WO2023248151A1 - Method of treating skin field cancerization with actinic keratoses - Google Patents

Abstract

The present disclosure relates to a method of treating skin field cancerization with actinic keratoses using N-[(1 S)-1-(5-fluoropyrimidin-2-yl)ethyl]-3-(5-isopropoxy-1H-pyrazol-3-yl)-3H-imidazo[4,5-b]pyridin-5-amine (hereinafter also referred to as "Compound A"), or a pharmaceutically acceptable salt thereof.

Description

METHOD OF TREATING SKIN FIELD CANCERIZATION WITH ACTINIC KERATOSES

TECHNICAL FIELD

[0001] The present disclosure provides a method of treating skin field cancerization with actinic keratoses using 7V-/(lS)-l-(5-fluoropyrimidin-2-yl)ethyl]-3-(5-isopropoxy-lJ/-pyrazol- 3-yl)-3J7-imidazo[4,5-Z>]pyridin-5-amine (hereinafter also referred to as “Compound A”), or a pharmaceutically acceptable salt thereof. Certain dosage regimens are also provided for treating skin field cancerization with actinic keratoses.

BACKGROUND

[0002] Growth factors are important signaling molecules that promote the growth, development and homeostasis of many cellular systems. Neurotrophins are growth factors which are responsible for central and peripheral neuronal growth, maturation and death. Neurotrophins activate cell surface receptors called tropomyosin-like receptors which in turn regulate intracellular kinases called tyrosine receptor kinases. The tropomyosin-related kinase (TRK) family of receptors includes TRKA, TRKB, TRKC and p75 and serve as high affinity cell surface receptors for the growth factors NGF, BDNF, NT3 and NT4, respectively. Inhibition of these receptors may lead to the modulation or inhibition of intracellular signaling cascades that regulate cell growth and proliferation, cellular communication between cells that regulate signaling, feedback mechanism and homeostasis. These growth factors have been implicated in the growth and proliferation of both neuronal and non-neuronal cells.

[0003] TRK inhibitors have the potential to be used in the treatment or prevention of various diseases including skin disorders, pain, inflammatory and immunological conditions. For example, neurotrophins are involved in the regulation of skin homeostasis, skin remodeling and response to various pathological conditions including infections, inflammation, and other skin insults. Specifically, neurotrophins have been implicated in certain dermatoses (see, e.g., Truzzi, et. al. (2011) Dermato-Endocrinology 3: 32; Botchkarev, et. al. (2006) J. Investigative Dermatology 126: 1719; Papoui, et. al. (2011) Neuropeptides, 45: 417). Compounds that can block the neurotrophin signaling cascade have been demonstrated to be beneficial in the treatment of skin disorders which rely on TRK signaling (see, e.g., Raychaudhuri, et. al. (2004) J. Investigative Dermatology 122:812; Raychauduri, et. al. (2004) Prog. Brain Res, 146: 133; Cranston, et. al. (2017) Trials, 18: 111).

[0004] The incidence of skin cancer has been rising over the years. Non-melanoma or keratinocyte carcinoma (squamous cell carcinoma and basal cell carcinoma) rates continue to increase by at least 5% per annum in the U.K. (Venables ZC, et al., “Epidemiology of basal and cutaneous squamous cell carcinoma in the U.K. 2013-15: a cohort study”, Br J Dermatol. 2019 Sep;181(3):474-482) and by 77% in the U.S (https://www.skincancer.org/skin-cancer- information/skin-cancer-facts/ accessed June 2022). As such they are the most common cancers in the U.K. and the most common cancers in white ethnic populations worldwide. This continued rise in basal (BCC) and squamous (SCC) carcinoma incidence occurs despite vigorous sun avoidance campaigning and has led to dramatic shifts in working patterns of dermatologists here in the U.K. and many other countries with Caucasian populations (Levell NJ, et al., “Basal cell carcinoma epidemiology in the U.K.: the elephant in the room”, Clin Exp Dermatol. 2013 Jun;38(4):367-9). Any person developing a keratinocyte carcinoma is at increased risk, 18-44%, of developing a subsequent SCC or BCC within 3 years, this rises to 81% risk if there have been two previous BCC (Marcil I, Stem RS. “Risk of developing a subsequent nonmelanoma skin cancer in patients with a history of nonmelanoma skin cancer: a critical review of the literature and meta-analysis”, Arch Dermatol. 2000 Dec;136(12): 1524- 30). Hence there is an urgent clinical need to improve prevention strategies to reduce the risk for developing keratinocyte carcinoma.

[0005] Actinic keratosis (AK) is a cutaneous condition that forms on skin damaged by chronic exposure to ultraviolet (UV) rays from the sun and/or indoor tanning. Actinic keratosis (also called “solar keratosis”) is a condition that results in thick, scaly or crusty patches of skin comprising dysplastic keratinolytic lesions.

[0006] In prospective studies 65% and 35% of all SCC and BCC, respectively, arise from a pre-existing actinic keratosis (Criscione VD et al., Department of Veteran Affairs Topical Tretinoin Chemoprevention Trial Group. “Actinic keratoses: Natural history and risk of malignant transformation in the Veterans Affairs Topical Tretinoin Chemoprevention Trial. Cancer” 2009 Jun 1;115(11):2523-30). Retrospectively, 72-82% pathological sections of SCC and 5-12% of BCC demonstrate actinic keratoses changes within the skin epidermis (Czarnecki D, et al., “The majority of cutaneous squamous cell carcinomas arise in actinic keratoses”, J Cutan. Med Surg. 2002; 6: 207- 209; Mittelbronn MA, et al., “Frequency of pre-existing actinic keratosis in cutaneous squamous cell carcinoma”, Int J Dermatol. 1998; 37: 677- 681; Frost CA, et al., “The prevalence and determinants of solar keratoses at a subtropical latitude”, Br J Dermatol. 1998; 139: 1033- 1039). In a pivotal study, after topical treatment with topical 5- flurouracil applied twice daily for 4 weeks to the face and scalp, within the 1-year follow-up window there was a 75% and 11% reduction in SCC and BCC (Weinstock MA, et al., Veterans Affairs Keratinocyte Carcinoma Chemoprevention Trial (VAKCC) Group. “Chemoprevention of Basal and Squamous Cell Carcinoma with a single course of Fluorouracil, 5%, cream: A Randomized Clinical Trial” JAMA Dermatol. 2018 Feb 1 ; 154(2): 167-174).

[0007J Actinic keratoses are one of the commonest diagnoses in dermatology. The risk of an individual actinic keratosis transforming into squamous cell carcinoma is relatively low, with an estimated rate of between 0.025% to 20% per year for an individual actinic keratosis based on a review of 62 studies (Quaedvlieg PJ, et al., “Actinic keratosis: how to differentiate the good from the bad ones?” Eur J Dermatol. 2006; 16: 335- 339). Studies have sought to try and identify further characteristics that pose the greatest risk for keratinocyte carcinoma development and therefore would benefit greatest from treatment. These features include: increased age (> 70 years of age), males, bald scalp, Fitzpatrick skin type 1/2, the presence of more than 10 actinic keratoses, the presence of multiple actinic keratoses with an area which is greater than 25% involved, and immunosuppression (Flohil et al., “Clinical comparison of actinic changes preceding squamous cell carcinoma vs. intraepidermal carcinoma in renal transplant recipients”, Clin Exp Dermatol. 2017 Dec;42(8):895-897).

[0008] The greatest risk of keratinocyte carcinoma development is when a patient has a confluent area of skin comprising multiple actinic keratoses (also called a “field cancerization” or “actinic neoplasia syndrome”), which similarly distinguishes treatments into those that treat an individual lesion and a those suitable for treatment of a “field” (de Berker D et al., British Association of Dermatologists' guidelines for the care of patients with actinic keratosis 2017. Br J Dermatol. 2017 Jan;176(l):20-43; and Weinstock, M. A., et al., VATTC Trial Group (2009). “Quality of life in the actinic neoplasia syndrome: The VA Topical Tretinoin Chemoprevention (VATTC) Trial”, Journal of the American Academy of Dermatology, 61(2), 207-215).

[0009] The concept and definition of skin field cancerization, a premalignant state of skin within which multiple keratinocyte carcinoma develop, has been adopted from the original work of Slaughter’s studies on oropharyngeal SCC (Slaughter DP, et al., “Field cancerization in oral stratified squamous epithelium”, Cancer. 1953;6:963-968; and Christensen SR. “Recent advances in field cancerization and management of multiple cutaneous squamous cell carcinomas”, FlOOORes. 2018;7:F1000 Faculty Rev-690). A Cochrane review determined that for field-directed treatments, diclofenac, 5-fluorouracil, and imiquimod are good options but these agents are associated with different side-effects and cosmetic results (Sinclair R, et al., “A review of actinic keratosis, skin field cancerisation and the efficacy of topical therapies”, Australas J Dermatol. 2021 May;62(2): 119-123). Primary care guidelines distinguish treatment based on “field” treatment (https://www.cochrane.org/CD004415/SKIN_interventions-actinic- keratoses) Gupta et al., Cochrane Db Syst Rev (2012), doi:10.1002/14651858. cd004415.pub2. 2. Gupta et al. Br J Dermatol 2013;169:250-9). In summary, treating areas with multiple actinic keratoses provides a possible approach to keratinocyte carcinoma prevention.

[0010] At the present time, there are a limited number of therapeutic treatments for AK. Furthermore, all of these treatments have some associated level of patient inconvenience and/or side effects. Surgical procedures, such as cryosurgery and surgical removal (curettage and desiccation), are used but are not generally suitable for patients who have one or more isolated lesions. Furthermore, these approaches are invasive and require to be performed in a clinical setting. These options can also lead to scar formation (surgery) or cause the affected area to lose its natural pigment (cryotherapy).

[0011] Photodynamic therapy (PDT) is another possible treatment option for AK and is suited for lesions located on the face and scalp. However, this treatment is not widely available and again requires a clinical setting. Reported side effects of PDT include scaling, pain, tenderness, itching, edema, ulceration, bleeding, and erosion.

[0012] There are a limited number of topical treatments to treat AK. For example, 5- fluorouracil, imiquimod, ingenol mebutate, diclofenac and tirbanibulin have been used for treatment of this condition. 5 -fluorouracil is an anti-metabolite, often used in cancer therapy, and is associated with several common side effects, including pain, burning, itching, redness, crusting, oozing and bleeding. Additionally, topical application of 5 -fluorouracil may cause abdominal pain, bloody diarrhea, vomiting, fever, stomatitis, and inflammation. Imiquimod activates immune cells through the toll-like receptor 7. In addition to the common local application related side effects of imiquimod (pain, burning, itching, irritation, and bleeding), systemic influenza-like symptoms may also occur. Ingenol mebutate causes irritation at the application site (redness, scaling, crusting, severe itching, and sometimes infection). Furthermore, the EMEA recommended suspending the marketing authorization of ingenol mebutate due to concerns about increased incidence rates of skin cancer in patients treated with ingenol mebutate versus vehicle or imiquimod. Diclofenac is a non-steroidal anti-inflammatory drug and side effects of its topical application include redness, itching, peeling or dry skin, irritation, swelling or a rash, as well as pain, burning at the application site, muscle, joint or back pain, and headache. Tirbanibulin is a microtubulin polymerization inhibitor and has been observed to induce skin reactions at the site of application, ranging from mild to severe erythema, flaking, ulceration and pain. Both diclofenac and tirbanibulin are less efficacious in actinic keratoses than imiquimod or 5 -fluorouracil. Although current treatments provide some benefit, they can severely damage adjacent, healthy skin. Furthermore, the available evidence for most topically administered agents supports a short- to medium-term benefit only and hence the need for ongoing surveillance with repeated treatments (Sinclair R, et al., “A review of actinic keratosis, skin field cancerisation and the efficacy of topical therapies”, Australas. J Dermatol. 2021 May;62(2): 119-123).

[0013] Therefore, there remains a need for further and improved treatments for skin field cancerization with actinic keratoses. Furthermore, it would be advantageous if treatments were able to offer reduced adverse side effects when compared to existing treatment options and could be applied outside a clinical setting, e.g., direct application by a patient at home, to large affected areas of skin.

SUMMARY OF INVENTION

[0014] N-[ ( 1 S)- 1 -(5 -fluoropyrimidin-2-y 1) ethyl ] -3 -(5 -i sopropoxy- 17/-py razol -3 -y 1 ) -377- imidazo[4,5-Z>]pyridin-5-amine, referred to as Compound A, is a potent and highly specific TRK inhibitor. The compound is disclosed, and its synthesis described in International Patent Application WO 2008/135785. Compound A has the following chemical structure:

Compound A.

[0015] The Applicants have discovered that Compound A, or a pharmaceutically acceptable salt thereof, can be used for the treatment of skin field cancerization with actinic keratoses.

[0016] As mentioned above, neurotrophins have been implicated in skin disorders. However, no TRK inhibitor has been previously found to be sufficiently efficacious for the treatment of skin field cancerization with actinic keratoses.

[0017] Compound A has clinically been evaluated in a Phase 1 trial in adults with recurrent glioblastoma multiforme (GBM). A summary of the study results was presented in Kreisl el. al. (2014) Neuro-Oncology 16:v79 - v95. In this study, up to 60 mg of the compound was administered daily. No early efficacy signals in GBM patients were observed and the drug program was subsequently discontinued by the clinical trial sponsor, AstraZeneca Pharmaceuticals.

[0018] In a first aspect, the present disclosure provides Compound A, or a pharmaceutically acceptable salt thereof:

Compound A; for use in treating skin field cancerization with actinic keratoses in a subject.

[0019] The present disclosure also provides a method of treating skin field cancerization with actinic keratoses, comprising the administration of Compound A, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0020] The present disclosure also provides the use of Compound A, or a pharmaceutically acceptable salt thereof, for the treatment of skin field cancerization with actinic keratoses.

[0021] The present disclosure also provides the use of Compound A, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of skin field cancerization with actinic keratoses.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : TRK receptor expression in normal skin, interfollicular and hair follicle epidermis.

[0023] Figure 2: TRK receptor expression in SCC. Increased expression within the basal layer is observed (upper panel). However, this pattern of cytoplasmic/membrane labelling is seen within supra-basal layers in areas of poor differentiation (lower panel).

[0024] Figure 3: TRK receptor expression in BCC. Basal layer expression was observed in BCC, occasionally extending to supra-basal layers. The labelling intensity remained comparable to the keratinocytes in the normal epidermis basal layer.

[0025] Figure 4: Frequency of TRK receptor positive keratinocytes in actinic keratoses (AK, also image right), normal skin, squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). ****p<0.01.

[0026] Figure 5: Intensity of TRK receptor labelling in normal skin vs AK skin samples.

[0027] Figure 6: Expansion of TRK receptor basal pattern labelling. ****p<0.01.

[0028] Figure 7: 5 days treatment with topical pharmaceutical composition comprising Compound A mesylate salt or vehicle of HPV8 C57BL/6 mouse back skin, then immunohistochemistry labelled for proliferation (ki67), apoptosis (Casp3) and pSTAT3 labelling.

[0029] Figure 8 : Fold change in gene expression of NTRKA in FVBN (n=4) compared to C57BL/6 mouse skin. Value of 10 represents a 10-fold increase in NTRKA expression compared to comparator. Significance p-values, ** <0.01

[0030] Figure 9: 9 days treatment with a topical pharmaceutical composition comprising Compound A mesylate salt or vehicle of HPV8 FVBN mouse back skin, then immunohistochemistry labelled for proliferation (ki67) and apoptosis (Casp3).

[0031] Figure 10: 7 days treatment with Compound A mesylate salt or vehicle of TPA treated HPV8 FVBN mouse back skin, examined for epidermal thickness and immunohistochemistry labelled for proliferation (ki67) and apoptosis (Casp3).

[0032] Figure 11: Cell proliferation of HaCaT and UWBCC1 cells with Compound A mesylate salt treatment. Results have been normalized to controls (100%).

[0033] Figure 12: Cell proliferation of MET1 and 2 cells with Compound A mesylate salt treatment. Results have been normalized to controls (100%).

[0034] Figure 13: Cell viability of HaCaT and UWBCC1 cells with Compound A mesylate salt treatment. Results have been normalized to controls (100%).

[0035] Figure 14: Cell viability of MET1 and 2 cells with Compound A mesylate salt treatment. Results have been normalized to controls (100%).

[0036] Figure 15: Apoptosis of HaCaT cells with Compound A mesylate salt treatment. Results have been normalized to controls.

[0037] Figure 16: Apoptosis of UWBCC1 cells with Compound A mesylate salt treatment. Results have been normalized to controls.

[0038] Figure 17: Apoptosis of MET1 cells with Compound A mesylate salt treatment. Results have been normalized to controls.

[0039] Figure 18: Apoptosis of MET2 cells with Compound A mesylate salt treatment. Results have been normalized to controls.

[0040] Figure 19: Microarray data showing differences in individual Trk receptor expression between normal skin, BCC, and SCC.

[0041] Figure 20: Immunofluorescence labelling of the hair follicle infundibulum.

[0042] Figure 21: BCC samples labelled with a pan-Tr receptor antibody.

[0043] Figure 22: SCC samples labelled with a pan-Tr receptor antibody.

[0044] Figure 23: SCC samples labelled with a pan-Tr receptor antibody.

[0045] Figure 24: Immunohistochemistry of actinic keratosis (AK), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) tissue sections with antibody that detects Trk fusion products.

DETAILED DESCRIPTION

[0046] In a first aspect, the present disclosure provides Compound A, or a pharmaceutically acceptable salt thereof:

Compound A; for use in treating skin field cancerization with actinic keratoses in subject.

[0047] The disclosed methods may be understood more readily by reference to the following detailed description.

Definitions [0048] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0049] As used herein, the singular forms “a,” “an,” and “the” include the plural.

[0050] Where an aspect or embodiment is said to comprise a stipulated item, it is to include the aspect or embodiment comprising one type of the feature, or a mixture of types of the stipulated feature. For example, said aspect or embodiment may comprise at least one type of the stipulated feature, such as at least two types, at least three types, or at least four types. Suitably, the aspect or embodiment comprises one type of the stipulated feature, two types of the stipulated features, three types of the stipulated features or four types of the stipulated features.

[0051] It is to be understood that concentrations, amounts, and other numerical data may be expressed or presented herein in range formats. It is to be understood that such range formats are used merely for convenience and brevity and include not just the numerical values explicitly recited as the end points of the range but also to include all the individual numerical values encompassed within that range as if each numerical value is explicitly recited. As an illustration, a numerical range of “1% w/w to 5% w/w” should be interpreted to include not just the explicitly recited values of 1% w/w to 5% w/w, but also include individual values within the indicated range. Thus, included in this numerical range are individual values such as 2, 3.5, and 4% w/w, etc. All ranges are inclusive and combinable.

[0052] It is to be appreciated that certain features of the methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0053] The term “about” when used in reference to numerical ranges, cut-offs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. As many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can, and often times will, vary among different experiments. The values used herein should not be considered unduly limiting by virtue of this inherent variation. Thus, the term “about” is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0.1% or less from the specified value. [0054] As used herein, “treating” and like terms refer to reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of symptoms and/or their underlying cause, delaying, preventing the recurrence and/or slowing the progression of the disease (field cancerization with actinic keratoses and/or SCC or BCC), and improving or remediating damage caused, directly or indirectly, by skin field cancerization with actinic keratoses.

[0055] As used herein, the phrase “therapeutically effective dose” or “therapeutically effective amount” refers to an amount of Compound A, or a pharmaceutically acceptable salt thereof, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. The therapeutically effective dose may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to cause a desired response in a subject. Such results include, but are not limited to, the reduction, remission, and/or regression of skin field cancerization with actinic keratoses or prevention of the development of the skin field cancerization with actinic keratoses, as determined by any means suitable in the art.

[0056] The term "subject", as used herein, includes humans, as well as non-human subjects such as cats, dogs, sheep, cattle, pigs, goats, non-human primates (including monkeys and apes), and the like. Conveniently, the subject is a human.

[0057] The term “pharmaceutically acceptable excipient” is used herein to refer to an essentially pharmacologically inert, non-toxic substance, e.g., that has been approved for inclusion in pharmaceutical products.

Therapeutic uses

[0058] Skin field cancerization with actinic keratoses may be defined as a confluent area of skin comprising two or more actinic keratoses, wherein the actinic keratoses present can be classified in respect to magnitude as mild, moderate or severe (for example using the Olsen grade scale of 1, 2 or 3). It is to be understood that the confluent area of skin can comprise a range of features matching any or all of the grades (e.g. Olsen grade scale of 1, 2 or 3) of actinic keratoses.

[0059] In one embodiment, the confluent area comprises more than one actinic keratosis lesion, for example at least two, five or ten lesions.

[0060] In one embodiment, the confluent area has an area of several centimetres or more. In one embodiment, the confluent area is greater than or equal to 2 cm², such as greater than or equal to 5, 10, 15 or 20 cm². In one embodiment, the confluent area is less than or equal to 25 cm², such as at less than or equal to 20, 15, 10 or 5 cm².

[0061] In one embodiment, the confluent area comprises actinic keratoses which are classified as Olsen Grade 1, 2 or 3, or a mixture thereof. The different grades of actinic keratosis are presented in Table 1 (Olsen et al., J Am Acad Dermatol. 1991 May; 24(5 Pt l):738-43).

Table 1: Olsen grades of actinic keratosis

[0062] In one embodiment, between 0.5 and 60% of the total skin surface of the subject is affected by skin field cancerization with actinic keratoses, such as between 0.5 and 20% or between 0.5 and 10%.

[0063] In one embodiment, the confluent area is a continuous area of solar damage skin.. Suitably, the continuous area of sun-damaged skin shows signs of erythema and or telangiectasia. Suitably, the continuous area of sun-damaged skin is greater than or equal to 1 cm², such as greater than or equal to 5, 10, 15 or 20 cm². Suitably, the continuous area of sundamaged skin is less than or equal to 25 cm² in area, such as at less than or equal to 20, 15, 10 or 5 cm². In one embodiment, the subject has more than one affected area, for example, more than one affected area of sun-damaged skin.

[0064] In one embodiment, the confluent area compri ses at least one clinical signs of photoageing.

[0065] In one embodiment, the subject has multiple actinic keratoses (i.e., greater than one) present in a given skin area, wherein the area of the multiple actinic keratoses is greater than 25% of the given skin area.

[0066] In one embodiment, the skin field cancerization with actinic keratoses is present on the face, ear, scalp, neck, back of hand, forearm, or lip (or a combination of any of these sites) of the subject. [0067] In one embodiment, the subject has a bald scalp.

[0068] In one embodiment, the subject is an immunocompetent adult.

[0069] In another embodiment, the subject is an immunocompromised adult. In one embodiment, the immunocompromised adult has received an organ transplant. In one embodiment, the immunocompromised adult is taking immunosuppressants.

[0070] In one embodiment, the subject has xeroderma pigmentosum or albinism.

[0071] In one embodiment, the subject is male.

[0072] In one embodiment, the subject is Caucasian.

[0073] In one embodiment, the subject has Fitzpatrick skin category of type I or II. The Fitzpatrick Phototype Scale (Arch Dermatol. 1988; 124(6): 869-871) is a numerical classification used to estimate the response of different types of skin to UV light. The Fitzpatrick Phototype scale categories are presented in Table 2.

Table 2: Fitzpatrick Phototype Scale

[0074] In one embodiment, the subject is aged 40 years or over, for example aged 50 years or over, 60 years or over, 70 years or over, or 80 years or over.

[0075] In one embodiment, the administration of Compound A, or a pharmaceutically acceptable salt thereof, reduces local skin reactions or other adverse side effects when compared to other treatments for skin field cancerization with actinic keratoses. In one embodiment, the administration of Compound A, or a pharmaceutically acceptable salt thereof, causes minimal or no local skin reaction, such as for example, vesiculation, pustulation, erosion, ulceration, redness, swelling, flaking, scaling, hard lumps, dryness, pus or blistering. In one embodiment, the administration of Compound A, or a pharmaceutically acceptable salt thereof, causes minimal or no other side effect, such as for example site pain, application site pruritus, application site irritation, application site swelling, application site burning sensation, application site infection, periorbital edema, nasopharyngitis, chills, sore throat, drooping eyes, puffy eyes, hypopigmentation, hyperpigmentation, or headache.

[0076] In addition to the beneficial effects seen in respect to the use of Compound A for the treatment skin field cancerization with actinic keratoses, the data disclosed in the Examples section, particularly Examples 5 and 6, also indicates increased TRK expression in differentiated skin cancers, including but not limited to basal cell carcinoma, squamous cell carcinoma/in situ squamous cell car cinoma/B owen’s disease. Furthermore, the data also shows that Compound A influences cell proliferation, apoptosis, and migration of keratinocytes cell lines from premalignant, SCC and BCC lesions. As such, in addition to its utility in the treatment skin field cancerization with actinic keratoses, Compound A is expected to also display beneficial effects in the treatment of differentiated skin cancers, including but not limited to basal cell carcinoma, squamous cell carcinoma/in situ squamous cell carcinoma/Bowen’s disease.

Compound A

[0077] Compound A, as referred to in the present disclosure, is A- (lS)-l-(5- fhioropyrimidin-2-yl)ethyl]-3-(5-isopropoxy-lJ7-pyrazol-3-yl)-3J7-imidazo[4,5-Z>]pyridin-5- amine:

[0078] Compound A is a nM inhibitor of TRKA, TRKB and TRKC.

[0079] As depicted above, Compound A is shown as a “free base”. In some embodiments, a pharmaceutically acceptable salt of Compound A is used. Pharmaceutically acceptable salts of Compound A include acid addition salts formed with inorganic acids or organic acids. Examples of acid addition salts include, but are not limited to, acetate, adipate, ascorbate, benzoate, besylate (benzenesulfonate), bicarbonate, bisulfate, butyrate, camphorate, camphorsulfonate, citrate, cyclohexyl sulfamate, esylate (ethanesulfonate), fumarate, glutamate, glycolate, hemisulfate, 2-hydroxy ethyl sulfonate, heptanoate, hexanoate, hydrochloride, hydroiodide, hydromaleate, lactate, maleate, mesylate (methanesulfonate), meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenyl acetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate, undecanoate, and 2,5-dimethylbenzene sulfonate.

[0080] In some embodiments a pharmaceutically acceptable salt of Compound A is selected from besylate, esylate, hydrochloride, mesylate, and tosylate. In some embodiments the pharmaceutically acceptable salt of Compound A is a mesylate salt. The mesylate salt of Compound A is depicted below:

[0081] The molecular weight of Compound A is 382.4 g/mol. In some embodiments the molar ratio of Compound A to mesylate in the salt form is 1 : 1. The molecular weight of the 1 :1 mesylate salt of Compound A is 478.5 g/mol.

[0082] Throughout the specification, unless specified otherwise, references to the amount of Compound A will be understood to refer to the amount of the parent compound on a free base basis, even if the compound is present as a salt of Compound A. Purely by way of example, reference to 10 mg of Compound A or a salt thereof, will be understood to refer to 10 mg of the free base, or a salt of Compound A with 10 mg of free base equivalent. In the context of the mono-mesylate salt of Compound A, 12.5 mg of the salt delivers 10 mg of Compound A (free base equivalent).

[0083] It is to be understood that, insofar as Compound A or a pharmaceutically acceptable salt may exist in tautomeric forms, the disclosure includes in its definition any such tautomeric form which possesses the herein mentioned activity/activities. Thus, the disclosure relates to all tautomeric forms of Compound A, or a pharmaceutically acceptable salt thereof, which inhibit TRK related activities in a human or animal. It is also to be understood that Compound A, or a pharmaceutically acceptable salt, thereof may exist in solvated as well as un-solvated forms such as, for example, hydrated forms. It is to be understood that the disclosure encompasses all such solvated or un-solvated forms.

[0084] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium are within the scope of this disclosure.

[0085] The synthesis of Compound A may be readily achieved by the methods described in PCT publication number WO2008/135785 the teachings of which are incorporated herein by reference in its entirety.

Routes of administration and doses

[0086] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein between 0.5 and 60% of the total skin surface of the subject is affected by skin field cancerization with actinic keratoses, such as between 0.5 and 20% or between 0.5 and 10%. In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein atleast 0.5% of the total skin surface of the subject is affected by skin field cancerization with actinic keratoses, such as at least 1% or 2.5%.

[0087] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein greater than or equal to 1 cm² of the total skin surface of the subject is affected by skin field cancerization with actinic keratoses, such as greater than or equal to 5, 10, 15 or 20 cm². In one embodiment, the affected area is less than or equal to 25 cm², such as at less than or equal to 20, 15, 10 or 5 cm².

[0088] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject topically, orally or subcutaneously. In a preferred embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject topically. In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is topically administered to the face, ear, scalp, back of hand, forearm, or lip of the subject.

[0089] In one embodiment, the treatment comprises topically applying Compound A, or a pharmaceutically acceptable salt thereof, to the affected area, wherein the affected area is between 0.5 and 60% of the total skin surface of the subject, such as between 0.5 and 20% or between 0.5 and 10%. In one embodiment, the treatment comprises topically applying Compound A, or a pharmaceutically acceptable salt thereof, to the affected area, wherein the affected area is at least 0.5% of the total skin surface of the subject, such as at least 1% or 2.5%.

[0090] In one embodiment, the treatment comprises topically applying Compound A, or a pharmaceutically acceptable salt thereof, to the affected area, wherein the affected area is greater than or equal to 1 cm², such as greater than or equal to 5, 10, 15 or 20 cm². In one embodiment, the affected area is less than or equal to 25 cm², such as at less than or equal to 20, 15, 10 or 5 cm².

[0091] A therapeutically effective dose refers to an amount of Compound A, or a pharmaceutically acceptable salt thereof, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. A daily dose in the range of 0.001-15 mg per kg body weight may be employed.

[0092] Effective doses of the topical composition of this disclosure will vary, as recognized by those skilled in the art, depending on the severity of the disease, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician. The administration of Compound A, or a pharmaceutically acceptable salt thereof, can continue for as long as necessary to treat the disorder, e.g., for one week, two weeks, one month, two months, three months, four months, six months, one year, two years, five years, ten years, or longer.

[0093] In one embodiment, after a course of treatment with Compound A, or a pharmaceutically acceptable salt thereof, there is at least 50%, such as at least 60% or 70%, clearance of the skin field cancerization with actinic keratoses in the subject. The clearance after treatment is relative to the skin field cancerization with actinic keratoses in the subject prior to the commencement of treatment with Compound A, or a pharmaceutically acceptable salt thereof. [0094] In one embodiment, between 0.005 and 50000 mg of Compound A, or a pharmaceutically acceptable salt thereof, is dosed per day, such as between 0.005 and 14000 mg.

[0095] In some embodiments, the total dose of Compound A, or a pharmaceutically acceptable salt thereof, administered topically to the skin of the subject in a single day ranges between 0.005 mg and 50 g, such as between 0.005 mg and 14 g, or between 0.05 mg and 10 g-

[0096] In a further aspect, Compound A, or a pharmaceutically acceptable salt thereof, is topically administered to the affected area of the subject’s skin at an application dose amount of from about 0.2 pg/cm²to about 120 pg/cm² skin surface.

[0097] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered once to a subject in need thereof. In another embodiment, Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject in need thereof at least once per week, such as at least two, three or four times per week. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject in need thereof once daily or twice daily.

[0098] In one embodiment, the time gap between individual administrations of Compound A, or a pharmaceutically acceptable salt thereof, is less than one day, for example 12 hours, or more than one day, for example two days.

[0099] In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is topically administered to the subject in need thereof for a duration of at least one week, such as at least two, four or six weeks.

[00100] In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is applied topically at least once per week, such as at least twice per week, for a duration of at least one week, such as, e.g. for a duration of two, four, six or eight weeks.

[00101] In some embodiments, Compound A, or a pharmaceutically acceptably salt thereof, is applied topically, and left on the subject’s skin for at least 1 hour, such as at least 2 hours.

[00102] In one embodiment, Compound A, or a pharmaceutically acceptably salt thereof, is administered to the subject until the skin field cancerization with actinic keratoses is fully treated, i.e., the skin field cancerization with actinic keratoses is clear from the affected area of the subject. The phrase "until the skin field cancerization with actinic keratoses is fully treated," as used herein, refers to the instance where the lesions on a subject suffering from skin field cancerization with actinic keratoses have substantially or completely disappeared from the treated area on the subject. In one embodiment, "substantially," in this context, refers to more than 50% (conveniently, more than 60%, 70%, 80%, 90%, 95% or 99%) of the lesions having disappeared from the treated area on the subject. Conveniently, "substantially" refers to more than 99% of the lesions have disappeared from the treated area on the subject.

[00103] In one embodiment, there is no reoccurrence of the skin field cancerization with actinic keratoses in the subject after completion of treatment with Compound A, or a pharmaceutically acceptable salt thereof, for at least 6 months, such as at least 12 months, 18 months, 24 months or 36 months.

Pharmaceutical Compositions

[00104] In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject in the form of a pharmaceutical composition. Suitably, the pharmaceutical composition comprises Compound A, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[00105] In some embodiments, the pharmaceutical composition is an oral, subcutaneous, or topical pharmaceutical composition. Preferably, the pharmaceutical composition is a topical pharmaceutical composition.

[00106] Topical pharmaceutical compositions are compositions that are applied directly to an external body surface, which includes the skin and membranes. In order for a topical composition containing an active pharmaceutical ingredient to be successfully used as a medicament, the composition once applied to an external body surface must remain at the site of application for a sufficient period of time in order for an efficacious dose of the active pharmaceutical ingredient to be delivered to the site of treatment.

[00107] Topical compositions may be presented in a number of formats. In some embodiments, the topical pharmaceutical composition is a cream, an ointment, a paste, a solution, a lotion, a gel, a foam (for example, a rigid foam), a spray solution for spraying onto the skin, a transdermal/topical patch, an adhesive strip, a solid or a semi-solid composition, or dissolvable microneedles. In some embodiments, the topical pharmaceutical composition is a cream. In some embodiments, the topical pharmaceutical composition is a cream based on an oil-in-water emulsion. [00108] In some embodiments, the topical pharmaceutical composition is well tolerated with no dermal irritation or only minimal erythema when applied to the skin.

[00109] In another embodiment, the pharmaceutical composition is a subcutaneous pharmaceutical injection. Examples of subcutaneous pharmaceutical injections include aqueous solutions, suspensions, oily solutions, emulsions, microemulsions, liposomes, microspheres, nanoparticles and implants. Advantages of subcutaneous administration include the rapid onset of action and systemic availability of the compound over time is reduced, since drug absorption from the skin is slow and sustained.

[00110] In another embodiment, the pharmaceutical composition is an oral pharmaceutical composition. Examples of oral pharmaceutical compositions include tablets, capsules, caplets, liquid dosage forms (such as emulsions, syrups, suspensions, liquids, or the like). Such compositions can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.

Combinations

[00111] Also contemplated herein are methods that include administering a second active agent both independently and via compositions that include a second active agent.

[00112] In an embodiment, the additional therapeutic agent is selected from one or more of the following agents: 5-flurouracil, imiquimod, diclofenac and tirbanibulin.

[00113] The treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti-tumor agents.

[00114] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ compounds of this invention, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically active agent within its approved dosage range.

[00115] Also contemplated herein are methods that include treating the subject with Compound A, or a pharmaceutically acceptable salt thereof, and with a surgical procedure (such as cryosurgery or surgical removal) or photodynamic therapy.

Kits

[00116] In one embodiment, the methods described herein provide kits for the treatment of skin field cancerization with actinic keratoses. These kits comprise a compound or composition described herein in a container and, optionally, instructions teaching the use of the kit according to the various methods and approaches described herein. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the topical pharmaceutical composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

[00117] Besides being useful for human treatment, compounds or compositions of the invention, may be useful for veterinary treatment of skin field cancerization with actinic keratoses in companion animals, exotic animals and farm animals, including mammals, rodents, and the like. Conveniently, such animals include horses, dogs, and cats. [00118] EXAMPLES

[00119] The following abbreviations have been used in the Examples:

AK Actinic Keratosis

BCC Basal cell carcinoma

SCC Squamous cell carcinoma

Example 1: Preparation of the mesylate salt of 7V- (lS)-l-(5-fluoropyrimidin-2-yl)ethyl]- 3-(5-isopropoxy-lZ/-pyrazol-3-yl)-3Z/-imidazo[4,5-/>]pyridin-5-amine (Compound A mesylate salt)

[00120] To a stirred solution of 2,6-dichloro-3-nitropyridine (1.0 eq.) and 5-propan-2-yloxy- lJT-pyrazol-3 -amine (1.1 eq.) in 2-MeTHF (10.0 vol.) triethyl amine (10.0 eq.) was added at 25-35°C under a nitrogen atmosphere. After addition, the reaction mixture was stirred at 25-35°C for 48 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction mixture was diluted with water (10 vol.) and the layers separated, and the organic layer was washed with 10.0 vol. of aqueous sodium chloride solution. Distillation of the solvent (2-Methyl-THF layer) at45-55°C under vacuum was carried out until 1-2 volumes remained in the round bottom flask, then co-distilled with 1-Butanol (2 x 2.5 volumes) and finally 10 volumes of 1 -butanol were added to the mass. The crude mass was taken to the next stage as such without purification.

[00121] To a stirred solution of the above crude mass (1.0 eq.) in 1-butanol [(S)-l-(5- Fluoropyrimidin-2-yl)ethanamine (1.1 eq.) and potassium carbonate (2.5 eq.) were added at 25-35°C, followed by 2.5 volumes of 1-butanol. Then the reaction mass was heated to 70-75°C for 24 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the solid suspension was filtered and washed with 1-butanol (2 x 2 volumes). The wet material was dried at 50-55°C for 12 hours and then slurried in 15 volumes of water. The solid suspension was stirred for 2 hours and filtered. The filtered cake was dried at 50-55°C until the solid met the desired moisture content (Limit: 1%). If the isolated purity did not meet the specification limit (Limit: NLT 95%) a purification in MTBE was performed.

[00122] To a solution of (S)-N6-(l-(5-fluoropyrimidin-2-yl)ethyl)-N2-(5-isopropoxy-lH- pyrazol-3-yl)-3-nitropyridine-2,6-diamine (1.0 eq.), 10%Pd/C (50% wet, 15 wt%), formamidine acetate (2.0 eq.) and ZnBn (0.2 eq.) in EtOAc (16.0 vol.), and MeOH (4.0 vol.) 4 - 6 kg/cm² of hydrogen gas was applied and the reaction mass was stirred at 65-70°C for 4 - 5 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction mixture was cooled to 25-30 °C and the catalyst was removed by filtration under a nitrogen atmosphere and it was washed with EtOAc. The obtained organic layer was washed with brine solution (3x5 volumes) and the organic layer was treated with SC-40 charcoal and filtered, washed with EtOAc.

[00123] To a stirred solution of the above organic layer a solution of methane sulfonic acid (1.08 eq.) in water (0.15 vol.) was added at 55-60°C. Then reaction mass was maintained at 55-60 °C for 4 hours. Then reaction mass was slowly cooled to 5-10°C and filtered to obtain crude methane sulfonic acid salt of Compound A.

[00124] The crude methane sulfonic acid salt of Compound A was added to 1 -butanol - water (3.3 eq and 0.7 eq) and heated to 50-55°C. Additional 5.5 eq 1-butanol was added and heated to 80-85°C and stirred for 2 hours. The reaction mixture was filtered through a 0.2 micron filter and cooled to 25-30°C over 12 hours. The slurry was cooled to 0-5°C, stirred for 6 hours and then filtered. The wet cake was washed with 1.9 eq butanol and dried at 50-55°C for 12 hours to obtain methanesulfonic acid salt of Compound A, Form B.

[00125] The methane sulfonic acid salt of Compound A was added to 1-butanol - water (3.3 eq and 0.7 eq) and heated to 50-55°C. Additional 5.5 eq 1-butanol was added and heated to 80- 85°C and stirred for 2 hours. The reaction mixture was filtered through a 0.2 micron filter and cooled to 25-30°C over 12 hours. The slurry was cooled to 0-5°C, stirred for 6 hours and then filtered. The wet cake was washed with 1.9 eq butanol and dried at 50-55°C for 12 hours to obtain methanesulfonic acid salt of Compound A, Form B.

[00126] Filtered mother liquors were combined. Methyl t-butyl ether (MTBE) (30 vol) was added, and the mixture was stirred for 30-45 minutes at 25-30°C. The precipitated solid was filtered under vacuum and washed with MTBE (2.0 vol.) and the wet cake was dried for 2-3 hours at 40-50°C to obtain the methane sulfonic acid salt of Compound A, Form A.

Alternative preparation of the methane sulfonic acid salt of Compound A, Form B

[00127] To a stirred solution of the methane sulfonic acid salt of Compound A (10 g, 1.0 eq.) in 1-Butanol (3.3 vol.), was added water (0.7 vol.) at 25 - 30°C and the temperature was raised to 60°C (at roughly 30°C the solid started to dissolve forming a thin pale brown slurry and dark brown solution by 45°C). 1-Butanol (5.5 vol.) was added drop wise into the reaction mass over a period of 20 minutes (Dark brown solution with small bits of suspension in it at 60°C).

[00128] The reaction mass was heated to 80-85°C and it was stirred at 80-85 °C for 1-2 hours.

[00129] The reaction mass was cooled to 58-62 °C and methane sulfonic acid salt of Compound A, Form B seed material (0.005 w/w) was added at 58-62 °C. The reaction mass was stirred for 4-6 hours at 58-62°C (Observation: Reaction mass becomes solid suspension).

[00130] The reaction mass was slowly cooled to 25-35 °C over a period of 10-12 hours. (Observation: Reaction mass is solid suspension).

[00131] Pre filtered 1-Butanol (1.9 vol.) was slowly added into the above RBF for a period of 1-2 hours at 25-35°C.

[00132] The reaction mass was stirred at 25-35°C for 2-3 hours.

[00133] The reaction mass was cooled to 0-5 °C (Observation: Reaction mass is solid suspension).

[00134] The reaction mass was stirred at 0-5°C for 5-6 hours and heated to 20 - 30°C and then stirred at 20-30°C for 12-14 hours. The reaction mass was further cooled to roughly 5°C and it was stirred at this temperature for 2 hours.

[00135] The solid was filtered under vacuum and it was washed with 1-Butanol (1.9 vol.) and the wet cake was washed with MTBE (1.9 vol.). The wet cake was dried at 12-14 hours at 50 °C to get methane sulfonic acid salt of Compound A, Form B.

Example 2: Trk Receptor expression in Actinic Keratosis [00136] Experiments were performed to determine whether actinic keratosis demonstrates an increase expression of Trk receptors.

Methods:

Immunohistochemistry of tissues

[00137] Pan-Trk staining for TrkA/B/C expression was performed on paraffin embedded tissues of actinic keratosis (n=98), normal skin (n=8), basal cell carcinoma (n=l) and squamous cell carcinoma (n=6) sections. Slides were heated at 60°C for 30 mins, then de-paraffinized with xylene (2 x 10 mins) and decreasing concentrations of ethanol (100 - 70%) for 5 mins each, followed by a 5 min wash in PBS. For antigen retrieval, samples were submerged in Tris- EDTA (pH9.0) and heated in a microwave for 20 mins in a pressure cooker and allowed to cool to room temperature gradually. A hydrophobic barrier was drawn around the tissue sample. Sections were then incubated in DAKO dual inhibition block for 10 min at room temperature, subsequently washed 3x5 min in PBS and then incubated in blocking buffer (10% donkey serum in PBS) for 1 hour at room temperature. A pan-Trk antibody (Santa Cruz trk (B-3) sc- 7268) was diluted in 5% donkey serum (1 :500 dilution) and incubated overnight at 4°C. No primary antibody was used as negative control. Samples were washed 4 x 5 mins in PBS Tween-20 (0.05%) before incubating with secondary antibody at room temperature for 1 hour. Samples were again washed 4 x 5 mins in PBS Tween-20 (0.05%) before incubating with ABC reagent (Vector Labs) for 30 mins at room temperature. Slides were washed 3x5 mins in PBS- Tween20 (0.05%) before staining with 3,3 '-Diaminobenzidine (DAB) for 30 seconds. Sections were submerged in haematoxylin and eosin solution for 1 min and once stain was considered strong enough, were washed under running water for 2 min. Sections were mounted using DPX solution and imaged. Images of tissue sections were captured with an Olympus slide scanner (Model VS200) at 40x magnification and processed using QuPath imaging software. Statistical analysis was undertaken using Graphpad v9 software.

Results:

[00138] We chose immunohistochemistry, rather than immunofluorescence, to visualize Trk receptor labelling within the tissue context for this study. We observed both cell membrane/cytoplasmic as well as perinuclear labelling patterns within normal skin and pathological samples. Within normal skin (n=8), Trk receptor expression is predominantly cell membrane/cytoplasmic in the basal layer and perinuclear labelling within the suprabasal layers (Figure 1). [00139] Likewise, SCC (n=6) similarly demonstrated basal layer and suprabasal layer pattern, with increased intensity of labelling within the basal layers (Figure 2). Often the basal layer pattern of Trk receptor labelling within SCC extended into neighboring suprabasal layers, most notably in less differentiated tumors (Figure 2). We labelled one human BCC samples with the pan-Trk receptor antibody (Figure 3). Where positive, labelling was predominantly within the cell cytoplasm within the tumor margin.

[00140] Actinic keratosis (AK) is the archetypal precancerous skin lesion. Characterized by the presence of intraepithelial dysplasia, clinically the lesions present as small red scaly lesions. These precursor lesions often predominate within areas that develop SCC. Often areas of skin containing multiple AK’s, referred to as skin field cancerization, has the potential to develop multiple independent SCCs. In individuals with skin field cancerization, clinical studies have shown that AK treatments can reduce the SCC incidence. Hence, AK treatment is an essential component in the management of a growing skin cancer problem.

[00141] Epidermal keratinocyte Trk receptor immunohistochemical expression was increased in AK lesions (n=98). The frequency of Trk receptor expression was greater in AK keratinocytes (81.97 +/- 12.52%, n=98) than in normal skin (30 +/- 16.01%, n=8) (Figure 4), and was comparable to that observed in SCC (71.65 +/- 10.48, n=6). As is appreciable in Figure 4, Trk labelling demonstrated greater intensity in AK compared to normal skin keratinocytes, the overall frequency of high (3+) versus low (1+) intensity labelling was skewed in AK (Figure 5). Thus, although the epidermal thickness and cell numbers in AK was greater than in normal skin, a greater fraction of keratinocytes expressed the Trk receptor with increased intensity.

[00142] As within the normal epidermis, the two types of expression pattens were evident in AK: cell membrane/cytoplasmic and perinuclear labelling. While in the normal epidermis, membrane/cytoplasmic labelling pattern was restricted to the basal cell layer, within AK lesions this extended into the suprabasal layer. In all cases, the uppermost suprabasal keratinocytes, both in normal skin and AK, continued to demonstrate perinuclear labelling. As Trk is a cell membrane receptor, one might assume that cells demonstrating membrane/cytoplasmic labelling pattern could be receptive to ligand binding. Since basal cell “crowding” and expansion AK has been described by pathologists, we next measured the thickness of the basal-type membrane/cytoplasmic labelling pattern in AK. As expected, basal cell expansion was increased within AK as was the basal -type membrane/cytoplasmic labelling pattern (72.8+/-47.5pm) compared to normal skin (29.7+/ -9.7). Even though AK frequently have an expanded epidermis (acanthosis), the relative thickness of basal-type membrane/cytoplasmic labelling pattern as a ratio of the overall epidermal thickness was greater in AK (54.36+/-9.79%) than in normal skin (34.48+/-4.25%, p<0.01) (Figure 6). Hence AK demonstrate increased Trk receptor immunohistochemical labelling compared to normal skin, with expansion of the basal layer pattern, with increased labelling intensity.

Conclusions:

[00143] The immunohistochemistry study has identified increased Trk receptor expression in actinic keratosis, with greater basal-type membrane/cytoplasmic labelling pattern.

Example 3: Topical compositions used in Example 4

[00144] The following pharmaceutical compositions which were used in the experiments described in Example 4.

Preparation of 0,01% cream formulation

[00145] Preparation of aqueous phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added Water (41.86g), polysorbate 80 (8g), methyl paraben (0.18g), and disodium edetate (0.05g). Heated to 65-70°C using a water bath. Mix all the solids at 200- 500 rpm until everything is dissolved. Reduce the temperature to 60°C ± 5°C and add Transcutol HP (5g). Cool the reaction mixture to 45°C ± 5°C and add Compound A mesylate salt (0.012g), heat and stir until dissolved.

[00146] Preparation of oil phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added octyl dodecanol (3g), oleyl alcohol (3g), cyclomethicone (3g), white petrolatum (15g), cetyl alcohol (3g), stearyl alcohol (3g), glyceryl monostearate Type II (7g), span 80 (3g), Butylated hydroxy Toluene (0.1g), sorbic acid (0.1g), and Propyl paraben (0.02g). The reaction mixture is heated to 65-70°C with stirring at 200-500 RPM until all the solids have dissolved and a clear solution is obtained.

[00147] Add the aqueous phase mixture to the oil phase mixture and stir for 25-30 min using overhead stirrer. Cool the reaction mixture to 25°C ± 5°C. When the reaction mixture reaches 35°C, add Trolamine (25% w/w, 0.1g) and stir for additional 5 min. Check pH of the product to make sure it is 5±1.

Preparation of 0,03% cream formulation

[00148] Preparation of aqueous phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added Water (46.5g), polysorbate 80 (8g), methyl paraben (0.18g), and disodium edetate (0.05g). Heated to 65-70°C using a water bath. Mix all the solids at 200-500 rpm until everything is dissolved. Reduce the temperature to 60°C ± 5°C and add Transcutol HP (5g). Cool the reaction mixture to 45°C ± 5°C and add Compound A mesylate salt (0.036g), heat and stir until dissolved.

[00149] Preparation of oil phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added octyl dodecanol (3g), oleyl alcohol (3g), cyclomethicone (3g), white petrolatum (15g), cetyl alcohol (3g), stearyl alcohol (3g), glyceryl monostearate Type II (7g), span 80 (3g), Butylated hydroxy Toluene (0.1g), sorbic acid (0.1g), and Propyl paraben (0.02g). The reaction mixture is heated to 65-70°C with stirring at 200-500 RPM until all the solids have dissolved and a clear solution is obtained.

[00150] Add the aqueous phase mixture to the oil phase mixture and stir for 25-30 min using overhead stirrer. Cool the reaction mixture to 25°C ± 5°C. When the reaction mixture reaches 35°C, add Trolamine (25% w/w, 0.1g) and stir for additional 5 min. Check pH of the product to make sure it is 5±1.

Preparation of 0, 1% cream formulation

[00151] Preparation of aqueous phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added Water (46.44g), polysorbate 80 (8g), methyl paraben (0.18g), and disodium edetate (0.05g). Heated to 65-70°C using a water bath. Mix all the solids at 200- 500 rpm until everything is dissolved. Reduce the temperature to 60°C ± 5°C and add Transcutol HP (5g). Cool the reaction mixture to 45°C ± 5°C and add Compound A mesylate salt (0.12g), heat and stir until dissolved.

[00152] Preparation of oil phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added octyl dodecanol (3g), oleyl alcohol (3g), cyclomethicone (3g), white petrolatum (15g), cetyl alcohol (3g) , stearyl alcohol (3g), glyceryl monostearate Type II (7g), span 80 (3g), Butylated hydroxy Toluene (0.1g), sorbic acid (0.1g), and Propyl paraben (0.02g). The reaction mixture is heated to 65-70°C with stirring at 200-500 RPM until all the solids have dissolved and a clear solution is obtained.

[00153] Add the aqueous phase mixture to the oil phase mixture and stir for 25-30 min using overhead stirrer. Cool the reaction mixture to 25°C ± 5°C. When the reaction mixture reaches 35°C, add Trolamine (25% w/w, 0.1g) and stir for additional 5 min. Check pH of the product to make sure it is 5±1.

Preparation of 0,3% cream formulation [00154] The pharmaceutical composition comprising 0.3% by weight of the Compound A mesylate salt was prepared in an analogous manner to the compositions detailed herein.

Preparation of 1% cream formulation

[00155] Preparation of aqueous phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added water (44.25 g), polysorbate 80 (4 g), sorbic acid (0.1 g), methyl paraben (0.18g), and disodium edetate (0.05 g). Heated to 65-70°C using a water bath. Mix until all the solids are dissolved. Add Compound A mesylate salt (1 g), heat and stir until dissolved.

[00156] Preparation of oil phase: In a 1 liter stainless steel container equipped with an IKA overhead stirrer is added oleyl alcohol (3 g), octyl dodecanol (5 g), cyclomethicone (5 g), dimethicone (3 g), white petrolatum (5 g), cetyl alcohol (4 g) , stearyl alcohol (4 g), glyceryl monostearate Type II (3 g), span 80 (4 g), Butylated hydroxy Toluene (0.1 g), and Propyl paraben (0.02 g). The reaction mixture is heated to 65-70°C with stirring until all the solids have dissolved.

[00157] Add the aqueous phase mixture to the oil phase mixture and stir for 15-20 min using a Silverson homogenizer. Cool the reaction mixture to below 30°C and add Transcutol HP (10 g, preheated to 40°C). Cool the reaction mixture and add Sepineo P 600 (3 g) and mix well. When the reaction mixture reaches 35°C, add Trolamine (25% w/w, 1.3 g) and stir for additional 5 minutes. Check pH of the product to make sure it is 5±1.

Preparation of 3% cream formulation

[00158] The pharmaceutical composition comprising 3% by weight of the Compound A mesylate salt was prepared in an analogous manner to the 1% composition detailed above and has the following composition - see Table 3.

Table 3: Pharmaceutical composition comprising 3% Compound A mesylate salt

Example 4: Testing of topical pharmaceutical compositions in an actinic keratosis mouse model

[00159] Actinic keratoses accumulate in fair skinned individuals from cumulative sun-light exposure causing DNA mutations within keratinocytes (https://www.pcds.org.uk/files/general/AK guidelines 2020-new-web-v01.pdf, accessed

September 2021). The risk of actinic keratoses is greater in immunosuppressed individuals and multiple studies have investigated the relationship with HPV virus reactivation (Reowert- Huber J, et al. “Actinic keratosis is an early in situ squamous cell carcinoma: a proposal for reclassification”, Br J Dermatol 2007; 156:8-12.; Frazer IH. “The actinic keratosis virome: can we prevent squamous cell carcinoma with a vaccine?” Curr Probl Dermatol 2015; 46:28-35). The HPV8 serotype is most commonly associated with actinic keratosis and squamous cell carcinoma development, and is particularly prevalent in immunosuppressed individuals (Lebwohl MG, Rosen T, Stockfleth E. “The role of human papillomavirus in common skin conditions: current viewpoints and therapeutic options”, Cutis 2010; 86:S1— 11; Quint K.D. et al., “Human Beta-papillomavirus infection and keratinocyte carcinomas”, J Pathol. 2015;235:342-354).

[00160] We have exploited the HPV8 mouse model, which spontaneously develops SCC, and determined that the skin phenotype mirrors the pathological findings in actinic keratosis (Arnold AW, Hofbauer GF. “Human papillomavirus and squamous cell cancer of the skinepidermodysplasia verruciformis-associated human papillomavirus revisited”, Curr Probl Dermatol. 2012;43:49-56; Lanfredini S, et al., “HPV8 Field Cancerization in a Transgenic Mouse Model Is due to Lrigl+ Keratinocyte Stem Cell Expansion”, J Invest Dermatol. 2017 Oct;137(10):2208-2216). Exposure of the mouse model accentuates the risk for SCC development (Schaper I.D. et al., “Development of skin tumors in mice transgenic for early genes of human papillomavirus type 8”, Cancer Res. 2005;65: 1394-1400). Therefore, the HPV8 mouse model is ideal for the study of actinic keratosis treatment, particularly in the context of field cancerization.

Animals:

[00161] All experiments undertaken under Home Office Project License.

Tissue processing. H&E labelling and Immunohistochemistry

[00162] Immunohistochemical staining for Ki67 (ab 16667, Abeam), cleaved caspase-3 (#9661, Cell Signaling) and phosphorylated-STAT3 (#9145, Cell Signaling) expression was performed on paraffin embedded tissues of mouse skin sections. Slides were heated at 60°C for 30 mins, then de-paraffinized with xylene (2 x 10 mins) and decreasing concentrations of ethanol (100 - 70%) for 5 mins each, followed by a 5 min wash in PBS. For antigen retrieval, samples were submerged in citrate buffer (pH6.0) and heated in a microwave for 20 mins in a pressure cooker and allowed to cool to room temperature gradually. A hydrophobic barrier was drawn around the tissue sample. Endogenous peroxidases were then inhibited using BLOXALL (Vector Laboratories, SP-6000) for 10 mins at room temperature before being washed 3 x 5 mins in PBS. Sections were then incubated in blocking buffer (10% goat serum in PBS) for 1 hour at room temperature. The antibodies, ki67, cleaved caspase-3 and phosphorylated STAT3 were diluted 1 :400 in 5% goat serum and incubated overnight at 4°C. A no primary antibody was used as a negative control. Samples were washed 3 x 5 mins in PBS Tween-20 (0.05%) before incubating with an anti-rabbit secondary antibody at room temperature for 1 hour. Samples were again washed 3 x 5 mins in PBS Tween-20 (0.05%) before staining with 3,3 ’-Di aminobenzidine (DAB). Following the DAB step, sections were washed 3 x 5 mins in PBS Tween-20 before being submerged in hematoxylin solution for 1 min and once stain was considered strong enough, were washed under running water for 2 mins. Finally, slides were dehydrated in a series of ethanol and xylene steps. Sections were mounted using DPX solution and imaged. Images of tissue sections were captured with an Axio Scan.Z 1 slide scanner (Zeiss) and processed using Olympus vs200 software. Statistical analysis was undertaken using Graphpad v9.

Results

Experiment 1. Determine the effect of topical pharmaceutical compositions comprising Compound A mesylate salt on the HPV8 mouse model (C57BL6 background)

[00163] The HPV8 mouse model was generated on a C57BL/6 background. The back of mice were shaved and treated daily for 5 days with vehicle or pharmaceutical compositions comprising Compound A mesylate salt 0.01%, 0.03%, 0.1%, 0.3%, 1%, or 3% (n=2/3 per dose). 24 hours after completion of treatment the back skin was collected and examined for epidermal thickness, level of proliferation and apoptosis. The mice remained healthy during treatment. But unfortunately there were no differences in epidermal thickness (data not shown) and level of proliferation, apoptosis and pSTAT3 expression (Figure 7).

[00164] A review of the literature revealed differences in NTRK expression in skin between mouse strains with C57BL/6 (low) to FVBN (high), which we confirmed by RT-PCR (Figure 8).

Experiment 2, Determine the effect of topical pharmaceutical compositions comprising Compound A mesylate salt on the HPV8 mouse model (FVBN background)

[00165] The HPV8 mouse model was generated on an FVBN background. The back of mice were shaved and treated daily for 9 days with vehicle or pharmaceutical compositions comprising 3% Compound A mesylate salt (n=6 per dose). 24 hours after completion of treatment the back skin was collected and examined for epidermal thickness, level of proliferation and apoptosis.

[00166] Within 9-days, topical treatment with the pharmaceutical composition comprising 3% Compound A mesylate salt was associated with a significant reduction in proliferation (31 ,9%+/-10% in vehicle treated mice vs 17.6%+/-6.6% in Compound A mesylate salt treated mice) and promoted keratinocyte apoptosis (0.09%+/-0.15% in vehicle treated mice vs 6.5%+/- 4.3% in Compound A mesylate salt treated mice) (Figure 9).

Experiment 3, Determine the effect of topical Compound A mesylate salt on the HPV8 mouse model of AK (FVBN background treated with TP A),

[00167] The HPV8 FVBN mouse model was used to generate actinic keratosis like skin changes by application of TPA. The back of mice were shaved and TPA was applied every three days for 3 treatment. After 24 hours vehicle or a topical composition comprising 3% Compound A mesylate salt % (n=6 per dose) was applied for a total of 7 days. 24 hours after completion of treatment the back skin was collected and examined on for epidermal thickness, level of proliferation and apoptosis. Within 7-days, topical treatment using Compound A mesylate salt 3% treatment led to a dramatic reduction in epidermal thickness, associated with reduced proliferation and increased keratinocyte apoptosis (Figure 10).

Conclusion

[00168] The HPV8 mouse model provides an important model for evaluation of the epidermal changes observed in human actinic keratosis, with epidermal thickening emanating from the hair follicle infundibulum. Here we show that topical treatment with Compound A mesylate salt can reverse these epidermal changes, by reducing epidermal proliferation and induction of apoptosis.

Discussion

[00169] These changes are in general very encouraging in this well characterized spontaneous model of actinic keratosis wherein we have demonstrated NTRK expression at levels similar to that in human actinic keratosis. Although not demonstrated, it may be assumed that these effects are independent of a gene fusion event. These in vivo changes in proliferation and apoptosis mirror the findings in vitro, which in turn support the proposed mechanism of action. Compound A mesylate salt clearly demonstrated rapid efficacy in reversing epidermal changes both through reduction in proliferation and also keratinocyte apoptosis.

Example 5: Effect of Compound A mesylate salt on cell proliferation, apoptosis, and migration of keratinocytes cell lines from premalignant, SCC and BCC lesions

Background

[00170] Trk receptor expression has been previously reported in 8 SCC and 7 of 8 BCC samples (Chen-Tsai CP, Colome-Grimmer M, Wagner RF Jr. “Correlations among neural cell adhesion molecule, nerve growth factor, and its receptors, TrkA, TrkB, TrkC, and p75, in perineural invasion by basal cell and cutaneous squamous cell carcinomas”, Dermatol Surg. 2004 Jul;30(7): 1009-16). [00171] We have since reviewed microarray data of normal skin, SCC and BCC and determined overexpression of NTRK1 in SCC (2.5 fold, n=5) and NTRK3 in BCC (1.7 fold, n=8) when compared to normal skin (n=7). This overexpression of Trk receptors was confirmed by immunofluorescence labelling using a panTrk antibody of tumor tissue sections of SCC (n=6) and BCC (n=3). An additional set of SCC (n=6) and BCC (n=l) tumor sections were labelled using pan-Trk antibody immunohistochemistry alongside actinic keratosis samples (n=98). All together the findings strongly point to Trk receptor expression in premalignant and malignant keratinocytes. Based on these studies on Trk receptors in keratinocyte carcinomas and more recently premalignant lesion (actinic keratosis), we next sought to determine the effect of Trk receptor antagonist Compound A mesylate salt on cell function.

Methods

Cell lines

[00172] The cell lines utilized in this project are summarized in Table 4.

[00173] All cell lines were thawed at 37°C in a water bath, transferred to a 15 mL falcon tube and 9 volumes of pre-warmed media supplemented with 10% fetal bovine serum (FBS). Cells were pelleted at 300-g for 5 min at room temperature (RT) and supernatant discarded. Cell pellets were re-suspended in 15 ml of aforementioned media and incubated at 37°C (5% CO2) in a T75 culture flask. Media was changed every 2-3 days, and cells were sub-cultured at high confluence (80-90%).

Proliferation assay

[00174] Cell lines outlined in Table 4 were seeded (5,000 cells/well) into white-sided 96 well clear flat-bottomed plates and allowed to adhere overnight. The following day, growth media was removed and replaced with growth media supplemented with compound and controls and left for 24 hours before proceeding with the assay. Reagents were prepared according to the manufacturer’s instructions (BrdU Cell Proliferation ELISA Kit (colorimetric) (abl26556)). After 24hr treatment, 20 pl of the prepared IX BrdU solution was added to each of the wells and allowed to incubate at 37°C, 5% CO2 for a further 24 hours. Assay was performed according to manufacturer’s instructions. The luminescent signal produced was measured using the CLARIOstar plate reader (BMG Labtech) by setting the absorbance wavelength to 450nm. Table 4: List of cell lines and media used

Viability assay

[00175] The Cell Titer Gio assay (Promega, Southampton, UK) is a homogenous method of determining the number of viable cells in culture based on the quantitation of the ATP present, which is an indicator of metabolically active cells. Cell lines outlined in Table 4 were seeded (5,000 cells/well) into white-sided 96 well clear flat-bottomed plates and allowed to adhere overnight. The following day, growth media was removed and replaced with growth media supplemented with compound and controls and left for 48 hours before proceeding with the assay. Experiment was performed by adding the single reagent (Cell Titer-Gio Reagent) directly to the cells cultured in media in a 1: 1 ratio (lOOpl or reagent to lOOpl media) and plate was placed on an orbital shaker for 2 min in order to lyse the cells, before being placed in a dark environment for 20 min at room temperature in order for the signal to stabilize. The Cell Titer-Gio reagent contains lysing reagents, which lyse the cells, and the enzyme Ultra-Gio rLuciferase, which requires ATP from the lysed cells to convert luciferin into oxyluciferin, to subsequently generate the luminescent output. Therefore, the amount of ATP was directly proportional to the number of cells present in culture. Following incubation, the luminescent signal produced was measured using the CLARIOstar plate reader (BMG Labtech) by setting the excitation/emission wavelengths to 560/590nm.

Apoptosis assay

[00176] The Annexin V apoptosis assay was used to determine the proportion of cells undergoing early or late apoptosis following drug treatment, by detecting/studying the externalization of phosphatidylserine, one of the earliest indicators of apoptosis. Cell lines outlined in Table 4 were seeded (40,000 cells/well) into 12 well plates and allowed to adhere overnight. The following day, growth media was removed and replaced with growth media supplemented with compound and controls and left for 48 hours before proceeding with the assay. On the day of analysis, media was removed from cultured cells, washed with PBS, trypsinised and pelleted. Supernatant was removed, and each sample/condition was resuspended in lOOpL of IxAnnexin V Binding Buffer (Biolegend) and 1 pL of Annexin V, Alexa Fluor 647 conjugate (ThermoFisher Scientific) was added to the suspension and incubated for 20 min at room temperature in a dark environment. Following incubation, a further 400 pL of Annexin V binding buffer was added to each sample to inactivate the reaction. Just before each sample was analysed, 1 pL of DAPI (20 pg/mL) was added. Samples/cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences), and were gated by firstly selecting the cell population using FSC-area/SSC-area, then obtaining a single cell population by removing doublets using FSC-area/FSC-height, before finally gating this single cell population based on the intensity of the far-red APC dye conjugated to Annexin V, and DAPI to identify cells that are either early or late apoptotic, and live or dead. Data was processed using FlowJo analysis software.

Results

[00177] We chose four cell lines to study: HaCaT keratinocytes (normal), MET-1 (human primary SCC), MET-2 (human SCC recurrence after MET-1 tumor excision), UWBCC1 (human BCC cell line). The normal keratinocyte (HaCaT) and BCC (UWBCC1 cell lines exhibited a dose dependent reduction in cell proliferation, using concentrations of Compound A mesylate salt (Figure 11). In contrast, the SCC cell lines MET-1 and MET-2 demonstrated much greater sensitivity at lower concentration of Compound A mesylate salt, but not in a dose dependent manner (Figure 12); suggesting additional effect of Compound A mesylate salt other than on cell proliferation.

[00178] We next assessed cell viability, once more HaCaT and UWBCC1 cells exhibited dose dependent loss of cell viability with increasing Compound A mesylate salt concentration (Figure 13). Intriguingly, the HaCaT cell line was more sensitive than the UWBCC1 cell line.

[00179] The MET-1 cell line was more sensitive than the MET -2 cell line to Compound A mesylate salt, with greater loss of viability even at low concentrations (Figure 14).

[00180] We next determined whether the loss of viability was related to apoptosis. Flow cytometric analysis of Compound A mesylate salt treated cells demonstrated dose dependent apoptosis within HaCaT, if we exclude one replicate (Figure 15). But no increase in cell death at this time point.

[00181] UWBCC1 cell line had no increase in apoptosis of dead cell numbers, with the exception of the highest concentration of Compound A mesylate salt (Figure 16).

[00182] Apoptosis was repeated greater and in a dose dependent manner in MET-1 (Figure 17). But MET-2 cells after Compound A mesylate salt treatment showed no change in apoptosis or dead cell numbers (Figure 18).

Conclusions

[00183] These in vitro experiments show that Compound A mesylate salt is efficacious at blocking proliferation and inducing selective apoptosis, leading to a loss of cell viability. In HaCaT and MET-1, Compound A mesylate salt at the concentrations studied predominantly blocks cell proliferation and induced apoptosis. In UWBCC1, Compound A mesylate salt predominantly stopped proliferation and did not induce apoptosis. Whereas in MET-2 SCC cell lines, Compound A mesylate salt blocked proliferation but the loss of cell viability could not be attributed to apoptosis.

Discussion

[00184] These findings suggest that SCC cell lines are sensitive to Compound A mesylate salt, in which even at low concentrations we observe a loss of cell viability from the combined effect of a block in cell proliferation and induction of apoptosis notably in MET-1. In contrast, Compound A mesylate salt in HaCaT and UWBCC1 cell lines blocks proliferation, wherein HaCaT also showed increased apoptosis. Extrapolating these findings, it may be hypothesized that both BCC and SCC cell lines may be sensitive to Trk receptor signaling to maintain cell viability, blocking signaling reproducibly led to loss of cell viability.

Example 6: Trk expression in BCC and SCC

Background

[00185] It has been previously reported that Trk receptor expression in SCC (N=8) and BCC (7 of 8) samples (Chen-Tsai CP, Colome-Grimmer M, Wagner RF Jr. “Correlations among neural cell adhesion molecule, nerve growth factor, and its receptors, TrkA, TrkB, TrkC, and p75, in perineural invasion by basal cell and cutaneous squamous cell carcinomas”, Dermatol Surg. 2004 Jul;30(7): 1009-16) is overexpressed in these keratinocyte carcinomas and the premalignant state actinic keratoses.

[00186] Quantitative approach using microarray analysis was also performed to confirm and quantify the upregulation of TRK receptor in SCC and BCC. Microarray analysis showed overexpression of NTRK1 in SCC (2.5-fold, n=5) and NTRK3 in BCC (1.7-fold, n=8) when compared to normal skin (n=7). This overexpression of Trk receptors was confirmed by immunofluorescence labelling using a pan-Trk antibody of tumor tissue sections of SCC (n=6) and BCC (n=3).

[00187] An additional set of SCC (n=6) and BCC (n=l) tumor sections were labelled using pan-Trk antibody immunohistochemistry alongside actinic keratosis samples (n=98).

Methods

Microarray

[00188] RNA for microarray was extracted and RNA quality was assessed as previously described. RNA was amplified, and cDNA was prepared using the Illumina TotalPrep RNA Amplification Kit, which is used to generate biotinylated amplified RNA for hybridization with Illumina Sentrix arrays. Samples were applied to the Illumina HumanHT-12 v4 Expression BeadChip, which provided genome-wide transcriptional coverage of well-characterized human genes. BCC (n=8), SCC (n=5), hair bearing normal skin (n=7) were applied to the chip using the Direct Hybridization assay protocol. A full protocol and reagent list can be found on the Illumina website. The labelled cRNA generated was hybridized to the BeadChip containing the complementary gene specific sequence. The cRNA samples were first preheated to 65°C for 5 mins and then allowed to cool to room temperature before adding the appropriate amount each cRNA sample into each hybridization tube (750ng worth of cRNA for 12-sample chip) and made up to 5 pL using nuclease free water. Finally, to each cRNA sample tube, 10 pL of HYB solution was added (15 pL total). Next 15 pL of the sample was loaded onto the ports of the BeadChip, which was loaded into a Hyb chamber insert. The BeadChip chamber lid was closed and placed in the Hyb chamber into a 58°C Illumina Hybridization Oven for at least 14 hr. Following incubation in the Hyb chamber the BeadChip was washed in E1BC solution before being blocked with Block El buffer for 10 mins. The final stage before scanning the BeadChip was detection of the signal, which involves the addition of Cy3 - SA to bind to the analytical probes that were hybridized to the BeadChip, which allowed for differential detection of signals upon scanning the BeadChip. Cy3 - Streptavidin was diluted 1 :1000 in Block El buffer and the BeadChip was incubated in the solution for 10 mins. After the incubation, the BeadChip was washed with fresh Wash E1BC buffer. After incubation, the BeadChip was dried by placing it in a centrifuge and spinning at 1,400 rpm for 4 min at room temperature. After the centrifugation was complete, the dried BeadChip was stored in a dark environment until ready to be scanned. The BeadChip was imaged in the iScan system.

Immunolabelling of tissues

[00189] Pan-Trk immunofluorescence staining for TrkA/B/C expression was performed on O.C.T embedded normal skin (n=l), basal cell carcinoma (n=3) and squamous cell carcinoma (n=4) sections. Slides were fixed with 4% paraformaldehyde for 10 minutes at room temperature, before being washed in PBS and subsequently permeabilized for a further 10 minutes at room temperature in PBS/Tween (0.25%). Sections were then blocked for nonspecific binding in PBS-3% BSA for 1 hour at room temperature and subsequently stained with a pan-trk antibody (Santa Cruz trk (B-3) sc-7268; 1 : 100 dilution) and cytokeratin-14 (1 :500 dilution) in PBS-3% BSA overnight at 4°C. Slides were then washed with PBS before incubating with the secondary antibodies, Goat anti-Mouse Alexa Fluor 488 (pan-trk; 1 :400 dilution), Goat anti-Chicken Alexa Fluor 647 (cytokeratin 14; 1 :400 dilution) and the nuclear stain DAPI (1 : 1000 dilution) for 1 hour at room temperature in PBS-3%BSA. Finally, sections washed with PBS and mounted using VECTASHIELD® Antifade Mounting Media (Vector

Laboratories). Images of tissue sections were captured with an Axio Scan.Zl slide scanner (Zeiss).

Immunohistochemistry of tissues

[00190] Pan-Trk immunofluorescence staining for TrkA/B/C expression was performed on paraffin embedded tissues of actinic keratosis (n=6), basal cell carcinoma (n=5) and squamous cell carcinoma (n=6) sections. Paraffin embedded sections were deparaffinized and re-hydrated in a series of xylene and ethanol washes. Slides were heated at 60°C for 30 mins, then deparaffinized with xylene (2 x 10 mins) and decreasing concentrations of ethanol (100 - 75%) for 5 mins each, followed by a 5 min wash in PBS. For antigen retrieval, samples were submerged in citrate buffer (pH6.0) and heated in a microwave for 10 mins in a pressure cooker and allowed to cool to room temperature gradually. A hydrophobic barrier was drawn around the tissue sample. Sections were then incubated in blocking buffer (10% donkey serum in PBS) for 1 hour at room temperature. A pan-Trk antibody (Santa Cruz trk (B-3) sc-7268) was diluted in 5% donkey serum (1 : 100 dilution) and incubated overnight at 4°C. No primary antibody was used as negative control. Samples were washed 4 x 5 mins in PBS Tween-20 (0.05%) before incubating with secondary antibody at room temperature. Samples were again washed 4 x 5 mins in PBS Tween-20 (0.05%) before staining with 3,3 ’-Diaminobenzidine (DAB). Sections were submerged in hematoxylin solution for 5 mins and once stain was considered strong enough, are washed under running water. Sections were then stained with eosin and washed before dehydrating in a series of ethanol and xylene. Sections were mounted using DPX solution and imaged. Images of tissue sections were captured with an Axio Scan.Zl slide scanner (Zeiss) and processed using Olympus vs200 software. Statistical analysis was undertaken using Graphpad v9.

Results

[00191] Microarray study identified differences in individual Trk receptor expression between normal skin, BCC and SCC. BCCs exhibited greater expression of NTRK3 compared to normal skin and SCC. In contrast, SCC had greater expression of NTRK1 compared to normal skin and BCC (see Figure 19 and Table 5). Although not reaching statistical significance, to suggest intra-sample variability, there appeared to be a consistent trend.

Table 5

[00192 ] Using only antibody controls for the pan-Trk receptors immunofluorescence labelling, we identified the hair follicle infundibulum as a possibly site expressing Trk receptors (see Figure 20). Similar to previous reports expression of Trk receptors was low/absent within the interfollicular epidermis.

[00193] We labelled three human BCC samples with the pan-Trk receptor antibody. Tumor labelling was absent in the nodular BCC (n=l) but variable low intensity labelling in the invasive BCCs (n=2) (see Figure 21). Where positive, labelling was predominantly within the cell cytoplasm but areas notably at the tumor margin also demonstrated strong nuclear labelling.

[001 4] Human cutaneous SCCs (n=6) were immunolabelled with pan-Trk receptor antibody (see Figures 22 and 23). All SCC samples demonstrated pan-Trk receptor antibody labelling, throughout the tumor mass. Well differentiated SCC (n=2) demonstrated both cytoplasmic and nuclear labelling; wherein the nuclear labelling was intense. The nuclear labelling was restricted to the areas of stratification within the center of the well differentiated SCC nodules. While cytoplasmic and membrane labelling was weaker and evident within the tumor periphery where keratinocytes are typically proliferating. In keeping with this the moderately differentiated SCC (n=4) samples pan-Trk receptor antibody was within the membrane and cytoplasm.

[00195] Immunohistochemistry with the antibody clone that detects Trk fusion products showed modified labelling in: actinic keratosis (n=6), BCC (n=5) and SCC (n=6) (Figure 24).

Conclusion

[00196] Our studies using immunohistochemistry (protein expression) and Microarray (gene expression) showed that Trk receptors are increased at both, protein and gene expression levels, in AK, SCC, and BCC and did not identify NTRK fusion.

Discussion

[00197] Similar to the study by Chen-Tsai et al our findings also show that BCC and SCC express Trk receptors. Whereas all SCC express Trk receptors but not all BCC. When present, Trk receptor expression was throughout the tumor, however labelling was weaker in positive BCCs. We observed similar cell membrane and cytoplasmic expression predominantly, with strong nuclear labelling within stratified cells within the tumor nodule. These demonstrated no evidence of perineural invasion and therefore we surmise Trk expression in keratinocyte carcinoma occurs independent of perineural invasion. (Chen-Tsai CP, Colome-Grimmer M, Wagner RF Jr. Correlations among neural cell adhesion molecule, nerve growth factor, and its receptors, TrkA, TrkB, TrkC, and p75, in perineural invasion by basal cell and cutaneous squamous cell carcinomas. Dermatol Surg. 2004 Jul;30(7): 1009-16.)

[00198] While earlier studies demonstrate a lack of Trk expression in the normal epidermis, our antibody labelling identified strong infundibulum Trk expression, consistent with our current studies that suggest these cells may give rise to SCC and also occasional BCC. Although earlier studies have identified TrkA positive cells within hair follicle melanocytes this is the first time Trk receptor expression has been documented in normal human skin keratinocytes.

[00199] We identified a novel difference in Trk receptor expression in BCC and SCC in our microarray data. While TrkA receptor is the dominant expressed strongly in SCC, it is not expressed in BCC. Instead BCC appear to only express the TrkC receptor..

[00200] NTRK fusion events appear not to be a prerequisite for keratinocyte carcinoma (SCC or BCC) development, if they do occur then this is likely to be a bystander effect rather than driver transformation. However, the process of carcinogenesis leads to over-expression of Trk receptors within tumor keratinocytes and their precursor lesions (actinic keratosis). Therefore, all keratinocytes tumors and their precursor lesion, actinic keratosis, may respond to Trk receptor blockade.

Claims

CLAIMS Compound A, or a pharmaceutically acceptable salt thereof:

Compound A; for use in treating skin field cancerization with actinic keratoses in a subject. The compound according to claim 1, wherein the skin field cancerization with actinic keratoses is present on the face, ear, scalp, neck, back of hand, forearm, or lip of the subject. The compound according to claim 1 or 2, wherein the subject is an immunocompetent adult. The compound according to any preceding claim, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject topically, orally or subcutaneously. The compound according to any preceding claim, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered to the subject topically. The compound according to claim 5, wherein Compound A, or a pharmaceutically acceptable salt thereof, is topically administered to the face, ear, scalp, neck, back of hand, forearm, or lip of the subject. The compound according to claims 5 or 6, wherein Compound A, or a pharmaceutically acceptable salt thereof, is applied topically at least once per week or at least twice per week. The compound according to claim 7, wherein Compound A, or a pharmaceutically acceptable salt thereof, is applied topically at least once per week, such as at twice per week, for a duration of at least four weeks. The compound according to claims 5 to 8, wherein Compound A, or a pharmaceutically acceptably salt thereof, is applied topically, and left on the subject’s skin for at least 1 hour, such as at least 2 hours. The compound according to any preceding claim, wherein the subject has a Fitzpatrick skin category of Type I or II. The compound according to any preceding claim, wherein the subject has more than one actinic keratosis lesion. The compound according to claim 11, wherein the more than one actinic keratoses lesion is in one continuous area of sun-damaged skin. The compound according to claim 12, wherein the sun-damaged skin shows signs of erythema and/or telangiectasia. The compound according to claims 12 to 14, wherein the continuous area of sundamaged skin is at less than or equal to 25 cm². The compound according to any preceding claim, wherein the skin field cancerization with actinic keratoses in the subject is present in a confluent area and the actinic keratoses are an Olsen grade 1, 2 or 3, or a mixture thereof. The compound according to claim 15, wherein the confluent area is less than or equal to 25 cm². The compound according to any preceding claim, wherein between 0.005 and 50000 mg of Compound A, or a pharmaceutically acceptable salt thereof, is dosed per day. The compound according to any preceding claim, wherein Compound A, or a pharmaceutically acceptable salt thereof, is topically administered to the affected area of the subject’s skin at an application dose amount of from about 0.2 to about 120 pg/cm² skin surface.