AU2005203044B2 - Method for scavenging radicals with urocanic acid, derivatives and analogues - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Method for scavenging derivatives and analogues radicals with urocanic acid, The following statement is a full description of this invention, including the best method of performing it known to us: METHOD FOR SCAVENGING RADICALS WITH UROCANIC ACID, DERIVATIVES AND ANALOGUES The invention relates to autioxidaflts or radical scavengers and their reaction products, Trans-uZ'ocaflic acid (trans-UCA) is a major ultraviolet (uv) absorbing component of the human epideryniR~- Absorption, of UV radiation from the TJV-C region (200 290 nm) into the tJV-A-I region (340 400 nm) causes photoisomerization of trans-ChA into cis-UCA in vivo as well as in vitro [1-31.

Because of this property, trans-UCA has been used as natural sunscreen agent This use had later been minimized since it became clear that photoproduct cis-UCA can mimic some of the effects of UJV on immunity, suggesting that this compound is an important mediator of UV-induced immunosuppressiofl [51 however, at the moment it is not clear what the main role of UCA or its mode of action is in the context of .immunomodulation. Although experiments in vivo supply evidence for the immuno- suppressive potential of cls-UCA (8- 12), it is remarkable that in a number of cell cultures (in vitro) suppression was not found (13-17). Similar levels of cis-UTCA can be induced by UV-A and UV-B, but nevertheless UV- B is more potent in suppressing contact hyp ersensitivity than UVA(18).

DyA The invention provides compounds and compositions for use in methods for scavenging radicals or for modulating the immune response. comprising urocanic acid or salts, derivatives, functional equivalents, and analogues thereof.

Said compounds, compositions and methods as provided by the invention are based on the novel insight that urocaflic acid isomers are radical scavengers and serve as natural antioxidants in the body, in particular in skin. UV exposure of the skin causes an increased level of oxidative stress with the inherent formation of reactive (hydroxyl) radicals.

it is shown herein that (salts of) urocanic acid isomers or functional equivalents such as imidazole equivalellt5 and imidazolone derivatives thereof, in particular physiologically (in the body) occurring imidazole compounds for example act as physiological antioxidants capable of efficiently protecting lipid phases of biological membranes and proteiflaceous substances in aqueous environments against the action of radicals such as hydroxyl, singlet oxygen or other reactive odd-electron species. These species can be generated from hydrogen peroxide upon UV irradiation, and from hydrogen peroxide in presence of metal ions the Fenton reaction. Both types of reaction can occur in the epidermis Under conditions of oxidative stress, enhanced by exposure to UV it is evident that UCA isomers will encounter the randomly produced hydroxyl radicals -in situ.

The invention thus provides in one embodiment a method for scavenging radicals in a substance comprising providing said substance with urocanic acid or a functional equivalent thereof, such as a salt or functionally related imidazole compound. Preferably, trans-urocanic acid or a functional equivalent thereof is used, being most active or being least immunosuppressive. Using urocanic acid or equivalents thereof as antioxidant or radical scavenger is advantageous over using other antioxidants, such as vitamin E, which are commonly not or only partly soluble in water, whereas urocanic acid or its analogues dissolve easily in aqueous solutions. Especially where said substance comprises a food product or cosmetic product, which are commonly water based, using urocanic acid or its functional equivalent as provided by the invention is advantageous over water insoluble antioxidants. Both isomers are water soluble hydroxyl radical scavengers and can be ased in the water phase of numerous emulsions. Furthermore, urocanic acid isomers, being natural components of the body, are essentially non-toxic, which additionally is advantageous when preparing a food product or cosmetic product.

Tn another or subsequent embodiment, the invention thus provides a method for scavenging radicals in a tissue, for 004673010 example subjective to oxidative stress, comprising providing said tissue with urocanic acid, e. g. the invention provides use of urocanic acid or equivalents thereof for the preparation of a pharmaceutical or cosmetic composition, for example for the treatment of oxidative stress, such as for example manifested in wrinkles and other signs of ageing Stissue, in particular skin. Oxidative stress in living (C organisms and their tissues, in particular the oxidation of Sproteins, has been implicated in the phenomenon of ageing, wrinkling, acute damage of proteins, ischemia reperfusion, atherosclerosis, and many chronic diseases, such as psoriasis, scleroderma, lupus erythematosus, allergic contact dermatitis, vitiligo, lichen planus and graftversus-host disease, or which treatment the invention now provides a pharmaceutical or cosmetic composition comprising urocanic acid or functional equivalent thereof. Such a composition is advantageously also used for immuno modulatory purposes. The invention thus provides a cosmetic composition comprising urocanic acid and an oxidation product thereof, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid or a cosmetic composition comprising an oxidation product thereof, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid.

In yet another embodiment, the invention provides use of an oxidation product of urocanic acid or equivalents thereof (such as salts or related imidazole compounds having similar effect) for the preparation of a pharmaceutical composition, in particular wherein said product is an photo oxidation product. Herein is used the novel insight that as a consequence of radical scavenging, epidermal UCA isomers are converted by reactive oxygen species (ROS) into oxidation products with biological i.e. immunomodulating 004673010 effects. In contrast to the photoisomerization of UCA, not much attention has as yet been given to the oxidation of UCA. In particular not to the reaction of UCA isomers with the very reactive hydroxyl radicals. Hydroxyl radicals can be generated from hydrogen peroxide upon UV irradiation, and from hydrogen peroxide in contact with reduced metal ions, n e.g. ferrous (Fe 2 ions. Both types of reaction can occur in CI the epidermis O Under conditions of. oxidative stress, enhanced by exposure to UV it is evident that UCA isomers will encounter the randomly produced hydroxyl radicals. We now provide the insight that it is in general not cis-urocanic acid per se that provides modulation or repression of immune responses, but oxidation products of urocanic acid, that for example have arisen after ultraviolet light (UV) exposure of for example skin. The invention thus provides use of urocanic acid and an oxidation product thereof, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid, for the preparation of a pharmaceutical composition for modulating the immune response of an animal.

Herein, urocanic acid scavenges radicals created by UV exposure, is thereby oxidised to for example imidazole containing urocanic acid derivatives, such as imidazole-4carboxyaldehyde, imidazole-4-acetic acid or imidazole-4carboxylic acid, which subsequently modulate, suppress or mitigate a mounting immune response of the body to the UV induced tissue damage. The invention thus provides use of an oxidation product of urocanic acid, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid, for the preparation of a pharmaceutical composition for mitigating a mounting immune response of the body.

004673010 By providing insight into this natural mechanism, we Sprovide insight in immune modulating mechanisms that are at work to keep (overly strong) immune responses, for example directed at UV exposure at bay. The invention thus provides use of a pharmaceutical composition comprising an oxidation product of urocanic acid for modulating immune responses i against various stimuli, thereby mimicking a, previously Sunknown, natural action of said product. Herewith the invention provides a method to modulate an immune response of an animal, for example a human being, comprising treating said animal with a pharmaceutical composition comprising an oxidation product of urocanic acid, for example wherein said product is an imidazole such as imidazole-4-carboxyaldehyde, imidazole-4-acetic acid or imidazole-4-carboxylic acid or an imidazolon derivative of urocanic acid such as 3- (4imidazolon-2yl)-acrylic acid and 3- acrylic acid. In particular the invention provides the use of one or more UCA photo-oxidation products as immuno modulator in various skin diseases, such as psoriasis or dermatitis.

The invention also provides use of an oxidation product of urocanic acid, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid, for the preparation of a pharmaceutical composition for the treatment of a skin disease.

Fu-r-thermore,--the-invent-ion-provides-a-pharmaceut±cal composition comprising urocanic acid or functional equivalent thereof for its radical scavenging properties, whereby said composition is additionally used as immuno modulator, optionally already comprising oxidation products having Simmune modulatory function.

The invention is further explained in the detailed S' description without limiting the invention thereto.

S' Detailed description Trans-UCA, cis-UCA, related imidazoles and non- CI imidazole compounds were tested with regard to their ability 0 10 to compete with deoxyribose to scavenge hydroxyl radicals. On Sexposure to hydroxyl radicals deoxyribose is degraded into malondialdehyde, which reacts with thiobarbituric acid to form a pink chromogen. Powerful hydroxyl-radical scavengers will compete with deoxyribose, resulting in a reduced amount of malondialdehyde (223. Ten compounds, UCA, UCA analogues, alanine and uric acid (Pig.l) were tested on their ability to scavenge hydroxyl radicals.

Method: the deoxyribose (dR) degradation test. The test was analogous to an earlier described method (22).

Briefly, the reactions were performed in 5 mL screw cap glass tubes in a final volume of 1.0 mL sodium phosphate buffer mM; pH containing 3.0 mM 2 deoxy-D-ribose, 0.5 mM hydrogen peroxide and one of the test compounds at graded concentrations. The reaction was started by the addition of 25 premixed disodium EDTA and ferrous iron solution (final concentrations 0.5 mM and 0.2 mM, respectively). The mixture was left for 15 minutes at room temperature. After addition of 1.0 mL 1 thiobarbituric acid in 50 mM NaOH and 0.75 mL 2,8 trichloroacetic acid, the tubes were heated for minutes in a boiling water bath. The pink color was read at 532 nm and reciprocal absorption values were plotted against the concentration of the test compound after subtraction of appropriate blanks. A series of six duplicate determinations from test compound dilutions was employed to constrUCt a graph slope for the calculation of a rate constant value. The mean, SD, number of rate constants and the percentage of inhibition of deoxyribose degradation, calculated for each test compound, are listed. R~esults. All second-order rate constants for reaction with hydroxyl radicals and, in addition, the percentage inhibition of deoxyribose degradation with equ-imolar concentrations (3 MM) of scavenger are summarized in Table 1. A typical graph with slopes to derive rate constants from is shown in Fig. 2 f or both UCA isomers. Trans-TCA and cis-TCA are substantially more NI powerful in scavenging hydroxyl radicals 0 and 7. 1 x 109 M .s ,respectively), than the other 1.-,-(5-)-suibstituted imidazoles, including L-histidine (2.6 x 100'. s")-L histidine, the precursor of UJCA, was included as a known moderate scavenger f22-24] with structural similarities to UCA. L-alanine was used as a known poor scavenger [22].

Tz-ans-MA was tested as a non-imidazole acrylic acid derivative, having a furan ring instead. This substitution yielded a very poor scavenging ability.

Other substituted imidazole analogues, dihydrourocanic acid or 3- (imidazol-4-yl) -propiolic acid and imidazole-4 -acetic acid, showed moderate scavenging ability.

comparable to histidine. Unsubstituted imnidazole and its 2methyl derivative appeared to be stronger scavengers than the UCA isomers. The well-.known hydroxyl radical scavenger uric acid showed an excellent abilitiy (27.8 x 109 MW 1 .5 1 ).4V Trans-UCA and. cis-UCA, two epidermal compounds, are good hydroxyl radical scavengers; their ability is less than that of uric acid, but ldrger- than that of the other substituted imidazoles, e.g. histidine.

Trans-UCA and cis.UCA are herein recognized as good hydroxyl radical scavengers. Both isomers occur in substantial concentrations in the epidermis, the latter in the UV-exposed skin. There is strong evidence for the occurrence of hydroxyl radicals in the epidermis, especially upon UV irradiation Normal human skin contains approximately 200 pM iron [26,27], predominantly corupiexed (NI to ferritin. The release of free ferrous ions by UV irradiation (281 and the presence of hydrogen peroxcide [29,301 are prerequisites for the generation of hydroxcyl radicals.

other reports indicate the UV-induced presence of hydzoxyl radicals indirectly since their effects on epidermal constituents could be neutralized with antioxidants [31, 32] UCA is an imidazole compound and several other irnidazole derivatives have already been shown to be good hydroxyl radical scavengers, e.g. histidine (22-241, histamine [331, histidine containing dipeptides [24,34], 0 cimetidine and other histamine (IH 2 receptor antagonists This study reveals that several other imidazoles show similar properties (Table 1) Hydroxyl radicals can react with the imnidazole ring to form imidazolone derivatives.

Their formation has led to the proposal to use the xidazolones of histidine and histamine as markers for oxidative stress [23,331. The importance of the imidazole ring in UCA molecules was also demonstrated in our experiments. The poor scavenging ability of trans-FAA, having a furan ring instead, was a remarkable contrast. Furthermore, the presence of the acrylic acid moiety in UCA molecules conjugated with the imidazole ring may account for its increased scavenging ability towards hydroxyl. radicals as compared to the other substituted imidazoles.

tnsubstituted imidazole and its 2-methyl derivative are stronger hydroxyl radical scavengers, accentuating that the presence of an imidazole ring is a prerequisite for sufficient hydroxyl radical scavenging ability. However, these compounds do not occur physiologically and are harmful oral rat 220 mg/kg for irnidazole and 1500 mg/kg for 2methylirnidazole).

!Tranzs-UCA and cis-UCA do occur physi~ologically, mainl~y in the epidermis, with relatively high concentrations. our findings point to a new physiological role for the UCA isomers, besides the suggested roles of trans-UCA as natural sunscreen agent and cis-UCA as immunosuppressant. Trans-UICA S' and cis-UCA may be major epidermal hydroxyl radical scavengers, providing a new view on the antioxidant status of C' the skin. The findings that 1. UCA isomers are good hydroxyl radical scavengers, though not as strong as uric acid, and that 2. the UCA isomers already occupy relatively high concentrations in the skin, create possibilities to apply the UCA isomers as non-toxic antioxidant additives in tood and Scosmetics in relatively high concentrations. Trans-UCA C (commercially available) should be preferred, because cis-UCA may exert immunosuppressive effects.

O In contrast to the photoisomerization of UCA, not much attention has as yet been given to the oxidation of UCA. In particular, the reaction of UCA isomers with the very reactive hydroxyl radicals should be explored. Hydroxyl radicals can be generated from hydrogen peroxide upon UV irradiation, and from hydrogen peroxide in contact with reduced metal ions, e.g. ferrous (Fe 2 ions. UV-A irradiation of trans-UCA or cis-UCA with hydrogen peroxide only results in UCA photoisomerization and not in UCA photooxidation. The lack of correlation between UV-A-induced cis-UCA formation and immunosuppression (18) may be another indication for a role of UCA-oxidation products in skin immunology. These compounds can either be formed in the presence of hydrogen peroxide upon UV-B' irradiation or by a Fenton reaction; both reaction types leading to comparable sets of oxidation products as determined by chromatographic .patterns. The common oxidizing species of both reaction types is most likely the hydroxyl radical. Starting the oxidation with trans-UCA or with cis-UCA yielded similar chromatographic patterns. In relation with hydroxyl radical scavenging of the UCA isomers, it should be noted that UCA isomers may as well interfere with UV-induced immunosuppression through scavenging of radical species.

The presence of the acrylic acid moiety in UCA molecules conjugated with the imidazole ring may account for its increased scavenging ability towards hydoxyl radical.s as compared to non-conjugated imidazoles, such as histidile and M histamine. It may also account for the diversity of the formed oxidation products.

Materials and methods Hig Perfo 9Mae Li ad- chromato raph (PLCY Trans-UCA and cis-UCA were separated from each other and from several UCA oxidation products on a 4.6 x 250 mm Alltima C,.

8 and a Luna reversed-phase column (Ailtech, Deerfield, 11 and Phenomenex, Torrence, CA, resp.) with a flow of 0.8 mL/min, d~livered by P-3S00 HPLC-pump~s (Pharmacia, Uppsala, Sweden) Sampler. of 20 to 260 ;1L were injected by a Promis II autosampler (spark HIolland, Emmnf, The Netherlands) and chromatographic data were recorded on an SP 4270 integrator (Spectra Physics, San Jose, CA) Peak area data from samples were only processed under identical HPLC circumstances. A UV-detector (Applied Biosystems, Model 759A, Foster City, CA) was set for 226 nm detection. Isocratic elution was performed with 10 mM amimonium formate buffer, containing 0.2 0.8 mM tetrabutylamonium(TA)formalte and 1.

acetonitrile (pH' Collected fractions were acidified with formic acid up to a final concentration of 100 mM and passed through solid phase extraction columns (JT Baker, Deventer, The Netherlands) in order to remove TBA.

Photooxidation A 1-cm quartz cuvette, filled with 1.4 mL sample, was placed in the parallel beam of a filtered 1000 W xenon arc lamp (Oriel, Stratford, CT). The samples were magnetically stirred during irradiation. To minimize infrared (heat) and visible radiation, the beam was passed through a water filter Nq (7 cm), reflected by a dichroic mirror and filtered through a 1-mm UGII filter. Short-wave cut off was achieved by passing the beam through WG280, WG305 or WG335 filters with 3 mm thickness each (Schott-Jena, Mainz, Germany). Xenon lamp emission filtered through WG280 included UV-C, UV-B and UV-A; through WG305 UV-1 and UV-A and through WG335 only UV-A was included. Two narrow bands in the UV-B and UV-A spectral regions were selected to monitor the xenon-arc emission. The probe of a calibrated EG&G 550 radiometer (Salem, MA, USA) was equipped with a neutral density filter and narrow band filter type UV-M-IL (Schott-Jena) with a transmission maximum of 21 at 303 nm and a half-width of 11.5 nm to monitor UV-B or with a type UV-PIL (Schott;Jena) with a transmission maximum of 46 at 363 nm and a half-width of 7.7 nm to monitor UV-A. Transmission spectra of the optical filters were checked on a Perkin Elmer Lambda 40 UV/VIS spectrometer (Norwalk, CT, USA).

Additional irradiations were performed with fluorescent tubes TLI2, used as a UV-B source, and TLIOR, used as a UV-A source (Philips, Eindhoven, The Netherlands), on samples that were magnetically stirred in small Petri dishes. The UV-B output was measured with an IL 443 phototherapy radiometer, fitted with a SEE 1240 silicon detector probe and the UV-A output with an IL 442A phototherapy radiometer with a SEn 115 detector probe (International Light, Newburyport, MA, USA).

Fenton oxidation.

SUCA isomers (10 or 40 AM) were oxidized with a hydroxyl-radical- generating system that consisted of various concentrations of ferrous ions (10 500 AM) and a fixed hydrogen peroxide concentration of 500 pM (the Fenton O reagent), either in a sodium phosphate (10 or 20 mM) medium r of pH 7.2, or in ultrapure water. In addition, two hydroxyl- CI radical-generating systems with copper ions were used, consisting of 50 pM Cu 2 with either 500 pM hydrogen peroxide or 5 mM ascorbic acid.

Synthesis of reference compound iicidazole-4-carboxaldehyde (4 -formylimidazole) 4- (Hydroxymethyl) imidazole-HCl (4 mmol) was stirred together with sodium bicarbonate (6 mmol) in 4 ml methanol for 1 hour at room temperature. The methanol was evaporated and the residue was extracted with a chloroform/methanol 1:1. After centrifugation at 3500 rpm for 5 minutes the supernatant was evaporated and the residue was taken up in 20 ml hot dioxane.

4.4 g manganese dioxide (activated; for synthesis) was added, followed by a reflux reaction for 2 hours. Manganese dioxide was removed by filtration and the filtrate was evaporated- Crystallization was carried out in methanol. The yield was mg of fine off-white crystals, 25 of maximum yield. The melting range was 168 169° C 173 175° Melting range of starting material was 108 1110 C and of the oxidation product imidazole-4-carboxylic acid 294 295° C. UV (water) (log e) 257 nm (3.85).

Results UCA isomers and hotooxidation The 0-0 bond of hydrogen peroxide can be cleaved by UV Sradiation to yield hydroxyl radicals. Because both UCA isomers could effectively scavenge hydroxyl radicals, it is to be expected that UCA will be degraded and/or converted into oxidation products. The ability of simulated solar UV radiation to convert trans-UCA in the presence of hydrogen peroxide into photooxidation products was tested in vitro and analyzed by reversed-phase HPLC analysis. Hydrogen peroxide eluted close to void volume and crans-UCA and cis-UCA eluted CI with markedly different elution times of 20 and 64 min (Fig.

3a-d). The unirradiated control sample did not show any interaction between trans-UCA and hydrogen peroxide (Pig.

3a). Exposing 80 AM trans-UCA in the absence of hydrogen peroxide at pH 7.2 to WG280-filtered xenon-arc emission (including UV-C and UV-B) resulted only in the formation of cis-UCA via the process of photoisomerization (Fig. 3b).

However, when trans-UCA was irradiated in the presence of 500 iM hydrogen peroxide under identical conditions, many additional peaks appeared in the chromatograms and both trans-UCA and cis-UCA peaks were strongly reduced (Fig. 3c), indicating a certain photochemical conversion or breakdown.

Eight main photooxidation products were recognized as new peaks based on retention times and were assigned in the chromatogram (Fig. 3c).

In contrast, when exposures were performed with simulated solar radiation from which both UV-C and UV-B were blocked out by a WG335 filter, virtually no photo-oxidation products were found (Fig. 3d). Only UCA photoisomerization was apparent, which is in accordance with earlier reports (2, The ratio of trans-UCA to cis-UCA photoisomerization was not affected by the degree of photooxidative breakdown.

Blocking out UV-C by the use of the WG305 filter showed intermediate results (Table This irradiation condition has the closest simulation with the spectral UV distribution CI of terrestrial solar radiation produced by an overhead sun on a bright day. Tests with the fluorescent lamps TL 12 (UV-B and UV-A; some UV-C) and TLIOR (UV-A) confirmed the above findings that UV-B and UV-C have photo-oxidative ability.

Although the UV-A dose of the fluorescent lamp was much higher than that of UV-B, the yield of UCA photo-oxidation products was much lower with Uv-A (Table The formation of M photo-oxidation products was quantified by summing the eight major peak areas (in arbitrary units; peaks A The degree of photo-oxidative breakdown, the yield of photooxidation products and the degree of UCA photoisomerization under different irradiation conditions were summarized in Table 2. Taking the various emissions of these UV sources into account, the photo-oxidative ability of UV radiation became substantial with wavelengths shorter than approximately 320 nm. Experiments with cis-UCA yielded similar results, except that cis-UCA/crans-UCA ratios were increased in this series (data not shown).

UC somers nFento oxidat ion In the next series of experiments we studied the Fenton oxidation of UCA, representing another natural oxidation process. !frans-UCA and cis-tICA isomers were Fenton oxidized by ferrous ions and hydrogen peroxide at physiulogical CccinLrdLiuas. The initial hydrogen peroxide concentration was 500 AM and the ferrous ion concentration was varied from 0 to 500 AM. In all Fenton- oxidation reactions the degree of UCA-isomer breakdown was calculated from their reduced peak areas. The oxidation reaction must CI have been completed within 2 minutes for all reaction conditions, because no ftirther breakdown was observed after) prolonged incubation. Hydrogen peroxide without Fe"1 had no effect on the UCA isomers at all; however, Fe3' without hydrogen peroxide resulted in a slow breakdown of U CA isomers after prolonged incubation (data not shown) The sequence order of addition of the two F~enton reagents did not markedly affect the UCA breakdown and yield of oxidation products, except at a low UCA concentration of yM. When Fe2 Was added after hydrogen peroxide, a larger breakdown and a smaller yield of Fenton- oxidation products were observed, whereas the reversed- sequence order gave opposite results (data not shown).

When the Fenton reaction was performed in water instead of phosphate buffer, the oxidative breakdown of trans-UCA was enhanced irrespective of the UCA concentration.

The turbidity seen in reactions performed in phoshate buffer mM) with high Fe'* concentration 100 uM) was probably due to the formation of insoluble iron phosphate, thereby reducing the free availability of Fe Table 3 summarizes the difference between water and phosphate medium for trans-UCA at an initial concentration of yM with respect to its breakdown and the formation of Fenton- oxidation products. Similarly to the photo-oxidation experiments, the peak areas of the a major oxidation products were summed. Comparable results were obtained with cis-UCA Z(data not shown), which finding is in accordance with the comparable rate constants of trans-UCA and cis-UCA in the deoxyribose degradation experiment (Table A close resemblance was observed between the chromatographic patterns of UCA Fenton oxidation products (not shown) and those of UCA photo-oxidation products. Three of them has been identified (vide infra).

When two other hydroxyl-radical-generating systems based on copper ions (cuh* were investigated with trans-UCA, the c-i combination of Cu 2 (50 pM) and ascorbic acid (5 mM) without hydrogen peroxide caused an almost complete breakdown of trans-UCA (3 left), whereas the system with Cu 2 (50 AM) and hydrogen peroxide (500 AM) showed little effect (88 trans-UCA left). Evaluation of the data was difficult with the ascorbate system, because several interfering peaks had occurred in the chromatograms, which were probably derived from ascorbic acid and its oxidation products. Both sytems are considered to be of minor importance for the situation in vivo, but these results indicate similarities in oxidative behaviour of the UCA isomers, independent of the nature of the hydroxyl-radical -generating system.

UCA isomers and Fenton oxidation.

In another series of experiments we studied the Fenton oxidation of UCA, representing another natural oxidation process. The initial hydrogen peroxide concentration was 500 VM in all experiments and the ferrous ion concentration was varied from 0 to 400 pM. Four sets of conditions were compared: 1. Fe 2 in phosphate buffer pH 7.2 2. Fe 2 in phosphate buffer plus EDTA 3. Fe 2 without buffer with a initial pH of 5.5 5.3 and 4. Cu 2 in phosphate buffer plus ascorbate. The degree of breakdown was similar for both UCA isomers. Table 3 shows oxidative breakdown of trans-UCA with hydrogen peroxide in increasing order: condition I 2 4 Z3.

The addition of Fe 2 at final concentrations of 100 400 pM in phosphate buffer caused a turbid solution of insoluble iron phosphate. Under this condition the smallest degree of breakdown was established. A limited availability of free Fea' is assumed to reduce the oxidutive breakdown of UCA. At the other hand, complexation of Fe to EDTA did not C cause a turbid reaction mixture and a larger breakdown was established (Table The largest breakdown was seen in the absence of phosphate buffer, with a less defined pH value of to 5.3 dependent on the UCA concentration (40, 100 or 250 PM). At the start of the Fenton reaction in the unbuffered medium, there was a rapid fall of the pH value from 5.1 to 3.4, with initial concentrations of trans-UCA, hydrogen peroxide and ferrous ions of 250, 500 and 400 pM, respectively. We attribute this effect to the unbuffered liberation of relatively strong acids, such as glyoxylic acid (GLX). Similar results of breakdown, though slightly less pronounced, were obtained with cis-UCA (Table This finding is in accordance with the comparable second order rate constants of trans-UCA and cis-UCA for hydroxyl radical scavenging Hydrogen peroxide without Fe 2 had no effect on the UCA isomers at all; however, Fe 2 without hydrogen peroxide resulted in a partial breakdown of the UCA isomers upon prolonged incubation of one day (data not shown).

The primary oxidation products formed are ImCHO and GLX.

Additional experiments in which ImCHO was used as starting material, a yield of virtually 100 ImCOOH was obtained after Fenton- or photooxidation. In UCA samples that were highly oxidized (containing 4 UCA) ImCOOH was the major 226 nm absorbing compound, while ImCHO concentration was largely reduced. An additional experiment demonstrated that under this oxidative condition the aldehyde (ImCHO) was oxidized to the carboxylic acid (ImCOOH) GLX was analyzed in lower amounts than ImCHO in all cases studied (Table 3), except for the Fenton oxidation of 40 AM UCA (Table 4, section 3.1 and Trans-UCA and cis-UCA in relatively high concentration of 250 M were broken down for 78 and respectively, by the unbuffered Fenton oxidation system.

Table 4 section 3 also shows that the yield of oxidation products was proportional with the initial UCA concentration.

Remarkably, the yield of ImCHO from cis-UCA was substantially larger than from trans-UA. In the phosphate buffered Fenton system a comparable breakdown and a comparable yield of oxidation products was recorded, irrespective of the initial UCA concentration range from 40 to 250 AM (Table 4, section 1, only results of 40 pM are shown). In the presence of EDTA, a larger breakdown and a higher yield of oxidation products (in particular ImCHO) resulted (Table 4, section This yield was raised as higher initial UCA concentrations were used. In the unbufffered system, the highest degree of' breakdown of all tested systems was recorded. The oxidation product yield was the largest of all systems when the initial UCA concentration was high (250 AM) (Table 4, section 3).

When another hydroxyl-radical-generating system, based on copper ions (Cu was investigated, the combination of Cu 2 ascorbic acid hydrogen peroxide caused a large breakdown of trans-UCA (Table 3) and a moderate yield of UCA oxidation products, in favor of ImCOOH. Without ascorbic acid, the system with CuL? (50 gM) and hydrogen peroxide (500 pM) showed little breakdown (88 trans-UCA left; data not shown). For the situation in vivo, one must remember that the epidermal copper content is lower than iron (29).

30. 2.3.4. UCA compared in Fenton and photooxidation A close resemblance was observed between the chromatographic patterns of UCA Fenton oxidation products and those of UCA photooxidation products (Fig.5) Also under photooxidation an oxidation inhibiting effect was seen in phosphate of pH 7.2, whereas the yield of oxidation products was in favor of ImCHO CI (Table 4, section 4 versus In photo- oxidation, the breakdown of cis-UCA was substantially decreased in comparison with the trans isomer (Table 4, section In Fenton oxidation, this effect was less pronounced. The data of Table 4 were given for air saturated solutions. Argonpurging of the solutions, prior to Fenton or photooxidation, enhanced UCA breakdown as well as tho yield of oxidation products, both by a factor 2 to 3. Heating (to 370 C) of argon-purged solutions slightly enhanced the yield of ImCHO.

The data of Table 4 indicate a discrepancy ('gap') between micromoles of UCA isomer broken down and micromoles of oxidation products formed. The smallest 'gap', though still 52 was found after the oxidation of cis-UCA in the unbuffered system (section Thin layer chromatography (TLC) gave more insight in the 'gap' products, that were not seen in reversed phase chromatography, using UV detection or fluorescence detection. TLC carried out on silica with the eluent isopropanol ammonia 25 (4 1) showed an array of elutable, partly overlapping fluorescent spots and a fluorescent spot at the start position (data not shown).

However, the initial weight of trans-UCA, introduced in a photooxidation experiment with extensive UCA breakdown 4 of each UCA isomer left over), was not lowered much 14 after severe photooxidation. This finding indicated a predominant formation of non-volatile, solid material in stead of gaseous compounds, such as CO 2 and water. The TLC pattern and the weighing experiment points to a possible hydroxyl radical initiated chain reaction of UCA, resulting in the formation of substances that may fill the above mentioned gap. These substances may not be fully detected under the chromatographic conditions used for the simultaneous determination of the UCA isomers, ImCHO and ImCOOH.

2.3.5. Inzhibition of contact hypersensitivity.

N The inhibitory effects of the UCA oxidation products Sare illustrated in Fig. 5. Maximum ear swelling response was Snormalized to 100 The largest reduction was obtained with the residue of severely photooxidized UCA (PO mix III) containing less than 4 residual cis-UCA. It resulted in only 19 ear swelling (81 reduction of swelling). Even a 0 tenfold dilution of that mix (0.2 g/1) reduced the ear swelling markedly (29 ear swelling), which is of similar C<I level as the effect of cis-UCA in a concentration of 1 g/l (31 ear swelling). Another remarkable effect was obtained by mixing the three identified imidazoles. When we tested one of the imidazoles alone (1 only a moderate effect was seen, however, when tested mixed together (1 g/l, each imidazole 0.33 a synergistic effect was observed (26 ear swelling). Glyoxylic acid and oxalic acid, as ammonium salts, did not exhibit significant inhibition of CHS.

UCA photo-oxidation on a preparative .scale Concentrations of trans-UCA and hydrogen peroxide were largely increased, as was the UV exposure, to obtain larger amounts of UCA photo-oxidation products as collected fractions from the reversed phase column for further analysis. A typical chromatogram is shown in Fig. 4. Four fractions, designated as R, 8, R, 10, R 14, Rt 17, were finally selected for identification (peak A, 1-3 in Fig.4).

Prior to analysis, tetrabutylammonium was removed by solid phase extraction on silica.

Identification R, 8 was identified as imidazole-4-carboxaldehyde (ImCHO). Its UV-spectrum was identical to the synthesized (see below) reference compound with an absorption maximum of 257 nm. Co-injection of R, 8 with synthesized imidazole-4carboxaldehyde resulted in a single chromatographic peak with Cq a retention time of 8.13 minutes. Further evidence is to be collected (peak A in Fig.4). The amount of ImCHO in the t photooxidized UCA sample was gradually reduced upon storage Sat 2 0

C.

R, 10 was identified as imidazole-4-acetic acid. Its UV-spectrum was identical with an absorption maximum of 213 nm. Mass spectrum was obtained with electrospray technique Sand the dry sample was treated with methanol/HC1 and n- O butanol/HC1 before analysis. A peak at mass 140 was obtained after methylation and at mass 183 after butylation.

SConsequently, the mass of the original compound was 126. Coinjection of R, 10 with commercially available imidazole-4acetic acid resulted in a single chromatographic peak with a retention time of 8.98 minutes (peak 1 in Fig.4).

R, 14 was identified as imidazole-4-carboxylic acid (ImCOOH). Its UV-spectrum was identical to the commercially obtained reference compound with an absorption maximum of 226 nm. Proton resonance (1H-NMR) analysis was done in D 2 0, showing imidazolic protons in a ratio 1:1 with shifts of 7.76 and 7.53 ppm. Mass spectrum was obtained with electrospray technique and the dry sample was treated with methanol/HCl and n-butanol/HC1 before analysis. A peak at mass 126 was obtained after methylation and at mass 169 after butylation.

Consequently, the mass of the original compound was 112. Coinjection of R. 14 with commercially available ImCOOH resulted in a single chromatographic peak with a retention time of 14.73 minutes (peak 2 in Fig.4). The amount of ImCOOH in the photooxidized UCA sample was gradually increased upon .storage at -20" C.

Synthesis of imidazole-4-carboxaldehvde (4-formylimidazole; FW 134.5) from 4- (hydroxymethyl) imidazole-HC1.

538 mg starting material (4 mmol) was dissolved in 4 ml methanol and 500 mg NaHCO, (6 mmol) was added. The tube 0 21 was occasionally stirred for 60 min, alternatively at 4° C Z and at warm water temperature. CO 2 was allowed to escape mfrom the glass tube. The mix was divided across several Eppendorf tubes and subjected to Speedvac treatment for 1 hour. Residues were white solids with light-yellow sirupy liquids. Chloroform/methanol mix 1;1 was added to the tubes 0 with subsequent gentle warming and stirring. NaHCO 3 was Sseparated by centrlfugation of the combined fractions at 3500 C1 rpm for 5 min. Clear supernatant was kept overnight at -20° C to allow the precipitation of additional NaHCO.. Then, the solution was cleared by filtration and evaporated to dryness with a Rotavapor device. The residue was taken up in 20 ml dioxane with magnetic stirring and 4.4 mg MnO 2 (activated; for synthesis) was added in the same flask. The residue may not have been dissolved completely in first instantion. The mix was refluxed for 2 hours on a paraffin oil bath. The warm solution was filtered and MnO 2 was washed once with warm dioxane. Dioxane was evaporated with the Rotavapor® yielding a white and yellow fine cristalline solid. Crystallization was carried out in methanol repeated times. Small volumes of methanol were required, because the residue dissolved well in methanol.

yield: 20 mg (lit: 475 mg) of fine off-white crystals.

167 168° C (lit: 173 175° C) 4-(hydroxymethyl)imidazole-HCl :108 111° C imidazole-4-carboxylic acid 294 2950 c (lit.: Battersby AR et al., J Chem Soc (Perkin I) 43 51, 1980) The results show that similar sets of several UCA oxidation products can be formed with UV irradiation and without (Fenton reaction type). Three products were identified so far. We assume that these compounds occur in n the upper layer of the epidermis as well and a method will be developed to determine UCA oxidation products in vivo. The CI simultaneous break-down of ImCHO and the gain of ImCOOH after photooxidation has led to our speculation that ImCHO is slowly oxidized to ImCOOH during storage. Many aldehydes are gradually oxidized to the corresponding carboxylic acids in contact with oxygen species.

Two phenomena out of the puzzling mechanism of cis-UCA induced immuno-suppression can be solved if UCA oxidation C products would have immunosuppressive properties. First, the pC abrogation of the immunosuppression by antioxidants (19-21) in the model of contact hyper-sensitivity measuring ear Sswelling response. In our scope, the formation of UCA oxidation products is prevented, because of neutralization of the hydroxyl radicals by the antioxidants. Second, the lack of correlation between cis-UCA formation by UV-B and UV-A No immunosuppression was found with UV-A irradiaton, despite the fact that cis-UCA was formed. In our scope, this finding may be explained as the inability of UV-A to photooxidize UCA. Consequently, no UCA photooxidation products are formed with UV-A (results section) and because of that immunosuppression would not occur. Our findings and the above assumptions may point to a important role for UCA (photo)oxidation products in the skin immune system.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as .elevant by a person skilled in the art.

LEGENDS to FIGURES.

Figure 1. Compounds tested in this study for hydroxyl radical scavenging ability. trans-UCA, cis-UCA, (c) L-histidine, dihydroUCA or 3-(imidazol-4-yl)propionic acid, imidazole acetic acid, 2-methylimidazole, (g) C imidazole, L-alanine, rans-2-furylacrylic acid and Cl uric acid.

CD SFigure 2. A determination of the second order rate constants of trans-UCA and cis-UCA with hydroxyl radicals.

The rate constant was derived from the slope of the line (k slope x ka x [dR] x where A0 is the absorbance, measured in the absence of hydroxyl radical scavenger. K. was taken as 3.1 x 10 MW'.s derived from pulse radiolysis studies and (dRi 3 mM. The rate constants in this particular set were 8.49 and 7.33 x 10' for trans-UCA and cis-UCA, respectively. The other scavengers were studied similarly.

Figure 3. Chromatograms of 80 pM trans-urocanic acid in 20 mM phoshate buffer pH 7.2. The initial concentration of hydrogen peroxide was 500 pM. Injection volume was 80 L.

a. with hydrogen peroxide; not irradiated, b. without hydrogen peroxide; irradiated with a WG280 filtered xenon-arc lamp, c. with hydrogen peroxide and irradiated as lb, d. with hydrogen peroxide and irradiated with a WG335 filtered xenonarc lamp. Peaks assigned with A H correspond with photooxidation products. Separation was performed on a Alltima column with UV detection at 210 nm. The eluent consisted of 10 mM sodium phosphate pH 7.2 with 1.0 mM tetrabutylammonium hydrogen sulphate. Further experimental conditions are described in the text.

Legends (cont.) P Figure 4. Comparable chromatographic patterns in the formation of UCA oxidation products from 80 AM trans-UCA and 500 AM hydrogen peroxide in water (no buffer). Left: after SFenton oxidation with 250 AM Fe7' and right:. after o photooxidation with 'full' UV, containing a UV-B dose of 32 SkJ.m- 2 The cis-UCA peak is missing after Fenton oxidation, tt 10 due to the absence of photoisomerization. Peak assignation (A G) was done as in Figure Ic. Peaks B,C and D refer to C imidazole-4-carboxaldehyde, imidazole-4-acetic acid and imidazole-4-carboxylic acid, respectively. Chromatographic conditions were identic'al to those applied in Figure 3.

Pigure 5. Inhibition of contact hypersensitivity as a reduction of ear swelling response from BALB/c mice. The positive control (no inhibition) was normalized to 100 Im-mix is a mix of the three identified imidazoles (see identifications) and POmix III is a mix of the three identified imidazoles among several other unidentified UCA oxidation products, obtained upon extensive photooxidation.

Rudimental trans- and cis-UCA are present in lower amounts than 3 (by weight). 1. Morrison, H. (1985) Photochemistry an~d photobiology of urocanic acid. Photodermatol oqy 2, 158 165.

2. Gibbs, M. Norval, N.J. Traynlor, M. Wolf, B.E.

Johnson and Crosby (1993) Action spectra for the trans to cis photoisomerisation of UCA in vitro and in mouse skin.

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Redziniac and D. Schmitt (1995) Cis-urocanic acid failed to affect in vitro human Langerhans cell allostirnulatory function. Photochem. Plzotobiol 62, 914 916.

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Ley (1994) Lack of correlation between suppression of contact hypersensitivity by UV radiation and photo- isomeri zation of epidermal urocanic acid in the hairless mouse. Photochem.

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27 19. Reeve M. Bosnic and E. Rozinova (1993) Carnosine protects from suppression of contact hypersensitivity by UV-B radiation or by cis-urocanic acid. Immunology 78, 99 104.

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Evatigneeva, E.A. Ageyeva and G.A. Zheltukhina (1994) L- Carnosine and carcinine act as natural antioxidants with hydroxyl-radical-scavenging ahd lipid-peroxidase activities.

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TABLE 1.

TM3 HYaOMY RADICAL SCAVENGING ASIITY I8OMRS MM~I RMLATED COMPOUNDS.

OF 1UROCAWZC ACID~

I=~RO=~

RlADICAkL

SCAVEMOMR

0jEh;.I96~j '.'s4jgAa Rag=~ CONSTAW1 DEGRADfATIONT [SCAVENGER] (DEOXYRIB 053] -3MM 6-1 1M1nAzUj-j15 trana-UrocaDnic acid cis-UZ!ocanic acid 8.0 0.9 8 7.4 0.6 6 64 4 34 L-Histidinfe 0.9 oihydrourocalic acid imidazole-4acetic acid 2 -Methyl imidazole 2.7 0.9 3 2.2 0.1. 3 11.7 2.6 5 76 OT1=R COMPROU)1D L-Alanine 0.2. 0.0 3 2 trans-2-FurylacyliO 3 <2 acid 0.1 uric acid 27.8 3.0 4 91 a. traris-2-furylacrylic acid was not tested in concentrations SlmM because of poor solubility.

b. n represents the number of slopes f rom which the rate was calculated.

c. 2.3-3.0 x 10' M' in literature [22] 2005203044 13 Jul 2005 TABLE 2.

UROCANIC ACID (UCA) ISOMERS after POTrOOXIDATION UV SPECTRAL CHARACERISTICS DOSE UCA YIELD OF PHOTOISOMERIZATION 1 RADIATION kJ.m 2 LEFT OVER PHOTOOXIDATION tran-:JCA CiS- SOURcE PRODUCTS UCA UV-B SD) 1' A.U. I SD)' tJV-A (2) Xe arc W0280 270 400 nm 31 43 11) 347 (1 59) 41 (t 2) 59 UV-C, Included Xe arc MG305 292 400 nm is 64 i± 6) 219 (1 14) 47. U 31 53 UV-B,-A included Xe arc W0335 320 400 nm 0 96 (t 5) 45 9) 60 2) 66 only UV-A included TL12 unfiltere 250 366 nm 3.6 90 20) 149 Ct 51) 41 (i 4) 59 d TL1OR unfiltere 320 440 nm 0 99 3) 16 51 84 7) 16 d 324 2005203044 13 Jul 2005 Initial concentration of trans-UCA or ciS-UCA is 40 pM and that of hydrogen peroxide 500 Standard Deviation of duplicate measurements.

113) Arbitrary Units derived from peak area integration. The peaks of 8 major products were Bummed.

This listing only applies to trans-urocanic acid as starting material.

Philips' fluorescent tubes. Different spectral distribution and radiometric measurements as compared to xenon-arc.

2005203044 13 Jul 2005 TABLE 3.

TRAN1]S-LJROCANIC ACID "'after FENTON OXIDATION TRANS-UROCANIC ACID LEFT YIELD of FENTON OXIDATION I[Fe 2 4i OVER PRODUCTS (14m) 96( S. A.U. in phosphate in water in phosphate in water buffer '"buffer .0 100 100 10 3) 97 77 10 194 11) 34) 100 94 4±6) 48 27 5) 272 6) 250 83 19 36 423 4 76) 500 78 4 49 4±9) 511 2005203044 13 Jul 2005 11 Initial trans-UCA concentration: 40 jiM.

12) Fe'* added before hydrogen peroxide.

[33 10 mM sodium phosphate buffer, pH 7.2 [41 Arbitrary Units derived from peak area-integration. The peaks of a major products were summed.

[51 Standard Deviation of duplicate measurements.

Claims (21)

1. Use of an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid, for the preparation of a pharmaceutical composition for Ce mitigating a mounting immune response of the body in which eC oxidative stress is implicated. o

2. Use of an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid, for the preparation of a pharmaceutical composition for the treatment of skin disease in which oxidative stress is implicated.

3. Use according to claim 2, wherein said skin disease comprises psoriasis, scleroderma, lupus erythematosus, dermatitis, vitiligo, lichen planus or graft-versus-host disease.

4. Use according to claim 2 or claim 3, wherein said skin disease comprises psoriasis or dermatitis.

5. Use according to any one of claims 1 to 4, wherein said imidazole-containing urocanic acid derivative comprises imidazole-4-carboxyaldehyde, imidazole-4-acetic acid or imidazole-4-carboxylic acid.

6. Use according to any one of claims 1 to 4, wherein said imidazolone-containing urocanic acid derivative comprises 3- (4-imidazolon-2yl)-acrylic acid or 3-(4-imidazolon-5-yl)- acrylic acid. COMS ID No: ARCS-220223 Received by IP Australia: Time 16:29 Date 2009-01-16 18/01/2009 16:27 FREEHILLS 03 10/12 500242091 0 0

7. Use of urocanic acid and an oxidation product thereof, l. wherein said oxidation product is an imidazole-containing .0 derivative or an imidazolone-containing derivative of urocanic acid, for the preparation of a pharmaceutical s composition for modulating the immune response of an animal. C

8. A pharmaceutical composition comprising urocanic acid and an Ci oxidation product thereof, wherein said oxidation product is o an imidazole-containing derivative or an imidazolone- C containing derivative of urocanic acid.

9. A cosmetic composition comprising urocanic acid and an oxidation product thereof, wherein said oxidation product is an imidazole-containing derivative or an imidazolone- containing derivative of urocanic acid.

A cosmetic composition comprising an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative or urocanic acid.

11. Use of urocanic acid and an oxidation product, wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid, thereof for the preparation of a cosmetic composition.

12. Use of an oxidation product of urocanic acid wherein said oxidation product is an imidazole or an imidazolone derivative of urocanic acid, for the preparation of a cosmetic composition.

13. Use of a oxidation product of urocanic acid, wherein said oxidation product is an imidazole or an imidazolone COMS ID No: ARCS-220223 Received by IP Australia: Time 16:29 Date 2009-01-16 16/01/2009 16:27 FREEHILLS 03 11/12 800242091 a> 36 derivative or urocanic acid substantially as hereinbefore described with reference to any of the examples. VO

14. A cosmetic composition comprising urocanic acid and an oxidation product thereof substantially as hereinbefore s described with reference to any of the examples.

A method of mitigating a mounting immune response of the Sbody in which oxidative stress is implicated comprising Sadministering a pharmaceutical composition comprising an oxidation product of urocanic acid, wherein said oxidation LO product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid.

16. A method of treatment of a skin disease in which oxidative stress is implicated comprising administering a pharmaceutical composition comprising an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid.

17. A method of modulating an immune response of an animal comprising administering a pharmaceutical composition comprising urocanic acid and an oxidation product thereof, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid.

18. A pharmaceutical composition comprising an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid when used in a method to mitigate a mounting immune response in the body. COMS ID No: ARCS-220223 Received by IP Australia: Time 16:29 Date 2009-01-16 16/01/2009 16:28 FREEHILLS 03 12/12 800242091 a> 37

19. A pharmaceutical composition comprising a urocanic acid and an oxidation product thereof, wherein said oxidation product D0 is an imidazole-containing derivative or an imidazolone- containing derivative of urocanic acid when used in a method to mitigate a mounting immune response in the body in which oxidative stress is implicated. Cg

20. Use of an oxidation product of urocanic acid, wherein said o oxidation product is an imidazole-containing derivative or 0i an imidazolone-containing derivative of urocanic acid, for the preparation of a pharmaceutical composition for mitigating a mounting immune response of the body, wherein said imidazole-containing urocanic acid derivative comprises imidazole-4-carboxyaldehyde or imidazole-4-carboxylic acid or wherein said imidazolone-containing urocanic acid 1i derivative comprises 3-(4-imidazolon-2yl)-acrylic acid or 3- acid.

21. A pharmaceutical composition comprising an oxidation product of urocanic acid, wherein said oxidation product is an imidazole-containing derivative or an imidazolone-containing derivative of urocanic acid, wherein said imidazole- containing urocanic acid derivative comprises imidazole-4- carboxyaldehyde or imidazole-4-carboxylic acid or wherein said imidazolone-containing urocanic acid derivative comprises 3-(4-imidazolon-2yl)-acrylic acid or 3-(4- imidazolon-5-yl)-acrylic acid. COMS ID No: ARCS-220223 Received by IP Australia: Time 16:29 Date 2009-01-16