Application of mitoxantrone hydrochloride liposome

Abstract

The invention provides application of mitoxantrone hydrochloride lipidosome to treatment of neuromyelitis optica pedigree diseases, and further provides a method for treating neuromyelitis optica pedigree diseases in an individual. The method comprises administering to the individual a therapeutically effective amount of a liposome of mitoxantrone hydrochloride or a pharmaceutical composition comprising a liposome of mitoxantrone hydrochloride. Animal and clinical test results show that the mitoxantrone hydrochloride liposome is safe and effective to neuromyelitis optica pedigree diseases.

Description

Uses of mitoxantrone hydrochloride liposomes

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202210856534.9 filed on July 20, 2022 and Chinese patent application No. 202210924224.6 filed on August 2, 2022, and the entire contents of the above applications are incorporated herein by reference for all purposes. Technical Field

[0003] The present application relates to the field of medicine, and in particular to the use of mitoxantrone hydrochloride liposomes, for example, in the treatment of neuromyelitis optica spectrum disorders. Background Art

[0004] Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune-mediated inflammatory demyelinating disease of the central nervous system characterized by simultaneous or sequential involvement of the optic nerve and spinal cord. Its pathogenesis is primarily linked to antibodies against aquaporin-4 (AQP4), mediated by autoantibodies, dominated by humoral immunity, and involving multiple immune cells and factors. NMOSD occurs in all age groups, but is most common in young adults, with an average age of onset of approximately 40 years. Most patients are positive for serum AQP4-IgG. Among AQP4-IgG-positive patients, the female to male ratio is as high as 4.7 to 11. Clinically, it is often characterized by severe optic neuritis and longitudinally extending long-segment transverse myelitis, often accompanied by characteristic NMO brain lesions (hypothalamus, corpus callosum, periventricular, or brainstem) or systemic autoimmune disease. NMOSD is a highly recurrent and disabling disease, with more than 90% of patients experiencing a multiphasic course, of which 40%-60% relapse within 1 year and approximately 90% relapse within 3 years. Among patients with a natural course, approximately 50% have severe residual visual or motor dysfunction within 5-10 years (Neuroimmunology Branch of the Chinese Society of Immunology, Huang Dehui, Wu Weiping, et al. Guidelines for the Diagnosis and Treatment of Neuromyelitis Optica Spectrum Disorders in China (2021 Edition). Chinese Journal of Neuroimmunology and Neurology, Vol. 28, No. 6, November 2021).

[0005] Evidence from pathogenesis, imaging, and clinical manifestations indicates that NMOSD is an independent disease entity distinct from multiple sclerosis (MS). Its treatment regimen is also different from that of MS, and some MS medications may exacerbate NMOSD symptoms (Ji Wei, Wang Limei, Tan Song. Progress in the differential diagnosis and treatment of laboratory indicators of neuromyelitis optica and multiple sclerosis. Chinese Journal of Practical Neurological Diseases, Volume 19, Issue 10, June 2016).

[0006] Currently, the commonly used clinical treatments for NMOSD are divided into acute attack treatment, sequential treatment (relapse prevention treatment), symptomatic treatment and rehabilitation treatment. The purpose of acute treatment is to reduce irreversible nerve damage, promote functional recovery, and reduce mortality. High-dose hormone pulse therapy, plasma exchange or immunoadsorption, and intravenous human immunoglobulin are often used; the purpose of sequential treatment is mainly to reduce the recurrence rate and reduce the severity of patient disability. Commonly used clinical drugs include monoclonal antibodies and immunosuppressants. First-line drugs include satralizumab, inalizumab, rituximab, azathioprine and mycophenolate mofetil; second-line drugs include tacrolimus, cyclosporine A, cyclophosphamide, methotrexate and mitoxantrone (Neuroimmunology Branch of the Chinese Society of Immunology, Huang Dehui, Wu Weiping, etc. Guidelines for the diagnosis and treatment of neuromyelitis optica spectrum disorders in China (2021 edition). Chinese Journal of Neuroimmunology and Neurology, Vol. 28, No. 6, November 2021).

[0007] Given the highly recurrent and disabling characteristics of NMOSD, a large number of NMOSD patients still cannot receive effective treatment, their condition is not effectively controlled, leaving them with severe physical disabilities and placing a heavy disease burden on their families and society. Therefore, there is an urgent need for early diagnosis of the disease, as well as drugs that effectively treat NMOSD and prevent NMOSD recurrence.

[0008] Mitoxantrone hydrochloride is an anthraquinone chemotherapy drug. The FDA has approved the indications of mitoxantrone hydrochloride injection for multiple sclerosis, prostate cancer and acute myeloid leukemia. In my country, it has been approved for patients with lymphoma, leukemia and breast cancer. The recommended dose for adults is 12-14 mg / m2 as a single drug. 2 , once every 3-4 weeks; or 4-8 mg / m 2 , once a day for 3-5 consecutive days, with an interval of 2-3 weeks; the combined dose is 5-10 mg / m 2 As an anthracycline, mitoxantrone has major adverse reactions such as cardiotoxicity, bone marrow suppression, and gastrointestinal reactions.

[0009] Among these, myelosuppression and gastrointestinal reactions can be addressed with appropriate medications, but cardiotoxicity often has serious consequences and is the most serious adverse reaction of anthracyclines. Clinical studies and practical observations have shown that anthracycline-induced cardiotoxicity is often progressive and irreversible, especially with initial use. Cardiotoxicity, a chronic, cumulative dose-limiting toxicity, is a common concern among clinicians. Therefore, although mitoxantrone is approved for breast cancer as an indication, authoritative clinical medication guidelines do not recommend it as a treatment for breast cancer.

[0010] Different tumors have different drug sensitivities. Existing studies have shown that the same drug may have different dosing regimens when used to treat different indications. For example, Doxil (liposomal doxorubicin hydrochloride) has been approved by the FDA for three indications: (1) ovarian cancer, with a recommended dose of 50 mg / m 2 , administered intravenously once every 4 weeks; (2) Kaposi's sarcoma, the recommended dose is 20 mg / m 2 , administered intravenously once every 3 weeks; (3) Multiple myeloma, the recommended dose is 30 mg / m 2 , administered intravenously on the fourth day after bortezomib. For example, Abraxane (paclitaxel for injection [albumin-bound]) is also approved by the FDA for three indications: (1) metastatic breast cancer: recommended dose 260 mg / m 2 , intravenous drip for 30 minutes, once every 3 weeks; (2) Non-small cell lung cancer: recommended dose 100 mg / m 2 , intravenous drip for 30 minutes, one course of treatment every 21 days, administered on the 1st, 8th and 15th days respectively; carboplatin is administered immediately after paclitaxel (albumin-bound) injection on the 1st day, and administered once every 21 days; (3) Pancreatic cancer: recommended dose 125 mg / m 2, intravenous drip for 30-40 minutes, with a cycle of 28 days, and one dose each on the 1st, 8th, and 15th days. Gemcitabine is given immediately after each dose of paclitaxel injection (albumin-bound). For example, the starting dose of AmBisome (liposomal 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) immunocompromised patients with visceral leishmaniasis: 4 mg / kg / day (days 1-5), 4 mg / kg / day (days 10, 17, 24, 31, and 38); the dosage and administration data are personalized according to the actual situation of the patient to achieve maximum efficacy and minimum toxicity or adverse reactions. It can be seen that the safe and effective doses of the same drug for different indications vary. The dosage and administration data should be personalized according to the specific disease and the actual situation of the patient to achieve maximum efficacy and minimum toxicity or adverse reactions, and to achieve safe and effective treatment of the disease. For mitoxantrone liposomes, a special dosage form different from ordinary injections, its absorption, distribution, and metabolism after entering the body are very complicated. For the treatment of different indications, especially in the treatment of different tumors, it is difficult to simply deduce from one indication to another.

[0011] Summary of the Invention

[0012] The present application provides the use of mitoxantrone hydrochloride liposomes in the treatment of neuromyelitis optica spectrum disorders. The inventors of the present application unexpectedly discovered in their research that mitoxantrone hydrochloride liposomes have a good therapeutic effect on neuromyelitis optica spectrum disorders and can be used to prepare related drugs.

[0013] In a first aspect, the present application provides the use of mitoxantrone hydrochloride liposomes in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

[0014] In a second aspect, the present application provides a method for treating neuromyelitis optica spectrum disorder in an individual, comprising administering to the individual a therapeutically effective amount of mitoxantrone liposomes or a pharmaceutical composition comprising a therapeutically effective amount of mitoxantrone liposomes.

[0015] In a third aspect, the present application provides mitoxantrone hydrochloride liposomes or a pharmaceutical composition comprising mitoxantrone hydrochloride liposomes, which is used to treat neuromyelitis optica spectrum disorder in an individual.

[0016] In some embodiments, the neuromyelitis optica spectrum disorder described in the first to third aspects is a highly relapsing neuromyelitis optica spectrum disorder. In some embodiments, the highly relapsing neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder that has failed other drug treatments.

[0017] In some embodiments, for highly recurrent neuromyelitis optica spectrum disorders that have failed other drug treatments, the other drugs are selected from first-line, second-line or higher-line drugs for neuromyelitis optica spectrum disorders, especially first-line, second-line or higher-line 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 neuromyelitis optica spectrum disorders, including but not limited to: FDA-approved glucocorticoids, monoclonal antibody drugs and immunosuppressants, etc.

[0018] In some embodiments, mitoxantrone hydrochloride liposomes are used as the sole active ingredient for treating neuromyelitis optica spectrum disorders or as the sole active ingredient for preparing a medicament or pharmaceutical composition for treating neuromyelitis optica spectrum disorders.

[0019] In some embodiments, the pharmaceutical composition or drug comprising mitoxantrone hydrochloride liposomes in the first to third aspects is in the form of an injectable formulation, including liquid injections, powders for injection, tablets for injection, etc. In a preferred embodiment, the pharmaceutical composition or drug comprising mitoxantrone hydrochloride liposomes is in the form of a liquid injection.

[0020] In some embodiments, when the pharmaceutical composition or drug comprising mitoxantrone hydrochloride liposomes is a liquid injection, the content of mitoxantrone in the drug or pharmaceutical composition is 0.5-5 mg / mL, calculated as mitoxantrone. In a preferred embodiment, the content of mitoxantrone in the drug is 1-2 mg / mL. In a more preferred embodiment, the content of mitoxantrone in the drug or pharmaceutical composition is 1 mg / mL.

[0021] In some embodiments, the therapeutically effective amount in the second aspect is 4-30 mg / m 2 , for example, 8-20 mg / m 2 or 12-30 mg / m 2 , or a dose within the range between any two of the above doses, for example, 8 mg / m 2 , 12mg / m 2 etc.

[0022] In some embodiments, the mitoxantrone hydrochloride liposomes, the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes, or the medicament of the present application are administered to a subject in need thereof by intravenous administration.

[0023] In some embodiments, the administration cycle of the mitoxantrone hydrochloride liposomes, the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes, or the drug of the present application is once every 12 weeks.

[0024] In some embodiments, the liposomes are used alone to treat a neuromyelitis optica spectrum disorder in a subject. In some embodiments, the therapeutically effective amount of the liposomes (as mitoxantrone) is 4-30 mg / m 2 , such as 8-20 mg / m 2 or 12-30 mg / m 2 , or a dose within the range between any two of the above doses, for example, 8 mg / m 2 or 12 mg / m 2 In some embodiments, the liposomes are administered intravenously.

[0025] In some embodiments, the liposomes are administered once every 12 weeks.

[0026] In the context of this application, the dosage is based on mitoxantrone unless otherwise specified.

[0027] In the context of the present application, the mitoxantrone hydrochloride liposomes can be prepared by methods known in the art, and can be prepared by any method disclosed in the prior art, for example, by the method disclosed in WO2008 / 080367A1.

[0028] In some embodiments, the drug, mitoxantrone hydrochloride liposome, or pharmaceutical composition comprising the mitoxantrone hydrochloride liposome in the first to third aspects above has one or more of the following properties:

[0029] (i) the particle size of the 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;

[0030] (ii) Mitoxantrone hydrochloride forms insoluble precipitates with multivalent counterions (e.g., sulfate, citrate, or phosphate) within the liposome;

[0031] (iii) the phospholipid bilayer in the mitoxantrone hydrochloride liposome contains a phospholipid having 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 soy lecithin, phosphatidylcholine, hydrogenated egg yolk lecithin, disalmitate lecithin, distearate lecithin or any combination thereof;

[0032] (iv) The phospholipid bilayer in the mitoxantrone hydrochloride liposomes contains hydrogenated soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine modified with polyethylene glycol 2000 (DSPE-PEG2000).

[0033] (iiv) The phospholipid bilayer in the mitoxantrone hydrochloride liposome contains hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoylphosphatidylethanolamine in a mass ratio of about 3:1:1. Mitoxantrone hydrochloride forms an insoluble precipitate with the multivalent acid ions in the liposome, and the particle size of the mitoxantrone hydrochloride liposome in the drug is about 60 nm.

[0034] (iiiv) Mitoxantrone hydrochloride liposomes are mitoxantrone hydrochloride liposomes with national medicine standard number H20220001.

[0035] In some embodiments, the particle size of the mitoxantrone hydrochloride liposomes described herein is about 30-80 nm, for example, any value between 30-80 nm. In some embodiments, the particle size is about 35-75 nm, preferably 40-70 nm, more preferably 40-60 nm, for example, 60 nm.

[0036] In some embodiments, the mitoxantrone hydrochloride liposomes described herein contain the active ingredient mitoxantrone and a phospholipid bilayer.

[0037] In some embodiments, the active ingredient mitoxantrone can form an insoluble precipitate with a multivalent counterion in the liposome. In some embodiments, the counterion is sulfate, citrate, or phosphate.

[0038] In some embodiments, the phospholipid bilayer comprises a phospholipid having a phase transition temperature (Tm) higher than body temperature, thereby the phase transition temperature of the liposome is higher than body temperature. In some embodiments, the phospholipid having a Tm higher than body temperature is phosphatidylcholine, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, disalmitate lecithin or distearate lecithin, or any combination thereof.

[0039] In some embodiments, the phospholipid bilayer contains hydrogenated soy lecithin, cholesterol, and distearoylphosphatidylethanolamine modified with polyethylene glycol 2000. In some embodiments, the mass ratio of hydrogenated soy lecithin, cholesterol, and distearoylphosphatidylethanolamine modified with polyethylene glycol 2000 in the phospholipid bilayer is 3:1:1.

[0040] In some embodiments, the particle size of the mitoxantrone hydrochloride liposomes described herein is about 60 nm, and the counter ion inside the liposomes is sulfate ion.

[0041] In some embodiments, the phospholipid bilayer of the mitoxantrone hydrochloride liposomes described herein comprises hydrogenated soy lecithin, cholesterol, and distearoylphosphatidylethanolamine modified with polyethylene glycol 2000 in a weight ratio of 3:1:1. The liposomes have a particle size of approximately 40-60 nm, and the counterion within the liposomes is sulfate. In some embodiments, the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone in the mitoxantrone hydrochloride liposomes described herein is 9.58:3.19:3.19:1.

[0042] In the context of this application, the preparation method of mitoxantrone liposomes is as follows: HSPC (hydrogenated soy lecithin), Chol (cholesterol), and DSPE-PEG2000 (distearoylphosphatidylethanolamine modified with polyethylene glycol 2000) are weighed in a mass ratio of (3:1:1) and dissolved in 95% ethanol to obtain a clear solution. The ethanolic solution of the phospholipids is mixed with a 300 mM ammonium sulfate solution and hydrated by shaking at 60-65°C for 1 hour to obtain heterogeneous multilamellar liposomes. The liposomes are then size-reduced using a microfluidizer. The resulting sample is diluted 200-fold with a 0.9% NaCl solution and analyzed using a NanoZS. The average particle size is approximately 60 nm, with a major peak concentrated between 40 and 60 nm. The ammonium sulfate in the blank liposome external phase is then removed using an ultrafiltration device and replaced with 290 mM sucrose and 10 mM glycine to form a transmembrane ammonium sulfate gradient. A mitoxantrone hydrochloride solution (10 mg / mL) was added to blank liposomes at a lipid-drug ratio of 16:1 and drug loading was performed at 60-65°C. After approximately one hour of incubation, gel exclusion chromatography demonstrated an encapsulation efficiency of approximately 100%. The weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone was 9.58:3.19:3.19:1. The osmotic pressure of the sucrose-glycine solution was close to physiological values.

[0043] It should be understood that the various 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 acid types that can replace glycine in the external phase used to form the transmembrane ammonium sulfate gradient include, but are not limited to, histidine, asparagine, glutamic acid, leucine, proline, and alanine. For another example, the mass ratio of HSPC, Chol, and DSPE-PEG2000 can be appropriately adjusted. For another example, for the lipid-drug ratio parameters in the preparation of a specific liposome drug formulation, those skilled in the art can design, test, and ultimately determine a suitable lipid-drug ratio to maximize drug loading while minimizing drug leakage. For the mitoxantrone hydrochloride liposome formulation of the present application, a wide range of lipid-drug ratios can be used, for example, as low as 2:1 or as high as 30:1, 40:1, or 50:1. A more suitable lipid-drug ratio can be about (15-20):1, for example, about 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Therefore, the several advantageous properties of the liposomal formulation of mitoxantrone hydrochloride described above are more important, and the methodologies for achieving these properties are diverse.

[0044] As used herein, the term "subject" refers to a mammal, such as a human, but may also be other mammals, such as livestock or laboratory animals.

[0045] The term "treatment" as used herein means administering the mitoxantrone hydrochloride liposomes or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) inhibiting a disease or disease state, i.e., curbing its development; and (ii) alleviating a disease or disease state, i.e., causing the disease or disease state to regress.

[0046] The term "therapeutically effective amount" means an amount of the mitoxantrone hydrochloride liposomes of the present application that (i) treats a particular disease, condition, or disorder, or (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder. The amount of the mitoxantrone hydrochloride liposomes of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the disclosure herein.

[0047] In some embodiments, the drugs, mitoxantrone hydrochloride liposomes or pharmaceutical compositions comprising the mitoxantrone hydrochloride liposomes in the above-mentioned first to third aspects, such as mitoxantrone hydrochloride liposome injection, have one or more of the following beneficial effects in the treatment of neuromyelitis optica spectrum disorders: safe and tolerable, low toxic and side effects, improved therapeutic efficacy, delayed recurrence of neuromyelitis optica spectrum disorders, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 shows the body weight analysis of animals with neuromyelitis optica. Data are expressed as Mean ± SEM. ***P < 0.001.

[0049] FIG2 shows representative images of AQP4 immunofluorescence staining, Bar: 200 μm.

[0050] Figure 3 shows the statistics of AQP4-positive cells. The data are expressed as Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explain the present application and should not be construed as limiting the scope of protection of the present application. All technologies implemented based on the above content of the present application are included within the scope of protection intended by the present application.

[0052] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0053] Example 1: Improvement of mitoxantrone liposomes on the NMOSD model induced by AQP4 antibody / complement combination in clinical patients

[0054] According to literature reports, direct injection of AQP4 antibodies and human complement from clinically diagnosed patients into the cerebral hemispheres or ventricles of mice can induce a NMOSD model (Saadoun S, Waters P, Bell BA, et al. Intra-cerebral injection of neuromyelitis optica immunoglobulin G and human complement produces neuromyelitis optica lesions in mice. Brain. 2010, 133:349-61). Therefore, this modeling method was selected in this example to study the therapeutic effect of mitoxantrone liposomes on NMOSD.

[0055] Experimental animals: Male CD-1 mice, 8 weeks old, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0056] Test drug: Mitoxantrone hydrochloride liposome injection 1.0 mg / ml (test sample).

[0057] Experimental methods: First, AQP4-IgG antibodies from clinical patient blood samples and normal human complement serum were extracted, purified, and verified. Then, 27 8-week-old CD-1 male mice were selected. After one week of adaptation, they were randomly divided into three groups according to body weight, namely, blank group, model group, and test article group (mitoxantrone liposome 15 mg / kg), with 9 animals in each group.

[0058] On days 1, 3, and 5, 2 μl of APQ4 antibody (concentration 18 mg / ml) plus 1 μl of human complement serum were injected into the lateral ventricle of each mouse in the model group and the test article group at a rate of 0.3 μl / min to induce the NMOSD model.

[0059] The blank group was a sham operation group. The animal modeling process was the same as that of the model group and the test article group, but no antibodies or complement were injected.

[0060] The animals in the test group received a single intravenous injection of 15 mg / kg of mitoxantrone hydrochloride liposome injection on the first day of modeling.

[0061] Animal health was monitored and body weights were recorded throughout the experiment. Three animals per group were euthanized via carbon dioxide on Day 7, Day 11, and Day 21 after model establishment. Brain tissue was collected and assessed for pathological changes using staining for AQP4, GFAP, and LFB. Pathological sampling sites were stained using 30 sections centered on the injection site to further characterize the extent of the lesion.

[0062] The steps required for the immunohistochemistry process and antibody information are described below:

[0063] 1. Perfusion-Fixation-Sugar Sedimentation-Slicing

[0064] 1) Mice were anesthetized with isoflurane.

[0065] 2) Use straight scissors to cut open the abdominal and thoracic cavities to expose the heart. First, flush out the blood with normal saline, and then perform initial fixation with paraformaldehyde.

[0066] 3) Cut off the head, carefully open the skull with forceps, remove the brain tissue and fix it in paraformaldehyde solution for 24 hours.

[0067] 4) The next day, the cells were taken out and placed in 20% sucrose solution for 24 hours.

[0068] 5) On the third day, the cells were taken out and placed in 30% sucrose solution for 24 hours.

[0069] 6) On the fourth day, remove the brain tissue and place it in a 35% sucrose solution for 24 hours. (The time or concentration may be increased as needed depending on the sugar content of the brain tissue.)

[0070] 7) The brain tissue was removed, embedded in OCT embedding medium, and cut into 16 μm brain slices using a freezing microtome.

[0071] 2. Immunofluorescence staining (AQP4, GFAP)

[0072] 1) Rewarm the slices for 30 minutes;

[0073] 2) Blocking: 10% serum + 0.3% TritonX-100 [approximately 50 μl per slide], room temperature for 1 hour;

[0074] 3) Shake the slides, add primary antibody (diluted in PBS), and incubate at 4°C overnight;

[0075] 4) Rewarm for 30 minutes;

[0076] 5) Wash the primary antibody (PBS), 5 min × 3 times;

[0077] 6) Add secondary antibody (diluted in PBS) in backlight at room temperature for 2 hours;

[0078] 7) Wash the secondary antibody (PBS), 5 min × 3 times;

[0079] 8) Add DAPI and incubate at room temperature for 10 minutes;

[0080] 9) Wash DAPI, 5 min × 3 times;

[0081] 10) Seal the slides with 70% glycerol to avoid air bubbles.

[0082] 3. Antibody information:

[0083] Primary Antibody

[0084] 1) AQP4: purchased from Abclonal, product number: A2887, batch number: 0073660402;

[0085] 2) GFAP: purchased from Huaan Biotechnology, catalog number: EM140707, batch number: HO0604;

[0086] Secondary Antibodies

[0087] 1) Goat Pab to Rabbit 594: purchased from abcam, catalog number: ab150080, batch number: GR3439696-1;

[0088] 2) Goat Pab to mouse 488: purchased from abcam, catalog number: ab150077, batch number: GR3403178-2

[0089] 4. Luxol fast blue stain (LFB)

[0090] Purchased from Solarbio, product number: G3242, batch number: 20220422. Operate according to the kit instructions.

[0091] Test results:

[0092] This study used CD-1 mice and established a neuromyelitis optica disease model by intracerebroventricular injection of AQP4 antibodies / complement from clinically diagnosed patients using brain stereotactic positioning. Related histological tests were also performed to evaluate the effects of the test compounds.

[0093] The results of animal body weight analysis are shown in Figure 1 and Table 1. Compared with the model group, the test group showed weight loss from day 7 to day 21 after model establishment and administration. The animals were in good condition, and no animals died due to drug toxicity.

[0094] Table 1 Note: Mean ± SEM, n = 3-9; compared with the model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0095] Representative images of AQP4 immunofluorescence staining and statistical results of positive cell counts are shown in Figures 2-3 and Table 2. On day 7 after modeling, the number of AQP4-positive cells in the striatum of the model group was significantly reduced compared to the blank and test article groups, and this decrease became increasingly significant over time, indicating that the NMOSD disease model was successfully established through stereotactic intracerebroventricular injection of AQP4 antibodies / complement from clinically diagnosed patients. The number of AQP4-positive cells in the test article group was higher than that in the model group from day 7 to day 21, with the difference reaching statistical significance on day 21 (P < 0.001), suggesting that mitoxantrone hydrochloride liposomes at 15 mg / kg can significantly inhibit the AQP4 deficiency in NMOSD and effectively improve the symptoms of NMOSD in mice.

[0096] Table 2 Note: Mean ± SEM, n = 3; compared with the model group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0097] The experimental results showed that mitoxantrone hydrochloride liposome injection (15 mg / kg) can significantly inhibit the loss of AQP4 in mice and improve histopathological changes such as astrocyte lesions and demyelination, indicating that mitoxantrone hydrochloride liposome injection can effectively improve the symptoms of NMOSD in mice and inhibit its occurrence and development.

[0098] Example 2 Phase II Clinical Study on the Efficacy and Safety of Mitoxantrone Hydrochloride Liposome Injection in the Treatment of Neuromyelitis Optica Spectrum Disorders (NMOSD)

[0099] This example is a randomized, double-blind, placebo-controlled Phase II study. The included subjects will receive mitoxantrone hydrochloride liposome injection. The purpose is to evaluate the safety and efficacy of mitoxantrone hydrochloride liposome injection in NMOSD subjects, explore the reasonable dose of mitoxantrone hydrochloride liposome injection for the treatment of neuromyelitis optica spectrum disorder (NMOSD), and provide a basis for later clinical development.

[0100] 1. Experimental Design

[0101] 1. Test process

[0102] The entire study period is approximately 60 weeks, including a screening period (up to 4 weeks), a treatment period (48 weeks), and a safety follow-up period (8 weeks). The investigator or designated personnel will contact all subjects by phone every 2 weeks starting from the start of the treatment period until the subject completes the trial (unless the time coincides with the study visit week). If the investigator suspects an NMOSD attack or impending attack, an assessment visit will be arranged within 72 hours.

[0103] Subjects diagnosed with NMOSD will be screened within 4 weeks (28 days) to determine their eligibility for the study based on the inclusion and exclusion criteria. AQP4-IgG serum status will be determined by a central laboratory. All subjects who meet the eligibility criteria will then be randomly assigned to a study center. Randomization (Day 1): Screened subjects will be randomly assigned in a 1:1:1 ratio to study group A (mitoxantrone hydrochloride liposomal injection 8 mg / m 2 ), experimental group B (mitoxantrone hydrochloride liposome injection 12 mg / m 2 ) and placebo groups. Successfully randomized subjects will receive mitoxantrone liposomes or placebo (once every 12 weeks). When the last enrolled patient relapses 48 weeks after randomization, the treatment period ends and the safety follow-up period begins. Safety follow-up will begin after the subject terminates the trial early or relapses, or when the treatment period is completed. An 8-week safety follow-up (including scale assessments, laboratory tests, and electrocardiograms) is required to monitor adverse events / serious adverse events in the subjects and collect information on concomitant medications or treatments related to NMO / NMOSD. During this period, at the investigator's discretion, subjects may receive standard treatment for NMO / NMOSD.

[0104] 2. Study duration

[0105] This study includes a 4-week screening period, a treatment period of up to 48 weeks, and a safety follow-up period of 8 weeks. The expected duration for each patient is approximately 60 weeks (excluding relapsed patients).

[0106] This study plans to enroll 45 subjects, and the entire study period is expected to be 24 months.

[0107] 2. Trial Population

[0108] Only subjects who meet all the following inclusion criteria and do not meet any exclusion criteria can be selected for this clinical study.

[0109] (1) Selection criteria:

[0110] Subjects must meet all of the following criteria:

[0111] 1) Applicants must be between 18 and 60 years old (inclusive) when signing the informed consent form, and both men and women are welcome;

[0112] 2) Patients who meet the 2015 international consensus diagnostic criteria for neuromyelitis optica spectrum disorder (NMOSD) and are AQP4-IgG positive or negative (test results within 24 weeks before signing the informed consent form are acceptable);

[0113] 3) experienced at least 2 episodes within 2 years before screening and / or had clinical evidence of at least one documented relapse (including the first episode) within 12 months before screening;

[0114] 4) EDSS ≤ 7.5 points at screening and baseline retest;

[0115] 5) The subject voluntarily signs the informed consent form and voluntarily completes the trial according to the protocol requirements

[0116] (II) Exclusion criteria:

[0117] 1. General security exclusions:

[0118] 1) Pregnant or lactating women; for patients of childbearing potential, the serum pregnancy test result is positive at screening, or they are unwilling to take reliable contraceptive measures (physical barriers [patient or partner] and spermicides, birth control pills, patches, injections), intrauterine devices or intrauterine contraceptive systems) and continue for at least 4 months after the last dose of the study drug. Males of childbearing potential should take effective contraceptive measures from the time they sign the informed consent form to 6 months after the last dose;

[0119] 2) Patients who had undergone any surgical procedure within 4 weeks before randomization and had evidence of other demyelinating diseases or progressive multifocal leukoencephalopathy (PML);

[0120] 3) Known active infection within 4 weeks before randomization (excluding nail bed fungal infection or dental caries);

[0121] 4) Infectious disease screening during the screening period: positive results for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCVAb), syphilis antibody, and human immunodeficiency virus (HIV antibody);

[0122] 5) Patients with a history of drug or alcohol abuse within 1 year before randomization (i.e., drinking more than 14 standard units of alcohol per week (1 unit = 360 ml of beer or 45 ml of 40% alcohol liquor or 150 ml of wine)) or mental disorders;

[0123] 6) Evidence of active tuberculosis (TB; excluding patients receiving drugs to prevent latent TB infection);

[0124] 7) evidence of active interstitial lung disease;

[0125] 8) Received any live vaccine or live attenuated vaccine within 6 weeks before randomization;

[0126] 9) History of malignant tumors in the past 5 years, including solid tumors, hematological malignancies, and carcinoma in situ (except for completely resected and cured basal cell carcinoma and squamous cell carcinoma of the skin or carcinoma in situ of the cervix);

[0127] 10) Subjects with a history of severe drug allergy, or allergy or intolerance to mitoxantrone and liposomes (shock, anaphylaxis);

[0128] 11) Patients with other chronic active immune system diseases other than NMOSD, or whose condition is stable but requires high-dose (dose determined by the investigator) glucocorticoid maintenance therapy: such as rheumatoid arthritis, scleroderma, Sjogren's syndrome, ulcerative colitis, genetic immunodeficiency, or drug-induced immunodeficiency; patients with only positive autoantibodies but no clinical manifestations can be included in the trial;

[0129] 12) The researcher determines that the subject has other diseases that make him or her unsuitable for participating in this study.

[0130] 2. Exclusion criteria related to previous or concurrent treatment:

[0131] 13) Those who have received any investigational medication within 3 months before randomization, or have participated in clinical research on medical devices and who are judged by the investigator to have an impact on the results of this trial;

[0132] 14) Patients with any of the following history of NMOSD treatment:

[0133] a. Previous treatment with mitoxantrone or anthracyclines, whole body irradiation, or bone marrow transplantation at any time;

[0134] b. Receipt of rituximab or any experimental B cell depleting agent within 6 months before randomization, unless the central laboratory determines that the subject's B cell count is above LLN;

[0135] c. Use of any of the following within 3 months before randomization: satelizumab, tocilizumab, inelizumab, eculizumab, alemtuzumab, cyclosporine, azathioprine, methotrexate, cyclophosphamide, tacrolimus, mycophenolate mofetil. Any other treatment for preventing relapse of multiple sclerosis (MS) (such as interferon, natalizumab, glatiramer acetate, fingolimod, teriflunomide, or dimethyl fumarate);

[0136] d. Receiving intravenous immunoglobulin (IVIG) treatment or plasma exchange (PE) treatment within 1 month before randomization;

[0137] e. Patients who are currently receiving oral corticosteroids at a dose higher than 30 mg / day during prednisolone conversion;

[0138] f. Use of anti-CD4 or cladribine within 2 years before randomization.

[0139] 3. Exclusion criteria related to laboratory tests:

[0140] 15) The presence of the following clinically significant diseases:

[0141] a. Echocardiographic ejection fraction (EF) less than 50% or below the lower limit of the laboratory test value of the research center; patients with a history of chronic congestive heart failure and NYHA cardiac function class III-IV;

[0142] b. Any of the following events occurred within 3 months before randomization: myocardial infarction, acute coronary syndrome, viral myocarditis, pulmonary embolism, stroke; coronary artery revascularization within 6 months;

[0143] c. Screening electrocardiogram examination indicates a QTc interval greater than 480ms (based on the Fridericia correction formula, where QTc = QT / RR^0.33), or a history of severe QTc interval prolongation.

[0144] 16) Those with the following abnormal laboratory test values ​​during the screening period:

[0145] a. Aspartate aminotransferase (AST) > 3 × upper limit of normal (ULN), alanine aminotransferase (ALT) > 3 × ULN, total bilirubin > 1.5 × ULN (unless caused by Gilbert syndrome);

[0146] b. Platelet count <50,000 / μL (or <50×10 9 / L), hemoglobin <9g / dL (or <90g / L), white blood cells <2.0×10 3 / μL, absolute neutrophil count <1.0×10 3 / μL;

[0147] c. Creatinine clearance (CLcr) <60 mL / min (calculated using the Cockcroft-Gault formula: [140 - age (years)] × [weight (kg)] × (0.85, if female) / [72 × serum creatinine (mg / dL)]) or receiving dialysis during the screening period.

[0148] (3) Exit / Termination Criteria

[0149] Subjects may decide to discontinue study treatment early at any time during the study without affecting their continued access to appropriate clinical care. Subjects should return all study medication. A subject's decision to withdraw treatment must be documented in the source documentation. Following the withdrawal of informed consent, patients will not be followed up for any reason. Patients who withdraw from the study will not be replaced.

[0150] If any of the following situations occurs, the investigator should arrange for the subject to terminate the study treatment early. After termination of treatment, the subject should be followed up for 8 weeks and the end-of-study (EOS) visit assessment should be completed as much as possible.

[0151] 1) The subject requests to withdraw from the trial or the subject is lost to follow-up;

[0152] 2) The subject is pregnant;

[0153] 3) The subject experiences a serious adverse event or an adverse event with a high risk of potential harm, and the investigator or sponsor determines that the subject should withdraw from treatment for safety reasons;

[0154] 4) Subjects have any of the following liver function abnormalities:

[0155] a. ALT or AST > 8 × ULN

[0156] b. ALT or AST > 5 × ULN for more than 2 weeks (without elevated bilirubin and / or fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and / or eosinophilia (>5%));

[0157] c. ALT or AST > 3 × ULN and total bilirubin > 2 × ULN or international normalized ratio [INR] > 1.5, with no cholestatic factors found;

[0158] d. ALT or AST > 3 × ULN accompanied by fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and / or eosinophilia (> 5%);

[0159] 5) During the study, if a subject is diagnosed with NMOSD recurrence according to the protocol-defined criteria, or develops cardiac toxicity (LVEF is below the lower limit or clinically significantly decreased), the investigator may decide to terminate the study treatment and withdraw from the study.

[0160] 6) The subject delays medication for more than 14 days, or there is a major protocol violation, and the researcher determines that the subject should withdraw from treatment for compliance or benefit considerations;

[0161] 7) Other researchers believe that the patient is not suitable to continue participating in this trial.

[0162] 3. Efficacy evaluation indicators

[0163] Primary endpoint: time to first relapse;

[0164] Time to first relapse is the time from randomization to the first onset of NMOSD. NMOSD relapse is defined as the presence of new symptoms or worsening of existing symptoms related to NMOSD, meeting at least one of the NMOSD flare criteria defined in the protocol.

[0165] Secondary endpoints:

[0166] ●Annualized relapse rate (ARR): The ARR at week 48 was calculated as the ratio of the sum of confirmed relapses for all participants divided by the sum of treatment time for all participants (in years).

[0167] Change from baseline in the Expanded Disability Status Scale (EDSS) score

[0168] Change from baseline in modified Rankin Scale (mRS) score

[0169] Change from baseline in T25FW score

[0170] Change from baseline in EQ-5D score

[0171] Change from baseline in low-contrast visual acuity (LCVA)

[0172] Change from baseline in pain NRS scores at five sites

[0173] Changes from baseline in serum glial fibrillary acid protein (sGFAP) and serum neurofilament light chain (NfL) levels

[0174] Serum AQP4 titer

[0175] IV. Research Results

[0176] For patients with neuromyelitis optica spectrum disorder, treatment with mitoxantrone hydrochloride liposome injection is safe and tolerable, with minimal toxic and side effects, which improves the effectiveness of treatment and delays the recurrence of neuromyelitis optica spectrum disorder.

[0177] The above is an exemplary description of the implementation methods of the technical solution of this application. It should be understood that the scope of protection of this application should not be unduly limited to these specific implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of this application should be included in the scope of protection of the claims of this application.

Claims

1. Use of mitoxantrone hydrochloride liposomes in the preparation of a medicament for treating neuromyelitis optica spectrum disorder; preferably, the neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder, and more preferably, the highly relapsing neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder that has failed to be treated with other drugs.

2. A method for treating a neuromyelitis optica spectrum disorder in a subject, comprising administering to the subject a therapeutically effective amount of mitoxantrone liposome hydrochloride or a pharmaceutical composition comprising a therapeutically effective amount of mitoxantrone liposome hydrochloride; preferably, the neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder, more preferably, the highly relapsing neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder that has failed other drug treatments.

3. Mitoxantrone hydrochloride liposomes or a pharmaceutical composition comprising mitoxantrone hydrochloride liposomes, for treating neuromyelitis optica spectrum disorder; preferably, the neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder, more preferably, the highly relapsing neuromyelitis optica spectrum disorder is a highly relapsing neuromyelitis optica spectrum disorder that has failed other drug treatments.

4. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the mitoxantrone hydrochloride liposomes are used as the sole active ingredient for treating neuromyelitis optica spectrum disorders, or the mitoxantrone hydrochloride liposomes are used alone for treating neuromyelitis optica spectrum disorders.

5. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the drug or pharmaceutical composition is in the form of an injectable, preferably a liquid injection, a powder for injection, or a tablet for injection; more preferably, the drug or pharmaceutical composition is a liquid injection.

6. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the active ingredient content of the drug or pharmaceutical composition is 0.5-5 mg / ml, preferably 1-2 mg / ml, more preferably 1 mg / ml, calculated as mitoxantrone.

7. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the particle size of the mitoxantrone hydrochloride liposomes is about 30-80 nm, for example, about 35-75 nm, preferably about 40-70 nm, more preferably about 40-60 nm, for example, about 60 nm.

8. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the active ingredient mitoxantrone in the mitoxantrone hydrochloride liposomes forms an insoluble precipitate with a multivalent counterion in the liposomes, and the multivalent counterion is, for example, sulfate, citrate or phosphate.

9. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes according to claim 3 or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes, wherein the phospholipid bilayer in the mitoxantrone hydrochloride liposomes contains a phospholipid having a phase transition temperature (Tm) higher than body temperature, so that the phase transition temperature of the liposomes is higher than body temperature, for example, the phospholipid is selected from hydrogenated soy lecithin, phosphatidylcholine, hydrogenated egg yolk lecithin, disalmitate lecithin, distearate lecithin or any combination thereof.

10. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposomes or the pharmaceutical composition comprising the mitoxantrone hydrochloride liposomes according to claim 3, wherein the mass ratio of hydrogenated soy lecithin, cholesterol and polyethylene glycol 2000-modified distearoylphosphatidylethanolamine is 3:1:1; and / or Preferably, the weight ratio of hydrogenated soy lecithin, cholesterol, polyethylene glycol 2000-modified distearoylphosphatidylethanolamine and mitoxantrone is 9.58:3.19:3.19:1; and / or Preferably, wherein the particle size is about 60 nm; and / or Preferably, the counter ion is a sulfate ion.

11. The method of claim 2, wherein the therapeutically effective amount is 4-30 mg / m 2 , preferably 8-20 mg / m 2 or 12-30 mg / m 2 , for example 8 mg / m 2 or 12 mg / m 2 and / or The administration method is intravenous administration; and / or The dosing cycle is once every 12 weeks.

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