Application of zinc pyrithione in preparation of medicine for treating retinoblastoma

Abstract

The invention discloses application of zinc pyrithione in preparation of a medicine for treating retinoblastoma, and relates to the technical field of biological medicines. On the basis of high-throughput drug screening, a small-molecule inhibitor zinc pyrithione (ZnPt) capable of efficiently inhibiting retinoblastoma (RB) cell proliferation is obtained, and the anti-tumor effect of ZnPt is verified through a series of in-vivo and in-vitro experiments. Furthermore, the invention also explores the anti-tumor action mechanism of the ZnPt, and finds that the ZnPt can play an anti-tumor role by inhibiting a glycolysis metabolic pathway. The research result of the invention provides a new strategy for developing clinical treatment of RB, and lays a foundation for precise treatment of a targeted metabolic pathway.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of zinc pyrithione in preparation of a drug for treating retinoblastoma. BACKGROUND

[0002] Retinoblastoma (RB) is the most common primary intraocular malignant tumor in infants, and is prone to occur in infants under 3 years old. It can manifest as single eye, double eyes or three lateral (double eyes and intracranial) lesions, and has a family genetic tendency. Statistics show that the number of RB patients in China ranks in the world, and most of the RB children are in the intraocular progression period (D period or E period) when diagnosed. With the enlargement of the tumor, the intraocular contents increase, leading to elevated intraocular pressure, which may be secondary to glaucoma. When the tumor further expands, it can spread along the optic nerve to the intracranial, or break through the sclera into the orbit, and even protrude outside the palpebral fissure to form a large mass, which seriously threatens the life of the child.

[0003] Chemotherapy is the main method for treating RB in the intraocular stage at present. According to the administration route, it can be divided into intravenous chemotherapy, ophthalmic artery interventional chemotherapy, vitreous cavity chemotherapy and periocular local chemotherapy. However, the current RB chemotherapy regimen is still mainly based on traditional cytotoxic drugs, which has problems such as large toxic side effects, high drug resistance rate, etc., affecting the long-term efficacy and success rate of eye preservation.

[0004] In recent years, with the continuous development of medical technology, tumor metabolic reprogramming has been recognized as the core feature of cancer cells adapting to the microenvironment and maintaining rapid proliferation, and has become an important target for anti-tumor therapy. In the field of RB tumor research, several targeted drugs have been proven to effectively inhibit the growth of RB tumors. For example, MDM2 inhibitors combined with topotecan can significantly induce RB cell death. MDM2 is the most important E3 ubiquitin ligase of p53, which can promote the degradation of p53, thereby inhibiting p53-mediated apoptosis; Combretastatin A-4 phosphate (CA-4P) as a tumor vascular targeting drug can significantly reduce the tumor volume in RB animal experiments after subconjunctival injection; Spleen associated tyrosine kinase (SYK) is an important molecule that maintains tumor cell survival, and silencing SYK can promote tumor cell apoptosis. However, metabolic targeted therapy still faces many challenges. First, tumor cells have strong metabolic plasticity and can escape single target inhibition through metabolic reprogramming; second, normal cells also rely on basal metabolism, and the selectivity of metabolic inhibitors is difficult to accurately control, which may cause toxicity problems; in addition, the tumor microenvironment is complex, and different types of tumors or different stages of the same tumor may exhibit different metabolic dependencies, making it difficult to establish a unified metabolic targeting strategy. Therefore, future research needs to further explore more specific and more precise metabolic intervention strategies to improve the effectiveness and safety of anti-tumor therapy. SUMMARY

[0005] The purpose of the present application is to provide the use of zinc pyrithione in the preparation of a medicament for treating retinoblastoma, to improve the effectiveness and safety of RB treatment, and to provide new treatment options for RB patients.

[0006] Based on the above, the present application first provides the use of zinc pyrithione in the preparation of a medicament for treating retinoblastoma.

[0007] Preferably, the chemical structure of the zinc pyrithione is as shown in the following formula (I):

[0008]

[0009] Preferably, the zinc pyrithione can inhibit the proliferation of retinoblastoma cells.

[0010] Preferably, the zinc pyrithione exerts its anti-tumor effect by inhibiting the glycolytic metabolic pathway of retinoblastoma cells.

[0011] Preferably, the retinoblastoma is retinoblastoma cell line Y79 or WERI-Rb1.

[0012] Preferably, the drug is used alone or in the form of a pharmaceutical composition.

[0013] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0014] Preferably, the dosage form of the drug comprises any one or more of injections, eye drops, tablets, capsules.

[0015] Preferably, the effective concentration of zinc pyrithione is 5mg / kg.

[0016] Compared with the prior art, the beneficial effects of the present application at least include:

[0017] The present application is based on high-throughput drug screening, and a new small molecule inhibitor zinc pyrithione (ZnPt) capable of efficiently inhibiting RB cell proliferation is obtained, and its anti-tumor effect on RB is verified through a series of in vitro and in vivo experiments, which can be used in the preparation of RB therapeutic drugs.

[0018] Further, the anti-tumor mechanism of ZnPt is also explored, and its pharmacodynamic mechanism is systematically analyzed in combination with the characteristics of RB metabolic heterogeneity, and the regulation effect of ZnPt on tumor metabolic pathways is revealed. Ultimately, it is found that ZnPt may mediate its anti-tumor effect by inhibiting the glycolysis metabolic pathway. In summary, the research results of the present application will provide a new strategy for the development of RB clinical treatment, and lay a foundation for precise treatment targeting metabolic pathways. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 High-throughput drug screening suggests that ZnPt has an inhibitory effect on RB cells, wherein:

[0020] A indicates that the HTS library is subjected to cell viability screening;

[0021] B indicates the results of combined analysis of inhibitors with effective inhibition rate >90% on Y79 and WERI-Rb1;

[0022] C indicates that ZnPt shows strong proliferation inhibition effect in Y79 and WERI-Rb1 cells.

[0023] Figure 2 In order to evaluate the in vitro and in vivo experimental results of ZnPt anti-RB tumor activity, wherein:

[0024] A indicates the half inhibitory concentration (IC50) of ZnPt on RB cell lines Y79 and WERI-Rb1;

[0025] B indicates the proliferation curve of Y79 and WERI-Rb1 cells treated with different concentrations of ZnPt;

[0026] C represents the soft agar colony formation experiment after Y79 treated with ZnPt concentration gradient;

[0027] D-F represent that ZnPt significantly inhibits the growth of RB tumor in nude mice.

[0028] Figures 3-4 The results of the analysis of the anti-tumor mechanism of ZnPt are as follows:

[0029] A represents that RNA-seq analysis shows that the glycolysis pathway is differentially enriched after Y79 cells are treated with ZnPt, and GSEA suggests that the glycolysis pathway is down-regulated;

[0030] B represents that the intracellular lactic acid content of RB cell lines Y79 and WERI-Rb1 is reduced after ZnPt treatment;

[0031] C represents that the hexokinase activity in RB cell lines Y79 and WERI-Rb1 is reduced after ZnPt treatment;

[0032] D represents that Seahorse experiment shows that glycolysis is reduced after Y79 cells are treated with ZnPt;

[0033] E represents that Seahorse experiment shows that glycolysis is reduced after WERI-Rb1 cells are treated with ZnPt.

[0034] Figure 5 The experimental results of retinoblastoma (RB) having glycolytic metabolic heterogeneity are as follows:

[0035] A represents the results of RB tissue metabolomics analysis, showing the expression distribution of sugar metabolism related metabolites;

[0036] B represents the comparison of glycolysis levels of RB cell lines Y79 and WERI-Rb1 by Seahorse analysis; wherein, Basal Glycolysis represents the baseline glycolysis level, Compensatory Glycolysis represents the compensatory glycolysis level, glycoPER represents the glycolytic Proton Efflux Rate, and mitoOCR is the mitochondrial Oxygen Consumption Rate;

[0037] C represents the comparison of glycolysis related protein levels of RB cell lines Y79 and WERI-Rb1;

[0038] D represents the comparison of glycolysis related protein levels of 3 RB-PDC samples. DETAILED DESCRIPTION

[0039] The technical solutions of the present application are further described below in combination with the drawings and examples.

[0040] As described above, chemotherapy is the main method for treating RB in the intraocular stage at present, but the current chemotherapy regimen for RB still mainly uses traditional cytotoxic drugs, which has problems such as great toxic side effects, high drug resistance rate, and the like, affecting long-term efficacy and success rate of eye preservation. Although in recent years, with the development of medical science and technology, a plurality of drugs targeting metabolic pathways have been confirmed to have inhibitory effects on RB, due to reasons such as strong metabolic plasticity of tumor cells and complex tumor microenvironment, the metabolic targeting therapeutic drugs still face challenges in effectiveness and safety, and the like.

[0041] To solve the above technical problems, the present application provides, through a large amount of research, an application of zinc pyrithione in preparation of a drug for treating retinoblastoma, the zinc pyrithione can inhibit proliferation of retinoblastoma cells, so that the purpose of effectively treating RB is achieved, and there is no obvious toxic side effect, and the use is safe.

[0042] Therefore, a first object of the present application is to provide an application of zinc pyrithione in preparation of a drug for treating retinoblastoma, the chemical structural formula of the zinc pyrithione is shown as the following formula (I):

[0043]

[0044] In some embodiments, the drug is used alone, and in other embodiments, the drug is used in the form of a pharmaceutical composition.

[0045] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical agent carriers: they are not essential active ingredients themselves, and have no excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. The pharmaceutically acceptable carrier in the composition can contain a liquid, such as water, phosphate buffer, ringer solution, physiological saline, balanced salt solution, glycerol or sorbitol, and the like. In addition, there can be auxiliary substances in these carriers, such as lubricants, flow aids, wetting agents or emulsifiers, pH buffering substances and stabilizers, such as albumin and the like.

[0046] In some embodiments, the dosage form of the drug includes any one or more of injections, eye drops, tablets, capsules.

[0047] Next, the research process of the present application is described in detail through specific examples and drawings.

[0048] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields. The materials and reagents used in the present application, if not specifically stated, can be obtained from commercial channels.

[0049] (I) High-throughput drug screening of drugs that can effectively kill RB cells

[0050] The present application uses RB cell lines Y79 and WERI-Rb1 to perform cell viability screening on 3075 inhibitors of known targets through a high-throughput drug screening platform, and counts the effectiveness and action proportion of the inhibitors. Figure 1 A).

[0051] Further combined analysis of the inhibitors with effective inhibition rate of Y79 and WERI-Rb1 > 90% screens out 13 potential effective compounds, including: Auranofin (Gold sodium thiomalate), ZnPt (Zinc Pyrithione, purchased from Selleck Company, S4075), SF1670 (PTEN inhibitor), PFK15 (a selective PFKFB3 inhibitor), b-AP15 (DUB inhibitor), LDN-212854 (bone morphogenetic protein inhibitor), Stattic (STAT3 inhibitor), Sanguinarine chloride (chloride of blood root alkali), BIX 01294 (a selective G9a histone methyltransferase inhibitor), JTC-801 (NOP antagonist), Gambogic Acid (gambogic acid), Ouabain (ouabain), CX-6258HCl (a selective PI3K inhibitor), and the like. Figure 1 B). Among them, ZnPt shows significant inhibition effect in two RB cell lines. Figure 1 C).

[0052] Subsequent further research experiments are carried out using ZnPt to evaluate the therapeutic effect and mechanism of ZnPt on RB through a series of in vivo and in vitro experiments.

[0053] (II) In vivo and in vitro experiments for evaluating the inhibition effect of ZnPt on RB

[0054] 1. In vitro experiment

[0055] IC50 determination of ZnPt obtained by the above screening, measuring its half inhibitory concentration on RB cell lines Y79, WERI-Rb1. In 96-well plates, RB cell lines Y79, WERI-Rb1 were inoculated respectively, different concentrations of ZnPt were added for 72h, then CCK8 was added, and the absorbance was detected after 3-4h of incubation in the dark. The results show that ZnPt can effectively inhibit the proliferation of RB cells at low concentration, and Y79 is more sensitive to ZnPt Figure 2 A and B of FIG. 12.

[0056] In addition, the anti-tumor activity of ZnPt was also evaluated by soft agar colony formation experiment. Specifically, Y79 cells were inoculated in 6-well plates at 5x10 4 cells per well, different concentrations (0, 50, 100, 200nM) of ZnPt were added, and the diameter of the spheres was observed and photographed after 3 weeks of culture. The results show that the size of Y79 cell spheres is significantly inhibited after ZnPt drug treatment Figure 2 C of FIG. 12.

[0057] 2. In vivo experiment

[0058] The anti-tumor activity of ZnPt was evaluated by in vivo experiment using the PDX model successfully constructed in the early stage. The PDX model is a patient-derived tumor transplantation model constructed by transplanting fresh human tumor biopsy into immunodeficient mice, and the tumor of the PDX model retains the characteristics of molecular, genetic, and histological heterogeneity of tumor patients, which is a powerful tool for preclinical drug screening. RB primary cells were injected into the subretinal space of the eye of nude mice by microinjection, and tumors were formed after one week. The mice were randomly divided into two groups, including the drug group (ZnPt) and the control group (Control). The drug group was injected intraperitoneally with ZnPt (5mg / kg, twice a day), and the control group was given the same volume of normal saline (NaCl). The tumor size was measured after 14 days of treatment, and the eyeball was removed and weighed to evaluate the anti-tumor effect of ZnPt.

[0059] The results show that compared with the control group, the tumor of the drug group treated with ZnPt is significantly reduced Figure 2 D and E of FIG. 13), and the weight of the eyeball is also significantly reduced Figure 2 F of FIG. 13), indicating that ZnPt has good anti-tumor effect in vivo.

[0060] (III) ZnPt exerts anti-RB effect by inhibiting glycolysis

[0061] To further understand the mechanism of ZnPt in exerting anti-tumor activity in RB, the present application observed the changes in KEGG pathway of Y79 cells treated with 100nM ZnPt for 24h by transcriptome sequencing analysis. The results show that the glycolysis metabolic pathway is significantly down-regulated Figure 3A).

[0062] Further, using lactic acid content detection kit (Solarbio, BC2235) and hexokinase activity detection kit (Solarbio, BC0740), the intracellular lactic acid content and hexokinase activity of the two RB cells Y79 and WERI-Rb1 after drug (i.e. ZnPt) treatment were detected according to the operation instruction of the kit instruction. The results show that after ZnPt treatment, the intracellular lactic acid content of the RB cells is significantly reduced Figure 3 B), and the hexokinase activity is also significantly reduced Figure 3 C). In addition, Seahorse metabolic analysis further confirms that the glycoPER of the two RB cells after ZnPt treatment is reduced Figure 4 D and E).

[0063] The above results show that ZnPt may exert its anti-tumor effect by inhibiting the glycolysis metabolic pathway.

[0064] (IV) Heterogeneity of RB glycolysis metabolism

[0065] In view of the fact that ZnPt mainly acts on the glycolysis pathway, we further explored the activity difference of the metabolic pathway in different RB patient samples. The present application first performed non-targeted metabolomics sequencing analysis on 56 RB tissues (from the sample library of the Department of Ophthalmology, Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine). The results showed that there was metabolic heterogeneity in the tumor tissues. Further attention to the glycometabolism metabolites found that the glycolysis in the tumor tissues was significantly different Figure 5 A).

[0066] The present application also performed Seahorse metabolic analysis experiment on RB cell lines Y79 (purchased from ATCC) and WERI-Rb1 (purchased from Chinese Academy of Sciences Typical Culture Collection Committee Cell Library) at 1×10 5 cells per well in a Seahorse XF96 plate to detect the extracellular acidification rate of the cells. The results show that the metabolic modes of the cell lines are also different, and the glycolysis activity of Y79 cells is higher, and the oxidative phosphorylation activity of WERI-Rb1 is stronger Figure 5 B). In addition, WB was used to detect the glycolysis-related protein levels in RB cell lines (Y79 and WERI-Rb1) for comparative analysis, and the results show that the expression levels of the glycolysis-related proteins (HK2, PKM2, ENO1, LDHA) in the RB cell lines Y79 and WERI-Rb1 are different Figure 5 C).

[0067] In addition, the present application also carries out protein extraction and WB detection on 3 different patient-derived RB fresh tissues through mechanical crushing, grinding, enzyme digestion, suspension culture and other operations. The results show that there are differences in the glycolysis-related protein levels of different RB primary cells Figure 5 D) It should be noted that the PDC01-derived patient is bilateral RB, right eye (enucleation eye) extraocular period, pre-treatment eye enucleation; the PDC02-derived patient is bilateral RB, left eye (enucleation eye) extraocular period cT4b, post-treatment eye enucleation; and the PDC03-derived patient is unilateral RB, left eye (enucleation eye) D period, post-treatment eye enucleation.

[0068] In summary, based on high-throughput drug screening, the present application obtains a new small-molecule inhibitor pyrithione zinc (ZnPt) capable of efficiently inhibiting RB cell proliferation, and through a series of in-vivo and in-vitro experiments, it is proved that the pyrithione zinc can significantly inhibit the proliferation of RB cells, achieving the purpose of effectively and safely treating RB.

[0069] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims

1. Application of zinc pyrithione in the preparation of a drug for treating retinoblastoma.

2. The use according to claim 1, characterized in that The chemical structural formula of the zinc pyrithione is shown in the following formula (I):

3. The use according to claim 1, characterized in that The zinc pyrithione can inhibit the proliferation of retinoblastoma cells.

4. The use according to claim 3, characterized in that The zinc pyrithione exerts its anti-tumor effect by inhibiting the glycolysis metabolic pathway of retinoblastoma cells.

5. The use according to claim 1, characterized in that The retinoblastoma cell line is retinoblastoma cell line Y79 or WERI-Rb1.

6. The use according to claim 1, wherein The drug is used alone or in the form of a pharmaceutical composition.

7. The use according to claim 1, wherein The drug further includes a pharmaceutically acceptable carrier.

8. The use according to claim 1, wherein The dosage form of the drug includes any one or more of injection, eye drops, tablets, and capsules.

9. The use according to claim 1, wherein The effective concentration of zinc pyrithione is 5 mg / kg.

Images