A method for treating cholangiocarcinoma with PTEN gene mutations and / or deletions.

Abstract

A method for treating cholangiocarcinoma with PTEN gene mutation and / or deletion, specifically, administering a therapeutically effective amount of a proteasome inhibitor of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof to a cholangiocarcinoma patient, preferably a cholangiocarcinoma with PTEN gene mutation and / or deletion; this method overcomes the problem of ineffectiveness of existing protease inhibitors in the treatment of solid tumors due to drug distribution issues, and shows good efficacy for cholangiocarcinoma, especially cholangiocarcinoma caused by PTEN gene mutation and / or deletion.

Description

A method for treating cholangiocarcinoma with PTEN gene mutation and / or deletion Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a method for treating cancer, and more specifically to the use of a proteasome inhibitor and its in vivo active ingredients in the preparation of a drug for treating bile duct cancer with PTEN gene mutation and / or deletion. Technical Background

[0002] Cholangiocarcinoma is a highly malignant disease with rapid progression. It often presents to clinical diagnosis in the advanced stage, often having already recurred and metastasized, resulting in a low operability rate. Conventional chemoradiotherapy and radiotherapy have very limited efficacy in cholangiocarcinoma, and targeted therapies and immunotherapies, which have demonstrated clear efficacy in other tumors, have rarely been successfully used in cholangiocarcinoma. A paper by Academician Wang Hongyang's research group, titled "PTEN status determines the chemosensitivity to proteasome inhibition in cholangiocarcinoma," reveals that nearly 40% of cholangiocarcinomas harbor a deletion in the crucial tumor suppressor gene PTEN. This mutation significantly increases proteasome activity, directly impacting the malignant behavior of tumor cells.

[0003] The ubiquitin-proteasome pathway (UPP) is the primary pathway for protein degradation in cells and is involved in many physiologically important cellular processes, including signal transduction, immune responses, the unfolded protein response, and cell cycle progression. This pathway is crucially involved in the pathogenesis of cardiovascular and cerebrovascular diseases, cancer, and neurodegenerative diseases. Proteasome inhibitors have been used as therapeutic drugs for diseases such as cancer.

[0004] In 2003, the first proteasome inhibitor, bortezomib, received FDA approval for the treatment of relapsed myeloma. In 2004, the drug was approved for marketing in the European Union for the treatment of multiple myeloma. In September 2005, the drug was introduced by Xi'an Janssen and first launched in Guangzhou, my country. On July 11, 2007, it was approved by the US FDA for the treatment of relapsed or refractory mantle cell lymphoma (MCL), becoming the only FDA-approved drug for the treatment of MCL. Velcade is priced at approximately 13,000 yuan per 3.5 mg dose in the Chinese market, and a single cycle of treatment costs approximately 40,000 yuan. This high cost is a significant financial burden for many patients. Furthermore, current clinical data shows that this type of drug also has a number of side effects, such as fatigue, nausea, diarrhea, and neuropathy.

[0005] Several other protease inhibitors have been developed as drugs or are in clinical trials and testing phases, including carfilzomib, ONX0912, CEP-18770, and MLN9708. MLN9708 is a second-generation small molecule proteasome inhibitor used to treat a variety of human malignant gliomas. After contact with aqueous solution and plasma, MLN9708 is rapidly hydrolyzed to MLN2238. MLN2238 is the biologically active form of MLN9708. MLN2238 is a dipeptide leucine boronic acid capped at the nitrogen end that inhibits the chymotrypsin (β5) hydrolysis site of the 20S proteasome, IC 50 The Ki value is 3.4 nM and 0.93 nM. MLN2238 is an effective, selective, reversible inhibitor of the proteasome, and this reversibility is time-dependent. However, proteasome inhibitors have drug distribution problems in the treatment of solid tumors. For example, clinical studies of bortezomib in solid tumor patients have failed to demonstrate any efficacy of the drug (Proteasome inhibitors in cancer therapy, Nat Rev Clin Oncol. 2017 Jul; 14(7): 417-433). Therefore, the development of a low-cost, low-toxic and highly effective proteasome inhibitor drug, the precise targeted treatment of cholangiocarcinoma based on molecular classification and PTEN mutation status, and the research on solutions that can solve the drug distribution problem and achieve drug efficacy in solid tumors are the key issues we need to address. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a new use of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides the use of a therapeutically effective amount of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating bile duct cancer. Preferably, the bile duct cancer is bile duct cancer with PTEN gene mutation and / or deletion.

[0009] The present invention also provides a method for treating bile duct cancer, comprising administering a therapeutically effective amount of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof to a bile duct cancer patient, wherein the bile duct cancer is preferably a bile duct cancer in which a PTEN gene mutation and / or deletion exists.

[0010] The structure of the proteasome inhibitor represented by formula (I) or formula (II) of the present invention is as follows:

[0011] In some embodiments of the present application, the bile duct cancer described herein includes intrahepatic bile duct cancer, hilar bile duct cancer, or distal bile duct cancer. In some embodiments, the bile duct cancer is intrahepatic bile duct cancer.

[0012] Cholangiocarcinoma of the present invention has PTEN gene mutation and / or deletion, wherein PTEN gene mutation and / or deletion can be PTEN gene transcription and protein abundance reduction, also can be PTEN gene complete deletion.Diagnosing whether PTEN gene has mutation and / or deletion can be conventional gene diagnosis method.Preferred is the application in cholangiocarcinoma with PTEN gene complete deletion.

[0013] The cholangiocarcinoma described in the present invention may be cholangiocarcinoma at different stages, such as early, middle, late or metastatic cholangiocarcinoma, or recurrent cholangiocarcinoma.

[0014] The application of the present invention is specifically to administer a therapeutically effective amount of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof to a patient in need of treatment. In the specific treatment of bile duct cancer, the dosage of the proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof can be appropriately selected according to the severity of the symptoms, age, gender, weight, and differences in patient sensitivity, administration method, administration time, administration interval, pharmaceutical preparation type, etc., for example, it can be 1 μg to 20g per day, which can be administered once a day or in multiple doses a day, for example, 2, 3 or 4 times.

[0015] In some specific embodiments of the present invention, the bile duct cancer patient of the present invention may have previously received chemotherapy, small molecule targeted therapy and / or radiotherapy and the disease has progressed; the chemotherapy previously received may be a chemotherapy method commonly used in the art, such as gemcitabine, 5-fluorouracil, paclitaxel, oxaliplatin, cisplatin and / or Tegafur treatment, and the small molecule targeted therapy may also be a commonly used small molecule targeted therapeutic agent in the art, such as sunitinib.

[0016] The proteasome inhibitors represented by Formula (I) or Formula (II) of the present invention, or pharmaceutically acceptable salts thereof, can be administered via a variety of routes, including but not limited to oral, intravenous, sublingual, intranasal, inhalation, and subcutaneous administration; in some specific examples, oral administration or injection. Formulations suitable for oral administration can be any dosage form suitable for oral administration known in the art, including but not limited to tablets, capsules, powders, granules, pills, pastes, powders, etc., preferably tablets or capsules. The oral formulations can be prepared by conventional methods using pharmaceutically acceptable carriers known in the art.

[0017] The present invention also provides a pharmaceutical composition for treating bile duct cancer with PTEN gene mutation and / or deletion, comprising a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0018] The present invention also provides use of the proteasome inhibitor represented by the above formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a 20S proteasome β5 subunit chymotrypsin-like inhibitor.

[0019] The present invention also provides use of the proteasome inhibitor represented by the above formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a β1 subunit caspase-like inhibitor.

[0020] The present invention also provides use of the proteasome inhibitor represented by the above formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a β2 subunit caspase-like inhibitor.

[0021] For the purposes of this application, unless otherwise stated, the following terms used in the specification and claims shall have the following meanings.

[0022] "Patient" refers to a mammal, preferably a human.

[0023] "Pharmaceutically acceptable" means that it is used to prepare a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes that which is acceptable for human pharmaceutical use.

[0024] "Therapeutically effective amount" means the amount of a compound that, when administered to a human for treating a disease, is sufficient to achieve control of the disease.

[0025] "Treatment" means any administration of a therapeutically effective amount of a compound, and includes:

[0026] (1) inhibiting the disease in a human being who is experiencing or exhibiting the pathology or symptomology of the disease (i.e., preventing further development of the pathology and / or symptomology), or

[0027] (2) Ameliorating a disease in a human who is experiencing or exhibiting the pathology or symptomology of the disease (i.e., reversing the pathology and / or symptomology).

[0028] The method for treating cholangiocarcinoma provided by the present invention overcomes the problem of ineffectiveness of existing protease inhibitors in treating solid tumors due to drug distribution problems, and shows good efficacy (tumor inhibition rate close to 60%) in patients with cholangiocarcinoma, especially cholangiocarcinoma caused by PTEN gene mutation and / or deletion. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples, based on common technical knowledge and customary means in the art. The following examples are only some preferred embodiments of the present invention and should not be regarded as limiting the present invention. It is obvious to those skilled in the art that several modifications can be made without departing from the scope of the present invention, and these modifications should also be regarded as the scope of protection of the present invention.

[0030] Example 1: Study on the in vitro antitumor activity and mechanism of action of Compound I and Compound II

[0031] Test 1: Inhibition of 20S proteasome and its isoforms by compounds

[0032] The inhibitory effects of compound I, active form compound II and MLN9708 on the activities of different proteasome subunits were studied by using purified human 20S proteasome and specific fluorescent substrates for each subunit. It was proved that compound I of the present invention can selectively inhibit the activity of 20S proteasome β5 subunit chymotrypsin-like (CT-L). 50 was 2.22±0.71nM; the inhibitory effect on β1 subunit caspase-like (CL) was second, IC 50 The inhibitory effect on β2 subunit trypsin-like (TL) was the weakest, IC 50 The inhibitory effect of active form compound II on the three subunits of 20S proteasome (β5, β1, β2) was comparable to that of compound I, IC 50 The IC values ​​of the reference drug MLN9708 for inhibition of the three subunits of the 20S proteasome (β5, β1, β2) were 2.61±0.75, 24.27±0.34, and 410.03±137.95 nM, respectively. 50 The results showed that compound II and compound I had similar activities and could selectively inhibit the activity of proteasome β5 subunit, and their inhibitory effects were stronger than those of reference drug MLN9708. 50 It is about 3.09-3.64 times lower than MLN9708 (as shown in Table 1).

[0033] Table 1 IC inhibition of 20S proteasome by compounds 50 Summary (n=3)

[0034] Experiment 2: Inhibition of proteasome CT-L activity by compounds in cholangiocarcinoma cells

[0035] Cholangiocarcinoma cells RBE, HuCCT1, and HCCC-9810 were incubated with Compound I and MLN9708 for 1 hour, respectively, and their inhibitory effects on intracellular 20S proteasome β5 subunit chymotrypsin-like (CT-L) activity were assessed. RBE cells showed intact PTEN gene expression, HuCCT1 cells showed reduced PTEN gene transcription and protein abundance (hemizygous deletion), and HCCC-9810 cells showed complete PTEN gene deletion (exons 6-9 deletion).

[0036] The results are shown in Table 2. Compound I and MLN9708 both have a certain inhibitory effect on the CT-L activity of the 20S proteasome in cholangiocarcinoma cells. Compound I has significantly better inhibitory activity against all three cell types than MLN9708, especially against the 20S proteasome CT-L activity in PTEN-completely deficient HCCC-9810 cells, with an IC 50 It is 23.09nM, significantly better than MLN9708.

[0037] The above results indicate that compound I can inhibit the activity of proteasome CT-L in cholangiocarcinoma cells, and the inhibitory intensity is significantly stronger than that of MLN9708.

[0038] Table 2 IC inhibition of proteasome CT-L activity in cholangiocarcinoma cells by compounds 50 Summarize

[0039] Experiment 3: Inhibition of the proliferation of cholangiocarcinoma cells by the compound

[0040] The CellTiter-Glo assay kit was used to detect the effects of compound I and MLN9708 on the proliferation of cholangiocarcinoma cells.

[0041] As shown in Table 3, Compound I has significantly improved inhibitory activity against HCCC-9810, HuCCT1, and RBE cells compared to MLN9708, and in particular has more significant selective proliferation inhibitory activity against cholangiocarcinoma cells with complete PTEN deficiency, with a selectivity of approximately 1.7 times compared to cells with complete PTEN expression.

[0042] Table 3 IC inhibition of compound I and MLN9708 on cholangiocarcinoma cell proliferation 50 Summary (n=3)

[0043] Example 2 Drug Distribution Study of Compounds

[0044] Thirty SD rats, half male and half female, weighing 195-217 g, were gavaged with 0.6 mg / kg of compound I at 5 sampling time points, with 6 rats in each time group. The sampling time points were 0 h before administration, and 5 min, 1 h, 4 h, and 24 h after administration.

[0045] After CO2 euthanasia, at least 1.0 mL of blood was collected by cardiac puncture, anticoagulated with EDTA-K2, mixed thoroughly, and placed on ice. A 100 μL portion of whole blood was added to an EP tube pre-filled with 200 μL of 0.5% formic acid and acetonitrile, with a ratio of whole blood to 0.5% formic acid and acetonitrile (v:v) of 1:2. Vortex to mix thoroughly. The whole blood sample was stored in an ultra-low temperature freezer. Within 30 minutes of blood collection (or any remaining blood sample), plasma was separated by centrifugation (11,000 rpm, 5 minutes, 2-8°C). A predetermined amount of plasma was added to an EP tube pre-filled with 0.5% formic acid and acetonitrile, with a ratio of plasma to 0.5% formic acid and acetonitrile (v:v) of 1:2 (e.g., 100 μL plasma + 200 μL of 0.5% formic acid and acetonitrile). Vortex to mix thoroughly. The plasma sample was stored in an ultra-low temperature freezer.

[0046] After blood samples were collected from the animals, the brain, heart, liver, spleen, lungs, and kidneys were collected; each tissue was rinsed separately with normal saline to avoid cross contamination, blotted dry with filter paper, and then placed in labeled tubes / ziplock bags (one tube / ziplock bag for each tissue). The samples were temporarily placed on ice before being stored in an ultra-low temperature freezer.

[0047] After thorough homogenization with a high-speed homogenizer to prepare tissue sample homogenates, the concentrations in plasma, whole blood, and tissue homogenates were determined by LC-MS / MS.

[0048] Results: After rats were gavaged with 0.6 mg / kg of Compound I, the concentrations of Compound II in their tissues are shown in Tables 4 to 7; the exposure levels of Compound II in different tissues and their comparison with plasma exposure levels are shown in Table 8.

[0049] Following oral administration of 0.6 mg / kg Compound I, the order of exposure to Compound II in male and female rats was spleen > liver > kidney > lung > heart > whole blood > plasma > brain. These results indicate that Compound II was more abundant in the liver, the target organ, following oral administration of 0.6 mg / kg Compound I. Liver tissue exposure was 36.9-51.0 times that of plasma, demonstrating significant clinical application value.

[0050] Table 4 Concentration of Compound II in various tissues 5 minutes after oral administration of 0.6 mg / kg Compound I to rats (ng / g or ng / mL) (BQL: below the limit of quantification: 5.00 ng / g or 0.300 ng / mL; NA: not applicable; / : insufficient homogenate or no sample, the same below)

[0051] Table 5 Concentration of Compound II in various tissues 1 hour after oral administration of 0.6 mg / kg Compound I to rats (ng / g or ng / mL)

[0052] Table 6 Concentration of Compound II in various tissues 4 hours after oral administration of 0.6 mg / kg Compound I to rats (ng / g or ng / mL)

[0053] Table 7 Concentration of Compound II in various tissues 24 hours after oral administration of 0.6 mg / kg Compound I to rats (ng / g or ng / mL)

[0054] Table 8 Exposure of Compound II in various tissues and plasma after oral administration of 0.6 mg / kg Compound I to rats (AUC 0-t , ng·h / g or ng·h / mL) (*: The software did not calculate this pharmacokinetic parameter; NA: Not Applicable.)

[0055] Example 3: In vivo antitumor activity study of compound I - human cholangiocarcinoma CC6679 subcutaneous xenograft (PDX) model

[0056] This research was commissioned by Crown Biotech (Beijing) Co., Ltd. (https: / / www.crownbio.cn / wp-content / uploads / 2023 / 01 / Crownbio_HuPrime_Factsheet_CN.pdf) Tumor tissue was harvested from CC6679 (PTEN frameshift mutation, p.N323KfsTer2) cholangiocarcinoma xenograft model mice, cut into 2-3 mm diameter tumor masses, and inoculated subcutaneously at the right anterior scapula of Balb / c nude mice. When the average tumor volume of the tumor-bearing mice reached approximately 104.46 mm 3 The mice were randomly divided into groups and given the drug. The doses of compound I were 1.0 mg / kg, 1.2 mg / kg and 1.0 / 1.5 mg / kg (1.0 mg / kg in the first week and 1.5 mg / kg in the next two weeks). After the start of the drug administration, the body weight and tumor size of the mice were measured twice a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0057] The experimental results are shown in Table 9. Compound I (1.0, 1.2, 1.0 / 1.5 mg / kg, ig, BIW, D1-19) dose-dependently inhibited the growth of subcutaneous transplanted tumors in nude mice, with tumor inhibition rates of 38.32%, 39.63%, and 52.32%, respectively. This shows that Compound I has a good therapeutic effect on cholangiocarcinoma with PTEN gene mutation and / or deletion, especially when Compound I was administered at 1.0 mg / kg in the first week and 1.5 mg / kg in the next two weeks, the tumor inhibition rate was close to 60%.

[0058] Table 9 Summary of the in vivo antitumor efficacy of compound I Note: TGI: 1–(Ti–T0) / (Ci–C0) × 100%. Dunnett's test was used for pairwise comparisons between all groups because Bartlett's test values ​​and Kruskal-Wallis test values ​​were >0.05; p values ​​were calculated based on absolute tumor volume.

Claims

1. Use of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer, wherein the cancer is bile duct cancer with PTEN gene mutation and / or deletion; 2. The use according to claim 1, characterized in that: The cholangiocarcinoma is hilar cholangiocarcinoma, intrahepatic cholangiocarcinoma or distal cholangiocarcinoma.

3. The use according to claim 1, characterized in that: The bile duct cancer is intrahepatic bile duct cancer.

4. The use according to claim 1, characterized in that: The PTEN gene mutation and / or deletion refers to a reduction in PTEN gene transcription and protein abundance, or a complete deletion of the PTEN gene.

5. The use according to claim 4, characterized in that: The PTEN gene mutation and / or deletion is a complete deletion of the PTEN gene.

6. The use according to claim 1, characterized in that: The cholangiocarcinoma is early, middle, late or metastatic cholangiocarcinoma, or recurrent cholangiocarcinoma.

7. The use according to claim 1, characterized in that: The administration route of the proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof is oral, intravenous, sublingual, intranasal, inhalation or subcutaneous; preferably oral or injection, more preferably oral.

8. Use of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a 20S proteasome β5 subunit chymotrypsin-like inhibitor; 9. Use of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a β1 subunit caspase-like inhibitor; 10. Use of a proteasome inhibitor represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a β2 subunit caspase-like inhibitor;

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