JPH04502611A - Compounds and methods for inhibiting collagen crosslinking - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Compounds and methods for inhibiting collagen crosslinking The present invention reduces cross-linking of hive collagen and/or damage to hive cell DNA. or methods for preventing it. In particular, the present invention relates to aging and/or UV irradiation. Certain compounds have the ability to reduce or prevent collagen cross-linking for periods of time following exposure to During the use of dipeptides and their analogues, the method of the present invention also involves the use of ultraviolet radiation. It can also be applied to reduce DNA damage.

Raw Butt Koyo I Oxidative stress and tissue damage caused by reactive oxygen species are associated with numerous diseases, including cancer and aging. (Halli-siell and Gutte ridge, 1985; Harsan, 1987; 5au1 etc., 1 987).

The concentration and production of highly reactive free radicals can be increased by UV radiation. is known. The reactive species are later converted into DNA, RNA, proteins and lipids. It is possible to react with natural defenses against reactive and potentially damaging species; It is believed that a number of molecules with anti-aging properties exist in tissues. (e.g. vitamin E, carnosine and ascorbic acid), However, the physiological role of carnosine and its analogues in vivo remains unclear. There is even.

In addition to being an efficient singlet oxygen scavenger, carnosine has been suggested to Other roles include neutralization of lactic acid (Davey).

1960), i-chelating agent (Brosyn, 1981), myosin light pan precipitate activator of the toxoid enzyme (Parker and Ring, 1980) and and enzyme regulators (Ikeda et al., 1980).

The amount of crosslinking in mature collagen increases during mammalian aging, whereas the amount of crosslinking in mature collagen increases. Decreasing maturation reduces the number of possible crosslinks (Ames, 1983). of other organizations ( Although the nature of the crosslinks (e.g. vagina) may differ, similar general trends exist (Am es, + 1983; Rattan et al., 19B2), for various species. The rate of change in the amount of crosslinking appears to reflect differences in lifespan for different species. . For example, mature cross-linked HHL (histidinohydroxylucinonol leuci The amount of phlegm) increases in a primary manner in human chicks up to the age of 40 years, whereas it increases in calf chicks. The amount of HHL levels off at 4 years of age (K.

Hen et al., 1988). Crosslinks occur before the collagen chains are oxidatively deaminated. The precursor lysine (Vizioli et al., 1983) or hydroxyl lysine (Bo ldyrev etc., 19B? ) arises from The generated aldehyde is then similar may be condensed with the remainder of It condenses with the xyllysine residue to form a Schiff compound. The degree of crosslinking of collagen is It is also increased by ultraviolet light. In this way, the cross-linking speed of collagen and the There is a correlation between the rate of aging of other tissues.

1 live group The present invention provides methods for treating skin with compositions containing suitable excipients in combination with active compounds. and the active compound is carnosine, homocarnosine, anserine, 3-methyl-histidine, L-alanyl-L-tyrosine, acyl homocarno Syn, acetyl carnosine, iodocarnosine, short carnosine, nitrate ace phosphorus, carbenoxylon, lunosine, its analogues, and two or more of the above cross-linking of skin collagen and/or selected from mixtures; The method comprises a method of reducing or preventing damage to skin cell DNA.

In a preferred embodiment of the invention, the active compound is naturally occurring (e.g. human Carnosine (p-alanyl-cytidine), which is found in the tissues of It is an anti-inflammatory compound.

Currently, the active compound is carnosine or homocarnosine by combination thereof. is preferred, most preferably carnosine.

In a further preferred embodiment of the invention, the active compound is bound to another molecule. and the molecule improves the composition's ability to penetrate into the skin, apply to the skin, and absorb tissue. It's like being able to do something.

Preferably, this other molecule is an amino acid or a peptide.

The method of the present invention generally involves topical application of the composition to the animal, but not limited to However, the composition can be injected subcutaneously or intramuscularly, or taken orally.

The concentration of active compound in the composition will depend on the route of administration and will be directed by your physician. However, the concentration of active compound may vary, for example in hif cream formulations. is expected to be within the range of 1 to 100 mg per -, with a preferred range of 3 to 2 It is 0■/d.

The method of the invention reduces cross-linking of collagen due to exposure to ultraviolet light or sunlight. It is believed that skin aging can be reduced by preventing or inhibiting skin aging. That person The method also shows that DNA damage as a result of ultraviolet radiation can be prevented. The method of the present invention has applicability in the prevention of skin cancer.

In addition to the active peptide molecules discussed in the main text, the compounds according to the invention include collagen In the compositions of the present invention, which may contain non-peptidic compounds capable of inhibiting or preventing the cross-linking of Such compounds that may be conveniently included in Nitol, reduced glutamine, selenium, uric acid, vitamin A1 vitamin C and vitamin An example is MinE.

Mok Hyun-soo 91 Yushi Ie In order to more clearly understand the essence of the invention, preferred embodiments thereof will now be described below. The description will be given with reference to examples.

Opening up rice fields Nibunkyu J] An example of carnosine's collagen cross-linking inhibitory activity is shown in Table 2. Expressing the effects of ultraviolet irradiation on reducible species, carnosine was produced by Sigma Chemical Co. Sigma Chewical Comp-any) or BDD H chemical It was obtained from BD HChemical Ltd. ChroIll ar HP L C grade acetonitrile Ma II I 1nkro-dt A obtained from U5tralia Pty, Ltd. and filtered all solvents before use. I degassed. KB (H')4 with a specific radioactivity of 50 to 70 curies/mmol is turned on. - Purchased from CE A France through the Stralia Atomic Energy Commission.

Dermal fibroblasts were cultured from primitive explants of tissue obtained from 5w1ss mice. Ta. Cells were cultured in Dulbecco's modified Eagles medium (Gibco). Supplemented with 10% tallow serum (Gtosystem IIs Pty, Ltd.), the second It was used between passage and third passage. Skin sections were also obtained from neonatal 5w1ss mice. Ta. Sections should be cut to remove as much subcutaneous material as possible and phosphated before the next experimental step. Rinsed briefly with buffered saline. All samples were treated with phosphoric acid prior to UV treatment. 37° for 1 h in various concentrations of Shiichiro Lunosine in salt-buffered saline (PBS). Incubated at C. Samples were collected in PBS prior to reduction with borohydride. Thoroughly wash at 40°C to remove contaminated material with minimal disruption of the collagen structure. Proteins, glycoproteins, and glycosaminoglycans were removed.

Open dishes of skin sections and skin fibroblasts were removed using a 24 watt germicidal UV lamp. and exposed to 920 W/am for 3 hours. Samples retained moisture through UV treatment .

Sample reduction with KB(H3)4 for 1 h in homogeneous PBS buffer at room temperature/borohydride. A 100:1 wet weight ratio of compounds was used. The reaction was stopped by the addition of 4M acetic acid and p H was lowered to 3.00. The sample is then soaked in distilled water until free of soluble radioactivity. Dialysis was performed at 4°C. The sample was incubated in 5 eqvana 1 grade 6N hydrochloric acid under nitrogen for 22 hours. Hydrolyzed at 110°C for a while. before each sample is then dissolved in distilled water. It was dried twice using a low-cleaning evaporator from distilled water. Melt ice before HPLC Neutralized with 0.5N NaOH.

Includes HPLC P3500HP LC pump, LCC500 programmer Pharmacia HP L C/F PLC device This was carried out using a heated column chamber.

First separation with Brownlees 25cmX0.46ctn amino-3ph Performed on an ERI 5 column at a temperature of 35°C and an eluent flow rate of 1.0 d/min. .

Brownlee 4.6X30nm amino 5ph in solvent line for column life eri5 play column guard cartridge, and Brownlee15X32 It was considerably extended by using anAnion Neevgvard. B The ro-wnlee column is Australian Activon 5cientif Supplied by icProduc-ts Co Pty, Ltd.

The gradient system consists of two solvents. Solvent A is pH 4, 3, 10n+M potassium phosphate HPLC containing buffer and solvent B diluted 50:7 (v/v) with water - Contained grade acetonitrile. Gradient program similar to prior art for amino acids Although it can be separated by using a program, it is possible to separate the collagen component by using a Fixed for separation.

Program 1 was as follows. i.e. 1.95% Solvent B for 5 minutes;2. Linear gradient 95% to 70% solvent B over 15 minutes; 3, linear gradient 70% to 5 0% Solvent B for 15 minutes; 4. 50% Solvent B for 10 minutes; 5. straight line slope 50% to 95% Solvent B over 5 minutes.

Fractions with a volume of 0.5 d were collected directly into 20-scintillation vials and ham PCS II high efficiency phase compound scintillating agent 5.0 relative was added. count It was carried out using a LKB 1215 Rack beta U instrument.

The mouth of your mouse topsoil ■Profession: Ultraviolet treatment, Indigo Ω polishing skin section Skin (external surface) 10000 Skin (external surface) + 24000 Skin (inner surface) 11000 Skin (inner surface) + 27000 skin cells Fibroblast 19000 Fibroblast + 38000 The sample of 2. was treated with normal light or ultraviolet light for 2.5 hours and then reduced with KB (Hz)a. The sample was prepared and hydrolyzed prior to HPLC.

deep red -fl ultraviolet treatment of carnosine addition of carnosine kdan U fallen skin section 15000 +23000 ＋　＋　14000 Skin cells 14000 +20000 + Low dose (2mM) 14000 High dose (10mM) 10000 Samples were treated as in Table 1 with or without protection by carnosine solution.

Real Mil- Primary cultures of mouse dermal fibroblasts (MDF) were isolated from newborn mouse skin and used for experiments. High glucose supplemented with lO% tallow serum for only 4 passages prior to The cells were continuously passed through DMEM medium. Prior to UV exposure, the cell layer is All traces of growth medium were removed by washing with PBS50d. MDF next 10 + PBS25M1 with or without IIM carnosine at 37 °C for 1 h. Both were incubated. All procedures were performed with minimal exposure to external light. .

At the end of the incubation period, expose the replicating cell population to UV light for 0.2, 4 or 6 min. exposed for a while. Following irradiation, peel the cell monolayer into a centrifuge tube containing PBS. Moved. After centrifugation, the supernatant was removed and the cell pellet was resuspended in PBS301d. . All samples were then washed at 4"C for 3 days. PBS was changed twice daily. Ta.

The sample was then reduced with B[3H]4 and acid hydrolyzed. Rotor the hydrolysis solution Li evaporator, neutralization and HPLC analysis (Smolenski et al., 1 983 Bioche + * J, , 213. pp. 523-532) before lyophilization. did. Collect 0.5W1 HPLC fraction directly into 20d scintillation vial Added 5m of Amersha+mP CS II high efficiency compound scintillating agent. added. Counting was performed using LK B 1215 Pack Beta ■. this The experimental results are shown in Figs. 1 to 6.

Mouse skin fibroblasts were scraped and washed with PBS. The separated cell suspension Processed for HPLC using the method described below.

The profile shown in Figure 1 is a depiction of a typical non-UV irradiated sample (control).

The range 40-90% consists of separate reducible collagen crosslinks. peak height The difference in size is an indication of the amount of a particular type of bridged amino acid complex present in the sample.

The profile shown in Figure 2 shows the isolated crosslinks after 2 minutes of exposure to UV A light. Depicting change. The main peak of number 55 disappears, and the peaks of number 83 to 87 disappear. There was a change in location.

These differences are due to the interaction of free radicals generated by the action of ultraviolet light on water molecules. It is speculated that this represents a modification of the cross-linked amino acid complex by the action.

As shown in Figure 3, the crosslinking profile that can be rapidly exposed to ultraviolet A light for 4 minutes is Fractions 55-75 showed a large increase in crosslinkable amino acid complexes.

As shown in Figure 4, after 6 minutes of exposure to ultraviolet A, the reduction that exists between 55 and 75 There were significant changes in the content of possible bridging amino acid complexes. This is probably 55 for both. Modification of fewer complex crosslinks causing accumulation in ~75 and possibly This is the result of the formation of entirely new crosslinks due to free radical interactions.

Figure 5 shows mouse skin fibroblasts exposed to UV light for 6 minutes in the presence of 10mM carnosine. The results obtained when exposed to line A are shown.

As seen there, the profile of the separated crosslinks compared to that shown in Figure 4. There were significant differences among them.

Figure 6 shows that MDF cells were exposed to UVA for 2 minutes or UVA for 6 minutes plus 10II. A comparison of the profiles produced when exposed to light in the presence of IM carnosine is shown.

Carnosine is exposed to ultraviolet A as seen by the decrease in the peak from fractions 55-75. It is clear from this overlap of profiles that MDF cells are protected from the effects of be.

belly 10 mM carnosine is present in the solution that soaks the MDF throughout the UV-A exposure. There was a 70% reduction in the formation of altered reducible crosslinks formed when this was illustrated by the appearance of the HPLC profile. i.e. 6 minutes of ultraviolet A exposure+ The profile of 1011M carnosine is similar to that of 2 minute UV-A exposure. was.

Implementation 1 , A Hi MRC-5 When human lung fibroblast cell line (MRC-5) was exposed to ultraviolet A light, was used to test the degree of protection that collagen provides to human collagen.

Fibroblasts are responsible for maintaining connective tissue in animals. They actively interstitial Collagen brobegutide is secreted into the space, and a part of it is combined with connective m-tissue collagen. and deposited in the extracellular matrix.

A similar experimental design to that used for MDF cells was used in these experiments. However, the UV-A exposure time was increased to 15 minutes. This is 6 minutes for MDF cells. This was done to increase the changes in collagen cross-linking that were evident with UV-A exposure. . The results of these experiments are shown in FIGS. 7-10.

In Figure 7, we scraped a population of MR(,-5) with a similar number of cells as the MDF population. Processed for HPLC. These cells were not treated with UV-A. Sunawa A control - the profile was similar to that produced by MDF cells. There was a difference. The main collagen crosslinking peaks were in the range of 40-90.

Figure 8 shows MRC-5 cells exposed to UV light for 15 minutes in the presence of 10mM carnosine. Shows the HPLC profile obtained when exposed to a control line ( 15 minutes of UV-A exposure similar to the profile in Figure 7) but without carnosine. It was a big difference from that of light (Figure 9).

Figure 9 shows the profile produced when MRC-5 cells were exposed to UV light for 15 minutes. shows. The range of fractions 40 to 90 represents newly formed crosslinked complexes in cytoplasmic collagen. This is a sign that the putative uptake of

Figure 1O from MRC-5 control and in the presence of 10n+M carnosine. Overlap of HPLC profiles separated from MRC-5 after exposure to UV-A for 15 minutes. shows the Furthermore, there are considerable similarities between the two types of profiles, and Carnosian This indicates that collagen protects collagen from free radical attack.

! bamboo Results from these experiments also show that carnosine inhibits collagen against UVA-induced crosslinking. supported the assumption that the This is the MRC-5 control HPLC profile Plasma of MRC-5 cells exposed to UV-A for 15 min in the presence of carnosine and carnosine. demonstrated by the remarkable similarities between the lophylls. In addition, in the absence of carnosine, ultraviolet Significant changes in the profiles obtained from MRC-5 cells exposed to line A Lunosine is extremely effective in its protection of collagen against cross-linking These experiments, which increased UV A exposure time by 150%, still showed that >70% protection was achieved with 10 mM carnosine.

1 difference” 1 work Dan! ”It1 Cunning Isometric melting has been widely used to determine the number of age-related changes in collagen. (Mitchel 1 and Rigby, 1975 BBA.

Direct, pp. 531-541), Robins and Balley (1975B iochemJ, 149, pp. 381-385) shows that the density of collagen crosslinking depends on time. It is constant in humans, but occurs with aging, and is produced from lysine and hydroxylysine. The unstable reducible allidimine bonds formed explain age-related changes in collagen. He proposed that the bond is converted into a thermally stable and irreducible bond.

This method of isometric melting demonstrates how UV light can alter collagen crosslinks. It was used for testing. Numerous other collagen aging studies (formerly tchel and and Rigby, 1975 BBA, pp. 393.531-541; Rigby and Mitchel], 1978, BBA, pp. 532.65-70 and BBA, [, pp. 62-68; Rigby et al., 1977, BBRC, n. For these experiments, rat tail membranes, which have been used for I used it.

This method is used more often than other methods as a direct measure of age-related crosslinking changes. It has the following advantages. This application relates to photoaging and specifically collagen cross-linking by ultraviolet light. This method allows quantitative measurement of the extent of UV crosslinking with time. and make it possible. Additionally, this method protects the legs against UV-induced free radicals. allows to determine the effect of carnosine and other antioxidants on .

Hayashi Fukigoyo U legislation The technique of isometric melting was applied to 7) Oboro to measure the effects of photoaging.

Ninety day old Sprague-Dawley rats were used in these experiments.

■Zabun lid The tail was removed, placed on ice, and transported to the work site. To prevent the legs from drying out The cells were carefully cultured on a saline-moistened surgical pad. Each key then The upper half of the measured and halved 0 fish was used for the experiment, while the lower half was used as a physical control. and stored at 4°C.

,and ' The experimental halves of the keys were incubated at 4°C in the appropriate test solution for 20 hours prior to UV exposure. Incubated. Following this exposure, the crotch was washed with PBS and kept at 4°C until analysis. Saved with.

Isometric device consisting of a strain meter - 5 hinkho transducer type UL - 100 gm was connected to the amplifier. After attaching the sample to the strain gauge, place the sample in a jacket containing PBS. Immersed in a Pyrex bath. The bath was kept in a Taeason W1 ring throughout the experiment. Heated with a water heater. FLUKE thermocouple type 280TX to measure temperature rise The crotch attachment was attached to the part next to the position. rcI DP force and temperature measurements 6002 pens were recorded simultaneously using a yard recorder.

The thighs were recut to a standard length of 3 cm before mounting on an isometric device for analysis. Tori The attached legs were then immersed in a PBS bath at 20°C and a tension of 1 g was applied. 15 After a relaxation period of 1 minute, turn the device and heat at a rate of 1”C/min until melting occurs. rose. Final measurements were then taken from the chart recorder.

To determine the exposure time required to produce a measurable change in the track , conducted a time course experiment. The results of this experiment are shown in FIG. 4 UV rays, I A tube and two ultraviolet lights! 180 minute exposure using B tube light source is reproducible It turns out that it gave results. This time was used for the next experiment.

Carnosine, homocarnosine and anserine were used to pre-treat the replicate calf did. These were then exposed to UV 1iAB for 180 minutes. Conclusions from these experiments The results are shown in Figure 12. In these experiments, carnosine and homocarnosine 10 and 100 mM effectively protected the crotch against UV-induced crosslinking. one On the other hand, no protective effect was observed at 10+mM of anserine; It is believed that high temperatures may provide a protective effect. (Unfortunately, Anserine is available. High concentrations were not tested due to lack of potential. ) Figure 13 compares a set of other dipeptides and tripeptides to carnosine. Results obtained when testing the ability to protect the crotch against UV-induced crosslinking shows. None of those tested protect keys against UV-induced crosslinking I understand. Two of the peptides contained histidine but remained inactive. Ta. These results indicate that carnosine probably protects against UV-induced collagen cross-linking. This suggests that it acts through its antioxidant properties.

Figure 14 is related to Sm measurement for glutathione 10+wM (IOG SH). A comparison with the thigh data from FIG. 12 is shown. Glutathione can be measured using this method. It seems to be even more effective when used. Anserine (IOA) is not produced at this concentration. Not used. Glutathione acts as a free radical scavenger in vivo at this concentration. It is important to note that this is not effective for the action of

! h Results from these experiments using isometric melting techniques demonstrate that carnosine is UV-induced Conclusive that collagen cross-links act effectively in rat tail key protection It was proved. The results for other dipeptides and tripeptides tested also indicate that It is also clear that the nosine effect is clear and occurs due to its antioxidant properties.

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HPLCproflla of mousedarma group blobtasts afler2’exposuretoυVA.

Figure, 2° HPLC profile of mouse flbroblms ave r　a　4’　UV　A　exposure.

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Fipure, 9° Figure, 10° FIGURE, it.

The r@duclon oτLIV Induced eollaaen 160’uVAφB rXGURt 12 Figure　13゜ 188°UVAB F engineering GυRE 14 Submission of translation of written amendment (Written amendment pursuant to the provisions of Article 184-8 of the Patent Law) March 28, 1991

Claims (14)

[Claims] 1. treatment of the skin with a composition containing suitable excipients in combination with an active compound. and the active compound is carnosine, homocarnosine, anserine, 3-methane, Tyl-L-histidine, L-alanyl-L-tyrosine, acyl homocarnosine, Acetylcarnosine, iodocarnosine, diiodocarnosine, anserine nitrate , carbenoxylon carnosine, analogs thereof and combinations thereof; cross-linking of collagen in the skin and/or DNA of skin cells, characterized by ways to reduce or prevent damage caused by 2. The active compound is carnosine or homocarnosine or a combination thereof. The method described in item 1 of the scope of the request. 3. 3. A method according to claim 2, wherein the active compound is carnosine. 4. The active compound is combined with another molecule that allows the composition to penetrate the skin. Claim no. The method according to any one of items 1 to 3. 5. 5. The method of claim 4, wherein the molecule is an amino acid or a peptide. 6. Any of claims 1 to 5, wherein the method comprises topical application of the composition to the skin. or the method described in item 1. 7. The composition includes bilirubin, carotenoids, mannitol, reduced glutathione, Select from selenium, uric acid, vitamin A, vitamin C, vitamin E and combinations thereof. 7. The method according to any one of claims 1 to 6, comprising a compound comprising: 8. treatment of the skin with a composition containing suitable excipients in combination with an active compound. and the active compound is carnosine, homocarnosine, anserine, 3-methane, Tyl-4-histidine, L-alanyl-L-tyrosine, acyl homocarnosine, Acetylcarnosine, iodocarnosine, diiodocarnosine, anserine nitrate , carbenoxylon carnosine, analogs thereof and combinations thereof. Reduces the cross-linking of collagen in the skin caused by exposure to ultraviolet light, which is characterized by How to reduce or prevent. 9. The active compound is carnosine or homocarnosine or a combination thereof. The method described in item 8 of the scope of the request. 10. 10. A method according to claim 9, wherein the active compound is carnosine. 11. The active compound is combined with another molecule that allows the composition to penetrate the skin. , the claims are such as to be improved with respect to skin application and tissue absorption. The method according to any one of items 8 to 10. 12. 12. The method of claim 11, wherein the molecule is an amino acid or a peptide. 13. Claims 8 to 12, wherein the method comprises topical application of the compound to the skin. The method described in any one of the above. 14. Composition includes bilirubin, carotenoids, mannitol, reduced glutathione, Consists of selenium, uric acid, vitamin A, vitamin C, vitamin E and combinations thereof Claims 8 to 13 comprising a compound selected from the group Method.