EP4419551A1

EP4419551A1 - O-glcnacase (oga) inhibitor combination therapy

Info

Publication number: EP4419551A1
Application number: EP22813018.3A
Authority: EP
Inventors: Kevin BIGLAN; Adam S. FLEISHER; Dustin James Mergott; Hugh N. NUTHALL
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2021-10-22
Filing date: 2022-10-21
Publication date: 2024-08-28
Also published as: AU2022371475A1; MX2024004514A; CN118139880A; IL312045A; JP2024539224A; WO2023070064A1; KR20240099297A; CA3235104A1

Abstract

The present invention provides methods of slowing progression of, treating, and/or preventing a disease characterized by amyloid beta deposits and/or a disease characterized by amyloid beta deposits and aberrant tau aggregation. Such methods comprise administering to a patient in need of such treatment an effective amount of an anti-N3pG Aβ antibody and an effective amount of an OGA inhibitor.

Description

O-GLCNACASE (OGA) INHIBITOR COMBINATION THERAPY

The present disclosure relates to combination of one or more O-GlcNAcase (“OGA”) inhibitors and one or more anti-N3pGlu Amyloid P (anti-N3pG AP) antibodies, and to methods of using the same for treatment of disorders characterized by i) amyloid beta (AP) deposits and/or ii) a combination amyloid beta (AP) deposits and tau-mediated neurodegeneration. Some aspects of the present disclosure are related to treating Alzheimer’s disease (AD).

Alzheimer’s disease is a devastating neurodegenerative disease that is pathologically characterized by amyloid beta deposits and/or aberrant tau aggregation. AD affects millions of people worldwide and a treatment for AD is one of the most significant unmet needs of society. A neuropathological hallmark of AD is the presence of intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Another pathological hallmark of AD is the presence of amyloid beta (AP) deposits. The interplay between tau and Ap pathology is still being deciphered. It is possible that Ap triggers tau pathology, with a more complex and synergistic interaction between Ap and tau manifesting at later stages and driving disease progression (Busche et al., Nature Neuroscience 23: 1183-93 (2020)).

OGA inhibitors that are brain penetrant are desired to provide treatments for tau-mediated neurodegeneration disorders, such as, Alzheimer’s disease. Antibodies targeting Ap (such as anti-N3pGlu Ap antibodies) have shown promise in removal of amyloid deposits in brains of subjects and are used/developed as a therapeutic for Alzheimer’s disease. The present disclosure provides i) certain compounds that are inhibitors of OGA in combination with anti-N3pGlu Ap antibodies and ii) associated methods of treating diseases mediated by aberrant tau and/or amyloid deposits, such as, AD.

The oligomerization of the microtubule-associated protein tau into filamentous structures such as paired helical filaments (PHFs) and straight or twisted filaments, which give rise to neurofibrillary tangles (NFTs) and neuropil threads (NTs), is one of the defining pathological features of Alzheimer’s disease and other tauopathies. The number of NFTs in the brains of individuals with Alzheimer’s disease correlates closely with the severity of the disease. This suggests that tau has a key role in neuronal dysfunction and neurodegeneration (Nelson et al., J Neuropathol Exp Neurol., 71(5), 362-381(2012)). Tau pathology has been shown to correlate with disease duration in PSP; cases with a more aggressive disease course have a higher tau burden than cases with a slower progression. (Williams et al., Brain, 130, 1566-76 (2007)). Recent studies (Yuzwa et al., Nat Chem Biol, 4(8), 483-490 (2008)) support the therapeutic potential of O-GlcNAcase (OGA) inhibitors to limit tau hyperphosphorylation and aggregation into pathological tau for the treatment of Alzheimer’s disease and related tau-mediated neurodegeneration disorders. Specifically, the OGA inhibitor Thiamet-G has been linked in slowing motor neuron loss in the JNPL3 tau mouse model (Yuzwa et al., Nat Chem Biol, 8, 393-399 (2012)) and to a reduction in tau pathology and dystrophic neurites in the Tg4510 tau mouse model (Graham et al., Neuropharmacology, 79, 307-313 (2014)). Accordingly, OGA inhibitors are recognized as a valid therapeutic approach to reduce the accumulation of hyperphosphorylated, pathological forms of tau, such as NFTs and NTs. U.S. Patent No. 9,120,781 discloses hexahydrobenzooxazole and hexahydrobenzothiazole derivatives which possess OGA inhibitory activity and are further disclosed as useful in treating diseases and disorders related to deficiency or overexpression of OGA, and/or accumulation or deficiency of 2- acetamido-2-deoxy-5B-D-glucopyranoside Q-GlcNAc). In addition, US 2016/0031871 discloses certain glycosidase inhibitors for treating Alzheimer’s disease.

Accumulation of amyloid-P peptide in the form of brain amyloid deposits is an early and essential event in Alzheimer’s disease leading to neurodegeneration and consequently the onset of clinical symptoms, such as, cognitive, and functional impairment (Selkoe, JAMA 283: 1615-7 (2000); Hardy et al., Science 297:353-6 (2002); Masters et al., Nat. Rev. Dis. Primers 1 : 15056 (2015); and Selkoe et al., EMBO Mol. Med. 8:595-608 (2016)). Amyloid beta is formed by the proteolytic cleavage of a larger glycoprotein called amyloid precursor protein (APP). APP is an integral membrane protein expressed in many tissues, but especially in neuron synapses. APP is cleaved by y-secretase to release the Ap peptide, which encompasses a group of peptides ranging in size from 37-49 amino acid residues. Ap monomers aggregate into various types of higher order structures including oligomers, protofibrils, and amyloid fibrils. Amyloid oligomers are soluble and may spread throughout the brain, while amyloid fibrils are larger and insoluble and can further aggregate to form amyloid deposits or plaques. The amyloid deposits found in human patients include a heterogeneous mixture of A0 peptides, some of which include N-terminal truncations and further may include N-terminal modifications such as an N-terminal pyroglutamate residue (pGlu). The amyloid deposits found in human patients include a heterogeneous mixture of A0 peptides. N3pGlu A0 (also referred to as N3pG Ap, N3pE AP, AP pE3-42, or APp3- 42) is a truncated form of AP peptide and is found only in amyloid deposits. N3pGlu AP lacks the first two amino acid residues at the N-terminus of human Ap and has a pyroglutamate which is derived from glutamic acid at the third amino acid position of Ap. Although N3pGlu AP peptide is a minor component of the deposited AP in the brain, studies suggest that N3pGlu Ap peptide has aggressive aggregation properties and accumulates early in the deposition cascade.

Antibodies to N3pGlu AP are known in the art. For example, U.S. Patent No. 8,679,498; U.S. Patent No. 8,961,972; US Patent No. 10,647,759; and US Patent No. 11,078,261 (which are hereby incorporated by reference in their entireties) disclose anti- N3pGlu AP antibodies, method of making the antibodies, antibody formulations, and methods of treating diseases, such as, Alzheimer’s disease with such antibodies.

Passive immunization by long term chronic administration of antibodies against Ap, including N3pGlu Ap, found in deposits has been shown to disrupt the Ap aggregates and promote the clearance of deposits in the brain in various animal models. Donanemab (disclosed in U.S. Patent No. 8,679,498) is an antibody directed at the pyroglutamate modification of the third amino acid of amyloid beta (N3pGlu AP) epitope that is present only in brain amyloid deposits.

The treatment and prevention strategy for donanemab includes targeting N3pGlu Ap specific to amyloid deposits in the population of early symptomatic AD patients with existing brain amyloid load. This rationale is based on the amyloid hypothesis of AD, which states that the production and deposition of Ap is an early and necessary event in the pathogenesis of AD. See, e.g., Selkoe, JAMA 283: 1615-1617 (2000), which is hereby incorporated by reference in its entirety. Donanemab has recently shown efficacy/potency in removal of amyloid deposits and in slowing of AD progression. See, e.g., Mintun et al., New England Journal of Medicine 384.18: 1691-1704 (2021), which is hereby incorporated by reference in its entirety.

A combination of an antibody that specifically binds anti-N3pG Ap and which reduces the amyloid beta in the brain of a human subject with an OGA inhibitor is desired to provide treatment for diseases, such as, AD. Such a combination allows for reduction in pathogenic tau species and tau aggregates as well as reduction of amyloid beta (AP). Such combination may also preferably be more effective than either molecule alone. For example, treatment with such combination may allow for use of lower doses of either or both molecules as compared to each molecule used alone, potentially leading to lower side effects (or a shorter duration of one or the other therapy) while maintaining efficacy. It is believed that the combination provided herein will not only reduce amyloid beta but also reduce aberrant tau, tau aggregation into pathological tau, and propagation thereof for the treatment of diseases, such as, AD.

In one aspect, the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta (AP) deposits. In some embodiments, the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and aberrant tau aggregation. This method comprises administering to the patient in need thereof an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl at position 2 of the OGA inhibitor is in cis configuration relative to the oxygen at position 4 on the piperidine ring:

In some embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4- [(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, N-[4-fluoro- 5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l- piperidyl]methyl]thiazol-2-yl]acetamide is characterized by a peak in the X-ray powder diffraction spectrum, at diffraction angle 2-theta of 12.1° in combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8°, with a tolerance for the diffraction angles of 0.2 degrees. Another aspect of the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta (AP) deposits comprising administering to the patient in need thereof an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and aberrant tau aggregation. In some embodiments, the methyl at position 2 of the OGA inhibitor is in cis configuration relative to the oxygen at position 4 on the piperidine ring: In some embodiments, the methyl at position 2 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 4 on the piperidine ring:

In some embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4- oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, the OGA inhibitor is characterized by a peak in the X-ray powder diffraction spectrum at diffraction angle 2- theta of 13.5° in combination with one or more peaks selected from the group consisting of 5.8°, 13.0°, 14.3°, 17.5°, 20.4°, 21.4°, and 22.2° with a tolerance for the diffraction angles of 0.2 degrees.

Another aspect of the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta (AP) deposits comprising administering to the patient in need thereof an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and aberrant tau aggregation. In some embodiments, the methyl at position 5 of the OGA inhibitor is in cis configuration relative to the oxygen at position 3 on the piperidine ring:

In some embodiments, the methyl at position 5 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 3 on the piperidine ring:

In some embodiments, the OGA inhibitor is l-(2-(((3R,5S)-l-((6-fluoro-2- methylbenzo[d]thiazol-5-yl)methyl)-5-methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H- pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one. In some embodiments, the OGA inhibitor is crystalline.

Yet another aspect of the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and/or aberrant tau aggregation, comprising administering to the patient in need thereof an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl at position 2 of the OGA inhibitor is in cis configuration relative to the oxygen at position 4 on the piperidine ring:

In some embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, N-[4-fluoro- 5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l- piperidyl]methyl]thiazol-2-yl]acetamide is characterized by a peak in the X-ray powder diffraction spectrum, at diffraction angle 2-theta of 12.1° in combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8°, with a tolerance for the diffraction angles of 0.2 degrees.

Yet another aspect of the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and/or aberrant tau aggregation, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl at position 2 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 4 on the piperidine ring:

In some embodiments, the methyl at position 2 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 4 on the piperidine ring: In some embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In embodiments, the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4- oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, the OGA inhibitor is characterized by a peak in the X-ray powder diffraction spectrum at diffraction angle 2- theta of 13.5° in combination with one or more peaks selected from the group consisting of 5.8°, 13.0°, 14.3°, 17.5°, 20.4°, 21.4°, and 22.2° with a tolerance for the diffraction angles of 0.2 degrees. Yet another aspect of the present disclosure is related to a method of treating a patient having a disease characterized by amyloid beta deposits and/or aberrant tau aggregation, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl at position 5 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 3 on the piperidine ring:

The present invention also provides a method of treating a cognitive or neurodegenerative disease, comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3pGlu Ap antibody in combination with an effective amount of an OGA inhibitor. The present invention further provides a method of treating clinical or pre-clinical AD comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3pGlu Ap antibody in combination with an effective amount of an OGA inhibitor. The present invention also provides a method of treating prodromal AD (sometimes also referred to as mild cognitive impairment, or MCI), mild AD dementia, moderate AD dementia and/or severe AD dementia, comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3pGlu Ap antibody in combination with an effective amount of an OGA inhibitor.

In some embodiments, the present disclosure provides a method of treating, preventing, or slowing functional/cognitive decline in a patient diagnosed with preclinical Alzheimer’s disease (AD) (also referred to as pre-symptomatic AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, Down’s syndrome, clinical cerebral amyloid angiopathy, or pre-clinical cerebral amyloid angiopathy. Such methods comprise administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor as disclosed herein. The present invention further provides a method of preventing memory loss, cognitive decline, or functional decline in clinically asymptomatic subjects with low or very low levels of Api-42 in the cerebrospinal fluid (CSF) and/or low or very low Ap deposits in the brain, comprising administering an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor as disclosed herein. In some embodiments, the clinically asymptomatic subjects are known to have an Alzheimer’s disease-causing genetic mutation. In the present disclosure, “clinically asymptomatic subjects known to have an Alzheimer’s diseasecausing genetic mutation” include patients known to have a PSEN1 E280A Alzheimer’s disease-causing genetic mutation (Paisa mutation), a genetic mutation that causes autosomal-dominant Alzheimer’s disease or are at higher risk for developing AD by virtue of carrying one or two APOE e4 alleles. In some embodiments, the present disclosure provides a method of treating, preventing, or slowing cognitive/functional decline in a patient known to have a PSEN1 E280A Alzheimer’s disease-causing genetic mutation (Paisa mutation), a genetic mutation that causes autosomal-dominant Alzheimer’s disease, or carrying one or two APOE e4 alleles, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor as disclosed herein.

In some embodiments, the present disclosure also provides a method of treating, preventing, or slowing cognitive/functional decline in a patient diagnosed with preclinical Alzheimer’s disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, and severe AD dementia, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor as disclosed herein. The present invention further provides a method of preventing memory loss or cognitive/functional decline in clinically asymptomatic patients with low levels of NFTs in the brain and/or low levels of amyloid deposits in the brain, comprising administering an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor as disclosed herein.

Another embodiment of the present invention provides a method for the prevention of the progression of mild cognitive impairment to AD, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor.

The present embodiments also provide an anti-N3pG Ap antibody, for use in simultaneous, separate, or sequential combination with an OGA inhibitor, for use in therapy.

The invention further provides a pharmaceutical composition comprising an anti- N3pG Ap antibody, with one or more pharmaceutically acceptable carriers, diluents, or excipients, in combination with a pharmaceutical composition of an OGA inhibitor, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

In addition, the invention provides a kit, comprising an anti-N3pG Ap antibody, and an OGA inhibitor. The invention further provides a kit comprising a pharmaceutical composition comprising an anti-N3pG Ap antibody (with one or more pharmaceutically acceptable carriers, diluents, or excipients) and a pharmaceutical composition comprising an OGA inhibitor (with one or more pharmaceutically acceptable carriers, diluents, or excipients). As used herein, a “kit” includes separate containers of each component, wherein one component is an anti-N3pG Ap antibody, and another component is an OGA inhibitor, in a single package. A “kit” may also include separate containers of each component, wherein one component is an anti-N3pG Ap antibody, and another component is an OGA inhibitor, in separate packages with instructions to administer each component as a combination.

The invention further provides the use of an anti-N3pG Ap antibody for the manufacture of a medicament for the treatment of AD, mild AD, prodromal AD, or for the prevention of the progression of mild cognitive impairment to AD, wherein the medicament is to be administered simultaneously, separately, or sequentially with an OGA inhibitor.

In some embodiments of the present disclosure, the anti-N3pG Ap antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR) and the LC comprises a light chain variable region (LCVR), said HCVR comprising complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and said LCVR comprising CDRs LCDR1, LCDR2 and LCDR3. In some embodiments, the anti-N3pG Ap antibodies of the present invention have the amino acid sequence of LCDR1 given by SEQ ID NO.5, the amino acid sequence of LCDR2 given by SEQ ID NO.6, the amino acid sequence of LCDR3 given by SEQ ID NO.7, the amino acid sequence of HCDR1 given by SEQ ID NO.8, the amino acid sequence of HCDR2 given by SEQ ID NO.9, and the amino acid sequence of HCDR3 given by SEQ ID NO.10.

In an embodiment, the present invention provides anti-N3pG Ap antibody comprising a LCVR and a HCVR, wherein the amino acid sequence of the LCVR is given by SEQ ID NO. l and the amino acid sequence of the HCVR is given by SEQ ID NO.2. In a further embodiment, the present invention provides anti-N3pG Ap antibody comprising a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of the LC is given by SEQ ID NO.3 and the amino acid sequence of the HC is given by SEQ ID NO.4.

In some embodiments, the anti-N3pG Ap antibodies of the present invention have the amino acid sequence of LCDR1 given by SEQ ID NO.15, the amino acid sequence of LCDR2 given by SEQ ID NO.16, the amino acid sequence of LCDR3 given by SEQ ID NO.17, the amino acid sequence of HCDR1 given by SEQ ID NO.18, the amino acid sequence of HCDR2 given by SEQ ID NO.19, and the amino acid sequence of HCDR3 given by SEQ ID NO.20. In an embodiment, the present invention provides anti-N3pG Ap antibody comprising a LCVR and a HCVR, wherein the amino acid sequence of the LCVR is given by SEQ ID NO.11 and the amino acid sequence of the HCVR is given by SEQ ID NO.12. In a further embodiment, the present invention provides an anti-N3pG Ap antibody comprising a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of the LC is given by SEQ ID NO.13 and the amino acid sequence of the HC is given by SEQ ID NO.14.

The anti-N3pG Ap antibodies of the present invention may be prepared and purified using known methods. For example, cDNA sequences encoding a HC of an anti-N3pG Ap antibody and cDNA sequences encoding a LC of the anti-N3pG Ap antibody may be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector may then be stably transfected into CHO cells. As one of skill in the art will appreciate, mammalian expression of antibodies will result in glycosylation, typically at highly conserved N-glycosylation sites in the Fc region. Stable clones may be verified for expression of an antibody specifically binding to amyloid deposits or N3pG Ap. Positive clones may be expanded into serum-free culture medium for antibody production in bioreactors. Media, into which an antibody has been secreted, may be purified by conventional techniques. For example, the medium may be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline. The column may be washed to remove nonspecific binding components. The bound antibody may be eluted, for example, by pH gradient, and antibody fractions may be detected using techniques such as by SDS-PAGE, and subsequently pooled. The antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, for example at -70 °C, or may be lyophilized.

The anti-N3pG Ap antibodies of the present invention bind human N3pG Ap (also referred to as N3pGlu AP). In an embodiment, the anti-N3pG Ap antibodies of the present invention bind a conformational epitope of human N3pG Ap. As used herein, an “antibody” is an immunoglobulin molecule comprising two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The CDRs are interspersed with regions that are more conserved, termed framework regions. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present invention is based on the following: Kabat numbering convention (Kabat, et al., Ann. NY Acad. Set. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242 (1991)), and North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)). Following the above methods, the CDRs of the antibodies of the present invention were determined.

The anti-N3pGlu Ap antibodies of the present invention include kappa LC and IgG HC. In a particular embodiment, the anti-N3pGlu Ap antibodies of the present invention are of the human IgGl isotype.

The antibodies of the present invention are monoclonal antibodies (“mAbs”). Monoclonal antibodies can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR- grafting, or combinations of such or other technologies known in the art. The monoclonal antibodies of the present invention are human or humanized. Humanized antibodies can be engineered to contain one or more human framework regions (or substantially human framework regions) surrounding CDRs derived from a non-human antibody. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT®) via their website: imgt.cines.fr, or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic 25 Press, 2001, ISBN 012441351. In another embodiment of the present invention, the antibody, or the nucleic acid encoding the same, is provided in isolated form. As used herein, the term “isolated” refers to a protein, peptide or nucleic acid that is not found in nature and is free or substantially free from other macromolecular species found in a cellular environment. “Substantially free”, as used herein, means the protein, peptide, or nucleic acid of interest comprises more than 80% (on a molar basis) of the macromolecular species present, preferably more than 90%, and more preferably more than 95%.

The anti-N3pGlu Ap antibody of the present invention or its combination with an OGA inhibitor is administered as a pharmaceutical composition. The pharmaceutical composition of the present invention can be administered to a patient at risk for, or exhibiting, diseases or disorders as described herein by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular). Subcutaneous and intravenous routes are preferred. In some embodiments, the pharmaceutical compositions of the present invention are administered by intravenous infusion.

The terms “treatment,” “treating” or “to treat” and the like include restraining, slowing, or stopping the progression or severity of an existing symptom, condition, disease, or disorder in a patient. The term “patient” or “subject” refers to a human.

The term “prevention” means prophylactic administration of the antibody of the present invention to an asymptomatic patient or a patient with pre-clinical Alzheimer’s disease to prevent onset or progression of the disease.

The terms “disease characterized by deposition of AP” or a “disease characterized by Ap deposits” are used interchangeably and refer to a disease that is pathologically characterized by Ap deposits in the brain or in brain vasculature of a subject. This includes diseases such as Alzheimer’s disease (AD), Down’s syndrome (DS), and cerebral amyloid angiopathy (CAA). A clinical diagnosis, staging or progression of Alzheimer’s disease can be readily determined by the attending diagnostician or health care professional, as one skilled in the art, by using known techniques and by observing results. This generally includes some form of brain plaque imaging, mental or cognitive assessment (e.g., Clinical Dementia Rating - summary of boxes (CDR-SB), Mini-Mental State Exam (MMSE) or Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog)) or functional assessment (e.g., Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL). The cognitive and functional assessment can be used to determine changes in a patient’s cognition (e.g., cognitive decline) and function (e.g., functional decline). “Clinical Alzheimer’s disease” as used herein is a diagnosed stage of Alzheimer’s disease. It includes conditions diagnosed as prodromal Alzheimer’s disease, mild Alzheimer’s disease dementia, moderate Alzheimer’s disease dementia, and severe Alzheimer’s disease dementia. The term “pre-clinical Alzheimer’s disease” is a stage that precedes clinical Alzheimer’s disease, where measurable changes in biomarkers (such as CSF Ap42 levels or deposited brain plaque by amyloid PET) indicate the earliest signs of a patient with Alzheimer’s pathology, progressing to clinical Alzheimer’s disease. This is usually before symptoms such as memory loss and confusion are noticeable. Pre-clinical Alzheimer’s disease also includes pre-symptomatic autosomal dominant carriers, as well as patients with higher risk for developing AD by virtue of carrying one or two APOE e4 alleles. The term “clinical AD” is the stage of disease where cognitive signs and symptoms are detectable.

In some embodiments, a subject is positive for amyloid deposits when amyloid is detected in the brain by methods such as, amyloid imaging with radiolabeled PET compounds or using a diagnostic that detects Ap or a biomarker for Ap. Exemplary methods that can be used in the present disclosure to measure the brain amyloid load/burden include, e.g., Florbetapir (Carpenter, et al., “The Use of the Exploratory IND in the Evaluation and Development of ¹⁸F-PET Radiopharmaceuticals for Amyloid Imaging in the Brain: A Review of One Company's Experience,” The Quarterly Journal of Nuclear Medicine and Molecular Imaging 53A.3X1 (2009), which is hereby incorporated by reference in its entirety); Florbetaben (Syed et al., “[¹⁸F]Florbetaben: A Review in P- Amyloid PET Imaging in Cognitive Impairment,” CNS Drugs 29, 605-613 (2015), which is hereby incorporated by reference in its entirety); and Flutemetamol (Heurling et al., “Imaging P-amyloid Using [¹⁸F] Flutemetamol Positron Emission Tomography: From Dosimetry to Clinical Diagnosis,” European Journal of Nuclear Medicine and Molecular Imaging 43.2: 362-373 (2016), which is hereby incorporated by reference in its entirety).

[¹⁸F]-Florbetapir can provide a qualitative and quantitative measurement of brain plaque load in patients, including patients with prodromal AD or mild AD dementia. For example, the absence of significant [¹⁸F]-florbetapir signal on a visual read indicates patients clinically manifesting cognitive impairment have sparse to no amyloid plaques. As such, [¹⁸F]-florbetapir also provides a confirmation of amyloid pathology. [¹⁸F]-Florbetapir PET also provides quantitative assessment of fibrillar amyloid plaque in the brain and, in some embodiments, can be used to assess amyloid plaque reductions from the brain by antibodies of the present disclosure.

Amyloid imaging with radiolabeled PET compounds can also be used to determine if Ap deposit in the brain of a human patient is reduced or increased (e.g., to calculate the percentage reduction in Ap deposit post treatment or to assess the progression of AD). A person of skill in the art can correlate the standardized uptake value ratio (SUVr) values obtained from amyloid imaging (with radiolabeled PET compounds) to calculate the % reduction in Ap deposit in the brain of the patient before and after treatment. The SUVr values can be converted to standardized centiloid units, where 100 is average for AD and 0 is average for young controls, allowing comparability amongst amyloid PET tracers, and calculation of reduction according to centiloid units (Klunk et al., “The Centiloid Project: Standardizing Quantitative Amyloid Plaque Estimation by PET,” Alzheimer ’s & Dementia 11.1 : 1-15 (2015) and Navitsky et al., “Standardization of Amyloid Quantitation with Florbetapir Standardized Uptake Value Ratios to the Centiloid Scale,” Alzheimer's & Dementia 14.12: 1565-1571 (2018), which are hereby incorporated by reference in their entireties). In some embodiments, the change in brain amyloid plaque deposition from baseline is measured by [¹⁸F]-florbetapir PET scan.

Cerebrospinal fluid or plasma-based analysis of P-amyloid can also be used to measure the amyloid load/burden for the purposes of the present disclosure. For example, AP42 can be used to measure brain amyloid (Palmqvist, S. et al., “Accuracy of Brain Amyloid Detection in Clinical Practice Using Cerebrospinal Fluid Beta-amyloid 42: a Cross-validation Study Against Amyloid Positron Emission Tomography. JAMA Neurol 71, 1282-1289 (2014), which is hereby incorporated by reference in its entirety). In some embodiments, the ratio of AP42/AP40 or AP42/AP38 can be used as a biomarker for amyloid beta (Janelidze et al., “CSF Abeta42/Abeta40 and Abeta42/Abeta38 Ratios: Better Diagnostic Markers of Alzheimer Disease,” Ann Clin Transl Neurol 3, 154-165 (2016), which is hereby incorporated by reference in its entirety).

In some embodiments, deposited brain amyloid plaque or Ap in CSF or plasma can be used to stratify subjects into groups and to identify which group of subjects is responsive to treatment/prevention of a disease (as described herein) using the antibodies or the methods described herein.

Tau levels in the brain of human subject can be determined using methods, such as, tau imaging with radiolabeled PET compounds (Leuzy et al., “Diagnostic Performance of RO948 Fl 8 Tau Positron Emission Tomography in the Differentiation of Alzheimer Disease from Other Neurodegenerative Disorders,” JAMA Neurology 77.8:955-965 (2020); Ossenkoppele et al., “Discriminative Accuracy of [¹⁸F]-flortaucipir Positron Emission Tomography for Alzheimer Disease vs Other Neurodegenerative Disorders,” JAMA 320, 1151-1162, (2018), which are hereby incorporated by reference in their entireties).

In some embodiments, the biomarker [¹⁸F]-florbtaucipir, which is a PET ligand, may be used for the purposes of the present disclosure. PET tau images can be, for example, quantitatively evaluated to estimate an SUVr (standardized uptake value ratio) by published methods (Pontecorvo et al., “A Multicentre Longitudinal Study of Flortaucipir (¹⁸F) in Normal Ageing, Mild Cognitive Impairment and Alzheimer's Disease Dementia,” Brain 142: 1723-35 (2019); Devous et al., “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir Fl 8,” Journal of Nuclear Medicine 59:937-43 (2018); Southekal et al., “Flortaucipir F 18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J. Nucl. Med. 59:944-51 (2018), which are hereby incorporated by reference in their entireties) and/or to visually evaluate patients, e.g., to determine whether the patient has an AD pattern (Fleisher et al., “Positron Emission Tomography Imaging With [¹⁸F]- flortaucipir and Postmortem Assessment of Alzheimer Disease Neuropathologic Changes,” JAMA Neurology 77:829-39 (2020), which is hereby incorporated by reference in its entirety). Lower SUVr values indicate less tau burden while higher SUVr values indicate a higher tau burden. In an embodiment, quantitative assessment by a flortaucipir scan is accomplished through an automated image processing pipeline as described in Southekal et al., “Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J. Nucl. Med. 59:944-951 (2018), which is hereby incorporated by reference in its entirety. In some embodiments, counts within a specific target region of interest in the brain (e.g., multiblock barycentric discriminant analysis or MUBADA, see Devous et al, “Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir Fl 8,” J. Nucl. Med. 59:937-943 (2018), which is hereby incorporated by reference in its entirety) are compared with a reference region wherein the reference region is, e.g., whole cerebellum, (wholeCere), cerebellar GM (cereCrus), atlas-based white matter (atlasWM), subjectspecific WM (ssWM, e.g., using parametric estimate of reference signal intensity (PERSI), see Southekal et al., “Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity,” J. Nucl. Med. 59:944-951 (2018), which is hereby incorporated by reference in its entirety). A preferred method of determining tau burden is a quantitative analysis reported as a standardized uptake value ratio (SUVr), which represents counts within a specific target region of interest in the brain (e.g., MUB DA,) when compared with a reference region (c.g, using PERSI).

In some embodiments, phosphorylated tau (P-tau; either phosphorylated at threonine 181 or 217) can be used to measure the tau load/burden for the purposes of the present disclosure (Barthelemy et al., “Cerebrospinal Fluid Phospho-tau T217 Outperforms T181 as a Biomarker for the Differential Diagnosis of Alzheimer's Disease and PET Amyloid-positive Patient Identification,” Alzheimer ’s Res. Ther. 12, 26 (2020); Mattsson et al., “Ap Deposition is Associated with Increases in Soluble and Phosphorylated Tau that Precede a Positive Tau PET in Alzheimer’s Disease,” Science Advances 6(16), (2020); which are hereby incorporated by reference their entireties). In a particular embodiment, antibodies directed against human tau phosphorylated at threonine at residue 217 can be used to measure the tau load/burden in a subject for the purposes of the present disclosure (see International Patent Application Publication No. WO 2020/242963, which is incorporated by reference in its entirety). The present disclosure includes, in some embodiments, the use of anti-tau antibodies disclosed in WO 2020/242963 to measure the tau load/burden in a subject. The anti-tau antibodies disclosed in WO 2020/242963 are directed against isoforms of human tau expressed in the CNS (e.g., recognizing the isoforms expressed in the CNS and not recognizing isoforms of human tau expressed exclusively outside the CNS). Such antibodies against isoforms of human tau expressed in the CNS can be used in a method of identifying/selecting a patient as one or more of: (i) having a disease disclosed herein; (ii) at risk for having a disease disclosed herein; (iii) in need of treatment for a disease disclosed herein; or (iv) in need of neurological imaging. A reduction or slowing of cognitive decline can be measured by cognitive assessments such as Mini -Mental State Exam (MMSE) or Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog). A reduction or slowing of functional decline can be measured by functional assessments such as Alzheimer’s Disease Cooperative Study- Activities of Daily Living (ADCS-ADL). A reduction or slowing of functional and cognitive decline can be measured by composite measures such as Clinical Dementia Rating - summary of boxes (CDR-SB) or Integrated Alzheimer's Disease Rating Scale (iADRS).

As used herein, “mg/kg” means an amount, in milligrams, of antibody or drug administered to a patient based on his or her bodyweight in kilograms. A dose is given one time. For example, a 10 mg/kg dose of antibody for a patient weighing 70 kg would be a single 700 mg dose of the antibody given in a single administration. A 20 mg/kg dose of antibody for a patient weighing 70 kg would be a single 1400 mg dose of the antibody given in a single administration. Similarly, a 40 mg/kg dose of antibody for a patient weighing 80 kg would be a 3200 mg dose of the antibody given at a single administration.

As used herein, the phrase “in combination with” refers to the administration of an anti-N3pGlu Ap antibody of the present invention, with another molecule (a “combination molecule,” such as an OGA inhibitor, symptomatic agent, or Ap antibody), simultaneously, or sequentially in any order, or any combination thereof. In some embodiments, the phrase “in combination with” refers to the administration of an anti-N3pGlu Ap antibody of the present invention, with an OGA inhibitor simultaneously, or sequentially in any order, or any combination thereof. The two molecules may be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The anti- N3pGlu Ap antibody can be administered prior to, at the same time as, or subsequent to administration of the OGA inhibitor, or in some combination thereof. Where the combination is administered at repeated intervals (e.g., during a standard course of treatment), the anti-N3pGlu Ap antibody can be administered prior to, at the same time as, or subsequent to, each administration of an OGA inhibitor, or some combination thereof, or at different intervals in relation to therapy with an OGA inhibitor, or in a single or series of dose(s) prior to, at any time during, or subsequent to the course of treatment with an OGA inhibitor.

In particular embodiments of the present invention, the antibodies and antibody fragments (e.g., anti-N3pG Ap antibodies), or the nucleic acids encoding same, may be provided in isolated form. As used herein, the term “isolated” refers to a protein, peptide, or nucleic acid that is not found in nature and which is free or substantially free from other macromolecular species found in a cellular environment. “Substantially free” as used herein, means the protein, peptide or nucleic acid of interest that comprises more than 80% (on a molar basis) of the macromolecular species present, preferably more than 90%, and more preferably more than 95%.

In some embodiments, the antibody of the present invention is expressed in cell cultures. Following expression and/or secretion of the antibodies and antibody fragments of the present invention, the medium is clarified to remove cells and the clarified media is purified using any of many commonly used techniques. Purified antibodies and antibody fragments may be formulated into pharmaceutical compositions according to well-known methods for formulating proteins and antibodies for parenteral administration, particularly for subcutaneous, intrathecal, or intravenous administration. The antibodies and antibody fragments may be lyophilized, together with appropriate pharmaceutically acceptable excipients, and later reconstituted with a water-based diluent prior to use. Alternatively, the antibodies and antibody fragments may be formulated in an aqueous solution and stored prior to use. In either case, the stored form and the injected form of the pharmaceutical compositions of the antibodies and antibody fragments will contain a pharmaceutically acceptable excipient or excipients, which are ingredients other than the antibodies and antibody fragments. Whether an ingredient is pharmaceutically acceptable depends on its effect on the safety and effectiveness or on the safety, purity, and potency of the pharmaceutical composition. If an ingredient is judged to have a sufficiently unfavorable effect on safety or effectiveness (or on safety, purity, or potency) to warrant it not being used in a composition for administration to humans, then it is not pharmaceutically acceptable to be used in a pharmaceutical composition of the antibody and antibody fragments. The novel combinations and methods of the present invention include OGA inhibitors that are brain penetrant. In some embodiments of the novel combinations and methods of the present invention, the OGA inhibitor comprises a compound of Formula I: or a pharmaceutically acceptable salt thereof.

In some embodiments, the OGA inhibitor of the novel combinations and methods of the present invention is a compound of Formula la: or a pharmaceutically acceptable salt thereof.

Certain configurations of Formula I, which comprise embodiments of the OGA inhibitor of the novel combinations and methods of the present invention, further include:

and pharmaceutically acceptable salts thereof.

The 5-methyl-l,2,4-oxadiazol-3-yl compound of Formula I wherein the methyl and oxygen substituents on the piperidine ring are in the cis or trans configuration, or pharmaceutically acceptable salt thereof, are included within the scope of the OGA inhibitor of the present novel combination. The present novel combination also contemplates all individual enantiomers and diasteromers, as well as mixtures of the enantiomers of 5-methyl-l,2,4-oxadiazol-3-yl compounds of the present invention, including racemates. Absolute configurations of 5-methyl-l,2,4-oxadiazol-3-yl compounds of the novel combinations and methods provided herein include:

N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l- piperidyl]methyl]thiazol-2-yl]acetamide, and pharmaceutically acceptable salts thereof; and

N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l- piperidyl]methyl]thiazol-2-yl]acetamide are particularly preferred.

5-Methyl-l,2,4-oxadiazol-3-yl OGA inhibitor compounds of the present invention, or salts thereof, may be prepared by a variety of procedures known to one of ordinary skill in the art. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention, or salts thereof. The products of each step below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents, unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following preparations, and examples are provided to further illustrate the invention. In addition, one of ordinary skill in the art appreciates that the compounds of Formulas la, lb, Ic, and Id may be prepared by using starting material with the corresponding stereochemical configuration which can be prepared by one of skill in the art. For example, the preparations below utilize starting materials with the configuration corresponding ultimately to Formula la.

The following Preparations and Examples are intended to illustrate but not to limit the invention, and that various modifications may be made by one of ordinary skill in the art. The following Examples and assays demonstrate that the anti-N3pG Ap antibodies of the present invention are useful for treating a disease characterized by deposition of Ap, such as of Alzheimer’s disease, Down’s syndrome, and CAA.

Preparation 1: Synthesis of tert-butyl N-(4-fluoro-5-formyl-thiazol-2-yl)carbamate.

Cesium fluoride (227 g, 1480 mmol) is added to a solution of tert-butyl N-(4- chloro-5-formyl-thiazol-2-yl)carbamate (38.8 g, 148 mmol; for preparation of tert-butyl N- (4-chloro-5-formyl-thiazol-2-yl)carbamate see for example, N. Masuda, et al., BioorgMed Chem, 12, 6171-6182 (2004)) in dimethyl sulfoxide (DMSO, 776 mL) at room temperature. The reaction mixture is stirred in a 145 °C heating block with an internal temperature of 133 °C for 48 hours, and the mixture is cooled in an ice-water bath. To the mixture is added saturated aqueous sodium bicarbonate solution (500 mL), saturated aqueous NaCl (500 mL) and ethyl acetate (500 mL). The mixture is stirred at room temperature for 10 minutes, and is filtered through diatomaceous earth, washing with ethyl acetate (500 mL). The filtrate is transferred to a separating funnel and the layers are separated, then the aqueous layer is extracted with ethyl acetate (1 L). The combined organics are washed with saturated aqueous NaCl (1 L), then the saturated aqueous NaCl layer is extracted with ethyl acetate (300 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue. The residue is passed through a pad of silica gel (330 g), eluting with 5% ethyl acetate in dichloromethane (1.5 L), and the filtrate is concentrated under reduced pressure to give a residue (24.2 g).

The residue (32.7 g of combined lots, 133 mmol) is dissolved in isopropanol (303 mL), filtered, and purified by SFC (Supercritical Fluid Chromatography) using an IC column (cellulose polysaccharide derivative: tris (3,5-dichlorophenylcarbamate, 30 x 250mm, 5pm) with 10% isopropyl alcohol (no additive) at 180 mL/minute with 3 mL injections. The product-containing fractions are concentrated to obtain the title compound (16.1 g, 48% yield). MS m/z 247.0 (M+H).

Preparation 2: Synthesis N-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (Method A).

In a jacketed vessel, zinc bromide (91.9 g, 408 mmol) is added in one portion to a mixture of tert-butyl N-(4-fluoro-5-formyl-thiazol-2-yl)carbamate (33.5 g, 136 mmol) and di chloromethane (503 mL) at room temperature. The reaction mixture is stirred overnight at an internal temperature of 37 °C, then the jacket temperature is set to -10 °C and tetrahydrofuran (111 mL) is added dropwise over 15 minutes, maintaining an internal temperature below 6 °C. The jacket temperature is then set to -30 °C and pyridine (110 mL, 1360 mmol) is added dropwise over 5 minutes, maintaining an internal temperature below 5 °C. The jacket temperature is set to 0 °C and acetic anhydride (116 mL, 1220 mmol) is added dropwise over 5 minutes. The reaction mixture is stirred overnight at an internal temperature of 37 °C, cooled to room temperature, and passed through a short pad of diatomaceous earth, eluting with tetrahydrofuran (500 mL). The filtrate is transferred to a flask and the mixture is concentrated under reduced pressure to give a residue, which is additionally concentrated from toluene (50 mL). To the residue is added a solution of citric acid monohydrate (57.2 g, 272 mmol) in water (400 mL) and 2-methyltetrahydrofuran (400 mL) and the mixture is stirred at 40 °C for 5 minutes. The mixture is passed through a short pad of diatomaceous earth, eluting with 2-methyltetrahydrofuran (100 mL). The filtrate is transferred to a separating funnel and the layers are separated. The aqueous layer is extracted with 2-methyltetrahydrofuran (2 x 250 mL) and the combined organic extracts are diluted with water (500 mL). To the mixture is added solid sodium bicarbonate portion wise over 5 minutes with stirring until gas evolution ceases. The mixture is transferred to a separating funnel and the layers are separated; the aqueous layer is extracted with 2- methyltetrahydrofuran (200 mL and 100 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which is diluted with 2-methyltetrahydrofuran (100 mL), and the mixture is passed through a short pad of silica gel (250 g), eluting with 2-methyltetrahydrofuran (2.5 L). The filtrate is concentrated under reduced pressure to give a residue which is suspended in a 1 : 1 mixture of dichloromethane and heptane (202 mL). The mixture is stirred at room temperature for 30 minutes and filtered. The filtered solid is collected and dried under vacuum at 40 °C for 2 hours to give the title compound (18.0 g, 70% yield). MS m/z 189.0 (M+H).

Alternative synthesis ofN-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (Method B).

Add dichloromethane (1325 g, 15.6 mol) to 2-amino-4-chlorothiazole-5- carbaldehyde (100 g, 0.61 mol) and pyridine (194.6 g, 2.46 mol), and cool to 0-5 °C. Add acetic anhydride (188.4 g, 1.85 mol) dropwise, maintaining the temperature at 0-5 °C. After addition is complete, adjust the temperature to 20-25 °C and stir for 41 hours. Concentrate under reduced pressure followed by addition of 35% aqueous HC1 (200 mL) and water (1.5 L), maintaining the temperature at less than 40 °C . Cool to 20-25 °C and stir for 18 hours. Filter the resulting mixture and wash the collected solid with water. Dry the solids at 60- 65 °C for 24 hours to provide N-(4-chloro-5-formylthiazol-2-yl)acetamide (75 g, 66% yield). Under an inert atmosphere, add sulfolane (1000 mL) to the N-(4-chloro-5- formylthiazol-2-yl)acetamide (50 g, 0.24 mol, prepared directly above), tetramethylammonium chloride (107.1 g, 0.98 mol), and cesium fluoride (370.6 g, 2.4 mol). Heat to 130 °C and stir for 23 hours. HPLC analysis shows 75% conversion with an in- situ yield of 45% of the title compound.

Alternative synthesis ofN-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (Method C).

Add 2-propanol (150 mL) to tetramethylammonium fluoride tetrahydrate (10.2 g, 109.0 mmol) and concentrate the mixture to about half volume under vacuum with internal temperature maintained at 70 °C to remove water. Add 2-propanol (200 mL) and concentrate the mixture to about half volume under vacuum. Repeat two more times. Add dimethylformamide (DMF, 200 mL) and concentrate to about half volume under vacuum. Add tetrahydrofuran (THF, 200 mL) and concentrate to about half volumes. Repeat two more times. Charge N-(4-chloro-5-formylthiazol-2-yl)acetamide (1.22 g, 6.0 mmol, prepared above in Method B) and DMF (12 mL). Heat to 110 °C and stir for 12 hours. Cool reaction mixture to 25 °C. Add 2-methyltetrahydrofuran (40 mL) and water (40 mL). Separate the resulting layers and extract the aqueous layer with 2-methyltetrahydrofuran (40 mL). The layers are separated, and the combined organic layers are washed with water (20 mL). The layers are separated, and the organic layer is concentrated under reduced pressure. Ethyl acetate (20 mL) and water (5 mL) are added, the resulting layers are separated, and the organic layer is concentrated to remove solvent. Ethyl acetate (2 mL) and heptane (2 mL) are added, and the resulting mixture is filtered. The filtered solids are dried under vacuum at 55 °C for 18 hours to give the title compound as a 93% mixture with N-(4-chloro-5-formylthiazol-2-yl)acetamide.

Preparation 3: Synthesis of tert-butyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l- carboxylate.

To a flask is added tert-butyl (2S)-2-methyl-4-oxo-piperidine-l -carboxylate (50 g, 234.4 mmol) and tetrahydrofuran (500 mL). The mixture is cooled to -65 °C under an atmosphere of nitrogen and a IM solution of lithium tri(sec-butyl)borohydride (304.77 mL, 304.8 mmol) in tetrahydrofuran is added dropwise over 45 minutes, maintaining an internal temperature below -60 °C. The reaction mixture is stirred at room temperature for 1 hour and cooled to -30 °C. To the reaction mixture is added a mixture of water (25.3 mL) and tetrahydrofuran (100.2 mL), maintaining an internal temperature below -20 °C. An aqueous solution of 30% weight/weight hydrogen peroxide (118.9 mL, 1.2 mol) in water (126.70 mL) is added dropwise over 1 hour, maintaining an internal temperature below 10 °C. To the mixture is added 5M aqueous HC1 solution (46.9 mL, 234.4 mmol) and methyl t-butyl ether (1 L) and the mixture is warmed to room temperature. The layers are separated, and the organic phase is stirred with a solution of sodium metabisulfite (222.8 g, 1.17 mol) in water (500 mL) for 10 minutes at room temperature. The layers are separated, and the organic phase is dried over magnesium sulfate and concentrated under reduced pressure. The residue is purified by flash chromatography over silica gel, eluting with 0- 50% methyl t-butyl ether/isohexane), and the product-containing fractions are combined and concentrated under reduced pressure to obtain the title compound (40.4 g, 78% yield). ES/MS m/z 238 (M+Na).

Alternative synthesis of tert-butyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l -carboxylate.

To a glass-lined reactor containing deionized water (460 L), and potassium dihydrogen phosphate (6.5 kg, 0.41 equiv) at 20 °C is charged DMSO (27.4 kg, 1.0 vol) and D-(+)-glucose monohydrate (28.9 kg, 1.25 equiv). The internal temperature is adjusted to 30 °C, and the pH of the reaction is adjusted to 6.9 by addition of an 8% solution of aqueous sodium hydroxide (15 L, 0.3 equiv). The reactor is charged with tert-butyl (2S)- 2-methyl-4-oxo-piperidine-l -carboxylate (24.9 kg, 1.0 equiv (99.1%ee)), and the mixture is agitated at 30 °C for 15 min. Ketoreductase (KRED-130, 250 g, 1% weight/weight), glucose dehydrogenase (GDH-101, 250 g, 1% weight/weight), and NADP sodium salt (63 g, 0.25% weight/weight) are charged directly to the reaction mixture via an open port. The mixture is maintained at a temperature of 30 °C and pH 7.0 ± 0.2 via addition of an 8% aqueous solution of NaHCO,. After stirring for 16.5 hours the reaction is charged with diatomaceous earth (12.5 kg, 50% weight/weight) and toluene (125 L, 5 vol). After stirring for 30 min at 30 °C, the mixture is transferred to another 2000 L reactor via an in-line GAF- filter (4 socket) over the period of 1 h. The mixture is allowed to stand 30 min without agitation, the layers are separated, and the aqueous layer is back-extracted with toluene (2 x 125 L). The combined organic layers are filtered (in-line GAF-filter), and the toluene mixture is washed with a 25% solution of aqueous sodium chloride solution (125 L, 5 vol) at 25 °C. The resulting toluene solution is azeotropically dried (partial vacuum, internal temp < 60 °C) to 0.10 weight/weight% water and cooled to 20 °C. The mixture is filtered out of the reactor via a cartridge filter into clean drums under positive nitrogen pressure. The reaction mixture is then transferred from the drums into a 500 L glass lined vessel and concentrated under vacuum (< 60 °C) to a target residual volume of about 56 L (2.25 vol). n-Heptane (169 kg, 10 vol) is charged at 40 °C, and the mixture is seeded with 25 g of tertbutyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l -carboxylate. The resulting thick slurry is diluted with additional n-heptane (25 L, 1 vol) and cooled to 16 °C over 4 hours. The product is isolated via centrifugation, washing with n-heptane (25 L per spin; 4 spins necessary), yielding 20.3 kg (81% yield; >99.9% ee) after drying for 11 hours in a tray dryer at 30 °C. ES/MS m/z 238 (M+Na).

Preparation 4: Synthesis of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol- 3-yl)methoxy]piperidine-l-carboxylate.

3-(Chloromethyl)-5-methyl-l,2,4-oxadiazole (43.5 g, 301 mmol) is added to a solution of tert-butyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l -carboxylate (29.5 g, 137 mmol) in acetonitrile (590 mL) at room temperature. The reaction mixture is stirred in an ice-water bath and sodium tert-butoxide (54.3 g, 548 mmol) is added in portions over 10 minutes, maintaining an internal temperature below 10 °C. The reaction mixture is stirred in an ice-water bath at an internal temperature of 5 °C for 9 hours, warmed slowly to room temperature, and is stirred overnight. The reaction mixture is cooled in an ice-water bath and saturated aqueous ammonium chloride solution (200 mL) is added over 5 minutes, maintaining an internal temperature below 10 °C during the addition. The mixture is then diluted with water (100 mL) and warmed to room temperature. The mixture is extracted with methyl Zc/V-butyl ether (2 x 300 mL) and the combined organic extracts are washed with saturated aqueous NaCl (300 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue. The residue is passed quickly through a pad of silica gel (300 g), eluting with methyl /c/V-butyl ether (1 L), and the filtrate is concentrated under reduced pressure to obtain the title compound (46.5 g, >99% yield). MS m/z 334.0 (M+Na). Alternative synthesis of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]piperidine-l-carboxylate. To a solution of tert-butyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l-carboxylate

(0.25g, 1.16 mmol) and 3-(chloromethyl)-5-methyl-l,2,4-oxadiazole (308 mg, 2.3 mmol) in N,N-dimethylformamide (3 mL) under nitrogen at 0 °C is added portion wise sodium tert-butoxide (0.35 g, 3.5mmol) over 5 minutes. The reaction mixture is stirred at room temperature for 10 minutes and at 40 °C for 12 hours. The reaction mixture is cooled to room temperature and quenched with water (10 mL). The layers are separated, and the aqueous phase is extracted with methyl tert-butyl ether (2 x 10 mL). The combined organic extracts are washed with a 5% aqueous solution of lithium chloride, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford the title compound (0.49 g, 81% yield, 60% purity) as a brown oil. MS m/z 334.0 (M+Na).

Preparation 5: Synthesis of 5-methyl-3-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]- 1,2,4-oxadiazole hydrochloride.

CIH A flask containing tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]piperidine-l -carboxylate (4.0 g, 12.9 mmol) is submerged in an ice- water bath. To this flask is added a 4 M solution of hydrochloric acid in 1,4-dioxane (25.9 mL, 104 mmol) dropwise over 5 minutes with stirring, maintaining an internal temperature below 20 °C during the addition. The reaction mixture is stirred at room temperature for 1 hour and is concentrated under reduced pressure to obtain the title compound (3.5 g, 92% yield based on 83% purity measured by NMR). MS m/z 212.0 (M+H).

Alternative synthesis of 5-methyl-3-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]-l,2,4- oxadiazole hydrochloride.

Add methanol (50 mL) to tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,2,4- oxadiazol-3-yl)methoxy]piperidine-l -carboxylate (12.9 g, 0.04 mol). The mixture is cooled to 0 °C. A 4M solution of hydrochloric acid in methanol (80 mL) is added dropwise to the cooled mixture, maintaining an internal temperature below 20 °C. The reaction mixture is stirred at room temperature for 18 hours. The mixture is concentrated to remove solvent. Acetone (10 mL) is added, and the mixture is stirred for 20 minutes. Tetrahydrofuran (40 mL) is added, and the mixture is stirred for 3 hours. The solid is collected by filtration under nitrogen and the filtered solid cake is rinsed with tetrahydrofuran. The filtered solid is dried under vacuum at 45 °C for 2 hours to obtain the title compound with 90% purity. Recrystallization using acetone can increase purity of title compound to 95%.

Preparation 6: Synthesis of 5-methyl-3-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]- 1,2,4-oxadiazole. To a solution of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]piperidine-l -carboxylate (0.49 g, 1.6 mmol) in dichloromethane (10 mL) under nitrogen is added trifluoroacetic acid (1.8 mL, 23 mmol). The mixture is stirred at room temperature for 3 hours. The mixture is concentrated under reduced pressure to afford a yellow oil. The residue is dissolved in methanol (5 mL) and poured onto a cation exchange cartridge, eluted sequentially with methanol (2x 10 mL) and a 2 M solution of ammonia in methanol (10 mL). The filtrate is concentrated under reduced pressure to give title compound (0.3 g, 91% yield). MS m/z 212.0 (M+H).

In other embodiments of the novel combinations and methods of the present invention, the OGA inhibitor a compound of Formula X: or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a compound of Formula Xa: or a pharmaceutically acceptable salt thereof.

Certain configurations of Formula X, which comprise embodiments of the OGA inhibitor of the novel combinations and methods of the present invention, further include:

The 5-methyl-l,3,4-oxadiazol-2-yl compound of Formula X wherein the methyl and oxygen substituents on the piperidine ring are in the cis or trans configuration, or pharmaceutically acceptable salt thereof, are included within the scope of the OGA inhibitor of the present novel combination. The present novel combination also contemplates all individual enantiomers and diasteromers, as well as mixtures of the enantiomers of 5-methyl-l,3,4-oxadiazol-2-yl compounds of the present invention, including racemates. Absolute configurations of 5-methyl-l,3,4-oxadiazol-2-yl compounds of the novel combinations and methods provided herein include:

N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l- piperidyl]methyl]thiazol-2-yl]acetamide, and pharmaceutically acceptable salts thereof, including with the free base, and including the crystalline form.

The 5-methyl-l,3,4-oxadiazol-2-yl compounds of the novel combinations and methods of the present invention, or salts thereof, may be prepared by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the schemes, preparations, and examples below. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention, or salts thereof. The products of each step below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations, and examples are provided to further illustrate the invention. In addition, one of ordinary skill in the art appreciates that the compounds of Formulas Xa, Xb, Xc, and Xd may be prepared by using starting material with the corresponding stereochemical configuration which can be prepared by one of skill in the art. For example, the preparations below utilize starting materials with the configuration corresponding ultimately to Formula Xa.

Preparation 7: Synthesis of 2-[[(2S,4S)-l-tert-butoxycarbonyl-2-methyl-4- piperidyl] oxy] acetic acid.

2-Chloro-l -morpholino-ethanone (59.4 g, 363 mmol) is added to a solution of tertbutyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l -carboxylate (52.1 g, 242 mmol) in acetonitrile (521 mL) at room temperature. The reaction mixture is stirred in an ice-water bath and sodium tert-butoxide (48.0 g, 484 mmol) is added in portions over 10 minutes, maintaining an internal temperature below 15 °C. The reaction mixture is stirred at room temperature for 2 hours and is added over 5 minutes to another flask containing saturated aqueous ammonium chloride solution (250 mL) and water (250 mL) with ice-water bath cooling, maintaining an internal temperature below 15 °C during the addition. The mixture is warmed to room temperature and extracted with methyl tert-butyl ether (2 x 500 mL), and the combined organic extracts are washed with saturated aqueous NaCl (300 mL). The combined organics are dried over sodium sulfate, filtered, and concentrated to give a residue, which is combined with 2-propanol (414 mL) and 2M aqueous sodium hydroxide solution (303 mL, 605 mmol) at room temperature. The reaction mixture is stirred in a 47 °C heating block overnight with an internal temperature of 45 °C. The reaction mixture is cooled to room temperature and concentrated to remove 2-propanol, and the mixture is diluted with water (50 mL). The mixture is extracted with methyl tert-butyl ether (250 mL), and the aqueous layer is cooled in an ice-water bath and acidified with acetic acid (55.6 mL, 968 mmol). The aqueous mixture is extracted with ethyl acetate (4 x 250 mL); the combined organic extracts are dried over sodium sulfate, filtered; and concentrated to give a residue, which is concentrated from toluene (3 x 100 mL) to give the title compound (79.8 g, >99% yield). MS m/z 272.0 (M-H).

Preparation 8: Synthesis of tert-butyl (2S,4S)-4-[2-(2-acetylhydrazino)-2-oxo- ethoxy]-2-methyl-piperidine-l-carboxylate.

Tetrahydrofuran (798 mL) is added to a flask containing 2-[[(2S,4S)-l-tert- butoxycarbonyl-2-methyl-4-piperidyl]oxy]acetic acid (79.8 g, 224 mmol) and the mixture is stirred in an ice-water bath with an internal temperature of 5 °C. To the mixture is added l,l'-carbonyldiimidazole (43.5 g, 268 mmol) in one portion and the reaction mixture is stirred at room temperature for 2 hours. An additional portion of 1, l'-carbonyldiimidazole (7.25 g, 44.7 mmol) is added and the reaction mixture is stirred at room temperature for 30 minutes. The reaction mixture is submerged in an ice-water bath and acetohydrazide (21.5 g, 291 mmol) is added in one portion. The reaction mixture is stirred at room temperature overnight. The reaction mixture is cooled with stirring in an ice-water bath and saturated aqueous sodium bicarbonate solution (500 mL) is added over 2 minutes, maintaining an internal temperature below 15 °C. The mixture is diluted with water (300 mL) and the resulting mixture is concentrated under reduced pressure to remove tetrahydrofuran. The resulting aqueous mixture is extracted with 2-methyltetrahydrofuran (4 x 500 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue which is combined with ethyl acetate (200 mL) and heptane (200 mL). The mixture is stirred at room temperature for 30 minutes, diluted with heptane (200 mL), stirred vigorously at room temperature for an additional 30 minutes, and is filtered. The filtered solid is dried under vacuum at 40 °C for 2 hours to give the first crop of the title compound (71.5 g). The filtrate is refiltered and the filtered solid is dried under a stream of nitrogen gas at room temperature for 15 minutes to give the second crop of the title compound (1.98 g). The majority of the first crop of product (71.1 g, 216 mmol) and the second crop of product (1.97 g, 5.98 mmol) are combined with /c/7-butyl methyl ether (731 mL) and the mixture is stirred in a 45 °C heating block for 30 minutes at an internal temperature 40 °C, cooled to room temperature over 1 hour with stirring, and filtered. The filtered solid is dried under vacuum at room temperature under a stream of nitrogen gas for 30 minutes to give the title compound (53.7 g, 71% yield). MS m/z 352.0 (M+Na).

Preparation 9: Synthesis of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol- 2-yl)methoxy]piperidine-l-carboxylate.

To a solution of tert-butyl (2S,4S)-4-hydroxy-2-methyl-piperidine-l-carboxylate (0.5 g, 2 mmol) in N,N-dimethylformamide (5 mL) under nitrogen at room temperature is added portion wise sodium tert-butoxide (920 mg, 9.3 mmol). The resulting reaction mixture is stirred at room temperature for 40 minutes. The reaction mixture is cooled to 0 °C and 2-(chloromethyl)-5-methyl-l,3,4-oxadiazole (416 mg, 3.1 mmol) is added. The resulting solution stirred at room temperature overnight. The reaction mixture is concentrated under reduced pressure and the resulting residue diluted with water. The mixture is extracted with 3 portions of ethyl acetate. The combined organic extracts are dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford a crude oil.

The residue is dissolved in dimethyl sulfoxide (to a total volume of 2 mL), and purified by prep-HPLC (Phenomenex Gemini -NX 10 Micron 30 x 100mm C-18), eluting with a gradient of 15% (CH3CN & Water with 10 mM ammonium bicarbonate adjusted to pH 9 with ammonium hydroxide) to 100% CH3CN over 7 minutes (50 mL/min, 1 injection, 204 nm) to afford the title compound (28 mg, 4% yield). MS m/z 312 (M+H).

Alternative synthesis of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol-2- yl)methoxy]piperidine-l-carboxylate.

To a flask is added tert-butyl (2S,4S)-4-[2-(2-acetylhydrazino)-2-oxo-ethoxy]-2- methyl-piperidine-1 -carboxylate (53.7 g, 163 mmol) and acetonitrile (537 mL) and the slurry is stirred at room temperature. To the mixture is added N,N-diisopropylethylamine (114 mL, 652 mmol) in one portion and p-toluenesulfonyl chloride (77.7 g, 408 mmol) in three portions over 5 minutes with water bath cooling. The reaction mixture is stirred at room temperature overnight, then is cooled in an ice-water bath. N',N'-dimethylethane- 1,2-diamine (21.8 g, 245 mmol) is added dropwise over 10 minutes, maintaining an internal temperature below 15 °C. The reaction mixture is stirred at room temperature for 30 minutes, and diluted with saturated aqueous citric acid solution (50 mL), ethyl acetate (500 mL) and water (450 mL) at room temperature. The layers are separated, and the organic layer is washed with a mixture of saturated aqueous citric acid solution (50 mL) and water (450 mL). The organic layer is washed with saturated aqueous sodium bicarbonate solution (500 mL) and the aqueous layer is then extracted with ethyl acetate (500 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue, which is passed through a short pad of silica gel (400 g), eluting sequentially with 25% ethyl acetate in heptane (2 x 500 mL fractions) and ethyl acetate (5 x 500 mL fractions). The product-containing fractions are concentrated to obtain the title compound (53.3 g, >99% yield). MS m/z 312.2 (M+H). Preparation 10: Synthesis of 5-methyl-5-[[(2S,4S)-2-methyl-4-pipendyl]oxymethyl]- 1,3,4-oxadiazole 2,2,2-trifluoroacetic acid.

To a solution of tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol-2- yl)methoxy]piperidine-l -carboxylate (27.5 mg, 0.09 mmol) in dichloromethane (3 mL) under nitrogen is added trifluoroacetic acid (0.035 mL, 0.45 mmol). The mixture is stirred at room temperature overnight. The mixture is concentrated under reduced pressure to afford the title compound (0.04 g, 84% yield). MS m/z 212.0 (M+H).

Preparation 11: Synthesis of 2-methyl-5-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]- 1,3,4-oxadiazole.

To a flask is added tert-butyl (2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol-2- yl)methoxy]piperidine-l -carboxylate (52.9 g, 170 mmol) and dichloromethane (265 mL) at room temperature. The reaction mixture is stirred in an ice-water bath at an internal temperature of 5 °C and trifluoroacetic acid (265 mL, 3500 mmol) is added dropwise over 5 minutes, maintaining an internal temperature below 10 °C. The reaction mixture is stirred at room temperature for 15 minutes and concentrated to give a residue, which is diluted with water (300 mL) and methyl tert-butyl ether (300 mL). The layers are separated, and the aqueous layer is stirred in an ice-water bath and basified with 50% aqueous sodium hydroxide solution (20 mL), maintaining an internal temperature below 10 °C during the addition. The mixture is extracted with dichloromethane (4 x 300 mL) and the combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give the title compound (30.5 g, 85% yield). MS m/z 212.2 (M+H).

In other embodiments of the novel combinations and methods of the present invention, the OGA inhibitor a compound of Formula XX: or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a compound of Formula XXa: or a pharmaceutically acceptable salt thereof.

and pharmaceutically acceptable salts thereof.

The 5-(methylpyrrolidin-3-yl)oxy compound of Formula XX wherein the methyl and oxygen substituents on the pyrrolidine ring are in the cis or trans configuration, or pharmaceutically acceptable salt thereof, are included within the scope of the OGA inhibitor of the present novel combination. The present novel combination also contemplates all individual enantiomers and diasteromers, as well as mixtures of the enantiomers of 5-(methylpyrrolidin-3-yl)oxy compounds of the present invention, including racemates. Absolute configurations of 5-(methylpyrrolidin-3-yl)oxy compounds of the novel combinations and methods provided herein include:

1 -(2-(((3R,5 S)- 1 -((6-fluoro-2-methylbenzo[d]thiazol-5-yl)methyl)-5 - methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one, and pharmaceutically acceptable salts thereof, including the free base, and including the crystalline form. The 5-(methylpyrrolidin-3-yl)oxy compounds of the novel combinations and methods of the present invention, or salts thereof, may be prepared by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the schemes, preparations, and examples below. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention, or salts thereof. The products of each step below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations, and examples are provided to further illustrate the invention. In addition, one of ordinary skill in the art appreciates that the compounds of Formulas XXa, XXb, XXc, and XXd may be prepared by using starting material with the corresponding stereochemical configuration which can be prepared by one of skill in the art. For example, the preparations below utilize starting materials with the configuration corresponding ultimately to Formula XXa.

Preparation 12: l-(2-chloro-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one

N,N-Diisopropylethylamine (DIPEA, 3.6 mL, 21 mmol) and acetyl chloride (0.4 mL, 6 mmol) are added dropwise to a 0 °C solution of 2-chloro-6,7-dihydro-5H- pyrrolo[3,4-b]pyridine hydrochloride (1.0 g, 5.2 mmol) in dichloromethane (DCM, 13 mL). The reaction mixture is stirred at room temperature for 24 hours. The resulting mixture is diluted with DCM (20 mL) and saturated aqueous NaHCO, (30 mL). The aqueous layer is extracted with DCM (2X in 30 mL). The combined organic extracts are dried over MgSCL, filtered, and concentrated under reduced pressure. The resulting residue is dissolved in DCM, adsorbed onto diatomaceous earth, and purified via flash chromatography over silica gel, eluted with a gradient of 50-100% acetone in hexanes to obtain the title compound after solvent evaporation of the desired chromatographic fractions (0.95 g, 92% yield). ES/MS m/z: 197 (M+H).

Preparation 13: tert-butyl (2S,4R)-4-((6-acetyl-6,7-dihydro-5H-pyrrolo[3,4- b]pyridin-2-yl)oxy)-2-methylpyrrolidine-l-carboxylate.

To a solution of tert-butyl (2S,4R)-4-hydroxy-2-methyl-pyrrolidine-l-carboxylate (0.41 g, 2.03 mmol), l-(2-chloro-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one (0.47 g, 2.36 mmol), and tetrahydrofuran (THF, 8 mL) at room temperature (RT) is portion wise added potassium tert-butoxide (KO-t-Bu, 0.45 g, 4 mmol) and the mixture is stirred at 50 °C for 4.5 hours. The reaction mixture is diluted with water (50 mL) and ethyl acetate (EtOAc, 50 mL). The aqueous layer is extracted with EtOAc (2X in 50 mL), and the combined organic extracts are dried over MgSCU, filtered, and concentrated under reduced pressure. The resulting residue is dissolved in DCM and purified via flash chromatography over silica gel, eluting with a gradient of 40-100% acetone in hexanes, to obtain the title compound after solvent evaporation of the desired chromatographic fractions (0.34 g, 47% yield). ES/MS m/z: 262 (M+H-C4H9).

Preparation 14: l-(2-(((3R,5S)-5-methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H- pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one hydrochloride.

HCI

To a solution of /c/7-butyl (2S,4R)-4-((6-acetyl-6,7-dihydro-5H-pyrrolo[3,4- b]pyridin-2-yl)oxy)-2-methylpyrrolidine-l -carboxylate (0.34 g, 0.94 mmol) in DCM (5.0 mL) a 4M solution of HCI in 1,4-dioxane (1.2 mL, 4.8 mmol) is added. The resulting mixture is stirred at RT for 3 hours. The resulting suspension is concentrated under reduced pressure, and the resulting residue is placed under vacuum for 1 hour to obtain the title compound (0.28 g, >99% yield). ES/MS m/z: 262 (M+H).

Preparation 15: N-(5-bromo-2,4-difluoro-phenyl)acetamide.

To a flask is added acetic anhydride (AC2O, 389 mL) with stirring in a heating block at about 61 °C (internal temperature 60 °C). To the flask is added 5-bromo-2,4- difluoroaniline (77.7 g, 374 mmol) portion wise over 30 minutes, maintaining an internal temperature below 65 °C during the addition. The reaction mixture is stirred in a heating block at about 61 °C for 10 minutes and cooled to RT to give a residue which is concentrated from toluene (4 x 200 mL) to give a pale brown/pink solid. The concentrated solid is suspended in heptane (80 mL) and the mixture is agitated on a rotary evaporator in a 50 °C water bath for 15 minutes at atmospheric pressure, cooled to RT, and filtered. The filtered solid is collected and dried under vacuum at 40 °C for 2 hours to obtain the title compound (89.6 g, 95% yield) as an off-white solid. ES/MS m/z: 250 (M+H).

Preparation 16: N-(5-bromo-2,4-difluoro-phenyl)thioacetamide.

To a solution of N-(5-bromo-2,4-difluoro-phenyl)acetamide (89.6 g, 358 mmol) in anhydrous acetonitrile (ACN, 896 mL) is added pyridin-l-ium-l-yl-[pyridin-l-ium-l- yl(sulfido)phosphinothioyl]sulfanyl-sulfido-thioxo-phosphane (68.2 g, 179 mmol, J. Org. Chem. 76, 1546-1553(2011)) at RT. The slurry is stirred in an 85 °C heating block overnight (internal temperature 80 °C), cooled to RT, and poured into a mixture of ice (200 g) and saturated aqueous NaCl (700 mL). The mixture is diluted with EtOAc (900 mL) stirred at RT for 10 minutes, the layers are separated, and the aqueous layer additionally extracted with EtOAc (900 mL). The combined organic extracts are washed with saturated aqueous NaCl solution (900 mL), dried over Na2SO4, and concentrated under reduced pressure to give the title compound as a dark brown oil, which is dissolved in DMF (953 mL) at RT, and used without additional purification.

Preparation 17: 5-bromo-6-fluoro-2-methyl-l,3-benzothiazole.

To a DMF solution of N-(5-bromo-2,4-difluoro-phenyl)thioacetamide is added sodium tert-butoxide (NaO-LBu, 42.6 g, 430 mmol) portion wise over 20 minutes with stirring, maintaining an internal temperature below 30 °C. The reaction mixture is stirred at RT for 5 minutes, stirred overnight in a 42 °C heating block (internal temperature 40 °C), and cooled to RT. The reaction mixture is added dropwise to a mixture of ice (250 g) and H2O (700 mL) over 5 minutes, maintaining an internal temperature below 20 °C. The mixture is stirred at RT for 10 minutes and filtered. The filtered solid is dried under vacuum at 40 °C overnight, and suspended in 50% MeOH/ELO (480 mL). The mixture is stirred in a 45 °C heating block for 15 min, cooled to RT, and filtered. The filtered solid is dried under vacuum at 40 °C for 72 hours to give a pale brown solid. The material is combined with EtOAc (700 mL) and the mixture is stirred at RT for 10 minuts, H2O (700 mL) is added, and the layers separated. The aqueous layer is extracted with EtOAc (700 mL), then the combined organic extracts are washed with saturated aqueous NaCl (700 mL), dried over MgSO4, and concentrated under reduced pressure to give the title compound (62.7 g, 71% yield) as a brown solid. 'H NMR (d₆-DMSO) 5: 2.82 (s, 3H), 7.57 (m, 1H), 8.12 (m, 1H).

Preparation 18: 6-fluoro-2-methyl-l,3-benzothiazole-5-carbaldehyde.

5-Bromo-6-fluoro-2-methyl-l,3-benzothiazole (100.9 g, 410 mmol) in DMF (1009 mL) is sparged with N2 for 5 minutes at RT with stirring. Potassium formate (52.3 g, 615.0 mmol), palladium(II) acetate (2.82 g, 12.30 mmol), 2-(di-ter/-butylphosphino)biphenyl (5.19 g, 17.2 mmol) and 1,1,3,3-tetramethylbutyl isocyanide (90.8 mL, 492.0 mmol) are added and the mixture is sparged with N2 for 30 minutes at RT with stirring. The reaction mixture is stirred overnight at an internal temperature of 65 °C, cooled to 20-25 °C, and 2M aqueous HC1 solution (820 mL) is added dropwise over 30 minutes, maintaining an internal temperature below 30 °C. The resulting mixture is stirred at 20-25 °C for 2 hours and diluted with EtOAc (1.5 L) and H2O (1 L). The layers are separated, and the organic layer is washed with 10% aqueous N-acetyl-cysteine solution (2 ^ 1 L), saturated aqueous ISfeCCL (750 mL x 2) and saturated aqueous NaCl (750 mL); the organic extract is dried over MgSCU and concentrated under reduced pressure to provide the first batch of crude material. The aqueous HC1 layer from the first extraction is further extracted with EtOAc (1 L, then 500 mL), and the combined organic extracts are washed with saturated aqueous NaCl (500 mL), dried over MgSO4, and concentrated under reduced pressure to provide the second batch of crude material. The combined aqueous N-acetyl-cysteine layers are then extracted with EtOAc (1 L, then 500 mL) and the combined organic extracts are washed sequentially with saturated aqueous Na2CO3 (500 mL) and saturated aqueous NaCl (500 mL); the combined organic extracts are dried over MgSO4 and concentrated under reduced pressure to provide the third batch of crude material. The three batches of crude material are combined in methyl tert-butyl ether (MTBE, 250 mL) and heptane (250 mL) and the resulting slurry is stirred at RT for 20 minutes. The resulting precipitate is filtered and washed with heptane (250 mL). The filtered solid is dried under vacuum at 45 °C to give a first batch of product. The filtrate is concentrated and the residue is purified by column chromatography over silica, eluting with a gradient of 0-100% EtOAc/heptane. The product-containing fractions are combined and concentrated to a volume of approximately 400 mL, the resulting slurry is stirred at RT for 15 minutes, filtered, and the filtered solid is washed with heptane (200 mL), to give a second batch of product. The first and second batches of product are combined with heptane (500 mL), slurried at RT, filtered, and the filtered solid is washed with heptane (250 mL). The filtered solid is dried under vacuum at 45 °C overnight to give the title compound (63.5 g, 79% yield). ES/MS m/z: 196 (M+H). It should be appreciated that individual isomers, enantiomers, and diastereomers of the OGA inhibitor compounds, provided herein as part of the novel combinations and methods of the instant invention, may be separated or resolved by one of ordinary skill in the art at any convenient point in the synthesis of compounds of the invention, by methods such as selective crystallization techniques or chiral chromatography (See for example, J. Jacques, et al., “Enantiomers, Racemates, and Resolutions," John Wiley and Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen, “Stereochemistry of Organic Compounds," Wiley - Interscience, 1994).

A pharmaceutically acceptable salt of the OGA inhibitor compounds of the invention can be formed, for example, by reaction of an appropriate free base of a compound of the invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. The formation of such salts is well known and appreciated in the art. See, for example, Gould, P.L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4: 427-435 (2000); and Berge, S.M., etal., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

Example 1: Synthesis of OGA Inhibitor N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide (Formula la).

This compound and its synthesis are disclosed in US Patent No. US 10,081,625, which is hereby incorporated by reference in its entirety. N-(4-Fluoro-5-formyl-thiazol-2-yl)acetamide (28.3 g, 150 mmol) is added to 5- methyl-3-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]-l,2,4-oxadiazole hydrochloride (48.7 g, 185 mmol, 94% purity) in ethyl acetate (707 mL) at room temperature. The reaction mixture is stirred at room temperature and N,N-diisopropylethylamine (34.1 mL, 195 mmol) is added dropwise over 1 minute, and sodium triacetoxyborohydride (98.5 g, 451 mmol) is added in one portion. The reaction mixture is stirred in a 31 °C heating block overnight with an internal temperature of 30 °C and cooled in an ice-water bath to an internal temperature of 5 °C. To the mixture is added 2 M aqueous hydrochloric acid solution (226 mL) over 15 minutes, maintaining an internal temperature below 10 °C. To the mixture is added water (250 mL) and the mixture is stirred at room temperature for 5 minutes. The layers are separated, and the organic layer is extracted with a mixture of 2 M aqueous hydrochloric acid solution (28 mL) in water (50 mL). The first aqueous layer is stirred in an ice-water bath and 50% aqueous sodium hydroxide solution (25.7 mL) is added dropwise over 10 minutes, maintaining an internal temperature below 10 °C. The mixture is diluted with saturated aqueous sodium bicarbonate solution (100 mL), stirred at room temperature for 10 minutes, and extracted with ethyl acetate (3 x 400 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue. The second aqueous layer from the extraction with aqueous hydrochloric acid is diluted with 2-methyltetrahydrofuran (200 mL) and the mixture is passed through a short pad of diatomaceous earth. The filtrate is transferred to a separating funnel and the layers are separated. The aqueous layer is stirred in an ice-water bath and 50% aqueous sodium hydroxide solution (3.15 mL) is added dropwise over 5 minutes, maintaining an internal temperature below 10 °C. The mixture is diluted with saturated aqueous sodium bicarbonate solution (10 mL), stirred at room temperature for 5 minutes, and extracted sequentially with ethyl acetate (3 x 40 mL) and 10% isopropanol in ethyl acetate (100 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue, which is combined with the residue from the first part of the workup. The combined residue is passed through a pad of silica gel (350 g), eluting with ethyl acetate (3.5 L), and the filtrate is concentrated to give a residue (45.8 g). The residue (47.5 g of combined lots, 123.9 mmol) is purified by flash chromatography over silica gel, eluting with 50-100% ethyl acetate in heptane. The product-containing fractions are concentrated to residue, which is suspended in a 1 : 1 mixture of methyl-Zc/V-butyl ether and heptane (448 mL). The mixture is stirred in a 46 °C heating block for 30 minutes at an internal temperature of 45 °C and cooled to room temperature over 2 hours with stirring. The mixture is filtered, washing the collected solid with a 1 : 1 mixture of methyl -tert-butyl ether and heptane (30 mL). The filtered solid is dried under vacuum at 40 °C overnight to give the title compound (28.5 g, 49% yield). MS m/z 384.0 (M+H). Optical rotation: [OC]D²⁰ = +33.4 ° (C=0.26, methanol).

Alternative synthesis of N-[4-jluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy ]-l -piperidyl methyl ]thiazol-2-yl acetamide.

To a solution of N-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (50 mg, 0.28 mmol) and 5-methyl-3-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]-l,2,4-oxadiazole (40 mg, 0.19 mmol) in di chloromethane (10 mL) under nitrogen are added N,N-diisopropylethylamine (0.1 mL, 0.57 mmol) and sodium triacetoxyborohydride (120 mg, 0.57 mmol). The reaction mixture is stirred at room temperature for 12 hours. The reaction mixture is poured into a saturated aqueous solution of sodium bicarbonate (10 mL). The layers are separated, and the aqueous phase is extracted with dichloromethane (2^ 10 mL). The combined organic extracts are dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford an orange oil. The residue is dissolved in methanol (to a total volume of 9.8 mL), filtered, and purified by prep-HPLC (Phenomenex Gemini -NX 10 Micron 50 x 150mm C-18), eluting with a gradient of 15% (CH3CN & Water with 10 mM ammonium bicarbonate adjusted to pH 9 with ammonium hydroxide) to 100% CH3CN over lOmin (110 mL/min, 1 injection, 271/204 nm) to obtain the title compound (20 mg, 28% yield). MS m/z 384.2 (M+H).

Example 1A: Crystalline N-[4-jluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol- 3-yl)methoxy ]-l-piperidyl methyl ]thiazol-2-yl acetamide.

Suspend crude N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,2,4-oxadiazol-3- yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide (29.9 g) in 448 mL of 50% methyl tert butyl ether in heptane at 46 °C for 30 minutes. Stir the mixture and cool to 19 °C over two hours before filtering following with a wash of 30 mL of 50% methyl tert butyl ether in heptane to provide the title compound (28.5 g, 95% yield).

X-Ray Powder Diffraction (XRPD) of Example 1A.

The XRPD patterns of crystalline solids are obtained on a Bruker D4 Endeavor X-ray powder diffractometer, equipped with a CuKa source (A = 1.54060 A) and a Vantec detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 29, with a step size of 0.0087° in 29 and a scan rate of 0.5 seconds/step, and with 0.6 mm divergence, 5.28 mm fixed anti-scatter, and 9.5 mm detector slits. The dry powder is packed on a quartz sample holder and a smooth surface is obtained using a glass slide. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. (See, e.g., The U. S. Pharmacopeia 38 - National Formulary 35 Chapter 941 Characterization of crystalline and partially crystalline solids by X-ray powder diffraction (XRPD) Official May 1, 2915). Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of ± 9.2 in 29 will consider these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of ° 29), typically the more prominent peaks. The crystal form diffraction patterns, collected at ambient temperature and relative humidity, are adjusted based on NIST 675 standard peaks at 8.85 and 26.77 degrees 2-theta.

A prepared sample of crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl- 1,2,4- oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide is characterized by an XRPD pattern using CuKa radiation as having diffraction peaks (2-theta values) as described in Table 1 below. Specifically, the pattern contains a peak at 12.1° in combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8° with a tolerance for the diffraction angles of 0.2 degrees.

Table 1 : X-ray powder diffraction peaks of crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl- 4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide, Example 1A,

Example 2: Synthesis of OGA Inhibitor N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide (Formula Xa).

This compound and its synthesis are disclosed in US Patent No. US 10,081,625, which is hereby incorporated by reference in its entirety.

To a solution of 2-methyl-5-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]-l,3,4- oxadiazole-2,2,2-trifluoroacetic acid (160 mg, 0.7 mmol) in ethyl acetate (1 mL) under nitrogen is added N,N-diisopropylethylamine (210 .L, 0.12 mmol) and the solution stirred for 5 minutes. N-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (40 mg, 0.12 mmol) is added and stirred for 5 minutes, sodium triacetoxyborohydride (55 mg, 0.25 mmol) is added, and the reaction mixture is warmed to 40 °C and stirred overnight. The mixture is concentrated under reduced pressure to afford a brown solid. The residue is taken up in dimethyl sulfoxide (to a total volume of 1 mL) and purified by prep-HPLC (Phenomenex Gemini- NX 10 Micron 30 x 100mm C-18), eluting with a gradient of 15% (CH3CN & water with 10 mM ammonium bicarbonate adjusted to pH 9 with ammonium hydroxide) to 100% CH3CN over 12 minutes (100 mL/min, 1 injection, 271/204 nm) to give title compound (7 mg, 14% yield). MS m/z 384.2 (M+H).

Alternative synthesis of crystalline N-[4-jluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4- oxadiazol-2-yl)methoxy ]-l-piperidyl methyl ]thiazol-2-yl acetamide.

Sodium triacetoxyborohydride (59.1 g, 279 mmol) is added to a mixture of 2- methyl-5-[[(2S,4S)-2-methyl-4-piperidyl]oxymethyl]-l,3,4-oxadiazole (23.3 g, 93.0 mmol), ethyl acetate (438 mL) and N,N-diisopropylethylamine (32.4 mL, 186 mmol) at room temperature. The reaction mixture is stirred in a 31 °C heating block for 15 minutes with an internal temperature of 30 °C, then N-(4-fluoro-5-formyl-thiazol-2-yl)acetamide (17.5 g, 93.0 mmol) is added portion wise over 5 minutes. The reaction mixture is stirred in a 31 °C heating block overnight with an internal temperature of 30 °C and cooled in an ice-water bath to an internal temperature of 5 °C. To the mixture is added 2M aqueous hydrochloric acid solution (140 mL) over 15 minutes, maintaining an internal temperature below 10 °C. The mixture is stirred at room temperature for 15 minutes, diluted with water (50 mL) and ethyl acetate (20 mL), and the layers are separated. The organic layer is extracted with a mixture of 2M aqueous hydrochloric acid solution (35 mL) in water (100 mL). The combined aqueous layers are stirred in an ice-water bath and 50% aqueous sodium hydroxide solution (19.5 mL) is added dropwise over 10 minutes, maintaining an internal temperature below 10 °C. The mixture is diluted with saturated aqueous sodium bicarbonate solution (50 mL) and extracted with 2-methyltetrahydrofuran (3 x 200 mL). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give a residue, which is purified by flash chromatography over silica gel, eluting with 0- 15% 2-propanol in dichloromethane. The product-containing fractions are concentrated to give a residue, which is concentrated from heptane (100 mL). The concentrated material is combined with 40% ethyl acetate in heptane (457 mL) and the mixture is stirred in a 50 °C heating block for 1 hour, cooled to room temperature, and filtered. The filtered solid is dried under vacuum at 40 °C for 1 hour to give a first crop of product (22.9 g). The filtrate is concentrated to give a residue, which is combined with 40% ethyl acetate in heptane (50 mL) and the mixture is stirred in a 50 °C heating block for 30 minutes, cooled to room temperature, and filtered. The filtered solid is combined with 50% ethyl acetate in heptane (33 mL) and the mixture is stirred in a 50 °C heating block for 1 hour, cooled to room temperature, and filtered. The filtered solid is dried under vacuum at 40 °C for 1 hour to give a second crop of product (2.50 g).

A combination of lots including the first and second crops of product (29.3 g) is combined with ethyl acetate (117 mL) and heptane (117 mL) at room temperature. The mixture is stirred in a 51 °C heating block for 30 minutes at an internal temperature of 50 °C and subsequently cooled to room temperature and filtered. The filtered solid is dried overnight at 40 °C under vacuum to obtain the title compound (26.7 g, 75% yield) as a crystalline solid. MS m/z 384.0 (M+H). Optical rotation: [OC]D²⁰ = +39° (C=0.2, methanol).

X-Ray Powder Diffraction (XRPD) of crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5- methyl-1, 3, 4-oxadiazol-2-yl)methoxy ]-l -piperidyl methyl ]thiazol-2-yl acetamide.

Crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol-2- yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide (218 mg) is dissolved in 1.25 mL of methanol at 60 °C for 5 minutes. The solution is cooled to ambient temperature with stirring for 20 minutes. The resulting solid is isolated by vacuum filtration. The final solid product is 163 mg or 75% yield. The XRPD patterns of crystalline solids are obtained on a Bruker D4 Endeavor X- ray powder diffractometer, equipped with a CuKa source (X = 1.54060 A) and a Vantec detector, operating at 35 kV and 50 mA. The sample is scanned between 4° and 40° in 29, with a step size of 0.0087° in 29 and a scan rate of 0.5 seconds/step, and with 0.6 mm divergence, 5.28 mm fixed anti-scatter, and 9.5 mm detector slits. The dry powder is packed on a quartz sample holder and a smooth surface is obtained using a glass slide. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. See, e.g. The U. S. Pharmacopeia 38 - National Formulary 35 Chapter <941> Characterization of crystalline and partially crystalline solids by X-ray powder diffraction (XRPD) Official May 1, 2915. Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of ± 9.2 in 29 will consider these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of ° 29), typically the more prominent peaks. The crystal form diffraction patterns, collected at ambient temperature and relative humidity, were adjusted based on NIST 675 standard peaks at 8.85 and 26.77 degrees 2-theta.

Thus, crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl-4-[(5-methyl-l,3,4-oxadiazol- 2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide is characterized by an XRPD pattern using CuKa radiation as having diffraction peaks (2-theta values) as described in Table 2. More specifically, the pattern preferably contains a peak at 13.5° in combination with one or more peaks selected from the group consisting of 5.8°, 13.9°, 14.3°, 17.5°, 29.4°, 21.4°, and 22.2° with a tolerance for the diffraction angles of 9.2 degrees.

Table 2: X-ray powder diffraction peaks of crystalline N-[4-fluoro-5-[[(2S,4S)-2-methyl- 4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2-yl]acetamide.

In vitro human OGA enzyme assay Generation of OGA proteins

The nucleotide sequence encoding full-length human O-GlcNAc-fi-N- acetylglucosaminidase (NM 012215) is inserted into pFastBacl (Invitrogen) vector with an N-terminal poly-histidine (HIS) tag. Baculovirus generation is carried out according to the Bac-to-Bac Baculovirus Expression system (Invitrogen) protocol. Sf9 cells are infected at 1.5 x 10⁶ cells/mL using 10 mL of Pl virus per Liter of culture and incubated at 28 °C for 48 hours. Cells are spun down, rinsed with PBS and the pellets stored at -80 °C.

The above OGA protein (His-OGA) is purified as follows: 4 L of cells are lysed in 200 mL of buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM Imidazol, 1 mM Dithiothreitol (DTT), 0.1% Triton™ X-100, 4 tablets of protease inhibitors (complete EDTA-Free, Roche) for 45 min at 4 °C. This cell lysate is then spun for 40 minutes at 16500 rpm at 4 °C, and supernatant incubated with 6 mL of Ni-NTA resin (nickel -nitrilotriacetic acid) for 2 hours at 4 °C.

Resin is then packed onto column and washed with 50 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM Imidazole, 0.1% Triton™ X-100, 1 mM DTT, followed by 50 mM Tris, pH 8.0, 150 mMNaCl, 10 mM Imidazol, 10% glycerol, 1 mM DTT. The proteins are eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 300 mM Imidazole, 10% glycerol, 1 mM DTT. Pooled His-OGA containing fractions are concentrated to 6 mL and loaded onto Superdex75 (16/60). The protein is eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM DTT. Fractions containing His-OGA are pooled and protein concentration measured with BCA (Bradford Colorimetric Assay).

OGA enzyme assay

The OGA enzyme catalyses the removal of O-GlcNAc from nucleocytoplasmic proteins. To measure this activity, Fluorescein di-N-acetyl-P-N-acetyl-D-glucosaminide (FD-GlcNAc, Kim, et al., Carbohydrate Research (2006), 341(8), 971-982) is used as a substrate at a final concentration of 10 pM (in the 96 well assay format) or 6.7 pM (in the 384 well assay format). This fluorogenic substrate becomes fluorescent upon cleavage by OGA, so that the enzyme activity can be measured by the increase in fluorescence detected at 535 nm (excitation at 485 nm).

The assay buffer is prepared to give a final concentration of 50 mM H2NaPO3-HNa2PO3, 0.01% bovine serum albumin and 0.01% Triton™ X-100 in water, at pH 7. The final enzyme concentration is 3 nM (in the 96 well assay format) or 3.24 nM (in the 384 well assay format). Both assay formats yield essentially equivalent results. Compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using ten point concentration response curves. Maximal compound concentration in the reaction mixture is 30 pM. Compounds at the appropriate concentration are pre-incubated with OGA enzyme for 30 minutes before the reaction is started by the addition of substrate. Reactions are allowed to proceed for 60 minutes at room temperature. Then, without stopping the reaction, fluorescence is read. IC50 values are calculated by plotting the normalized data vs. log of the compound and fitting the data using a four-parameter logistic equation.

The compound of Example 1 was tested essentially as described above and exhibited an IC50 of 1.97 nM ± 1.22 (n=9). This data demonstrates that the compound of Example 1 inhibits OGA enzyme activity in vitro.

The compound of Example 2 was also tested essentially as described above and exhibited an IC50 of 2.13 nM ± 0.89 (n=5). This result demonstrates that the compound of Example 2 inhibits OGA enzyme activity in vitro.

Whole cell assay for measuring the inhibition of OGA enzyme activity

Cell Plating'.

Utilizing standard conditions known in the art, TRex-293 cells modified for inducible expression of the P301 S-1N4R form of the microtubule associated protein tau are generated and maintained in growth media, consisting of DMEM High Glucose (Sigma# D5796), supplemented with 10% Tetracyclin-free Fetal Bovine Serum (FBS, Sigma F2442), 20 mM HEPES, 5 pg/mL Blasticidin (Life Technologies# Al 1139-03) and 200 pg/mL Zeocin (Life Technologies# R250-01). For the experiments, cells are plated in growth media at 10,000-14,000 cells per well in a Corning Biocoat (356663) 384 well plate coated with poly-D-Lysine and incubated 20-24 hours in a cell incubator at 37 °C/5% CO2. Experiments are performed without inducing Tau expression. Compound treatment:

Compounds to be tested are serially diluted 1/3 in pure DMSO using ten-point concentration response curves and further diluted in growth media. 20-24 h after plating, cells are treated with test compound in growth media; maximal compound concentration is 15 pM (0.15% DMSO). The maximum inhibition is defined by replicate measurements of 15 pM Thiamet G and the minimum inhibition is defined by replicate measurements of 0.15% DMSO treatment. The cells are returned to the incubator at 37 °C/5% CO2 for 20- 24 hours. Compounds are tested in duplicates within each plate.

Immunostaining:

After 20-24 hours of compound treatment, the media is removed from the assay plate and 25 pL of 3.7% Formaldehyde solution (Sigma# F 1635) in DPBS (Sigma #D8537; Dulbecco’s phosphate buffered saline) is added to each well and incubated for 30 minutes. The cells are then washed once with DPBS and then permeabilized with 0.1% Triton™ X- 100 (Sigma# T9284). After 30 minutes, cells are washed twice with DPBS and then blocking solution(l% BSA/DPBS/0.1% Triton™ X-100) is added to each well and incubated for 60 minutes. The blocking solution is removed and a 0.40-0.33 pg/mL solution of O-GlcNAc Protein antibody (RL2 clone, Thermo, MAI 072) in blocking solution is added to the cells and allowed to sit overnight at 2-8 °C. The next day, the cells are washed twice with DPBS and the secondary antibody, Alexa Fluor 488 goat anti -mouse IgG (Life Technologies # Al 1001) at 2 pg/mL in DPBS is added to each well and allowed to sit at room temperature for 90 minutes. The secondary antibody is removed, cells washed twice with DPBS and a solution of DAPI (Sigma #D9564; 4',6-diamidino-2-phenyindole, dilactate) and RNase (Sigma, R6513) in DPBS at a concentration of 1 and 50 pg/mL, respectively, is added to each well. The plate is sealed, incubated for one hour and analyzed on an Acumen eX3 hci (TTP Labtech). All the incubations and washing steps described above are done at room temperature, except for the primary antibody.

Analysis and Results: The plates are analyzed on an Acumen eX3 instrument using a 488 and 405 nm excitation lasers and two emission filters FL2 (500-530 nm) and FL1 (420-490 nm). The FL2 filter is the signal corresponding to the Q-GlcNAc Protein antibody (RL2 clone) and the FL1 filter is the signal corresponding to the cell nuclei (DAPI). The ratio Total FL2/Total FL1 (Total fluorescence of each well without object or population selection) is used for data analysis. The data are normalized to a maximum inhibition as referenced by a 15 pM treatment of Thiamet G and a minimum inhibition as achieved by a 0.15% DMSO treatment. The data are fitted with a non-linear curve fitting application (4-parameters logistic equation) and IC50 values are calculated and reported.

The compound of Example 1 was tested essentially as described above and exhibited an IC50 of 21.9 nM ± 7.3 (n=5). This data demonstrates that the compound of Example 1 inhibits OGA enzyme activity in a cellular assay.

The compound of Example 2 was also tested essentially as described above and exhibited an IC50 of 22.6 nM ± 7.3 (n=3). This result demonstrates that the compound of Example 2 inhibits OGA enzyme activity in a cellular assay.

A Single Ascending Dose Study in Healthy Subjects to Assess the Safety and Pharmacokinetics of the Compound of Example 1

A Phase 1, single-center, subject- and investigator-blind, single-ascending dose, placebo-controlled, crossover, randomized study is performed to evaluate safety, tolerability, and pharmacokinetics (PK) of the compound of Example 1 in healthy subjects. The study is conducted in 2 alternating cohorts (cohorts 1 and 2) in up to 3 study periods across 6 dose levels. Subjects are randomized to 1 of 3 treatment sequences in each cohort, with each sequence including 2 doses of compound of Example 1 and 1 placebo dose over the 3 study periods in a complete crossover manner. The clinical study design is summarized in Table 3. Table 3. Clinical Study Design.

After an overnight fast of at least 8 hours, oral capsules are administered with approximately 240 mL of room-temperature water in the morning of each dosing day in a sitting position. Doses of 0.15 mg to 2 mg are administered as an oral solution of compound of Example 1 via an oral dosing syringe with water similar to oral capsule dosing. Tables 4a and 4b summarize the treatment regimens.

Table 4a. Treatment Regimens for Compound of Example 1.

Table 4b. Treatment Regimens for Placebo.

¹ HPMC = hypromellose The capsules containing the compound of Example 1 are prepared extemporaneously. Oral doses of 0.15 mg to 2 mg of the compound of Example 1 are prepared extemporaneously as drug in solution. For any specific cohort/dosing period, the total number of capsules administered is the same for all subjects, regardless of whether assigned to placebo or the compound of Example 1. However, the number of capsules may vary between dosing periods and cohorts. This is similarly the case for extemporaneously prepared oral dosing solutions to maintain the blind. The compound of Example 1 is supplied in the form of free base with no inactive ingredients for extemporaneous preparation. A matching volume of oral solution vehicle without compound of Example 1 is used as placebo for the doses of 0.15 mg to 2 mg.

Venous blood samples of approximately 3 mL each are collected to determine the plasma concentrations of compound of Example 1. Concentrations of compound of Example 1 are assayed using a validated liquid chromatography with tandem mass spectrometry method. PK parameter estimates for compound of Example 1 are calculated using standard noncompartmental methods of analysis. Plasma concentrations of compound of Example 1 are summarized by administered dose of compound of Example 1 and the approximate time of PK blood sample collection. Following the procedure essentially as described above, PK data for a single ascending dose of the compound of Example 1 in healthy subjects are set forth in Table 5 and Table 6.

Table 5. Mean plasma concentrations for a single ascending dose study following oral administration of the compound of Example 1 in healthy subjects.

Table 6. PK parameters of compound of Example 1 following oral administration in healthy subjects¹.

'Data presented as geometric mean (%CV geometric mean) unless noted otherwise

² Amount of compound of Example 1 administered

³N = number of subjects

⁴CL/F = apparent clearance calculated after oral administration ⁵AUC(0-CO) = area under the concentration versus time curve from time zero to infinity

⁶Cmax = maximum observed drug concentration

⁷V_Z/F = apparent volume of distribution after oral administration

⁸ti/2 = terminal half-life (geometric mean (min - max))

⁹tmax = time of maximum observed drug concentration; median (min - max) ^loAeO-24 = amount of compound of Example 1 excreted in urine up to 24 hours post dose ⁿCLr = renal clearance

¹²N = 5

¹³N = 3

¹⁴NC = not calculated The data discloses that the AUC(o-oo) and Cmax increase approximately dose- proportionally over the 0.6-mg to 16-mg dose range for the compound of Example 1 The median tmaxis about 1 hour and ti/2 is about 6 hours for the compound of Example 1.

Assessment of Brain O-GlcNAcase Enzyme Occupancy after Single Oral Doses of the Compound of Example 1 as Measured by Positron Emission Tomography with the Radioligand [¹⁸F]LSN3316612 in Healthy Subjects

A single-center, open-label, nonrandomized, positron emission tomography (PET) study demonstrating brain penetration and target engagement of brain O-GlcNAcase (OGA) after single oral doses of 0.25 mg, 1 mg, and 5 mg of N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide (compound of Formula I) is carried out by one of ordinary skill in the art essentially as set forth below. [¹⁸F]LSN3316612 is a positron-emitting radiopharmaceutical for in vivo imaging of OGA in the brain and is used to evaluate target engagement of compounds which inhibit OGA. The preparation and use of ¹⁸F- LSN3316612 as a PET radioligand is known in the art, for example as described by S. Lu, et. al, Science Translational Medicine , 12, (2020). This single-dose PET study using the [¹⁸F]LSN3316612 tracer assesses the brain OGA enzyme occupancy (EO) across a suitable range of doses that have been demonstrated to be safe and well tolerated.

Healthy subjects are assigned to 1 of 4 cohorts with 4 subjects in each cohort completing the study. All subjects undergo one baseline PET scan and two post-dose PET scans. A baseline PET scan is performed from up to approximately 14 days before dosing of the compound of Example 1. Overall, each subject receives a single dose of the compound of Example 1 and 3 administrations of the [¹⁸F]LSN3316612 PET tracer. Dosing of the compound of Example 1 occurs after completion of the baseline PET scan. Scans are conducted at approximately 2- and 24-hours post-dose for the 0.25 mg and 5 mg doses of the compound of Example 1, and at approximately 2 and 24 hours or 30 and 54 hours post-dose for the 1 mg dose of the compound of Example 1. Dynamic PET data of the brain are acquired over 120 min immediately following tracer injection. EO is summarized by the compound of Example 1 dose and approximate scanning time. The compound of Example 1 is administered orally in capsule formulation for doses >3 mg. For doses lower than 3 mg, the compound of Example 1 is weighed into a suitable container and dissolved in an appropriate volume of degassed Sprite® or diluent.

[¹⁸F]LSN3316612 is produced in the clinical site radiochemistry facility from the nonradioactive precursor on the day of each PET scan. [¹⁸F]LSN3316612 injection is a clear solution for intravenous injection formulated in normal saline containing ethanol, sterile water for injection, and sodium ascorbate. [¹⁸F]LSN3316612 is delivered in normal saline (0.9% NaCl) formulated with the intent to contain approximately 3.3% (v/v) ethanol (EtOH) and sodium ascorbate (4.67 mg/mL). [¹⁸F]LSN3316612 is administered intravenously over a 3 -minute infusion period using an infusion pump followed by a 10- mL saline flush. Prior to the PET imaging, subjects have an intravenous catheter (for radiotracer infusion) inserted according to standard clinical practice. Each subject receives a single injection of [¹⁸F]LSN3316612 at each imaging visit. The radiopharmaceutical is injected intravenously at a dose of approximately 5 mCi (not more than 6 mCi), with a maximum mass dose of 10 pg and maximum volume of 10 mL.

For each PET scan, the radiochemistry laboratory synthesizes the radioligand from the precursor according to PET unit production protocol known in the art, such as that described by Lee, J., Liow, J., Paul, S. et al. “PET Quantification of Brain O-GlcNAcase With [¹⁸F]LSN3316612 in Healthy Human Volunteers,” EJNMMI Res 10, 20 (2020).

Arterial blood samples are collected from all subjects during each PET scan to measure radioactivity to provide input for the PET tracer kinetic analysis. Venous blood samples are collected following dosing of compound of Example 1 to measure plasma concentrations of compound of Example 1 using a validated liquid chromatography with tandem mass spectrometry assay.

The primary imaging outcome for the [¹⁸F]LSN3316612 PET is the total distribution volume (V_T) which is determined in regions where OGA is present, including cortical regions, regions of basal ganglia, thalamus, and cerebellum. Analysis uses the decay-corrected time activity data in the different brain regions. Imaging data is analyzed with 2-tissue compartment model with arterial input function to determine V_T. OGA EO after a single dose of the compound of Example 1 is obtained using graphical analysis according to the occupancy plot:

VT(Baseline) - VT(Dosing) = Occupancy * (VT(Baseline) - VND), where VT(Baseline) and VT (Dosing) are the total distribution volumes in several regions obtained at baseline and after compound of Example 1 administration, respectively. The occupancy is determined as the slope of the linear regression of the plot, and the nondisplaceable volume of distribution VND as the x-intercept.

Following the procedure essentially as described above, the target engagement of brain OGA after single oral doses of 0.25 mg, 1 mg, and 5 mg of compound of Example 1 is set forth in Tables 7a-7c.

Table 7a: Brain OGA Occupancy for the 0.25 mg dose of Compound of Example 1.

Table 7b: Brain OGA Occupancy for the 1 mg dose of Compound of Example 1.

Table 7c: Brain OGA Occupancy for the 5 mg dose of Compound of Example 1.

The data provided in Tables 7a-7c above discloses a plasma concentrationdependent change in brain OGA EO with EO for the 5 mg dose of the compound of Example 1 exceeding 90% EO at 24 hours post dose. The 1 mg dose of the compound of Example 1 was found to be 80.6% EO at 24 hours post dose and 30.3% EO at 54 hours post dose. The 0.25 mg dose of the compound of Example 1 was found to be 46% EO at 24 hours post dose.

Utilizing the PK data from the single ascending dose study of the compound of Example 1 in healthy subjects and the PET study demonstrating brain penetration and target engagement of brain OGA after single oral doses of the compound of Example 1 as disclosed above, the low doses and dosage regimens for treating a neurodegenerative disease, including AD and other neurodegenerative tauopathies, with a compound of Example 1 are set forth below:

A total dose of the compound of Example 1 of 0.25 mg/day to 5 mg/day.

A total dose of the compound of Example 1 of 0.1 mg/day to 3 mg/day.

A total dose of the compound of Example 1 of 0.25 mg/day to 3 mg/day.

A total dose of the compound of Example 1 of 0.1 mg/day to 2 mg/day. A total dose of the compound of Example 1 of 0.25 mg/day to 2 mg/day.

A total dose of the compound of Example 1 of 0.1 mg/day to 1 mg/day.

A total dose of the compound of Example 1 of 0.25 mg/day to 1 mg/day.

A total dose of the compound of Example 1 of 5 mg/day.

A total dose of the compound of Example 1 of 3 mg/day.

A total dose of the compound of Example 1 of 2 mg/day.

A total dose of the compound of Example 1 of 1 mg/day.

A total dose is the compound of Example 1 of 0.25 mg/day.

A total dose of the compound of Example 1 of 0.1 mg/day.

It is preferred that the total daily dose of compound of Example 1 administered is in one unit dose.

It is further preferred that the total daily dose of the compound of Example 1 administered is in two unit doses. It is preferred that the total daily dose of the compound of Example 1 is administered in two unit doses wherein each dose contains equal amounts of the compound of Example 1. It is preferred that when the total daily dose of the compound of Example 1 is administered in two unit doses, the administration of each unit dose is separated by at least 8 hours.

In addition, total doses of the compound of Example 1 in a pharmaceutical composition are set forth below:

A total dose of the compound of Example 1 is 0.25 mg to 5 mg.

A total dose of the compound of Example 1 is 0.1 mg to 3 mg.

A total dose of the compound of Example 1 is 0.25 mg to 3 mg.

A total dose of the compound of Example 1 is 0.1 mg to 2 mg.

A total dose of the compound of Example 1 is 0.25 mg to 2 mg.

A total dose of the compound of Example 1 is 0.1 mg to 1 mg.

A total dose of the compound of Example 1 is 0.25 mg to 1 mg.

A total dose of the compound of Example 1 is 3 mg.

A total dose of the compound of Example 1 is 2 mg.

A total dose of the compound of Example 1 is 1 mg.

A total dose of the compound of Example 1 is 0.25 mg. In addition, it is preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in one unit dose.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in one unit dose wherein the single unit dose is administered once per day.

It is also preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in two unit doses.

It is preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in two unit doses with each unit dose containing equal amounts of the compound of Example 1.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in two unit doses wherein each dose is administered in one day.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of Example 1, or pharmaceutically acceptable salt thereof is contained in two unit doses with each unit dose containing equal amounts of the compound of Example 1 wherein each dose is administered within one day, preferably separated by at least 8 hours.

Example 3: Expression and Purification of Engineered anti-N3pGlu AP Antibodies

Antibodies to N3pGlu A0 are known in the art. For example, U.S. Patent No. 8,679,498; U.S. Patent No. 8,961,972; US Patent No. 10,647,759; and US Patent No. 11,078,261 (which are hereby incorporated by reference in their entireties) disclose anti- N3pGlu A0 antibodies, method of making the antibodies, antibody formulations, and methods of treating diseases, such as, Alzheimer’s disease with such antibodies. The amino acid sequences for exemplary anti-N3pG Ap antibodies are provided in Table 8 below. Table 8: Anti-N3pGlu Aft Antibody SEQ ID NOs.

Anti-N3pGlu A0 antibodies of the present invention can be made and purified essentially as follows. For antibody I, an appropriate host cell, such as HEK 293 EBNA or CHO, is either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC, such as SEQ ID NO: 22, and LC, such as SEQ ID NO: 21. Clarified media, into which the antibody has been secreted, is purified using any of many commonly-used techniques. For example, the medium may be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound antibody is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected, such as by SDS-PAGE, and then are pooled. Further purification is optional, depending on the intended use. The antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The purity of antibody I after these chromatography steps is greater than 99%. The product may be immediately frozen at -70 °C or may be lyophilized.

Antibody II can be expressed and purified essentially as follows. A glutamine synthetase (GS) expression vector containing the DNA sequence encoding the HC amino acid sequence of SEQ ID NO: 24, and the DNA sequence encoding the LC amino acid sequence of SEQ ID NO: 23 is used to transfect a Chinese hamster ovary cell line (CHO) by electroporation. The expression vector encodes an SV Early (Simian Virus 40E) promoter and the gene for GS. Post-transfection, cells undergo bulk selection with 0-50 pM L-methionine sulfoximine (MSX). Selected bulk cells or master wells are then scaled up in serum-free, suspension cultures to be used for production. Clarified medium, into which the antibody has been secreted, is applied to a Protein A affinity column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed with 1 M NaCl to remove nonspecific binding components. The bound N3pGlu A0 antibody is eluted, for example, with sodium citrate at pH (approx.) 3.5 and fractions are neutralized with 1 M Tris buffer. Anti-N3pGlu A0 antibody fractions are detected, such as by SDS-PAGE or analytical size-exclusion and are pooled. Anti-N3pGlu A0 antibody II is concentrated in either PBS buffer at pH 7.4 or 10 mM sodium citrate buffer, 150 mM NaCl at pH around 6. The final material can be sterile filtered using common techniques. The purity of anti-N3pGlu A0 antibody II is greater than 95%. Anti- N3pGlu A0 antibody II of the present invention may be immediately frozen at -70 °C or stored at 4 °C for several months.

Example 4: Binding Affinity and Kinetics of anti-N3pGlu A|3 antibodies.

The binding affinity and kinetics of anti-N3pGlu Ap antibody of the present invention (Antibody I or Antibody II) to pE3-42 Ap peptide is measured by surface plasmon resonance using BIACORE® 3000 (GE Healthcare). The binding affinity is measured by capturing the anti-N3pGlu Ap antibody via immobilized protein A on a BIACORE® CMS chip, and flowing pE3-42 Ap peptide, starting from 100 nM in 2-fold serial dilution down to 3.125 nM. The experiments are carried out at 25 °C in HBS-EP buffer (GE Healthcare BR100669; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4).

For each cycle, the antibody is captured with 5 pL injection of antibody solution at a 10 pg/mL concentration with 10 pL/min. flow rate. The peptide is bound with 250 pL injection at 50 pL/min, and then dissociated for 10 minutes. The chip surface is regenerated with 5 pL injection of glycine buffer at pH 1.5 at 10 pL/mL flow rate. The data is fit to a 1 : 1 Langmiur binding model to derive k_on, koff, and to calculate KD. Following procedures essentially as described above, the following parameters (shown in Table 9) are observed.

Table 9: Binding affinity and kinetics.

These data demonstrate that the antibodies of the present invention bind pE3-42 Ap.

Example 5: Ex Vivo Target Engagement

To determine ex vivo target engagement on brain sections from a fixed PDAPP brain, immunohistochemical analysis is performed with an exogenously added anti- N3pGlu Ap antibodies of the present invention (Antibody I or Antibody II). Cryostat serial coronal sections from aged PDAPP mice (25-month-old) are incubated with 20 pg/mL of an exemplified anti-N3pGlu Ap antibody of the present invention. Secondary HRP reagents specific for human IgG are employed and the deposited plaques are visualized with DAB- Plus (DAKO). Biotinylated murine 3D6 antibody followed by Step-HRP secondary is used as a positive control. The positive control antibody (biotinylated 3D6) labeled significant quantities of deposited Ap in the PDAPP hippocampus, and the anti-N3pGlu Ap antibodies (Antibody I or Antibody II) labeled a subset of deposits. These histological studies demonstrated that the anti-N3pGlu Ap antibodies of the present invention engaged deposited Ap target ex vivo.

Example 6: In Vivo Target Engagement Studies

The ability of anti-N3pGlu Ap antibodies of the present disclosure to cross the blood-brain-barrier and bind to deposited plaque in vivo is measured. Aged PDAPP transgenic mice (18.5 to 32 months of age) are given intraperitoneal injections with anti- N3pGlu AP antibody (e.g., Antibody I or II) or negative control IgG. Six mice per group receive one 40 mg/kg injection of the antibody on day 1 and on day 3. In vivo target engagement is determined on day 6, when mice are sacrificed, and brains are collected for histochemical analyses.

The extent of in vivo target engagement is quantified as the percent area positive for the in vivo anti-N3pGlu Ap antibody engagement normalized to the total plaque area as defined by an exogenous control antibody immunostaining on sister sections (TE Ratio). The TE Ratio is generated by measuring the percent of area bound by the antibody and normalizing the value against the total percent of area of possible target (total deposited Ap visualized by exogenous immunohistochemistry with a positive control antibody (3D6) on a sister section).

Following procedures essentially as described above, both anti-N3pGlu Ap antibodies I and II are found to engage the deposited plaque. These results demonstrate that the anti-N3pGlu antibodies I and II when administered peripherally can cross the blood-brain barrier and engage the intended target of deposited Ap.

Example 7: Preparation of OGA inhibitor l-(2-(((3R,5S)-l-((6-fluoro-2- methylbenzo[d]thiazol-5-yl)methyl)-5-methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H- pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one.

This compound and its synthesis are disclosed in US Patent No. US 10,752,362, which is hereby incorporated by reference in its entirety.

To a solution of 6-fluoro-2-methyl-l,3-benzothiazole-5-carbaldehyde (0.19 g, 0.95 mmol) and l-(2-(((3R,5S)-5-methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H-pyrrolo[3,4- b]pyridin-6-yl)ethan-l-one hydrochloride (0.28 g, 0.94 mmol) in DCM (9 mL) is added DIPEA (0.45 mL, 2.6 mmol). The resulting solution is stirred at RT for 40 minutes. To the solution is added sodium triacetoxyborohydride (NaBH(OAc)3, 0.65 g, 3.04 mmol). The resulting solution is stirred at RT for 17 hours. The reaction mixture is quenched slowly with saturated aqueous NaHCOs (5 mL). The aqueous layer is extracted with DCM (2 x 5 mL). The combined organic extracts are dried over MgSCL, filtered, and concentrated under reduced pressure. The resulting residue is dissolved in DCM and purified via flash chromatography over silica gel, eluting with a gradient of 40-100% acetone in hexanes, to obtain the title compound after solvent evaporation of the desired chromatographic fractions (0.27 g, 65% yield). ES/MS m/z: 441 (M+H); [OC]D²⁰ = +101.4° (c = 0.2, MeOH).

Alternative preparation of OGA inhibitor l-(2-(((3R,5S)~ l-((6-fluoro-2- methylbenzo[d]thiazol-5-yl)methyl)-5-methylpyrrolidin-3-yl)oxy)-5, 7-dihydro-6H- pyrrolo[ 3, 4-b ]pyridin-6-yl)ethan-l-one:

Preparation of 4-methylbenzenesulfonic acid;(3R,5S)-5-methylpyrrolidin-3-ol.

To a flask is added Zc/V-butyl (2S,4R)-4-hydroxy-2-methyl-pyrrolidine-l- carboxylate (53.0 g, 263 mmol) and 2-propanol (265 mL) at room temperature. The mixture is stirred at room temperature (internal temperature 20 °C) and p-toluenesulfonic acid monohydrate (60.1 g, 316 mmol) is added in one portion. The reaction mixture is stirred in a 62 °C heating block overnight, then is cooled to room temperature and concentrated to approximately 150 mL total volume. The mixture is diluted with methyl Zc/V-butyl ether (MTBE, 530 mL) and the mixture is stirred vigorously at room temperature for 30 minutes and then is filtered under flow of N2 gas. The filtered solid is dried under vacuum at 40 °C for 2 hours to provide 4-methylbenzenesulfonic acid;(3R,5S)-5- methylpyrrolidin-3-ol (67.6 g, 93% yield) as a white solid. ES/MS m/z: 102 (M+H).

Preparation of (3R,5S)-l-[(6-fluoro-2-methyl-l,3-benzothiazol-5-yl)methyl]-5- methyl-pyrrolidin-3-ol.

To a flask is added 4-methylbenzenesulfonic acid; (3R,5S)-5-methylpyrrolidin-3-ol (61.9 g, 226 mmol), EtOAc (850 mL), and 6-fluoro-2-methyl-l,3-benzothiazole-5- carbaldehyde (42.5 g, 216 mmol) at room temperature. The reaction mixture is stirred in an ice-water bath (internal temperature 3 °C) and triethylamine (60.1 mL, 431 mmol) is added in one portion. The reaction mixture is stirred in an ice-water bath for 30 minutes, then sodium triacetoxyborohydride (91.4 g, 431 mmol) is added in one portion. The reaction mixture is stirred in an ice-water bath for 10 minutes, then at room temperature for 2 hours (internal temperature 20 °C). The reaction mixture is stirred in an ice-water bath and 15% aqueous KHSO4 solution (650 mL) is added over 5 minutes, maintaining an internal temperature below 15 °C during the addition. The mixture is stirred vigorously at room temperature for 1 hour, then saturated aqueous citric acid solution (100 mL) is added and the mixture is stirred at room temperature for 5 minutes, then the layers are separated. The aqueous layer is washed with EtOAc (400 mL), then the aqueous layer is stirred in an ice-water bath and solid NazCOs (80 g) is added portion wise over 10 minutes with vigorous stirring until pH = 10 (measured by pH paper). The aqueous layer is then extracted with EtOAc (3 *400 mL). The combined organics are dried over Na2SO4 and concentrated to give a residue that is crushed into a fine powder using a pestle and mortar, then is combined with 25% MTBE/heptane (280 mL). The mixture is stirred vigorously in a 45 °C heating block for 1 hour, then at room temperature for 1 hour and then is filtered to give the first batch of filtered solid. The filtrate is concentrated, then the residue is combined with 25% MTBE/heptane (40 mL) and the mixture is stirred vigorously at room temperature for 30 minutes and then is filtered to give the second batch of filtered solid. The first and second batches of filtered solids are combined and the mixture is ground up with a spatula, then is dried under vacuum at room temperature overnight to provide (3R,5S)-l-[(6-fluoro-2- methyl-l,3-benzothiazol-5-yl)methyl]-5-methyl-pyrrolidin-3-ol (53.3 g, 87% yield) as a cream-colored solid. ES/MS m/z: 281 (M+H).

Alternative Preparation of OGA inhibitor l-(2-(((3R,5S)-l-((6-fluoro-2- methylbenzo[d]thiazol-5-yl)methyl)-5-methylpyrrolidin-3-yl)oxy)-5,7-dihydro-6H- pyrrolo[3,4-b]pyridin-6-yl)ethan-l-one.

To a flask is added (3R,5S)-l-[(6-fluoro-2-methyl-l,3-benzothiazol-5-yl)methyl]- 5-methyl-pyrrolidin-3-ol (26.9 g, 95.0 mmol), l-(2-chloro-5,7-dihydropyrrolo[3,4- b]pyridin-6-yl)ethanone (22.1 g, 109 mmol), cesium carbonate (92.8 g, 285 mmol), MorDalPhos (1.76 g, 3.80 mmol), palladium(II)(pi-cinnamyl) chloride dimer (984 mg, 1.90 mmol) and toluene (538 mL) at room temperature. N2 gas is bubbled through the mixture at room temperature with stirring for 30 minutes, then the reaction mixture is stirred in an 86 °C heating block overnight (internal temperature 80 °C). The reaction mixture is cooled to room temperature and diluted with EtOAc (269 mL) and diatomaceous earth (27 g) is added. The mixture is stirred at room temperature for 5 minutes, then is filtered through diatomaceous earth, washing with EtOAc (200 mL). The filtrate is concentrated to give a residue, which is dissolved in EtOAc (100 mL) and the mixture is passed through a short pad of silica gel (300 g), eluting with EtOAc (2 L) and then with 20% isopropanol/EtOAc (IPA/EtOAc, 2 L). The IPA/EtOAc fraction is concentrated to give a residue, which is dried under vacuum at room temperature for 1 hour to give the title compound (42.1 g, 88% yield, 88% purity by mass) as a pale brown foam.

The foam is combined with another lot of similar purity and the combined material (46.0 g, 92.3 mmol) is combined with MTBE (230 mL) and heptane (230 mL) at room temperature. The mixture is stirred vigorously in a 45 °C heating block for 1 hour, then at room temperature for 30 minutes and then is filtered. The filtered solid is combined with EtOAc (400 mL) and SiliaMetS® Thiol (40 g) is added. The mixture is agitated on a rotary evaporator at room temperature for 1 hour, then is filtered. The filtrate is concentrated to give a residue, which is combined with 25% EtOAc/heptane (400 mL) and the mixture is stirred vigorously in a 50 °C heating block for 1 hour, then at room temperature for 10 minutes, then is filtered, keeping aside the first batch of filtrate. The filtered solid is combined with 35% EtOAc/heptane (400 mL) and the mixture is stirred vigorously in a 50 °C heating block for 1 hour, then at room temperature for 10 minutes, then is filtered, keeping aside the second batch of filtrate. The filtered solid is combined with EtOAc (500 mL) and 15% aqueous KHSO4 solution (500 mL). The mixture is stirred vigorously at room temperature for 15 minutes, then is transferred to a separating funnel and the layers are separated, leaving a rag layer in the organics. The organic layer is further extracted with 15% aqueous KHSO4 solution (100 mL), leaving a rag layer in the organics. The rag layer is removed from the organics and is diluted with CH2Q2 (100 mL) and 15% aqueous KHSO4 solution (100 mL) and the layers are separated. The combined aqueous layers are stirred in an ice-water bath and solid Na2CO3 (100 g) is added portion wise over 5 minutes with stirring (pH measured as 10 by pH paper). The mixture is extracted with CH2CI2 (2 x 500 mL) and the combined organics are dried over Na2SO4 and concentrated to give the first batch of crude product. The first and second batches of filtrates from the filtrations are combined and concentrated, then the residue is combined with EtOAc (100 mL) and 15% aqueous KHSO4 solution (100 mL). The mixture is stirred vigorously at room temperature for 15 minutes, then is transferred to a separating funnel and the layers are separated. The aqueous layer is stirred in an ice-water bath and solid NazCOs (15 g) is added portion wise over 5 minutes with stirring (pH measured as 10 by pH paper). The mixture is extracted with CH2CI2 (2 x 100 mL) and the combined organics are dried over Na2SO4 and concentrated to give a residue which is combined with 25% EtOAc/heptane (80 mL) and the mixture is stirred vigorously in a 50 °C heating block for 30 minutes, then at room temperature for 10 minutes, then is filtered to give the second batch of crude product. The two batches of crude product are combined with 25% EtOAc/heptane (400 mL) and the mixture is stirred vigorously in a 50 °C heating block for 30 minutes, then at room temperature for 10 minutes, then is filtered. The filtered solid is dried under vacuum at room temperature 3 days to provide the final title compound (37.4 g, 90% yield) as a white solid. ES/MS m/z: 441 (M+H). Optical rotation [a]D2o = +104.0° (c = 0.2, MeOH).

The compound of Example 7 was tested essentially as described above for the in vitro human OGA enzyme assay and exhibited an IC50 of 0.214 nM ± 0.037 (n=4). This data demonstrates that the compound of Example 7 inhibits OGA enzyme activity in vitro. The compound of Example 7 was tested essentially as described above for the whole cell assay for measuring the inhibition of OGA enzyme activity and exhibited an IC50 of 70.5 nM + 0.002 (n=2). This data demonstrates that the compound of Example 7 inhibits OGA enzyme activity in a cellular assay.

Example 8: In Vivo Combination Study

The following Example demonstrates how a study could be designed to verify that the combination of OGA inhibitor of the present disclosure, in combination with an anti- N3pGlu A0 antibody of the present disclosure, may be useful for treating a disease characterized by amyloid deposits and/or aberrant tau aggregation, such as AD. It should be understood however, that the following descriptions are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

In order to evaluate the impact of tau hyperphosphorylation and aggregation reduction by exemplified OGA inhibitors, and the amyloid beta deposit reduction exemplified by anti-N3pGlu A0 antibody, in a combination therapy as described herein, delay in disease progression may be assessed by biomarkers and/or cognitive and functional decline assessment using validated rating scales.

Patients may be divided into treatment groups consisting of double-blinded placebo and combination therapy groups. Combination therapy groups are administered an effective amount of an OGA inhibitor, in combination with an effective amount of an anti-N3pGlu A0 antibody. Monotherapy groupings (monotherapy group of OGA inhibitor at the same dosage as the OGA inhibitor in the combination group; and monotherapy group of anti- N3pGlu A0 antibody at the same dosage as the anti-N3pGlu A0 antibody in the combination therapy group) may be included to further elucidate the contributions of each individual molecule to the disease modification. Moreover, treatment groups may be characterized based on a diagnosis of pre-clinical or clinical AD or based on a diagnosis that the patient (although asymptomatic for AD) possesses an AD disease-causing genetic mutation. For example, groups may include one or more of: (a) asymptomatic but AD- causing genetic-mutation positive; (b) prodromal AD; (c) mild AD dementia; (d) moderate AD dementia; and (e) severe AD dementia. Each treatment group may receive the respective treatment (e.g., every four weeks for the anti-N3pGlu A0 antibody and daily for the OGA inhibitor) for a treatment period of 9 months to 18 months.

Following the treatment period, AD neurodegeneration may be assessed through one or more of the following biomarker assessments: (a) amyloid PET imaging; (b) phosphorylated tau (P-tau; either phosphorylated at threonine 181 or 217) (c) Tau PET imagining (assessment of NFT accumulation); (d) volumetric MRI (assessment of neuroanatomical atrophy); (e) FDG-PEGPET imagining (assessment of hypometabolism); (f) florbetapir perfusion PET imagining (assessment of hypometabolism); (g) CSF tau concentration (assessment of neurodegeneration); and/or (i) CSF phosphorylated-Tau concentration (assessment of neurodegeneration). Additionally, one or more validated rating scales assessing the cognitive and functional decline of each treatment group may be applied, for example ADAS-cog, MMSE, CDR-SB, ADCS-ADL, and Functional Activities Questionnaire (FAQ).

This study may show that the combination therapy of an OGA inhibitor of the present invention and an anti-N3pGlu A0 antibody of the present invention may result in reduction of A0 plaque and limit tau hyperphosphorylation and intraneuronal aggregation into pathological tau, such as NFTs, for the treatment of diseases such as AD.

SEQUENCES

SEQUENCE ID NO:1 LCVR of Antibody I

DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAV SKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEI K

SEQUENCE ID NO:2 HCVR of Antibody I

QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINP GSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQ GTTVTVSS

SEQUENCE ID NO:3 LC of Antibody I

DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAV SKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQUENCE ID NO:4 HC of Antibody I

QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINP GSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG SEQUENCE ID NO:5 LCDR1 of Antibody I

KSSQSLLYSRGKTYLN

SEQUENCE ID NO:6 LCDR2 of Antibody I

AVSKLDS

SEQUENCE ID NO:7 LCDR3 of Antibody I

VQGTHYPFT

SEQUENCED ID NO:8 HCDR1 of Antibody I

GYDFTRYYIN

SEQUENCE ID NO:9 HCDR2 of Antibody I

WINPGSGNTKYNEKFKG

SEQUENCE ID NO: 10 HCDR3 of Antibody I

EGITVY

SEQUENCE ID NO: 11 LCVR of Antibody II

DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLE

SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK

SEQUENCE ID NO: 12 HCVR of Antibody II

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYN GFD YWGQGTL VT VS S

SEQUENCE ID NO: 13 LC of Antibody II

DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLE SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQUENCE ID NO: 14 HC of Antibody II

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYN GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG

SEQUENCE ID NO: 15 LCDR1 of Antibody II

RASQSLGNWLA

SEQUENCE ID NO: 16 LCDR2 of Antibody II

YQASTLES

SEQUENCE ID NO: 17 LCDR3 of Antibody II

QHYKGSFWT

SEQUENCE ID NO: 18 HCDR1 of Antibody II

AASGFTFSSYPMS

SEQUENCE ID NO: 19 HCDR2 of Antibody II

AISGSGGSTYYADSVKG

SEQUENCE ID NO:20 HCDR3 of Antibody II AREGGSGSYYNGFDY

SEQUENCE ID NO:21 LC DNA sequence of Antibody I gatattgtgatgactcagactccactctccctgtccgtcacccctggacagccggcctccatctcctgcaagtcaagtcagagcct cttatatagtcgcggaaaaacctatttgaattggctcctgcagaagccaggccaatctccacagctcctaatttatgcggtgtctaaa ctggactctggggtcccagacagattcagcggcagtgggtcaggcacagatttcacactgaaaatcagcagggtggaggccga agatgttggggtttattactgcgtgcaaggtacacattacccattcacgtttggccaagggaccaagctggagatcaaacgaactg tggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttct atcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagga cagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgc gaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc

SEQ ID NO: 22 HC DNA Sequence of Antibody I caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcagtgaaggtttcctgcaaggcatctggttacgac ttcactagatactatataaactgggtgcgacaggcccctggacaagggcttgagtggatgggatggattaatcctggaagcggta atactaagtacaatgagaaattcaagggcagagtcaccattaccgcggacgaatccacgagcacagcctacatggagctgagc agcctgagatctgaggacacggccgtgtattactgtgcgagagaaggcatcacggtctactggggccaagggaccacggtcac cgtctcctcagcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagcacctctgggggcacagcggccct gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacacct tcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagaccta catctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatg cccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctg caccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggacgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgccccccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQUENCE ID NO:23 LC DNA Sequence of Antibody II

Gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtc ttggtaactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctatcaggcgtctactttagaatctggg gtcccatcaagattcagcggcagtggatctgggacagagttcactctcaccatcagcagcctgcagcctgatgattttgcaacttat tactgccaacattataaaggttctttttggacgttcggccaagggaccaaggtggaaatcaaacggaccgtggctgcaccatctgt cttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagc acctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc

SEQUENCE ID NO:24 HC DNA Sequence of Antibody II gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacct ttagcagctatcctatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtag cacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacag cctgagagccgaggacacggccgtatattactgtgcgagagaggggggctcagggagttattataacggctttgattattgggg ccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagcacctc tgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg accagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagc agcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatc ttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaaccc aaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggacg agctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaat gggcagccggagaacaactacaagaccacgccccccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggt

Claims

WE CLAIM:

1. A method of treating a patient having a disease characterized by amyloid beta

(AP) deposits, comprising administering to the patient in need thereof an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the disease characterized by amyloid beta (AP) deposits is selected from a group consisting of preclinical Alzheimer’s disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, Down’s syndrome, clinical cerebral amyloid angiopathy, and pre-clinical cerebral amyloid angiopathy.

3. The method of claim 1, wherein the methyl at position 2 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 4 on the piperidine ring:

4. The method of claim 3, wherein the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

85

5. The method of claim 4, wherein the OGA inhibitor is crystalline.

6. The method of claim 5, wherein the compound is characterized by a peak in the X-ray powder diffraction spectrum, at diffraction angle 2-theta of 12.1° in combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8°, with a tolerance for the diffraction angles of 0.2 degrees.

7. The method of claim 1, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of: i) LCDR1 is given by SEQ ID NO: 5, the amino acid sequence of LCDR2 is given by SEQ ID NO: 6, the amino acid sequence of LCDR3 is given by SEQ ID NO: 7, the amino acid sequence of HCDR1 is given by SEQ ID NO: 8, the amino acid sequence of HCDR2 is given by SEQ ID NO: 9, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 10; or ii) LCDR1 is given by SEQ ID NO: 15, the amino acid sequence of LCDR2 is given by SEQ ID NO: 16, the amino acid sequence of LCDR3 is given by SEQ ID NO: 17, the amino acid sequence of HCDR1 is given by SEQ ID NO: 18, the amino acid sequence of HCDR2 is given by SEQ ID NO: 19, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 20.

8. The method of claim 7, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of: i) the LCVR is given by SEQ ID NO: 1 and the amino acid sequence of the HCVR is given by SEQ ID NO: 2; or

86 ii) the LCVR is given by SEQ ID NO: 11 and the amino acid sequence of the HCVR is given by SEQ ID NO: 12.

9. The method of claim 8, wherein the anti-N3pG Ap antibody comprises a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of: i) the LC is given by SEQ ID NO: 3 and the amino acid sequence of the HC is given by SEQ ID NO: 4; or ii) the LC is given by SEQ ID NO: 13 and the amino acid sequence of the HC is given by SEQ ID NO: 14.

10. A method of treating a patient having a disease characterized by amyloid beta (AP) deposits, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula: or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the disease characterized by amyloid beta (AP) deposits is selected from a group consisting of preclinical Alzheimer’s disease, clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, Down’s syndrome, clinical cerebral amyloid angiopathy, or pre-clinical cerebral amyloid angiopathy.

12. The method of claim 10, wherein the methyl at position 2 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 4 on the piperidine ring:

13. The method of claim 10 wherein the methyl at position 2 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 4 on the piperidine ring:

14. The method of claim 12 wherein the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

15. The method of claim 13 wherein the OGA inhibitor is N-[4-fhioro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

16. The method of claim 14 wherein the OGA inhibitor is crystalline.

17. The method of claim 15 wherein the OGA inhibitor is characterized by a peak in the X-ray powder diffraction spectrum at diffraction angle 2-theta of 13.5° in combination with one or more peaks selected from the group consisting of 5.8°, 13.0°, 14.3°, 17.5°, 20.4°, 21.4°, and 22.2 ° with a tolerance for the diffraction angles of 0.2 degrees.

18. The method of claim 10, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the

88 LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of: i) LCDR1 is given by SEQ ID NO: 5, the amino acid sequence of LCDR2 is given by SEQ ID NO: 6, the amino acid sequence of LCDR3 is given by SEQ ID NO: 7, the amino acid sequence of HCDR1 is given by SEQ ID NO: 8, the amino acid sequence of HCDR2 is given by SEQ ID NO: 9, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 10; or ii) LCDR1 is given by SEQ ID NO: 15, the amino acid sequence of LCDR2 is given by SEQ ID NO: 16, the amino acid sequence of LCDR3 is given by SEQ ID NO: 17, the amino acid sequence of HCDR1 is given by SEQ ID NO: 18, the amino acid sequence of HCDR2 is given by SEQ ID NO: 19, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 20.

19. The method of claim 18, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of i) the LCVR is given by SEQ ID NO: 1 and the amino acid sequence of the HCVR is given by SEQ ID NO: 2; or ii) the LCVR is given by SEQ ID NO: 11 and the amino acid sequence of the HCVR is given by SEQ ID NO: 12.

20. The method of claim 19, wherein the anti-N3pG Ap antibody comprises a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of: i) the LC is given by SEQ ID NO: 3 and the amino acid sequence of the HC is given by SEQ ID NO: 4; or ii) the LC is given by SEQ ID NO: 13 and the amino acid sequence of the HC is given by SEQ ID NO: 14.

89

21. A method of treating a patient having a disease characterized by amyloid beta (AP) deposits and aberrant tau aggregation, comprising administering to the patient in need thereof an effective amount of an anti-N3pG AP antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of formula: or a pharmaceutically acceptable salt thereof.

22. The method of claim 21, wherein the disease characterized by amyloid beta (AP) deposits and aberrant tau aggregation is selected from a group consisting of preclinical Alzheimer’s disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, Down’s syndrome, clinical cerebral amyloid angiopathy, and pre-clinical cerebral amyloid angiopathy.

23. The method of claim 21, wherein the methyl at position 2 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 4 on the piperidine ring:

24. The method of claim 23, wherein the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,2,4-oxadiazol-3-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

25. The method of claim 24, wherein the OGA inhibitor is crystalline.

90

26. The method of claim 25, wherein the compound is characterized by a peak in the X-ray powder diffraction spectrum, at diffraction angle 2-theta of 12.1° in combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8°, with a tolerance for the diffraction angles of 0.2 degrees.

27. The method of claim 21, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of: i) LCDR1 is given by SEQ ID NO: 5, the amino acid sequence of LCDR2 is given by SEQ ID NO: 6, the amino acid sequence of LCDR3 is given by SEQ ID NO: 7, the amino acid sequence of HCDR1 is given by SEQ ID NO: 8, the amino acid sequence of HCDR2 is given by SEQ ID NO: 9, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 10; or ii) LCDR1 is given by SEQ ID NO: 15, the amino acid sequence of LCDR2 is given by SEQ ID NO: 16, the amino acid sequence of LCDR3 is given by SEQ ID NO: 17, the amino acid sequence of HCDR1 is given by SEQ ID NO: 18, the amino acid sequence of HCDR2 is given by SEQ ID NO: 19, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 20.

28. The method of claim 27, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of: i) the LCVR is given by SEQ ID NO: 1 and the amino acid sequence of the HCVR is given by SEQ ID NO: 2; or ii) the LCVR is given by SEQ ID NO: 11 and the amino acid sequence of the HCVR is given by SEQ ID NO: 12.

91

29. The method of claim 28, wherein the anti-N3pG Ap antibody comprises a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of: i) the LC is given by SEQ ID NO: 3 and the amino acid sequence of the HC is given by SEQ ID NO: 4; or ii) the LC is given by SEQ ID NO: 13 and the amino acid sequence of the HC is given by SEQ ID NO: 14.

30. A method of treating a patient having a disease characterized by amyloid beta (AP) deposits and aberrant tau aggregation, comprising administering to a patient in need of such treatment an effective amount of an anti-N3pG Ap antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula: or a pharmaceutically acceptable salt thereof.

31. The method of claim 30, wherein the disease characterized by amyloid beta (AP) deposits and aberrant tau aggregation is selected from a group consisting of preclinical Alzheimer’s disease, clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, Down’s syndrome, clinical cerebral amyloid angiopathy, and pre-clinical cerebral amyloid angiopathy.

32. The method of claim 30, wherein the methyl at position 2 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 4 on the piperidine ring:

33. The method of claim 30 wherein the methyl at position 2 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 4 on the piperidine ring:

34. The method of claim 32 wherein the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

35. The method of claim 33 wherein the OGA inhibitor is N-[4-fluoro-5-[[(2S,4S)-2- methyl-4-[(5-methyl-l,3,4-oxadiazol-2-yl)methoxy]-l-piperidyl]methyl]thiazol-2- yl]acetamide.

36. The method of claim 34 wherein the OGA inhibitor is crystalline.

37. The method of claim 35 wherein the OGA inhibitor is characterized by a peak in the X-ray powder diffraction spectrum at diffraction angle 2-theta of 13.5° in combination with one or more peaks selected from the group consisting of 5.8°, 13.0°, 14.3°, 17.5°, 20.4°, 21.4°, and 22.2 ° with a tolerance for the diffraction angles of 0.2 degrees.

38. The method of claim 30, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of: i) LCDR1 is given by SEQ ID NO: 5, the amino acid sequence of LCDR2 is given by SEQ ID NO: 6, the amino acid sequence of LCDR3 is given by SEQ ID NO: 7, the amino acid sequence of HCDR1 is given by SEQ ID NO: 8, the amino acid sequence of HCDR2 is given by SEQ ID NO: 9, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 10; or ii) LCDR1 is given by SEQ ID NO: 15, the amino acid sequence of LCDR2 is given by SEQ ID NO: 16, the amino acid sequence of LCDR3 is given by SEQ ID NO: 17, the amino acid sequence of HCDR1 is given by SEQ ID NO: 18, the amino acid sequence of HCDR2 is given by SEQ ID NO: 19, and the amino acid sequence of HCDR3 is given by SEQ ID NO: 20.

39. The method of claim 38, wherein the anti-N3pG Ap antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of i) the LCVR is given by SEQ ID NO: 1 and the amino acid sequence of the HCVR is given by SEQ ID NO: 2; or ii) the LCVR is given by SEQ ID NO: 11 and the amino acid sequence of the HCVR is given by SEQ ID NO: 12.

40. The method of claim 39, wherein the anti-N3pG Ap antibody comprises a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of: i) the LC is given by SEQ ID NO: 3 and the amino acid sequence of the HC is given by SEQ ID NO: 4; or ii) the LC is given by SEQ ID NO: 13 and the amino acid sequence of the HC is given by SEQ ID NO: 14.

1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing: a. X forming part of the international application as filed. b. furnished subsequent to the international filing date for the purposes of international search (Rule 13ter1 (a)). accompanied by a statement to the effect that the sequence listing does not go beyond the disclosure in the international application as filed.

With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this report has been established to the extent that a meaningful search could be carried out without a WIPO Standard ST.26 compliant sequence listing.

3. Additional comments:

Form PCT/ISA/210 (continuation of first sheet (1)) (July 2022)