RU2786476C2 - Composition containing antibody against abeta protofibrils and inhibitor of beta-secretase bace1 for treatment of alzheimer's disease - Google Patents

Abstract

FIELD: pharmaceutics; medicine.

SUBSTANCE: object 1 is a method for the treatment of Alzheimer’s disease, including administration to a subject who needs it of a combination of therapeutically effective amount of an antibody against Aβ protofibrils and therapeutically effective amount of N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazine-7a-yl)-4-fluorphenyl]-5-difluormethylpyrazine-2-carboxamide and/or its pharmaceutically acceptable salt. Object 2 is a combination for use in the treatment of Alzheimer’s disease, containing an antibody against Aβ protofibrils and N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazine-7a-yl)-4-fluorphenyl]-5-difluormethylpyrazine-2-carboxamide and/or its pharmaceutically acceptable salt.

EFFECT: reduction in a level of Aβ42 in the brain and increase in power of electroencephalograms in ranges α1 (11-13 Hz) and β1 (13-22 Hz) with administration of a combination.

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Description

[0001] This application claims priority to U.S. Provisional Application No. 62/413961, filed October 27, 2016, and U.S. Provisional Application No. 62/415165, filed October 31, 2016, incorporated herein by reference.

[0002] Alzheimer's disease affects 1 in 9 older people and is the cause of dementia in more than 5.2 million people in the United States and more than 30 million people worldwide. Currently, there is no remedy for the cure or prevention of this serious disease. Histologically, the disease is distinguished by senile plaques found mainly in the association zone of the cerebral cortex, the limbic system, and the basal ganglia. The main component of these plaques is amyloid beta peptide (Aβ).

[0003] Aβ exists in various conformational states—monomers, oligomers, protofibrils, and insoluble fibrils. The details of the mechanistic relationship between the onset of Alzheimer's disease and Aβ production are unknown. However, several anti-Aβ antibodies and beta-secretase inhibitors (BACE1) are currently in clinical trials as potential therapeutic agents for Alzheimer's disease.

[0004] The present invention relates to combined treatment methods for treating, preventing and/or delaying the onset and/or progression of Alzheimer's disease, comprising administering a therapeutically effective amount of an anti-Aβ protofibril antibody and a therapeutically effective amount of a beta-secretase inhibitor. In some embodiments, the combination treatment inhibits the production of Aβ and/or toxic oligomeric Aβ. In some embodiments, the combination treatment reduces Aβ and/or toxic Aβ oligomeric protofibrils in the brain.

[0005] The invention relates to methods, combined treatment, pharmaceutical compositions and kits for the treatment, prevention and/or delay of the onset and/or development of Alzheimer's disease using a combination of antibodies against Aβ protofibrils and N-[3-((4aS,5R,7aS )-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazine -2-carboxamide or a pharmaceutically acceptable salt thereof.

[0006] Within the scope of the invention, an anti-Aβ protofibril antibody comprises (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. Assigning amino acids to each domain generally matches the definitions in SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (Kabat, et al., 5th ed., U.S. Department of Health and Human Services, NIH Publication No.91-3242, 1991, hereinafter referred to as the "Kabat article" ).

[0007] In some embodiments, the anti-Aβ protofibril antibody comprises a human constant region.

[0008] In some embodiments, the human anti-Aβ protofibril constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE, and any allelic variant thereof, as described in Kabat. Any one or more of these sequences may be used in the present invention. In some embodiments, the heavy chain constant region is selected from IgG1 and allelic variants thereof. The amino acid sequence of the human IgG1 constant region is known in the art and is shown in SEQ ID NO: 3.

[0009] In some embodiments, the human anti-Aβ constant region comprises a light chain constant region selected from κ-λ chain constant regions and any allelic variants thereof, as described in the Kabat article. Any one or more of these sequences may be used in the present invention. In some embodiments, the light chain constant region is selected from the κ chain constant region and allelic variants thereof. The amino acid sequence of the human κ chain constant region is known in the art and is shown in SEQ ID NO: 4.

[00010] In some embodiments, the anti-Aβ protofibril antibody is BAN2401. mAb158 is a mouse monoclonal antibody raised against target protofibrils and BAN2401 is a humanized IgG1 mAb158 monoclonal. mAb158 is described in WO2007/108756A1 and Journal of Alzheimer's Disease 43 (2015) 575-588.

[00011] BAN2401 is a humanized monoclonal antibody containing (a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and (b) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2. The full length sequence of BAN2401 is shown in SEQ ID NO: 5.

[00012] BAN2401 is believed to selectively bind, neutralize and eliminate soluble toxic Aβ aggregates (protofibrils) that are believed to contribute to the neurodegenerative process in Alzheimer's disease. As such, BAN2401 exhibits an immunomodulatory effect that can suppress the progression of Alzheimer's disease. BAN2401 is currently in phase II clinical trials.

[00013] N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3] thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazin-2-carboxamide (referred to herein as "compound X") corresponding to the chemical formula (1) below, or a pharmaceutically acceptable salt thereof, is an enzyme inhibitor, beta-amyloid precursor protein 1 (BACE1). See, for example, US Pat. No. 8,158,620 and US Pat. No. 8,426,584. Compound X is also known as E2609, or it may also be referred to as elenbecestat. By inhibiting BACE, Compound X may decrease Aβ peptides in the brain, potentially improving symptoms and/or slowing the progression of Alzheimer's disease.

[00014] In some embodiments, compound X is in free base form.

[00015] In some embodiments, the invention relates to methods for preventing, treating and/or delaying the onset and/or progression of Alzheimer's disease, and includes administering to a subject in need thereof a therapeutically effective amount of BAN2401 and a therapeutically effective amount of Compound X.

[00016] In some embodiments, the subject is a subject considered to be at risk of developing Alzheimer's disease, for example, a subject having at least one family member diagnosed with Alzheimer's disease.

[00017] In some embodiments, the subject is a subject diagnosed as having at least one genetic mutation associated with Alzheimer's disease.

[00018] In some embodiments, the subject is a subject having at least one mutant or abnormal gene associated with Alzheimer's disease (e.g., an APP mutation, a presenilin mutation, and/or an ApoE4 allele) but has not been diagnosed with Alzheimer's disease.

[00019] In some embodiments, the subject is a subject not identified as being genetically predisposed to developing Alzheimer's disease.

[00020] Brief Description of the Drawings

[00021] FIG. 1 shows the amount of Aβ in Tg2576 mouse brain extracts.

[00022] FIG. 2 shows the effects of combined treatment in the α1 and β1 frequency ranges on the EEG recording of Tg2576 hetero mice.

[00023] As used herein, the term "prophylaxis" includes, but is not limited to, the inhibition and/or prevention of one or more biochemical changes, histological changes, and/or behavioral symptoms associated with Alzheimer's disease. Symptoms and pathological changes associated with Alzheimer's disease include, but are not limited to, cognitive decline, increased amyloid plaque formation, soluble Aβ peptide circulating in body fluids, accumulation of Aβ peptide in the brain, and abnormalities in memory, decision tasks, speech, computation, visual-spatial perception, reasoning ability and behavior.

[00024] In the context of the invention, the term "treatment" means an approach to achieve beneficial and/or desired results, including, as non-limiting examples, clinical results. Non-limiting examples of beneficial and/or desirable outcomes include one or more of the following: inhibition and/or suppression of amyloid plaque formation, reduction, removal and/or elimination of amyloid plaques, improvement in cognition and/or reversal of cognitive decline, sequestration of soluble Aβ peptide circulating in biological fluids, reduction of Aβ-peptide (including soluble and deposited) in tissue (for example, brain), inhibition and/or reduction of accumulation of Aβ-peptide in the brain, inhibition and/or reduction of toxic effects of Aβ-peptide in tissue (eg, brain), reduction in brain atrophy, reduction in one or more symptoms resulting from disease (eg, abnormalities in memory, problem solving, speech, computation, visuospatial awareness, reasoning and behaviour, inability to take care of oneself) , improving quality of life, reducing the dose of one or more other drugs drugs needed to treat the disease, delay the progression of the disease, alter the underlying pathological process and/or the course of the disease, and/or prolong survival.

[00025] Within the scope of the invention, the term "treatment" is used to describe the implementation of the method after the onset of symptoms of Alzheimer's disease, while the term "prophylaxis" is used to describe the implementation of the method before the onset of symptoms, for example, in patients at risk of developing Alzheimer's disease.

[00026] Within the scope of the invention, patients at risk of developing Alzheimer's disease may or may not have a detectable disease and may or may not exhibit a detectable disease prior to using the treatments provided herein. The term "at risk" means that the subject has one or more measurable parameters (risk factors) that correlate with the development of Alzheimer's disease. These risk factors include, but are not limited to, age, gender, race, diet, history of previous illnesses, presence of disease precursors, genetic (ie, hereditary) factors, and exposure to environmental factors. As non-limiting examples, subjects at risk of developing Alzheimer's disease include subjects with a family history of Alzheimer's disease, subjects whose risk is determined by analysis of genetic or biochemical markers, subjects with a positive blood test for any of the signaling proteins present in blood plasma, and /or cerebrospinal fluid ("CSF"), known as prognostic factors in the clinical diagnosis of Alzheimer's disease.

[00027] As used herein, the term "delaying" the development of Alzheimer's disease means delaying, preventing, slowing, inhibiting, stabilizing and/or postponing the development of the disease, and/or slowing the progression of, or altering the underlying pathological process and/or course of the disease once it has developed. This delay may be of varying duration depending on the medical history and/or the subject being treated. As will be appreciated by one of skill in the art, a sufficient or significant delay may actually include prophylaxis in the sense that the subject does not develop the disease. A method that "delays" the progression of Alzheimer's disease is a method that reduces the likelihood of developing the disease in a specified time period and/or reduces the severity of the disease in a specified time period compared to not using the method, including stabilizing one or more symptoms resulting from the disease (e.g., anomalies in memory, problem solving, speech, computation, visual-spatial perception, reasoning and behavior, inability to take care of oneself). Such a comparison may be based on clinical studies, usually using a sufficient number of subjects to achieve a statistically significant result. The development of Alzheimer's disease can be determined by standard clinical methods, such as routine neurological examination, patient questioning, neuroimaging, determination of changes in the levels of specific proteins in serum or cerebrospinal fluid (for example, amyloid peptides and/or tau protein), imaging of amyloid or tau protein in brain through computed tomography (CT), magnetic resonance imaging (MRI) and/or positron emission tomography (PET). Within the scope of the invention, the term "development" may also refer to the progression of the disease, which may initially be indeterminate, and may include onset, recurrence, worsening, and/or onset.

[00028] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of a compound or pharmaceutical composition sufficient to produce the desired therapeutic effect, including, but not limited to, preventing and/or delaying the onset and/or development at least one disease. A therapeutically effective amount may vary depending on the intended use, the subject to be treated (including, for example, its body weight and age), the disease and its severity, the route and time of administration, the desired effect (for example, reduction of side effects), the dosage regimen used. and composition and delivery system (if necessary). In some embodiments, a "therapeutically effective amount" of a drug used in combination with at least one other therapeutic agent may be the same or different (below or higher) than a "therapeutically effective amount" of a drug used alone (i.e., .e with monotherapy).

[00029] In some embodiments, the described methods of combination treatment may include lower doses of one or more individual therapeutic agents than the doses that would be needed if the individual therapeutic agents were administered separately (i.e., with monotherapy with BAN2401 and compound X ). This dose reduction may result in a reduction in one or more side effects associated with therapy. For example, in some embodiments, the same or greater therapeutic benefit is achieved using a smaller amount (e.g., a lower dose or less frequent administration) of BAN2401, Compound X, or both in combination treatment than the amounts typically used for monotherapy. . As a further example, in some embodiments, the use of a small amount of BAN2401, Compound X, or both results in a reduction in the number, severity, frequency, and/or duration of one or more side effects associated with the compounds. As non-limiting examples, combination treatment may include, compared to doses typically used for monotherapy: (i) a lower dose of Compound X and a lower dose of BAN2401; (ii) a lower dose of BAN2401 and the same dose of Compound X; (iii) a lower dose of compound X and the same dose of BAN2401.

[00030] In some embodiments, the combination treatments described may include higher doses of the individual therapeutics than would be needed if the individual therapeutics were administered alone (i.e., BAN2401 and Compound X monotherapy). For example, in some embodiments of combination treatment methods, the dose of one of the drugs (BAN2401 and Compound X) is lower than the dose typically used for monotherapy while the other drug is administered at the same or higher dose than dose generally used for monotherapy. As non-limiting examples, combination treatment may include (i) a higher dose of Compound X and a lower dose of BAN2401, or (ii) a higher dose of BAN2401 and a lower dose of Compound X. In some cases, increasing the dose of one of the drugs while decreasing the dose of the other may have one or both of the advantage of reducing drug side effects using a lower dose and the advantage of achieving the same or greater therapeutic benefit than monotherapy. As a further example, in some embodiments, the combination treatment may comprise, compared to doses typically used for monotherapy, (i) a higher dose of Compound X and a higher dose of BAN2401, (ii) a higher dose of BAN2401 and the same a dose of Compound X; or (iii) a higher dose of Compound X and the same dose of BAN2401.

[00031] In some embodiments, the described combination allows to reduce the severity of one or more symptoms associated with Alzheimer's disease by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 % or more than 95% compared with the corresponding symptom in the same subject before treatment or compared with the corresponding symptom in other subjects not subjected to combined treatment. For example, in some embodiments, administration of a combination of BAN2401 and Compound X results in a reduction in cognitive decline, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 % or more than 95% compared to control.

[00032] In some embodiments, combinations of BAN2401 and Compound X may be administered to a subject in a single dosage form and/or via separate administration of each active agent.

[00033] In some embodiments, BAN2401 and Compound X may be formulated into a tablet, pill, capsule, or solution. You can select the composition of BAN2401 and compound X accordingly. In some embodiments, BAN2401, Compound X, or both, are formulated in a parenteral solution. In some embodiments, BAN2401 and Compound X may be formulated into separate regions of a capsule or separate capsule-shaped tablets enclosed in a capsule. In some embodiments, BAN2401 and Compound X can be formulated as isolated tablet layers.

[00034] In some embodiments, the invention relates to a pharmaceutical composition for the treatment, prevention and/or delay of the onset and/or development of Alzheimer's disease, comprising: a therapeutically effective amount of BAN2401, a therapeutically effective amount of Compound X, and at least one pharmaceutically acceptable carrier.

[00035] In some embodiments, BAN2401 and Compound X can be administered as separate compositions and optionally in different forms, such as separate tablets or solutions. For example, in some embodiments, Compound X is administered as once daily oral tablets and BAN2401 is administered as an injection. As a further non-limiting example, BAN2401 and Compound X are administered separately as oral tablets. Also as a further non-limiting example, BAN2401 and Compound X are administered separately as injections.

[00036] In some embodiments, if BAN2401 and Compound X are administered as separate compositions, the invention relates to:

- a pharmaceutical composition for use in combination with compound X for the treatment, prevention and/or delay of the onset and/or development of Alzheimer's disease, containing a therapeutically effective amount of BAN2401 and at least one pharmaceutically acceptable carrier; and

- a pharmaceutical composition for use in combination with BAN2401 for the treatment, prevention and/or delay of the onset and/or development of Alzheimer's disease, containing a therapeutically effective amount of Compound X and at least one pharmaceutically acceptable carrier.

[00037] In some embodiments, the present invention relates to a kit containing a first pharmaceutical composition containing a therapeutically effective amount of BAN2401, a second pharmaceutical composition containing a therapeutically effective amount of Compound X, and instructions for use in the treatment, prevention and/or delay of onset and/ or development of Alzheimer's disease.

[00038] In some embodiments, BAN2401 and Compound X may be administered simultaneously. In some embodiments, BAN2401 and Compound X may be administered sequentially. In some embodiments, BAN2401 and compound X may be administered at intervals. The duration of the periods between administrations of BAN2401 and compound X can be adjusted to achieve the desired therapeutic effect. In some embodiments, BAN2401 and Compound X may be administered only a few minutes apart. In some embodiments, BAN2401 and Compound X may be administered several hours apart (eg, about 2, 4, 6, 10, 12, 24, or 36 hours apart). In some embodiments, it may be preferable to administer multiple doses of one of BAN2401 and Compound X between administrations of the other therapeutic agent. For example, one therapeutic agent may be administered 1 hour and then again 11 hours after another therapeutic agent is administered. In some embodiments, the therapeutic effects of each of BAN2401 and Compound X should overlap for at least a portion of the time period such that the overall therapeutic effect of the combination treatment can be partially attributed to the combined or synergistic effect of the combination treatment.

[00039] The dose of BAN2401 and Compound X may depend on a number of factors, including the pharmacodynamic characteristics of each agent, the route of administration, the health status of the patient being treated, the desired amount of treatment, the nature and type of concomitant therapy, if any, the frequency of administration, and the nature of the desired effect . In some embodiments, BAN2401 may be administered at a dose ranging from about 0.001 mg/kg body weight per day to about 200 mg/kg body weight per day. In some embodiments, BAN2401 may be administered at a dose ranging from 0.001 mg/kg body weight per day to 200 mg/kg body weight per day. In some embodiments, Compound X may be administered at a dose in the range of 5 mg/day to 100 mg/day, 10 mg/day to 75 mg/day, 5 mg/day to 50 mg/day, or 15 mg/day up to 50 mg / day. In some embodiments, Compound X may be administered at a dose ranging from about 5 mg/day to about 100 mg/day, from about 10 mg/day to about 75 mg/day, from about 5 mg/day to about 50 mg/day or from about 15 mg/day to about 50 mg/day. In some embodiments, Compound X may be administered at a dose of 5 mg/day, 10 mg/day, 15 mg/day, 20 mg/day, 25 mg/day, 30 mg/day, or 50 mg/day.

[00040] In some embodiments, BAN2401 can be administered at a dose in the range of 2.5 mg/kg to 10 mg/kg, or 5 mg/kg to 10 mg/kg. In some embodiments, BAN2401 is administered at a dose of 10 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of 5 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of 2.5 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of 5 mg/kg every month. In some embodiments, BAN2401 is administered at a dose of 10 mg/kg every month.

[00041] In some embodiments, BAN2401 may be administered at a dose in the range of about 2.5 mg/kg to about 10 mg/kg, or about 5 mg/kg to about 10 mg/kg. In some embodiments, BAN2401 is administered at a dose of approximately 10 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of approximately 5 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of approximately 2.5 mg/kg every 2 weeks. In some embodiments, BAN2401 is administered at a dose of approximately 5 mg/kg every month. In some embodiments, BAN2401 is administered at a dose of approximately 10 mg/kg every month.

[00042] In some embodiments, each of BAN2401 and Compound X can be administered according to an administration schedule, an example of which is shown in Table 1:

Compound X 5 mg/day 15 mg/day BAN2401 2.5 mg/kg/2 weeks 5 mg/kg/2 weeks 5 mg/kg/month 10 mg/kg/2 weeks 10 mg/kg/month

[00043] In some embodiments, each of BAN2401 and Compound X can be administered according to an administration schedule, an example of which is shown in Table 2:

Compound X 25 mg/day 50 mg/day BAN2401 2.5 mg/kg/2 weeks 5 mg/kg/2 weeks 5 mg/kg/month 10 mg/kg/2 weeks 10 mg/kg/month

[00044] In some embodiments, a fixed dose of 50 mg of Compound X is administered. In some embodiments, the frequency of BAN2401/placebo infusions is every 2 or 4 weeks and Compound X is administered once a day orally in the form of at least one tablet in the maximum tolerated dose.

[00045] In some embodiments, dosage ranges may vary depending on the age and body weight of the subject being treated and the intended route of administration. In some embodiments, the dosage is selected to improve and/or maintain efficacy and improve at least one of safety and tolerability. In some embodiments, the implementation of the dose is chosen so as to reduce at least one side effect and at the same time improve and/or maintain efficiency.

[00046] In some embodiments, the combinations and methods of the invention can inhibit the production of Aβ and/or toxic oligomeric Aβ. In some embodiments, the combination and methods of the invention can reduce Aβ and/or toxic Aβ oligomeric protofibrils in the brain.

[00047] In some embodiments, the combinations and methods of the invention may result in improved therapeutic efficacy compared to monotherapy using either of the components alone (ie, BACE1 inhibitor alone or anti-Aβ protofibril antibody alone). In some embodiments, the combinations and methods of the invention may result in improved safety as well as equal efficacy (with dose reduction thus resulting in reduced adverse events) compared to monotherapy using any of the components alone.

[00048] In some embodiments, the combinations and methods of the invention may correspond to monotherapy. The combinations and methods of the invention may provide greater choice for tailoring multicomponent treatment regimens to the needs of the individual patient.

[00049] In some embodiments, combination treatment may result in a greater reduction in monomeric Aβ, protofibrils/oligomeric Aβ, or both, compared to monotherapy using either component alone.

[00050] In some embodiments, the combination treatment may result in a greater reduction in the number and/or area of Aβ plaque formation in the brain compared to monotherapy using either component alone.

[00051] In some embodiments, the combination treatment may result in improved memory impairment and/or inhibition of hyperlocomotion compared to monotherapy using any of the components alone.

[00052] In some embodiments, the combination treatment may result in an improvement in abnormal neuronal viability and/or abnormal synaptic function compared to monotherapy using either of the components alone.

[00053] In some embodiments, the implementation of the combination treatment may lead to improvement in dysfunction of the cortical network compared with monotherapy using any of the components alone.

[00054] In some embodiments, the implementation of the combination treatment may lead to an improvement in increased and/or abnormal neuroinflammatory response compared to monotherapy using any of the components alone.

[00055] In some embodiments, the implementation of the combination treatment can lead to inhibition of the formation of pathology associated with Aβ and/or tau protein, compared with monotherapy using any of the components alone.

[00056] In some embodiments, the combination treatment may result in improved neuronal and/or glial cell viability compared to monotherapy using either component alone.

[00057] In some embodiments, the implementation of the combination treatment can lead to the inhibition of altered gene expression under the influence of pathological Aβ compared with monotherapy using any of the components alone.

[00058] EXAMPLES

[00059] Example 1: Evaluation of in vivo combination treatment in Tg2576 mice in a biochemical study

[00060] Dose

[00061] To study the effects of combined treatment with mAb158 ("Compound A") and Compound X, Tg2576 hetero mice over 11 months of age were treated with Compound A alone (12 mg/kg/week, n=19), Compound X alone (3 mg /kg/day, n=19) or a combination of compounds A and X (n=19). PBS was used as vehicle solution for compound A, and 0.5% methylcellulose solution containing 5% 1 N HCl ("MC solution") was used as vehicle solution for compound X. Tg2576 mice in the control group (n= 20) and wild-type mice (n=15) were treated with both vehicle solutions, PBS (6 ml/kg/week) and MC solution (10 ml/kg/day). The dose of compound A was administered weekly and intraperitoneally, and the dose of compound X was administered once a day and orally. Tg2576 hetero mice in the Compound A or Compound X group were also treated with MC solution or PBS solution, respectively. The duration of administration was 3 months. Mice were sacrificed to obtain brain, CSF and plasma samples. The brain was dissected into separate hemispheres after reperfusion. One cerebral hemisphere was frozen in liquid nitrogen, and the other hemisphere was fixed in 10% phosphate buffered formalin. Frozen brain samples were used to measure Aβ species.

[00062] Measurement of Aβ in the brain

[00063] Frozen Tg2576 mouse cerebral hemispheres were homogenized in Tris buffered saline (TBS, Sigma) supplemented with a mixture of protease inhibitors (cOmplete, Roche) and the homogenized TBS solution was centrifuged at 100,000×g for 1 hour at 4°C . The supernatant (soluble extract) and precipitate were separated from the TBS solution after centrifugation, and the precipitates were successively homogenized using 70% formic acid (as an insoluble extract). For each brain, Aβ concentrations in the soluble and insoluble extract were measured using an Aβ ELISA (human/rat Aβ(40)/(42) ELISA kit, Wako). Statistical differences in Aβ40/42 levels in each extract in the vehicle control group and combination treatment group were analyzed primarily by Student's t-test using GraphPad Prism (GraphPad Software, Inc.). Given the significant differences between the vehicle control group and the combination treatment group, comparison of the combination treatment group and the Compound A group (individually) or the Compound X group (individually) was performed using Dunnett's test.

[00064] Figure 1 shows the measurement of Aβ42 in soluble and insoluble Tg2576 mouse brain extracts.

[00065] Combination treatment with Compound A and Compound X resulted in a significant reduction in Aβ42 levels compared to vehicle administration (p=0.042). In the case of the insoluble extract, the combined treatment resulted in a significant decrease in the level of Aβ42 compared to the vehicle (p=0.035). Statistical differences between the control group and the combination treatment group and between the combination treatment group and the compound X group are indicated in the figure, where * indicates p<0.05 and ^# indicates p<0.1.

[00066] Example 2: Evaluation of In Vivo Combination Treatment in Tg2576 Mice in Biochemical, Pathological and EEG Studies

[00067] Dose

[00068] To study the effects of combined treatment with Compound A and Compound X, Tg2576 mice over 11 months of age were treated with Compound A alone (12 mg/kg/week, n=7), Compound X alone (3 mg/kg/day, n =8) or a combination of compounds A and X (n=9). Compound A was dosed once a week and intraperitoneally, and Compound X was dosed daily and orally. PBS solution was used to administer Compound A and 0.5% methylcellulose solution containing 5% 1 N HCl (MC solution) was used to administer Compound X. Tg2576 hetero mice in the control group (n=8) received both vehicle solutions, PBS (6 ml/kg/week) and MC solution (10 ml/kg/day). Mice in the Compound A group or Compound X group were also injected with MC solution or PBS solution, respectively. The duration of administration was 3 months. In the final week of dosing, mice were subjected to functional evaluation by analysis of electroencephalogram (EEG) records.

[00069] EEG analysis

[00070] Under inhalation anesthesia using isoflurane, Tg2576 mice were kept in a stereotaxic machine and their skulls were exposed for implantation of recording electrodes for EEG and electromyography (EMG). Four EEG electrodes were placed in the skull. Two of the electrodes were placed in positions on the right side [anteroposterior axis (AP)=1.1 mm/lateral (L)=1.3 mm and AP=-4.0 mm/L=1.3 mm], and the remaining two the electrode was placed on the left side [AP=1.1 mm/L=-1.3 mm and AP=-4.0 mm/L=-1.3 mm]. Then, EMG electrodes for recording the myoelectric potential were implanted in the right and left skeletal muscles of the neck.

[00071] EEG and EMG were recorded in mice placed in cages for measuring EEG and EMG. Real-time EEG and EMG signals were acquired from electrodes implanted in Tg2576 mice, amplified using a three-channel biopotential recording system (Pinnacle Technology, Inc.) and recorded to a hard disk using data acquisition software (Sirenia Acquisition, Pinnacle Technology, Inc. ).

[00072] EEG spectral power analysis was performed using SleepSign software (KISSEI COMTEC CO., LTD.). Recorded EEG data for each mouse was analyzed using Fast Fourier Transform (FFT) to obtain the EEG power level (raw EEG power values) for each frequency.

[00073] Raw EEG power values were divided into 8 frequency ranges from 1 to 100 Hz. 8 frequency ranges were arranged in the following order: δ (1-4 Hz), θ (4-8 Hz), α1 (8-11 Hz), α2 (11-13 Hz), β1 (13-22 Hz), β2 ( 22-30 Hz), γ1 (30-48 Hz) and γ2 (52-100 Hz). In each group, the EEG power was summarized in each frequency range and compared between groups.

[00074] Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.). First, a statistical analysis of the EEG powers between the vehicle-treated control group and the combination treatment group was performed using Student's t-test in each frequency range of δ, θ, α1, α2, β1, β2, γ1 and γ2. In the case where significant differences were observed for the EEG power in the frequency range in the first statistical analysis, the EEG power differences between the combination treatment group and the Compound A group or the Compound X group were analyzed by one-way analysis of variance (ANOVA) and then Fisher's least significant difference method. In the combination treatment group, a statistically significant increase in EEG powers in α1 and β1 was observed compared to the vehicle control group. Further statistical analysis showed that the EEG powers in the combination treatment group were significantly higher than in the Compound A group (p=0.0009 in α1 and 0.0106 in β1, respectively) and tended to be higher than in the Compound X group. (p=0.0512 in α1).

[00075] Resting EEG features in AD patients are known to be characterized by an increase in diffuse delta and theta activity as well as a decrease in posterior alpha and beta activity, and an increase in EEG slow wave power, together with a decrease in alpha activity, is associated with a decrease in cognitive performance in MCI compared with healthy subjects (Electroencephalogram and Alzheimer's Disease: Clinical and Research Approaches (A. Tsolaki, et al., International Journal of Alzheimer's Disease, 2014); Electroencephalographic Rhythms in Alzheimer's Disease (R. Lizio et al., International Journal of Alzheimer's Disease, 2011. The effects of combination treatment in the presented mouse model of AD suggest that combination treatment may improve EEG abnormalities in patients with AD.

[00076] Figure 2 shows the effects of combined treatment on EEG recordings in the α1 and β1 frequency bands in Tg2576 hetero mice. The figure shows the statistical differences between the vehicle control group and the combination treatment group and between the combination treatment group and the compound A group, where * indicates p<0.05 and ^# and ^## indicate p<0.1 and p<0.05, respectively.

[00077] Example 3: Compositions

[00078] In some embodiments, Compound X can be formulated into a solid dosage form as described in Example 1 of WO2016/056638. In particular, 2500 mg of pharmacological compound, 2285 mg of lactose (DFE Pharma Corp.), 330 mg of low substituted hydroxypropyl cellulose (LH21 type, Shinetsu Chemical Co., Ltd.) and 165 mg of hydroxypropyl cellulose (SL type, Nippon Soda Co., Ltd.) were mixed in a mortar. An appropriate amount of aqueous ethanol (30% w/w) was added to the resulting mixture, followed by wet granulation in a mortar. After drying the obtained granules using a constant temperature bath, the granules were separated by size using a 1 mm sieve. 33 mg of low substituted hydroxypropylcellulose (type LH21, Shinetsu Chemical Co., Ltd.) and 11 mg of sodium stearyl fumarate (JRS Pharma Corp.) were added per 1056 mg of separated granules and mixed in a vessel. The resulting mixture was compressed at 9 kN using a single-die tableting machine to obtain tablets with a diameter of 6.5 mm and a weight of 110 mg.

[00079] On the other hand, BAN2401 can be formulated in a conventional manner in a liquid dosage form containing, for example, sodium citrate, sodium chloride, and polysorbate 80 with a pH of approximately 5. In some embodiments, the formulation may contain 10 mg/ml BAN2401, 25 mM citrate sodium, 125 mm sodium chloride and 0.2% (wt./vol.) polysorbate 80 and have a pH of 5.7.

[00080] Example 4: Placebo-controlled, double-blind, 24-month, double sham, factorial design study to evaluate the safety and efficacy of Compound X, BAN2401 and the combination of Compound X and BAN2401 in subjects with early Alzheimer's disease

[00081] Study design

[00082] This study is a multicenter, double-blind, placebo-controlled, double sham, factorial design study in subjects with early Alzheimer's disease. The study includes a double sham design using placebo matching BAN2401 intravenous infusions and Compound X oral tablets to allow complete masking in the monotherapy and co-administration groups. All subjects were subjected to intravenous infusions, and they were orally administered tablets.

[00083] The dose and frequency of administration for BAN2401/placebo infusions corresponded to the highest dose and frequency of the well-tolerated regimens selected for the design of the phase 3 study based on the results of the BAN2401-G000-201 extension study, which currently uses 2- and 4-week regimens of administration. Compound X was administered once daily as oral tablets at the maximum tolerated dose from the ongoing study. A total of 3064 subjects were randomized into 4 study arms, all of whom were offered currently approved and stable treatment for Alzheimer's disease or no treatment:

- Arm A (Placebo): Placebo given daily by mouth and intravenous (IV) infusion every 2 (or 4) weeks,

- Group B (compound X monotherapy): Compound X administered orally daily and placebo administered as an IV infusion every 2 (or 4) weeks,

- Group C (BAN2401 monotherapy): BAN2401 given as an IV infusion every 2 (or 4) weeks and placebo given orally daily,

- Group D (co-administration): BAN2401 administered as an IV infusion in 2 doses 2 (or 4) weeks apart, together with placebo administered daily orally until the scheduled 3rd dose of BAN2401. Starting with the 3rd dose of BAN2401, BAN2401 is administered as an IV infusion every 2 (or 4) weeks with Compound X administered orally daily.

[00084] Subjects were randomized at a fixed ratio of 1:1:1:1 to the 4 study groups. Subject randomization was stratified by ApoE genotype, concomitant Alzheimer's drug therapy, and severity of Alzheimer's disease at the time of randomization (i.e., mild cognitive impairment due to Alzheimer's disease/prodromal versus mild dementia).

[00085] Inclusion Criteria

[00086] Diagnostics

[00087] Mild Alzheimer's Cognitive Impairment - Intermediate Probability of MCI Associated with Alzheimer's/Prodromal Alzheimer's:

1. Meeting the National Institute on Aging and Alzheimer's Association (NIA-AA) core clinical criteria for mild cognitive impairment due to Alzheimer's disease - an intermediate likelihood of an association of MCI with Alzheimer's disease;

2. Clinical Dementia Rating Scale (CDR) score of 0.5 and Memory Box CDR score of 0.5 or higher at screening and at baseline; and

3. Subject's history of memory decline with gradual onset and slow progression over the past 1 year prior to screening.

[00088] Mild dementia in Alzheimer's disease:

1. Meeting the NIA-AA core clinical criteria for probable dementia in Alzheimer's disease; and

2. CDR scores of 1.0 and Memory Box scores of 0.5 or higher at screening and at baseline.

[00089] Key inclusion criteria that all subjects must meet:

1. A positive biomarker for brain amyloid pathology, as indicated by at least one of the following:

a) evaluation of imaging agent capture in the brain on PET; an archived amyloid-positive PET scan may be used if performed within 12 months of screening, provided that the central PET reading team considers the scan and its results to be acceptable

b) CSF analysis for Aβ(1-42)

2. Mini-Mental Status Scale scores of 22 or higher at screening and at baseline.

[00090] Investigational treatment

[00091] Treatment includes a double sham design using placebo matching intravenous infusions of BAN2401 and Compound X oral tablets to allow complete masking in the monotherapy and co-administration groups. All subjects were subjected to intravenous infusions, and they were orally administered tablets.

[00092] The dose and frequency of administration for BAN2401/placebo infusions is selected from the most effective and well-tolerated treatment regimen based on the results of the ongoing BAN2401-G000-201 study, which currently uses 2- and 4-week regimens.

[00093] Compound X is administered once daily as oral tablets at the maximum dose believed to be optimally safe and effective based on ongoing study data.

[00094] Compound X is supplied as a 50 mg tablet. The placebo tablets corresponding to Compound X have an identical appearance. Each subject is administered one tablet of Compound X or placebo to be administered orally daily ("QD") in the morning with food.

[00095] The BAN2401 drug product is formulated as a sterile, non-pyrogenic liquid for intravenous administration. Each vial contains 5 ml of a 100 mg/ml BAN2401 solution in isotonic buffer. BAN2401 is administered in normal saline via IV infusion. BAN2401 must be administered using an infusion set containing a 0.22 µm finish inline filter. BAN2401 is administered on a mg/kg basis or placebo. All subjects were infused every 2 weeks or monthly.

[00096] For example, BAN2401 is administered at 2.5 mg/kg/2 weeks, 5 mg/kg/2 weeks, 10 mg/kg/2 weeks, 5 mg/kg/month, or 10 mg/kg/month.

[00097] In previous studies using BAN2401/placebo, infusion reactions have been common AEs, typically occurring on the first infusion, and can be avoided or minimized with prophylactic drugs administered prior to subsequent infusions. Thus, the initiation of Compound X in the co-administration group is delayed to be administered on the same day as the 3rd intravenous infusion of BAN2401 to avoid possible confusion in the definition of adverse events in BAN2401 related infusion reactions and adverse events. associated with co-administration.

[00098] To ensure complete masking, in the case of subjects assigned to group D (co-administration), the first two infusions of BAN2401 are administered intravenously, while placebo tablets are administered to subjects orally QD. At the time of the third infusion, compound X was administered orally to the subject QD and continued for the duration of the study.

[00099] Performance evaluation

[000100] The CDR/Clinical Sum of Dementia Rating Scale, Brief Mental Status Rating Scale, Functional Questionnaire Scorecard (FAQ), and Alzheimer's Modified Severity Rating Scale-Cognitive Subscale (ADAS-Cog14) are well-known clinical tools for use in assessment Alzheimer's disease.

[000101] Disease progression is defined as an increase from baseline of at least 0.5 on the CDR scale at 2 consecutive visits at which the CDR is assessed. In the case of subjects with a CDR of 0.5 at baseline, disease progression is indicated by a CDR of 1 or higher. In the case of subjects with a CDR of 1.0 at baseline, disease progression is indicated by a CDR of 2 or higher.

--->

SEQUENCE LIST

<110> EISAI R&D MANAGEMENT CO., LTD. (EISAI R&D MANAGEMENT CO., LTD.)

<120> COMPOSITION CONTAINING ANTIBODY AGAINST ABETA PROTOFIBRIL AND INHIBITOR

BACE1 BETA-SECRETASES FOR THE TREATMENT OF ALZHEIMER'S DISEASE

<130> 08061.28-304

<140>

<141>

<150> 62/415.165

<151> 2016-10-31

<150> 62/413.961

<151> 2016-10-27

<160> 6

<170>PatentIn version 3.5

<210> 1

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 2

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 2

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 3

<211> 330

<212> Protein

<213> Homo sapiens

<400> 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 4

<211> 107

<212> Protein

<213> Homo sapiens

<400> 4

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 5

<211> 454

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

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Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

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Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

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Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

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Ser Leu Ser Pro Gly Lys

450

<210> 6

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<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 6

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

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85 90 95

Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (9)

1. A method of treating Alzheimer's disease comprising administering to a subject in need a combination of a therapeutically effective amount of an anti-Aβ protofibril antibody and a therapeutically effective amount of N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5 ,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazine-2-carboxamide and/or a pharmaceutically acceptable salt thereof, where the antibody contains (a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and (b) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2. 2. The method of claim 1, wherein the antibody is administered every week. 3. The method of claim 1, wherein (a) the heavy chain constant region further comprises the amino acid sequence of SEQ ID NO: 3, and (b) the light chain constant region further comprises the amino acid sequence of SEQ ID NO: 4. 4. The method according to claim 1, where N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][1,3]thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazine-2-carboxamide is in free base form. 5. Combination for use in the treatment of Alzheimer's disease containing an antibody against Aβ protofibrils and N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H- furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazine-2-carboxamide or a pharmaceutically acceptable salt thereof, where the antibody contains (a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and (b) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2. 6. The combination of claim 5, wherein (a) the heavy chain constant region further comprises the amino acid sequence of SEQ ID NO: 3, and (b) the light chain constant region further comprises the amino acid sequence of SEQ ID NO: 4. 7. The combination according to claim 5, where N-[3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][1,3]thiazin-7a-yl)-4-fluorophenyl]-5-difluoromethylpyrazine-2-carboxamide is in free base form.

Images