FABP4 as a therapeutic target in skin diseases

Abstract

Provided are methods of regulating proliferation and/or differentiation of keratinocytes and immune cells, more specifically to methods of treating pathologies characterized by hyperproliferative keratinocytes or inflammatory skin diseases by administration of FABP4-inhibitor.

Claims

1. A method for treating or preventing a skin disease in a subject in need thereof having, in the skin thereof, keratinocytes or inflammatory cells that overexpress FABP4, said method comprising administering to said subject a therapeutically effective amount of at least one FABP4 inhibitor or a pharmaceutical composition thereof, wherein said skin disease is characterized by the presence, in the skin, of keratinocytes or inflammatory cells overexpressing FAPB4, and wherein said at least one FABP4 inhibitor each independently is selected from the group consisting of: ##STR00090## wherein R.sup.1 and R.sup.2 each is H or halogen; and R.sup.3 is H or C.sub.1-4-alkyl; ##STR00091## wherein R.sup.1 is H or halogen, R.sup.2 and R.sup.3 each is H or C.sub.1-4-alkyl; and Ar is phenyl or 2-theinyl; ##STR00092## wherein R is H, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl or C.sub.1-4-carboxylic acid; and X is O or NH; ##STR00093## wherein n is an integer of 0-4; ##STR00094## wherein X is halogen, C.sub.1-4alkyl or C.sub.1-4alkoxy; ##STR00095## wherein X is halogen, C.sub.1-4alkyl or C.sub.1-4alkoxy; ##STR00096## wherein n is an integer of 0-2; ##STR00097## wherein R is C.sub.1-6-alkyl, C.sub.1-4-haloalkyl, C.sub.1-4-alkyl, CF.sub.3, CONH.sub.2 or halogen; and n is an integer of 1-2; ##STR00098## wherein X is O or NOH; ##STR00099## wherein n is an integer of 1-2; ##STR00100## wherein R.sup.1 is H or C.sub.1-4alkyl; R.sup.2 is H, C.sub.1-4alkyl, C.sub.1-4alkoxy, halogen, NO.sub.2 or NH.sub.2; and R.sup.3 is H, C.sub.1-4alkyl, C.sub.1-4alkoxy, halogen or NO.sub.2; ##STR00101## wherein X is selected from the group consisting of ##STR00102## wherein R is C.sub.1-4alkyl, phenyl or fluorophenyl; ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## or a salt, stereomer or hydrate thereof.

2. The method of claim 1, further comprising administering a PPARγ agonist to the subject.

3. The method of claim 2, wherein said PPARγ agonist is administered concomitantly with said at least one FABP4 inhibitor.

4. The method of claim 2, wherein said FABP4 inhibitor and said PPARγ agonist are administered sequentially.

5. The method of claim 1, wherein said FABP4 inhibitor is also a FABP5 inhibitor.

6. The method of claim 1, wherein said skin disease is selected from the group consisting of psoriasis, dermatitis (atopic, seborrheic, contact), eczema, parapsoriasis, lichen planus, lichen plano-pilaris, Pityriasis lichenoides et varioliformis acuta, Pityriasis lichenoides chronica, Pityriasis rubra pilaris, Pityriasis rosea, graft-versus-host disease, histiocytoses, drug-induced eruptions, autoimmune connective tissue diseases, rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, scarring alopecia, cutaneous T cell lymphoma (CTCL), actinic keratosis, squamous cell carcinoma, basal cell carcinoma, nevus, lichen simplex chronicus, xerosis, keratosis, keratoderma, pruritus, a burn, a scar, a callus, and a keloid.

7. The method of claim 1, wherein the skin disease is an inflammatory skin disease.

8. The method of claim 1, wherein said at least one FABP4 inhibitor is administered topically, orally, by inhalation, nasally, transdermally, ocularly or parenterally into the circulatory system of a subject.

9. The method of claim 6, wherein the skin disease is psoriasis.

10. The method of claim 6, wherein the skin disease is CTCL.

11. The method of claim 6, wherein the skin disease is lichen planus/lichen planopilaris.

12. The method of claim 6, wherein the skin disease is dermatitis.

13. The method of claim 1, wherein said at least one FABP4 inhibitor each independently is a compound of the formula: ##STR00111## wherein R.sup.1 and R.sup.2 each is H or halogen; and R.sup.3 is H or C.sub.1-4-alkyl; or ##STR00112## wherein R.sup.1 is H or halogen; R.sup.2 and R.sup.3 each is H or C.sub.1-4-alkyl; and Ar is phenyl or 2-thienyl, or a salt, stereomer, or hydrate thereof.

14. The method of claim 13, wherein said at least one FABP4 inhibitor is (2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy)-acetic acid (BMS309403), or a salt, stereomer, or hydrate thereof.

15. The method of claim 2, wherein the PPARγ agonist is selected from the group consisting of thiazolidinedione, pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, rhodamine, and a non-steroidal anti-inflammatory drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIGS. 1A-1F show expression of FABP4 and PPARγ proteins in immunohistochemistry staining of human skin: healthy controls skin (FIGS. 1A-1B), psoriasis-involved skin (FIGS. 1C-1D), and dermatitis-involved skin (FIGS. 1E-1F). Tissue sections were stained with anti FABP4 (FIGS. 1A, 1C, 1E) or anti PPARγ (FIGS. 1B, 1D, 1F) antibodies. Original magnification ×100.

(3) FIGS. 2A-2C show expression of FABP4 protein in immunohistochemistry staining of human skin: normal human skin (FIG. 2A), epidermis of psoriasis-involved skin (FIG. 2B), and dermis of psoriasis-involved skin (FIG. 2C). Tissue sections were stained with anti-FABP4 antibody. Original magnification ×200.

(4) FIGS. 3A-3B show expression of PPARγ protein in immunohistochemistry staining of human skin: normal human skin (FIG. 3A), and psoriasis-involved skin (FIG. 3B). Tissue sections were stained with anti-PPARγ antibody. Original magnification ×200.

(5) FIGS. 4A-4B are immunohistochemistry staining of FABP4 protein in human skin: normal skin (FIG. 4A), and human cutaneous T cell lymphoma (CTCL) skin lesions (FIG. 4B). Tissue sections were stained with anti-FABP4 antibody. Original magnification ×200.

(6) FIG. 5 is an immunoblot (Western Blotting) showing levels of keratinocyte differentiation markers K1 and K5 in primary murine keratinocytes overexpressing FABP4. The keratinocytes were infected with a lentiviral vector expressing FABP4 tagged with GFP (FABP4-GFP), lentiviral vector expressing GFP (GFP), or left untreated (no infection).

(7) FIG. 6 shows Psoriasis Area Severity Index (PASI) scoring of the animals from the in vivo experiment at various time points. *p<0.05; ***p<0.001 compared to Vehicle group (using Two-way ANOVA followed by Bonferroni post-hoc test).

(8) FIG. 7 shows erythema severity scoring of the animals from the in vivo experiment at various time points. ***p<0.001 compared to Vehicle group (using Two-way ANOVA followed by Bonferroni post-hoc test).

(9) FIG. 8 shows skin thickness severity scoring of the animals from the in vivo experiment at various time points. **p<0.01; ***p<0.001 compared to Vehicle group (using Two-way ANOVA followed by Bonferroni post-hoc test).

(10) FIG. 9 shows scaling severity scoring of the animals from the in vivo experiment at various time points. *p<0.05, **p<0.01; ***p<0.001 compared to Vehicle group (using Two-way ANOVA followed by Bonferroni post-hoc test).

(11) FIGS. 10A-10F are representative pictures of mice on Day 7 of the in vivo experiment: Naïve (FIG. 10A), Vehicle (FIG. 10B), BMS 5 mg/kg (FIG. 10C), BMS 15 mg/kg (FIG. 10D), BMS 30 mg/kg (FIG. 10E), and Cortisone acetate (FIG. 10F).

DETAILED DESCRIPTION OF EMBODIMENTS

(12) The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

Example 1

Tissue Expression Analysis of FABP4, FABP5, and PPARγ in Human Psoriatic Skin Lesions

(13) Punch biopsies (4 mm in diameter) were obtained from disease-involved skin of patients with psoriasis (n=10). Biopsies were further obtained from involved skin of patients with chronic dermatitis (n=5). Normal skin was obtained from patients after surgical reduction of redundant skin (n=10). The tissues were fixed in formalin and embedded in paraffin. For histopathological examination with light microscopy, the sections were stained with hematoxylin and eosin (H&E) and observed by a pathologist who confirmed the diagnosis. In addition, for immunohistochemical analysis, the sections were stained with the following antibodies: FABP4 (Rabbit polyclonal anti FABP4 antibody, PAB12276, Abnova), FABP5 (Rabbit polyclonal anti FABP5 antibody, SC-50379, Santa Crus) and PPARγ (mouse monoclonal anti-PPARγ antibody, E-8, Santa Crus), all diluted 1:50. The paraffin-embedded and cryopreserved tissues were processed according to a standard protocol.

(14) FIG. 1C shows high expression levels of FABP4 as detected in psoriatic skin lesions compared to normal skin (FIG. 1A), both in the epidermis and the dermis. This finding was observed in all the psoriasis-involved biopsies that were tested (100 percent prevalence). To the best of the inventors' knowledge, this is the first time that the expression of FABP4 in keratinocytes and dermal cells in psoriatic lesions has been reported. Without wishing to be bound by theory, this finding may suggest that FABP4 overexpression is related to the dysregulation of keratinocyte proliferation and differentiation in psoriasis. In addition, this observation supports a role of FABP4 in promoting inflammation by its action in dermal immune cells, which is another significant feature of psoriasis.

(15) Increased level of FABP4 in both keratinocytes and inflammatory cells in the dermis were also observed in skin biopsies from patients with chronic dermatitis, as shown in FIG. 1E, although less than in psoriasis (FIG. 1C).

(16) Moreover, a negative correlation between FABP4 and PPARγ expression was observed in keratinocytes and immune cells: while in normal skin PPARγ was detected throughout the epidermis and in few cells in the dermis, as shown in FIG. 1B, in psoriatic lesions the expression of PPARγ was significantly reduced and was displayed only in the uppermost layers of the epidermis, and not detected in dermal cells (FIG. 1D). PPARγ expression was also reduced in dermatitis skin biopsies, as shown in FIG. 1F. FABP5 was expressed only in keratinocytes from psoriatic skin, as previously reported ([16], not shown).

(17) Psoriatic skin is characterized by a hyperproliferative epidermis comprised from multiple layers of keratinocytes, resulting in increased thickness of the skin. At higher magnification, FABP4 was demonstrated throughout the hyperplastic epidermis in psoriatic lesions, wherein staining was mostly cytoplasmic in the basal layers and nuclear in the upper layers, as shown in FIG. 2B. FABP4 expression in the dermis was predominantly observed in macrophages, lymphocytes and endothelial cells, in a cytoplasmic pattern, as shown in FIG. 2C. PPARγ expression in psoriatic skin was markedly reduced in the epidermis, appeared only in the uppermost layers, and was undetected in the dermis compared as to normal skin (see FIG. 3B versus 3A).

(18) This suggests that the tendency to develop skin diseases having an inflammatory component may be detected by evaluating FABP, and particularly FABP4, expression in dermis and epidermis skin samples. It also suggests that FABP4 overexpression, and PPARγ downregulation, are linked to the pathogenesis of psoriasis.

Example 2

Tissue Expression Analysis of FABP4 in Human Cutaneous T Cell Lymphoma (CTCL) Skin Lesions

(19) CTCL is a group of neoplasms of skin-homing T cells. Mycosis fungoides (MF) represents the most common type of CTCL and accounts for approximately 50% of all primary cutaneous lymphomas. Although malignant in nature, MF has a protracted clinical course with inflammatory, dermatitis-like presentation [8].

(20) The expression of FABP4 among patients with MF was studied by performing an immunohistochemical analysis of lesional skin. Samples from 5 patients with MF were tested and compared to normal human skin. Punch biopsies (6 mm in diameter) were obtained from patients with MF, from disease-involved skin. Normal skin was obtained from patients after surgical reduction of redundant skin. Tissue sections were processed and stained with anti-FABP4 antibody as described in Example 1.

(21) High expression levels of FABP4 were observed in MF lesions compared to normal skin with all tested patients, as shown in FIGS. 4B and 4A, respectively. FABP4 expression was confined to the infiltrating T lymphocytes in the epidermis and the dermis. A strong cytoplasmic stain was observed in the dermal infiltrating cells, as well as in the malignant cells which penetrated the lower epidermis, which is the hallmark of MF.

(22) Thus, due to the high expression of FABP4 protein in the malignant CTCL T-lymphocytes, detection of overexpression can be used to determine a patient's propensity towards development of CTCL or allow for early detection thereof.

(23) Many dermatologic diseases (including psoriasis, dermatitis, and CTCL) are mediated by lymphocytes, predominantly T lymphocytes [17]. FABP4 expression has been reported in a restricted repertoire of cell types, only in macrophages, adipocytes and endothelial cells. In immunohistochemistry analysis performed by the present inventor, FABP4 was found to be expressed in inflammatory cells in the dermis, in both macrophages and lymphocytes. FABP4 expression in lymphocytes has not been reported so far, nor expression thereof in dermal cells in general, thus these findings are of high relevance in dermatological diseases.

Example 3

Overexpressing FABP4 in Primary Murine Keratinocytes

(24) The introduction of FABP4 into keratinocytes was suggested to create a hyperproliferative state with impaired differentiation as seen in psoriasis. Differentiation was assessed by measuring the expression of two keratinocyte differentiation markers K1 and K5. K5 is a keratin whose level does not change during keratinocyte differentiation and therefore serves as a loading control, while K1 is induced during normal keratinocyte differentiation.

(25) For assessing the effect of FABP4 overexpression in mouse primary keratinocytes, cells were infected with a lentiviral vector construct containing the FABP4 gene. The FABP4 lentiviral vector, named FABP4-T2A-EGFP, was constructed with the FABP4 gene under the CMV promotor, and contained the green fluorescence protein (GFP) gene as a reporter of expression. The presence of a T2A residue at the N-terminus of the FABP4 protein precludes FABP4 detection by anti-FABP4 commercial antibodies, due to interference with their binding. Therefore, anti-GFP antibodies for validation of infection and FABP4 overexpression were used instead.

(26) For evaluating expression of K1 and K5 differentiation markers in primary murine keratinocytes with or without FABP4 expression, primary keratinocytes were prepared from neonatal mice as previously described [18], and infected with FABP4-T2A-EGFP vector. Two internal controls were used: cells which were not infected with the lentivirus and cells infected with the EGFP vector lacking the FABP4 gene. The cells were grown in differentiation media containing three different calcium concentrations. Elevation of extracellular calcium from low (0.05 mM) to medium (0.12 mM) or high (1 mM) concentration induced differentiation of keratinocytes.

(27) Expression of keratinocyte differentiation markers K1 and K5 in transfected and non-transfected cells is demonstrated in FIG. 5: infection with the lentivirus expressing FABP4-T2A-EGFP reduced the level of K1 both in medium and high calcium conditions compared to non-infected cells and GFP-infected cells. The level of K5 did not change during keratinocyte differentiation in absence of infection, as expected. The observation that FABP4 overexpression reduces K1 suggests that FABP4 interferes with the normal differentiation process of keratinocytes and promotes a hyperproliferative state, resembling psoriasis.

Example 4

Inhibition of FABP4 In Vivo in Imiquimod-Induced Psoriasis Model in Mice

(28) As already explained, psoriasis is a chronic inflammatory skin disorder. It occurs when the immune system mistakes the skin cells as a pathogen, and sends out faulty signals that speed up the proliferation of skin cells. Imiquimod (IMQ) is a potent immune activator that induces and exacerbates psoriasis when applied topically. Daily application of IMQ on mouse back skin induces inflamed scaly skin lesions resembling plaque type psoriasis [19, 20]. IMQ-induced psoriasis in mice has been long used as a model for human psoriasis.

(29) The efficacy of oral administration of a FABP4-inhibitor to treat psoriasis in the IMQ-induced model in mice was tested.

(30) BMS309403 (2-[[2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)[1,1′-biphenyl]-3-yl]oxy]-acetic acid, Formula I) was used as a FABP4-inhibitor:

(31) ##STR00089##

(32) The test was carried out using Balb/c mice (Envigo RMS (Israel) Ltd), average (±SD) body weight at study initiation was 18.2±0.81 g. Animals were fed ad libitum a commercial rodent diet (Teklad Certified Global 18% Protein Diet, Harlan cat #2018SC). Animals had free access to sterilized and acidified drinking water (pH between 2.5 and 3.5).

(33) The tests included six groups of 3-10 mice per group, as follows: one naïve (no IMQ) group; one Vehicle control group, the vehicle formulation being 10% 1-methyl-2-pyrrolidone and 5% Cremophor EL in water for injection (WFI); one treated group that received Cortisone acetate as a positive control—one tablet (25 mg each, Rekah Pharm) was triturated with a mortar. The powder was dissolved in 2.5 ml WFI yielding 10 mg/ml. The compound was administrated per-os at 12.5 mg/kg BW (body weight) mouse of the compound once daily for six days starting the first day of IMQ application (Day 1); and three treated groups that received BMS (i.e. Test Item) at three doses—5, 15 and 30 mg/kg. BMS powder (Cayman Chemical) was dissolved in ethanol to create a 30 mg/ml solution. This stock solution was diluted in the Vehicle to create three different concentrations 0.5, 1.5 and 3 mg/ml. A new aqueous solution was prepared in each day. BMS was administrated per-os once daily for six days starting the first day of IMQ application (Day 1).

(34) IMQ-induction: A daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals) was applied to animals from all the groups except for the naïve group on the shaved back for six consecutive days, translated to a daily dose of 3.125 mg of the active compound.

(35) Experiment conditions: All animals except for the naïve group received IMQ cream (Aldara™ 5% cream, #3 Pharmaceuticals) applied on the back skin daily for six consecutive days starting on Day 1 (˜60 mg/mouse). The Test Item, Vehicle and Cortisone acetate were administered per-os daily for 6 days. During the study morbidity and mortality, body weight (BW), clinical signs, and Psoriasis Area and Severity Index (PASI) scoring were performed and representative pictures were taken. The animals were sacrificed on Day 9.

(36) Scoring was performed by a trained observer in a “blinded” way, i.e., being unaware of the treatment. To score the severity of inflammation of the back skin, the clinical PASI scoring was employed. Erythema, scaling, and thickening were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative score (erythema plus scaling plus thickening), PASI scoring, served as a measure of the severity of inflammation (scale 0-12).

(37) Mortality & morbidity: During both experiments, no animal died or was found in morbid conditions. No abnormal clinical signs were observed in any of the animals during the study. No effect of the treatment on body weight was detected.

(38) Scoring severity of skin inflammation: The average score of the severity of inflammation of the back skin, i.e. the PASI, is presented in FIG. 6. Each parameter, namely erythema severity, skin thickness, and scaling, was measured individually (FIGS. 7-9, respectively). According to the PASI scoring, all IMQ treated groups developed considerable skin reaction, as compared to the naïve group, notably on Days 5 and 7 of the study. On Day 7 significant amelioration of the skin inflammation by the BMS treatment was observed. The middle dose of BMS (15 mg/kg) seemed to have the highest impact on the inflammation severity. This dose exhibited the best results on the tested parameters—erythema, skin thickness and scaling. Representative pictures taken at Day 7 are shown in FIGS. 10A-10F.

(39) In summary, all IMQ treated groups developed considerable skin reaction, as compared to the naïve group, notably on Days 5 and 7 of the study. On Day 7 significant amelioration of the effect was observed in the BMS treated groups for all test parameters, i.e. inflammation severity, erythema, skin thickness and scaling. The experiment was repeated tree times with similar results.