Application of BCL2 inhibitor in treatment of congenital megathema

Abstract

The invention relates to an application of a BCL2 inhibitor in the preparation of a medicine or a medical device for treating a congenital megaterium nevi (GCMN), in particular to an application of a BCL2 inhibitor in the preparation of a medicine or a medical device for treating the GCMN. When the BCL2 inhibitor acts on GCMN, it is verified that the BCL2 inhibitor has the effects of reversing the anti-apoptosis reaction of GCMN and inducing GCMN apoptosis to achieve the treatment effect, and a good killing effect is achieved on GCMN primary cells of multiple patients and the animal experiment level, and it is indicated that the BCL2 inhibitor is high in killing effect on megaevus cells, has specificity, has small influence on other normal cells, and has good clinical application prospects. The effects of precise treatment and killing are achieved, and the awkward situation that the GCMN lacks an effective treatment method is solved.

Description

Technical Field

[0001] The invention relates to a protein inhibitor, in particular to a protein inhibitor related to cell apoptosis, and application of the protein inhibitor in preparing medicine or medical device for treating giant congenital nevus. Background Art

[0002] Giant congenital melanocytic nevi (GCMN) is a skin disease caused by abnormal proliferation of melanocytes. Children with this disease have large areas of melanosis at birth, and the main lesion is expected to have a maximum diameter of >40 cm in adulthood. The clinical diagnosis is defined as giant congenital nevi. The skin at the lesion site also shows epidermal thickening, abnormal skin appendages, varying degrees of hair hyperplasia and nodule formation.

[0003] In addition, most children also have scattered satellite nevi of varying sizes and numbers. In some cases, the lesions may affect the central nervous system, leading to poor development of the nervous system and neurocutaneous melanosis (NCM). According to statistics, the incidence of GCMN in newborns is about 1 / 50,000, and the incidence of malignant transformation to melanoma is 4% to 8%.

[0004] In summary, GCMN not only seriously affects the patient's mental health, but its potential lifelong malignant transformation also threatens the patient's life safety. Therefore, research on drug treatment of GCMN has important clinical significance.

[0005] At present, no drug has been approved for the treatment of GCMN, and only animal and cell experiments have been conducted. In order to study the potential drug targets for GCMN, several foreign research teams have used first-generation or second-generation sequencing technology to detect target gene mutations in giant nevi. The results showed that 50% to 70% of GCMN patients had NRAS somatic mutations. Since GCMN patients have a high NRAS mutation rate, the currently recognized theory in academia is that the occurrence and development of GCMN is closely related to NRAS mutations. NRAS mutations can cause the continuous activation of the MAPK and PI3K-AKT signaling pathways, and inhibitors of these pathways, such as MEK inhibitors, ERK inhibitors, and AKT inhibitors, have become the focus of research. They have all played a certain role in the treatment of GCMN at the cellular and animal levels, but they cannot completely kill GCMN cells. There have also been clinical case reports abroad that the use of the MEK inhibitor trametinib for palliative treatment of seriously ill GCMN patients has a good immediate effect, but it is easy to develop drug resistance, the long-term effect is poor, and most patients ultimately have a poor prognosis.

[0006] In addition, the targeted research and treatment of the nevus cell biomarker SOX10 have only been studied at the preclinical stage, and it cannot specifically kill nevus cells without damaging normal melanocytes, and the side effects may be relatively large. Therefore, there is currently no effective targeted treatment method for congenital giant hairy nevus, and the research on its drug treatment still needs to be further explored and improved. Summary of the Invention

[0007] An object of the present invention is to provide an active substance for treating congenital giant nevus to inhibit BCL2 protein.

[0008] Another object of the present invention is to provide the use of a BCL2 inhibitor in the preparation of a drug for treating congenital giant nevus.

[0009] Another object of the present invention is to provide the use of a composition in the preparation of a drug for treating congenital giant nevus.

[0010] Another object of the present invention is to provide the use of a medical device containing a BCL2 inhibitor in the preparation of a drug for treating congenital giant nevus.

[0011] The present invention is proposed based on an important phenomenon found in the GCMN study: the anti-apoptotic response, in which the anti-apoptotic related pathways are significantly up-regulated in GCMN, and the apoptotic related pathways are significantly down-regulated. In the GCMN tissues of multiple patients, it was also found that BCL2 is widely highly expressed in GCMN cells, but not expressed in keratinocytes, fibroblasts and normal epidermal melanocytes in skin tissues. These findings indicate that GCMN cells have obvious anti-apoptotic activity, enabling them to continuously survive in skin tissues and avoid apoptosis and being cleared. This phenomenon is also consistent with the clinical manifestations of GCMN, that is, the GCMN lesions can persist on the skin of patients and never regress throughout life.

[0012] An active substance for treating congenital giant nevus according to the present invention is an inhibitor of BCL2 (protein). Active substances that exert an inhibitory effect on 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).

[0013] Use a BCL2 inhibitor to inhibit the anti-apoptotic response of GCMN, induce its apoptosis, and achieve the purpose of treating GCMN. In addition, since only GCMN highly expresses BCL2 specifically in tissues, this therapy will be able to specifically kill GCMN cells while avoiding damaging normal cells in other skin tissues.

[0014] Preferably, the active substance for treating congenital giant congenital nevus in the present invention is venetoclax, which has the strongest killing effect on primary GCMN cells.

[0015] As used herein, "pharmaceutically acceptable salts" refers to those salts that are suitable for contact with the tissues of humans and lower animals within the scope of reasonable medical judgment, without undue toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art, such as: hydrochloride salts, sulfonate salts, sulfate salts, phosphate salts, citrate salts, mesylate salts, amino acid salts, sodium salts, potassium salts, calcium salts, ammonium salts, and other salts suitable for use as medicaments.

[0016] Mix the BCL2 inhibitor shown in the present invention as an active ingredient with other excipients to prepare a drug (preparation) for treating congenital giant congenital nevus.

[0017] These pharmaceutical excipients can be those conventionally used in various preparations, such as: but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants, etc.; or they can be selected for use to adapt to the said substance, such as: emulsifying agents, solubilizing agents, bacteriostatic agents, analgesics, and antioxidants, etc. Such 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, etc., thereby improving the metabolism of various compounds in vivo and further enhancing the administration effect of the composition.

[0018] In aqueous solution injections, the excipients generally include isotonic agents and buffers, as well as necessary emulsifying agents (such as: Tweeen-80, Pluronic, and Poloxamer, etc.), solubilizing agents, and bacteriostatic agents, etc. In addition, it also includes other pharmaceutically acceptable pharmaceutical excipients, such as: antioxidants, pH regulators, and analgesics, etc.

[0019] The excipients for preparing oral liquid preparations generally include solvents, as well as necessary flavoring agents, bacteriostatic agents, emulsifying agents, and coloring agents, etc.

[0020] Excipients for preparing tablets generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, and mannitol, etc.), 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, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, and cross-linked sodium carboxymethyl cellulose), and lubricants (such as magnesium stearate, colloidal silica, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate, etc.), etc.

[0021] Excipients for preparing emulsions generally include water, oil (such as fatty acids), emulsifiers, as well as necessary preservatives and flavoring agents, etc.

[0022] Excipients for preparing granules are similar to those for tablets, but the granulation process is different. According to needs, the prepared granules are mixed with glidants and then filled into capsules to obtain capsule agents.

[0023] Various excipients and compounds are made into dosage forms that are beneficial for drug delivery, such as: but not limited to aqueous injection solutions, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsule agents, aerosols, sprays, powder aerosols, sustained-release agents, and controlled-release agents, etc. In addition, excipients used to achieve specific drug delivery purposes or methods, such as: sustained-release drug delivery, controlled-release drug delivery, and pulsed drug delivery, etc., are also used, such as: but not limited to gelatin, albumin, chitosan, polyethers, and polyester-based polymer materials, such as: but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers, etc. The so-called "beneficial for drug delivery" mainly includes: but not limited to improving the therapeutic effect, enhancing the bioavailability, reducing the toxic and side effects, and improving the patient compliance, etc.

[0024] Combining the BCL2 inhibitor of the present invention with other excipients, such as: chemical coupling, to further improve the drug efficacy of the compound, reduce the toxic effects, and extend the dosing cycle, etc. These excipients are usually polymers, such as: polyesters, polyethers, and polyamides, etc.

[0025] Drug-containing medical devices made by combining the BCL2 inhibitor with medical devices have also been relatively common, such as: dressings containing the BCL2 inhibitor. And they are mixed with biocompatible and biodegradable materials to make microneedles and their microneedle arrays, or loaded into metal microneedles to make microneedle chips. When the microneedles penetrate the skin, the BCL2 inhibitor is released into the dermis layer to achieve precise treatment.

[0026] For the first time, the present invention uses BCL2 inhibitors to treat GCMN, attempts to reverse its anti-apoptotic response, induces apoptosis of GCMN to play a therapeutic role, and has achieved good killing effects at the levels of primary GCMN cells of multiple patients, animal experiments, and transgenic animal models. It is suggested that BCL2 inhibitors have strong and specific killing effects on giant nevus cells, have little impact on other normal cells, and play a role in precise treatment and killing to solve the dilemma of the lack of effective treatment methods for GCMN. Description of the Drawings

[0027] Figure 1 Functional enrichment analysis of differentially expressed genes in giant nevi. Among them, the most significantly enriched in giant nevi is the anti-apoptotic pathway (at the boxed line).

[0028] Figure 2 Lesion changes of a patient with congenital giant nevus over 10 years.

[0029] Figure 3 Volcano plot of differentially expressed genes between giant nevus tissues and adjacent normal skin analyzed by RNA-seq; among them, the points to the left of the dashed line represent genes significantly down-regulated in giant nevi, the points to the right of the dashed line represent genes significantly up-regulated in giant nevi, and the BCL2 gene related to cell survival and anti-apoptosis is significantly up-regulated in giant nevi and has a large Log2(Fold Change) value (marked in the figure).

[0030] Figure 4 Immunohistochemical staining results of a tissue chip of giant nevi with a large sample size (36 cases).

[0031] Figure 5 Results of using the CCK8 technique to detect the killing effects of common BCL2 inhibitors on primary cells of 5 different genotypes of giant nevi.

[0032] Figure 6 Results of morphological and apoptotic ratio changes after BCL2 inhibitor (Venetoclax) and MEK inhibitor (Trametinib) intervened in giant nevus cells.

[0033] Figure 7 Results of the comparison of the killing effects of BCL2 inhibitor and MEK inhibitor on cells of giant nevus patients.

[0034] Figure 8 The BCL2 inhibitor showed significant killing effects in giant nevus cells of different genotypes (at the boxed line)

[0035] Figure 9Results of the intervention of BCL2 inhibitor (Venetoclax) and MEK inhibitor (Trametinib) on normal melanocytes and fibroblasts. Among them, A is the morphological result diagram of each group of inhibitors, and B is the statistical chart of cell viability of each group of inhibitors.

[0036] Figure 10 HE pathological result diagram after soaking and culturing giant nevus tissue blocks with various culture media.

[0037] Figure 11 HE pathological results after injecting drugs into the giant nevus PDX model with various inhibitors.

[0038] Figure 12 Phenotype diagrams of tissues such as hair, paw, tail, and skin of transgenic giant nevus mice with Nras Q61K mutation; among them, WT is a normal wild-type C57 mouse, and NrasQ61K is a transgenic giant nevus mouse with NRAS mutation constructed on the background of C57 mice.

[0039] Figure 13 Phenotype change result diagram of transgenic giant nevus mice without shaving hair treated with drug injection.

[0040] Figure 14 Phenotype change result diagram of transgenic giant nevus mice with shaving hair treated with drug injection.

[0041] Figure 15 Pathological manifestation result diagram of the mouse tail and hair follicles of transgenic giant nevus mice after drug injection treatment.

[0042] Figure 16 Pathological manifestation result diagram of the skin of transgenic giant nevus mice after drug injection treatment. Detailed implementation manners

[0043] The technical solutions 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 solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

[0044] Example 1 Establishment of anti-apoptotic state and target screening of congenital giant nevus

[0045] To solve the dilemma of the lack of effective drug treatment methods for congenital giant nevus (i.e., giant nevus), the inventors previously performed RNA-seq on giant nevus samples and adjacent normal skin to screen potential targets. We found that the apoptosis and inflammation pathways were most significantly downregulated in giant nevi, such asFigure 1 As shown, the most significantly enriched pathway in giant nevi is the anti-apoptotic pathway (at the boxed area). It was also found that the cell cycle was inhibited, suggesting that giant nevi are not only in a state of cell cycle arrest, but also have very active anti-apoptotic activity, manifested as "low proliferation, low apoptosis", indicating that giant nevi have a strong anti-apoptotic response, which also explains why congenital giant nevi can survive continuously in human skin.

[0046] As Figure 2 shown, giant nevi show a growth arrest state after birth, the lesions do not grow, and they survive continuously for ten years after birth without regression (the lightened areas in the figure are due to dermabrasion treatment), and can persist in human skin throughout life. These phenomena suggest that there are very likely important mechanisms for giant nevi and nevi to maintain their survival and evade apoptosis.

[0047] Since the anti-apoptotic activity of nevi is the most significant, we further analyzed the RNA-seq data of giant nevus tissues with three different gene mutations (including NRAS mutation, BRAF mutation, and wild type) to clarify the RNA expression levels of their anti-apoptotic and apoptotic genes, and screen for targets that reverse the anti-apoptotic response of giant nevus cells and promote their apoptosis. We found that the anti-apoptotic gene BCL2 was the most significantly upregulated ( Figure 3 ).

[0048] In addition, at the protein level, as Figure 4 shown, IHC of giant nevus tissues showed that AEC staining was used to label BCL2, and the results of tissue microarray staining showed that BCL2 was expressed in nevus cells in the superficial and deep dermis, and was widely strongly positive in all detected patient tissues, and this phenomenon was universal. And BCL2 was also widely highly expressed in giant nevus tissues with different genotypes (BRAF fusion gene, BRAF mutation, NRAS mutation, and wild type), suggesting that the anti-apoptotic response mediated by BCL2 is a common feature of giant nevi and is not affected by genotype.

[0049] Since BCL2 is generally highly expressed in multiple giant nevus patients and is an important gene mediating the anti-apoptotic response, BCL2 was finally selected as the target for targeting giant nevus cells.

[0050] Example 2 The BCL2 inhibitor has a good targeted killing effect on congenital giant nevus cells

[0051] Five most commonly used BCL2 inhibitors were used to intervene in primary giant nevus cells, and the CCK8 assay was used to detect cell viability. We found that: at the same concentration, the killing effect on giant nevus cells of different patients (n = 5: NRAS mutation = 2, BRAF mutation = 1, BRAF fusion gene mutation = 1, wild type = 1), the killing effect of Venetoclax was the most significant ( Figure 5 at the boxed area).

[0052] The most studied MEK inhibitor (Trametinib) in congenital giant nevi was used as the control drug for drug intervention experiments on giant nevus cells (n = 9). We found that at the same concentration (10 μmol), compared with the MEK inhibitor and the DMSO group, the BCL2 inhibitor caused nuclear shrinkage and rupture, and there were a large number of cell debris ( Figure 6 ), and the cell morphology of the MEK group and the DMSO group was normal; in terms of the apoptosis ratio, the BCL2 inhibitor caused a total of about 84% cell apoptosis ( Figure 6 the sum of the two boxed lines), much higher than 40% of the MEK inhibitor group.

[0053] As Figure 7 shown, the comparison of the effects of the BCL2 inhibitor and the MEK inhibitor on killing the cells of patients with giant nevi (n = 9, NRAS mutation = 3, BRAF mutation = 3, wild type = 3). The BCL2 inhibitor (Venetoclax) compared with the traditional targeted drug MEK inhibitor (Trametinib) for giant nevi, its killing effect at a concentration of 2.5 μM was already very significant, and it could inhibit 85% of the giant nevus activity, and at a high concentration (10 μM) it reached more than 90%, and its killing effect was much better than that of the MEK inhibitor. The average IC 50 value of BCL2 was about 1.072 μM, while the average IC 50 value of the MEK inhibitor > 10 μM.

[0054] To confirm that the BCL2 inhibitor has a good killing effect on giant nevus cells with different gene mutations, we intervened on the giant nevus cells with NRAS mutation, BRAF mutation and wild type with the BCL2 inhibitor and the MEK inhibitor respectively, and found that BCL2 has a good killing effect on giant nevi with different gene mutations ( Figure 8 ), however, the MEK inhibitor only has a significant killing effect on giant nevus cells with specific gene mutations (BRAF mutation).

[0055] We also further verified the targeting and safety of the BCL2 inhibitor. The results are shown in Figure 9 . At the same concentration (10 μM), neither the BCL2 inhibitor, the MEK inhibitor nor the DMSO group had a significant killing effect on normal melanocytes and fibroblasts. The cell morphology was normal, there was no nuclear shrinkage or rupture, and there was no large amount of cell debris. Cell viability assays showed that the IC 50All were greater than 10 μM. The above results suggest that BCL2 has a specific killing effect on giant nevus cells and has less impact on normal cells. No obvious killing reaction was seen in normal melanocytes and fibroblasts after adding the BCL2 inhibitor, indicating that the BCL2 inhibitor can specifically kill giant nevus cells. The traditional drug, the MEK inhibitor, also had no significant effect on normal melanocytes and fibroblasts.

[0056] The above results suggest that the BCL2 inhibitor has a good and specific killing effect on congenital giant nevi, and this phenomenon exists in the giant nevus cells of multiple patients with different gene mutations. The targeted killing of the BCL2 inhibitor is a common feature in giant nevi.

[0057] Example 3: The BCL2 inhibitor has a good targeted killing effect on congenital giant nevus tissue blocks

[0058] HE pathological manifestations after soaking and culturing giant nevus tissue blocks with culture media without drugs, with the BCL2 inhibitor (Venetoclax), and with the MEK inhibitor (Trametinib).

[0059] In this example, the culture medium soaking and culturing method was used to observe the killing effect of drugs on giant nevus tissue blocks. The culture medium was MELM medium, and the BCL2 inhibitor and the MEK inhibitor were added to the culture medium at a concentration of 10 μM each. The control group was the DMSO group. As Figure 10 shown, compared with the control group and the MEK inhibitor group, the BCL2 inhibitor group induced the disintegration of nevus cell nests, nuclear shrinkage, disordered morphological structure, and increased cell debris, indicating a good killing effect. The BCL2 inhibitor group significantly killed the nevus cells and cell nests in the giant nevus tissue blocks.

[0060] In addition, tissue blocks of congenital giant nevi were implanted subcutaneously into the neck skin of C-NKG immunodeficient mice (purchased from Cyagen Biosciences) to construct a PDX model of giant nevi. After the tissue blocks were completely viable for two months, drug injection into the tissue blocks was carried out, divided into the DMSO group, the MEK inhibitor group, and the BCL2 inhibitor group. The results were as Figure 11 shown. The BCL2 inhibitor group reduced the nevus cells in the superficial and deep dermis of the tissue blocks, the melanin faded, and the nevus cell nests disappeared, while a large number of nevus cells and nevus cell nests still remained in the MEK group and the DMSO group. These results demonstrate the effective killing effect of the BCL2 inhibitor on giant nevi in the PDX model.

[0061] Example 4: The BCL2 inhibitor has a good killing effect in the transgenic animal model of congenital giant nevus

[0062] NRAS Q61K mutation is the most common mutation type in congenital giant nevi. Accordingly, in this example, a mouse model with Tyr-Nras Q61K mutation was constructed, which showed the phenotype of congenital giant nevi, including significant melanin deposition in the skin, palms and tails, darker hair, etc. As Figure 12 shown, compared with the phenotype of normal C57 mice, the hair color of the whole body was deepened, and extensive melanotic lesions similar to giant nevi could be seen on the palms, tails and skin.

[0063] Local injection of drugs was performed on the nevus skin lesions of transgenic mice with congenital giant nevi. The groups included the DMSO group, the MEK inhibitor group, the BCL2 inhibitor group, etc. The results Figure 13 showed that after drug intervention, the mice in the BCL2 inhibitor group showed the phenomena of hair turning white and the pigment of the mouse tail becoming lighter and white, which were not observed in the MEK inhibitor group and the DMSO group.

[0064] As Figure 14 shown, after drug intervention, the skin nevus lesions of the mice in the BCL2 inhibitor group regressed, while there were no obvious changes in the MEK inhibitor group and the DMSO group.

[0065] The results of HE staining are as Figure 15 and Figure 16 shown. The nevus cells and melanin in the mouse tail skin (dermis) and hair follicles significantly regressed after BCL2 inhibitor intervention. However, a large number of nevus cells and melanin still remained in the MEK inhibitor group and the DMSO group. The above results showed that the BCL2 inhibitor could cause the regression of giant nevus lesions and hair turning white in a mouse animal model simulating the state of human giant nevi, and its killing effect was good.

Claims

1. Use of a BCL2 inhibitor in the preparation of a drug or medical device for treating giant congenital nevus.

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

3. The use according to claim 1, characterized in that The BCL2 inhibitors include venetoclax and pharmaceutically acceptable salts thereof.

4. The use according to claim 3, characterized in that The venetoclax is venetoclax hydrochloride.

5. The use according to claim 1, characterized in that The drug has a BCL2 inhibitor as the only active ingredient and is also added with pharmaceutical excipients.

6. The use according to claim 1, characterized in that The medical device is an auxiliary material, with the BCL2 inhibitor as the only active ingredient.

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

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