WO2020080957A1

WO2020080957A1 - Methods of treating or preventing skin conditions

Info

Publication number: WO2020080957A1
Application number: PCT/NZ2019/050135
Authority: WO
Inventors: Olivier Serge Gasser; Katherine Jane PARATA; Karmella NAIDOO
Original assignee: Malcorp Biodiscoveries Limited
Priority date: 2018-10-19
Filing date: 2019-10-17
Publication date: 2020-04-23

Abstract

The present invention is concerned with methods and compositions useful for the treatment or prevention of skin conditions or skin disease including, for example, pruritis and atopic dermatitis. In certain aspects, the methods and compositions described herein target MR1-restricted T cells, and in particular classical mucosal-associated invariant T (MAIT) cells, by administering non-agonistic MR1 ligands in both prophylactic and therapeutic regimes as a means to target the disease pathology of atopic dermatitis. The non-agonistic MR1 ligands described herein include, without limitation, 6-formylpterin (6FP), acetyl-6-formylpterin (Ac6FP), 3-formylsalicylic acid (FSA), luteolin, genistein, 5-amino-6-(2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU) and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU) as well as combinations thereof, and in certain examples, may be formulated as topical compositions for administration to a patient requiring treatment. In other aspects, the compositions described herein may be administered in conjunction with UV light, in particular UVB light, as a combination treatment to target MR1-restricted T cells, including MAIT cells.

Description

METHODS OF TREATING OR PREVENTING SKIN CONDITIONS

FIELD OF THE INVENTION

The present invention relates generally to methods for treating or preventing inflammatory skin conditions, including methods of treating or preventing pruritus (itch) and atopic dermatitis, as well as in the prevention of allergic disease immunologically associated with various inflammatory skin conditions including (e.g.) asthma which occurs as a consequence of allergic march.

BACKGROUND

A number of different skin diseases are associated with significant pruritus and therefore lead to bacterial translocation via pruritus-scratch cycles. By way of example, atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease¹, characterized by highly pruritic lesions². AD is associated with significant direct and indirect costs, including health care³ and diminished quality of life⁴. New Zealand has a high prevalence of AD (15-20% of 6- 14 year-olds), as documented by the International Study of Asthma and Allergies in Childhood (ISAAC)⁵. The etiology of AD is multifactorial but centers around the loss of skin barrier function, invasion of skin resident bacteria and chronic immune activation. Indeed, while bacterial colonization of the skin, and the associated microbial-immune crosstalk, has been established as a major determina nt of cutaneous homeostasis⁶, this mutua lism is rapidly revoked upon microbial penetration through the epithelial surface, which often leads to chronic skin inflammation such as AD⁶.

Current first line treatments for pruritus and/or AD include topical emollients and topical immunosuppressive medications⁷. Topical corticosteroids are most commonly used to reduce inflammation, followed by topical calcineurin inhibitors and others. For the treatment of steroid -refractory disease, therapies include UVA and UVB phototherapy, systemic immunosuppressants, systemic immune-modulating biologies (e.g. monoclonal anti-IL-4Ra antibodies)⁷. Therapies in development or currently in clinical trials include anti-Th2 and anti- Th22 cell signalling drugs, anti-pruritic drugs (targeting the IL-3 l/IL-31 receptor pathway and N K1 R) and anti-inflammatory agents targeting Janus Activating Kinase (JAK) pathways, either topically or systemically⁷.

In addition both UVA and UVB phototherapy are also used for the treatment of pruritus and atopic dermatitis^{8- 11}.

However, drawbacks of the current treatments include the significant side effects of topically and systemically administered immunosuppressants. Emollients can have a steroid sparing effect, but do not block the disease. When used improperly (a common issue with topical drugs with dose-dependent toxicities), topical corticosteroids can lead to local and systemic adverse events¹²'¹³. Topical corticosteroid use is also associated with anxiety of use on the side of patients and parents. Topical ca lcineurin inhibitor use might be associated with the development of malignancy¹⁴. Systemic immunosuppressants are associated with severe side effects, commonly lead ing to their discontinuation. Systemic corticosteroids are not advised for long term disease management.

Accordingly, there is a need in the art for new methods of treating skin diseases and/or conditions caused by or associated with bacterial invasion and/or infection . It is an object of the invention to provide a method of treating and/or preventing a skin disease or condition caused by or associated with bacterial invasion and/or infection in a subject in need thereof and/or to at least provide the public with a useful choice.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

SUMMARY OF INVENTION

In one aspect the present invention provides a method for treating or preventing a skin disease or condition comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In another aspect the present invention provides a method for treating or preventing pruritus comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In another aspect the present invention provides a method for treating or preventing atopic dermatis comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In another aspect the present invention provides a method for preventing an allergic disease that is immunologically associated with atopic dermatitis comprising administering a non-agonistic MR1 ligand to a subject.

In another aspect the present invention provides a method for preventing asthma in a subject previously disposed to atopic dermatitis comprising administering a non-agonistic MR1 ligand to a subject.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand to treat or prevent a skin disease or condition in a subject in need thereof.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand to treat or prevent pruritus in a subject in need thereof.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand to treat or prevent atopic dermatis in a subject in need thereof. In another aspect the present provides a use of a non-agonistic M R1 ligand to prevent an allergic disease in a subject in need thereof, wherein the allergic disease is immunologically associated with atopic dermatitis.

In another aspect the present invention provides a use of non-agonistic MR1 ligand to prevent asthma in a subject previously disposed to atopic dermatitis.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing a skin disease or condition in a subject in need thereof.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing pruritus in a subject in need thereof.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing atopic dermatitis in a subject in need thereof.

In another aspect the present invention provides a use of a non-agonistic MR1 ligand in the manufacture of a medicament for preventing an allergic disease in a subject, wherein the allergic disease is immunologically associated with atopic dermatitis.

In another aspect the present invention a use of a non-agonistic MR1 liga nd in the manufacture of a medicament for preventing asthma in a subject previously disposed to atopic dermatitis.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing a skin disease or condition in a subject in need thereof.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing pruritis in a subject in need thereof.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing atopic dermatitis in a subject in need thereof.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in preventing an allergic disease in a subject who has previously contracted atopic dermatitis.

In another aspect the present invention provides a non-agonistic MR1 liga nd for use in preventing asthma in a subject previously disposed to atopic dermatitis.

In another aspect the present invention provides a pharmaceutical composition comprising a non-agonistic MR1 ligand for use in the treatment or prevention of a skin disease or condition.

In another aspect the present invention provides a pharmaceutical composition comprising a non-agonistic MR1 ligand for use in the treatment or prevention of pruritis.

In another aspect the present invention provides a pharmaceutical composition comprising a non-agonistic MR1 ligand for use in the treatment or prevention of atopic dermatitis. In another aspect the present invention provides a pharmaceutical composition comprising a non-agonistic MR1 ligand for use in the prevention of an a llergic disease in a subject who has previously contracted atopic dermatitis.

In another aspect the present invention provides a pharmaceutical composition comprising a non-agonistic MR1 ligand for use in the prevention of asthma in a subject previously disposed to atopic dermatitis.

In another aspect the present invention provides a method for treating or preventing a skin disease or condition comprising administering a combination treatment comprising a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof.

In another aspect the present invention relates to a method of treating or preventing pruritus comprising administering a combination treatment comprising a non-agonistic MR1 ligand precursorand UV light to a subject in need thereof.

In another aspect the present invention relates to a method of treating or preventing atopic dermatitis comprising administering a combination treatment comprising a non- agonistic MR1 ligand precursor and UV light to a subject in need thereof.

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent a skin disease or condition .

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent pruritus.

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent atopic dermatitis.

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing a skin disease or condition, wherein the medicament is formulated for use in a prophylactic of therapeutic regime with UV light.

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing pruritus, wherein the medicament is formulated for use in a prophylactic of therapeutic regime with UV light.

In another aspect the present invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing atopic dermatitis, wherein the medicament is formulated for use in a prophylactic of therapeutic regime with UV light.

In another aspect the present invention relates to a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing a skin disease or condition.

In another aspect the present invention relates to a precursor non-agonistic MR1 ligand precursor and UV light for use in treating or preventing pruritus. In another aspect the present invention relates to a precursor non-agonistic MR1 ligand precursor and UV light for use in treating or preventing atopic dermatitis.

Various examples of the different aspects of the invention as discussed above are also set out below in the detailed description of the invention, although the invention is not limited thereto.

Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF FIGURES

The invention will now be described by way of example only and with reference to the Figures in which:

FIGURE 1 shows MR1 dependence of atopic dermatitis. C57BI/6 and MR1-/- mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. (A) Ear thickness at each treatment timepoint and at endpoint. (B) Trans-epidermal water loss (TEWL) at endpoint. (C-E) Ear tissue sections stained with haematoxylin &. eosin (H&E) at day 19 (scale bar = lOOpm). (C) Representative H&.E stained sections. (D) epidermal and (E) dermal thickening quantified by Image J. Each data point represents an individual ear. Plots show mean ± SEM. Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test., ****p <0.001.

FIGURE 2 shows bacterial colonization of the skin is necessary to induce atopic dermatitis. C57BI/6 and MR1-/- mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. An antibiotic cocktail of polymyxin B, bacitracin and neomycin (5000U/mL, 400U/mL and 3.5mg/mL, respectively) was topically applied daily from day -5. (A) Schema of treatment regimen. (B) Ear thickness at endpoint. (C) TEWL at endpoint. (D) ELISA quantification of TSLP serum levels at day 9. ND = not detected. Plots show mean ± SEM. Each data point represents an individual ear. Data represent 3 independent experiments. Statistics were calculated using one-way ANOVA with Tukey post test. *** 0.01< P <0.001, ****P <0.001, NS = non-significant.

FIGURE 3 shows prophylactic use of non-agonistic MR1 ligands reduced atopic dermatitis pathology. C57BI/6 mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. 6-FP or Ac-6-FP was applied daily from day 0 until day 18. (A) Schema of treatment regimen. (B-C) Ear thickness at (B) each treatment timepoint and (C) endpoint. (D) TEWL at endpoint. (E-F) Tissue sections were stained with H&E and (E) epidermal and (F) dermal thickening quantified by Image J analysis. Plots show mean ± SEM. Each data point represents an individual ear. Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test. *P <0.05, **P <0.01, *** 0.01< P <0.001, ****P <0.001, NS = non-significant.

FIGURE 4 shows therapeutic use of non-agonistic MR1 ligand reduced atopic dermatitis and promotes wound healing. C57BI/6 and MR1-/- mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. Ac-6-FP was topically applied from day 9 until day 18. (A) Schema of treatment regimen. (B-C) Ear thickness at each treatment timepoint (B) and endpoint (C). (D) TEWL at endpoint. (E) Expression of Furin, MmplO and Mmp25, as assessed by q-PCR. (F) Ear tissue sections were stained for CD31 (endothelial cell marker indicating neovascularization), vimentin and keratin 14/Ki67 (to detect proliferating keratinocytes) and area of CD31 and vimentin, and percentage of Ki- 67+ K14+ cells determined in each high-power field (HPF). Each data point represents an individual ear. Plots show mean ± SEM. Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test. **P <0.01, *** 0.01< P <0.001, ****P <0.001, NS = non-significant.

FIGURE 5 shows therapeutic activity of structurally distinct non-agonistic MR1 ligands. C57BI/6 mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. Non-agonistic MR1 ligands were topically applied from day 9 until day 18. (A) Schema of treatment regimen. (B-C) Endpoint ear thickness after treatment with Ac- 6-FP, 3-formylsalicylic acid (3-FSA), luteolin and genistein (B) as well as 5-(2- oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU) and 5-amino-6- (2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU) (C). (D) TEWL at endpoint following treatment with 5-OP-(2-deoxy)RU and 5-A-(2-deoxy)RU. Each data point represents an individual ear. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Dunnett's post-test comparing ear thickness or TEWL observed with MC903-only with the values obtained after therapeutic treatment with the different MAIT cell antagonists. *P <0.05, **P <0.01, *** 0.01< P <0.001, ****p <0.001, NS = non-significant.

FIGURE 6 shows MAIT cells control eosinophil activation in atopic dermatitis, (a-c) C57BI/6, iPHIL and MR1-/- mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. Ac-6-FP was applied daily from day 9 until day 18. Assessment of eosinophil (A) frequency and (B) activation in skin tissue by flow cytometry. (C) Ear tissue was stained with anti-SiglecF antibodies to detect and quantify eosinophils in the skin (number per high-power field; HPF). (D-H) iPHIL mice received diptheria toxin intraperitoneally alongside topical treatment of Ac-6-FP as described above. (D) Schema of treatment regimen. (E) Ear thickness at each treatment timepoint and (F) endpoint. (G) TEWL at endpoint. (H) Epidermal and dermal thickening. Each data point represents an individual ear. Plots show mean ± SEM . Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test. *P <0.05, **P <0.01, *** 0.01 < P <0.001, ****P <0.001, NS = non-significant.

FIGURE 7 shows MAIT cells control eosinophil activation in whole blood.

Whole human blood was incubated for 18 hours with the agonistic MR1 ligand and MAIT cell- specific activator 5-amino-6-D-ribitylaminouracil (5-A-RU) and MAIT cell as well as eosinophil activation assessed by flow cytometry. Frequency of activated MAIT cells (CD69+) and eosinophils (CD69+ or HLA-DR+) following whole blood activation with 5-A-RU in the presence or absence of blocking anti-MRl antibodies or Ac-6-FP are shown in the upper and lower panel, respectively. GM-CSF was used as a positive control for eosinophil activation. Data represent three independent experiments. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Dunnett's post-test comparing the level of cellular activation observed with 5-A-RU-only with the ones observed upon MR1 or cytokine blockade. *** 0.01< P <0.001, ****p <0.001.

FIGURE 8 shows therapeutic use of non-agonistic MR1 ligand, or genetic deletion of MR1, reduces IL-4 production and itch, (a-d) C57BI/6 or MR1-/- mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. Ac-6-FP was applied daily from day 9 until day 18. IL-4 levels were evaluated at endpoint in ear tissue by (A) ELISA and (B) q-PCR. Itching/scratching events were quantified by time-lapse videography and expressed as itching events per mouse within a 30-minute timeframe in (C) MR1-/- mice and (D) Ac-6-FP-treated wild-type mice. Each data point represents an individual ear or mouse. Plots show mean ± SEM. Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test. *P <0.05, *** 0.01< P <0.001, ****P <0.001.

FIGURE 9 shows topical application of folic acid, the pro-form of a non- agonistic MR1 ligand, enhances the therapeutic efficacy of phototherapy against atopic dermatitis. (A-C) C57BI/6 or MR1-/- mice were topically administered MC903 for 18 days. Ears were exposed to 500mJ/cm2 UV-B on the day following MC903 application with the last UV-B exposure on day 17. (A) Ear thickness, (B) TEWL, and (C) itch behavior at endpoint. (D-H) C57BI/6 mice were topically administered lnmol MC903 or EtOH (vehicle control) for 19 days. Ears were treated with either 6S-tetrahydrofolic acid (THF, which releases 6-FP upon photolysis) or 5-methyltetrahydrofolic acid (5MTHF, which does not release 6-FP upon photolysis) on the days following MC903 application and prior to phototherapy (500mJ/cm2 UV-B exposure). (D) Schema of treatment regimen. (E-H) Endpoint measurements of (E) ear thickness, (F) TEWL, (G) epidermal and (H) dermal thickening. Each data point represents an individual ear. Plots show mean ± SEM. Data represent three independent experiments. Statistics were calculated using one-way ANOVA with Tukey post-test. *P <0.05, **P <0.01, ****p <0.001, NS = non-significant. FIGURE 10 shows non-agonistic MR1 ligands block the allergic march from the skin to the lung. To study the allergic march, C57BI/6 or MR1-/- mice were sensitized with house dust mite (HDM) extract by topical application onto the skin, challenged intranasally 1 week later and eosinophils quantified in bronchoalveolar lavage (BAL) fluid. Data representative of 3 independent experiments. *** p<0.001, **** p<0.0001.

FIGURE 11 shows chemical structure of non-agonistic MR1 ligands. (A) 6- formylpterin (6-FP); (B) acetyl-6-formylpterin (Ac-6-FP); (C) 3-formylsalicylic acid (FSA); (D) luteolin; (E) genistein; (F) 5-amino-6-(2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU); and (G) 5-(2-oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU).

DETAILED DESCRIPTION OF THE INVENTION Definitions:

The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in microbiology, molecular biology, pharmacology, immunology, and biochemistry can be found in ¹⁵-²⁰.

It is also believed that practice of the present invention can be performed using standard microbiological, molecular biology, pharmacology and biochemistry protocols and procedures as known in the art, and as described, for example in ¹⁶ _' ²°-²⁴ and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.

As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic measures which reduce, alleviate, ameliorate, manage, prevent, restrain, stop or reverse the etiology (symptoms) of at least one disease or condition caused by or associated with microbial infection and/or invasion of the skin. The subject may show observable or measurable (statistically significant) decrease in one or more of the symptoms associated with or related to the microbial infection and/or invasion as known to those skilled in the art, as indicating improvement. In some examples, the microbial infection and/or invasion is bacterial infection and/or invasion.

The term "effective amount" as used herein means an amount effective to protect against, delay, reduce, stabilize, improve or treat a skin disease and/or condition as known in the art, and/or as described herein. In particular, an "therapeutically effective amount" of a non-agonistic MR1 ligand or a non-agonistic MR1 ligand precursor as described, is an amount that is sufficient to achieve at least a lessening of the symptoms associated with a microbial invasion and/or infection of the skin that is being or is to be treated or that is sufficient to achieve a reduction in microbial growth, or that is sufficient to increase microbial susceptibility to other therapeutic agents or to natural immune clearance.

In some examples, an effective a mount is an amount sufficient to achieve a statistically different result as compared to an untreated control.

The term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For exa mple, "about 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.

The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

The term "consisting essentially of" as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention .

The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed . These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

The term "non-agonistic MR1 ligand" as used herein mea ns any compound or molecule that is capable of inhibiting classical mucosal-associated invariant T (MAIT) cells and/or which is capable of activating non-classical MAIT cells and atypical MR1 restricted T cells.

The term "MRl-restricted T cell" is intended to cover any MRl-restricted T cell including a classical MAIT cell, non-classical MAIT cell and an atypical MRl-restricted T cell.

The term "precursor" as used in the context of "non-agonistic MR1 ligand precursor" is intended to mean any non-actived form of a non-agonistic MR1 ligand . An advantage in administering a non-agonistic MR1 ligand precursor is that the half life of the compound is significantly extended meaning it may remain in situ for extended periods of time (e.g . hours, days, weeks, months), and only following activation (e.g. by UV light) is it transformed to a prophylactically or therapeutically effective drug .

Detailed Description:

As discussed previously herein, the etiology of AD is multifactorial, with current theories considering that the combination of the loss of skin barrier function, invasion of skin resident bacteria and chronic immune activation are central to the condition. However, the primary signalling cascade that triggers the body's response via sentinel cells that signal bacterial intrusion and initiate potentially host-detrimental defence mechanisms, has not been identified .

Surprisingly, the inventors describe herein their determination that bacterial invasion of the skin is primarily sensed by MRl-restricted T cells such as mucosal-associated invariant T (MAIT) cells. More importantly, and unexpectedly, the inventors have determined that this host defence mechanism (i .e., M Rl-restricted T cell activation and the subsequent inflammatory cascade) is critically implicated in atopic dermatitis (AD) . Based on this determination, the inventors provide a potent method of inhibiting disease progression, both prophylactically and therapeutically, using non-agonistic MR1 ligands. In some examples the non-agonistic MR1 ligands include, but are not limited to, 6-formylpterin (6FP), acetyl-6- formylpterin (Ac6FP), 3-formylsalicylic acid (FSA), luteolin, genistein, 5-amino-6-(2-deoxy)- D-ribitylaminouracil (5-A-(2-deoxy)RU), and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D- ribitylaminouracil (5-OP-(2-deoxy)RU). In other examples according to the present invention, the non-agonistic M R1 ligands include certain MR1 ligands described in Keller et al⁶¹, which is incorporarted herein by reference.

Classical MRl-restricted MAIT cells express a semi-invariant T cell receptor that specifically recognizes vitamin (vit)B2 (riboflavin) metabolites derived from bacteria or yeast²⁵ in the context of MHC-related molecule 1 (MR1)²⁶'²⁷. MAIT cells have been extensively studied at their predicted mucosal homing sites, such as the gut and the lung^{28 29}, but very little is known regarding their function in the skin³⁰'³¹. What the inventors have unexpectedly determined is that the specificity of MAIT cells are unique attributes among skin-resident cells and that their activation is one of the first events that occurs upon skin barrier disruption.

The surprising role of MAIT cells in skin diseases and conditions, including pruritus, is elegantly investigated herein via the inventors' use of a new calcipotriol (MC903)-driven model of atopic dermatitis³².

M R1 knock-out (MR1KO; MR1-/-) mice are resistant to atopic dermatitis development.

Through the use a calcipotriol (MC903)-driven mouse model, the inventors have surprisingly found that the absence of MAIT cells in MRl-/-mice²⁹ strongly limits tissue pathology in the MC903 model of atopic dermatitis. In particular, the inventors have identified a surprisingly significant reduction in ear thickness, a surrogate marker of skin inflammation, and trans-epidermal water loss (TEWL) (Figs. 1A-B). Furthermore, histopathology in the MC903-treated MR1-/- mice revealed a decrease in epidermal and dermal thickening (Figs. 1C-E).

Skin resident microbes are necessary for atopic dermatitis tissue pathology, further indicating that microbial immune recognition is central to skin inflammation.

Based on the result that MR1-/- mice are resistant to atopic dermatitis development, the inventors investigated the role of MC903-induced pruritus and scratching for enabling bacterial translocation across the skin epithelium and recognition of bacterial vitB2 metabolites by MAIT cells, resulting in an inflammatory cascade. Surprisingly, the inventors have determined that topical antibiotic treatment of the skin of wild-type mice has an effect that is very similar to that observed in MR1 deficiency. The antibiotic cocktail comprised polymyxin B, bacitracin and neomycin (5000U/mL, 400U/mL and 3.5mg/mL, respectively; all in distilled water; 20mI_ applied per ear). Both ear thickness and TEWL were markedly reduced following the co-administration of MC903 and antibiotics (Figs. 2A-C). Furthermore, topical antibiotic treatment of MR1-/- mice did not further decrease disease severity, suggesting that both treatment modalities operate via the same mode of action. Thymic stromal lymphopoietin (TSLP) levels in serum were unaffected by topical antibiotic treatment (Fig 2D), indicating that this additional topical application did not impair cutaneous absorption of MC903 and subsequent release of TSLP.

Non-agonistic MR1 ligation blocks atopic dermatitis disease progression and promotes wound healing MAIT cell activation was then blocked prophylactically by topical application of the non-agonistic MR1 ligands 6FP and Ac6FP (Fig 3A). The topical co-administration of MC903 and either 6FP or Ac6FP resulted in substantial decrease in ear thickness as well as TEWL (Figs. 3B-D). Histopathology of skin tissue revealed a reduced epidermal and dermal thickening (Figs. 3E-F).

The prophylactic efficacy of non-agonistic MR1 ligation indicated that MRl-restricted T cells participate in disease initiation. To address whether non-agonistic MR1 ligation could potentially be used therapeutically (i.e. to block the progression of established disease), Ac6FP was applied topically (daily) from day 9. As shown in Figures 4A-D, topical application of Ac6FP resulted in equally reduced skin inflammation and improved barrier integrity. In addition, it was observed that the wound healing genes that are typically associated with extracellular matrix remodelling (i.e. Furin, MmplO and Mmp25 )³³ were upregulated in 6FP/MC903-treated skin, as detected by qPCR (Fig 4E).

Accelerated wound healing and associated tissue remodelling was confirmed by confocal microscopy (images not shown). In particular, upregulation of vimentin, a cytoskeletal protein crucial for cutaneous wound healing due to its supporting role for tissue integrity³⁴, increase in CD31⁺ endothelial cells³⁵ indicating neovascularization and angiogenesis, and an increase in proliferating keratinocytes (Ki-67⁺keratinl4⁺ cells) (Fig 4F) were observed.

The inventors then sought to confirm that the therapeutic activity of non-agonistic MR1 ligands was not confined to the MAIT cell antagnosists 6FP and Ac6FP. The inventors repeated the experiments described in Figure 4 using structurally dissimilar MAIT cell antagonists (or non-agonistic MR1 ligands; refer to disclosure below) 3-formylsalicylic acid (FSA), luteolin, ginstein, 5-amino-6-(2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU), and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU) in the same experiment as Ac6FP. These data are presented in Figure 5, and demonstrate that FSA, luteolin and ginstein had a similar effect to Ac6FP on skin inflammation as measured by ear thickness (Fig. 5B), while effect was less significant using 5-amino-6-(2-deoxy)-D- ribitylaminouracil (5-A-(2-deoxy)RU), and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D- ribitylaminouracil (5-OP-(2-deoxy)RU) (Fig. 5C). However, both 5-A-(2-deoxy)RU and 5-OP- (2-deoxy)RU demonstrated improved barrier activity relative to the non-treatment control with significantly reduced levels of trans-epidermal water loss (Fig. 5D).

In addition, the absence or antagonism of MAIT cells significantly reduced IL-4 production at the level of protein secretion and gene expression as measured by ELISA and qPCR (Fig 8A-B). Consistent with the involvement of IL-4 in driving pruritus in the skin³⁶, the absence or non-agonistic ligation of MR1 was associated with less pruritus (Fig 8C-D).

Finally, the data presented Figs. 6 and 7 demonstrate that the disease apthology of atopic dermatitis is mechanistically linked to eosinophil activation. The initial animal model data (Fig. 6) was confirmed by pre-clinical human data using whole blood (Fig. 7).

An important consideration having regard to the data presented herein is the classification of T cells against which MAIT cell antagonists/non-agonistic MR1 ligands demonstrate prophylactic and therapeutic activity for atopic dermatitis.

MRl-restricted T cells have been categorized into several subsets, including classical MAIT cells, non-classical MAIT cells as well as atypical MRl-restricted T cells⁵¹. Importantly, the non-agonistic MR1 ligands 6FP and Ac6FP have been shown to inhibit the activation of classical MAIT cells, but to also promote activation of non-classical MAIT cells and atypical MRl-restricted T cells⁵² _' ⁵³. Thus, Applicants cannot completely rule out that the therapeutic activities reported in the Examples/Figures referred to above occur, at least partly, via the activation of non-classical MAIT cells or other atypical MR1 restricted T cells. Nevertheless, the therapeutic activities observed with the structurally distinct non-agonistic MR1 ligands luteolin, genistein and 3-formylsalicylic acid⁵⁴, as well as the 2-deoxy modifications of 5-A-RU and 5-OP-RU⁵⁵ (Fig. 5), suggest that the antagonism of classical MAIT cells is a major function of non-agonistic MR1 ligands in the context of atopic dermatitis, since these compounds have not yet been shown to activate any non-classical or atypical MR1 restricted T cell subset. As such, the term "non-agonistic MR1 ligand" as used throughout this specification is intended to mean any compound or molecule that is capable of inhibiting activation of classical MAIT cells (i.e. a MAIT cell antagonist; MCA) and/or that is capable of activating non-classical MAIT cells or atypical MRl-restricted T cells.

Accordingly, in one aspect the present invention provides a method for treating or preventing a skin disease or condition comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In another aspect the present invention provides a method for treating or preventing pruritis comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In yet another aspect the present invention provides a method for treating or preventing atopic dermatitis comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

It is important to note that a person skilled in the relevant art may refer to atopic dermatitis as eczema, and that the terms "atopic dermatitis" and "eczema" may be used interchangeably throughout this specification. Indeed, the denomination of atopic dermatitis has evolved over time, and may be also referred to as "atopic eczema" or even "dermatitis syndrome". For example, refer to https://www.worldallerQv.org/education-and- proqrams/education/allerqic-disease-resource-center/professionals/eczema-e-atopic- eczema-ae-and-atopic-dermatitis-ad .

In certain examples according to these and other aspects of the present invention, the non-agonistic MR1 ligands includes, but is not limited to, 6-formylpterin (6-FP), acetyl-6- formylpterin (Ac-6-FP), 3-formylsalicylic acid (FSA), luteolin, genistein, 5-amino-6-(2-deoxy)- D-ribitylaminouracil (5-A-(2-deoxy)RU) and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D- ribitylaminouracil (5-OP-(2-deoxy)RU). In other examples, the non-agonistic MR1 ligand is a non-agonistic MR1 ligand described in Keller et al.⁶¹

The various structures for these compounds are illustrated in Figure 11. An important point here is that the various non-agonistic MR1 ligands are structurally dissimilar, yet demonstrate similar levels of activity in the various animal models used. For example, the data presented in Figure 5A show FSA, luteolin and genistein had similar activity profiles to Ac6FP in an animal model of atopic dermatitis (MC903; as measured by ear thickness) relative to a no treatment control. Similarly, the data presented in Figure 5C show a reduced level of trans-epidermal water loss in the MC903 animal model when treated with 5-A-(2-deoxy)RU and 5-OP-(2-deoxy)RU relative to a no treatment control.

Atopic dermatitis typically occurs in early life, and predisposes to asthma later in life through a process called the allergic or atopic ma rch⁶'⁷-⁸. The mechanism driving the pathogenic progression of allergic disease from the skin to the lung is currently unknown, although a sequential involvement of the innate and adaptive immune system has been proposed⁹. No therapeutic strategy has to date been able to prevent the allergic march¹⁰. The data presented in Fig. 10 demonstrates that the non-agonistic MR1 ligand Ac6FP blocked the allergic march from the skin to the lungs, providing prima facie evidence that a non-agonistic MR1 ligand could be used to prevent the development of asthma as an immunologically related allergic disease in patients having atopic dermatitis.

Accordingly, in another aspect the present invention provides a method for preventing an allergic disease that is immunologically associated with atopic dermatitis comprising administering a non-agonistic MR1 ligand to the subject.

In one example, the allergic disease would be predisposed to developing in a non-skin organ. In another example the orga n is the lungs.

In one example, the allergic disease is selected from asthma, allergic rhinitis, food allergy, eosinophilic esophagitis, drug allergy, vaccine allergy, insect venom allergy (i.e. systemic anaphylaxis upon bee sting), plant allergy (i .e. systemic reaction upon contact with poison ivy) .

Accordingly, in another aspect the present invention provides a method for preventing asthma in a subject disposed to atopic dermatitis comprising administering a non-agonistic MR1 ligand to the subject.

The term 'disposed' as used herein (e.g . ) in the context of atopic dermatitis means the subject previously had one or more disease characteristics of atopic dermatitis, or still currently has one or more disease characteristics of atopic dermatitis, such as skin barrier dysfunction. The characteristics of atopic dermatitis referred to herein may include both clinical and sub-clinical symptoms of the disease.

In one example according to these and other aspects of the present invention, administration comprises administration of a therapeutically or prophylactically effective amount of the non-agonistic MR1 ligand.

In one example administration comprises contacting at least one MR1- restricted T cell in a subject with the non-agonistic MR1 ligand .

In one example, administration comprises contacting at least one tissue that comprises at least one MRl-restricted T cell in a subject with the non-agonistic MR1 ligand.

In one example, the MRl-restricted T cell is selected from a classical MAIT cell, a non- classical MAIT cell or an atypical MRl-restricted T cell, as well as combinations thereof. Preferably the MRl-restricted T cell is a classical MAIT cell.

In one example at least one tissue is epidermis tissue.

In one example at least one tissue is dermis tissue.

In one example the non-agonistic MR1 ligand is a photolytic breakdown product of folic acid .

In one example the non-agonistic MR1 liga nd is a photo-derivative of folic acid that forms in vivo in a mammal under UV light. In one example the skin disease or condition is dermatitis, preferably atopic dermatitis or non-atopic dermatitis.

In one example the skin disease or condition is atopic dermatitis (AD).

In one example a therapeutically effective amount comprises about 0.001 mg to about 10 mg of the non-agonistic MR1 ligand.

In one example a therapeutically effective amount comprises 0.001 mg to 10 mg of the non-agonistic MR1 ligand.

In one example a therapeutically effective amount is at least 0.001 mg, preferably at least 0.1 mg, preferably at least 1 mg, preferably at least 5 mg, preferably at least 10 mg of the non-agonistic MR1 ligand.

In one example a therapeutically effective amount is about 0.001 mg, preferably 0.01 mg, preferably about 0.1 mg, preferably about 1 mg, preferably about 5 mg, preferably about 10 mg of the non-agonistic MR1 ligand.

In one example a therapeutically effective amount is 0.001, preferably 0.01 mg, preferably 0.1 mg, preferably 1 mg, preferably 5 mg, preferably 10 mg of the non-agonistic MR1 ligand.

In one example a prophylactically effective amount comprises about 0.001 mg to about 10 mg of the non-agonistic MR1 ligand.

In one example a prophylactically effective amount comprises 0.001 mg to 10 mg of the non-agonistic MR1 ligand.

In one example a prophylactically effective amount is at least 0.001 mg, preferably at least 0.01, preferably at least 0.1 mg, preferably at least 1 mg, preferably at least 5 mg, preferably at least 10 mg of the non-agonistic MR1 ligand.

In one example a prophylactically effective amount is about 0.001 mg, preferably about 0.01, preferably about 0.1 mg, preferably about 1 mg, preferably about 5 mg, preferably about 10 mg of the non-agonistic MR1 ligand.

In one example a prophylactically effective amount is 0.001, preferably 0.01 mg, preferably 0.1 mg, preferably 1 mg, preferably 5 mg, preferably 10 mg of the non-agonistic MRl ligand.

In one example, administration is topical administration.

In one example topical administration comprises administration of a topical composition. In one example the topical composition is a pharmaceutical composition.

As will be understood by a person skilled in the art, a number of different types of topical compositions, including but not limited to topical compositions that are pharmaceutical compositions, can be prepared including peroral, parenteral, intravenous, vaginal, or rectal compositions (but not limited thereto) as described herein, and by following the guidelines for topical application, for example, according to conventional formulation practice, see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopaedia of Pharmaceutical Technology". Cosmetic, hair care and skin care compositions may also be prepared as topical compositions. It is believed that the preparation of such topical compositions is also within the skill in the art.

In one example the composition is in the form of, or is formulated as, a topical composition. In one example the topical composition is also a pharmaceutical composition as described herein.

In one example topical administration is to an animal or part of an animal, preferably a mammal, preferably a human. In one example topical administration to an animal comprises administration to the skin.

In some examples, topical administration is onto or close to an effected area of the body.

In some examples, topical administration is onto an exterior part of the body. In one example the exterior part of the body is the skin or a part thereof.

Topical administration may be by simple application of a topical composition such as by smearing a cream, ointment, lotion or gel comprising the non-agonistic MR1 ligand onto or around an area to be treated, or from which at least one MRl-restricted T cell is to be targeted, or using a soap, detergent, disinfectant or shampoo for the same purpose. Alternatively, it may involve the use of an applicator or device suitable for enhancing the establishment of contact between the topical composition and the substrate, particularly skin, to which it is applied such as by the use of occlusive dressings or plasters comprising the composition or by way of a brush to apply a soap or shampoo. By way of non-limiting example, a composition of the invention may be impregnated or distributed onto pads, plasters, strips, gauze, sponge materials or cotton or wool pieces. In some examples, topical administration comprises spraying or misting an area to be treated.

In some examples, the topical composition comprises or consists essentially of about 0.001% to about 10% by weight (w/w) of an non-agonistic MR1 ligand, preferably about 0.01% to about 5%, preferably about 0.1% to about 1% w/w of an non-agonistic MR1 ligand.

In some examples, the topical composition comprises or consists essentially of 0.001% to 10% by weight (w/w) of an non-agonistic MR1 ligand, preferably 0.01% to 5%, preferably 0.1% to 1% w/w of an non-agonistic MR1 ligand.

In some examples, the topical composition comprises or consists essentially of less than 10% by weight (w/w) of an non-agonistic MR1 ligand, preferably less than 5%, preferably less than 1%, preferably less than 0.1%, preferably less than 0.01%, preferably less than 0.001% of an non-agonistic MR1 ligand.

In one example the topical composition is applied from once to 10 times. In some examples the topical composition is applied at least once, preferably at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times preferably at least ten times. In one example the topical composition is applied once, preferably twice, three times, four times, five times, six times, seven times, eight times, nine times preferably ten times.

The number of a pplications may be determined by the skilled person based on the disclosure provided herein and common general knowledge, and will include consideration of the extent and/or the type, severity and localisation of the skin disease or condition being treated .

In some examples the topical composition is applied hourly, daily, weekly, monthly or yearly.

In one example the topical composition is applied from once to 10 times hourly, daily, weekly, monthly or yearly. In some examples the topical composition is applied at least once, preferably at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, preferably at least ten times hourly, daily, weekly, monthly or yearly.

In one example the topical composition is applied once, preferably twice, three times, four times, five times, six times, seven times, eight times, nine times preferably ten times hourly, daily, weekly, monthly or yearly.

For topical pharmaceutical applications, a topical composition as described herein may be formulated in accordance with conventional pharmaceutical practice with pharmaceutical excipients conventionally used for topical applications. The nature of the vehicle employed in the preparation of any particular composition will depend on the method intended for administration of that composition.

Vehicles other than water can be used in topical compositions and can include solids or liquids such as emollients, solvents, humectants, thickeners a nd powders. A skilled person will appreciate that such vehicles may be used as appropriately in other compositions described herein that comprise the antibacterial combination as described herein including pharmaceutical and cosmetic compositions, as well as disinfectant compositions. Examples of each of these types of vehicles, which can be used singly or as mixtures of one or more vehicles, are as follows.

Emollients, such as stearyl alcohol, glyceryl, monoricinoleate, glyceryl monostearate, propane- 1, 2-diol, butane- 1, 3-diol, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl a lcohol, cetyl palmitate, dimethylpolysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate.

Solvents, such as water, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, tetrahydrofuran, vegetable and animal oils, glycerol, ethanol, propanol, propylene glycol, and other glycols or alcohols, fixed oils; humectants or moistening agents, such as glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin.

Powders, such as chalk, talc, kaolin, starch and derivatives thereof, gums, colloidal silicon dioxide, sodium polyacrylate, chemically modified magnesium aluminium silicate, hydrated aluminium silicate, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate.

Gelling or swelling agents, such as pectin, gelatin and derivatives thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose or oxidised cellulose, cellulose gum, guar gum, acacia gum, ka raya gum, tragacanth gum, bentonite, agar, alginates, carbomer, gelatine, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, xanthan gum.

Polymers, such as polylactic acid or polyglycolic acid polymers or copolymers thereof, paraffin, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone.

Surfactants, such as non-ionic surfactants, e.g . glycol and glycerol esters, macrogol ethers a nd esters, sugar ethers and esters, such as sorbitan esters, ionic surfactants, such as amine soaps, metallic soaps, sulfated fatty alcohols, alkyl ether sulfates, sulfated oils, and ampholytic surfactants and lecitins; buffering agents, such as sodium, potassium, aluminium, magnesium or calcium salts (such as the chloride, ca rbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartrate) .

For topical applications, the pH of a composition of the invention may be about 3 to about 9, preferably about 4 to about 8, preferably about 5, about 6 or about 7. In some examples the pH of a composition of the invention is between 3 a nd 9, preferably between, 4 and 8, between 5 and 8, between 6 and 8, preferably 7. Depending on the intended use of the composition, buffering agents as described herein and as known in the art may be used by the skilled person to obtain the pH desired.

In some examples, a topical composition as described herein is formulated as a soap, detergent, body wash, shampoo, lotion, ointment, tooth paste or foam spray.

In one example a topical composition as described herein is formulated as, or is in the form of a coating.

In one example the subject is an animal or part thereof.

In one example the animal is a mammal.

In one example the mammal is selected from the group consisting of canines, felines, bovines, ovines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids.

In one example the mammal is selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, hippopotami and humans.

In one example the part of the animal is the, skin or hide, preferably human, cow, deer, sheep or horse, skin or hide.

In one example contacting is to an animal or part thereof, and the skin disease or condition is dermatitis, preferably atopic or non-atopic dermatitis.

In one example contacting is for a sufficient time to treat or prevent the skin disease or condition.

In one example sufficient time is at least 1 minute, preferably at least 5 min, preferably at least 10 min, preferably at least 20 min, preferably at least 30 min, preferably at least 40 min, preferably at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 5 hours, preferably at least 12 hours.

In one example sufficient time is about 1 minute, preferably about 5 min, preferably about 10 min, prefera bly about 20 min, preferably about 30 min, preferably about 40 min, preferably about 1 hour, preferably about 2 hours, preferably about 3 hours, preferably about 5 hours, preferably about 12 hours.

In one example contacting comprises directly or indirectly applying the non-agonistic MR1 liga nd or a composition, including pharmaceutical compositions, comprising the non- agonistic MR1 ligand to the subject or part thereof. In one example applying is directly applying. In another example applying is indirectly applying .

In one exa mple applying comprises applying the non-agonistic MR1 ligand or composition comprising the non-agonistic MR1 ligand to the subject or part thereof at least two times.

In one example applying is applying as a coating or partial coating .

In some examples, applying comprises applying the combination or composition at least lx, or 2x, or 3x, or 4x, or 5x, or 6x, or 7x, or 8x, or 9x, preferably at least lOx. In some examples applying is applying between lx and lOx.

In one example applying is applying at least lx per day (lx/d), at least 2x/d, at least 3x/d, at least 4x/day, at least 5x/day, at least 6x/day, at least 7x/day, at least 8x/day, at least 9x/day, at least lOx/day.

In one example applying is applying lx per day ( lx/d), preferably 2x/d, 3x/d, 4x/day, 5x/day, 6x/day, 7x/day, 8x/day, 9x/day, preferablylOx/day.

In one example, administration is loca l or systemic administration. In one example, administration is intranasal, epidermal, and transderma l, oral or parenteral. In one example oral administration comprises aerosol delivery.

In one example aerosol delivery is by spray.

In one example the oral dosage form is a liquid dosage form. In one example the liquid dosage form is aqueous suspension, an aqueous solution, a non-aqueous suspension or a non-aqueous solution.

In one example the oral dosage from comprises an additional ingredient selected from the group consisting of thickeners, flavoring agents, diluents, emulsifiers, dispersing aids and binders.

In one example, parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.

In one example, administration is transient administration. In one example transient administration comprises administration of an non-agonistic MR1 ligand or a composition comprising an non-agonistic MR1 ligand as described herein for a sufficient period of time to provide a treatment or achieve a therapeutic result.

A particular and effective dosage regime according to a method of treating a skin disease or condition as described herein will be dependent on severity of the disease and/or condition to be treated and on the responsiveness of the treated subject to the course of treatment. An effective treatment may last from several hours to several days to several months or longer, or until a n acceptable therapeutic outcome is effected or assured or until an acceptable reduction of the infection is observed .

An optimal dosing schedule(s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations a nd dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of the non-agonistic MR1 ligand or composition comprising the non-agonistic MR1 ligand as described herein. In general, dosage is from 0.0001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.

In another aspect the invention relates to a method of treating or preventing pruritus comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

In another aspect the invention relates to a method of preventing a n allergic disease that is immunologically associated with atopic dermatitis comprising administering a non- agonistic MR1 ligand to a subject.

In another aspect the present invention relates to a method of preventing asthma in a subject disposed to atopic dermatitis comprising administering a non-agonistic MR1 ligand to the subject.

Specifically contemplated as examples of this aspect of the invention that is a method of treating or preventing pruritus, or preventing an allergic disease that is immunologically associated with atopic dermatitis, or preventing asthma in a subject disposed to atopic dermatitis are all of the examples set out herein relating to the aspect of the invention that is a method of treating or preventing a skin disease or condition, including all examples set out and encompassed therein.

In another aspect the present invention relates to the use of an non-agonistic MR1 ligand to treat or prevent a skin disease or condition .

In another aspect the present invention relates to the use of an non-agonistic MR1 ligand to treat or prevent pruritus.

In another aspect the present invention relates to the use of an non-agonistic MR1 ligand to treat or prevent atopic dermatitis.

In another aspect the present invention relates to the use of an non-agonistic MR1 ligand to prevent an allergic disease that is immunologically associated with atopic dermatitis. In one example the allergic disease is asthma.

Accordingly, in another aspect the present invention relates to the use of an non- agonistic MRl ligand to prevent asthma in a subject disposed to atopic dermatitis.

Specifically contemplated as examples of these aspects of the invention that are the use of an non-agonistic MRl ligand to treat or prevent a skin disease or condition, or to treat or prevent pruritus, or to treat or prevent atopic dermatitis, or to prevent an allergic disease that is immunologically associated with atopic dermatitis, are all of the examples set out herein relating to the aspects of the invention that are a method of treating or preventing a skin disease or condition, or of treating or preventing pruritus, or of treating or preventing atopic dermatitis, or of preventing an allergic disease that is immunologically associated with atopic dermatitis, including all examples set out and encompassed therein.

In another aspect the present invention provides a use of an non-agonistic MRl ligand in the manufacture of a medicament for treating or preventing a skin disease or condition in a subject in need thereof.

In another aspect the present invention provides a use of an non-agonistic MRl ligand in the manufacture of a medicament for treating or preventing pruritis in a subject in need thereof.

In another aspect the present invention provides a use of an non-agonistic MRl ligand in the manufacture of a medicament for treating or preventing atopic dermatitis in a subject in need thereof.

In another aspect the present invention provides a use of an non-agonistic MRl ligand in the manufacture of a medicament for preventing an allergic disease immunologically associated with atopic dermatitis in a subject disposed to atopic dermatitis.

In another aspect the present invention provides a use of an non-agonistic MRl ligand in the manufacture of a medicament preventing astham in a subject disposed to atopic dermatitis. In one example the medicament comprises an effective amount of the non- agonistic MRl ligand . In one example the effective amount is a therapeutically or prophylactically effective amount. In one example the medicament comprises at least one additional active agent. In one example the at least one additional active agent is an antibiotic. In one example the at least one additional active agent is a penetration enhancer.

In one example the medicament comprises an effective amount of the additional active agent. In one example the effective amount of the at least one additional active agent is a therapeutically or prophylactically effective amount.

In one example the medicament consists essentially of an effective amount of the non- agonistic MR1 ligand and an additional active agent. In one example the effective amount of the additional active agent is a therapeutically or prophylactically effective amount.

In one example the medicament is formulated for administration, or is in a form for administration, to a subject in need thereof.

In other examples the medicament is in a form for, or is formulated for intranasal, epidermal, transdermal, oral or parenteral administration. In one example parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. Preferably the medicament is formulated for topical administration, or is in the form of a topical composition, or when administered, is administered topically.

In one example the medicament is in a form for, or is formulated for, parenteral administration in any appropriate solution, preferably in a sterile aqueous solution which may also contain buffers, diluents and other suitable additives.

In one example the medicament formulated for, or is in a form for oral administration selected from the group consisting of a powder, a granule, an aqueous suspension, an aqueous solution, a non-aqueous suspension, a non-aqueous solution, a gel, a slurry, an ointment, a cream, a spray, a capsule, a pill, a lozenge, and a tablet.

When administered orally, the addition of one or more of the following may be desirable: thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders.

In one example the medicament is formulated for, or is in a form for topical or direct administration selected from the group consisting of transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders. Conventional carriers, particularly pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be used as required or desired in this example.

In one example, the direct administration is direct application or local application. In one example direct or local application comprises application of the medicament in combination with a delivery reagent or additional active agent.

A person skilled in the art will be able to choose the appropriate mode of administration of the medicament with reference to the literature and as described herein. By way of non- limiting example, a topical application would be preferred for the treatment and prevention of atopic dermatitis.

In one example the medicament is for, is formulated for, or is in a form for administration separately, simultaneously or sequentially with an additional active agent.

By way of non-limiting example, one additional active agent that may be included in the composition of, or for use in the invention, is an antibiotic that is, or is suspected of being effective against a target cell, particularly a target bacterial cell. In one example the target bacterial cell is a Gram-negative bacterial cell or a Gram positive bacteria l cell, preferably a Gram-negative bacterial cell.

In one example the medicament comprises an non-agonistic MR1 ligand as described herein and an antibiotic, wherein the medicament is for, is formulated for, or is in a form for separate, simultaneous or sequential administration of the non-agonistic MR1 ligand and the antibiotic in the combination to a subject.

In one example the medicament is formulated for application to an animal or part thereof. In one example the medicament is in a form for application to an animal or part thereof. In one example the medicament is formulated for administration to an animal . In one example the medicament is in a form for administration to an animal.

Specifically contemplated herein as examples of these aspects of the invention that are the use of an non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing a skin disease or condition, or for treating or preventing pruritus, or for treating or preventing atopic dermatitis, are a ll of the examples set out herein relating to the aspects of the invention that are a method for treating or preventing a skin disease or condition, or for treating or preventing pruritus, or for treating or preventing atopic dermatitis, including all examples set out and encompassed therein.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing a skin disease or condition.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing pruritis.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing atopic dermatitis.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in treating or preventing an allergic disease immunologically associated with atopic dermatitis.

In another aspect the present invention provides a non-agonistic MR1 ligand for use in preventing asthma in a subject disposed to atopic dermatitis.

Specifically contemplated herein as examples of these aspects of the invention that are an non-agonistic MR1 ligand for use in treating or preventing a skin disease or condition, or for use in treating or preventing pruritis, or for use in treating or preventing atopic dermatitis, or for use in treating or preventing an allergic disease immunologically associated with atopic dermatitis, or for preventing asthma in a subject disposed to atopic dermatitis, are all of the examples set out herein relating to the aspects of the invention that are the use of an non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing a skin disease or condition, or for treating or preventing pruritus, or for treating or preventing atopic dermatitis, as well as a method for treating or preventing a skin disease or condition, or for treating or preventing pruritus, or for treating or preventing atopic dermatitis, including all examples set out and encompassed therein.

Phototherapy methods:

Both UVA and UVB phototherapy are currently being used for the treatment of atopic dermatitis^{8 11}. Several mechanisms of action of UV light therapy have been published. UV light induces the apoptosis (induced cell death) of pathogenic T and B cells as well as mast cells, but the extent and specific cell death mechanism depends on the source of the UV light (i.e. UVA versus UVB light)²⁸ _' ³⁷. The source of UV light also impacts levels of TNF-a in human skin³⁸. UVB increases TNF-a levels while UVA diminishes TNF-a production. Both treatment modalities increase local IL-10 and cis-urocanic production, both associated with immunomodulatory activity³⁸. UVA has also been shown to downregulate IFN-g and ICAM-1, leading to decreased lymphocyte recruitment³⁹'⁴⁰. Another hypothesis is that phototherapy exerts its activity through the production of vitamin D⁴¹ _' ⁴², but the data to support this hypothesis are equivocal. Finally, more recent observations indicate that the UV light driven photoconversion nitric oxide stores in skin drives the differentiation of CD25⁺foxp3⁺ regulatory T cell with skin-homing characteristics⁴³.

The non-agonistic MR1 ligand 6FP is a photo-derivative of folic acid that readily forms in vitro under UVA and UVB light⁴⁴'⁴⁵. Studies in psoriasis patients showed that folic acid/vitB9 blood levels are lowered by phototherapy⁴⁶, likely reflecting photolysis of folic acid. 6FP was fortuitously identified as MR1 ligand due to its presence in cell culture media, but it has not yet been associated with any physiological relevance⁴⁷.

Additionally, and quite unexpectedly, the inventors have found that UVB treatment of a lmg/mL solution of folic acid with narrow band UVB dose-dependently reduces AD pathology when applied topically onto the ears of MC903 treated mice (Figs. 9A-C).

Without wishing to be bound by theory the inventors believe that they have identified a novel mechanism of action of phototherapy whereby inhibition of MR1- restricted T cell activation occurs as a consequence of the photoconversion of folic acid into 6FP both ex vivo and in vivo.

Accordingly, in another aspect the invention provides a method for treating or preventing a skin disease or condition comprising administering a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof. In another aspect the present invention provides a method for treating or preventing pruritis comprising administering a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof.

In another aspect the present invention provides a method for treating or preventing atopic dermatitis comprising administering a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof.

In one example administration comprises contacting at least one MR1- restricted T cell in a subject with the non-agonistic MR1 ligand precursor.

In one example administration comprises contacting at least one MAIT cell in a subject with the non-agonistic MR1 ligand precursor.

In one example, administration comprises contacting at least one tissue that comprises at least one MAIT cell in a subject with the non-agonistic MR1 liga nd precursor.

In one example at least one tissue is epidermis tissue.

In one example at least one tissue is dermis tissue.

In one example the non-agonistic MR1 ligand precursor is folic acid.

In one example, administration of the non-agonistic MR1 ligand precursor is by is topical administration .

In one example topica l administration comprises administration of a topical composition . In one example the topical composition is a pharmaceutical composition .

In one example, administration of the non-agonistic MR1 ligand precursor is by oral administration.

In one example oral administration comprises aerosol delivery.

In one example oral administration comprises application of a liquid, gel, cream, ointment, lotion or slurry.

In one example oral administration comprises delivery of an oral dosage form.

In one example the oral dosage form is a solid oral dosage form.

In one example the solid oral dosage form comprises a powder, a granule, a tablet, a pill, a capsule or a lozenge or combination thereof.

In one example the oral dosage form is a liquid dosage form.

In one example the liquid dosage form is aqueous suspension, an aqueous solution, a non-aqueous suspension or a non-aqueous solution.

Also specifically contemplated herein as formulation and administration examples of the combination method for treating or preventing a skin disease or condition comprising administering a non-agonistic MR1 liga nd precursor and UV light, are all of the formulation and administration examples set out herein for the methods for treating or preventing a skin disease or condition, or for treating or preventing pruritis, or for treating or preventing atopic dermatitis (including the corresponding uses) using an non-agonistic MR1 ligand alone. For example, a skilled worker recognizes that the examples described herein that relate to the formulation and administration of an non-agonistic MR1 ligands or a composition comprising an non-agonistic MR1 ligand, or the use of an non-agonistic MR1 ligand in the manufacture of a medicament, are also specifically contemplated as examples of formulating and administering a non-agonistic MR1 ligand precursor.

In one example the topical composition comprises lug/mL folic acid w/w.

In one example the topical composition comprises 1% to 10% folic acid, preferably 2% to 9%, preferably 3% to 8%, preferably 4% to 7%, preferably 5% to 6%, preferably 5% folic acid w/w.

In one example the topical composition comprises about 1%, preferably about 2%, preferably about 3%, preferably about 4%, preferably about 5%, preferably about 6%, preferably about 7%, preferably about 8%, preferably about 9% preferably about 10% folic acid.

In one example the topical composition comprises 1%, preferably 2%, preferably 3%, preferably 4%, preferably 5%, preferably 6%, preferably 7%, preferably 8%, preferably 9%, preferably 10% folic acid.

In one example administering the UV light is by direct illumination of a subject, or of a tissue of a subject, that comprises at least one MAIT cell.

In one example administering the UV light is by direct illumination of the subject.

In one example administering the UV light is by direct illumination of at least one tissue comprising at least one MAIT cell.

In one example the UV light is from an artificial source.

In one example the UV light is UVA or UVB.

In one example the UV light has a wavelength of about 10 nm to about 400 nm, preferably of 280 nm to 400 nm.

In one example the UV light is UVA.

In one example the UVA light has a wavelength of about 315 nm to about 400 nm, preferably of 315 nm to 400 nm. This includes, without limitation, a wavelength that is about 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 and about 400 nm, and preferably a wavelength that is 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 and 400 nm.

In one example the UV light is UVB.

In one example the UVB light has a wavelength of about 280 nm to about 315 nm, preferably 280 nm to 315 nm. This includes, without limitation, a wavelength that is about 280, 285, 290, 295, 300, 305, 310 and about 315 nm, and a wavelength that is preferably 280, 285, 290, 295, 300, 305, 310 and 315 nm. In one example direct illumination provides an effective dose of UV.

In one example the effective dose of UV is about 50%, preferably is 50% of the minimal erythema dose (MED) for the subject to be treated.

In one example the effective dose is about 100 mJoule/cm² to about 1200 mJoule/cm², preferably about 150 mJoule/cm² to about 1100 mJoule/cm², preferably about 200 mJoule/cm² to about 1000 mJoule/cm², preferably about 250 mJoule/cm² to about 900 mJoule/cm², preferably about 300 mJoule/cm² to about 800 mJoule/cm², preferably about 350 mJoule/cm² to about 700 mJoule/cm², preferably about 400 mJoule/cm² to about 600 mJoule/cm², preferably about 450 mJoule/cm² to about 550 mJoule/cm², preferably about 500 mJoule/cm².

In one example the effective dose is 100 mJoule/cm² to 1200 mJoule/cm², preferably 150 mJoule/cm² to 1100 mJoule/cm², preferably 200 mJoule/cm² to 1000 mJoule/cm², preferably 250 mJoule/cm² to 900 mJoule/cm², preferably 300 mJoule/cm² to 800 mJoule/cm², preferably 350 mJoule/cm² to 700 mJoule/cm², preferably 400 mJoule/cm² to 600 mJoule/cm², preferably 450 mJoule/cm² to 550 mJoule/cm², preferably 500 mJoule/cm².

In one example the effective dose is about 100 mJoule/cm², preferably about 200 mJoule/cm², preferably about 300 mJoule/cm² , preferably about 400 mJoule/cm², preferably about 500 mJoule/cm², preferably about 600 mJoule/cm², preferably about 700 mJoule/cm² , preferably about 800 mJoule/cm², preferably about 900 mJoule/cm², preferably about 1000 mJoule/cm², preferably about 1100 mJoule/cm², preferably about 1200 mJoule/cm².

In one example the effective dose is 100 mJoule/cm², preferably 200 mJoule/cm², preferably 300 mJoule/cm² , preferably 400 mJoule/cm², preferably 500 mJoule/cm², preferably 600 mJoule/cm², preferably 700 mJoule/cm² , preferably 800 mJoule/cm², preferably 900 mJoule/cm², preferably 1000 mJoule/cm², preferably 1100 mJoule/cm², preferably 1200 mJoule/cm².

Specifically contemplated herein as examples of this aspect of the present invention that is a combination method for treating or preventing a skin disease or condition comprising administering a non-agonistic MR1 ligand precursor and UV light, are all of the examples set out herein relating the aspect of the invention that is a method for treating or preventing a skin disease or condition comprising administering an non-agonistic MR1 ligand, including all examples set out and encompassed therein that relate to to formulation and/or administration of the non-agonistic MR1 ligand.

In another aspect the invention relates to a method of treating or preventing pruritus comprising administering a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof.

Specifically contemplated herein as examples of this aspect of the invention that is a combination method for treating or preventing pruritus are all of the examples set out herein relating the aspect of the invention that is a method of treating or preventing a skin disease or condition, including all examples set out and encompassed therein.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent a skin disease or condition.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent pruritus.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent atopic dermatitis.

Specifically contemplated herein as examples of these aspects of the invention that are the use of a non-agonistic MR1 ligand precursor and UV light to treat or prevent a skin disease or condition, or to treat or prevent pruritus, including atopic dermatitis, a re all of the examples set out herein relating the aspects of the invention that are methods for treating or preventing a skin disease or condition, or for treating or preventing pruritus, or for treating or preventing atopic dermatitis, including all examples set out and encompassed therein.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing a skin disease or condition, wherein the medicament is used in combination with UV light.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing pruritis, wherein the medicament is used in combination with UV light.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing atopic dermatitis, wherein the medicament is used in combination with UV light.

In one example the non-agonistic MR1 ligand precursor is formulated for administration to a subject or to a tissue comprising at least one MRl-restricted T cell with UV light. In one example, the MRl-restricted T cell is a classical MAIT cell.

In one example the non-agonistic MR1 liga nd precursor is to be administered to a subject or to a tissue comprising at least one MRl-restricted T cell, including classical MAIT cells, with UV light.

In one example the non-agonistic MR1 ligand precursor, when adiminstered, is administered to a subject or to a tissue comprising at least one MR- 1 restricted T cell, including classical MAIT cells, with UV light.

In one example the non-agonistic M R1 ligand precursor and the UV light are to be administered in a method comprising administering the non-agonistic MR1 ligand precursor, optionally waiting a predetermined period of time, administering the UV light.

In one example the method comprises first determining the MED of the subject to be treated . Specifically contemplated herein as examples of this aspect of the invention is the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament to treat or prevent a skin disease or condition, are all of the examples set out herein relating the aspects of the invention that are combination methods of treating or preventing a skin disease or condition, and all of the examples set out herein relating the aspects of the invention that are the use of an non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing a skin disease or condition, including all examples set out and encompassed therein. In one example, the skin disease or condition is atopic dermatitis.

In another aspect the invention relates to the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament for treating or preventing pruritus.

Specifically contemplated herein as examples of this aspect of the invention that is the use of a non-agonistic MR1 ligand precursor in the manufacture of a medicament to treat or prevent pruritus, are all of the examples set out herein relating the aspect of the invention that is the use of a non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing a skin disease or condition, including all of the examples set out herein relating the aspects of the invention that are the use of a n non-agonistic MR1 liga nd in the manufacture of a medicament for treating or preventing a skin disease or condition, including all examples set out and encompassed therein.

In another aspect the invention relates to a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing a skin disease or condition.

In another aspect the invention relates to a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing pruritus.

In another aspect the invention relates to a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing atopic dermatitis.

Specifically contemplated herein as examples of these aspects of the invention that are a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing a skin disease or condition, and a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing pruritus, and a non-agonistic MR1 ligand precursor and UV light for use in treating or preventing atopic dermatitis, are all of the examples set out herein relating to the aspects of the invention that are the use of a non-agonistic MR1 liga nd in the manufacture of a medicament for treating or preventing a skin disease or condition or for treating or preventing puritis, or for treating or preventing atopic dermatitis, including all examples set out and encompassed therein.

Various aspects of the invention will now be illustrated in non-limiting ways by reference to the following examples. EXAMPLES

MATERIAL AND METHODS:

Mice: Specific pathogen-free C57BL/6 mice (wild-type) a nd MR1-/- were used between 6-8 weeks of age and sex-matched for each experiment. Mice were housed at the Malaghan Institute of Medical Research Biomedical Research Unit and all experimental procedures were approved by the Victoria University Animal Ethics Committee a nd performed according to the institutional guidelines.

Topical applications on the skin to induce AD, apply non-agonistic MR1 ligands and to block skin resident microbes: Mice were anaesthetized using i. p. ketamine/xylazine. MC903 (calcipotriol; Cayman Chemicals, Michigan USA) was dissolved in 100% ethanol (EtOH) and topically applied on mouse ears (l nmol in 20mI ear). As vehicle control, the same volume of EtOH was applied . Topical application was 3 times a week for 19 days³². Acetyl-6-formylpterin (Ac6FP) or 6-formylpterin (6FP) (Cayman chemicals, Michigan USA) were dissolved in 100% ethanol (EtOH), at lmg/mL (5mM) and topically applied daily from day 0 (prophylactically) and from day 9 (therapeutica lly) until day 18 (Figure 3 and 4, respectively) . An antibiotic cocktail comprising of polymyxin B, bacitracin and neomycin (5000U/ml_, 400U/mL a nd 3.5mg/mL, respectively; a ll in distilled water; 20pL applied per ear) (Sigma Aldrich, Auckland New Zealand) dissolved in distilled water. This cocktail was applied topically daily from day -5 until day 16 (Figure 2) .

Photolysis of folic acid products: Folic acid (Sigma Aldrich, Auckland New Zealand) and 6S tetrahydrofolic acid (Cayman chemicals, Michigan USA) were dissolved in 70% and 100% EtOH, respectively, at 5mM . This was topically applied to the skin following MC903 treatment 3 times a week. The DermaPal handheld phototherapy unit (Daavlin, Netherlands) was used for UV exposure. It emits narrowband ultraviolet B (UVB) light at wavelengths of 311-312nm and runs at 6.1 mW/cm². For the ex vivo experiments the folic acid itself was exposed to narrowband UVB for either 2 or 8 minutes. The resulting solution was then topically applied to the skin. For the in vivo experiments the skin was exposed to narrowband UVB at 500mJ/cm² directly after topical application of the 6S tetrahydrofolic acid (Figure 6).

Ear thickness measurements: Ear thickness was measured, while mice were sedated with a digital caliper on each treatment day just prior to topical application of MC903 (Figure 1- 4, 6) .

Transepidermal water loss (TEWL) measurements: TEWL was measured while mice were sedated, using the DERMALAB™ TEWL probe (Cortex Technology Denmark) at endpoint. TEWL was measured on both ears of the mice, at room temperature and results were recorded when TEWL regions stabilized. Two readings from each ear were taken and averaged for each mouse (Figure 1-4) .

Histological evaluation and quantification : Ear tissue was fixed in 4% PFA (Life Technologies, Auckland New Zealand) for 24 hours, thereafter processed, paraffin embedded and sectioned at 4pm. Haematoxylin & Eosin (H&E), staining was conducted on ear sections. H&E stained tissue was evaluated at 20X magnification and measurements of acanthosis (epidermal thickening) and dermal thickening were quantified by Image J analysis. Images of the tissue sections were taken on an Olympus Brightfield (BX51) microscope (Figure 1, 3) .

Confocal microscopy: Ears tissue was cryosectioned at 8pm and fixed in 100% cold acetone for 3 minutes. Thereafter, tissue was rinsed in PBS and incubated with the respective antibodies as listed herein; purified keratin 14 (Poly9060; Biolegend) secondary AF647 IgG (Jackson Immuno Research), Ki-67 (SolA15) eF450 (eBioscience), anti-CD31 (390) BV421(Biolegend), anti-vimentin (091D3) AF488 (Biolegend) and SiglecF (E50-2440) PE CF594 (BD). Slides were rinsed in PBS, mounted and visualized on a FV1200 confocal microscope. Images were obtained at both 40X and 20X. These images were subsequently assessed a nd quantified using Image J analysis (Figure 4).

Measurement of cytokines in tissue: Ear samples were homogenized in PBS containing protease inhibitors (HaltTM protease inhibitor; Thermo Scientific, Auckland New Zealand) and 5mM EDTA. Tissue lysates were used to perform ELISAs to detect cytokine production⁴⁹. TSLP (R & D systems), and IL-4 (prepared in house) ELISA kits were used. Tissue concentrations are expressed relative to total protein using a BCA™ protein assay kit (Thermo Scientific, Auckland New Zealand) (Figure 2, 5) .

Scratching events: Mice were recorded via time-lapse videography and itch events determined and quantified . This pathophysiological measurement was obtained from video observation of the relevant treatment groups for 40 minutes prior to experimental endpoint and this was conducted as per specifications in Lee et a/⁵⁰ (Figure 5).

Quantitative Real Time PCR (Q-PCR): A quantitative real time PCR was performed from cDNA of mouse ear tissue using Qiagen DNeasy Mini Kit (Applied Biosystems, Foster City, CA USA) as per manufactures protocol. RNA to cDNa conversion was carried out using Applied Biosciences High - Capacity RNA-to cDNA™ Kit. TaqMan® gene expression master mix (Applied Biosystems, Foster City, CA USA) and TaqMan® primers for mmplO, mmp25 and furin were utilized to conduct q-PCR analysis from ear tissue. Applied Biosystems software was used to determine the cycle threshold (CT) value for each primer. ACT values were calculated by normalizing samples to GAPDH control and comparing with the EtOH- treated tissue (figure 4, 5).

INDUSTRIAL APPLICABILITY

The methods and uses of the invention find use in the treatment and/or prevention of pruritus/scratching and in the treatment and/or prevention of skin diseases and or conditions, particularly dermatitis. REFERENCES

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Claims

CLAIMS:

1. A method of treating or preventing atopic dermatitis comprising administering a non- agonistic MR1 ligand to a subject in need thereof.

2. A method of preventing asthma in a subject disposed to atopic dermatitis comprising administering a non-agonistic MR1 ligand to the subject.

3. A method of treating or preventing pruritis comprising administering a non-agonistic MR1 ligand to a subject in need thereof.

4. The method of any one of claims 1 to 3 wherein the non-agonistic MR1 ligand is selected from 6-formylpterin (6FP), acetyl-6-formylpterin (Ac6FP), 3-formylsalicylic acid (FSA), luteolin, genistein, 5-amino-6-(2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU) and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU) or a combination thereof.

5. The method of any one of claims 1 to 4 wherein:

a. administering comprises contacting at least one MRl-restricted T cell in a subject or at least one tissue that comprises at least one MRl-restricted T cell in a subject with the non-agonistic MR1 ligand; and

b. the at least one tissue is dermis or epidermis.

6. A pharmaceutical composition comprising an effective amount of a non-agonistic MR1 ligand for use in treating atopic dermatitis in a subject in need thereof, or for use in treating or preventing pruritis in a subject, or for use in preventing asthma in a subject disposed to atopic dermatitis.

7. The pharmaceutical composition of claim 6, wherein the non-agonistic MR1 ligand is selected from 6-formylpterin (6FP), acetyl-6-formylpterin (Ac6FP), 3-formylsalicylic acid (FSA), luteolin, genistein, 5-amino-6-(2-deoxy)-D-ribitylaminouracil (5-A-(2-deoxy)RU) and 5-(2-oxopropylideneamino)-6-(2-deoxy)-D-ribitylaminouracil (5-OP-(2-deoxy)RU) or a combination thereof.

8. The pharmaceutical composition of claim 6 or claim 7 formulated for topical administration.

9. Use of non-agonistic MR1 ligand in the manufacture of a medicament for treating or preventing atopic dermatitis in a subject, or for treating or preventing pruritis in a subject, or for preventing asthma in a subject disposed to atopic dermatitis.

10. The use of claim 9 wherein the medicament comprises at least one antibiotic.

11. The use of claim 9 or claim 10 wherein the medicament is in a form for, or is formulated for, topical administration.

12. The use of any one of claims 9 to 11 wherein the medicament comprises a non- agonistic MR1 ligand and an antibiotic, wherein the medicament is for, is formulated for, or is in a form for separate, simultaneous or sequential administration of the non-agonistic MR1 ligand and the antibiotic in combination to a subject.

13. A method for treating or preventing atopic dermatitis, the method comprising administering a combination treatment comprising a non-agonistic MR1 ligand precursor and UV light to a subject in need thereof.

14. The method of claim 13 wherein administering comprises contacting at least one MR1- restricted T cell in a subject or at least one tissue that comprises at least one MRl-restricted T cell in a subject with the non-agonistic MR1 ligand.

15. The method of claim 13 or claim 14 wherein :

a . the at least one tissue is dermis or epidermis;

b. the non-agonistic MR1 ligand precursor is folic acid ;

c. administering the UV light is by direct illumination of a subject, or of a tissue of a subject, that comprises at least one MRl-restricted T cell; d . UV light is from an artificial source; and/or

e. UV light is UVA or UVB or a combination of UVA and UVB.