Use of BCL2 inhibitor in treatment of giant congenital melanocytic nevi

Abstract

Use of a BCL2 inhibitor in the preparation of a drug or medical instrument for treating giant congenital melanocytic nevi (GCMN). The use of the BCL2 inhibitor in GCMN has been validated to exert a therapeutic effect by means of reversing anti-apoptotic response of GCMN and inducing apoptosis of GCMN. In addition, good killing effects have been achieved in both primary GCMN cells from multiple patients and animal experiments, indicating that the BCL2 inhibitor has a strong and specific killing effect on giant nevus cells while exerting minimal impact on other normal cells, thereby achieving precise therapeutic and killing effects to address the predicament of the lack of effective treatment methods for GCMN.

Description

Application of BCL2 inhibitors in the treatment of congenital giant nevi Technical Field

[0001] This invention relates to a protein inhibitor, and more particularly to a protein inhibitor associated with apoptosis, and its use in the preparation of drugs or medical devices for treating congenital giant nevi. Background Technology

[0002] Giant congenital melanocytic nevi (GCMN) is a skin disease caused by abnormal proliferation of melanocytes. Affected children are born with extensive blackening of the skin, and the main lesion is clinically defined as having a maximum diameter >40 cm in adulthood. The affected skin also exhibits epidermal thickening, abnormal skin appendages, varying degrees of hair hyperplasia, and nodule formation.

[0003] In addition, most affected children also have scattered satellite nevi of varying sizes and numbers throughout the body. Even more serious cases can involve the central nervous system, leading to neurocutaneous melanosis (NCM). Statistics show that the incidence of GCMN in newborns is approximately 1 in 50,000, with a malignant transformation rate of 4% to 8%.

[0004] In summary, GCMN not only severely impacts patients' mental health, but its lifelong potential for malignant transformation also poses a threat to their lives. Therefore, research on drug treatment for GCMN has significant clinical implications.

[0005] Currently, no drugs have been approved for the treatment of GCMN; only animal and cell experiments have been conducted. To investigate potential drug targets for GCMN, several international research teams have used first- or second-generation sequencing technology to detect target gene mutations in giant nevi. The results show that 50%–70% of GCMN patients have NRAS somatic mutations. Due to the high NRAS mutation rate in GCMN patients, the generally accepted theory is that the occurrence and development of GCMN are inextricably linked to NRAS mutations. NRAS mutations can cause sustained activation of the MAPK and PI3K-AKT signaling pathways. Inhibitors of these pathways, such as MEK inhibitors, ERK inhibitors, and AKT inhibitors, have become a focus of research. They have all shown some efficacy in treating GCMN at the cellular and animal levels, but they cannot completely eradicate GCMN cells. There have also been clinical case reports abroad showing that palliative treatment with the MEK inhibitor trametinib for severely ill GCMN patients has shown promising immediate results, but resistance is easily developed, leading to poor long-term efficacy and ultimately a poor prognosis for most patients.

[0006] Furthermore, research on targeted therapy using the nevus cell biomarker SOX10 has only been conducted in the preclinical stage, and it cannot specifically kill nevus cells without damaging normal melanocytes, potentially leading to significant side effects. Therefore, there is currently no effective targeted treatment for congenital giant congenital melanocytic nevi, and research on its drug therapy still needs further in-depth exploration and refinement.

[0007] Summary of the Invention

[0008] One object of the present invention is to provide an active substance for treating congenital giant nevi by inhibiting BCL2 protein.

[0009] Another object of the present invention is to provide the application of a BCL2 inhibitor in the preparation of a drug for treating congenital giant nevi.

[0010] Another object of the present invention is to provide the use of the composition in the preparation of a medicament for treating congenital giant nevi.

[0011] Another object of the present invention is to provide the application of a medical device containing a BCL2 inhibitor in the preparation of a drug for treating congenital giant nevi.

[0012] This invention is based on an important phenomenon discovered in GCMN research: anti-apoptotic response. Anti-apoptotic pathways are significantly upregulated in GCMN, while apoptosis-related pathways are significantly downregulated. Furthermore, BCL2 was found to be widely and highly expressed in GCMN cells from multiple patients, but not in keratinocytes, fibroblasts, or normal epidermal melanocytes in skin tissue. These findings indicate that GCMN cells possess significant anti-apoptotic activity, enabling them to survive persistently in skin tissue and avoid apoptosis and clearance. This phenomenon is also consistent with the clinical manifestations of GCMN, namely, GCMN lesions can persist on the patient's skin throughout life without regression.

[0013] This invention discloses an active substance for treating congenital giant nevi, which is a BCL2 (protein) inhibitor. Active substances that inhibit BCL2 include: Venetoclax (CAS: 152459-95-5, trade name: Venetoclax), ABT-263 (CAS: 923564-51-6, trade name: Navitoclax), ABT-737 (CAS: 852808-04-9), GX15-070 (CAS: 803712-79-0, trade name: Obatoclax), BGB-11417 (CAS: 2383086-06-2, trade name: Sonrotoclax), APG-2575 (CAS: 2180923-05-9, trade name: Lisaftoclax), and TW-37 (CAS: 877877-35-5).

[0014] By using BCL2 inhibitors, the anti-apoptotic response of GCMN is suppressed, inducing its apoptosis and achieving the goal of treating GCMN. Furthermore, since only GCMN specifically highly expresses BCL2 in tissues, this therapy will be able to specifically kill GCMN cells while avoiding damage to normal cells in other skin tissues.

[0015] Preferably, the active substance in the present invention for treating congenital giant nevi is Venecra, which has the strongest killing effect on primary GCMN cells.

[0016] In this article, "pharmaceutically acceptable salts" refers to salts that, within the bounds of reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and whose benefits / risks are proportionate to a reasonable ratio. Pharmaceutically acceptable salts are well known in the art, such as hydrochlorides, sulfonates, sulfates, phosphates, citrates, methanesulfonates, amino acid salts, sodium salts, potassium salts, calcium salts, ammonium salts, and other salts suitable for medicinal use.

[0017] The BCL2 inhibitor shown in this invention is mixed with other excipients to prepare a drug (formulation) for treating congenital giant nevi.

[0018] These pharmaceutical excipients can be those commonly used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for use to be compatible with the substances in the formulation, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the drug delivery effect of the composition.

[0019] In aqueous injections, excipients generally include isotonic agents and buffer solutions, as well as necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer), solubilizers, and antibacterial agents. In addition, they may include other pharmaceutically acceptable excipients, such as antioxidants, pH adjusters, and analgesics.

[0020] Excipients used in the preparation of oral liquid formulations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers, and coloring agents.

[0021] Excipients used in tablet manufacturing generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0022] The excipients used in the preparation of emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0023] The excipients used to produce granules are similar to those used for tablets, but the granulation process is different. Depending on the requirements, the produced granules are mixed with a gliding agent and then encapsulated to obtain capsules.

[0024] Various excipients and compounds are used to formulate dosage forms that facilitate drug delivery, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release formulations, and controlled-release formulations. Furthermore, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release, controlled-release, and pulsatile administration, including, but not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The main manifestations of "facilitating drug delivery" include, but are not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0025] The BCL2 inhibitor of this invention can be combined with other excipients, such as through chemical coupling, to further improve the efficacy of the compound, reduce toxicity, and prolong the dosing cycle. These excipients are typically polymers, such as polyesters, polyethers, and polyamides.

[0026] Drug-eluting medical devices combining BCL2 inhibitors with medical devices are already quite common, such as dressings containing BCL2 inhibitors. Microneedles and microneedle arrays are also being created by mixing BCL2 inhibitors with biocompatible and biodegradable materials, or by embedding them in metal microneedles to create microneedle chips. When the microneedles pierce the skin, they release the BCL2 inhibitor into the dermis, achieving precise treatment.

[0027] This invention is the first to use BCL2 inhibitors to treat GCMN, attempting to reverse its anti-apoptotic response and induce apoptosis in GCMN to achieve a therapeutic effect. It has achieved good killing effects in primary GCMN cells of multiple patients, animal experiments, and transgenic animal models, suggesting that BCL2 inhibitors have a strong and specific killing effect on giant nevus cells, with little impact on other normal cells, thus playing a precise therapeutic and killing role, in order to solve the dilemma of lacking effective treatments for GCMN. Attached Figure Description

[0028] Figure 1 shows the functional enrichment analysis of differentially expressed genes in giant nevi. Among them, the anti-apoptotic pathway was the most significantly enriched in giant nevi (within the box).

[0029] Figure 2 shows the changes in the lesion of a patient with a congenital giant nevus over 10 years.

[0030] Figure 3 is a volcano plot of differentially expressed genes between giant nevus tissue and normal skin adjacent to the nevus, analyzed by RNA-seq. The dots to the left of the dashed line represent genes that are significantly downregulated in giant nevi, and the dots to the right of the dashed line represent genes that are significantly upregulated in giant nevi. The BCL2 gene, which is related to cell survival and anti-apoptosis, is significantly upregulated in giant nevi and has a large Log2 (Fold Change) value (marked in the figure).

[0031] Figure 4 shows the immunohistochemical staining results of tissue microarrays for a large sample size (36 cases) of giant nevi.

[0032] Figure 5 shows the results of using CCK8 technology to detect the killing effect of common BCL2 inhibitors on primary cells of 5 different genotypes of giant nevi.

[0033] Figure 6 shows the changes in morphology and apoptosis ratio of giant nevus cells after intervention with BCL2 inhibitor (Venetoclax) and MEK inhibitor (Trametinib).

[0034] Figure 7 shows the comparison of the effects of BCL2 inhibitors and MEK inhibitors on killing cells in patients with giant nevi.

[0035] Figure 8 shows that BCL2 inhibitors exhibited significant killing effects in giant nevus cells of different genotypes (within the box).

[0036] Figure 9 shows the results of BCL2 inhibitor (Venetoclax) and MEK inhibitor (Trametinib) intervention on normal melanocytes and fibroblasts. In the figure, A is the morphological results of each inhibitor group, and B is the statistical graph of cell activity of each inhibitor group.

[0037] Figure 10 shows the HE pathological results after immersing and culturing giant nevus tissue blocks in various culture media.

[0038] Figure 11 shows the HE pathological results of the PDX model of giant nevus after injection of various inhibitors.

[0039] Figure 12 shows the phenotypic characteristics of NrasQ61K mutant giant nevus transgenic mice in the hair, paw, tail, and skin tissues; where WT is a normal wild-type C57 mouse, and NrasQ61K is an NRAS mutant giant nevus transgenic mouse constructed using C57 mice as a background.

[0040] Figure 13 shows the phenotypic changes in transgenic mice with unshaved giant nevi treated with drug injection.

[0041] Figure 14 shows the phenotypic changes in transgenic mice treated with drug injection for shaving giant nevi.

[0042] Figure 15 shows the pathological results of the tail and hair follicles of the giant nevus transgenic mouse after drug injection treatment.

[0043] Figure 16 shows the pathological manifestations of the skin of transgenic mice with giant nevi after drug injection treatment. Detailed Implementation

[0044] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0045] Example 1: Establishment of the anti-apoptotic status of congenital giant nevus and target screening

[0046] To address the lack of effective drug treatments for congenital giant nevi (GNs), the inventors previously performed RNA-seq on GN samples and adjacent normal skin to screen for potential targets. We found that apoptosis and inflammation pathways were most significantly downregulated in GNs, as shown in Figure 1. The most significantly enriched pathway in GNs was the anti-apoptotic pathway (highlighted in the box). We also found cell cycle inhibition, suggesting that GNs are not only in a state of cell cycle arrest, but also exhibit highly active anti-apoptotic activity, characterized by "low proliferation and low apoptosis." This indicates a strong anti-apoptotic response in GNs, which explains why congenital giant nevi can persist in human skin.

[0047] As shown in Figure 2, giant nevi exhibit growth arrest after birth, with the lesions failing to grow and persisting for up to ten years without regression (the faded areas in the figure are due to dermabrasion treatment). They can remain in the skin throughout a person's life. These phenomena suggest that giant nevi and moles likely possess important mechanisms for maintaining their survival and escaping apoptosis.

[0048] Since the anti-apoptotic activity of melanocytic nevi was the most significant, we further analyzed RNA-seq data from three different gene mutations (including NRAS mutation, BRAF mutation, and wild-type) of giant nevi to clarify the RNA expression levels of anti-apoptotic and apoptotic genes, and to screen for targets that could reverse the anti-apoptotic response of giant nevi cells and promote their apoptosis. We found that the expression of the anti-apoptotic gene BCL2 was most significantly upregulated (Figure 3).

[0049] Furthermore, at the protein level, as shown in Figure 4, IHC analysis of giant nevus tissue revealed that AEC staining labeled BCL2. Tissue microarray staining results showed that BCL2 was expressed in both superficial and deep dermal nevus cells, and was widely and strongly positive in all patient tissues tested, indicating a universal phenomenon. Moreover, BCL2 was also widely and highly expressed in giant nevus tissues of different genotypes (BRAF fusion gene, BRAF mutation, NRAS mutation, and wild type), suggesting that BCL2-mediated anti-apoptotic response is a common feature of giant nevi and is not affected by genotype.

[0050] Because BCL2 is widely overexpressed in many patients with giant nevus and is an important gene mediating anti-apoptotic responses, BCL2 was ultimately chosen as the target for giant nevus cells.

[0051] Example 2: BCL2 inhibitors showed good targeted killing effect on congenital giant nevus cells.

[0052] We intervened with primary giant nevus cells using the five most commonly used BCL2 inhibitors and used the CCK8 assay to detect cell viability. We found that, at the same concentration, Venetoclax showed the most significant killing effect on giant nevus cells from different patients (n=5: NRAS mutation=2, BRAF mutation=1, BRAF fusion gene mutation=1, wild type=1) (Figure 5, boxed area).

[0053] The MEK inhibitor (Trametinib), which has been most extensively studied in congenital giant nevi, was used as a control in a drug intervention experiment on giant nevus cells (n=9). We found that at the same concentration (10 μmol), the BCL2 inhibitor caused cell nucleus shrinkage and rupture, with a large amount of cell debris, compared to the MEK inhibitor and DMSO groups (Figure 6). The cells in the MEK and DMSO groups had normal cell morphology. In terms of apoptosis rate, the BCL2 inhibitor induced approximately 84% apoptosis (the sum of the two boxes in Figure 6), which was much higher than the 40% in the MEK inhibitor group.

[0054] Figure 7 shows a comparison of the killing effects of BCL2 inhibitors and MEK inhibitors on cells from patients with giant nevi (n=9, NRAS mutation=3, BRAF mutation=3, wild-type=3). Compared to the traditional targeted drug for giant nevi, the MEK inhibitor (Trametinib), the BCL2 inhibitor (Venetoclax) showed a significant killing effect at a concentration of 2.5 μM, inhibiting 85% of the activity of giant nevi, and reaching over 90% at higher concentrations (10 μM). Its killing effect is far superior to that of MEK inhibitors. The average IC50 of BCL2... 50 The value was approximately 1.072 μM, while the average IC50 of MEK inhibitors was... 50 Value >10μM.

[0055] To confirm that BCL2 inhibitors have a good killing effect on giant nevus cells with different gene mutations, we treated giant nevus cells with NRAS mutation, BRAF mutation and wild type with BCL2 inhibitor and MEK inhibitor respectively. We found that BCL2 has a good killing effect on giant nevus cells with different gene mutations (Figure 8), but MEK inhibitor only significantly kills giant nevus cells with a specific gene mutation (BRAF mutation).

[0056] We further verified the targeting and safety of the BCL2 inhibitor. The results are shown in Figure 9. At the same concentration (10 μM), the BCL2 inhibitor, MEK inhibitor, and DMSO groups did not significantly kill normal melanocytes or fibroblasts. Cell morphology was normal, with no nuclear shrinkage or rupture, and no large amounts of cell debris were observed. Cell viability assays showed that the IC50 values ​​of the BCL2 inhibitor and MEK inhibitor were relatively high. 50All values ​​were greater than 10 μM. These results suggest that BCL2 has a specific killing effect on giant nevus cells, with minimal impact on normal cells. No significant killing response was observed in normal melanocytes and fibroblasts after the addition of the BCL2 inhibitor, indicating that the BCL2 inhibitor can specifically kill giant nevus cells. In contrast, the traditional drug MEK inhibitor also had no significant effect on normal melanocytes and fibroblasts.

[0057] The above results suggest that BCL2 inhibitors have a good and specific killing effect on congenital giant nevi, and this phenomenon exists in the cells of giant nevi from multiple patients with different gene mutations. The targeted killing effect of BCL2 inhibitors is a common feature in giant nevi.

[0058] Example 3: BCL2 inhibitors showed good targeted killing effect on congenital giant nevus tissue masses.

[0059] HE pathological features of giant nevus tissue blocks after soaking and culturing in a culture medium containing drug-free BCL2 inhibitor (Venetoclax) and MEK inhibitor (Trametinib).

[0060] In this embodiment, the immersion culture method was used to observe the killing effect of the drug on the giant nevus tissue block. The culture medium was MELM medium, and BCL2 inhibitor and MEK inhibitor were added to the culture medium at a concentration of 10 μM. The control group was the DMSO group. As shown in Figure 10, compared with the control group and the MEK inhibitor group, the BCL2 inhibitor group induced the disintegration of nevus cell nests, cell nucleus shrinkage, disordered morphology and structure, and increased cell debris, indicating a good killing effect. The BCL2 inhibitor group significantly killed nevus cells and cell nests in the giant nevus tissue block.

[0061] In addition, tissue blocks of congenital giant nevi were implanted under the skin of the necks of C-NKG immunodeficient mice (purchased from Cyagen Biosciences) to construct a PDX model of giant nevi. After two months of complete survival of the tissue blocks, drugs were injected into the tissue blocks, divided into DMSO group, MEK inhibitor group, and BCL2 inhibitor group. The results are shown in Figure 11. In the BCL2 inhibitor group, the number of nevus cells in the superficial and deep dermis of the tissue block decreased, melanin faded, and nevus cell nests disappeared, while the MEK group and DMSO group still had a large number of nevus cells and nevus cell nests. These results demonstrate the effective killing effect of BCL2 inhibitors on giant nevi in ​​the PDX model.

[0062] Example 4: BCL2 inhibitors showed good killing effect in a transgenic animal model of congenital giant nevus.

[0063] The NRAS Q61K mutation is the most common mutation type in congenital giant nevi. Based on this, this embodiment constructed a Tyr-Nras Q61K mutant mouse, which exhibited the phenotype of congenital giant nevi, including significant melanin deposition on the skin, palms, and tail, and darkened hair. As shown in Figure 12, compared with the phenotype of normal C57 mice, the mouse showed darker hair all over its body, and extensive melanin lesions resembling giant nevi were visible on the palms, tail, and skin.

[0064] Local injections of drugs were administered to the skin lesions of congenital giant nevi in ​​transgenic mice. The groups included the DMSO group, the MEK inhibitor group, and the BCL2 inhibitor group. As shown in Figure 13, mice in the BCL2 inhibitor group exhibited whitening of hair and a fading of tail pigmentation after drug intervention, a phenomenon not observed in the MEK inhibitor group or the DMSO group.

[0065] As shown in Figure 14, the skin moles in the mice in the BCL2 inhibitor group regressed after drug intervention, while no significant changes were observed in the MEK inhibitor group and the DMSO group.

[0066] The HE staining results are shown in Figures 15 and 16. Nevus cells and melanin in the dermis and hair follicles of the mouse tail significantly regressed after BCL2 inhibitor intervention, while a large number of nevus cells and melanin remained in the MEK inhibitor group and the DMSO group. These results indicate that BCL2 inhibitors can induce regression of giant nevus lesions and whitening of hair in a mouse animal model simulating human giant nevus, demonstrating a good killing effect.

Claims

1. The application of a BCL2 inhibitor in the preparation of a drug or medical device for treating congenital giant nevi.

2. The application according to claim 1, characterized in that... The BCL2 inhibitors include one or more of Veneclare, ABT-263, ABT-737, GX15-070, BGB-11417, APG-2575 and TW-37, and their pharmaceutically acceptable salts.

3. The application according to claim 1, characterized in that... The BCL2 inhibitors mentioned include Veneclare and its pharmaceutically acceptable salts.

4. The application according to claim 3, characterized in that... The Veneclare mentioned is Veneclare hydrochloride.

5. The application according to claim 1, characterized in that... The drug has a BCL2 inhibitor as its sole active ingredient and also contains pharmaceutical excipients.

6. The application according to claim 1, characterized in that... The medical device described herein is an excipient with BCL2 inhibitor as the sole active ingredient.

7. The nucleic acid chain according to claim 5, characterized in that... The medical device described is a microneedle containing a BCL2 inhibitor as the sole active ingredient.

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