Pharmaceutical composition for resisting keloid, application and preparation method

Abstract

The invention relates to the technical field of medicine for resisting keloid. The invention discloses a pharmaceutical composition for resisting keloid, application and a preparation method. The pharmaceutical composition comprises a therapeutically effective amount of nicotinamide N-methyltransferase (NNMT) inhibitor and a pharmaceutically acceptable carrier. According to the invention, an NNMT inhibitor is taken as a core, and keloids are treated through metabolism-epigenetic regulation. NAD / SAM balance can be recovered, fibroblast activation, collagen deposition and chronic inflammation are inhibited from the source, and the anti-fibrosis and anti-inflammatory dual effects are achieved. The scheme adopts a local delivery system such as a microneedle and gel, has the advantages of strong targeting property, low toxicity, relapse prevention, clear mechanism and the like, and provides a root intervention strategy for skin fibrosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for treating keloids, and more particularly to a pharmaceutical composition, its uses, and a method for preparing an anti-keloid drug. Background Technology

[0002] Currently, the mainstream treatments and research directions for keloid scars mainly include the following: Injectable and topical corticosteroids, which soften scars by inhibiting fibroblast proliferation and collagen synthesis, such as triamcinolone and hydrocortisone. However, these drugs easily cause skin atrophy, pigmentation, and vasodilation, and have a high recurrence rate after discontinuation, making it difficult to continuously regulate cellular metabolism. Physical and radiation therapies, such as radiation irradiation, cryotherapy, laser therapy, and pressure therapy, are often used to reduce scar thickness, but these methods are highly invasive and cannot cure the condition; re-stimulation by mechanical stress often leads to recurrence. Antimetabolites and interventional injections, such as 5-fluorouracil and bleomycin, block ECM accumulation by inhibiting cell division or protein synthesis, but they lack specific target regulation, affecting normal cells and inducing adverse reactions. Signaling pathway inhibitors or anti-TGF-β drugs: In recent years, studies have reported that TGF-β / Smad signaling is a key pathway in keloid formation, and various antagonists have been tried, such as Smad3 inhibitors and epigenetic drugs. However, these drugs mostly work indirectly and cannot inhibit the sustained activation of fibroblasts at the energy metabolism level. Regarding anti-inflammatory and antioxidant agents, some studies have attempted to use natural products such as quercetin and resveratrol to reduce inflammatory responses, but their efficacy depends on long-term use and is limited.

[0003] In summary, most existing technologies focus on downstream signal or symptom control, failing to address the metabolic driving mechanism of keloid formation and resulting in a high recurrence rate.

[0004] Lack of intervention targeting primary metabolic pathways: Current technologies mainly focus on TGF-β, Smad, or mechanical stimulation, neglecting upstream metabolic abnormalities. Recent research has found that nicotinamide N-methyltransferase (NNMT) is overexpressed in keloid tissue, driving SAM depletion and methyl donor imbalance, thereby enhancing fibroblast epigenetic activation. This core pathway lacks effective drug regulation. While traditional hormone and radiotherapy can temporarily alleviate scar appearance, recurrence rates often exceed 50% after discontinuation, primarily due to the failure to correct cellular metabolic states. Antimetabolites and glucocorticoids lack skin targeting, easily leading to atrophy, pigmentation changes, and even systemic side effects, making long-term use unsuitable. Existing research largely remains at the single-factor validation level, lacking a systematic integration of metabolomics, transcriptomics, and animal experiments, failing to clarify the holistic regulatory pathway of scar metabolism and epigenetic abnormalities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses an anti-keloid drug composition with nicotinamide N-methyltransferase (NNMT) inhibitor as the core active ingredient. It also discloses the uses and preparation method of this composition.

[0006] This invention discloses a pharmaceutical composition for treating keloids, comprising:

[0007] A therapeutically effective dose of a nicotinamide N-methyltransferase (NNMT) inhibitor and a pharmaceutically acceptable carrier.

[0008] Furthermore, NNMT inhibitors are small molecule inhibitors or nucleic acid inhibitors;

[0009] Small molecule inhibitors are selected from at least one of 1-methylnicotinamide analogs, aminopyrimidine derivatives, or pharmaceutically acceptable salts thereof;

[0010] Nucleic acid inhibitors are selected from at least one of small interfering RNA and antisense RNA.

[0011] Furthermore, the concentration of the small molecule inhibitor in the composition is from 1 μM to 100 μM;

[0012] The concentration of nucleic acid inhibitors in the composition is from 1 nM to 100 nM.

[0013] Furthermore, nucleic acid inhibitors are delivered via a carrier system, which may be a liposome nanocarrier or polymer nanoparticles.

[0014] Furthermore, the pharmaceutically acceptable carrier is selected from at least one of liposomes, microneedle patches, gels, injections, creams, or sprays;

[0015] Furthermore, the carrier is a sustained-release carrier, which can achieve targeted release into the dermis.

[0016] Furthermore, the pharmaceutical composition is a gel or a microneedle patch;

[0017] When it is a microneedle patch, it contains an array of soluble microneedles.

[0018] Furthermore, the pharmaceutical composition also includes an auxiliary active ingredient selected from at least one of anti-inflammatory agents, antioxidants, and collagenase inhibitors;

[0019] Co-active ingredients include quercetin, resveratrol, or triamcinolone.

[0020] Furthermore, the mass ratio of the auxiliary active ingredient to the NNMT inhibitor is 1:10 to 1:1.

[0021] This invention discloses the use of a pharmaceutical composition for treating keloids, comprising:

[0022] Use of a nicotinamide N-methyltransferase inhibitor in the preparation of a medicament for treating keloids, wherein the medicament is a pharmaceutical composition according to any one of the above.

[0023] This invention discloses a method for preparing an anti-keloid pharmaceutical composition, which includes the following steps:

[0024] A therapeutically effective amount of an NNMT inhibitor is mixed with a pharmaceutically acceptable carrier and a stable formulation is formed by lyophilization, encapsulation, or emulsification.

[0025] The beneficial effects of this invention are:

[0026] To address the aforementioned technical deficiencies, this invention proposes an innovative strategy centered on NNMT inhibition as the core of metabolic-epiocular regulation. By blocking the nicotinamide methylation pathway, it restores the dynamic balance of NAD⁺ / SAM, reversing fibroblast activation and abnormal ECM accumulation from the metabolic source, thereby achieving radical anti-scar treatment.

[0027] This solution has the following advantages:

[0028] Addressing the root causes of scar recurrence from a metabolic perspective;

[0029] The medication can be applied topically or delivered via microneedles, with low toxicity and side effects;

[0030] It also has both anti-fibrotic and anti-inflammatory effects;

[0031] The mechanism was clearly established and the effects were significant, as verified through multi-omics and animal models.

[0032] This invention focuses on the metabolic-epiocular regulatory mechanism of keloid formation, proposing a comprehensive intervention strategy centered on the targeted inhibition of nicotinamide N-methyltransferase (NNMT). Through the synergistic regulation of metabolic and signaling pathways, this strategy can systematically solve the following main technical problems:

[0033] (1) Problems of persistent activation and abnormal proliferation of fibroblasts. Current treatments have failed to reverse the hyperactive metabolic state of cells, leading to long-term scar hyperplasia. This regimen blocks the persistent activation and hyperproliferative phenotype of fibroblasts at the source by inhibiting NNMT activity and reducing SAM consumption and energy flux.

[0034] (2) Problems of excessive collagen deposition and abnormal ECM accumulation during wound healing. NNMT-induced metabolic imbalance leads to enhanced collagen I / III synthesis. This treatment effectively inhibits ECM deposition and restores the normal structure of dermal tissue by regulating the NAD⁺ / SAM balance and metabolic remodeling.

[0035] (3) Local chronic inflammation and excessive chemotaxis of immune cells in scar tissue. NNMT inhibition can significantly downregulate the Cytokine-cytokinereceptor interaction and Wnt signaling pathways, reduce the expression of inflammatory factors such as CCL2, CXCL6, and CX3CL1, block the positive feedback loop of inflammation-fibrosis, and exert a dual effect of anti-inflammatory and anti-fibrotic action.

[0036] (4) High recurrence rate and short-lived efficacy. Existing hormone and radiotherapy only suppress symptoms for a short period, and the retention of cellular metabolic memory leads to recurrence. This regimen achieves fibroblast phenotype reversal through metabolic-epimetic dual-layer intervention, resulting in a long-lasting anti-scarring and recurrence-preventing effect.

[0037] (5) The problems of significant toxic side effects and poor targeting of antimetabolites. Traditional chemotherapy drugs lack skin specificity and damage normal tissues. This invention uses selective NNMT inhibitors in combination with local delivery systems (gels, microneedles, liposomes) to improve drug targeting and reduce systemic toxicity.

[0038] (6) Lack of systematic mechanism verification and quantifiable indicators. Previous studies lacked multi-omics joint verification. This protocol establishes a joint system of "metabolicome + transcriptome + single-cell sequencing + ATAC-seq + animal model" to achieve quantitative analysis at the molecular-cellular-tissue level and clarify the NNMT-mediated metabolic-epiocular regulatory mechanism.

[0039] (7) Problems of low drug delivery efficiency and difficulty in industrialization. Traditional topical drugs have poor permeability. This plan simultaneously develops local sustained-release and microneedle patch formulations of NNMT inhibitors to achieve targeted release and sustained action in the dermis, thereby improving the feasibility of clinical translation.

[0040] General Explanation:

[0041] This application proposes a comprehensive solution through triple regulation of metabolism, epigeneity, and pharmacology, establishing a complete pathway from upstream metabolic reprogramming and inflammatory signal inhibition to downstream phenotypic reversal. It systematically addresses core technical challenges in keloid treatment, including metabolic abnormalities, persistent inflammation, fibrosis deposition, high recurrence rates, low safety, unclear mechanisms, and poor formulation stability, providing a new and precise intervention approach for the prevention and treatment of skin fibrosis. Attached Figure Description

[0042] Figure 1 To reveal abnormal amino acid and nicotinamide metabolism in keloid tissue through integrated transcriptomic and metabolomic analysis.

[0043] Figure 2 The pharmacological inhibition of NNMT in vivo inhibits the growth of keloid xenograft tumors and inflammatory signals.

[0044] Figure 3 The image shows that NNMT is mainly expressed in fibroblasts and is significantly upregulated in keloid tissue.

[0045] Figure 4 Mapping of NNMT inhibition that can reprogram the fibroblast transcriptome and attenuate extracellular matrix and cytokine signaling.

[0046] Figure 5 This diagram illustrates how NNMT promotes the expression of pro-inflammatory chemokines and the recruitment of inflammatory cells.

[0047] Figure 6 A diagram of the NNMT network for inhibiting chromatin remodeling accessibility and suppressing AP-1-related transcriptional networks. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present invention, the technical solutions in the specific embodiments of the present invention will be clearly and completely described below.

[0049] This invention proposes an anti-keloid drug based on nicotinamide N-methyltransferase (NNMT) inhibition and its applications, belonging to the field of skin fibrosis regulation. Its core idea is to reverse the pathological activation of fibroblasts through multi-layered regulation of metabolism, epigeneity, and signaling, thereby preventing keloid formation and recurrence from the metabolic root cause.

[0050] like Figures 1-6 As shown, this protocol first identifies NNMT as a key metabolic target in keloids through metabolomics and single-cell sequencing; subsequently, it uses compounds or nucleic acid-based NNMT inhibitors to block its catalytic pathway, and then verifies its role in tissue remodeling and inflammation suppression using animal models. The overall structure includes:

[0051] (1) Active pharmaceutical ingredient: at least one NNMT inhibitor, such as small molecule NNMTi (1-methylnicotinamide analog, aminopyrimidine derivative) or nucleic acid inhibitor (siNNMT, asRNA).

[0052] (2) Carrier system: used for skin-targeted delivery, selected from liposomes, microneedle patches, gels or injection solutions.

[0053] (3) Preparation method of composition: freeze drying, encapsulation and sustained release technology are used to improve the stability of drug efficacy and delivery efficiency.

[0054] For example, 50 mg of the active ingredient and 200 mg of PLGA were dissolved in 5 mL of dichloromethane to form an oil phase, and 50 mL of 1% w / v PVA solution was used as the aqueous phase. The mixture was emulsified by ultrasonication (50% power) for 2 min and stirred at room temperature for 4 h to evaporate the organic solvent. The resulting particles were centrifuged and washed, and trehalose was added to 5% w / v before being dispensed. The mixture was pre-frozen at -80°C for 4 h and lyophilized to a residual moisture content of approximately 1.5%. Then, it was spray-coated in a fluidized bed with 2% w / v HPMC solution, with a target coating amount of 10% w / w. The resulting composition released approximately 70% cumulatively in PBS (pH 7.4) over 72 h.

[0055] Explanation of main working principle

[0056] 1. Metabolic layer mechanism ( Figure 1 , Figure 2 ):

[0057] In keloid tissue, overexpression of NNMT leads to abnormal nicotinamide methylation metabolism, excessive consumption of SAM, and disruption of NAD⁺ metabolic balance. NNMT inhibitors can block this methylation pathway, restore NAD⁺ / SAM homeostasis, and thereby reduce the sustained activation of the TGF-β / Smad and AP-1 pathways, achieving metabolic reprogramming.

[0058] 2. Cell signaling regulation mechanisms ( Figure 4 , Figure 5 ):

[0059] Gene expression profiles and signaling pathway enrichment analysis showed that after NNMT inhibition, pro-inflammatory pathways such as Cytokine-receptor, Wnt, and MAPK were significantly downregulated, and the expression of chemokines such as CCL2, CXCL6, and CX3CL1 was inhibited, thereby weakening inflammatory stimulation and immune cell migration and blocking the positive feedback loop of inflammation-fibrosis.

[0060] 3. Epigenetic and Transcriptional Regulatory Mechanisms ( Figure 6 ):

[0061] ATAC-seq analysis showed that NNMT inhibition significantly reduced chromatin openness and AP-1 (FOS / JUN) binding activity in fibroblasts, thereby decreasing the expression of ECM-related genes (COL1A1, COL3A1, FN1) at the transcriptional level. This resulted in phenotypic reversal of fibroblasts without exogenous stimulation.

[0062] 4. Organizational and phenotypic validation ( Figures 2-3 ):

[0063] In a mouse model of human scar xenograft, NNMT inhibitor treatment for 21 days reduced scar volume by more than 60%. Pathological sections showed that the collagen bundle arrangement changed from dense to loose, accompanied by a significant decrease in the expression of α-SMA and Ki-67. Immunofluorescence double staining showed that the colocalization signal of NNMT and α-SMA was significantly weakened.

[0064] Scar volume measurement: The longest diameter (L), longest vertical diameter (W), and height (H) of the scar were measured using vernier calipers. The volume was calculated using the ellipsoid volume formula: V = π·L·W·H / 6. Scar volume (mean ± SD, unit mm^3).

[0065] Action Relationship Explanation

[0066] The operational relationships of this technical solution are illustrated in the diagram below:

[0067] (1) NNMT inhibition - nicotinamide methylation pathway blockade - NAD⁺ level restoration - energy metabolism homeostasis reconstruction;

[0068] (2) Restoration of metabolic homeostasis - inactivation of TGF-β / Smad & AP-1 pathway - downregulation of ECM protein;

[0069] (3) Decreased chemokines - reduced inflammatory infiltration - normalized tissue remodeling;

[0070] (4) Stable tissue microenvironment - reduced scar volume and prevention of recurrence.

[0071] This series of actions constitutes a synergistic mechanism of "metabolic signaling-epiotranscription-tissue remodeling," enabling systematic intervention in the entire process of scar formation.

[0072] Implementation method:

[0073] Example 1: When a small molecule NNMT inhibitor (concentration 1~10μM) was loaded into a liposome gel and cultured human scar fibroblasts in vitro for 72h, α-SMA expression decreased by 55% and COL1A1 decreased by 60%.

[0074] Example 2: Subcutaneous injection of siNNMT-nanocarrier into a human scar transplant mouse model, once a week for three weeks, resulted in a 63% reduction in scar volume and a 70% decrease in inflammatory cell infiltration score.

[0075] Example 3: A pharmaceutical composition for treating keloids, formulated as follows: each dose contains 0.5 mg of siNNMT (approximately 2 mg / kg), 200 mg of PLGA, 1% w / v of PVA, 5% w / v of trehalose, and 2% w / v of HPMC. It is administered subcutaneously to the area around the scar once a week for three consecutive weeks. The composition is prepared by dissolving 50 mg of siNNMT and 200 mg of PLGA in 5 mL of dichloromethane as the oil phase. The oil phase is then added dropwise to 50 mL of 1% PVA aqueous phase and sonicated (50% power, 2...). Emulsify (min), stir at room temperature for 4 h to evaporate the solvent, centrifuge at 12,000 g three times, add 5% trehalose, freeze at -80°C for 4 h and lyophilize (primary -30°C for 10 h, secondary -20°C for 6 h, residual moisture ≈ 1.5%), and spray-coat with 2% HPMC solution in a fluidized bed (1 mL / min, air inlet 30-35°C) until the coating is 10% w / w; quality control targets: particle size 150-250 nm, PDI < 0.2, encapsulation efficiency ≥ 70%, resolubility after lyophilization ≥ 90%. In the NOD-SCID human scar xenograft mouse model (n=8 per group), subcutaneous injections were administered weekly starting from baseline on day 7 post-transplantation. After three weeks, scar volume (V=π·L·W·H / 6) and histological parameters were measured. On day 21, the scar volume in the siNNMT group decreased from baseline 120±10 mm^3 to 44±7 mm^3 (a decrease of approximately 63%, p<0.01), and the inflammatory cell infiltration score decreased from 3.0±0.2 to 0.9±0.3 (a decrease of approximately 70%, p<0.01). Masson staining showed a significant decrease in collagen content, a reduction in CD45 / α-SMA positive cells, and a significant decrease in 1-MNA levels and NNMT expression. Safety assessments showed no abnormalities in body weight, complete blood count, or liver and kidney function. Skin irritation was assessed in mice / guinea pigs (n=6-10 per group) after local injection or application according to OECD / ISO standards, with erythema / edema (Draize score 0-4). Example results showed an average erythema score of 0.3±0.2 at 72h and no obvious epidermal / dermal damage, which was judged as no or mild irritation.

[0076] This invention:

[0077] 1. Using the NNMT metabolic-epiota axis as an anti-scarring target;

[0078] 2. A new approach to phenotypic reversal through metabolic remodeling;

[0079] 3. A unified technical platform for multi-omics validation and delivery systems has been established.

[0080] Therefore, the technical solution of this invention has a clear structure, a clear mechanism, and traceable action, and can systematically solve the problem of metabolic regulation and mechanism verification in the field of keloid treatment.

[0081] This invention:

[0082] I. Blocking the metabolic drivers of scar formation at the root

[0083] Technical effects: By inhibiting NNMT activity and blocking the nicotinamide methylation pathway, SAM consumption is significantly reduced and NAD⁺ homeostasis is maintained, eliminating the high-energy metabolism and pathological activation state of fibroblasts from the metabolic source.

[0084] Cause analysis: NNMT is overexpressed in scar tissue, causing cells to remain in a state of high methylation and high activation. This approach reduces the methylation rate by using NNMTi, restoring the NAD⁺ / SAM ratio to normal, thereby breaking the metabolically abnormal fibrosis cycle.

[0085] II. Highly effective inhibition of fibroblast proliferation and ECM deposition

[0086] Technical results: In in vitro experiments, the expression levels of α-SMA, COL1A1 / 3 and FN1 decreased significantly by more than 50%, and the tissue ECM density was significantly reduced.

[0087] Cause analysis: NNMT inhibition leads to the disappearance of TGF-β / Smad, AP-1 and MAPK pathway activity, and a decrease in the transcriptional level of related fiber genes; at the same time, the reduced metabolic energy supply limits the ECM synthesis rate, thus achieving structural anti-fibrosis.

[0088] III. Long-term anti-relapse and phenotypic stabilizing effects

[0089] Technical results: In animal models of scarring, there was no significant recurrence within 30 days after treatment; the tissue repair morphology was stable, and inflammatory cells no longer infiltrated.

[0090] Cause analysis: NNMT inhibition not only affects transient signaling events but also alters cellular epigenetic memory, enabling fibroblasts to restore their homeostatic phenotype. By reducing the activity of the H3K27ac promoter and its binding capacity to AP-1, cellular phenotype "re-education" is achieved, resulting in a lasting therapeutic effect.

[0091] IV. Reducing inflammatory response and immune infiltration

[0092] Technical effects: After treatment, the expression of inflammatory factors such as CCL2, CXCL6, and CX3CL1 decreased by 70%; the infiltration of macrophages and neutrophils in the tissue was significantly reduced.

[0093] Cause analysis: NNMT inhibition unblocks the metabolic reprogramming of pro-inflammatory cells, reducing lactate accumulation and ROS generation; at the same time, it blocks the Cytokine-receptor signaling loop, alleviating chronic inflammation through both metabolic and signaling pathways.

[0094] V. Improve drug safety and local targeting

[0095] Technical effect: No obvious erythema, atrophy or systemic toxicity occurred after local administration.

[0096] Cause analysis: This invention uses a liposome or microneedle targeted delivery system to focus the NNMT inhibitor on the dermal target area; the drug load is stable and the release is controllable, thereby reducing system exposure.

[0097] VI. Enhance drug efficacy stability and formulation convertibility

[0098] Technical effects: The NNMT inhibitor formulation retains 90% of its activity after accelerated stability testing at 40°C; and its sustained release time in vivo reaches 72 hours.

[0099] Analysis of the reasons: The combination of sustained-release gel and multilayer lipid nanocarrier structure can prevent the oxidative degradation of NNMT inhibitors, improve skin permeability and stability, and provide a technical basis for industrial production.

[0100] VII. Formation of quantifiable metabolic-epimetic-morphological linkage mechanisms

[0101] Technical results: Multi-omics analysis showed that NNMT inhibition was linearly negatively correlated with NAD⁺ level, SAM dosage, epigenetic openness, gene expression and scar area (r=-0.82 to -0.90).

[0102] Cause analysis: This scheme establishes a multi-layered data closed-loop validation system of molecules, cells, and tissues, making the mechanism of drug efficacy clear and quantifiable, and improving the comparability and evaluability between scientific research and clinical practice.

[0103] 8. Significantly improves the quality and appearance of tissue remodeling.

[0104] Technical results: Histological examination showed that the scar collagen bundles changed from dense to uniform; skin elasticity and smoothness were restored to near normal levels; subjective scar score decreased by 65%.

[0105] Cause analysis: NNMT inhibits the balanced rate of fiber synthesis and degradation, promotes signal exchange between normal fibroblasts and keratinocytes, improves the dermal-epidermal interface structure, and achieves appearance improvement from the perspective of tissue regeneration.

[0106] IX. Promotional Value in Terms of Application

[0107] Technical benefits: The drug can be flexibly formulated into topical, local injection, or microneedle patch forms, making it easy to use in clinical practice; it can be extended to multiple scenarios such as wound repair, surgical scars, and post-burn fibrosis.

[0108] Analysis of the reasons: Since NNMT is a common target of metabolism in multiple tissues, this technology has the potential for versatility and modular upgrades, and can be used in combination with existing drugs or independently.

[0109] This invention achieves a bottom-up mechanistic innovation in scar treatment at the metabolic regulation level. Compared to traditional methods that only inhibit signaling pathways or end symptoms, this approach precisely targets the NNMT, a core metabolic-epiocular node, transforming anti-scar treatment from symptomatic control to intervention at the root cause of the pathology. This results in: significantly improved efficacy (scar inhibition rate ≥60%); long-lasting recurrence prevention; safety, low toxicity, and skin-targeting properties; and feasibility for production and commercial application.

[0110] Figure label name:

[0111] Figure 1 Comprehensive transcriptomic and metabolomic analyses revealed abnormal amino acid and nicotinamide metabolism in keloid tissue.

[0112] (A) Pathway enrichment analysis of differentially expressed metabolites between keloid tissue and normal control tissue. Significantly affected metabolic pathways included histidine metabolism, alanine / aspartate / glutamate metabolism, nicotinic acid and nicotinamide metabolism, and glutamine / glutamate metabolism. The size and color of each bubble represent the degree of pathway influence and statistical significance (-log), respectively. 10 p).

[0113] (B) The heatmap shows the relative levels of related metabolites in the above pathways. Red indicates an increase, and blue indicates a decrease. Compared with the control, aspartic acid, glutamate, nicotinamide, and related metabolites were significantly increased in keloid tissue.

[0114] (C, E, G) Volcano plots show differentially expressed genes involved in histidine metabolism (C), alanine / aspartate / glutamate metabolism (E), and nicotinic acid / nicotinamide metabolism (G) pathways.

[0115] (D, F, H) Violin plots show the FPKM values ​​of key genes associated with corresponding metabolic pathways: ALDH1B1, ALDH3A1, and ASPA (D) in the histidine metabolic pathway; GPT, GOT1, ABAT, GLS2, and ASPA (F) in the alanine / aspartate / glutamate metabolism; and NNMT, ENPP3, and AOX1 (H) in the nicotinic acid / nicotinamide metabolism. The data indicate that these metabolism-related genes are significantly upregulated in keloid tissue compared to normal controls, suggesting that amino acid and nicotinamide metabolism has undergone metabolic reprogramming in scar fibroblasts.

[0116] Figure 2 Pharmacological inhibition of .NNMT suppresses the growth of keloid xenograft tumors and inflammatory signals in vivo.

[0117] (A) Schematic diagram of a mouse model of human keloid xenograft, which received either PBS control or NNMT inhibitor (NNMTi) treatment.

[0118] (B) Quantitative analysis of xenograft tumor volume showed that the NNMTi treatment group had a significant reduction in volume (p<0.01, unpaired Student's test).

[0119] (C) Histological analysis of xenograft tissues. H&E staining showed reduced cell density and collagen deposition in the NNMTi group; Ki-67 and α-SMA immunohistochemical staining showed decreased fibroblast proliferation and myofibroblast activation. Scale bar = 100 μm.

[0120] The (DF) volcano plot shows the distribution of differentially expressed genes in the three pathways mentioned above: cytokine-receptor interactions, the Wnt signaling pathway, and cancer-related transcriptional dysregulation pathways (D, E, F).

[0121] Figure 3 .NNMT is primarily expressed in fibroblasts and is significantly upregulated in keloid tissue.

[0122] (A) UMAP map obtained from single-cell RNA-seq analysis of human keloids and normal skin tissue, showing the ten major cell populations identified.

[0123] (B) UMAP visualization of NNMT expression levels across all cell populations.

[0124] (C) The violin plot shows the distribution of NNMT expression among different cell types, indicating that it is mainly enriched in fibroblasts.

[0125] (D) Immunohistochemical staining of NNMT in normal skin and keloid (KD) tissue shows significant enhancement of NNMT in the dermal layer of the scar. Scale bar: Top image 200μm, bottom image 100μm.

[0126] (E) Immunofluorescence co-staining of NNMT (green) and α-SMA (magenta) in normal and keloid tissues. DAPI (blue) marks cell nuclei. Combined image shows NNMT co-localization with α-SMA-positive fibroblasts. Scale bar = 50 μm.

[0127] Figure 4 NNMT inhibition can reprogram the fibroblast transcriptome and attenuate extracellular matrix and cytokine signaling.

[0128] (A, C)GO enrichment scatter plots show the biological processes most significantly enriched in human keloid fibroblasts after treatment with 10 μM (A) or 100 μM (C) NNMT inhibitors compared to controls. Items related to cytokine activity, chemokine signaling, and extracellular structure and tissue were significantly enriched.

[0129] (B, D) Heatmaps show the expression patterns of GO-related genes in the extracellular space that are differentially expressed between the NNMTi treatment group and the control group.

[0130] (E) Venn diagrams show extracellular space-related genes that were significantly affected in both the NNNMTi_10μM and NNNMTi_100μM groups.

[0131] (F) FPKM expression values ​​of representative extracellular and cytokine genes (CCL2, CXCL2, TNFAIP2, SEMA4B, VIM, SFN, LAMC1, WNT7B), confirming consistent downregulation after NNMT inhibition. Data are presented as mean ± standard error (*p<0.05, **p<0.01).

[0132] Figure 5 NNMT promotes the expression of pro-inflammatory chemokines and the recruitment of inflammatory cells.

[0133] (A, B)qRT-PCR was used to detect the expression levels of chemokine genes (CX3CL1, CCL2, CCL5) in scar fibroblasts after treatment with NNMT inhibitors (10 μM, 100 μM; A) or NNMTsiRNA transfection (B). GAPDH was used as an internal control.

[0134] (C, D) ELISA was used to determine the levels of CCL2 and CXCL1 secreted in the culture supernatant after NNMTi treatment (C) or NNMT knockdown (D).

[0135] (E, F) correlation scatter plots show a positive correlation between NNMT expression and CX3CL1(E) or CCL2(F) in human and mouse genomic data.

[0136] (G) Immunofluorescence staining of scar xenograft tissues shows the macrophage marker CD62 (red) and cell nuclei (DAPI, blue) after PBS control and NNMTi treatment. Macrophage infiltration was significantly reduced after NNMT inhibition. Scale bar = 50 μm.

[0137] (H) Quantitative analysis of CD62⁺ (THP-1-like) cell counts in xenograft tissues. Data are presented as mean ± standard error; *p<0.05, **p<0.01.

[0138] Figure 6 .NNMT inhibits chromatin remodeling accessibility and suppresses AP-1-related transcriptional networks.

[0139] (A) The heatmap and aggregate plot of ATAC-seq signal intensity show the difference in global distribution of chromatin accessibility in scar fibroblasts between NNMT inhibition treatment (NNMTi) and PBS control.

[0140] (B) Volcano plot of differential ATAC-seq peaks, with red and blue dots representing genomic regions where accessibility is increased or decreased after NNMT inhibition, respectively.

[0141] (C) Example of gene browsing trajectory: Chromatin accessibility of representative genes (CXCL1, CCL5, CCL2) associated with fibrosis is reduced after NNMT suppression.

[0142] (D) The results of transcription factor binding motif enrichment analysis showed that the accessibility of promoter / enhancer regions containing AP-1 family (FOS, JUN, BATF, ATF3), Smad and TCF binding sites was significantly reduced after NNMT inhibition.

[0143] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A pharmaceutical composition for treating keloids, characterized in that, include: A therapeutically effective dose of a nicotinamide N-methyltransferase (NNMT) inhibitor and a pharmaceutically acceptable carrier.

2. The anti-keloid pharmaceutical composition according to claim 1, characterized in that: NNMT inhibitors are small molecule inhibitors or nucleic acid inhibitors; Small molecule inhibitors are selected from at least one of 1-methylnicotinamide analogs, aminopyrimidine derivatives, or pharmaceutically acceptable salts thereof; Nucleic acid inhibitors are selected from at least one of small interfering RNA and antisense RNA.

3. The anti-keloid pharmaceutical composition according to claim 2, characterized in that: The concentration of the small molecule inhibitor in the composition is from 1 μM to 100 μM; The concentration of nucleic acid inhibitors in the composition is from 1 nM to 100 nM.

4. The anti-keloid pharmaceutical composition according to claim 2, characterized in that: Nucleic acid inhibitors are delivered via a carrier system, which can be a liposome nanocarrier or a polymer nanoparticle.

5. The anti-keloid pharmaceutical composition according to claim 1, characterized in that: Pharmaceutically acceptable carriers are selected from at least one of liposomes, microneedle patches, gels, injections, creams, or sprays; Furthermore, the carrier is a sustained-release carrier, which can achieve targeted release into the dermis.

6. The anti-keloid pharmaceutical composition according to claim 5, characterized in that: The drug composition is in the form of a gel or microneedle patch; When it is a microneedle patch, it contains an array of soluble microneedles.

7. The anti-keloid pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition also includes an auxiliary active ingredient selected from at least one of anti-inflammatory agents, antioxidants, and collagenase inhibitors; Co-active ingredients include quercetin, resveratrol, or triamcinolone.

8. The anti-keloid pharmaceutical composition according to claim 7, characterized in that: The mass ratio of the auxiliary active ingredient to the NNMT inhibitor is 1:10 to 1:

1.

9. The use of a pharmaceutical composition for treating keloids, characterized in that, include: Use of a nicotinamide N-methyltransferase inhibitor in the preparation of a medicament for treating keloids, the medicament being a pharmaceutical composition according to any one of claims 1 to 8.

10. A method for preparing an anti-keloid pharmaceutical composition, used to prepare an anti-keloid pharmaceutical composition according to any one of claims 1 to 8, characterized in that, Includes the following steps: A therapeutically effective amount of an NNMT inhibitor is mixed with a pharmaceutically acceptable carrier and a stable formulation is formed by lyophilization, encapsulation, or emulsification.

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