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1. WO2004022023 - REPAIR OF DNA MUTAGENIC DAMAGE

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[ EN ]

REPAIR OF DNA MUTAGENIC DAMAGE

The present invention relates to the use of equol, dehydroequol and isoflav-3-ene and isoflavan compounds in promoting repair of DNA mutagenic damage.

Metallothioneins (MT) are proteins synthesised or over expressed in response to DNA damaging agents e.g. UNR (Hansen et al 1997). In most of the studies in animals and tissue cultures, high does of radiation were used to induce MT, and therefore, it is difficult to extrapolate these results to low level or repeated exposures to UNR in humans (Cai et al 1999). Induced synthesis of MT is considered as one of the mechanisms involved in the adaptive response to low dose UNR exposure, and increased levels of MT appear to be associated with protection from UNR, possibly mediated through scavenging of ROS in the skin (Hanada, et al 1992). As well, MT is implicated in protecting against the immunosuppressive effects of UNR on cell-mediated responses as demonstrated in MT=I and II knockout mice (Reeve, et al 2000). UNR induces immunohistochemically detectable MT in keratinocytes and dermal fibroblasts concurrently with the photoconduction of p53, which suggests the these protein systems are protective and complimentary in function. MT is detectable in dermal fibroblasts from 2 hours post-UN (Anstey, et al l996).

Equol, dehydroequol, isofla-3-ene and isoflavan compounds and methods for producing the same are described in copending International Patent Application PCT/AU03/00427 and WO 98/08503 which are incorporated herein by reference.

UN exposed skin causes damage in DΝA which may give rise to carcinogenesis. The most common tumour in humans is the basal cell carcinoma (BCC) followed by squamous cell carcinoma (SCC), and more rarely malignant melanoma.

It has now been found by the applicant that compounds of the present invention, when applied to the skin, result in elevation of metallothioneins production in the skin, particularly the basal layer of irradiated skin.

As mentioned above, metallothioneins affect and promote repair of DNA mutagenic damage of skin subject to UN exposure.

In accordance with the present invention there is provided use of equol, dehydroequol, isoflav-3-ene or isoflavan structures for protecting skin from DΝA mutagenic damage associated with UN exposure.

In another aspect there is provided use of equol, dehydroequol, isoflav-3-ene or isoflavan structures for the over expression of metallothioneins in the skin, particularly the basal layer of skin.

In accordance with another aspect of this invention there is provided a method for protecting skin from UN induced DΝA mutagenic damage which comprises applying to skin a composition containing one or more of equol, dehydroequol, isoflav-3-ene, or isoflavan compounds in admixture with a dermally acceptable carrier.

Isoflav-3-ene and isoflavan compounds may be represented by the general formula (II)


in which
Ri, R2, R3 and R4 are independently hydrogen, hydroxy, OR9, OC(O)Rι0, OS(O)Rιo,

CHO, C(O)Rιo, COOH, CO20, CO R11R12, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or R3 and Rt are as previously defined, and Ri and R2 taken together with the carbon atoms to which they are attached form a five-membered ring selected from


Ri and R4 are as previously defined, and R2 and R3 taken together with the carbon atoms to which they are attached form a five-membered ring selected from


Ri and R2 are as previously defined, and R3 and R4 taken together with the carbon atoms to which they are attached form a five-membered ring selected from


and
wherein
R5, R6 and R7 are independently hydrogen, hydroxy, OR9, OC(O)Rιo, OS(O)Rι0, CHO,

C(O)Rιo, COOH, CO2Ri0, CONRπ2, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo,

Rg is hydrogen, hydroxy, alkyl, aryl, amino, thio, NRnRι , CONR11R12, C(O)R13 where

3 is hydrogen, alkyl, aryl, arylalkyl or an amino acid, or CO24 where Rχ4 is hydrogen, alkyl, haloalkyl, aryl or arylalkyl, R9 is alkyl, haloalkyl, aryl, arylalkyl, C(O)Rι3 where Rι3 is as previously defined, or

Si(Rι5)3 where each Rι5 is independently hydrogen, alkyl or aryl,
R10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an amino acid, alkylamino or dialkylamino,
Rπ is hydrogen, alkyl, arylalkyl, alkenyl, aryl, an amino acid, C(O)Rι3 where R13 is as previously defined, or CO24 where Rι4 is as previously defined,
R12 is hydrogen, alkyl or aryl, or
Rπ and Rι2 taken together with the nitrogen to which they are attached comprise pyrrolidinyl or piperidinyl,
the drawing "— " represents either a single bond or a double bond, preferably a double bond,
T is independently hydrogen, alkyl or aryl, and
X is O, NRι2 or S, preferably O,
including pharmaceutically acceptable salts and derivatives thereof.

Equol corresponds to the formula (II) when Ri, R2, R , R4, R6, R7 and R8 are hydrogen, R5 is hydroxy, X is O, and "™" is a single bond. Dehydroequol corresponds to formula (II) when Ri, R2, R3, R , Re, R7 and R8 are hydrogen, R5 is hydroxy, X is O and "— " is a double bond.

Dermally acceptable carriers and lotions are well known in the art, and are described for example in Remington's Pharmaceutical Sciences, Gennaro A. 18th Ed., Mack Publishing Co., Easton, PA, 1990, pp. 1492-1517. Any derrnatologically acceptable carrier can be used in the compositions of the invention. As used herein, "derrnatologically acceptable carrier" refers to vehicles, diluents, carriers, which can include adjuvants, additives, or excipients, known for use in dermatological compositions. The compositions of the invention include, but are not limited to, creams, ointments, solutions, sticks, wipes, cleansers and/or gels. The compounds of the present invention may be simply mixed, admixed or blended with suitable carriers to give compositions suitable for application to the skin. Dermally acceptable carriers may include one or more sunscreen agents. Sunscreens include those materials commonly used to block ultraviolet light. Illustrative compounds include the derivatives of cinnamate, PABA, and salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trademarks, Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen employed can vary depending upon the degree of protection desired from the sun's UN irradiation.

In a prefened embodiment one or more compounds of the formula (II) are formulated into cosmetic preparations. Examples of cosmetic formulations include creams, gels, powders, pastes, cakes and the like. Typically such cosmetics may be referred to as "make-up", and/or foundation (typically used to provide a smooth, even appearance to skin and as a base for coloured cosmetics).

Compounds of the formula (II) may be used in the compositions in an amount from 0.001% to 100%, preferably from 0.1% to 20%, most preferably from 0.1% to 10% w/w. For example, compositions may comprise 1 μm to 500 mmol equol or other compounds of the formula (II), such as 20 μm to 400 μm. The remainder of the composition will comprise one or more derrnatologically acceptable carriers and excipients as are well known in the art. One or more compounds may be utilised in the compositions, with equol and dehydroequol being particularly preferred. Compositions may be administered topically to the skin before, during and/or after sun exposure. Typically, doses of between about 1 to 500 mg per day, with doses between 2 to 100 mg per day being preferred.

In accordance with another aspects of this invention there is provided a method for the treatment, or amelioration or preventing the formation of skin cancer, such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma, which comprises applying to the skin of a subject a composition containing one or more of equol, dehydroequol, or an isoflav-3-ene or isoflavan compounds of the general formula (II).

In another aspect of this invention there is provided a method for increasing metallothionein production in the skin, such as the basal layer of skin, which comprises applying to skin one or more of equol, dehydroequol, isoflav-3-ene or isoflavan compound in association with a dermally acceptable carrier.

The applicant has further found that the compounds according to this invention promote DNA repair. The promotion of DNA repair may be by one or more of increasing the rate of repair of cyclobutane pyrimidine dimers (CPDs), promoting DNA repair by decreasing P53 expression, and/or by promoting the formation of metallothionein (MT).

The formation of CPD is considered to be an important lethal and mutagenic consequence of UNR exposure (Mitchell et al, 1989; Liardet et al, 2000). Animal models have demonstrated an inverse relationship between epidermal CPD repair and skin carcinogenesis (Young et al, 1996). The P53 protein (TP53) is expressed after DΝA damage by UN irradiation. P53 is a transcription factor which blocks cellular progression from Gl to S phase, thus preventing replication of damaged DΝA (Campbell et al, 1993). The P53 protein may act as a tumour promoting agent (Murphey et al, 2001).

This invention will be described with reference to the following, non-limiting examples.

Example 1
The effect of equol on the induction of CPD was examined in the skin of hairless mice (a standard model for human dermatological investigations) exposed to solar simulated ultraviolet radiation (SSUN). At various time points after SSUN, dorsal skin was excised, fixed for 6hr in a standard fixing medium (HistoChoice , Amersco Inc, Solon, Ohio, USA), processed and paraffin-embedded. Pyrimidine dimers were detected immunohistochemically using citric acid antigen retrieval and the H3 anti-pyrimidine dimer antibody. The number of dimer-positive cells was counted manually in 30 fields per mouse, at 40x magnification.

When equol lotion (containing 20 μM equol) was applied daily for 7 days prior to and following irradiation with 1 x 3MED of SSUN, the effect of equol was to reduce the initial induction of dimers, and to enhance the rate of their repair, as evidenced by a reduced number of dimers at 24 hr (Table 1).

Table 1: Induction of epidermal CPD-positive cells following UN irradiation

Time of collection Treatment CPD +ve cells/linear cm
Normal skin 0
lhr post-SSUN Vehicle + ssuv 300 ± 18
equol + SSUN 238 ± 22
Vehicle + SSUN 340 ± 55
24hr post-SSUN
equol + SSUN 167 + 17

Application of equol immediately after SSUN exposure (and continuing for 5d) resulted in significantly reduced dimers at 1 day post-inadiation (a significant reduction of 23%), and at 2d (a significant reduction of 42% -data not shown).

When equol lotion (20μM) was applied for both 7 days prior and 5 days after SSUN exposure, the reduction in CPD numbers was evident immediately and at 1, 24 and 48 hours after (p < 0.05; 54%, 50% and 26% reduction in the number of CPD respectively) compared with the control group (vehicle alone).

Example 2
Equol was applied to the skin of five human volunteers immediately after, and at 4 hours and 6 hours post-UN irradiation. A control lotion was also used containing no equol. Twenty-four hours after UN irradiation, skin biopsies were taken and MT production was measured using immunohistochemistry.

Table 2 shows the counts of cells in the basal epidermis and superficial dermis that demonstrated positive staining for MT. Approximately half of the cells in the basal epidermis constitutively expressed MT at baseline, whereas almost none of the cells in the more superficial layers of the epidermis expressed MT. At 24 hrs after exposure to 2.5 MED SSUN, there were apparent differences in the expression of MT in the basal layers of the epidermis between sections treated with equol and those treated with DMSO in base lotion (vehicle). In all 5 participants, the expression of MT was higher in the skin treated with equol, with the magnitude of the difference ranging from +4% to +21%.

Table 2: Proportion of cells staining positively for MT in the epidermis of five human volunteers, by treatment group
Total epidermis Upper epidermis Basal epidermis

Subject treatment neg pos % neg pos % neg pos %

ΝO1DWH Baseline 303 201 40 99 0 0 204 201 50 lO mins 255 179 41 72 0 0 183 179 49

DMSO 282 185 40 70 0 0 212 185 47 equol 303 382 56 185 2 1 118 380 76

ΝO3PPA Baseline 227 109 32 97 0 0 130 109 46 lO mins 231 237 51 77 4 5 154 233 60

DMSO 317 236 43 96 4 4 221 232 51 equol 270 271 50 82 0 0 188 271 59

NO6MED Baseline 420 413 50 169 0 0 251 413 62 lO mins 437 565 56 168 1 1 269 564 68

DMSO 440 442 50 130 6 4 310 436 58 equol 315 539 63 16 8 10 239 531 69

N13PDO Baseline 267 217 45 112 0 0 155 217 58 lO mins 468 703 60 270 10 4 198 693 78

DMSO 465 405 47 144 0 0 321 405 56 equol 323 527 62 169 5 3 154 522 77

N14GBO Baseline 270 127 32 113 0 0 157 127 45 lO mins 381 242 39 247 0 0 134 242 64

DMSO 276 217 44 111 4 3 165 213 56 equol 225 234 ' 51 68 1 1 157 233 60

Note: "Baseline" refers to the skin sections from the punch biopsy taken prior to exposure to 2.5 MED SSUN.
"10 mins" refers to the skin sections from the punch biopsy taken 10 mins after exposure to

2.5 MED SSUN. The skin was not treated with either DMSO in base lotion (vehicle) or equol at 200 μM.
"DMSO" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to

2.5 MED SSUN. The skin was from the grid treated with DMSO in base lotion (vehicle).

"Equol" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to 2.5 MED SSUN. The skin was from the grid treated with equol at 200 μM.

The increase of MT immunoreactivity in basal and suprabasal keratinocytes of recently UN-exposed individuals was highest in skin that had been treated with equol.

Example 3
The skin biopsies from the five human volunteers from Example 2 were tested for cyclobutane pyrimidine dimer formation using immunohistochemistry.

Table 3 presents the counts and percentages of cells staining positively with an antibody directed against CPD. These data demonstrate that, as expected, there were essentially no CPD-positive cells in the epidermis prior to irradiation with 2.5 MED. However, skin sections taken from all of the participants 10 mins after UN exposure showed high levels of DΝA damage, with the proportion of positively-staining cells ranging from 36% (participants Ν01DWH and Ν03PPA) to 87% (participant N14GBO).

Skin sections taken 24 hrs after UN exposure showed substantially lower levels of CPD damage in all subjects. For 4 out of 5 participants, the skin sections treated with equol lotion had proportionally less CPD-positive cells than the skin sections treated with DMSO in base lotion (vehicle).

Table 3: Proportion of cells staining positively for CPDs in the epidermis of five human volunteers, by treatment group
Total epidermis Upper epidermis Basal epidermis

Subject treatment neg pos % neg pos % neg pos %

NO1DWH Baseline 345 0 0 134 0 0 211 0 0

10 mins 162 107 40 64 51 44 98 56 36

DMSO 231 105 31 81 47 37 150 58 28 equol 164 39 19 70 23 25 94 16 15

NO3PPA Baseline 309 0 0 104 0 0 205 0 0

10 mins 204 191 48 56 106 65 148 85 36

DMSO 179 25 12 55 20 27 124 5 4 equol 349 18 5 70 17 20 279 1 0

NO6MED Baseline 309 0 0 90 0 0 219 0 0 lO mins 136 364 73 19 198 91 117 166 59

DMSO 339 65 16 112 59 35 227 6 3 equol 279 92 25 98 71 42 181 21 10

N13PDO Baseline 205 0 0 60 0 0 145 0 0

10 mins 69 195 74 20 94 82 49 101 67

DMSO 105 68 39 78 51 40 27 17 39 equol 213 94 31 79 50 39 134 44 25

N14GBO Baseline 255 0 0 98 0 0 157 0 0 lO mins 34 389 92 0 157 100 34 232 87

DMSO 240 131 35 93 69 43 147 62 30 equol 188 85 31 63 44 41 125 41 25

Note: "Baseline" refers to the skin sections from the punch biopsy taken prior to exposure to 2.5 MED SSUN. "10 mins" refers to the skin sections from the punch biopsy taken 10 mins after exposure to 2.5 MED SSUN. The skin was not treated with either DMSO in base lotion (vehicle) or equol at 200 μM.
"DMSO" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to 2.5 MED SSUN. The skin was from the grid treated with DMSO in base lotion (vehicle). "Equol" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to 2.5 MED SSUN. The skin was from the grid treated with lotion containing equol at 200 μM.

When data from all participants were pooled, it can be seen that skin sections treated with equol had moderately lower levels of CPD damage at 24 hours.

Example 4
The skin biopsies from the five human volunteers from Example 2 were tested for P53 staining following UN irradiation. Results are shown in Table 4.

Table 4: Proportion of cells staining positively for p53 in the epidermis of five human volunteers, by treatment group
Total epidermis Upper epid lermis Basal
epidermis

Subject treatment neg pos % neg pos % neg pos %

ΝO1DWH Baseline 261 0 0 115 0 0 146 0 0 lO mins 343 1 0 154 1 1 189 0 0

DMSO 187 12 6 79 4 5 108 8 7 equol 270 94 26 109 48 31 161 46 22

ΝO3PPA Baseline 274 2 1 112 1 1 162 1 1 lO mins 316 2 1 114 2 2 202 0 0

DMSO 223 55 20 81 31 28 142 24 14 equol 337 87 21 165 72 30 172 15 8

NO6MED Baseline 412 1 0 134 0 0 278 1 0 lO mins 402 3 1 153 1 1 249 2 1

DMSO 462 133 22 165 77 32 297 56 16 equol 500 50 9 250 19 7 250 31 11

N13PDO Baseline 325 0 0 141 0 0 184 0 0 lO mins 304 0 0 140 0 0 164 0 0

DMSO 222 45 17 109 8 7 113 37 25 equol 287 13 4 147 4 3 140 9 6

N14GBO Baseline 321 0 0 149 0 0 172 0 0 lO mins 292 4 1 185 2 1 107 2 2

DMSO 217 190 47 106 106 50 111 84 43 equol 227 76 25 109 35 24 118 41 26

Note: "Baseline" refers to the skin sections from the punc ;h biops ιy taken j . rior to expos ure to 2.5 MED SSUN.
"10 mins" refers to the skin sections from the punch biopsy taken 10 mins after exposure to 2.5 MED SSUN. The skin was not treated with either DMSO in base lotion (vehicle) or equol at 200 μM.
"DMSO" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to 2.5 MED SSUN. The skin was from the grid treated with DMSO in base lotion (vehicle).

"Equol" refers to the skin sections from the punch biopsy taken 24 hrs after exposure to 2.5 MED SSUN. The skin was from the grid treated with equol at 200 μM.

As expected, there were essentially no cells in the epidermis expressing p53 prior to irradiation with 2.5 MED for any of the participants. Similarly, skin sections taken from participants 10 mins after UN exposure showed negligible levels of p53 expression, in accordance with the literature.

Skin sections taken 24 hrs after UN exposure showed substantially higher levels of p53 expression in all subjects. The percentage of p53 expression in upper and/or basal epidermis was reduced in four out of five equol treated subjects. For example, in subjects Ν13PDO and N14GBO the percentage of p53 staining was reduced significantly (generally more than 50%) compared with vehicle controls.

Conclusions
The biomarkers assessed in these experiments were selected based on their biological associations with skin cancer (which is directly associated with UV-induced DNA mutagenic damage).

UN-induced oxidative damage is now recognised as a potentially important causal factor in skin cancer. MTs are molecules with anti-oxidant properties that are specifically induced in response to UN exposure. This study found consistent evidence that human skin treated with equol, and it is believed other compounds of the formula (II), induce more MT than skin treated with base lotion.

CPDs are the earliest indicator of molecular damage following exposure to UN radiation, and if not repaired, lead to fixed mutations in the DΝA of skin cells. Thus one mechanism of action of a post-exposure treatment would be to increase the rate of repair of these lesions. The experiments conducted here suggest that CPD repair may be enhanced by topical equol compositions, and other compositions containing one or more compounds of the formula (II).

P53 is clearly an important regulatory gene that is commonly mutated in epidermal skin cancers. Moreover, in normal skin cells, p53 is up-regulated following UN exposure to prevent mitosis until DΝA damage is repaired. Equol modulated the expression of p53 in this study causing a reduction in the number of cells in the upper or basal epidermis expressing p53 for four of five subjects.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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