Combination therapy for alzheimer's disease

Abstract

This invention relates to the treatment of Alzheimer's disease in human patients, the treatment comprising administering an anti-Aβ antibody component and co-administering edaravone, the anti-Aβ antibody component being selected from anti-Aβ antibodies, Aβ-binding fragments of Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized Aβ antibodies.

Description

Technical Field

[0001] This invention relates to the treatment of Alzheimer's disease. In particular, this invention relates to the treatment of Alzheimer's disease by administering a combination of the following: (i) an anti-β-amyloid (Aβ) antibody component selected from anti-Aβ antibodies, Aβ-binding fragments of anti-Aβ antibodies, vectorized anti-Aβ antibodies and Aβ-binding fragments of vectorized anti-Aβ antibodies, and (ii) edaravone. Background Technology

[0002] Alzheimer's disease is well known to be associated with extracellular plaque deposits of β-amyloid peptide. β-amyloid protein (“Aβ”) is a peptide produced by the metabolism of amyloid precursor protein (APP). Several Aβ peptide isoforms exist (e.g., Aβ1-40, Aβ1-42, and pGlu Aβ3-42), and they exhibit different aggregation tendencies. Through fibrillation, soluble aggregates (oligomers) can transform into insoluble deposits with a β-sheet structure. These deposits are also known as amyloid plaques and are primarily composed of fibrillary amyloid protein.

[0003] Pathological Aβ accumulation is known to induce synaptic dysfunction and neurodegeneration. Aβ deposition can damage nerve cells through a variety of biological mechanisms, including oxidative stress, altered calcium homeostasis, pathological tau protein accumulation, and pro-apoptosis.

[0004] Based on evidence of Aβ's neurotoxicity and the potential adverse effects of Aβ load in the brain detected in humans by positron emission tomography (PET), Aβ has been a primary target for experimental therapies. Advances in passive anti-amyloid immunotherapy research include the identification of antibodies that promote microglia activation, catalyze depolymerization, and enhance the flow of Aβ from cerebrospinal fluid to plasma, thereby reducing Aβ's neurotoxicity.

[0005] Anti-Aβ antibodies used in Aβ immunotherapy are typically humanized IgG (e.g., IgG1, IgG2, IgG4) monoclonal antibodies that can bind to different epitopes and conformations of β-amyloid protein, such as monomers, oligomers, protofibrils, or fibrils. Anti-Aβ antibodies derived from lamas and sharks have also been proposed for the treatment of Alzheimer's disease.

[0006] Recent Phase 2 and 3 trials of third-generation anti-amyloid immunotherapies support their clinical efficacy in reducing brain Aβ burden and preventing cognitive decline. Recent trial data suggest these agents represent some of the first effective disease-modifying therapies for Alzheimer's disease and have prompted the FDA to recently grant accelerated approval to two anti-amyloid monoclonal antibodies, aducanumab and lecanemab. However, the clinical efficacy of these agents is modest and associated with amyloid-associated imaging abnormalities (ARIA).

[0007] He et al. Inhibitory effects of edaravone in β-amyloid-induced neurotoxicity in rats [Inhibitory effect of edaravone in β-amyloid-induced neurotoxicity in rats], Biomed Research Int, April 2, 2014, 1-7) Through a study of A β The study investigated the effects of edaravone on ADL (Alzheimer's disease) by measuring induced voltage-gated calcium channel currents in hippocampal CA1 pyramidal neurons (1-40), learning and memory behavior tests, the number of surviving cholinergic neurons in the basal forebrain, and acetylcholine levels in the hippocampus of this AD rat model. β Its role in an induced Alzheimer's disease rat model. Results showed that edaravone inhibited A in a dose-dependent manner. β 1 40 induced I Increased calcium levels. Treatment with edaravone significantly improved A. β 1 40. Induced learning and memory performance. Compared with non-edaravone-treated A β Compared with groups 1-40, administration increased the content of choline acetyltransferase-positive cells in the basal forebrain and acetylcholine in the hippocampus.

[0008] Jiao et al. (Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits. Proc Natl Acad Sci USA. April 21, 2015; 112(16):5225-30) reported that edaravone has a potent ability to inhibit Aβ accumulation and attenuate Aβ-induced oxidation in vitro. When administered via intraperitoneal injection before or after the onset of Aβ deposition, edaravone significantly reduced Aβ deposition, alleviated oxidative stress, mitigated downstream pathological changes, including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescued behavioral deficits in APPswe / PSI mice.

[0009] Ren et al. Edaravone exerts brain protective function by reducing the expression of AQP4, APP and Aβ proteins [Edaravone exerts neuroprotective function by reducing the expression of AQP4, APP, and Aβ proteins], Open Life Sci. 2019; 14: 651–65) investigated the changes in aquaporin 4 (AQP4), β-amyloid precursor protein (APP), and β-amyloid protein (Aβ) in the brain tissue of rats after cerebral ischemia-reperfusion injury (CIRI) and evaluated the effect of edaravone. The model group showed significant neurological deficits, with improved neurological function scores compared to the control group; while the edaravone group showed improved neurological deficits, as neurological function scores were lower than those in the model group. The levels of Aβ, APP, and AQP4 in the model group were significantly higher than those in the control and edaravone groups.

[0010] Fessel Cure of Alzheimer's Dementia in Many Patients by Using Intranasal Insulin to Augment an Inadequate Counter-Reaction, Edaravone to Scavenge ROS, and 1 or 2 Other Drugs to Address Affected Brain Cells [Many patients with Alzheimer's disease have been cured by using intranasal insulin to enhance the inadequate compensatory response, edaravone to clear ROS, and one or two other drugs to treat the affected brain cells], J. Clin. Med. 2023, 12, 3151. (https: / / doi.org / 10.3390 / jcml2093151) This indicates that Alzheimer's disease (AD) can be cured through the following combination therapy. First, intranasal insulin is used to enhance the body's natural compensatory response to the changes in brain cell types that lead to dementia. Second, edaravone is used to reduce free radicals, which increase in AD and are a cause of the disease. Third, one or two of pioglitazone, fluoxetine, and lithium are used, which act on the functionally altered brain cell types that lead to dementia.

[0011] Ramanan et al. Anti-amyloid therapies for Alzheimer disease: finally, Good news for patients [Anti-amyloid therapy for Alzheimer's disease: the final good news for patients], Molecular Neurodegeneration (2023) 18:42 https: / / d0i.0rg / l 0.1186 / si 3024-023-00637-0) According to reports, in a recent phase 3 trial for early symptomatic Alzheimer's disease (AD), lencanezumab and donepezumab slowed cognitive and functional decline and positively altered disease-specific biomarkers.

[0012] EP-A 3 290 525 describes a method for screening drugs and therapeutic targets for the treatment of Alzheimer's disease, including screening for drugs that can increase Aβ. 42 Drugs and therapeutic targets secreted by cells.

[0013] Mounting evidence suggests that Alzheimer's disease does not have a single pathogenic mechanism. Combination therapy is considered one of the most promising options for treating multifactorial diseases such as Alzheimer's. Summary of the Invention

[0014] The inventors unexpectedly discovered that co-administration of edaravone significantly improved the efficacy of anti-β-amyloid (anti-Aβ) antibody therapy (immunotherapy) for Alzheimer's disease.

[0015] Accordingly, a first aspect of the invention relates to an anti-Aβ antibody component for use in treating Alzheimer's disease in human patients, said anti-Aβ antibody component being selected from anti-Aβ antibodies, Aβ-binding fragments of Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized Aβ antibodies, wherein the treatment comprises administering the anti-Aβ antibody component and co-administering edaravone.

[0016] Not wanting to be bound by theory, the inventors of this invention hypothesize that anti-Aβ antibodies and edaravone produce a synergistic effect by acting on different pathways involved in the progression of Alzheimer's disease.

[0017] Another aspect of the invention relates to a pharmaceutical kit comprising a package containing one or more first dose units comprising (i) an anti-Aβ antibody component selected from anti-Aβ antibodies, Aβ-binding fragments of Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized Aβ antibodies, and (ii) one or more second dose units comprising edaravone.

[0018] Detailed description of the invention A first aspect of the invention relates to an anti-β-amyloid (Aβ) antibody component for use in treating Alzheimer's disease in human patients, said anti-Aβ antibody component being selected from anti-Aβ antibodies, Aβ-binding fragments of anti-Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized anti-Aβ antibodies, wherein the treatment comprises administration of the anti-Aβ component and co-administration of edaravone.

[0019] As used in this article, the term "treatment" encompasses: (a) To prevent the disease from occurring in subjects who have been identified as susceptible to the disease but have not yet been diagnosed with the disease; (b) Suppress the disease, such as stopping or slowing its progression (including maintenance therapy); (c) Alleviating the disease, for example, causing it to subside; and (d) Prolonged survival compared to expected survival without treatment.

[0020] Unless otherwise stated, the term "anti-Aβ antibody" as used herein refers to an antibody capable of binding to an epitope or conformation of Aβ and capable of reducing the accumulation of Aβ deposits in and / or removing Aβ deposits from the human brain. Examples of Aβ conformations that antibodies can bind to include monomeric, oligomeric, and fibrillary conformations of Aβ.

[0021] As used herein, the term "vectored anti-Aβ antibody" refers to a viral or non-viral vector that produces anti-Aβ antibodies in vivo. Similarly, as used herein, the term "Aβ-binding fragment of a vectored anti-Aβ antibody" refers to a viral or non-viral vector that produces such a fragment in vivo.

[0022] Unless otherwise stated, the term "immunotherapy" as used herein refers to the administration of an anti-Aβ antibody or an Aβ-binding fragment of an anti-Aβ antibody and / or treatment with an anti-Aβ antibody or an Aβ-binding fragment of an anti-Aβ antibody.

[0023] As used herein, the term "antibody gene therapy" refers to the administration of a vectorized antibody and / or treatment with a vectorized antibody. Unlike immunotherapy, antibody gene therapy typically requires only a single administration of a vectorized anti-Aβ antibody or an Aβ-binding fragment of a vectorized anti-Aβ antibody.

[0024] As used herein, the term “edaravone” refers to the substance 3-methyl-1-phenyl-2-pyrazolin-5-one, including its prodrug and pharmaceutically acceptable salt.

[0025] Whenever this article refers to the dosage of edaravone, the dosage refers to the amount of edaravone equivalent administered. Here, "edaravone equivalent" refers to the amount of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) contained in a given amount of edaravone salt.

[0026] As used herein, the term "co-administration of edaravone" refers to the simultaneous administration of the anti-Aβ antibody component with the administration of edaravone. Co-administration can be achieved by administering edaravone and the anti-Aβ antibody component separately or by administering a formulation containing both edaravone and the anti-Aβ antibody component.

[0027] This invention covers the treatment of human subjects with Alzheimer's disease and subjects identified as susceptible to this neurodegenerative disease. The treatment of this invention is preferably applied to human subjects already suffering from Alzheimer's disease.

[0028] The anti-Aβ antibodies used according to the present invention are preferably humanized IgG (e.g., IgG1, IgG2, IgG4) monoclonal antibodies capable of binding to different epitopes and conformations (e.g., monomeric, oligomeric, or fibrillary conformations) of β-amyloid protein. Examples of suitable anti-Aβ antibodies include: bapineuzumab, solanezumab, gantenerumab, crenezumab, ponezumab, solanezumab, lencanezumab, BAN2401, donanemab, and adunazab. The present invention covers the use of the Aβ-binding fragments of these antibodies as well as the use of these antibodies or fragments in vectorized forms.

[0029] The anti-Aβ antibody is preferably selected from the group comprising: barpinizumab, sorazizumab, gantelinumab, crifenizumab, pokonnetumab, sorazizumab, lencanemab, adunatumab, donepemumab, and combinations thereof. More preferably, the anti-Aβ antibody is selected from lencanemab, adunatumab, donepemumab, and combinations thereof.

[0030] The vector used for vectorized anti-Aβ antibodies or Aβ-binding fragments of vectorized Aβ antibodies is preferably a viral vector, and more preferably an adeno-associated virus (AAV)-based vector. The use of AAV-based vectors offers the advantage of efficiently delivering Aβ antibodies or fragments thereof in the brain.

[0031] The treatment of the present invention may be suitably performed using a prodrug or edaravone. Examples of edaravone prodrugs can be found in WO2019 / 213335, WO 2020 / 060092, WO 2021 / 107686 and CN115073518A.

[0032] The edaravone used according to the present invention is preferably selected from 3-methyl-1-phenyl-2-pyrazolin-5-one and its pharmaceutically acceptable salts. Most preferably, the edaravone used is 3-methyl-1-phenyl-2-pyrazolin-5-one.

[0033] Treatment according to the invention preferably includes oral, intraoral, or intravenous administration of edaravone. Examples of intraoral administration include sublingual, buccal, and sublipal administration.

[0034] Preferably, edaravone is administered at a dose of 10-800 mg, more preferably 20-400 mg, and even more preferably 50-200 mg.

[0035] According to particularly preferred embodiments, edaravone is administered at a daily dose of 10-800 mg, more preferably 20-400 mg, and even more preferably 50-200 mg.

[0036] Edaravone is preferably administered intravenously at a dose of 0.2-5 mg edaravone / kg body weight / day, more preferably 0.3-3 mg edaravone / kg body weight / day, and most preferably 0.4-2 mg edaravone / kg body weight / day.

[0037] Edaravone is preferably administered orally at a dose of 0.3-15 mg edaravone / kg body weight / day, more preferably 0.4-10 mg edaravone / kg body weight / day, and most preferably 1-5 mg edaravone / kg body weight / day.

[0038] In the treatment of this invention, edaravone is preferably administered at a dose sufficient to achieve a plasma concentration of 250-2,500 ng·h / mL, more preferably 500-1,500 ng·h / mL.

[0039] Preferably, edaravone is administered at least once a day, and most preferably once a day.

[0040] According to one embodiment, edaravone is administered in the form of an aqueous solution of edaravone.

[0041] According to another embodiment, edaravone is administered orally or intraorally in the form of solid dose units. More preferably, edaravone is administered orally.

[0042] In one embodiment of the invention, treatment includes administration of an anti-Aβ antibody or an Aβ-binding fragment thereof. According to a particularly preferred embodiment, treatment includes administration of an anti-Aβ antibody or an Aβ-binding fragment thereof and co-administration of edaravone for a period of at least 4 weeks, preferably at least 8 weeks, more preferably at least 18 weeks, and even more preferably at least 40 weeks. In some embodiments, treatment includes administration of an anti-Aβ antibody or an Aβ-binding fragment thereof and co-administration of edaravone until the patient's death. The combined administration period may be interrupted by one or more holiday periods.

[0043] Anti-Aβ antibodies or their Aβ-binding fragments can be administered intravenously, subcutaneously, or intramuscularly. According to a preferred embodiment, the anti-Aβ antibody or Aβ-binding fragment is administered intravenously.

[0044] Typically, anti-Aβ antibodies or their Aβ-binding fragments are administered at doses of 10-1,200 mg / μg, preferably 30-1,000 mg, and more preferably 40-800 mg.

[0045] According to preferred embodiments, the anti-Aβ antibody or its Aβ-binding fragment is administered at an amount of 0.5-40 μg / kg body weight, more preferably 1-30 mg μg / kg body weight, and most preferably 2-20 mg μg / kg body weight.

[0046] The synergistic interaction between the anti-Aβ antibody component and edaravone enables treatment with reduced doses or frequency of anti-Aβ antibody administration.

[0047] According to a preferred embodiment, the combination therapy of the present invention comprises intravenous administration of adunatumab once every 4 weeks for a period of more than 1 year, wherein the dose of adunatumab administered during this period does not exceed 6 mg / kg, more preferably not more than 4 mg / kg.

[0048] According to another preferred embodiment, the combination therapy of the present invention includes intravenous administration of adunatumab every 5-8 weeks for a period of more than 1 year, wherein the dose of adunatumab administered during this period does not exceed 10 mg / kg, more preferably not more than 8 mg / kg.

[0049] According to a preferred embodiment, the combination therapy of the present invention comprises intravenous administration of lencanemab once every 2 weeks for a period of more than 20 weeks, wherein the dose of lencanemab administered during this period does not exceed 6 mg / kg, more preferably not more than 4 mg / kg.

[0050] According to another preferred embodiment, the combination therapy of the present invention comprises intravenous administration of lencanizumab once every 3-5 weeks for a period of at least 20 weeks, wherein the dose of lencanizumab administered during this period does not exceed 12 mg / kg, more preferably not exceeding 10 mg / kg.

[0051] Preferably, in the treatment according to the invention, the patient is given an anti-Aβ antibody or its Aβ-binding fragment at least once every 2-8 weeks, more preferably at least once every 2-6 weeks.

[0052] In a preferred embodiment, the anti-Aβ antibody or its Aβ-binding fragment is administered in the form of an aqueous solution of the antibody or fragment.

[0053] In another embodiment of the invention, the treatment comprises administering a carrier-coated anti-Aβ antibody or an Aβ-binding fragment of a carrier-coated anti-Aβ antibody. The carrier-coated anti-Aβ antibody or the Aβ-binding fragment of a carrier-coated Aβ antibody is preferably administered intravenously.

[0054] The treatment of the present invention preferably includes a single administration of a vectorized anti-Aβ antibody or a vectorized Aβ-binding fragment of the antibody.

[0055] The Alzheimer's disease treated according to the present invention is preferably selected from mild Alzheimer's disease, early Alzheimer's disease, pre-Alzheimer's disease, mild Alzheimer's dementia, or mild cognitive impairment caused by Alzheimer's disease.

[0056] Aβ immunotherapy carries a significant risk of developing amyloid-associated imaging abnormalities (ARIA). So-called ARIA events can manifest as cerebral edema following immunotherapy, which is detected as abnormal on MRI. ARIA encompasses two types of MRI signal abnormalities: ARIA-edema / effusion (ARIA-E) and ARIA-hemosiderin deposition / microbleeds (ARIA-H). ARIA-E refers to extravasation of fluid leading to interstitial vasogenic edema or sulcal effusion in the pia mater / subpia mater. ARIA-H refers to microbleeds (mH) or large hemorrhages observed as low-signal hemosiderin deposition. Data suggest that ARIA events, particularly vascular leakage (such as microbleeds), may be associated with vascular Aβ clearance induced by anti-β-amyloid (Aβ) antibody therapy (Zago et al. Vascular alterations in PDAPP mice after anti-Aβ immunotherapy: Implications for amyloid-related imaging abnormalities. Alzheimers Dement. 2013 Oct;9(5 Supplement):S105-15).

[0057] According to a preferred embodiment of the invention, co-administration of edaravone achieves a reduction in amyloid-associated imaging abnormalities (ARIA). The reduction in ARIA events can be clinically determined, for example, by MRI.

[0058] According to a particularly preferred embodiment, the treatment according to the invention includes the step of determining the presence of ARIA events in patients who have received administration of the anti-Aβ antibody component prior to initiating co-administration of edaravone.

[0059] Unlike the treatment of EP-A 3 290 525, the treatment of Alzheimer's disease of the present invention preferably does not include the administration of antibodies against BO / EFR3 / EFR3A / EFR3B, antibodies against PI4KIIIα, antibodies against TTC7, antibodies against PI4P, or antibodies against [other substances]. rbo / Efr3 / Efr3a / Efr3b Genes possess specific repressive polypeptides, which... PI4KIIIα / PI4KA Genes possess specific inhibitory peptides or small molecule compounds that inhibit the phosphokinase activity of PI4KIIIα protein.

[0060] Another aspect of the invention relates to a pharmaceutical kit comprising a package containing one or more first dose units comprising (i) an anti-Aβ antibody component selected from anti-Aβ antibodies, Aβ-binding fragments of anti-Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized anti-Aβ antibodies, and (ii) one or more second dose units comprising edaravone.

[0061] The kit preferably contains 1 to 4, more preferably 1 to 3, even more preferably 1 or 2, and most preferably one first dose unit.

[0062] The first dose unit is preferably an aqueous solution of the anti-Aβ antibody component, more preferably a sterile aqueous solution of the anti-Aβ antibody component, and even more preferably a sterile isotonic aqueous solution of the anti-Aβ antibody component.

[0063] The first dose unit preferably comprises an aqueous solution containing at least 10 mg / mL, more preferably 20-250 mg / mL, and most preferably 40-160 mg / mL of anti-Aβ antibody or its Aβ-binding fragment.

[0064] The first dose unit preferably comprises 1-50 mL, more preferably 1.3-20 mL, and most preferably 1.5-10 mL of an aqueous solution of anti-Aβ antibody or its Aβ-binding fragment.

[0065] The anti-Aβ antibody is preferably selected from the group comprising: barpinizumab, sorazizumab, gantelinumab, crifenizumab, pokonnetumab, sorazizumab, lencanemab, adunatumab, donepemumab, and combinations thereof. More preferably, the anti-Aβ antibody is selected from lencanemab, adunatumab, donepemumab, and combinations thereof.

[0066] In a preferred embodiment, the kit contains a plurality of second dose units. More preferably, the kit contains at least 7, more preferably at least 14 second dose units. Preferably, the total number of second dose units in the kit does not exceed 56, more preferably not more than 28.

[0067] In one embodiment of the present invention, the second dosage unit is preferably an aqueous solution of edaravone, and more preferably a sterile aqueous solution of edaravone.

[0068] The second dosage unit preferably comprises an aqueous solution containing at least 0.5 mg / mL, more preferably 0.8-5 mg / mL, and most preferably 1-2.5 mg / mL of edaravone.

[0069] The second dose unit preferably contains 10-200 mL, more preferably 20-150 mL, and most preferably 25-100 mL of an aqueous solution of edaravone.

[0070] In another embodiment of the invention, the second dosage unit is a solid dosage unit suitable for oral or intraoral administration. Preferably, the solid dosage unit contains 5-400 mg, more preferably 10-200 mg, and even more preferably 20-150 mg of edaravone.

[0071] Preferably, the packaging includes instructions for administering the first and second dose units to a human patient. The instructions may be in the form of, for example, an insert, a package insert, or a graphic symbol for electronic access (such as a barcode, QR code, etc.).

[0072] The instructions for use preferably specify that the first dose unit should be administered intravenously, subcutaneously, or intramuscularly, with intravenous administration being the most preferred.

[0073] Preferably, the instructions for use indicate that the first dose unit is administered at least once every 4 weeks, more preferably at least once every 2 weeks.

[0074] The instructions for use preferably specify that the second dose unit be administered intravenously, orally, or orally, with intraoral or oral administration being the most preferred method.

[0075] Preferably, the instructions for use indicate that the second dose unit will be administered at least once a day, and most preferably once a day.

[0076] According to a particularly preferred embodiment, the first dose unit is an aqueous solution containing 10-1,200 mg of an anti-Aβ antibody component, preferably an anti-Aβ antibody or its Aβ-binding fragment, and the second dose unit is a solid dose unit containing 5-400 mg of edaravone.

[0077] The drug kit of the present invention preferably does not contain antibodies against RBO / EFR3 / EFR3A / EFR3B, antibodies against PI4KIIIα, antibodies against TTC7, antibodies against PI4P, or antibodies against [other specific antibodies]. rbo / Efr3 / Efr3a / Efr3b Genes possess specific repressive polypeptides, which... PI4KIIIα / PI4KA The dosage unit of a gene-specific inhibitory polypeptide or a small molecule compound that inhibits the phosphokinase activity of the PI4KIIIα protein.

[0078] The invention is further illustrated by the following non-limiting examples.

[0079] Example Example 1 To evaluate the potential of edaravone's cross-protective activity against pathological Aβ in an in vitro AD model. The in vitro AD model consisted of cell cultures of primary mouse neurons incubated with brain tissue components from human Alzheimer's disease (AD) patients.

[0080] plan Preparation of immune-depleted brain tissue components from AD patients Brain extracts were prepared and immunodepleted using lencanemab, aducanamab, donepemab, or a control. Brain tissue components were biochemically characterized, and toxicity assays were performed in primary mouse neuron cultures using an automated high-content microscope. Detailed protocols are described below.

[0081] Post-mortem human brain tissue was used from the Netherlands Brain Bank. The type of material selected was freshly frozen tissue blocks (approximately 3 grams of gray matter w / w). A total of ten tissue blocks were used; five from AD donors and five from age-matched non-dementia controls (without β-amyloid pathological changes). Brain tissue from the temporal, frontal, and parietal lobes was used, and gray matter was selectively separated.

[0082] To confirm the presence of AD pathological changes in brain tissue, the levels of Aβ and p-tau were measured using immunohistochemistry (IHC). Five-fold sections were cut from each tissue block. Co-pathological extent was assessed using IHC: mouse primary mAbs with N-terminal aa 1–16 (clone IC16, internal), 4G8, and AT8 (phosphate-p-phospho-taupSer 202 + Thr 205) were used for Aβ and p-tau, respectively; and Dako Envision goat anti-mouse / rabbit-HRP K5007 was used as a secondary antibody. Visual examination and semi-quantitative assessment using pathological scoring were used as readouts.

[0083] Brain tissue extracts were prepared to generate in vitro AD and control cell phenotypes. The methods used were based on those of Hong et al. (Acta Neuropathologica [Neurological Journal] (2018)) and optimized and validated in the Wiep Scheper laboratory (Sandberg et al., Alzheimers Res Ther [Alzheimer's Disease Research and Treatment]. 2022). Human brain tissue samples were homogenized in artificial cerebrospinal fluid (aCSF), centrifuged at 22,000 g for 110 min, and the supernatant was collected for subsequent analysis. Whole brain extracts (supernatant) were dialyzed using a Slide-Al-Lyzer dialysis kit (2K MWO), aliquoted, rapidly frozen, and stored at -80°C.

[0084] Based on previous research (Sandberg et al., 2022), only the H component was isolated. The H components from all AD or control brain tissues were combined to obtain the following components: combined H component controls, and combined H component AD cases (2 samples).

[0085] For immunodepletion, brain extract samples (500 μl) were incubated overnight at 4°C with 1 μg mAb under continuous rotation. The next day, 50 μl of 10% Protein G-agarose beads in PBS was added, and the mixture was incubated at 4°C under rotation for 4 h. Brain extracts were immunodepleted with lencanemab, adunatumab, donepemab, a positive control (4G8), or a negative isotype control, matching the levels of lencanemab, adunatumab, and donepemab. Subsequently, the samples were centrifuged at 10,000 g for 10 min at 4°C to remove the beads. The supernatant was collected, rapidly frozen in liquid nitrogen, and stored at -80°C until use.

[0086] To characterize the Aβ profile in different obtained brain tissue components, the following measurements were performed: • The total protein content of the brain extract was measured using the Pierce BCA Protein Kit, according to the manufacturer's protocol.

[0087] •Aβ 40 and Aβ 42 MSD Immunoassay: Aβ was measured using the MesoScale Discovery (MSD) platform according to the manufacturer's protocol, using the β-amyloid peptide group 1 kit (6E10: Aβ42, Aβ40, Aβ38). 40 and Aβ 42 concentration.

[0088] Preparation of neuronal cell cultures Following an optimized protocol (Sandberg et al., 2022), wild-type mouse embryonic neurons from day 18.5 were used for in vitro cell culture. Briefly, the cortex of E18.5 wild-type mice was dissected, digested with trypsin, washed, and plated at a cell density of 15 K / well in 96-well plates with a black background in Neurobasal medium.

[0089] Determine the optimal edaravone dosage To establish the optimal edaravone dose for testing, embryonic neurons (without any brain extract) were treated with menadione (4 h) to induce reactive oxygen species (ROS) in the cells. Next, neurons were incubated with different doses of edaravone or the mediator (30 min / 1 h), after which the ROS response was measured: during the last 30 min of incubation, cells were loaded with CellROX green reagent (5 μM). Fluorescence was measured in the dapi ROI. Technique replicated: 3 times, all within one plate. The dose of edaravone used for further testing will be selected based on the ROS response.

[0090] Determine the combined effect of anti-Aβ antibody and edaravone Using different test settings, control and immune-depleted brain tissue components were inoculated onto mouse neurons to establish an in vitro AD model.

[0091] Test setup 1: Immune-depleted brain tissue components were incubated with edaravone. First, control and immune-depleted brain tissue components were pre-incubated with the optimal dose of edaravone or the appropriate medium. Then, the pre-incubated brain tissue components were added to neuronal cell cultures.

[0092] Test setup 2: Adding edaravone to an in vitro AD model Control and immune-depleted brain tissue components were added to the cell culture. Additionally, the optimal dose of edaravone or a carrier was added.

[0093] Neuron morphology and activity: To assess neuronal phenotype, high-content automated microscopy, including neurite growth / length / dendriticization and synaptic properties, was performed on a Cell Insight CX7 HCS platform. Neurons were incubated with primary antibodies diluted in blocking solution at 4°C for 16–17 h. The primary antibodies used were vesicle glutamate transporter 1 (vGlut 1) for presynapses and microtubule-associated protein 2 (Map 2) for dendrites. After washing with PBS for 5 min, the nuclei were stained with 4′,6-diamidinyl-2-phenylindole (DAPI) diluted in PBS (1:1000). Finally, the plates were stored in PBS at 4°C until microscopic analysis.

[0094] High-content automated microscopy was then performed to obtain 25 images per well. Images were analyzed using Columbus 2.5 software (PerkinElmer). Nuclei were detected in the DAPI channel, and the nucleus count was used as a measure of neuronal viability / loss. Map2-based regions of interest (ROIs) were used to determine the number of neurite segments, branches (node ​​type 1), and terminals. The number of synapses was measured in the vGlut1 channel within the Map2-based ROI. The values ​​of all analyzed morphological features were normalized relative to the number of nuclei and expressed as a percentage change compared to control conditions.

[0095] Oxidative stress assay: To determine ROS levels, cells were loaded with CellROX green reagent (5 μM) during the last 30 min of incubation. Fluorescence was measured in the dapi ROI.

[0096] Evaluation of the combined effect The data obtained in this way showed that, compared with the non-AD control, treatment with AD brain extract caused a significant increase in the loss of dendritic segments and branches, as well as the number of vGlut1 points at the presynapse of each neuron, demonstrating the neurotoxicity of AD brain extract.

[0097] The data further showed that, compared with non-immunodepleted AD brain extract, immunization with anti-Aβ antibody significantly reduced the loss of dendritic segments and branches as well as presynapses, demonstrating the neuroprotective effect associated with Aβ depletion.

[0098] The data also showed that in immune-depleted AD brain tissue fractions treated with edaravone, the loss of dendritic segments and branches as well as presynapses was further reduced compared with untreated immune-depleted AD brain extracts, demonstrating that edaravone has an enhancing effect when combined with immune depletion using anti-Aβ antibodies.

[0099] Example 2 In animal models (ADPP mice), vascular alterations associated with anti-Aβ immunotherapy (with or without edaravone) were assessed by quantifying leptomeningeal Vaβ, capillary Aβ, and microbleeds, as described by Zago et al. Vascular alterations in PDAPP mice after anti‐Aβ immunotherapy: Implications for amyloid‐related imaging abnormalities [Vascular changes in PDAPP mice following anti-Aβ immunotherapy: significance for amyloid-related imaging abnormalities]. Alzheimers Dement, 2013;9(5S). Three different anti-Aβ antibodies were tested: lencanemab, adunatumab, or donenetumab.

[0100] In particular, Aβ-deposited corticopiemal vessels were assessed by counting the number of all amyloid-containing vessels in the brain slices. Similar to corticopiemal vessels, Aβ-deposited capillaries were assessed. Microbleeds were quantified to determine the presence, amount, location, and intensity of hemosiderin staining throughout the brain slices.

[0101] The results showed that, compared with the sample representing anti-Aβ immunotherapy (without edaravone), the combination of anti-Aβ immunotherapy and edaravone administration significantly reduced Aβ deposition in cortical leptomeningeal vessels and capillary deposition in brain slices. Furthermore, microbleeds were significantly reduced when edaravone was combined with immunotherapy. These results support the use of combination therapy to reduce the ARIA effect in patients undergoing anti-Aβ therapy.

[0102] Example 3 The potential of edaravone anti-Aβ antibody cross-protective activity against pathological Aβ was evaluated in the in vitro AD model described in Example 1.

[0103] Postmortem human brain tissue was obtained from a Dutch brain bank as fresh frozen tissue blocks (approximately 3 g white matter w / w). Each tissue sample was sectioned for neuropathological confirmation in either AD or non-AD (control) cases, followed by the preparation of brain extracts. A total of ten tissue blocks were used; five from AD donors and five from age-matched non-dementia controls (without β-amyloid pathological changes).

[0104] To confirm the presence of AD pathological changes in brain tissue, the levels of Aβ and p-tau were measured using immunohistochemistry (IHC). Five-fold sections were cut from each tissue block. Co-pathological severity was assessed using IHC: mouse primary mAbs with N-terminal aa 1–16 (clone IC16, internal), 4G8, and AT8 (phosphate-p-phospho-taupSer 202 + Thr 205) were used for Aβ and p-tau, respectively; and Dako Envision goat anti-mouse / rabbit-HRP K5007 was used as a secondary antibody. Visual examination and semi-quantitative assessment of pathological scoring were used as readouts. Results confirmed the presence of AD pathological changes in 5 AD donors and the absence of AD pathological changes in 5 non-dementia controls.

[0105] Brain tissue extracts were prepared to generate in vitro AD and control cell phenotypes. The methods used were based on those of Hong et al. (Hong et al., Acta Neuropathologica [Neurological Journal] (2018)), optimized and validated in the Wiep Scheper laboratory (Sandberg et al., Aβ42 oligomer-specific antibody ALZ-201 reduces the neurotoxicity of Alzheimer's disease brain extracts[ALZ-201, an Aβ42 oligomer-specific antibody, reduces the neurotoxicity of brain extracts from Alzheimer's disease patients], Alzheimers Res Ther [Alzheimer's Disease Research and Treatment]. (2022) 14:196 https: / / doi.org / 10.1186 / s13195-022-01141-1). Brain extracts (supernatant) were dialyzed using a Slide-Al-Lyzer dialysis kit (2K MWCO) (Thermo Fisher Scientific), aliquoted, rapidly frozen, and stored at -80°C. The total protein content of the brain extracts was measured using the Pierce BCA protein kit according to the manufacturer's protocol (Thermo Fisher Scientific). Following the manufacturer's protocol (Meso Scale Diagnostics), the concentrations of Aβ40 and Aβ42 were determined using the MesoScale Discovery platform and the Amyloid-β Peptide Mechanism 1 kit (4G8: Aβ42, Aβ40, Aβ38). Only the H fraction was isolated. The H fractions obtained from AD brain tissue were combined. Similarly, the H fractions obtained from control brain tissue were also combined.

[0106] For immune depletion, brain extract samples (500 μl) were incubated overnight at 4°C with 1 μg mAb under continuous rotation. The next day, 50 μl of 10% Protein G agarose beads (Abcam) in PBS was added, and the mixture was incubated at 4°C under rotation for 4 h. Subsequently, the sample was incubated at 4°C with 100... g Centrifuge for 5 min to remove beads. Collect the supernatant, rapidly freeze in liquid nitrogen, and store at -80°C until use. Immunohistolysis of brain extracts was performed using three different anti-Aβ antibodies: lencanemab (purchased from Selleckchem), donepemab (purchased from Selleckchem), and 4G8 (purchased from BioLegend).

[0107] Preparation of neuronal cell cultures Wild-type mouse embryos obtained by cesarean section from pregnant females at timed mating were used for primary neuronal culture. The cortex was dissected in Hanks buffered saline (HBSS; Sigma-Aldrich) containing 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer (Gibco) (Hanks-HEPES) and digested at 37°C for 20 min with the addition of 0.25% trypsin (Gibco). Digested tissues were washed three times in Hanks-HEPES and then dispersed by grinding in fire-polished Pasteur pipettes in Dalberg Modified Eagle Medium (DMEM) supplemented with 4.5 g / L glucose and UltraGlutamine I (Lonza), 10% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin / strepmycin (Pennisex; Fisher Scientific), and 1% non-essential amino acid solution (Fisher Emergo) (DMEM+). Dissociated cells were rotated to sedimentation, resuspended, and plated in NB (Fisher Scientific) supplemented with 2% B-27 (Life Technologies), 18 mM HEPES, 0.25% glutamax (Fisher Scientific), and 0.1% Pennisex (NB+). The resuspended cells were seeded at a density of 12.5 K / well in 96-well plates with a black substrate (Greiner Bio-One BV) and cultured at 37°C and 5% CO2.

[0108] Determine the optimal edaravone dosage and its compatibility with neuron culture. Experimental studies were conducted in parallel to determine the optimal edaravone concentration. All readouts were optimized for a 96-well format. Mouse neurons (without any brain extract) were treated with different edaravone concentrations (1, 3, 10, 30, and 100 µM) and mediators (30 min / 1 h). Neuronal cells were treated with menadione (33, 66, and 100 uM) for 4 h to induce reactive oxygen species (ROS).

[0109] The effect of edaravone on ROS response was measured by loading cells with CellROX green reagent (5 μM; Thermo Fisher Scientific) during the last 30 minutes of incubation. The procedure used was described by Batenburg et al. Intraneuronal tau aggregation induces the integrated stress response in astrocytes[Tau protein aggregation in neurons induces integrated stress response in astrocytes], Journal of Molecular Cell Biology, Vol. 14, No. 10, October 2022, mjac071 https: / / doi.org / 10.1093 / jmcb / mjac071 Fluorescence was measured in the dapi (nuclear) region of interest (ROI). Edaravone showed no neurotoxicity at the tested concentrations. The optimal edaravone concentration for inhibiting ROS without neurotoxicity was 100 µM.

[0110] Determine the combined effect of anti-Aβ antibody and edaravone The combined H brain tissue components were added to mouse neuronal cells on day 16 in vitro (DIV) along with edaravone (100 µM) or a mediator, as described by Sandberg et al. Aβ42 oligomer-specific antibody ALZ- 201 reduces the neurotoxicity of Alzheimer's disease brain extracts [ALZ-201, an Aβ42 oligomer-specific antibody, reduces the neurotoxicity of brain extracts from Alzheimer's disease patients], Alzheimers Res Ther [Alzheimer's Disease Research and Treatment]. (2022) 14:196 https: / / doi.org / 10.1186 / s13195-022-01141-1). Cells were fixed at DIV 17 by replacing half the volume of culture medium with 1.85% paraformaldehyde (PFA; Merck Millipore) in PBS at pH 7.4 for 10 min. Subsequently, the culture medium was removed, PFA was added, and the medium was replaced with 3.7% PFA, and the incubation period was 10 min at room temperature.

[0111] The results obtained from the oxidative stress assay are shown in Figure 1 These results indicate that treatment with AD brain extract induced a significant increase in ROS levels in neuronal cells compared to treatment with non-AD brain extract (control), demonstrating the neurotoxicity of AD brain extract compared to brain extract from healthy subjects (control). The ROS levels observed in these cell cultures were equivalent to those produced by the strong oxidative stress inducer menadione.

[0112] Data showed that when edaravone was added, ROS levels were further reduced in immune-depleted AD brain tissue components.

[0113] In this experiment, the beneficial effect of edaravone was observed at abnormally high ROS concentrations. A more pronounced beneficial effect is expected at ROS levels corresponding to those observed better in the brains of AD patients.

Claims

1. An anti-β-amyloid (Aβ) antibody component for use in treating Alzheimer's disease in human patients, said anti-Aβ antibody component being selected from anti-Aβ antibodies, Aβ-binding fragments of anti-Aβ antibodies, vectorized anti-Aβ antibodies, and Aβ-binding fragments of vectorized anti-Aβ antibodies, wherein the treatment comprises administration of the anti-Aβ component and co-administration of edaravone.

2. The anti-Aβ antibody component for use according to claim 1, wherein the treatment comprises administering the anti-Aβ antibody component and co-administering edaravone over a period of at least 4 weeks.

3. The anti-Aβ antibody component for use according to claim 1 or 2, wherein the anti-Aβ antibody is selected from barpinizumab, sorazizumab, gantenerumab, crifenizumab, ponozumab, sorazizumab, lencanemab, adunatumab, donenetumab, and combinations thereof.

4. The anti-Aβ antibody component for use according to claim 3, wherein the anti-Aβ antibody is lencanezumab.

5. The anti-Aβ antibody component for use according to claim 3, wherein the anti-Aβ antibody is adunatumab.

6. The anti-Aβ antibody component for use according to claim 3, wherein the anti-Aβ antibody is donepemab.

7. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein the anti-Aβ antibody component is administered intravenously, subcutaneously, or intramuscularly.

8. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein the patient administers the anti-Aβ antibody component at least once every 4 weeks.

9. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein the treatment comprises oral, intraoral, or intravenous administration of edaravone.

10. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein edaravone is administered at a dose of 10-800 mg.

11. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein edaravone is administered at least once daily.

12. The anti-Aβ antibody component for use according to any of the preceding claims, wherein co-administration of edaravone achieves a reduction in amyloid-associated imaging abnormalities (ARIA).

13. The anti-Aβ antibody component for use according to any one of the preceding claims, wherein the treatment does not include administration of antibodies against RBO / EFR3 / EFR3A / EFR3B, antibodies against PI4KIIIα, antibodies against TTC7, antibodies against PI4P, or antibodies against... rbo / Efr3 / Efr3a / Efr3b Genes possess specific repressive polypeptides, which... PI4KIIIα / PI4KA Genes possess specific inhibitory peptides or small molecule compounds that inhibit the phosphokinase activity of PI4KIIIα protein.

14. A pharmaceutical kit comprising a package containing one or more first dose units comprising (i) an anti-Aβ antibody component selected from anti-Aβ antibodies, an Aβ-binding fragment of an anti-Aβ antibody, a vectorized anti-Aβ antibody, and an Aβ-binding fragment of a vectorized anti-Aβ antibody, and (ii) one or more second dose units comprising edaravone.

15. The pharmaceutical kit of claim 14, wherein the package contains instructions for use for administering the first dose unit and the second dose unit to a human patient.

16. The pharmaceutical kit according to claim 14 or 15, wherein the first dose unit is an aqueous solution containing 10-1,200 mg of an anti-Aβ antibody component, and the second dose unit is a solid dose unit containing 5-400 mg of edaravone.

17. The pharmaceutical kit according to any one of claims 14-16, wherein, This kit does not contain antibodies against RBO / EFR3 / EFR3A / EFR3B, PI4KIIIα, TTC7, or PI4P. rbo / Efr3 / Efr3a / Efr3b Genes possess specific repressive polypeptides, which... PI4KIIIα / PI4KA The dosage unit of a gene-specific inhibitory polypeptide or a small molecule compound that inhibits the phosphokinase activity of the PI4KIIIα protein.

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