HK1018885B - Method of inhibiting photoaging of skin - Google Patents

Description

Method for inhibiting skin photoaging

Technical Field

The invention belongs to the field of light protection. More particularly, the present invention relates to the use of inhibitors of Matrix Metalloproteinase (MMP) production and/or activity to inhibit photoaging of intact skin.

Background

Photoaging is the term used to describe the change in appearance and function of skin after repeated exposure to sunlight. The Ultraviolet (UV) component of sunlight, especially the mid-UV (called UVB, wavelength at 290-320nm) is the main inducing factor for photoaging. The amount of UVB exposure required to cause photoaging is currently unknown. However, repeated exposure to UVB at intensities that cause erythema and tanning is often accompanied by photoaging. Photoaging is clinically characterized by rough skin, wrinkling, mixed pigmentation, yellowish coloration, laxity, telangiectasia, nevus formation, purpura and tumors that are prone to bruising, atrophy, appearance of areas of fibrotic depigmentation, and ultimately premalignant and malignant tumors. Photoaging typically occurs in skin areas that are routinely exposed to sunlight, such as the face, ears, bald areas of the scalp, neck and hands.

There are methods for preventing photoaging of unaged skin and treating aged skin, and sunscreens (sunscreens) are commonly used to prevent photoaging of skin sites that are routinely exposed to sunlight. Sunscreens are topical preparations which absorb, reflect or scatter UV light. Some are based on opaque particulate materials such as zinc oxide, titanium oxide, clays and ferric chloride. These opaque adjuncts are considered by many to be cosmetically undesirable because these formulations are visibly noticeable and can clog pores. Other sunscreens contain chemicals such as para-aminobenzoic acid (PABA), oxybenzone, dihydroxybenzone, ethylhexyl-methoxycinnamamide and butyl methoxydibenzoylmethane, which are light-transmitting and colorless because they do not absorb light in the visible wavelengths. While these clear sunscreens may be more cosmetically acceptable, they are still relatively short lived and are easily washed off or washed away by perspiration. In addition, all sunscreens inhibit the production of vitamin D.

Rieger in Cosmetic and Toiletries (1993) 108: 43-56 the role of Reactive Oxygen Species (ROS) in UV-induced skin aging is reviewed. It is reported that topical application of known antioxidants to the skin reduces the presence of ROS on the skin, thereby reducing photodamage.

Retinoids have been used to slow the effects of photoaging in sun damaged skin. U.S. Pat. No.4,877,805 describes the treatment of photodamaged skin for the intervention of slowing the progression of photoaging. The patent states that there is no opportunity for treatment before the onset of aging effects. In this sense, the applicant is aware that there is no suggestion in the prior art of using retinoids to prevent photoaging of intact skin.

MMPs are a family of enzymes that play a major role in the physiological and pathological destruction of connective tissue. More than 10 of these enzymes have been identified. They are indicated by numbers (MMP-1, MMP-2, etc.) and by common names. They appear to be identical in several structural and functional properties, but different in tissue substrate specificity. They include interstitial collagenases (MMP-1) and PMN collagenases (MMP-8) that degrade collagen types I, II, III, VII, VIII, IX and gelatin; collagen type IV/gelatinase of 72kDa (MMP-2) and 92kDa (MMP-9) that degrades IV, V, VII, X, XI, gelatin, elastin, and fibronectin; stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11) which degrade fibronectin, PG core protein, collagen types IV, V, IX, and X, laminin, and elastin; PUMP-1(MMP-7) that degrades collagen type IV, gelatin, laminin, fibronectin, and PG core protein; and metalloelastase (MMP-12) that degrades elastin and fibronectin.

The expression of MMP gene is induced by transcription factors AP-1 and NF- κ B. See p.angel et al, Cell (1987) 49: 729-: AP-1 and NF-. kappa.B activities are mediated by cytokines (e.g., interleukins IL-1, IL-6 and TNF. alpha.), growth factors (TGF. alpha., bFGF) and external environmental influences (e.g., oxidizing agents, heat and ultraviolet radiation). AP-1 Induction and production of the jun proteins (C9-jun, jun-B and jun-D) and fos proteins (C-fos, fos-B, fra-1 and fra-2) that make up AP-1 are mediated by a number of molecules (e.g., RAC, CDC42, MEKK, JNK, RAS, RAF, MEK and ERK). AP-1 and NF-. kappa.B are known to be activated in mammalian cells exposed to UV. Devary et al, Science (1993) 262: 1442-1445.M.Wlaschek et al, Photochemistry and Photobiology (1994)59 (5): 550-556 also reports that fibroblasts under UVA irradiation cause IL-1 and IL-6 mediated MMP-1 induction, which may contribute to collagen loss in photoaging.

In addition, MMP inhibitors or transcription factors that affect MMP gene expression are also known. Hill et al, Biochem J (1995) 308: 167- & 175 describe two MP inhibitors, CT1166 and RO 31-7467. R. Gowravaram et al, J Med Chem (1995) 38: 2570-2581 describes the development of a series of hydroxamates which inhibit MMP and describes the thiols, phosphonates, phosphinates, aminophosphonates and N-carboxyalkyl compounds as known MMP inhibitors. The article states that MMP inhibitors include a zinc-chelating moiety and peptide fragments that bind to a small group of MMP-specific capsules. Hodgson in Biotechnology (1995) 13: the clinical use of several MMP inhibitors including Galardin (Galardin), Batimastat (Batimastat) and Marimastat (Marimastat) was reviewed in 554- & 557. Other MMP inhibitors include succinimides (J.G.Conway et al, J Exp Med (1995) 182: 449-.

Disclosure of the invention

The present invention is based on the following findings of the applicant: UVB irradiation rapidly potentiates AP-1 and NF- κ B and induces MMPs in exposed skin. The increased MMP activity produced by UVB irradiation degrades connective tissue proteins in the skin. These lesions, if not repaired completely, can lead to sun burn marks that accumulate during repeated UVB irradiation and can also cause photoaging.

Accordingly, applicants have prevented the photoaging of undamaged skin caused by skin exposure to UVB by applying an inhibitor of MMP inducible by UVB in an amount sufficient to inhibit the induction and/or activity of MMP inducible by UVB to the skin prior to the skin exposure to UVB. Surprisingly, this occurs at UVB doses below those that cause erythema and at UVB doses that cause erythema.

Another aspect of the present invention is the use of an inhibitor of MMP induction or activity inducible by UVB in the manufacture of a medicament for preventing the photoaging of undamaged skin due to repeated UVB irradiation.

Brief description of the drawings

FIG. 1 is a flow diagram showing the path of UVB-induced MMP production.

FIGS. 2a-d, 3a-b, 4a-d and 5a-e are graphs of the results of the tests described in the examples below.

Modes for carrying out the invention

The present invention is useful for inhibiting (i.e., slowing or preventing) photoaging of intact skin, i.e., the effect that skin does not exhibit photoaging. Thus, the treatment of the present invention should be performed on skin (e.g., skin on the head, neck, hands and arms that are routinely exposed to sunlight) before these skin show a warning sign of photoaging. Since repeated exposure to UVB at doses below those that cause erythema can lead to photoaging, the present invention should be performed on skin exposed to these low doses. In this respect, 30 to 50mJ/cm²The UVB dose of (a) causes erythema in most people with white skin. Thus, the present invention will prevent exposure to doses below this range (typically at about 5mJ/cm equivalent to several minutes of solar exposure)²Above) skin photoaging.

In the present invention, the method of preventing or suppressing photoaging is: inhibit UVB-induced degradation of the extracellular matrix of the skin by MMPs. It is achieved by applying MMP inhibitors to the skin which is to be exposed to sunlight. In this regard, the term "MMP inhibitor" refers to those agents that directly or indirectly inhibit (i.e., slow or eliminate) the expression of MMPs in the skin that can be induced by UVB or inhibit the enzymatic activity of these MMPs. By "indirectly inhibit" is meant binding to one or both of the transcription factors Ap-1 and NF- κ B and/or to one or more molecules involved in the three kinase cascades that induce Jum and fos in the skin, thereby reducing or eliminating the expression of MMPs that can be induced by UVB.

FIG. 1 is a schematic representation of MMP expression pathways inducible by UVB. As shown in FIG. 1, UVB irradiation produces Reactive Oxygen Intermediates (ROIs) that activate the activity of AP-1 and NF-. kappa.B, while activated AP-1 and NF-. kappa.B induce cytokines and growth factors. The interaction of cytokines and growth factors with their receptors triggers the small GTP-binding proteins RAC/CDC42 and RAS. These proteins activate the three cascade of kinases necessary for the production of the jun and fos proteins, which constitute AP-1. AP-1 induces the expression of certain MMPs. Agents that prevent photoaging may act on MMPs, transcription factors AP-1 and NF- κ B, and/or one or more molecules comprised in the three kinase cascades shown in fig. 1. Aspirin and E5510(T. Fujimmori et al, Jpn J Pharmacol (1991)55 (1): 81-91) described) inhibit NF-. kappa.B activation. Farnesyl transferase inhibitors such as B-581(A.M. Garcia et al J Biol Chem (1993)268 (25): 18415-18), BZA-5B (M.B. Dalton et al Cancer Res (1995) 3295-3304), farnesyl acetate and (. alpha. -hydroxyfarnesyl) phosphate act on RAS, inhibiting ERK cascade activation; and geranyl transferase inhibitors and lisofline inhibit JNK cascade activation. Compounds such as SB202190(J.C.Lee et al, Natrue (1994) 372: 739-. Retinoids (such as those disclosed in U.S. Pat. No.4,877,805) and dissociated retinoids specific for AP-1 antagonism (such as those described by Fanjul et al, Nature (1994) 372: 104-110), glycocortin, and vitamin D3 are directed against AP-1. In addition to retinol, other retinoids include natural and synthetic analogs of vitamin A (retinol), vitamin A aldehyde (retinal), retinoids (retinoic acid, including all-trans and 13-cis retinoic acid), and other compounds described in EP379367A2, and finally MMP can be inhibited by BB2284 (described in A.J.H.Gearing et al Natrue (1994) 370: 555-.

It is desirable to topically apply one or more of these MMP inhibitors to skin that will be exposed to sunlight. For these applications, they are usually formulated as emulsions, gels, ointments, sprays or lotions. The inhibitors can be formulated with customary carriers which meet the pharmacological and cosmetic requirements. Examples of such vectors are described in U.S. Pat. No.4,877,805 and European patent publication EP0586106A 1. As noted, there may be one or more inhibitors in a given formulation. For example, a combination of inhibitors acting on two or more different molecules involved in effecting MMP degradation of the skin can be used. The preparation may also contain additives such as emollients, skin penetration enhancers, pigments and fragrances. In addition, the formulation may contain absorbent particles (e.g., polymeric beads) that allow for sustained release of the inhibitor onto the skin. The concentration of inhibitor in the formulation (by weight) is typically 0.01 to 10%, more typically 0.1 to 1%. Typically about 50mg of the formulation is applied per square centimeter of skin.

It is desirable to apply the inhibitor prior to exposure of the intact skin to sunlight. The administration schedule (i.e., daily, weekly, etc.) will depend primarily on the lifetime of the inhibitors (e.g., metabolism in the skin, half-life) and the target molecules for which they act. In addition, it is affected by bathing in sea, perspiration and the degree of sun exposure. Typically, the inhibitor is administered daily.

The invention is further described below by way of examples. However, these examples are not intended to limit the present invention.

Examples molecular-based determination of UVB-induced photoaging high UVB dose-induced MMPs

The change in MMP-1, MMP-3, MMP-9, and MMP-2, mRNA, protein, and enzyme activity over time after UVB irradiation was determined as follows.

The subject was a caucasian adult (approximately equal in number of males and females) with mild to moderate pigmentation. At 24 hours post-irradiation, the UVB dose (minimum erythemal dose, i.e., "MED") that caused only a perceptible reddening was determined for each subject. All subjects had 1MED ranging from 30-50mJ/cm². The buttocks of the subject were irradiated with an Ultralite Panelite lamp containing 4F 36T12 ERE-VHO UVB tubes. The intensity of the radiation was monitored with an IL443 phototherapeutic radiometer and an SED240/UVB/W photodetector. UVB output at 48cm from the light source of 0.5mW/cm². At 8, 16, 24, 48 and 72 hours after irradiation, the skin was removed from 4 sites (one was unirradiated and 3 were irradiated) of each subject with a keratome. Tissues were flash frozen and frozen using g.j.fisher et al, J Invest dermaltol (1991) 96: 699 Nort as described in 707Total RNA was isolated and analyzed by southern blotting. The band intensity was quantified using a PhosphorImager. The MMP transcript background values were normalized to the value of the control gene 36B 4. The results of these experiments are shown in FIG. 2a (MMP-1), 2b (MMP-3), 2c (MMP-9), and 2d (MMP-2). Results are mean ± SEM (8, 16, 48 and 72 hours n 6, no UVB control and 24 hours n 17) expressed as the fold increase of normalized values relative to non-irradiated skin. The bands appearing in the figure are a composite of several individuals.

As shown in FIGS. 2a-d, induction of MMP-1, MMP-3 and MMP-9 reached a maximum (6-60 fold) in 16-24 hours, returning to near baseline in 48-72 hours. MMP-2 mRNA was detected, but only 1.6-fold increase was observed 24 hours after irradiation. The time-dependent profiles of MMP-1 and MMP-9 induction and activity elicited by 2MED UVB were parallel to those observed for their mRNA. Neither MMP-2 nor activity was induced.

Northern analysis of UVB-treated skin with a probe specific for MMP-3 (stromelysin I) gave the same results as with the full-length MMP-3 probe (FIG. 2b), whereas hybridization with a probe specific for MMP-10 (stromelysin II) gave no signal. This indicates that, among the stromelysins, UVB mainly induces stromelysin I. Low dose UVB induction of MMPs

As described above, the subject is exposed to UVB at a dose of 0.01-2 MED. Skin samples (6mm cylinders) were obtained in full thickness from the treated and untreated sites 24 hours after irradiation. Samples were incubated in 20mM Tris HCl (pH7.6), 5mM CaCl₂Homogenized and centrifuged at 3000 Xg for 10 min. The following assays were performed with the supernatant: MMP-1 and MMP-9 proteins were determined by Western blotting (100. mu.g/lane) using chemiluminescence detection, as described by C.L.Hu et al (Anal Biochem (1978) 88: 638-³Hydrolysis of H-fibrillar collagen (100. mu.g/assay) and activity was determined by zymographic analysis of gelatin according to the method of M.S. Hibbs et al (J Biol Chem (1985) 260: 2493-. MMP-1 and MMP-9 antibodies used were described in J Cell Biol (1989) 109: 877 Biochem J (1989)258 from 889 and G.Murphy et al: 463-472 are described. Knots for these testsSee fig. 3a and 3b for the results.

In FIG. 3a, MMP-1 protein values are indicated by open bars, while MMP-1 activity values are indicated by cross-hatched bars. FIG. 3a is an inset of a typical Western blot of two subjects, respectively. The larger 54kDa band is the intact MMP-1 and the smaller 45kDa band is the proteolytically activated form of MMP-1.

In FIG. 3b, MMP-9 protein values are indicated by open bars, while MMP-9 activity values are indicated by cross-hatched bars. FIG. 3b inset is a typical Western blot (left panel) and a typical zymogram (right panel). The multiple bands on the zymogram are proteolytically activated forms of MMP-9.

The intensity of the bands was quantified by laser densitometry. Results are given as mean ± SEM of n ═ 10.

As shown in FIGS. 3a and 3b, the induction of MMP-1 and MMP-9 proteins and activities is dose dependent, as MMP changes in both protein and activity are reflected in each other. MMP-1 was induced by all UVB doses tested, while MMP-9 was induced by doses ≧ 0.1 MED. Induction reached a maximum at 1MED, and was approximately half the maximum at 0.1 MED. A 0.1MED corresponds to a summer exposure to sunlight for 2-3 minutes, which does not cause any perceived skin redness. Low dose UVB induces AP-1 and NF-kB

The subject is irradiated and a tissue sample is removed as described above. Nuclear extracts were prepared according to the method of G.J.Fisher et al (J Biol Chem (1994) 269: 20629-20635). Contains (1) to⁸Cell biopsies (approximately 200mg wet weight) produced on average 500. mu.g of nuclear extracted protein. According to the method of G.J.Fisher et al (same as above)³²P-labeled DMA probes containing the same and mutated DNA binding sequences of AP-1 and NF-. kappa.B were subjected to electrophoretic migration shift assays (8. mu. nuclear extraction of protein). Antibodies against the supratranslocation band were obtained using Santa Cruz biotechnology. The Jun and fos antibodies have broad reactivity against all Jun and fos families of proteins, respectively. NF-. kappa.B antibodies are specific for p 65/RelA. The results of these tests are shown in FIGS. 4a, 4b, 4c and 4d (NS refers to the non-specific example). The insets in these figures are typical AP-1 andNF-. kappa.B blocked complexes. + Compet refers to the addition of a 100-fold excess of unlabeled probe; mut means mutated³²And (3) a P probe.

FIG. 4a is AP-1 and NF- κ B bound to unirradiated and irradiated (4 hours after 2MED UVB irradiation) skin. As shown in FIG. 4a, the binding of both transcription factors to their DNA response elements is specific, which is manifested by a loss of blocked complex with the mutated labeled probe. Antibody translocation indicates that the specific AP-1 and NF-. kappa.B blocked complexes observed with extracts of UVB irradiated skin contain jun and fos proteins and Rel A protein, respectively.

FIGS. 4B and 4c are the induction of AP-1 and NF- κ B binding under 2MED UVB irradiation, respectively. The results are expressed as mean ± SEM of n ═ 9. As shown, induction by both factors occurred within 15 minutes.

FIG. 4d is a graph of the dose dependence induced by AP-1 (shown by open bars) and NF- κ B (shown by cross-hatching). DNA binding was measured 30 minutes after irradiation. As shown, both factors induced half-maximal at about 0.1MED and maximal at 1 MED. The dose-dependence of UVB induction by these factors closely corresponds to the values reported above for MMP-1 and MMP-9, consistent with the involvement of these transcription factors in the UVB-induced increase in both MMPs. Inhibition of UVB induction of AP-1, MMP-1 and MMP-9

Fisher et al (J Invest Dermatol (1991) 96: 699-707) apply (300mg/6 cm) all-trans retinoic acid (t-RA) and its vehicle (70% ethanol and 30% propylene glycol) or clobetasol propionate 0.05% Glucocorticosteroid (GC) and its vehicle (2% propylene glycol + 2% sorbitan monooleate-sorbitan dioleate in white petrolatum) to subjects²Skin) for 48 hours. The treated skin site was then irradiated with 2MED UVB. The resulting skin was subjected to AP-1 measurement 30 minutes after irradiation or MMP measurement 24 hours after irradiation as described above. AP-1 assay and MMP-1 and MMP-9 assays were performed as described previously. To determine whether t-RA caused changes in UVB-induced skin redness, 0.1% t-RA and its load were usedSubjects were treated for 24 hours. Using 10-80mJ/cm²The UVB irradiated treated area, and after 24 hours, the degree of skin redness was determined using a Minolta colorimeter. The results of these tests are shown in FIGS. 5a, 5b, 5c and 5 e.

FIG. 5a shows the result of AP-1 assay. As shown, pre-treatment of the skin with t-RA reduced UVB-induced AP-1DNA binding by about 70%.

FIGS. 5b and 5c are the results of MMP-1 and MMP-9 assays. As shown, t-RA pretreatment reduced UVB-induced MMP-1 and MMP-9mRNA, protein, and activity by 50-80%.

FIG. 5d is a test of the effect of t-RA pretreatment on skin redness. As shown, t-RA does not reduce UVB-induced skin redness, although t-RA absorption overlaps with the UVB range (t-RA λ max ═ 351 nm). This suggests that the observed reduction in AP-1 and MMP induction is specific and not due to absorption of UVB by t-RA.

Figure 5e is the effect of pre-treating skin with GC. As shown, GC pretreatment reduced MMP-1 and MMP-9 activity to a similar extent to that of the t-RA pretreatment.

The documents mentioned in the above description are hereby expressly incorporated herein by reference.

Claims (11)

1. A method of inhibiting photoaging of undamaged human skin from exposure to ultraviolet b (uvb) radiation, comprising: providing an inhibitor of at least one of: (a) MMP activity inducible by UVB irradiation in the skin, (B) one or both of the transcription factors AP-1 and NF- κ B, and (c) at least one of a GTP-binding protein or a kinase involved in the activation and/or production of jun or fos proteins constituting AP-1; and applying to the skin, prior to skin exposure, the inhibitor in an amount sufficient to inhibit UVB-inducible MMP production or activity, one or both of AP-1 and NF- κ B, or at least one of a GTP-binding protein or a kinase involved in the activation and/or production of jun or fos proteins comprising AP-1.

2. The method of claim 1, wherein said method inhibits photoaging induced by exposure to UVB doses below the minimum required to cause redness of the skin.

3. The method of claim 2, wherein the UVB dose is about 5mJ/CM²The above.

4. The method of claim 1, wherein the inhibitor inhibits the activity of at least one of AP-1 and NF- κ B.

5. The method of claim 1, wherein the inhibitor inhibits an MMP activity inducible by UVB.

6. The method of claim 1, wherein the inhibitor inhibits a kinase necessary for the production of a GTP-binding protein or a jun or fos protein.

7. The method of claim 4, wherein the inhibitor inhibits AP-1 and the inhibitor is a retinoid, glucocorticoid or vitamin D3.

8. The method of claim 4, wherein the inhibitor inhibits NF- κ B, and the inhibitor is glucocorticoid, aspirin, or E5510.

9. The method of claim 5, wherein the inhibitor is TIMP, galanin, batimastat, marimastat, or a hydroxamate.

10. The method of claim 6, wherein the inhibitor is a farnesyl transferase inhibitor, a geranylgeranyl transferase inhibitor, SB202190, or PD 98059.

11. Use of an inhibitor of matrix metalloprotease induced by ultraviolet B radiation in the manufacture of a medicament for preventing photoaging of intact human skin.