CN116687927A

CN116687927A - Application of hydroxychloroquine, combined medicine containing hydroxychloroquine and application of combined medicine

Info

Publication number: CN116687927A
Application number: CN202310679005.0A
Authority: CN
Inventors: 宋尔卫; 龚畅; 林群; 罗晴
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2023-06-08
Filing date: 2023-06-08
Publication date: 2023-09-05

Abstract

The application relates to the technical field of medicines, in particular to application of hydroxychloroquine, and a combined medicine containing hydroxychloroquine and application thereof. Studies in accordance with the present application demonstrate that hydroxychloroquine significantly reduces the neutral particle toxicity caused by high doses of CDK4/6 inhibitors and has a synergistic effect on the anti-cancer effect of CDK4/6 inhibitors. Based on this, the present application further proposes a new strategy for cancer treatment based on the combination of hydroxychloroquine and a high dose of CDK4/6 inhibitor drug, which will promote the use of hydroxychloroquine and a high dose of CDK4/6 inhibitor in clinical treatment of cancer, greatly shortening the time from discovery to clinical conversion of the drug.

Description

Application of hydroxychloroquine, combined medicine containing hydroxychloroquine and application of combined medicine

Technical Field

The application relates to the technical field of medicines, in particular to application of hydroxychloroquine, and a combined medicine containing hydroxychloroquine and application thereof.

Background

Breast cancer is the most common malignancy in women worldwide, with about 70% of breast cancer patients being hormone receptor (ER) positive/human epidermal growth factor receptor 2 (human epidermal growth factor receptor, her2) negative (er+/her2-), and with er+/her2-advanced breast cancer patients, the clinical guidelines recommend a combination of a cyclic dependent protein kinase (cyclin dependent kinase, CDK) 4/6 inhibitor with endocrine therapy, which can significantly extend the progression-free survival (PFS) of the patient. However, 80-90% of patients inevitably develop primary or secondary resistance. The current clinical guidelines recommend that grade II recommended treatment regimens for CDK4/6 inhibitors after drug resistance be effective only for initial treatment of 20% -40% of patients, other clinically alternative treatment regimens also include endocrine therapy in combination with PI3K inhibitors or ADC drugs, but the clinical needs are still not fully met in efficacy, and new, poorly controlled and poorly managed toxic reactions may occur.

Thus, exploring new mechanisms of CDK4/6 inhibitor resistance in er+/HER 2-advanced breast cancer patients and finding potential highly effective low-toxicity therapeutic regimens is a current clinical problem that is urgently addressed. In preclinical animal experiments, the high doses (75 mg/kg/day and 150 mg/kg/day) of the CDK4/6 inhibitor palbociclib are far higher than the current clinical recommended doses (25 mg/kg/day, corresponding to the clinical dose of 125mg/day used by patients), the antitumor effect is remarkable, and in ovarian cancer human tumor xenograft models, other dose groups (25 mg/kg/day and 50 mg/kg/day) only can partially inhibit tumor growth, and only the high dose (100 mg/kg/day) of the CDK4/6 inhibitor can achieve the effect of completely inhibiting tumor growth.

However, with respect to CDK4/6 inhibitors, there is dose-limiting toxicity (mainly neutropenia), even though CDK4/6 inhibitor-induced neutropenia is reversibly restored after withdrawal, several studies have shown that with increasing doses of CDK4/6 inhibitors, the occurrence and extent of neutropenia is much higher, making the recommended doses currently in clinical use far below the optimal anti-tumor dose in animal pharmacokinetic models, which may be one of the reasons for poor clinical therapeutic effects or drug resistance. In order to maximize the anti-tumor efficacy of the CDK4/6 inhibitor and to overcome the dose limiting toxic side effect neutropenia of the CDK4/6 inhibitor, high doses of CDK4/6 inhibitor are contemplated in combination with other agents.

Hydroxychloroquine is used as a traditional antimalarial drug, is mainly used for treating rheumatic immune diseases at present, has antitumor activity, and the antitumor effect is not only limited to preclinical mechanism research, and successful in-vivo and in-vitro preclinical research results provide theoretical basis for developing a plurality of clinical experiments. Several clinical trials are underway, including hydroxychloroquine alone or in combination with chemotherapy, in pancreatic cancer, glioblastoma multiforme, advanced solid tumors and melanoma, metastatic colorectal cancer, multiple myeloma, sarcoma and chemotherapy. In glioblastoma multiforme patients, radiation therapy with hydroxychloroquine was combined with temozolomide at a maximum tolerated dose of 600mg/d. In patients with metastatic colorectal cancer, the results of vorinostat combined with oral hydroxychloroquine 400mg/d and 600mg/d show that the drug is well tolerated and safe and effective.

Disclosure of Invention

The present application aims to solve the technical problems in the prior art described above. Therefore, the application provides application of hydroxychloroquine, and combined medicine containing hydroxychloroquine and application thereof. Hydroxychloroquine can significantly reduce the neutral particle toxicity caused by high doses of CDK4/6 inhibitors and has a synergistic effect on the anticancer effect of CDK4/6 inhibitors. Based on this, the present application further proposes a new strategy for cancer treatment based on the combination of hydroxychloroquine and a high dose of CDK4/6 inhibitor drug, which will promote the use of hydroxychloroquine and a high dose of CDK4/6 inhibitor in clinical treatment of cancer, greatly shortening the time from discovery to clinical conversion of the drug.

In a first aspect of the application there is provided the use of hydroxychloroquine in the manufacture of a medicament for reducing the resistance of a CDK4/6 inhibitor.

In some embodiments of the application, the hydroxychloroquine reduces CDK4/6 inhibitor resistance by reducing neutrophil toxicity caused by high doses of the CDK4/6 inhibitor.

In some preferred embodiments of the application, the hydroxychloroquine reduces neutrophil toxicity caused by high doses of CDK4/6 inhibitor by alleviating the reduction in neutrophil count caused by high doses of CDK4/6 inhibitor.

In some preferred embodiments of the application, the hydroxychloroquine reduces neutrophil toxicity caused by high doses of a CDK4/6 inhibitor by reducing neutrophil extracellular trap (neutrophil extracellular traps, NETS) formation.

The proposed dose of CDK4/6 inhibitor is one of the reasons for poor clinical treatment effect or drug resistance of cancers, and the research of the application finds that the cancer inhibiting effect of high dose of CDK4/6 inhibitor is obviously increased, but the high dose of CDK4/6 inhibitor can promote the formation of neutrophils NETS and lead to the reduction of the number of neutrophils, thereby causing a series of toxic side effects; hydroxychloroquine can relieve the reduction of the number of neutrophils by reducing the formation of neutrophils NETS, so that the neutrophils are protected, the treatment effect of the CDK4/6 inhibitor-resistant cancer is finally exerted, the occurrence of toxic and side effects is avoided, and no organic damage is caused to a tested object.

In some embodiments of the application, the CDK4/6 inhibitor is selected from at least one of abemaciclib, ribociclib, palbociclib, dalpiciclib.

In the present application, the subject to which hydroxychloroquine and CDK4/6 inhibitor are administered is a mammal, such as a human, bovine, equine, feline, canine, rodent (e.g., mouse), or primate. The effective dose conversion of different animals can be based on equivalent dose conversion relation of experimental animals and human in the field (generally, the guidance of drug administration such as FDA, SFDA and the like can be seen, and the like can also be seen in Huang Jihan, etc., the equivalent dose conversion between animals and human bodies in pharmacological tests, chinese clinical pharmacology and therapeutics, 2004;9 (9): 1069-1072 "), and the unit weight dose of human can be deduced from the dose of experimental animals.

In some embodiments of the application, the subject to which the CDK4/6 inhibitor is administered is a rodent (e.g., a mouse); correspondingly, the high dose is 75-150 mg/kg daily, specifically 75mg/kg daily when the CDK4/6 inhibitor is palbociclib and 125mg/kg daily when the CDK4/6 inhibitor is abemaciclib; the recommended dose is 25-62.5 mg/kg daily, specifically 25mg/kg daily of palbociclib when the CDK4/6 inhibitor is palbociclib and 62.5mg/kg daily of abemaciclib when the CDK4/6 inhibitor is abemaciclib.

In a second aspect of the application there is provided a combination of a therapeutically effective amount of hydroxychloroquine and a high dose of a CDK4/6 inhibitor as active ingredients.

In some embodiments of the application, the subject to which hydroxychloroquine is administered is a rodent (e.g., a mouse), and the therapeutically effective amount is, correspondingly, 20 to 100mg/kg per day.

In some embodiments of the application, the subject to which the CDK4/6 inhibitor is administered is a rodent (e.g., a mouse); correspondingly, the high dose is 75-150 mg/kg of daily administration dose; in particular, when the CDK4/6 inhibitor is palbociclib, the daily dosage of palbociclib is 75mg/kg; when the CDK4/6 inhibitor is abelmoschi, the daily dosage of abelmoschi is 125mg/kg.

In some embodiments of the application, it further comprises pharmaceutically acceptable excipients.

In some embodiments of the application, wherein said hydroxychloroquine and said CDK4/6 inhibitor may be administered simultaneously, separately or sequentially.

For example, hydroxychloroquine and a CDK4/6 inhibitor may be administered simultaneously to a subject in need of treatment, separately to a subject in need of treatment, or sequentially to a subject in need of treatment, e.g., hydroxychloroquine is administered first, followed by CDK4/6 inhibitor at intervals; CDK4/6 inhibitors may also be administered first, followed by hydroxychloroquine at intervals.

In some preferred embodiments of the application, wherein said hydroxychloroquine and said CDK4/6 inhibitor are administered simultaneously.

Through a great deal of research and exploration, the application finds out an FDA approved drug for reducing the toxic and side effects of a high-dose CDK4/6 inhibitor, namely hydroxychloroquine. The results of the study show that hydroxychloroquine can significantly relieve neutropenia caused by high-dose CDK4/6 inhibitors and can enhance the anti-tumor effect of CDK4/6 inhibitors. The application compares the difference of anti-tumor effect of hydroxychloroquine and CDK4/6 inhibitor when singly used and combined in animals through a tumor xenograft (Patient-derived tumor xenograft, PDX) model of patients with breast cancer recommended dose CDK4/6 inhibitor sensitivity and primary drug resistance. The results show that the synergistic effect of the drug combination greatly improves the inhibition of breast cancer by the CDK4/6 inhibitor alone, can reverse the mice resistant to the recommended dose of the CDK4/6 inhibitor, and does not cause obvious toxic or side effect.

In a third aspect, the application provides the use of a combination as described above for the manufacture of a medicament for the prophylaxis and/or treatment of cancer.

In some embodiments of the application, the combination drug is capable of inhibiting tumor volume and thereby preventing and/or treating cancer.

In some embodiments of the application, the combination is used to prevent and/or treat cancer by inhibiting cancer cell activity.

In some embodiments of the application, the combination is used to prevent and/or treat cancer by inhibiting cancer cell growth.

In some embodiments of the application, the cancer is selected from at least one of bladder cancer, breast cancer, colon cancer, kidney cancer, epidermoid cancer, liver cancer, lung cancer, esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer, and skin cancer.

In some preferred embodiments of the application, the cancer is breast cancer; further, the breast cancer is at least one selected from ER-positive breast cancer, her 2-positive breast cancer, PI3K mutant breast cancer, primary breast cancer, lymph node negative breast cancer, breast invasive ductal carcinoma, and non-endometrium-like breast cancer.

In particular, the combined medicine of the application has better treatment effect on ER+HER2-advanced breast cancer.

The beneficial effects are that:

the application firstly provides the effect of hydroxychloroquine in reducing the toxic and side effects of the high-dose CDK4/6 inhibitor, further provides a novel strategy based on the combination of hydroxychloroquine and the high-dose CDK4/6 inhibitor, and clarifies the action mechanism of the novel strategy, and has important significance for the application of hydroxychloroquine and the high-dose CDK4/6 inhibitor in clinical treatment of cancers. It is well known that drug research takes an average of 8-10 years from compound molecule to actual clinical running, and requires a great deal of manpower and material resources to support, and the time cost and the economic cost are huge. The scheme of the application can realize the new use of the old medicine and can greatly shorten the time from the medicine discovery to the clinical transformation.

Definition:

in the present application, the term "palbociclib" is palbociclib.

In the present application, the term "abemaciclib" is abbe-cili.

In the present application, the term "CDK4/6 inhibitor resistance" means that the tumor cells are able to resist the action of the CDK4/6 inhibitor at the recommended dose (20-50 mg/kg), resulting in tumor resistance.

In the present application, the term "pharmaceutically acceptable" means that there is no substantial toxic effect when used in the usual dosage amounts, and thus can be approved by the government or by an international organization equivalent thereto or has been approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.

In the present application, the "pharmaceutically acceptable excipients" that are useful in combination medicaments may be any conventional carrier or excipient in the pharmaceutical formulation arts, and the choice of a particular excipient will depend on the mode of administration or type and state of disease used to treat a particular patient. Methods of preparing suitable combinations for particular modes of administration are well within the knowledge of those skilled in the pharmaceutical arts. Examples of pharmaceutically acceptable excipients include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions, such as sodium; and/or nonionic surfactants such as tween (tm), polyethylene glycol (PEG), and pluronic stm. Such excipients include, for example, binders, fillers, disintegrants, lubricants in tablets, preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, and the like in liquid formulations.

In the present application, the term "combination" has its ordinary meaning. In addition, the "combination drug" of the present application may be present or provided in the form of a health product, a functional food, a food additive, or the like. The combination according to the application can be prepared by extracting, separating and purifying the active ingredients of the raw materials of the combination according to the application, optionally mixed with one or more pharmaceutically acceptable carriers or excipients, by means of conventional techniques in the pharmaceutical field, in particular in the field of formulations, and then forming the desired dosage form. The combination according to the application is a pharmaceutical formulation which may be suitable for oral administration, a pharmaceutical formulation (e.g. solution) suitable for parenteral injection (e.g. intravenous injection, subcutaneous injection), a pharmaceutical formulation suitable for topical administration (e.g. ointment, patch or cream), or a pharmaceutical formulation suitable for rectal administration (e.g. suppository) etc. Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, granules, pellets, elixirs, and the like, without limitation thereto. These formulations may contain, in addition to the active ingredient, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (e.g. silica, talc, stearic acid or its magnesium, calcium and polyethylene glycols). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrants (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, colorants, flavoring agents, sweeteners, etc. The tablets may be prepared according to conventional mixing, granulating or coating methods.

In the present application, the term "effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

Drawings

The application is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a graph showing the results of a high dose of CDK4/6 inhibitor resulting in a reduction in neutrophil count; wherein, # Pal-S refers to palbociclib-sensitive PDX mice, # Pal-R refers to palbociclib-primary drug resistant PDX mice, # Abe-S refers to abemaciclib-sensitive PDX mice, # Abe-R refers to abemaciclib-primary drug resistant PDX mice, # P < 0.001, ANC refers to neutrophil absolute count (Absolute Neutrophil Count, ANC).

FIG. 2 is a graph showing the results of hydroxychloroquine in alleviating the reduction in neutrophil count caused by high doses of CDK4/6 inhibitor; wherein, # Pal-S refers to palbociclib-sensitive PDX mice, # Pal-R refers to palbociclib-primary drug resistant PDX mice, # Abe-S refers to abemaciclib-sensitive PDX mice, # Abe-R refers to abemaciclib-primary drug resistant PDX mice, and P < 0.001.

FIG. 3 is a graph showing the results of HE staining of mice with hydroxychloroquine; wherein, # Pal-R refers to palbociclib primary drug resistant PDX mice, and # Abe-R refers to abemaciclib primary drug resistant PDX mice.

FIG. 4 is a graph of the results of hydroxychloroquine to protect neutrophils by reducing neutrophil NETS formation; wherein, # Pal-R refers to palbociclib primary drug resistant PDX mice, and # Abe-R refers to abemaciclib primary drug resistant PDX mice.

FIG. 5 is a graph showing analysis of the results of tumor volumes in mice of each group; wherein, # Pal-S refers to palbociclib-sensitive PDX mice, # Pal-R refers to palbociclib-primary drug resistant PDX mice, # Abe-S refers to abemaciclib-sensitive PDX mice, # Abe-R refers to abemaciclib-primary drug resistant PDX mice, and P < 0.001.

FIG. 6 is a graph showing the results of CCK8 detection for each group of cells; wherein Pal-SC refers to palbociclib-sensitive PDX mouse mammary gland primary cells, pal-RC refers to palbociclib-primary drug-resistant PDX mouse mammary gland primary cells, abe-SC refers to abemociclib-sensitive PDX mouse mammary gland primary cells, abe-RC refers to abemociclib-primary drug-resistant PDX mouse mammary gland primary cells, and P < 0.001.

FIG. 7 is a graph showing the results of cloning experiments for each group of cells; wherein CDK4/6i refers to CDK4/6 inhibitor, pal-SC refers to palbociclib-sensitive PDX mouse mammary gland primary cell, pal-RC refers to palbociclib-primary drug-resistant PDX mouse mammary gland primary cell, abe-SC refers to abemaciclib-sensitive PDX mouse mammary gland primary cell, and Abe-RC refers to abemaciclib-primary drug-resistant PDX mouse mammary gland primary cell.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.

Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.

The application selects two CDK4/6 inhibitors palbociclib and abemaciclib for experiments.

Experimental animals: the experimental animal sources of the application are female NOD/SCID mice of 4-6 weeks old and NSG mice are purchased from experimental animal houses of university of Zhongshan. Animal experiments are carried out in the animal experiment center of Zhongshan university, and experimental animals strictly follow the guidelines for management and use of experimental animals.

Example 1

A PDX model of breast cancer CDK4/6 inhibitor sensitivity and primary drug resistance is constructed, and the type of toxic and side effects caused by high-dose CDK4/6 inhibitor in animals is studied, wherein the specific operation is as follows:

1. ER+/HER 2-advanced breast cancer patients of primary IV from the center were collected, and puncture specimens were obtained before untreated and stored in DMEM medium.

2. Fresh tumor tissue from breast cancer patients was kept in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin.

3. Cutting it into 1-2 mm ³ Size, based on tumor mass, was inoculated into fat pads of 1-2 immunodeficient NOD/SCID mice and labeled as P0 generation.

4. After the tumor is formed, periodically measuring the long diameter and the short diameter of the tumor by using a vernier caliper, and until the volume of the subcutaneously transplanted tumor reaches 180-200mm ³ At this time, 5 groups of random data were obtained.

palbociclib groups were: (1) NC group-0.5% methyl suspension (0.5% CMC), (2)palbociclib 12.5mg/Kg/day, (3)palbociclib 25mg/Kg/day (clinical recommended dose, pal) ^RD ) (4)palbociclib 75mg/Kg/day group (high agent)Quantity, pal ^HD ) (5)palbociclib 150mg/Kg/day (maximum tolerated dose in mice).

The abelmoschlib groups are: (1) NC group-0.5% methyl suspension (0.5% CMC), (2) abemacliclib 31.25mg/Kg/day, (3)abemaciclib 62.5mg/Kg/day (clinically recommended dose, abe) ^RD ) (4) abelmacicalib 125mg/Kg/day (high dose, abe) ^HD ) (5)abemaciclib 250mg/Kg/day (maximum tolerated dose in mice).

5. Peripheral blood of mice was collected every 3 days during treatment for analysis of blood cells, which showed that high doses of CDK4/6 inhibitor resulted in a significant reduction in neutrophil numbers (see fig. 1).

Example 2

1. A PDX model was constructed as in example 1.

2. Grouping of CDK4/6 inhibitors with hydroxychloroquine is:

palbociclib treatment group: (1) NC group-0.5% methyl suspension (0.5% CMC), (2) palbociclib25mg/Kg/day (Pal) ^RD )、③palbociclib 75mg/Kg/day(Pal ^HD ) (4) hydroxychloroquine 60mg/Kg/day (HCQ), (5)palbociclib 25mg/Kg/day+hydroxychloroquine 60mg/Kg/day (Pal) ^RD +HCQ), (6) palbociclib75 mg/Kg/day+hydroxychloroquine 60mg/Kg/day (Pal) ^HD +HCQ)。

The abelmoschlib treatment groups were: (1) NC group-0.5% methyl suspension (0.5% CMC), (2. Abelmacocilib 62.5mg/Kg/day (Abe) ^RD )、③abemaciclib125mg/Kg/day(Abe ^HD ) (4) hydroxychloroquine 60mg/Kg/day (HCQ), (5)abemaciclib 62.5mg/Kg/day+hydroxychloroquine 60mg/Kg/day (Abe) ^RD +HCQ), (6)abemaciclib 125mg/Kg/day+hydroxychloroquine 60mg/Kg/day (Abe) ^HD +HCQ)。

3. Peripheral blood collected from mice every 3 days during treatment was analyzed and the results showed that high doses of CDK4/6 inhibitor resulted in a significant decrease in neutrophil count and hydroxychloroquine was able to reverse the decrease in neutrophil count caused by high doses of CDK4/6 inhibitor (see fig. 2).

4. Mice were sacrificed after the end of the treatment period and HE stained for hydroxychloroquine was found to be free of organic lesions (see fig. 3).

5. The mice peripheral blood neutrophils were isolated and tested for NETS formation at the end of the treatment cycle, and it was found that high doses of CDK4/6 inhibitor resulted in increased neutrophil NETS formation and hydroxychloroquine was able to alleviate NETS formation by high doses of CDK4/6 inhibitor (see FIG. 4).

6. Tumor volumes were measured and recorded every 3 days during treatment, and mice were sacrificed for recording of photographs after the end of the treatment period (specifically, 21 days for mice in the # Pal-S and # Pal-R groups and 28 days for mice in the # Abe-S and # Abe-R groups), which indicated that hydroxychloroquine combined with the high dose CDK4/6 inhibitor reversed the resistance of the recommended dose CDK4/6 inhibitor (see fig. 5).

Example 3

1. Inoculating cells: cells (purchased from ATCC) were seeded in 96-well plates at a density of 2000-4000 cells/well/100. Mu.L.

2. After the next day after the cells had attached, the drugs were diluted with fresh medium and added to the above-described cell-plated plates at 100 μl per well, and the group was as follows:

(1) vector + drug untreated group; (2) vector+pal ^RD /Abe ^RD ；③Vector+Pal ^HD /Abe ^HD ；④P62 ^KO +drug untreated group; (5) p62 ^KO +Pal ^RD /Abe ^RD ；⑥P62 ^KO +Pal ^HD /Abe ^HD ；⑦ATG5 ^KO +drug untreated group; (8) ATG5 ^KO +Pal ^RD /Abe ^RD ；⑨ATG5 ^KO +Pal ^HD /Abe ^HD 。

At least three wells are provided for each concentration to reduce experimental error, and a control group (no cells added) and a blank group (no cells added and no drugs added, only medium).

3. The above culture plate was placed at 37℃with 5% CO ₂ Is incubated for 72h in the incubator.

4. On the fourth day, dissolved CCK-8 working solution (20. Mu.L/well) was added and incubation was continued for 3h.

5. The OD value of each hole is detected by an enzyme label instrument, and the detection wavelength is 450nm.

6. From the OD value readings, cell viability was calculated and calculated using Grapad Prism 8.

The results show that hydroxychloroquine and CDK4/6 inhibitor have strong synergism on ER+/HER 2-mammary gland primary cells, and especially when hydroxychloroquine and CDK4/6 inhibitor are combined at high dose, the synergism is obvious, and the drug resistance of CDK4/6 inhibitor at recommended dose can be reversed (see figure 6).

Example 4

1. The primary cells of mammary gland were inoculated into 6-well plates according to 400 per well, 1800. Mu.L of culture medium was added and mixed well, and 3 compound wells were set per group.

2. The next day, after cell attachment, the drug was diluted with fresh medium and added to the plated plates at 200 μl per well. The grouping situation is as follows:

At least three complex wells are provided for each concentration to reduce experimental error.

4. After 10 days, the clone grows to a diameter of 1-2 mm ³ At this time, the culture was terminated, and the supernatant was discarded and washed 2 times with PBS.

5. 2mL of methanol was added to each well and the mixture was allowed to stand for 15 minutes.

6. The fixative was removed and stained with 0.5mL crystal violet stain for 15 minutes.

7. Washing off the dyeing liquid with clear water, and airing at room temperature.

8. The pictures were taken and the number of clones per well was counted.

The results show that hydroxychloroquine has synergistic anti-tumor effects with high doses of CDK4/6 inhibitor, and can completely reverse the situation that breast primary cells are resistant to recommended doses of CDK4/6 inhibitor (see FIG. 7).

The application adopts single-use and combined withdrawal medicines to observe the anti-tumor curative effect and toxic and side effects of a breast cancer recommended dose CDK4/6 inhibitor sensitive and drug-resistant PDX mouse, and finds that hydroxychloroquine can relieve the neutropenia caused by a high dose of CDK4/6 inhibitor, and the combination of the high dose of CDK4/6 inhibitor can reverse the drug resistance of the recommended dose. In vitro experiments further find that hydroxychloroquine can sensitize CDK4/6 inhibitor to efficacy, and completely inhibit proliferation of primary cells of mammary gland in a short period of time. Not only is the synergistic and toxicity-reducing effects of hydroxychloroquine combined with a high-dose CDK4/6 inhibitor verified from in vitro and in vivo experiments, but also the mechanism of the hydroxychloroquine for reducing the toxicity of neutral granules and the mechanism of the synergistic anti-tumor effect of the hydroxychloroquine and the high-dose CDK4/6 inhibitor are importantly clarified. Based on the above, the combination of the two can be used as a new anti-tumor drug combination development strategy.

The embodiments of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

Claims

1. Use of hydroxychloroquine in the manufacture of a medicament for reducing resistance to a CDK4/6 inhibitor.

2. The use according to claim 1, wherein the hydroxychloroquine reduces CDK4/6 inhibitor resistance by reducing the neutrophil toxicity of a high dose CDK4/6 inhibitor;

preferably, the high dose is 75-150 mg/kg of daily administration.

3. The use according to claim 1, wherein the CDK4/6 inhibitor is selected from at least one of abemaciclib, ribociclib, palbociclib, dalpiciclib.

4. A combination comprising a therapeutically effective amount of hydroxychloroquine and a high dose of a CDK4/6 inhibitor as active ingredients.

5. The combination according to claim 4, wherein the therapeutically effective amount is 20 to 100mg/kg per day; and/or, the high dose is 75-150 mg/kg of daily administration dose.

6. The combination according to claim 4, wherein the CDK4/6 inhibitor is selected from at least one of abemaciclib, ribociclib, palbociclib, dalpiciclib.

7. The combination according to claim 4, further comprising a pharmaceutically acceptable adjuvant.

8. The combination according to claim 4, wherein the hydroxychloroquine and the CDK4/6 inhibitor are administered simultaneously, separately or sequentially.

9. Use of a combination according to any one of claims 4 to 8 for the manufacture of a medicament for the prophylaxis and/or treatment of cancer.

10. The use according to claim 9, wherein the cancer is selected from at least one of bladder cancer, breast cancer, colon cancer, kidney cancer, epidermoid cancer, liver cancer, lung cancer, esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer and skin cancer;

preferably, the cancer is breast cancer.