Method to modulate pigmentation process in the melanocytes of skin

Abstract

The invention describes a method to modulate melanosome maturation process in melanocytes. Further, it describes a method to reversibly alter the process of pigmentation in the human epidermal skin. The method targets melanosome maturation and its transfer to keratinocytes, which are key processes involved in the pigmentation for its potential use in predictable and controlled regulation of pigmentation in human skin.

Claims

1. A method to alter stage I and II melanosome maturation in normal epidermal melanocytes of a subject by contacting the normal epidermal melanocytes with IFN-, wherein the normal epidermal melanocytes are treated with 300 to 500 U/mI of IFN- for 8 days, wherein the IFN- increases expression of IFN- pathway components selected from the group consisting of interferon gamma receptor 1 (IFNGR1), janus kinase 1 (JAK1), and signal transducer and activator of transcription 1 (STAT1).

2. The method as claimed in claim 1 wherein IFN- alters the levels of tyrosinase (TYR), dopachrome tautomerase (TYRP2/DCT), cappuccino protein (CNO), Hermansky-Pudlak syndrome 4 protein homolog (HPS4), G-protein coupled receptor 143 (GPR143), oculocutaneous albinism 2 gene (OCA2), solute carrier family 7 member 11 gene (SLC7A11), and mucolipin 3 gene (MCOLN3).

3. The method as claimed in claim 1 wherein the transfer of melanosome to keratinocytes is altered by targeting components of the IFN- pathway selected from the group comprising of interferon gamma (IFNG), interferon gamma receptor 1 (IFNGR1), janus kinase 1 (JAK1), and signal transducer and activator of transcription 1 (STAT1).

4. The method as claimed in claim 1, wherein the normal epidermal melanocytes are treated with 300 U/ml of IFN-.

5. The method as claimed in claim 1, wherein the normal epidermal melanocytes are treated with 400 U/ml of IFN-.

6. The method as claimed in claim 1, wherein the normal epidermal melanocytes are treated with 500 U/ml of IFN-.

7. A method of treating skin pigmentation in normal epidermal melanocytes of a subject by contacting the normal epidermal melanocytes with IFN-, wherein the normal epidermal melanocytes are treated with 300 to 500 U/ml of IFN- for 8 days, wherein the IFN- increases expression of IFN- pathway components selected from the group consisting of interferon gamma receptor 1 (IFNGR1), janus kinase 1 (JAK1), and signal transducer and activator of transcription 1 (STAT1).

8. The method as claimed in claim 7 wherein IFN- is administered to the subject by topical delivery or by sub-cutaneous delivery.

9. The method as claimed in claim 8 wherein the topical delivery or sub-cutaneous delivery is in a dosage form selected from the group comprising, a powder, a solution, an emulsion, a fluid emulsion, a suspension, a fluid suspension, a semi-solid, an ointment, a paste, a cream, a gel, a jelly, a foam, an implant, an injection, a patch and a spray.

10. The method as claimed in claim 7 useful for treating skin disorders selected from the group comprising of hyperpigmentation, hypopigmentation, uneven pigmentation, post inflammatory pigmentation, sun-damaged skin, pigmented birthmark, acanthosis nigricans and solar lentiges, age-related pigmentation changes, indeterminate leprosy, tuberculoid leprosy, borderline leprosy, leprematous leprosy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

(1) Figure Legend 1

(2) Electron micrographs of primary normal human melanocytes were treated with 100 U/ml IFN- using transmission electron microscope. (a) Control (untreated) melanocyte (b) treated with 100U/ml IFN-. Melanocytes were fixed, permeabilized and the melanosomes probed using HMB45 antibody for melanosomal matrix (green) and melanin (red) (c) control (d) 100 U/ml IFN- treated melanocytes. Quantified image normalized fluorescence intensity of (e) HMB45 and (f) melanin staining of melanocytes.

(3) Figure Legend 2

(4) (a) Primary human melanocytes treated with 100 U/ml IFN- followed by microarray analysis of regulated genes represented as heat-map. Some of the conspicuously downregulated genes include DCT, Mlan A and Trpl. Notably many genes involved in immune functions were upregulated by the treatment. (b) Real time PCR analysis of regulated genes under two different concentrations of IFN- (100 U/ml and 200 U/ml). (c) Western blot analysis of DCT, phosphorylated STAT-1, phosphorylated GSK-3 from control, MSH (6 nM) or IFN- (100 U/ml) treated melanocytes.

DETAILED DESCRIPTION OF THE INVENTION

(5) IFN- stalls the maturation of melanosomes by altering the levels of key proteins involved in melanosome formation and maturation, like TYR, TYRP1, and DCT/TYRP2, CNO, HPS4, GPR143, OCA2, SLC7A11 and MCOLN3, thus decreasing pigmentation in normal human melanocytes. Modulation of pigmentation protein levels by IFN- can be useful in attaining hypopigmented states.

(6) Another determinant of skin pigmentation is the transfer of mature (Stage III and IV) melanosomes from melanocytes to keratinocytes. Our in-vitro studies using melanocyte-keratinocyte co-culture models, demonstrates that that IFN- decreases this transfer process, altering the number of melanosomes in keratinocytes, leading to decreased apparent pigmentation.

(7) As detailed in Example 5 later, this mechanism of IFN- mediated hypopigmentation may be apparent in several hypopigmentary conditions of skin, like tuberculoid leprosy, where localised concentrations of IFN- can inhibit melanosomes maturation and transfer in the melanocytes of the lesional skin. Targeting this pathway would thus be of therapeutic importance for the treatment of hypopigmentary conditions. Alternately, mimicking this pathway would promote controlled pigmentation of skin by targeting melanosome maturation in melanocytes and the transfer of melanosomes to keratinocytes.

(8) IFN- can be formulated as a composition using a variety of physiologically acceptable carriers, excipients or diluents. The composition can be, for example, in a cream, gel or lotion for topical use or can be present as a liquid. The composition can also be present in dosage unit form that could be administered through sub-cutaneous infusion if necessary.

(9) Since TGF- has previously been shown to inhibit melanin synthesis in B16 melanoma cells as well as human primary melanocytes (Martinez-Esparza et al., 2001; Nishimura et al.; Nishimura et al., 2010) Therefore it was important to determine whether IFN- acted via a mechanism involving TGF-. It was reported that TGF- causes reduction in pigmentation by reducing the level of MITF transcription factor. However IFN- and its downstream effector proteins achieve hypopigmentation despite having unaltered levels of MITF. These findings indicate that IFN- mediated hypopigmention process is different from that brought about by TGF-.

(10) The generalizability of the hypopigmenting functions of IFN- is demonstrated by its inhibitory effect on a overall pigmentation process involved in normal human melanocytes. IFN- is locally produced in the epidermis in several conditions (Sarra et al., 2011). Others have previously demonstrated that keratinocyte-derived factors, which are also locally produced, influence melanocyte behaviour, like melanocyte proliferation, dendricity, and the amount of melanin produced (Gordon et al., 1989). Thus taken together, the data provided herein indicate that local administration of IFN- could be used for predictably alter pigmentation in human epidermal cells.

(11) The present disclosure provides details of how IFN- can be used to reversibly modulate melanosome maturation occurring in stage I and II in epidermal melanocytes. This disclosure makes it obvious that derivatives, mimics or antagonists of IFN- can also be used to reversibly modulate melanosome maturation occurring in stage I and II in epidermal melanocytes. The present specifications therefore do not limit the scope of the invention to IFN-, and instead extends the scope to derivatives, mimics and antagonists of IFN-.

(12) Accordingly, the present invention is directed towards a method to reversibly alter melanosome maturation occurring in stage I and II in an epidermal melanocytes in a subject by contacting or withdrawing IFN- and its derivative, mimic, antagonist thereof with said melanocytes in a controlled manner,

(13) and wherein the IFN- and derivative, mimic, antagonist thereof modulate the expression of IFN- pathway components selected from a group comprising IFNGR1, JAK1 and STAT1.

(14) In yet another aspect of the invention IFN-, or its derivative, or its mimic, or an antagonist, or components of IFN- pathway are contacted with melanocytes in the range of 100-500 U/ml for a period in the range of 1 to 10 days.

(15) In yet another aspect of the invention IFN- or its derivative, or a mimic or an antagonist of IFN- reversibly alters the levels of TYR, and TYRP2/DCT, CNO, HPS4, GPR143, OCA2, SLC7A11 and MCOLN3.

(16) In further aspect of the invention the transfer of melanosome to keratinocytes is altered by targeting components of IFN- pathway selected from the group comprising of IFNG, IFNGR1, JAK1 and STAT1.

(17) In an additional aspect of the invention the use of IFN- and its components and its agonists and antagonists in modulating pigmentation in humans for clinical or cosmaceutical purposes.

(18) In yet another aspect of the invention a method of treating skin pigmentation by administering to a subject IFN- or its derivative, or a mimic or antagonist of IFN-.

(19) In further aspect of the invention IFN- or its derivative, or a mimic or an antagonist of IFN- is administered to the subject by topical delivery or by sub-cutaneous delivery.

(20) In yet another aspect of the invention the topical delivery or sub-cutaneous delivery is in a dosage form selected from the group comprising, a powder, a solution, an emulsion, a fluid emulsion, a suspension, a fluid suspension, a semi-solid, an ointment, a paste, a cream, a gel, a jelly, a foam, an implant, an injection, a patch and a spray.

(21) In an additional aspect the invention is useful for treating skin disorders selected from the group comprising of hyperpigmentation, hypopigmentation, uneven pigmentation, post inflammatory pigmentation, sun-damaged skin, pigmented birthmark, acanthosis nigricans and solar lentiges, age-related pigmentation changes, indeterminate leprosy, tuberculoid leprosy, borderline leprosy, leprematous leprosy.

(22) The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1

(23) Cultures of normal human epidermal melanocytes (NHEM) (procured from Lonza, or established from epidermis) were maintained in M254 medium containing PMA-free supplements (Invitrogen), at 37 C., in 5% CO.sub.2. To study the effect of IFN- on melanocytes, early passage cultures of melanocytes were grown in the presence or absence of recombinant human IFN- (Peprotech) at a dose of 100 U/ml and the cells were observed after 8 days of treatment. Decreased pigmentation could be observed in the cell pellets treated with IFN-, confirming the presence of a mechanism for the induction of hypopigmentation (FIG. 1). To understand the molecular targets of human IFN- (henceforth referred to simply as IFN-) in melanocytes and the mechanism by which the cells are hypopigmented further studies were carried out.

(24) Melanosome maturation status in the cells and visible pigmentation are tightly linked together. The process of melanin synthesis is sequestered in the late stages of melanosome maturation and requires a proper targeting of multiple proteins to early stages of melanosomes for initiation. The status of melanosomal receptivity for the synthesis of melanin and the content of melanin within each melanosome could therefore be considered as cellular endpoint of pigmentation program. Since melanin is an electron dense substance, visualization of melanosomes is classically carried out using electron microscopy. The technique allows identification of distinct stages of melanosomes I to IV based on the organelle architecture and melanin content. IFN- treated cells (as mentioned above) were subjected to electron microscopy and confocal imaging using HMB45 and melanin antibody. Electron microscopic evaluation was carried out on control and IFN- treated cells. Analysis of the micrographs suggested that fewer stage III and IV melanosomes were detected in IFN- treated cells, whereas several could be distinctly identified in control cells. For finer and quantitative analysis of the status of melanosomes immuno-cytochemical approach was adopted. Control and IFN- treated primary melanocytes were stained with Human melanoma Black 45 (HMB45) from (Dako Cytomation) and anti-melanin antibody (rabbit polyclonal raised against synthetic melanin, raised in the laboratory). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Invitrogen) and the cells observed under confocal microscope. Recurrent observations indicated that lesser fluorescence signals were obtained from IFN- treated cells. For comparative quantitation, processing and imaging of control and IFN- treated cells was carried out under identical conditions. The settings used for imaging like background correction, signal to noise ratio etc are arbitrary and were kept constant. Mean fluorescence intensity of DAPI was subtracted from HMB45 or melanin signals from each cell for normalization. Comparison of the normalized intensities indicated that IFN- treated cells had significantly less melanin content as well as melanosome matrix as detected by HMB45 antibody.

Example 2

(25) In order to understand the molecular changes induced by IFN- on melanocytes a microarray based study was carried out on two independent primary melanocyte cultures. Cells were treated with 100 U/ml of IFN- for 8 days (as mentioned in example 1) and total RNA was isolated and subjected to microarray. Microarray was performed using Illumina whole genome chip Human WG-6. RNA labelling was performed using Illumina total prep RNA labelling kit (Ambion). Subsequent hybridization and washing steps were carried out as per manufacturer's instructions. The data was average normalized; background subtracted and analyzed using Bead-studio software (Illumina). Typical of an immune cytokine IFN- elicited a gene expression profile suggestive of an immune response in melanocytes. However the pattern of gene expression also included suppression of certain pigmentation genes (FIG. 2). This confirmed that the hypopigmentory role of IFN- is due to the suppression of pigmentation genes by affecting the maturation of melanosomes. Regulation of chosen genes was confirmed by real-time PCR. In primary human melanocytes the regulation of multiple IFN- downstream genes were observed and is tabulated in table 1.

(26) TABLE-US-00001 TABLE 1 List of genes regulated by IFN- in primary human melanocytes significantly regulated in primary melanocytes based on microarray DCT, TSPAN10, TSPAN9, SERPINF1, KIF1B, SOX10, MYO18A, MLANA, CREB1, MC1R, DCTNI, CNO, TYRP1, HPS4, GPR143, ID2, JARID2, OCA2 CHMP2A, JARID1B, SLC7A11, MCOLN3, IFT2, IFTM1, IRF1, IRF3, IRF4, IRF7, IRF9, OAS1, PSMB8, PSMB9, STAT1, STAT6, TNFRSF19, TNFSF12, IFNGR1, IFNGR2, MAPKAP1, LOC402221, PDK4, C19ORF28, FARSA, TSPAN4, TYSND1, DEAF1, PKN1, WHSC1L1, ACP1, RASSF1, FAM76B, XL, PGAM4, WDR26, PIK3CD, FEN1, HS.31532, NR1H2, DNAJB12, FEZ1, PWWP2B, TNFAIP1, SH3BGRL3, TCIRG1, PCMTD2, LOC643357, MYO1B, HS.374257, LOC648249, BIN1, HS.12876, PRMT1, JAK 1, JAK 2, HS.231861, BRD8, LOC347544, BANF1, GYPC, AGXT2L2, MORF4L1, MRRF, ACTB, OAS3, H1FX, UBE2G1, FAM10A4, C22ORF29, HLA, DRB4, TMEM69, C8ORF33, RCE1, HLAH, ARL6IP6, LOC402251, CYBA, HLAB, AHSA1, ERP29, ADAT3, PPT2, SCAND1, TNC, RPL32, IFIT3, GRN, CTTN, LOC648210, ARPC4, TBCC, CTH, HLAF, RIPK5, TAP2, GMPR, APRT, ACLY, TMED10, TUBA1A, AGPAT1, LOC407835, KIAA0460, PDIA6, PHF20L1, MRPL12, VAMP5, UBB, ARMCX1, BEX4, LOC441775, FAM152B, NAT5, OBFC2B, RAB4A, LOC440589, PHF5A, LRP11, TNPO1, STEAP1, LOC649150, CXORF40B, PTTG1IP, LOC284821, HP1BP3, CAPNS1, RNH1, HCG4, HS.406790, EEF1A1, HLA.DPA1, POLRMT, LOC388654, CPEB2, IFI27, GAA, ARFGEF1, RAB7L1

Example 3

(27) To verify the pathway and elucidate the components of the pathway that mediate the hypopigmenting effect of IFN- we resorted to a siRNA based testing of protein functions. Primary human melanocytes were cultured in serum free media as described above (in example 1) and were transfected with 100 nM of siRNA (a mixture of 4 targeting siRNA from Dharmacon) that would target the component mRNA of interest using lipid based transfection reagent (Cellfectin II from Invitrogen). After the transfection the cells were trypsinized and plated in wells and were either treated with IFN- or left untreated as control. After 48 h of treatment, cells were lysed and total RNA was prepared from the cells and levels of the targeting gene transcript as well as that of DCT was measured by real-time PCR. Targeting siRNA to JAK-1, STAT-1 and IFNGR-1 substantially decreased the mRNA level of the cognate genes and concomitantly abrogated the IFN- mediated suppression of DCT transcript. This series of experiment further validated that the components of the pathway are potentially excellent targets for achieving altered pigmentation.

Example 4

(28) IFN- is known to function in a species-specific manner. To verify whether the hypopigmentation observed in melanocytes is specifically brought about by IFN-; melanocytes were treated with mouse IFN- (100U/ml) and human IFN- (100U/ml) for 8 days. Mouse IFN- did not hypopigment human melanocytes confirming that the effect was specific and not a generic effect of peptide addition. When the human IFN- treated hypopigmented melanocytes were grown in the absence of the IFN- in M254 only, the cells were found to regain their pigmentation levels after 5 days, indicating reversibility of IFN- mediated hypopigmentaion in melanocytes. This supports IFN- to be an important component in mediating physiological reversible pigmentation changes.

Example 5

(29) To probe for the causative role of IFN- in skin hypopigmenting conditions, RNA was isolated from the lesional hypopigmented and non-lesional epidermis of subjects with lepramatous leprosy. cDNA was synthesised and real-time PCR analysis performed for comparing gene expression changes between in pigmentation (TYR, TYRP1, TYRP2, MITF) genes and genes known to be upregulated by IFN- (PSMB8, PSMB9, HLA-DRB1, HLA-A, HLA-B, HLA-C, IFNGR1, IRF1). The expression levels of pigmentation genes including DCT (except MITF) was found to be less in the lesional skin of leprosy subjects, consistent with the visible hypopigmentaion. Concordant increase in IFN- gene signatures was also observed. Electron microscopic analysis of the non-lesional versus lesional skin also indicated the decrease in the number of mature (Stage III and IV) melanosomes in the lesional skin as compared to non-lesional skin. These results are concordant with our in vitro observations that IFN- stalls melanosomes in early stage of maturation, thereby leading to visible hypopigmentation. This study demonstrates that in diseased states this pathway could be operative to cause visible changes in human skin pigmentation.

Example 6

(30) To understand the effect of IFN- on transfer of melanosomes from melanocytes to keratinocytes, cocultures of melanocytes and normal human epidermal keratinocytes (from Lonza or established from foreskin derived epidermis) were established in a ratio of 1:2. The cultures were maintained in 1:2 M254 and KSFM (Invitrogen) respectively in the presence or absence of IFN- (100U/ml) for 8 days. At the end of 8 days, keratinocytes were separated from melanocytes by differential trypsinization method and processed for electron microscopic analysis. Multiple micrographs of keratinocytes with melanosomes were obtained and analyzed for the number of melanosomes per micrograph. The cytoplasmic area of keratinocytes was calculated with image analysis software. Number of melanosomes/area of keratinocytes was observed to be decreased in keratinocytes which were grown in the presence of IFN-, indicating that IFN- inhibits the process of melanosome transfer to keratinocytes in addition to its effect on melanosome maturation.

(31) TABLE-US-00002 TABLE 2 Prior art in relation to current claims Major outcome of S. No Title Reference their study Relevant results Novelty in our patent 1 Upregulation of the J Invest Increase in the The study correlates These studies are performed IFN-gamma- Dermatol. expression of interferon gamma with mice which do not have stimulated genes in 2005 interferon (IFN)- signatures with epidermal pigmentation and the development of May; gamma-stimulated pigmentation, apparently contradicts our delayed pigmented 124(5): 1053-61. genes was detected however the study. spots on the dorsal in delayed involvement is in the skin of F1 mice of pigmented lesions. development of HR-1 HR/De. Authors claim that delayed pigmented IFN-gamma may spots. play a pivotal role in the development of delayed pigmented spots in the mouse model of solar lentiges. 2 Interferon-gamma Am J IFN-gamma may MART1, and GP100 This study does not reveal any reduces Pathol. enhance antigen expression is new findings related to the melanosomal 2002 inflammatory decreased by IFN- pigmentation in the melanoma antigen expression February; responses yet in melanoma cells cells studied. and recognition of 160(2): 521-8. hamper effective Whereas our study divulges melanoma cells by recognition of information about cytotoxic T cells. melanoma cells by pigmentation regulation by the immune system interferon gamma in melanocytes through melanosome maturation regulation 3 Interferon-gamma Br J Interferon gamma Follicular This study focusses on is a potent inducer Dermatol. causes catagen like melanogenesis is follicular cells which are of catagen-like 2005 changes in human switched off in cells distinct from melanocytes. changes in cultured April; scalp follicular cells treated with Moreover the effect of human anagen hair 152(4): interferon gamma interferon gamma herein follicles 623-31. requires cell cycle changes and pigmentation is a consequence. no evidence for melanosome maturation alterations are observed. 4 Antitumor activities J Invest Interferon beta Interferon alpha There was no information on of interferon alpha, Dermatol. causes potent anti- increases melanin the melanosome maturation beta, and gamma 1990 proliferative effect synthesis and and the process of and their December; on human Interferon beta pigmentation, which is a combinations on 95 (6Suppl): melanoma cells. decreases melanin concerted effect of several human melanoma 231S-237S. Cell surface synthesis distinct pathways. Our study cells in vitro: antigens (HLA-DR significantly. demonstrates the effect of changes of and DQ) are altered Melanin synthesis interferon gamma on many of proliferation, upon interferon decreased observed these independent biochemical melanin synthesis, gamma treatment with Interferon pathways. and gamma but the data immunophenotype. was not statistically significant

ADVANTAGES OF THE INVENTION

(32) 1) Application potential of this invention is immense in its ability to accomplish controlled hypopigmentation of the skin as it targets the entire process of melanosome maturation.

(33) 2) Small molecules inhibitors or antibody based neutralization of this pathway would be useful in treating pigmentation disorders like melasma, post-inflammatory hyperpigmentation, sun burn, UV induced hyperpigmentation, nevus, acanthosis nigricans etc.

(34) 3) Non-clinical, cosmetic application of the pathway could also be of use in modulating the skin tone in individuals

(35) 4) As interferon functions without affecting the levels of MITF pigmentation this strategy would be useful in conditions that increase pigmentation through MITF

(36) 5) IFN- or its derivative thereof can be formulated as a composition, for example, in a cream, gel or lotion for topical use or can be present as a liquid. The composition can also be present in dosage unit form that could be administered through sub-cutaneous infusion if necessary.

REFERENCES

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