WO2021180184A1

WO2021180184A1 - Use of mitoxantrone hydrochloride liposomes

Info

Publication number: WO2021180184A1
Application number: PCT/CN2021/080282
Authority: WO
Inventors: 李春雷; 张熙; 林红梅; 李桂霞; 李萌萌; 王凤霞; 王世霞; 金鑫
Original assignee: 石药集团中奇制药技术（石家庄）有限公司
Priority date: 2020-03-12
Filing date: 2021-03-11
Publication date: 2021-09-16
Also published as: CN115279344A

Abstract

A use of mitoxantrone hydrochloride liposomes in the preparation of a drug for the treatment of liver cancer. The liver cancer is preferably advanced liver cancer, and liver cancer on which first-line and/or second-line drug treatment fails or results in intolerance. A method for treating liver cancer, the method comprises: giving a therapeutically effective amount of mitoxantrone hydrochloride liposomes to patients with liver cancer. Animal test results show that mitoxantrone hydrochloride liposomes have better efficacy and lower toxicity. Preliminary clinical research results show that the mitoxantrone hydrochloride liposomes may effectively treat advanced liver cancer and have good safety and tolerability.

Description

Use of Mitoxantrone Hydrochloride Liposome

Cross-references to related patent applications

This application claims the priority of the prior application whose patent application number is 202010168792.9 and the invention title is "Uses of Mitoxantrone Hydrochloride Liposomes" filed with the State Intellectual Property Office of China on March 12, 2020. The full text of this earlier application is incorporated into this application by reference.

This patent application quotes Chinese patent application 200610102339.8 filed on December 29, 2006 and PCT application WO2008/080367A1 filed on December 29, 2007, the full disclosure of which is incorporated into this application by reference.

Technical field

The invention belongs to the technical field of anti-tumor drugs, and specifically relates to the use of mitoxantrone hydrochloride liposomes in the preparation of drugs for treating hepatocellular carcinoma, especially advanced hepatocellular carcinoma.

Background technique

Primary liver cancer (PLC) is one of the most common malignant tumors and ranks third in morbidity and mortality worldwide. Hepatocellular carcinoma (HCC, sometimes referred to as "liver cancer" hereinafter) is the main pathological type, accounting for about 90%. The progression of hepatitis B or C virus infection to liver cirrhosis and further progression to liver cancer is considered to be a trilogy of liver cancer progression. my country is a major country with hepatitis B and a high incidence of liver cancer. In 2015, the incidence of HCC in my country ranked first in the world.

At present, the staging of liver cancer is mainly based on the Barcelona staging system. The five stages of the system are 0 (very early stage), A (early stage), B (middle stage), C (late stage), and D (terminal stage). Stage 0 or A can choose surgical resection, local ablation or liver transplantation. Stage B tumors are confined to the liver, and hepatic artery chemoembolization (TACE) therapy can be selected. However, more than 80% of patients were found to be at an advanced stage and lost the opportunity for local treatment, and the 5-year survival rate was only 18%. For patients who progress or do not meet the conditions of local treatment, systemic treatment is generally chosen. The current FDA-approved treatments for advanced HCC are the first-line molecular targeted drugs sorafenib, lenvatinib, and second-line PD-1 inhibitors, regorafenib, etc., but their effectiveness is still limited, and due to drug tolerance Reasons such as poor sex and high economic costs are far from benefiting all patients. It can be seen that in the treatment of liver cancer, especially in the treatment of advanced liver cancer, there are huge unmet clinical needs, and there is an urgent need to research and develop more effective treatment methods.

Mitoxantrone hydrochloride is an anthraquinone antitumor drug. The FDA-approved indications are multiple sclerosis, prostate cancer and acute myeloid leukemia. Clinical studies have shown that it is effective for malignant lymphoma, breast cancer and acute myeloid leukemia, lung cancer, melanoma, soft tissue sarcoma, multiple myeloma, and liver cancer. , Colorectal cancer, kidney cancer, prostate cancer, endometrial cancer, testicular cancer, ovarian cancer and head and neck cancer have certain curative effects. Due to serious side effects, such as bone marrow suppression, white blood cell and thrombocytopenia (dose-limiting toxicity), palpitations, premature beats, and electrocardiogram abnormalities, the clinical dose of mitoxantrone hydrochloride is limited, and combination drugs are often required, such as Combined with cisplatin and fluorouracil to treat HCC. However, previous studies have shown that the effectiveness of these combination regimens is not high. For example, in a 2004 study by Tsai-Shen Yang et al., 63 patients with advanced HCC were intravenously administered 80 mg/m ² cisplatin, 6 mg/m ² mitoxantrone, and 450 mg/m ² fluorouracil. The results confirmed that the overall effect of the program was not ideal, and only some patients benefited. How to further improve the anti-tumor efficacy without causing serious side effects is a difficult problem facing the clinical application of mitoxantrone hydrochloride.

Liposomes are a new form of drug delivery. Studies have shown that it can change the distribution of encapsulated drugs in the body, so that the drugs are mainly accumulated in tissues and organs such as liver, spleen, lung and bone marrow, thereby increasing the therapeutic index of the drug, reducing the therapeutic dose of the drug and reducing the toxicity of the drug. These characteristics make the application of liposome-loaded drugs in the research of anti-tumor drugs more and more important. Some researchers have conducted research on mitoxantrone liposome preparations. For example, the Chinese patent application 200610102339.8 filed on December 29, 2006 and the PCT application WO2008/080367A1 filed on December 29, 2007, disclose a mitoxantrone liposome, the disclosure of which is hereby incorporated in its entirety as refer to. Studies have shown that compared with mitoxantrone free drug, liposome preparations have lower toxicity and can achieve better anti-tumor efficacy at lower doses. Mitoxantrone liposomes seem to provide a potential therapeutic method for the treatment of liver cancer, especially advanced liver cancer.

However, it is well known in the art that patients with advanced liver cancer often suffer severely impaired liver function, and blood platelets decrease due to hypersplenism. Mitoxantrone is mainly metabolized in the liver, and has serious bone marrow suppression toxicity, which causes leukopenia and thrombocytopenia in a dose-dependent manner. Mitoxantrone liposomes further accumulate the drug in the liver, spleen, and bone marrow. Whether this drug aggregation phenomenon will increase the metabolic burden of the liver, spleen, and bone marrow, aggravate the adverse drug reactions, and affect the efficacy of the drugs is a problem that needs to be solved urgently.

In addition, existing studies have shown that the safe and effective dose of the same drug in the treatment of different indications may be quite different. For example, Doxil (doxorubicin hydrochloride liposome) has three indications approved by the FDA, namely: (1) ovarian cancer, the recommended dose is 50mg/m ² , intravenously administered once every 4 weeks; (2) For Kaposi's sarcoma, the recommended dose is 20 mg/m ² , intravenously administered once every 3 weeks; (3) For multiple myeloma, the recommended dose is 30 mg/m ² , intravenously administered on the fourth day after the administration of bortezomib . Another example is Abraxane (paclitaxel for injection [albumin-binding type]). The three indications approved by the FDA have different dosing regimens: (1) Metastatic breast cancer: the recommended dose of 260mg/m ² , intravenous drip 30 minutes, once every 3 weeks; (2) non-small cell lung cancer: the recommended dose of 100mg/m ² , intravenous drip for 30 minutes, a course of treatment every 21 days, respectively on the first day, the eighth day and the 15th day Medicine; on the first day, paclitaxel for injection (albumin-binding) is administered immediately after administration, once every 21 days; (3) Pancreatic cancer: the recommended dose is 125mg/m ² , intravenous drip for 30-40 minutes , Take 28 days as a cycle, and administer once on the first, eighth, and fifteenth days. Paclitaxel for injection (albumin-bound type) will be given gemcitabine immediately after each administration. Another example is AmBisome (Amphotericin B liposomes for injection). The starting doses for the following indications are: (1) Empirical treatment: recommended dose 3mg/kg/day; (2) systemic fungal infection (Aspergillus, Candida, Cryptococcus): the recommended dose is 3～5mg/kg/day; (3) Cryptococcal meningitis in HIV-infected persons: the recommended dose is 6mg/kg/day (days 1-5), 3mg/kg/day (Day 4, 21); Patients with visceral leishmaniasis with low immune function: 4mg/kg/day (days 1-5), 4mg/kg/day (days 10, 17, 24, 31, 38) . It can be seen that the safe and effective doses of the same drug for different indications are different. The dosage and dosing data are individually formulated according to the specific disease and the actual situation of the patient, in order to achieve the maximum efficacy and minimum toxicity or adverse reactions, and achieve the effect of safe and effective treatment of the disease.

In summary, although mitoxantrone has previous experience in combination therapy for liver cancer, it is difficult to provide a reliable reference for the safety and efficacy of mitoxantrone alone due to its poor effectiveness due to dosage and other reasons. Although the inventors have carried out exploratory research on mitoxantrone liposomes in solid tumors and lymphomas in the early stage, hepatocellular carcinoma is different from other solid tumors. Mitoxantrone drugs are mainly metabolized by the liver, and the liposomes carry The drug will further accumulate the drug in the liver, spleen, and bone marrow, which will lead to a significant increase in the metabolic load of the liver, spleen, and bone marrow with impaired functions. Whether this will affect the efficacy and safety of clinical medication is unpredictable. Moreover, for mitoxantrone liposomes, a special dosage form that is different from ordinary injections, its absorption, distribution, and metabolism after entering the body are very complicated. It is suitable for the treatment of different indications, especially in the treatment of different tumors. It is difficult to simply deduce from one indication to another. Therefore, it is necessary to conduct a systematic study on whether mitoxantrone liposomes are suitable for the treatment of liver cancer, especially advanced liver cancer, to clarify its safe and effective dosage, and to provide a reference for clinical treatment.

Summary of the invention

The invention provides the use of mitoxantrone hydrochloride liposomes in the preparation of drugs for treating liver cancer.

Preferably, the liver cancer is advanced liver cancer, or liver cancer that has failed or is intolerant to first-line and/or second-line drug treatment.

Preferably, the mitoxantrone hydrochloride liposome is used as the sole active ingredient to prepare a medicine for treating liver cancer.

Preferably, the medicine is in the form of injection, including liquid injection, powder for injection, tablet for injection, and the like. When the drug is a liquid injection, based on mitoxantrone, the drug contains an active ingredient of 0.5-5 mg/ml, preferably 1-2 mg/ml, more preferably 1 mg/ml.

The present invention also provides a method for treating liver cancer, which includes the following steps: administering a therapeutically effective amount of mitoxantrone hydrochloride liposomes to patients with liver cancer.

Preferably, in terms of mitoxantrone, the therapeutically effective amount (in terms of mitoxantrone) refers to 8-30 mg/m ² , more preferably 12-20 mg/m ² or 16-30 mg/m ² . Specific examples are 12 mg/m ² , 14 mg/m ² , 16 mg/m ² , 18 mg/m ² , and 20 mg/m ² . Preferably, the total dose (calculated as mitoxantrone) of the liposome administered to each patient does not exceed 200 mg/m ² , preferably does not exceed 160 mg/m ² , further preferably does not exceed 140 mg/m ² , and more It is preferably not more than 120 mg/m ² .

Preferably, the administration mode of the present invention is intravenous administration. Preferably, the administration cycle is once every 4 weeks. Preferably, for each intravenous administration, the instillation administration time of the liposome pharmaceutical preparation is 30 min-120 min, preferably 60 min-120 min, more preferably 90±15 min.

The present invention also provides a mitoxantrone hydrochloride liposome, which is used to treat liver cancer in patients. Preferably, the liver cancer is advanced liver cancer, or liver cancer that has failed or is intolerant to first-line and/or second-line drug treatment.

In some embodiments, the liposome is in the form of injection, including liquid injection, powder for injection, tablet for injection, and the like. When the drug is a liquid injection, based on mitoxantrone, the liposome contains the active ingredient (calculated as mitoxantrone) 0.5-5 mg/ml, preferably 1-2 mg/ml, more preferably 1 mg/ml .

In some embodiments, the liposomes are used alone to treat liver cancer in patients.

In some embodiments, the therapeutically effective amount of the liposome (calculated as mitoxantrone) is 8-30 mg/m ² , more preferably 12-20 mg/m ² or 16-30 mg/m ² . Specific examples are 12 mg/m ² , 14 mg/m ² , 16 mg/m ² , 18 mg/m ² , and 20 mg/m ² .

In some embodiments, the liposome is administered intravenously. Preferably, the administration cycle is once every 4 weeks. Preferably, for each intravenous administration, the infusion time of the liposome is 30 min-120 min, preferably 60 min-120 min, and more preferably 90±15 min.

In some embodiments, the total dose of mitoxantrone received by the patient does not exceed 200 mg/m ² , preferably does not exceed 160 mg/m ² , further preferably does not exceed 140 mg/m ² , and more preferably does not exceed 120 mg/m ² .

In the context of the present invention, the "total administered dose of mitoxantrone" or "total administered dose (in terms of mitoxantrone)" refers to all mitoxantrone drugs received by the patient, including the The invented mitoxantrone hydrochloride liposomes, mitoxantrone hydrochloride injection and other mitoxantrone preparations are the sum of the doses of mitoxantrone.

In the context of the present invention, unless otherwise specified, the dosage is calculated as mitoxantrone.

In the context of the present invention, the mitoxantrone hydrochloride liposomes can be prepared by conventional methods in the art, and can be mitoxantrone hydrochloride liposomes prepared by any method disclosed in the prior art, for example, WO2008 /080367 Prepared by the method disclosed in A1, the content of the patent disclosure is incorporated herein by reference in its entirety.

In the context of the present invention, the mitoxantrone hydrochloride liposome has a particle size of about 30-80nm and contains: 1) the active ingredient mitoxantrone, which can form with multivalent counter ions in the liposome The precipitate that is difficult to dissolve, 2) the phospholipid bilayer contains phospholipids with a phase transition temperature (Tm) higher than body temperature, so that the phase transition temperature of liposomes is higher than body temperature. The phospholipid with a Tm higher than body temperature is phosphatidylcholine, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, distearic acid lecithin, distearic acid lecithin, or any combination thereof, and the particle size is about 35- 75nm, preferably about 40-70nm, more preferably about 40-60nm, particularly preferably about 60nm. The phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, the mass ratio is 3:1:1, the particle size is about 60nm, and the counterion For the sulfate ion. Preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, the mass ratio is 3:1:1, and the particle size is About 40-60 nm, the counter ion is sulfate ion, and the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the liposome is 9.58:3.19:3.19:1.

In the context of the present invention, the preparation method of the mitoxantrone hydrochloride liposome is as follows: HSPC (hydrogenated soy lecithin), Chol (cholesterol) and DSPE-PEG2000 (polyethylene glycol 2000 modified distearyl Acylphosphatidylethanolamine) is weighed at a mass ratio of 3:1:1 and dissolved in 95% ethanol to obtain a clear solution (ie, an ethanol solution of phospholipids). The ethanol solution of phospholipids was mixed with 300 mM ammonium sulfate solution, and the mixture was shaken and hydrated at 60-65° C. for 1 hour to obtain heterogeneous multilamellar liposomes. Afterwards, a microfluidic device was used to reduce the particle size of the liposomes. After the obtained sample was diluted 200 times with NaCl solution with a concentration of 0.9%, it was detected with NanoZS. The average particle size of the particles was about 60nm, and the main peak was concentrated between 40-60nm. Afterwards, an ultrafiltration device was used to remove the ammonium sulfate in the outer phase of the blank liposome, and the outer phase was replaced with 290 mM sucrose and 10 mM glycine to form a transmembrane ammonium sulfate gradient. According to the ratio of lipid-drug ratio 16:1, mitoxantrone hydrochloride solution (10 mg/mL) was added to the blank liposomes, and the drug was loaded at 60-65°C. After incubating for about 1 hour, gel exclusion chromatography can prove that the encapsulation efficiency is about 100%. The product obtained from this prescription was named PLM 60. The weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in PLM60 is 9.58:3.19:3.19:1, and the osmotic pressure of the sucrose glycine solution is close to the physiological value.

In the context of the present invention, the first-line and second-line drugs for the treatment of liver cancer refer to the first-line and second-line drugs approved by the drug administration of China or foreign countries (such as the United States, the European Union, Japan, South Korea, etc.) for the treatment of liver cancer, including but Not limited to: FDA-approved first-line molecular targeted drugs for the treatment of advanced HCC, sorafenib, lenvatinib, etc., second-line drugs PD-1 inhibitors, regofini, etc.

The animal test results of the present invention show that the mitoxantrone hydrochloride liposome can effectively treat liver cancer, and has better curative effect and lower toxicity than ordinary mitoxantrone hydrochloride injection. Preliminary clinical research results show that mitoxantrone hydrochloride liposomes can effectively treat advanced liver cancer, ^{and the doses of 12mg/m 2} and 16mg/m ² are safe and well tolerated.

Specific implementation method

Example 1 The inhibitory effect of mitoxantrone hydrochloride liposomes on H22 solid tumors

Select ascites-type tumor-bearing mice inoculated with H22 tumor cells for 7 days, necked and sacrificed, draw milky white viscous ascites, dilute with normal saline injection, and adjust the number of tumor cells to 5×10 ⁶ cells/mL after dilution. The tumor cell suspension was inoculated into the forelimb armpit subcutaneous male KM mice inoculated with a volume of 0.2 mL, containing approximately 10 ⁶ tumor cells.

After 4 days of inoculation, the tumors grew to ^{about 100-300mm 3} and were randomly divided into: ①Mit-Lipo (mitoxantrone hydrochloride liposomes) group: Mitoxantrone hydrochloride liposomes (PLM60) 1 2, 4, 6, and 8 mg/kg dose groups, ②Mit-Inj (common mitoxantrone injection) group: 2, 4, and 6 mg/kg dose groups of common mitoxantrone injection, and ③5% glucose injection solvent Control group. Each group was given a single intravenous administration. After the administration, the animals were bred normally. On the 9th day after the administration, the mice were sacrificed by removing their necks, and the tumor masses were dissected and weighed. Calculate the tumor inhibition rate (%). The experimental results were analyzed by SPSS 19.0, One-way ANOVA. The test results are shown in Table 1.

Table 1 Antitumor effect of PLM 60 on H22 solid tumors (n=10)

^** P<0.01, compared with the solvent control group; ^△△ P<0.01, compared with the same dose of Mit-Inj.

The experimental results showed that compared with the solvent control group, each administration group of Mit-Lipo could significantly inhibit tumor growth in a dose-dependent manner (P<0.01). Compared with the same dose of Mit-Inj, the inhibitory effect of Mit-Lipo 2, 4 and 6mg/kg groups on H22 solid tumors was significantly stronger (P<0.01). The body weight of the animals in each treatment group was reduced after the administration. Except for the Mit-Inj 6mg/kg dose group, the body weight of the animals in the other treatment groups returned to a positive increase at the end of the experiment. The experimental results fully demonstrate that Mit-Lipo has significantly better anti-tumor effects and lower toxicity.

Example 2 Inhibition of Mitoxantrone Hydrochloride Liposomes on Human Hepatocarcinoma BEL-7402 Transplanted Tumor

Note: Currently, the first-line treatment drug for advanced HCC approved by the FDA is sorafenib, so in Examples 2 and 3 of the present invention, sorafenib tosylate is used as a positive control drug.

Test animals: female NU/NU mice, provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number 1100111911079092.

Test method: NU/NU mice were subcutaneously inoculated with human liver cancer BEL-7402 cells to construct a xenograft tumor model. After the animals are inoculated with tumor cells, they are administered in groups ^{after the tumor grows to about 100-200 mm 3.} During the experiment, the animal body weight and tumor volume were monitored twice a week, and the tumor volume (TV), relative tumor volume (RTV), and relative tumor volume growth rate (T/C) values were calculated. After the experiment, the tumor weight was weighed and the tumor weight was calculated. Inhibition rate.

Related calculation formula: Tumor volume (TV)=1/2×a×b ² , where a and b represent the length and short diameter of the tumor respectively. RTV=TV _t /TV ₀ . TV ₀ is the tumor volume measured when the drug is divided into cages (that is, D ₀ ), and TV _t is the tumor volume at each measurement. T/C(%)=(TRTV/CRTV)×100% (TRTV: RTV of the administration group; CRTV: RTV of the solvent control group). Tumor weight inhibition rate (%) = (1-tumor weight in the administration group/tumor weight in the solvent control group) × 100%.

Test drugs Mit-lipo (mitoxantrone hydrochloride liposome (PLM60)), Sorafenib (sorafenib tosylate). Mit-lipo is diluted with 5% dextrose injection (5% INJ GS) to a suitable concentration, Sorafenib is dissolved in a mixture of castor oil and absolute ethanol (volume ratio is 1:1), vortexed and ultrasonicated to a uniform solution, and then Add ultrapure water (castor oil: absolute ethanol: ultrapure water volume ratio 1:1:6), and dilute to a suitable concentration.

Test results: Compared with the solvent control group, a single intravenous administration of Mit-lipo 20, 10, and 5 mg/kg can significantly inhibit the growth of human liver cancer BEL-7402 transplanted tumor in a dose-dependent manner (P<0.05), the specific results See Table 2.

The tumor weight inhibition rates of Mit-lipo 20, 10, 5 mg/kg and Sorafenib 30 mg/kg groups were 63.0%, 46.3%, 32.4%, and 60.1%, respectively; Mit-lipo 20 mg/kg single intravenous administration and Sorafenib 30 mg/kg Oral intragastric administration for 14 consecutive days has the same inhibitory effect on human liver cancer BEL-7402 transplantation tumor (tumor weight inhibition rate 63.0% vs 61.0%, P = 0.7840); compared with the solvent control group, Mit-lipo 20, 10, A single intravenous administration of 5 mg/kg can significantly reduce the tumor weight of transplanted tumors (P<0.01). The specific results are shown in Table 3.

Table 2 Effect of tumor volume of transplanted tumor of human liver cancer BEL-7402 (n=7)

^* P<0.05, ^** P<0.01, ^*** P<0.001, compared with the solvent control group. D0: day of administration; D16: 16 days after administration; RTV: relative tumor volume; T/C%: relative tumor volume growth rate.

Table 3 Influence of tumor weight of human liver cancer BEL-7402 transplanted tumor (n=7)

^** P<0.01, ^*** P<0.001, compared with the solvent control group.

Example 3 The inhibitory effect of mitoxantrone hydrochloride liposomes on human liver cancer SMMC-7721 transplanted tumor

Test animals: female NU/NU mice, provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number 1100112011003025.

Test drugs: Mit-lipo (mitoxantrone hydrochloride liposome (PLM60)), Sorafenib (sorafenib tosylate). Mit-lipo is diluted with 5% dextrose injection (5% INJ GS) to a suitable concentration, Sorafenib is dissolved in a mixture of castor oil and absolute ethanol (volume ratio is 1:1), vortexed and ultrasonicated to a uniform solution, and then Add ultrapure water (castor oil: absolute ethanol: ultrapure water volume ratio 1:1:6), and dilute to a suitable concentration.

Test method: NU/NU mice were subcutaneously inoculated with human liver cancer SMMC-7721 cells to construct a xenograft tumor model. After the animals are inoculated with tumor cells, they are administered in groups ^{after the tumor grows to about 100-200 mm 3.} During the experiment, the animal body weight and tumor volume were monitored twice a week, and the tumor volume (TV), relative tumor volume (RTV), and relative tumor volume growth rate (T/C) values were calculated. After the experiment, the tumor weight was weighed and the tumor weight was calculated. Inhibition rate.

Test results: Compared with the solvent control group, a single intravenous administration of Mit-lipo 15, 10, and 5 mg/kg can significantly inhibit the growth of human liver cancer SMMC-7721 transplanted tumor in a dose-dependent manner (P<0.01), the specific results See Table 4.

The tumor weight inhibition rates in the Mit-lipo 15, 10, 5 mg/kg and Sorafenib 30 mg/kg groups were 77.7%, 59.6%, 48.0%, and 51.2%, respectively. A single intravenous administration of 5 mg/kg of Mit-lipo and 30 mg/kg of Sorafenib for 14 consecutive days have the same inhibitory effect on human liver cancer SMMC-7721 xenograft tumors (tumor weight inhibition rate 48.0% vs 51.2%, P = 0.7241 ); Compared with the solvent control group, a single intravenous administration of Mit-lipo 15, 10, and 5 mg/kg can significantly reduce the tumor weight of the transplanted tumor (P<0.01). The specific results are shown in Table 5.

Table 4 The effect of human liver cancer SMMC-7721 transplanted tumor tumor volume (n=8)

^** P<0.01, ^*** P<0.001, compared with the solvent control group. D0: the day of administration; D23: the 23rd day after administration; RTV: relative tumor volume; T/C (%): relative tumor volume proliferation rate.

Table 5 The effect of human liver cancer SMMC-7721 transplanted tumor weight (n=8)

^*** P<0.001, compared with the solvent control group.

Example 4 Clinical study of mitoxantrone hydrochloride liposome injection in the treatment of advanced liver cancer

This is a single-arm, open, single-center phase Ib dose escalation study. Subjects included will receive mitoxantrone hydrochloride liposome injection treatment, aiming to evaluate mitoxantrone hydrochloride liposome injection Safety, tolerability and efficacy in subjects with advanced liver cancer.

1. Experimental design

1. Test procedure

This study is a dose-escalation phase study. The main purpose is to determine the MTD (maximum tolerated dose) or RP2D (recommended dose for phase 2 study) of Mitoxantrone Hydrochloride Liposome Injection. Approximately 10-18 subjects will be included in this phase. Subjects will receive drug treatment on the first day of each cycle, one treatment cycle every 4 weeks, and administration on the first day of each cycle until the subject’s disease progresses, Untolerable toxicity, completion of 6 treatment cycles, withdrawal of informed consent, death, or the investigator's decision to withdraw from the study (whichever occurs first), for subjects who have completed 6 treatment cycles If the treatment is still beneficial and tolerable, the investigator and the sponsor can determine whether the treatment can be continued.

The test procedure for each subject is arranged as follows: a 4-week screening period, a total of 4 weeks of DLT (dose limiting toxicity) observation period after the first administration, extended administration period and follow-up period.

After the subjects sign the informed consent form and complete all baseline checks during the screening period, the subjects who meet the selection criteria will enter different dose groups from low to high in the order of entry, and receive mitoxantrone hydrochloride liposome injections Liquid treatment. All subjects collected PK (pharmacokinetics) blood samples at different time points before and after the administration according to the protocol, and completed the relevant inspections stipulated in the protocol during the entire trial period of the treatment period to observe safety and tolerability.

2. Dose escalation group settings

Test dose grouping

There are three dose groups of 12mg/m ² , 16mg/m ² and 20mg/m ² , the dose climbing is carried out according to the 3+3 principle, while taking into account the exposure of patients to lower ineffective doses as much as possible, the 12mg/m ² group is included in 1 Three subjects were included in each of the remaining two dose groups.

3. Extended dosing after completing the DLT observation period

After completing the 4-week DLT observation period of the first cycle, if the investigator evaluates that the patient is tolerable and the continued treatment may have clinical benefits, the subject can continue to be given the study drug treatment according to the established cycle, and the dose and the first The dosage is the same for 1 cycle. Until disease progression (PD) occurs, 6 treatment cycles are completed, death, intolerable toxicity, completion of the scheduled treatment cycle, the investigator's decision or the subject voluntarily withdraws from the treatment (whichever occurs first), for the completion of 6 Subjects who are administered for one treatment cycle, if the treatment is still beneficial and tolerable, can be determined by the investigator and the sponsor after discussing whether the treatment can be continued.

Extend the dosing period at least once every 2 weeks for safety follow-up (at least weekly blood test), at the end of the second cycle, the end of the fourth cycle and every 8 weeks (every two cycles) after the tumor efficacy evaluation (such as The tumor-related symptoms are aggravated, and the investigator considers it necessary to shorten the time for tumor evaluation).

4. Research end time

This study will end 8 weeks after the last subject's last medication or when the disease progresses, whichever is later.

2. Test population:

Subjects who meet all the following selection criteria and do not have any exclusion criteria can be selected for this clinical study.

(1) Selection criteria:

Participants must meet all of the following conditions:

1. Age 18-70 when signing the informed consent form, regardless of gender;

2. ECOG physical performance (PS) score 0-1;

3. According to the judgment of the investigator, the expected survival period is ≥12 weeks;

4. Advanced hepatocellular carcinoma diagnosed by histopathology and/or cytology, reaching locally advanced (unresectable) or distant metastasis, and at least one measurable focus without local treatment (defined according to the RECIST 1.1 standard );

5. Child-Pugh liver function classification: Grade A or B (≤7 points), total bilirubin ≤1.5 times the upper limit of normal, albumin ≥28g/L, Child-Pugh score of albumin and bilirubin Only one of the indicators can be worth 2 points;

6. Previous first-line and second-line systemic treatment failures are defined as: a) disease progression during treatment or after the last administration (according to the RECIST 1.1 version standard); b) intolerance to systemic anti-liver cancer treatment regimens.

7. Barcelona liver cancer clinical staging (BCLC) stage C and stage B that are ineffective by interventional, ablation and other local treatments, without contraindications to intravenous chemotherapy;

8. The functions of vital organs meet the following requirements:

a) Routine blood test: white blood cell count ≥3.0×10 ⁹ /L, absolute neutrophil value ≥1.5×10 ⁹ /L (without G-CSF and other supportive treatment), platelet ≥75×10 ⁹ /L (without infusion Supportive therapy such as platelets or TPO), hemoglobin ≥80g/L (no need for blood transfusion therapy or supportive therapy such as erythropoietin);

b) Blood biochemistry: AST/ALT≤5 times the upper limit of normal value, AST/ALT≤3 times the upper limit of normal value ); Serum creatinine ≤ 1.5 times the upper limit of normal;

c) Coagulation function: prothrombin time (PT) prolonged ≥ 4s or activated partial thromboplastin time (APTT) ≤ 1.5 times the upper limit of normal;

9. Women with childbearing potential should agree to use contraceptive measures (such as intrauterine device [IUD], contraceptives or condoms) during the study period and 6 months after the end of the study; 14 before the first study drug administration The blood pregnancy test is negative within one day and must be a non-lactating subject; male subjects should agree to use contraceptive measures during the study period and within 6 months after the end of the study period;

10. Subjects voluntarily join the study, and sign an informed consent form, have good compliance and cooperate with follow-up.

(2) Exclusion criteria:

Subjects who meet any of the following conditions are not allowed to enter this clinical study:

1. Past anti-tumor clinical research and surgical treatment history meet any of the following:

In the treatment period of other interventional clinical studies within 4 weeks before the first medication;

Have received a major surgical operation within 4 weeks before the first medication or have not fully recovered from any previous invasive operation;

2. The subject’s previous anti-tumor treatment history meets one of the following conditions:

a. Previously received mitoxantrone or mitoxantrone liposome treatment;

b. Previously received doxorubicin or other anthracycline therapy, including local administration during interventional surgery, the total cumulative dose of doxorubicin>360mg/m ² (conversion of other anthracycline drugs: 1mg doxorubicin equivalent In 2mg epirubicin);

3. The previous history of anti-tumor treatment meets any of the following:

a. The subject has received any targeted therapy (including sorafenib, lenvatinib, and regorafenib) or anti-liver cancer treatments within 2 weeks before the first administration of the study drug;

b. Received any systemic anti-cancer therapy (including chemotherapy, radiotherapy, immunomodulators, hormone therapy, etc.) other than targeted therapy within 4 weeks before the first medication;

c. Any local treatment (including but not limited to percutaneous ethanol injection, radiofrequency or microwave or cryoablation, hepatic artery chemoembolization, hepatic artery perfusion, etc.) within 4 weeks before the first administration

d. The study drug has received blood transfusion, blood products (except plasma/human albumin used to control ascites, etc.) or drugs that stimulate blood cell production (such as granulocyte colony stimulating factor G-CSF) within 2 weeks before the first medication.

4. The subject has known intrahepatic cholangiocarcinoma, sarcomatoid HCC, mixed cell carcinoma, liver cancer and fibrolamellar cell carcinoma caused by autoimmune hepatitis;

5. The toxicity of the previous anti-tumor therapy has not recovered (NCI-CTC AE 5.0 version, >1 grade), but hair loss and pigmentation, previous chemotherapy-related neurotoxicity (allowed ≤2 level) and the investigator's judgment do not affect the safety of the study drug Except for other adverse reactions;

6. There is a history of active bleeding within 6 months before screening, including portal hypertension leading to esophageal and gastric venous bleeding; or the investigator believes that there is a clear gastrointestinal bleeding tendency (such as esophageal and gastric varices with bleeding risk, local Active ulcer lesions);

7. Severe liver cirrhosis; ascites requiring clinical intervention; hepatic encephalopathy;

8. Untreated active hepatitis B (HBsAg positive and HBV DNA> ¹⁰³ copies/ml);

9. Human immunodeficiency virus (HIV) infection (HIV positive); hepatitis C virus (HCV) antibody is positive, and HCV-RNA is higher than the upper limit of the normal value of the detection unit;

10. Uncontrollable or serious heart and cardiovascular and cerebrovascular diseases, including any of the following:

■ Myocardial infarction, congestive heart failure (NYHA grade ≥ 2), unstable angina pectoris, cerebrovascular accident, myocardial and pericardial diseases occurred within 6 months;

■High blood pressure that is difficult to control (defined as multiple measurements of systolic blood pressure ≥140mmHg or diastolic blood pressure ≥90mmHg under drug control);

■Severe uncontrolled arrhythmia requiring medical treatment;

■Complete left bundle branch block, right bundle branch block + left anterior branch block (double bundle branch block), degree II and degree III atrioventricular block, PR interval> 250 milliseconds;

■Severe ECG abnormalities with clinical significance;

■Electrocardiogram (ECG) examination in the research center during the screening period, QTc>470 milliseconds;

■Detected by echocardiography, left ventricular ejection fraction <50%;

■Arterial or venous thrombosis or embolism events (subjects with transient ischemic attack, lacunar infarction of no clinical significance can be included), deep vein thrombosis or pulmonary embolism occurred within 6 months before the start of study treatment . For subjects with known tumor thrombosis or deep vein thrombosis, those who remain stable after anticoagulant treatment for ≥ 4 weeks are eligible for inclusion;

11. Within 2 weeks before the first use of the study drug, there is any uncontrollable active infection that will prevent the subject from receiving the study drug;

12. Past medical history of liver transplantation;

13. Subjects with brain metastases, meningeal metastases, spinal cord compression, or mental disorders, in the case of treatment, asymptomatic and stable (the study drug has lasted for at least 4 weeks before the first administration of the study drug does not require hormone therapy), allowing brain metastasis Enrollment of subjects;

14. Serious, non-healing wounds, ulcers or fractures;

15. Suffered from any other malignant tumors within 5 years (except for skin basal cell carcinoma, cervical carcinoma in situ and other malignant tumors that were effectively controlled without any treatment within 5 years);

16. According to the judgment of the investigator, the subject has a serious or uncontrolled systemic disease, which is not suitable for entry into this study or affects the compliance of the protocol, or significantly interferes with the correct evaluation of the safety, toxicity, and effectiveness of the study drug.

(3) Withdrawal/termination criteria

Subjects can withdraw from the study treatment and evaluation without any reason at any stage of the study. Subjects must withdraw if:

■The subject withdrew the informed consent and asked to withdraw;

■ Participated in other clinical studies during the research process.

If the subject cannot participate in the set research evaluation, the researcher must clarify the reason and record it as completely and accurately as possible.

3. Research results

The clinical study enrolled a total of eight cases of hepatocellular carcinoma, wherein the 12mg / m ² dose group 1 cases, 16mg / m ² dose group 7 cases. All DLT observations have been completed so far, and no DLT incident has occurred. It shows that the mitoxantrone hydrochloride liposome injection is safe and well tolerated in the treatment of advanced liver cancer.

In the treatment field of liver cancer, chemotherapy has been difficult to achieve breakthroughs, and it has been at a stagnant stage for a long time. At present, there is no guideline recommendation for the third-line research of advanced liver cancer, so there is no alternative treatment for patients with first-line and second-line resistance. The inventor of the present invention expects to use rice Toxantrone liposomes, after the drug enters the human body through intravenous infusion, it has the effects of slow release, targeting, attenuation, and synergism. Not only the dosage is higher than that of ordinary injections, but it is also a single-drug treatment, which can improve the effectiveness and at the same time Reduce the chance of adverse reactions.

This plan will fill the gap in the third-line treatment of liver cancer, lay the foundation for the subsequent combination of drugs to impact the first-line and second-line treatments, change the traditional treatment model of liver cancer, that is, excessive dependence on TACE, and look forward to similar high-efficiency and low-toxic chemotherapeutics in the field of advanced solid tumors Applications.

Claims

The use of mitoxantrone hydrochloride liposomes in the preparation of medicines for treating liver cancer.
The use of mitoxantrone hydrochloride liposome as the sole active ingredient in the preparation of a medicine for the treatment of liver cancer.
The use according to claim 1 or 2, wherein the liver cancer is selected from the group consisting of advanced liver cancer, liver cancer that has failed or is intolerant to first-line and/or second-line drug treatment.
The use according to any one of claims 1 to 3, wherein the medicine is an injection dosage form, including liquid injection, powder for injection, tablet for injection, etc.; preferably, the medicine is a liquid injection.
The use according to claim 4, characterized in that, based on mitoxantrone, the medicine contains the active ingredient 0.5-5 mg/ml, preferably 1-2 mg/ml, more preferably 1 mg/ml.
The use according to any one of claims 1 to 5, wherein the mitoxantrone hydrochloride liposome has a particle size of about 30-80 nm and contains: 1) the active ingredient mitoxantrone, which can be combined with lipid The multivalent counter ions in the plastids form precipitates that are difficult to dissolve. 2) The phospholipid bilayer contains phospholipids with a phase transition temperature (Tm) higher than body temperature, so the phase transition temperature of liposomes is higher than body temperature. The phospholipids with Tm higher than body temperature are phosphatidylcholine, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, distearic acid lecithin, distearic acid lecithin, or any combination thereof;

Or preferably, the particle size of the liposome is about 35-75 nm, preferably about 40-70 nm, more preferably about 40-60 nm;

Or preferably, the phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, the mass ratio is 3:1:1, and the particle size is about 60nm, The counter ion is sulfate ion;

Or preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, with a mass ratio of 3:1:1, and the particle size It is about 40-60 nm, the counter ion is sulfate ion, and the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the liposome is 9.58:3.19:3.19:1.
A method for the treatment of liver cancer, comprising the steps of: administering a therapeutically effective amount of mitoxantrone hydrochloride liposomes to patients with liver cancer; or preferably, the liver cancer is selected from the group consisting of advanced liver cancer, first-line and/or second-line drug treatment failure or intolerance Affected liver cancer;

Or preferably, the liposome is used alone to treat liver cancer in patients;

Or preferably, the liposomes are in the form of injections, including liquid injections, powders for injection, tablets for injections, etc.; when they are liquid injections, based on mitoxantrone, the liposomes contain 0.5 to 0.5 of the active ingredient. 5mg/ml, preferably 1-2mg/ml, more preferably 1mg/ml;

Or preferably, the liposomes are administered intravenously; preferably, for each intravenous administration, the liposome instillation administration time is 30min-120min, preferably 60min-120min, more preferably 90min. ±15min;

Or preferably, the administration cycle of the liposome is once every 4 weeks;

Or preferably, based on mitoxantrone, the therapeutically effective amount is 8-30 mg/m 2 , more preferably 12-20 mg/m 2 or 16-30 mg/m 2 ; for example, 12 mg/m 2 , 14 mg/m 2 m 2 , 16mg/m 2 , 18mg/m 2 , 20mg/m 2 ;

Or preferably, based on mitoxantrone, the total dose of the liposome pharmaceutical preparation administered to each patient does not exceed 200 mg/m 2 , preferably does not exceed 160 mg/m 2 , and further preferably does not exceed 140 mg/m 2 , More preferably not more than 120mg/m 2 .
The method according to claim 7, wherein the particle size of the mitoxantrone hydrochloride liposome is about 30-80nm, and it contains: 1) the active ingredient mitoxantrone, which can be as much as the amount in the liposome. The valence counterion forms a precipitate that is difficult to dissolve. 2) The phospholipid bilayer contains phospholipids with a phase transition temperature (Tm) higher than body temperature; the phospholipids with a Tm higher than body temperature are phosphatidylcholine, hydrogenated soy lecithin, and hydrogenated egg yolk egg. Phospholipids, distearic acid lecithin or distearic acid lecithin or any combination thereof;

Or preferably, the particle size of the mitoxantrone hydrochloride liposome is about 35-75nm, preferably 40-70nm, further preferably 40-60nm, particularly preferably 60nm;

Or preferably, the phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, the mass ratio is 3:1:1, and the particle size is about 60nm, The counter ion is sulfate ion;

Or preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, with a mass ratio of 3:1:1, and the particle size It is about 40-60 nm, the counter ion is sulfate ion, and the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the liposome is 9.58:3.19:3.19:1.
A mitoxantrone hydrochloride liposome, which is used to treat liver cancer in patients; or preferably, the liver cancer is advanced liver cancer, or liver cancer that has failed or is intolerant to first-line and/or second-line drug treatment;

Or preferably, the liposome is used alone to treat liver cancer in patients;

Or preferably, the liposomes are in the form of injections, including liquid injections, powders for injection, tablets for injections, etc.; when they are liquid injections, based on mitoxantrone, the liposomes contain 0.5 to 0.5 of the active ingredient. 5mg/ml, preferably 1-2mg/ml, more preferably 1mg/ml;

Or preferably, the liposomes are administered intravenously; preferably, for each intravenous administration, the liposome instillation administration time is 30min-120min, preferably 60min-120min, more preferably 90min. ±15min;

Or preferably, the administration cycle of the liposome is once every 4 weeks;

Or preferably, based on mitoxantrone, the therapeutically effective amount is 8-30 mg/m 2 , more preferably 12-20 mg/m 2 or 16-30 mg/m 2 ; for example, 12 mg/m 2 , 14 mg/m 2 m 2 , 16mg/m 2 , 18mg/m 2 , 20mg/m 2 ;

Or preferably, based on mitoxantrone, the total dose of the liposome pharmaceutical preparation administered to each patient does not exceed 200 mg/m 2 , preferably does not exceed 160 mg/m 2 , and further preferably does not exceed 140 mg/m 2 , More preferably not more than 120mg/m 2 .
The mitoxantrone hydrochloride liposome of claim 9, wherein the particle size of the mitoxantrone hydrochloride liposome is about 30-80 nm, and it contains: 1) the active ingredient mitoxantrone, which It can form insoluble precipitates with multivalent counter ions in liposomes. 2) The phospholipid bilayer contains phospholipids with a phase transition temperature (Tm) higher than body temperature; the phospholipids with Tm higher than body temperature are phosphatidylcholine, hydrogenated Soy lecithin, hydrogenated egg yolk lecithin, distearic acid lecithin or distearic acid lecithin or any combination thereof;

Or preferably, the particle size of the mitoxantrone hydrochloride liposome is about 35-75nm, preferably 40-70nm, further preferably 40-60nm, particularly preferably 60nm;

Or preferably, the phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, the mass ratio is 3:1:1, and the particle size is about 60nm, The counter ion is sulfate ion;

Or preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearoylphosphatidylethanolamine, with a mass ratio of 3:1:1, and the particle size It is about 40-60 nm, the counter ion is sulfate ion, and the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the liposome is 9.58:3.19:3.19:1.