Use of Mitoxantrone Hydrochloride Liposomes

Abstract

The present invention provides the use of mitoxantrone hydrochloride liposomes for treating neuromyelitis optica spectrum disorder, and also provides a method for treating an individual's neuromyelitis optica spectrum disorder, the method comprising administering to the individual a therapeutically effective amount of mitoxantrone hydrochloride liposomes or a pharmaceutical composition comprising mitoxantrone hydrochloride liposomes. The results of animal tests and clinical trials have shown that mitoxantrone hydrochloride liposomes are safe and effective against neuromyelitis optica spectrum disorder.

Description

Cross-reference to Related Applications

[0001] 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, the entire contents of which are incorporated herein by reference and used for all purposes.

Technical Field

[0002] The present invention relates to the field of medicine, and specifically to the use of mitoxantrone hydrochloride liposomes, for example, the use of mitoxantrone hydrochloride liposomes in the treatment of neuromyelitis spectrum disorders.

Background Art

[0003] Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune-mediated central nervous system inflammatory demyelinating disease mainly characterized by simultaneous or consecutive involvement of the optic nerve and spinal cord. Its etiology (pathogenesis) is mainly associated with antibodies against water channel protein 4 (aquaporin-4, AQP4), mediated by autoantibodies, dominated by humoral immunity, and involves various immune cells and factors jointly. NMOSD can be seen in all age groups, but is more common in young and middle-aged adults, with an average onset age of about 40 years, and most patients are serum AQP4-IgG positive. Among AQP4-IgG positive patients, the ratio of women to men is as high as (4.7 - 11):1. Clinically, severe optic neuritis and longitudinally extensive transverse myelitis are the main clinical features, and NMO-characteristic brain lesions (hypothalamus, corpus callosum, periventricular or brainstem) and systemic autoimmune diseases are often accompanied. NMOSD is a disease with high recurrence (high recurrence rate) and high disability (high disability-causing characteristics). More than 90% of patients have a multiphase disease course, among which 40% - 60% relapse within 1 year, and about 90% relapse within 3 years. Among patients with the natural history, about 50% have severe visual or motor dysfunction remaining within 5 - 10 years (Chinese Society of Immunology, Neuroimmunology Branch, Huang Dehui, Wu Weiping, et al., "Diagnosis and Treatment Guidelines for Neuromyelitis Optica Spectrum Disorder in China (2021 Edition)", Chinese Journal of Neuroimmunology and Neurology, Vol. 28, No. 6, November 2021).

[0004] Evidence from etiology, imaging, and clinical manifestations shows that NMOSD is an independent disease entity different from multiple sclerosis (MS), and its treatment plan is also different from that of MS. It has been shown that certain MS treatment drugs may exacerbate the condition of NMOSD (Ji Wei, Wang Limei, Tan Song, "Laboratory Index Differential Diagnosis and Treatment Progress of Neuromyelitis Optica and Multiple Sclerosis", Chinese Journal of Practical Nervous Diseases, Vol. 19, No. 10, June 2016).

[0005] Currently, the NMOSD treatments commonly used clinically are divided into acute exacerbation treatment, sequential treatment (relapse prevention treatment), symptomatic treatment, and rehabilitation treatment. The purpose of acute-phase treatment is to reduce irreversible nerve damage, promote its functional recovery, and reduce the fatality rate. High-dose hormone shock therapy, plasma exchange or immunoadsorption, and intravenous injection of human immunoglobulin are commonly used. The purpose of sequential treatment is mainly to reduce the relapse rate and alleviate the severity of the patient's disability. Clinically commonly used drugs include monoclonal antibodies and immunosuppressants. The first-choice drugs include satralizumab, ine bilizumab, rituximab, azathioprine, and mycophenolate mofetil. The second-choice drugs include tacrolimus, cyclosporine A, cyclophosphamide, methotrexate, and mitoxantrone (Chinese Society of Neuroimmunology, Huang Dehui, Wu Weiping, etc., "Diagnosis and Treatment Guidelines for Neuromyelitis Optica Spectrum Disorders in China (2021 Edition)", "Chinese Journal of Neuroimmunology and Neurology", Vol. 28, No. 6, November 2021).

[0006] In view of the characteristics of NMOSD showing high recurrence and high disability, a large number of NMOSD patients still cannot receive effective treatment, cannot effectively control the disease condition, leave severe physical dysfunction, and bring a heavy burden of the disease to the family and society. Therefore, the early diagnosis of this disease and the development of effective treatments for NMOSD and drugs for preventing NMOSD recurrence are urgent tasks.

[0007] Mitoxantrone hydrochloride is an anthraquinone chemotherapy drug. The FDA has approved that the indications of the general injection of mitoxantrone hydrochloride are multiple sclerosis, prostate cancer, and acute myeloid leukemia. In China, it has been approved for application to patients with lymphoma, leukemia, breast cancer, etc. The recommended dose is 12 - 14 mg / m 2 for adults as a single agent, administered once every 3 - 4 weeks, or 4 - 8 mg / m 2 administered once a day for 3 - 5 consecutive days, with an interval of 2 - 3 weeks. The combined dose is 5 - 10 mg / m 2It is. Mitoxantrone, as an anthracycline agent, its main side effects (adverse effects) are mainly cardiotoxicity, myelosuppression, and gastrointestinal reactions, etc.

[0008] Among them, myelosuppression and gastrointestinal reactions can be resolved by administering appropriate drugs, but cardiotoxicity often leads to serious consequences and is the most serious side effect of anthracycline agents. According to both clinical research and practical observations, most of the cardiotoxicity caused by anthracycline agents is progressive and irreversible, and it has been shown that heart damage is particularly likely to be caused during the first use of anthracycline agents. Chronic dose cumulative limiting toxicity - cardiotoxicity is a common concern for clinicians. Therefore, although the indication of breast cancer has been approved, mitoxantrone is not recommended as a treatment method for breast cancer by authoritative clinical dosing guidelines.

[0009] Drug sensitivity varies depending on the differences in tumors. Existing research has shown that when the same drug treats different indications, its dosing schedule may be different. For example, Doxil (Doxil, doxorubicin hydrochloride liposome) has three indications approved by the FDA: (1) ovarian cancer (recommended dose 50 mg / m 2 , administered intravenously once every 4 weeks), (2) Kaposi's sarcoma (recommended dose 20 mg / m 2 , administered intravenously once every 3 weeks), (3) multiple myeloma (recommended dose 30 mg / m 2 , administered intravenously on the 4th day after bortezomib administration). As another example, Abraxane (Abraxane, paclitaxel for injection [albumin-bound]) also has: (1) metastatic breast cancer (recommended dose 260 mg / m 2 , intravenous infusion for 30 minutes, administered once every 3 weeks), (2) non-small cell lung cancer (recommended dose 100 mg / m 2, intravenous infusion for 30 minutes, administered on the 1st, 8th, and 15th days with a 21-day treatment course as one cycle. Here, after the administration of paclitaxel for injection (albumin-bound) on the 1st day, carboplatin is immediately administered, and it is administered once every 21 days), (3) pancreatic cancer (recommended dose 125 mg / m 2 , intravenous infusion for 30 - 40 minutes, with a 28-day cycle, administered once each on the 1st, 8th, and 15th days. After the administration of paclitaxel for injection (albumin-bound) each time, gemcitabine is immediately administered). Three indications have been approved by the FDA. Further, as another example, the starting doses of AmBisome (Ambisome, liposomal amphotericin B for injection) are (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, 21)), (4) visceral leishmaniasis patients with immunodeficiency (4 mg / kg / day (days 1 - 5), 4 mg / kg / day (days 10, 17, 24, 31, 38)). Dosage and administration data are individualized according to the actual situation of the patient in order to achieve the maximum drug efficacy and the minimum toxicity or side effects. From this, it is obvious that when treating different indications, even the same drug has different safe and effective dosages. In order to achieve the maximum drug efficacy and the minimum toxicity or side effects and obtain a safe and effective therapeutic effect, the setting of dosage and administration data needs to be individualized based on the specific disease type and the actual situation of the patient. For mitoxantrone liposome, a specific dosage form different from ordinary injections, the absorption, distribution, and metabolism after entering the body are very complex, and it is difficult to simply derive one indication from another in the treatment of different indications, especially in the treatment of different tumors.

Summary of the Invention

[0010] The present invention provides the use of mitoxantrone hydrochloride liposomes in the treatment of neuromyelitis optica spectrum disorder. The inventors of the present invention surprisingly discovered during the research that mitoxantrone hydrochloride liposomes have excellent therapeutic effects against neuromyelitis optica spectrum disorder and can be used in the manufacture of related drugs.

[0011] According to a first aspect, the present invention provides the use of mitoxantrone hydrochloride liposomes in the manufacture of a medicament for treating neuromyelitis optica spectrum disorder.

[0012] According to a second aspect, the present invention provides a method for treating an individual's neuromyelitis optica spectrum disorder, the method comprising administering to the individual a therapeutically effective amount of mitoxantrone hydrochloride liposomes, or a pharmaceutical composition comprising a therapeutically effective amount of mitoxantrone hydrochloride liposomes.

[0013] According to a third aspect, the present invention provides mitoxantrone hydrochloride liposomes or a pharmaceutical composition comprising mitoxantrone hydrochloride liposomes for treating an individual's neuromyelitis optica spectrum disorder.

[0014] In some embodiments, the neuromyelitis optica spectrum disorder described in the above first to third aspects is a highly recurrent neuromyelitis optica spectrum disorder. In some embodiments, the highly recurrent neuromyelitis optica spectrum disorder is a highly recurrent neuromyelitis optica spectrum disorder that has failed other drug treatments.

[0015] In some embodiments, for the highly recurrent neuromyelitis optica spectrum disorder that has failed other drug treatments, the other drugs are the first-choice drug, second-choice drug or a drug of second-choice or higher for the treatment of neuromyelitis optica spectrum disorder, in particular, selected from the first-choice drug, second-choice drug or a drug of second-choice or higher for the treatment of neuromyelitis optica spectrum disorder approved by the drug regulatory authorities in China or outside China (such as the United States, the European Union, Japan, South Korea, etc.), including but not limited to FDA-approved glucocorticoids, monoclonal antibody drugs and immunosuppressants.

[0016] In some embodiments, mitoxantrone hydrochloride liposomes are used for the treatment of neuromyelitis optica spectrum disorder as the sole active ingredient, or are used for the manufacture of a medicament or pharmaceutical composition for the treatment of neuromyelitis optica spectrum disorder as the sole active ingredient.

[0017] In some embodiments, the pharmaceutical composition or medicament containing the mitoxantrone hydrochloride liposomes described in the above first to third aspects is in an injectable form including liquid injections, powder for injection, tablets for injection, etc. In a preferred embodiment, the pharmaceutical composition or medicament containing mitoxantrone hydrochloride liposomes is a liquid injection.

[0018] In some embodiments, when the pharmaceutical composition or medicament containing mitoxantrone hydrochloride liposomes is a liquid injection, the content of mitoxantrone in the medicament or pharmaceutical composition is 0.5 to 5 mg / mL based on mitoxantrone. In a preferred embodiment, the content of mitoxantrone in the medicament is 1 to 2 mg / mL. In a more preferred embodiment, the content of mitoxantrone in the medicament or pharmaceutical composition is 1 mg / mL.

[0019] In some embodiments, the therapeutically effective amount described in the above second aspect is 4 to 30 mg / m based on mitoxantrone 2 , for example 8 to 20 mg / m 2 or 12 to 30 mg / m 2 , and is also a dose within the range between any two of the above doses, for example 8 mg / m 2 , 12 mg / m 2 and so on.

[0020] In some embodiments, the form of administering the mitoxantrone hydrochloride liposomes, the pharmaceutical composition or medicament containing mitoxantrone hydrochloride liposomes according to the present invention to an individual in need thereof is intravenous administration.

[0021] In some embodiments, the administration cycle of the mitoxantrone hydrochloride liposomes, pharmaceutical composition or drug comprising the mitoxantrone hydrochloride liposomes according to the present invention is to administer once every 12 weeks.

[0022] In some embodiments, the liposomes are used alone to treat an individual's neuromyelitis spectrum disorder. In some embodiments, the therapeutically effective amount of the liposomes (based on mitoxantrone) 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 or 12 mg / m 2 . In some embodiments, the administration form of the liposomes is intravenous administration.

[0023] In some embodiments, the administration cycle of the liposomes is to administer once every 12 weeks. The doses described in the context of this specification are calculated based on mitoxantrone unless otherwise specified.

[0024] The mitoxantrone hydrochloride liposomes described in the context of this specification may be manufactured by known methods in the art, for example, mitoxantrone hydrochloride liposomes manufactured using any method disclosed in the art such as the method described in WO2008 / 080367A1.

[0025] In some embodiments, the drug, mitoxantrone hydrochloride liposomes or pharmaceutical composition comprising mitoxantrone hydrochloride liposomes described in the above first to third aspects (i) has the property that 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 (ii) The property that mitoxantrone hydrochloride forms an insoluble precipitate with polyvalent counterions (e.g., sulfate, citrate, or phosphate) within liposomes, (iii) The property that the phospholipid bilayer in mitoxantrone hydrochloride liposomes contains phospholipids having a phase transition temperature (Tm) higher than body temperature, whereby the phase transition temperature of the liposomes is higher than body temperature, and the phospholipids are selected from, for example, the hydrogenated soy lecithin, phosphatidylcholine, hydrogenated egg yolk lecithin, bispalmitate lecithin (also called dipalmitoyl lecithin), distearate lecithin, or any combination thereof, (iv) The property that the phospholipid bilayer in mitoxantrone hydrochloride liposomes contains hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine (DSPE-PEG2000), (iiv) The property that the phospholipid bilayer in mitoxantrone hydrochloride liposomes contains hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine in a mass ratio of about 3:1:1, mitoxantrone hydrochloride forms an insoluble precipitate with polyvalent acid ions within the liposomes, and the particle size of the mitoxantrone hydrochloride liposomes in the drug is about 60 nm, and (iiiv) The property that the mitoxantrone hydrochloride liposomes are mitoxantrone hydrochloride liposomes with the Chinese drug approval number H20220001 having one or more properties selected from the group consisting of.

[0026] 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.

[0027] In some embodiments, the mitoxantrone hydrochloride liposomes described herein comprise mitoxantrone, which is the active ingredient, and a phospholipid bilayer. In some embodiments, mitoxantrone, which is the active ingredient, can form a poorly soluble precipitate with polyvalent counterions within the liposomes. In some embodiments, the counterions are sulfate, citrate, or phosphate.

[0028] In some embodiments, the phospholipid bilayer comprises phospholipids with a phase transition temperature (Tm) higher than body temperature such that the phase transition temperature of the liposomes is higher than body temperature. In some embodiments, the phospholipids with a Tm higher than body temperature are phosphatidylcholine, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, dipalmitoyl lecithin, or distearoyl lecithin, or any combination thereof.

[0029] In some embodiments, the phospholipid bilayer comprises hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine. In some embodiments, the mass ratio of hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine in the phospholipid bilayer is 3:1:1. In some embodiments, the particle size of the mitoxantrone hydrochloride liposomes described herein is about 60 nm, and the counterion in the liposomes is sulfate ion.

[0030] In some embodiments, the phospholipid bilayer of the mitoxantrone hydrochloride liposomes described herein contains hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine with a mass ratio of 3:1:1, the particle size of the liposomes is about 40-60 nm, and the counter ion in the liposomes is sulfate ion. In some embodiments, the weight ratio of HSPC, Chol, DSPE-PEG2000, and mitoxantrone in the mitoxantrone hydrochloride liposomes described herein (HSPC:Chol:DSPE-PEG2000:mitoxantrone) is 9.58:3.19:3.19:1.

[0031] The method for manufacturing mitoxantrone liposomes described in the context of this specification is as follows. HSPC (hydrogenated soy lecithin), Chol (cholesterol), and DSPE-PEG2000 (polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine) are weighed at a mass ratio of (3:1:1), 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 with shaking at 60 - 65 °C for 1 hour to obtain heterogeneous multivesicular liposomes. Then, the particle size of the liposomes is reduced using a microfluidic device. After diluting the obtained sample 200-fold with a 0.9% NaCl solution and detecting it with NanoZS, the average particle size of the particles is about 60 nm, and the main peak is concentrated in the range of 40 - 60 nm. Then, using an ultrafiltration device, ammonium sulfate in the external phase of the blank liposomes is removed, and the external phase is replaced with 290 mM sucrose and 10 mM glycine to form a transmembrane ammonium sulfate gradient. At a lipid-to-drug ratio of 16:1, a mitoxantrone hydrochloride solution (10 mg / mL) is added to the blank liposomes, and the drug is loaded at 60 - 65 °C. After incubating for about 1 hour, it is demonstrated using gel exclusion chromatography that the encapsulation efficiency is about 100%. Among them, the weight ratio of HSPC:Chol:DSPE-PEG2000:mitoxantrone is 9.58:3.19:3.19:1, and the osmotic pressure of the sucrose·glycine solution is close to the physiological value.

[0032] It should be understood that the multiple technical details and parameters in the above exemplary manufacturing methods can be adjusted and determined within a reasonable range by those skilled in the art. For example, amino acid species that can be substituted for 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. As another example, the mass ratios of HSPC, Chol, and DSPE-PEG2000 can be appropriately adjusted. Also, for example, regarding the lipid-to-drug ratio parameter in the manufacture of a specific liposomal drug formulation, those skilled in the art can design, test, and ultimately obtain an appropriate lipid-to-drug ratio to increase the drug loading amount as much as possible while reducing the drug leakage amount. In the mitoxantrone hydrochloride liposomal formulation according to the present invention, the available lipid-to-drug ratio has a wide range. For example, it may be as low as 2:1, or as high as 30:1, 40:1, or 50:1. A more appropriate lipid-to-drug ratio may be about (15 - 20):1, for example, about 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. Therefore, some advantageous properties of the above mitoxantrone hydrochloride liposomal formulation are more important, and the methodologies for achieving these properties are diverse.

[0033] As used herein, the term "individual" refers to mammals such as humans, but may also be other mammals such as domestic animals and laboratory animals. As used herein, the term "treatment" means administering the mitoxantrone hydrochloride liposome or formulation according to the present invention to improve or eliminate a disease or one or more symptoms associated with the disease, and includes (i) inhibiting the disease or disease state, i.e., inhibiting its occurrence and progression, and (ii) alleviating the disease or disease state, i.e., reducing the disease or disease state.

[0034] The term "therapeutically effective amount" means the dosage of mitoxantrone hydrochloride liposomes according to the present invention for (i) treating a specific disease, condition or disorder, or (ii) reducing, ameliorating or eliminating one or more symptoms of a specific disease, condition or disorder. The amount of mitoxantrone hydrochloride liposomes according to the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the dosage form, and the age of the mammal being treated, but can be routinely determined by those skilled in the art based on their own knowledge and the disclosure of this specification.

[0035] In some embodiments, the agent, mitoxantrone hydrochloride liposomes or a pharmaceutical composition containing mitoxantrone hydrochloride liposomes described in the above first to third aspects, for example, mitoxantrone hydrochloride liposome injection solution, has one or more beneficial effects such as safety, tolerability, low toxic side effects, improved therapeutic efficacy, and delayed recurrence of neuromyelitis spectrum disorder in the treatment of neuromyelitis spectrum disorder.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0037] Hereinafter, embodiments of the present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. All technologies realized based on the above content of the present invention are included within the scope to be protected by the present invention. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be produced by known methods.

Example

[0038] Improvement effect of mitoxantrone liposomes on the NMOSD model induced by the combined use of AQP4 antibody / complement from clinical patients According to the literature report, the NMOSD model can be induced by directly injecting AQP4 antibody and human complement from clinically diagnosed patients into the mouse cerebral hemisphere or ventricle (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, in this example, the above-described modeling method was selected to examine the therapeutic effect of mitoxantrone liposomes on NMOSD.

[0039] Experimental animals: Male CD-1 mice, 8 weeks old, purchased from Jinan Pengyue Experimental Animal Breeding and Rearing Co., Ltd. Test drugs: Mitoxantrone hydrochloride liposome injection 1.0 mg / mL (test article).

[0040] Test method: First, AQP4-IgG antibodies were extracted, purified, and verified from blood samples of clinical patients, and normal human complement serum (human complement serum) was also extracted, purified, and verified. Then, 27 8-week-old male CD-1 mice were selected. After one week of acclimation, they were randomly divided into three groups according to body weight: a blank group (blank control group), a model group, and a test article group (mitoxantrone liposomes at 15 mg / kg), with 9 animals in each group.

[0041] Into the lateral ventricles of each mouse in the model group and the test article group, 2 μL of APQ4 antibody (concentration 18 mg / mL) and 1 μL of human complement serum were injected at a rate of 0.3 μL / min on the 1st, 3rd, and 5th days to induce an NMOSD model. 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. The animals in the test article group were intravenously injected with 15 mg / kg of mitoxantrone liposome injection once on the first day of modeling.

[0042] During the test process, the health status of the animals was monitored, and the body weights of the animals were recorded. After the completion of modeling, on the 7th day (Day7), 11th day (Day11), and 21st day (Day21), 3 animals were taken from each group and euthanized with carbon dioxide, and then the brain tissues were collected. The pathological changes in the mouse brain tissues were evaluated by AQP4, GFAP, and LFB staining. Pathological collection site: The stained area was centered on the injection site, and a total of 30 sections were made to further explore the lesion range.

[0043] The procedures and antibody information required for the immunohistochemistry process are as follows. 1. Perfusion - fixation - sugar sedimentation (sucrose gradient centrifugation) - sectioning 1) The mice were anesthetized by inhalation with isoflurane. 2) The abdomen and chest were incised with straight scissors to expose the heart. First, it was perfused with physiological saline to wash away the blood, and then perfused with paraformaldehyde for initial fixation. 3) The head was cut off, the skull was carefully removed with forceps, the brain tissue was taken out, and placed in a paraformaldehyde solution for fixation for 24 hours. 4) The next day, the brain tissue was taken out and placed in a 20% sucrose solution for 24 hours. 5) On the third day, the brain tissue was taken out and placed in a 30% sucrose solution for 24 hours. 6) On the fourth day, the brain tissue was taken out and placed in a 35% sucrose solution for 24 hours. (The time and concentration were appropriately increased according to the sugar sedimentation situation of the brain tissue.) 7) The brain tissue was taken out, embedded with OCT embedding agent, and 16-μm brain sections were prepared using a cryostat microtome.

[0044] 2. Immunofluorescence staining (AQP4, GFAP) 1) Slide rewarming for 30 minutes. 2) Blocking: 10% serum + 0.3% Triton X-100 (about 50 μL per slide), at room temperature for 1 hour. 3) Rotate the slide, add the primary antibody (diluted with PBS), and leave it overnight at 4°C. 4) Rewarming for 30 minutes. 5) Wash with the primary antibody (PBS), 5 minutes × 3 times. 6) Add the secondary antibody (diluted with PBS) in the dark and leave it at room temperature for 2 hours. 7) Wash with the secondary antibody (PBS), 5 minutes × 3 times. 8) Add DAPI and leave it at room temperature for 10 minutes. 9) Wash with DAPI, 5 minutes × 3 times. 10) Seal the slide with 70% glycerol, avoiding air bubbles.

[0045] 3. Antibody information: 3.1. Primary antibody 1) AQP4: Purchased from Abclonal, product number: A2887, lot number: 0073660402. 2) GFAP: Purchased from Hua'an Biotech, product number: EM140707, lot number: HO0604. 3.2. Secondary antibody 1) Goat Pab to Rabbit 594: Purchased from abcam, product number: ab150080, lot number: GR3439696-1. 2) Goat Pab to mouse 488: Purchased from abcam, product number: ab150077, lot number: GR3403178-2. 4. Luxol fast blue stain (LFB) staining Purchased from Solarbio, product number: G3242, lot number: 20220422. Operated according to the kit instructions.

[0046] Test results: In this study, CD-1 mice were selected, and AQP4 antibody / complement from clinically diagnosed patients was injected into the lateral ventricle by stereotactic fixation to establish a neuromyelitis optica disease model, and then related histological examinations were carried out to evaluate the effect of the test compound.

[0047] The results of animal body weight analysis are shown in Figure 1 and Table 1. Compared with the model group, in the test article group, weight loss was observed from day 7 to day 21 after modeling administration, the animal condition was good, and there was no animal death due to drug toxicity.

[0048]

Table 1

[0049] Representative figures of AQP4 immunofluorescence staining and statistical results of the number of positive cells are shown in Figures 2 - 3 and Table 2. On the 7th day after model establishment, compared with the blank group and the test article group, the number of AQP4 positive cells in the striatum of the animals in the model group was significantly decreased, and this decrease became more and more significant with the passage of time, indicating that the establishment of the NMOSD disease model by injecting AQP4 antibody / complement from clinically diagnosed patients into the lateral ventricle by stereotactic fixation was successful. 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, and the difference was statistically significant on day 21 (P < 0.001), suggesting that mitoxantrone hydrochloride liposome 15 mg / kg can significantly inhibit AQP4 deletion in NMOSD mechanistically and effectively improve the symptoms of mouse NMOSD.

[0050]

Table 2

[0051] From the test results, mitoxantrone hydrochloride liposome injection (15 mg / kg) significantly inhibited the deletion of AQP4 in mice, improved histopathological changes such as astrocyte lesions and myelin loss, indicating that mitoxantrone hydrochloride liposome injection can effectively improve the symptoms of NMOSD in mice and inhibit its occurrence and development.

Example

[0052] Phase II Clinical Study on the Efficacy and Safety of Mitoxantrone Hydrochloride Liposome Injection in the Treatment of Neuromyelitis Optica Spectrum Disorder (NMOSD) This example is a Phase II study with randomization, double-blind, and placebo parallel control. The selected subjects will receive treatment with mitoxantrone hydrochloride liposome injection. The purpose is to evaluate the safety and efficacy of mitoxantrone hydrochloride liposome injection in NMOSD subjects, explore the appropriate dosage of mitoxantrone hydrochloride liposome injection for the treatment of neuromyelitis optica spectrum disorder (NMOSD), and provide a basis for later clinical development.

[0053] I. Test Design 1. Test Procedure The study period is approximately 60 weeks in total, including a screening period (up to 4 weeks), a treatment period (48 weeks), and a safety follow-up period (8 weeks). The researcher or designee will contact all subjects by phone every two weeks after the start of the treatment period until the subject completes the test (except when that time coincides with the week of the study visit). If the researcher suspects that the subject is about to develop or has developed NMOSD, an evaluation visit will be scheduled within 72 hours.

[0054] Subjects diagnosed with NMOSD will be screened within 4 weeks (28 days), and eligibility for the study will be determined based on inclusion and exclusion criteria. The AQP4-IgG serum status will be determined by the central laboratory. Then, all subjects meeting the eligibility criteria will be randomly assigned to the study centers. Randomization (Day 1): Subjects who pass the screening will be randomly assigned to the test group A (mitoxantrone hydrochloride liposome injection 8 mg / m 2 ), test group B (mitoxantrone hydrochloride liposome injection 12 mg / m 2 ) and the placebo group at a ratio of 1:1:1. Subjects who succeed in randomization will receive treatment with mitoxantrone hydrochloride liposome or placebo (administered once every 12 weeks). 48 weeks after the last eligible patient is randomly grouped or when recurrence occurs, the treatment period ends and the safety follow-up period begins. The safety follow-up investigation is initiated when the subject terminates the study early, after recurrence, or at the end of the treatment period of the study. It is necessary to conduct an 8-week safety follow-up investigation (such as scale evaluation, laboratory tests, and electrocardiogram tests), monitor the adverse events / serious adverse events of the subjects, and collect information on concomitant medications or treatments related to NMO / NMOSD. During that period, at the discretion of the researcher, the subject can receive standard treatment for NMO / NMOSD.

[0055] 2. Study Duration This study includes a screening period (4 weeks), a treatment period (up to 48 weeks), and a safety follow-up period (8 weeks). The duration of each patient (excluding recurrence patients) is expected to be approximately 60 weeks. A total of 45 subjects are expected to be enrolled in this study, and the total duration of this study is expected to be 24 months.

[0056] II. Subjects of the Test: Only subjects who meet all the following inclusion criteria and have no exclusion criteria can be enrolled in this clinical study.

[0057] (I) Inclusion Criteria: Subjects must meet all of the following criteria: 1) The age at the time of signing the informed consent is 18 to 60 years old (including the upper and lower limits), and both men and women are eligible. 2) Meeting the international consensus diagnostic criteria for neuromyelitis optica spectrum disorder (NMOSD) in 2015, and being a patient with positive or negative AQP4-IgG (all test results within 24 weeks before signing the informed consent are acceptable). 3) Having experienced at least two attacks within two years before screening and / or having clinical evidence of at least one recurrence (including the first attack) recorded within 12 months before screening. 4) The EDSS at screening and baseline retest is ≤ 7.5 points. 5) The subject must voluntarily sign the informed consent and voluntarily complete the test in accordance with the protocol requirements. It is necessary to meet these requirements.

[0058] (II) Exclusion criteria: 1. Exclusion items for general safety: 1) Being a pregnant or lactating woman. For patients with a possibility of pregnancy, the serum pregnancy test result at screening is positive, or there is no intention to use reliable contraceptive methods (physical barriers [patient or partner] and spermicides, contraceptive pills, patches, injections, intrauterine contraceptive devices or intrauterine contraceptive systems), and it should be ensured to last for at least 4 months after the last dose of the drug under study. Reproductive men should take effective contraceptive measures within 6 months after the last dose after signing the informed consent. 2) Having evidence of other demyelinating diseases or progressive multifocal leukoencephalopathy (PML) after undergoing any surgery within 4 weeks before randomization. 3) Having an active infection (excluding onychomycosis or dental caries) known to be present within 4 weeks before randomization. 4) Screening period: Positive for infectious disease screening, hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCVAb), syphilis antibody, and human immunodeficiency virus (HIV antibody). 5) History of drug or alcohol abuse within 1 year prior to randomization (i.e., drinking more than 14 standard units per week (1 unit = 360 mL of beer, or 45 mL of 40% alcohol spirits, or 150 mL of wine)) or having a mental disorder. 6) Evidence of active tuberculosis (TB; excluding patients receiving drug treatment to prevent latent TB infection). 7) Evidence of active interstitial lung disease. 8) Receiving any live vaccine or live attenuated vaccine within 6 weeks prior to randomization. 9) Having a history of malignant tumors (excluding completely resected and cured basal cell carcinoma and squamous cell carcinoma of the skin or cervical intraepithelial neoplasia) including solid tumors, hematological malignancies, and in situ carcinomas within the past 5 years. 10) Whether the subject has a history of severe drug allergy, or allergy or intolerance (shock, allergic reaction) to mitoxantrone and liposomes. 11) Patients with other chronic active immune system diseases other than NMOSD, or patients with diseases that are stable but require high-dose (dose determined by the researcher) glucocorticoid maintenance therapy (e.g., rheumatoid arthritis, scleroderma, Sjögren's syndrome, ulcerative colitis, hereditary immunodeficiency, or drug-induced immunodeficiency), and patients with only positive autoantibodies but no clinical symptoms can be enrolled in the study. 12) Having other diseases or conditions that, in the judgment of the researcher, make the subject ineligible to participate in this study.

[0059] 2. Exclusion criteria related to previous treatment or concomitant treatment: 13) Receiving treatment with any investigational product within 3 months prior to randomization, or participating in a clinical study of a medical device, which may, in the judgment of the researcher, affect the results of this trial. 14) The presence of a history of NMOSD treatment in any of the following: a. A history of treatment with mitoxantrone or anthracycline agents, total body irradiation, or bone marrow transplantation at any time. b. Rituximab or any investigational B-cell depleting agent was administered within 6 months prior to randomization, unless the central laboratory determines that the subject's B-cell count exceeds the LLN. c. Use of any of satralizumab, tocilizumab, inebilizumab, eculizumab, alemtuzumab, cyclosporine, azathioprine, methotrexate, cyclophosphamide, tacrolimus, mycophenolate mofetil within 3 months prior to randomization. Any other treatment for preventing relapses of multiple sclerosis (MS) (e.g., interferon, natalizumab, glatiramer acetate, fingolimod, teriflunomide, or dimethyl fumarate). d. Received intravenous immunoglobulin (IVIG) injection treatment or plasma exchange treatment (PE) within 1 month prior to randomization. e. Patients who have received treatment by oral administration of corticosteroid agents at a dose exceeding 30 mg / day in terms of prednisone equivalent. f. Use of anti-CD4 or cladribine within 2 years prior to randomization.

[0060] 3. Exclusion criteria regarding laboratory tests: 15) The presence of any of the following clinically significant diseases: a. The cardiac ejection fraction (EF) detected by echocardiogram is less than 50% or less than the lower limit of the laboratory test values of the research center, and there is a history of chronic congestive heart failure and has a cardiac function NYHA class III-IV. b. Patients who have experienced any of the events of myocardial infarction, acute coronary syndrome, viral myocarditis, pulmonary embolism, or stroke within 3 months prior to randomization, or have received coronary artery revascularization within 6 months. c. The electrocardiogram during the screening period shows a QTc interval > 480 ms (according to the Fridericia correction formula, QTc = QT / RR^0.33), or has a history of severe QTc interval prolongation.

[0061] 16) Those with the following abnormalities in the laboratory test values during the screening period a. Aspartate aminotransferase (AST) > 3 × upper limit of normal value (ULN), Alanine aminotransferase (ALT) > 3 × ULN, Total bilirubin > 1.5 × ULN (except in cases due to Gilbert's syndrome), b. Platelet count < 50,000 / μL (or < 50×10 9 / L), Hemoglobin < 9 g / dL (or < 90 g / L), White blood cells < 2.0×10 3 / μL, Absolute neutrophil count < 1.0×10 3 / μL, c. Creatinine clearance (CLcr) < 60 mL / min (calculated by the Cockcroft - Gault formula: [140 - age (years)] × [body weight (kg)] × (0.85, for example, in the case of women) / [72 × serum creatinine (mg / dL)]), or those who received dialysis during the screening period.

[0062] (III) Withdrawal / Ending Criteria If the subject decides to end the research and treatment prematurely at any point during the research process, it will not affect the continuation of receiving appropriate clinical treatment. The subject should return all research drugs. The subject's decision to withdraw from the treatment must be recorded in the source file. After the informed consent is withdrawn, the patient will not be followed up for any reason. A patient who withdraws from the research cannot be replaced.

[0063] In any of the following situations, the researcher should arrange for the subject to end the research treatment prematurely, conduct a follow - up investigation for 8 weeks after the end of the treatment, and complete the End of Study (EOS) visit assessment as fully as possible. 1) If the subject requests to withdraw from the trial or the subject becomes untraceable, 2) If the subject is pregnant, 3) If a serious adverse event or an adverse event with a relatively high risk occurs to the subject and the researcher or sponsor determines that treatment should be discontinued from the perspective of safety, 4) If the subject shows any of the following liver function abnormalities, a. When ALT or AST > 8 × ULN, b. When ALT or AST > 5 × ULN and persists for more than 2 weeks (without elevation of bilirubin and / or fatigue, nausea, vomiting, pain or tenderness in the right upper abdomen, fever, rash and / or eosinophilia (>5%)), c. When ALT or AST > 3 × ULN, total bilirubin > 2 × ULN or international normalized ratio [INR] > 1.5 and no cholestatic factors are observed, d. When ALT or AST > 3 × ULN, accompanied by fatigue, nausea, vomiting, pain or tenderness in the right upper abdomen, fever, rash and / or eosinophilia (>5%), 5) During the process of the study, for subjects who are definitely diagnosed with a recurrence of NMOSD or subjects who develop cardiotoxicity (LVEF is below the lower limit or clinically significantly decreased) based on the criteria specified in the protocol, if the researcher determines that the study and treatment should be terminated and the subject should withdraw from the study, 6) If the subject delays drug administration for more than 14 days or a major protocol violation occurs and the researcher determines that the subject should withdraw from treatment from the perspective of compliance or benefit, 7) Otherwise, if the researcher determines that the patient is inappropriate to continue participating in this trial.

[0064] III. Evaluation Indicators of Treatment Effect Primary Evaluation Items: Time to the first recurrence The time to the first recurrence refers to the time until the subject first experiences an NMOSD attack randomly. The definition of an NMOSD recurrence is that there is a new symptom related to NMOSD or a worsening of an existing symptom, and it meets at least one of the NMOSD attack criteria in the recurrence definition of the protocol.

[0065] Secondary Evaluation Items: Annualized relapse rate (ARR): The ARR at week 48 is calculated as the ratio of the total number of confirmed relapses among all participants divided by the total treatment time (in years) of all participants. · Change in the Expanded Disability Status Scale (EDSS) score relative to baseline · Change in the modified Rankin Scale (mRS) score relative to baseline · Change in the T25FW score relative to baseline · Change in the EQ-5D score relative to baseline · Change in the low contrast visual acuity score (LCVA) relative to baseline · Change in the pain NRS score at 5 sites relative to baseline · Change in the serum glial fibrillary acidic protein (sGFAP) and serum neurofilament light chain protein NfL levels relative to baseline · Serum AQP4 titer

[0066] IV. Research Results For patients with neuromyelitis optica spectrum disorder, after treatment with mitoxantrone hydrochloride liposome injection, it is safe and tolerable, has low toxic side effects, improves the effectiveness of treatment, and delays the relapse of neuromyelitis optica spectrum disorder.

[0067] As described above, the embodiments of the present invention have been exemplarily explained. It should be understood that the protection scope of the present invention should not be unduly limited to these specific embodiments. Any changes, equivalent substitutions, improvements, etc. made by those skilled in the art within the scope of the spirit and principle of the present invention shall be included within the protection scope described in the claims of the present invention.

Claims

1. Use of mitoxantrone hydrochloride liposomes in the manufacture of a medicament for treating neuromyelitis spectrum disorder. Preferably, the neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder, and more preferably, the highly recurrent neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder that has failed other drug treatments.

2. Comprising administering to an individual a therapeutically effective amount of mitoxantrone hydrochloride liposomes, or a pharmaceutical composition comprising a therapeutically effective amount of mitoxantrone hydrochloride liposomes, Preferably, the neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder, More preferably, the highly recurrent neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder that has failed other drug treatments, A method for treating an individual's neuromyelitis spectrum disorder.

3. Used for treating neuromyelitis spectrum disorder, Preferably, the neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder, More preferably, the highly recurrent neuromyelitis spectrum disorder is highly recurrent neuromyelitis spectrum disorder that has failed other drug treatments, Mitoxantrone hydrochloride liposomes or a pharmaceutical composition comprising mitoxantrone hydrochloride liposomes.

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

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

6. The active ingredient content of the agent or pharmaceutical composition is 0.5 to 5 mg / mL, preferably 1 to 2 mg / mL, more preferably 1 mg / mL, based on mitoxantrone. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposome or pharmaceutical composition containing the mitoxantrone hydrochloride liposome according to claim 3.

7. The particle size of the mitoxantrone hydrochloride liposome is about 30 to 80 nm, such as about 35 to 75 nm, preferably about 40 to 70 nm, more preferably about 40 to 60 nm, such as about 60 nm. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposome or pharmaceutical composition containing the mitoxantrone hydrochloride liposome according to claim 3.

8. Mitoxantrone, which is the active ingredient in the mitoxantrone hydrochloride liposome, forms a poorly soluble precipitate with polyvalent counterions in the liposome, such as sulfate, citrate, or phosphate. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposome or pharmaceutical composition containing the mitoxantrone hydrochloride liposome according to claim 3.

9. The phospholipid bilayer in the mitoxantrone hydrochloride liposome contains a phospholipid having a phase transition temperature (Tm) higher than body temperature, whereby 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, dipalmitoyl lecithin, distearoyl lecithin, or any combination thereof. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposome or pharmaceutical composition containing the mitoxantrone hydrochloride liposome according to claim 3.

10. The mass ratio of the hydrogenated soy lecithin, cholesterol, and polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine is 3:1:1, and / or Preferably, the weight ratio of the hydrogenated soy lecithin, cholesterol, polyethylene glycol 2000-modified distearoyl phosphatidylethanolamine, and mitoxantrone is 9.58:3.19:3.19:1, and / or Preferably, the particle size is about 60 nm, and / or Preferably, the counterion is sulfate ion. The use according to claim 1, the method according to claim 2, or the mitoxantrone hydrochloride liposome or pharmaceutical composition containing the mitoxantrone hydrochloride liposome according to claim 3.

11. The therapeutically effective amount is 4 to 30 mg / m based on mitoxantrone ２ , preferably 8 to 20 mg / m ２ or 12 to 30 mg / m ２ , for example 8 mg / m ２ or 12 mg / m ２ and / or The administration form is intravenous administration, and / or The administration cycle is to administer once every 12 weeks, The method according to claim 2.

Images