Use of melanocyte stimulating hormone α-MSH in treatment of liver cancer

Abstract

Provided is a use of α-MSH or a promoter thereof in liver cancer; specifically, provided is a use of α-MSH, an analog thereof or a promoter thereof for preparing a pharmaceutical composition or preparation for: (i) inhibiting the growth of liver cancer cells, and / or (ii) treating liver cancer. The pharmaceutical composition or preparation can induce apoptosis of liver cancer cells, reducing the number of liver cancer cells and thus achieving an anti-tumor effect.

Description

Application of melanocyte stimulating hormone alpha-MSH in treating liver cancer TECHNICAL FIELD

[0001] The present application relates to the technical field of alpha-MSH application, in particular to the application of melanocyte stimulating hormone alpha-MSH in treating liver cancer. BACKGROUND

[0002] Liver cancer is one of the diseases that cause the heaviest medical burden in China, seriously affecting the survival period and quality of life of patients. According to the latest survey data, about 950,000 people worldwide suffered from liver cancer in 2020, and about 830,000 people died of liver cancer. Under the condition that the incidence and mortality rate remain unchanged, it is estimated that there will be 1.4 million people suffering from liver cancer and 1.3 million people dying of liver cancer in 2040.

[0003] Due to the insidious onset of liver cancer, it is often discovered late, easy to metastasize, and often resistant. At the time of first diagnosis, only less than 30% of liver cancer patients are suitable for radical treatment, and drug anti-tumor treatment is the only choice for patients with advanced liver cancer. The representative drugs for inhibiting liver cancer currently include sorafenib, lenvatinib, PD-1, etc. Most of the liver cancer drugs have not been approved or are still in the clinical trial stage, and their effects are not very ideal.

[0004] Sorafenib (Nexavar) is an oral multi-target tyrosine kinase inhibitor approved by FDA in 2007, which is the only approved first-line drug for targeted treatment of hepatocellular carcinoma. Its targets include RAF / MEK / ERK signaling pathway, vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). Lenvatinib (E7080) is also a multi-target kinase inhibitor, which was approved by FDA in 2015 for the treatment of other tumors, and can inhibit multiple targets such as VEGFR2 and VEGFR3, FGFR and PDGFR. Current big data shows that these drugs have some effect, but only for some patients, even so, they cannot significantly improve the prognosis of liver cancer patients. Therefore, the currently used liver cancer treatment drugs have very limited efficacy.

[0005] Therefore, there is an urgent need in the art to develop a liver cancer drug with good treatment effect. SUMMARY

[0006] The purpose of the present application is to provide the use of alpha-MSH or its promoters for preparing a composition or a preparation for: (i) inhibiting the growth of liver cancer cells; and / or (ii) treating liver cancer.

[0007] In a first aspect, the present application provides the use of alpha-MSH or its promoters for preparing a pharmaceutical composition or a preparation for:

[0008] (i) inhibiting the growth of hepatocarcinoma cells; and / or

[0009] (ii) treating hepatocarcinoma.

[0010] In another preferred embodiment, the α-MSH comprises an α-MSH protein, a nucleic acid (DNA or mRNA) encoding an α-MSH protein, or a vector (expression vector, viral vector) expressing an α-MSH protein.

[0011] In another preferred embodiment, the pharmaceutical composition or preparation is administered to a subject whose plasma α-MSH expression is significantly decreased.

[0012] In another preferred embodiment, the significant decrease means that the concentration of α-MSH in the plasma is ≤ 950 pg / ml, preferably ≤ 925 pg / ml, more preferably ≤ 850 pg / ml.

[0013] In another preferred embodiment, the significant decrease means that the relative decrease ((S0-S1) / S0) of the concentration of α-MSH in the plasma (S1) compared to the reference value (S0) is ≥ 5%, preferably ≥ 10%, more preferably ≥ 15%.

[0014] In another preferred embodiment, the reference value means the concentration of α-MSH in the plasma of a healthy person.

[0015] In another preferred embodiment, the reference value means the concentration of α-MSH in the plasma of a healthy person.

[0016] In another preferred embodiment, the plasma α-MSH expression in the subject is significantly decreased.

[0017] In another preferred embodiment, the concentration of α-MSH in the plasma of the subject is ≤ 950 pg / ml, preferably ≤ 925 pg / ml, more preferably ≤ 850 pg / ml.

[0018] In another preferred embodiment, the relative decrease ((Z0-Z1) / Z0) of the concentration of α-MSH in the plasma of the subject (Z1) compared to the reference value (Z0) is ≥ 5%, preferably ≥ 10%, more preferably ≥ 15%.

[0019] In another preferred embodiment, the pharmaceutical composition or preparation is also used for one or more of the following purposes:

[0020] (u1) promoting apoptosis of hepatocarcinoma cells;

[0021] (u2) inhibiting the clonogenicity of hepatocarcinoma cells;

[0022] (u3) inhibiting the growth of hepatocarcinoma transplanted tumors;

[0023] (u4) reducing the volume of hepatocarcinoma tumors.

[0024] In another preferred embodiment, the pharmaceutical composition or formulation has an inhibition rate of liver cancer tumor of ≥ 80%, preferably ≥ 85%, more preferably ≥ 90%.

[0025] In another preferred embodiment, the liver cancer cell is selected from the group consisting of PLC802, Huh7, 97H, 97L, or a combination thereof.

[0026] In another preferred embodiment, the concentration of α-MSH in the pharmaceutical composition or formulation is 0.001 nM to 500 mM, preferably 0.01 nM to 50 mM, more preferably 0.1 nM to 5000 nM.

[0027] In another preferred embodiment, the liver cancer tumor size of the subject is 10 to 2000 mm 3 .

[0028] In another preferred embodiment, the subject is a human or a non-human mammal, such as a rodent.

[0029] In another preferred embodiment, when the subject is a mouse, the tumor size is 50 to 2000 mm 3 , preferably 100 to 1500 mm 3 , more preferably 100 to 1000 mm 3 .

[0030] In another preferred embodiment, when the subject is a human, the tumor size is 50 to 2000 mm 3 , preferably 100 to 1500 mm 3 , more preferably 100 to 1000 mm 3 .

[0031] In another preferred embodiment, the dose of the pharmaceutical composition or formulation administered to the subject is 0.001 to 5000 mg / kg, preferably 0.01 to 1000 mg / kg, more preferably 0.1 to 100 mg / kg.

[0032] In a second aspect of the present application, a pharmaceutical composition is provided, comprising:

[0033] (c1) an active ingredient, the active ingredient comprising α-MSH or a promoter thereof; and

[0034] (c2) a pharmaceutically acceptable carrier;

[0035] The pharmaceutical composition is used for (i) inhibiting the growth of liver cancer cells; and / or (ii) treating liver cancer.

[0036] In another preferred embodiment, the pharmaceutical composition further comprises an additional drug for treating liver cancer.

[0037] In another preferred embodiment, the additional drug for treating liver cancer comprises a chemotherapeutic drug, a targeted drug, a traditional Chinese medicine, or a combination thereof.

[0038] In another preferred embodiment, the chemotherapeutic drug is selected from the group consisting of fluorouracil, doxorubicin, cisplatin, mitomycin, gemcitabine, capecitabine, irinotecan, or a combination thereof.

[0039] In another preferred embodiment, the targeted drug is selected from the group consisting of sorafenib, sunitinib, bevacizumab, erlotinib, or a combination thereof.

[0040] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of huachansu capsule, kanglaite injection, cidan capsule, sodium cantharidate vitamin B6 injection, or a combination thereof.

[0041] In another preferred embodiment, the pharmaceutical composition is further used for one or more of the following purposes:

[0042] (u1) promoting apoptosis of liver cancer cells;

[0043] (u2) inhibiting clonogenicity of liver cancer cells;

[0044] (u3) inhibiting growth of liver cancer xenografts;

[0045] (u4) reducing tumor volume of liver cancer.

[0046] In a third aspect, the present application provides a method for inhibiting growth of liver cancer cells in vitro, comprising the steps of:

[0047] contacting α-MSH or an enhancer thereof with liver cancer cells, thereby inhibiting growth of the liver cancer cells.

[0048] In another preferred embodiment, the concentration of α-MSH is 0.1-5000 nM.

[0049] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0050] In another preferred embodiment, the liver cancer cells are selected from the group consisting of hepatocellular carcinoma cells PLC802, Huh7, 97H, 97L, or a combination thereof.

[0051] In a fourth aspect, the present application provides a method for treating and / or preventing liver cancer, comprising the steps of:

[0052] administering α-MSH, an analog thereof, or an enhancer thereof to a subject, thereby treating and / or preventing liver cancer.

[0053] In another preferred embodiment, the expression of α-MSH in the plasma of the subject is significantly decreased.

[0054] In another preferred embodiment, said significant decrease means that the relative decrease ((S0-S1) / S0) of the concentration of α-MSH in the plasma (S1) of said subject compared to the reference value (S0) is ≥ 5%, preferably ≥ 10%, more preferably ≥ 15%.

[0055] In another preferred embodiment, said reference value means the concentration of α-MSH in the plasma of a healthy person.

[0056] In another preferred embodiment, said reference value means the concentration of α-MSH in the plasma of a healthy person.

[0057] In another preferred embodiment, the expression of α-MSH in the plasma of said subject is significantly decreased.

[0058] In another preferred embodiment, the concentration of α-MSH in the plasma of said subject is ≤ 950 pg / ml, preferably ≤ 925 pg / ml, more preferably ≤ 850 pg / ml.

[0059] In another preferred embodiment, said significant decrease means that the relative decrease ((Z0-Z1) / Z0) of the concentration of α-MSH in the plasma (Z1) of said subject compared to the reference value (Z0) is ≥ 5%, preferably ≥ 10%, more preferably ≥ 15%.

[0060] In another preferred embodiment, said significant decrease means that the relative decrease ((S0-S1) / S0) of the concentration of α-MSH in the plasma (S1) of said subject compared to the reference value (S0) is ≥ 5%, preferably ≥ 10%, more preferably ≥ 15%.

[0061] In another preferred embodiment, said subject has a liver cancer tumor size of 10 to 2000 mm 3 .

[0062] In another preferred embodiment, said subject is a human or a non-human mammal, such as a rodent.

[0063] In another preferred embodiment, when said subject is a mouse, the tumor size is 50 to 2000 mm 3 , preferably 100 to 1500 mm 3 , more preferably 100 to 1000 mm 3 .

[0064] In another preferred embodiment, when said subject is a human, the tumor size is 50 to 2000 mm 3 , preferably 100 to 1500 mm 3 , more preferably 100 to 1000 mm 3 .

[0065] It should be understood that, in the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 shows the effect of different doses of melanocyte stimulating hormone α-MSH on the colony formation of liver cancer cells.

[0067] Figure 2 shows the effect of different doses of melanocyte stimulating hormone α-MSH on the survival of liver cancer cells.

[0068] Figure 3 shows the effect of melanocyte stimulating hormone α-MSH on the growth of subcutaneous ectopic transplanted tumors of liver cancer in mice.

[0069] Figure 4 shows the effect of melanocyte stimulating hormone α-MSH on the treatment of early liver cancer.

[0070] Figure 5 shows the therapeutic effect of melanocyte stimulating hormone α-MSH on overloading liver cancer transplanted tumors.

[0071] Figure 6 shows the effect of other related neuroendocrine peptides on the growth of liver cancer cells.

[0072] Figure 7 shows the changes in the concentration of α-MSH in the plasma of liver cancer patients. DETAILED DESCRIPTION

[0073] Through extensive and in-depth research, and after a large number of experiments and screening, it is first discovered that melanocyte stimulating hormone α-MSH can effectively treat liver cancer in patients expressing low levels of α-MSH in the plasma. On this basis, the present application is completed.

[0074] TERMS

[0075] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have the meanings generally understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present application will only be limited by the appended claims.

[0076] As used herein, the term "comprise" or its variants such as "comprises" or "comprising", etc., is understood to include the stated elements or components, without excluding other elements or components.

[0077] As used herein, the term "subject" or "subject in need thereof refers to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

[0078] As used herein, the terms "liver cancer cell 1", "PLC8024" are used interchangeably, and both refer to PLC8024 liver cancer cells.

[0079] As used herein, the terms "liver cancer cell 2", "Huh7" are used interchangeably, and both refer to Huh7 liver cancer cells.

[0080] As used herein, the terms "liver cancer cell 3", "97H" are used interchangeably, and both refer to 97H liver cancer cells.

[0081] As used herein, the terms "liver cancer cell 4", "97L" are used interchangeably, and both refer to 97L liver cancer cells.

[0082] As used herein, the terms "normal liver cell 1", "WRL68" are used interchangeably, and both refer to normal liver cells.

[0083] As used herein, the terms "normal liver cell 2", "LO2" are used interchangeably, and both refer to normal liver cells.

[0084] α-MSH

[0085] Melanocyte stimulating hormone is a neuroendocrine peptide, mainly including α-MSH, β-MSH, γ-MSH, etc. The Chinese name of α-MSH is α-melanocyte stimulating hormone, and the chemical structural formula is: 77 H 109 N 21 O 19 S, the natural sequence is: SYSMEHFRWGKPV (SEQ ID NO: 1). In order to protect the integrity of the α-MSH polypeptide, the sequence of the artificially synthesized α-MSH polypeptide is modified at both ends of the carboxyl and amino group.

[0086] Studies have shown that α-MSH is a neuroendocrine polypeptide secreted by the hypothalamic-pituitary neuroendocrine axis in the brain, which has very important physiological regulation function, and liver tissue and liver cancer cells do not express α-MSH.

[0087] The terms "α-MSH", "α-melanocyte stimulating hormone" and "active ingredient of the present application" are used interchangeably, and all refer to a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1.

[0088] α-MSH can be obtained in the following ways:

[0089] (a1) an isolated or purified polypeptide;

[0090] (a2) the cell body of the engineered bacteria expressing α-MSH, its lysate or its supernatant;

[0091] (a3) the live bacteria of the engineered bacteria expressing α-MSH;

[0092] (a4) commercially available;

[0093] (a5) or a combination thereof.

[0094] Subject

[0095] The subject to which α-MSH is administered refers to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

[0096] The subject of the present application is one expressing low level of α-MSH in plasma, which means the concentration of α-MSH in the plasma is ≤ 850 pg / ml.

[0097] In another preferred embodiment, the subject expressing low level of α-MSH in plasma means that the concentration of α-MSH in the plasma of the subject (S1) is compared with the standard value (S0), and the ratio (S1 / S0) is ≤ 3 / 4, preferably ≤ 2 / 3, more preferably ≤ 1 / 2.

[0098] In another preferred embodiment, the reference value refers to the concentration of α-MSH in the plasma of a healthy person, which is 800-1300 pg / ml.

[0099] In another preferred embodiment, the tumor volume of the subject, for example, the tumor volume of a mouse, is 1-2000 mm 3 , preferably 50-1500 mm 3 , more preferably 100-1000 mm 3 , for example, 100 mm 3 , 300 mm 3 , 1000 mm 3 . (relative to the tumor volume of a human, 1-2000 cm 3 )

[0100] Composition or preparation

[0101] The composition or preparation provided by the present application preferably contains 0.1-99 wt% of α-MSH, and the rest is pharmaceutically acceptable carriers, diluents or solutions or salt solutions.

[0102] The active ingredient of the present application is α-MSH, or its promoters. In addition, it can also be used in combination with other therapeutic agents. The other therapeutic agents can be any drug ingredients capable of preventing and / or treating liver cancer.

[0103] If necessary, one or more pharmaceutically acceptable carriers can be added to the pharmaceuticals of the present application. The carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption accelerators, surfactants, adsorptive carriers, lubricants, etc. that are conventionally used in the pharmaceutical field.

[0104] The compositions or formulations of the present application can be in various forms such as tablets, injections, capsules, powders, syrups, solutions, suspensions, and aerosols, etc., and can be present in suitable solid or liquid carriers or diluents and suitable sterilized instruments for injection or instillation.

[0105] The various dosage forms of the compositions of the present application can be prepared according to the conventional methods in the pharmaceutical field. The unit dose of the formulation usually contains 0.05-1000 mg of the active ingredient of the present application, and preferably, the unit dose of the formulation contains 1 mg-500 mg of the active ingredient of the present application.

[0106] The pharmaceutical compositions of the present application can be clinically used for mammals including humans and animals, and can be administered through oral, nasal, dermal, pulmonary, or gastrointestinal routes, etc. The most preferred is oral administration. The most preferred daily dose is 0.01-400 mg / kg of body weight, administered at one time or 0.01-200 mg / kg of body weight administered in several times. Regardless of the administration method, the optimal dose for an individual should be determined depending on the specific treatment. Generally, the administration should start from a small dose and the dose should be gradually increased until the optimal dose is found.

[0107] The pharmaceuticals or inhibitors of the present application can be administered in various ways, for example, by injection, spraying, nasal instillation, eye instillation, penetration, absorption, physical or chemical mediated methods into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped with other substances and introduced into the body.

[0108] Typically, the active ingredient of the present application or the composition containing the same can be administered in unit dose form, and the administration route can be enteral or parenteral such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0109] The administration form can be a liquid form, a solid form or a semi-solid form. The liquid form can be a solution (including true solution and colloidal solution), an emulsion (including O / W type, W / O type and multiple emulsion), a suspension, an injection (including water injection, powder injection and infusion), eye drops, nose drops, lotion and liniment, etc.; the solid form can be a tablet (including ordinary tablet, enteric-coated tablet, chewable tablet, dispersible tablet, effervescent tablet, oral disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a drop, a suppository, a film, a patch, an aerosol (powder) spray, a spray, etc.; the semi-solid form can be an ointment, a gel, a paste, etc.

[0110] The active ingredient of the present application can be prepared into a common preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various micro-particle administration systems.

[0111] In order to prepare the active ingredient of the present application into a tablet, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0112] The tablet can be further prepared into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a double-layer tablet and a multi-layer tablet.

[0113] In order to prepare the administration unit into a capsule, the active ingredient of the present application can be mixed with diluents and glidants, and the mixture can be directly placed into a hard capsule or a soft capsule. Alternatively, the active ingredient can be first mixed with diluents, binders and disintegrants to prepare granules or pellets, and then placed into a hard capsule or a soft capsule. The various diluents, binders, wetting agents, disintegrants and glidants used for preparing the tablet of the present application can also be used for preparing the capsule of the present application.

[0114] To prepare an injection of the active ingredient of the present application, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and an appropriate amount of a solubilizer, a co-solvent, a pH adjustor, an osmotic pressure adjustor commonly used in the art can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjustor can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; and the osmotic pressure adjustor can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a supporting agent.

[0115] In addition, if necessary, a coloring agent, a preservative, a flavoring agent, a taste corrector or other additives can also be added to the pharmaceutical preparation.

[0116] The active ingredient or composition of the present application can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0117] When the active ingredient of the present application has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0118] The main advantages of the present application include:

[0119] (1) α-MSH can induce apoptosis of hepatocarcinoma cells, reduce the number of hepatocarcinoma cells and thus achieve an anti-tumor effect.

[0120] (2) α-MSH can improve the tumor.

[0121] (3) α-MSH has no toxic side effects on normal cells.

[0122] (4) The present application provides a new method and new idea for treating hepatocarcinoma and improving the prognosis of patients with hepatocarcinoma.

[0123] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0124] Example 1

[0125] 1.1 Method

[0126] Effect of melanocyte stimulating hormone α-MSH ((T7813, Targetmol manufacturer) on hepatocarcinoma cell colony formation. 1 × 10 3The cells were seeded at a density of 1 x 105cells per well in 6-well plates and incubated overnight. The next day, the cells were treated with α-MSH at a final concentration ranging from 0 to 1000 nM.

[0127] 1.2 Results

[0128] As shown in Figure 1, α-MSH treatment reduced the colony formation of four hepatoma cell lines (PLC8024, Huh7, 97H, 97L) in a dose-dependent manner, but had little effect on the colony formation of normal liver cells (WRL68, LO2).

[0129] The results show that α-MSH only has an inhibitory effect on hepatoma cells, but has no effect on normal cells.

[0130] Example 2

[0131] 2.1 Methods

[0132] Effect of melanocyte stimulating hormone α-MSH on the survival of hepatoma cells. 1 x 105cells were seeded in 6-well plates and incubated overnight. The next day, the cells were treated with α-MSH at a final concentration ranging from 0 to 1000 nM. After 3 days of treatment, the cells were stained with crystal violet. 3

[0133] 2.2 Results

[0134] As shown in Figure 2, α-MSH treatment had a certain killing effect on hepatoma cells, promoting the death (apoptosis) of hepatoma cells. However, it had little effect on the growth of normal liver cell clones.

[0135] Example 3

[0136] 3.1 Methods

[0137] Effect of melanocyte stimulating hormone α-MSH on the growth of subcutaneous ectopic transplanted tumors in mice. 5 x 105hepatoma cells were subcutaneously inoculated into nude mice. After 3 weeks, when the tumors grew to about 300 mm 6 3 in diameter, the tumor-bearing mice were randomly divided into experimental and control groups (injected with saline). The experimental group was treated with intraperitoneal injection of α-MSH polypeptide for two months, once every other day, with a dose of 10 mg / kg per injection.

[0138] 3.2 Results

[0139] As shown in Figure 3, the tumor volume and weight of the mice in the α-MSH experimental group were significantly smaller than those in the control group.

[0140] The above results show that α-MSH polypeptide can significantly inhibit the growth of hepatoma and reduce the volume of hepatoma, with an inhibition rate (calculated based on the tumor volume before and after injection of the drug) of up to 80-90%. ​​

[0141] Example 4

[0142] 4.1 Method

[0143] Effect of melanocyte stimulating hormone α-MSH on early stage hepatocarcinoma treatment. 5x10 6 Hepatocarcinoma cells were subcutaneously inoculated in nude mice, and when the volume of the hepatocarcinoma transplanted tumor in the mice reached 100mm 3 left, treatment was started, and the treatment method was intraperitoneal injection, once every other day, with an injection amount of 20mg / kg, and a cycle of one month.

[0144] 4.2 Results

[0145] As shown in Figure 4, treatment with the α-MSH polypeptide when the volume of the hepatocarcinoma transplanted tumor was small, could significantly slow down the growth rate of the hepatocarcinoma, effectively treat the tumor, and even make the transplanted tumor disappear.

[0146] The results show that the α-MSH polypeptide is effective for early stage hepatocarcinoma treatment, and can inhibit the growth of early stage hepatocarcinoma tumors.

[0147] Example 5

[0148] 5.1 Method

[0149] Effect of melanocyte stimulating hormone α-MSH on treatment of overloaded hepatocarcinoma transplanted tumors. When the tumor in the mice reached 1000mm 3 left, treatment was started, and the treatment method was intraperitoneal injection, once every other day, with an injection amount of 20mg / kg, and a cycle of one month.

[0150] 5.2 Results

[0151] As shown in Figure 5, the tumor volume of the late stage treatment group mice was significantly smaller than that of the control group.

[0152] The results show that the α-MSH polypeptide still has good treatment effect on hepatocarcinoma transplanted tumors with a larger volume.

[0153] The above in vitro and in vivo animal test results show that the α-MSH polypeptide can promote cell death and inhibit the growth of hepatocarcinoma transplanted tumors, thereby achieving the purpose of treating hepatocarcinoma. The present application also opens up a new use of the α-MSH polypeptide for treating hepatocarcinoma.

[0154] Example 6

[0155] 6.1 Method

[0156] Effect of γ-melanocyte stimulating hormone γ-MSH (HY-P4985, MCE), β-MSH, and α-melanocyte stimulating hormone α-MSH (T7813, Targetmol) on hepatocarcinoma cell colony formation. 1x10 3The number of cells was counted, and after overnight adhesion, 0, 100, 1000 nM of γ-MSH, β-MSH and α-MSH were added to the culture medium respectively.

[0157] 6.2 Results

[0158] As shown in Figure 6, high concentration of α-MSH treatment reduced the clone formation of two hepatoma cells (PLC8024, Huh7), but β-MSH and γ-MSH had little effect on the clone formation of the two hepatoma cells.

[0159] The results showed that α-MSH had an inhibitory effect on hepatoma cells, while β-MSH and γ-MSH had no effect.

[0160] Example 7

[0161] 7.1 Method

[0162] Alpha melanocyte-stimulating hormone ELISA kit Human Alpha Melanocyte-stimulating hormone Enzyme Immunoassay Kit (EIA-aMSH, RayBiotech) was used to detect the expression level of plasma α-MSH in clinical hepatoma patients and normal healthy controls.

[0163] According to the manufacturer's instructions, first label 7 microtubes with the following concentrations: 1000 pg / ml, 250 pg / ml, 62.5 pg / ml, 15.6 pg / ml, 3.9 pg / ml, 1 pg / ml and 0 pg / ml to calibrate and draw the standard curve, and the dilution ratio of the sample stock solution was 2 times. The following steps were followed (1) Add 100 μl of anti-Alpha MSH to each well. Incubate at room temperature for 1.5 hours or at 4°C overnight. (2) Add 100 μl of diluted sample (or standard) to each well. Incubate at room temperature for 2.5 hours or at 4°C overnight. (3) Add 100 μl of prepared streptavidin solution. Incubate at room temperature for 45 minutes. (4) Add 100 μl of TMB one-step substrate reagent to each well. Incubate at room temperature for 30 minutes. (5) Add 50 μl of stop solution to each well. Immediately read the absorbance value at 450 nm.

[0164] 7.2 Results

[0165] As shown in Figure 7, the plasma α-MSH level in hepatoma patients was significantly lower than that in normal healthy controls, with statistical significance.

[0166] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that embodiments can be practiced without the specific details that are set forth herein. Further, it should be noted that, in this application, "exemplary" or "for example" is used on a purely illustrative basis to aid the reader in understanding the application. Any process steps, or sequences of steps, or examples, or embodiments, or materials, or components, or compounds, or elements described herein are understood to be illustrative only and not limiting.

Claims

1. Use of α-MSH or a promoter thereof, characterized in that, A pharmaceutical composition or formulation for use in: (i) inhibiting growth of a liver cancer cell; and / or (ii) treating a liver cancer.

2. Use according to claim 1, characterized in that, The pharmaceutical composition or formulation is administered to a subject whose plasma expression of α-MSH is significantly decreased.

3. Use according to claim 2, characterized in that, The significant decrease means that the concentration of α-MSH in the plasma is ≤ 950 pg / ml.

4. The use according to claim 2, characterized in that, The significant decrease means that the relative decrease ((S0-S1) / S0) of the concentration of α-MSH in the plasma (S1) compared to a reference value (S0) is ≥ 5%.

5. The use according to claim 1, characterized in that, The pharmaceutical composition or formulation is also for one or more of the following uses: (u1) promoting apoptosis of a liver cancer cell; (u2) inhibiting clonogenicity of a liver cancer cell; (u3) inhibiting growth of a liver cancer xenograft; (u4) reducing tumor volume of a liver cancer.

6. The use according to claim 1, characterized in that, The concentration of α-MSH in the pharmaceutical composition or formulation is 0.001 nM to 500 mM.

7. The use according to claim 2, characterized in that, The liver cancer tumor size of the subject is 10-2000mm 3 .

8. A pharmaceutical composition, characterized by, Comprising: (c1) an active ingredient comprising α-MSH or a promoter thereof; and (c2) a pharmaceutically acceptable carrier; The pharmaceutical composition is for use in (i) inhibiting growth of a liver cancer cell; and / or (ii) treating a liver cancer. The pharmaceutical composition further comprises an additional drug for treating a liver cancer.

9. The pharmaceutical composition of claim 8, wherein Comprising the step of:

10. A method for inhibiting the growth of liver cancer cells in vitro, characterized in that, contacting α-MSH or a promoter thereof with a liver cancer cell, thereby inhibiting growth of the liver cancer cell. Comprising the step of:

11. A method for treating and / or preventing liver cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-10. administering α-MSH, an analogue thereof or a promoter thereof to a subject, thereby treating and / or preventing a liver cancer. The subject's plasma expression of α-MSH is significantly decreased.

12. The method of claim 11, wherein, The concentration of α-MSH in the subject's plasma is ≤ 950 pg / ml.

13. The method of claim 11, wherein, ​

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