Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
  • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
  • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
  • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
  • C07D333/14—Radicals substituted by singly bound hetero atoms other than halogen
  • C07D333/16—Radicals substituted by singly bound hetero atoms other than halogen by oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/37—Esters of carboxylic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
  • A61K8/4926—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having six membered rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/4986—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with sulfur as the only hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C67/00—Preparation of carboxylic acid esters
  • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
  • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
  • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/14—The ring being saturated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

• the invention relates to the method of preparation of 2-oxoglutarate ester, a preparation containing 2-oxoglutarate ester and a use thereof for supporting the biosynthesis of collagen.
• Prolin is essential for the biosynthesis of collagen. Thereafter, post-translationally by means of prolyl-4- hydrox lase, prolin gives rise to hydroxyprolin which is necessary for the formation of collagen triple helix and for the mechanical properties thereof. Incomplete hydroxylation of prolin residues X-Pro-Gly in the repeating amino acid motives leads to errors in the synthesis of the collagen triple helix (Myllyharju, 2003). 2-oxoglutarate, the intermediate of the rebs cycle, which is the substrate for prolyl-4-hydroxylase, is essential for the hydroxylation of prolin, together with Fe 2+ ions and vitamin C. Therefore, the presence of said substances is crucial for collagen biosynthesis.
• the deficiency of said solution is the use of the non-modified molecule which, due to its hydrophilic character, has difficulties with getting over biological barriers and must be combined with substances (e.g. propylenglycol and ethanol), which facilitate the penetration of the non-modified molecule and thus enhance the biological availability thereof.
• substances e.g. propylenglycol and ethanol
• these substances function in a way where they destroy the natural protective function of biological barriers, which is undesirable.
• the cells react to the external addition of l on- modified 2-oxoglutarate just by a slight and statistically insignificant increase of the production of collagen.
• the esterified 2-oxoglutarate is a substance which is known and the effect thereof on pseudohypoxia control in cells has been disclosed (MacKenzie et al; 2007).
• the use of 2- oxoglutarate monoesters for the prevention of pseudohypoxia of cells is disclosed in US2009005437 (WO2006/016143). It is known that 2-oxocarbox lic acids are generally instable, therefore ali up-to-date known processes of preparation of 2-oxoglutaric acid esters disclosed in the literature have been performed without any significant heating, in some cases even with cooling (WO2006/016143).
• this invention makes use of ester ification of at least one carboxyl of 2-oxoglutarate by means of alcohol.
• the ester is, as the transport form, stable enough in physiological conditions for an easy penetration of the molecule through the physiological barriers.
• the present enzymes After entering the intracellular space it is quickly hydrolysed by the present enzymes to give the biologically active 2- oxoglutarate and biocompatible alcohols.
• esters of 2-oxoglutarate have a significant procollagen effect in cells exposed to oxidation stress due to e.g. age, inflammation, UV exposition, harmful chemical substances etc. Such effect is comparable to the effects of growth hormones.
• R 1 and R 2 are identical or different and are independently selected from the group comprising hydrogen, C]-C 3 o alkyl, C2-C30 alkenyl, C 4 -C 3 o cycloalkyl, C4-C30 cycloalkenyl, O -C30 alkylaryl or Ci-C 30 alkylheteroary!, In which R ! and R 2 optionally contain one or more identical or different heteroatoms selected from the group comprising N, O, S; and wherein R 1 and R 2 are not hydrogens at the same time; or pharmaceutically acceptable salts thereof;
• 2-oxoglutarate ester defined above is used for the production of a medical device, medicinal preparation, veterinary preparation, veterinary technical device, veterinary medicinal preparation or preparation serving as a component of media for cultivation and storage of cells, for tissue engineering or for biotechnological applications for the stimulation of collagen biosynthesis.
• the preparations mentioned above may be used for stimulation of collagen biosynthesis in vivo in connective tissues, as well as . in biotechnological applications and in cell and tissue engineering.
• the amount of the 2-oxoglutarate ester defined above in the preparation according to the invention is within the range of 0.01 %wt. to 10 %wt. with respect to the total weight of the preparation.
• the preparation according to the invention contains physiologically acceptable additives selected from the group including acrylates/C10-C30 alkylacrylate crossl inked polymer, panthenol, glycerin, methyl paraben, fatty acid esters of olive oil and sorbitan/cetearyl alcohol, sorbitan monolaurate, olive oil, glycerylmonostearate, squalene, butylparaben, macadam oil, hyaluronic acid, its derivatives and salts, alantoin, sodium polyacrylate, cetearylalcohol, coconut butter, isododecane, hydroxypropyl methylcellulose, sodium salt of carboxy methyl cellulose, xylitol, manitol, erythritol, vaseline
• the preparation according to the invention is in the form of a solution, emulsion, suspension, gel, ointment, cream or dressing material.
• the preparation according to the invention is preferably used for stimulation of collagen biosynthesis in cells exposed to the oxidation stress, containing collagen.
• the oxidation stress is a non-equilibrium between the formation of the reactive oxygen (in other words also free radicals), which is formed as a by-product of oxygenation and metabolism, and the ability of the organism of fast degradation and detoxication of reactive intermediate products. It is involved in many diseases, such as atherosclerosis, Parkinson disease, heart failure or heart attack. Long lasting oxidation stress is, inter alia, the cause of skin ageing, the skin ceils being characterized by an increased error rate in post-translation modification of collagen. However, such error rate may also occur in any cells containing collagen e.g. by means of UV exposition, inflammation and other harmful substances, as mentioned above.
• the preparation according to the invention is selected from the group including medical device, medicinal preparation, veterinary preparation, veterinary technical device, veterinary medicinal preparation or preparation serving as a component of media for cultivation and storage of cells, for tissue engineering and for biotechno logical applications.
• 2-oxoglutarate ester defined above is preferably prepared by a method, the subject- matter of which being 2-oxoglutaric acid reacting with an alcohol according to the general formula R3-OH, wherein R 3 is independently selected from the group including C1 -C30 alkyl, C2-C30 alkenyl, C 4 -C 30 cycloalkyl, C 4 -C 30 cycloalkenyl, C i-C 30 alkylaryl or Ci -C 30 alkylheteroaryl optionally containing one or more identical or different heteroatoms selected from the group including N, O, S; above the melting point of 2-oxoglutaric acid, preferably at temperatures 113.6 °C to 125 °C, more preferably at 120 °C.
• the ester ificat ion reaction is carried out for 0.5 to 2 hours, preferably for 1 hour.
• the preferred amount of the alcohol in the reaction mixture is within the range of 2 to 3 molar equivalents with respect to the molar amount of 2-oxoglutaric acid, more preferably 2.4 molar equivalents with respect to the molar amount of 2-oxoglutaric acid.
• the method of preparation of 2-oxoglutarate ester according to the invention is, as opposed to the processes known up to now, totally different. Since there is no need of neither solvents, catalysts, nor activating agents, the resulting mixture does not have to be isolated or purified, which facilitates the preparation itself very much and reduces the cost of the prepared material.
• the active substance which is used for the production of the preparation according to the invention is the crude reaction mixture. The effectivity of the support of the collagen production was observed both in cells of juvenile donors, and in cells of senescent donors. The cells of senescent donors (age above 50 years) served as a model of cells exposed to the oxidation stress in which an increased error rate of post-translation modification of collagen is generally known.
• Fig. 1 represents the viability of the cells of 3T3 line in the presence of 5 various batches of hexyl-2-oxoglutarate (batch OG50 to OG54 prepared according to Example 1) having the concentration of 50 - 500 ⁇ g / ml.
• a test with neutral red was used for the measurement of cell viability.
• Fig. 2 represents the cell viability of 3T3 line in the presence of 2-phenylethyl-2-oxoglutarate (batch OG47 prepared according to Example 2) having the concentration 50 - 500 g I ml.
• the control was the solvent alone (d i m ethyls u fox ide; DMSO).
• DMSO dimethyl sulfoxide
• Fig. 3 represents the influence of hexyl ester of 2-oxoglutarate prepared according to Example I on in vitro production of collagen by human dermal fibroblasts (NHDF) from child donors and donors older than 50years.
• NHDF human dermal fibroblasts
• Fig. 4 represents the influence of 2-phenylethyl ester of 2-oxoglutarate prepared according to Example 2 on in vitro collagen production by human dermal fibroblasts (NHDF).
• Fig. 5 represents the influence of 2-phenylethyl ester of 2-oxoglutarate prepared according to Example 2 on in vitro collagen production by human gingival fibroblasts (HGF).
• esterification degree nE /nf , defines how many ester groups fall on one molecule of the initial 2-oxogIutaric acid in average.
• nE molar amount of all ester hydrogens (alkyl-CH 2 -0-CO-), expressed by integral of the signals in ⁇ NMR spectrum within the interval 4.0 to 4.5 ppm
• nK molar amount of all aipha-keto hydrogens (-O-CO-CO-CH2-) expressed by integral of the signals in ⁇ NMR spectrum within the interval 3.0 to 3.3 ppm
• the cosmetic preparation was prepared by mixing the individual components identified below together.
• the cosmetic preparation was prepared by mixing the individual components identified below together.
• composition of the cosmetic preparation in the form of a gel
• D-panthenol 0.3 % 1 1) glycerin, copolymer of glyceryl aery late/acrylic acid,
• the cosmetic preparation was prepared by mixing the individual components identified below together.
• dimethicone (silicone oil) 0.5 %
• Fig. 1 shows the cell viability (fibroblasts of 3T3 line) in the presence of hexylester of 2-oxoglutaraie (50 - 500 g / ml) prepared according to Example I .
• Fig. 2 shows cell viability (fibroblasts of 3T3 line) in the presence of 2-phenylethyl ester of 2-oxoglutarate (50 - 500 ⁇ g/ml) prepared according to Example 2. The viability was measured by the standard neutral red method. The results document that the tested esters are not cytotoxic.
• Fig. 3 shows the test results of the effect of 2-oxoglutarate hexylester (1000 ⁇ g / ml) prepared according to Example 1 on in vitro production of the total collagen by human dermal fibroblasts (NHDF) obtained from human donors (age 50 - 65 years - senescent cells) and also (age 8-1 1 years - juvenile cells).
• Fig. 4 shows the test results of the effect of 2- phenylethyl ester of 2-oxoglutarate (10 - 1000 ⁇ g / ml) prepared according to Example 2 on in vitro production of the total collagen by human dermal fibroblasts (NHDF) obtained from human donors (age 50 - 65 years).
• Fig. 3 shows the test results of the effect of 2-oxoglutarate hexylester (1000 ⁇ g / ml) prepared according to Example 1 on in vitro production of the total collagen by human dermal fibroblasts (NHDF) obtained from human donors (age 50 - 65 years).
• Example 5 shows the test results of the effect of 2-phenylethyl ester of 2-oxoglutarate (10 - 1000 ⁇ g / ml) prepared according to Example 2 on in vitro production of the total collagen by human gingival fibroblasts (HGF) obtained from human donors (females, 50 - 60 years).
• HGF gingival fibroblasts
• the cells were seeded in 96-wells culture panels in seeding density of 10 000 cells/well and they were cultivated for at least 12 hours in the medium (DMEM) with 10% of fetal bovine serum. Thereafter, the medium was changed for DMEM containing proline (400 mg/1) and vitamin C (trisodium 2-phospho-L-ascorbate; 480 mg/1). After further 20-24 hours, the medium was changed for DMEM with the tested substances (exposition of 24 hours). The total production of collagen was tested in the medium by the method of specific coloration of collagen by the saturn red solution F3B200 in picric acid (Walsh et al. ⁇ 1992; Heng et al. 2006).
• Vandenheuvel FA Structure of membranes and role of lipids therein. Adv Lipid Res. 1971 ;9: 161-248.

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