EA043417B1 - LONG-ACTING GLP-1r AGONIST AS A THERAPY FOR THE TREATMENT OF NEUROLOGICAL AND NEURODEGENERATIVE PATHOLOGICAL CONDITIONS - Google Patents

Description

Related applications

This USC 35 119(e) application claims the benefit of provisional application US Patent No. 62/387,319, filed December 23, 2015, which is incorporated herein by reference in its entirety.

Application for federal financial support for research or development

The present invention was made with government support in the form of grants R21 CA 198243 and P50 CA103175 from the National Institutes of Health (NIH) of the United States. The government has certain rights to this invention.

BACKGROUND OF THE INVENTION

Neurodegenerative disease includes a range of conditions caused by the gradual loss of neuronal structure or function, including neuronal death. A variety of neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington's chorea (XX), arise from neurodegenerative processes.

Parkinson's disease (PD) is a progressive, late-onset neurodegenerative disease that affects approximately one million Americans and 7 to 10 million people worldwide. Although dopamine replacement reduces symptomatic motor dysfunction, its effectiveness decreases as the disease progresses, leading to unacceptable side effects such as severe motor fluctuations and dyskinesias. Moreover, this palliative therapeutic approach does not address the underlying mechanisms of the disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and accounts for more than 80% of dementia cases worldwide. It leads to a gradual decline in mental abilities, behavioral skills, functional decline and decreased learning ability. Currently approved medications, such as acetylcholinesterase inhibitors, provide only symptomatic improvement and do not alter the disease process. A number of new strategies, including amyloid- and τ-protein-based therapies, are in clinical trials, but none of the drugs had demonstrated clear efficacy in humans at the time of the invention described herein.

As life expectancy increases, more and more people develop these common, debilitating neurological disorders. Prior to the invention described herein, there was no proven neuroprotective therapy that could treat the disease or stop the progression of the disease in humans. Thus, there is a significant unmet need for therapeutic strategies to treat this relentlessly progressive chronic disease.

The essence of the invention

The present invention is based, at least in part, on the production of long-acting GLP-1r (glucagon-like peptide-1 receptor) agonists with neuroprotective and disease-modifying effects on the central nervous system. A long-acting, subcutaneously administered exenatide was developed that, despite its large molecular weight, has high biological activity in the brain and significant neuroprotective and disease-modifying effects in mouse models of the central nervous system, such as neurodegenerative diseases.

Designed peptide drugs fused to high molecular weight carriers to increase half-life, such as immunoglobulin Fc, albumin or PEG, do not cross the blood-brain barrier (BBB) well (Pardridge W.M. 2005 NeuroRx 2(1):3-14), unless the molecule has been re-engineered to target BBB receptors such as transferrin receptor (Yu Y. J. et al., 2014. Sci. Transl. Med. 6(261): 261ra154). The present invention describes PEGylated exenatide (NLY001) with significantly improved half-life and mean retention time in non-human primates compared to BYETTA® exenatide (exendin-4 with a 2-hour half-life) and liraglutide (which has a half-life of 13 h). PEGylated exenatide retains its biological activity by site when a polyethylene glycol (PEG) molecule is site-specifically attached to the exenatide (WO 2013002580, incorporated herein by reference). Due to its increased half-life and potency, this chemical compound is suitable for clinical dosing at weekly, bimonthly, or monthly frequency. This dosing frequency is an improvement over the current dosing frequency of other drugs: twice daily (exenatide, BYETTA®) or once daily (liraglutide, VICTOZA®). However, as with peptide drugs fused to high molecular weight carriers, this PEGylated exenatide was not expected to be effective in neurodegenerative diseases such as Parkinson's disease (PD) or Alzheimer's disease (AD) when administered systemically. As described in the present invention, the unexpected discovery was that subcutaneous administration of a long-acting GLP-1r agonist, PEGylated exenatide, has a clear effect on Parkinson's disease and Alzheimer's disease in clinical

- 1 043417 relevant transgenic animal models of PD and AD due to effective targeted effects on activated microglia and reactive astrocytes in the brain. The present invention describes the use of a long-acting GLP-1r agonist for the treatment of PD and AD, as well as other neurodegenerative pathological conditions such as Huntington's chorea and amyotrophic lateral sclerosis (ALS), diseases caused by the pathogenesis of microglial activation.

Compositions and methods have been identified for selectively blocking or inverting the activation of microglia and reactive astrocytes, which are key cells involved in the onset and/or progression of neurodegenerative diseases, to stop the initiation of a cascade of neurotoxic pathways. This method involves administering a long-acting GLP-1r agonist to the subject.

In one embodiment, a method of treating or preventing a neurodegenerative disease in a mammalian subject includes administering a long-acting GLP-1r agonist capable of crossing the BBB and activating GLP-1r in the PD brain in a continuous manner without interruption of action and without adverse toxicity. For example, the long-acting GLP-1r agonist, NLY001, accumulates efficiently in the brains of PD and AD models and, importantly, exhibits slow internalization of GLP-1r compared to short-acting GLP1r agonists. Sustained activation of GLP-1r in the brain helps maximize the synergistic anti-inflammatory and neuroprotective properties of the GLP-1r agonist.

In one embodiment of the invention, a method of protecting neuronal cells acts by blocking gliosis (activation of microglia and astrocytes) and releasing toxic molecules from activated microglia and reactive astrocytes by targeting GLP-1r activation in activated microglia.

Thus, the present invention provides methods for treating a neurodegenerative disease or disorder, comprising administering to a subject suffering from or at risk of developing the neurodegenerative disease or disorder a pharmaceutically effective amount of a composition containing a long-acting GLP-1r agonist to relieve one or more symptoms of the neurodegenerative disease or disorder. diseases or disorders. Suitable long-acting GLP-1r agonist includes a PEGylated GLP-1r analog, an Fc-fused GLP-1 analog, an albumin-fused GLP-1 analog, or derivatives thereof. In an illustrative aspect, the long-acting GLP-1r agonist comprises a PEGylated analogue of exenatide. In some cases, the neurodegenerative disease or disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, spinocerebellar ataxia type 1 (SCA1) and prion disorder. Optionally, the neurodegenerative disorder is Parkinson's disease and Alzheimer's disease. In an exemplary embodiment of the invention, the methods further include identifying a patient suffering from or at risk of developing a neurodegenerative disease or disorder, such as Parkinson's disease or Alzheimer's disease.

Preferably, the long-acting GLP-1r agonist blocks the activation of resident innate immune cells. For example, innate immune cells include microglia and/or astrocytes. In some cases, abnormally aggregated proteins are inhibited from activating immune cells by activating GLP-1r. For example, abnormally aggregated proteins include α-synuclein, β-amyloid, or τ protein. The amount of long-acting GLP-1r agonist effectively inhibits the secretion of inflammatory and/or neurotoxic mediators secreted by activated innate immune cells.

In some cases, the GLP-1r agonist is administered in an amount effective to reduce inflammatory or neurotoxic mediators selected from the group consisting of TNF-α, IL-1a, Π-1β, IFN-γ, IL-6 and C1q, compared with appropriate controls. In one aspect, the GLP-1r agonist is administered in an amount effective to reduce the cell population of activated microglia and reactive astrocytes.

In one embodiment of the invention, levels of TNF-α, IL-1a, IL-1e, IL-6 or C1q and/or activated microglia and reactive astrocytes in the brain of a subject are reduced, maintained or restored to normal values compared to matched controls.

In one embodiment of the invention, levels of abnormal brain protein deposition in a subject, such as α-synuclein (α-syn) (Lewy body) and amyloid plaques and τ protein, are reduced, maintained, or restored to normal values compared to matched controls.

In one embodiment of the invention, the treatment attenuates or restores motor impairment, improves memory function and/or increases life expectancy in the subject compared to a matched control.

Suitable routes of administration include oral administration, intravenous administration,

- 2 043417 local administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intrauterine administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intracavitary administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration , transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration and subcutaneous administration. A typical route of administration involves subcutaneous administration.

In some cases, the composition is administered in a form selected from the group including pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, suspensions, gels, creams, pastes, films, patches and vapors.

In one aspect, the compositions described herein, for example, PEGylated exenatide, are administered approximately 1-4 times per month. For example, the compositions are administered approximately once a week or twice a week. Alternatively, the compositions are administered approximately every two weeks. In other cases, the compositions are administered once a month. In other cases, the compositions are administered once every two months. In other aspects, the compositions are administered approximately 1-3 times every 6 months. Alternatively, the compositions of the present invention are administered approximately 1-12 times per year, for example once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months or once every 12 months.

Preferably, the compositions described herein, for example, PEGylated exenatide, have an increased in vivo half-life, for example, from at least 2 hours to 200 hours. For example, PEGylated exenatide has an in vivo half-life of 2, 6, 12, 20, 24, 36. 48, 60, 72, 88, 100, 112, 124, 150, 175 or 200 hours. In an exemplary embodiment, the compositions of the present invention have an in vivo half-life of 88 hours in non-human primates.

The compositions described herein are administered at a dose of 0.001 to 100 mg/kg, for example 0.001, 0.01, 0.1, 0.5, 1.0, 2.0, 4, 5, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg. For example, the compositions are administered at a dose of 0.2 to 20 mg/kg in mouse models. In humans, the compositions can be administered at a dose of 0.001 to 10 mg/kg.

In one aspect, the compositions described herein are administered to a subject over a period of time ranging from 1 to 20 years, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20 years old. If necessary, the compositions described herein are administered for 10 years. In one aspect, the effect of the treatment lasts for at least 1 year.

Preferably, the compositions described herein protect against α-synuclein-associated dopaminergic neuron loss. In one aspect, the compositions described herein protect against amyloid-β and/or τ protein toxicity in neurons in Alzheimer's disease. For example, the compositions described herein protect against amyloid plaques and τ protein-associated neuronal loss. In one aspect, the compositions described herein improve motor, cognitive, and memory skills in a subject compared to a control. In another aspect, the compositions described herein protect synapses and/or synaptic functions, enhance neurogenesis, reduce apoptosis, protect neurons from oxidative stress, reduce plaque formation, and prevent a chronic inflammatory response in a subject compared to a control.

The present invention also relates to compositions containing long-acting glucagon-like peptide 1 receptor (GLP-1r) agonists and one or more polyethylene glycol (PEG) molecules or derivatives thereof. In some cases, the long-acting GLP-1r agonist includes a PEGylated GLP-1 analog or derivative thereof. In some cases, the long-acting GLP-1r agonist includes an Fc-fused GLP-1 analog, an albumin-fused GLP-1 analog, or derivatives thereof. In some cases, the GLP-1r agonist is an exendin-4 analogue and/or derivatives thereof. In some cases, the exendin-4 analogue is a peptide. For example, this peptide contains the sequence

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 1).

In one aspect, the PEG molecule or derivative thereof is selected from the group consisting of linear PEG, branched PEG, star PEG, comb PEG, dendrimeric PEG, PEG succinimidyl propionate, PEG N-hydroxysuccinimide, PEG propionaldehyde, PEG maleimide, linear methoxy polyethylene glycol (m PEG) , branched mPEG, star mPEG, comb mPEG, dendrimer mPEG, mPEG succinimidyl propionate, mPEG Nhydroxysuccinimide, mPEG propionaldehyde and mPEG maleimide. In some cases, a branched PEG molecule or derivative thereof includes monomeric, dimeric and/or trimeric PEG molecules or derivatives thereof. In some cases, the PEG molecule or derivative thereof is a trimeric methoxy polyethylene glycol maleimide.

In another aspect, the PEG molecule comprises a PEG molecule with a mass of at least 1000 daltons. In some cases, the PEG molecule contains a PEG molecule weighing in the range of 1000 to 1000000

- 3 043417 dalton. In other examples, the PEG molecule contains a PEG molecule with a mass ranging from 10,000 to 500,000 daltons. In other cases, the PEG molecule contains a PEG molecule with a mass ranging from 2,0000 to 250,000 daltons. In other aspects, the PEG molecule comprises a PEG molecule with a mass ranging from 30,000 to 100,000 daltons. Alternatively, the PEG molecule contains a PEG molecule with a mass ranging from 40,000 to 80,000 daltons. In an exemplary embodiment of the invention, the PEGylated exenatide described herein is NLY001, exenatide PEGylated with a 50,000 dalton PEG molecule.

Definitions.

Unless specifically stated otherwise or otherwise apparent from the context, as used herein, the term is roughly understood to mean the range of normal tolerance in the art, for example, within 2 standard deviations from the mean. The term can be roughly understood as 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05 or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values presented here are modified by the term approximately.

The term agent means any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide or fragments thereof.

The term change means a change (increase or decrease) in the levels of expression or activity of a gene or polypeptide detected by methods known standard in the art, such as those described herein. In the present invention, the term change includes at least a 1% change in expression levels, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 , 70, 80, 90 or 100% change in expression levels. For example, the change includes at least a 5-10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

The term improvement means to reduce, suppress, attenuate, reduce, stop or stabilize the development or progression of a disease.

The term binding to a molecule means physical-chemical affinity with that molecule.

The transitional term containing, which is synonymous with the terms including, comprising or characterized, is open-ended and does not exclude additional, unmentioned elements or method steps. In contrast, the transition phrase consisting of excludes any element, step or ingredient not specified in the claims. A transitional phrase essentially consisting of limits the scope of the claims to those materials or steps, and to those materials or steps that do not materially affect the essential and novel feature(s) of the claimed invention.

The term control or standard means a standard of comparison. As used herein, the term altered relative to a control sample or subject means having a level that is statistically different from a sample from a normal, untreated, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more humans. Methods for selecting and testing control samples are known to those skilled in the art. The analyte may be a naturally occurring substance that is characteristically expressed or produced by a cell or organism (eg, antibody, protein) or a substance produced by a reporter construct (eg, β-galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change may vary. The determination of statistical significance depends on the choices of those skilled in the art, such as the number of standard deviations from the mean that constitutes a positive result.

The term detection refers to the identification of the presence, absence or quantity of an analyte to be detected.

As used herein, the term diagnosis refers to the classification of a pathology or symptom, determining the severity of the pathology (eg, grade or stage), monitoring the progression of the pathology, predicting the outcome of the pathology, and/or determining the prospects for recovery.

The terms effective amount and therapeutically effective amount of a composition or composition component mean a sufficient amount of the composition or component, alone or in combination, to provide the desired effect. For example, by effective amount is meant the amount of a chemical compound, alone or in combination, that is required to provide relief from the symptoms of a disease, such as prostate cancer, compared to an untreated patient. The effective amount of the active chemical compound(s) used to implement the present invention for the therapeutic treatment of a disease varies depending on the route of administration, age, body weight and general health of the subject. Ultimately, the treating physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is called the effective amount.

The term fragment means part of a polypeptide or nucleic acid molecule. This part preferably contains at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the total length of the reference

- 4 043417 nucleic acid or polypeptide molecules. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 10oO nucleotides or amino acids.

The terms isolated, purified or biologically pure refer to a material that is, to varying degrees, free of the components that normally accompany it when in its native state. Emphasis refers to the degree of separation from the original source or environment. Purification means a degree of separation that is higher than isolation.

The term long-acting GLP-1r agonist means a GLP-1r agonist that is effective for at least one hour, at least six hours, at least twelve hours, at least one day, at least two days , at least one week, at least two weeks, at least one month, or at least two months.

The term marker means any protein or polynucleotide having a change in expression level or activity that is associated with a disease or disorder.

The term modulation means change (increase or decrease). Such changes are detected by known standard methods, such as those described herein.

Neurodegenerative disease or disorder is a general term for the progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's chorea, occur as a result of neurodegenerative processes. Such diseases are incurable, leading to progressive degeneration and/or death of neuronal cells.

The term normal amount refers to the normal amount of the complex in an individual who has not been diagnosed with a disease or disorder. The amount of a molecule can be measured in a test sample and compared to a normal control level using methods such as control limits, discrimination limits, or cutoff values to determine cutoff points and abnormal values (eg, for prostate cancer). The term normal control level means the level of one or more proteins (or nucleic acids) or combined protein indices (or combined nucleic acid indices) that are typically found in a subject not known to have prostate cancer. Such normal reference levels and cut-off points may vary depending on whether the molecule is used alone or in a composition combining other proteins into an index. Alternatively, the normal reference level may represent a database of protein structures in previously studied subjects who have not developed a disease or disorder over a clinically relevant time horizon.

The detected level may be the same as the control level or cutoff level or threshold level, or may be increased or decreased compared to the control level or cutoff level or threshold level. In some aspects, a control subject is a matched control of the same species, sex, ethnicity, age group, smoking status, body mass index (BMI), current therapeutic regimen status, medical history, or a combination thereof, but different from the diagnosis subject in that the control the subject does not suffer from the disease or is not at risk of developing the disease.

Compared to the control level, the detected level may be increased. As used herein, the term increased relative to a level (eg, expression level, biological activity level, etc.) refers to any % increase above the control level. The increased level may be at least or about a 1% increase at least, or about a 5% increase at least, or about a 10% increase at least, or about a 15% increase at least at least or about a 20% increase at least or about a 25% increase at least or about a 30% increase at least or about a 35% increase at least or about an increase of at least 40%, or approximately a 45% increase, at least, or approximately a 50% increase, at least, or approximately a 55% increase, at least, or approximately a 60% increase, at least or about 65% increase at least or about 70% increase at least or about 75% increase at least or about 80% increase at least or about 80% increase an 85% increase, at least or about a 90% increase, or an at least or approximately 95% increase over the control level.

Compared to the control level, the detected level may be reduced. As used herein, the term downregulated (eg, expression level, biological activity level, etc.) refers to any % reduction below the control level. The reduced level may be at least or about a 1% reduction at least, or about a 5% reduction at least, or about a 10% reduction at least, or about a 15% reduction, at least at least or about 20% reduction at least or about 25% reduction at least or about 30% reduction at least or about 35% reduction at least or about 35% reduction by 40%,

- 5 043417 at least, or approximately 45% reduction, at least, or approximately 50% reduction, at least, or approximately 55% reduction, at least, or approximately 60% reduction, at least , or about a 65% reduction at least, or about a 70% reduction at least, or about a 75% reduction at least, or about an 80% reduction at least, or about an 85% reduction %, at least or about 90% reduction or at least or about 95% reduction compared to the control level.

The term pharmaceutically acceptable carrier is recognized in the art and includes a pharmaceutically acceptable material, composition or carrier suitable for administering the chemical compounds of the present invention to mammals. Carriers include a liquid or solid filler, diluent, excipient, solvent or encapsulating material that is involved in carrying or transporting the respective agent from one organ or body part to another organ or body part. Each carrier must be acceptable in terms of compatibility with the other ingredients of the composition and not cause harm to the patient.

Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyhydric alcohols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical preparations.

The term protein or polypeptide or peptide means any chain of more than two naturally occurring or artificial amino acids, regardless of post-translational modification (eg, glycosylation or phosphorylation), constituting all or part of a naturally occurring or non-naturally occurring polypeptide or peptide as described herein.

A purified or biologically pure nucleic acid or protein contains substantially no other materials such that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. Thus, a nucleic acid or peptide of the present invention is purified if it contains substantially no cellular material, viral material or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when synthesized chemically. Purity and uniformity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term purified can mean that the nucleic acid or protein produces substantially a single band on an electrophoresis gel. For a protein that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

The term substantially pure means a nucleotide or polypeptide that has been separated from the components that accompany it in nature. Generally, nucleotides and polypeptides are substantially pure when they are at least 60, 70, 80, 90, 95 or even 99% by weight free of proteins and naturally occurring organic molecules with which they are naturally associated .

The value ranges given here are intended to be abbreviations for all values within that range. For example, the range 1 to 50 is assumed to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, as well as all intermediate decimal values between the above integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. With respect to subranges, nested subranges that start from any endpoint of the range are specifically considered. For example, a nested subrange of the example range 1 to 50 may contain 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20 and from 50 to 10 in the other direction.

The term decrease means a negative change of at least 1%, for example at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least by 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95% or by at least 99%.

- 6 043417

The term reference means a standard or control condition.

A reference sequence is a specific sequence used as a basis for sequence comparison or gene expression comparison. The reference sequence may be a subset or an integer of the specified sequence; for example, a segment of a full-length cDNA or gene sequence or a full-length cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid is typically at least about 40 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 or about 500 nucleotides or any whole next to or between them.

As used herein, the term obtaining, as in obtaining an agent, includes the synthesis, purchase or other acquisition of an agent.

The term subject means a mammal, including without limitation a human or non-human mammal such as a cow, horse, dog, sheep or cat. The subject is preferably a mammal in need of treatment, for example a subject who has been diagnosed with a disease or predisposition thereto.

A mammal is any mammal, such as a human, primate, mouse, rat, dog, cat, horse, as well as livestock or animals raised for food, such as cattle, sheep, pigs, chickens and goats. In a preferred embodiment of the invention, the mammal is human.

The term substantially identical means a polypeptide or nucleic acid molecule having at least 50% identity with a reference amino acid sequence (eg, any of the amino acid sequences described herein) or nucleic acid sequence (eg, any of the nucleotide sequences described herein). Preferably, such a sequence has at least 60%, more preferably 80% or 85%, and more preferably 90, 95 or even 99% identity at the amino acid or nucleotide level with the sequence used for comparison.

As used herein, the term sample refers to a biological sample obtained for in vitro evaluation purposes. With respect to the methods described herein, the sample or specimen obtained from the patient may preferably include any body fluid or tissue. In some embodiments, the body fluid includes, but is not limited to, blood, blood plasma, serum, lymph, breast milk, saliva, mucus, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous fluid body or urine obtained from the subject. In some aspects, the sample is a composite panel of at least two components selected from a blood sample, a blood plasma sample, a blood serum sample, and a urine sample. In exemplary aspects, the sample includes blood or a fraction thereof (eg, plasma, serum, leukapheresis fraction). Preferred samples are whole blood, serum, plasma, or urine. The sample may also be a partially purified fraction of tissue or body fluid.

The reference sample may be a normal fluid sample obtained from a donor without a disease or condition, or a normal tissue sample obtained from a subject with a disease or condition. The reference sample may also be obtained from an untreated donor or a cell culture not treated with the active agent (eg, without treatment or administration of vehicle alone). The reference sample may also be collected at time zero before the cell or subject is exposed to the agent or therapeutic intervention to be tested, or at the start of a prospective study.

The term specific binding means a chemical compound or antibody that recognizes and binds to a polypeptide of the present invention, but which does not recognize or bind substantially to other molecules in a sample, such as a biological sample, that naturally includes the polypeptide of the present invention.

As used herein, the term subject includes all members of the animal kingdom that may be susceptible to the disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. These methods are also applicable to companion animals such as dogs and cats, as well as farm animals such as cows, horses, sheep, goats, pigs and other domestic and wild animals.

A subject who has or is believed to have a particular disease, condition or syndrome, has a sufficient number of risk factors, or has a sufficient number or combination of signs or symptoms of the disease, condition or syndrome

- 7 043417 syndrome such that a person skilled in the art would be able to diagnose or suspect that the subject is suffering from a disease, condition or syndrome. Methods for identifying subjects suffering or suspected of suffering from pathological conditions associated with heart disease, neurodegenerative disorders and the like are within the capabilities of those skilled in the art. Subjects suffering or suspected of suffering from a particular disease, condition or syndrome are not necessarily two different groups.

As used herein, the term sensitive to or prone to or predisposed to or at risk of developing a particular disease or condition refers to an individual who, based on genetic, environmental, medical and/or other risk factors, is more likely to develop a disease or condition than the general population. populations. The increase in the likelihood of developing the disease may represent an increase of approximately 10, 20, 50, 100, 150, 200% or more.

The term substantially identical means a polypeptide or nucleic acid molecule having at least 50% identity with a reference amino acid sequence (eg, any of the amino acid sequences described herein) or nucleotide sequence (eg, any of the nucleotide sequences described herein). Preferably, such a sequence has at least 60%, more preferably 80 or 85%, and more preferably 90, 95 or even 99% identity at the amino acid or nucleotide level with the sequence used for comparison.

As used herein, the terms treat, treatment process, treatment and the like refer to the administration of an agent or composition to a symptomatic individual suffering from an adverse condition, disorder or disease in order to achieve a reduction in the severity and/or frequency of symptoms, elimination of symptoms and/or their cause and/or alleviation, reduction or elimination of damage. It is understood that, although not impossible, treatment of a disorder or condition does not require complete elimination of the disorder, condition, or associated symptoms.

The term PEGylation refers to the process of either covalently or non-covalently attaching or fusing polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as a drug, therapeutic protein or vesicle.

The terms prevent, prevent, prevent, prophylactic treatment and the like refer to the administration of an agent or composition to a clinically asymptomatic individual who is at risk of developing, susceptible to or predisposed to a particular adverse condition, disorder or disease and, therefore, they refer to preventing the onset of symptoms and/or or their root cause.

In some cases, the composition of the present invention is administered orally or systemically. Other routes of administration include rectal, topical, intraocular, buccal, intravaginal, intra-articular, intracerebroventricular, intratracheal, nasal, transdermal, intra-implant or parenteral routes. The term parenteral includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. However, they may be preferable in emergency situations. Compositions containing the composition of the present invention can be added to a physiological fluid such as blood. Oral administration may be preferred for prophylactic treatment due to its convenience to the patient as well as the dosing schedule. Parenteral routes of administration (subcutaneous or intravenous) may be preferred for more acute illness or for therapy in patients who cannot tolerate enteral administration due to gastrointestinal intolerance, intestinal obstruction, or other underlying critical illnesses. Inhalation therapy may be most appropriate in the case of pulmonary vascular disease (eg, pulmonary hypertension).

Pharmaceutical compositions can be formulated into kits or pharmaceutical systems for use in cell cycle arrest in rapidly dividing cells, such as cancer cells. Kits or pharmaceutical systems in accordance with this aspect of the present invention comprise packaging, such as a box, carton, tube, directly containing one or more containers, such as vials, tubes, ampoules, bottles, syringes or pouches. The kits or pharmaceutical systems of the present invention may also contain appropriate instructions for use of the kit.

Unless specifically stated otherwise or is apparent from the context, the term or used herein is understood to be inclusive. Unless specifically stated otherwise or is clear from the context, the singular terms used herein refer to both the singular and the plural.

Unless specifically stated otherwise or otherwise apparent from the context, the term used herein

- 8 043417 is roughly understood as the range of normal tolerance in the art, for example, within 2 standard deviations from the mean. The term can be roughly understood as 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05 or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values presented here are modified by the term approximately.

The description of a list of chemical groups in any definition of a variable in the present invention includes definitions of that variable as any single group or combination of listed groups. The description of an embodiment for a variable or aspect in the present invention includes this embodiment as any one embodiment or in combination with any other embodiments or parts thereof.

A therapeutically effective amount is an amount sufficient to produce beneficial or desired results, including clinical results. An effective amount may be administered in one or more administrations.

Any compositions or methods presented herein may be combined with one or more of any other compositions or methods presented herein.

Other features and advantages of the present invention will be apparent from the following description of preferred embodiments and the claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Registrations in Genbank and NCBI indicated by the registration number given here are incorporated into the present invention by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the event of a conflict, this description, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting.

Brief description of the figures

In fig. Figure 1 shows the pharmacokinetic (PK) profiles of NLY001, a PEGylated exenatide. In fig. Figure 1 shows the PK profiles of subcutaneously administered exenatide and NLY001 in cynomolgus monkeys (n=2). t1/2: half-life, ALT: average retention time. The half-lives of exenatide and Oleadin are 2.7 ± 0.9 hours and 88.0 ± 10.9 hours, respectively. The average retention time of exenatide and Oleadin is 2.5 ± 0.3 hours and 114.0 ± 17.5 hours, respectively.

Fig. 2 is a series of histograms showing GLP-1r mRNA expression profiles in (Fig. 2A) α-synuclein PFF-activated primary microglial cells (n=4) and human post-mortem brain tissues from healthy individuals and patients with (Fig. 2B) disease Parkinson's (n=10) and (Fig. 2C) Alzheimer's disease (n=6). *P<0.05, ***P<0.001.

In fig. Figure 3 shows a schematic representation of activated microglia-neuron co-culture experiments. Primary microglia were activated with α-synuclein with or without NLY001, and microglia were co-cultured with primary neurons for 72 h, followed by neuronal death assay.

Fig. 4A and 4B are diagrams showing a timeline that depicts the experimental strategy in complemented mouse models of PD, (FIG. 4A) α-synuclein PFF-induced PD models, and (FIG. 4B) A53T α-synuclein transgenic models of PD.

Fig. 5 is a series of photomicrographs showing that treatment with NLY001 in mice with α-synuclein PFF-induced PD significantly inhibits α-synuclein accumulation in the brain. In fig. Figure 5 shows immunostaining with an antibody selective for α-synuclein LB (brown, arrow) in brain tissue.

In fig. Figure 6 shows Kaplan-Meier survival curve analysis for transgenic (Tg) hA53T α-synuclein PD mice with PBS or NLY001. Mice were treated with vehicle (PBS) or NLY001 at 6 months of age.

In fig. Figure 7 shows a timeline depicting the experimental strategy for mouse models of AD.

Fig. 8 is a graph showing the results of the Morris water maze (MWM) test in NLY001 3xTg-BA-treated mice ± SKO, n=7 mice per groups. ^### P <0.001 compared with the WT+PBS group. **P<0.01, **P<0.001 compared with 3xTg-BA+PBS.

Fig. 9 is a series of images showing representative video observations of wild-type (WT) and 3xTg-BA mice treated with vehicle (PBS) or NLY001.

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Detailed Description of the Invention

Neurodegenerative disease includes a range of conditions caused by the gradual loss of neuronal structure or function, including neuronal death. A variety of neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington's chorea (XX), arise from neurodegenerative processes.

Parkinson's disease (PD) is a progressive, late-onset neurodegenerative disease that affects approximately one million Americans and 7 to 10 million people worldwide. Although dopamine replacement reduces symptomatic motor dysfunction, its effectiveness decreases as the disease progresses, leading to unacceptable side effects such as severe motor fluctuations and dyskinesias. In addition, this palliative therapeutic approach does not address the underlying mechanisms of the disease (Nagatsua T. and M. Sawadab, Parkinsonism Relat Disord, 2009. 15 Suppl 1: p. S3-8).

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and accounts for more than 80% of dementia cases worldwide. It leads to a gradual decline in mental abilities, behavioral skills, functional decline and decreased learning ability (Anand R et al., Neuropharmacology, 2014. 76 Pt A:27-50). Currently approved medications, such as acetylcholinesterase inhibitors, provide only symptomatic improvement and do not alter the disease process. A number of new strategies, including amyloid- and τ-protein-based therapies, are in clinical trials, but none have yet shown clear efficacy in humans.

As life expectancy increases, more and more people develop these common, debilitating neurological disorders. Prior to the invention described herein, there was no proven neuroprotective therapy that could treat the disease or stop the progression of the disease in humans. Thus, there is a significant unmet need for therapeutic strategies to treat this relentlessly progressive chronic disease.

Prior to the invention described herein, the pathogenic mechanisms underlying neurodegenerative disorders were complex and largely unknown. Among the many factors associated with the pathology of neurodegenerative disorder, microglia and neuroinflammation are believed to be among the main causes of this disorder (Sanchez-Guajardo et al., Neuroscience, 2015. 302:4758). The predominant negative role of activated microglia has been discussed in the context of PD and AD. As disease progresses in the brain, quiescent microglia become activated and cause neuroinflammation and neuronal damage directly or through activation of astrocytes through the release of toxic molecules, including proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1a (IL-1a) ), IL-1e, IL-6 or C1q (Hirsch E.C. et al., Lancet Neurol, 2009. 8:382-397; Saijo K. et al., Cell, 2009. 137:47-59; Farber K. et al., J. Neurosci. Res., 200. 87(3): 644652). By their nature, activated microglia and reactive astrocytes are a major initial target for neurodegenerative diseases. Therefore, the development of a highly selective agent that can block microglial activation and stop the release of toxic molecules without side toxicity could lead to significant therapeutic effects in neurodegenerative diseases. However, the lack of reliable ways to selectively interact with activated microglia without causing side effects in the brain makes this strategy difficult.

Prior to the invention described herein, there was a need for therapies that prevent, arrest and/or attenuate neurodegenerative diseases. Thus, it is an object of the present invention to provide compositions and methods for the treatment and prevention of neurodegenerative diseases without adverse toxicity. Another object of the present invention is to provide compositions and methods for blocking or reducing microglial activation in neurodegenerative diseases while normal cells remain undamaged. Another object of the present invention is to provide compositions and methods for blocking or reducing reactive astrocytes in neurodegenerative diseases while normal cells remain undamaged. Another object of the present invention is to provide compositions and methods for protecting neurons from activated microglia and or reactive astrocytes in neurogenerative diseases while normal cells remain undamaged.

Accordingly, the present invention is based, at least in part, on the development of compositions and methods for the treatment of Parkinson's disease (PD) and Alzheimer's disease (AD) using long-acting GLP-1r (glucagon-like peptide-1 receptor) agonists having high biological activity in the brain. In particular, as described here, PEGylated forms of GLP-1 analogues (eg, exenatide) have disease-modifying effects, with longer half-lives compared to existing drugs. This allows for less frequent dosing and provides better adherence to treatment in patients with, or at risk of developing, PD and/or AD.

- 10 043417

Subjects may have PD or AD, or be at risk of developing these diseases in the presence or absence of one or more other non-neurological conditions. Non-neurological conditions include type 1 diabetes, type 2 diabetes, proliferative diseases such as cancer, autoimmune diseases and other local or systemic diseases such as inflammation and infection.

As described herein, the present invention includes an injectable, eg once-weekly or once-monthly, peptide-based drug with disease-modifying activity in PD and AD. Exenatide, a US Food and Drug Administration-approved peptide (BYETTA®) and glucagon-like peptide-1 receptor agonist (GLP-1r), has recently been studied in a number of patients with PD, with results demonstrating improvements in motor and cognitive function that indicates the potential of this drug in the treatment of PD (Aviles-Olmos I., et al., J. Clin. Invest., 2013. 123(6): p. 2730-6; Simuni T. and P. Brundin, J. Parkinsons Dis., 2014. 4(3):p.345-7). As detailed below, the rationale for the use of this glucagon-like peptide-1 (GLP-1r) agonist is based on experimental studies demonstrating GLP-1r agonist-mediated neuroprotective effects that promote function-beneficial neuroplasticity in animal models of neurodegeneration. Clinical results of exenatide demonstrated improvements in motor and cognitive function even one year after drug administration (Aviles-Olmos, I., et al., J. Clin. Invest., 2013. 123(6): p. 2730-6; Simuni, T . and P. Brundin, J. Parkinsons Dis., 2014. 4(3): p. 345-7). The ability of exenatide to treat PD is being investigated in a phase 2 clinical trial. However, exenatide has a short half-life (t1/2 in humans is 2 hours or less) and must be administered twice daily by subcutaneous injection, which is inconvenient and difficult for patients with PD , especially in the later stages. The compositions described herein provide similar pharmacological benefits to exenatide in PD with a reduced dosing frequency, for example, once monthly dosing.

Exenatide (exendin-4) is a peptide GLP-1r agonist that facilitates insulin release in type 2 diabetes and is currently marketed under the trade name BYETTA® for the treatment of type 2 diabetes (Meier J.J., Nat. Rev. Endocrinol ., 2012. 8(12): p. 728-42). This peptide controls insulin release in a glucose-dependent manner and is therefore safe for non-diabetic patients. Exenatide also slows down a number of neurodegenerative processes (Holscher C. J. Endocrinol., 2014. 221(1): p. T31-41). In preclinical models, exenatide crosses the blood-brain barrier (BBB), protects memory formation in AD or motor activity in PD, protects synapses and synaptic function, enhances neurogenesis, reduces apoptosis, protects neurons from oxidative stress, and reduces plaque formation and chronic inflammatory response. in the brain of mouse models of AD and PD. In a recent clinical trial, patients with moderately advanced PD who received exenatide for 12 months showed improvements in motor and cognitive function, and these effects continued for up to 12 months after treatment stopped (Aviles-Olmos I., et al., J Clin. Invest., 2013. 123(6): p. 2730-6; Simuni T. and P. Brundin, J. Parkinsons Dis., 2014. 4(3): p. 345-7).

Short-acting GLP-1r agonists (exenatide and liraglutide) exhibit neuroprotective effects in an acute animal model of toxin-induced PD and AD (Holscher S., J. Endocrinol., 2014. 221(1): p. T31-41). Of note, none of the GLP-1r agonists have anti-PD efficacy in clinically relevant genetic animal models of α-synuclein-related PD. In AD, short-acting GLP-1r agonists have demonstrated neuroprotective properties in toxin-induced AD models, but their anti-AD effects in AD genetic transgenic (Tg) mice are controversial. For example, liraglutide showed anti-PD efficacy in toxin-induced models, but failed to demonstrate similar effects in genetic AD Tg mouse models after long-term treatment (Hansen H.H. et al., PLoS One. 2016, 11(7):e0158205). Typically, chemical compounds with proven effectiveness only in toxin-induced models of neurodegenerative diseases often have not demonstrated such effectiveness in clinical trials. It was recently reported that liraglutide failed to change cognitive outcomes in AD patients after long-term treatment. Taken together, this means that an alternative GLP-1r agonist with high therapeutic efficacy in clinically relevant models associated with pathology in PD (α-synuclein PD phenotypes) or AD (amyloid and τ phenotypes) is needed to ensure successful clinical trials in patients . The compositions described herein provide potent anti-PD and anti-AD therapeutic efficacy of a long-acting GLP-1r agonist in α-synuclein-linked and 3xTg-AD models of PD, which are believed to represent closely related models of the neurodegenerative process of PD and AD , respectively.

Exenatide, like other peptide drugs, is by nature short-lived and unstable in the bloodstream and therefore requires frequent injections. Although PEGylation is the gold standard method for extending the half-life of protein drugs (Harris J.M. and R.B. Chess, Nat. Rev. Drug Discov., 2003. 2(3): p. 214-21), it is generally not used

- 11 043417 to small molecule peptide drugs, since conjugation with a large molecule PEG often reduces the biological activity of the peptide (for example, up to 1% of the biological activity compared to the native peptide). As detailed herein, a unique PEGylation technology has been developed to enhance potent and long-acting peptides, which increases the half-life of short-acting peptides in the bloodstream while maintaining the therapeutic activity of exenatide (patent publications WO 2013002580 and US 20130217622, incorporated herein by reference ). NLY001 is a long-acting form of exenatide using this PEGylation technology and is being investigated as a treatment for type 2 diabetes administered once weekly, twice monthly, or once monthly (Figure 1), with promising results compared to others GLP-1r agonists available on the market.

NLY001, as a long-acting PEGylated form of exenatide, has an extended half-life (88 hours in primates). The long-acting properties of NLY001 are designed using a unique half-life extension technology that still allows the formulation to remain specific for the same target and maintain the same mechanism of action as exenatide. In addition to facilitating insulin release in patients with type 2 diabetes by stimulating GLP-1r, the GLP-1r agonist peptide exenatide delays numerous neurodegenerative processes (Holscher S., J. Endocrinol., 2014. 221(1): p. T31- 41). This peptide controls insulin release in a glucose-dependent manner and is therefore safe for non-diabetic patients.

As a long-acting exenatide-based therapy, NLY001 provides an improved drug delivery route compared to exenatide while maintaining its pharmacological effects. For example, as detailed below, similar to exenatide, NLY001 improves motor and cognitive function in PD. Unlike exenatide therapy known prior to the invention described herein, NLY001 is administered to patients by injection once or twice a month, preventing multiple daily injections and improving treatment adherence.

Because NLY001 contains a high molecular weight polyethylene glycol (PEG, 50,000 Da) conjugated to a low molecular weight exenatide peptide (~4000 Da), the pharmacological efficacy similar to exenatide in PD and AD was completely unexpected due to the likely inability to cross the blood-brain barrier (BBB) (Pardridge W.M. , NeuroRx, 2005. 2(1):p.3-14). As detailed below, subcutaneously administered NLY001 was unexpectedly found to accumulate in the brains of animal models of PD and AD at significantly higher levels and exhibits clear beneficial effects in several recently established animal models of PD (see also Luk K. S. et al ., Science, 2012. 338(6109):p. 949-53) and animal models of AD. As detailed below, the results show that NLY001 administration protects against α-synuclein preformed fibrils (PFF)-induced dopaminergic neuron loss, reduces PFF-induced Lewy body pathology, inhibits PFF-induced reduction in terminal striatal dopamine receptor density, and restores PFF-induced behavioral dysfunction. , and also increases the life expectancy of Tg BP models. Importantly, NLY001 significantly blocked microglial activation and reduced the formation of reactive astrocytes in the brain. Taken together, the results detailed below clearly demonstrate that NLY001 has beneficial neuroprotective/disease-modifying effects against alpha-synuclein PFF-induced behavioral dysfunction. Similarly, in TG models of AD, treatment with NLY001 improved memory and reduced amyloid aggregation and τprotein formation, hallmarks of AD. Similar to PD studies, NLY001 demonstrated significant inhibition of microglial activation and reduction of reactive astrocyte populations in AD brain.

The anti-PD efficacy of long-acting GLP-1r agonists, such as GLP-1 peptide analogues carrying a high molecular weight carrier to increase half-life, was previously unknown. The results described here demonstrate the anti-PD and anti-AD effects of the long-acting GLP-1r agonist in a number of complemented animal models (preformed in mouse models of fibril-induced α-synucleinopathy, A53T α-synuclein Tg mouse models and 3xTg AD mouse models), and also clarify the mechanisms of its action. Long-acting exenatide is revolutionizing the treatment of PD and AD, combined with significantly improved patient compliance with once-weekly or monthly treatment regimens. The introduction of exenatide therapy with significantly less frequent injections is an effective treatment option for sick patients and their families.

The pathogenesis of PD is due to: 1) neuroinflammation and neurotoxicity caused by activated microglia and reactive astrocytes, 2) mitochondrial dysfunction, 3) synaptic dysfunction, and 4) lower levels of neurotrophic factor. Although the mechanisms of exenatide's neuroprotective effects are unclear, increasing evidence suggests that it regulates/delays some or all of these processes that contribute to the neurodegenerative process in PD. Therefore, how

- 12 043417 described in detail below, NLY001 provides beneficial neuroprotective effects in these pathways as well. As described below, the recently established preformed fibril (PFF)-induced α-synuclein mouse model of PD and the A53T Tg mouse model of PD have abnormal levels of reactive oxygen species (ROS), leading to increased oxidative stress and subsequently mitochondrial dysfunction, and recapitulating PD as a pathology Lewy bodies through the cell-to-cell signal transmission pathway. All of these processes contribute to the disease process in PD. As detailed here, since administration of NLY001 in complemented mouse models of PD protects against α-synuclein-related PD pathology, NLY001 participates in and manipulates these pathways.

The etiology of neurodegenerative diseases, including PD and AD, is now largely well defined. There is evidence of increased immune activation in neurodegenerative diseases. Most studies have focused on the role of microglia and astrocytes, resident innate immune cells of the brain, in the pathology of PD and AD (Sanchez-Guajardo V. et al., Neuroscience. 2015. 302:47-58, Perry V.H. et al., Nat. Rev Neurol 2014 10:217-224). In response to neurodegeneration and the accumulation of abnormally aggregated proteins such as α-synuclein and β-amyloid, quiescent microglia become activated and release various cytokines and neurotoxic molecules, including TNF-α, IL-1a, IL-1e, IL-6, and C1q. , which stimulate its proliferation and activate astrocytes (A1 astrocytes). Therefore, such inflammatory mediators released from activated microglia or reactive astrocytes induced by activated microglia cause neuronal damage and contribute to the progression of neurodegenerative diseases. Thus, activated microglia can be described as a major primary negative cause in neurodegenerative diseases. Inhibiting microglial activation without side toxicity is a logical strategy to prevent, arrest, and/or reverse the neurodegeneration process. However, prior to the invention described here, the lack of translational methods specifically targeting activated microglia hampered this strategy. For example, clinical trials of various anti-inflammatory drugs, including NSAIDs (de Jong D. et al., PLoS ONE. 2008. 23:e1475), rosiglitazone (Gold M. et al. Dement Geriatr Cogn Disord. 2010. 30:131-146) , statins (Feldman H.H. et al., Neurology. 2010. 74:956-964) and prednisone (Aisen P.S et al., Neurology. 2000. 54:588-593), failed to slow the progression of AD. Disappointing clinical trial results may be due to limited BBB passage and/or insufficient suppression of key proinflammatory and neurotoxic cytokines.

These studies describe a unique strategy to selectively interact with and block the activation of microglia and astrocytes and the release of inflammatory and neurotoxic molecules from activated resident innate immune cells; which prevents, stops and/or attenuates the progression of neurodegenerative diseases. Surprisingly, microglia activated by abnormally aggregated proteins were found to activate GLP-1r, and a long-acting GLP-1r agonist associated with activated microglia significantly inhibited the release of toxic molecules, including TNF-α, IL-1a, IL-1e, IL-1. 6 and C1q and protect neurons. Surprisingly, using the GLP-1r internalization assay, it was found that the long-acting GLP-1r agonist exhibits slow GLP-1r internalization, reduces the recycling rate of GLP-1r compared to the short-acting GLP-1r agonists (exenatide and liraglutide), and thus , can continuously activate GLP-1r and induce GLP-1r signaling in the brain. Patients treated with a short-acting GLP-1r agonist experienced a break in action, which reduces the therapeutic effect of chronic treatment. In contrast, NLY001 has the ability to cross the BBB and activate GLP-1r in the brain in a continuous manner without interruption of action and without side toxicity. This unique property of a long-acting GLP1r agonist is critical to maximizing the synergistic anti-inflammatory and neuroprotective properties of a GLP-lr agonist in neurodegenerative diseases.

Parkinson's disease.

Parkinson's disease (PD, also known as idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or shaking palsy) is a degenerative disorder of the central nervous system primarily affecting the motor system. The motor symptoms of Parkinson's disease are caused by the death of dopamine-producing cells in the substantia nigra of the midbrain. The causes of this cell death are poorly understood. At the very beginning of the disease, the most obvious symptoms are related to movement. These include tremors, stiffness, slowness of movement, and difficulty walking and gait. Problems with mental and behavioral functioning may occur later, along with dementia, which usually occurs in the later stages of the disease, and depression, the most common psychiatric symptom. Other symptoms include problems with touch, sleep and emotions. Parkinson's disease is more common in older adults, with most cases occurring after age 50; when this disease occurs in young people, it is called early-onset PD.

The general name for the main motor symptoms is parkinsonism or parkinsonism syndrome.

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This disease can be either primary or secondary. Primary Parkinson's disease is called idiopathic (without a known cause), although some atypical cases are genetic in origin, and secondary parkinsonism is due to known causes such as toxins. The pathology of the disease is characterized by accumulation of Lewy body protein in neurons and insufficient dopamine synthesis and activity in some parts of the midbrain. The areas of the brain where Lewy bodies are located are often associated with the severity and extent of an individual's symptoms. Diagnosis of typical cases is primarily based on symptoms, with tests such as neuroimaging used for confirmation.

Diagnosis of Parkinson's disease involves a doctor taking a medical history and performing a neurological examination. There are no laboratory tests that can clearly identify the disease, but brain scans are sometimes used to rule out disorders that may present with similar symptoms. Patients can be given levodopa, and relief of motor impairment usually confirms the diagnosis. The discovery of Lewy bodies in the midbrain at autopsy is usually considered evidence that the person had Parkinson's disease. The progression of the disease over time may indicate that it is not Parkinson's disease, and some experts recommend periodic testing of the diagnosis. Other causes that can secondary cause parkinsonism include Alzheimer's disease, multiple cerebral infarctions, and drug-induced parkinsonism. Parkinson plus syndromes such as progressive supranuclear palsy and multiple system atrophy should be excluded. Anti-Parkinson's disease medications are generally less effective at controlling symptoms in Parkinson's plus syndromes. A higher rate of progression, early cognitive dysfunction or postural instability, minimal tremor or symmetry at disease onset are more likely to indicate Parkinson's plus disease rather than PD itself. Genetic forms are usually classified as PD, although the terms familial Parkinson's disease and familial parkinsonism are used for disease entities with an autosomal dominant or recessive inheritance pattern.

The Parkinson's Disease Society Brain Bank criteria include slow movement (bradykinesia) as well as stiffness, resting tremor, or postural instability. Other possible causes of these symptoms must be excluded before diagnosing PD. Finally, the onset or development of PD requires the presence of three or more of the following: unilateral onset, tremor at rest, progression over time, asymmetry of motor symptoms, response to levodopa for at least five years, a clinical course lasting at least ten years and the appearance of dyskinesias caused by the consumption of excessive amounts of levodopa. The accuracy of diagnostic criteria assessed at autopsy is 75-90%, with specialists such as neurologists having the highest rates. Brain scans of people with PD using computed tomography (CT) and conventional magnetic resonance imaging (MRI) usually show normal brain function. However, these methods are useful in ruling out other diseases that may be secondary causes of parkinsonism, such as basal ganglia tumors, vascular pathology, and hydrocephalus. A special MRI technique, diffusion MRI, has been reported to be useful in distinguishing between typical and atypical parkinsonism, although its precise diagnostic value is still under investigation. Dopaminergic function in the basal ganglia can be measured using various radiopharmaceuticals in PET and SPECT. Examples are ioflupane ( ¹²³ I) (trade name DaTSCAN) and iomethopane (Dopascan) for SPECT or fluorodeoxyglucose ( ¹⁸ F) and DTBZ for PET. A pattern of decreased dopaminergic activity in the basal ganglia may aid in the diagnosis of PD.

Treatment, typically L-DOPA and dopamine agonists, relieves early symptoms of the disease. As the disease progresses and the loss of dopaminergic neurons continues, these drugs eventually become ineffective in treating symptoms while causing complications marked by involuntary squirming movements. Surgery and deep brain stimulation have been used to reduce motor symptoms as a last resort in severe cases when drugs are ineffective. Although dopamine replacement improves symptomatic motor dysfunction, its effectiveness decreases as the disease progresses, leading to unacceptable side effects such as severe motor fluctuations and dyskinesias. In addition, there is no therapy that stops the progression of the disease (Lang A.E. and A.M. Lozano, N. Engl. J. Med., 1998. 339(15): p. 1044-53; Lang A.E. and A.M. Lozano, N. Engl. J. Med., 1998. 339(16): p. 1130-43). Moreover, this palliative therapeutic approach does not address the underlying mechanisms of the disease (Nagatsua T. and M. Sawadab, Parkinsonism Relat Disord, 2009. 15 Suppl 1: p. S3-8).

The term parkinsonism is used for a motor syndrome whose main symptoms are resting tremor, stiffness, slowness of movement and postural instability. Parkinsonism syndromes can be divided into four subtypes depending on their origin: primary or idiopathic, secondary or acquired, hereditary parkinsonism and

- 14 043417 Parkinson plus syndromes or multiple systemic degeneration. Typically classified as a movement disorder, PD also has several non-motor symptoms, such as sensory deficits, cognitive impairment, or sleep problems. Parkinson's plus diseases are primary parkinsonisms that have additional features. These include multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration and dementia with Lewy bodies.

In terms of pathophysiology, PD is considered a synucleopathy due to the abnormal accumulation of alpha-synuclein protein in the brain in the form of Lewy bodies, unlike other diseases such as Alzheimer's disease where τ protein accumulates in the brain in the form of neurofibrillary tangles. However, there is clinical and pathological overlap between tauopathies and synucleopathies. The most typical symptom of Alzheimer's disease, dementia, occurs in the later stages of PD, while neurofibrillary tangles are common in the brains of patients with PD. Dementia with Lewy bodies (DLB) is another synucleopathy that shares similarities with PD, and especially with the subgroup of cases of PD with dementia. However, the relationship between PD and DLB is complex and needs to be clarified. They may be parts of the same disease, or they may be separate diseases.

Mutations in certain genes have been shown to cause PD. These genes encode α-synuclein (SNCA), parkin (PRKN), leucine repeat-rich kinase 2 (LRRK2 or dardarin), PTEN-induced putative kinase 1 (PINK1), DJ-1, and ATP13A2. In most cases, people with these mutations will develop PD. However, with the exception of LRRK2, they represent only a small number of PD cases. The most widely studied PD-related genes are SNCA and LRRK2. Mutations in genes including SNCA, LRRK2, and glucocerebrosidase (GBA) have been found to be risk factors for sporadic PD. Mutations in GBA are known to cause Gaucher disease. Comprehensive genomic studies that look for mutant alleles with low penetrance in sporadic cases have now yielded many positive results.

The role of the SNCA gene is important in PD because α-synuclein protein is a major component of Lewy bodies. Histopathology (microscopic anatomy) of the substantia nigra and several other brain regions shows neuronal loss and the presence of Lewy bodies in many of the remaining nerve cells. Neuronal loss is accompanied by the death of astrocytes (star-shaped glial cells) and activation of microglia (another type of glial cell). Lewy bodies are a key pathological feature of PD.

Alzheimer's disease.

Alzheimer's disease (AD) accounts for 60 to 70% of dementia cases. It is a chronic neurodegenerative disease that often begins slowly but gradually worsens over time. The most common early symptom is short-term memory loss. As the disease progresses, symptoms include speech problems, mood swings, loss of motivation, confusion, behavioral problems and poor self-care management. Gradually, body functions are lost, eventually leading to death. Although the rate of progression may vary, the average life expectancy after diagnosis is three to nine years. The cause of Alzheimer's disease is poorly understood. About 70% of the risk is believed to be genetic, involving many genes. Other risk factors include a history of head injury, hypertension, or depression. The disease process is associated with plaques and tangles in the brain.

Alzheimer's disease is characterized by the loss of neurons and synapses in the cerebral cortex and some subcortical areas. This loss results in widespread atrophy of the affected areas, including degeneration in the temporal lobe and parietal lobe, as well as parts of the frontal cortex and cingulate cortex. It has been hypothesized that Alzheimer's disease is a protein-related (proteopathy) disease caused by the accumulation of abnormally folded Ab and τ proteins in the brain. The plaques are made up of small peptides 39-43 amino acids long called amyloid beta (also spelled A-beta or Av). β-amyloid is a fragment of a larger protein called amyloid precursor protein (APP), a transmembrane protein that crosses the neuronal membrane. BPA is critical for neuronal growth, survival, and recovery from injury. In Alzheimer's disease, an unknown process causes BPA to be broken down into smaller fragments by enzymes through proteolysis. One of these fragments results in the formation of beta-amyloid fibrils, which form clumps that are deposited on the outside of the neuron in dense formations known as senile plaques.

The probable diagnosis is based on medical history and cognitive tests with medical imaging and blood tests to rule out other possible causes. Initial symptoms are often mistaken for normal aging. Studying brain tissue is necessary for an accurate diagnosis. Alzheimer's disease is diagnosed through a complete medical evaluation. There is more than one clinical test that can determine whether a person has Alzheimer's disease. Several tests are usually done to rule out any other cause of dementia. The only one

- 15 043417 The definitive diagnostic method is the examination of brain tissue obtained by biopsy or autopsy. Tests (such as blood tests and brain imaging) are used to rule out other causes of symptoms associated with dementia. Laboratory tests and screening include: complete blood count; electrolyte panel; metabolic panel screening; thyroid function tests; Vitamin B-12 folate levels; tests for syphilis and, depending on history, for antibodies to human immunodeficiency; Analysis of urine; electrocardiogram (ECG); chest x-ray; computed tomography (CT); and electroencephalogram (EEG). Lumbar puncture can also be informative for the general diagnosis.

There are no medications or supplements that reduce the risk. No treatments stop or reverse the progression of this disease, although some may temporarily relieve symptoms.

GLP-1 agonists (eg Exenatide).

Exenatide (marketed as BYETTA®, bidureon) is a glucagon-like peptide-1 agonist (GLP-1 agonist) drug belonging to the group of incretin mimetics approved in April 2005 for the treatment of type 2 diabetes mellitus. Exenatide in its BYETTA® form is administered by subcutaneous injection (under the skin) into the abdomen, thighs, or arm any time within 60 minutes before the first and last meals of the day. The once-weekly injection was approved on January 27, 2012 under the brand name bidureon. This drug is manufactured by Amylin Pharmaceuticals and was commercialized by Astrazeneca.

Exenatide is a synthetic version of exendin, a hormone found in the saliva of the Arizona dragonfly. It has biological properties similar to human glucagon-like peptide-1 (GLP-1), a regulator of glucose metabolism and insulin secretion. According to the instructions for use, exenatide enhances glucose-dependent insulin secretion from pancreatic β-cells, suppresses inappropriately increased glucagon secretion and slows gastric emptying, although its mechanism of action is still being studied.

Exenatide is a 39-amino acid peptide insulin secretagogue with glucoregulatory effects. The peptide sequence of exenatide is as follows:

H(His)G(Gly)E(Glu)G(Gly)T(Thr)F(Phe)T(Thr)

S(Ser)D(Asp)L(Leu)S(Ser)К(Lys)Q(Gin)M(Met)E(Glu)E(Glu)E(Glu)A(Al

a) V (Vai) R (Arg) L (Leu) F (Phe) I (He) E (Glu) W (Trp) L (Leu) K (Lys) N (Asn) G ( Gly)G(Gly) P(Pro)S(Ser)S(Ser)G(Gly)A(Ala)P(Pro)P(Pro)P(Pro)S(Ser) (SEQ ID NO: 1). Exenatide was approved by the US Food and Drug Administration on April 28, 2005 for patients whose diabetes is not well controlled by other oral medications. This drug is administered subcutaneously twice daily using a prefilled pen syringe.

The incretin hormones GLP-1 and glucose-dependent insulinotropic peptide (GIP) are produced by L and K endocrine cells of the intestine after food intake. GLP-1 and GIP stimulate insulin secretion from the beta cells of the islets of Langerhans in the pancreas. Only GLP-1 induces insulin secretion in the diabetic state; however, GLP-1 by itself is not effective as a drug for the clinical treatment of diabetes because it has a very short half-life in vivo. Exenatide has 50% amino acid sequence homology with GLP-1 and has a longer half-life in vivo. Thus, it has been tested for its ability to stimulate insulin secretion and lower blood glucose levels in mammals and has been found to be effective in diabetic conditions. It has also been shown in rodent studies to increase the number of beta cells in the pancreas.

Commercially, exenatide is produced by direct chemical synthesis. Historically, exenatide was discovered as exendin-4, a protein naturally secreted in saliva and concentrated in the tail of the Arizona quillfish. Exendin-4 has extensive homology and similar function to mammalian GLP-1, but has the therapeutic advantage of being resistant to DPP-4 degradation (which degrades GLP-1 in mammals), allowing for an extended pharmacological half-life. The biochemical characteristics of exendin-4 led to the consideration and proposal of exenatide as a strategy for the treatment of diabetes mellitus. Subsequent clinical trials led to the discovery that exenatide also had desirable glucagon and appetite suppressant effects.

In its twice-daily dosage form, BYETTA®, exenatide rapidly increases insulin levels (within approximately 10 minutes after administration), with insulin levels decreasing significantly over the next one to two hours. A dose taken after a meal has much less effect on blood sugar levels than before a meal. The effect on blood sugar decreases after 6-8 hours. In the form of BYETTA®, this medicine is available in two doses: 5 and 10 mcg. Treatment often begins with a dose of 5 mcg, which is increased if side effects are not significant. In its once-weekly form, bidureon works

- 16 043417 effect does not depend on the time between injections and meals. Bidureon has the advantage of providing 24-hour blood sugar lowering action, while BYETTA® has the advantage of providing better control of the rise in blood sugar that occurs immediately after a meal. According to the US Food and Drug Administration for bidureon, bidureon reduces blood sugar in the form of glycosylated hemoglobin by an average of 1.6%, while BYETTA® reduces it by an average of 0.9 %. Both BYETTA® and bidureon have similar beneficial effects on weight loss. According to the US Food and Drug Administration for bidureon, levels of nausea are lower in patients taking bidureon compared to patients taking BYETTA®.

In some embodiments, the present invention is based on the use of other (non-exatide) types of long-acting GLP-1 agonists for the treatment of PD and AD. In some cases, the long-acting GLP-1 agonist includes an Fc-fusion of GLP-1 (eg, dulaglutide, eppeglenatide) or a derivative thereof. In some cases, the long-acting GLP-1 agonist includes an albumin-fused GLP-1 (eg, albiglutide) or a derivative thereof. An example of a GLP-1 Fc-fusion composition used for the treatment of PD or AD is dulaglutide. Dulaglutide is a glucagon-like peptide-1 receptor agonist (GLP-1 agonist) for the treatment of type 2 diabetes that can be administered once weekly. Dulaglutide consists of GLP-1 (7-37) covalently linked to the Fc fragment of human IgG4, thereby protecting the GLP-1 moiety from inactivation by dipeptidyl peptidase 4. GLP-1 is a hormone that is involved in normalizing blood glucose levels (glycemia ). GLP-1 is normally secreted by L cells in the gastrointestinal mucosa in response to food intake. Dulaglutide binds to glucagon-like peptide-1 receptors, slowing gastric emptying and increasing insulin secretion by pancreatic β-cells. At the same time, this chemical compound reduces the increased secretion of glucagon by inhibiting pancreatic α-cells, which is known to be undesirable for a diabetic patient. An example of a GLP-1 albumin fusion composition used for the treatment of PD or AD is albiglutide. Albiglutide is a glucagon-like peptide-1 agonist (GLP-1 agonist) drug used to treat type 2 diabetes. It is a dimer of dipeptidyl peptidase-4-resistant glucagon-like peptide-1 fused to human albumin. Albiglutide has a half-life of four to seven days.

Polyethylene glycol (PEG).

Polyethylene glycol (PEG) is a polyether chemical compound with many uses from industrial production to medicine. The structure of PEG (note the repeat element in parentheses): H-(O-CH ₂ -CH ₂ ) _p -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. The abbreviations PEG, PEO or POE refer to an oligomer or polymer of ethylene oxide. The three names are chemically synonymous, but historically PEG is preferred in the biomedical field, while PEO is more common in the field of polymer chemistry. Because different applications require different lengths of polymer chains, the term PEG generally refers to oligomers and polymers with a molecular weight less than 20,000 g/mol, PEO to polymers with a molecular weight above 20,000 g/mol, and POE to a polymer of any molecular weight, PEG and PEO are liquids or fusible solids depending on their molecular weight. PEG is produced by polymerization of ethylene oxide and is commercially available in a wide range of molecular weights from 300 g/mol to 1,000,000 g/mol. Although PEG and PEO with different molecular weights are used for different purposes and have different physical properties (eg, viscosity), due to the influence of chain length, their chemical properties are almost identical. Various forms of PEG are also available depending on the initiator used for the polymerization process. The most common initiator is monofunctional PEG methyl ester or methoxypoly(ethylene glycol), abbreviated mPEG. Lower molecular weight PEG is also available as purer oligomers called monodisperse, homogeneous, or discrete. Recently, high purity PEG has been shown to be crystalline, allowing the crystal structure to be determined by X-ray diffraction. Because purification and separation of pure oligomers is difficult, the price for this type of quality is often 10-1000 times higher than polydisperse PEG.

PEGs are also available in different geometries. Branched PEGs have three to ten PEG chains emanating from a central backbone. Star-shaped PEGs have 10 to 100 PEG chains emanating from a central backbone. Comb PEGs have multiple PEG chains typically grafted onto a polymer backbone. The numbers that are often included in the names of a PEG indicate its average molecular weight (for example, a PEG with n=9 would have an average molecular weight of approximately 400 daltons and would be designated PEG 400. Most PEGs include molecules with a molecular weight distribution (i.e. they are polydisperse.) The size distribution can be characterized statistically by its weight-average molecular weight (Mw) and its average number

- 17 043417 molecular weight (Mn), the ratio of which is called the polydispersity index (Mw/Mn). Mw and Mn can be measured using mass spectrometry.

PEGylation is the formation of a covalent bond of a PEG structure with another larger molecule, such as a therapeutic protein, which is then called PEGylated protein. PEGylated interferon a2a or -2b is commonly used for injection to treat hepatitis C infection. PEG is soluble in water, methanol, ethanol, acetonitrile, benzene, and dichloromethane and insoluble in diethyl ether and hexane. It is combined with hydrophobic molecules to produce nonionic surfactants. PEGs contain potential toxic impurities such as ethylene oxide and 1,4-dioxane. Ethylene glycol and its esters are nephrotoxic when applied to damaged skin.

Polyethylene glycol (PEG) and related polymers (PEG phospholipid constructs) are often sonicated for biomedical applications. However, PEG is very sensitive to sonolytic degradation, and PEG degradation products can be toxic to mammalian cells. Therefore, it is necessary to evaluate the potential degradation of PEG to ensure that the final material does not contain unknown contaminants that could introduce artifacts into the experimental results.

PEG and methoxy polyethylene glycols vary in consistency from liquid to solid depending on molecular weight, as indicated by the number next to the name. They are used commercially for many purposes, including as surfactants, in foods, cosmetics, pharmaceuticals, biomedicine, as dispersing agents, as solvents, in ointments, in suppositories, as excipients in tablets and in as laxatives. Some specific groups are laromacrogols, neoxynols, octoxins and poloxamers.

Polyethylene glycol is produced by reacting ethylene oxide with water, ethylene glycol or ethylene glycol oligomers. This reaction is catalyzed by acid or base catalysts. Ethylene glycol and its oligomers are preferred as starting materials instead of water because they allow the creation of polymers with low polydispersity (narrow molecular weight distribution). The length of the polymer chain depends on the ratio of reagents.

HOCH2CH2OH+p(CH2CH2O)^HO(CH2CH2O)p+ 1H

Depending on the type of catalyst, the polymerization mechanism can be cationic or anionic. Polymerization of ethylene oxide is an exothermic process.

Polyethylene oxide or high molecular weight polyethylene glycol is synthesized by suspension polymerization. During the polycondensation process, it is necessary to keep the growing polymer chain in solution. The reaction is catalyzed by organoelement compounds of magnesium, aluminum or calcium. To prevent coagulation of polymer chains from solution, chelating agents such as dimethylglyoxime are used. To produce low molecular weight polyethylene glycol, alkaline catalysts such as sodium hydroxide (NaOH), potassium hydroxide (KOH) or sodium carbonate (Na2CO ₃ ) are used.

PEG is used as an excipient in many pharmaceutical products. Low molecular weight variants are used as solvents in oral liquids and soft capsules, while solid variants are used as ointment bases, tablet binders, film coatings and lubricants. PEG is also used to lubricate eye drops.

Polyethylene glycol has low toxicity and is used in a variety of products. This polymer is used as a lubricating coating for a variety of surfaces in aqueous and non-aqueous environments. Since PEG is a flexible, water-soluble polymer, it can be used to create very high osmotic pressures (on the order of tens of atmospheres). It is also less likely to interact specifically with biological chemicals. These properties make PEG one of the most useful molecules for generating osmotic pressure in biochemistry and biomembrane experiments, particularly when using the osmotic stress method.

PEGylation (also often written as PEGylation) is the process of either covalently or non-covalently attaching or fusing polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as a drug, therapeutic protein or vesicle, which are then referred to as PEGylated (PEGylated) . PEGylation is typically achieved by incubating a reactive PEG derivative with the target molecule. Covalent attachment of a PEG to a drug or therapeutic protein can mask the agent from the host immune system (reduced immunogenicity and antigenicity) and increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulation time in the bloodstream, reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

PEGylation is the process of attaching polymer chains of PEG to molecules,

- 18 043417 most often to peptides, proteins and antibody fragments, which can improve the safety and effectiveness of many therapeutic agents. It produces changes in physicochemical properties, including changes in conformation, electrostatic binding, hydrophobicity, etc. These physical and chemical changes increase systemic retention of the therapeutic agent. In addition, it may influence the binding affinity of the therapeutic moiety to cellular receptors and may alter absorption and distribution patterns.

PEG is a particularly attractive polymer for conjugation. Specific characteristics of PEG molecules relevant to pharmaceutical applications are water solubility, high mobility in solution, lack of toxicity and low immunogenicity, easy excretion from the body and altered distribution in the body.

PEGylation process.

The first step of PEGylation is the proper functionalization of the PEG polymer at one or both ends of the molecule. PEGs that are activated at each end by the same reactive molecule are called homobifunctional, while if the functional groups present are different, then the PEG derivative is called heterobifunctional or heterofunctional. Reactive or activated derivatives of the PEG polymer are ready for the PEG to be attached to the desired molecule.

General PEGylation processes for protein conjugation can be roughly classified into two types, liquid-phase batch process and fed-column batch process. A simple and common batch process involves mixing the reagents together in a suitable buffer solution, preferably at a temperature of 4 to 6°C, followed by separation and purification of the desired product using a suitable method based on its physicochemical properties, including size exclusion chromatography (SEC), ion exchange chromatography (IEC), hydrophobic interaction chromatography (HC) and membranes or aqueous two-phase systems.

The selection of an appropriate functional group for a PEG derivative is based on the type of available reactive group on the molecule that will bind to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and C-terminal carboxyl group can also be used as a site-specific site by conjugation with aldehyde functional polymers. Methods used to prepare first-generation PEG derivatives typically involve reacting the PEG polymer with a group that reacts with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. In the second generation of PEGylation chemistry, more effective functional groups such as aldehydes, esters, amides, etc. became available for conjugation.

As the application of PEGylation becomes more advanced and sophisticated, the need for heterobifunctional PEGs for conjugation increases. These heterobifunctional PEGs are widely used for coupling two molecules where a hydrophilic, flexible and biocompatible spacer is required. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and Nhydroxysuccinimide esters. Third generation PEGylating agents, where the polymer is branched, Y-shaped or comb-shaped, have demonstrated reduced viscosity and no accumulation in organs. The unpredictability of the clearance time of PEGylated compounds may lead to the accumulation of high molecular weight chemicals in the liver, resulting in the formation of inclusion bodies with unknown toxicological consequences. In addition, changes in chain length may result in unexpected clearance times in vivo.

PEGylation of GLP-1r Agonist (NLY001, PB-119, LY2428757) As described below, the yield of exendin-4 analog (eg, exenatide) or GLP-1 analog can be increased by selective PEGylation, and the therapeutic effect of drugs can be enhanced. This technology increases molecular weight, protects the metabolic site, and inhibits the immunogenicity site, increasing half-life and in vivo stability and reducing immunogenicity. In addition, renal excretion of PEG-bound peptides and proteins is reduced due to the increase in molecular weights of the peptides and proteins by PEG, so PEGylation has the benefit of increasing both pharmacokinetic and pharmacodynamic effects.

Moreover, the polyethylene glycol or a derivative thereof according to the present invention is a linear or branched type of molecule, and in the case of a branched type, a dimeric or trimeric type may be used, and more preferably, a trimeric type may be used. Specifically, the polyethylene glycol derivative is, for example, methoxy polyethylene glycol succinimidyl propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, or several branched types of these derivatives. Preferably, the polyethylene glycol derivative is linear methoxy polyethylene glycol maleimide, branched methoxy polyethylene glycol maleimide or trimeric methoxy polyethylene glycol maleimide, and is more preferably three

- 19 043417 dimensional methoxypolyethylene glycol maleimide.

As described herein, once an exendin-4 analog (eg, exenatide) has been prepared and PEGylated with polyethylene glycol or a derivative thereof, the molecular structure of the analog can be confirmed by mass spectroscopy, liquid chromatography, X-ray diffraction analysis, polarimetry, and by comparison of calculated values and measured values of representative elements constituting PEGylated exenatide.

When the composition of the present invention is used as a medicine, a pharmaceutical composition containing an exendin-4 analogue (for example, exenatide) PEGylated with polyethylene glycol or a derivative thereof can be administered after being formulated in various forms for oral or non-oral administration, as in the clinical application below, but not limited to.

For example, compositions for oral administration exist in the form of tablets, granules, hard/soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, lozenges, etc., and these compositions include, in addition to the active ingredient, diluents ( for example: lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), slip modifiers (for example, silicon dioxide, talc, stearate and its magnesium or calcium salt and/or polyethylene glycol). Tablets may also include binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and may include disintegrants such as starch, agar, alginic acid or sodium salt thereof or boiling mixture and/or absorbents, dyes, flavors and sweeteners, if necessary.

A pharmaceutical composition containing an exendin-4 analog PEGylated with polyethylene glycol or a derivative thereof can be administered non-orally, and this administration is carried out by subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or local administration.

An exendin-4 analog (eg, exenatide) PEGylated with polyethylene glycol or a derivative thereof can be prepared as a liquid or suspension by mixing it with a stabilizer or buffer in water to form a non-oral composition and can be packaged in an ampoule or bottle for administration. The composition is sterilized and/or adjuvants such as antiseptics, stabilizers, hydrators or emulsifying stimulants, salts and/or buffers that regulate osmotic pressure, and other substances useful for treatment, and this composition can be prepared using conventional methods mixing, granulating or coating.

The human dose of a pharmaceutical composition containing an exendin-4 analogue (eg, exenatide) PEGylated with polyethylene glycol or a derivative thereof according to the present invention may vary depending on the age, body weight, gender, route of administration, health status and disease level of the patients , and may be administered orally or non-orally at the discretion of the physician or pharmacist, with a preferred dose of 0.01 to 200 mg/kg per day.

Blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a highly selective permeability barrier that separates circulating blood from brain extracellular fluid (ECF) in the central nervous system (CNS). The blood-brain barrier is formed by endothelial cells of the brain, which are connected by tight junctions with extremely high electrical resistance. Astrocytes are essential for creating the blood-brain barrier. The blood-brain barrier allows the passage of lipid-soluble molecules, water, and some gases by passive diffusion, as well as the selective transport of molecules such as amino acids and glucose that are critical for neuronal function. The blood-brain barrier is found in all capillaries in the brain and consists of tight junctions around the capillaries that are not found in the normal bloodstream. Endothelial cells limit the diffusion of microscopic objects (eg, bacteria) and large or hydrophilic molecules into the cerebrospinal fluid (CSF), while allowing the diffusion of small hydrophobic molecules (eg, O ₂ , CO ₂ , hormones). Barrier cells actively transport metabolic products such as glucose across the barrier with specific proteins.

This barrier results from selectivity of tight junctions between endothelial cells in the vessels of the central nervous system, which restrict the passage of solutes. At the interface between the blood and the brain, endothelial cells are held together by these tight junctions, which are composed of small subunits, often biochemical dimers, that are transmembrane proteins such as occludin, claudins, junctional adhesion molecule (JAM), or ESAM. Each of these transmembrane proteins is anchored in endothelial cells by another protein complex that includes zo-1 and associated proteins.

The blood-brain barrier is formed by the capillary endothelium of the brain and excludes ~100% of high-molecular-weight neurotherapeutic drugs and more than 98% of all small-molecule drugs from the brain. Overcoming the challenge of delivering therapeutic agents to specific areas

- 20 043417 brain represents a major challenge for the treatment of most brain disorders. In its neuroprotective role, the blood-brain barrier acts to prevent the delivery of many potentially important diagnostic and therapeutic agents to the brain. Therapeutic molecules and antibodies that might otherwise be effective in diagnosis and therapy did not cross the BBB in sufficient quantities. Mechanisms for targeted drug delivery to the brain include either across or beyond the BBB. Methods of delivering drugs/dosage forms across the BBB involve its disruption by osmotic means; biochemically through the use of vasoactive substances such as bradykinin; or even localized high-intensity focused ultrasound (HIFU). Other methods used to cross the BBB may involve the use of endogenous transport systems, including carrier-mediated transporters such as glucose and amino acid carriers; receptor-mediated transcytosis for insulin or transferrin; and blocking active efflux transporters such as p-glycoprotein. However, vectors targeting BBB transporters such as the transferrin receptor have been found to remain captured by capillary brain endothelial cells rather than being transported across the BBB into the brain parenchyma. Methods for delivering drugs beyond the BBB include intracerebral implantation (eg, using needles) and extended distribution with convection. In addition, mannitol can be used to bypass the BBB.

It is well known that peptide or protein biologic drugs that do not have the ability to interact with blood-brain barrier receptors such as the transferrin receptor do not cross the BBB. Due to the high molecular weight of the PEGylated exenatide analog or GLP-1 analog and the lack of ligands that interact with blood-brain barrier receptors, the compositions described herein have surprisingly been found to cross the blood-brain barrier and treat PD and AD.

The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the drawings, are incorporated herein by reference.

Examples

Example 1: Activated microglia activate GLP-1r, and GLP-1r agonists selectively block microglial activation and inhibit the release of several neurotoxic molecules.

Activated microglia are one of the initiators of neurodegenerative diseases. Activation of microglia leads to increased production of pro-inflammatory and neurotoxic mediators such as TNF-α, IL-1α, IL-1e, IL-6 and C1q. As a result, these inflammatory mediators directly or through astrocyte activation induce neuroinflammation and neuronal damage. For example, TNF-α is expressed at very low levels by various brain cells, including neurons, but when microglia and astrocytes are activated by pathogens or damaged, they express and release high levels of TNF-α. TNF-α produced by activated microglia has been reported to be necessary and sufficient to initiate apoptosis in neuronal cells (Guadagno J. et al., Cell Death and Disease. 2013. 4:e538), and there is evidence that TNF-α contributes contribution to various brain pathologies such as PD, AD, multiple sclerosis and other neurodegenerative diseases. Prior to the invention described herein, there were no clinically proven, reliable methods to selectively interact with and influence microglial and astrocyte activation in humans.

Results.

NLY001, a long-acting GLP-1r agonist, was found to target activated microglia transformed from quiescent microglia and simultaneously inhibit multiple inflammatory and neurotoxic mediators in neurodegenerative diseases. When primary microglia were activated by abnormally aggregated proteins, such as α-synuclein preformed fibrils (PFF), the level of GLP-1r mRNA expression was increased in activated microglia (Fig. 2A, Fig. 2B, and Fig. 2C). Brain tissues from PD and PD patients exhibit increased GLP-1r activity compared to healthy brain tissues (Figure 2A, Figure 2B, and Figure 2C). Importantly, when microglia were treated with α-synuclein PFF (1 μg/ml) and NLY001 (1 μM) for 6 h, NLY001 blocked microglial activation and significantly reduced the release of several inflammatory mediators, including TNF-α, ΓΕ-1α, IL -1e and IL-6. In in vivo experiments, it was found that subcutaneous administration of NLY001 simultaneously inhibits the levels of TNF-α, IL-1α, IL-III, IL-6 and C1q (Table 1). This result indicates that a long-acting GLP-1r agonist is able to selectively interact with activated microglia through activated GLP-1r and simultaneously suppress the release of multiple inflammatory and toxic mediators that may cause neuronal damage in neurodegenerative diseases.

- 21 043417

Table 1. mRNA levels (relative fold) of TNF-α, IL-1α, IL-Ιβ, and IL-6 in normal and α-synuclein PFF activated primary microglia from mice treated or not treated with NLY001. ±SD, n=3 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni post hoc test for multiple group comparisons.

mRNA FSB α-synuclein PFFs FSB NLY001 (1 µM) FSB NLY001 (1 µM) TNF-a 1+0.07 1.9+0.27 113.2+18.9*** 11.3+3, 15 ^### IL-6 1+0.02 1.6+0.04 70.9+0.75*** 12.3+2.38 ^### IL-Ιβ 1+0.02 1.6+0.02 506.5+13.58** 57+1.58 ^### IL-la 1+0.02 1.5+0.02 276.8+4*** 142.2+4.1 « ^#

***P<0.001 compared to the control group (FSB), P<0.001 compared to the PFF+FSB group.

Example 2: NLY001 protects neurons from microglia-mediated neuronal death.

Materials and methods.

To determine the effect of NLY001 on α-synuclein PFF-induced microglia-induced neurotoxicity, NLY001 was tested to test whether it protects primary neurons from α-synuclein PFF-activated microglia-mediated neuronal death. To do this, microglial cells were activated with wasp-synuclein PFF (1 μg/ml) with or without NLY001 (1 μM) for 6 h, and then washed from the culture medium. Primary neurons were then co-cultured with activated microglia for 72 h as described in Fig. 3. Neuronal toxicity was assessed by propidium iodide staining.

Results.

As shown in table. 2, neurons cocultured with microglia activated by α-synuclein PFFs had increased rates of cell death. In contrast, when neurons were co-cultured with microglial cells treated with PFF and NLY001, they experienced significantly reduced levels of neuronal death. These results suggest that NLY001 may protect neuronal cells from transformed microglial cells or astrocytes activated by abnormally aggregated proteins during the progression of neurodegenerative diseases.

Table 2. Analysis of neuronal cell death during co-culture of primary microglia and primary cortical neurons. ±SD, n=4 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni's post hoc test for multiple __________________________group comparison____________________

mRNA At rest microglia+neurons PFF-activated microglia + neurons FSB NLY001 (1 µM) FSB NLY001 (1 µM) Death cells (%) 8.7+0.8 8.6+1.6 46.1+9.6*** 13, 8+4, 8 ^###

###^ ***P<0.001 compared to the control group (FSB), P<0.001 compared to the PFF+FSB group.

Example 3 Long-acting GLP-1r agonist NLY001 constitutively activates GLP-1r through delayed GLP-1r internalization compared to short-acting GLP-1r agonists.

The effectiveness of short-acting GLP-1r agonists in clinically relevant, transgenic mouse models of neurodegenerative diseases is unclear. For example, liraglutide (after daily injection) had no effect on β-amyloid plaque burden in 2xTd AD models after long-term treatment (Hansen H.H. et al., PLoS One. 2016. 11(7):e0158205). In PD, no GLP-1r agonists (short-acting or long-acting) have demonstrated efficacy in clinically relevant Td or mutant models of PD. In general, short-acting GLP-1r agonists have proven effective in acute, toxin-induced models of PD and AD, but not in chronic, transgenic models. A short-acting GLP-1r agonist may exhibit reduced or absent

- 22 043417 current effectiveness in chronic models of PD or AD. Historically, these compounds have only been shown to be effective in toxin-induced neurodegenerative models and have not been shown to be effective in most clinical trials. In contrast, as described in the Examples, NLY001 clearly demonstrated strong anti-PD and anti-AD efficacy in multiple and complemented chronic models of PD and AD.

Unlike the short-acting GLP-1r agonists, NLY001 is a long-acting GLP-1r agonist and surprisingly crosses the BBB and exhibits high accumulation in the brain in neurodegenerative disease (Table 7). It has been suggested that short-acting GLP-1r agonists are not effective in chronic Tg models of neurodegenerative disease due to their lack of ability to continuously activate GLP-1r in the brain. In addition to the increased plasma half-life of NLY001, which can continuously deliver active GLP-1 ligand to the brain, at the molecular level, NLY001 was found to significantly slow down the internalization of GLP-1r, allowing for increased release of GLP-1 compared to short-acting agonists. GLP-1r (Table 3). Thus, combined with its long plasma half-life and ability to cross the BBB, NLY001 can continuously activate GLP1r and induce GLP-1 signaling in target cells in the brain without interruption of action compared to short-acting GLP-1r agonists .

The internalization properties of GLP-1r short-acting and long-acting GLP1r agonists were examined using the PathHunter express GLP1RA activated GPCR internalization assay reagent kit (DisoverRX, CA). Briefly, this set of reagents detects the interaction by inhibiting the activated receptor through enzyme and fragment complementation, as described in the instructions for use. Internalization assay cells using the activated GPCR PathHunter express were plated in a 96-well plate (105 cells per well) and stimulated with two short-acting GLP-1r agonists (exenatide and liraglutide) and a long-acting GLP-1r agonist, NLY001, at a concentration of 10 ^-12 -10 ^-6 M. After stimulation, signal detection was carried out in accordance with the protocols recommended by the manufacturer. As shown in table. 3, NLY001 demonstrated a 10- to 20-fold delay in GLP-1r internalization in EC ₅₀ units for agonist stimulation (nM) compared to the short-acting GLP1r agonists exenatide and liraglutide.

Table 3. Internalization of human GLP-1r by short-acting and long-acting GLP-1r agonists. ±SD, n=4 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni post hoc test for multiple group comparisons.

Short-acting GLP-lr agonists Long acting GLP-lr agonist GLP-lr agonist exenatide liraglutide NLY001 EU50 for stimulation agonist 6.5+2.8 3.9+2.9 60+2, 6 « ^#

^### P < 0.001 compared with short-acting GLP-1r agonists.

Example 4: NLY001 reduces α-synuclein-associated gliosis (microglia and astrocyte activation), inhibits α-synuclein aggregation and ameliorates Lewy body/Lewy neuritis pathology, and reduces motor cell deficits in mice with α-synuclein PFF-induced PD.

Materials and methods.

Animals.

All experimental procedures were followed in accordance with the guidelines of the National Institutes of Health Laboratory Animal Husbandry Guide for the Care and Use of Animals, which were approved by the Johns Hopkins Institutional Animal Care and Use Committee. Mice with α-synuclein PFF-induced PD were generated (Luk, K.S., et al., Science, 2012. 338(6109):p. 949-53). For stereotactic injection of α-synuclein PFFs, 12-week-old male mice were anesthetized with xylazene and ketamine. An injection cannula (26.5 gauge) was inserted stereotactically into the striatum (anterior to posterior, 3.0 mm from bregma, mediolateral, 0.2 mm, dorsoventral, 2.6 mm) unilaterally (right hemisphere). The infusion was performed at a rate of 0.2 μl per minute, and 2 μl of α-synuclein PFFs (5 μg/ml in PBS) or the same volume of PBS was injected into the mice. The scalp was closed by suturing, and wound healing and recovery from surgery were observed. For stereological analysis, animals were perfused and fixed intracardially with ice-cold PBS and then 4% paraformaldehyde 6 months after striatal injection of α-synuclein PFFs. Brains were removed and processed for immunohistochemistry or immunofluorescence. Behavioral test was performed 6 months after unilateral striatal injection of α

- 23 043417 synuclein PFFs. Treatment with NLY001 (3 mg/kg) was administered after a one-month unilateral striatal injection of α-synuclein PFFs, twice weekly, as described in FIG. 4A.

Beneficial neuroprotective effects of NLY001 against α-synuclein PFF-induced behavioral disorders.

Four different behavioral tests were performed on mice treated with vehicle (PBS) or NLY001 6 months after injection of α-synuclein PFFs.

Vertical rod test. Animals were acclimatized in the behavioral test room for 30 minutes. The rod was made of a metal rod 76.2 cm high with a diameter of 9 mm and wrapped in a gauze bandage. Briefly, mice were placed on the top of the rod (7.62 cm from the top of the rod), head up. The total time required for the animal to reach the base of the rod was determined. Before actual testing, mice were trained for two consecutive days, and each training session consisted of three test trials. On the day of testing, mice were assessed in three sessions and the total time was recorded. The maximum time to stop the test and record the results was 30 s. Results were expressed in total time (seconds). Administration of α-synuclein PFFs resulted in a significant increase in the time to reach the base of the rod, while treatment with NLY001 resulted in a reduction in α-synuclein PFF-induced behavioral impairment to the level observed in healthy mice (Table 4).

Horizontal rotarod test: For the horizontal rotarod test, mice were placed on the accelerating cylinder of the rotarod and the time the animals remained on the rod was measured. The speed was slowly increased from 4 to 40 rpm over 5 min. The test ended if the animal fell from the rod or clung to the device and made 2 consecutive turns on it without attempting to walk on the rod. Animals were trained 3 days before testing. Motor skill test data are presented as percentage of mean duration (3 trials) on the rotarod compared to control. Treatment with NLY001 significantly improved rotarod test performance compared with PBS-treated PFF-induced PD models (Table 4).

Test cylinder. Spontaneous movement was measured by placing the animals in a small transparent cylinder (height, 15.5 cm, diameter, 12.7 cm). Spontaneous activity was recorded for 5 min. The number of touches of the front paws, rearing of the hind paws and acts of grooming were measured. Recording files were reviewed and scored in slow motion by an examiner blinded to mouse type and NLY001 treatment status. Mice with α-synuclein PFF-induced PD showed deficits in forelimb use in the cylinder test, while PD mice treated with NLY001 had less deficits in motor function with balanced use of both forelimbs (Table 4).

Amphetamine-induced stereotypic rotation: Mice were intraperitoneally injected with 5 mg/kg amphetamine (Sigma-Aldrich). Mice were placed in a 20 cm diameter white paper cylinder and observed for 30 min. The behavior of the mice was recorded at three-minute intervals between 20 and 30 minutes after amphetamine administration. For each mouse, total ipsilateral rotations (clockwise) were calculated from the video recordings during a one-minute session. Injection of α-synuclein increased amphetamine-induced rotational behavior by 7-fold, indicating loss of dopamine neurons. In contrast, NLY001 prevented amphetamine-induced rotation, indicating that dopamine neurons are functional (Table 4).

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Table 4. Behavioral test in healthy mice and α-synuclein PFF-induced PD models. ±SD, n=10 animals per group. For statistical analysis, two-way analysis of variance was used, followed by a post hoc test ____________Bonferroni for multiple group comparison____________

Test Healthy FSB (subcutaneous) Healthy NLY001 (subcutaneous) PFF FSB (subcutaneous) PFF NLY001 (subcutaneous) Rotating rod (time to fall, sec) 143.60+8.28 156.66+9.39 67.75+6.48 115.05+7.34“ Test vertical rod (Descent time, sec) 11.87+1.25 9.91+0.82 22.69+1.17 13.7+1.07° Test cylinder (Touch the front paws for 5 minutes) 21.83+1.35 21.54+2.10 14.50+2.08 22.54+2.05° Amphetamine test 1.57+0.21 1.42+0.23 7.13+0.27* 2.29+0.55"^#

* **P<0.001 compared with the control group (PFF), ^## P<0.01, ^### P<0.001 compared with the group receiving PFF injection.

NLY001 rescues dopaminergic (DA) neurons and ameliorates Lewy body pathology in mice with α-synuclein PFF-induced PD.

Accumulation of pathological α-synuclein is associated with degeneration of DA neurons. As described here, the potential of systemically administered NLY001 to protect against DA neuronal loss caused by inoculation of α-synuclein PFFs was examined. Mice were sacrificed, and loss of DA neurons was measured by counting the number of tyrosine hydroxylase (TH)-positive and Nissl-positive neurons in the SNpc using objective stereology. In addition, the relative density of TH-positive fibers in the striatum (STR) was analyzed by measuring optical density. Immunostaining of SNpc and STR sections and quantification of TH-positive stained DA neurons and fiber density showed significant loss of dopaminergic neurons in PFF-treated mice compared with PBS-treated controls. In contrast, administration of NLY001 significantly protected against PFF-induced loss of TH neurons (Table 5).

Table 5. ΝΝΥ0 01 rescues DA neurons in mice with α-synuclein PFF-induced PD.

±SD, n=10 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni post hoc test for _________________ multiple group comparison ________________

Test Healthy FSB (subcutaneous) Healthy NLY001 (subcutaneous) PFF (BI) FSB (subcutaneous) PFF (BI) NLY001 (subcutaneous) TH neurons in SNps ( ^x104 ) 5.62+0.51 5.77+0.62 2.90+0.37 4.54+0.27“ Nisslpo surviving cells (xO ⁴ ) 7.22+0.56 7.41+0.20 4.31+0.26 6.53+0.44" Relative fiber density in STR 1.00+0.07 0.972+0.02 0.60+0.06 0.87+0.04"

-25 043417 * **P<0.001 compared with the control group (PBS), ^## P<0.01 compared with the group receiving PFF injection.

The ability of NLY001 to reduce the spread of Lewy body/Lewy neuritis (LB/LN)-like pathology induced by inoculation of α-synuclein PFFs was then examined. After treatment as described above, deposits of hyperphosphorylated α-synuclein, a marker of human LB/LN, were visualized at the injection site (STR) and substantia nigra (SN) using the p-Syn ^Ser129 antibody. pSyn ^Ser129 positive neurons showed a significant increase in LB/LN-like pathology in the striatum and SN in PFF-treated mice compared with PBS-treated controls. As shown in FIG. 5, NLY001 reduces LB/LN pathology in PD brain.

NLY001 inhibits gliosis in PD brain by reducing α-synuclein-associated activation of microglia and astrocytes.

Microglia and astrocytes from the SNpc region were stained with anti-Iba-1 antibodies (1:1000, Wako) or anti-GFAP antibodies (1:2000, Dako) followed by incubation with biotin-conjugated anti-rabbit antibodies and ABC reagents. Sections were then developed using SigmaFast DAB peroxidase substrate (Sigma-Aldrich, St. Louis, MO, USA). The number of microglia and astrocyte density in the SNpc region were measured using ImageJ software. In PD models, populations of Iba-1-positive cells (activated microglia) and GFAP-positive cells (reactive astrocytes) were greatly increased. Treatment with NLY001 significantly blocked microglial activation and reduced the formation of reactive astrocytes in PFF-induced PD models (Table 6).

Table 6. NLY001 blocks α-synuclein-associated activation of microglia and astrocytes in mice with PFF-induced PD. ±SD, n=10 animals per group. For statistical analysis, two-way analysis of variance was used, followed by a post hoc ________Bonferroni test for multiple group comparisons_______

Test Healthy FSB (subcutaneous) Healthy NLY001 (subcutaneous) PFF (BI) FSB (subcutaneous) PFF (BI) NLY001 (subcutaneous) Relative intensity 1.00+0.06 0.94+0.04 1.92+0.13*** 1.31+0.06" # GFAP Relative intensity 1La-1 1.00+0.11 0.98+0.09 2.66+0.31*** 1.55+0.14" # Microglia density (cells/ ^mm2 ) 26, 50+4.0 0 24.21+4.55 138.22+11.21* 9y 9y 55.12+6.14 ###

***P<0.001 compared with the control group (PBS), ^### P<0.001 compared with the group receiving PFF injection.

Example 5. NLY001 reduces α-synuclein-associated gliosis (activation of microglia and astrocytes), inhibits α-synuclein aggregation and alleviates LB/LN pathology and increases lifespan in A53T Tg PD mice.

Animals.

A53T α-synuclein transgenic mice (A53T) were obtained from Jackson Lab (B6; PrnpSNCA*A53T, PMID: 12084935). Mice were crossed with C57BL/6 mice (Jackson Lab) and bred for the present study. NLY001 and PBS were administered subcutaneously (3 mg/kg, twice weekly) to wild-type (WT) and A53T PD mice from 6 months of age until 10 months of age and the date of death, as described in Fig. 4B.

In the brains of mice with PD, NLY001 accumulates in significantly higher quantities compared to the brains of WT mice.

Mice were sacrificed at 10 months of age and NLY001 concentrations in the brain (cerebellum and hemisphere) were measured using an immunoassay as described above. NLY001 was extracted from brain tissue using a C-18 SEP-Column (Phoenix Pharmaceuticals, Inc.) and analyzed using the Exendin-4 ELISA reagent kit (Phoenix Pharmaceuticals Inc.).

Unexpectedly, subcutaneously administered NLY001 penetrated the BBB and accumulated to a significantly greater extent (10-30 times) in the brains of mice with PD (A53T) compared to the brains of healthy WT mice (Table 7).

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Table 7. Accumulation of NLY001 in the brain. ±SD, n=6-8 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni post hoc test for multiple group comparisons.

Brain DT FSB (subcutaneous) DT NLY001 (subcutaneous) А53Т NLY001 (subcutaneous) Cerebellum (pg per mg brain tissue) 0.00+0.00 28.48+1.41 961.29+202.73*** Hemisphere (pg per mg brain tissue) 3.00+0.73 40.48+2.6 445.55+22.95***

* **P<0.001 compared with the control group WT+NLYOOl.

NLY001 treatment increases body weight in PD models.

Mouse models of progressive PD typically exhibit a decrease in body weight. In the clinic, weight loss in PD is an important issue. PD complicated by weight loss leads to a deterioration in the overall treatment outcome and a decrease in quality of life. Approved GLP-lr agonists for diabetic/obese patients are known to effectively reduce body weight during chronic treatment. Liraglutide has been shown to be effective for long-term weight loss in type 2 diabetes, and high-dose liraglutide was recently approved as an anti-obesity drug (Sexenda). Unlike other GLP-lr agonists, NLY001 does not reduce body weight in models of neurodegenerative diseases, but increases body weight, attenuating disease progression (Table 8).

Table 8. Body weight of WT mice and A53T PD models at the age of 12 months treated with PBS or NLY001. ±RMS, n=20 animals per group

Mice DT FSB (subcutaneous) DT NLY001 (subcutaneous) A53T FSB (subcutaneous) A53T NLY001 (subcutaneous) Weight body (g) 37.94+0.56 38.76+1.96 27.01+0.28*** 33.60+0.50 ^# ##

* **P<0.001 compared to the WT control group, ^### P<0.001 compared to the A53T+PBS group.

NLY001 increases lifespan in A53T α-synuclein Tg mice.

A53T α-synuclein Tg mice have a shortened lifespan due to degeneration of neurons in the brainstem and spinal cord, leading to limb paralysis, autonomic dysfunction and premature death (Lee M.K. et al., Proc. Natl. Acad. Sci. USA 2002. 99(13):8969-8973). In most affected animals, the disease quickly led to death, probably due to inability to feed and dehydration. In this study, A53T mice exhibited premature lethality between 1 and 14 months of age. Moreover, treatment with NLY001 significantly increased the lifespan of A53T (Fig. 6 and Table 9).

Table 9. Median survival calculated using GraphPad Prism 6. ±SD, n=20 animals per group

Mice DT FSB (subcutaneous) DT NLY001 (subcutaneous) A53T FSB (subcutaneous) А53Т NLY001 (subcutaneous) Median survival time (months) No death no death 12, 9 17.1###

^{# ##} P<0.001 compared with the A53T+PBS group.

NLY001 inhibits gliosis in PD brain and reduces α-synuclein aggregation in A53T asynucleic Tg mice with PD.

Microglia and astrocyte activation were analyzed as described above. The number of microglia and astrocyte density in the brain were measured using ImageJ software. In A53T Tg PD models, populations of Iba-1-positive cells (activated microglia) and GFAP-positive cells (reactive astrocytes) were greatly increased. Treatment with NLY001 significantly blocked microglial activation and reduced the formation of reactive astrocytes in A53T Tg PD models (Table 10). It is important to note that the relative protein expression of α-synuclein, α-synuclein aggregation and

-27043417 β-actin was analyzed by immunoblotting in the detergent-insoluble fraction of brainstem obtained from 10-month-old A53T Tg mice and age-matched control mice from the same litter. Mice were treated with PBS or NLY001. In addition, the formation of ubiquitin-positive inclusions in the brainstem of A53T Tg mice was analyzed by immunohistochemical images of p-a-synuclein. As seen in PFF-induced PD mice, NLY001 significantly blocks α-synuclein aggregation in A53T Tg mice with PD. The results are shown in table. eleven.

Table 10. NLY001 blocks α-synuclein-associated activation of microglia and astrocytes in A53T α-synuclein Tg mice with PD. ±SD, n=7 animals per group. For statistical analysis, two-way analysis of variance was used, followed by Bonferroni post hoc test for multiple group comparisons.

Test DT FSB (subcutaneous) DT NLY001 (subcutaneous) A53T FSB (subcutaneous) А53Т NLY001 (subcutaneous) Relative GFAP intensity 1.00+0.04 0.98+0.08 1.95+0.05 1.24+0.03 ^### Relative intensity of Iba-1 1.00+0.11 0.98+0.09 2.66+0.31 1.55+0.14### Microglia density (cells/ ^mm2 ) 29.10+3.0 0 27.33+2.89 141.33+8, 49*** 50.66+5.06###

***P<0.001 compared to the control group (PBS), ^## P<0.01, ^### P<0.001 compared to the A53T+PBS group.

Table 11. NLY001 reduces the expression of α-synuclein and ubiquitin in A53T α-synuclein Tg mice with PD. ±SD, n=3 animals per group. For statistical analysis, two-way analysis of variance was used, followed by a post hoc ________Bonferroni test for multiple group comparisons________

Test A53T FSB (subcutaneous) А53Т NLY001 (subcutaneous) Relative amount of a- 1.00+0.10 0.48+0.06 ^# « synuclein ^p ~ ^ser129 Relative quantity α-synuclein aggregation 1.00+0.04 0.43+0.05### Relative intensity of ubiquitin 1.00±0.08 0.52±0.06###

^{# ##} P<0.001 compared with the A53T+PBS group.

Example 6: NLY001 reduces Alzheimer's-like pathology and memory impairment in 3xTg AD mice.

Materials and methods.

Animals: 3xTg AD mice were obtained from Jackson Lab. These commonly used mice have three mutations: Swedish AAP, MART 3P01L and PSEN1 M126V, which are associated with familial Alzheimer's disease. 3xTg mice have pathology in the form of both plaques and tangles. Amyloid-β deposition is progressive and appears intracellularly as early as three to four months of age, while extracellular deposits appear at six months of age in the frontal cortex and become more extensive at twelve months. 6-month-old male 3xTg AD mice were used in this study. NLY001 and PBS were administered subcutaneously (1 and 10 mg/kg, twice weekly) to wild-type (WT) control and 3xTg AD mice after 7 months of age for 5 months as described in FIG. 7.

NLY001 improves memory in 3xTg AD mice. Two different behavioral tests (Webster S.J. et al., Front Genet. 2014. 5:88) were performed on mice treated with vehicle (PBS) or NLY001.

Morris water maze test (MWM): The Morris water maze is a white circular pool (100 cm in diameter and 35 cm in height) with a uniform inner surface. This circular pool was filled with water and a non-toxic, water-soluble white dye. The pool was divided into four quadrants of equal area. The platform (8 cm in diameter and 10 cm in height) was placed in the center of one of the quadrants of the pool and was submerged 1 cm below the surface of the water so that it was invisible at water level. The pool was located in the test room, in which

- 28 043417 there were various noticeable visual landmarks. The position of each floating mouse from the starting position to the platform was monitored by a video surveillance system (ANY-maxe system, Wood Dale, IL, USA). One day before the start of the experiment was devoted to training mice to swim for 60 s in the absence of a platform. Mice were then given three test sessions each day for five consecutive days at 15-min intervals, and escape latency was recorded. This parameter was averaged for each test session and for each mouse. Once the mouse reached the platform, it was allowed to remain on it for 10 s. If the mouse did not find the platform within 60 s, it was placed on the platform for 10 s and then removed from the pool by the experimenter. On day 6, a search test was carried out, which involved removing the platform from the pool. This test was carried out with a time limit of 60 s. The mouse's entry point into the pool and the location of the escape platform remained constant between trials 1 and 2 but changed each day thereafter.

As shown in FIG. 8 and 9, 3xTg mice treated with PBS had learning defects compared to wild-type mice. On days 4 and 5, PBS 3xTg-treated mice took longer to find the hidden platform than their wild-type littermates. In contrast, NLY001-treated 3xTg AD mice had significantly improved performance compared with PBS-treated 3xTg mice, indicating that NLY001 treatment alleviated the spatial learning impairment in 3xTg mice. To assess memory performance in spatial learning, retrieval tests were administered on day 5. 3xTg mice treated with NLY001 spent significantly more time searching for the platform in the target quadrant compared to 3xTg BA mice treated with PBS (Table 12). NLY001 treatment had no effect on swimming speed and distance.

Table 12. Retrieval swim test in 3xTg AD mice treated with NLY001. ±RMS, n=7 mice per group

Search DT ZxTg BA test FSB NLY001 10 mg/kg FSB NLY001 1 mg/kg NLY001 10 mg/kg Sailing time in target quadrant (sec) 26.26±5.49 27.02±4.19 17.52±9.63 24.49±6.17# 32.66±7.1 6 ## # Swimming speed (m/s) 0.21±0.02 0.19±0.02 0.20±0.03 0.20±0.0 2 0.20±0.02

* P<0.05 compared with WT+PBS, #P<0.05, ^### P<0.001 compared with the 3xTg BA+PBS group.

Passive avoidance test. The effects of NLY001 on learning/memory were assessed using a graded passive avoidance procedure in which animals learn to avoid electrical shock by suppressing their natural preference for being in the dark. Testing began with training in which the mouse was placed in a lighted chamber; When the mouse moved into the dark chamber, it received a mild (0.25 mA/1 s) electric shock to its paws. This initial latency to enter the dark (electric shock) chamber served as the baseline value. During search tests, 24 h after training, the mouse was again placed in the light chamber, and the latency to return to the dark chamber was measured as a measure of passive avoidance fear. Treatment with NLY001 significantly improved learning in 3xTg AD mice assessed by the passive avoidance test compared to 3xTg AD mice treated with PBS (Table 13).

Table 13. Learning assessed using the passive avoidance test in WT mice and 3xTg AD mice treated with PBS or NLY001. Error bars represent mean ± SD, n=7 mice per group

Passive avoidance DT ZxTg BA FSB NLY001 10 mg/kg FSB NLY001 1 mg/kg NLY001 10 mg/kg Time to go dark 213.3±93.3 242.3±96.7 148.0±1 25.2* 242.1±91.5# 228.9±12 1.7 ^#

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camera (sec)

* P <0.05 compared with WT+PBS, #P<0.05 compared with the 3xTg BA+PBS group.

NLY001 accumulates in the brains of AD mice in significantly greater quantities compared to the brains of WT mice.

As described for PD models, mice were sacrificed after the study, and whole brain NLY001 concentrations were measured using an immunoassay as described above. NLY001 was extracted from brain tissue using C-18 SEP-Column and analyzed using the Exendin-4 ELISA reagent kit. As proven in PD models, subcutaneously administered NLY001 penetrated the BBB and accumulated in the brain of 3xTg AD mice in an amount two to five times greater than in the brain of healthy WT mice.

NLY001 inhibits gliosis in AD brain by reducing microglial and astrocyte activation.

Microglia and astrocytes from fixed brain tissues were stained with anti-Iba-1 or anti-GFAP antibodies, followed by incubation with biotin-conjugated anti-rabbit antibodies and ABC reagents as described above. In AD models, populations of Iba-1-positive cells (activated microglia) and GFAP-positive cells (reactive astrocytes) are greatly increased. Treatment with NLY001 significantly blocked microglial activation and reduced reactive astrocyte formation in 3xTg AD models. In addition, it was also confirmed that GLP-1r is highly expressed on Iba-1-positive cells (activated microglia) but not on MAP2-positive cells (neurons). These results support in vitro findings that a long-acting GLP-1r agonist blocks gliosis and stops the release of inflammatory and neurotoxic molecules by binding to GLP-1r expressed on resident innate immune cells, including microglia in the brain.

NLY001 treatment reduces the expression of inflammatory and neurotoxic molecules in the brain of 3xTg AD mice.

To further confirm that the anti-AD efficacy of NLY001 in 3xTg mice is due to inhibition of the release of inflammatory and neurotoxic molecules secreted by activated microglia and reactive astrocytes, brain tissue homogenates were analyzed by real-time PCR for TNF-α, IL-1e, IFN -γ, IL-6 and C1q. Expression levels of inflammatory markers were found to be significantly higher in 3xTg mice compared to WT mice. Consistent with the results of the in vitro cell study, 3xTg mice treated with NLY001 showed a significant decrease in the expression of inflammatory and neurotoxic markers, as shown in Table 1. 14.

Table 14. Effects of NLY001 in 3xTg AD mice. The mRNA levels of TNF-α, IL-1p, IFN-γ, IL-6 and C1q in the brain were analyzed by real-time PCR. ±RMS, n=5 mice per group

mRNA DT ZxTg BA FSB NLY001 10 mg/kg FSB NLY001 1 mg/kg NLY001 10 mg/kg TNF-a 1.0±0.5 1.0±0.4 3.7±3, I* 1.8±0.5 ^# 2.0±0.7 ^# IL-Ιβ 1.0±0.4 0.9±0.5 2.9±1.4*** 1.4±0.5 ^## 1.6±0.9 ^## IFN-γ 1.0±0.7 0.6±0.3 5.3±5.7** 1.4±0.5 ^# 1.6±0.8 ^## IL-6 1.0±0.4 0.8±0.1 1.9±0.4* 1.4±0.4 1.1±0.3 ^# Clq 1.0±0.3 1.0±0.1 1.6±0.3* 1.2±0.3 1.1±0.2 ^#

*P<0.05, **P<0.01, ***p<0.001 compared to DT+PBS, #P<0.05, ##P<0.01 compared to the 3xTg BA+PBS group .

Equivalents.

Those skilled in the art will be aware that they will be able to determine, by no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be covered by the following claims.

Other embodiments of the invention.

Although the present invention has been described in terms of its detailed description, the foregoing description is intended to be illustrative and does not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

The patent and scientific literature referenced herein describes knowledge that is available to those skilled in the art. All US Patents and published or unpublished US Patent applications cited herein are incorporated herein by reference. Everything is published-

Claims (27)

The foreign patents and patent applications cited herein are incorporated herein by reference. Data provided herein from the Genbank and NCBI databases, indicated by accession number, are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. While the present invention has been particularly shown and described with reference to preferred embodiments thereof, those skilled in the art will appreciate that various changes in form and detail may be made thereto without departing from the scope of the invention. covered by the attached claims. CLAIM 1. A method of treating a neurodegenerative disease or disorder associated with microglia and/or astrocyte activation, comprising parenteral administration to a subject suffering from or at risk of developing a neurodegenerative disease or disorder associated with microglia and/or astrocyte activation, a pharmaceutically effective amount of a composition containing an agonist long-acting GLP-1r, which is the GLP-1 analog exenatide stably conjugated to polyethylene glycol (PEG) or a derivative thereof, to relieve one or more symptoms of a neurodegenerative disease or disorder, wherein the long-acting GLP-1r agonist has an in vivo half-life of at least 36-200 hours in non-human primates and administered no more than twice per week. 2. The method of claim 1, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising a long-acting GLP-1r agonist to block activation of resident innate immune cells. 3. The method according to claim 2, wherein the abnormally aggregated proteins are inhibited from activating immune cells by activating GLP-1r. 4. The method of claim 2, wherein the amount of the long-acting GLP-1r agonist effectively inhibits the secretion of inflammatory and/or neurotoxic mediators secreted by activated innate immune cells. 5. The method according to claim 2, wherein the innate immune cells are microglia and/or astrocytes. 6. The method according to claim 3, wherein the abnormally aggregated proteins are α-synuclein, β-amyloid or τ protein. 7. The method of claim 2, wherein the long-acting GLP-1r agonist is administered in an amount effective to reduce inflammatory or neurotoxic mediators selected from the group consisting of TNF-α, IL-1a, IL-1e, IFN-γ, IL -6 and C1q, compared with the corresponding control. 8. The method of claim 5, wherein the effective amount of a GLP-1r agonist reduces the cell population of activated microglia and reactive astrocytes. 9. The method according to claim 1, wherein the neurodegenerative disease is Parkinson's disease. 10. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease. 11. The method according to claim 1, wherein the neurodegenerative disease is Huntington's chorea. 12. The method according to claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis. 13. The method according to claim 1, wherein the long-acting GLP-1r agonist contains a PEGylated analogue of exenatide. 14. The method according to claim 1, where the composition is administered subcutaneously. 15. The method according to claim 1, where the composition is administered in a form selected from the group consisting of pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, suspensions, gels, creams, pastes, films, patches and vapors. 16. The method according to claim 1, where the composition is administered 1-4 times a month. 17. The method according to claim 1, where the composition is administered once a week. 18. The method according to claim 1, where the composition is administered every two weeks. 19. The method of claim 1, wherein the composition is administered approximately once a month. 20. The method according to claim 1, where the composition is administered once every two months. 21. The method according to claim 1, where the composition is administered 1-3 times every 6 months. 22. The method according to any one of claims 1 to 21, wherein the long-acting GLP-1r agonist has an in vivo half-life of 48 to 200 hours in non-human primates. 23. The method according to claim 1, where the composition is administered to a person at a dose of 0.001 to 100 mg/kg. 24. The method according to claim 23, where the composition is administered to a person at a dose of 0.001 to 10 mg/kg. 25. The method of claim 1, wherein the composition is administered to the subject for about 10 years. 26. The method according to claim 1, where the effect of treatment lasts at least one year. 27. The method of claim 1, wherein the method protects against α-synuclein-associated dopaminergic neuron loss. -