WO2023105482A1 - Inhibiteurs du cycle de l'acide citrique et du transport du lactate pour la prévention et/ou le traitement de troubles cutanés - Google Patents

Inhibiteurs du cycle de l'acide citrique et du transport du lactate pour la prévention et/ou le traitement de troubles cutanés Download PDF

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WO2023105482A1
WO2023105482A1 PCT/IB2022/061996 IB2022061996W WO2023105482A1 WO 2023105482 A1 WO2023105482 A1 WO 2023105482A1 IB 2022061996 W IB2022061996 W IB 2022061996W WO 2023105482 A1 WO2023105482 A1 WO 2023105482A1
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skin
lactate
formulation
macrophages
citric acid
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PCT/IB2022/061996
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English (en)
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Uttkarsh AYYANGAR
Srikala RAGHAVAN
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Institute For Stem Cell Science And Regenerative Medicine (Instem)
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Priority to EP22903713.0A priority Critical patent/EP4444319A1/fr
Publication of WO2023105482A1 publication Critical patent/WO2023105482A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4995—Pyrazines or piperazines forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/548—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Citric acid cycle and lactate transport inhibitors for prevention and/or treatment of skin disorders
  • 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.
  • 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.
  • EB Epidermolysis bullosa
  • EB Epidermolysis bullosa
  • 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.
  • 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.
  • IL-4R interleukin-4 receptor
  • 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.
  • 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).
  • TCA tricarboxylic acid cycle
  • lactate transport inhibitors Some other possible uses of TCA (tricarboxylic acid cycle) cycle and/or lactate transport inhibitors have been studied in the literature.
  • 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.
  • 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.
  • 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.
  • 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).
  • Panther http://www.pantherdb.org/
  • NGS Next Generation Sequencing
  • 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.
  • FIGS 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.
  • FIGS 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.
  • FIG 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.
  • TCA-OXPHOS tricarboxylic acid- oxidative phosphorylation
  • 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 17 illustrates increased glycolysis and decreased TCA in epidermal compartment in [31 cKO skin.
  • Figure 17D illustrates western blot for Hexokinase 2 (hk2) in epidermal and dermal compartment of KO skin.
  • 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.
  • 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 19B illustrates immunostaining of HIFla in E18.5 cKO and WT skin.
  • 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-
  • Figure 21 illustrates epidermis is an exporter while macrophages are importers of lactate metabolite in [31 cKO skin.
  • 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 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 21G illustrates quantification of basement membrane (EAM332) degradation and Figure 21H illustrates MMP9 expression in cKO skin compared to WT.
  • 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 22D shows that expression of GLUT1 (red) does not colocalize with F4/80 (green) in dermal macrophages in bl cKO skin.
  • 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 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
  • Figure 24F illustrates schematic showing Syrosingopine dose schedule in imiquimod induced mice model for psoriasis.
  • Figure 24M illustrates model summarizing the key findings of the study.
  • 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.
  • Figure 25 shows that inhibition of epidermal ROS and HIFla leads to reduction in macrophage pro-remodelling fate acquisition.
  • Figure 25B illustrates quantification of basement membrane spread
  • 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
  • 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.
  • Figure 26 illustrates lactate mediated cross-talk in imiquimod induced psoriatic skin samples.
  • Figure 26B illustrates increase in epidermal thickness and number of proliferating epidermal cells (Ki67) in imiquimod treated mice compared to Vaseline treated controls.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the other active agent in the combination is selected from the group consisting of antibiotics, antihistamines, steroids, fluoxetine and sertraline and tricyclic antidepressants.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 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.
  • 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.
  • 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.
  • 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).
  • Example 2 Study to demonstrate that psoriatic skin phenocopies pl KO skin metabolically
  • 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
  • macrophages overexpress a wide range of markers associated with pro-remodelling M2 fate acquisition (Figure 3c).
  • MMP matrix metalloproteinases
  • COL collagens
  • FN fibronectins
  • Example 4 Study of epidermis and macrophages in pl KO skin
  • 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.
  • Example 5 In vivo study of macrophages to understand metabolic states
  • Example 6 Oxidative stress induced HIF-la activation and stabilization augment the metabolic changes observed in epidermis of itgpi KO skin
  • 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).
  • immunostaining was performed. A decrease in expression of GLUT1 and LDHa was observed ( Figure 8b).
  • Example 7 Study for mechanism of HIFla activation and stabilization in the skin
  • ROS reactive oxygen species
  • Example 8 Study to demonstrate that loss of HIF driven glycolysis and TCA cycle in macrophages rescue ECM disruption in itgpi KO skin
  • glycolysis and TCA cycle were inhibited globally using 2-deoxy D-glucose (2DG) (500mg/kg), pyromellitic acid (lOmg/kg) and UK-5099 (lOmg/kg).
  • 2DG 2-deoxy D-glucose
  • pyromellitic acid lOmg/kg
  • UK-5099 lOmg/kg
  • Epidermis primarily expresses glucose transporters and drives glycolysis and macrophages drive TCA.
  • global inhibition of glycolysis and TCA will specifically block glycolysis in KO epidermis and macrophages in the dermis, respectively.
  • Example 9 Study of association of TCA cycle with MMP generation and staining the skin with nile red for studying changes in skin barrier
  • Example 11 Staining skin with MCT1 to study if macrophages express lactate importers
  • Example 12 Abrogation of epidermal-macrophage lactate crosstalk decreases severity of psoriasis
  • 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.).
  • mice 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).
  • T cells CD4+, CD8+
  • neutrophils neutrophils
  • macrophages F4/80+
  • monocytes GDI IB
  • Example 13 Sterile inflammation in embryonic skin is associated with increased glycolysis in the epidermis
  • TCA cycle tri-carboxylic acid cycle
  • Example 14 ROS mediated HIF1 stabilization augments glycolysis in the embryonic skin epidermis during sterile inflammation
  • HIFla Hypoxia inducible factor
  • the NGS analysis suggested enrichment in pathways associated with response to hypoxia ( Figure 18A).
  • 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).
  • ROS reactive oxygen species
  • 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
  • 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.
  • 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).
  • 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).
  • Example 16 Inhibition of lactate transport from epidermis to macrophages inhibits sterile inflammation and psoriatic skin disease
  • 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.
  • psoriasis development in imiquimod induced mice model of psoriasis a study was conducted.
  • 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.
  • 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.
  • Integrin [31 cKO animals were generated by crossing ITG
  • 31fl/+ 1 KRT14-Cre males were generated by crossing KRT14-Cre homozygous males (GDI background) with ITG
  • 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.
  • SPF2 Specific pathogen free 2
  • 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.
  • SPF2 Specific pathogen free 2
  • mice 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.
  • SPF2 Specific pathogen free 2
  • 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 a C.
  • 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.
  • 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).
  • Table 3 Sequence of forward and reverse primers used for preparing cDNA from RNA obtained from KO and control skin compartments
  • 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:
  • 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.
  • the subject matter has been described herein with reference to certain preferred embodiments thereof, other embodiments are possible.
  • 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.

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Abstract

La présente invention concerne des inhibiteurs du cycle de l'acide citrique et du transport du lactate. L'invention concerne également des combinaisons ou des formulations comprenant des inhibiteurs du cycle de l'acide citrique et/ou du transport du lactate pour la prévention et/ou le traitement de troubles cutanés.
PCT/IB2022/061996 2021-12-10 2022-12-09 Inhibiteurs du cycle de l'acide citrique et du transport du lactate pour la prévention et/ou le traitement de troubles cutanés WO2023105482A1 (fr)

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