Metformin reverses scarring in ciciatricial alopecia and promotes hair growth

Abstract

Methods for treating cicatricial alopecia, including central centrifugal cicatricial alopecia (CCCA) by administering a low-dose metformin are disclosed.

Description

METFORMIN REVERSES SCARRING IN CICIATRICIAL ALOPECIAAND PROMOTES HAIR GROWTHCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 562,739, filed March 8, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Central Centrifugal Cicatricial Alopecia (CCCA) is a cicatricial, or scarring, alopecia that is associated with abnormal hair follicle fibrosis and an elevated risk of Type 2 diabetes. Current treatments target inflammation, but the underlying fibrotic processes remain unaddressed, rendering hair loss permanent in a majority of cases. There are, however, no FDA-approved treatments for scarring alopecia.SUMMARY

[0003] In some aspects, the presently disclosed subject matter provides a method for treating cicatricial alopecia in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of metformin to treat the cicatricial alopecia. In certain aspects, the cicatricial alopecia comprises central centrifugal cicatricial alopecia (CCCA). In particular aspects, the CCCA is refractory CCCA.

[0004] In some aspects, the metformin is administered orally. In certain aspects, the metformin is administered at a dosage of metformin of about 500 mg. In certain aspects, the metformin comprises an extended-release formulation of metformin. In certain aspects, the metformin comprises an immediate-release formulation of metformin.

[0005] In certain aspects, the metformin is administered once or twice daily. In certain aspects, the metformin is administered over a time period of at least six months.

[0006] In certain aspects, the method further comprises administering metformin with one or more additional therapeutic agents. In particular aspects, the one or more additional therapeutic agents are selected from a topical high-potency steroid, an intralesional steroid injection, an oral tetracycline, and topical or oral minoxidil.

[0007] In certain aspects, administering the metformin to the subject further promotes hair growth.

[0008] In certain aspects, administering the metformin upregulates pathways and genes associated with kcratinization, epidermis development, and hair cycle. In particular aspects, the genes associated with kcratinization, epidermis development, and hair cycle include one or more KRTAPs.

[0009] In certain aspects, administering the metformin downregulates pathways and genes associated with fibrosis. In particular aspects, the genes associated with fibrosis include MMP7 and COL6A1.

[0010] In certain aspects, the method further comprises reducing Thl7-inllammation, epithelial- mesenchymal transition pathway-expression, and fibrosis markers.

[0011] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0013] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0014] FIG. 1A, FIG. IB, and FIG. 1C show results before and after treatment with metformin, 500 mg, daily for six months or more. Patients at baseline with evidence of decreased density in areas of follicular dropout on the scalp, characteristic of central centrifugal cicatricial alopecia with improvement after metformin was added to treatment. Each patient was taking metformin treatment for at least 6 months between images without other changes to their regimen. FIG. 1 A, Patient 2, before treatment and eight months after treatment; FIG. IB, Patient 4, before treatment and six months after treatment; and FIG. 1C, Patient 8, before treatment and seven months after treatment.

[0015] FIG. 2A, FIG. 2B, and FIG. 2C show differential gene analysis of metformin in central centrifugal cicatricial alopecia. FIG. 2A, Heatmap of entire gene signature (n = 16 655) of patients before and after metformin treatment. Distinct gene differentiation patterns emerged in scalpbefore and after treatment. FIG. 2B, Heatmap subset of all differentially expressed genes, clustered using hierarchical clustering. FIG. 2C, Volcano plot displaying fold change and -log 10 P value for all genes. Differentially expressed genes are labeled.

[0016] FIG. 3A and FIG. 3B show gene set and pathway analysis of metformin in central cicatricial alopecia. FIG. 3A, Top upregulated and downregulated pathways enriched in gene set enrichment analysis. The blue line indicates P = .05. FIG. 3B, Gene set variation analysis (GSVA) scores before and after metformin treatment using 8 distinct gene sets representing helper T cell 1 (TH1), TH2, TH17, TH22, epithelial-mesenchymal transition (EMT), keratin-associated protein (KRTAP), lipid metabolism, and adenosine monophosphate kinase (AMPK) signaling pathways. Differential expression was noted in pathways representing AMPK signaling, EMT, and TH17. The dots represent individual values for each patient before and after metformin. The midline indicates the median value, the box indicates the lower quartile to upper quartile, and the whiskers reflect the highest and lowest values.aP < .05.DETAILED DESCRIPTION

[0017] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0018] In some embodiments, the presently disclosed subject matter provides a method for treating cicatricial alopecia in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of metformin to treat the cicatricial alopecia. In certain embodiments, the cicatricial alopecia comprises central centrifugal cicatricial alopecia (CCCA). In particular embodiments, the CCCA is refractory CCCA. As used herein, the term “refractory” means the CCCA is resistant to treatment or cure.

[0019] In some embodiments, the metformin is administered orally. In certain embodiments, the metformin is administered at a dosage of metformin of about 500 mg. A daily dose of metformin can range from about 500 mg to about 500 mg to 2550 mg, including about 500 mg, 1000 mg,1500 mg, 2000 mg, and 2550 mg. In representative embodiments, a low dose of metformin, e.g., 500 mg, is administered to the subject.

[0020] In certain embodiments, the metformin comprises an extended-release formulation of metformin. An extended-release formulation of metformin can be in the form of a salt, e.g., 500 mg of metformin hydrochloride as the active ingredient and active ingredients including one or more sodium carboxymethyl cellulose, hypromellose, microcrystalline cellulose, magnesium stearate, and combinations thereof.

[0021] In certain embodiments, the metformin comprises an immediate-release formulation of metformin. In immediate-release formulations of metformin, metformin is released within 1-2 hours after administration. A drawback of the immediate-release formulation is that it results in high drug concentrations in the gastrointestinal tract (potentially with undesired adverse effects).

[0022] To counteract such common gastrointestinal side-effects, metformin slow-release (SR) and extended-release (XR) were introduced in 2004, allowing once-daily dosage. Such formulations release the active drug via hydrated polymers, which expand after the uptake of fluid.

[0023] In certain embodiments, the metformin is administered once or twice daily. In certain embodiments, the metformin is administered over a time period of at least six months, including one week, two weeks, three weeks, one month, two months, three months, four months, five months, and six months.

[0024] In certain embodiments, the method further comprises administering metformin with one or more additional therapeutic agents. In particular’ embodiments, the one or more additional therapeutic agents are selected from a topical high-potency steroid, an intralesional steroid injection, an oral tetracycline, and topical or oral minoxidil.

[0025] In certain embodiments, administering the metformin to the subject further promotes hair growth.

[0026] In certain embodiments, administering the metformin upregulates pathways and genes associated with keratinization, epidermis development, and hair cycle. In particular embodiments, the genes linked to keratinization, epidermis development, and hair cycle include one or more KRTAPs.

[0027] In certain embodiments, administering the metformin downregulates pathways and genes associated with fibrosis. In particular embodiments, the genes associate with fibrosis include MMP7 and COL6A1.

[0028] In certain embodiments, the method further comprises reducing Thl7-inflammation, cpithclial-mcscnchymal transition pathway-expression, and fibrosis markers.

[0029] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0030] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0031] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration,progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0032] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly metformin and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0033] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0034] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at differenttimes (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0035] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0036] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0037] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0038] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0039] Qa / QA + QB / QB = Synergy Index (SI)

[0040] wherein:

[0041] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0042] Qais the concentration of component A, in a mixture, which produced an end point;

[0043] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0044] Qb is the concentration of component B, in a mixture, which produced an end point.

[0045] Generally, when the sum of QH / QA and QB / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0046] As provided herein, metformin can be formulated into a solid dosage form and administered orally. The metformin may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the ail. In other embodiments, the metformin can be administered in an immediate release formulation. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0047] Pharmaceutical preparations of metformin for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0048] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / - 15%, + / -10%, -+7-5%, -+7-4%, -+7-3%, + / - 2%, and + / -!%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0049] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0050] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0051] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1

[0052] LOW-DOSE METFORMIN AND PROFIBROTIC SIGNATURE IN CENTRAL CENTRIFUGAL CICATRICIAL ALOPECIA

[0053] Overview

[0054] Central centrifugal cicatricial alopecia (CCCA) is a scarring alopecia predominantly affecting Black female individuals. Current conventional treatments target inflammation but not the underlying fibrotic processes, often leading to permanent hair loss. This Example investigatesthe associations of low-dose oral metformin, an antidiabetic medication with antifibrotic properties, with clinical symptoms and scalp gene expression patterns in patients with CCCA.

[0055] More particularly, this Example investigates whether low-dose oral metformin leads to clinical and molecular signs of improvement in patients with treatment-refractory central centrifugal cicatricial alopecia (CCCA). In this retrospective clinical case series and transcriptomic analysis included patients treated at a single tertiary academic medical center between January 2023 and March 2024. All patients had biopsy-confirmed CCCA refractory to standard treatments. Transcriptomic analysis was performed on patients with previously banked, paired scalp biopsies before and after treatment with adjuvant metformin for at least 6 weeks.

[0056] In this Example, twelve black female participants were included in the study, and transcriptomic analysis was performed in four participants. The participants with CCCA were treated with daily metformin. Extended-release metformin, 500 mg, once daily was added to participants’ baseline CCCA treatment regimens. Clinical assessments included pruritus, inflammation, scalp resistance, and hair regrowth. Gene expression profiling via bulk RNA sequencing analysis evaluated differential gene expression and pathway enrichment.

[0057] After at least six months of metformin treatment, nine participants experienced improvement in disease, including scalp pain, inflammation, and / or pruritus, and six demonstrated clinical evidence of hair regrowth. The addition of metformin led to reversal of many prominent gene pathways previously identified in CCCA. Transcriptomic analysis revealed upregulation of pathways and genes (keratin-associated proteins [KRTAPs]) involved in keratinization, epidermis development, and the hair cycle (absolute log2-fold change > 4), with concomitant downregulation of fibrosis-related pathways and genes (eg, MMP7. COL6A 1) (fold change >1.5; all false discovery rate <.05). Gene set analysis showed reduced expression of helper T cell 17 and epithelial-mesenchymal transition pathways and elevated adenosine monophosphate kinase signaling and KRTAPs after metformin treatment.

[0058] In this Example, a case series of patients with treatment-refractory CCCA, low-dose oral metformin was associated with symptomatic improvement and dual modulation of gene expression, stimulating hair growth pathways while suppressing fibrosis and inflammation markers. These results indicate that low-dose oral metformin may reverse the fibrotic transcriptional signature in CCCA and promote hair regrowth, suggesting its potential as a targetedtherapy for this scarring alopecia and provide a rationale for future clinical trials studying metformin as a targeted therapy for CCCA and other cicatricial alopecias.

[0059] Introduction

[0060] Cicatricial or scarring alopecias are a group of chronic inflammatory hair disorders characterized by permanent hair loss resulting from destruction of the hair follicle and replacement with fibrous scar tissue. These disfiguring conditions substantially impair patients’ quality of life. Central centrifugal cicatricial alopecia (CCCA) is the most common form of primary lymphocytic cicatricial alopecia and predominantly affects Black female individuals. Patients with CCCA have an increased risk of systemic comorbidities, particularly type 2 diabetes (T2D). Ali et al., 2022.

[0061] Current treatments for CCCA, including topical glucocorticoids, intralesional triamcinolone injections, and oral tetracycline antibiotics, primarily target inflammatory aspects of the disease but do not address the underlying processes of fibrosis and scarring that ultimately lead to hair loss. Whiting and Olsen, 2008. Gene expression profiling of CCCA-affected scalps has revealed upregulation of profibrotic transcripts, Aguh et al., 2018, sharing molecular signatures with abnormal scarring disorders, such as idiopathic pulmonary fibrosis and systemic sclerosis. In cases where fibrosis persists amid low-grade inflammation, decreased vascularity of fibrotic tissues may further impair drug delivery, diminishing the effectiveness of anti-inflammatory therapies. While medications like topical and oral minoxidil can be used concurrently if there is suspicion for concomitant female pattern hair loss, these agents do not directly target the core pathogenesis of CCCA and have limited efficacy. Cardoso et al., 2021. Thus, a strategic focus on interventions targeted at reversing fibrosis is warranted. Wynn, 2008.

[0062] Metformin is an antidiabetic drug that upregulates adenosine monophosphate kinase (AMPK), enhancing insulin sensitivity. Preclinical studies have demonstrated metformin’s antifibrotic effects via IL10-mediated inhibition of transforming growth factor 0 signaling and collagen production. Rena et al., 2017; Wu et al., 2021. Furthermore, metformin blocks the angiotensin II pathway leading to fibroblast-to-myofibroblast transition. Jeon et al., 2021; Moon et al., 2021. With its approval for treating T2D, a comorbidity enriched in patients with CCCA, metformin represents a promising repurposed therapeutic candidate. Cardoso et al., 2021; Gadre et al., 2023. A preliminary case series has demonstrated potential clinical utility of topical metformin for CCCA, but the role of systemic oral metformin in cicatricial alopecias remains unexplored. Gu et al., 2021; Araoye et al., 2020.

[0063] Scope

[0064] This Example presents, a ease series evaluating the clinical associations of low-dose extended-release oral metformin, 500 mg, daily in 12 patients with treatment-refractory CCCA. To evaluate the impact of metformin on gene expression, bulk RNA sequencing was performed on a subset of 4 patients who had existing scalp biopsies both before and after treatment with metformin.

[0065] Methods

[0066] Study Participants

[0067] This Example represents a retrospective case series of twelve participants seen at the Johns Hopkins alopecia clinic with biopsy-confirmed CCCA that had been clinically refractory to conventional therapies. Given the prevalence of CCCA among Black patients, demographic data, including sex and race, were collected through patient self-reporting. All participants had remained taking their baseline CCCA treatment regimen for at least six months with stagnant or worsening clinical symptoms prior to adding oral extended-release metformin, 500 mg, once daily as an adjunct treatment. Duration between pretreatment biopsy and initiation of metformin was within 1 week. Medical records were reviewed for changes in clinical symptoms (pruritus, inflammation, pain, scalp resistance, and hair regrowth) following metformin initiation. Scalp resistance is a subjective measurement reflecting the amount of force required to inject the scalp, with 0 being normal (as would be expected in nonscarring alopecia, like alopecia areata), 1 being mild resistance, and 2 being significant resistance, oftentimes leading to bending of the needle on insertion. A total of ten of twelve patients received baseline hemoglobin Aicscreening prior to starting metformin, of whom 3 were in the prediabetic range. One patient (patient 12), who was treated with low-dose metformin by her primary care physician 1 week prior to evaluation, had been diagnosed with T2D and was taking low-dose metformin as monotherapy. This study was reviewed and approved by the Johns Hopkins Institutional Review Board. Oral informed consent was obtained from all participants, and written informed consent was obtained from all patients who received biopsies. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.

[0068] Tissue Sampling

[0069] A total of four of twelve patients had existing scalp biopsy samples in the alopecia tissue repository prior to and after at least six weeks of sustained oral metformin treatment (range, 6 to31 weeks after treatment start). Biopsy samples were originally obtained from areas of active disease on the scalp vertex.

[0070] Sample Processing and Sequencing

[0071] Fresh tissue samples were immediately submerged in a 2-mL tube containing RNA / a / cr (QIAGEN) stabilization reagent. Tissue was stored at 4 °C for 24 hours and transferred afterward to a freezer for long-term storage at -80 °C. Total RNA was later extracted using the RNA extraction RNeasy Kit (QIAGEN) according to vendor protocols. Pretreatment and posttreatment tissue samples were subsequently processed in a single batch. RNA purity and concentration were measured using NanoDrop (Thermo Fisher Scientific). The RNA library preparation and subsequent transcriptome sequencing were outsourced to Novogene. mRNA was extracted and purified using polyT oligo-attached magnetic beads. Following fragmentation, the first strand of cDNA was synthesized with random primers, while the second strand of cDNA was generated using deoxyuridine triphosphate for directional and deoxythymosine triphosphate for nondirectional libraries. Library integrity and size distributions were assessed through Qubit (Thermo Fisher Scientific) and real-time polymerase chain reaction. The quantified libraries were pooled and sequenced on the HiSeq 2500 platform (Illumina). Percentage error rate, guanine- cytosine distribution, and sequence read filtering was performed for quality control (Table 1). Genes with an average cohort count less than 10 were systematically excluded from subsequent analyses to enhance result validity and statistical reliability.

[0073] Differential Gene Expression Analysis and Statistical Analysis

[0074] Differential gene expression analysis comparing premetformin and postmetformin samples was performed using the DESeq2 R package, version 1.40.2 (R Project for Statistical Computing), which uses a Wald test. The Benjamini-Hochberg procedure was used to control for false discovery rate (FDR). Genes with an adjusted FDR less than 0.05 and an absolute log2-fold change of 0.5 or greater were considered differentially expressed.

[0075] Gene Set and Pathway Analysis

[0076] Gene set enrichment analysis (GSEA) was conducted using the local version of the GSEA analysis tool (Broad Institute) to evaluate enrichment of Gene Ontology molecular function, cellular component, and biological process pathways in the set of differentially expressed genes. Pathways with an adjusted 2-tailed P < .05 were considered significantly enriched.

[0077] In addition, gene set variation analysis (GSVA) using the GSVA package version 1.50.0 generated pathway enrichment scores for each sample. GSVA gene sets were curated from the literature of relevant genes associated with known pathways. Prior to GSVA, raw counts underwent variance-stabilizing transformation using DESeq2 version 1.40.2. The limma package version 3.58.1 was used to perform moderated 2-tailed t tests to assess statistical differences in GSVA scores between the premetformin and postmetformin samples.

[0078] Results

[0079] Clinical Improvements and Hair Regrowth

[0080] The study included 12 Black female patients with treatment-refractory CCCA. Eight patients experienced noticeable improvement in clinical symptoms after metformin treatment, including symptoms of scalp pain, scalp resistance, pruritus, and inflammation (Table 2), and two patients noted continued worsening of symptoms since metformin initiation. Six patients demonstrated clinical evidence of hair regrowth after sustained treatment for at least six months, although one patient experienced subsequent regression months following metformin discontinuation (FIG. 1).Table 2. Patient Cohort Characteristics at Baseline and Following Therapy With Daily Extended-Release Metformin, 500 mgFfctient,No; sex; Treatments prior to Pretreatment Posttreatment Improvement Time to symptom age,y metformin treatment symptoms3symptoms noted improvement, mo Notes1; Female; Clobetasol, 0.05%, ointment None None No NA discontinued all treatments50sbprior to initial biopsy; no clinicalCompounded minoxidil, 7%, and regrowthRA, 0.025%Fluocinolone, 0.01%, oilTable 2. Patient Cohort Characteristics at Baseline and Following Therapy With Daily Extended-Release Metformin, 500 mgRrtient,No; sex; Treatments prior to Pretreatment Posttreatment Improvement Time to symptom age,y metformin treatment symptoms3symptoms noted improvement, mo Notes2; Female; Compounded metformin, 10%; Scalp None Yes 5 Was being treated with oral30sbclobetasol, 0.05% resistance minoxidil at time of initial biopsy but discontinued on day of Doxycycline, 100 mg, twice daily metformin initiation; clinical appearance of regrowth at 6 mo3; Female; Compounded metformin, None None No NA Had discontinued all treatments30sb10%, in lipoderm cream at time of initial biopsy; no clinical regrowth noted Fluocinolone, 0.01%, oil4; Female; Clobetasol, 0.05%, ointment3Pruritus None Yes 1 Was taking oral minoxidil,50sbclobetasol, and ILK at time ofDoxycycline, 100 mg, twice daily initial biopsy; clinical appearance of regrowth at 6 mo5; Female; Clobetasol, 0.05%, ointment Pruritus Pruritus Yes 3 No clinical regrowth40sCompounded RA, 0 025%, minoxidil, 7%, and fluocinolone, 0.01%, oil6; Female; Doxycycline, 100 mg, twice daily Pruritus, Pruritus Yes 6 All symptoms noted in first50s scalp pain, (decreased) follow-up after metforminCompounded minoxidil, 8%, and and scalp and scalp pain started, which resolved after 3RA, 0.025%cresistance mo more of metformin use; no clinical regrowth7; Female; Clobetasol, 0.05%, ointment Scalp Scalp No NA Slight worsening of scalp50s resistance resistance resistance noted between visits;Compounded metformin, no clinical regrowth10%, in lipoderm cream8; Female; Clobetasol, 0 05%, ointment Pruritus None Yes 3 Clinical appearance of regrowth50s at 6 moFinasteride, 2.5 mgILKC9; Female; Compounded metformin, 10%, None Pruritus and No NA Noted clinical worsening of30s and clobetasol, 0.05%cscalp symptoms after starting oral metforminILKCresistance10; Female; Clindamycin Pruritus and None Yes 6 Clinical appearance of regrowth30s erythema at 6 moClobetasol, 0.05%, ointmentCompounded RA, 0 025%, minoxidil, 8%, and TAC11; Female; Clobetasol, 0.05%, ointment Scalp None Yes 4 Clinical appearance of regrowth at40Sresistance 6mosubsequent regressionCompounded metformin, 20%, noted 3 mo after discontinuation and clobetasol, 005%, ointment3°f metforminDoxycycline, 100 mg, twice daily12; Female; Fluocinolone, 0.01%, oil None None Yes NA Clinical appearance of regrowth50sdat 6 moCompounded minoxidil, 8%, RA,0.025%, and TAC ointment

[0081] Abbreviations: ILK, intralesional triamcinolone acetonide; NA, not applicable; RA, retinoic acid; TAC, triamcinolone acetonide.

[0082] Truritus and scalp pain were patient reported. Scalp resistance is a subjective measurement reflecting the amount of force required to inject the scalp during ILK administration.

[0083] bRNA sequencing performed on banked samples before and after addition of metformin to treatment regimen. Baseline samples taken after patients had been taking treatment regimens for at least 6 months.

[0084] cSimultaneous usage during metformin treatment period.

[0085] dPatient was given immediate-release metformin, 500 mg, tablets twice daily by primary care physician.

[0086] Differential Gene Expression Analysis

[0087] In the transcriptomic analysis of 16,655 genes, we identified 34 genes that were upregulated and 8 genes that were downregulated posttreatment (FDR less than 0.05; absolute log2-fold change greater than 0.5). Gene set analysis revealed enriched upregulated pathways related to keratinization, epidermis development, and hair cycle (FDR less than 0.001). Upregulated genes were associated with insulin regulation (SGK1: fold change, 0.51; FDR = 0.0288), immune processes (IGHGh fold change, 0.66; FDR = 0.0064; IGHG2'. fold change, 0.83; FDR = 0.0007; NFIL3: fold change, 0.60; FDR = 0.0400), and calcium signaling (S100P'. fold change, 0.94; FDR = 0.0424). Notably, 23 different hair keratin-associated proteins (KRTAPs) were upregulated after treatment, encompassing members of the KRTAP9, KRTAP4, KRTAP3, KRTAP2, and KRTAP1 families (all fold change >4) (FIG. 2A-FIG. 2C; Table 3).

[0088] Enriched downregulated pathways included those associated with extracellular matrix organization, collagen fibril organization, and collagen metabolism (all fold change >1.5). Prominently downregulated genes included matrix metalloproteinase 7 (MMP7 fold change, -1.76; FDR = 0.0018), collagen VI alpha 1 (COL6A1- fold change, -0.55; FDR = 0.0179), and dermcidin (DCD; fold change, -1.1; FDR = 9.78 x 106) (FIG. 3A).

[0089] GSVA

[0090] GSVA quantified posttreatment expression within established pathways. Notably, there was a reduction in the mean posttreatment expression of gene sets associated with helper T cell 17 (TH1 ; mean [SD] GSVA score: pretreatment, 0.17 [0.11]; posttreatment, -0.11 [0.23]; P = .O4) and epithelial-mesenchymal transition (mean [SD] GSVA score: pretreatment, 0.22 [0.21]; posttreatment, -0.20 [0.24]; P = .03). Conversely, an AMPK signaling gene set (mean [SD] GSVA score: pretreatment, -0.10 [0.13]; posttreatment, 0.11 [0.08]; P= .02) and a gene set comprising all KRTAPs (mean [SD] GSVA score: pretreatment, -0.49 [0.14]; posttreatment, 0.45 [0.28]; P < .001) exhibited elevated expression following metformin treatment (FIG. 3B).

[0091] Discussion

[0092] This Example found that, in patients with treatment-refractory CCCA, low-dose oral metformin was associated with downregulation of fibrotic processes and concurrent upregulation of keratinocyte proliferation and hair cycle-related processes. Notably, despite patients already receiving standard therapies, baseline sequencing still showed expression patterns similar to those in untreated affected scalps, Cardoso et al., 2021, a pathogenic state that was largely reversed with metformin (Table 4). Additionally, most patients experienced symptomatic improvement after initiating metformin treatment. Of the six patients who showed clinical signs of regrowth, half had not taken minoxidil during six months of metformin usage, suggesting that metformin may help with regrowth in select patients. One patient noted disease worsening within three months of treatment discontinuation, which may reflect the need for long-term treatment in patients.

[0093] Previously defined gene pathways characteristic of affected scalp in patients with CCCA by Aguh et al., 2018.

[0094] bRelative to pretreatment values.

[0095] Antifibrotic Effects of Metformin

[0096] Beyond its effects on glycemic control, metformin exhibits antifibrotic activity, particularly through prolifcrator-activatcd receptor a-dcpcndcnt upregulation of AMPK and subsequent downregulation of transforming growth factor P expression. Rena et al., 2017; Wu et al., 2021.

[0097] While these patients were treated with low-dose metformin, 500 mg, AMPK signaling was still upregulated in scalp tissue, consistent with prior translational studies demonstrating that low- dose metformin is enough to activate AMPK and lower blood glucose in patients. Yajima et al., 2004; Liu et al., 2022. However, while low-dose metformin may be sufficient to target fibrosis in patients without diabetes, it is likely inadequate to improve insulin resistance in susceptible individuals. Baseline hemoglobin Aiclevels could help identify those requiring additional monitoring and higher-dose titration. Clinically, metformin has shown benefits in treating fibrotic conditions, like scleroderma and metabolic dysfunction-associated steatotic liver disease. Kim et al., 2022; Zhang et al., 2024. In a murine model of idiopathic pulmonary fibrosis, metformin reversed fibrosis by deactivating myofibroblast activity and inducing apoptosis. Kheirollahi et al., 2019.

[0098] Metformin as a Therapeutic Agent for CCCA

[0099] The potential use of metformin as a therapeutic agent for CCCA has been previously explored. The current findings build on a prior case series demonstrating hair regrowth with topical metformin in patients with CCCA. Araoye et al., 2020. A retrospective analysis also found increased odds of clinical improvement, as assessed by a central scalp alopecia photographic severity scale, in patients with CCCA who were incidentally taking metformin. Onamusi et al., 2023. Furthermore, transcriptomic profiling has revealed unique differential expression of the AMPK signaling pathway in CCCA-affected scalps compared with other scarring alopecias, like frontal fibrosing alopecia and lichen planopilaris. Wang et al., 2022. Lastly, epidemiologic studies have identified an association between CCCA and T2D, with CCCA conferring an increased diabetes risk (hazard ratio, 1.68; 95% CI, 1.38-2.06) in African American women, although the underlying mechanisms remain unclear'. Roche et al., 2022.

[0100] Effects of Metformin on the Immune System

[0101] Metformin’s observed impact on immune pathways, such as the TH17 axis, may be mediated through AMPK-dependent inhibition of the mammalian target of rapamycin pathway, which plays a crucial role in suppressing Tul, Tu2, and TH17 cell differentiation and mitigatingproduction of inflammatory cytokines, such as interferon-y and IL-17. Duan et al., 2019. Metformin’s anti-inflammatory effects may also stem from its enhancing effect on mitochondrial metabolism and mitophagy, and treatment in patients with T2D was associated with reduced levels of IL-6, tumor necrosis factor-a, and reactive oxygen species, de Maranon et al., 2022.

[0102] While the precise role of the TH17 axis in CCCA pathogenesis remains unclear, immunohistochemistry studies demonstrate that IL- 17-positive cells are markedly elevated in scalp tissue of patients with lichen planopilaris or discoid lupus erythematosus, 2 of the most frequent causes of primary cicatricial alopecia. Shahidi Dadras et al., 2022. Furthermore, the highest cellular infiltration was noted at perifollicular and perivascular regions. Given these observations, TH17 may be explored as a potential treatment target in cicatricial alopecias.

[0103] Other dermato logic conditions, such as hidradenitis suppurativa and scleroderma, are canonically characterized by Tul7 dysregulation and IL-17-mediated inflammation. Moran et al., 2017; Balanescu et al., 2017. In both conditions, metformin treatment has demonstrated antiinflammatory effects, immune-metabolic reprogramming, and suppression of TH17 signaling. Moon et al., 2021; Hambly et al., 2023; Petrasca et al., 2023. These observations establish therapeutic precedents and carry implications for other dermatological diseases that could potentially benefit from metformin treatment.

[0104] Transcriptomic Trends Associated With Metformin Treatment

[0105] The observed downregulation of fibrotic pathways was reflected in decreased expression of key genes previously implicated in CCCA, including MMP7 and COL6A1. MMP7 is a small protease involved in extracellular matrix degradation and a prospective marker of systemic fibrotic diseases. Elevated plasma MMP7 levels correlate with declines in lung function in idiopathic pulmonary fibrosis and with liver stiffness in metabolic dysfunction-associated steatotic liver disease. Bauer et al., 2017; Irvine et al., 2021. Moreover, previous microarray analyses identified aberrant MMP7 upregulation, among other MMPs, in CCCA-affected scalp tissue compared with nonlesional tissue, supporting its relevance as a potential biomarker of CCCA activity. Cardoso et al., 2021; Jamerson et al., 2022.

[0106] COL6A1, an integral component of type VI collagen, serves as an abundant extracellular matrix component with pronounced deposition in hair follicles. In vitro studies have elucidated that cellular knockout of collagen VI results in diminished cellular focal adhesion, heightened activation of the phosphoinositide 3-kinase / protein kinase B signaling pathway, and, ultimately,fibroblast apoptosis. Castagnaro et al., 2018. Genetic variants in collagen VI manifest in diverse kcratin-rclatcd disorders, including abnormal scarring, follicular hyperkeratosis, and, more recently, an alopecia scalp phenotype. Ritter and Wine, 2022; Lee et al., 2022; Starace et al., 2023. Murine studies have demonstrated that lack of COL6 A 1 promotes increased wound-induced hair regrowth. Chen et al., 2015. Lastly, DCD, an antibiotic peptide secreted by sweat glands, Schittek et al., 2001, was also downregulated in our cohort. Notably, in mouse keratinocytes, DCD-derived peptides induce mast-cell activation, Che et al., 2022, a process that has been histologically and translationally characterized in all forms of cicatricial alopecia. Wang et al., 2022; Almodovar- Real et al., Look- Why et al., 2023.

[0107] In contrast, we also observed prominent upregulation of multiple KRTAPs, which play a key structural role in conferring rigidity to the hair shaft. Prior studies have substantiated the relevance of KRTAP dysregulation in diverse alopecic conditions. Downregulation of KRTAPs has been observed in the hair cortex of bald harlequin mice. Hintze et al., 2021. Furthermore, in transcriptomic studies, KRTAPs were markedly downregulated in patients with androgenetic alopecia, Mirmirani et al., 2015, as well as in those with alopecia areata who carry the CCHCR risk allele. Oka et al., 2020. Consistent with our findings, KRTAP downregulation was previously identified in active CCCA scalp tissue. Malki et al., 2019.

[0108] In prior studies comparing lesional with nonlesional tissue in CCCA scalps, fibrotic processes were enriched among upregulated genes, whereas hair-related processes were enriched among downregulated genes in lesional scalps (Table 4). Aguh et al., 2018; Moon et al., 2019. Notably, our findings reveal that oral metformin appears to reverse these aberrant baseline expression pathways typically observed in untreated lesions, with metformin treatment being associated with downregulated fibrotic processes and upregulated hair growth and cycling pathways. The magnitude of differential gene expression between nonlesional and lesional scalps (approximately 800 genes with nominal P < .05) is comparable with that between baseline lesional and metformin-treated lesional scalps (approximately 530 genes with nominal P < .05), Aguh et al., 2018, suggesting metformin elicits transcriptomic changes of similar scale to the inherent biological distinction between nonlesional and lesional states.

[0109] Clinical Considerations and Dosing Strategies

[0110] Metformin should be considered in patients who have not responded to standard therapies or are not candidates for adjunct therapies, such as oral minoxidil. Often, younger women will notdisplay signs of concomitant androgenetic alopecia, rendering minoxidil less effective. While results in this case scries arc preliminary, metformin is already frequently used in those with prediabetes or harbingers of insulin resistance, such as polycystic ovarian syndrome or acanthosis nigricans, and should be similarly considered for patients with CCCA experiencing the aforementioned comorbidities. Flory and Lipska, 2019. This case series, however, suggests possibly even patients with CCCA without prediabetes can benefit from treatment. Given its association with insulin resistance, we recommend routine testing of patients with CCCA for insulin resistance via hemoglobin Aiclevels (normal, <5.7%) or homeostatic model assessment of insulin resistance (HOMA-IR; normal, <2). Whiting and Olsen, 2008.

[0111] In this Example, patients benefited from low-dose metformin, 500 mg, once daily, although 1 patient received immediate-release tablets of metformin, 500 mg, twice daily. Extended-release formulation is thought to lead to fewer gastrointestinal adverse effects than immediate-release tablets and, combined with its less frequent dosing, may improve compliance. Flory and Lipska, 2019. Potential adverse effects of metformin include gastrointestinal upset, hypoglycemia lactic acidosis in patients with kidney impairment, or congestive heart failure. Weight loss and enhanced fertility are also reported as unintended effects but, in our experience, patients have either a neutral or positive reaction to these effects and, in some cases, are more eager to try the medication as a result. Overall, the medication was well-tolerated in our cohort.

[0112] Note that this Example has certain limitations that do not impact it patentability, including the small sample size, retrospective design, lack of placebo control group, and single-center setting, which limit the generalizability of our findings. Additionally, while we implemented a graded systematic assessment of clinical symptoms, no validated activity or severity scale currently exists for CCCA. Our transcriptomic analysis is constrained by single posttreatment sampling without parallel sampling of nonlesional tissue for comparison. Longer-term repeated biopsies at multiple time points and at various dosing strategies, along with biopsies from healthy scalp regions, may clarify evolving transcriptional trends across treatment duration and elucidate metformin’s effects relative to nonlesional baseline states. Larger prospective, multicenter controlled trials are needed to validate these results and further delineate metformin’s mechanism of action and clinical applications in cicatricial alopecias and other dermatologic disorders.

[0113] Summary

[0114] In this retrospective case series of patients with treatment-refractory CCCA, adjuvant low- dosc oral metformin was associated with symptomatic improvement, clinical evidence of hair regrowth, and modulation of gene expression profiles. Transcriptomic analyses revealed metformin upregulated hair growth pathways while downregulating fibrotic and TH 17 inflammatory pathways at a low daily dose of 500 mg. The appearance of hair regrowth seen in some patients challenges the notion that cicatricial alopecias inexorably lead to permanent hair loss. Metformin’s ability to concomitantly target fibrosis and inflammation provides a plausible mechanism for its therapeutic effects in CCCA and other fibrosing alopecia disorders. However, larger prospective, placebo-controlled randomized clinical trials are needed to rigorously evaluate metformin’s efficacy and optimal dosing for treatment of cicatricial alopecias.REFERENCES

[0115] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

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[0160] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A method for treating cicatricial alopecia in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of metformin to treat the cicatricial alopecia.

2. The method of claim 1, wherein the cicatricial alopecia comprises central centrifugal cicatricial alopecia (CCCA).

3. The method of claim 2, wherein the CCCA is refractory CCCA.

4. The method of any one of claims 1 to 3, wherein the metformin is administered orally.

5. The method of any one of claims 1 to 4, wherein the metformin is administered at a dosage of metformin of about 500 mg.

6. The method of any one of claims 1 to 5, wherein the metformin comprises an extended release formulation of metformin.

7. The method of any one of claims 1 to 5, wherein the metformin comprises an immediate release formulation of metformin.

8. The method of any one of claims 1 to 7, wherein the metformin is administered once or twice daily.

9. The method of any one of claim 1 to 8, wherein the metformin is administered over a time period of at least six months.

10. The method of any one of claims 1 to 9, further comprising administering metformin with one or more additional therapeutic agents.

11. The method of claim 10, wherein the one or more additional therapeutic agents arc selected from a topical high-potency steroid, an intralesional steroid injection, an oral tetracycline, and topical or oral minoxidil.

12. The method of any one of claims 1 to 11, wherein administering the metformin to the subject further promotes hair growth.

13. The method of any one of claims 1 to 12, wherein administering the metformin upregulates pathways and genes associated with keratinization, epidermis development, and hair cycle.

14. The method of claim 13, wherein the genes linked to keratinization, epidermis development, and hair cycle include one or more KRTAPs.

15. The method of any ones of claims 1 to 14, wherein administering the metformin downregulates pathways and genes associated with fibrosis.

16. The method of claim 15, wherein the genes associate with fibrosis include MMP7 and COL6A1.

17. The method of any one of claims 1 to 16, further comprising reducing Thl7- inflammation, epithelial-mesenchymal transition pathway-expression, and fibrosis markers.

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