiency of certain molecules. A typical example is the accumulation of glucose, cholesterol and / or triglycerides above normal levels.
• Increased levels of glucose, cholesterol and / or triglycerides both at the systemic level (eg, increase in plasma levels) and at the cellular level (eg, in cell membranes) is associated with alterations in cellular signaling that leads to dysfunctions at various levels, and that are normally due to errors in the activity of certain enzymes or the control of said proteins.
• hypercholesterolemia high levels of cholesterol
• hypertriglyceridemia high levels of triglycerides
• Other important metabolic diseases are diabetes and insulin resistance, characterized by problems in controlling glucose levels.
• These metabolic diseases are involved in the appearance of other pathological processes, such as cancer, hypertension, obesity, arteriosclerosis, etc. Another pathological process related to the previously described metabolic diseases has been identified and that could constitute per se a new metabolic disease, which is the metabolic syndrome.
• a neoplasm is defined as an abnormal mass of tissue that occurs when cells multiply more than they should or are not destroyed at the appropriate time.
• Neoplasms are benign (not cancer) or malignant (cancer).
• the term "neoplasia" is equivalent to that of "tumor.”
• cancer There are multiple types of cancer, including, for example, cancer of the oral cavity and pharynx, cancer of other digestive organs, cancer of other respiratory organs, cancer of bone and articular cartilage, melanoma and other malignant neoplasms of the skin, cancer from mesothelial tissues and soft tissues, cancer of genital organs, cancer of the urinary tract, cancer of the eye, brain and other regions of the nervous system, cancer of the thyroid and other endocrine glands, neuroendocrine malignancies, cancer of lymphoid, hematopoietic and related tissues, carcinomas in situ, benign tumors, neoplasms of uncertain behavior, polycythemia vera and myel
• Lipid modification of the cell membrane can be used as a strategy for the prevention or treatment of multiple types of cancer.
• the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, bile duct cancer, neuroblastoma, colon cancer, gastric cancer, liver cancer, glioblastoma, non-Hodgkin lymphoma, kidney cancer, esophageal cancer, stomach cancer, cervical cancer, or lymphoma tumors.
• the cancer is selected from the group consisting of lung cancer, brain cancer, glioma, glioblastoma, breast cancer, leukemia, liver cancer, endometrial cancer, and pancreatic cancer. Most preferably, the cancer is selected from the group consisting of lung cancer, brain cancer, breast cancer, leukemia, liver cancer, and pancreatic cancer.
• cardiovascular disease is defined as a group of diseases or disorders of the heart and blood vessels.
• Said cardiovascular diseases are selected from the group consisting of: ischemic stroke, acute rheumatic fever, chronic heart diseases, hypertensive disease, ischemic heart disease, pericarditis, endocarditis, valve disorders, cardiomyopathy, tachycardia, heart failure, amyloid angiopathy, diseases and cerebrovascular disorders, sequelae of cerebral hemorrhage, sequelae of cerebral infarction, sequelae of cerebrovascular diseases, diseases of arterioles and capillaries; diseases of veins, vessels and lymph nodes.
• pathology of the skin and subcutaneous tissue refers, for the purposes of the present invention, to pathologies of the dermal tissue among which are: bullous disorders, dermatitis, eczema, papulosquamous disorders, disorders of the skin appendages, postoperative complications , urticaria and erythema.
• Inflammatory processes include a wide spectrum of pathologies that are characterized by the presence of inflammation.
• said inflammatory processes are selected from the group consisting of: cardiovascular inflammation; inflammation caused by tumors; inflammation of rheumatoid origin; respiratory inflammation; acute and chronic inflammation; inflammatory hyperalgesia; and edema and swelling caused by trauma or burns.
• digestive pathology refers, for the purposes of the present invention, to diseases of the oral cavity and salivary glands; diseases of the esophagus, stomach and duodenum; appendix diseases; non-infectious enteritis and colitis; diseases of the peritoneum and retroperitoneum; liver diseases; disease of the gallbladder, bile ducts and pancreas.
• a musculoskeletal and connective tissue disease refers to pathologies of muscles, joints and bones that may or may not have an autoimmune origin.
• Said musculoskeletal and connective tissue diseases are selected from the group consisting of: arthropathies, connective tissue disorders, muscle and soft tissue disorders; Synovial membrane and tendon disorder; osteopathies and chondropathies.
• genitourinary pathology refers, for the purposes of the present invention, to glomerular diseases; tubulo-interstitial kidney diseases; acute kidney failure; chronic kidney disease; lithiasis; and inflammatory and non-inflammatory disorders of the renal tract.
• the present invention also relates to a nutraceutical composition
• a nutraceutical composition comprising at least a first compound selected from the group consisting of: a compound of formula (II), or a nutraceutical acceptable salt, or ester thereof:
• composition optionally comprises a second compound of formula (I), or a nutraceutically acceptable salt, or ester thereof:
• the present invention also relates to a nutraceutical composition
• a nutraceutical composition comprising at least one first compound selected from the group consisting of: a nutraceutically acceptable salt or ester of a compound of formula (II):
• composition optionally comprises a nutraceutically acceptable salt or ester of a compound of formula (I):
• the present invention also relates to a nutraceutical composition
• a nutraceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a nutraceutical acceptable salt or ester thereof.
• said composition optionally comprises a compound of formula (I), or a nutraceutically acceptable salt or ester thereof, as described above, for use in the prevention of a disease or pathology.
• the present invention also relates to a method of preventing a disease or pathology, wherein said method comprises administering to a subject an effective amount of a nutraceutical composition comprising at least one first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a nutraceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I), or a nutraceutically acceptable salt or ester thereof, as described above.
• said disease or pathology is selected from the group that consists of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a pathology of the skin and subcutaneous tissue; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease.
• m 0 and, each of the embodiments of the present invention described, including those embodiments referring to compounds of formula (II), or of formula (III), pharmaceutical and nutraceutical compositions comprising them, their first and second Medical uses, methods of induction of neuroregeneration and / or prevention of neurodegeneration, or methods of prevention and / or treatment of a disease or pathology, as well as the use and in vitro method of determining the efficacy of a treatment, are refer to a compound selected from the group consisting of a compound of formula (II), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
• m 0 and, each of the embodiments of the present invention described, including those embodiments referred to compounds of formula (II), or of formula (III), pharmaceutical and nutraceutical compositions comprising them, their first and foremost second medical uses, the methods of induction of neuroregeneration and / or prevention of neurodegeneration, or the methods of prevention and / or treatment of a disease or pathology, as well as the use and the in vitro method of determining the efficacy of a treatment, refer to a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II):
• m 0 and, each of the embodiments of the present invention described, including those embodiments referring to compounds of formula (II), or of formula (III), pharmaceutical and nutraceutical compositions comprising them, their first and second medical uses, the methods of induction of neuroregeneration and / or prevention of neurodegeneration, or the methods of prevention and / or treatment of a disease or pathology, as well as the use and in vitro method of determining the efficacy of a treatment , refer to a compound selected from the group consisting of: a compound of formula (II), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
• m 0 and, each of the embodiments of the present invention described, including those embodiments referring to compounds of formula (II), or of formula (III), pharmaceutical and nutraceutical compositions comprising them, their first and second medical uses, the methods of induction of neuroregeneration and / or prevention of neurodegeneration, or the methods of prevention and / or treatment of a disease or pathology, as well as the use and in vitro method of determining the efficacy of a treatment , refer to a compound selected from the group consisting of: a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II):
• said salt is a sodium salt
• said ester is an ethyl ester
• the pharmaceutical and nutraceutical compositions described in the present application comprise a compound of formula (I), together with a compound of formula (II) or a compound of formula (III), in a concentration between 0.01% to 99.99% w / w, preferably the composition comprises 10% to 80% w / w, or even more preferably in a concentration between 20% to 80% w / w.
• compositions described in the present application comprise a prodrug of formula (I) together with a compound of formula (II) or a compound of formula (III), wherein said combination is in a ratio of the range between 0.01: 100 to 100: 0.01, preferably 1: 5 to 5: 1 and even more preferably 1: 2 to 2: 1.
• the pharmaceutical or nutraceutical compositions of the invention can be presented in vials, ampoules, powders, capsules, tablets, sachets, solutions, syrups, ointments, creams, emulsions, gels, patches, controlled release formulations, suppositories, ovules. , etc.
• the formulations are useful to be administered, inter alia, by oral, sublingual, gastroenteric, rectal, parenteral (intravenous, intraarterial, intramuscular and subcutaneous), respiratory, topical (ophthalmic, otic, transdermal) routes.
• the route of administration can be easily determined by a person skilled in the art.
• compositions of the present invention may be in the form of a gastro-resistant composition to avoid degradation of its components by the low pH of the gastric environment.
• the composition of the invention further includes one or more additional components or excipients, such as diluents, antioxidants, sweeteners, gelling agents, flavors, fillers, or other carriers, such as anhydrous colloidal silica and glyceryl monostearate.
• additional components or excipients such as diluents, antioxidants, sweeteners, gelling agents, flavors, fillers, or other carriers, such as anhydrous colloidal silica and glyceryl monostearate.
• Such compositions can be in the form of a capsule, sachet, paper, or other container. In producing the compositions, conventional techniques for the preparation of pharmaceutical compositions can be used.
• the compounds disclosed hereinbefore can be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier that can be in the form of a blister, capsule, envelope, paper, or other container.
• a carrier When the carrier is a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound.
• Suitable diluents are water, saline solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin , gum arabic, stearic acid or lower alkyl cellulose ethers, silicic acid, fatty acids, fatty acid amines, monoglycerides and diglycerides of fatty acids, fatty acid esters of pentaerythritol, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone.
• the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax.
• sustained release material such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax.
• Such compositions can also include wetting agents, antioxidants, emulsifying and suspending agents, preserving agents, sweetening agents, and flavoring agents.
• Compositions of the invention may be formulated so as to provide rapid, sustained, or delayed release of the compounds disclosed herein after administration to the patient using procedures well known in the art.
• compositions can be solid compositions or liquid solutions.
• said composition is a solid composition that can comprise 20-80%, of the compound of formula (I) and / or of the compound of formula (II) or of formula (III), 20- 80% of a diluent, 0.1-20% of an antioxidant, 0.01-10% of a sweetener, 0.1-20% of a gelling agent and 0.01-10% of a flavoring.
• said composition is a solution for oral administration comprising 20 to 80% of the compound of formula (I) and / or of the compound of formula (II) or of formula (III), 20 to 80% diluent, 0.1 to 20% antioxidant, 0.01 to 10% of a sweetener, 0.1 to 20% of a gelling agent and 0.01 to 10% of a flavoring.
• compositions can be sterilized and mixed, if desired, with auxiliary agents, emulsifiers, salt to influence the osmotic pressure, buffers and / or coloring substances and the like, which do not react deleteriously with the compounds previously disclosed in the document. present document.
• FIG. 1 A Illustrative diagram of the cellular metabolism of 2-hydroxydocosahexaenoic acid (DHA-H) giving rise to (6Z, 9Z, 12Z, 15Z, 18Z) -heneicosa-6,9,12,15,18-pentaenoic acid (HPA ) by a-oxidation.
• DHA-H requires activation by an Acyl-CoA synthetase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg 2+ ).
• DHA-H-CoA it would be subject to the activity of 2-hydroxyfitanoyl-CoA Nase (2-hydroxyacyl-CoA Nase 1, HACL1), which would lead to the formation of an intermediate polyunsaturated aldehyde that should contain 5 or 6 double bonds.
• HACL1 activity is dependent on thiamine pyrophosphate (TPP) and Mg 2+ , and can be inhibited by a competitive antagonist (eg, oxythiamine).
• TPP thiamine pyrophosphate
• Mg 2+ Mg 2+
• the enzyme aldehyde dehydrogenase would be responsible for the conversion of the intermediate aldehyde into HPA in a process dependent on NAD + (Nicotinamide Adenine Dinucleotide).
• DHA-H is metabolically transformed into HPA by a-oxidation in HEK293T cells.
• Intracellular levels of DHA-H (B1 and B3) and HPA (B2 and B4) are represented on the ordinate axis (nmoles / mg of protein), versus concentration of treatment with the sodium salt of DHA-H (mM) for 24 hours (B1 and B2) or incubation time (h) with a constant concentration of the sodium salt of DHA-H of 30 pM (B3 and B4), including untreated controls (C), on the abscissa axis.
• the black bars represent the result in cells without additional stimulus
• the blank bars represent the result after simultaneous treatment with 1 mM oxythiamine
• the striped bars represent the result after treatment with 10 mM oxythiamine.
• Intracellular levels of DHA are represented on the ordinate axis (nmol / mg protein), versus concentration of treatment with the sodium salt of DHA-H (pM) for 24 hours (C1) or incubation time (h) with a constant concentration of the sodium salt of DHA-H of 30 pM (C2), including untreated controls (C), on the abscissa.
• Treatment with the sodium salt of DHA-H had no significant effect on DHA levels, neither as a function of concentration nor of incubation time.
• the bars represent the mean ⁇ standard error, and the statistical analysis was done using one-way ANOVA and the Tukey multiple evaluation test.
• FIG. 1 Mice treated with the sodium salt of DHA-H show dose-dependent accumulation of HPA in the brain, with DHA-H being undetectable in the brain.
• Brain levels of HPA (A1) or DHA (A2) are plotted on the ordinate axis (nmoles / mg protein), versus treatment doses with the sodium salt of DHA-H (A1) and the sodium salt of DHA (A1 and A2) (mg / kg).
• A1 ⁇ WT animals; or 5xFAD.
• A2 the black bars refer to WT animals and the blank bars refer to 5xFAD.
• HPA and DHA levels were determined in the brain of WT and 5xFAD mice after chronic administration of the sodium salt of DHA-H (4 months; 5 doses / week LV; between 3 and 7 months of age; sacrifice at 7 months).
• DHA levels did not vary significantly between experimental conditions (A2). Data are shown as mean ⁇ standard error, and statistical analysis was done by one-way ANOVA and Tukey multiple evaluation test: * p ⁇ 0.05 compared to control (vehicle-treated mice).
• Each point in the graphs represents the mean ⁇ standard error for each pathology / treatment condition: ⁇ WT + vehicle, T WT + DHA-H 20 mg / kg; ⁇ WT + DHA-H 200 mg / kg; or 5xFAD + vehicle; D 5xFAD + DHA-H 5mg / kg; V 5xFAD + DHA-H 20mg / kg; ⁇ 5xFAD + DHA-H 50 mg / kg; Or 5xFAD + DHA-H 200mg / kg.
• FIG. 3 A. Mice treated with the sodium salt of DHA-H show tumor accumulation of HPA, being DHA-H undetectable, in xenographic tumors of U118 cells.
• the levels of DHA (black bars) and HPA (open bars) (pmol / mg tissue) in the tumor are plotted, on the ordinate axis, against the treatment condition (vehicle and DHA-H 200 mg / kg) on the abscissa axis.
• the 3-month-old NUDE (immunosuppressed) mice were injected subcutaneously with 7.5.10 6 U118 cells (human glioblastoma multiforme grade IV).
• Tumor growth was allowed subcutaneously for 10 days before the start of oral treatments (vehicle or sodium salt of DHA-H 200 mg / kg), which were maintained for 42 days until sacrifice. Lipid analysis of the xenographic tumors revealed the absence (undetectable levels) of DHA-H. The bars represent the mean ⁇ standard error for each treatment condition.
• B. Tumor HPA levels inversely correlate with tumor size in xenographic models. Tumor size (cm 3 ) is plotted, on the ordinate axis, against HPA levels (pmol / mg of tissue) in the tumor, on the abscissa axis, for two treatment conditions: o vehicle and ⁇ DHA-H sodium salt 200 mg / kg.
• DHA-H is a prodrug that is metabolically transformed into HPA by ⁇ -oxidation in U118 cells.
• the intracellular levels of DHA black bars
• DHA-H white bars
• HPA striped bars
• Control C
• 150 mM DHA-H sodium salt 48 h
• Both DHA-H and HPA were increased in cells treated with the sodium salt of DHA-H.
• the cell viability (% of the control -C- without oxythiamine) on the ordinate axis, against the treatment conditions: Control (C- black bars) and the sodium salt of DHA-H 150 mM, 48 h (white bars) in the presence and absence of treatment simultaneously with 1 mM oxythiamine, on the abscissa axis.
• Control C- black bars
• the sodium salt of DHA-H 150 mM, 48 h white bars
• Treatment with DHA-H on U118 cells significantly reduces culture viability, while treatment with oxythiamine (alone) has no effect on cell viability.
• the treatment with the sodium salt of DHA-H is done simultaneously with oxythiamine, the anti-proliferative effect of this compound is significantly decreased compared to the effect without oxythiamine.
• the bars represent the mean ⁇ standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: * p ⁇ 0.05 when compared with the control (C); # p ⁇ 0.05 when the effect of DHA-H is compared in the presence and absence of oxythiamine.
• FIG. 5 A. Viability of U118 cells in culture after treatment with the sodium salt of DHA-H, the sodium salt of DHA and HPA.
• Cell viability (% of the control without treatment) is represented on the ordinate axis, against the different treatment conditions, on the abscissa axis: Control (black bar), DHA-H sodium salt (150 mM, 48h - white bar), DHA (150 mM, 48 h - hatched bar) and HPA (150 mM, 48 h - grid bar).
• Control black bar
• DHA-H sodium salt 150 mM, 48h - white bar
• DHA 150 mM, 48 h - hatched bar
• HPA 150 mM, 48 h - grid bar
• HPA levels (nmol / mg protein) are represented on the ordinate axis, against the treatment conditions, on the abscissa axis: DHA-H sodium salt (150 mM, 48 h - black bar) and HPA (5-150 mM, 48 hr - open bars).
• Administration of 150mM of the sodium salt of DHA-H results in HPA levels equivalent to those generated by the treatment with HPA itself at 5 mM.
• Treatment with 150 mM HPA results in intracellular levels of HPA markedly higher than those generated by the same concentration of prodrug administration. The bars represent the mean ⁇ standard error.
• DHA-H and DHA levels in HEK293T cells in the presence (C1) or absence (C2) of culture medium The levels of DHA-H ( abscissa axis. The lipid concentration in the culture medium is 30 mM and the culture plates were incubated up to 72 h. In the presence of cell culture (C1), DHA levels in the medium, they decreased significantly at 48 and 72 h, as a consequence of the DHA uptake by the cells, while the levels of DHA-H remained unchanged up to 72 h. In the absence of cell culture (C2), the levels of both DHA and DHA-H remained constant over time. The bars represent the mean ⁇ standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: * p ⁇ 0.05 when compared with the control.
• FIG. 6 A, B and C: Chronic treatment with HPA acid or its prodrug, DHA-H, prevents the cognitive decline typical of Alzheimer's disease in the murine transgenic model (5xFAD).
• the cognitive evaluation was carried out using the 8-arm Radial Maze test. The animals received treatment between 3 and 7 months of age and the test was performed during the last month of treatment. During this test the total errors committed during the test (A), the errors of the reference memory (RME; Working Memory Errors) (B) and the errors of the working memory (WME; Reference Memory Errors) were taken into account. (C).
• Each column represents the mean ⁇ SEM of the errors during the last week of the radial maze test. The black columns represent the errors made by the WT mice.
• the blank columns represent the errors made by the 5xFAD transgenic mice treated with the vehicle (5% ethanol).
• the striped columns represent the errors made by 5xFAD mice treated with DHA-H (20 mg / kg / day).
• Checkered columns represent errors made by HPA (20 mg / kg / day) treated 5xFAD mice.
• the results show a cognitive improvement of the 5xFAD mice treated with DHA-H and with HPA in a similar way.
• the bars represent the mean ⁇ standard error for each treatment condition and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: * p ⁇ 0.05 when compared with the healthy control (WT) and # p ⁇ 0.05 when compared to the control 5xFAD condition (vehicle treated).
• FIG. 7 Tumor growth is inhibited in vivo in the presence of sodium salt treatment of HPA or its prodrug, DHA-H, in xenographic models.
• the size of the tumor (cm 3 ) is represented, on the ordinate axis, against the days of treatment elapsed, on the abscissa axis.
• To induce xenographic tumors in 3-month-old NU DE (immunosuppressed) mice 7.5.10 6 grade IV human glioblastoma cells (U-118 MG) were inoculated subcutaneously on both sides of the dorsal flank of the animal (8- 12 weeks old, 30-35 g). After 10 days, the tumors became visible with a volume of approximately 0.1 cm 3 .
• the animals were randomly divided into groups with a similar mean tumor volume and received daily oral treatments for 42 days: o vehicle (no treatment control), A DHA-H (200 mg / kg / day) and ⁇ HPA (200 mg / kg / day).
• B HPA and the prodrug, DHA-H, reduce xenographic tumor volume significantly relative to the no treatment control.
• the volume of the tumors induced 42 days after the start of the treatments is plotted on the ordinate versus the treatment conditions on the abscissa.
• the individualized data of the animals participating in the study are represented: o vehicle (control without treatment), A DHA-H (200 mg / kg / day) and ⁇ HPA (200 mg / kg / day).
• the bars represent the mean ⁇ standard error for each treatment condition and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: * p ⁇ 0.05 when compared with the control condition.
• FIG. 8 Illustrative diagrams of the cellular metabolism of 2-hydroxylated polyunsaturated fatty acids (prodrugs, PUFA-H) giving rise via a-oxidation to their corresponding non-hydroxylated metabolites, the latter having one less carbon atom than the initial molecule.
• the hydroxylated fatty acid requires activation by an Acyl-CoA synthetase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg 2+ ).
• This PUFA-H-CoA would be subject to the activity of 2-hydroxyacyl-CoA Nase (HACL, isoforms 1 or 2 depending on the cell type), which would lead to the formation of an intermediate polyunsaturated aldehyde.
• HACL 2-hydroxyacyl-CoA Nase
• HACL activity is dependent on thiamine pyrophosphate (TPP) and Mg 2+ , and can be inhibited by a competitive antagonist, such as oxythiamine.
• TPP thiamine pyrophosphate
• the enzyme aldehyde dehydrogenase would be responsible for the conversion of the intermediate aldehyde into the final fatty acid in a process dependent on NAD + (Nicotinamide Adenine Dinucleotide).
• NAD + Nicotinamide Adenine Dinucleotide.
• A Scheme of the cellular conversion of 2-hydroxy-linoleic acid (LA-H) giving rise to (8Z, 11Z) -heptadeca-8,11-dienoic acid (HDA).
• FIG. 9 Amplified regions of the different chromatograms obtained by gas chromatography with flame ionization detector (GC-FID) when treating HEK293T cells with the corresponding prodrug:
• A. (1) control (vehicle) and (2) LA-H (100 mM, 24 h).
• the white arrow indicates the chromatographic peak of the LA-H parent molecule, the black arrow indicates the chromatographic peak corresponding to the HDA metabolite. HDA formation is inhibited in the presence of 10 mM oxythiamine.
• the white arrow indicates the chromatographic peak of the ALA-H parent molecule
• the black arrow indicates the chromatographic peak corresponding to the metabolite HTA w-3.
• the formation of HTA w-3 is inhibited in the presence of 10 mM oxythiamine.
• the white arrow indicates the chromatographic peak of the GLA-H parent molecule
• the black arrow indicates the chromatographic peak corresponding to the metabolite HTA w-6.
• the formation of HTA oü-6 is inhibited in the presence of 10 mM oxythiamine.
• the white arrow indicates the chromatographic peak of the parent molecule ARA-H
• the black arrow indicates the chromatographic peak corresponding to the metabolite NTA.
• NTA formation is inhibited in the presence of 10 mM oxythiamine.
• E. (1) control (vehicle) and (2) EPA-H (100 pM, 24 h).
• the white arrow indicates the chromatographic peak of the EPA-H parent molecule
• the black arrow indicates the chromatographic peak corresponding to the metabolite NPA.
• NPA formation is inhibited in the presence of 10 mM oxythiamine.
• the white arrow indicates the chromatographic peak of the HPA parent molecule
• the black arrow indicates the chromatographic peak corresponding to the HPA metabolite.
• the formation of HPA is inhibited in the presence of 10 mM oxythiamine.
• FIG. 10 Treatment with HPA and other odd-chain polyunsaturated fatty acids prevent excitotoxicity-induced neuronal death.
• Neuronal culture was obtained by differentiation from human SH-SY5Y neuroblastomas using retinoic acid and BDNF (Brain Derived Neurotrophic Factor).
• Neuronal death by excitotoxicity was induced by adding NMDA (10 mM) and calcium / glycine (530 pM / 10 mM) to the culture medium for 1 hour.
• FIG. 11 Illustrative diagram of the cellular metabolism of 2-OHOA (LAM561) giving rise to 8Z-heptadecenoic acid (C17: 1n9), by a-oxidation.
• 2-OHOA requires activation by an Acyl-CoA ligase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg2 +).
• the 20HOA-CoA would be subject to the activity of 2-hydroxyphytanoyl-CoA Nase (2-hydroxyacyl-CoA Nase 1, HACL1), which would lead to the formation of an aldehyde intermediate monounsaturated.
• HACL1 activity is dependent on thiamine pyrophosphate (TPP) and Mg 2+ , and can be inhibited by a competitive antagonist (eg, oxythiamine).
• TPP thiamine pyrophosphate
• a competitive antagonist eg, oxythiamine.
• aldehyde dehydrogenase would be responsible for the conversion of the intermediate aldehyde into 8Z-heptadecenoic in a process dependent on NAD + (Nicotinamide Adenine Dinucleotide).
• FIG. 12 Analysis of the fatty acid composition in U-118 MG glioma cells.
• A Representative chromatograms showing the fatty acid composition in U-118 MG cells incubated in the presence of 400 mM of 20HOA sodium salt or no treatment (Control) for 24 h, determined by gas chromatography. Retention times (min): C17: 1n-9 (10.12), OA (13.01), 20HOA (16.87) and C17: 0 margaric acid as internal control (10.81).
• the black bar corresponds to the concentration of each fatty acid in the control and the white bar corresponds to the concentration of the fatty acid after treatment with the sodium salt of 20HOA.
• the columns show the mean ⁇ SEM of three independent experiments expressed in nmol and normalized per mg of protein. Statistical significance is determined with a Student's t test (*** p ⁇ 0.001 with respect to the control).
• Figure 13 Analysis of the fatty acid composition in different glioma and non-tumor cell lines after treatment with the sodium salt of 20HOA.
• Representative chromatograms showing the composition of fatty acids (left) and quantification of different fatty acids identified in the chromatograms (C17: 0, OA, 20HOA and C17: 1n-9) (right) in glioma cells: (A) and (B ) U-251 MG; (C) and (D) SF-295; and non-tumor: (E) and (F) MRC-5 (human fibroblasts); (G) and (H) mouse astrocytes, after treatment in the absence (control) or presence of 20HOA sodium salt (400 pM, 24 hours) determined by gas chromatography analysis.
• the black bar corresponds to the concentration of each fatty acid in the control
• the white bar corresponds to the concentration of the fatty acid after treatment with the sodium salt of 20HOA.
• Margaric acid C17: 0 is included as an internal control.
• the columns show the mean ⁇ SEM of three independent experiments expressed in nmol and normalized per mg of protein. Statistical significance is determined with a Student's t test (** p ⁇ 0.01, *** p ⁇ 0.001 with respect to the control).
• FIG. 14 Effect of the sodium salt of 20HOA, OA and sodium salt of C17: 1n-9 on the viability and cell proliferation of glioma cells. Viability curves of different glioma cell lines (A1-A3) U-118 MG; (B1-B3) U-251 MG; and (C1-C3) SF-295 treated with increasing doses of 20HOA sodium salt (0-1000 pM) (A1, B1 and C1); OA (0-300 pM) (A2, B2 and C2); and C17: 1n-9 sodium salt (0-300 pM) (A3, B3 and C3) for 72 hours. Viability was determined by crystal violet staining. Each value represents the mean ⁇ SEM of three independent experiments with at least three biological replicates, expressed as a percentage with respect to the cells treated with vehicle (100%).
• Figure 15 Effect of the sodium salt of 20H0A, sodium salt of C17: 1n-9 and OA on the viability and cell proliferation of non-tumor cells.
• Non-tumor cell viability curves (A1-A3) MRC-5 (human fibroblasts); and (B1-B3) mouse astrocytes treated with increasing doses of 20H0A sodium salt (0-1000 mM) (A1 and B1); OA (0-300 pM) (A2 and B2); and C17: 1n-9 sodium salt (0-300 pM) (A3 and B3) for 72 hours.
• Viability was determined by crystal violet staining. Each value represents the mean ⁇ SEM of three independent experiments with at least three biological replicates, expressed as a percentage with respect to the cells treated with vehicle (100%).
• FIG. 16 Analysis of the effect of different fatty acids on proliferation and death markers in different cell lines. Immunoblots representative of the effect of fatty acids (200 pM OA, 200 pM C17: 1n-9 sodium salt, and 400 pM 20HOA sodium salt) on various proteins involved in 20HOA-regulated cell signaling and death pathways in cells of glioma: (A) U-118 MG; (B) U-251 MG; and (C) SF-295; and non-tumor: (D) MRC-5 (human fibroblasts); and (E) mouse astrocytes, after 72h of treatment.
• fatty acids 200 pM OA, 200 pM C17: 1n-9 sodium salt, and 400 pM 20HOA sodium salt
• Figure 17 Analysis of the fatty acid composition in U-118 MG glioma cells after inhibition of ⁇ -oxidation and effect of oxythiamine on the cell survival of U-118 MG glioma cells.
• A Quantification of 20HOA and C17: 1n-9 fatty acids in U-118 MG cells treated with 400 pM of 20HOA for 24 hours, preincubated with increasing doses (1-10 mM) of oxythiamine (a-oxidation inhibitor ) for 90 minutes, determined by gas chromatography. Results are shown as the mean ⁇ SEM of three independent experiments expressed in nmol and normalized per mg of protein.
• Results are represented as the mean cell count ⁇ SEM from three independent experiments. Statistical significance is determined with a Student's t test (*** p ⁇ 0.001 with respect to the absence of 20HOA and oxythiamine, Control-0; and $$ p ⁇ 0.01, $$$ p ⁇ 0.001 with respect to the treatment with 20HOA without preincubation with oxythiamine).
• Figure 18 Effect of the metabolite C17: 1n-9 on the action of 20HOA.
• Cell viability was determined by counting by trypan blue vital exclusion staining. Results are represented as the mean cell count ⁇ SEM from three independent experiments. Statistical significance is determined with a Student's t test (** p ⁇ 0.01 and *** p ⁇ 0.001 with respect to the absence of 20HOA and oxythiamine; and $ p ⁇ 0.05 with respect to treatment with only 20HOA).
• FIG. 19 Analysis of the effect of the metabolite C17: 1n-9 on the action of 20HOA on proliferation and death markers in different cell lines by inhibiting its formation by oxythiamine.
• Figure 20 Analysis of the fatty acid composition in rat plasma after 24 hours of treatment with sodium salt of 20HOA.
• A Representative chromatograms showing the fatty acid composition in rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) after acute treatment with 20HOA (2mg / Kg, 24 hours) determined by gas chromatography.
• Margaric acid C17: 0 was quantified as an internal control in the chromatogram.
• B Quantification of the fatty acids 20HOA and C17: 1n-9 identified in the chromatograms. The results are shown as the mean ⁇ SEM of 4 animals and expressed in nmol and normalized by ml of plasma.
• Figure 21 Analysis of the fatty acid composition in rat plasma after 15 days of treatment with sodium salt of 20HOA.
• A Representative chromatograms showing the fatty acid composition in rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) after chronic treatment with 20HOA (2mg / Kg, 15 days) determined by gas chromatography.
• Margaric acid C17: 0 was quantified as an internal control.
• B Quantification of the fatty acids 20HOA and C17: 1n-9 identified in the chromatograms. The results are shown as the mean ⁇ SEM of 4 animals, expressed in nmol and normalized per ml of plasma.
• Figure 22 Analysis of the fatty acid composition of xenographic tumors of immunosuppressed mice.
• A Representative chromatograms showing fatty acid composition in xenographic tumors originating from U-118 MG glioblastoma cells in mice treated orally and daily with 20H0A sodium salt (200 mg / kg, 42 days) determined by chromatography. of gases.
• B Quantification of fatty acids OA and C17: 1n-9 identified in the chromatograms.
• Margaric acid C17: 0 was quantified as an internal control. The white bar corresponds to the concentration of each fatty acid in the control, and the black bar corresponds to the concentration of the fatty acid after treatment with the sodium salt of 20H0A.
• results are shown as the mean ⁇ SEM of at least 7 tumors xenographic and expressed in nmoles and normalized per g of tissue. Statistical significance is determined with a Mann-Whitney test (*** p ⁇ 0.01 with respect to the control).
• Figure 24 Analysis of fatty acid composition in human patients with advanced glioma.
• A Representative chromatogram of the fatty acid composition of a glioma patient responding to treatment with sodium salt of 20HOA (12 g / day, 21 days) and determined in plasma samples obtained at different times of treatment (0, 4 and 360 hours, 15 days) by gas chromatography.
• B Quantification of the fatty acids 20HOA and C17: 1n-9 identified in the chromatograms of responding and non-responding patients to treatment with 20HOA in plasma samples obtained at different times of the first day of treatment (0, 1, 2, 4 , 6, 8 hours) and on days 8 (192 hours), 15 (360 hours), 21 (504 hours) and the first day of the second treatment cycle (574 hours).
• Example 1 Fatty acids, reactants and organic solvents 1.1. DHA, DHA-H and HPA
• DHA sodium salt of docosahexaenoic acid; C22: 6 n-3
• DHA-H sodium salt of 2-hydroxy-drocosahexaenoic acid; 20H-C22: 6 n-3
• EPA-H sodium salt of 2- hydroxy-eicosapentaenoic acid
• ARA-H sodium salt of 2-hydroxy-arachidonic acid
• GLA-H sodium salt of 2-hydroxy-gamma (g) -I ⁇ hoI ⁇ h ⁇ oo acid
• ALA-H sodium salt of 2- hydroxy-alpha (a) -linolenic
• LA-H (2-hydroxy-linoleic acid
• HPA sodium salt of (6Z, 9Z, 12Z, 15Z, 18Z) - heneicose-6,9,12,15,18 -pentaenoic acid
• NTA sodium salt of acid (4Z, 7Z, 10Z, 13Z
• Margaric acid (C17: 0) was purchased from Sigma-Aldrich and heneicosapentaenoic acids (HPA free acid; C21: 5 n-3) and (4Z, 7Z, 10Z, 13Z, 16Z) -nonadeca- 4,7,10 , 13,16-pentaenoic (free acid NPA; C19: 5 w-3) were purchased from Cayman Chemicals (Michigan, USA).
• the salt is obtained under an acid-base reaction, a liquid-liquid extraction is carried out with MTBE / HCl and the pH is adjusted with NaOMe to obtain the sodium salt of HPA with good yields.
• a similar procedure can be carried out for the synthesis of HDA, HTA w-3, HTA w-6, NTA and NPA, adjusting the starting substrate.
• the lipid compounds sodium salt of 20H0A, sodium salt of OA and sodium salt of C17: 1n-9 were purchased from Medalchemy, SL (Spain).
• Example 2 Compositions with DHA-H and HPA
• Example soft capsule oral formulation Example 3: Cell tests with DHA-H and HPA
• HEK293T cells were cultured in Dulbecco's Modified Medium (DMEM; Dubelcco's Modified Eagle's Medium, Biowest, France), supplemented with 10% FBS (Fetal Bovine Serum; Gibco, Thermo-Fisher), 2 mM L-GIn, 25 mM D (+) - glucose, 1mM sodium pyruvate and penicillin / streptomycin.
• DMEM Dulbecco's Modified Medium
• FBS Fetal Bovine Serum
• mouse neuroblastoma N2a cells were maintained in a 1: 1 (v: v) mixture of DMEM and Opti-Mem (Gibco, Thermo-Fisher), supplemented with 5% FBS and penicillin / streptomycin. Both cell lines were incubated in an atmosphere of 5% C02 at 37 ° C.
• HEK293T cells were incubated with DHA-H and DHA at 10, 30 and 100 mM for 24 hours, and at 30 pM for 6, 48 and 72 hours. These cells were also incubated with LA-H, ALA-H, GLA-H, ARA-H and EPA-H at 100 pM for 24 hours. HEK293T cells were also incubated with oxythiamine in the presence of DHA-H under the same conditions, at final oxythiamine concentrations of 1 and 10 mM. HEK293T cells were removed from the plates by pipetting with cold phosphate buffered saline (PBS).
• PBS cold phosphate buffered saline
• Cells were recovered by centrifugation (1000 xg, 10 min at 4 ° C) and washed twice with cold PBS before being frozen at -80 ° C.
• 90 mm diameter plates were filled with 11 ml of complete cell culture medium containing 30 pM DHA-H or DHA in the presence or absence of HEK293T cells. adhered (5.10 5 cells / plate). The plates were incubated as described above and 1 ml aliquots were collected from the plates at 0, 6, 24, 48 and 72 hours. Aliquots of cell culture medium were immediately centrifuged at 1000 xg for 10 min at 4 ° C to remove any cell suspension and cell-free aliquots were stored at -20 ° C.
• the U-118 MG, MIA-PaCa 2 and A549 cell lines were obtained from the European Cell Culture Collection (ECACC) through Sigma-Aldrich Co (St Louis, MO) and maintained in RPMI culture medium (Roswell Park Memorial Institute) (U-118 MG and A549) or DMEM (MIA-PaCa 2) supplemented with 10% FBS (Gibco, Thermo-Fisher), in an atmosphere of 5% CO2 at 37 ° C.
• the U-118 MG, MIA-PaCa 2 and A549 cells were treated under the conditions described in the description of the assay carried out to obtain the results of Table 4, optionally in the presence or absence of oxythymine (1 or 10 mM).
• Cell survival was analyzed in a Burker chamber using trypan blue vital exclusion staining (Scharlab) or by means of the cell proliferation kit II (Roche). Briefly, cells were seeded in 96 - well plates at a density of 3 c 10 3 cells per well 24 h before the treatment, and then cultured in the presence or absence of compounds of interest at the concentrations and for the times indicated in figures. After different times, viable cells on the plate were determined by adding XTT according to the manufacturer's instructions. Cells were incubated at 37 ° C in 5% CO2 until a constant color developed and absorbance at 495 nm was recorded using a 650 nm reference wavelength microplate reader (FLUOstar Omega, BMG LABTECH , Germany).
• FLUOstar Omega BMG LABTECH , Germany
• SH-SY5Y human neuroblastoma cells were maintained in DMEM-F12 (Invitrogen) supplemented with 10% FBS (Sigma), penicillin / streptomycin (PAA), non-essential amino acids (Sigma), and 2 mM L-GIn (Sigma ).
• the differentiation of these cells to a neuronal phenotype was carried out following a standard procedure. Briefly, the cells were seeded on plates pretreated with poly-L-Lysine and 24 h later, the medium was replaced by fresh medium supplemented with 10 mM retinoic acid (Sigma).
• the cells were then incubated in the dark for 5 days and the medium was replaced with serum-free medium and supplemented with 50 ng / ml human brain derived neurotrophic factor (hBDNF; Alomone Labs; Tel Aviv, Israel). Finally, the cells were incubated for 6 days to complete the differentiation.
• the neurons were treated for 24 h with the compounds HDA, HTA w-3, HTA w-6, NTA, NPA and HPA, at 1, 3 and 10 pm, for 24 hours, before induction of excitotoxicity with NMDA (n -Methyl-D-Aspartate, 10 mM, Sigma) in a medium containing glycine (530 pM, Sigma) and calcium (10 mM, Sigma).
• DHA-H treatment results in high cellular levels of HPA, compared to levels of the prodrug in cell cultures (Figure 1B).
• Figure 1B intracellular levels of DHA-H and HPA in HEK293T cells under treatment with DHA-H are shown. The accumulation of both compounds is evident as a function of the treatment concentration or the incubation time, but the levels of HPA are significantly higher than those of the prodrug after 24 hours of incubation and 30 pM of treatment.
• the increase in HPA levels is inhibited in the presence of concomitant treatment of oxythiamine (partial inhibition with 1 mM and almost total with 10 mM), a competitive antagonist of 2-hydroxyacyl-CoA Nase (see figure 1A). In this sense, it has also been possible to verify that endogenous levels of DHA (the non-hydroxylated native form) are not altered by this treatment with DHA-H ( Figure 1C).
• Figure 8 shows that this same metabolic pathway is valid for other 2-hydroxylated polyunsaturated fatty acids, used as prodrugs, such as LA-H, ALA-H, GLA-H, ARA-H and EPA-H , giving rise to HDA, HTA w-3, HTA w-6, NTA, NPA, respectively (chromatograms shown in figure 9). All of these metabolites have demonstrated therapeutic activity, as shown in Figure 10 and Table 4 below:
• the antitumor activity of the different metabolites described in Figures 8 and 9 was determined by direct treatment with these molecules (HDA or C17: 2 w-6, HTA w-3 or C17: 3 w-3, HTA oo-6 or C17 : 3 w-6, NTA or C19: 4 w-6, NPA or C19: 5 oo-3 and HPA or C21: 5 w-3) in tumor cell cultures, on which the IC50 value was determined for each one of these compounds (Inhibitory Concentration 50: concentration of compound under study that induces the death of 50% of the tumor cell population).
• the cell cultures used correspond to different types of cancer: U118-MG (grade IV human glioblastoma), MIA-PaCa 2 (pancreatic carcinoma) and A549 (small cell lung adenocarcinoma).
• U118-MG grade IV human glioblastoma
• MIA-PaCa 2 pancreatic carcinoma
• A549 small cell lung adenocarcinoma
• the different compounds showed variable IC50 values on the different tumor lines, which demonstrate the selectivity of some of them to induce the selective death of certain types of tumor cells.
• Example 4 In vivo tests with DHA-H and HPA.
• the 5xFAD model of Alzheimer's disease is a double transgenic PS1 / APP mouse harboring five human mutations associated with familial AD (Tg6799 line): Swedish (K670N / M671L), Florida 151 (1716V) and London (V717I) in APP; and the M146L and L286V clinical mutations in human PS1. Both transgenes are expressed under the control of the Thy-1 promoter and mice show cognitive decline from 4 months of age (Oackley et al., 2006, Neurosci 26 (40), 10129-10140. Doi: 10.1523 / jneurosci .1202-06.2006).
• the 5xFAD and wild type (WT) transgenic animals were obtained from Jackson Laboratories (USA) and maintained in a B6 / SJL genetic background by crossing heterozygous transgenic mice with B6 / SJL WT (F1) broodstock.
• the animals were housed at a controlled temperature of 22 ° C ( ⁇ 2 ° C) and a humidity of 70%, in a 12h-12h light-dark cycle, with free access to a standard laboratory diet (Panlab A03, Barcelona, Spain).
• WT and 5xFAD transgenic male mice received DHA-H (or DHA) orally, dissolved in 5% ethanol, at a daily dose of 5, 20, 50 and 200 mg / kg, or just the vehicle.
• these animals have also been treated with HPA (20 mg / kg) and DHA-H (20 g / kg) to compare the effect of both compounds in this model.
• HPA 20 mg / kg
• DHA-H 20 g / kg
• mice were maintained on a normal diet (and treatment) for a further week, after which they were anesthetized with an intraperitoneal injection of ketamine / xylazine (100 / 10mg / kg) and infused intracardiacly. with 50 ml of heparinized saline solution. The brains of the animals were immediately removed and dissected midline on a cold surface.
• NUDE (Swiss) Crl NU (lco) -Foxn1 nu mice (8-12 weeks old, 30-35 g, Charles River Laboratories, Paris, France) were kept in a thermostatic cabinet (28 ° C, EHRET, Labor -U-Pharmatechnik) with sterile air flow at 40-60% relative humidity and 12-hour dark / light cycles.
• Their diet consisted of a standard diet with chow (Labdiet 22% rat-mouse offspring, Sodispan) ad libitum.
• the mice were sacrificed by cervical dislocation and the xenographic tumors were dissected and frozen in liquid nitrogen and at -80 ° C. All the protocols used were approved by the Bioethics Committee of the University of the Balearic Islands, and comply with the guidelines national and international on animal welfare.
• the spatial behavior test was performed as previously described, with some modifications (Fiol-Deroque et al., 2013, Biogerontology 14 (6), 763-775. Doi: 10.1007 / s10522-013-9461 -4). All animals were isolated and subjected to caloric restriction until reaching 80-85% of normal body weight, and they were kept under these conditions one week before starting the test and until the end of it. After food restriction and 3 days before the start of the tests, the animals were trained twice a day in the eight-arm radial maze test (LE766 / 8, Panlab SL, Spain) for 3 days.
• Each mouse was placed in the center of the maze and allowed to search for the reward, a 45 mg food pellet (Dustless Precision Pellets, Bio-Serv, USA), located at the end of each arm.
• a 45 mg food pellet Dustless Precision Pellets, Bio-Serv, USA
• Each session ended when the animal managed to find all eight primed arms or failed to complete all arms in 10 minutes.
• the movement of each animal was recorded with a digital video tracking system (LE 8300 with Sedacomv1.3 software, Panlab, SL, Spain) and after training, the experimental paradigm began. In all experimental sessions (1 session per day), only four arms were primed compared to the training protocol, and each session ended when the animals managed to find all four primed arms or failed after 10 minutes.
• Performance was evaluated taking into account: (1) the time to perform the test; (2) the number of working memory errors (WME, re-entry into a previously visited primed arm); (3) the number of reference memory errors (RME, input on an unprimed arm); and (4) the total number of errors (WME + RME).
• WME working memory errors
• RME reference memory errors
• WME + RME total number of errors
• Example 5 Extraction of lipids and transmethylation of fatty acids relative to examples 3 and 4
• the HEK293T or U-118 MG cells used in the previous examples were used with a cold hypotonic buffer (1 mM EDTA, 20 mM Tris-HCl [pH 7.4]) by pipetting up and down.
• Cell lysates were subjected to ultrasound pulses (4 cycles, 10 s / cycle, 10W) before lipid extraction.
• tissue from each animal was homogenized in cold PBS at 1:10 (p: v) in the presence of protease inhibitors (Roche), using a blade homogenizer (Polytron PT3100). Homogenates were sonicated, aliquoted, and stored at -80 ° C. Only one aliquot of each sample, containing about 8 mg protein / aliquot, was subjected to lipid extraction. The protein content before lipid extraction was determined by a modified Lowry method (Bio-rad DC Protein Assay).
• Margaric acid (C17: 0) was added to lipid-extracted samples as an internal standard and lipids were extracted with chloroform: methanol (Eggers and Schwudke, 2016). Briefly, 0.75 volumes of the aqueous phase (which already contained the biological sample) were mixed with 2 volumes of chloroform and 1 volume of methanol. This mixture was vortexed for 1 minute and centrifuged at 1000 xg for 10 minutes. The lower organic phase was collected and washed with 1 ml of PBS: methanol (1: 1, v: v), and the resulting organic phase was dried under argon flow.
• the film containing the extracted lipids was transmethylated by incubating the lipid mixture for 90 minutes at 100 ° C in 3 ml of methanol: acetyl chloride (10: 1, v: v), under an argon atmosphere (Christie, 1993).
• the resulting fatty acid methyl esters (FAMEs) were extracted with hexane, adding 3 ml of H2O and 1 ml of hexane to the transmethylation reaction, and thoroughly vortexing the mixture. After centrifugation at room temperature (1000 xg for 10 min), the upper phase containing the FAMEs was collected and the remaining volume was washed twice with 1 ml of hexane.
• hexane phases were combined, evaporated under argon flow and resuspended in 60 ml of hexane (for the analysis of cell samples, cell culture medium and blood plasma) or in 200 ml (for the analysis of brain samples).
• the isolated FAME were subjected to a second derivatization with trimethylsilyl (Alderson et al., 2004, J Biol Chem 279 (47), 48562-48568.
• the anti-proliferative effect of HPA on a culture of U-118 MG cells was also studied, in comparison with the administration of the prodrug DHA-H and the native form of DHA.
• the anti-proliferative effect on U-118 MG is much superior for HPA with respect to DHA-H and DHA (see figure 5A).
• this effect can be explained by differences in intracellular levels of HPA, induced by DHA-H and HPA (see Figure 5B).
• Figure 5C shows that the uptake of the hydroxylated form of DHA is hindered compared to that of the non-hydroxy analog.
• Example 6 In vitro tests with 20H0A and C17: 1n-9.
• C17: 1n-9 sodium salt solutions were used at a concentration of 200 pM for 24 or 72 hours.
• a stock aliquot was started at 100mM.
• the corresponding milligrams of the lipid compound (powder) were dissolved in absolute ethanol and autoclaved distilled water (1: 1 volume, normally an aliquot of 1 ml is prepared, so 500 ml of ethanol and 500 ml of water are added.
• the solution is introduced 10 min in the culture oven at 37 ° C so that the lipid compound dissolves and is subsequently subjected to stirring.
• the fatty acid composition of lipid membranes was analyzed in other glioma cell lines (U-251 MG and SF-295) in comparison with non-tumor cells, human fibroblasts (MRC-5) and primary cultures of mouse astrocytes, after its incubation in the absence or presence of sodium salt of sodium salt of 20HOA (400 mM, 24 hours) by gas chromatography, No significant change in the amount of OA was observed after treatment with sodium salt of 20HOA in any of the lines cell phones analyzed ( Figure 13).
• the glioma cells (U-251 MG and SF-295) showed a significant increase in their levels of C17: 1n-9; accumulating 97.42% and 108.03% more than 20H0A (19.16 ⁇ 0.53 versus 9.21 ⁇ 0.41 and 18.38 ⁇ 1.97 versus 9.31 ⁇ 1.44 nmol / mg nmol / mg, respectively) (Figure 13B and 13D, Table 5).
• the detected levels of 20HOA were significantly higher than those of its metabolite C17: 1n-9.
• Table 5 Levels of 20HOA and C17: 1n-9 fatty acids in different glioma and non-tumor cell lines after treatment with 20HOA. Values of the quantification of 20HOA and C17: 1n-9 fatty acids in different glioma lines, U-118 MG, U-251 MG and SF-295 (top) and non-tumor, MRC-5 and astrocytes, (bottom) after treatment with 20HOA (400 mM for 24 hours) determined by gas chromatography. The results correspond to the mean ⁇ SEM of three independent experiments expressed in nmol and normalized per mg of protein.
• IC50 corresponds to the amount of a compound necessary to reduce cell viability in vitro by 50%, as well as its effect on the regulation of proteins involved in the mechanism of action of 20HOA.
• glioma cell lines U-118 MG, U-251 MG and SF-295
• non-tumor cells MRC-5 and astrocytes
• the IC50 was determined using the crystal violet staining technique.
• the IC5 0 values of the sodium salt of 20H0A were 432.75 ⁇ 10.77, 429.96 ⁇ 9.67 and 399.14 ⁇ 11.47 mM in the glioma cells U-118 MG, U-251 MG and SF-295, respectively (Table 6).
• the IC5 0 of 20H0A were in both cases 1000 pM.
• IC5 0 values were 222.04 ⁇ 9.09, 220.35 ⁇ 7.93 and 248.85 ⁇ 6.02 pM in U-118 MG, U-251 MG and SF-295 glioma cells, respectively.
• C17: 1n-9 induced an antiproliferative effect, in a very similar way, both in glioma and non-tumor cells.
• the 20HOA treatment only affected the viability of the different glioma cell lines, without affecting the viability of the non-tumor cells.
• the IC5 0 values of 20HOA were 1.90, 1.95 and 1.60 times higher than those of its metabolite C17: 1n-9 in U-118 MG, U-251 MG and SF-295 glioma cells, respectively (Table 6).
• the IC5 0 values of 20HOA were 1.92, 1.80 and 1.56 times higher than those of its non-hydroxy analog OA.
• the fact that C17: 1n-9 has shown greater antiproliferative potency may be due to the fact that it has a greater capacity for accumulation in cells than 20HOA.
• IC50 values of different cell lines after treatment with 20HOA, OA and C17 1n-9. Summary of the IC5 0 of the glioma cell lines (U-118 MG, U-251 MG and SF-295) and non-tumor cells (MRC-5 and astrocytes), calculated from the results obtained in Figures 16 and 17. The IC5 0 values obtained correspond to the mean of three independent experiments and calculated by means of a dose-response equation using the statistical program GraphPad Prism 6.0 (sigmoid model).
• the effect of the metabolite C17: 1n-9 on different signaling pathways that are altered by the effect of 20HOA was analyzed.
• the different glioma cell lines U-118 MG, U-251 MG and SF-295) and non-tumor cells (MRC-5 and mouse astrocytes) were treated with doses close to the IC5 0 of each of the compounds. (200pM of C17: 1 n.9, 200pM of OA or 400pM of 20HOA) for 72 hours and its effect on different proteins of signaling by Western Blot.
• oxythiamine chloride was used, which inhibits the enzyme 2-hydroxyphytanoyl-CoA Nase (HACL1, key enzyme in a-oxidation) , Among other functions.
• HACL1 2-hydroxyphytanoyl-CoA Nase
• the U-118 MG glioma cells were first pre-incubated with 1 or 10 mM oxythiamine for 90 minutes, then they were treated with 400 mM of the sodium salt of 20HOA for 24 hours and the fatty acids were analyzed by gas chromatography. .
• the effect of preincubation with oxythiamine on cell survival and proteins regulated by 20HOA was studied.
• the cell survival of different glioma and non-tumor cell lines treated with 20HOA (400 mM, 72 hours) and pre-incubated or not with 2 mM oxythiamine (90 minutes) was analyzed by counting the cells with the staining of vital exclusion with trypan blue.
• the proteins modulated by 20HOA were studied by Western-blot. In glioma cells, a significant decrease in cell survival was observed after incubation with 2 mM oxythiamine for 72 hours.
• Oxythiamine induced 18.51 ⁇ 0.58% and 17.35 ⁇ 0.63% of cell death in U-251 MG and SF-295 cells, respectively ( Figure 18A and 18B). These results support the in vitro antitumor effect of oxythiamine on glioma cells. Treatment of cells with 20HOA induced 23.22 ⁇ 1.32% and 23.97 ⁇ 1.25% cell death in U-251 MG and SF-295, respectively. After combination with 2 mM oxythiamine, there was a significant recovery in cell viability of 12% (14.07 ⁇ 1.62% of death in U-251 MG cells) and of 17.25% (10.85 ⁇ 0.58% of death) in cells. SF-295. On the contrary, in In non-tumor cells, none of the treatments tested produced an effect on cell survival (Figure 18C and 18D).
• 20H0A has an antiproliferative activity when its metabolism in C17: 1n-9 is inhibited, the formation of the metabolite C17: 1n-9 has a great impact on the mechanism of action of 20H0A, enhancing its antiproliferative effect, and confirms that the 20H0A is also a prodrug that gives rise to an active metabolite 17: 1 n-9.
• Example 7 In vivo tests with 20H0A and C17: 1n-9.
• mice The pharmacokinetic profile of 20H0A and its metabolite C17: 1n-9 was studied in animal plasma.
• rats were used as an experimental animal model. Rats have a higher blood volume than mice, being the most suitable model to study the effect of the continuous administration of the maximum tolerated dose of 20H0A (2 g / Kg) defined in preclinical studies.
• mice were euthanized and the tumors were extracted, the lipids were processed to detect the fatty acids 20HOA and C17: 1n-9 by gas chromatography.
• the fatty acid 20HOA was not detected in the xenographic tumors of mice treated with this compound, since no peak was observed in the retention time corresponding to 20HOA ( Figure 22A).
• the 20HOA metabolite the fatty acid C17: 1n-9 (0.25 ⁇ 0.04 nmol C17: 1n-9 / g tissue), was detected in the tumors of mice treated with 20HOA ( Figure 22B).
• the detection and quantification of the fatty acids 20H0A and C17: 1n-9 was carried out in plasma samples from 8 patients who responded, or not, to treatment with 12 g / day of sodium salt of 20H0A for, at least, 1 cycle of 3 weeks in clinical phase I / IIA of 20H0A (MIN-001-1203). Plasma samples were obtained at different times (0, 2, 4, 6, 8 hours and after 8, 15, 21 and 28 days after treatment with 20H0A) and, later, they were transferred for fatty acid analysis using the technique gas chromatography.

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