EP4444319A1 - Citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders - Google Patents

Abstract

The present disclosure relates to citric acid cycle and lactate transport inhibitors. The invention also relates to combinations or formulations comprising at least one of citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders.

Description

Citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders

FIELD OF THE INVENTION

The present invention relates to citric acid cycle and lactate transport inhibitors. The invention also relates to combinations or formulations comprising at least one of citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders.

BACKGROUND OF THE INVENTION

Skin disorders vary greatly in symptoms and severity. Skin disorders can be temporary or permanent and can be painless or painful. Some skin disorders have situational causes, while others are genetic. Also, some skin conditions are minor whereas others can be life-threatening.

Epidermolysis bullosa (EB) is a genetic skin disorder and is a group of rare medical conditions that result in easy blistering of skin and mucous membranes. Minor trauma or friction can cause occurrence of painful blisters. Severity of EB can range from mild to fatal (Bardhan et al., 2020 "Epidermolysis bullosa". Nature Reviews Disease Primers. 6 (1): 78. doi:10.1038/s41572-020-0210-0. ISSN 2056-676X. PMID 32973163. S2CID 221861310). More than 300 mutations have been identified for this condition (Koshida, et al., 2013. "Hallopeau- Siemens dystrophic epidermolysis bullosa due to homozygous 5818delC mutation in the COL7A gene". Pediatr Int. 55 (2): 234-7. doi:10.1111/j,1442-200X.2012.03638.x. PMID 23679163. S2CID 24238328).

Psoriasis is a common chronic skin disease that causes red, itchy scaly patches. It tends to go through cycles, flaring for a few weeks or months, then subsiding for a while or going into remission (https://www.mayoclinic.org/diseases-conditions/psoriasis/symptoms- causes/syc-20355840). Psoriasis is an immune-mediated disease characterized by inflammation caused by dysfunction of the immune system. Inflammation caused by psoriasis can impact other organs and tissues in the body. The visible signs of the inflammation comprise raised plaques and scales on the skin. The overactive immune system speeds up skin cell growth which causes the plaques and/or scales on skin. Normal skin cells completely grow and shed (fall off) in a month, however, with psoriasis the skin cells grow in only about three or four days. The skin cells with psoriasis, instead of shedding, starts piling up on surface of the skin (https://www.psoriasis.org/about- psoriasis/).

EP2475371A1 discloses pharmaceutical compositions comprising A3 adenosine receptor agonist for treatment of psoriasis. US20170333557A1 discloses administration of interleukin-4 receptor (IL-4R) antagonists to treat or prevent atopic dermatitis in a patient in need thereof. EP1889608B1 discloses a hydrogel comprising a biocompatible polymer, a polyalcohol and a medicinal plant extract, for atopic dermatitis. US9878038B2 discloses methods of treating atopic dermatitis with IL-31 monoclonal antibodies. EP2504012B1 discloses oleogel for use in wound healing, containing a nonpolar liquid and a triterpene- containing powder as an oleogel forming agent.

However, the available literature for treatment of various skin disorders lacks disclosure for use of citric acid cycle and lactate transport inhibitors.

Syrosingopine is a drug, derived from reserpine which is used (since about 1960) to treat hypertension (Shelburne PF, Orgain ES (March 1963). "Comparison of syrosingopine and reserpine in the treatment of ambulatory hypertensive patients". The American Journal of the Medical Sciences. 245: 304-10. doi:10.1097/00000441-196303000-00013).

Pyromellitic acid is a tetracarboxylic acid that is benzene substituted by four carboxy groups at positions 1, 2, 4 and 5, respectively. It is a member of benzoic acids and a tetracarboxylic acid (https://pubchem.ncbi.nlm.nih.gov/compound/Pyromellitic-acid).

Some other possible uses of TCA (tricarboxylic acid cycle) cycle and/or lactate transport inhibitors have been studied in the literature.

Benjamin et al., 2016 (Syrosingopine sensitizes cancer cells to killing by metformin. Science Advances. 2 (12): Sci Adv. 2016 Dec; 2(12): el601756. doi:10.1126/sciadv,1601756) discloses that a combination of the diabetes drug metformin and syrosingopine kills tumor cells in blood samples from leukaemia patients. The combination of metformin and syrosingopine reduce or eliminate tumours in mice with malignant liver cancer.

Benjamin et al., 2018 (Cell Rep. 2018 Dec ll;25(ll):3047-3058.e4. doi: 10.1016/j.celrep.2018.11.043) discloses dual inhibition of lactate transporters MCT1 (monocarboxylate transporters) and MCT4. The document discloses that syrosingopine, an anti-hypertensive drug, act as dual MCT1 and MCT4 inhibitor which prevents lactate and H+ efflux.

US20210087561A1 discloses inhibitors of lactate transporters, sodium-coupled monocarboxylate transporter 2 (SLC5A12), for use in treatment of inflammatory diseases. .

WO2011123788A1 relates to a method of inhibiting survival of a proliferating, quiescent, or hypoxic cancer cell comprising contacting the proliferating, quiescent, or hypoxic cancer cell with an effective amount of an agent that inhibits a protein involved in lactate transport or enzymatic conversion in the proliferating, quiescent, or hypoxic cancer cell.

Contreras-Baeza et al., 2019 (J. Biol. Chem. 2019 Dec 27; 294(52):20135-20147. doi: 10.1074/jbc.RA119.009093. Epub 2019 Nov 12) discloses that MCT4 is a H+-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or warburg effect. Tan et al., 2015 (J. Biol. Chem. 2015 Jan 2; 290(l):46-55. doi: 10.1074/jbc.M114.603589. Epub 2014 Nov 18) discloses that MCT4 is required for glycolytic reprogramming and inflammatory response in macrophages.

The current treatment strategies to treat inflammatory skin disorders include use of biologies against inflammatory cytokines and systemic anti-inflammatory drugs which have several side effects due to prolonged systemic immunosuppression.

There exists a need for pharmaceutical agents either alone or in combination or formulations for prevention and/or treatment of skin diseases. The agents, combination or formulations should be capable of exhibiting better therapeutic outcomes even at advanced stages of skin diseases. The agents, combination or formulations should be able to treat at least one of the skin diseases, but not limited to, atopic dermatitis, psoriasis and epidermolysis bullosa.

To overcome the limitations of treatment and/or prevention strategies known in the field of art, the inventors of present invention provide citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders. The present invention also provides combination or formulations comprising at least one of citric acid cycle and lactate transport inhibitors and uses thereof. DESCRIPTION OF THE DRAWINGS

Figure 1A illustrates transcriptomic analysis of differentially expressed genes from the epidermal compartments using Panther (http://www.pantherdb.org/) post Next Generation Sequencing (NGS).

Figures IB and ID illustrate qPCR analysis and immunostaining of KO (knockout) and control skin with glucose transporter (GLUT1) and glycolytic enzymes (Lactate dehydrogenase, LDHa).

Figure 1C illustrates lactate concentration quantification in the epidermis showing increased lactate concentration in the KO skin.

Figures IE and IF illustrate qPCR and immunostaining of the lactate transporter MCT4 (Monocarboxylic acid transporter 4) in the KO skin.

Figures 1G and 1H illustrate transcriptomic expression of genes in the NGS analysis of the macrophages isolated from the KO skin.

Figures II and IJ illustrate NGS analysis and immunostaining of MCT1 (Monocarboxylic acid transporter 1).

Figures IK and IL illustrate status of extracellular matrix (stained by using antibody against laminin 332 (Lam5) post inhibition of TCA cycle using intraperitoneal pyromellitic acid and lactate crosstalk using syrosingopine (syrosingopine blocks MCT1 and MCT4).

Figure 2A illustrates staining of psoriatic skin with GLUT1 and MCT4.

Figure 2B illustrates back skin condition in C57BL/6JNcbs [2019] strain mice models injected subcutaneously for about 5 days with DMSO control and syrosingopine.

Figure 2C illustrates reduced epidermal thickness and area in mice models injected with syrosingopine.

Figure 2D illustrates immune cell burden by staining DMSO control and syrosingopine treated back skin with monocyte (GDI IB), macrophage (F4/80), T cells (CD4) and neutrophils (Gr-1).

Figure 2E illustrate that syrosingopine treatment led to a decrease in MMP9 expression in skin.

Figure 3a illustrates method of NGS analysis of epidermis, fibroblasts and macrophages. Figures 3b and 3c illustrate epidermal response to loss of ECM attachment: augments an immune activating and recruiting response by synthesizing cytokines, chemokines and DAMPs (damage associated molecular patterns).

Figures 3d and 3e illustrate that tissue resident and recruited macrophages respond to inflammation by generating matrix remodelling enzymes to remodel the ECM and ECM molecules itself, respectively.

Figure 3f illustrates association of ECM remodelling properties of macrophages by depleting macrophages using CSF1R and observing ECM rescue.

Figure 3g illustrate loss of MMP9 synthesis at the dermal-epidermal junction upon macrophage depletion using CSF1R (Colony stimulating factor 1 receptor) antibody.

Figure 4a illustrates NGS analysis of KO epidermis showing augmentation of glycolytic and lipolytic pathways.

Figure 4b illustrate epidermis has higher expression of GLUT1 and LDHa.

Figure 4c illustrates lipolysis in skin by nile red staining in KO compared with WT skin.

Figures 4d and 4e illustrate qPCR validation of glycolysis and TCA.

Figure 5 illustrates validation of lipolysis, lipogenesis and glutaminolysis pathway associated with KO epidermis NGS data by qPCR.

Figure 6 illustrates pathways associated with metabolism in NGS analysis of macrophages using Panther (http://www.pantherdb.org/).

Figure 7a illustrates NGS data for HIF (Hypoxia inducible factor) expression when compared with other transcription factors associated with glycolysis upregulation.

Figure 7b illustrates HIF associated pathways shown in gene ontology analysis in the KO epidermis.

Figures 7c and 7d illustrate validation of the NGS data by HIF expression transcriptionally and by immunostaining, respectively.

Figure 7e illustrates validation of HIF downstream genes transcriptionally.

Figure 7f demonstrates validation of HIF downstream genes by immunostaining in the KO skin.

Figure 8a illustrates staining HIF targets which were reduced by inhibiting HIF translation using intraperitoneal YC-1 compound. Figure 8b illustrates HIF reduced expression of glucose transporter GLUT1 and LDHa expression in skin.

Figures 8c and 8d illustrate ROS associated pathways in the KO epidermis alongside several genes associated with antioxidant response to ROS species, respectively.

Figure 8e illustrates DHE staining of back skin from WT and KO skin.

Figure 9a illustrates inhibition of glycolysis (using 2-deoxy-D-glucose/ 2DG) and TCA cycle (using pyromellitic acid/ PA) reduces ECM degradation, MMP9 staining and MMP activity in the KO back skin.

Figure 9b illustrates loss of macrophage M2 polarization signatures (Arginase 1 - ARG1) upon treatment with 2DG and PA treated.

Figures 9c and 9d illustrate that inhibition of HIF and TCA cycle (by using UK-5099) also leads to rescue of ECM.

Figure 10 illustrates macrophage polarization and ECM disruption status in etomoxir treated KO skin.

Figures 10a, 10b, 10c and lOd indicate expression of macrophage M2 marker CD206 in the WT and KO skin post treatment with etomoxir.

Figures lOe, lOf, 10g and lOh indicate MMP activity in the WT and KO skin post treatment with etomoxir. There was no appreciable reduction in the MMP activity.

Figures lOi, lOj, 10k, and 101 indicate expression of MMP9 in the KO skin and associated ECM disruption.

Figure 11a illustrates MMP9 staining of back skin in different metabolic treatments in WT.

Figure lib illustrates nile staining of back skin in different metabolic treatments.

Figure 11c illustrates results of dye-exclusion assay.

Figure lid illustrates reduction in weights of animals in different metabolic treatments.

Figure 12a illustrates results of lactate assay demonstrated by lactate concentrations in KO epidermis when compared with WT littermate controls.

Figure 12b illustrates transcriptional expression of lactate exporter MCT4 in KO epidermis. Figure 12c shows experimental results of immunostaining demonstrating membrane localization in KO skin compared to WT skin.

Figure 12d illustrates that macrophages overexpress MCT importers.

Figure 12e illustrates reduction in MMP9 expression in the KO skin treated with syrosingopine compared to DMSO treated control skin which is further associated with reduction in the ECM degradation status.

Figure 13a illustrates that 5-day imiquimod treatment progressively induces psoriatic lesions in 2-month-old C57BL/6JNcbs [2019] strain mice.

Figure 13b illustrates association of induction of psoriatic lesions with increase in epidermal thickness and area.

Figure 13c illustrates association of induction of psoriatic lesions with immune cell burden.

Figure 13d illustrates enhanced expression of MMP9 in macrophages in psoriatic skin.

Figure 14a illustrates that imiquimod treated skin shows enhanced GLUT1 and MCT4 expression demonstrating increase in glycolysis and lactate export.

Figure 14b illustrates subcutaneous syrosingopine treatment reduce psoriatic plaques.

Figure 14c illustrates reduction of epidermal thickness and area by syrosingopine treatment.

Figure 14d illustrates reduction of immune cell burden in skin by syrosingopine treatment.

Figure 14e illustrates that syrosingopine treatment reduces MMP9+ macrophages in the psoriatic skin.

Figure 15 illustrates skin resident embryonic macrophages TCA-OXPHOS (tricarboxylic acid- oxidative phosphorylation) in inflammation which in turn is supported by lactic acid production from epidermis in [31 KO and psoriatic skin.

Figure 16 illustrates NGS data summary and validation of glycolysis and TCA cycle gene expression in epidermal compartment in [31 cKO skin.

Figure 16A shows metabolic pathways upregulated in GSEA analysis of epidermal compartment in E18.5 cKO skin compared to WT. Figure 16B shows relative transcript expression (from NGS) of glucose transporters and glycolytic genes in the epidermis of cKO and WT skin (N=2).

Figure 16C shows qPCR validation of glucose transporter GLUT1 and glycolytic genes in the epidermis of cKO compared to WT (N=4).

Figure 16D shows quantification of temporal change in the expression of GLUT1 in cKO and WT (N=2).

Figure 16E shows relative transcript expression (from NGS) of TCA cycle enzyme genes in the epidermis of cKO compared to WT (N=2).

Figure 16F shows qPCR validation of TCA cycle enzyme genes in the epidermal compartment compared to WT (N=3). Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 17 illustrates increased glycolysis and decreased TCA in epidermal compartment in [31 cKO skin.

Figure 17A and Figure 17B show epidermal compartment of cKO skin showing increased expression of GLUT1 (N=3) and LDHa (N=2), respectively; which are quantified in Figure 17C.

Figure 17D illustrates western blot for Hexokinase 2 (hk2) in epidermal and dermal compartment of KO skin.

Figure 17E illustrates reduced expression of CS and IDH1 in epidermis of cKO as compared to WT (N=2) which are quantified in Figure 17F.

Figure 17G shows schematic illustration of glycolysis and TCA cycle changes as suggested by transcriptomic, proteomic and metabolomic approaches in the cKO skin compared to WT. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 18 illustrates increased HIFla target expression in epidermal compartment of the [31 cKO skin.

Figure 18A illustrates GSEA analysis of cKO epidermis showing upregulated HIFla signalling and response to hypoxia at E18.5 compared to WT.

Figure 18B illustrates qPCR validation of increase in transcription of downstream targets of HIFla in the epidermis normalised to WT (N=3). Figure 18C illustrates epidermis of cKO skin showing increased expression of KRT14 (Figure 18C, top), COX2 (Figure 18C, bottom) compared to WT (N=3), which is quantified in Figure 18E.

Figure 18D illustrates epidermis of cKO skin showing decrease in expression of HIFla targets KRT14 (Figure 18D, top), COX2 (Figure 18D, bottom) after treatment with chetomin compared to controls (N=3) which is quantified in Figure 18F. Schematic showing dose schedule of various drugs used in the study in pregnant dams carrying cKO and WT embryos (G). Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 19 illustrates increased HIF and ROS expression in epidermal compartment in [31 cKO skin.

Figure 19A illustrates quantification of temporal change in the expression epidermal HIFla in cKO and WT skin (N=2).

Figure 19B illustrates immunostaining of HIFla in E18.5 cKO and WT skin. Figure 19C illustrates expression of GLUT1, LDHa in chetomin treated cKO and DMSO treated cKO controls (N=2) which is quantified in Figure 19E.

Figure 19F illustrates quantification of temporal change in the expression epidermal 8-OHdG expression in cKO and WT skin (N=2). Figure 19G illustrates immunostaining of 8- OHdG in E16.5 cKO and WT skin (N=2).

Figure 19H and Figure 191 illustrate GLUT1 and LDHa expression in NAG (N-acetyl cysteine) treated cKO and PBS treated cKO controls (N=3) which is quantified in Figure 19J.

Figure 19K illustrates schematic showing glycolysis regulation by HIFla and its regulation by ROS in cKO skin. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 20 illustrates increased ROS response in epidermal compartment of [31 cKO skin.

Figure 20A illustrates GSEA analysis of cKO epidermis showing upregulated ROS response at E18.5 compared to WT.

Figure 20B illustrates cKO skin showing no significant change in the expression of 8-

OHdG at E18.5 compared to WT (N=2). Figure 20C illustrates qPCR validation of increase in transcription of both ROS source and scavenger genes, in E18.5 cKO epidermis compared to WT (N=3).

Figure 20D illustrates epidermis of cKO skin showing decrease in expression of 8OHdG (Figure 20D, top), HIFla (Figure 20D, bottom) upon NAC treatment compared to controls (N=2) which is quantified in Figure 20F.

Figure 20E illustrates epidermis of cKO skin showing decrease in the expression of KRT14 (Figure 20E, top), COX2 (Figure 20E, bottom) upon NAC treatment compared to controls (N=3) which is quantified in Figure 20G. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 21 illustrates epidermis is an exporter while macrophages are importers of lactate metabolite in [31 cKO skin.

Figure 21A illustrates increased membrane expression of lactate exporter MCT4 in epidermis of E17.5 and E18.5 cKO skin compared to WT (N=3).

Figure 21B illustrates increased expression of TCA cycle enzymes CS and IDH1 in macrophage compartment in cKO skin as compared to controls (N=2) which is quantified in Figure 21C.

Figure 21D illustrates schematic showing the compartment separation of glycolysis and TCA cycle in epidermal and macrophages compartment in cKO skin compared to WT.

Figure 21E illustrates increased membrane expression of lactate importer MCT1 in macrophages in the KO skin compared to WT (N=2, n=30-40 cells each) (Figure 21E, top) Scale bar: 20um.

Figure 21E also illustrates increased basement membrane (EAM332) disruption in cKO skin compared to WT (Figure 21E, middle).

Figure 21E illustrates increase in expression of MMP9 in the cKO skin as compared to WT (Figure 12E, bottom).

Figure 12F illustrates decrease in nuclear to cellular ratio of expression of MCT1 in macrophages in KO and WT skin (N=2, n=30-40 cells each).

Figure 21G illustrates quantification of basement membrane (EAM332) degradation and Figure 21H illustrates MMP9 expression in cKO skin compared to WT. Figure 211 illustrates schematic representation of epidermis as a potential source and macrophages as potential sink for lactate metabolite in cKO skin. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant

Figure 22 shows that macrophages upregulate TCA cycle genes and downregulate glycolysis in the [31 cKO skin.

Figure 22A illustrates metabolic pathways upregulated in GSEA analysis of macrophage compartment in E18.5 cKO skin compared to WT.

Figure 22B illustrates relative transcript expression (from NGS) of major glucose transporters, glycolytic enzyme and TCA cycle enzyme genes in the macrophages in cKO skin compared to WT (N=2).

Figure 22C illustrates qPCR validation of glycolytic genes in cKO skin macrophages compared to WT (N=3).

Figure 22D shows that expression of GLUT1 (red) does not colocalize with F4/80 (green) in dermal macrophages in bl cKO skin.

Figure 22E illustrates qPCR validation of TCA cycle enzyme gene expression in cKO skin macrophages compared to WT (N=3).

Figure 22F illustrates schematic showing macrophages as potential sinks for lactate as it can be converted to pyruvate to drive the TCA cycle. Scale bars: 50 pm.

Figure 23 shows that macrophages upregulate TCA cycle genes and downregulate glycolysis in the [31 cKO skin.

Figure 23A illustrates qPCR validation of TCA cycle enzyme genes in cKO skin fibroblasts compared to WT (N=3).

Figure 23B illustrates metabolic pathways upregulated in GSEA analysis of fibroblast in E18.5 cKO skin compared to WT.

Figure 23C illustrates LAM332 staining suggesting no obvious difference in the basement membrane spread in the cKO skin treated with etomoxir compared to controls which is quantified in Figure 23D. Scale bars: 50 pm.

Figure 24 shows that inhibition of lactate transport using small molecule inhibitor reduces pro-remodelling fate acquisition in dermal macrophages. Figure 24A illustrates decreased MMP9 expression and basement membrane (LAM332) remodelling in cKO skin treated with AZD3965, Syrosingopine compared to

DMSO control (N=3).

Figure 24B, Figure 24C, Figure 24D and Figure 24E illustrate quantification of MMP9 expression in cKO skin treated with AZD3965, Syrosingopine and DMSO treated control cKO skin (N=3).

Figure 24F illustrates schematic showing Syrosingopine dose schedule in imiquimod induced mice model for psoriasis.

Figure 24G illustrates decrease in epidermal plaques in Syrosingopine treated C57B6/J mice compared to 5% DMSO treated control in imiquimod induced mice model of psoriasis (N=3).

Figure 24H illustrates reduction of epidermal thickening and number of proliferative cells (Ki67, red) in Syrosingopine treated C57B6/J mice compared to 5% DMSO treated control in imiquimod induced mice model of psoriasis (N=3).

Figure 241 and Figure 24J illustrate quantification of number of Ki67 positive cells in epidermal compartment and epidermal thickness in Syrosingopine and control treated mice treated with topical imiquimod (N=3).

Figure 24K illustrates quantification of MMP9 expression in dermal compartment in Syrosingopine and control treated mice treated with topical imiquimod (N=3).

Figure 24L illustrates reduction in expression of MMP9 (Figure 24L, top), number of macrophages (F4/80) (Figure 24L, middle), and number of monocytes (GDI IB) (Figure 24L, bottom) in Syrosingopine treated psoriatic mice compared to controls (N=3).

Figure 24M illustrates model summarizing the key findings of the study. In sterile inflammatory conditions in skin, early augmentation of ROS-HIFla axis leads to enhanced glycolysis and lactate generation in the epidermal compartment. The macrophage compartment in the dermis acts as sink of lactate released by the epidermal compartment, which is then utilized as substrate for driving TCA cycle which, in turn, is necessary for proremodelling fate switch macrophages. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant. Figure 25 shows that inhibition of epidermal ROS and HIFla leads to reduction in macrophage pro-remodelling fate acquisition.

Figure 25A illustrates decreased MMP9 expression and basement membrane (LAM332) remodelling in cKO skin treated with TCA cycle inhibitor, PA and glycolysis inhibitor, 2DG, compared with PBS treated control cKO skin (N=3).

Figure 25B illustrates quantification of basement membrane spread and Figure 25C illustrates MMP9 expression in PA and 2DG treated cKO and control treated cKO skin (N=3).

Figure 25D illustrates reduction in basement membrane spread (Figure 25D, left) and MMP9 expression (Figure 25D, right) in cKO skin treated with UK-5099 compared to controls.

Figure 25E illustrates quantification of basement membrane spread and

Figure 25F illustrates MMP9 expression in cKO skin treated with UK-5099 compared to controls.

Figure 25G, Figure 25H, Figure 251, Figure 25J and Figure 25K illustrate reduction in basement membrane spread in basement membrane spread (Figure 25G, Figure 25H, Figure 251) and MMP9 expression (Figure 25G, Figure 25J, Figure 25K) in cKO skin treated with chetomin and NAG compared to controls. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

Figure 26 illustrates lactate mediated cross-talk in imiquimod induced psoriatic skin samples.

Figure 26A illustrates increase in epidermal plaques upon induction of psoriasis using topical imiquimod for 5 days (N=3).

Figure 26B illustrates increase in epidermal thickness and number of proliferating epidermal cells (Ki67) in imiquimod treated mice compared to Vaseline treated controls.

Figure 26C illustrates quantification of epidermal area in imiquimod treated mice compared to Vaseline treated controls (N=3).

Figure 26D illustrates increase in macrophage (F4/80) (Figure 26D, top) and monocytes (CD11B) (Figure 26D, bottom) in imiquimod treated mice compared to Vaseline treated controls (N=3). Figure 26E illustrates increase in MMP9 expression in imiquimod treated mice compared to Vaseline treated control (N=3) which is quantified in Figure 26F.

Figure 26G illustrates increase in expression of GLUT1 (Figure 26G, top) and membrane localization of MCT4 (Figure 26G, bottom) in Imiquimod treated mice compared to Vaseline treated controls (N=3).

Figure 26H illustrates increase in expression of CS (Figure 26H, top), IDH1 (Figure 26H, middle) and MCT1 (Figure 26H, bottom) in the macrophages in imiquimod treated mice compared to Vaseline treated controls (N=3) Scale bars: 20 pm.

Figure 261 illustrates quantification of GLUT1 expression in epidermis of imiquimod treated mice and Vaseline treated controls (N=3).

Figure 26J illustrates quantification of expression of CS, IDH1 and MCT1 in macrophages in imiquimod treated mice compared to Vaseline treated controls (J). All experiments done for this figure are analysed at Day 6 of imiquimod treatment. Scale bars: 50 pm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant.

SUMMARY OF THE INVENTION

Metabolic factors have been shown to play supportive, instructive and permissive roles, essential for regulating fate decision in immune cells. The acquired metabolic state depends on the functional requirements of the immune cells and local availability of carbon and nitrogen sources. Innate immune cells especially macrophages have been shown to exhibit remarkable functional and metabolic flexibility, capable of acquiring distinct Ml and M2 fates, in vitro. While stimulation with IFN-y, LPS and TNF, polarize macrophages to a pro-inflammatory Ml state, stimulation with cytokines such as IL-4 and IL-13 polarize macrophages to a pro-remodelling and anti-inflammatory M2 state. Simultaneously, these Ml and M2 macrophages have been shown to be associated with distinct metabolic states where Ml macrophages have preferential dependence on glycolysis and M2 macrophages on TCA and oxidative phosphorylation (OXPHOS). While the distinct metabolic states, associated with macrophage polarization, have been clearly described in vitro, metabolic states of polarized tissue resident macrophages in vivo is not well understood. This is in part due to a wide range of metabolites received from the micro-niches, which is in turn complicated by tissue specific metabolic diversity. It can be expected that macrophages in different tissues depend on distinct sources of carbon and nitrogen due to cooperative metabolic interaction among niche factors to support their homeostatic function.

Using skin as in vivo model system (in vivo model system employed is C57BL/6JNcbs [2019] strain at animal care and resource centre, INSTEM, Bangalore and ItgBl KO mice available at the animal care and resource center (ACRC) at INSTEM Bangalore.), the inventors of present invention illustrate that metabolic crosstalk between macrophages and different skin compartments dictate acquisition of distinct metabolic and, in turn, functional states in the resident macrophages.

Unlike adults, the embryonic skin comprises mainly of resident macrophages and completely lacks adaptive immune arm. These tissue resident macrophages get recruited to the skin during early embryonic development at primitive and definitive haematopoiesis. Interestingly, this recruitment is concomitant with development of skin epithelia and appendages which raises speculation that these macrophages have a developmental role which remains to be understood. This allows the inventors of present invention a rather simplified system to understand the crosstalk between different skin compartments with the macrophages necessary for regulating macrophage fates.

The inventors of present invention have shown that macrophages during skin development exist in M2 (CD206+ RELMA+ MERTK+ 2ARG1+) state that maintains an immunosuppressive and pro-remodelling environment, both necessary for normal skin development (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005). Indeed, macrophage depletion during early embryogenesis disrupts normal skin development (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005). Naturally, acquisition of said state is a resultant of a rather unexplored metabolic crosstalk between macrophages and skin compartments which dictates overall fate decisions of different cell types in the tissue.

Previously published work from inventors' lab (Kurbet et al., 2016 https://pubmed.ncbi.nlm.nih.gov/27653694) shows that conditional KO (knockout) of integrin [31 from epidermis in embryos incites a sterile inflammatory response associated with increased immune infiltration. Inflammation occurring in absence of external pathogens and tissue barrier breach is termed as sterile inflammation. Inability of the tissues to resolve sterile inflammation leads to progression of diseases such as cancer and rheumatoid arthritis (Chen, G. Y. and Nunez, G., 2010. Sterile inflammation: Sensing and reacting to damage. Nature Reviews Immunology vol. 10). Tissue resident and recruited immune cells are key drivers of this inflammatory response (Zindel, J. and Kubes, P., 2020. DAMPs, PAMPs, and LAMPs in Immunity and Sterile Inflammation. Annual Review of Pathology: Mechanisms of Disease vol. 15). Interestingly, transcriptomic data from the macrophages isolated from [31 KO skin show that they have an exacerbated extra-cellular matrix (ECM) remodelling phenotype characterized by upregulation of multiple remodelling enzymes such as MMPs (matrix metalloproteinases) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs). This in turn exacerbates disruption of the basement membrane. Indeed, depletion of macrophages from the skin rescues the ECM phenotype (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005). Thus, skin resident macrophages in the KO condition have an altered phenotype that can be expected to be supported by an altered metabolic state. Using integrin [31 KO skin as a model for sterile inflammation, the inventors of present invention conducted experiments to understand how tissue resident macrophages switch to an alternate metabolic state to support an alternate function and how this alternation in turn changes the metabolic state of the niche cells during inflammation.

Taken together, skin resident macrophages have distinct functional states in homeostasis and inflammation. The inventors of present invention conducted experiments to identify the metabolic states of embryonic skin resident macrophages and its niche in homeostasis and inflammation. This aids in understanding the metabolic crosstalk between different skin compartments and macrophages is essential for bringing about normal skin development and inflammatory state.

The invention provides TCA cycle and lactate transport inhibitors for prevention and/or treatment of one or more of skin disorders. The invention also provides a method of using TCA cycle and lactate transport inhibitor to treat skin disorder which includes and is not limited to atopic dermatitis (AD), psoriasis and epidermolysis bullosa (EB). The disclosure provides long term topical use of the TCA cycle and lactate transport inhibitors for reducing activation of the innate immune system which is associated with inflammatory burden in skin. The invention provides combination and formulations of the TCA cycle and lactate transport inhibitors for prevention and/or treatment of one or more of the skin disorders even at advanced stages of the disorders.

The inventors employed Cre-loxP mediated integrin [31 KO model (10.1083/jcb.150.5.1149 - Generated by Dr. Srikala Raghavan. In the current study, the mice used is K14 Cre-driven integrin [31 KO mice available with ACRC at INSTEM, Bangalore) to recapitulate disease physiology associated with EB. Psoriasis and AD is studied in imiquimod induced mice model system (C57BL/6JNcbs [2019] strain available at the animal care and resource center (ACRC) at INSTEM Bangalore, treated with imiquimod, vaseline treatment is used as control). NGS data from epidermal compartment shows increased expression of genes associated with glycolytic pathway which in turn leads to enhanced generation of lactate getting transported to the epidermal compartment through MCT4. Further, the NGS analysis of the macrophages from the KO skin shows down-regulation of the glycolytic pathway and upregulation of the TCA cycle with lactate being imported through MCT1. The inventors of present invention conducted experiments to find out whether inhibition of TCA cycle and lactate crosstalk reduced ECM disruption. Inhibition experiments were carried out using intraperitoneal pyromellitic acid (for TCA cycle) and syrosingopine (MCT1 and MCT4 blocker for lactate crosstalk), which showed decrease in skin basement membrane disruption by reducing production of matrix re-modelling enzymes by the macrophages in the KO skin.

In some aspects, the present invention relates to TCA cycle and lactate transport inhibitors for use in the prevention and/or treatment of one or more of skin disorders.

An aspect of present invention relates to a combination and formulations comprising at least one of the TCA cycle and lactate transport inhibitors.

In another aspect, the present invention relates to a method of treatment and/or prevention of one or more of skin disorders by the TCA cycle and lactate transport inhibitors.

A further aspect of the present invention relates to a method of treatment and/or prevention of one or more of skin disorders by the combination and formulations comprising the TCA cycle and lactate transport inhibitors. In another aspect, the present invention relates to use of the TCA cycle and lactate transport inhibitors, formulations and combination thereof for the treatment and/or prevention of one or more of the skin disorders.

In yet another aspect of present invention, the developed pharmaceutical combination and/or formulations demonstrate long-term use without demonstrating severe side-effects. The combination and/or formulations of the invention are effective even at advanced stage of the skin disorders. The combination and/or formulations of the invention can be formulated into any pharmaceutically acceptable dosage forms. The combination and/or formulations of the invention can be formulated into a topical dosage form selected from the group consisting of rapid release, immediate-release or slow-release. The combination and/or formulations of the invention can be formulated as topical, intravenous, subcutaneous, controlled release, delayed-release, a combination of immediate and controlled release, nano-encapsulation formulations, creme formulation, gel formulation or as ointments.

In one of the embodiments, the present invention provides a pharmaceutical formulation for treating and/or preventing skin disorders, said formulation comprising at least one of citric acid cycle, lactate transport inhibitor and one or more pharmaceutically acceptable excipients wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875%, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v.

In another embodiment, the present invention provides that citric acid cycle and lactate transport inhibitors in the formulation are selected from the group consisting of 2- deoxy-D-glucose, pyromellitic acid, syrosingopine, UK-5099, etomoxir, chetomin, N-acetyl cysteine and AZD3965.

In a yet another embodiment, the present invention provides that the formulation treats and/or prevents the skin disorders which are at least one of atopic dermatitis, psoriasis, epidermolysis bullosa, alopecia areata, acne, atopic dermatitis, hidradenitis suppurativa, ichthyosis, pachyonychia congenita, vitiligo, scleroderma, rosacea and pemphigus. In a still another embodiment, the present invention provides that said one or more suitable pharmaceutically acceptable excipients in the formulation are selected from the group consisting of suitable carriers, diluents, vehicles, disintegrant, swelling agent, antioxidant, buffer, bacteriostatic agent, emollient, emulsifier, plasticizer, penetration enhancer, preservative, cryoprotectant, neutralizer, fragrance additives, dispersants, surfactants, binders and lubricants.

In still another embodiment, the present invention provides that the formulation is suitable for topical dosage form selected from the group consisting of rapid release, immediate-release or slow-release.

In a further embodiment, the present invention provides that the formulation is suitable for topical, intravenous, subcutaneous, controlled release, delayed-release, a combination of immediate and controlled release, nano-encapsulation formulations, creme formulation, gel formulation or as ointments mode of administration.

In a further embodiment, the present invention provides that the formulation prevents and/or treats one or more symptoms of psoriasis selected from hyperplasia, parakeratosis, red patches of skin covered with thick, silvery scales, small scaling spots, dry cracked skin that may bleed and/or itch, itching, burning or soreness, thickened pitted or ridged nails, swollen and stiff joints.

In one of the embodiments, the present invention provides a pharmaceutical combination for treating and/or preventing skin disorders, said combination comprising at least one of citric acid cycle, lactate transport inhibitor and one or more other active agent wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875%, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v.

In another embodiment, the present invention provides that the combination treats and/or prevents skin disorders which are at least one of atopic dermatitis, psoriasis, epidermolysis bullosa, alopecia areata, acne, atopic dermatitis, hidradenitis suppurativa, ichthyosis, pachyonychia congenita, vitiligo, scleroderma, rosacea and pemphigus. In another embodiment, the present invention provides that the other active agent in the combination is selected from the group consisting of antibiotics, antihistamines, steroids, fluoxetine and sertraline and tricyclic antidepressants.

In a yet another embodiment, the present invention provides a method of preparing pharmaceutical formulation or combination for treating and/or preventing skin disorders comprising the steps: a) adding required quantity of at least one of citric acid cycle and lactate transport inhibitors at suitable conditions to one or more pharmaceutically acceptable excipients to obtain a mixture; b) subjecting the mixture obtained in step 'a' to suitable conditions to obtain the formulation or combination in desired dosage form.

In a further embodiment, the present invention provides citric acid cycle and lactate transport inhibitors alone or in combination in an amount of from 0.01 gm/kg tolOOO gm/kg for treating and/or preventing skin disorders.

In a still another embodiment, the present invention provides a method of treating and/or preventing skin disorders in an individual, comprising administering to the individual a formulation or a combination comprising citric acid cycle and lactate transport inhibitors in an amount of from 0.01 gm/kg tolOOO gm/kg.

In another embodiment, the present invention provides use of a formulation or combination comprising citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said formulation or combination.

In a yet another embodiment, the present invention provides a kit comprising a pharmaceutical formulation and/or combination as claimed in, any one of claims 1 to 6 and instructions for administration of the pharmaceutical formulation and/or combination to a subject in need of treatment and/or prevention of skin disorders.

The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention. Other embodiments, aspects, advantages, and features will be readily apparent to those skilled in the art from the following detailed description of the invention. DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical agents along with combination and formulations thereof for treatment and/or prevention of skin diseases.

The inventors of present invention surprisingly found that TCA cycle and lactate transport inhibitors are capable of treatment and/or prevention of skin diseases. The present invention accordingly provides a pharmaceutical formulation for treating and/or preventing skin disorders, said formulation comprising at least one of citric acid cycle, lactate transport inhibitor and one or more pharmaceutically acceptable excipients wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% % w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875% of sterile PBS, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v of 5%DMSO in sterile PBS.

The present invention also provides a pharmaceutical combination for treating and/or preventing skin disorders, said combination comprising at least one of citric acid cycle, lactate transport inhibitor and one or more other active agent wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875% of sterile PBS, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v of 5%DMSO in sterile PBS.

The citric acid cycle and lactate transport inhibitors in the formulation and combination of present invention are selected from, but are not limited to, 2-deoxy-D- glucose, pyromellitic acid, syrosingopine, UK-5099, etomoxir, chetomin, N-acetyl cysteine and AZD3965.

The present invention also provides citric acid cycle and lactate transport inhibitors alone or in combination in an amount of from 0.01 gm/kg tolOOO gm/kg for treating and/or preventing skin disorders.

The present invention further provides a method of treating and/or preventing skin disorders in an individual, comprising administering to the individual a formulation or a combination comprising citric acid cycle and lactate transport inhibitors in an amount of from 0.01 gm/kg tolOOO gm/kg. The citric acid cycle and lactate transport inhibitors of present invention along with formulation and combination thereof are capable of treating and/or preventing skin disorders which are, but not limited to, one or more of atopic dermatitis, psoriasis, epidermolysis bullosa, alopecia areata, acne, atopic dermatitis, hidradenitis suppurativa, ichthyosis, pachyonychia congenita, vitiligo, scleroderma, rosacea and pemphigus.

The TCA cycle and lactate transport inhibitors of present invention demonstrate enhanced therapeutic outcomes even at advanced stages of skin diseases, for instance but not limited to, atopic dermatitis, psoriasis and epidermolysis bullosa.

The present invention provides long term topical use of TCA cycle and lactate transport inhibitors for reducing activation of the innate immune system which is associated with inflammatory burden in skin. These inhibitors have not been used for treating skin disorders. The inhibitors and combination or formulations thereof can be used for long term clinical treatment of chronic skin diseases.

Model system for study: The inventors of present invention have used cre-loxP mediated integrin [31 KO (knockout) model to recapitulate disease physiology associated with epidermolysis bullosa. Loss of integrin bl from epidermis in the embryonic skin leads to detachment of epidermis from the dermis which is similar to EB. Previous work from the lab (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005) suggests that skin resident macrophages are associated with increased skin basement membrane disruption. Psoriasis and AD is studied in imiquimod induced mice model system (C57BL/6JNcbs [2019] mice model available at the animal care and resource centre, INSTEM, Bangalore).

Few examples for representative purpose without limiting scope of the invention are illustrated below:

Example 1: Transcriptomic analysis of cell compartments using NGS

The experiment was conducted to elucidate that metabolic pathways associated with epidermis and macrophages functional states in the integrin [31 KO skin. The NGS data from epidermal compartment demonstrated increase in expression of genes associated with the glycolytic pathway (Figure 1A). This was also validated by qPCR analysis and immunostaining of the KO and the control skin with glucose transporter (GLUT1) and glycolytic enzymes (Figure IB, Figure 1C). The study showed that glycolysis lead to enhanced generation of lactate. Lactate concentration quantification in the epidermis showed increased lactate concentration in the KO skin (Figure ID). As lactate accumulation in cells leads to acidosis, the inventors carried further experiment to demonstrate that lactate is exported from the epidermal compartment. MCT4 has been shown to be associated with lactate export. qPCR and immunostaining of the KO skin shows increased expression and peripheral localization of the MCT4 transporter (Figure IE, Figure IF). NGS analysis of the macrophages from the KO skin shows down regulation of the glycolytic pathway and upregulation of the TCA cycle (Figure 1G, Figure 1H). TCA cycle can directly be supported through lactate as it can be converted directly to pyruvate and then acetyl CoA. The inventors conducted NGS analysis and immunostaining of MCT1 to demonstrate that macrophages expressed lactate importer MCT1. NGS analysis and immunostaining of MCT1 showed that macrophages express MCT1 and can utilize lactate from external sources (Figure II, Figure 1J). The inventors conducted experiments to demonstrate that inhibition of TCA cycle and lactate crosstalk reduced ECM disruption. Inhibition of TCA cycle using intraperitoneal pyromellitic acid and lactate crosstalk using syrosingopine, which blocks MCT1 and MCT4, shows decreased skin basement membrane disruption by reducing production of matrix remodelling enzymes by the macrophages in the KO skin (Figure IK, Figure IL).

Example 2: Study to demonstrate that psoriatic skin phenocopies pl KO skin metabolically

To demonstrate that psoriatic skin phenocopies [31 KO skin metabolically, the skin isolated from imiquimod induced mice model (C57BL/6JNcbs [2019] strain at the Animal Care and Resource centre, INSTEM, Bangalore) stained with GLUT1 and MCT4. The experimental results demonstrated increase in psoriatic skin phenocopies illustrating that psoriatic epidermis indeed augments glycolysis and can lead to lactate generation and release (Figure 2A). To demonstrate that inhibiting lactate release from epidermis lead to loss lactate mediated crosstalk between the two compartments and targeting this pathway lead to inhibition of psoriatic phenotype, further experiments were conducted. To inhibit lactate transport, a 5-day IP injection of syrosingopine alongside imiquimod treatment was conducted. Surprisingly considerable reduction in plaquing of the backskin was noticed (Figure 2C) alongside reduction in epidermal area and thickness (Figure 2D). The inventors conducted experiments to study if syrosingopine reduce immune cell burden. Stained DMSO control and syrosingopine treated back skin with monocyte (CD11B), macrophage (F4/80), T cells (CD4), and neutrophils (Gr-1) were observed. Surprisingly a remarkable decrease in the immune cell burden in skin was observed (Figure 2E). Finally, to establish that syrosingopine treatment leads to a decrease in MMP expression in skin further experiments were conducted which demonstrated dramatic decrease in the MMP expression (Figure 2F).

Example 3: Study of metabolic states of embryonic skin epidermis, fibroblasts and resident macrophages in homeostasis and inflammation and metabolic crosstalk of macrophages in homeostasis and inflammation with their niche

To understand crosstalk between different skin compartments in WT (wild type) and KO (Knockout -inflamed) in integrin pi KO embryonic skin, NGS analysis of epidermis, FACS sorted fibroblasts (CD45- cells in dermis) and macrophages (F4/80+ cells in dermis) was conducted (Figure 3a). Interestingly, epidermal signatures mainly comprised of wide range of inflammatory cytokines, chemokines and damage associated molecular patterns (DAMPs) indicating that epidermis acquires a pro-inflammatory role and contributes towards inflammation by recruiting and activating more immune cells from circulation (Figure 3b). On the other hand, the resident and recruited macrophages overexpress a wide range of markers associated with pro-remodelling M2 fate acquisition (Figure 3c). In line with this, macrophages also express a wide range of matrix metalloproteinases (MMP), which are enzymes capable of digesting ECM proteins, and ECM molecules itself such as collagens (COL) and fibronectins (FN) (Figure 3d, Figure 3e). The data indicated that macrophages in Itg i KO skin acquire an M2/ pro-remodelling phenotype. Indeed, removal of macrophages from the skin reduced MMP9, collagen 1 and fibronectin expression (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005) which leads to reduced disorganization of ECM (Figure 3f, Figure 3g). Interestingly, no significant difference between the WT and KO fibroblasts was observed which indicate that mice embryonic skin fibroblasts are relatively quiescent to inflammatory cues (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005). Overall, the NGS data showed that during inflammation, the epidermal keratinocytes and dermal macrophages acquire distinct functional states. In vitro pro-remodelling macrophages have been shown to be CD206+, ARG1+ and RELMA+ and hence the expression of CD206, ARG1 and RELMA in both WT and KO skin was observed. All F4/80+ macrophages were CD206+ARG1+RELMA+ in both WT and KO, however, the expression of ARG1 and RELMA was enhanced in the KO skin (Bhattacharjee et al., 2021. https://doi.org/10.3389/fimmu.2021.718005).

Conclusions: The above conduced experiments allowed inventors to make two important conclusions that macrophages during the course of skin development maintain a ground M2 state that must be required to (1) facilitate a pro-remodelling environment for skin appendage development and (2) maintain an anti-inflammatory state as racking up an inflammatory response will be restrictive to the growth of the organism. In the itgpi KO, however, they have an enhanced M2 marker expression which is associated with excess remodelling. This state is different from WT skin resident macrophages which have a basal level remodelling phenotype and thus the M2 macrophages in itgpi KO skin are addressed as alternatively activated pro-remodelling macrophages (M2a) from here onwards in the disclosure.

Example 4: Study of epidermis and macrophages in pl KO skin

Metabolic pathways support and facilitate fate acquisition in cells. Therefore, the inventors conducted experiments to demonstrate that epidermis and macrophages in pl KO skin are associated with distinct metabolic states. The pl KO skin is B6;129- ItgbltmlEfu/JNcbs strain from the Animal Care and Resource Centre, INSTEM, Bangalore. The metabolic modulations in the epidermis and macrophages accompany the pro- inflammatory and pro-remodelling fate acquisition, respectively. The inventors employed the NGS data analysis to understand possible metabolic pathways in both the compartments. Interestingly, within the epidermal compartment several pathways associated with carbohydrate, glucose and lipid metabolism were upregulated (Figure 4a). Upon analysis, it was noticed that the genes were primarily involved in driving the glycolytic and fatty acid oxidation pathway. qPCR analysis of enzymes involved in glycolytic and fatty acid oxidation pathways and immunostaining of skin with GLUT1 (glucose transporter), LDHa, nile red demonstrated remarkable increase in glycolysis and lipid oxidation pathway in epidermis (Figure 4b, Figure 4c, Figure 4d, Figure 5). The TCA cycle metabolic pathways were also probed. Interestingly, there was a decrease in transcript levels of enzymes associated with the TCA cycle demonstrating a decrease in utilization of TCA-OXPHOS for energy and metabolic intermediate generation in epidermis (Figure 4e). A decrease in the transporters and enzymes involved in Glutaminolysis pathway was noticed which demonstrate reduced compensation of Glutaminolysis pathway towards TCA cycle (Figure 5).

Interestingly, on the other hand, the NGS data from the macrophages in itg [31 KO skin showed downregulation of glucose transporters, and enzymes catalysing glycolysis, glutaminolysis and fatty acid oxidation (Figure 6). However, no change in the enzyme catalysing the TCA cycle was observed, except IDH1 (necessary for alpha-KG synthesis) which was upregulated, which demonstrate that the TCA cycle is intact. Additionally, upregulation of PDP1 gene was observed which is necessary for activating PDH enzyme required for acetyl-CoA synthesis. Taken together, macrophages seem to increase flux into the TCA cycle. This is in line with certain reports on M2 polarized macrophages in vitro that have indicated they have an intact TCA cycle, which is otherwise broken in Ml macrophages. In line with the observation the total lipid content in macrophages was fairly reduced in the KO macrophages compared to the WT.

Example 5: In vivo study of macrophages to understand metabolic states

To establish in vivo (B6;129-ItgbltmlEfu/JNcbs mice model) metabolic states of macrophages, the inventors stained the macrophages with enzymes associated with TCA cycle (IDH1, OGDH), glycolysis (HK2, PFK2) and lipid oxidation (CPTla). 2NBDG injection was employed to show reduced uptake of glucose in KO macrophages and increased uptake in epidermis.

Example 6: Oxidative stress induced HIF-la activation and stabilization augment the metabolic changes observed in epidermis of itgpi KO skin

The inventors conducted experiments to understand what is causing augmentation of glycolytic response in the itgpi KO epidermis. Glycolysis pathway has been shown to be transcriptionally under the control of HIFla, P53, OCTI, and MYC signalling, therefore the NGS data was analysed to detect any changes in the gene expression. Interestingly, the NGS data reflected an increase in the transcription of HIF-la while there was no change in the expression of other factors (Figure 7a). Additionally, NGS analysis using KEGG (kyoto encyclopedia of genes and genomes) and PANTHER (protein annotation through evolutionary relationship) suggested increase in pathways involving HIFla such as 'response to hypoxia' and 'HIF signalling pathway' (Figure 7b). The NGS results were validated by using immunostaining. An increase in expression and nuclear localization of HIFla in epidermis of itgPl KO was observed (Figure 7c, Figure 7d). The HIFla activation was further validated by checking for classical genes downstream of HIF signalling pathway. An increase in HIF target genes was observed in epidermis, such as krtl4, vegfa, timpl, ptgs2 and fnl (Figure 7e). Immunostaining data was used to validate this by showing increase protein expression of KRT14, PTGS2 and FN1 (Figure 7f).

The inventors treated pregnant dams mouse models (B6;129-ItgbltmlEfu/JNcbs mice strain available with the Animal Care and Resource Centre at INSTEM, Bangalore) with HIFla translation inhibitor to demonstrate that HIFla augmentation leads to overexpression of glycolytic genes, YC-1. The skin was stained with KRT14 and FN1 for confirming a definite inhibition. A decrease in the expression of the KRT14 and FN1 was observed (Figure 8a). To study if there is a decrease in the glycolytic gene expression, immunostaining was performed. A decrease in expression of GLUT1 and LDHa was observed (Figure 8b).

Conclusions: The experimental data of the studies conducted demonstrate that the augmentation of the glycolytic response was primarily due to HIFla stabilization.

Example 7: Study for mechanism of HIFla activation and stabilization in the skin

The inventors conducted experiments to understand mechanism of HIFla activation and stabilization in the skin. Apart from hypoxia, reactive oxygen species (ROS) has been shown to stabilize HIFla by unknown mechanisms. Interestingly, ROS has been shown to be generated in pi KO keratinocytes and loss of ROS scavengers due to ECM detachment has been shown to be a possible mechanism. Several pathways associated with ROS generation getting upregulated in the KO epidermis NGS data were observed (Figure 8c). This demonstrates that epidermal detachment due to loss of itgPl might lead to generation of ROS. The upregulated genes primarily comprised of both ROS generators and scavengers (Figure 8d). DHE staining was conducted developmentally at E16.5, 17.5 and E18.5. Interestingly, initial increase in ROS species in the KO from E16.5 to El 7.5 followed by reduction in E18.5 was observed (Figure 8e). The reduction in E18.5 is due to scavenging of ROS species by glycolysis flux towards pentose phosphate pathway which generates NADPH and glutathione ROS scavengers.

Example 8: Study to demonstrate that loss of HIF driven glycolysis and TCA cycle in macrophages rescue ECM disruption in itgpi KO skin

In the above conducted experiments, the inventors have demonstrated that loss of itgPl expression in skin leads to ECM degradation phenotype. The ECM degradation is a result of excess remodelling by skin resident and recruited macrophages. Consistently, depletion of macrophages using CSF1R antibody leads to reduction in the ECM degradation phenotype. Additional experiments were conducted to demonstrate that inhibition of epidermal or macrophage metabolic state leads to abrogation of the attainment of said functional states in both the compartments.

In the previous conducted experiments mentioned above the inventors have demonstrated that epidermis acquires a pronounced glycolytic state whereas the resident macrophages downregulate glycolysis and activate TCA cycle in the absence of itgbl. Therefore, glycolysis and TCA cycle were inhibited globally using 2-deoxy D-glucose (2DG) (500mg/kg), pyromellitic acid (lOmg/kg) and UK-5099 (lOmg/kg). Epidermis primarily expresses glucose transporters and drives glycolysis and macrophages drive TCA. Thus, global inhibition of glycolysis and TCA will specifically block glycolysis in KO epidermis and macrophages in the dermis, respectively. Interestingly, a reduction in the amount of ECM degradation in 2DG, PA and UK-5099 treated embryonic skin was observed (Figure 9a). Since both the compartments also expressed several markers associated with lipid metabolism, lipid metabolism was inhibited by using etomoxir (50mg/kg and lOOmg/kg) (Figure 10). Interestingly, no reduction was observed in ECM degradation which was further accompanied with no reduction in MMP9 expression in etomoxir treated skin (Figure 10g, lOh, lOi, lOj, 10k). Furthermore, there was no reduction in the expression of M2 marker CD206 in KO skin treated with etomoxir (Figure 10c, lOd). ECM degradation in 2DG, PA and UK-5099 treated sets also lead to a remarkable reduction in MMP9 staining and MMP activity in the back skin of 2DG, PA and UK-5099, but not etomoxir treated skin (Figure 9a). Furthermore, reduction in arginase 1 expression in macrophages in 2DG and PA treated skin was observed (Figure 9b). Since epidermal metabolic state is driven by HIFla, further experiment was conducted to confirm if HIF inhibition also leads to a rescue in the ECM phenotype. A reduction in the ECM degradation in YC-1 treated skin was observed (Figure 9c). Overall, perturbing epidermal and macrophage metabolic states are sufficient to override MMP activity and resultant ECM degradation in [31 KO skin.

To understand whether a particular metabolic pathway could be associated with MMP production and activity, the inventors analysed back skin obtained from 2DG, PA, UK-5099, and etomoxir treated WT skin. Contrary to KO skin, an upregulation of MMP9 staining in WT 2DG treated skin was observed while there was further reduction in MMP9 expression in PA treated skin overall demonstrating that TCA cycle was specifically associated with MMP generation and activity (Figure 11a).

Example 9: Study of association of TCA cycle with MMP generation and staining the skin with nile red for studying changes in skin barrier

Since downregulated glycolysis was specifically associated with macrophage compartment, it was concluded that recapitulating KO macrophage phenotype, specifically, glycolysis loss was sufficient to increase MMP generation from macrophages. A positive correlation between TCA cycle and MMP synthesis was established. The association of TCA cycle and MMP synthesis is not known in the field of art and has a potential for therapeutic target in sterile inflammatory conditions such as cancer, fibrosis, arthritis etc. Barrier assay was performed to indicate whether treatment with metabolic inhibitors cause barrier dysfunction with all the metabolic pathway treatments (Figure lib, Figure 11c). Metabolic pathway inhibitors do no cause barrier defects as nile red staining clearly shows presence of lipid rich flattened cornified envelope (Figure lib). Interestingly, the barrier is intact despite reduction in weights in the animals in the metabolic treatments (Figure lid). This indicates that metabolic pathways are dispensable for barrier dysfunction. This can be due to augmentation of compensatory pathways which fulfil barrier requirements. Example 10: Analysis of macrophage polarization states in KO with different metabolic treatments

Experiments were conducted to study effect of macrophage and epidermis specific deletion of metabolic pathways and to study epidermally derived lactate feed TCA cycle in macrophages in [31 KO skin. Macrophage polarization in the KO skin was visualized by staining the skin with MMP9.

The inventors have previously established (as shown in Figure 9) that loss of epidermal metabolic state (glycolysis) leads to abrogation of macrophage phenotype in the dermis. Further experiments were conducted to understand how these two compartments in the skin might be crosstalking during inflammation.

Several published papers (Rabinowitz, J.D., Enerback, S. Lactate: The ugly duckling of energy metabolism. Nat Metab 2, 566-571 (2020). https://doi.org/10.1038/s42255-020-0243- 4) have reported that glycolysis essentially culminates into the generation and secretion of lactate. Additionally, in certain cancer model systems, lactate derived from cancer has been shown to polarize macrophages into a tumour promoting pro-remodelling macrophages. Therefore, to confirm if lactate is generated and released from epidermis which drives macrophage pro-remodelling state, the lactate concentration in epidermis taken from WT and KO embryonic skin was quantified. Higher lactate concentrations in KO epidermis was observed when compared with WT littermate controls (Figure 12a). Lactate exporters MCT2 and MCT4 have been shown to release lactate under conditions of high lactate concentration in cells to escape deleterious effects of high acidic environment in cells (https://doi.Org/10.1016/j.molmet.2019.07.006). Therefore, expression of MCT2 and MCT4 in NGS data was observed from epidermis and found MCT4 to be specifically upregulated which was validated (Figure 12b). Cell membrane localization of lactate transporter MCT4 was observed by immunostaining in the KO skin demonstrating that it is capable of releasing lactate upon accumulation (Figure 12c).

Example 11: Staining skin with MCT1 to study if macrophages express lactate importers

Interestingly, upregulation of several lactate transporters in macrophages in NGS data was observed (Figure 12d). This indicates that the lactate in the microenvironment can be imported by macrophages. Using immunostaining of lactate importer MCT1 it was demonstrated that macrophages can uptake lactate from their microenvironment. In order to understand if the two compartments are crosstalking using lactate, MCT4 and MCT1 in skin were inhibited. Furthermore, inhibition of lactate transport between epidermis and macrophages using syrosingopine led to reduction in the expression of MMP9 which was associated with reduction in basement membrane disruption (Figure 12e).

Example 12: Abrogation of epidermal-macrophage lactate crosstalk decreases severity of psoriasis

Experiments were conducted to study inhibition of MCT4 using syrosingopine and MCT1 using 4 hydroxy cinnamate. The experiments were conducted to study if prevention of lactate mediated crosstalk between two compartments abrogates [31 KO phenotype in the skin.

In integrin pi KO skin, under conditions of inflammation, epidermis acquires a pro- inflammatory role which is supported by augmentation of glycolysis which leads to the generation of lactate. Lactate is released and taken up by macrophages which use it to drive TCA cycle, which in turn supports acquisition of pro-ECM remodelling state in macrophages.

To study whether this crosstalk between the two compartments can be used as a target for drug therapy for inflammatory skin diseases, hyperproliferative epidermis was used. Since hyper-proliferative cells are associated with glycolysis and provide a rapid source for ATP and metabolites for lipids, amino acids and nucleotides. Additionally, hyperproliferative epidermis in psoriatic lesions is related with enhanced glycolysis. Therefore, experiments were conducted to study if psoriatic skin phenotype phenocopies pi KO skin phenotype. In mice, psoriasis is typically induced by topical application of imiquimod (doi: 10.4049/jimmunol.0802999.). Upon application of imiquimod and control skin with vaseline for about 5 days, the mice (C57BL/6JNcbs [2019] strain mice available at the Animal Care and Resource Center (ACRC) at INSTEM Bangalore) were successfully induced with psoriasis as shown by appearance of plaque lesions and increased skin redness (Figure 13a). Histological analysis of the skin demonstrates epidermal thickening, hyperproliferation and increased epidermal area (Figure 13b). As psoriatic skin is associated with increased immune cell burden, several immune cells such as T cells (CD4+, CD8+), neutrophils (Grl+), macrophages (F4/80+) and monocytes (GDI IB) were observed and found them to be substantially increased (Figure 13c). To understand if indeed macrophages were proremodelling in phenotype, the inventors stained the skin with MMP9 and found a dramatic increase in MMP9+ macrophages in the skin suggesting that macrophages are indeed proremodelling in polarization state (Figure 13d).

To understand if psoriatic skin phenocopies pi KO skin metabolically, the inventors stained the skin with GLUT1 and MCT4 and found it to be increased in the psoriatic skin suggesting that psoriatic epidermis indeed augments glycolysis and might lead to lactate generation and release (Figure 14a). To understand, if inhibiting lactate release from epidermis would lead to loss lactate mediated crosstalk between the two compartments and whether targeting this pathway would lead to inhibition of psoriatic phenotype, the inventors did 5-day IP injection of syrosingopine, alongside imiquimod treatment. Interestingly, considerable reduction in plaquing of the backskin was observed (Figure 14b) alongside reduction in epidermal area and thickness (Figure 14c). To demonstrate that syrosingopine reduce immune cell burden, DMSO control and syrosingopine treated back skin were stained with monocyte (GDI IB), macrophage (F4/80), T cells (CD4), and neutrophils (Gr-1). Indeed, a remarkable decrease in the immune cell burden in skin was observed (Figure 14d). Finally, a dramatic decrease in the MMP expression indicated that syrosingopine treatment led to a decrease in MMP expression in skin (Figure 14e).

Overall, in the pl KO and psoriatic skin the epidermal compartment augments glycolysis and leads to release of lactate in the dermis, which in turn is taken up by tissue resident and recruited macrophages. Macrophages utilize lactate to drive TCA cycle is absolutely necessary to generate MMP molecules which are required for ECM remodelling (Figure 15).

Example 13: Sterile inflammation in embryonic skin is associated with increased glycolysis in the epidermis

As reported previously, loss of integrin betal (itgpl) from the epidermal compartment in mouse embryonic skin, leads to augmentation of an inflammatory response characterized by increased macrophage cell infiltration and pro-remodeling fate acquisition (Kurbet, A. S. et al., 2016 and Bhattacharjee, O. et al., 2021). To gain insights into the metabolic states of macrophages and its niche cells, the inventors of present invention employed previously generated NGS data from the epidermis, macrophages and fibroblasts of WT and itg|31cKO embryonic skin and identified differentially expressed metabolic pathways in cKO epidermis (Bhattacharjee, O. et al., 2021). Pathway analysis and qPCR validation suggested an increase in expression of genes associated with the glucose uptake and glycolysis metabolism in the cKO epidermis (Figure 16A to Figure 16C). Immunostaining analysis of the cKO skin with GLUT1 (Glucose transporter 1) and LDHa (Lactate dehydrogenase A) and western blot analysis of HK2 (Hexokinase 2) further corroborated these results (Figure 17A to Figure 17D). Notably, temporal analysis of cKO epidermis suggested increase in membrane GLUT1 expression at E18.5 (Figure 16D).

Glycolytic end products pyruvate and lactate feed directly into the tri-carboxylic acid cycle (TCA cycle) as part of central carbon respiration chain. The expression of TCA cycle genes in the epidermal compartment from WT and cKO epidermis was examined. Interestingly, NGS and qPCR analysis suggested a general reduction in all the TCA cycle enzyme transcripts in the cKO epidermis (Figure 16E and Figure 16F). IDH1 (Isocitrate dehydrogenase 1) and CS (Citrate Synthase) immunostaining suggested a similar reduction in the TCA cycle enzyme expression cKO epidermis (Figure 17G to Figure 171). Furthermore, steady state metabolomics confirmed increase in glycolytic intermediate metabolites and glycolysis end product lactate, and decrease in TCA cycle metabolites in E18.5 cKO epidermis compared to WT (Figure 17E, Figure 17F and Figure 17J). Taken together, the results indicate augmentation of glycolysis that leads to enhanced lactate generation in the cKO the epidermal compartment (Figure 17K).

Example 14: ROS mediated HIF1 stabilization augments glycolysis in the embryonic skin epidermis during sterile inflammation

The inventors conducted further studies to identify the molecular events that initiated the metabolic reprogramming in the cKO epidermal compartment. Hypoxia inducible factor (HIFla) regulates glycolysis under both hypoxic and normoxic conditions (McGettrick, A. F. and O'Neill, L. A. J., 2020. The Role of HIF in Immunity and Inflammation. Cell Metabolism vol. 32). Notably, the NGS analysis suggested enrichment in pathways associated with response to hypoxia (Figure 18A). Consistent with this, an increase in the expression of HIFla and its downstream targets in the cKO epidermis compared to the WT (Figure 19A and 19B, Figure 18B, Figure 18C, Figure 18E) is observed. Temporal HIFla expression analysis suggested augmentation of HIFla expression in the KO skin as early as embryonic day E17.5 (Figure 19 A).

To ensure if increased HIFla expression is directly associated with glycolytic upregulation in the epidermal compartment, pregnant dams harbouring WT and cKO embryos were treated with inhibitor of HIFla dependent transcription, chetomin (3mg/kg) (Kung, A. L. et al., 2004. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell 6) (Figure 18G). The inhibition of HIFla using chetomin not only reduced the expression of HIFla targets - COX2 and KRT14, but also the expression of GLUT1 and LDHa in the in the cKO epidermis compared to DMSO treated cKO controls (Figures 19C to 19E. Figure 18D, Figure 18F). These results suggested that glycolytic programming in the epidermal compartment is augmented due to enhanced epidermal HIFla expression and stabilization.

In order to identify the mechanism of HIFla stabilization in the cKO epidermis further studies were conducted. Several studies have identified a role for reactive oxygen species (ROS) generated during trauma induced injury and tumors in stabilizing HIFla response (Mittal, M., et al., 2014. Reactive oxygen species in inflammation and tissue injury. Antioxidants and Redox Signaling vol. 20; Dunnill, C. et al., 2017. Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int. Wound J. 14). Notably, GSEA analysis of the cKO epidermis suggested enrichment in pathways associated with an active response to increased ROS (Figure 20A). Temporal analysis of cKO and control skin using 8- OHdG (ROS indicator), suggested increase in ROS expression at embryonic day E16.5 cKO epidermis that quenches by E18.5 (Figures 19F, 19G, Figure 20B). It was concluded that ROS burden in the KO skin might be counterbalanced by increased expression of ROS scavengers in the cKO epidermis. Consistent with this, an increase in the expression of ROS scavengers in E18.5 cKO epidemis (Figure 20C) was observed and validated. This suggested that the upregulation of ROS in the cKO epidermis could be an early response to the loss of epidermal [31 which is eventually quenched through upregulation of ROS scavengers. Further studies were conducted in order to identify possible sources of ROS in the epidermal compartment. qPCR analysis of different ROS sources suggested a global increase in genes associated with ROS generation through both oxidative and non-oxidative mechanisms (Figure 20C). This increase can be attributed to enhanced epidermal stress due to detachment from the extracellular matrix (ECM), the basement membrane. Notably, ROS increase upon ECM detachment has been reported previously in cancer cells during metastasis (Mason, J. A. et al., 2016. Oncogenic Ras differentially regulates metabolism and anoikis in extracellular matrix-detached cells. Cell Death Differ. 23; Davison, C. A. et al., 2013. Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix. Cancer Res. 73; Schafer, Z. T. et al., 2009. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461).

Since temporal analysis suggested that ROS augmentation precedes HIFla stabilization, it was concluded that ROS might be the potential activator for HIFla signalling in the cKO epidermis. To address this, pregnant dams harbouring WT and cKO embryos were treated with the ROS scavenger, N-acetyl cysteine (NAC) (Zafarullah, M. et al., 2003. Molecular mechanisms of N-acetylcysteine actions. Cellular and Molecular Life Sciences vol. 60) (Figure 18G). Interestingly, NAC treatment led to reduced expression of 8-OHdG and HIFla in the cKO epidermal compartment compared to the PBS controls (Figures 20D, 20F). Immunostaining analysis further suggested a significant reduction in expression of HIFla targets - KRT14, COX2, GLUT1 and LDHa in the cKO epidermis treated with NAC compared to the controls (Figures 19H, 191, 19J and Figures 20E, 20G). The temporal analysis combined with pathway inhibition results suggested that an early ROS-HIFla axis augments glycolytic metabolism in the cKO epidermis (Figure 19K).

Example 15: Macrophages are potential sinks for lactate during sterile inflammation in embryonic skin

As described previously, enhanced glycolysis in the epidermal compartment led to lactate accumulation. Further studies were conducted in order to understand the fate of epidermally generated lactate. Recent in vitro studies suggested that increase in lactate accumulation within the cells lead to increased membrane localization of lactate exporter MCT4 (monocarboxylic acid transporter 4) (Benjamin, D. et al., 2018. Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells. Cell Rep. 25; Kirk, P. et al., 2000. CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J. 19; Miranda-Goncalves, V. et al., 2013. Monocarboxylate transporters (MCTs) in gliomas: Expression and exploitation as therapeutic targets. Neuro. Oncol. 15). Immunostaining of the cKO epidermis at El 7.5 and E18.5 showed an increase in the membrane expression of MCT4 transporters (Figure 21A). These data, in conjunction with metabolomics (Figure 17E), suggest enhanced lactate release from the cKO epidermal compartment.

It was therefore concluded that the dermal fibroblasts and/or macrophages may serve as potential 'sinks' for epidermally derived lactate during sterile inflammation. NGS analysis of the macrophage compartment suggested an increase in the pathways associated with glucose deprivation (Figure 22A, Figure 22B). This suggested a reduction in the glucose dependent metabolic program in the macrophages in the cKO skin. Notably, qPCR validation of the glycolytic genes from sorted macrophage population suggested no change in the genes associated with the glycolytic pathway (Figure 22C). The macrophages in the cKO skin did not express GLUT1 (Figure 22D). Interestingly, it was observed and subsequently validated that there is an increase in the genes associated with the TCA cycle in macrophages isolated from KO skin compared to the controls (Figure 22B, Figure 22E). Immunostaining the F4/80+ve macrophages from the WT and itg|31 cKO skin with TCA cycle enzymes - CS (citrate synthase) and IDH1 (Isocitrate Dehydrogenase 1) further corroborated the qPCR and NGS data (Figure 21B, Figure 21C).

Unlike the epidermal and macrophage compartment, no change was observed in the expression of enzymes associated with TCA cycle in CD45-ve fibroblasts (Figure 23A). This suggested that macrophages in the dermal compartment might be the key drivers of the TCA cycle. Notably, metabolomic analysis of the dermal compartment suggested a decrease in glycolysis metabolites and increase in TCA cycle metabolites. (Figure 21D, Figure 21E). Taken together, our analysis suggested that macrophages reduce dependence on glycolysis and instead augment TCA cycle in the KO skin. In addition, our results suggest a clear compartment-specific differentiation in glycolysis versus TCA cycle activity between the epidermis and macrophages, respectively, in the inflamed cKO skin (Figure 21F). It was concluded that since macrophages have decreasing dependence on the glycolytic metabolic pathway, they can utilize epidermally derived lactate as an alternative fuel to drive the TCA cycle (Figure 24F) (Hui, S. et al., 2017. Glucose feeds the TCA cycle via circulating lactate. Nature 551; Faubert, B. et al., 2017. Lactate Metabolism in Human Lung Tumors. Cell 171). Immunostaining analysis suggested increased membrane localization of lactate importer, MCT1, in the macrophage compartment (Figure 21G and Figure 21H). This suggested that macrophages can act as potential sinks for lactate which in turn can be utilized to drive TCA in the macrophage compartment. Notably, membrane expression of MCT1 correlated with increased generation of MMP9 and basement membrane disruption (LAM332) in the KO skin underpinning a possible role for epidermally derived lactate in driving macrophage polarization during sterile inflammation (Figure 21G, Figure 211 and Figure 21J).

Since both the epidermis and macrophage compartments in the itg[31 cKO express lactate transporters, it was concluded that epidermally derived lactate might be sufficient to drive macrophage metabolic program and in turn its effector function. To test this, the inventors of present invention treated dams harbouring WT and cKO embryos with the MCT1 inhibitor, AZD396524 (Figure 18G). Interestingly, inhibition of lactate uptake by dermal macrophages in itg|31 cKO animals led to a significant decrease in the expression of MMP9 which further resulted in a remarkable reduction in ECM degradation compared to DMSO treated controls (Figure 24A to Figure 24C). Additionally, inhibition of MCT4 (expressed in epidermis) using the MCT1/4 blocker, syrosingopine (SYRO) (Benjamin, D. et al., 2018. Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells. Cell Rep. 25) with the identical treatment paradigm resulted in a similar reduction in the MMP9 generation and ECM degradation compared to the controls (Figure 24A, Figure 24D, Figure 24E). These results underpin an important role for epidermal derived lactate in driving macrophage effector function during sterile inflammation in the KO skin. These results can further be extrapolated to sterile inflammatory disorders where increased lactate and MMP levels are associated with exacerbation of the diseased condition. Example 16: Inhibition of lactate transport from epidermis to macrophages inhibits sterile inflammation and psoriatic skin disease

Further studies were conducted to ensure if the augmentation of the observed metabolic pathway in the macrophages was sufficient to cause basement membrane disruption in the KO skin. To address this, pregnant dams were treated with the small molecule inhibitors of the TCA cycle, pyromellitic acid (PA) (Polanski, R. et al., 2014. Activity of the monocarboxylate transporter 1 inhibitor azd3965 in small cell lung cancer. Clin. Cancer Res. 20), a fumarase inhibitor, and UK-5099 (Halestrap, A. P., 1975. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem. J. 148), a pyruvate uptake inhibitor (Figure 18G). Interestingly, treatment with PA and UK-5099 lead to a significant reduction in the basement membrane disruption and MMP9 expression in KO skin compared to the controls (Figure 25A to Figure 25F). These results suggested that perturbation of macrophage intrinsic metabolic program was sufficient to inhibit macrophages effector function viz. MMP9 synthesis and concomitant basement membrane disruption itg|31 cKO skin. Additionally a positive correlation between lactate uptake and augmenting TCA cycle by macrophages is established in driving macrophage effector function.

To further strengthen this, the inventors of present invention treated pregnant dams with glycolysis inhibitor, 2DG (2 deoxy D glucose) (Laszlo, J., Humphreys, S. R. and Goldin, A., 1960. Effects of glucose analogues (2-deoxy-d-glucose, 2-deoxy-d-galactose) on experimental tumors. J. Natl. Cancer Inst. 24) using identical treatment paradigm. It was concluded that since lactate transport inhibition led to reduction in macrophage proremodelling fate acquisition in the KO skin, inhibition of lactate source i.e., glycolysis in the epidermal compartment, should have similar consequences. Remarkably, blocking glycolysis with 2DG lead to a significant reduction in the generation of MMP9 and basement membrane disruption by macrophages (Figure 25A to Figure 25C). On the other hand, inhibition of fatty acid metabolism, which is primarily augmented by the fibroblast compartment using etomoxir (Zhou, Y. P. and Grill, V. E., 1994. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J. Clin. Invest. 93) did not lead to reduction in macrophage mediated ECM degradation (Figure 23B, Figure 23C). This suggested that the metabolic program augmented by the epidermal compartment, and not fibroblasts, have a direct consequence in regulating the effector functions of the dermal macrophages.

To test if inhibition of ROS and HIFla, the upstream regulators or glycolysis, led to similar macrophage phenotype further studies were conducted. It was observed that a significant reduction expression of MMP9 and extent of ECM disruption in NAC and chetomin treated KO skin compared to the controls (Figure 25G, Figure 25H, Figure 25J, Figure 25K).

Finally, to understand the therapeutic applicability of findings in the cKO skin more studies were conducted. Previous studies on imiquimod induced mice model of psoriasis suggested increase in glucose utilization, glycolytic intermediates, and lactate in the epidermal compartment (Zhang, Z. et al., 2018. Differential glucose requirement in skin homeostasis and injury identifies a therapeutic target for psoriasis article. Nat. Med. 24,). It was therefore hypothesized that lactate generated as a result of increased glycolysis in psoriatic epidermis might potentially drive psoriatic skin disease by augmenting fate changes in the macrophage compartment. Analysis of the mice skin post 5 days of imiquimod treatment suggested increased epidermal hyperproliferation and thickening which was concomitant with increased monocyte and macrophage burden (Figure 26A, Figure 26B, Figure 26C, Figure 26D). In addition, increased MMP9 expression was observed in the dermal compartment (Figure 26E, Figure 26F). Notably, increased hyper-proliferation in the epidermal compartment was associated with increased expression of GLUT1 and MCT4 (Figure 26G, Figure 261). This suggested that the epidermal compartment in the imiquimod induced mice model of psoriasis was a potential exporter of lactate. Additionally, macrophages show increased expression of TCA cycle enzymes, CS and IDH1 and lactate importer MCT1 (Figure 26H, Figure 26J). This suggested that macrophages in the psoriatic skin potentially import lactate to drive TCA cycle which, in turn is necessary for their pro-remodelling fate switch. To test whether inhibition of lactate transport between epidermis and macrophages prevents macrophage polarization and in turn, psoriasis development in imiquimod induced mice model of psoriasis a study was conducted. Treatment of mice with Syrosingopine lead to dramatic reduction in epidermal hyperproliferation, monocyte-macrophage burden and MMP9 expression in the psoriatic skin compared to the controls (Figure 24F, Figure 24G, Figure 24H, Figure 241, Figure 24J, Figure 24K). These results establish lactate mediated epidermal - macrophage crosstalk as an important driver of the psoriatic skin disease.

Overall, using small molecule metabolic pathway inhibition studies, it is shown that perturbation of lactate transportation between epidermal and macrophage compartment and pathways leading to lactate synthesis leads to inhibition of macrophage proremodelling fate acquisition, thereby enhancing damage and inflammation in the KO skin (Figure 241).

Discussion

In several skin disorders, the epidermal compartment has been shown to augment glycolytic program however, the functional consequence of this metabolic reprogramming is not well understood (Zhang, Z. et al., 2018. Differential glucose requirement in skin homeostasis and injury identifies a therapeutic target for psoriasis article. Nat. Med. 24; Choi, S. Y. et al., 2020. 2-deoxy-d-glucose ameliorates animal models of dermatitis. Biomedicines 8). Additionally, in sterile inflammatory diseases such as rheumatoid arthritis, while macrophages have been shown to be the associated with disease progression (Chen, G. Y. and Nunez, G., 2010. Sterile inflammation: Sensing and reacting to damage. Nature Reviews Immunology vol. 10) the key molecular events that drive macrophages activation is not well understood. The study of the present invention suggests that under such conditions, glycolytic cell types may influence macrophage activation through enhanced lactate generation and release. Hence, lactate metabolism may be an important target to treat sterile inflammatory diseases.

Increased uptake of lactate by macrophages is important to drive their functional stares. Interestingly, recent studies have suggested that extra-cellular lactate can act as source of carbons for generating acetyl-CoA that changes acetylation states in tumour associated macrophages31. In addition, TCA cycle metabolites can also be used as epigenetic adducts which can cause major changes in the transcriptional output of the immune cells (Martinez-Reyes, I. and Chandel, N. S., 2020. Mitochondrial TCA cycle metabolites control physiology and disease. Nature Communications vol. 11). It is speculated that lactate and TCA cycle intermediates might contribute to changes in the epigenetic landscape of macrophages that, in turn, may lead to enhanced MMP synthesis. The hypothesis needed further investigation.

Overall, the study of present invention identifies a lactate mediated crosstalk that drives sterile inflammation and psoriatic skin disease. The ability of lactate transport inhibitors to block the progression of psoriasis in mouse models provides an exciting avenue to identify additional "druggable" metabolic pathways to treat sterile inflammatory diseases.

Animal study

Integrin [31 cKO animals (mixed background) were generated by crossing ITG|31fl/+ 1 KRT14-Cre males with ITG|31fl/fl (C57B6J background) females. ITG|31fl/+ 1 KRT14-Cre males were generated by crossing KRT14-Cre homozygous males (GDI background) with ITG|31fl/fl (C57B6J background). Since integrin [31 cKO embryos are neonatally lethal, all the experiments reported in the current study have been done on embryos extracted from euthanized dams at specific embryonic stages.

Pregnant dams containing the KO and the WT embryos were housed at NCBS/inStem ACRC (Animal Care and Resource Centre) facility. Handling, breeding and euthanization of animals were done in accordance with the guidelines and procedures approved by the InStem IACUC (Institutional Animal Care and Use Committee). All experimental and breeder cages were maintained in SPF2 (Specific pathogen free 2) facility with standard ventilation, temperature (21 degree Celsius), 12-hour light and dark cycle, and sterilized food and water.

Example 17: Drug Treatments for pl KO animals

The pregnant dams containing the WT and integrin [31 cKO embryos were treated with small molecule inhibitors of specific metabolic pathways. All animals were treated for 3 days starting from E15.5. Embryos were extracted on E18.5 and analysed. In control experiments, pregnant dams were treated with the vehicles such as sterile PBS or 5% DMSO. The details of the drugs and treatment schedule are given in Table 1 below. Table 1: Details of the drugs and treatment schedule of pregnant dams containing WT and integrin [31 cKO embryos

Example 18: Imiquimod induced mice model for psoriasis C57B6/J mice back skin was shaved and about 12.5 mg of commercially available 5% imiquimod (Glenmark) was applied topically daily on the shaved back skin. Vaseline was used as control for the above experiment. After 5 days of daily imiquimod or Vaseline application animals were euthanized as per guidelines and procedures approved by the inStem IACUC (Institutional Animal Care and Use Committee). The back skin was collected for further analysis. All experimental and breeder cages were maintained in SPF2 (Specific pathogen free 2) facility with standard ventilation, temperature (21 degree Celsius), 12-hour light and dark cycle, and sterilized food and water.

Example 19: Drug Treatments for Imiquimod animal models

For lactate transport inhibition experiments about 12.5 mg of Imiquimod (Glenmark) was applied on the back skin of the mice induce psoriasis for a total of 5 days. From the third day onwards, animals were treated with intraperitoneal doses of Syrosingopine (SML-1908, SIGMA) at about 10 mg/kg concentration. The control animals were treated with 5% DMSO in sterile 1XPBS. Both male and female C57B6/J were used in these experiments. After 5 days, mice were euthanized as per guidelines and procedures approved by the inStem IACUC (Institutional Animal Care and Use Committee). The back skin was collected for further analysis. All experimental and breeder cages were maintained in SPF2 (Specific pathogen free 2) facility with standard ventilation, temperature (about 21 °C), 12-hour light and dark cycle, and sterilized food and water.

Example 20: Immunostaining

Embryos extracted from euthanized pregnant dams were frozen in tissue freezing media (OCT) and about 10-micron cryosections were collected on charged glass slides and stored in -80 ^aC. For immunostaining, cryosections were thawed in room temperature (RT) for about 5 minutes and fixed in acetone (Merck) for about 5 minutes at about -20 °C or about 4% paraformaldehyde (Sigma) at room temperature for about 10 minutes. Paraformaldehyde fixed sections were permeabilized using permeabilization solution - 1XPBS plus 0.2-0.5% Triton X-100 (Sigma) for about 10 minutes at RT. Fixed and permeabilized sections were blocked using about 5%NDS (normal donkey serum, Abeam) in permeabilization solution. This was followed by addition of primary antibodies diluted in block. Details of the antibody dilutions and catalogue information are given in Table 2 below. Primary antibody staining was carried overnight in about 4 °C or about 2 hours in RT. After washing with 1XPBS, secondary antibody staining was carried for about 45 minutes at RT. Nucleus was stained using 1XDAPI (Sigma). The slides were covered with Moviol (Sigma), mounted and sealed. All images were taken in FV3000 5 Laser confocal microscope. Table 2: Details and source of materials employed for conducting studies Western Blotting

Snap-frozen epidermis and dermis obtained from WT and KO skin were pulverized using sterilized pestles. Homogenized tissue was then suspended in RIPA lysis buffer containing lXprotease inhibitor cocktail. Protein extraction was facilitated using multiple freeze-thaw cycles followed by centrifugation at maximum speed for about 15 minutes at about 4 °C. Protein concentration in the supernatant was measured using BCA assay (Promega). All protein isolate concentrations were normalised using RIPA-PIC buffer. About 50 pg of protein was loaded onto PAGE (8%) and electrophoresed, and transferred onto PVDF membrane (BioRad). Blocking of the membrane was done using about 5%BSA (Sigma). Primary antibody staining was done overnight at about 4 °C. After washing with 0.1%TBST and secondary antibody (HRP conjugated) were added for about an hour at RT. Unbound secondary antibodies were washed using about 0.1%TBST and the blots were developed using ECL substrate (Thermo).

RNA Extraction and Real Time PCR

Total RNA was extracted using Trizol (Thermo) for epidermal tissue and Trizol LS (Thermo) for sorted fibroblast and macrophages. Visualization of RNA pellet was facilitated by using about 1 pL of Glycoblue (Thermo). Air-dried RNA was re-suspended in nuclease free water (Thermo). Equal amounts of RNA obtained from KO and control skin compartments were used to prepare cDNA by using SSIII RT cDNA synthesis kit (Invitrogen). SYBR green (2X) master mix (Invitrogen) was used for real time PCR. Delta Ct method was used to quantify the relative changes in the level of transcripts. 18S was used as an endogenous control. The list of primers used for preparing cDNA from RNA obtained from KO and control skin compartments are given in Table 3 below.

Table 3: Sequence of forward and reverse primers used for preparing cDNA from RNA obtained from KO and control skin compartments

Example 21: RNA Sequencing Analysis and data availability

RNA sequencing used in the report has been done previously (Bhattacharjee et al., 2020). The data sets obtained from the report are submitted in NCBI with reference ID:

SRP324814 (PRJNA739149). The data can additionally be accessed from the link: https://datavie .ncbi.nlm.nih. gov/object/PRJNA739149?reviewer=lklobv7agr78somt2toldvk jvu. Statistical Analysis

All the statistical analysis in the present invention was done in GraphPad Prism version 9.0.0. Two tailed students t-test has been performed across all graphs with multiple biological replicate.

The surprising, remarkable and novel citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders as set forth in the present application accurately describe the efficacy and utility of these inhibitors to restore healthy functioning in humans and treat the conditions and disorders in humans as identified and described in this patent application. Although the subject matter has been described herein with reference to certain preferred embodiments thereof, other embodiments are possible. For illustrative purpose, the citric acid cycle and lactate transport inhibitors for treatment and/or prevention of psoriasis, atopic dermatitis and epidermolysis bullosa have been specified in description. However, those skilled in the art would appreciate that scope of the invention would extend to use of other citric acid cycle and lactate transport inhibitors to treat and/or prevent other skin disorders known in the field of art. It will be obvious to those skilled in the art to make various changes, modifications and alterations to the invention described herein. To the extent that these various changes, modifications and alteration do not depart from scope of the present invention, they are intended to be encompassed therein.

Claims

49

We Claim:

1. A pharmaceutical formulation for treating and/or preventing skin disorders, said formulation comprising at least one of citric acid cycle, lactate transport inhibitor and one or more pharmaceutically acceptable excipients wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% % w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875%, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v.

2. The formulation as claimed in claim 1, wherein said citric acid cycle and lactate transport inhibitors are selected from the group consisting of 2-deoxy-D-glucose, pyromellitic acid, syrosingopine, UK-5099, etomoxir, chetomin, N-acetyl cysteine and AZD3965.

3. The formulation as claimed in claim 1 or 2, wherein the skin disorders are at least one of atopic dermatitis, psoriasis, epidermolysis bullosa, alopecia areata, acne, atopic dermatitis, hidradenitis suppurativa, ichthyosis, pachyonychia congenita, vitiligo, scleroderma, rosacea and pemphigus.

4. The formulation as claimed in any one of claim 1 to 3, wherein said one or more suitable pharmaceutically acceptable excipients are selected from the group consisting of suitable carriers, diluents, vehicles, disintegrant, swelling agent, antioxidant, buffer, bacteriostatic agent, emollient, emulsifier, plasticizer, penetration enhancer, preservative, cryoprotectant, neutralizer, fragrance additives, dispersants, surfactants, binders and lubricants.

5. The formulation as claimed in any one of claim 1 to 4, wherein said formulation is suitable for topical dosage form selected from the group consisting of rapid release, immediate-release or slow-release.

6. The formulation as claimed in any one of claim 1 to 4, wherein said formulation is suitable for topical, intravenous, subcutaneous, controlled release, delayed-release, a combination of immediate and controlled release, nano -encapsulation formulations, creme formulation, gel formulation or as ointments mode of administration. 50 The formulation as claimed in any one of claim 1 to 6, wherein the formulation prevents and/or treats one or more symptoms of psoriasis selected from hyperplasia, parakeratosis, red patches of skin covered with thick, silvery scales, small scaling spots, dry cracked skin that may bleed and/or itch, itching, burning or soreness, thickened pitted or ridged nails, swollen and stiff joints. A pharmaceutical combination for treating and/or preventing skin disorders, said combination comprising at least one of citric acid cycle, lactate transport inhibitor and one or more other active agent wherein said citric acid cycle inhibitor is present in 0.0001 to 15% w/v, said lactate transport inhibitor is present in 0.0001 to 15% w/v, preferably wherein said citric acid cycle inhibitor is present in 0.0125 to 1.875%, said lactate transport inhibitor is present in 0.0125 to 0.1875 % w/v. The combination as claimed in claim 8, wherein the skin disorders are at least one of atopic dermatitis, psoriasis, epidermolysis bullosa, alopecia areata, acne, atopic dermatitis, hidradenitis suppurativa, ichthyosis, pachyonychia congenita, vitiligo, scleroderma, rosacea and pemphigus. The combination as claimed in claim 8 or 9, wherein said citric acid cycle and lactate transport inhibitors are selected from the group consisting of 2-deoxy-D-glucose, pyromellitic acid, syrosingopine, UK-5099, etomoxir, chetomin, N-acetyl cysteine and AZD3965. The combination as claimed in any one of claim 8 to 10, wherein said other active agent is selected from the group consisting of antibiotics, antihistamines, steroids, fluoxetine and sertraline and tricyclic antidepressants. A method of preparing pharmaceutical formulation or combination for treating and/or preventing skin disorders comprising the steps: a) adding required quantity of at least one of citric acid cycle and lactate transport inhibitors at suitable conditions to one or more pharmaceutically acceptable excipients to obtain a mixture; b) subjecting the mixture obtained in step ‘a’ to suitable conditions to obtain the formulation or combination in desired dosage form. 51

13. Citric acid cycle and lactate transport inhibitors alone or in combination in an amount of from 0.01 gm/kg tolOOO gm/kg for treating and/or preventing skin disorders. 14. A method of treating and/or preventing skin disorders in an individual, comprising administering to the individual a formulation or a combination comprising citric acid cycle and lactate transport inhibitors in an amount of from 0.01 gm/kg tolOOO gm/kg.

15. Use of a formulation or combination comprising citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of said formulation or combination.

16. A kit comprising a pharmaceutical formulation and/or combination as claimed in any one of claims 1 to 11 and instructions for administration of the pharmaceutical formulation and/or combination to a subject in need of treatment and/or prevention of skin disorders.