WO2025221208A1 - Use of mitoxantrone hydrochloride as a therapeutic drug for treating neurodegenerative disorders - Google Patents

Abstract

The present invention relates to the use of mitoxantrone hydrochloride as a therapeutic drug for the treatment of neurodegenerative disorder in a subject, wherein the mitoxantrone hydrochloride is an inhibitor of β-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject.

Description

USE OF MITOXANTRONE HYDROCHLORIDE AS A THERAPEUTIC DRUG FOR

TREATING NEURODEGENERATIVE DISORDERS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore application No. 10202401 126W filed April 17, 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Various embodiments of this disclosure may relate to the use of mitoxantrone hydrochloride as a therapeutic drug for treating neurodegenerative disorders.

BACKGROUND

[0003] Alzheimer's disease (AD) is the most common neurodegenerative disease. It accounts for majority of dementia cases and affects millions of people worldwide. Parkinson’s disease (PD) is the second most common neurodegenerative disease. There are an estimated 7- 10 million people with Parkinson’s disease worldwide. The prevalence of Parkinson’s disease is estimated to be 0.041% among individuals in their 40s, increasing to 1 .9% among those over the age of 80.

[0004] Parkinson’s disease is pathologically characterized by the presence of misfolded alpha-sy nuclein (aSyn) in Lewy bodies, causing a progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN).

[0005] However, to date, there is no disease-modifying therapy for Parkinson’s disease, which may be partially attributed to its complex and still poorly understood pathogenesis, involving genetic factors, environmental toxins, and free radicals. Current drugs and therapies can only relieve the symptoms but are unable to slow disease progression. [0006] Genetically, the loss of DA neurons in the substantia nigra compacta (SNc) is believed to be the cause of the motor and non-motor symptoms of Parkinson’s disease. Dopamine replacement therapy can relieve symptoms but does not halt disease progression. Over the past two decades, several genes have been identified as being associated with Parkinson’s disease, including SNCA, Parkin/PRKN, DJ-l/Park7, PINK1, and LRRK2. Although therapies targeting these pathological pathways have become an area of interest since their discovery, no effective treatment has been developed to slow down disease progression or reverse the underlying pathology.

[0007] It is therefore desirable to provide an alternative drug and therapeutical method for the treatment of Parkinson’s disease or other neurodegenerative disorders, which seeks to address at least one of the problems described hereinabove or provides an alternative to existing drugs and treatments.

SUMMARY

[0008] In a first aspect, the present disclosure provides a use of mitoxantrone hydrochloride in the manufacture of a pharmaceutical composition for the treatment of neurodegenerative disorder in a subject, wherein the mitoxantrone hydrochloride is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject.

[0009] In a second aspect, the present disclosure provides a method of treating a neurodegenerative disorder. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof, wherein the mitoxantrone hydrochloride is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject. [0010] Tn a third aspect, the present disclosure provides a pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative disorder, wherein the mitoxantrone hydrochloride or the pharmaceutically acceptable salt thereof is an inhibitor of p-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in a subject.

[0011] In some embodiments, the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In the drawings, reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings.

FIG. 1 illustrates a fluorescence-based biosensor system for screening inhibitors from an FDA- approved library.

FIG. 2A are Western blots showing MH inhibits P-amyloid precursor protein (APP) and LRRK2 (leucine-rich repeat kinase 2) expression levels.

FIGs. 2B to 2C are graphs showing results of APP and LRRK2 expression levels inhibited by MH. The data are presented as the mean ± SD; n=3 per group. **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

FIG. 3A is a table showing the docking scores of predicted binding sites of MH against LRRK2. FIG. 3B illustrates the predicted binding poses of MH against binding site 1 (in the kinase domain) of LRRK2. FTGs. 4A to 4C are Western blots and graphs showing MH inhibits endogenous LRRK2 protein level in different cell lines: (4A) shows in SH-SY5Y cells; (4B) in LRRK2^WT and LRRK2^G2019S expressed HEK293T cells; and (4C) in LRRK2^G2019S patient iPSC-derived DA neurons The cells were treated with MH at 0, 0.1, 0.25, 1, and 4 pM for 24 hrs before collected for western blot analysis. N=3.

FIGs. 5A to 5D are Western blot and graphs showing MH ameliorates DA neuronal loss in the 6-OHDA PD mouse model. 6-OHDA (4 pg) was stereotaxically injected into a 4-month-old C57 mice on the left striatum (day 0), and MH (1 mg/kg) was administered via intraperitoneal (i.p.) on day 8 for 5 days of treatments before (5C) Cylinder test, and (5D) Pole test. (5 A) shows a Western blot analysis of brain and tissues samples that were harvested from the mice. (5B) shows Western blotting images of relative tyrosine hydroxylase (TH) expression in left and right striata. The data are mean ± SD; n=3 per group. *P < 0.01 and **P < 0.05 by one-way ANOVA with Tukey’s post hoc test.

FIGs. 6A to 6E are Western blot and graphs showing the results of MH treatment on DA neuronal loss and motor behavioural performance in the 6-OHDA + LRRK2^G2019S PD mouse model. 6-OHDA (2 pg) was stereotaxically injected into a 4-month-old LRRK2^G2019S on the left striatum (day 0), and MH (1 mg/kg) was administered via i.p. on day 8 for 5 days of treatments before (6C) Cylinder test, (6D) Pole test, and (6E) Rotarod test. (6A) shows a Western blot analysis of brain and tissues samples that were harvested from the mice. (6B) shows Western blotting images of relative TH expression in left and right striata. The data are mean ± SD; n=3 per group. */' < 0.01 and **P < 0.01 by one-way ANOVA with Tukey’s post hoc test.

FIGs. 7A to 7D are Western blot and graphs showing ex vivo treatment of healthy human PBMCs with drug MH decreased both pS935-LRRK2 and pT73-RablO in a dose-dependent manner. Healthy human peripheral blood mononuclear cells (PBMCs) (n=2 from 2 healthy donors) were treated with 0, 0.25, 1, 4, and 10 pM of drug MH for 1 hour before protein extraction. Total protein was used for western blot analysis and the results are shown in FIG. 7A Statistical analysis of western blot analysis of FIG. 7 A for pS935-LRRK2/LRRK2 is shown in FIG. 7B, and pThr73-RablO/RablO in FIG. 7C. The drug potencies for pS935-LRRK2 and pT73-RablO were plotted in FIG. 7D. pLRRK2: IC50=4.2 uM, pRablO IC50=10.1 uM.

FIGs. SA to 8D are grapah showing the results of motor behavior performance in PD mice after MH treatment. 6-OHDA (4 pg) was stereotaxic ally injected into 2-month-old C57 mice on the left striatum (day 0), and MH was administered via i.p. injection (0.5 mg/kg/day for 5 days, total dose=2.5mg/kg) or oral gavage (1 mg/kg/day for 5 days, total dose=5mg/kg) from day 8 to day 13 before (8 A) Rotation Test, (8B) Cylinder test, (8C) Rotarod Test, and (8D) Pole test after 2 weeks, 6 weeks, and 12 weeks post drug treatment. The data are mean ± SD; n=4 per group. *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test.

FIGs. 9A to 9H are graphs showing the body weight and vital organ index in mice before and after MH treatment. 2-month-old 6-OHDA PD mice were treated with MH i.p injection at 2.5mg/kg or oral gavage at 5 mg/kg, total dose). FIG. 9A is a graph showing the body weights measured before and after 2, 6, and 12 weeks of drug treatment. After 12 weeks, mice were sacrificed and vital organ to body weight ratios (index) were measured. FIG. 9B shows the measurements taken for Brain, (9C) for Heart, (9D) for Liver, (9E) for Thymus, (9E) for Spleen, (9G) for Lung, and (9H) for Kidney. The data are mean ± SD, n=4 per group. ns=non- significant. *P < 0.05 by one-way ANOVA with Tukey’s post host test.

FIGs. 10A to 10C are images of hematoxylin and eosin (H&E) staining showing the results of morphological changes or tissue damage in the heart in mice after MH treatment 6-0HDA- induced PD mice were sacrificed after 12 weeks of drug MH treatment via i.p. injection (0.5 mg/kg for 5 days, totally 2.5mg/kg) or oral gavage (1 mg/kg for 5 days, totally 5mg/kg). Morphology of heart tissues was examined by H&E staining. No morphological changes or tissue damage was observed for both treatment groups. N=3 mice per group were used for H&E staining. Representative images (10A), (10B) and (IOC) were shown. Scale bar = 50 pm. ns=non-significant.

FIGs. HA to HD show images of Sirius red staining and graph showing the results to detect cardiac fibrosis in the heart of mice after MH treatment. 6-OHDA-induced PD Mice were sacrificed after 12 weeks of drug MH treatment via i.p. injection (0.5 mg/kg for 5 days, totally 2.5mg/kg) or oral gavage (1 mg/kg for 5 days, totally 5mg/kg). Sirius red cardiac staining was conducted to detect cardiac fibrosis in heart tissues. No significant difference in cardiac fibrosis was observed among all 3 groups. N=3 mice per group, and 6 images/areas per mice, a total of 18 images were used for quantification of the collagen to skeletal muscle ratio Representative images were shown. Scale bar = 50 pm. ns=non-significant.

FIGs 12A to 12D show images of DAB immunohistochemistry staining and graph showing the results to detect inflammation in heart tissue in mice after MH treatment. 6-OHDA-induced PD Mice were sacrificed after 12 weeks of drug MH treatment via i.p. injection (0.5 mg/kg for 5 days, totally 2.5mg/kg) or oral gavage (1 mg/kg for 5 days, totally 5mg/kg). DAB immunohistochemistry staining was performed to detect inflammation status of heart tissues by using CD68 antibody. No significant difference in CD68+ expression area was observed among all 3 groups. N=3 mice per group, and 6 images/areas per mice, a total of 18 images were used for quantification of relative CD68+ areas. Representative images were shown. Scale bar = 50 pm. ns=non-significant

DESCRIPTION

[0013] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0014] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0015] In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.

[0016] In the context of various embodiments, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance, e g. within 10% of the specified value.

[0017] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0018] As used herein, “comprising” means including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. [0019] As used herein, “consisting of’ means including, and limited to, whatever follows the phrase “consisting of’. Thus, use of the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0020] As used herein, mitoxantrone hydrochloride or “MH” of the present invention can be identified with CAS Number: 70476-82-3, with molecular weight of 517.4 g/mol, and having chemical formula: C22H30Q2N4O6. [0021] As used herein, the term “subject” refers to a mammal A subject therefore refers to, for example, mice, cow, pigs, dogs, cats, and the like. When the subject is a human, the subject may be referred to herein as a patient.

[0022] As used herein, the term “APP” refers to |3-amyloid precursor protein, a transmembrane protein that is the precursor to the amyloid-beta (A0) peptide.

[0023] As used herein, the term “LRRK2” refers to leucine-rich repeat kinase 2, is a protein encoded by the LRRK2 gene.

[0024] As used herein, the term "in vitro" is intended to encompass experiments with cells in culture, outside of a living organism, whereas the term "in vivo" is intended to encompass experiments conducted within living organisms.

[0025] As used herein, the term “ex vivo” is intended to encompass experiments involving tissues, organs, or cells maintained outside their native organism under controlled laboratory conditions.

[0026] As used herein, the term “neurodegenerative disorder” refers to disease or condition that damage and destroy part of the nervous system leading to the impaired function of a subject’s nervous system. Neurodegenerative disorders that may be treated using the composition and methods described herein include those involving APP dysregulation, LRRK2 activity, or related pathways. Examples of such neurodegenerative disorders include, but are not limited to, Alzheimer’s disease, Parkinson’s disease, Lewy Body Dementia, Frontotemporal Dementia, Multiple System Atrophy, Progressive Supranuclear Palsy, Corticobasal Degeneration, and dementia associated with Down Syndrome.

[0027] The terms “treating” or “treatment” refers to any indication of healing or amelioration of a disease or condition; alleviating or diminishing symptoms or making a condition more tolerable to the patient; slowing the rate of degeneration or decline. For example, treatment mentioned herein may treat Alzheimer's or Parkinson’s disease by restoring neuronal loss, restoring motor deficit in the patient.

[0028] As used herein, the term “inhibition”, “inhibit” or “inhibiting” may refer to decreasing the activity of a protein from functioning normally or preventing the production of a protein. Inhibition includes, at least in part, partially or totally blocking activity of a protein, inactivating enzymatic activity or reducing the amount of a protein.

[0029] In a first aspect, the present disclosure relates to a use of mitoxantrone hydrochloride in the manufacture of a pharmaceutical composition for the treatment of neurodegenerative disorder in a subject, wherein the mitoxantrone hydrochloride is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject

[0030] In one embodiment, the mitoxantrone hydrochloride described herein reduces the LRRK2 protein levels in vitro by at least 20% compared to untreated control. In some embodiments, the mitoxantrone hydrochloride described herein reduces the LRRK2 protein levels by at least 20% in vivo. In other embodiments, the mitoxantrone hydrochloride described herein is formulated to modulate the LRRK2 protein levels by at least 20% in an ex vivo cellbased assay.

[0031] In one embodiment, the mitoxantrone hydrochloride described herein reduces the APP protein levels in vitro by at least 20% compared to untreated control. In some embodiments, the mitoxantrone hydrochloride described herein reduces the APP protein levels by at least 20% in vivo. In other embodiments, the mitoxantrone hydrochloride described herein is formulated to modulate the APP protein levels by at least 20% in an ex vivo cell-based assay. [0032] In some embodiments, the mitoxantrone hydrochloride described herein binds to one or more binding sites of LRRK2 selected from the group consisting of Alal904, Argl957, Lysl996, Leu2001, and Thr2035 , thereby inhibiting LRRK2 kinase activity. In some embodiments, the mitoxantrone hydrochloride binds to two or more binding sites of LRRK2 selected from the group consisting of Alal904, Argl957, Lysl996, Leu2001, and Thr2035. In yet other embodiments, the mitoxantrone hydrochloride binds to the Alai 904 binding site, optionally in combination with none, one, or more of the other binding sites selected from the group consisting of Argl957, Lysl996, Leu2001, and Thr2035. In yet other embodiments, the mitoxantrone hydrochloride binds to the Thr2035 binding site, optionally with none, one, or more of the other binding sites selected from the group consisting of Alal904, Argl957, Lysl996 and Leu2001.

[0033] In various embodiments, the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease, Alzheimer’s disease, Lewy Body Dementia, Frontotemporal Dementia, Multiple System Atrophy, Progressive Supranuclear Palsy, Corticobasal Degeneration, and dementia associated with Down Syndrome. In some embodiments, the neurodegenerative disorder is Parkinson’s disease or Alzheimer’s disease.

[0034] In various embodiments, the treatment promotes neuronal regeneration in a subject.

[0035] In various embodiments, the treatment alleviates motor deficits in a subject.

[0036] In various embodiments, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients, carriers or diluents. In certain embodiments, the excipients are selected from the group consisting of binders, fillers, stabiliser, solubilizers, and/or flavouring agents.

[0037] In various embodiments, the pharmaceutical composition disclosed herein can be in the form of a tablet, a film coated tablet, a capsule, a gel cap, a caplet, a pellet, or the like, or in the form of a liquid.

[0038] In some embodiments, the pharmaceutical composition may be formulated for oral, intravenous, intranasal, subcutaneous or other suitable routes of administration, depending on the formulation and clinical context. [0039] n a second aspect, the present disclosure provides a method of treating neurodegenerative disorders in a subject. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof, wherein the mitoxantrone hydrochloride is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject.

[0040] In various embodiments, the pharmaceutical composition comprising the mitoxantrone hydrochloride may be administered to a subject orally, intravenously, intranasally, subcutaneously, or via other suitable routes of administration, depending on the formulation and clinical context.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative disorder, wherein the mitoxantrone hydrochloride or the pharmaceutically acceptable salt thereof is an inhibitor of p-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in a subject.

[0042] Therapeutic Applications

[0043] In some embodiments, the pharmaceutical composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0044] The pharmaceutical composition may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment.

[0045] Dosage Regimes [0046] Tn some embodiments, the pharmaceutical composition is administered according to a dosage regimen that may vary depending on factors such as the subject’s weight, age, sex, clinical condition, biomarker response, and treatment response. The dosage and/or frequency of administration may be adjusted based on one or more of patient weight, biomarker response, or tolerability. For example, the pharmaceutical composition may be administered once daily, twice daily, every other day, once every 5 days, weekly, biweekly, triweekly or monthly. In some embodiments, the dose may range from 0.5 to 3 mg/kg, 0.5 to 2.5 mg/kg or 1 to 2 mg/kg. In some embodiments, the dose may range from 6 to 14 mg/m², 7 to 13 mg/m², 7 to 12 mg/m², or 7 to 11 mg/m², 8 to 13 mg/m², 8 to 12mg/m² or 8 to l lmg/m². The doses may be lower or titrated upward depending on therapeutic response and observed side effects. In some embodiments, treatment may be admini stered in cycles, for example, 5 days per cycle, or 5 days on treatment followed by 2 to 3 days off, or 3 to 4 weeks on treatment followed by 1 to 2 weeks off, to optimize efficacy. In various embodiments, the pharmaceutical composition may be administered to a subject orally, intravenously, intranasally, subcutaneously, or via other suitable routes, depending on the formulation and clinical context. The treatment duration may range from a single administration to chronic or long-term administration over weeks, months, or years. In some exemplary embodiments, the pharmaceutical composition is administered intravenously at a dose of 8 to 12 mg/m² every 2 to 4 weeks, or orally at 1 to 2 mg/kg daily for 5 days per cycle.

[0047] Currently, there are several potential therapies for Parkinson’s disease, including (i) Medications: drugs that increase dopamine levels in the brain, such as levodopa and dopamine agonists, can help relieve specific PD symptoms; (ii) Deep Brain Stimulation (DBS): DBS is a surgical procedure in which electrodes are implanted in the brain to deliver electrical impulses to the brain, which can help reduce Parkinson’s symptoms; (iii) Gene Therapy: Gene therapy involves introducing new genetic material into cells to correct or replace defective genes (such as VM202 and AXO-Lenti-PD to deliver GDNF), which is under development and may cause ethical issues; and (iv) Stem Cell Therapy: Stem cell therapy involves using stem cells to generate new brain cells that produce dopamine, which is still experimental and requires further research. However, all these potential therapies are either still under development or can only relieve specific PD symposiums and are not capable of slowing disease progression or providing a cure

[0048] The drug, mitoxantrone hydrochloride, as disclosed herein, targets the LRRK2 expression and neurotoxicity, and inhibits APP protein levels, making it a suitable therapeutic agent for the treatment of PD and AD. In addition, mitoxantrone hydrochloride has been shown to be safe and well -tolerated in humans. Preliminary data demonstrate that it rescues motor deficits in a preclinical mouse model ofPD.

[0049] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the disclosure. One skilled in the art will recognize that the examples set out below are not an exhaustive list of the embodiments of this disclosure.

EXAMPLES

Example 1: Materials and Methods

[0050] APP and LRRK2 Inhibitors

[0051] With reference to FIG. 1, a fluorescence-based biosensor system for screening APP and LRRK2 inhibitors from an FDA-approved library is illustrated.

[0052] Step 1 : The step involves the construction of APP inhibitors detection vector pCopGFP-BSD-pAPP (101). A puromycin (PURO) expression cassette, a blasticidin expression cassette (BSD), a CopGFP expression cassette, and a mCherry driven by a 1463 bp human APP promoter were cloned into a pGL3-basic vector. Vector sequence was confirmed by Sanger sequencing. [0053] Step 2: The step involves generation of stable cell lines (102) The pCopGFP-BSD- pAPP vector was transfected into SH-SY5Y cells by Lipofectamine LTX (ThermoFisher) following manufacturer’s instruction. 2 days after transfection, cells were selected with 2.5 pg/rnl Puromycin for 2 weeks with medium changes every 3 days until all cells are positive for GFP signal.

[0054] Step 3: The step involves large scale screening (103). pCopGFP-BSA-pAPP stable SH-SY5Y cells were seeded in 96 well plates (black plates with clear bottom) and 10 pM of each drug from the FDA-approved drug library (ApexBio, #L1021). mCherry signal and GFP signal were read (105) at Oh, 2h, and 24h post drug treatment (104). The drugs that reduced mCherry signal without affecting GFP signals were selected as the APP inhibitor candidates (106).

[0055] Step 4: The step involves validation (107) of the shortlisted candidate drugs. Naive SH-SY5Y cells were seeded into cell culture plates and the shortlisted candidate drugs were added with various concentrations. After 24 hours, the cells were collected for qPCR and western blot analysis for APP and LRRK2 protein levels. The best drugs, which inhibit both APP and LRRK2 protein levels without affecting cell viability were selected as the hit-to-lead drugs (108).

[0056] Western Blot Analysis

[0057] Western blot analysis was performed according to the method described in Zhang 2022, et al., Sei. Signal., 15, eabk3411 (2022), DOI: 10.1126/scisignal.abk3411 (which is incorporated herein by reference and hereinafter referred to as “Zhang 2022”). Briefly, cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail. 20 pg of protein lysates were separated on SDS-PAGE gels and transferred to poly vinylidene difluoride (PVDF) membranes. The blots were blocked with TBST (trisbuffered saline-Tween 20) supplemented with 5% skim milk and incubated with primary antibodies against LRRK2, APP or P-actin overnight at 4°C The membranes were washed in TBST and then incubated with secondary horseradish peroxidase (HRP)-conjugated antibody and developed using an ECL detection kit and captured with a ChemiDoc Imaging System. The images were then quantified with ImageJ and plotted with GraphPad Prism for graphs.

[0058] Cylinder Test

[0059] The cylinder test is to measure the vertical movement of mice. Each mouse was placed in an empty cylinder and a 5-minute timer was simultaneously started. When mice are placed in a new environment, they tend to stand on their hind legs to better smell the surroundings (a behavior known as rearing). In this test, the number of rears were recorded for each mouse.

[0060] Pole Test

[0061] The pole test is a behavioral test that measures coordination and motor function in mice. A 50 cm pole with a stand was placed in the cage. Extra bedding was placed in the cage to protect the falling mice from the pole. The pole test was performed on 3 consecutive days. For the first 2 days, mice were placed on top of the pole and trained to walk down the pole. The training can be performed a few times until mice can walk down the pole by themselves. For the third day, the timings of each mouse walking down the pole for two rounds were recorded for analysis.

[0062] Rotarod Test

[0063] The Rotarod test measures coordination and motor skill learning. Mice were placed on a barrel-shaped platform that rotated slowly at first (4 rpm), which then gradually accelerated over 5 min (to 40 rpm). Each mouse had to balance for as long as possible before falling off. There is a pressure plate below the apparatus, which stops the time recording when the mouse has activated it. The time taken for each mouse to fall was measured here. The longer the duration, the better the coordination and motor skill learning. [0064] Rotation Test

[0065] A video recorder is set up on a tripod facing downward above the laboratory floor. Each mouse is placed in standard laboratory beakers and allowed to explore the environment for 20 min. Mice are inj ected with 5 mg/kg amphetamines (i.p.) and placed back into the beaker. The investigator will then record peak rotation over 20 min immediately following apomorphine injection and 20 min after amphetamine injection. These recordings then can be analyzed post hoc, and full rotations can be counted using fast-forward video playback reporting net rotations over the period (No. of clockwise rotations - No. of anticlockwise rotations).

[0066] Mice Body Weight and Organ Index Measurement

[0067] Mice’s body weight was measured using a scale. Mice were anesthetized with a mixture of ketamine and xylazine hydrochloride. Mice were then transcardiac perfused with PBS. Mice were dissected and the weights of vital organs including brain, heart, liver, thymus, spleen, lung, and kidney were measured. The organ index is calculated as the organ to body weight ratio. The body weight and organ index were analyzed and plotted using GraphPad Prism.

Example 2: LRRK2 Mutation

[0068] Among known genetic contributors to PD, pathogenic LRRK2 mutations are currently recognized to be the most common, accounting for up to 40% of familial cases in certain populations. Previously, the inventors demonstrated that mutant LRRK2^G2019S promotes the processing of APP into its transcriptionally active form, the APP intracellular domain (AICD) (Chen et al., Sci. Signal., 10, eaam6790 (2017), DOI: 10. l l 26/scisignal.aam6790, which is incorporated herein by reference and hereinafter referred to as “Chen 2017”). Specifically, the study showed that: (1) LRRK2 stimulates the transcriptional activity of the AICD (see Fig.

3 of Chen 2017); (2) AICD enhances LRRK2^G2019S-mediated neurotoxicity in vitro (see Fig. 5 of Chen 2017); (3) AICD enhances LRRK2^{G20l 9S}-mediated neurotoxicity in vivo (see Fig. 6 of Chen 2017); and (4) an LRRK2 inhibitor restores dopamine neuronal loss in PD patient- derived dopaminergic (DA) neurons and in PD mouse models (see Fig. 7 of Chen 2017).

[0069] The inventors further demonstrated that LRRK2-APP/AICD are linked in a self- perpetuating cycle (Zhang 2022). The LRRK2 accumulation induced by this cycle further activates a series of vicious cellular responses, including excessive mitophagy activity, impaired mitochondrial function, increased alpha-synuclein (which leads to the formation of Lewy body in PD), and decreased tyrosine hydroxylase (TH, a marker of dopaminergic (DA) neuron).

[0070] All the above events result in the DA neuron loss and eventually neurodegeneration. Remarkably, the inventors have discovered that a small molecule drug named “Itanapraced” can break this vicious cycle and restore dopamine neuronal loss (Zhang 2022). Itanapraced is the only known AICD inhibitor, which shows beneficial effects for mild cognitive impairment (MCI). Itanapraced binds to AICD and blocks its nuclear translocation thus stopping the feedforward cycle. The findings revealed that: (1) AICD promotes LRRK2 transcriptional expression (see Fig. 1(A) and (B) of Zhang 2022); (2) AICD overexpression promotes LRRK2-mediated neurotoxicity in vivo (see Fig. 4 of Zhang 2022); (3) Inhibition of AICD by itanapraced prevents LRRK2-induced neurotoxicity in vitro and in vivo (see Fig. 5 of Zhang 2022); (4) Itanapraced rescues LRRK2-mediated mitochondrial dysfunction in vitro and in vivo (see Fig. 6 of Zhang 2022); and (5) Itanapraced ameliorates DA neuronal loss in the 6-OHDA PD mouse model (see Fig. 8 of Zhang 2022).

[0071] The findings further show that LRRK2 and the APP/AICD are reciprocally linked and that inhibitors of APP/AICD and LRRK2 are potential therapeutic treatment for PD.

Example 3: High Throughput Screening for LRRK2 Inhibitors

[0072] The fluorescence-based biosensor system as shown in FIG. 1 was used to conduct high throughput screening (HTs) using APP reporter to identify small molecular compounds from FDA-approved libraries that could inhibit APP. The FDA-approved drug library contains 1971 FDA-approved drugs, targeting different disease-related pathways. The SH-SY5Y cells were treated using IOJIM of each library compound for various time points. Based on the in vitro cell-based assay and in vivo PD mouse model testing, mitoxantrone hydrochloride has been identified as a compound that has the best inhibition effect on APP and LRRK2 expression levels.

Example 4: Validation of APP and LRRK2 Inhibitors by Cell-based Assay

[0073] Cell-based assay, including Western Blot is conducted on 49 shortlisted candidate compounds. Mitoxantrone hydrochloride (MH) is shortlisted as the hit-to-lead compound. Then, naive SH-SY5Y cells were seeded into 6-well cell culture plates. At 70-80% confluency, 0, 0.25, 1, and 4uM of drug MH was added into cells and incubated for 24 hours before collected for western blot lysis.

[0074] FIGs. 2A to 2C show that MH inhibits APP expression as a low dose from 0.25pM. Given the link between APP and LRRK2, the inhibition effect of MH on LRRK2 expression is shown in FIG. 2C, whereby MH was added to SH-SY5Y cells for 24 hours and collected for Western blot analysis.

Example 5: Binding Sites Prediction by Computational Modeling

[0075] The human LRRK2 protein structure (PDB ID: 7LI4) was downloaded from PDB. Schrodinger’s SiteMap was employed for the prediction of potential binding sites within the LRRK2 protein. The SiteMap analysis predicted four potential binding pockets within the LRRK2 protein. These pockets were ranked based on their Dscore and druggability score.

[0076] Computational modeling predicts four binding sites of MH against LRRK2, of which the Kinase Domain (KD) is shown to have the most potential to bind LRRKs against its kinase activity. FIG. 3A shows the docking scores of predicted binding sites of MH against LRRK2. FIG. 3B shows the predicted binding poses of MH against binding site 1 (in the kinase domain) of LRRK2. MH formed hydrogen bonds with G1886, H1998 and N1999 and dihydroxyanthraquinone ring of MH occupied the hydrophobic region within the predicted binding pocket. The hydrophobic region within the predicted binding pocket is formed by V1893, A1904, M1947, L1949, and L2001 residues.

Example 6: In vitro Inhibition of LRRK2 Protein Levels by MH

[0077] To investigate the MH inhibition of LRRK2 protein levels in various cell lines, several cell lines, such as SH-SY5Y cells, LRRK2^WT and LRRK2^G2019S, human LRRK2^G2019S iPSC-derived DA neurons, were selected for experiments.

[0078] For SH-SY5Y cells, naive SH-SY5Y cells were seeded into 6-well cell culture plates. At 70-80% confluency, 0, 0.1, 0.25, 1, and 4pM of drug MH was added into cells and incubated for 24 hours before collected for western blot analysis (FIG. 4A).

[0079] For LRRK2^WT and LRRK2^G2019S, plasmids carrying LRRK2^WT and LRRK2^G2019S were transfected into HEK293T cells using Lipofectamine LTX kits following manufacturer’s instructions. 48 hours post-transfection, 0, 0.1, 0.25, 1, and 4pM of drug MH was added into cells and incubated for 24 hours before collected for western blot analysis (FIG. 4B).

[0080] For human LRRK2^G2019S iPSC-derived DA neurons, LRRK2^G2019S patient iPSCs were differentiated into DA neurons as previously described (Zhang 2022). 0.0, 0.1 , 0.25, 1 .0, and 4.0 pM of drug MH was added into the DA neurons and incubated for 24 hours before collected for western blot analysis (FIG. 4C).

[0081] FIGs. 4A to 4C show that MH effectively inhibits endogenous LRRK2 protein levels in SH-SY5Y cells, in LRRK2^WT and LRRK2^G2019S overexpressed HEK293T cells, and in human LRRK2^G2019S iPSC-derived DA neurons.

Example 7: In vivo MH treatment on 6-OHDA neurotoxin lesion-induced PD mouse model [0082] Given that MH showed significant inhibition of LRRK2 in vitro, the rescue effect in

6-OHDA neurotoxin lesion-induced PD mouse model was further examined. [0083] The effect was tested in 6-hydroxydopamine (6-OHDA)-toxin-induced PD mouse model because the 6-OHDA mouse model has been commonly used to test drug effects, including neuroprotection against DA neuronal loss in PD.

[0084] 6-OHDA (4 pg) was stereotaxically injected into 4-month-old C57 mice on the left striatum (day 0), and MH (1 mg/kg) was administered via i.p. on day 8 for 5 days of treatments before Cylinder test and Pole test were conducted. Brain tissues were then harvested for western blot. FIGs 5A and 5B show that MH can restore TH (tyrosine hydroxylase) level which is reduced by 6-OHDA lesion, indicating the ability of MH to rescue neuronal loss. FIGs 5C and 5D show that MH can rescue motor deficits in cylinder and pole tests, which further supports MH’s role as a LRRK2 inhibitor for the treatment of PD.

Example 8: In vivo MH treatment on 6-OHDA + LRRK2^G2<I19S PD Mouse Model

[0085] The effect of MH in the 6-OHDA + LRRK2^G2019S PD mouse model was further investigated.

[0086] 6-OHDA (2 pg) was stereotaxically injected into 4-month-old LRRK2^G2019S on the left striatum (day 0), and MH (1 mg/kg) was administered via i.p. on day 8 for 5 days of treatments before Cylinder test, Pole test, and Rotarod test were conducted

[0087] The brain tissues of the mice were harvested for western blot. As shown in FIGs 6A to 6D, MH ameliorates DA neuronal loss and improves motor behavioural performance in the 6-OHDA + LRRK2^G2U19S PD mouse model. Inhibition of APP and LRRK2 can restore neuronal loss and motor behavioural deficits in PD and AD mouse models, which strongly supports MH’s therapeutic potential as a novel APP and LRRK2 inhibitor for the treatment of both sporadic and familial PD, and AD.

Example 9: Ex vivo MH Treatment of Healthy Human PBMCs

[0088] The cellular potency of drug MH was further assessed in human PBMCs using S935- LRRK2 and T73-Rab as measures for LRRK2 kinase activity (Chen 2017, Zhang 2022; Jennings et a!., Sci. Transl. Med., 2022, DOI: 10.1 126/scitranslmed.abj2658). Healthy human peripheral blood mononuclear cells (PBMCs) (n=2 from 2 healthy donors) were treated with 0, 0.25, 1.0, 4.0, and 10 pM of drug MH for 1 hour before protein extraction Total protein was used for western blot analysis. Ex vivo treatment of healthy human PBMCs with drug MH decreased both pS935-LRRK2 and pT73-Rabl0 in a dose-dependent manner as shown in FIGs 7A to 7D. The results show that both pS935-LRRK2 and pT73-Rab10 responded well with drug MH treatment, which is in a dose-dependent manner (FIGs 7A to 7C). 1 hour treatment of 10 pM drug MH significantly decreased pS935-LRRK2 level to 30.7% (p=0.036) (FIGs 7A and 7B) and pT73-Rabl0 level to 38.0% (p=0.0027) (FIGs 7A and 7C) as compared to vehicle treatment. The half-maximal inhibitory concentration (IC50) of drug MH for pS935-LRRK2 is 4.2 pM and IC50 for pT73-Rab10 is 10.1 pM (FIG. 7D) These results illustrate that drug MH has a high potency to inhibit LRRK2 in human PBMCs, which further supports the utility of drug MH as a LRRK2 inhibitor in the pharmaceutical treatment of LRRK2 -associated PD.

Example 10: Assessment of MH Treatment on PD Mice

[0089] To determine the effectiveness of MH treatment on PD mice, 6-OHDA (4 pg) was stereotaxi cal ly injected into 2-month-old C57 mice on the left striatum (day 0), and MH was administered via i.p. injection (0.5 mg/kg/day for 5 days, total dose=2.5mg/kg) or oral gavage (1 mg/kg/day for 5 days, total dose=5mg/kg) from day 8 to day 13 before Rotation test, Cylinder test, Rotarod Test, and Pole test were conducted after 2 weeks, 6 weeks, and 12 weeks post drug treatment. FIGs 8A to 8D show that drug MH treatment improves motor behavior performance in PD mice.

Example 11: Effect of MH Treatment on Body Weight and Vital Organ Index in PD Mice [0090] To show the effect of MH treatment on the body weight and vital organ index in PD mice, the following protocol was conducted. [0091] 6-OHDA (4 pg) was stereotaxi cal ly injected into 2-month-old C57 mice on the left striatum (day 0), and MH was administered via i.p. injection (0.5 mg/kg/day for 5 days, total dose=2.5mg/kg) or oral gavage (1 mg/kg/day for 5 days, total dose=5mg/kg) from day 8 to day 13 before behavioural tests.

[0092] The mice’s body weight was measured using a scale. The body weights were measured before and after 2, 6, and 12 weeks of drug treatment. After 12 weeks, the mice were sacrificed. The mice were anesthetized with a mixture of ketamine and xylazine hydrochloride, followed by trans-cardiac perfusion with PBS. After perfusion, the mice were dissected, and the weights of vital organs - including brain, heart, liver, thymus, spleen, lung, and kidney - were measured. The organ index was calculated as the ratio of organ weight to body weight. Body weight and organ index data were analyzed and plotted using GraphPad Prism The results are as shown in FIGs 9A to 9H. The results show that drug MH treatment does not affect body weight and vital organ index in mice.

Example 12: Effect of MH Treatment on Heart Tissues of PD Mice

[0093] 6-OHDA-induced PD mice were sacrificed after 12 weeks of drug MH treatment via i.p. injection (0.5 mg/kg for 5 days, totally 2.5mg/kg) or oral gavage (1 mg/kg for 5 days, totally 5mg/kg). Morphology of heart tissues was examined by hematoxylin and eosin (H&E) staining. No morphological changes or tissue damage was observed for both treatment groups. FIGs 10A to 10C show that treatment with drug MH does not induce morphological changes or tissue damage in the hearts of mice.

[0094] To investigate the possibility of cardiac fibrosis, Sirius red cardiac staining was conducted to detect cardiac fibrosis in heart tissues. No significant difference in cardiac fibrosis was observed among all 3 groups. N=3 mice per group, and 6 images/areas per mice. A total of 18 images were used for quantification of the collagen to skeletal muscle ratio. FIGs. HA to HD show that treatment with drug MH does not induce cardiac fibrosis in the hearts of mice. 1 [0095] Further, to investigate the possibility of inflammation, DAB immunohistochemistry staining was performed to detect inflammation status of heart tissues by using CD68 antibody. No significant difference in CD68+expression area was observed among all 3 groups FIGs. 12A to 12D show that treatment with drug MH does not induce inflammation in the heart tissue of mice.

[0096] Tn summary, the above Examples demonstrate that mitoxantrone hydrochloride (MH) is an inhibitor of APP and LRRK2. The inhibition of APP and LRRK2 promotes neuronal regeneration and alleviates motor behavioural deficits in mouse models of PD and AD, which strongly supports mitoxantrone hydrochloride’s therapeutic potential as an APP and LRRK2 inhibitor for the treatment of both sporadic and familial neurodegenerative disorders, including PD and AD.

[0097] Although embodiments of the invention have been shown and described, the invention is not limited to the described embodiments. Instead, it would be appreciated by those skilled in the art that various modifications and variations can be made to the embodiments of the invention without departing from the scope of the invention, the scoop of which is set forth in the following claims.

Claims

1. Use of mitoxantrone hydrochloride in the manufacture of a pharmaceutical composition for the treatment of neurodegenerative disorder in a subject, wherein the mitoxantrone hydrochloride is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject.

2. The use of claim 1 , wherein the mitoxantrone hydrochloride reduces the LRRK2 protein levels in vitro by at least 20% compared to untreated control.

3. The use of claim 1 , wherein the mitoxantrone hydrochloride reduces the LRRK2 protein levels by at least 20% in vivo.

4. The use of claim 1, wherein the mitoxantrone hydrochloride is formulated to modulate the LRRK2 protein levels by at least 20% in an ex vivo cell-based assay.

5. The use of any one of claims 1 to 4, wherein the mitoxantrone hydrochloride binds to one or more binding sites of LRRK2 selected from the group consisting of Alal904, Argl957, Lysl996, Leu2001, and Thr2035, for inhibiting LRRK2 kinase activity.

6. The use of claim 1, wherein the mitoxantrone hydrochloride reduces the APP protein levels in vitro by at least 20% compared to untreated control.

7. The use of claim 1, wherein the mitoxantrone hydrochloride reduces the APP protein levels by at least 20% in vivo.

8. The use of claim 1 , wherein the mitoxantrone hydrochloride is formulated to modulate the APP protein levels by at least 20% in an ex vivo cell-based assay.

9. The use of claim 1, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease.

10. The use of claim 1 , wherein the treatment promotes neuronal regeneration in the subj ect.

11. The use of claim 1 , wherein the treatment alleviates motor deficits in the subject.

12. A method of treating a neurodegenerative disorder, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof, wherein the mitoxantrone hydrochloride is an inhibitor of |3-amyloid precursor protein (APP) and leucine- rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in the subject.

13. The method of claim 12, wherein the mitoxantrone hydrochloride reduces the LRRK2 protein levels by at least 20% in vivo.

14. The method of claim 12, wherein the mitoxantrone hydrochloride binds to one or more binding sites of LRRK2 selected from the group consisting of Alal904, Argl957, Lysl996, Leu2001, and Thr2035, for inhibiting LRRK2 kinase activity.

15. The method of claim 12, wherein the mitoxantrone hydrochloride reduces the APP protein levels by at least 20% in vivo.

16. The method of claim 12, wherein the pharmaceutical composition or the pharmaceutically acceptable salt thereof is administered orally or intravenously.

17. The method of claim 12, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease.

18. The method of claim 12, wherein the treatment promotes neuronal regeneration in the subject.

19. The method of claim 12, wherein the treatment alleviates motor deficits in the subject.

20. A pharmaceutical composition comprising mitoxantrone hydrochloride or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative disorder, wherein the mitoxantrone hydrochloride or the pharmaceutically acceptable salt thereof is an inhibitor of P-amyloid precursor protein (APP) and leucine-rich repeat kinase 2 (LRRK2), capable of inhibiting APP and LRRK2 protein levels in a subject.

21. The pharmaceutical composition of claim 20, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease.