CA2219976A1 - An in vivo and in vitro model of cutaneous photoaging - Google Patents
An in vivo and in vitro model of cutaneous photoaging Download PDFInfo
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Abstract
A transgenic mouse capable of expressing human elastin promoter ois provided.
Mouse fibroblast cultures derived from this transgenic mouse are also provided. In addition methods of identifiying compounds capable of inhibiting cutaneous photodamaged with this transgenic mouse or fibroblast cultures derived from these mice are provided.
Mouse fibroblast cultures derived from this transgenic mouse are also provided. In addition methods of identifiying compounds capable of inhibiting cutaneous photodamaged with this transgenic mouse or fibroblast cultures derived from these mice are provided.
Description
W 096137237 PCTrUS96107337 AN IN VIVO AND IN VIT~O MODEL OF CUTANEOUS PHOTOAGING
R~ ~GROUND OF THE lN V ~-r~ ~lON
Cutaneous aging results from both intrinsic chronological aging and extrinsic sun-exposure. Montagna et al., J. Am.
5 Acad. Dermatol. 1989, 21:907-918; Kligman, L.H., Aging and the Skin. Raven Press, New York, 1989, pp. 331-346; Taylor et al., J. Am. Acad. Dermatol. 1990, 221:1-15. The majority of changes associated with an aged appearance result from chronic sun-damage. Warren et al., J. Am. Acad. Dermatol. 1991, 25:751-10 760; Frances, C. and Robert, L., Int. J. Dermatol. 1984,23:166-179. Dramatic alterations of the superficial dermis ~.~n~p~ny the deep wrinkles and laxity ~ - in photoaged skin. The major histopathologic alteration of photoaged skin is the A~-cl ~lation of material which, on routine 15 histopathologic ~x~ in~tion, has the st~;ning characteristics of elastin and is, thus, termed solar elastosis.
T ~nohistochemical st~ining has shown the poorly-formed fibers comprising solar elastosis to be composed of elastin (Chen et al., J. Invest. Dermatol. 1986, 87:334-337; Mera et al., Br. J.
20 Dermatol. 1987, 117:21-27) fibrillin (Chen et al., J. Invest.
Dermatol. 1986, 87:334-337; Dahlback et al., J. Invest.
Dermatol. 1990, 94:284-291; Bernstein et al., J. Invest.
Dermatol. 1994, 103:182-186) and versican, the normal components of elastic fibers (Zimmerman et al., J. Cell. Biol.
25 1994, 124:817-825). A coordinate increase in elastin, fibrillin and versican mRNAs has been demonstrated in fibroblasts derived from photodamaged skin, as compared to fibroblasts derived from normal skin from the same individuals.
W 096/37237 PCTrUS96/07337 Bernstein et al., J. Invest. Dermatol. 1994, 103:182-186.
Elevated elastin mRNA levels in sun-damaged skin result from enhanced elastin promoter activity, as shown by transient transfections of fibroblasts with a DNA construct composed of 5 the human elastin p o,.-~er linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Bernstein et al., J.
Invest. Dermatol . 1994, 103:182-186.
A transgenic mouse line which expresses the human elastin promoter/CAT construct has now been developed to further study 10 the role of elastin promoter activation in cutaneous photoaging. These mice express hl ~n elastin promoter activity in a tissue-specific and developmentally regulated ~nne~.
Promoter activity can be studied in this model as a function of small increases in ultraviolet radiation, ~~o~trating the 15 sensitivity of the assay. In addition quantitative data can be obt~ after only a single exposure to ultraviolet radiation.
This transgenic mouse and fibroblasts derived from this mouse are useful as in vivo and in vitro models to study cutaneous photoaging and in the identification of agents which may 20 protect against photoA~~ge.
SUMMARY OF THE lN V ~ ON
An object of the present invention is to provide a transgenic mouse capable of expressing human elastin promoter.
Another object of the present invention is to provide 25 mouse fibroblast cultures derived from a transgenic mouse capable of expressing human elastin promoter.
Method of identifying compounds capable of inhibiting cutaneous photodamage using either the transgenic mouse or fibroblasts derived from these mice are also provided.
30 DETAILED DESCRIPTION OF THE Ihv~NllON
Profound changes take place in the superficial dermis as a result of chronic sun-exposure. The major alteration is the deposition of massive amounts of abnormal elastic material, termed solar elastosis. It has been shown that solar elastosis 35 is ~ccompanied by elevations in elastin and fibrillin mRNAs and elastin pl- -~er activity. A transgenic mouse model useful as both an in vivo and in vltro model for studying cutaneous photoaging, and for testing c-~ A ounds that may inhibit 3 cutaneous photodamage has now been developed. Using this 5 transgenic mouse line, which expresses the human elastin promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene in a tissue-specific and developmentally regulated ~nn~, it is now possible to investigate the effects of ultraviolet A (UVA) and ultraviolet B (UVB) on h~ ~ elastin 10 promoter activity in vivo and in vitro.
In mice a single dose of WB (491.4 mJ/cm2) resulted in up to an 8.5-fold increase in promoter activity, while a more modest 1.8-fold increase was measured with WA (38.2 J/cm2).
In addition, in vitro studies revealed over a 30-fold increase 15 in elastin promoter activity in response to WB (5.5 mJ/cm2), while no change was measured in response to W A (2.2 J/cm2).
These results confirm the role of WB in elastin promoter activation in photoaging and WA as a contributing factor. In vitro results suggest a direct stimulatory effect of W on the 20 dermal fibroblasts, in addition to any contribution by pro-inflammatory cytokines from other inflammatory and resident cells of the dermis.
In the present invention, a transgenic mouse model has been developed which permits the investigation of human elastin 25 promoter activity in response to ultraviolet irradiation both in vivo by direct irradiation of mouse skin, and in vitro by irradiation of dermal fibroblasts grown from skin explants.
Previous studies investigating the effect of ultraviolet radiation on photoaging in ~ni~l models have measured elastic 30 fiber damage, solar elastosis, skin wrinkling, and skin sagging. The similarity between the mouse and human action spectra for development of edema and erythema in response to ultraviolet radiation suggests that mouse models may accurately mimic human responses to ultraviolet radiation. For example, 35 wrinkling of mouse skin mimics human wrinkling which occurs in chronically photodamaged skin. These changes take place more rapidly in mouse skin, permitting more rapid evaluation of WO 96/37237 PCT~US96/07337 ultraviolet radiation effects than may be possible in humans.
However, differences in several parameters measured in previous studies as also apparent. For example, mice do not develop erythema in a m-nn~ similar to hl~m-n~ and the elastic fiber 5 alterations in mice in response to ultraviolet radiation are ~ualitatively different from those occurring in chronically sun-damaged human skin. In addition, the numerous dermal cysts present in hairless mice proliferate in response to ultraviolet radiation, a response which has no clinical correlate in human 10 skin. Skin sagging, a response in certain mice to high dose W A, does not appear to have a histopathologic correlate in mice and may not have a clinical correlation to a human response. Finally, mouse skin is relatively thin in comparison to hl-m-n skin, thus, permitting better penetration of 15 ultraviolet radiation. This may, in part, account for the accentuated response of mouse skin to ultraviolet radiation.
However, the decreased thickness of mouse skin may also exaggerate the potential effects of W B as compared to WA, relative to h~ -n~, WB penetrates only the most superficial 20 dermis in hl ~n~, whereas in mice, the relative proportion of the dermis exposed to W B is much greater. Utili7in~ the human elastin promoter in the transgenic mouse model of the present, is believed to more accurately reflect the h~ " response to ultraviolet radiation.
The approximate time of -xi ~1 ~l~.. ,~er activation and the duration of promoter elevation after a single exposure of W A or WB radiation was determined. Mice were treated with a single dose of 245.7 mJ/cm2 of WB or 38.2 J/cm2 of WA.
Elastin promoter activity, as measured by CAT assay, was 30 ~x;m~l 24 hours after WB exposure with a 4.6-fold increase over controls. CAT activity remained elevated 72 hours after irradiation at nearly 2 times control levels. By 96 hours, the activity fell to below one-third that of controls. After WA
irradiation, CAT activity was m~xim~l 12-24 hours after light 35 exposure, ~o~trating a more modest increase of less than twice that of controls. This increase persisted until 48 hours after WA exposure. By 72 hours after WA exposure, CAT
activity fell to one-third that of controls. To determine the earliest increase in CAT activity following WB irradiation, mice were harvested 1, 2, 3 and 6 hours following WB exposure.
A 20% increase in CAT activity was measured 1 hour following 5 exposure while a 70% elevation was measured 2 hours after exposure.
Since CAT activity decreases as a function of age, time course experiments underestimated relative CAT activity for time points after the initial 12 hour time point. Controls for 10 time course experiments were harvested at the 12 hour time point. If the decrease in endogenous CAT activity is considered, CAT activity rem~;ne~ at, or near, ~ l levels until 72 hours after light exposure for WB-treated mice, and until 48 hours in W A-treated mice. By 72 hours after WA
15 exposure and 96 hours after WB exposure, CAT activity fell to one-third that of controls sacrificed 12 hours after light exposure. Untreated mice sacrificed 72 and 96 hours after 4-5 day old controls also ~ strated baseline CAT activity which was one-third that of the younger control mice. Thus, baseline 20 endogenous CAT activity in mice 8-9 days old decreases to one-third that of 5 day old mice from the same litter.
The dose-response relationship for elastin promoter activity in W B-treated mice was observed after only a single dose of WB. Other in Vt vo models of photoaging require 25 numerous treatments over a much longer period of time to ~mo~trate a measurable effect. Experimentally produced elastosis in mice was first produced by Sams et al. using very large amounts of ultraviolet radiation. J. Invest. Dermatol.
1964, 43:467-471. In these studies, one group of mice received 30 1,040 human ini ~1 erythema doses (MEDs) over 3 months from a bank of fluorescent tubes, while another group received 13,000 MEDs given over 52 weeks in 260 treatments. Elastosis was d~ ~-ctrated by histochemical st~in;ng for elastin and, in irradiated mice, demonstrated an increased elastin staining.
35 Since this initial report, a number of researchers have used murine models of cutaneous photoaging evaluating the production of dermal elastosis. Sams et al., J. Invest . Dermatol . 1964, W 096/37237 PCTrUS96/07337 43:467-471; Nakamura, K. and Johnson, W.C., J. I~vest.
Dermatol. 1968, 51:253-258; Berger et al., Arch. Dermatol. Res.
1980, 269:39-49; Kligman, L.H., Arch. Dermatol. Res. 1982, 272:229-238; Kligman et al., J. Invest. Dermatol. 1982, 78:181-5 189; Poulsen et al., Br. J. Dermatol. 1984, 110:531-538;
Kligman et al., J. Invest. Dermatol. 1985, 84:272-276; Bissett et al., Photochem. Photobiol. 1987, 46:367-376; Bissett et al., Photochem. Photob~ol. 1989, 50:763-769; Wulf et al., Photodermatology 1989, 6:44-51; Kligman, L.H. and Sayre, R.M., Photochem. Photob~ol. 1991, 53:237-242; and Moran, M. and Granstein, R.D., J. Invest. Dermatol. 1994, 103:797-800. The number of treatments with ultraviolet radiation in these studies ranges from 36 to 260 given over 13 to 62 weeks. This is in contrast to the single doses used in the current study.
15 Thus, the total dose of WB used to produce measurable solar elastosis using the same WB lamps in the present study, ranged from 6- (Bissett et al., Photochem. Photobiol. 1987, 46:367-376) to almost 40-fold (Poulsen et al., Br. J. Dermatol. 1984, 110:531-538) larger than the largest dose used with the model 20 of the present invention.
The elevation of the elastin promoter in response to WB
and WA was also determined to be dose dependent. Increasing doses of ultraviolet radiation were A~mi n~ stered and skin harvested 24 hours after light exposure. In response to UVB
25 irradiation at 30.7, 122.8 and 491.4 mJ/cmZ, CAT activity increased to 1.7-, 4.1- and 8.5-fold greater than controls, respectively. In response to WA irradiation, a more modest increase in CAT activity was seen. Doses of 9.5 and 38.2 J/cm2 resulted in increases of 1.6- and 1.7-fold over controls, 30 respectively.
In addition to requiring substantially shorter treatment times and doses of ultraviolet radiation, the transgenic mouse model of the present invention yields quantitative data. With the exception of Kligman and Sayre, Photochem. Photob~ol. 1991, 35 53:237-242, who used an image analysis ~ys~e~ to quantify elastosis, the parameters used to assess degree of elastosis in the prior art were evaluated subjectively.
W 096/37237 PCT~US96/07337 The effect of ultraviolet radiation on CAT activity in v~tro was also determined. Early passage fibroblasts derived from skin explants of the transgenic mice were irradiated and harvested for determination of CAT activity 24 hours later.
5 Doses of WB ranged from 0.7 to 10.9 mJ/cm2, with the highest doses resulting in over a 30-fold increase in CAT activity.
Promoter activity, as measured by CAT assay, peaked at a dose of 5.5 mJ/cm2, which corresponded with a treatment time of 40 seconds. CAT activity re~~ elevated at about 30-fold with 10 increasing WB doses, eventually resulting in a decrease in CAT
activity which corresponds with cell death. In contrast, WA
doses of up to 2.2 J/cm2, which corresponds with a light treatment time of over 18 minutes, did not increase CAT
activity in vitro. Longer treatment times resulted in cell 15 death.
The ability of treatment with a combination of 8-methoxypsoralen (8-MOP) and WA, referred to as P WA, was also found to upregulate elastin promoter activity in this model.
Treatment of skin di~A~ with P WA results in clinical 20 alterations in treated skin similar to those observed in chronically photoA~-ged skin. PW A-treated patients develop non-melanoma skin ~-~nc~s, pigmentary alterations and wrinkling characteristics of sun-induced changes. Fibroblast cultures treated with 8-MOP or WA alone exhibited no significant change 25 in CAT activity as compared to untreated controls. However, PW A-treated cell cultures demonstrated 2.6-, 13.2- and 2.0-fold increases in CAT activity in response to 1 J/cm2 of WA
with 8-MOP doses of 0.3, 1.0, and 3.0 ~g/ml of 8-MOP, respectively. Although the highest dose of 8-MOP (3 ~g/ml) 30 were expected to generate a greater number of photoadducts than lower doses, the induction of elastin promoter activity decreased significantly at this dose presumably due to decreased cell viability. Protein assays demonstrated a dose-dependent reduction in total protein harvested indicative of 35 cell death at the highest 8-MoP doses. Mice treated with 8-MOP
or WA alone did not exhibit a significant change in CAT
activity, as compared to untreated controls. However, PWA-W 096137237 PCTrUS96/07337 treated mice demonstrated a 3.1-fold increase in CAT activity demonstrating in vivo activation of the elastin promoter in response to P WA as well.
The transgenic mouse model of the present invention 5 provides a rapid, quantitative means of measuring human elastin pl~ ~er activity in response to single doses of ultraviolet radiation. ~nhA~ CAT activity was demonstrated in response to both WA and W B. Further, the ability to study the e~fects of ultraviolet radiation both in vivo and in vitro enables 10 further investigation of the ~S - h~n;~ responsible for elastin ~ er activation by ultraviolet radiation. This model also provides a tool for the rapid evaluation of sunscreens and other compounds thought to alter the effects of solar radiation. In addition, this model can be used to study the 15 effects of treatments such as psoralen on a specific gene as in the studies performed with PWA treatment.
Methods of identifying co...~ounds capable of inhibiting cutaneous photoAF ~ge using the mo~l~ of the present invention are also provided. In one emboAi~ent a test ~-t~ound is 20 applied to the skin of a transgenic mouse capable of expressing hl -n elastin promoter. The transgenic mouse is then exposed to ultraviolet radiation, either WB or WA and human elastin ~ er activity in the mouse is determined. Alternatively, the transgenic mouse is exposed to 8-MOP followed by WA. The 25 hl -n elastin promoter activity is then compared to that in transgenic mice also exposed to an equivalent dose of ultraviolet radiation which were not treated with the test compound to determine whether or not the test compound provided protection against the ultraviolet radiation. In another 30 ~mbo~i~?nt, fibroblast cells derived from a transgenic mouse capable of expressing human elastin promoter are treated with a test compound. The treated fibroblast cells are then exposed to WB radiation or 8-MOP followed by WA radiation and human elastin promoter activity in the fibroblast cells is 35 deter~;ne~. This activity is compared to fibroblast cells from the transgenic mice exposed to the same dose of WB radiation or 8-MOP followed by WA radiation but which were not treated W 096/37237 PCTrUS96/07337 g _ with the test compound to determine if the test compound provided protection against the exposure.
The following nonlimiting examples are provided to ~ further illustrate the present invention.
Example 1: Transgenic mice expressing the h - elastin ~ ~ -L~l A homozygous line of transgenic mice expressing the 5.2-kb hl ~n elastin promoter linked to a CAT reporter gene was 10 used. Hsu-Wong et al., J. Biol. Chem. 1994, 269:18072-18075.
These mice express the human elastin promoter in a tissue-specific and developmentally regulated l~nn~ . Mice four or five days old were used since at this age, visible hair growth is not yet present.
15 Example 2: Fibroblast Cultures Fibroblast cultures were established from the skin of transgenic mice by explanting tissue sp~ n~ onto the tissue culture plastic dishes and allowing cells to migrate to the surrolln~ing area. The primary cultures were maint~;n~ in 20 Dlllh~-o's modified Eagle's (DME) medium supplemented with 10%
fetal calf serum, 1 mM L-glutAmin~ and antibiotics at 37~C.
The primary cell cultures were passaged by trypsinization and the subcultures in passages 2 or 3 were utilized for radiation experiments. After exposure to ultraviolet radiation, the 25 cells were incubated in DME medium supplemented with 10% fetal calf serum for 24 hours, then harvested for determination of CAT activity as described in Example 3.
Example 3: CAT Assay To measure the expression of the human elastin 30 promoter/CAT reporter gene construct in the skin of transgenic mice and in fibroblast cultures established from these ~n;~-l S, CAT activity was determined. For extraction of the CAT from skin, the speci~ns were homogenized in 0.25 Tris-HCl, pH 7.5, using a tissue homogenizer (Brinkmann Instruments, Inc.
35 Westbury, NY). The homogenates were centrifuged at 10,000 X g =
W 096/37237 PCT~US~GJ'U73~7 for 15 minutes at 4~C and the protein ~n~c~ntration in the supernatant determined by a ~- ?~cial protein assay kit (Bio-Rad Laboratories, Ric-h~on~, CA). Aliquots of the supernatant cont~; n i ng 100 ~g of protein were used for assay of CAT
5 activity by incubation with tl~C] chlor~mr~en;col in accordance with well-known procedures. The acetylated and non-acetylated forms of radioactive chlor~mph~n;col were separated by thin-layer chromatography and CAT activity was deter~; ne~ by the radioactivity in the acetylated forms as a percent of the total 10 radioactivity in each sample.
Example 4: W Sources For ~m;~;stration of WB radiation, a closely spaced array of seven West;nghouse FS-40 sunlamps was used which delivered uniform irradiation at a distance of 35 cm.
15 Irradiating with W A was performed using seven Sylvania FR40T12 PWA lamps in the above mentioned array, filtered through window glass of 2 mm thi~-kn~c to 1~ ,v~ wavelengths below 320 nm. The energy output at 35 cm was measured with a Solar Light model 3D WA and W B detector (Solar Light ComrAny, 20 Philadelphia, PA). The output of FX-40 sunlamps was 23.4 units/hour of WB at 38 cm, where each unit is equivalent to 21 mJ/cm2 of erythema effective energy. The output for FR40T12 PWA lamps filtered through window glass was 2.02 mW/cm2, with no detectable WB radiation.
25 Example 5: Irradiation Mice were placed under the center of the light array and restrained with Adh~;ve tape, exposing their dorsal surfaces to the ultraviolet radiation at a distance of 35 cm from the fluorescent tubes. Untreated control mice were restrained in 30 a similar r~nn~, To determine the time of m~i m~ 1 promoter activation and the duration of elevated promoter activity following WA and WB irradiation, time course experiments were carried out. Doses were selected in accordance with amounts showing moderate promoter activation. Mice were irradiated for 35 one-half hour with WB (dose of 245.7 mJ/cm2) or with WA for -W O 96/37237 PCT~US96/07337 5.2 hours (dose of 38.2 J/cm~). Irradiated skin was then harvested over the next 72 to 96 hours for determination of CAT
activity. Control mice were sacrificed at the first time point (12 hours after irradiation). To determine the earliest 5 response of CAT activity to WB irradiation, mice were harvested 1, 2, 3 and 6 hours after W B exposure. Unirradiated mice were harvested 24, 48, 72 and 96 hours after control mice to determine the fall in endogenous CAT activity over time.
To determine the dose/response relationship for UVB, 10 doses of 30.7, 122.8 and 491.4 mJ/cm2 were ~in;stered over 0.06, 0.25 and 1 hours, respectively. The dose/response relationship for W A was determined utilizing doses of 9.5 and 38.2 J/cm2, ~ nistered over 1.3 and 5.2 hours, respectively.
For each experiment, only mice from the same litter were used.
15 Following light exposure, mice were returned to the mother for 24 hours and then sacrificed and skin harvested for determination of CAT activity. At least 2 mice were used for each dose or time point in each experiment.
Fibroblast cultures as described above were exposed for 20 5, 10, 20, 40 and 80 s~con~ of WB correspo~ing to doses of 0.7, 1.4, 2.7, 5.5 and 10.9 mJ/cm2, respectively. Cultures were exposed to WA for 2.3, 4.6, 9.2 and 18.4 minutes correspon~ing to doses of 0.3, 0.6, 1.1 and 2.2 J/cm2. To prevent light absorption by tissue culture medium, just prior 25 to irradiation, tissue culture medium was removed from cells and replaced with a thin layer o~ phosphate buffered saline (PBS) sufficient to cover the cells. Control unirradiated cells were also placed in PBS. Medium was replaced in all dishes i ~iately after the last light dose was administered.
30 Only fibroblasts from mice in the same litters were used for any given experiment and utilized in the first few passages.
Two dishes of cells were used for each time point.
Example 6: P WA Treatment In vitro: Fibroblast cultures obtained as described in 35 Example 2 were divided into 6 groups and treated as follows.
Control cells received no WA or 8-MOP. WA controls received CA 022l9976 l997-ll-24 W 096/37237 PCTrUS96/07337 1 J/cm2 of WA without prior incubation with 8-MOP. 8-MOP
controls received the highest dose of 8-MOP (3 ~g/ml) but no WA. P WA-treated cells were pr~in~l~hated with either 0.3, 1.0 or 3.0 ~g/ml of 8-MOP in phosphate buffered saline (PBS) and 5 then exposed to 1 J/cm2 of W A. 8-MOP was ~;n;stered by diluting 8-MOP in PBS to the desired concentration. Before application to cultures, tissue culture medium was removed and the cells were rinsed twice with PBS. Cells were incubated in 10 cm diameter dishes with 3 ml of 8-MOP for 12.5 minutes. WA
10 controls were placed in equal amounts of PBS for 12.5 minutes prior to irradiation with WA. After treatment, cells were cleansed twice with PBS and tissue culture medium was replaced in all ~;chec Only fibroblasts from mice representing the same litter were used for any given experiment, and utilized in 15 passages 2-3. Two dishes of cells were used for each time point. Cells were harvested for determination of CAT activit~
24 hours after phototreatment, because ~~;~~1 promoter activation occurs 24 hours after irradiation.
In vivo: Mice were placed under the center of the light 20 array and restrained with adhesive tape, exposing their dorsal surfaces to WA at a distance of 38 cm from the fluorescent tubes. Unirradiated control mice were restrained in a similar ~nne~. For each experiment, only mice from the same litter were used. 8-MOP-treated mice received 25 ~1 of an ethanolic 25 solution cont~n~ng 2 mg/ml of 8-MOP, applied twice to their backs 15 and 7.5 minutes before WA (10 J/cm2) exposure. 8-MOP
control mice received identical topical applications of 8-MOP.
PWA-treated mice received both 8-MOP and WA. Following phototreatment, the backs of the mice were rinsed twice with 30 70~ isopropyl alcohol pads to remove excess 8-MOP. Mice were sacrificed and skin harvested for determination of CAT activity 24 hours after phototreatment.
R~ ~GROUND OF THE lN V ~-r~ ~lON
Cutaneous aging results from both intrinsic chronological aging and extrinsic sun-exposure. Montagna et al., J. Am.
5 Acad. Dermatol. 1989, 21:907-918; Kligman, L.H., Aging and the Skin. Raven Press, New York, 1989, pp. 331-346; Taylor et al., J. Am. Acad. Dermatol. 1990, 221:1-15. The majority of changes associated with an aged appearance result from chronic sun-damage. Warren et al., J. Am. Acad. Dermatol. 1991, 25:751-10 760; Frances, C. and Robert, L., Int. J. Dermatol. 1984,23:166-179. Dramatic alterations of the superficial dermis ~.~n~p~ny the deep wrinkles and laxity ~ - in photoaged skin. The major histopathologic alteration of photoaged skin is the A~-cl ~lation of material which, on routine 15 histopathologic ~x~ in~tion, has the st~;ning characteristics of elastin and is, thus, termed solar elastosis.
T ~nohistochemical st~ining has shown the poorly-formed fibers comprising solar elastosis to be composed of elastin (Chen et al., J. Invest. Dermatol. 1986, 87:334-337; Mera et al., Br. J.
20 Dermatol. 1987, 117:21-27) fibrillin (Chen et al., J. Invest.
Dermatol. 1986, 87:334-337; Dahlback et al., J. Invest.
Dermatol. 1990, 94:284-291; Bernstein et al., J. Invest.
Dermatol. 1994, 103:182-186) and versican, the normal components of elastic fibers (Zimmerman et al., J. Cell. Biol.
25 1994, 124:817-825). A coordinate increase in elastin, fibrillin and versican mRNAs has been demonstrated in fibroblasts derived from photodamaged skin, as compared to fibroblasts derived from normal skin from the same individuals.
W 096/37237 PCTrUS96/07337 Bernstein et al., J. Invest. Dermatol. 1994, 103:182-186.
Elevated elastin mRNA levels in sun-damaged skin result from enhanced elastin promoter activity, as shown by transient transfections of fibroblasts with a DNA construct composed of 5 the human elastin p o,.-~er linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Bernstein et al., J.
Invest. Dermatol . 1994, 103:182-186.
A transgenic mouse line which expresses the human elastin promoter/CAT construct has now been developed to further study 10 the role of elastin promoter activation in cutaneous photoaging. These mice express hl ~n elastin promoter activity in a tissue-specific and developmentally regulated ~nne~.
Promoter activity can be studied in this model as a function of small increases in ultraviolet radiation, ~~o~trating the 15 sensitivity of the assay. In addition quantitative data can be obt~ after only a single exposure to ultraviolet radiation.
This transgenic mouse and fibroblasts derived from this mouse are useful as in vivo and in vitro models to study cutaneous photoaging and in the identification of agents which may 20 protect against photoA~~ge.
SUMMARY OF THE lN V ~ ON
An object of the present invention is to provide a transgenic mouse capable of expressing human elastin promoter.
Another object of the present invention is to provide 25 mouse fibroblast cultures derived from a transgenic mouse capable of expressing human elastin promoter.
Method of identifying compounds capable of inhibiting cutaneous photodamage using either the transgenic mouse or fibroblasts derived from these mice are also provided.
30 DETAILED DESCRIPTION OF THE Ihv~NllON
Profound changes take place in the superficial dermis as a result of chronic sun-exposure. The major alteration is the deposition of massive amounts of abnormal elastic material, termed solar elastosis. It has been shown that solar elastosis 35 is ~ccompanied by elevations in elastin and fibrillin mRNAs and elastin pl- -~er activity. A transgenic mouse model useful as both an in vivo and in vltro model for studying cutaneous photoaging, and for testing c-~ A ounds that may inhibit 3 cutaneous photodamage has now been developed. Using this 5 transgenic mouse line, which expresses the human elastin promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene in a tissue-specific and developmentally regulated ~nn~, it is now possible to investigate the effects of ultraviolet A (UVA) and ultraviolet B (UVB) on h~ ~ elastin 10 promoter activity in vivo and in vitro.
In mice a single dose of WB (491.4 mJ/cm2) resulted in up to an 8.5-fold increase in promoter activity, while a more modest 1.8-fold increase was measured with WA (38.2 J/cm2).
In addition, in vitro studies revealed over a 30-fold increase 15 in elastin promoter activity in response to WB (5.5 mJ/cm2), while no change was measured in response to W A (2.2 J/cm2).
These results confirm the role of WB in elastin promoter activation in photoaging and WA as a contributing factor. In vitro results suggest a direct stimulatory effect of W on the 20 dermal fibroblasts, in addition to any contribution by pro-inflammatory cytokines from other inflammatory and resident cells of the dermis.
In the present invention, a transgenic mouse model has been developed which permits the investigation of human elastin 25 promoter activity in response to ultraviolet irradiation both in vivo by direct irradiation of mouse skin, and in vitro by irradiation of dermal fibroblasts grown from skin explants.
Previous studies investigating the effect of ultraviolet radiation on photoaging in ~ni~l models have measured elastic 30 fiber damage, solar elastosis, skin wrinkling, and skin sagging. The similarity between the mouse and human action spectra for development of edema and erythema in response to ultraviolet radiation suggests that mouse models may accurately mimic human responses to ultraviolet radiation. For example, 35 wrinkling of mouse skin mimics human wrinkling which occurs in chronically photodamaged skin. These changes take place more rapidly in mouse skin, permitting more rapid evaluation of WO 96/37237 PCT~US96/07337 ultraviolet radiation effects than may be possible in humans.
However, differences in several parameters measured in previous studies as also apparent. For example, mice do not develop erythema in a m-nn~ similar to hl~m-n~ and the elastic fiber 5 alterations in mice in response to ultraviolet radiation are ~ualitatively different from those occurring in chronically sun-damaged human skin. In addition, the numerous dermal cysts present in hairless mice proliferate in response to ultraviolet radiation, a response which has no clinical correlate in human 10 skin. Skin sagging, a response in certain mice to high dose W A, does not appear to have a histopathologic correlate in mice and may not have a clinical correlation to a human response. Finally, mouse skin is relatively thin in comparison to hl-m-n skin, thus, permitting better penetration of 15 ultraviolet radiation. This may, in part, account for the accentuated response of mouse skin to ultraviolet radiation.
However, the decreased thickness of mouse skin may also exaggerate the potential effects of W B as compared to WA, relative to h~ -n~, WB penetrates only the most superficial 20 dermis in hl ~n~, whereas in mice, the relative proportion of the dermis exposed to W B is much greater. Utili7in~ the human elastin promoter in the transgenic mouse model of the present, is believed to more accurately reflect the h~ " response to ultraviolet radiation.
The approximate time of -xi ~1 ~l~.. ,~er activation and the duration of promoter elevation after a single exposure of W A or WB radiation was determined. Mice were treated with a single dose of 245.7 mJ/cm2 of WB or 38.2 J/cm2 of WA.
Elastin promoter activity, as measured by CAT assay, was 30 ~x;m~l 24 hours after WB exposure with a 4.6-fold increase over controls. CAT activity remained elevated 72 hours after irradiation at nearly 2 times control levels. By 96 hours, the activity fell to below one-third that of controls. After WA
irradiation, CAT activity was m~xim~l 12-24 hours after light 35 exposure, ~o~trating a more modest increase of less than twice that of controls. This increase persisted until 48 hours after WA exposure. By 72 hours after WA exposure, CAT
activity fell to one-third that of controls. To determine the earliest increase in CAT activity following WB irradiation, mice were harvested 1, 2, 3 and 6 hours following WB exposure.
A 20% increase in CAT activity was measured 1 hour following 5 exposure while a 70% elevation was measured 2 hours after exposure.
Since CAT activity decreases as a function of age, time course experiments underestimated relative CAT activity for time points after the initial 12 hour time point. Controls for 10 time course experiments were harvested at the 12 hour time point. If the decrease in endogenous CAT activity is considered, CAT activity rem~;ne~ at, or near, ~ l levels until 72 hours after light exposure for WB-treated mice, and until 48 hours in W A-treated mice. By 72 hours after WA
15 exposure and 96 hours after WB exposure, CAT activity fell to one-third that of controls sacrificed 12 hours after light exposure. Untreated mice sacrificed 72 and 96 hours after 4-5 day old controls also ~ strated baseline CAT activity which was one-third that of the younger control mice. Thus, baseline 20 endogenous CAT activity in mice 8-9 days old decreases to one-third that of 5 day old mice from the same litter.
The dose-response relationship for elastin promoter activity in W B-treated mice was observed after only a single dose of WB. Other in Vt vo models of photoaging require 25 numerous treatments over a much longer period of time to ~mo~trate a measurable effect. Experimentally produced elastosis in mice was first produced by Sams et al. using very large amounts of ultraviolet radiation. J. Invest. Dermatol.
1964, 43:467-471. In these studies, one group of mice received 30 1,040 human ini ~1 erythema doses (MEDs) over 3 months from a bank of fluorescent tubes, while another group received 13,000 MEDs given over 52 weeks in 260 treatments. Elastosis was d~ ~-ctrated by histochemical st~in;ng for elastin and, in irradiated mice, demonstrated an increased elastin staining.
35 Since this initial report, a number of researchers have used murine models of cutaneous photoaging evaluating the production of dermal elastosis. Sams et al., J. Invest . Dermatol . 1964, W 096/37237 PCTrUS96/07337 43:467-471; Nakamura, K. and Johnson, W.C., J. I~vest.
Dermatol. 1968, 51:253-258; Berger et al., Arch. Dermatol. Res.
1980, 269:39-49; Kligman, L.H., Arch. Dermatol. Res. 1982, 272:229-238; Kligman et al., J. Invest. Dermatol. 1982, 78:181-5 189; Poulsen et al., Br. J. Dermatol. 1984, 110:531-538;
Kligman et al., J. Invest. Dermatol. 1985, 84:272-276; Bissett et al., Photochem. Photobiol. 1987, 46:367-376; Bissett et al., Photochem. Photob~ol. 1989, 50:763-769; Wulf et al., Photodermatology 1989, 6:44-51; Kligman, L.H. and Sayre, R.M., Photochem. Photob~ol. 1991, 53:237-242; and Moran, M. and Granstein, R.D., J. Invest. Dermatol. 1994, 103:797-800. The number of treatments with ultraviolet radiation in these studies ranges from 36 to 260 given over 13 to 62 weeks. This is in contrast to the single doses used in the current study.
15 Thus, the total dose of WB used to produce measurable solar elastosis using the same WB lamps in the present study, ranged from 6- (Bissett et al., Photochem. Photobiol. 1987, 46:367-376) to almost 40-fold (Poulsen et al., Br. J. Dermatol. 1984, 110:531-538) larger than the largest dose used with the model 20 of the present invention.
The elevation of the elastin promoter in response to WB
and WA was also determined to be dose dependent. Increasing doses of ultraviolet radiation were A~mi n~ stered and skin harvested 24 hours after light exposure. In response to UVB
25 irradiation at 30.7, 122.8 and 491.4 mJ/cmZ, CAT activity increased to 1.7-, 4.1- and 8.5-fold greater than controls, respectively. In response to WA irradiation, a more modest increase in CAT activity was seen. Doses of 9.5 and 38.2 J/cm2 resulted in increases of 1.6- and 1.7-fold over controls, 30 respectively.
In addition to requiring substantially shorter treatment times and doses of ultraviolet radiation, the transgenic mouse model of the present invention yields quantitative data. With the exception of Kligman and Sayre, Photochem. Photob~ol. 1991, 35 53:237-242, who used an image analysis ~ys~e~ to quantify elastosis, the parameters used to assess degree of elastosis in the prior art were evaluated subjectively.
W 096/37237 PCT~US96/07337 The effect of ultraviolet radiation on CAT activity in v~tro was also determined. Early passage fibroblasts derived from skin explants of the transgenic mice were irradiated and harvested for determination of CAT activity 24 hours later.
5 Doses of WB ranged from 0.7 to 10.9 mJ/cm2, with the highest doses resulting in over a 30-fold increase in CAT activity.
Promoter activity, as measured by CAT assay, peaked at a dose of 5.5 mJ/cm2, which corresponded with a treatment time of 40 seconds. CAT activity re~~ elevated at about 30-fold with 10 increasing WB doses, eventually resulting in a decrease in CAT
activity which corresponds with cell death. In contrast, WA
doses of up to 2.2 J/cm2, which corresponds with a light treatment time of over 18 minutes, did not increase CAT
activity in vitro. Longer treatment times resulted in cell 15 death.
The ability of treatment with a combination of 8-methoxypsoralen (8-MOP) and WA, referred to as P WA, was also found to upregulate elastin promoter activity in this model.
Treatment of skin di~A~ with P WA results in clinical 20 alterations in treated skin similar to those observed in chronically photoA~-ged skin. PW A-treated patients develop non-melanoma skin ~-~nc~s, pigmentary alterations and wrinkling characteristics of sun-induced changes. Fibroblast cultures treated with 8-MOP or WA alone exhibited no significant change 25 in CAT activity as compared to untreated controls. However, PW A-treated cell cultures demonstrated 2.6-, 13.2- and 2.0-fold increases in CAT activity in response to 1 J/cm2 of WA
with 8-MOP doses of 0.3, 1.0, and 3.0 ~g/ml of 8-MOP, respectively. Although the highest dose of 8-MOP (3 ~g/ml) 30 were expected to generate a greater number of photoadducts than lower doses, the induction of elastin promoter activity decreased significantly at this dose presumably due to decreased cell viability. Protein assays demonstrated a dose-dependent reduction in total protein harvested indicative of 35 cell death at the highest 8-MoP doses. Mice treated with 8-MOP
or WA alone did not exhibit a significant change in CAT
activity, as compared to untreated controls. However, PWA-W 096137237 PCTrUS96/07337 treated mice demonstrated a 3.1-fold increase in CAT activity demonstrating in vivo activation of the elastin promoter in response to P WA as well.
The transgenic mouse model of the present invention 5 provides a rapid, quantitative means of measuring human elastin pl~ ~er activity in response to single doses of ultraviolet radiation. ~nhA~ CAT activity was demonstrated in response to both WA and W B. Further, the ability to study the e~fects of ultraviolet radiation both in vivo and in vitro enables 10 further investigation of the ~S - h~n;~ responsible for elastin ~ er activation by ultraviolet radiation. This model also provides a tool for the rapid evaluation of sunscreens and other compounds thought to alter the effects of solar radiation. In addition, this model can be used to study the 15 effects of treatments such as psoralen on a specific gene as in the studies performed with PWA treatment.
Methods of identifying co...~ounds capable of inhibiting cutaneous photoAF ~ge using the mo~l~ of the present invention are also provided. In one emboAi~ent a test ~-t~ound is 20 applied to the skin of a transgenic mouse capable of expressing hl -n elastin promoter. The transgenic mouse is then exposed to ultraviolet radiation, either WB or WA and human elastin ~ er activity in the mouse is determined. Alternatively, the transgenic mouse is exposed to 8-MOP followed by WA. The 25 hl -n elastin promoter activity is then compared to that in transgenic mice also exposed to an equivalent dose of ultraviolet radiation which were not treated with the test compound to determine whether or not the test compound provided protection against the ultraviolet radiation. In another 30 ~mbo~i~?nt, fibroblast cells derived from a transgenic mouse capable of expressing human elastin promoter are treated with a test compound. The treated fibroblast cells are then exposed to WB radiation or 8-MOP followed by WA radiation and human elastin promoter activity in the fibroblast cells is 35 deter~;ne~. This activity is compared to fibroblast cells from the transgenic mice exposed to the same dose of WB radiation or 8-MOP followed by WA radiation but which were not treated W 096/37237 PCTrUS96/07337 g _ with the test compound to determine if the test compound provided protection against the exposure.
The following nonlimiting examples are provided to ~ further illustrate the present invention.
Example 1: Transgenic mice expressing the h - elastin ~ ~ -L~l A homozygous line of transgenic mice expressing the 5.2-kb hl ~n elastin promoter linked to a CAT reporter gene was 10 used. Hsu-Wong et al., J. Biol. Chem. 1994, 269:18072-18075.
These mice express the human elastin promoter in a tissue-specific and developmentally regulated l~nn~ . Mice four or five days old were used since at this age, visible hair growth is not yet present.
15 Example 2: Fibroblast Cultures Fibroblast cultures were established from the skin of transgenic mice by explanting tissue sp~ n~ onto the tissue culture plastic dishes and allowing cells to migrate to the surrolln~ing area. The primary cultures were maint~;n~ in 20 Dlllh~-o's modified Eagle's (DME) medium supplemented with 10%
fetal calf serum, 1 mM L-glutAmin~ and antibiotics at 37~C.
The primary cell cultures were passaged by trypsinization and the subcultures in passages 2 or 3 were utilized for radiation experiments. After exposure to ultraviolet radiation, the 25 cells were incubated in DME medium supplemented with 10% fetal calf serum for 24 hours, then harvested for determination of CAT activity as described in Example 3.
Example 3: CAT Assay To measure the expression of the human elastin 30 promoter/CAT reporter gene construct in the skin of transgenic mice and in fibroblast cultures established from these ~n;~-l S, CAT activity was determined. For extraction of the CAT from skin, the speci~ns were homogenized in 0.25 Tris-HCl, pH 7.5, using a tissue homogenizer (Brinkmann Instruments, Inc.
35 Westbury, NY). The homogenates were centrifuged at 10,000 X g =
W 096/37237 PCT~US~GJ'U73~7 for 15 minutes at 4~C and the protein ~n~c~ntration in the supernatant determined by a ~- ?~cial protein assay kit (Bio-Rad Laboratories, Ric-h~on~, CA). Aliquots of the supernatant cont~; n i ng 100 ~g of protein were used for assay of CAT
5 activity by incubation with tl~C] chlor~mr~en;col in accordance with well-known procedures. The acetylated and non-acetylated forms of radioactive chlor~mph~n;col were separated by thin-layer chromatography and CAT activity was deter~; ne~ by the radioactivity in the acetylated forms as a percent of the total 10 radioactivity in each sample.
Example 4: W Sources For ~m;~;stration of WB radiation, a closely spaced array of seven West;nghouse FS-40 sunlamps was used which delivered uniform irradiation at a distance of 35 cm.
15 Irradiating with W A was performed using seven Sylvania FR40T12 PWA lamps in the above mentioned array, filtered through window glass of 2 mm thi~-kn~c to 1~ ,v~ wavelengths below 320 nm. The energy output at 35 cm was measured with a Solar Light model 3D WA and W B detector (Solar Light ComrAny, 20 Philadelphia, PA). The output of FX-40 sunlamps was 23.4 units/hour of WB at 38 cm, where each unit is equivalent to 21 mJ/cm2 of erythema effective energy. The output for FR40T12 PWA lamps filtered through window glass was 2.02 mW/cm2, with no detectable WB radiation.
25 Example 5: Irradiation Mice were placed under the center of the light array and restrained with Adh~;ve tape, exposing their dorsal surfaces to the ultraviolet radiation at a distance of 35 cm from the fluorescent tubes. Untreated control mice were restrained in 30 a similar r~nn~, To determine the time of m~i m~ 1 promoter activation and the duration of elevated promoter activity following WA and WB irradiation, time course experiments were carried out. Doses were selected in accordance with amounts showing moderate promoter activation. Mice were irradiated for 35 one-half hour with WB (dose of 245.7 mJ/cm2) or with WA for -W O 96/37237 PCT~US96/07337 5.2 hours (dose of 38.2 J/cm~). Irradiated skin was then harvested over the next 72 to 96 hours for determination of CAT
activity. Control mice were sacrificed at the first time point (12 hours after irradiation). To determine the earliest 5 response of CAT activity to WB irradiation, mice were harvested 1, 2, 3 and 6 hours after W B exposure. Unirradiated mice were harvested 24, 48, 72 and 96 hours after control mice to determine the fall in endogenous CAT activity over time.
To determine the dose/response relationship for UVB, 10 doses of 30.7, 122.8 and 491.4 mJ/cm2 were ~in;stered over 0.06, 0.25 and 1 hours, respectively. The dose/response relationship for W A was determined utilizing doses of 9.5 and 38.2 J/cm2, ~ nistered over 1.3 and 5.2 hours, respectively.
For each experiment, only mice from the same litter were used.
15 Following light exposure, mice were returned to the mother for 24 hours and then sacrificed and skin harvested for determination of CAT activity. At least 2 mice were used for each dose or time point in each experiment.
Fibroblast cultures as described above were exposed for 20 5, 10, 20, 40 and 80 s~con~ of WB correspo~ing to doses of 0.7, 1.4, 2.7, 5.5 and 10.9 mJ/cm2, respectively. Cultures were exposed to WA for 2.3, 4.6, 9.2 and 18.4 minutes correspon~ing to doses of 0.3, 0.6, 1.1 and 2.2 J/cm2. To prevent light absorption by tissue culture medium, just prior 25 to irradiation, tissue culture medium was removed from cells and replaced with a thin layer o~ phosphate buffered saline (PBS) sufficient to cover the cells. Control unirradiated cells were also placed in PBS. Medium was replaced in all dishes i ~iately after the last light dose was administered.
30 Only fibroblasts from mice in the same litters were used for any given experiment and utilized in the first few passages.
Two dishes of cells were used for each time point.
Example 6: P WA Treatment In vitro: Fibroblast cultures obtained as described in 35 Example 2 were divided into 6 groups and treated as follows.
Control cells received no WA or 8-MOP. WA controls received CA 022l9976 l997-ll-24 W 096/37237 PCTrUS96/07337 1 J/cm2 of WA without prior incubation with 8-MOP. 8-MOP
controls received the highest dose of 8-MOP (3 ~g/ml) but no WA. P WA-treated cells were pr~in~l~hated with either 0.3, 1.0 or 3.0 ~g/ml of 8-MOP in phosphate buffered saline (PBS) and 5 then exposed to 1 J/cm2 of W A. 8-MOP was ~;n;stered by diluting 8-MOP in PBS to the desired concentration. Before application to cultures, tissue culture medium was removed and the cells were rinsed twice with PBS. Cells were incubated in 10 cm diameter dishes with 3 ml of 8-MOP for 12.5 minutes. WA
10 controls were placed in equal amounts of PBS for 12.5 minutes prior to irradiation with WA. After treatment, cells were cleansed twice with PBS and tissue culture medium was replaced in all ~;chec Only fibroblasts from mice representing the same litter were used for any given experiment, and utilized in 15 passages 2-3. Two dishes of cells were used for each time point. Cells were harvested for determination of CAT activit~
24 hours after phototreatment, because ~~;~~1 promoter activation occurs 24 hours after irradiation.
In vivo: Mice were placed under the center of the light 20 array and restrained with adhesive tape, exposing their dorsal surfaces to WA at a distance of 38 cm from the fluorescent tubes. Unirradiated control mice were restrained in a similar ~nne~. For each experiment, only mice from the same litter were used. 8-MOP-treated mice received 25 ~1 of an ethanolic 25 solution cont~n~ng 2 mg/ml of 8-MOP, applied twice to their backs 15 and 7.5 minutes before WA (10 J/cm2) exposure. 8-MOP
control mice received identical topical applications of 8-MOP.
PWA-treated mice received both 8-MOP and WA. Following phototreatment, the backs of the mice were rinsed twice with 30 70~ isopropyl alcohol pads to remove excess 8-MOP. Mice were sacrificed and skin harvested for determination of CAT activity 24 hours after phototreatment.
Claims (2)
1. A method of identifying compounds capable of inhibiting cutaneous photodamage comprising:
(a) applying a test compound to skin of a transgenic mouse capable of expressing human elastin promoter;
(b) exposing the transgenic mouse to UVB radiation, UVA
radiation, or 8-methoxypsoralen followed by UVA radiation; and (c) measuring human elastin promoter activity in the transgenic mouse.
(a) applying a test compound to skin of a transgenic mouse capable of expressing human elastin promoter;
(b) exposing the transgenic mouse to UVB radiation, UVA
radiation, or 8-methoxypsoralen followed by UVA radiation; and (c) measuring human elastin promoter activity in the transgenic mouse.
2. A method of identifying compounds capable of inhibiting cutaneous photodamage comprising:
(a) contacting fibroblast cells derived from a transgenic mouse capable of expressing human elastin promoter with a test compound;
(b) exposing the fibroblast cells to UVB radiation, or 8-methoxypsoralen followed by UVA radiation; and (c) measuring human elastin promoter activity in the fibroblast cells.
(a) contacting fibroblast cells derived from a transgenic mouse capable of expressing human elastin promoter with a test compound;
(b) exposing the fibroblast cells to UVB radiation, or 8-methoxypsoralen followed by UVA radiation; and (c) measuring human elastin promoter activity in the fibroblast cells.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/449,826 | 1995-05-24 | ||
| US08/449,826 US5648061A (en) | 1995-05-24 | 1995-05-24 | In vivo and in vitro model of cutaneous photoaging |
| US28095P | 1995-06-16 | 1995-06-16 | |
| US60/000,280 | 1995-06-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2219976A1 true CA2219976A1 (en) | 1996-11-28 |
Family
ID=26667437
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2219976 Abandoned CA2219976A1 (en) | 1995-05-24 | 1996-05-21 | An in vivo and in vitro model of cutaneous photoaging |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0831931A4 (en) |
| JP (1) | JPH11507807A (en) |
| CA (1) | CA2219976A1 (en) |
| WO (1) | WO1996037237A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6689936B1 (en) | 1997-12-17 | 2004-02-10 | The General Hospital Corp. | Method for evaluating a compound for its effect on skin |
| EP1875927B1 (en) * | 1997-12-17 | 2011-10-26 | The General Hospital Corporation | Methods for evaluating a compound for its effect on skin |
| DE69837221T2 (en) * | 1997-12-17 | 2007-12-20 | The General Hospital Corp., Boston | METHOD OF EVALUATING COMPOUNDS RELATED TO THEIR EFFECT ON THE SKIN |
| AU760969B2 (en) * | 1999-02-23 | 2003-05-22 | Charles River Laboratories, Inc. | System and method for evaluating agents which prevent oxidative damage |
| US6753146B1 (en) * | 1999-02-23 | 2004-06-22 | Eric F. Bernstein | System and method for evaluating agents which prevent oxidative damage |
| CA2362585A1 (en) * | 1999-02-24 | 2000-08-31 | Eric F. Bernstein | An in vivo and in vitro model for cutaneous photoaging and oxidative damage |
| US7163679B1 (en) | 1999-05-06 | 2007-01-16 | The General Hospital Corporation | Versican and epithelial-mesenchymal interaction |
-
1996
- 1996-05-21 WO PCT/US1996/007337 patent/WO1996037237A1/en not_active Application Discontinuation
- 1996-05-21 EP EP96920342A patent/EP0831931A4/en not_active Withdrawn
- 1996-05-21 JP JP8535812A patent/JPH11507807A/en not_active Abandoned
- 1996-05-21 CA CA 2219976 patent/CA2219976A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0831931A1 (en) | 1998-04-01 |
| EP0831931A4 (en) | 2001-09-26 |
| JPH11507807A (en) | 1999-07-13 |
| WO1996037237A1 (en) | 1996-11-28 |
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