Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
• • 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
  • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
  • A61K8/4946—Imidazoles or their condensed derivatives, e.g. benzimidazoles
• • 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
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/52—Stabilizers
  • A61K2800/522—Antioxidants; Radical scavengers

Definitions

• the present invention relates to a method for reducing or preventing collagen cross-linking in skin and/or damage to skin cell DNA.
• the present invention relates to the use of specific dipeptides and analogues thereof which have the capability of decreasing or preventing collagen cross-linking either during aging and/or following exposure to UV radiation.
• the method of the present invention is also applicable for decreasing DNA damage due to UV radiation.
• Oxidative stress and tissue damage by active oxygen species has been suggested as the basis for a number of diseases including cancer and aging (Halliwell and Gutteridge, 1985; Harman, 1987; Saul et al, 1987).
• camosine In addition to being an efficient singlet-oxygen scavenger, include neutralization of lactic acid (Davey, I960), a copper chelator (Brown, 1981), an activator of myosin ATPase (Parker and Ring 1980) and a regulator of enzymes (Ikeda et al 1980) .
• Cross-links arise from precursor lysine (Vizioli et al, 1983) or hydroxylysine (Boldyrev et al 1987) residues in collagen chains which are oxidatively deaminated.
• the aldehydes produced then condense with with similar residues to give aldols or with adjacent lysine or hydroxylysine residues to give Schiff-based compounds.
• the degree of cross-links in collagen also increases on UV irradiation. Thus there is a correlation between the rate of collagen cross-links and the rate of aging of skin and possibly other tissues.
• the present invention consists in a method for reducing or preventing collagen cross-linking in skin and/or damage to skin cell DNA comprising treating the skin with a composition comprising a suitable excipient in combination with an active compound, the active compound being selected from the group consisting of camosine, homocamosine, anserine, 3-methyl-L-histidine, L-alanyl-L-tyrosine, acyl homoca osine, acetyl camosine, iodo camosine, di-iodo ca osine, anserine nitrate carbenoxylone camosine, analogs thereof and mixtures of two or more of the foregoing.
• an active compound being selected from the group consisting of camosine, homocamosine, anserine, 3-methyl-L-histidine, L-alanyl-L-tyrosine, acyl homoca osine, acetyl camosine, iodo camosine, di-i
• the active compound is a naturally occurring (e.g. found in human tissue) anti-oxidant compound such as carnosine (/2-alanyl-L-histidine) .
• the active compound is camosine or homocamosine on a combination thereof and most preferably camosine.
• the active compound is linked to another molecule, which molecule is such that the composition is improved in regard to skin permeation, skin application and tissue absorption. It is preferred that this other molecule is an amino acid or peptide.
• the method of the present invention will generally involve topical application of the composition to the skin, however, the composition may be injected subcutaneously or intramuscularly or may be taken orally.
• concentration of the active compound in the composition will depend upon the route of administration and may be at the direction of a physician, however, it is expected that the concentration of the active compound would be in the range of 1 to 100 mg per ml of a skin cream formulation for instance, the preferred range would be from 3 to 20 mg/ml.
• the method of the present invention will reduce aging of the skin by decreasing or preventing collagen cross-linking due to exposure to UV radiation or sunlight. Given that the method is also capable of preventing DNA damage as a result of UV radiation, the method of the present invention has applicability in the prevention of skin cancer.
• composition according to the present invention may include, in addition to the active peptide molecules discussed above, a non-peptide compound which can inhibit or prevent cross-linking of collagen.
• a non-peptide compound which can inhibit or prevent cross-linking of collagen.
• Such compounds which may be advantageously included in the present composition include bilirubin, carotenoids, mannitol, reduced glutathione, selenium, uric acid, vitamin A, vitamin C and Vitamin E.
• L-carnosine is available from the Sigma Chemical
• HPLC grade acetonitrile was obtained through Mallinkrodt Australia Pty. Ltd. and all solvents were filtered and
• KB(H ) 4 was purchased from CEA
• Dermal fibroblasts were cultured from primary explants of tissue derived from Swiss mice. The cells were maintained throughout in Dulbecco's Modified Eagles Medium (Gibco) and supplemented with 10% foetal bovine serum (Cytosystems Pty. Ltd.) and used between second and third passages. Skin sections were also obtained from neonatal Swiss mice. Sections were trimmed of as much subcutaneous material as possible and rinsed briefly in phosphate-buffered saline before following experimental procedure. All samples were incubated in varying concentrations of L-carnosine in phosphate-buffered saline (PBS) for 1 hour at 37°C prior to UV treatment.
• PBS phosphate-buffered saline
• the gradient system comprised two solvents.
• Solvent A contained 10 mM-potassium phosphate buffer, pH 4.3
• solvent B contained HPLC-grade acetonitrile diluted 50:7 (v/v) with water.
• a ino acids could be separated by using a gradient programme similar to that of the prior art, but the programme was modified for separation of reduced collagen components.
• Programme 1 was as follows: 1.95% solvent B for 5 min; 2, linear gradient 95% - 70% solvent B over 15 in; 3, linear gradient 70% - 50% solvent B over 15 min; 4, 50% solvent B for 10 min; 5, linear gradient 50% - 95% solvent -B over 5 min.
• MDF mouse dermal fibroblasts
• the replicate cell populations were exposed to 0,2,4 or 6 minutes of UV A light. Following irradiation the cell monolayers were scraped and transferred to centrifuge tubes containing
• the profile shown in Figure 1 is a representation of a typical non ultraviolet irradiated sample (CONTROL) .
• the fractions in region 40 to 90 consist of isolated reducible collagen crosslinks.
• the difference in peax height is indicative of the amount of a particular type of crosslinking amino acid complex present in the sample.
• the profile shown in Figure 2 depicts the alteration in isolated crosslinks after a 2 minute exposure to UV A light.
• the major peak at fraction no. 55 has disappeared and there has been a positional shift of the peak at fraction. 83 to 87.
• FIG. 4 shows the results obtained when mouse dermal fibroblasts are exposed for 6 minutes to UV A in the presence of 10 mM camosine. As can be seen there is a marked difference in the profile of isolated crosslinks in comparison to that shown in Figure 4.
• Figure 6 shows a comparison of profiles produced when MDF cells were exposed to 2 minutes UV A or to 6 minutes UV A but in the presence of 10 mM ca osine. It is obvious from this overlay of profiles that camosine has protected the MDF cells from the effects of UV A as seen by the decrease in peaks from fractions 55-75. Summary
• Fibroblasts are responsible for the maintenance of connective tissue in animals. They actively secrete collagen propeptides into the interstitial spaces, a proportion of which are deposited into the extracellular matrix as connective tissue collagen.
• Figure 9 shows the profile produced when MRC-5 cells were exposed to 15 minures UV A light. The region from fractions 40 through 90 are extensively increased indicating a putative incorporation of newly produced crosslinking complexes into the cellular collagen.
• Figure 10 shows an overlay of HPLC profiles isolated from the MRC-5 control and from the MRC-5 after 15 minutes exposure to UV A in the presence of 10 mM camosine. Again, there is a great deal of similarity between the two profiles indicating that camosine is protecting the collagen from free radical attack. Summary
• Isometric melting has been widely used to determine a number of age related changes in collagen (Mitchell and Rigby, 1975 BBA, 3!93, 531-541). Robins and Bailey (1975 Biochem J., 149, 381-385) have proposed that the density of collagen crosslinks is constant with time but as ageing occurs, the labile reducible aldimine bonds formed from lysine and hydroxylysine, are converted to a thermally stable, non-reducible bond which accounts for the age related -collagen changes .
• the experimental half of the tendon was incubated at 4°C in the appropriate test solution for 20 hours prior to UV exposure. Following this exposure the tendons were washed in PBS and kept at 4°C until analysed.
• The- isometric apparatus consisted of a strain gauge - Shinkho transducer type UL-lOOgm connected to an amplifier. After attachment to the strain gauge the sample was immersed in a jacketed pyrex bath containing PBS. During the experiments the bath was heated with a Tamson circulating water heater. To measure the temperature increase a FLUKE thermocouple model 80TK was fixed to a region next to the tendon attachment site. Measurements of force and temperature were recorded simultaneously with an ICI DP600 dual pen chart recorder.
• the tendon was re-cut to a standard length of 3cm before attachment to the isometric apparatus.
• the attached tendon was then immersed into the bath of PBS at 20°C and a tension of 1 gram applied.
• Figure 13 shows the results obtained when a set of other di and tri peptides were examined for their ability to protect tendons against UV induced crosslinking in comparison to camosine. It was found that none of those tested protected tendons against UV induced crosslinking. Two of the peptides contained histidine and still were inactive. These results suggest that camosine is probably acting via its antioxidant property to protect against UV induced collagen crosslinking.
• Figure 14 shows comparison of tendon data from Figure 12 with Sm measurements for glutathione at 10 mM (10GSH). Glutathione appears more effective using this method of measurement. Anserine (10A) is not working at this concentration. It is important to note that glutathione would not be available to act as free radical scavenger in vivo at this concentrations. Summary

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• Proteomics, Peptides & Aminoacids (AREA)
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• 1989
  • 1989-09-28 EP EP19890910999 patent/EP0436611A4/en not_active Withdrawn
  • 1989-09-28 WO PCT/AU1989/000422 patent/WO1990006102A1/en not_active Ceased

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