Application of Hedera oleifera saponin A1 in the preparation of drugs for the prevention and / or treatment of seborrheic keratosis or its secondary lesions

Abstract

This invention relates to the application of hedera saponin A1 in the preparation of drugs for the prevention and / or treatment of seborrheic keratosis or its secondary lesions. Through molecular-level experiments, this invention verifies that hedera saponin A1 can directly bind to the AKT1 protein, inhibiting AKT1-induced upregulation of pro-apoptotic proteins, thereby initiating cell apoptosis. Compared with existing technologies, this invention is a non-invasive treatment, painless, non-invasive, and leaves no scars after healing. Furthermore, this invention has the dual efficacy of "treating seborrheic keratosis" and "chemopreventing malignant transformation." In addition, hedera saponin A1 has a significant killing effect on seborrheic keratosis lesion cells, exhibiting a killing effect on the epidermal layer of isolated seborrheic keratosis lesion tissue, but without inducing significant apoptosis in dermal cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of ivy saponin A1 in the preparation of medicaments for the prevention and / or treatment of seborrheic keratosis or its secondary lesions. Background Technology

[0002] Seborrheic keratosis (SK) is the most common benign epidermal tumor in humans. Although traditionally considered benign, recent clinicopathological studies indicate a significant risk of malignant transformation. Epidemiological data show that approximately 1.4% to 7% of SK lesions can transform in situ into squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) [Muzic, JG, et al. (2018)]. The incidence of malignant neoplasms arising in seborrheic keratosis . Mayo Clinic Proceedings], [Noiles, K., & Vender, R. (2008). Are all seborrheic keratoses benign? [Journal of Cutaneous Medicine and Surgery], and it is often found that some malignant tumors are hidden inside SK lesions.

[0003] Current treatment methods mainly involve physical destruction (cryotherapy, laser therapy, electrocautery), which carries risks such as trauma, pain, scarring, and the inability to rule out potential malignant lesions through histopathology.

[0004] FDA-approved topical medications from SK use 40% hydrogen peroxide solutions (such as Eskata). These solutions utilize the strong oxidizing properties of high-concentration hydrogen peroxide for chemical corrosion. The mechanism is non-specific, causing damage to normal skin and posing risks of pain, erythema, and pigmentation. Furthermore, they cannot target carcinogenic pathways.

[0005] Studies have shown that abnormal activation of the PI3K / AKT / mTOR signaling pathway is a core driver of SK pathogenesis. More than 90% of SK patients carry FGFR3 or PIK3CA mutations, leading to persistent phosphorylation of downstream AKT1 kinase and inhibiting normal apoptosis of keratinocytes [Hafner, C., et al. (2007). Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a frequency of 24% [Journal of Investigative Dermatology]. Therefore, blocking the AKT pathway is an ideal strategy for clearing SK and preventing its malignant transformation.

[0006] However, most existing AKT inhibitors are oral anti-tumor drugs with significant systemic toxicity (hyperglycemia, rash), making them unsuitable for treating benign skin diseases. Furthermore, there are currently no topical medications targeting the core mechanism of SK pathogenesis (AKT pathway abnormalities), and physical therapies and corrosive agents lack molecular-level specificity. Existing therapies only focus on removing visible skin lesions and cannot block the malignant transformation process of cells at the signaling pathway level.

[0007] Traditional Chinese medicine ingredients are mild, safe, and have few side effects, making them uniquely effective and advantageous in treating many immune-related skin diseases.

[0008] Patent CN114796293B discloses the application of ivy saponin C, specifically its application in the treatment of skin diseases such as psoriasis. It is the first time that ivy saponin C has been disclosed to have a protective effect on imiquimod-induced mice, increasing the body weight of mice after imiquimod induction, reducing PASI scores, improving skin erythema, reducing the degree of scaling infiltration, improving pathological changes in skin lesions, and exerting anti-inflammatory and immunomodulatory functions, which may become a potential drug for the treatment of skin diseases.

[0009] Patent WO2025092276A1 discloses a series of derivatives of Pulsatilla chinensis saponin B4, their preparation methods, and their applications in the preparation of anti-inflammatory and immunomodulatory drugs. Specifically, it discloses that these derivatives can be used to treat atopic dermatitis, eczema, psoriasis, inflammatory bowel disease, etc. Through detailed description of improving the lipid solubility and efficacy of B4 through structural modification, and by providing extensive in vitro and in vivo experimental data, it demonstrates that the derivatives possess superior anti-inflammatory activity compared to the original compound B4.

[0010] Hederin A1 is a large molecular weight saponin (~897 Da), belonging to the pentacyclic triterpenoid saponin class. Due to its large molecular weight (~897 Da), far exceeding the ideal threshold for passive absorption through the skin (500 Da), it is traditionally believed that it is difficult to penetrate the stratum corneum to exert local pharmacological effects. However, as an extract of natural plants (such as Chinese ivy or Pulsatilla chinensis), it has in vitro cytotoxicity against some malignant tumor cell lines (such as lung cancer A549 and liver cancer HepG2) [Rooney, S., et al. (2022). α-Hederin: A review of its pharmacology, toxicity, and pharmacokinetics. Phytotherapy Research], [Cheng, L., et al. (2020). α-Hederin induces apoptosis of lung cancer cells via the PI3K / AKT / mTORsignaling pathway. Journal of Cellular Physiology]. Therefore, there are no reports on the use of ivy saponin A1 for topical treatment of seborrheic keratosis, nor are there any reports on its ability to induce apoptosis in benign skin lesions by specifically binding to AKT1. Summary of the Invention

[0011] Current treatments for seborrheic keratosis primarily employ physical methods (cryotherapy, laser therapy, electrocautery) and hydrogen peroxide cauterization, lacking specific targeted drugs. Currently, there are no topical medications targeting the core pathogenesis of SK (AKT pathway abnormalities). Both physical therapies and corrosive agents lack molecular-level specificity; existing treatments only focus on removing visible lesions and cannot block the malignant transformation process of cells at the signaling pathway level. Existing AKT inhibitors are mostly oral antitumor drugs with significant systemic toxicity (hyperglycemia, rash), making them unsuitable for treating benign skin diseases. Furthermore, geranium saponins, as large molecular weight saponins (~897 Da), are traditionally considered to have difficulty penetrating the stratum corneum to exert local therapeutic effects.

[0012] The purpose of this invention is to overcome the problems of existing SK treatment methods, such as trauma, pain, easy scarring, and inability to target carcinogenic pathways, and to provide the application of ivy saponin A1 in the preparation of drugs for the prevention and / or treatment of seborrheic keratosis or its secondary lesions.

[0013] This invention utilizes ivy saponin A1 as an AKT1 kinase inhibitor, which penetrates diseased tissue through local administration and specifically induces SK cell apoptosis.

[0014] The objective of this invention can be achieved through the following technical solutions: This invention provides the use of ivy saponin A1 in the preparation of a medicament for the prevention and / or treatment of seborrheic keratosis or its secondary lesions.

[0015] Preferably, the use of ivy saponin A1 in the preparation of a medicine for treating or alleviating seborrheic keratosis or its secondary lesions.

[0016] Preferably, the combination of ivy saponin A and a transdermal penetration enhancer is used in the preparation of a medicament for the prevention and / or treatment of seborrheic keratosis or its secondary lesions.

[0017] Preferably, the transdermal penetration enhancer includes, but is not limited to, dimethyl sulfoxide (DMSO), azone, propylene glycol, or a surfactant.

[0018] Preferably, the amount of ivy saponin A1 in the drug is 0.5wt% - 5.0wt%.

[0019] Preferably, the use of ivy saponin A1 in the preparation of drugs that directly bind to AKT1 protein.

[0020] Preferably, the use of ivy saponin A1 in the preparation of drugs that initiate apoptosis by inhibiting AKT1 and thus upregulating pro-apoptotic proteins.

[0021] Preferably, the use of ivy saponin A1 in the preparation of medicaments for the prevention and / or treatment of seborrheic keratosis or its secondary lesions, in the form of an AKT1 specific inhibitor.

[0022] In one embodiment of the present invention, secondary lesions of seborrheic keratosis include inflammation and infection, hyperplasia and hypertrophy of skin lesions caused by seborrheic keratosis.

[0023] In one embodiment of the present invention, the drug is a topical preparation.

[0024] Preferably, the dosage form of the drug is an ointment, cream, gel, or patch.

[0025] This invention involves applying a drug containing ivy saponin A1 to the surface of SK skin lesions. The drug enters the basal layer and spinous layer of the epidermis with the help of a transdermal penetration enhancer, binds to abnormally activated AKT1, and induces apoptosis of diseased cells in batches.

[0026] The scheme provided in this application utilizes ivy saponin A1 as an AKT1 kinase inhibitor, which penetrates diseased tissue through local administration and specifically induces SK cell apoptosis.

[0027] The specific implementation principles include: (1) Molecular target: Experiments have shown that hedera saponin A1 can directly bind to AKT1 protein (KD value is 5.8 μM).

[0028] (2) Biological effects: Inhibition of AKT1 leads to upregulation of the pro-apoptotic protein (Cleaved-Caspase 3), thereby initiating cell apoptosis.

[0029] This application is the first to discover and verify the application of ivy saponins in the treatment of seborrheic keratosis, realizing the "repurposing of an old drug".

[0030] This application clarifies the mechanism of action of this compound as an "AKT1-specific inhibitor" in skin lesions.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. Precise and non-invasive, superior to physical therapy: Compared with laser and cryotherapy, the proposed treatment is non-invasive, painless, woundless, and leaves no scars after healing, making it particularly suitable for patients with multiple skin lesions and facial lesions.

[0032] 2. Mechanism-driven, potential cancer prevention: Unlike hydrogen peroxide corrosion, this invention works by blocking carcinogenic pathways. Given that the AKT pathway is also a driving force behind the transformation of SK into squamous cell carcinoma, this drug has the dual effects of "treating SK" and "chemopreventing malignant transformation."

[0033] 3. High safety: Hedera saponin A1 has a significant killing effect on SK diseased cells, and has a killing effect on the epidermal layer of SK isolated diseased tissue, but does not cause obvious apoptosis in dermal cells. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the binding kinetics and affinity (SPR) analysis of hederacolchiside A1 and the target protein AKT1. In diagram a, the binding analysis of the positive control drug Capivasertib with recombinant human AKT1 protein is shown, with the left side showing the multi-cycle binding kinetics sensor diagram and the right side showing the steady-state affinity fit curve. In diagram b, the binding analysis of hederacolchiside A1 with recombinant human AKT1 protein is shown, with the left side showing the multi-cycle binding kinetics sensor diagram and the right side showing the steady-state affinity fit curve.

[0035] Figure 2A schematic diagram illustrating the in vitro killing effect of ivy saponin A1 on SK primary cells; where a represents cell morphology observation under an inverted microscope; and b represents statistical analysis of cell viability.

[0036] Figure 3 This diagram illustrates the clearance effect of hederacolchiside A1 on isolated SK tissue fragments; where a represents histopathological observation using H&E staining; b represents TUNEL immunofluorescence staining for apoptosis detection, with blue indicating DAPI-labeled cell nuclei and red indicating TUNEL-positive apoptotic cells; and c represents statistical analysis of the TUNEL-positive cell rate. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0038] Example 1: Molecular-level verification (SPR binding experiment) Methods: Surface plasmon resonance (SPR) detection was performed using the Biacore system. Recombinant human AKT1 protein was immobilized on the chip surface. Capivasertib (a known AKT1 inhibitor) was used as a positive control. The binding kinetics of hederacolchiside A1 at different concentrations (20 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.25 μM, 0.08 μM) to AKT1 were detected.

[0039] result: Figure 1 In the SPR multi-cycle dynamics sensing diagrams on the left of a and b: different colors represent response signals at different concentrations, specifically within... Figure 1 a and b are marked on the right.

[0040] Figure 1 a represents the binding curve of Capivasertib (an AKT1-positive compound) to the AKT1 protein. The KD value was found to be 3.19 uM after fitting with a steady-state affinity model. This result is consistent with previously reported data, indicating the reliability of the experiment.

[0041] Figure 1 b shows the binding curve of ivy saponin A1 to AKT1 protein. The equilibrium dissociation constant (KD) of ivy saponin A1 is 5.8 μM, calculated by fitting the steady-state affinity model.

[0042] Conclusion: Experiments confirmed that ivy saponin A1 can directly and specifically bind to the AKT1 protein. Furthermore, under the same experimental system, its binding affinity is on the same order of magnitude as that of the known AKT inhibitor Capivasertib, indicating its significant target-binding ability.

[0043] Example 2: In vitro cytotoxic effect of ivy saponin A1 on SK primary cells Methods: Primary keratinocytes derived from SK patients were isolated and cultured, and divided into a blank control group (DMSO) and an experimental group (hederone A1 was dissolved in DMSO to obtain a stock solution, which was then added to the culture medium to a final concentration of 5 μM). After 48 hours of drug treatment, cell morphology changes were observed under an optical microscope, and cell viability was detected using the Cell Viability Assay (CCK-8 method). Statistical differences were analyzed using t-tests.

[0044] result: Morphological observation: like Figure 2 As shown in a, before the drug was added, the cells in the DMSO group and the ivy saponin A1 group (5μM) grew densely in a cobblestone pattern, with regular morphology and strong refractive properties. 48 hours after drug administration, the cells in the DMSO group still exhibited dense growth in a cobblestone pattern, with regular morphology and strong refractive properties; the cells in the hedera saponin A1 group (5 μM) showed significant morphological changes, including rounding, shrinking, breaking, and large-scale shedding of cells, with a significant decrease in the number of adherent cells.

[0045] Conclusion: At a concentration of 5 μM, hedera saponin A1 can induce apoptosis in SK cells.

[0046] Survival rate statistics: like Figure 2 As shown in b, the bar chart data shows that, compared with the DMSO control group (100%), the cell survival rate of the group treated with hedera saponin A1 (5 μM) dropped sharply to <5%.

[0047] Statistical analysis showed that the treatment group with hygrophila saponin A1 was significantly different from the control group (P<0.0001).

[0048] Conclusion: At a concentration of 5 μM, ivy saponin A1 exhibits potent cytotoxic activity, producing a strong killing effect on SK diseased cells at micromolar concentrations.

[0049] Example 3: Validation of the ex vivo tissue model Methods: Fresh SK tissue blocks obtained from clinical resection were used to establish an air-liquid interface culture model (this model is a known technique, using Transwell chambers as supports. The tissue blocks were placed on the microporous membrane of the chamber, with the culture medium below the membrane providing nutrients, and the cell surface above the membrane in direct contact with the air). The experimental group was topically treated daily with a DMSO solution (1 mM concentration) containing hedera oleifera saponin A1, while the control group was treated with DMSO solvent. The treatment lasted for 48 hours.

[0050] result: H&E (Historical and Morphological Assessment): like Figure 3 As shown in Figure a, the SK tissue block in the DMSO solvent control group maintained the complete pathological structure, showing significant hyperkeratosis and acanthosis with tightly packed cells; after 48 hours of local application of 1mM ivy saponin A1 to the experimental group, the tissue morphology changed significantly, and the overall thickness of the epidermis became thinner.

[0051] Safety observation: It is noteworthy that, in H&E staining, the dermal collagen fiber structure (pink area) beneath the ivy saponin A1 treatment group remained intact, with no obvious nonspecific necrosis or inflammatory infiltration, suggesting that the drug has good safety for dermal tissue while effectively destroying the diseased epidermis.

[0052] Validation of apoptosis mechanism (TUNEL): like Figure 3 As shown in b, TUNEL immunofluorescence staining detected cell apoptosis. Blue represents DAPI-labeled cell nuclei, and red represents TUNEL-positive apoptotic cells. Compared with the DMSO group, the epidermis of the hedera saponin A1 treatment group showed extensive and strong red fluorescence signals, and the apoptosis signal was significantly enhanced, indicating that the drug successfully induced large-scale cell apoptosis at the tissue level. In addition, red fluorescence was also widely distributed throughout the spinous layer and basal layer, indicating that the drug successfully penetrated the tissue and initiated the apoptosis program in the core area of ​​the lesion. like Figure 3 As shown in Figure c, statistical analysis of the TUNEL-positive cell rate revealed that the TUNEL-positive cell rate increased to over 60%, and the apoptosis rate in the hedera oleifera saponin A1 treatment group was significantly higher than that in the control group, confirming its effectiveness in clearing SK lesions through a pro-apoptotic mechanism. TUNEL fluorescence staining further confirmed that the aforementioned tissue damage was mediated by apoptosis.

[0053] Statistical analysis: The t-test results showed that the difference between the two groups was statistically significant (**P<0.01).

[0054] Conclusion: With solvent assistance, hedera saponin A1 can effectively penetrate tissues and induce apoptosis clearance in SK tissues.

[0055] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of ivy saponin A1 in the preparation of drugs for the prevention and / or treatment of seborrheic keratosis or its secondary lesions.

2. The application according to claim 1, the use of ivy saponin A1 in the preparation of a medicament for treating or alleviating seborrheic keratosis or its secondary lesions.

3. The application according to claim 1, characterized in that, The use of the combination of ivy saponin A and transdermal penetration enhancer in the preparation of drugs for the prevention and / or treatment of seborrheic keratosis or its secondary lesions.

4. The application according to claim 3, characterized in that, The transdermal penetration enhancer is selected from one of dimethyl sulfoxide, azone, propylene glycol, or a surfactant.

5. The application according to claim 1, characterized in that, The amount of ivy saponin A1 in the drug is 0.5wt% - 5.0wt%.

6. The application according to claim 1, characterized in that, Application of Hedera styracifolium saponin A1 in the preparation of drugs that directly bind to AKT1 protein to treat or alleviate seborrheic keratosis or its secondary lesions.

7. The application according to claim 1, characterized in that, Application of Hedera oleifera saponin A1 in the preparation of drugs that induce apoptosis by inhibiting AKT1, thereby initiating apoptosis to treat or alleviate seborrheic keratosis or its secondary lesions.

8. The application according to claim 1, characterized in that, Secondary complications of seborrheic keratosis include inflammation and infection, hyperplasia and hypertrophy of skin lesions caused by seborrheic keratosis.

9. The application according to claim 1, characterized in that, The drug is a topical preparation.

10. The application according to claim 1, characterized in that, The dosage form of the drug is ointment, cream, gel, or patch.

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