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Title:
METHOD OF PREPARATION OF 2-OXOGLUTARATE ESTER, PREPARATION CONTAINING 2-OXOGLUTARATE ESTER AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2014/180455
Kind Code:
A1
Abstract:
The invention relates to a method of preparation of 2-oxoglutarate ester, a preparation containing 2-oxoglutarate ester and use thereof for supporting collagen biosynthesis.

Inventors:
KLEIN, Pavel (Dolni Dobrouc 444, Dolni Dobrouc, 56102, CZ)
PRAVDA, Martin (Podhomoli 1543, Chocen, 56501, CZ)
BUFFA, Radovan (Kukorelliho 1495/2, Humenne, 06601, SK)
MATEJKOVA, Ilona (Dukelska 307, Usti nad Orlici, 56201, CZ)
VELEBNY, Vladimir (Sadova 1466, Zamberk, 56401, CZ)
Application Number:
CZ2014/000050
Publication Date:
November 13, 2014
Filing Date:
May 05, 2014
Export Citation:
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Assignee:
CONTIPRO BIOTECH S.R.O. (Dolní Dobrouč 401, Dolní Dobrouč, 561 02, CZ)
International Classes:
C07C67/08; A61K8/37; A61K8/49; A61Q19/00; C07C69/716; C07D295/088; C07D333/16
Domestic Patent References:
WO2006016143A12006-02-16
WO2006016143A12006-02-16
Foreign References:
EP0303523A11989-02-15
EP1541174A12005-06-15
KR101219294B12013-01-08
US20090005437A12009-01-01
KR20080011543A2008-02-05
Other References:
J CELL BIOCHEM., vol. 98, no. 2, 2006, pages 409 - 20
VANDENHEUVEL FA: "Structure of membranes and role of lipids therein", ADV LIPID RES., vol. 9, 1971, pages 161 - 248
MYLLYHAR U J: "Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis", MATRIX BIOL., vol. 22, no. 1, 2003, pages 15 - 24
SON ED; CHOI GH; KIM H; LEE B; CHANG IS; HWANG JS: "Alpha-ketoglutarate stimulates procollagen production in cultured human dermal fibroblasts, and decreases UVB-induced wrinkle formation following topical application on the dorsal skin of hairless mice", BIOL PHARM BULL., vol. 30, no. 8, 2007, pages 1395 - 9
MACKENZIE ED; SELAK MA; TENNANT DA; PAYNE LJ; CROSBY S; FREDERIKSEN CM; WATSON DG: "Gottlieb E: Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells", MOL CELL BIOL., vol. 27, no. 9, 2007, pages 3282 - 9
BENSON, H. A. E.: "Current Drug Delivery", vol. 2, 2005, BENTHAM SCIENCE PUBLISHERS LTD., article "Transdermal Drug Delivery: Penetration Enhancement Techniques", pages: 23 - 33
ROSENBLAT G; PERELMAN N; KATZIR E; GAL-OR S; JONAS A; NIMNI ME; SORGENTE N: "Neeman I: Acylated ascorbate stimulates collagen synthesis in cultured human foreskin fibroblasts at lower doses than does ascorbic acid", CONNECT TISSUE RES., vol. 37, no. 3-4, 1998, pages 303 - 11
WALSH BJ; THORNTON SC; PENNY R: "Breit SN: Microplate reader-based quantitation of collagens", ANAL BIOCHEM., vol. 203, no. 2, September 1992 (1992-09-01), pages 187 - 90
GOTTLIEB E; SELAK MA; MACKENZIE ED; WATSON DG, ALPHA-KETOGLUTARATES AND THEIR USE AS THERAPEUTIC AGENTS
SON ED; LEE SH; KIM HK; PARK CM; HWANG JS; CHANG IS, ANTI-WRINKLE COSMETIC COMPOSITION CONTAINING ALPHA-KETO LUTARATE
Attorney, Agent or Firm:
DVORAKOVA, Martina (Kania, Sedlak Smol, Mendlovo namesti 1a Brno, 603 00, CZ)
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Claims:
CLAIMS

1. A method of preparation of 2-oxoglutarate ester according to the general formula I

wherein

R1 and R2 are identical or different and are independently selected from the group comprising hydrogen, C1-C30 alkyl, C2-C30 alkenyl, C4-Cjo cycloalkyl, C4-C30 cycloalkenyl, C1-C30 alkylaryl or C1-C30 alkylheteroaryl in which R1 and R2 optionally contain one or more identical or different heteroatoms selected from the group comprising N, O, S; and wherein R1 and R2 are not hydrogens at the same time; or pharmaceutically acceptable salts thereof,

characterised by that 2-oxoglutaric acid reacts with alcohol according to the general formula R3OH, wherein R3 is independently selected from the group comprising C1-C30 alkyl, C2-C30 alkenyl, C4-C3o cycloalkyl, C4-C3o cycloalkenyl, C1-C30 alkylaryl or C1 -C30 alkylheteroaryl optionally containing one or more identical or different heteroatom selected from the group comprising N, O, S; above the melting point of 2-oxoglutaric acid.

The method of preparation of 2-oxoglutarate ester according to the general formula Γ according to claim 1, characterised by that RJ is selected from the group comprising hexyl, octyl, phenylethyi.

The method according to any of claims 1 and 2, characterised by that 2-oxoglutaric acid reacts with the alcohol at the temperature of 1 13.6 °C to 125 °C.

4. The method according to claim 3, characterised by that the temperature is 120 °C.

5. The method according to any of claims I to 4, characterised by that 2-oxoglutaric acid reacts with the alcohol for 0.5 to 2 hours.

6. The method according to claim 5, characterised by that 2-oxoglutaric acid reacts with the alcohol for 1 hour.

7. The method according to any of claims 1 to 6, characterised by that the amount of 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.

8. The method according to claim 7, characterised by that the amount of alcohol in the reaction mixture is 2.4 molar equivalents with respect to the molar amount of 2- oxoglutaric acid.

9. 2-oxoglutarate ester prepared by the method of any claim 1 to 8 for use in cosmetics, biotechnology applications or in tissue engineering for stimulation of collagen biosynthesis.

10. 2-oxoglutarate ester according to claim 9 for use for stimulation of collagen biosynthesis in cells exposed to oxidation stress.

1 1. Use of 2-oxoglutarate ester prepared by the method of any claim 1 to 8 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 biotechno logical applications for stimulation of collagen biosynthesis.

12. The use of 2-oxoglutarate ester according to claim 1 1 for stimulation of collagen biosynthesis in cells exposed to oxidation stress.

1 3. 2-oxoglutarate ester according to the general formula I

wherein

R1 and R2 are identical or different and are independently selected from the group comprising hydrogen, C1 -C30 alkyl, C2.-C30 alkenyl, C4-C30 cycloalkyl, C4-C30 cycloalkenyl, O-C30 alkylaryl or C1-C30 alkylheteroaryl in which R1 and R2 optionally contain one or more identical or different heteroatoms selected from the group comprising N, O, S; and wherein RJ and R" are not hydrogens at the same time; or pharmaceutically acceptable salts thereof, for use in cosmetics, biotechnological applications or tissue engineering for stimulation of collagen synthesis.

14. 2-oxoglutarate ester according to claim 13 for use in stimulation of collagen biosynthesis in cells esposed to oxidation stress.

15. A preparation for stimulation of collagen biosynthesis characterized in that it contains 2- oxoglutarate ester according to claim 13.

16. The preparation according to claim 15 characterized in that the amount of 2- oxoglutarate ester is within the range of 0.01 %wt. to 10 %wt. with respect to the weight of the preparation.

17. The preparation according to any of claims 15 or 16, characterized in that it further contains physiologically acceptable substances.

18. The preparation according to claim 17, characterized in that the physiologically acceptable substance is selected from the group comprising acrylates/C !0-C30 alkylacrylate crosslinked polymer, panthenol, glycerin, methylparaben, esters of olive oil fatty acids and sorbitan/cetearyl alcohol, sorbitan monolaurate, olive oil, glyceryl monostearate, squaiene, butylparaben, macadam oil, hyaluronic acid, derivatives and salts thereof, alantoin, sodium polyacrylate, cetearyl alcohol, coconut butter, isododecan, hydroxypropyl methylceUulose, sodium salt of carbo ymethyl cellulose, xylitol, manitol, erythriioi, vaseline, liquid paraffin, propylenglycoi, stearomacrogol 100, stearo macro go [ 1050, cetylalcohol, light liquid pai'affin, propylparaben, cocoa oil, medium saturated triacylglycerols, benzylacohol, sorbitans and polysorbates, alligator pear butter, ceteareth- 20, cetyl alcohol, shea butter, sorbitan stearate, span 20, 60, 80 stearic acid, stearyl palmitate, xanthan gum, 1 ,3-butanediol, almond oil, aloe vera, dimethicon, izopropylmyristate, polyethylengiycols, grape seed oil or isohexadecane.

19. The preparation according to any of claims 15 to 18, characterized in that it is in the form of a solution, emulsion, suspension, gel, ointment, cream or dressing material.

20. The preparation according to any of claims 15 to 19 for use in stimulation of collagen biosynthesis in cells exposed to oxidation stress.

21. The preparation according to any of claims 15 to 20, characterized in that it is selected from the group comprising 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 and for biotechnological applications.

22. A use of 2-oxoglutarate ester according to claim 13 or 14 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 lor biotechnological applications for the stimulation of collagen biosynthesis.

23. The use of 2-oxoglutarate ester according to claim 22 for stimulation of collagen biosynthesis in cells exposed to oxidation stress.

Description:
Method of preparation of 2-oxoglutarate ester, preparation containing 2-oxoglutarate ester and use thereof

Field of the Art

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.

Prior Art

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 use of native 2-oxoglutarate as the means of stimulation of collagen biosynthesis is known, wherein the positive effect of the substance was evidenced experimentally in vitro on the culture of fibroblasts and in vivo on hairless mice which were long UVB irradiated. External application of 1% 2-oxoglutarate together with propylen glycol and ethanol lead in the experimental animal to the reduction of consequences of actinic ageing of skin, including suppression of wrinkle formation (Son et al., 2007). Biological availability and effectivity of transdermal penetration of transdermal penetration of 2-oxoglutarate is herein achieved using propylen glycol-ethanol carrier which impairs the natural function of skin barrier by disrupting the surface structures thereof, which is disadvantageous. The Korean patent KR121 294 discloses a cosmetic composition effective in smoothing out wrinkles, containing non-modified 2-oxogiutarate and optionally a-lipoic acid.

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. However, these substances function in a way where they destroy the natural protective function of biological barriers, which is undesirable. Moreover, 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).

Summary of the invention

For hydro phobizat ion of the molecule in order to make the molecule more biologically available for the cells, without the necessity of using further additives, 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. However, after entering the intracellular space it is quickly hydrolysed by the present enzymes to give the biologically active 2- oxoglutarate and biocompatible alcohols.

Moreover, compared to the effect of the simple 2-oxoglutarate, it was surprisingly found out that the 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.

The drawbacks of the prior art are overcome by the ester of 2-oxoglutarate according to the general formula I,

wherein

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;

for use in medicine, veterinary medicine, cosmetics, biotechnological applications or in tissue engineering for the stimulation of collagen biosynthesis, especially in cells exposed to oxidation stress.

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. Further, 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, liquid paraffin, propylenglycol, stearomacrogol 100, stearomacrogol 1050, cetylalcohol, light liquid paraffin, propylparaben, cocoa oil, medium saturated triacyl glycerols, benzylacohol, sorbitans and polysorbates, butter pear butter, ceteareth-20, cetyl alcohol, shea butter, sorbitan stearate, span 20, 60, 80 stearic acid, stearyl palmitate, xanthan gum, 1 ,3-butanediol, almond oil, aloe vera, dimethicon, isopropylmyristate, polyethylenglycols (e.g. PEG-100 stearate, PEG 400), oil from grape stones or isohexadecane, which are commonly used for preparations especially in the field of cosmetics, medicine, veterinary medicine, tissue engineering and in biotechnological applications.

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.

According to a preferred embodiment of the method of the invention, 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.

Detailed Description of the Drawings

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 μg / ml. For the measurement of cell viability, a test with neutral red was used.

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). For the measurement of cell viability, a test with neutral red was used.

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.

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).

Examples

The following examples of the embodiments of the invention should be understood as merely illustrative, without limiting the scope of the invention and the required protection in any way. SE = 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 Ή 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 Ή NMR spectrum within the interval 3.0 to 3.3 ppm

Ή NMR spectra measured in deuterated chloroform with a device having the frequency of 500 MHz, chemical shifts were calibrated with respect to the inner standard of deuterated 3- trimefhyl-silyl propane acid.

Example 1

Preparation of hexylesters of 2-oxoglutarate.

2-Oxogjutaric acid (1 g) was melted at the temperature of 1 15 °C. Then 2 equivalents of 1 -hexanol were slowly added and the mixture was stirred for 2 hours at the temperature of 1 15 °C. The resulting product (20 mg) was then dissolved in deuterated trichloromethane (0.8 ml) and analysed by means ofΉ NMR spectroscopy, SE = 1.4.

Example 2

Preparation of 2-phenethylesters of 2-oxoglutarate.

2-Oxoglutaric acid (lg) was melted at the temperature of 1 15 °C. Then 2 equivalents of 2-phenyIethyl alcohol were slowly added and the mixture was stirred for 0.5 hour at the temperature of 125 °C. The resulting product (20 mg) was then dissolved in deuterated trichloromethane (0.8 ml) and analysed by means ofΉ NMR spectroscopy. SE = 1.9 .

Example 3

Preparation of octylesters of 2-oxoglutarate.

2-Oxoglutaric acid (I g) was melted at the temperature of 1 15 °C. Then 2 equivalents of 1 -octanol were slowly added and the mixture was stirred for 1 hour at the temperature of 125 °C. The resulting product (20 mg) was then dissolved in deuterated trichloromethane (0.8 ml) and analysed by means of L H NMR spectroscopy, SE = 1.5.

Example 4

Preparation of 4-(thiophene-2-yl) butyl esters of 2-oxoglutarate.

2-Oxoglutaric acid (lg) was melted at the temperature of 1 15 °C. Then 3 equivalents of 4-(thiophene-2-yl)butan-l-ol were slowly added and the mixture was stirred for 1 hour at the temperature of 120 °C. The resulting product (20 mg) was then dissolved in deuterated chloroform (0.8 ml) and analysed by means ofΉ NMR spectroscopy, SE = 1.4 . Example 5

Preparation of 4-(l -methylpiperidiniitm-l-yl) butyl esters of 2-oxogiutarate.

2-Oxoglutaric acid (lg) was melted at the temperature of 1 15 °C. Then 2 equivalents of4-(l -methylpiperidinium-l -y!)butan-l -ol were slowly added and the mixture was stirred for 1 hour at the temperature of 120 °C. The resulting product (20 mg) was then dissolved in deuterated chloroform (0.8 ml) and analysed by means of ! H NMR spectroscopy, SE = 1.3.

Example 6

Preparation of oct-7-ene-l-yl esters of 2-oxogiutarate.

2-Oxoglutaric acid (lg) was melted at the temperature of 1 15 °C. Then 2.6 equivalents of oct-7-ene- l-ol were slowly added and the mixture was stirred for 1 hour at the temperature of 120 °C. The resulting product (20 mg) was then dissolved in deuterated chloroform (0.8 ml) and analysed by means ofΉ NMR spectroscopy, SE = 1.6.

Example 7

Preparation of 4-(cyclohex- l -ene-l -yl)butyl esters of 2-oxoglutarate.

2-Oxoglutaric acid (lg) was melted at the temperature of 1 15 °C. Then 2.8 equivalents of 4-(cyclohex-l -ene-l -yI)butan- l-ol were slowly added and the mixture was stirred for 1 hour at the temperature of 120 °C The resulting product (20 mg) was then dissolved in deuterated chloroform (0.8 ml) and analysed by means ofΉ NMR spectroscopy, SE = 1 .7.

Ex mple 8

Preparation of 4-cyclohexylbutyl esters of 2-oxoglutarate.

2-Oxoglutaric acid (lg) was melted at the temperature ofl 15 °C. Then 2.4 equivalents of 4-cyclohexylbutan-l-ol were slowly added and the mixture was stirred for 1 hour at the temperature of 120 °C. The resulting product (20 mg) was then dissolved in deuterated chloroform (0.8 ml) and analysed by means of L H NMR spectroscopy, SE = 1.5.

Example 9

The cosmetic preparation was prepared by mixing the individual components identified below together. The composition of the cosmetic preparation in the form of a cream

1) triglycerides of octanoic acid and decanoic acid

(Triglyceride, MakingCosmetics, USA)

2) glyceryl monostearate, PEG 100 stearate

(CreamMaker Blend, Making Cosmetics, USA)

3) sodium aery i ate, copolymer of acrylo l dimethyl taurate,

isohexadecane, polysorbate 80

(GelMaker EMU, Making Cosmetics, USA)

4) tocopheryl acetate

5) glycerin

6) hexylester of 2-oxoglutaric acid

7) benzylalcohol, DHA

8) perfume

9) distilled water

Example 10

The cosmetic preparation was prepared by mixing the individual components identified below together.

The composition of the cosmetic preparation in the form of a gel

wt./wt.

1 ) sodium hyaluronan 0.7 %

2) distilled water 88.25 %

3) PEG-40 hydrogenated castor oil 0.6 %

4) ethylalcohol 96% 2 %

5) perfume 0.05 %

6) 1,3-butandiol 2 %

7) sodium lactate, sodium salt of pyrolidoncarbonic acid,

glycine, fructose, urea, niacinamide, inositol,

sodium benzoate, lactic acid

(Lactil, Evonik) 3 %

9) disodium salt of EDTA 0.05 %

10) D-panthenol 0.3 % 1 1) glycerin, copolymer of glyceryl aery late/acrylic acid,

propylene glycol

(Lubrajel MS, Ashland) 2 %

hexyiester of 2-oxogIutaric acid in PEG400 ( 1%) 1%

methylchloro isothiazolinone, methyl isothiazolinone

(Isocii PC, Lonza AG) 0.05%

Example 11

The cosmetic preparation was prepared by mixing the individual components identified below together.

The composttion of the cosmetic preparation in the form of a cream t./wt.

1 ) Steareth-2 (PEG-2 stearylether) 2 %

2) Steareth-21 (PEG-21 stearylether) 2 %

3) PPG- 15 stearylether 9 %

4) palmitic acid, stearic acid

(Cutina FS 45, Cognis) 1 .5 %

5) dimethicone (silicone oil) 0.5 %

6) iso propyl myristate 3 %

7) triglycerides of octanoic acid and decanoic acid

(Triglyceride, MakingCosmetics, USA) 2 %

8) squalene 3 %

9) cetylalcohol 2 %

10) glycerylstearate 1 %

1 1) hexyiester of 2-oxogIutaric acid 10 %

12) glycerin 4 %

13) D-panthenoI 0.5 %

14) distilled water 59.25 %

15) methylchloro isothiazolinone, methyl isothiazolinone

(isocii PC, Lonza AG) 0.05 %

16) perliime 0.2 % Example 12

Cell via ility test

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 μg/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.

Example 13

Comparative in vitro test of the effects of hexylester of 2-oxoglutarate (Fig. 3) and 2- phenylethyl (Fig. 4 and 5) on the production of total collagen

Fig. 3 shows the test results of the effect of 2-oxoglutarate hexylester (1000 μg / 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 μg / 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. 5 shows the test results of the effect of 2-phenylethyl ester of 2-oxoglutarate (10 - 1000 μg / 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).

In order to monitor the collagen production, 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).

All experiments on cell cultures were repeated at least 3x, while keeping the above described experimental conditions. The obtained data were evaluated statictically by variance analysis, Scheffe's test was used for comparing the individual groups (multiple range test). The results illustrate that 2-oxoglutarate hexylester prepared according to Example 1 and 2- oxoglutarate 2-phenylethyl ester prepared according to Example 2 increased significantly the total collagen production by human dermal fibroblasts. 2-phenylethyl ester of 2-oxog!utarate prepared according to Example 2 increased significantly the total collagen production by human gingival fibroblasts. The surprising effect is apparent not only when compared to the control (non-effected cells), but also when compared to the non-modified 2-oxoglutarate.

According to the published experimental data (Son et al. 2007), in case of senescent cells no statistically significant pro-collagen effect of the non-modified 2-oxoglutarate was proven. Moreover, as seen in Fig. 3, the effect of 2-oxoglutarate and 2-oxoglutarate ester on collagen production in cells of juvenile donors is statistically comparable.

The used literature

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