HK40116442A - Application of mitoxantrone hydrochloride liposome in treatment of bone and soft tissue sarcoma - Google Patents

Description

Use of mitoxantrone hydrochloride liposomes in the treatment of bone and soft tissue sarcomas

This invention claims priority to an earlier application filed on April 16, 2021, with the China National Intellectual Property Administration, patent application number 202110410490.2, entitled "Use of Mitoxantrone Hydrochloride Liposomes". The entire contents of that earlier application are incorporated herein by reference.

This patent application references PCT application WO2008/080367A1, filed on December 29, 2007, the full text of which is incorporated herein by reference.

This invention is a divisional application filed with the China National Intellectual Property Administration on April 15, 2022, with patent application number 202210414741.9 and title "Use of Mitoxantrone Hydrochloride Liposomes".

Technical Field

This invention belongs to the field of antitumor therapy, specifically relating to the use of mitoxantrone hydrochloride liposomes, such as their use in the preparation of liposomes for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma.

Background Technology

Urothelial carcinoma is a multifocal malignant tumor originating from the urothelial tract, including renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer, and is the most common urinary system tumor. Globally, in 2018, bladder-derived tumors ranked 12th in incidence among all solid tumors, accounting for 2.1% of all cancer deaths (Freddie, Bray, Jacques, Ferlay, Isabelle, & Soerjomataram, et al. (2018). Global cancer statistics 2018: global cancer estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians.). In China, in 2015, bladder-derived tumors ranked outside the top 10 in incidence among all cancers; they ranked 7th in men (6.2/100,000) and outside the top 10 in women. The standardized mortality rate ranks 14th among all cancers, at 1.96 per 100,000 people (Zheng Rongshou, Sun Kexin, Zhang Siwei, et al. Analysis of the prevalence of malignant tumors in China in 2015. Chinese Journal of Oncology, January 2019, Vol. 41, No. 1). The male-to-female ratio of bladder cancer is approximately 3:1 (Freddie, Bray, Jacques, Ferlay, Isabelle, & Soerjomataram, et al. (2018). Global cancer statistics 2018: global cancer estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians.).

For first-line treatment of unresectable locally advanced or metastatic urothelial carcinoma, if cisplatin is tolerated, cisplatin-containing chemotherapy regimens such as GC (gemcitabine + cisplatin) and MVAC (methotrexate + vincristine + doxorubicin + cisplatin) are the preferred options. For patients who cannot tolerate cisplatin, carboplatin-containing regimens, such as gemcitabine + carboplatin, can be considered. Other patients can use PD-1 inhibitors, such as pembrolizumab or atezolizumab, and chemotherapy drugs such as gemcitabine + paclitaxel or gemcitabine monotherapy. PD-1 inhibitors are preferred for patients expressing PD-L1. For second-line treatment, current treatment options include immunotherapy, targeted therapy, ADC drugs, and chemotherapy. There is currently no standard treatment for third-line and above treatment; appropriate chemotherapy, targeted therapy, or biological therapy should be selected based on the previous line of treatment.

In summary, patients who have failed platinum-based chemotherapy and PD-1 inhibitor therapy lack effective subsequent treatment options, and there is an urgent need to explore new drugs.

Breast cancer ranks first in incidence and fifth in mortality among malignant tumors in Chinese women, seriously threatening the health of Chinese women (Zheng Rongshou, Sun Kexin, Zhang Siwei, et al. Analysis of the prevalence of malignant tumors in China in 2015 [J]. Chinese Journal of Oncology, 2019, 41(1): 19-28.). In 2015, there were approximately 304,000 new cases of breast cancer in Chinese women, with an incidence rate of 45.29/100,000 and more than 70,000 deaths. Approximately 3% to 10% of new breast cancer cases have distant metastases at the time of diagnosis, and 30% of early-stage cases develop into advanced breast cancer. The 5-year survival rate of advanced breast cancer is only 20% (National Cancer Quality Control Center Breast Cancer Expert Committee. Guidelines for the Standardized Diagnosis and Treatment of Advanced Breast Cancer in China (2020 Edition)).

In inoperable locally advanced, recurrent, or metastatic breast cancer, chemotherapy is an indispensable treatment option for patients who are hormone receptor-negative, HER-2 positive, have short disease-free survival (<2 years), rapid tumor growth, significant symptoms, extensive visceral metastases, are hormone receptor-positive, or have failed endocrine therapy. For patients who have never received chemotherapy (including adjuvant chemotherapy), first-line chemotherapy should be based on anthracyclines and/or taxanes. For metastatic breast cancer that has failed anthracycline therapy or is close to the cumulative dose, and has never previously used taxanes, first-line chemotherapy should prioritize taxane-based regimens or taxane monotherapy. Patients who have received adjuvant taxane therapy and have been relapsed for more than 1 year can be given taxanes again. Among other drug options, those not used in adjuvant therapy or during the treatment phase should be given priority. For recurrent metastatic breast cancer that has failed both anthracycline and taxane preoperative/adjuvant therapy, there is currently no standard chemotherapy regimen. Drugs that can be considered include capecitabine, vinorelbine, gemcitabine, platinum-based drugs, eribulin, utedelon, another type of taxane (such as albumin-bound paclitaxel), and doxorubicin liposomes. Monotherapy or combination therapy can be considered. Therefore, the research and exploration of new drugs for second-line treatment still need to continue.

Classic osteosarcoma is the most common primary malignant tumor of bone, with an annual incidence of approximately 2-3 per million, accounting for only 0.2% of all human malignant tumors. Osteosarcoma can occur at any age, but it is most common in adolescents. Approximately 75% of patients are diagnosed between the ages of 15 and 25, with a median age of 20. Onset before age 6 or after age 60 is relatively rare. The disease is more prevalent in males than females, with a ratio of approximately 1.4:1, a difference particularly pronounced before age 20. About 20% of patients have clear lung metastases at initial diagnosis, while the remaining 30% develop lung metastases within one year of diagnosis. Before the 1970s, surgery was the only treatment for osteosarcoma, primarily involving amputation, with an average 5-year survival rate of only 19.7%. In the late 1970s, a treatment regimen of neoadjuvant chemotherapy + surgery + adjuvant chemotherapy was developed, significantly improving the 5-year survival rate to 60-80% (Niu Xiaohui. Current status and prospect of comprehensive treatment of osteosarcoma in China [J]. Chinese Journal of Anatomy and Clinical Medicine, 2019(01):1-5.), which is still in use today. However, the survival rate of patients with metastatic or recurrent osteosarcoma has hardly changed in the past 30 years, with an overall 5-year survival rate of about 20% (Meyers P A, Healey J H, Chou A J, et al. Addition of pamidronate to chemotherapy for the treatment of osteosarcoma [J]. Cancer, 2011, 117(8):1736-1744.).

There are numerous chemotherapy regimens for osteosarcoma internationally, but no single, universally accepted regimen exists. However, due to similar drug types and dosage intensities, their efficacy is generally similar. Doxorubicin, cisplatin, ifosfamide, and high-dose methotrexate are listed as first-line chemotherapy drugs for osteosarcoma, and the same drugs are used in neoadjuvant chemotherapy. Each patient should be treated with two or more drugs at sufficient dosage intensity. Compared to two-drug combination regimens, the 5-year EFS (time-free survival) rate for three-drug combination regimens is 58% and 48%, respectively, and the 5-year OS rate is 70% and 62%, respectively. For osteosarcoma patients who have failed first-line chemotherapy, since there are currently no second-line treatments with significant overall survival benefits, participating in clinical trials offers an opportunity to obtain better efficacy or the latest treatment options.

In summary, chemotherapy is currently an important clinical treatment method besides surgery. Patients with advanced or unresectable osteosarcoma who fail first-line chemotherapy have a poor prognosis. Medical evidence for targeted and immunotherapy for osteosarcoma is still insufficient, and seeking or adopting new cytotoxic or targeted therapies presents an opportunity for second-line treatment.

Soft tissue sarcoma refers to a group of malignant tumors originating from non-epithelial extraosseous tissues, accounting for approximately 0.8% of all human malignant tumors. The annual incidence rate in the United States is about 3.4/100,000, in Europe it is 4-5/100,000, and in my country it is about 2.38/100,000 (Siegel R L, Miller K D, Fuchs H E, et al. Cancer Statistics, 2021[J]. CA: A Cancer Journal for Clinicians, 2021, 71(1).). Patients of any age can be affected. In my country, the male-to-female ratio of patients is close to 1:1. The incidence rate increases significantly with age. According to the age-adjusted incidence rate, the incidence rate at age 80 is about 8 times that at age 30 (Burningham Z, Hashibe M, Spector L, et al. The epidemiology of sarcoma[J]. Clin Sarcoma Res, 2012, 2(1):14). Soft tissue sarcomas primarily originate from the mesoderm, with some originating from the neuroectoderm. They mainly consist of tissues such as muscle, fat, fibrous tissue, blood vessels, and peripheral nerves. The most common sites of occurrence are the extremities (approximately 53%), followed by the retroperitoneum (19%), trunk (12%), and head and neck (11%). Based on tissue origin, they can be classified into 12 major categories, and further subdivided into more than 50 subtypes based on different morphologies and biological behaviors. Common subtypes include undifferentiated pleomorphic sarcoma, liposarcoma, leiomyosarcoma, and synovial sarcoma. Rhabdomyosarcoma is the most common soft tissue sarcoma in adolescents and children.

Soft tissue sarcomas primarily present as a gradually growing, painless mass, exhibiting strong insidiousness. The disease course can range from months to years. As the tumor gradually enlarges and compresses nerves or blood vessels, pain, numbness, and even limb edema may occur. In some cases, the mass may rapidly increase in size within a short period, accompanied by increased skin temperature and regional lymph node enlargement. The possibility of an increased tumor grade should be considered. Soft tissue sarcomas are highly malignant, characterized by a short disease course, early hematogenous metastasis, and a high recurrence rate after treatment. The most common site of metastasis for limb sarcomas is the lung, while retroperitoneal and gastrointestinal sarcomas most frequently metastasize to the liver. The overall five-year survival rate for soft tissue sarcomas is approximately 60-80%.

Soft tissue sarcomas are treated with a comprehensive strategy primarily based on surgery. Radiotherapy, chemotherapy, or concurrent chemoradiotherapy are considered among the most effective treatments. With the development of combined cancer treatment models, preoperative treatment + surgery + chemotherapy has gradually become the norm. Its advantages include reducing tumor size, increasing limb-sparing opportunities, improving local control rates, reducing the risk of distant metastasis and recurrence, and improving patient survival rates. Chemosensitivity is a crucial factor in deciding whether to administer chemotherapy for soft tissue sarcomas. Anthracycline-based regimens are the first choice for neoadjuvant and adjuvant therapy. However, in patients with high-grade soft tissue sarcomas, 40%-50% experience local recurrence postoperatively, and >50% develop distant metastasis. Chemotherapy used for patients with metastatic or recurrent tumors that cannot be completely resected is palliative chemotherapy, aimed at shrinking and stabilizing the tumor to alleviate symptoms, prolong survival, and improve quality of life.

For unresectable tumors that have metastasized or recurred, palliative chemotherapy needs to be tailored to the individual patient. For patients with metastatic non-polymorphic rhabdomyosarcoma, chemotherapy regimens should be selected according to the high-risk group, alternating between VAC/VI/VCD/IE. For nonspecific soft tissue sarcomas, doxorubicin and ifosfamide are cornerstone drugs. Currently, there is no universally accepted second-line chemotherapy regimen for nonspecific soft tissue sarcomas.

In conclusion, soft tissue sarcomas are highly malignant, and for patients who have failed first-line treatment, the types of second-line drugs available are limited, and their efficacy is also limited. Participating in clinical trials is one of the recommended options. Improving the treatment of soft tissue sarcoma patients and exploring new drugs have become urgent clinical needs.

Mitoxantrone hydrochloride is an anthraquinone chemotherapy drug. The FDA has approved mitoxantrone hydrochloride injection for multiple sclerosis, prostate cancer, and acute myeloid leukemia. In China, it is approved for patients with lymphoma, leukemia, and breast cancer. The recommended dose is 12-14 mg/ ^m² as monotherapy in adults every 3-4 weeks; or 4-8 mg/ ^m² once daily for 3-5 days, with an interval of 2-3 weeks; the combination dose is 5-10 mg/ ^m² once daily. As an anthraquinone drug, the main adverse reactions of mitoxantrone are cardiotoxicity, myelosuppression, and gastrointestinal reactions. While myelosuppression and gastrointestinal reactions can be resolved with appropriate medication, cardiotoxicity often has serious consequences and is the most serious adverse reaction of anthraquinone drugs. Clinical studies and practical observations show that cardiotoxicity caused by anthraquinone drugs is mostly progressive and irreversible, especially when used for the first time. Chronic dose-cumulative limiting toxicity—cardiotoxicity—is a common concern for clinicians. Therefore, although mitoxantrone has been approved for breast cancer, it is not recommended as a treatment for breast cancer by authoritative clinical guidelines. Furthermore, mitoxantrone has never been approved for urothelial carcinoma or bone and soft tissue sarcoma.

Different tumors have different drug sensitivities. Existing studies have shown that the dosing regimen of the same drug may differ when treating different indications. For example, Doxil (liposome doxorubicin hydrochloride) has been approved by the FDA for three indications: (1) ovarian cancer, with a recommended dose of 50 mg/ ^m² , administered intravenously every 4 weeks; (2) Kaposi's sarcoma, with a recommended dose of 20 mg/ ^m² , administered intravenously every 3 weeks; and (3) multiple myeloma, with a recommended dose of 30 mg/ ^m² , administered intravenously on the fourth day after bortezomib administration. For example, Abraxane (paclitaxel for injection [albumin-bound]) has been approved by the FDA for three indications: (1) Metastatic breast cancer: recommended dose 260 mg/ ^m² , intravenous infusion over 30 minutes, once every 3 weeks; (2) Non-small cell lung cancer: recommended dose 100 mg/ ^m² , intravenous infusion over 30 minutes, every 21 days as one cycle, administered on day 1, day 8 and day 15 respectively; carboplatin is given immediately after the administration of paclitaxel for injection (albumin-bound) on day 1, and once every 21 days; (3) Pancreatic cancer: recommended dose 125 mg/ ^m² , intravenous infusion over 30-40 minutes, every 28 days as one cycle, administered once on day 1, day 8 and day 15, gemcitabine is given immediately after each administration of paclitaxel for injection (albumin-bound). For example, the starting dose of AmBisome (liposome amphotericin B for injection) for the following indications is: (1) empirical treatment: recommended dose 3 mg/kg/day; (2) systemic fungal infections (Aspergillus, Candida, Cryptococcus): recommended dose 3-5 mg/kg/day; (3) cryptococcal meningitis in HIV-infected patients: recommended dose 6 mg/kg/day (days 1-5), 3 mg/kg/day (days 4 and 21); (4) visceral leishmaniasis patients with immunodeficiency: 4 mg/kg/day (days 1-5), 4 mg/kg/day (days 10, 17, 24, 31, and 38). Dosage and administration data should be individualized according to the patient's actual situation to achieve maximum efficacy and minimum toxicity or adverse reactions. It can be seen that the safe and effective dose of the same drug for different indications varies. Dosage and administration data should be individualized according to the specific disease and the patient's actual situation to achieve maximum efficacy and minimum toxicity or adverse reactions, and to achieve safe and effective treatment of the disease. Mitoxantrone liposomes, a special dosage form unlike ordinary injections, have a very complex absorption, distribution, and metabolism after entering the body. It is also difficult to simply extrapolate from one indication to another for the treatment of different indications, especially for the treatment of different tumors.

Therefore, it is necessary to conduct a systematic study on whether mitoxanone liposomes are suitable for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma, to determine their safe and effective dosage, and to provide a reference for clinical treatment.

Summary of the Invention

This invention provides the use of mitoxantrone hydrochloride liposomes in the preparation of medicaments for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma.

Preferably, the urothelial carcinoma is locally advanced or metastatic urothelial carcinoma; more preferably, the urothelial carcinoma is locally advanced or metastatic urothelial carcinoma that has failed treatment with platinum-based chemotherapy and/or PD-1 inhibitors; or, locally advanced or metastatic urothelial carcinoma that has failed treatment with platinum-based chemotherapy but refuses PD-1 inhibitor treatment; or, locally advanced or metastatic urothelial carcinoma that is intolerant to cisplatin.

Preferably, the breast cancer is HER-2 negative breast cancer; more preferably, the breast cancer is locally advanced or recurrent/metastatic HER-2 negative breast cancer; or, hormone receptor negative HER-2 negative breast cancer; or, hormone receptor positive but unsuitable for endocrine therapy or resistant to endocrine therapy HER-2 negative breast cancer; or, HER-2 negative breast cancer that has failed anthracycline and/or taxane therapy; preferably, the HER-2 negative breast cancer includes breast cancer with immunohistochemical HER-2 0 or 1+, and immunohistochemical HER-2 2+ needs to be confirmed negative by in situ hybridization.

Preferably, the bone and soft tissue sarcoma is an advanced bone and soft tissue sarcoma, and more preferably, it is a metastatic or locally advanced bone and soft tissue sarcoma that has failed at least one first-line treatment.

Preferably, mitoxantrone hydrochloride liposomes are used as the sole active ingredient in the preparation of drugs for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma.

Preferably, the drug is an injectable dosage form, including liquid injections, injectable powders, injectable tablets, etc.

Preferably, the drug is a liquid injection.

According to an embodiment of the present invention, when the drug is a liquid injection, the drug contains 0.5-5 mg/ml of mitoxantrone, preferably 1-2 mg/ml, more preferably 1 mg/ml, based on mitoxantrone.

The present invention also provides a method for treating urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma, wherein the method comprises administering a therapeutically effective amount of mitoxantrone hydrochloride liposomes to the patient in need of treatment.

This invention also provides the use of mitoxantrone hydrochloride liposomes in the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma.

Preferably, the urothelial carcinoma is locally advanced or metastatic urothelial carcinoma; more preferably, the urothelial carcinoma is locally advanced or metastatic urothelial carcinoma that has failed platinum-based chemotherapy and/or PD-1 inhibitor treatment, or locally advanced or metastatic urothelial carcinoma that has failed platinum-based chemotherapy but refuses PD-1 inhibitor treatment, or locally advanced or metastatic urothelial carcinoma that is intolerant to cisplatin.

Preferably, the breast cancer is HER-2 negative breast cancer; more preferably, the breast cancer is locally advanced or recurrent/metastatic HER-2 negative breast cancer; or, hormone receptor negative HER-2 negative breast cancer; or, hormone receptor positive but unsuitable for endocrine therapy or resistant to endocrine therapy HER-2 negative breast cancer; or, HER-2 negative breast cancer that has failed anthracycline and/or taxane therapy; preferably, the HER-2 negative breast cancer includes breast cancer with immunohistochemical HER-2 0 or 1+, and immunohistochemical HER-2 2+ needs to be confirmed negative by in situ hybridization.

Preferably, the bone and soft tissue sarcoma is an advanced bone and soft tissue sarcoma, and more preferably, it is a metastatic or locally advanced bone and soft tissue sarcoma that has failed at least one first-line treatment.

Preferably, the therapeutically effective dose, calculated as mitoxantrone, refers to a mitoxantrone dose of 8-30 mg/ ^m² , more preferably 12-20 mg/ ^m² or 16-30 mg/ ^m² . Specifically, for example, 12 mg/ ^m² , 14 mg/ ^m² , 16 mg/ ^m² , 18 mg/ ^m² , and 20 mg/ ^m² .

Preferably, the administration method of the present invention is intravenous administration. Preferably, the administration cycle is once every 3 weeks. Preferably, the treatment for the patient is 6-8 cycles. Preferably, for each intravenous administration, the infusion time of the liposomal drug preparation is 30 min-120 min, more preferably 60 min-120 min, and even more preferably 60 ± 15 min.

The present invention also provides a mitoxantrone hydrochloride liposome for the treatment of patients with urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma.

In some embodiments, the liposomes are injectable dosage forms, including liquid injections, injectable powders, injectable tablets, etc. When the drug is a liquid injection, the liposomes contain 0.5-5 mg/ml of mitoxantrone, preferably 1-2 mg/ml, more preferably 1 mg/ml, based on mitoxantrone.

In some implementations, the liposomes are used alone to treat patients with urothelial carcinoma, breast cancer, or bone and soft tissue sarcoma.

In some embodiments, the therapeutically effective amount of the liposome (calculated as mitoxantrone) is 8-30 mg/ ^m² , more preferably 12-20 mg/ ^m² or 16-30 mg/ ^m² . Specifically, for example, 12 mg/ ^m² , 14 mg/ ^m² , 16 mg/ ^m² , 18 mg/ ^m² , and 20 mg/ ^m² .

In some embodiments, the liposomes are administered intravenously. Preferably, the dosing cycle is once every 3 weeks. Preferably, the treatment is administered to the patient for 6-8 cycles. Preferably, each intravenous administration of the liposomes takes 30-120 minutes, more preferably 60-120 minutes, and even more preferably 60 ± 15 minutes.

In the context of this invention, unless otherwise specified, the dosage is expressed in mitoxantrone.

In the context of this invention, the mitoxantrone hydrochloride liposomes can be prepared using methods known in the art, and can be mitoxantrone hydrochloride liposomes prepared by any method disclosed in the prior art, such as the method disclosed in WO2008/080367A1.

In some implementations, the drug or mitoxanone hydrochloride liposomes have one or more of the following properties:

(i) The particle size of mitoxantrone hydrochloride liposomes is about 30-80 nm, for example about 35-75 nm, about 40-70 nm, about 40-60 nm or about 60 nm;

(ii) Mitoxantrone hydrochloride forms an insoluble precipitate with multivalent counterions (such as sulfate, citrate or phosphate) in the lipid body;

(iii) The phospholipid bilayer of the mitoxantrone hydrochloride liposome contains phospholipids with a phase transition temperature (Tm) higher than body temperature, so that the phase transition temperature of the liposome is higher than body temperature. For example, the phospholipid is selected from hydrogenated soybean lecithin, phosphatidylcholine, hydrogenated egg yolk lecithin, dispalmitoyl lecithin, distearate lecithin, or any combination thereof.

(iv) The phospholipid bilayer of mitoxantrone hydrochloride liposomes contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearate phosphatidylethanolamine (DSPE-PEG2000).

(iiv) The phospholipid bilayer of the mitoxantrone hydrochloride liposomes contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000-modified distearate phosphatidylethanolamine in a mass ratio of approximately 3:1:1. The mitoxantrone hydrochloride forms an insoluble precipitate with the polyvalent anions in the liposomes, and the mitoxantrone hydrochloride liposomes in the drug have a particle size of approximately 60 nm.

(iiiv) Mitoxantrone hydrochloride liposomes are liposomes with the national drug approval number H20220001.

In the context of this invention, the mitoxantrone hydrochloride liposomes, having a particle size of about 30-80 nm, contain: 1) the active ingredient mitoxantrone, which can form an insoluble precipitate with the multivalent counterion within the liposomes; and 2) a phospholipid bilayer containing phospholipids with a phase transition temperature (Tm) higher than body temperature, thus the liposomes have a phase transition temperature higher than body temperature. The phospholipids with a Tm higher than body temperature are phosphatidylcholine, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, dispalmitoyl lecithin, or distearate lecithin, or any combination thereof. The particle size is about 35-75 nm, preferably 40-70 nm, more preferably 40-60 nm, and particularly preferably 60 nm. Preferably, the phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000-modified distearate phosphatidylethanolamine in a mass ratio of 3:1:1, the particle size is about 60 nm, and the counterion is sulfate ions. Preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearate phosphatidylethanolamine in a mass ratio of 3:1:1, the particle size is about 40-60 nm, the counterion 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 this invention, the preparation method of mitoxantrone liposomes is as follows: HSPC (hydrogenated soybean lecithin), Chol (cholesterol), and DSPE-PEG2000 (distearate phosphatidylethanolamine modified with polyethylene glycol 2000) are weighed at a mass ratio of (3:1:1) and dissolved in 95% ethanol to obtain a clear solution. The ethanol solution of phospholipids is mixed with a 300 mM ammonium sulfate solution and hydrated by shaking at 60-65°C for 1 h to obtain heterogeneous multi-compartment liposomes. The particle size of the liposomes is then reduced using a microfluidic apparatus. The obtained sample is diluted 200 times with a 0.9% NaCl solution and detected by NanoZS; the average particle size is approximately 60 nm, with the main peak concentrated between 40-60 nm. The ammonium sulfate in the outer phase of the blank liposome is then removed using an ultrafiltration device, and the outer phase is replaced with 290 mM sucrose and 10 mM glycine to form a transmembrane ammonium sulfate gradient. Mitoxantrone hydrochloride solution (10 mg/mL) was added to blank liposomes at a lipid-to-drug ratio of 16:1, and drug loading was performed at 60-65℃. After incubation for approximately 1 hour, gel size exclusion chromatography confirmed an encapsulation efficiency of approximately 100%. The weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone was 9.58:3.19:3.19:1, and the osmotic pressure of the sucrose-glycine solution was close to physiological values.

It should be understood that many technical details and parameters in the above exemplary preparation methods can be adjusted and determined by those skilled in the art within a reasonable range. For example, the amino acids that can be replaced by glycine in the outer phase used to form the transmembrane ammonium sulfate gradient include, but are not limited to, histidine, asparagine, glutamic acid, leucine, proline, and alanine. Furthermore, the mass ratio of HSPC, Chol, and DSPE-PEG2000 can be appropriately adjusted. Moreover, for the lipid-to-drug ratio parameter in the preparation of specific liposome drug formulations, those skilled in the art can design, test, and ultimately determine a suitable lipid-to-drug ratio to maximize drug loading while minimizing drug leakage. For the mitoxantrone hydrochloride liposome formulation of this application, a wide range of lipid-to-drug ratios can be used, such as as low as 2:1 or as high as 30:1, 40:1, or 50:1. A more suitable lipid-to-drug ratio can be approximately (15-20):1, such as approximately 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Therefore, the several advantageous properties of the mitoxantrone hydrochloride liposome formulations described above are more important, and there are various methodologies for achieving these properties.

The first-line, second-line, or higher-line drugs for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma described in this invention refer to drugs approved by drug regulatory authorities in China or abroad (such as the United States, the European Union, Japan, South Korea, etc.) for the treatment of urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma, including but not limited to: FDA-approved taxanes, PD-1 inhibitors, ifosfamide, etc.

Beneficial effects

During the applicant's research, it was unexpectedly discovered that mitoxantrone hydrochloride liposomes have better therapeutic effects on urothelial carcinoma, breast cancer, and bone and soft tissue sarcoma, and can be used to prepare related drugs.

Detailed Implementation

The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

The following abbreviations are used in this invention:

CR: Complete remission, specifically defined according to the internationally recognized "Research Criteria for Evaluating the Efficacy of Solid Tumors (RECIST 1.1)".

PR: Partial remission, specifically defined according to the internationally accepted "Research Criteria for the Evaluation of Treatment Efficacy in Solid Tumors (RECIST 1.1)".

SD: Stable disease, specifically defined according to the internationally recognized "Research Criteria for Evaluating the Efficacy of Solid Tumors (RECIST 1.1)".

PD: Disease progression, specifically defined according to the internationally recognized "Research Criteria for Evaluating the Efficacy of Solid Tumors (RECIST 1.1)".

Objective response rate (ORR) = (CR + PR) / total number of evaluable cases * 100%.

Disease control rate (DCR) = (CR + PR + SD) / total number of evaluable cases * 100%.

To investigate the efficacy of mitoxantrone hydrochloride liposomes in treating urothelial carcinoma, breast cancer, and bone and soft tissue sarcomas, the inventors conducted a clinical study. Mitoxantrone hydrochloride liposomes effectively treated urothelial carcinoma, breast cancer, and bone and soft tissue sarcomas, showing better efficacy and fewer adverse reactions compared to conventional mitoxantrone hydrochloride injections. The mitoxantrone hydrochloride liposome injection used in the following examples was provided by CSPC Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd. (National Drug Approval Number H20220001).

Example 1: A Phase II clinical trial evaluating the efficacy and safety of mitoxanone hydrochloride liposome injection for the treatment of unresectable locally advanced or metastatic urothelial carcinoma.

This is an open-label, multicenter phase II study in which participants will receive mitoxantrone hydrochloride liposome injection to evaluate the efficacy and safety of mitoxantrone hydrochloride liposome injection in unresectable locally advanced or metastatic urothelial carcinoma.

I. Experimental Design

1. Test Procedure

Forty patients with unresectable locally advanced or metastatic urothelial carcinoma were enrolled. The study population primarily consisted of patients who had failed platinum-based chemotherapy and PD-1 inhibitor therapy (including those not receiving PD-1 inhibitor therapy); those intolerant to cisplatin required systemic therapy. The study included a screening period, a treatment period, and a follow-up period. The screening period was 28 days, and eligible patients entered the treatment period. During the treatment period, patients received mitoxantrone hydrochloride liposome injection 20 mg/ ^m² as monotherapy, every 3 weeks (q3w), with the first dose administered on the first day of each cycle, for a total of 6 cycles. After the treatment period, patients entered the follow-up period. The follow-up period included safety monitoring (28 days after the last dose) and survival monitoring (every 6 weeks after the last dose). Safety evaluation was performed from the first dose to 28 days after the last dose, and a safety evaluation was performed before each cycle to determine whether to administer the drug in the next cycle. Throughout the study, tumor evaluation was performed every two cycles according to RECIST 1.1 criteria until disease progression, death, or receipt of new anti-tumor therapy.

2. Duration of the study

This study included a 4-week (28-day) screening period, a 6-cycle (18-week) treatment period, a final safety follow-up, a 4-week (28-day) follow-up after the end of treatment, and a survival follow-up every 6 weeks thereafter, with each patient lasting approximately 12 months.

This study enrolled 40 participants, and the entire study period lasted 18–24 months.

II. Trial population:

Subjects who meet all of the following inclusion criteria and have no exclusion criteria are eligible to be enrolled in this clinical study.

(I) Selection Criteria

1) Participants voluntarily participate in the study and sign an informed consent form;

2) Age ≥ 18 years old, gender not limited;

3) Urothelial carcinoma confirmed by histology;

4) Subjects who have failed platinum-based chemotherapy regimens and PD-1 inhibitors (including those who do not receive PD-1 inhibitor therapy); those who are intolerant to cisplatin need to undergo systemic treatment;

5) At least one evaluable lesion meeting the definition of RECIST 1.1 was present at baseline;

6) ECOG score 0-1;

7) The subjects' laboratory test values meet the following requirements:

• Absolute neutrophil count (ANC) ≥ 1.5 x ^10⁹ /L (no G-CSF white blood cell boosting therapy received within 1 week prior to laboratory test);

• Hemoglobin (Hb) > 9.0 g/dL (no red blood cell transfusion received within 1 week prior to laboratory test);

• Platelet count ^≥75x10⁹ /L (no platelet transfusion received within 1 week prior to laboratory test);

Creatinine ≤ 1.5x ULN;

• Total bilirubin ≤1.5x ULN (≤3x ULN for subjects with liver metastases);

• Alanine aminotransferase (AST)/aspartate aminotransferase (ALT) ≤3x ULN (≤5x ULN for subjects with liver metastases);

• Coagulation function: Prothrombin time (PT) and international normalized ratio (INR) ≤ 1.5 x ULN (INR ≤ 3 is allowed for those receiving anticoagulant therapy such as warfarin);

8) Subjects and their partners shall use effective contraception during the trial period and for 6 months after the last dose (e.g., combined hormone (including estrogen and progestin) combined with ovulation suppression, progestin contraception combined with ovulation suppression, intrauterine device, intrauterine hormone-releasing system, bilateral vasectomy, bilateral tubal ligation, avoidance of sexual intercourse, etc.); female subjects shall have negative urine or blood HCG (except for menopausal and hysterectomy);

(II) Exclusion Criteria

1) Severe allergy to mitoxantrone or liposomal drugs;

2) Subjects with brain or meningeal transfers;

3) Previous allogeneic organ transplantation or allogeneic bone marrow transplantation;

4) Survival period < 12 weeks;

5) Patients with active hepatitis B (HBsAg or HBcAb positive and HBV DNA ≥ 2000 IU/mL), active hepatitis C (HCV antibody positive and HCV RNA higher than the lower limit of the research center's detection value), or HIV antibody positive;

6) Having an active bacterial, fungal, or viral infection requiring intravenous infusion within one week prior to the first dose of the drug;

7) Patients who have received any anti-tumor treatment within 4 weeks prior to the first dose (2 weeks prior to the dose for traditional Chinese medicine or proprietary Chinese medicine);

8) Has received treatment with other clinical trial drugs within 4 weeks prior to the first dose;

9) Individuals who have undergone major surgery within 3 months prior to the first dose, or who plan to undergo major surgery during the study period;

10) Presence of grade 1 toxicity in previous antitumor treatments (excluding hair loss, pigmentation, or other toxicities that were considered to pose no safety risk to subjects in the study);

11) A serious thrombotic or embolic event has occurred within the past 6 months, such as cerebrovascular accident (including transient ischemic attack), pulmonary embolism, etc.

12) Had other malignant active tumors within the past 3 years; except for locally curable cancers that have been cured, such as basal or squamous cell skin cancer or prostate, cervical or breast cancer in situ;

13) Abnormal cardiac function, including:

• Long QTc syndrome or QTc interval > 480 ms;

• Complete left bundle branch block, second- or third-degree atrioventricular block (excluding those treated with pacemaker implantation);

• Severe, uncontrolled cardiac arrhythmias requiring medication;

• History of chronic congestive heart failure, NYHA class ≥ 3;

• Cardiac ejection fraction below 50% within 6 months;

• Heart valve disease with CTCAE > grade 2;

• Uncontrollable hypertension (defined as systolic blood pressure >150 mmHg or diastolic blood pressure >100 mmHg on multiple measurements under medication control);

• History of myocardial infarction, unstable angina, severe pericardial disease, or electrocardiographic evidence of acute ischemic or severe conduction abnormalities within 6 months prior to screening.

14) Has received doxorubicin or other anthracycline treatment, and the cumulative dose of doxorubicin exceeds 350 mg/ ^m² (Anthracycline equivalent dose calculation: 1 mg doxorubicin = 2 mg epirubicin = 2 mg pirarubicin = 0.5 mg daunorubicin = 0.45 mg mitoxantrone; except for doxorubicin liposomes);

15) Pregnant or breastfeeding women;

16) Suffering from any serious and/or uncontrollable disease, or other diseases that, in the investigator's opinion, may affect a patient's participation in this study (including, but not limited to, uncontrolled diabetes, kidney disease requiring dialysis, severe liver disease, life-threatening autoimmune diseases and bleeding disorders, substance abuse, neurological diseases, etc.).

17) Other situations where researchers deem it unsuitable for participation.

(III) Criteria for Withdrawal from Treatment and Research

Subjects must discontinue treatment with the investigational drug if any of the following conditions occur during the study:

1) The subject experienced intolerable toxicity, and the researchers believed that the risks of continuing treatment with the investigational drug outweighed the benefits;

2) Comorbidities that occur during treatment do not preclude continued medication;

3) Disease progression as assessed by imaging;

4) Clinical assessment shows disease progression or significant protocol violation, or subject compliance is poor, and the investigator determines that continuing treatment with the investigational drug will not be beneficial;

5) The subject becomes pregnant;

6) Death;

7) Meets any one of the criteria for exiting the research.

All subjects who withdraw from treatment must continue to be followed up according to the study plan, except for subjects who stop treatment due to death or meet any of the following withdrawal criteria.

Participants have the right to withdraw from the study at any time for any reason. Participants will be withdrawn from the study process if any of the following occurs:

1) Loss to follow-up;

2) The participant or their family (when the participant is unable to make a decision) requests to withdraw from the trial;

3) Research terminated;

4) Others.

III. Research Results

Subjects who have failed platinum-based chemotherapy regimens and PD-1 inhibitors (including those not receiving PD-1 inhibitors), as well as those intolerant to cisplatin who have undergone systemic therapy, lack effective subsequent treatment options and urgently need new drugs. The inventors of this invention use mitoxantrone liposomes. After intravenous infusion, the drug exhibits sustained-release, targeted, toxicity-reducing, and synergistic effects. Compared to conventional mitoxantrone injections, liposomal formulations allow for higher doses, improving efficacy while reducing the likelihood of adverse reactions. Ideal therapeutic effects can be achieved with monotherapy alone.

This study demonstrates that, in at least four subjects (two who received two cycles of treatment and two who received one cycle), two had stable disease (SD) (one who received one cycle of treatment and one who received two cycles of treatment), with a disease control rate (DCR) of 50% (2/4). Mitoxantrone liposomes showed good efficacy in patients with urothelial carcinoma, particularly those who had failed platinum-based chemotherapy and PD-1 inhibitor therapy. Continued treatment in the two subjects assessed as having SD resulted in even better efficacy.

This plan will improve the treatment options for urothelial carcinoma, lay the foundation for combination therapy to challenge first- and second-line treatments, change the traditional treatment model for urothelial carcinoma, and look forward to the application of similar highly effective and low-toxicity chemotherapy drugs in the field of advanced solid tumors.

Example 2: Phase II clinical trial of mitoxantrone hydrochloride liposome injection for the treatment of HER-2 negative advanced breast cancer

This is an open-label, multicenter phase II study in which participants will receive mitoxantrone hydrochloride liposome injection to evaluate the safety and efficacy of mitoxantrone hydrochloride liposome injection for the treatment of HER-2 negative advanced breast cancer.

I. Experimental Design

1. Test Procedure

This study consisted of a screening period, a treatment period, and a follow-up period. The screening period was 28 days, after which qualified subjects entered the treatment period. During the treatment period, subjects received mitoxanone hydrochloride liposome injection at 20 mg/ ^m² , with each cycle lasting 3 weeks, for a total of 6 cycles. After completing 6 cycles, the investigators and sponsors decided whether to continue treatment based on the subject's benefit and risk. Follow-up included a post-treatment visit (28 days after the last dose) and survival follow-up (every 6 weeks after the post-treatment visit). All subjects were required to undergo survival follow-up via telephone or in person until their death.

Tumor assessment: According to RECIST 1.1 criteria, tumor assessment is performed every 2 cycles during the treatment period and every 6 weeks during the follow-up period until disease progression or the initiation of new anti-tumor therapy.

Safety assessment: Before and after each treatment cycle, vital signs, physical examination, weight, ECOG performance status, 12-lead electrocardiogram, echocardiography, and laboratory tests were performed. Complete blood count was performed on days 8 and 15 of each cycle.

2. Duration of the study

This study included a 4-week (28-day) screening period, a 6-cycle (18-week) treatment period, a follow-up visit at the end of treatment (28 days after the last dose), and survival follow-up every 6 weeks until the patient was lost to follow-up or died. The duration of each patient's treatment was approximately 12 months.

This study enrolled 73 participants and lasted approximately 36 months.

II. Trial population:

Subjects who meet all of the following inclusion criteria and have no exclusion criteria are eligible to be enrolled in this clinical study.

(I) Selection Criteria:

Subjects must meet all of the following criteria:

1) Participants voluntarily participate in the study and sign an informed consent form;

2) Age 18 to 75 (inclusive);

3) HER-2 negative breast cancer confirmed by histopathology (including HER-2 0 or 1+ by immunohistochemistry; HER-2 2+ by immunohistochemistry must be confirmed negative by in situ hybridization).

4) Inoperable locally advanced or recurrent/metastatic breast cancer that has progressed after first-line or higher chemotherapy;

5) Hormone receptor-negative or hormone receptor-positive breast cancer that is unsuitable for endocrine therapy or resistant to endocrine therapy;

6) Subjects who have previously received at least one anthracycline and one taxane (subjects who have not previously used anthracyclines due to high-risk factors for cardiotoxicity may be accepted) and have received a maximum of four chemotherapy regimens;

7) The baseline must have at least one measurable lesion (RECIST 1.1 criteria);

8) ECOG score: 0-2 points;

9) Good organ function (no blood transfusion or growth factor support therapy received within 2 weeks prior to first use of the investigational drug), including:

• Absolute neutrophil count (ANC) ≥ 1.5 × ^10⁹ /L;

• Hemoglobin (Hb) ≥ 90 g/L;

• Platelet count ≥ 90 × ^10⁹ /L;

Creatinine ≤ 1.5 times the upper limit of normal;

• Total bilirubin ≤ 1.5 times the upper limit of normal;

• Alanine aminotransferase (AST) and aspartate aminotransferase (ALT) ≤ 3 times the upper limit of normal (≤ 5 times the upper limit of normal for patients with liver metastases);

10) The pregnancy test result is negative, and the subjects of reproductive age promise to use effective contraception or abstain from sex for 6 months from the start of the study to the last dose of medication in the study;

11) Has good compliance and is willing to cooperate with follow-up visits.

(II) Exclusion Criteria:

Subjects meeting any of the following criteria will be excluded from this trial:

1) Severe allergy to mitoxantrone or liposomes;

2) Had other malignant tumors within the past 3 years, excluding cervical carcinoma in situ, basal cell carcinoma of the skin, or squamous cell carcinoma that has been cured;

3) Brain metastases and membrane transfusions in subjects;

4) Subjects with active hepatitis B (HBsAg positive and HBV DNA ≥ 2000 IU/mL), active hepatitis C (HCV antibody positive and HCV RNA higher than the lower limit of the research center's detection value), or HIV antibody positive;

5) Expected survival time < 3 months;

6) The cumulative dose of anthracyclines previously received is converted to doxorubicin > 350 mg/ ^m2 (Anthracycline equivalent dose calculation: 1 mg doxorubicin = 2 mg epirubicin = 2 mg pirarubicin = 2 mg daunorubicin = 0.5 mg demethoxydaunorubicin = 0.45 mg mitoxantrone, except doxorubicin liposomes);

7) The toxicity of previous antitumor treatment has not recovered to ≤ Grade 1 (excluding hair loss, pigmentation, or other toxicities that were considered to pose no safety risk to the subjects in the study);

8) Abnormal cardiac function includes:

• Long QTc syndrome or QTc interval > 480 ms;

• Complete left bundle branch block, second- or third-degree atrioventricular block;

• Severe, uncontrolled cardiac arrhythmias requiring medication;

• History of chronic congestive heart failure and NYHA class ≥ 3;

• Cardiac ejection fraction below 50% within 6 months prior to screening;

• Heart valve disease with CTCAE grade ≥ 3;

• History of myocardial infarction, unstable angina, severe ventricular arrhythmia, severe pericardial disease, or electrocardiographic evidence of acute ischemic or active conduction abnormalities within 6 months prior to screening.

9) Uncontrollable hypertension (under medication control, multiple measurements show systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥100 mmHg);

10) Having an active bacterial, fungal, or viral infection requiring intravenous infusion therapy within one week prior to the first dose;

11) The patient has received any anti-tumor treatment (including chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy, endocrine therapy) within 4 weeks prior to the first dose, received immunomodulatory agents as adjuvant therapy for malignant tumors within 2 weeks prior to the first dose, or received any anti-tumor traditional Chinese medicine (excluding tonifying traditional Chinese medicine and symptom-relieving traditional Chinese medicine) within 2 weeks prior to the first dose.

12) Individuals who have received other investigational drugs within 4 weeks prior to the first dose;

13) Individuals who have undergone major surgery within 12 weeks prior to the first dose, or who plan to undergo major surgery during the study period;

14) Deep vein thrombosis or arterial embolism within the past 6 months, including but not limited to superior/inferior vena cava thrombosis, lower extremity deep vein thrombosis, and pulmonary embolism;

15) Breastfeeding women;

16) Suffering from any serious and/or uncontrollable disease, or other diseases that, in the investigator's opinion, may affect a patient's participation in this study (including but not limited to uncontrolled diabetes, kidney disease requiring dialysis, severe liver disease, life-threatening autoimmune diseases and bleeding disorders, drug abuse, neurological diseases, etc.).

17) Other situations where researchers deem it unsuitable for participation.

(III) Criteria for Withdrawal from Treatment and Research

Subjects must discontinue treatment with the study drug if any of the following conditions occur during the study:

1) The subject experienced intolerable toxicity, and the researchers believed that the risks of continuing to receive the investigational drug treatment outweighed the benefits;

2) Disease progression as assessed by imaging;

3) Clinical assessment shows disease progression or significant protocol violation, or subject compliance is poor, and the investigator determines that continuing to receive the investigational drug treatment will not be beneficial;

4) The subject becomes pregnant;

5) Death;

6) Meet any one of the criteria for exiting the research.

All subjects who withdraw from treatment must continue to be followed up according to the study plan, except for subjects who stop treatment due to death or meet any of the following withdrawal criteria.

Participants have the right to withdraw from the study at any time for any reason. Participants will be withdrawn from the study process if any of the following occurs:

1) Loss to follow-up;

2) The subject withdraws informed consent or the subject or their family requests to withdraw from the trial;

3) Research terminated;

4) Others.

III. Research Results

New treatments are urgently needed for advanced breast cancer that has failed or recurred in patients who have previously received anthracycline and taxane therapy.

The study of this invention showed that, among at least 20 evaluable cases, 6 achieved partial response (PR) (1 after 7 cycles of treatment, 3 after 6 cycles, 1 after 5 cycles, and 1 after 4 cycles), and 8 achieved stable disease (SD) (2 after 6 cycles, 2 after 5 cycles, 1 after 4 cycles, 1 after 3 cycles, and 2 after 2 cycles). The objective response rate (ORR) was 30% (6/20), and the disease control rate (DCR) was 70% (14/20).

Mitoxantrone liposomes showed good efficacy in breast cancer patients, especially those with advanced breast cancer who had failed or relapsed after prior anthracycline and taxane therapy. Further treatment in 14 subjects who were rated as PR or SD resulted in even better efficacy.

In previous clinical studies, the inventors included mitoxantrone hydrochloride monotherapy for recurrent/metastatic breast cancer. Each group consisted of 30 patients. The experimental group received mitoxantrone hydrochloride liposome injection at 20 mg/ ^m² , with each cycle lasting 4 weeks. The control group received mitoxantrone hydrochloride injection at 14 mg/ ^m² , with each cycle lasting 4 weeks. Results: In the experimental group, 4 patients achieved partial response (PR), 11 achieved stable disease (SD), with an objective response rate (ORR) of 13.3% (4/30) and a disease control rate (DCR) of 50% (15/30). In the control group, 2 patients achieved PR, 7 achieved SD, with an ORR of 6.7% (2/30) and a DCR of 30% (9/30).

The inventors of this invention use mitoxantrone liposomes. After the drug is administered intravenously to the human body, it has the effects of sustained release, targeting, toxicity reduction, and synergistic effect. Not only is the dosage higher than that of ordinary injections, but it is also a single-drug treatment, which can improve the effectiveness and reduce the probability of adverse reactions.

Example 3: Phase Ib clinical trial of mitoxantrone hydrochloride liposome injection for the treatment of advanced bone and soft tissue sarcoma that has failed at least one first-line therapy.

This is an open-label, multicenter phase Ib study in which enrolled participants will receive mitoxantrone hydrochloride liposome injection to evaluate the safety and efficacy of mitoxantrone hydrochloride liposome injection in patients with metastatic or unresectable bone and soft tissue sarcomas who have failed at least one prior line of therapy.

I. Experimental Design

1. Test Procedure

The study included a screening period, a treatment period, and a follow-up period.

The screening period was 28 days, and qualified subjects entered the treatment period. During the treatment period, subjects received mitoxanone hydrochloride liposome injection 20 mg/ ^m² on day 1, with each treatment cycle lasting 3 weeks (q3w), for a total of 6 cycles. For subjects who completed 6 cycles of treatment, the investigator and sponsor jointly determined whether to continue treatment based on the subject's risk and benefit. Follow-up included safety monitoring (28±7 days after the last dose) and survival monitoring (every 6 weeks after the last dose).

Safety evaluations were conducted from the first dose to 28 days after the last dose. Safety evaluations were also conducted before each cycle of administration to determine whether to administer the drug in the next cycle. Tumor evaluations were performed according to the RESIST 1.1 criteria throughout the study. Evaluations were conducted every two cycles during the treatment period and every 6 weeks during the follow-up period, until disease progression, death, or receipt of new anti-tumor therapy.

2. Duration of the study

This study included a 4-week (28-day) screening period, a 6-cycle (18-week) treatment period, a final safety follow-up, a 4-week (28-day) follow-up after the end of treatment, and a survival follow-up every 6 weeks thereafter, with each patient lasting approximately 12 months.

This study enrolled no fewer than 50 participants, and the entire study period was 24–36 months.

II. Trial population:

Subjects who meet all of the following inclusion criteria and have no exclusion criteria are eligible to be enrolled in this clinical study.

(I) Selection Criteria:

Subjects must meet all of the following criteria:

1) Participants voluntarily participate in the study and sign an informed consent form;

2) Age ≥ 18 years old, gender not limited;

3) Pathologically confirmed osteosarcoma or non-specific soft tissue sarcoma (soft tissue sarcoma types do not include: sarcomas that are highly sensitive to chemotherapy, such as embryonal/alveolar rhabdomyosarcoma and Ewing sarcoma; sarcomas that are extremely insensitive to chemotherapy, such as alveolar soft tissue sarcoma and extraosseous myxoid chondrosarcoma; and sarcomas that require special treatment, such as gastrointestinal stromal tumors and aggressive fibromatosis).

4) Metastatic or unresectable osteosarcoma or soft tissue sarcoma that has failed at least one previous line of treatment;

5) The baseline shows at least one measurable lesion that meets the definition of RECIST 1.1;

6) ECOG score 0-1;

7) Laboratory test values must meet the following standards:

• Absolute neutrophil count (ANC) ≥ 2.0 x ^10⁹ /L (no G-CSF white blood cell boosting therapy received within 1 week prior to laboratory test);

• Hemoglobin (Hb) ≥ 110 g/L (no red blood cell transfusion within 1 week prior to laboratory test);

• Platelet count ≥100 x ^10⁹ /L (no platelet transfusion within 1 week prior to laboratory test);

Creatinine ≤ 1.5x ULN;

• Total bilirubin ≤1.5x upper limit of normal (ULN) (≤3.0x ULN for subjects with liver metastases);

• Alanine aminotransferase (AST)/aspartate aminotransferase (ALT) ≤3.0x ULN (≤5.0x ULN for subjects with liver metastases);

• Coagulation function: Prothrombin time (PT) and international normalized ratio (INR) ≤ 1.5 x ULN (INR ≤ 3 is allowed for those receiving anticoagulant therapy such as warfarin)

8) Female subjects had negative urine or blood HCG (except for menopausal and hysterectomy subjects);

9) The subjects and their partners agree to use effective contraception during the trial and for 6 months after the last dose (e.g., combined hormone (including estrogen and progestin) combined with ovulation suppression, progestin contraception combined with ovulation suppression, intrauterine device, intrauterine hormone-releasing system, bilateral tubal ligation, vasectomy, avoidance of sexual intercourse, etc.).

(II) Exclusion Criteria:

Subjects meeting any of the following criteria will be excluded from this trial:

1) Severe allergy to mitoxantrone or liposomal drugs;

2) Brain or meningeal metastasis;

3) Previous allogeneic organ transplantation or allogeneic bone marrow transplantation;

4) Expected survival time < 12 weeks;

5) Chronic hepatitis B (HBsAg or HBcAb positive and HBV DNA ≥ 1000 IU/mL), hepatitis C (HCV antibody positive and HCV RNA quantitative detection higher than the lower limit of the research center's detection value), HIV antibody positive;

6) Presence of grade 1 toxicity from previous antitumor treatments (excluding hair loss, pigmentation, or other toxicities that were considered to pose no safety risk to subjects in the study);

7) Having an active bacterial, fungal, or viral infection requiring systemic treatment within one week prior to the first dose;

8) Received local or systemic antitumor treatment within 4 weeks prior to the first dose (2 weeks prior to the dose for traditional Chinese medicine or proprietary Chinese medicine);

9) Has received treatment with other clinical trial drugs within 4 weeks prior to the first dose;

10) Severe thrombosis within the past 6 months, including pulmonary embolism, visceral embolism, deep vein thrombosis, etc.;

11) Having other malignant active tumors within the past 3 years, excluding locally curable cancers such as basal or squamous cell skin cancer, superficial bladder cancer or prostate cancer in situ, cervical cancer or breast cancer;

12) Abnormal cardiac function, including:

• Long QTc syndrome or QTc interval > 480 ms;

• Complete left bundle branch block, second- or third-degree atrioventricular block (excluding those treated with pacemaker implantation);

• Severe, uncontrolled cardiac arrhythmias requiring medication;

• History of chronic congestive heart failure, with NYHA class ≥ 3;

• Left ventricular ejection fraction less than 50% within the past 6 months;

• Heart valve disease with CTCAE grade ≥ 3;

• Uncontrollable hypertension (defined as systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥100 mmHg on multiple measurements under medication control);

• A history of myocardial infarction, unstable angina, severe pericardial disease, or electrocardiographic evidence of acute ischemic or severe active conduction abnormalities within 6 months prior to screening.

13) Has received doxorubicin or other anthracycline treatment, and the cumulative dose of doxorubicin exceeds 400 mg/ ^m² (anthracycline equivalent dose calculation: 1 mg doxorubicin = 2 mg epirubicin = 2 mg daunorubicin = 0.5 mg demethoxydaunorubicin = 0.45 mg mitoxantrone); or the cumulative dose of doxorubicin liposomes exceeds 900 mg/ ^m² .

14) Pregnant or breastfeeding women;

15) Suffering from any serious and/or uncontrollable disease, or other diseases that, as determined by the investigator, may affect participation in this study (including, but not limited to, uncontrolled diabetes, kidney disease requiring dialysis, severe liver disease, life-threatening autoimmune diseases and bleeding disorders, substance abuse, neurological diseases, etc.).

16) Other situations where researchers deem it unsuitable for participation.

(III) Criteria for Withdrawal from Treatment and Research

Subjects must discontinue treatment with the investigational drug if any of the following conditions occur during the study:

1) The subject experienced intolerable toxicity, and the researchers believed that the risks of continuing treatment with the investigational drug outweighed the benefits;

2) Comorbidities that occur during treatment do not preclude continued medication;

3) Disease progression as assessed by imaging;

4) Clinical assessment shows disease progression or significant protocol violation, or subject compliance is poor, and the investigator determines that continuing treatment with the investigational drug will not be beneficial;

5) The subject becomes pregnant;

6) Death;

7) Meet any of the exit criteria for the study.

All subjects who withdraw from treatment must continue to be followed up according to the study plan, except for subjects who stop treatment due to death or meet any of the following withdrawal criteria.

Participants have the right to withdraw from the study at any time for any reason. Participants will be withdrawn from the study process if any of the following occurs:

1) Loss to follow-up;

2) The participant or their family requests to withdraw from the trial;

3) Research terminated;

4) Others.

III. Research Results

For patients with bone and soft tissue sarcomas who have no chance of surgical cure or who have metastatic bone and soft tissue sarcomas, the efficacy of second-line treatments is often unsatisfactory after first-line standard therapy. Therefore, improving treatment has always been a challenging clinical issue, and new treatments are urgently needed. In the escalation study (HE071-01), mitoxantrone liposomes showed some efficacy in patients with advanced soft tissue sarcomas in the 12 mg/ ^m² and 14 mg/ ^m² groups. The inventors of this invention use mitoxantrone liposomes, which, after intravenous infusion, have sustained-release, targeted, toxicity-reducing, and synergistic effects. Not only is the dosage higher than that of ordinary injectable drugs, but it is also a monotherapy, which can improve efficacy while reducing the probability of adverse reactions.

The study of this invention shows that among evaluable subjects, of the 3 subjects with osteosarcoma (2 who received 2 cycles of treatment and 1 who received 1 cycle of treatment), 1 subject was evaluated as SD (receiving 1 cycle of treatment), with a DCR of 33.3% (1/3).

In the soft tissue sarcoma category, among 12 subjects (1 treated for 6 cycles, 2 treated for 5 cycles, 2 treated for 4 cycles, 1 treated for 3 cycles, and 6 treated for 2 cycles), 8 subjects were evaluated as having stable disease (SD) (1 treated for 6 cycles, 2 treated for 5 cycles, 2 treated for 4 cycles, 1 treated for 3 cycles, and 2 treated for 2 cycles; among them, 3 subjects showed a decrease in tumor volume), with a disease control rate (DCR) of 66.7% (8/12).

Mitoxantrone liposomes showed good efficacy in patients with osteosarcoma and soft tissue sarcoma. Nine subjects with stable disease (SD) who continued treatment achieved even better results.

The embodiments of the technical solution of the present invention have been described above by way of example. It should be understood that the protection scope of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the protection scope of the claims of this application.

Claims (10)

1. Use of mitoxantrone hydrochloride liposomes in the preparation of drugs for the treatment of bone and soft tissue sarcomas. 2. The use of mitoxantrone hydrochloride liposomes as the sole active ingredient in the preparation of drugs for the treatment of bone and soft tissue sarcomas. 3. The use as described in claim 1 or 2, characterized in that, The bone and soft tissue sarcoma is advanced bone and soft tissue sarcoma, and more preferably metastatic or locally advanced bone and soft tissue sarcoma that has failed at least one first-line treatment. 4. The use as described in claim 1 or 2, wherein the drug is an injectable dosage form, including liquid injection, powder for injection, and tablet for injection. 5. The use as described in claim 1, wherein the drug is a liquid injection; And/or, based on mitoxantrone, the drug contains 0.5-5 mg/ml of the active ingredient. 6. The use as described in claim 1, characterized in that, based on mitoxantrone, the drug contains 1-2 mg/ml of the active ingredient. 7. The use according to any one of claims 1-6, characterized in that the mitoxantrone hydrochloride liposome has one or more of the following properties: (i) The particle size of mitoxantrone hydrochloride liposomes is approximately 30-80 nm; (ii) Mitoxantrone hydrochloride forms an insoluble precipitate with the multivalent counterions in the lipid body; (iii) The phospholipid bilayer of mitoxantrone hydrochloride liposomes contains phospholipids with a phase transition temperature higher than body temperature, thus the phase transition temperature of the liposomes is higher than body temperature. (iv) The phospholipid bilayer of mitoxantrone hydrochloride liposomes contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearate phosphatidylethanolamine; (iiv) The phospholipid bilayer of the mitoxantrone hydrochloride liposomes contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearate phosphatidylethanolamine in a mass ratio of about 3:1:1. The mitoxantrone hydrochloride forms an insoluble precipitate with the multivalent counterion in the liposomes, and the mitoxantrone hydrochloride liposomes in the drug have a particle size of about 60 nm. 8. The use according to claim 7, characterized in that the mitoxantrone hydrochloride liposomes have a particle size of about 30-80 nm and contain: 1) the active ingredient mitoxantrone, which forms an insoluble precipitate with the multivalent counterion in the liposomes; 2) the phospholipid bilayer contains phospholipids with a phase transition temperature higher than body temperature; the phospholipids with a phase transition temperature higher than body temperature are phosphatidylcholine, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, dispalmitoyl lecithin, or distearate lecithin, or any combination thereof. 9. The use according to claim 7, wherein the mitoxantrone hydrochloride liposomes have a particle size of about 35-75 nm, preferably 40-70 nm, more preferably 40-60 nm, and particularly preferably 60 nm. 10. The use according to claim 7, characterized in that the phospholipid bilayer contains hydrogenated soybean lecithin, cholesterol and polyethylene glycol 2000 modified distearate phosphatidylethanolamine in a mass ratio of 3:1:1, the particle size is about 60 nm, and the counterion is sulfate ion; Preferably, the phospholipid bilayer of the liposome contains hydrogenated soybean lecithin, cholesterol, and polyethylene glycol 2000 modified distearate phosphatidylethanolamine in a mass ratio of 3:1:1, the particle size is about 40-60 nm, the counterion is sulfate ion, and the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the liposome is 9.58:3.19:3.19:1. Preferably, the multivalent counterion is selected from sulfate, citrate, or phosphate. Preferably, the phospholipid is selected from hydrogenated soybean lecithin, phosphatidylcholine, hydrogenated egg yolk lecithin, distearate lecithin, distearate lecithin, or any combination thereof.