Combination treatment of alzheimer's disease

Abstract

The invention relates to the treatment of Alzheimer's disease in a human patient, said treatment comprising administration of an anti-Aβ antibody component and co-administration of edaravone, the anti-Aβ antibody component being selected from anti-Aβ antibody, an Aβ- binding fragment of an Aβ antibody, a vectorised anti-Aβ antibody and a vectorised Aβ- binding fragment of an Aβ antibody.

Description

[0001] COMBINATION TREATMENT OF ALZHEIMER’S DISEASE

[0002] Field of the Invention

[0003] The present invention relates to the treatment of Alzheimer’s disease. In particular, the invention relates to the treatment of Alzheimer’s disease by administering a combination of (i) an anti-amyloid-beta (Ap) antibody component selected from anti-Ap antibody, an Ap-binding fragment of an anti-Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an anti-Ap antibody, and (ii) edaravone.

[0004] Background of the Invention

[0005] It is well established that Alzheimer’s disease is associated with extracellular plaque deposits of the p-amyloid peptide. Amyloid beta (“Ap”) is a peptide generated from the metabolism of amyloid precursor protein (APP). Several Ap peptide alloforms exist (e.g. Api-40, Api-42 and pGlu AP3-42), and they present a variable tendency to aggregate. Through fibrillogenesis, soluble aggregates (oligomers) may transition into insoluble deposits having a p-pleated sheet structure. These deposits are also referred to as amyloid plaques and are composed of predominantly fibrillar amyloid.

[0006] Pathological Ap accumulation is known to induce synaptic dysfunction and neurodegeneration. Ap deposition can damage nerve cells through a variety of biological mechanisms including oxidative stress, alterations in calcium homeostasis, pathological tau accumulation and pro-apoptosis.

[0007] Ap has been the main target of experimental therapies, based on evidence of the neurotoxic effects of Ap, and of the potential adverse effects of brain Ap burden detected in humans in vivo by positron emission tomography (PET). Progress on passive anti-amyloid immunotherapy research includes identification of antibodies that facilitate microglial activation, catalytical disaggregation, and increased flow of Ap from cerebrospinal fluid to plasma, thus decreasing the neurotoxic effects of Ap.

[0008] The anti-Ap antibodies used in Ap immunotherapy are typically humanized IgG (e.g. lgG1 , I gG2 , lgG4) monoclonal antibodies capable of binding to different epitopes and conformations of amyloid-p such as monomer, oligomer, protofibril or fibril conformations. Also anti-Ap antibodies from lama and shark have been proposed for use in the treatment of Alzheimer’s. Recently completed phase 2 and 3 trials of 3rdgeneration anti-amyloid immunotherapies are supportive of their clinical efficacy in reducing brain Ap burden and preventing cognitive decline. Data from recent trials implicate these agents as the first effective disease-modifying therapies against Alzheimer’s disease and has led to the US FDA recent approval of two antiamyloid monoclonal antibodies, aducanumab and lecanemab, under an accelerated approval pathway. The clinical effects of these agents are modest, however, and associated with amyloid-related imaging abnormalities (ARIA).

[0009] He et al. (Inhibitory effects of edaravone in / 3-amyloid-induced neurotoxicity in rats, Biomed Res Int, 2 April 2014, 1-7) investigated the effects of edaravone in an A^-induced rat model of Alzheimer’s disease by studying A / ?1-40-induced voltage-gated calcium channel currents in hippocampal CA1 pyramidal neurons, learning and memory behavioural tests, the number of surviving cholinergic neurons in the basal forebrain, and the acetylcholine level in the hippocampus in this rat model of AD. The results showed that the A / ?1-40-induced increase of / Ca can be inhibited by edaravone in a dose-dependent manner. Treatment with edaravone significantly improved A / ?1-40-induced learning and memory performance. Choline acetyltransferase positive cells in basal forebrain and acetylcholine content in the hippocampus were increased by the administration of as compared with the non-edaravone treated A 1-40 group.

[0010] Jiao et al. (Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits. Proc Natl Acad Sci U S A. 2015 Apr 21 ;112(16):5225-30) report that edaravone has a potent capacity of inhibiting Ap aggregation and attenuating A -induced oxidation in vitro. When given before or after the onset of Ap-deposition via i.p. injection, edaravone substantially reduced Ap deposition, alleviated oxidative stress, attenuated the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescued the behavioural deficits of APPswe / PSI mice.

[0011] Ren et al. (Edaravone exerts brain protective function by reducing the expression of AQP4, APP and A proteins, Open Life Sci. 2019; 14: 651-65) investigated the changes of aquaporin-4 (AQP4), p-amyloid precursor proteins (APP) and p-amyloid (Ap) in brain tissues of rats after cerebral ischemia reperfusion injury (CIRI), and evaluated the effect of edaravone. The neurological deficits in the model group were obvious and the neurological score increased compared to the control group, while the neurological deficits of the edaravone group were improved as the neurological score decreased compared to the model group. The model group had significantly higher levels of Ap, APP and AQP4 than the control group and edaravone group.

[0012] 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, J. Clin. Med. 2023,12, 3151. suggests that curing AD may be achieved by combination therapy as follows. First, with intranasal insulin to augment the body's natural counter reaction to the changes in brain cell-types that produced the dementia. Second, with edaravone to decrease free radicals, which are increased and causal in AD. Third, with one or two drugs from among pioglitazone, fluoxetine, and lithium, which address the brain celltypes whose changed functions cause the dementia.

[0013] Ramanan et al. (Anti-amyloid therapies for Alzheimer disease: finally, good news for patients, Molecular Neurodegeneration (2023) 18:42 htps: / / dOi.Org / l 0.1186 / si 3024-023-00637-0) report that in recent phase 3 trials for early symptomatic AD, lecanemab and donanemab led to slowing of cognitive and functional decline, and positive alterations on disease-specific biomarkers.

[0014] EP-A 3 290 525 describes a method for screening medicine and therapeutic target for treating Alzheimer’s disease, the method comprises screening medicine and therapeutic target capable of increasing A 42 secretion from cells

[0015] There is increasing evidence showing that Alzheimer’s disease has no single pathogenic mechanism. Combination therapy is seen as one of the most promising options to treat multifactorial diseases like Alzheimer’s disease.

[0016] Summary of the invention

[0017] The inventors have surprisingly found that the efficacy of anti-amyloid-beta (anti-A ) antibody treatment (immunotherapy) of Alzheimer’s disease is significantly improved by coadministration of edaravone.

[0018] Accordingly, a first aspect of the invention relates to an anti-Ap antibody component for use in the treatment of Alzheimer’s disease in a human patient, said anti-Ap antibody component being selected from anti-Ap antibody, an Ap-binding fragment of an Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an Ap antibody, wherein the treatment comprises administration of the anti-Ap antibody component and co-administration of edaravone.

[0019] Without wishing to be bound by theory, the inventors hypothesize that via addressing different pathways involved in Alzheimer’s disease progression, together anti-Ap antibodies and edaravone generate a synergistic effect.

[0020] Another aspect of the invention relates to a pharmaceutical kit comprising a package holding one or more first dosage units comprising (i) an anti-Ap antibody component selected from anti-Ap antibody, an Ap-binding fragment of an Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an Ap antibody, and (ii) one or more second dosage units comprising edaravone.

[0021] Detailed description of the invention

[0022] A first aspect of the invention relates to an anti-amyloid-beta (Ap) antibody component for use in the treatment of Alzheimer’s disease in a human patient, said anti-Ap antibody component being selected from anti-Ap antibody, an Ap-binding fragment of an anti-Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an anti-Ap antibody, wherein the treatment comprises administration of the anti-Ap component and coadministration of edaravone.

[0023] The term “treatment” as used herein encompasses:

[0024] (a) preventing the disease from occurring in a subject who has been identified as being predisposed to develop the disease, but has not yet been diagnosed as having it;

[0025] (b) inhibiting the disease, e.g. halting or slowing down its progression (includes maintenance therapy);

[0026] (c) relieving the disease, e.g. causing regression thereof; and

[0027] (d) prolonging survival as compared to expected survival if not receiving treatment.

[0028] The term “anti-Ap antibody” as used herein, unless indicated otherwise, refers an antibody that is capable of binding to an epitope or conformation of Ap and that is capable of reducing build-up of Ap deposits in the human brain and / or of removing Ap deposits from the human brain. Examples of conformations of Ap to which the antibody may bind include monomer, oligomer and fibril conformations of Ap. The term “vectorised anti-Ap antibody” as used herein refers to a viral or non-viral vector that produces anti-Ap antibody in vivo. Likewise, the term “vectorised Ap-binding fragment of an anti-Ap antibody” as used herein refers to a viral or non-viral vector that produces such a fragment in vivo.

[0029] The term “immunotherapy” as used herein, unless otherwise specified, refers to administration of and / or treatment with anti-Ap antibody or an Ap-binding fragment of an anti-Ap antibody.

[0030] The term “antibody gene therapy” as used herein, refers to administration of and / or treatment with a vectorised antibody. Unlike immunotherapy, antibody gene therapy typically requires only a single administration of the vectorised anti-Ap antibody or the vectorised Ap-binding fragment of an anti-Ap antibody.

[0031] The term "edaravone" as used herein refers to the substance 3-methyl-1 -phenyl-2-pyrazolin- 5-one, including prodrugs and pharmaceutically acceptable salts thereof.

[0032] Whenever reference is made herein to an administered dose of edaravone, the dose refers to the amount of edaravone equivalent that is administered. Here "edaravone equivalent" refers to the quantity of edaravone (3-methyl-1 -phenyl-2-pyrazolin-5-one) that is contained in a given quantity of edaravone salt.

[0033] The term “co-administration of edaravone” as used herein refers to the administration of edaravone alongside the administration of the anti-Ap antibody component. Co-administration may be achieved by separately administering edaravone and the anti-Ap antibody component or by administering a formulation containing both edaravone and the anti-Ap antibody component.

[0034] The present invention encompasses treatment of human subjects who are suffering from Alzheimer’s disease as well as subjects who have been identified as being predisposed to develop this neurodegenerative disease. The present treatment is preferably applied in human subjects that are already suffering from Alzheimer’s disease.

[0035] The anti-Ap antibodies employed in accordance with the invention are preferably humanized IgG (e.g. lgG1 , lgG2, lgG4) monoclonal antibodies capable of binding to different epitopes and conformations of amyloid-p (such as monomer, oligomer or fibril conformations). Examples of suitable anti-Ap antibodies include: bapineuzumab, solanezumab, gantenerumab, crenezumab, ponezumab, solanezumab, lecanemab, BAN2401 , donanemab and aducanumab. The present invention encompasses the use of Ap-binding fragments of these antibodies and the use vectorised versions of these antibodies or fragments.

[0036] The anti-Ap antibody is preferably selected from the group comprising bapineuzumab, solanezumab, gantenerumab, crenezumab, ponezumab, solanezumab, lecanemab, aducanumab, donanemab and combinations thereof. More preferably, the anti-Ap antibody is selected from lecanemab, aducanumab, donanemab and combinations thereof.

[0037] The vector used in the vectorised anti-Ap antibody or vectorised Ap-binding fragment of an Ap antibody preferably is a viral vector, more preferably an adeno-associated virus-based vector. The use of an adeno-associated virus-based vector offers the advantage that it enables effective delivery of Ap antibodies or fragments thereof in the brain.

[0038] The present treatment may suitably employ a prodrug or edaravone. Examples of prodrugs of edaravone can be found in WO 2019 / 213335, WO 2020 / 060092, WO 2021 / 107686 and ON 115073518A.

[0039] The edaravone employed in accordance with the present invention is preferably selected from 3-methyl-1-phenyl-2-pyrazolin-5-one and pharmaceutically acceptable salts thereof. Most preferably, the edaravone used is 3-methyl-1-phenyl-2-pyrazolin-5-one.

[0040] The treatment according to the present invention preferably comprises peroral, intraoral or intravenous administration of edaravone. Examples of intraoral administration include sublingual administration, buccal administration and sublabial administration.

[0041] Preferably, edaravone is administered in a dose of 10-800 mg, more preferably 20-400 mg, even more preferably 50-200 mg.

[0042] According to a particularly preferred embodiment, edaravone is administered in a daily dose of 10-800 mg, more preferably a daily dose of 20-400 mg, even more preferably a daily dose of 50-200 mg.

[0043] Edaravone is preferably administered intravenously in an amount of 0.2-5 mg edaravone per kg of bodyweight per day, more preferably of 0.3-3 mg edaravone per kg of bodyweight per day and most preferably of 0.4-2 mg edaravone per kg of bodyweight per day. Edaravone is preferably administered orally in an amount of 0.3-15 mg edaravone per kg of bodyweight per day, more preferably of 0.4-10 mg edaravone per kg of bodyweight per day and most preferably of 1-5 mg edaravone per kg of bodyweight per day.

[0044] In the present treatment, edaravone is preferably administered in a dose that is sufficient to achieve a plasma concentration of 250-2,500 ng.h / mL, more preferably of 500-1 ,500 ng.h / mL.

[0045] Preferably, edaravone is administered at least once daily, most preferably once daily.

[0046] According to one embodiment, edaravone is administered in the form of an aqueous edaravone solution.

[0047] According to another embodiment, edaravone is administered perorally or intraorally in the form of a solid dosage unit. More preferably, edaravone is administered intraorally.

[0048] In one embodiment of the invention the treatment comprises administration of an anti-Ap antibody or an A -binding fragment thereof. According to a particularly preferred embodiment, the treatment comprises administration of the anti-Ap antibody or Ap-binding fragment thereof and co-administration of edaravone during 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, the treatment comprises administration of the anti-Ap antibody or the Ap- binding fragment thereof and co-administration of edaravone until the patient’s death. The periods of combined administration may be interrupted by one or more holiday periods.

[0049] The anti-Ap antibody or the Ap-binding fragment thereof may be administered intravenously, subcutaneously or intramuscularly. According to a preferred embodiment, the anti-Ap antibody or the Ap-binding fragment is administered intravenously.

[0050] Typically, the anti-Ap antibody or the Ap-binding fragment thereof is administered in dose of 10-1 ,200 mg pg, preferably in dose of 30-1 ,000 mg, more preferably in dose of 40-800 mg.

[0051] According to a preferred embodiment, the anti-Ap antibody or the Ap-binding fragment thereof is administered in an amount of 0.5-40 pg per kg of body weight, more preferably in an amount of 1-30 mg pg per kg of body weight and most preferably in an amount of 2-20 mg pg per kg of body weight. The synergistic interaction between the anti-Ap antibody component and edaravone enables a treatment in which the dose or dose frequency of anti-Ap antibody is reduced.

[0052] In accordance with one preferred embodiment, the present combination treatment comprises intravenous administration of aducanumab once every 4 weeks during a period of more than 1 year, during which period the administered dose of aducanumab not exceed 6 mg / kg, more preferably does not exceed than 4 mg / kg.

[0053] In accordance with another preferred embodiment, the present combination treatment comprises intravenous administration of aducanumab once every 5-8 weeks during a period of more than 1 year, during which period the administered dose of aducanumab not exceed 10 mg / kg, more preferably does not exceed than 8 mg / kg.

[0054] In accordance with one preferred embodiment, the present combination treatment comprises intravenous administration of lecanemab once every 2 weeks during a period of more than 20 weeks, during which period the administered dose of lecanemab not exceed 6 mg / kg, more preferably does not exceed than 4 mg / kg.

[0055] In accordance with another preferred embodiment, the present combination treatment comprises intravenous administration of lecanemab once every 3-5 weeks during a period of at least 20 weeks, during which period the administered dose of lecanemab not exceed 12 mg / kg, more preferably does not exceed than 10 mg / kg.

[0056] Preferably, in the treatment according to the invention, the patient is administered anti-Ap antibody or the A -binding fragment thereof at least once every 2-8 weeks, more preferably at least once every 2-6 weeks.

[0057] In a preferred embodiment, the anti-Ap antibody or the Ap-binding fragment thereof is administered in the form of an aqueous solution of the antibody or the fragment.

[0058] In another embodiment of the invention the treatment comprises administration of a vectorised anti-Ap antibody or a vectorised Ap-binding fragment of an anti-Ap antibody. The vectorised anti-Ap antibody or vectorised Ap-binding fragment of an Ap antibody is preferably administered intravenously.

[0059] The present treatment preferably comprises a single administration of the vectorised anti-Ap antibody or vectorised Ap-binding fragment of that antibody. The Alzheimer’s disease that is treated in accordance with the invention is preferably selected from mild Alzheimer’s disease, early Alzheimer’s disease, prodromal Alzheimer’s disease, mild Alzheimer’s disease dementia, or mild cognitive impairment due to Alzheimer’s disease.

[0060] Ap immunotherapy has been associated with a significant risk of appearance of amyloid- related imaging abnormalities (ARIA). The so-called ARIA events can be manifested as brain edema after immunotherapy and detected as MRI abnormalities. ARIA covers two classes of MRI signal abnormalities: ARIA-oedema / effusion (ARIA-E) and ARIA- haemosiderosis / microhaemorrhages (ARIA-H). ARIA-E refers to the extravasation of fluid resulting in interstitial vasogenic oedema or sulcal effusion in the leptomeningeal / subpial space. ARIA-H refers to microhaemorrhages (mH) or macrohaemorrhages observed as hypointense haemosiderin deposition. Data have suggested that ARIA events, in particular vascular leakage such as microhaemorrhage may be related to the removal of vascular Ap caused by anti-amyloid-beta (Ap) antibody therapy (Zago et al. Vascular alterations in PDAPP mice after anti-Ap immunotherapy: Implications for amyloid-related imaging abnormalities. Alzheimers Dement. 2013 Oct;9(5 Suppl):S105-15).

[0061] According to a preferred embodiment of the invention, co-administration of edaravone achieves a reduction in Amyloid-related Imaging Abnormalities (ARIA). Reduction in ARIA events can be clinically determined, e.g., by MRI.

[0062] According to a particularly preferred embodiment, the treatment according to the invention comprises the step of determining the presence of ARIA events in the patient undergoing administration of the anti-Ap antibody component, prior to the start of co-administration of edaravone.

[0063] The present treatment of Alzheimer’s disease, unlike the treatment of EP-A 3 290 525 preferably does not comprise administration of antibodies against BO / EFR3 / EFR3A / EFR3B, antibodies against PI4Kllla, antibodies against TTC7, antibodies against PUP, inhibitory polypeptides specific to rbo / Efr3 / Efr3a / Efr3b gene, inhibitory polypeptides specific to PI4Kllla / PI4KA gene or small molecule compounds inhibiting phosphokinase activity of PI4KI I la protein.

[0064] Another aspect of the invention relates to a pharmaceutical kit comprising a package holding one or more first dosage units comprising (i) an anti-Ap antibody component selected from anti-Ap antibody, an Ap-binding fragment of an anti-Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an anti-Ap antibody, and (ii) one or more second dosage units comprising edaravone.

[0065] The kit preferably contains 1 to 4, more preferably 1 to 3, even more preferably 1 or 2 and most preferably one first dosage unit.

[0066] The first dosage unit preferably is an aqueous solution of the anti-Ap antibody component, preferably a sterile aqueous solution of the anti-Ap antibody component, even more preferably a sterile isotonic aqueous solution of the anti-Ap antibody component.

[0067] The first dosage unit preferably comprises an aqueous solution comprising at least 10 mg / mL, more preferably 20-250 mg / mL and most preferably 40-160 mg / mL of an anti-Ap antibody or an Ap-binding fragment thereof.

[0068] The first dosage unit preferably comprises 1-50 mL, more preferably 1.3-20 mL and most preferably 1.5-10 mL of an aqueous solution of an anti-Ap antibody or an Ap-binding fragment thereof.

[0069] The anti-Ap antibody is preferably selected from the group comprising bapineuzumab, solanezumab, gantenerumab, crenezumab, ponezumab, solanezumab, lecanemab, aducanumab, donanemab and combinations thereof. More preferably, the anti-Ap antibody is selected from lecanemab, aducanumab, donanemab and combinations thereof.

[0070] In a preferred embodiment, the kit contains a plurality of the second dosage units. More preferably, the kit contains at least 7, more preferably at least 14 of the second dosage units. Preferably, the total number of second dosage units in the kit does not exceed 56, more preferably it does not exceed 28.

[0071] In one embodiment of the invention, the second dosage unit preferably is an aqueous solution of edaravone, preferably a sterile aqueous solution of edaravone.

[0072] The second dosage unit preferably comprises an aqueous solution comprising at least 0.5 mg / mL, more preferably 0.8-5 mg / mL and most preferably 1-2.5 mg / mL edaravone.

[0073] The second dosage unit preferably comprises 10-200 mL, more preferably 20-150 mL and most preferably 25-100 mL of an aqueous solution of edaravone. In another embodiment of the invention, the second dosage unit is a solid dosage unit that is suitable for peroral or intraoral administration. Preferably the solid dosage unit contains 5- 400 mg, preferably 10-200 mg, even more preferably 20-150 mg of edaravone.

[0074] Preferably, the package comprises instructions for administering the first and second dosage units to human patients. Instructions are, for instance, in the form of an insert, a medicine leaflet or a graphic sign for electronic access (such as a bar code, QR code, and the like).

[0075] The instructions preferably indicate that the first dosage unit is to be administered intravenously, subcutaneously or intramuscularly, most preferably intravenously.

[0076] Preferably, the instructions indicate that the first dosage unit is to be administered at least once every 4 weeks, more preferably at least once every 2 weeks.

[0077] The instructions preferably indicate that the second dosage unit is to be administered intravenously, perorally or intraorally, most preferably intraorally or perorally.

[0078] Preferably, the instructions indicate that the second dosage unit is to be administered at least once daily, most preferably once daily.

[0079] According to a particularly preferred embodiment, the first dosage unit is an aqueous solution comprising 10-1,200 mg of the anti-Ap antibody component, preferably of an anti-Ap antibody or an Ap-binding fragment thereof, and the second dosage unit is a solid dosage unit comprising 5-400 mg of edaravone.

[0080] The pharmaceutical kit of the present invention preferably does not comprise a dosage unit comprising an antibody against RBO / EFR3 / EFR3A / EFR3B, an antibody against PI4KII la, an antibody against TTC7, an antibody against PUP, an inhibitory polypeptide specific to rbo / Efr3 / Efr3a / Efr3b gene, an inhibitory polypeptide specific to PI4Kllla / PI4KA gene or a small molecule compound inhibiting phosphokinase activity of PI4KII la protein.

[0081] The invention is further illustrated by the following non-limiting examples.

[0082] EXAMPLES

[0083] Example 1 The effect of edaravone on anti-Ap antibody’s potential for cross-protective activity against pathological A is evaluated in an in vitro AD model. The in vitro AD model consists of a cell culture of primary mouse neurons incubated with brain fractions of human Alzheimer’s Disease (AD) patients.

[0084] Protocol

[0085] Preparation Immunodepleted brain fractions from AD patients

[0086] Brain extracts are prepared and immunodepleted using lecanemab, aducanumab or donanemab or control. Fractions are biochemically characterised, and toxicity assays are performed in primary mouse neuronal cultures using automated high-content microscopy. The detailed protocol is described below.

[0087] Postmortem human brain tissue is used from the Netherlands Brain Bank. The type of material that is selected is fresh-frozen tissue blocks (approximately 3 grams of grey matter w / w). Ten tissue blocks are used in total; 5 from AD donors and 5 from age-matched non-demented controls (without amyloid beta pathology). Brain tissue from the temporal, frontal and parietal lobe is used, and grey matter is selectively isolated.

[0088] In order to confirm that AD pathology is present or absent in the brain tissues, the levels of Ap and p-tau are measured using immunohistochemistry (IHC). Of each tissue block, 5x amount of sections is cut. The extent of co-pathology is assessed using IHC, using murine primary mAbs with N-terminal a. a. 1-16, (clone IC16, in-house), 4G8, and AT8 (phospho-PHF-tau pSer202+Thr205) for Ap and p-tau, respectively, and the Dako Envision goat anti- mouse / rabbit-HRP K5007 as a secondary antibody. Visual examination and semi-quantitative assessment of pathology score is used as readouts.

[0089] Brain tissue extracts are prepared to generate the in vitro AD and control cellular phenotypes. The methodology used is based on Hong et al (Hong et al, Acta Neuropathologica (2018), optimized and validated in the Wiep Scheper lab (Sandberg et al, Alzheimers Res Ther. 2022). Human brain tissue samples are homogenized in artificial cerebrospinal fluid (aCSF), centrifuged at 22,000 g for 110 min, and the supernatant is collected for subsequent analysis. Whole-brain extracts (supernatant) are be dialyzed using Slide-AI-Lyzer dialysis cassettes, 2K MWO, aliquoted, snap-frozen, and stored at -80 °C.

[0090] Based on previous studies (Sandberg et al, 2022), only the H fraction is isolated. H Fractions are pooled from all AD or control brain tissue, resulting in the following fractions: H fraction controls pooled, H fraction AD cases pooled (2 samples). For immunodepletion, a sample of brain extract (500 pl) is incubated with 1 pg mAb overnight at 4 °C under continuous rotation. The following day, 50 pl of 10% Protein G-sepharose beads in PBS are added and the mixture is incubated for 4 h at 4 °C under rotation. Brain extracts are immunodepleted with either lecanemab, aducanumab, donanemab, positive control (4G8) or negative isotype control to match lecanemab, aducanumab and donanemab. Subsequently, samples are centrifuged at 10 000g for 10 min at 4 °C to remove the beads. Supernatants are collected, snap-frozen in liquid nitrogen, and stored at -80 °C until use.

[0091] To characterize Ap profiles in the different obtained brain fractions the below assays are performed:

[0092] • Total protein content of the brain extracts is measured using the Pierce BCA Protein Kit according to the manufacturer’s protocol.

[0093] • MSD Immunoassay for A 40 and A 42: The A 40 and A 42 concentrations are determined with the MesoScale Discovery (MSD) platform using the Amyloid-p Peptide Panel 1 Kit (6E10: A 42, A 40, A 38) according to the manufacturer’s protocol.

[0094] Preparation of neuronal cell culture

[0095] Embryonic day 18.5 wild-type mouse embryonic neurons are used for in vitro cell culture, according to optimized protocols (Sandberg et al, 2022). In short, E18,5 wild type mouse cortices are dissected, digested using trypsin, washed, and plated in Neurobasal culture medium in black-based, 96-well plates at a cell density of 15K per well.

[0096] Determining optimal edaravone dose

[0097] To establish the optimal edaravone dose for testing, embryonic neurons (without any brain extracts) are treated with Menadione (4 hours) to induce Reactive Oxygen Species (ROS) in the cells. Next, the neurons are incubated with different doses of edaravone or vehicle (30 minutes / 1 hour), after which the ROS response is measured: cells are loaded with CellROX green reagent (5 pM) during the last 30 min of incubation. Fluorescence is determined in dapi ROI. Technical replicates: 3, all within 1 plate. The dose of edaravone for further testing will is selected based on the ROS response.

[0098] Determining combined effect of anti- AB antibodies and edaravone

[0099] The control and immunodepleted brain fractions are seeded on the mouse neurons establishing an AD in vitro model, using different test settings.

[0100] Test setting 1: Incubation of immunodepleted brain fractions with edaravone The control and immunodepleted brain fractions are preincubated with the optimum edaravone dose or vehicle first. Thereafter, the preincubated brain factions are added to the neuronal cell culture.

[0101] Test setting 2: Addition of edaravone to the AD in vitro model

[0102] The control and immunodepleted brain fractions are added to the cell culture. In addition, the optimum edaravone dose or vehicle is added.

[0103] Neuronal morphology and viability:

[0104] To assess neuronal phenotype, high-content automated microscopy is performed on a Cell Insight CX7 HCS platform including neurite outgrowth / length / arborization, synaptic properties. Neurons are incubated with primary antibodies diluted in blocking solution 16-17 h at 4°C. The primary antibodies used are vesicular glutamate transporter 1 (vGlutl) for presynaptic and microtubule-associated protein 2 (Map2) for dendrite. After one 5-min wash with PBS, nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI) diluted in PBS (1 :1000). Finally, the plates are stored in PBS at 4°C until microscopic analysis.

[0105] High-content automated microscopy is then performed to obtain 25 images per well. Images are analysed using Columbus 2.5 software (PerkinElmer). Nuclei are detected in the DAPI channel, nuclear count are used as measure of neuronal viability / loss. A Map2-based region of interest (ROI) is used to determine the number of neurite segments, branches (nodes type 1) and extremities. The number of synapses is measured in the vGlutl channel within the Map2-based ROI. The values of all analysed morphological characteristics are normalized to the number of cell nuclei and represented as percentage change compared to the control condition.

[0106] Oxidative stress assay:

[0107] To determine ROS levels, cells are loaded with CellROX green reagent (5 pM) during the last 30 min of incubation. Fluorescence is determined in dapi ROI.

[0108] Evaluation of combined effect

[0109] The data so obtained show that, compared to non-AD control, treatment with AD brain extracts caused a significantly increased loss of dendrite segments and branches, as well as in the number of presynaptic vGlutl puncta per neuron, demonstrating neurotoxicity of AD brain extracts. The data further show that immunodepletion of AD brain extracts with anti-Ap antibody significantly reduces the loss of dendrite segments and branches, as well as presynapses, when compared to AD-non immunodepleted brain extracts demonstrating a neuroprotective effect associated with the depletion of A .

[0110] The data additionally show that in the immunodepleted AD brain fractions treated with edaravone, the loss of dendrite segments and branches as well as presynapses is further attenuated compared to non-treated immunodepleted AD brain extracts, demonstrating an enhancing effect of edaravone when combined with immunodepletion with an anti-Ap antibody.

[0111] Example 2

[0112] Vascular changes associated with anti-Ap immunotherapy with or without edaravone in an animal model (ADPP mice) is performed by leptomeningeal Vap quantification, capillary Ap quantification and microhaemorrhage, as described in Zago et al. Vascular alterations in PDAPP mice after anti-A / 3 immunotherapy: Implications for amyloid -related imaging abnormalities. Alzheimers Dement, 2013;9(5S). Three different anti-Ap antibodies are tested: lecanemab, aducanumab or donanemab.

[0113] In particular, cortical leptomeningeal vessels for Ap deposition are assessed by counting the number of all amyloid-containing vessels in the brain sections. Capillaries for Ap deposition are assessed similarly to cortical leptomeningeal vessels. Microhaemorrhage quantification determines the presence, amount, location and intensity of hemosiderin staining across brain sections.

[0114] The results show that anti-Ap immunotherapy combined with administration of edaravone significantly reduces Ap deposition in cortical leptomeningeal vessels and capillary deposition of the brain sections, as opposed to samples representing anti-Ap immunotherapy without edaravone. Also, microhaemorrhage is significantly reduced when edaravone is combined with immunotherapy. The results support a combination therapy for reducing ARIA effects in patients subject to anti-Ap therapy.

[0115] Example 3

[0116] The effect of edaravone on anti-Ap antibody’s potential for cross-protective activity against pathological Ap was evaluated in the in vitro AD model that is described in Example 1. Postmortem human brain tissue was obtained as a fresh-frozen tissue block (approximately 3g of white matter w / w) from the Netherlands Brain Bank. Each tissue sample was used for sectioning for neuropathological confirmation of AD or non-AD (controls) followed by preparation of brain extracts. Ten tissue blocks were used in total; 5 from AD donors and 5 from age-matched non-demented controls (without amyloid beta pathology).

[0117] In order to confirm that AD pathology is present or absent in the brain tissues, the levels of A and p-tau were measured using immunohistochemistry (I HC). Of each tissue block, 5x amount of sections was cut. The extent of co-pathology was assessed using I HC, using murine primary mAbs with N-terminal a. a. 1-16, (clone 1016, in-house), 4G8, and AT8 (phospho-PHF-tau pSer202+Thr205) for Ap and p-tau, respectively, and the Dako Envision goat anti- mouse / rabbit-HRP K5007 as a secondary antibody. Visual examination and semi-quantitative assessment of pathology score was used as readouts. The results confirmed the presence of AD pathology in the 5 AD donors and the absence of AD pathology in the 5 non-demented controls.

[0118] Brain tissue extracts were prepared to generate the in vitro AD and control cellular phenotypes. The methodology used is based on Hong et al (Hong et al, Acta Neuropathologica (2018), optimized and validated in the Wiep Scheper lab (Sandberg et al, A / 342 oligomerspecific antibody ALZ-201 reduces the neurotoxicity of Alzheimer’s disease brain extracts, Alzheimers Res Ther. (2022) 14:196 https: / / doi.org / 10.1186 / s13195-022-01141-1). The brain extracts (supernatant) were dialyzed using Slide-AI-Lyzer dialysis cassettes, 2K MWCO (Thermo Fisher Scientific), aliquoted, snap-frozen and stored at -80 °C. Total protein content of the brain extracts was measured using the Pierce BCA Protein Kit according to the manufacturer’s protocol (Thermo Fisher Scientific). The Ap40 and Ap42 concentrations were determined with the MesoScale Discovery platform using the Amyloid-p Peptide Panel 1 Kit (4G8: Ap42, Ap40, Ap38) according to the manufacturer’s protocol (Meso Scale Diagnostics). Only the H fractions were isolated. The H fractions obtained from the AD brain tissues were pooled. Likewise, the H fractions obtained from the control brain tissues were pooled.

[0119] For immunodepletion, a sample of brain extract (500 pl) was incubated with 1 pg mAb overnight at 4 °C under continuous rotation. The following day, 50 pl of 10% Protein G- sepharose beads (Abeam) in PBS were added and the mixture was incubated for 4 h at 4 °C under rotation. Subsequently, samples were centrifuged at 100g for 5 min at 4 °C to remove the beads. Supernatants were collected, snap-frozen in liquid nitrogen, and stored at -80 °C until use. Brain extracts were immunodepleted with three different anti-Ap antibodies, i.e. lecanemab (ex Selleckchem), donanemab (ex Selleckchem), and 4G8 (ex BioLegend). Preparation of neuronal cell culture

[0120] Embryonic day 18.5 wild-type mouse embryos obtained by Caesarean section of pregnant females from timed mating were used for primary neuronal cultures. Cortices were dissected in Hanks Buffered Salt Solution (HBSS; Sigma-Aldrich) containing 10 mM 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer (Gibco) (Hanks-HEPES) and digested by addition of 0.25% trypsin (Gibco) for 20 min at 37 °C. Digested tissue was washed three times in Hanks-HEPES and subsequently triturated with fire-polished Pasteur pipettes in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g / L glucose and UltraGlutamine I (Lonza) supplemented with 10% heat-inactivated foetal bovine serum (HI-FBS), 1% penicillin / streptomycin (pen-strep; Fisher Scientific) and 1% non-essential amino acid solution (Fisher Emergo) (DMEM+). Dissociated cells were spun down, re-suspended and plated in Neurobasal culture medium (NB; Fisher Scientific) supplemented with 2% B-27 (Life Technologies), 18 mM HEPES, 0.25% glutamax (Fisher Scientific) and 0.1% pen-strep (NB+). The re-suspended cells were plated in black-based, 96-well plates (Greiner Bio-One BV) at a cell density of 12,5K per well and cultured at 37 °C and 5% CO2.

[0121] Determining optimal edaravone dose and compatibility with neuronal culture

[0122] A pilot study to determine the optimal edaravone concentration was performed in parallel. All read-outs were optimized for 96-well format. Mouse neurons (without any brain extracts) were treated with different edaravone concentrations (1 , 3, 10, 30 and 100pM) and vehicle (30 minutes / 1 hour). The neuronal cells were treated with menadione (33, 66 and 100uM) for 4 hours to induce Reactive Oxygen Species (ROS).

[0123] The effect of edaravone on ROS response was measured by loading the cells with CellROX green reagent (5 pM; Thermo Fisher) during the last 30 min of incubation. The procedure used is described by Batenburg et al. (Intraneuronal tau aggregation induces the integrated stress response in astrocytes, Journal of Molecular Cell Biology, Volume 14, Issue 10, October 2022, Fluorescence was determined in nuclear (dapi) ROI. Edaravone was not neurotoxic in none of the tested concentrations. The optimal edaravone concentration for suppressing ROS while not being neurotoxic was found to be 100 pM.

[0124] Determining combined effect of anti- AB antibodies and edaravone

[0125] The pooled H brain fractions were added to the mouse neuronal cells day in vitro (DIV) 16 together with edaravone (100 pM) or vehicle as described in Sandberg et al. (A / 342 oligomerspecific antibody ALZ-201 reduces the neurotoxicity of Alzheimer’s disease brain extracts, Alzheimers Res Ther. (2022) 14:196 https: / / doi.org / 10.1186 / s13195-022-01141-1) . Cells were fixed on DIV 17 by incubation with 1.85% paraformaldehyde (PFA; Merck Millipore) in PBS at pH 7.4 for 10 min by replacing half the volume of the media with 3.7% PFA in PBS. Subsequently, the medium plus PFA were removed and substituted with 3.7% PFA for 10 min at RT.

[0126] The results obtained from the oxidative stress assay are shown in Figure 1 . These results show that, compared to treatment with non-AD brain extracts (control), treatment with AD brain extracts caused a substantial increase of the ROS levels in the neuronal cells, demonstrating neurotoxicity of AD brain extracts compared to brain extracts of healthy subjects (control). The levels of ROS observed in these cell cultures are equivalent to the ROS levels produced by the strong oxidative stress inducer menadione.

[0127] The data show that in the immunodepleted AD brain fractions ROS levels were further reduced when edaravone was added.

[0128] In this experiment the favourable effect of edaravone was observed at unusually high ROS concentrations. The favourable effect is expected to be more pronounced at ROS levels that correspond better with the ROS levels observed in the brains of AD patients

Claims

CLAIMS1. An anti-amyloid-beta (Ap) antibody component for use in the treatment of Alzheimer’s disease in a human patient, said anti-Ap antibody component being selected from anti- Ap antibody, an Ap-binding fragment of an anti-Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an anti-Ap antibody, wherein the treatment comprises administration of the anti-Ap antibody component and coadministration of edaravone.

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

3. The anti-Ap antibody component for use according to claim 1 or 2, wherein the anti-Ap antibody is selected from bapineuzumab, solanezumab, gantenerumab, crenezumab, ponezumab, solanezumab, lecanemab, aducanumab, donanemab and combinations thereof.

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

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

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

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

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

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

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

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

12. The anti-Ap antibody component for use according to any one of the preceding claims, wherein co-administration of edaravone achieves a reduction in Amyloid-related Imaging Abnormalities (ARIA).

13. The anti-Ap antibody component for use according to any one of the preceding claims, wherein the treatment does not comprise administration of antibodies against RBO / EFR3 / EFR3A / EFR3B, antibodies against PI4KI I la, antibodies against TTC7, antibodies against PI4P, inhibitory polypeptides specific to rbo / Efr3 / Efr3a / Efr3b gene, inhibitory polypeptides specific to PI4Kllla / PI4KA gene or small molecule compounds inhibiting phosphokinase activity of PI4KII la protein.

14. A pharmaceutical kit comprising a package holding one or more first dosage units comprising (i) an anti-Ap antibody component selected from anti-Ap antibody, an Ap- binding fragment of an anti-Ap antibody, a vectorised anti-Ap antibody and a vectorised Ap-binding fragment of an anti-Ap antibody, and (ii) one or more second dosage units comprising edaravone.

15. Pharmaceutical kit according to claim 14, wherein the package comprises instructions for administering the first and second dosage units to human patients.

16. Pharmaceutical kit according to claim 14 or 15, wherein the first dosage unit is an aqueous solution comprising 10-1 , 200 mg of the anti-Ap antibody component and the second dosage unit is a solid dosage unit comprising 5-400 mg of edaravone.

17. Pharmaceutical kit according to any one of claims 14-16, wherein the kit does not comprise a dosage unit comprising an antibody against RBO / EFR3 / EFR3A / EFR3B, an antibody against PI4KI I la, an antibody against TTC7, an antibody against PI4P, an inhibitory polypeptide specific to rbo / Efr3 / Efr3a / Efr3b gene, an inhibitory polypeptidespecific to PI4Kllla / PI4KA gene or a small molecule compound inhibiting phosphokinase activity of PI4KII la protein.

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