Methods for delivering neuroprotective polypeptides to the central nervous system - Patents.com

Abstract

The present disclosure provides a method for delivering a neuroprotective polypeptide to at least a portion of a subject's central nervous system (CNS). The method includes administering a therapeutically effective amount of a neuroprotective polypeptide to the subject's systemic circulatory bloodstream using a controlled-release preparation or device that provides sustained release of the neuroprotective polypeptide, wherein the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled-release neuroprotective preparation or sustained release of the neuroprotective polypeptide increases delivery of the neuroprotective polypeptide across the subject's blood-brain barrier (BBB) ​​to at least a portion of the CNS compared to an immediate-release preparation of the neuroprotective polypeptide. The present disclosure also discloses a method for treating a subject with a CNS-related disorder or reducing at least one symptom of a CNS-related disorder in a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 410,748, filed October 20, 2016, which is incorporated by reference in its entirety.

[0002] Government funding The work supporting this application was conducted by the United States Government as represented by the Secretary of Health and Human Services. The United States Government has certain rights in this invention.

[0003] Incorporation by Reference In accordance with 37 C.FR § 1.52(e)(5), the entire sequence information contained in the electronic file (Filename: 1568390_100WO2_Sequence_Listing_ST25.txt, Size: 24.4KB, Created: September 3, 2017 using PATENT-In 3.5 and Checker 4.4.0) is incorporated herein by reference.

[0004] background Field The present disclosure relates to compositions containing neuroprotective therapeutic polypeptides, such as, for example, glucagon-like peptide-1 (GLP-1), exendin-4, and / or peptide analogs thereof. In particular, the present disclosure relates to methods for maintaining steady-state levels of neuroprotective therapeutic polypeptides in plasma, thereby facilitating delivery across the blood-brain barrier (BBB) ​​to the brain, for treating neurodegenerative conditions. [Background technology]

[0005] Background technology Glucagon-like peptide-1 (GLP-1), a hormone normally secreted by intestinal neuroendocrine cells in response to food, has been suggested as a novel treatment for type 2 diabetes (Gutniak et al., 1992; Nauck et al., 1993). GLP-1 increases insulin release by beta cells, an effect also seen in long-term type 2 diabetes patients (Nauck et al., 1993). GLP-1 stimulates endogenous insulin secretion, and this stimulation disappears when blood glucose levels fall, making GLP-1 treatment superior to insulin therapy (Nauck et al., 1993; Elahi et al., 1994). GLP-1 promotes normoglycemia by increasing insulin release and synthesis, inhibiting glucagon release, and reducing gastric emptying (Nauck et al., 1993; Elahi et al., 1994; Wills et al., 1996; Nathan et al., 1992; De Ore et al., 1997). GLP-1 is a post-translational modification product of proglucagon. The sequence of GLP-1 and its active fragments GLP-1(7-37) and GLP-1(7-36)amide are well known in the art (Fehmann et al., 1995). GLP-1 has been proposed as a therapeutic agent in the treatment of diabetes, but it has a short biological half-life (De Ore et al., 1997), even when administered as a bolus subcutaneous dose (Ritzel et al., 1995). Degradation of GLP-1 (and GLP-1(7-36)amide) is due in part to the enzyme dipeptidyl peptidase (DPP 1V), which cleaves the polypeptide between amino acids 8 and 9 (alanine and glutamic acid).

[0006] Exendin-4 is a polypeptide produced in the salivary glands of the Gila monster lizard (Goke et al., 1993). The amino acid sequence of exendin-4 is well known in the art (Fehmann et al. 1995). Although exendin-4 is a non-mammalian specific gene product whose expression appears to be restricted to the salivary glands (Chen and Drucker, 1997), it shares 52% amino acid sequence identity with GLP-1 and interacts with the GLP-1 receptor in mammals (Goke et al., 1993; Thorens et al., 1993). In vitro, exendin-4 has been shown to stimulate insulin secretion by insulin-producing cells and is more potent at causing insulin release by insulin-producing cells than administration of an equimolar amount of GLP-1. Furthermore, exendin-4 potently stimulates insulin release and reduces plasma glucose levels in both rodents and humans, and has a longer duration of action than GLP-1. However, exendin-4 is not naturally occurring in mammals, and therefore may have some antigenic properties in mammals that GLP-1 does not. Summary of the Invention [Problem to be solved by the invention]

[0007] In addition to the decreased insulin production in diabetes, peripheral neuropathy and diabetes are interrelated. 20-30 percent of all diabetic patients eventually develop peripheral neuropathy. Furthermore, it has been reported that patients with Alzheimer's disease are at increased risk of developing heart disease, stroke, hypertension, and diabetes (Moceri et al., 2000; Ott et al., 1999). Thus, diabetes is also associated with neurodegenerative diseases.

[0008] GLP-1 receptors are present in the rodent (Jin et al. 1988, Shughrue et al. 1996, Jia et al. 2015) and human (Wei and Mojsov 1995, Satoh et al. 2000) brains. The chemical makeup of this distribution seems to be largely restricted to the hypothalamus, thalamus, brainstem, lateral septum, subfornical organ, and area postrema, and generally, large numbers of peptide receptors are present in all of these periventricular regions. However, specific binding sites for GLP-1 have also been detected, albeit at lower densities, throughout the caudate putamen, cerebral cortex, and cerebellum (Campos et al. 1994, Calvo et al. 1995, and Goke et al. 1995). For example, Lu et al. (2014) reported that GLP-1 receptor expression is found in the amygdala, cerebellum, frontal cortex, hippocampus, hypothalamus, midbrain, medulla, pons, striatum, thalamus, and temporal cortex of Mustela putorius furo (ferret). The brain expression level of GLP-1 receptor is not affected by aging.

[0009] Furthermore, GLP-1 has been shown to be involved in cognition and behavior. (During et al. 2003). In fact, many studies suggest that GLP-1 receptor agonists are a new treatment for neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, traumatic brain injury, stroke, and peripheral neuropathy. However, for use in the treatment of central nervous system (CNS)-related diseases, a major obstacle is the delivery of drugs to the CNS through the blood-brain barrier (BBB). For example, the half-life of GLP-1 is short, at 1-2 minutes, and the GLP-1-transferrin fusion protein (GLP-1-Tf), which was created to make it resistant to inactivation, has a half-life of approximately 2 days, which is longer than that of GLP-1, but it has not yet achieved BBB crossing. Kim et al. (2010) and Martin et al. (2012).

[0010] Exendin-4 has also been shown to improve the results of the rotarod test more than GLP-1-Tf (Martin et al. 2012), and although it is known that exendin-4 enters the brain from the blood, the influx rate is not sufficient (Kastin AJ and Akeerstrom V, International Journal of Obesity (2003) 27, 313-318). In addition, exenatide was administered once a day for 7 days to the MPTP Parkinson's disease mouse model with post-treatment, and no neuroprotective effect was observed (Liu et al. 2015).

[0011] Thus, there is a need in the art for methods of treating neurodegenerative conditions and for methods of maintaining steady state levels of neuroprotective GLP-1 receptor agonists in plasma, thereby facilitating and enhancing drug delivery across the BBB to the central nervous system. [Means for solving the problem]

[0012] overview In one embodiment of the present disclosure, a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject is provided. The method includes administering a therapeutically effective amount of a neuroprotective polypeptide to the systemic circulatory bloodstream of a subject using a controlled release preparation or device that provides sustained release or sustained delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation or sustained release of the neuroprotective polypeptide increases the delivery and / or uptake of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to a fast release preparation of the neuroprotective peptide.

[0013] In another aspect of the present disclosure, a method is provided for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method includes administering a therapeutically effective amount of a neuroprotective polypeptide to the subject's systemic circulatory bloodstream using a controlled release preparation or device that provides sustained release or sustained delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation or device increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject by passing through the blood-brain barrier (BBB) ​​compared to a fast release preparation of the neuroprotective polypeptide.

[0014] In one aspect of the present disclosure, a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject is provided. The method includes administering a controlled release neuroprotective preparation to the systemic circulatory bloodstream of the subject. In one embodiment, the controlled release neuroprotective preparation comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin (e.g., exendin-4), or a therapeutically effective GLP-1 or exendin analog (e.g., exendin-4 analog), the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin (e.g., exendin-4), or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to a fast release preparation of the neuroprotective polypeptide.

[0015] In certain embodiments, the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide.

[0016] In other embodiments, sustained release preparations include depot preparations for sustained release of the neuroprotective polypeptide.

[0017] In a specific embodiment, the time-release preparation comprises a composition for the sustained release of a neuroprotective polypeptide.

[0018] In additional embodiments, the controlled release preparation further comprises a biodegradable polymer having a particular viscosity and coating material, such that the bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time.

[0019] In a specific embodiment, the controlled release neuroprotective preparation comprises a neuroprotective polypeptide. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0020] In one embodiment, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in a subject.

[0021] In further embodiments, the CNS-related condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy (CTE), motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0022] In one particular embodiment, administration of the controlled release neuroprotective preparation comprises an injection of the controlled release neuroprotective preparation.

[0023] In additional embodiments, the injection of the controlled release neuroprotective preparation is a subcutaneous injection. For example, the administration of the controlled release neuroprotective preparation may include a subcutaneous injection of the controlled release neuroprotective preparation.

[0024] In another embodiment, administration of the controlled release neuroprotective preparation results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0025] In other embodiments, administration of the controlled release neuroprotective preparation cumulatively increases the concentration of the neuroprotective polypeptide in the subject's cerebrospinal fluid (CSF), brain, or a combination thereof.

[0026] In specific embodiments, the concentration of the neuroprotective polypeptide in the CSF is in the range of about 5 to about 400 pg / mL (eg, about 10 to about 400 pg / mL).

[0027] In a further embodiment, a method is provided for treating a subject with a CNS-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method comprises administering a therapeutically effective amount of a controlled release neuroprotective preparation to the subject into the subject's systemic circulatory bloodstream.The controlled release neuroprotective preparation comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin (such as exendin-4), or a therapeutically effective GLP-1 or exendin (such as exendin-4) analog, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin (e.g., exendin-4), or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide through the BBB to at least a portion of the CNS of the subject compared to a fast release preparation of the neuroprotective polypeptide.

[0028] In certain embodiments, the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide.

[0029] In other embodiments, sustained release preparations include depot preparations for sustained release of the neuroprotective polypeptide.

[0030] In additional embodiments, the time-release preparation comprises a composition for sustained release of the neuroprotective polypeptide.

[0031] In additional embodiments, the controlled release preparation further comprises a biodegradable polymer having a particular viscosity and coating material, such that the bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time.

[0032] In a specific embodiment, the controlled release neuroprotective preparation comprises a neuroprotective polypeptide. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0033] In a specific embodiment, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in the subject.

[0034] In one embodiment, the CNS condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron disease (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0035] In a further embodiment, administering the controlled release neuroprotective preparation comprises injecting the controlled release neuroprotective preparation into the subject.

[0036] In certain embodiments, the injection of the controlled release neuroprotective preparation into the subject is a subcutaneous injection.

[0037] In additional embodiments, administration of the controlled release formulation results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0038] In some embodiments, administration of the controlled release preparation cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0039] In a further embodiment, a method for delivering neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject is provided.The method includes providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery and / or uptake of the neuroprotective polypeptide through the blood-brain barrier (BBB) ​​of the subject to at least a portion of the central nervous system (CNS) compared to a fast release preparation of the neuroprotective peptide.

[0040] In yet another embodiment, a method is provided for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method comprises providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide through the blood-brain barrier (BBB) ​​of the subject to at least a portion of the central nervous system (CNS) compared to the immediate release preparation of the neuroprotective polypeptide.

[0041] In one embodiment, providing sustained release of the neuroprotective polypeptide(s) comprises administering the polypeptide(s) via a device (e.g., a pump, a mini-pump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini-infusion pump).

[0042] In certain embodiments, the neuroprotective polypeptide(s) are administered at a rate of about 1 pM / kg / min to about 30 pM / kg / min (e.g., about 3 pM / kg / min to about 17.5 pM / kg / min).

[0043] In other embodiments, administration of a controlled release neuroprotective preparation or providing sustained delivery of a neuroprotective polypeptide alleviates at least one symptom of at least one CNS-related condition in a subject.

[0044] In specific embodiments, the CNS-related condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0045] In one embodiment, administration of a controlled release formulation or providing sustained release of the neuroprotective polypeptide results in a steady state concentration of the neuroprotective polypeptide in the plasma within the range of about 50 to about 4500 pg / mL.

[0046] In certain embodiments, administration of a controlled release preparation or providing sustained release of a neuroprotective polypeptide cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0047] In yet another embodiment, the concentration of the neuroprotective polypeptide in the CSF is in the range of about 5 to about 400 pg / mL.

[0048] In some embodiments, the ratio of the concentration of the polypeptide in the CFS at steady state to the concentration of the polypeptide in plasma at steady state is within the range of about 0.1% to about 5%.

[0049] In further embodiments, the neuroprotective polypeptide is selected from the group consisting of SEQ ID NOs: 1-55. For example, the neuroprotective polypeptide may comprise an amino acid sequence selected from SEQ ID NOs: 1-55.

[0050] In other embodiments, the exendin-4 analog is represented by Formula I, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa27 Xaa28 -Z1 (Chemical formula I) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg; Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, or a linear or branched alkanoyl having 1 to 10 carbon atoms; Xaa22 is Ala, Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn, and Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; Xaa39 is Ser, or Tyr (e.g., Ser); and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27, and Xaa28 are Ala; and When Xaa1 is His, Arg, or Tyr, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0051] In other embodiments, the exendin-4 analog is represented by formula II, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala X1-Z1 (Chemical formula II) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg, Xaa21 is Ala, Leu, or Lys-NHε-R (wherein R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloarylene-alkanoyl); Xaa22 is Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; X1 is Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloalkylalkanoyl; Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, and N-alkylalanine; Xaa39 is Ser or Tyr; and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, and Xaa26 are Ala; and When Xaa1 is His, Arg, Tyr, or 4-imidazopropionyl, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0052] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. [Brief description of the drawings]

[0053] [Figure 1A] FIG. 1 is a graph showing the time-dependent plasma levels of exenatide (exendin-4) following a single subcutaneous dose of 2 mg / kg sustained-release exenatide (PT302) in adult (9 week old) male Sprague-Dawley rats. [Figure 1B] FIG. 1 is a graph showing the time-dependent plasma levels of exenatide (exendin-4) following a single subcutaneous dose of 2.4 mg / kg, 4.8 mg / kg, or 9.6 mg / kg of sustained release exenatide (PT302) in adult (9 week old) male Sprague-Dawley rats. [Diagram 2] 1 is a graph showing the time-dependent plasma levels of exendin-4 following a single subcutaneous injection of 4.0 mg / kg sustained-release exenatide (PT304) in adult (9 week old) male Sprague-Dawley rats. [Diagram 3] FIG. 1 is a graph showing time-dependent exendin-4 plasma levels over a 10-week period in male Sprague-Dawley rats administered either PT302 or PT304 at the indicated amounts and times. [Figure 4A] The experimental design of Example 2 is outlined below: Sprague-Dawley rats were administered PT302 prior to unilateral 6-OHDA lesioning of the medial forebrain bundle. [Figure 4B] FIG. 1 is a graph showing methamphetamine-induced (meth-induced) rotation behavior in 6-OHDA rats pretreated with PT302. [Figure 4C] 1 is a graph showing plasma levels of exendin-4 in 6-OHDA rats pretreated with PT302. [Figure 5A] The experimental design of Example 3 is outlined below. Sprague-Dawley rats were given unilateral 6-OHDA lesions of the medial forebrain bundle followed by administration of PT302. [Figure 5B] FIG. 13 is a graph showing meth-induced rotation behavior in 6-OHDA rats post-treated with PT302. [Figure 5C]Representative microscopic images of tyrosine hydroxylase (TH) immunohistochemistry in the striatum of rats with unilateral 6-OHDA lesions of the medial forebrain bundle and administration of vehicle (control) (rats #866, #883, and #886) and PT302 (rats #881, #875, and #882). Plasma concentrations of exendin-4 (ng / ml) are shown for each animal. [Figure 5D] FIG. 5C is a graph showing the quantification of TH immunoreactivity observed in the immunohistochemistry study described in and associated with FIG. 5C. [Figure 5E] Representative microscopic images of TH immunohistochemistry of the substantia nigra in rats with unilateral 6-OHDA lesions of the medial forebrain bundle and treated with vehicle (control) (rats #866, #883, and #886) and PT302 (rats #881, #875, and #882). Plasma exendin-4 concentrations (ng / ml) are shown for each animal. [Figure 5F] FIG. 5B is a graph showing the quantification of TH immunoreactivity observed in the immunohistochemistry described in and associated with FIG. 5E. [Figure 5G] Representative microscopic images of TH+ neurons observed in the uninjured side of the rat brain. [Figure 5H] Representative microscopic images of TH+ neurons observed in the uninjured side of the rat brain. [Figure 5I] Representative microscopy images showing the absence of TH+ neurons in the substantia nigra on the lesioned side of the brain in rats with unilateral 6-OHDA lesion of the medial forebrain bundle. [Figure 5J] Representative images of the brains of 6-OHDA rats treated with PT302. [Figure 5K] Representative images of the brains of 6-OHDA rats treated with PT302. [Figure 5L] Representative images of the brains of 6-OHDA rats treated with PT302. [Figure 5M]FIG. 1 is a graph showing the significant correlation observed between normalized TH immunoreactivity and exendin-4 plasma levels (in the striatum). [Figure 5N] 1 is a graph showing the significant correlation observed between exendin-4 plasma levels and TH immunoreactivity in the substantia nigra. [Figure 6A] 1 shows an outline of the experimental design for Example 4. [Figure 6B] Exendin-4 and PT302 treatment reduce rotational behavior in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. [Figure 6C] Representative microscopic images of the substantia nigra of control (sham), 6-OHDA, exendin-4-treated, and PT302-treated 6-OHDA rats. [Figure 6D] FIG. 6B is a graph showing quantified data from FIG. 6C. [Figure 7A] 1 shows an outline of the experimental design for Example 5. [Figure 7B] PT302 significantly reduced the turning behavior of unilaterally 6-OHDA-lesioned rats, but exendin-4 did not reduce the turning behavior of unilaterally 6-OHDA-lesioned rats. [Figure 8A] 1 shows an outline of the experimental design for Example 6. [Figure 8B] FIG. 1 is a graph showing that PT302, but not exendin-4, significantly increases the latency of MPTP-treated mice to fall off the wire. [Figure 9A] 1 shows an outline of the experimental design for Example 8. [Figure 9B] 1 is a graph showing that sustained release exenatide significantly increased novel object recognition in mice with mild traumatic brain injury (mTBI). [Figure 10A] 1 is a graph showing that exendin-4 plasma levels are sustained for 7 days after PT302 administration and are dose-dependent. [Figure 10B]FIG. 13 is a graph showing that no significant difference in exendin-4 plasma levels was observed between normal / control and mTBI-treated mice from PT302 administration. [Figure 10C] 1 is a graph showing that three different doses of sustained release exenatide (PT302, 0.024, 0.12, and 0.6 mg / kg) were able to accumulate in plasma and be maintained in plasma in a time-dependent manner. [Figure 11A] FIG. 13 shows that sustained release exenatide (PT302, 0.024, 0.12, and 0.6 mg / kg) increased novel object preference in mTBI mice compared to untreated mTBI mice that exhibited visual memory deficits and spent less time near the novel object. Assessment was performed using a novel object recognition paradigm 7 days after mTBI. [Figure 11B]

[0023] Figure 1 shows that sustained release exenatide (PT302, 0.024, 0.12, and 0.6 mg / kg) ameliorated mTBI spatial memory deficits observed in naive mTBI mice. Assessment was performed by Y-maze 7 days after mTBI. [Figure 11C] FIG. 11B is a graph showing that the results in FIGS. 11A and 11B were not the result of anxiety-like behavior. [Figure 12A] FIG. 13 shows that sustained release exenatide (PT302, 0.12 and 0.6 mg / kg) increased novel object preference in mTBI-induced mice compared to untreated mTBI-induced mice, which exhibited visual memory deficits and spent less time near the novel object. Assessment was performed using a novel object recognition paradigm 30 days after mTBI. [Figure 12B]

[0023] Figure 1 shows that sustained release exenatide (PT302, 0.12 and 0.6 mg / kg) ameliorated mTBI spatial memory deficits observed in naive mTBI-induced mice. Assessment was performed by Y-maze 30 days after mTBI. [Figure 12C]FIG. 12C is a graph showing that the results in FIGS. 12A and 12B were not the result of anxiety-like behavior. [Figure 13A] Representative images showing that sustained release exenatide (PT302, 0.6 mg / kg) was able to prevent the neuronal loss observed in the cortex, CA3, and dentate gyrus of mTBI-induced mice. [Figure 13B] 13B is a graph showing quantification of cerebral cortex data associated with the representative images in FIG. 13A. The graph shows that sustained release exenatide (PT302, 0.6 mg / kg) was able to prevent the neuronal loss observed in the cerebral cortex of mTBI-induced mice. [Figure 13C] 13B is a graph showing quantification of CA3 data associated with the representative images of FIG. 13A. The graph shows that sustained release exenatide (PT302, 0.6 mg / kg) was able to prevent the neuronal loss observed in the CA3 region of the hippocampus in mTBI-induced mice. [Figure 13D] 13B is a graph showing quantification of dentate gyrus data associated with the representative images of FIG. 13A. The graph shows that sustained release exenatide was able to prevent the neuronal loss observed in the dentate gyrus region of the hippocampus in mTBI-induced mice. [Figure 14A] Representative images of Fluoro-Jade® C staining, a marker of neuronal loss due to brain injury, are shown in control (CTRL) and mTBI-treated mice given vehicle or sustained release exenatide (0.6 mg / kg or 0.12 mg / kg (PT302)). [Figure 14B] 14B is a graph showing quantification of data from the hippocampal CA1 region associated with the representative images from FIG. 14A, demonstrating that administration of sustained release exenatide (PT302) at 0.6 mg / kg significantly reduced the large increase in neuronal loss caused by traumatic brain injury in CA1 of untreated mTBI-induced mice. [Figure 14C]14B is a graph showing quantification of data from the hippocampal CA3 region associated with the representative images in FIG. 14A, demonstrating that administration of sustained release exenatide (PT302) at 0.6 mg / kg or 0.12 mg / kg significantly reduced the large increase in neuronal loss caused by traumatic brain injury in CA3 of untreated mTBI-induced mice. [Figure 14D] 14B is a graph showing quantification of dentate gyrus data associated with the representative images from FIG. 14A. The large increase in neuronal loss resulting from traumatic brain injury in the dentate gyrus of untreated mTBI-induced mice was significantly reduced by administration of sustained release exenatide (PT302) at 0.6 mg / kg or 0.12 mg / kg. [Figure 14E] 14B is a graph showing quantification of cerebral cortex data associated with the representative images in FIG. 14A, demonstrating that administration of sustained release exenatide (PT302) at 0.6 mg / kg significantly reduced the large increase in neuronal loss resulting from traumatic brain injury in the cortex of untreated mTBI-induced mice. [Figure 15A] Representative images are shown of ionized calcium-binding adaptor molecule 1 (IBA1) staining, a marker of microglial activation and neuroinflammation (resulting from traumatic brain injury), in control and mTBI-induced mice given vehicle or sustained-release exenatide (PT302) (0.6 mg / kg or 0.12 mg / kg). [Figure 15B] 15B is a graph showing quantification of CA1 data associated with the representative images from FIG. 15A. The graph shows that the proinflammatory cytokine TNF-α was significantly increased in IBA1+ cells in CA1 of untreated mTBI-induced mice, which was significantly reduced by administration of sustained release exenatide (PT302) at 0.6 mg / kg or 0.12 mg / kg. [Figure 15C]15B is a graph showing quantification of CA3 data associated with the representative images of FIG. 15A. The graph shows that the proinflammatory cytokine TNF-α was significantly increased in IBA1+ cells in CA3 of untreated mTBI-induced mice, which was significantly reduced by administration of sustained release exenatide (PT302) at 0.6 mg / kg or 0.12 mg / kg. [Figure 15D] 15B is a graph showing quantification of dentate gyrus data associated with the representative images from FIG. 15A. The pro-inflammatory cytokine TNF-α was significantly increased in IBA1+ cells in the dentate gyrus of untreated mTBI-induced mice, which was significantly reduced by administration of sustained release exenatide (PT302) at 0.6 mg / kg or 0.12 mg / kg. [Figure 15E] 15B is a graph showing quantification of cerebral cortex data associated with the representative images of FIG. 15A, showing that the proinflammatory cytokine TNF-α was significantly increased in IBA1+ cells in the cortex of untreated mTBI-induced mice, which was significantly reduced by administration of sustained release exenatide at 0.6 mg / kg or 0.12 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Detailed Description The present disclosure is based on the surprising and unexpected enhancement of delivery of the active ingredients described herein (e.g., GLP-1, exendin, and bioactive analogs or derivatives of GLP-1 and exendin). The subject matter described herein will be more readily understood by reference to the detailed description of the preferred embodiments of the present disclosure set forth below and the examples contained therein, as well as by reference to the drawings and accompanying text.

[0055] Prior to disclosing and describing the present compounds, compositions, articles, devices, and / or methods, it is to be understood that this disclosure is not limited to specific synthetic methods, specific treatment regimens, or specific purification procedures, which can, of course, vary. It is also to be understood that the purpose of using the terminology herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0056] All publications, patent applications, patents and other prior art documents mentioned in this specification are incorporated herein by reference in their entirety.For example, U.S. Patent No. 8,853,160, U.S. Patent No. 8,278,272, U.S. Patent No. 7,576,050, U.S. Patent No. 9,155,702, International Patent Publication No. WO / 2003 / 011892, and Gu et al. (Clinical Therapeutics.36(1):101-114(2014)) are incorporated herein by reference in their entirety for all purposes.

[0057] As used in the specification and appended claims, the singular forms "a," "an," and "the" include plural references unless the context indicates otherwise. Thus, for example, reference to a "polypeptide" includes a mixture of polypeptides, and reference to a "pharmaceutical carrier" includes a mixture of two or more pharmaceutical carriers.

[0058] As used herein, ranges may be expressed as "about" a particular value to "about" another particular value. When a range is expressed in this manner, in another embodiment, the range is included from the particular value to the other particular value. Similarly, when a value is expressed as an approximation, preceded by "about," it is understood that the particular value constitutes another embodiment. It is further understood that, for each range, each endpoint has meaning relative to the other endpoint and also independently of the other endpoint. As used herein, "about" refers to a given value ±10%.

[0059]

[0023] Many terms that may be referenced in this specification and in the claims that follow are defined to have the following meanings.

[0060] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur. The description includes both cases where such event or circumstance occurs and cases where it does not occur.

[0061] Throughout this specification, "subject" means an individual. In certain embodiments, the subject is a mammal, such as a primate. In a specific embodiment, the subject is a human. Thus, a "subject" may include domesticated animals, such as cats and dogs, farm animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.).

[0062] The terms "polypeptide" and "peptide" are generally used interchangeably unless otherwise specified. Unless otherwise specified, both "polypeptide" and "peptide" can refer to natural or unnatural amino acids joined by peptide bonds.

[0063] The term "steady state" is used in its usual sense in pharmacokinetics. Briefly, the steady state concentration, for example in plasma or cerebrospinal fluid (CSF), is the concentration in plasma and CSF when the rate at which the neuroprotective polypeptide(s) is administered is equal to the rate at which the neuroprotective polypeptide(s) is eliminated from the subject's body. Determining the steady state concentration of the neuroprotective polypeptide(s) of the present disclosure can be routinely performed by those skilled in the art.

[0064] An "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that is substantially free of materials with which the polypeptide is normally associated in nature or in culture. A polypeptide according to the present disclosure may be obtained, for example, by extraction from a natural source (e.g., a mammalian cell) if such a source is available, by expression (e.g., in a cell or in a cell-free translation system) from a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the polypeptide. A polypeptide may also be obtained by cleavage of a full-length polypeptide. When a polypeptide is a fragment of a larger naturally occurring polypeptide, an isolated polypeptide is shorter than the full-length naturally occurring polypeptide prior to fragmentation, and does not include the full-length naturally occurring polypeptide prior to fragmentation.

[0065] The present disclosure relates to a method of delivering a neuroprotective polypeptide to at least a portion of a subject's central nervous system (CNS), comprising administering a controlled release neuroprotective preparation to the subject's systemic circulatory bloodstream, the controlled release neuroprotective preparation comprising at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin (e.g., exendin-4), or a therapeutically effective GLP-1 or exendin analog (e.g., an exendin-4 analog), the neuroprotective polypeptide binds to and activates a receptor that binds at least one of GLP-1, exendin (e.g., exendin-4), or a combination thereof, and the controlled release neuroprotective preparation increases delivery of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​to at least a portion of the central nervous system (CNS) of the subject compared to an immediate release preparation of the neuroprotective polypeptide.

[0066] As shown in Figures 1A, 1B, 2, and 3, the controlled release neuroprotective preparations better maintain plasma levels of exenatide in animal models. Additionally, Figures 7B and 8B show that the controlled release preparations of exenatide are preparations and / or treatments with enhanced neuroprotective and neuroregenerative effects compared to exenatide alone.

[0067] The present disclosure relates to a method for delivering a neuroprotective polypeptide to at least a portion of a subject's central nervous system (CNS), which may include administering a therapeutically effective amount of a neuroprotective polypeptide to the subject's systemic circulatory bloodstream using a controlled release preparation or device that provides sustained release or delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprising at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation, or sustained release of the neuroprotective polypeptide, increases the delivery and / or uptake of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​of the subject to at least a portion of the central nervous system (CNS) compared to a fast release preparation of the neuroprotective peptide.

[0068] The present disclosure also relates to a method for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method may include administering a therapeutically effective amount of a neuroprotective polypeptide to the subject's systemic circulatory bloodstream using a controlled release preparation or device that provides sustained release or sustained delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation or device increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to a fast release preparation of the neuroprotective polypeptide.

[0069] In certain embodiments, the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide. The sustained release preparation may include a depot preparation for sustained release of the neuroprotective polypeptide. For example, the sustained release preparation may include a composition for sustained release of the neuroprotective polypeptide (described in detail below). In additional embodiments, the controlled release preparation further includes a biodegradable polymer having a particular viscosity and a coating material, and the bioavailability and sustained release of the neuroprotective polypeptide at an effective concentration continues for a period of time. In additional embodiments, the controlled release preparation further includes a biodegradable polymer having a particular viscosity and a coating material, and the bioavailability and sustained release of the neuroprotective polypeptide at an effective concentration continues for a period of time without an initial burst of the active ingredient (e.g., without an initial burst, such as a harmful initial burst, of the active ingredient). In a specific embodiment, the controlled release neuroprotective preparation is a depot preparation for sustained release of the neuroprotective polypeptide. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0070] In one embodiment, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in a subject.For example, the CNS-related condition may be selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron disease (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0071] Administration of the controlled release neuroprotective preparation may include injection of the controlled release neuroprotective preparation. For example, the controlled release neuroprotective preparation may be injected subcutaneously.

[0072] In another embodiment, administration of the controlled release neuroprotective preparation results in a plasma concentration of the neuroprotective polypeptide in a stable state within the range of about 50 to about 4500 pg / mL. For example, the plasma concentration of the neuroprotective polypeptide in the stable state may be about 50 to about 4500 pg / mL, about 50 to about 4250 pg / mL, about 50 to about 4000 pg / mL, about 50 to about 3750 pg / mL, about 50 to about 3500 pg / mL, about 50 to about 3250 pg / mL, about 50 to about 3000 pg / mL, about 50 to about 2750 pg / mL, about 50 to about 2500 pg / mL, about 50 to about 2250 pg / mL, about 50 to about 2000 pg / mL, about 50 to about 1750 pg / mL, about 50 to about 1500 pg / mL, about 50 to about 2 ... 0 to about 1250 pg / mL, about 50 to about 1000 pg / mL, about 50 to about 750 pg / mL, about 50 to about 500 pg / mL, about 50 to about 250 pg / mL, about 250 to about 4500 pg / mL, about 250 to about 4250 pg / mL, about 250 to about 4000 pg / mL mL, about 250 to about 3750 pg / mL, about 250 to about 3500 pg / mL, about 250 to about 3250 pg / mL, about 250 to about 3000 pg / mL, about 250 to about 2750 pg / mL, about 250 to about 2500 pg / mL, about 250 to about 2250 pg / mL, Approximately 250 to approximately 2000pg / mL, approximately 250 to approximately 1750pg / mL, approximately 250 to approximately 1500pg / mL, approximately 250 to approximately 1250pg / mL, approximately 250 to approximately 1000pg / mL, approximately 250 to approximately 750pg / mL, approximately 250 to approximately 500pg / mL, approximately 500 to approximately Approximately 4500pg / mL, approximately 500 to approximately 4250pg / mL, approximately 500 to approximately 4000pg / mL, approximately 500 to approximately 3750pg / mL, approximately 500 to approximately 3500pg / mL, approximately 500 to approximately 3250pg / mL, approximately 500 to approximately 3000pg / mL, approximately 500 to approximately 27 50pg / mL, about 500 to about 2500pg / mL, about 500 to about 2250pg / mL, about 500 to about 2000pg / mL, about 500 to about 1750pg / mL, about 500 to about 1500pg / mL, about 500 to about 1250pg / mL, about 500 to about 1000p g / mL, about 500 to about 750 pg / mL, about 750 to about 4500 pg / mL, about 750 to about 4250 pg / mL, about 750 to about 4000 pg / mL, about 750 to about 3750 pg / mL, about 750 to about 3500 pg / mL, about 750 to about 3250 pg / mL,About 750 to about 3000 pg / mL, about 750 to about 2750 pg / mL, about 750 to about 2500 pg / mL, about 750 to about 2250 pg / mL, about 750 to about 2000 pg / mL, about 750 to about 1750 pg / mL, about 750 to about 1500 pg / mL, about 750 to about 1250 pg / mL, about 750 to about 1000 pg / mL, about 1000 to about 4500 pg / mL, about 1000 to about 4250 pg / mL, about 1000 to about 4000 pg / mL, about 1000 to about 3750 pg / mL, about 1000 to about 3500 pg / mL, about 1000 to about 3250 pg / mL, about 1000 to about 3000 pg / mL, about 1000 to about 2750 pg / mL, about 1000 to about 2500 pg / mL, about 1000 to about 2250 pg / mL, about 1000 to about 2000 pg / mL, about 1000 to about 1750 pg / mL, about 1000 to about 1500 pg / mL, about 1000 to about 1250 pg / mL, about 1250 to about 4500 pg / mL, about 1250 to about 4250 pg / mL, about 1250 to about 4000 pg / mL, about 1250 to about 3750 pg / mL, about 1250 to about 3500 pg / mL, about 1250 to about 3250 pg / mL, about 1250 to about 3000 pg / mL, about 1250 to about 2750 pg / mL, about 1250 to about 2500 pg / mL, about 1250 to about 2250 pg / mL, about 1250 to about 2000 pg / mL, about 1250 to about 1750 pg / mL, about 1250 to about 1500 pg / mL, about 1500 to about 4500 pg / mL, about 1500 to about 4250 pg / mL, about 1500 to about 4000 pg / mL, about 1500 to about 3750 pg / mL, about 1500 to about 3500 pg / mL, about 1500 to about 3250 pg / mL, about 1500 to about 3000 pg / mL, about 1500 to about 2750 pg / mL, about 1500 to about 2500 pg / mL, about 1500 to about 2250 pg / mL, about 1500 to about 2000 pg / mL, about 1500 to about 1750 pg / mL, about 1750 to about 4500 pg / mL, about 1750 to about 4250 pg / mL, about 1750 to about 4000 pg / mL, about 1750 to about 3750 pg / mL, about 1750 to about 3500 pg / mL, about 1750 to about 3250 pg / mL, about 1750 to about 3000 pg / mL, about 1750 to about 2750 pg / mL, about 1750 to about 2500 pg / mL, about 1750 to about 2250 pg / mL, about 1750 to about 2000 pg / mLAbout 2000 to about 4500 pg / mL, about 2000 to about 4250 pg / mL, about 2000 to about 4000 pg / mL, about 2000 to about 3750 pg / mL, about 2000 to about 3500 pg / mL, about 2000 to about 3250 pg / mL, about 20 00 to about 3000 pg / mL, about 2000 to about 2750 pg / mL, about 2000 to about 2500 pg / mL, about 2000 to about 2250 pg / mL, about 2250 to about 4500 pg / mL, about 2250 to about 4250 pg / mL, about 2250 to about 4000pg / mL, about 2250 to about 3750pg / mL, about 2250 to about 3500pg / mL, about 2250 to about 3250pg / mL, about 2250 to about 3000pg / mL, about 2250 to about 2750pg / mL, about 2250 to about 250 0 pg / mL, about 2500 to about 4500 pg / mL, about 2500 to about 4250 pg / mL, about 2500 to about 4000 pg / mL, about 2500 to about 3750 pg / mL, about 2500 to about 3500 pg / mL, about 2500 to about 3250 pg / mL mL, about 2500 to about 3000 pg / mL, about 2500 to about 2750 pg / mL, about 2750 to about 4500 pg / mL, about 2750 to about 4250 pg / mL, about 2750 to about 4000 pg / mL, about 2750 to about 3750 pg / mL, About 2750 to about 3500 pg / mL, about 2750 to about 3250 pg / mL, about 2750 to about 3000 pg / mL, about 3000 to about 4500 pg / mL, about 3000 to about 4250 pg / mL, about 3000 to about 4000 pg / mL, about 300 It may be in the range of 0 to about 3750 pg / mL, about 3000 to about 3500 pg / mL, about 3000 to about 3250 pg / mL, about 3250 to about 4500 pg / mL, about 3250 to about 4250 pg / mL, about 3250 to about 4000 pg / mL, about 3250 to about 3750 pg / mL, about 3250 to about 3500 pg / mL, about 3500 to about 4500 pg / mL, about 3500 to about 4250 pg / mL, about 3500 to about 4000 pg / mL, or about 3500 to about 3750 pg / mL.

[0073] As described above, the concentration of the neuroprotective polypeptide in the plasma is maintained at a steady state, thereby cumulatively increasing the concentration of the neuroprotective polypeptide in the subject's cerebrospinal fluid (CSF), brain, or a combination thereof. For example, the concentration of the neuroprotective polypeptide in the CSF may be in the range of about 5 to about 400 pg / mL or about 10 to about 400 pg / mL. That is, the concentration of the neuroprotective polypeptide in the CSF is about 10 to about 350 pg / mL, about 10 to about 300 pg / mL, about 10 to about 250 pg / mL, about 10 to about 200 pg / mL, about 10 to about 150 pg / mL, about 10 to about 100 pg / mL, about 10 to about 50 pg / mL, about 50 to about 400 pg / mL, about 50 to about 350 pg / mL, about 50 to about 300 pg / mL, about 50 to about 250 pg / mL, about 50 to about 200 pg / mL, about 50 to about 150 pg / mL, about 50 to about 100 pg / mL, about 100 to about 400 pg / mL, about 100 to about 350 pg / mL, about 100 to about 300 pg / mL, about 100 to about 250 pg / mL, about 100 to about 200 pg / mL, about 100 to about 150 pg / mL, about 150 to about 400 pg / mL, about 150 to about 350 pg / mL, about 1 50 to about 300 pg / mL, about 150 to about 250 pg / mL, about 150 to about 200 pg / mL, about 200 to about 400 pg / mL, about 200 to about 350 pg / mL, about 200 to about 300 pg / mL, about 200 to about 250 pg / mL, about 250 to about 400 pg / mL, about 250 to about 350 pg / mL, about 250 to about 300 pg / mL, about 300 to about 400 pg / mL, about 300 to about 350 pg / mL, or about 350 to about 400 pg / mL.

[0074] In any aspect or embodiment described herein, the ratio of the concentration of the polypeptide in the CSF at steady state to the concentration of the polypeptide in plasma at steady state may be in the range of about 0.1% to about 5%. For example, the ratio of the concentration of the polypeptide in the CSF at steady state to the concentration of the polypeptide in plasma at steady state may be at least about 0.1%, at least about 0.3%, at least about 0.5%, at least about 0.7%, at least about 0.9%, at least about 1%, at least about 1.1%, at least about 1.2%, at least about 1.5 ... may be about 2.0%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5%.

[0075] In any aspect or embodiment described herein, the percent change in plasma concentration level of the neuroprotective polypeptide at steady state (i.e., the percent change after reaching the steady state) may be about 80% or less (e.g., about 50% or less). For example, when re-administration is performed after the steady state plasma level and / or concentration is reached, the percent change in plasma concentration of the neuroprotective polypeptide may be about 80% or less (e.g., about 50% or less) (e.g., the plasma concentration of the neuroprotective polypeptide may reach a plateau about 2, about 3, about 4, about 5, or about 6 weeks after the first administration).For example, the percent change in plasma concentration of a neuroprotective polypeptide at steady state after reaching steady state can be, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, , about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, or about 28 days after administration), about 80% or less, about 79% or less, about 78% or less, about 77% or less, about 76% or less, about 75% or less, about 74% or less, about 73% or less, about 72% or less, about 71% or less, about 70% or less, about 69% or less, about 68% or less, about 67% or less, about 66% or less, about 65% or less, about 64% or less, about 63% or less, about 65% or less, about 66% or less, about 67% or less, about 68% or less, about 68% or less, about 69 ... 2% or less, 61% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less Lower, about 47% or less, about 46% or less, about 45% or less, about 44% or less, about 43% or less, about 42% or less, about 41% or less, about 40% or less, about 39% or less, about 39% or less, about 37% or less, about 36% or less, about 35% or less, about 34% or less, about 33% or less, about 32% or less, about 31% or less, about 30% or less, about 29% or less, about 28% or less, about 27% or less, about 26% or less, about 25% or less, about 24% or less, about 23% or less, about 22% or less, about 21% or less, about 20% or less, about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, or about 5% or less.

[0076] In any aspect or embodiment described herein, the preparation is administered once every about 7 to about 28 days (e.g., about 7 to about 21 days or about 7 to about 14 days). For example, the preparation may be administered multiple times, with the administration interval being about 7 to about 28 days (e.g., once every about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, or about 28 days) (e.g., a subsequent administration is administered about 7 to about 28 days after the preceding administration). The preparation, in any aspect or embodiment described herein, may be administered multiple times, where the administration of the preparation is at intervals of about 7 to about 28 days (e.g., about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 21 days (i.e., once every about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 26, about 27, or about 28 days).

[0077] In a further embodiment, a method is provided for treating a subject having a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method comprises administering a therapeutically effective amount of a controlled release neuroprotective preparation to the subject into the subject's systemic circulatory bloodstream, the controlled release neuroprotective preparation comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin (e.g., exendin-4), or a therapeutically effective GLP-1 or exendin (e.g., exendin-4) analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin (e.g., exendin-4), or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to the neuroprotective polypeptide alone.

[0078] In a specific embodiment, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in a subject, for example, the CNS condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0079] In further embodiments, administering the controlled release neuroprotective preparation comprises injecting the controlled release neuroprotective preparation into the subject. For example, administering the controlled release neuroprotective preparation comprises subcutaneously injecting the controlled release neuroprotective preparation into the subject. In additional embodiments, the controlled release preparation further comprises a biodegradable polymer having a particular viscosity and a coating material, whereby the bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time. In any aspect or embodiment described herein, the controlled release preparation provides bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time without an initial burst (e.g., a deleterious initial burst) of the active ingredient. In a specific embodiment, the controlled release neuroprotective preparation provides a neuroprotective polypeptide at effective concentrations without an initial burst (e.g., a deleterious initial burst) of the active ingredient. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0080] In additional embodiments, administration of the controlled release preparation results in a steady state concentration of the neuroprotective polypeptide in plasma within the ranges described above, for example, between about 50 and about 4500 pg / mL.

[0081] In some embodiments, administration of the controlled release preparation cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0082] In a further aspect, the present disclosure provides a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject, comprising providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery and / or uptake of the neuroprotective polypeptide through the blood-brain barrier (BBB) ​​of the subject to at least a portion of the CNS compared to a fast release preparation of the neuroprotective peptide.

[0083] In yet another embodiment, the present disclosure provides a method for treating a subject with a CNS-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method includes providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide through the BBB to at least a portion of the CNS of the subject compared to a fast release preparation of the neuroprotective polypeptide. In any aspect or embodiment described herein, providing sustained release of the neuroprotective polypeptide(s) includes administering the neuroprotective polypeptide(s) using a device (e.g., a pump, a mini-pump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini-infusion pump, etc.).

[0084] The neuroprotective polypeptide(s) (e.g., in a controlled release neuroprotective formulation) may be administered at a rate of about 0.5 pM / kg / min to about 35 pM / kg / min (e.g., about 3 pM / kg / min to about 17.5 pM / kg / min). For example, the neuroprotective polypeptide(s) may be administered at a concentration of from about 0.5 pM / kg / min to about 35 pM / kg / min, from about 0.5 pM / kg / min to about 35 pM / kg / min, from about 0.5 pM / kg / min to about 32.5 pM / kg / min, from about 0.5 pM / kg / min to about 30 pM / kg / min, from about 0.5 pM / kg / min to about 27.5 pM / kg / min, from about 0.5 pM / kg / min to about 25 pM / kg / min, from about 0.5 pM / kg / min to about 22.5 pM / kg / min, from about 0.5 pM / kg / min to about 20 pM / kg / min, about 0.5pM / kg / min to about 17.5pM / kg / min, about 0.5pM / kg / min to about 15pM / kg / min, about 0.5pM / kg / min to about 12.5pM / kg / min, about 0.5pM / kg / min to about 10pM / kg / min, about 1.5pM / kg / min ~ approx. 35pM / kg / min, approx. 1.5pM / kg / min ~ approx. 35pM / kg / min, approx. 1.5pM / kg / min ~ approx. 32.5pM / kg / min, approx. 1.5pM / kg / min ~ approx. 30pM / kg / min, approx. 1.5pM / kg / min ~ approx. 27.5pM / kg / min , about 1.5pM / kg / min to about 25pM / kg / min, about 1.5pM / kg / min to about 22.5pM / kg / min, about 1.5pM / kg / min to about 20pM / kg / min, about 1.5pM / kg / min to about 17.5pM / kg / min, about 1.5pM / kg / min ~15pM / kg / min, approx. 1.5pM / kg / min ~ approx. 12.5pM / kg / min, approx. 1.5pM / kg / min ~ approx. 10pM / kg / min, approx. 2.5pM / kg / min ~ approx. 35pM / kg / min, approx. 2.5pM / kg / min ~ approx. 35pM / kg / min, approx. 2. 5pM / kg / min ~ approx. 32.5pM / kg / min, approx. 2.5pM / kg / min ~ approx. 30pM / kg / min, approx. 2.5pM / kg / min ~ approx. 27.5pM / kg / min, approx. 2.5pM / kg / min ~ approx. 25pM / kg / min, approx. 2.5pM / kg / min ~ approx. 22 .5pM / kg / min, about 2.5pM / kg / min to about 20pM / kg / min, about 2.5pM / kg / min to about 17.5pM / kg / min, about 2.5pM / kg / min to about 15pM / kg / min, about 2.5pM / kg / min to about 12.5pM / kg / min, about 2.5pM / kg / min ~ approx. 10pM / kg / min, approx. 5pM / kg / min ~ approx. 35pM / kg / min, approx. 5pM / kg / min ~ approx. 35pM / kg / min, approx. 5pM / kg / min ~ approx. 32.5pM / kg / min, approx. 5pM / kg / min min ~ approx. 30pM / kg / min, approx. 5pM / kg / min ~ approx. 27.5pM / kg / min, approx. 5pM / kg / min ~ approx. 25pM / kg / min, approx. 5pM / kg / min ~ approx. 22.5pM / kg / min, approx. 5pM / kg / min ~ approx. 20p M / kg / min, about 5pM / kg / min to about 17.5pM / kg / min, about 5pM / kg / min to about 15pM / kg / min, about 5pM / kg / min to about 12.5pM / kg / min, about 5pM / kg / min to about 10pM / kg / min , about 10pM / kg / min to about 35pM / kg / min, about 10pM / kg / min to about 35pM / kg / min, about 10pM / kg / min to about 32.5pM / kg / min, about 10pM / kg / min to about 30pM / kg / min, about 10 pM / kg / min ~ approx. 27.5pM / kg / min, approx. 10pM / kg / min ~ approx. 25pM / kg / min, approx. 10pM / kg / min ~ approx. 22.5pM / kg / min, approx. 10pM / kg / min ~ approx. 20pM / kg / min, approx. 10p M / kg / min ~ approx. 17.5pM / kg / min, approx. 15pM / kg / min ~ approx. 35pM / kg / min, approx. 15pM / kg / min ~ approx. 35pM / kg / min, approx. 15pM / kg / min ~ approx. 32.5pM / kg / min, approx. It may be administered at a rate of about 15 pM / kg / min to about 30 pM / kg / min, about 15 pM / kg / min to about 27.5 pM / kg / min, about 15 pM / kg / min to about 25 pM / kg / min, about 20 pM / kg / min to about 35 pM / kg / min, about 20 pM / kg / min to about 35 pM / kg / min, about 20 pM / kg / min to about 32.5 pM / kg / min, about 20 pM / kg / min to about 30 pM / kg / min, or about 25 pM / kg / min to about 35 pM / kg / min.

[0085] In any aspect or embodiment described herein, the controlled release preparation or neuroprotective polypeptide(s) may be administered by a device. For example, the device may be an implantable device that contains and delivers the controlled release preparation or neuroprotective polypeptide(s). The device may be a pump, a mini-pump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini-infusion pump. In any aspect or embodiment described herein, the device may be an implantable device that can provide the controlled release preparation or neuroprotective polypeptide(s) at a constant flow rate, an adjustable flow rate, or a programmable flow rate. For example, osmotic delivery devices such as those described in U.S. Pat. No. 8,298,561 B2 or U.S. Pat. No. 8,940,316 B2, both of which are incorporated herein by reference in their entirety.

[0086] The term "osmotic delivery device" as used herein refers to a device used to deliver one or more beneficial agents (e.g., neuroprotective polypeptide(s) or controlled release preparations) to a subject, the device having, for example, a container (e.g., made of a titanium alloy) having an internal cavity containing a suspension formulation (e.g., containing a neuroprotective polypeptide(s) or controlled release preparation) and an osmotic agent preparation. A piston assembly disposed within the internal cavity separates the osmotic agent preparation from the suspension preparation. A semi-permeable membrane is disposed at a first distal end of the container adjacent to the osmotic agent preparation, and a flow regulator is disposed at a second distal end of the container adjacent to the suspension preparation (the flow regulator defines a delivery hole through which the suspension preparation is released from the device). The osmotic delivery device or osmotic pump may be implanted within the subject's body, for example subcutaneously (eg, in the inner, outer, or back of the upper arm, or in the abdominal area).

[0087] The neuroprotective polypeptide(s) or controlled release preparations described herein may be administered by a device to provide sustained delivery of the neuroprotective polypeptide(s) or controlled release preparations over an extended period of time, such as weeks, months, or up to about a year. The device may be an implantable device, capable of delivering the neuroprotective polypeptide(s) or controlled release preparations at a desired flow rate for a desired period of time. The introduction of the neuroprotective polypeptide(s) or controlled release preparations into the implantable drug delivery device may be performed by conventional techniques.

[0088] The neuroprotective polypeptide(s) or controlled release preparations may be delivered, for example, using an osmotically, mechanically, electromechanically, or chemically driven device. In any aspect or embodiment described herein, the neuroprotective polypeptide(s) or controlled release preparations are delivered at a flow rate that is therapeutically effective for a subject in need of treatment with the neuroprotective polypeptide(s).

[0089] The neuroprotective polypeptide(s) or controlled release preparation may be delivered for a period ranging from more than about one week to about one year or more (e.g., from about one month to about one year or more, or from about three months to about one year or more). The device may have a container with at least one hole through which the neuroprotective polypeptide(s) or controlled release preparation is delivered. The neuroprotective polypeptide(s) or controlled release preparation may be stored in the container. In any aspect or embodiment described herein, the device is an osmotic delivery device, which may be an implantable device, and the delivery of the neuroprotective polypeptide(s) or controlled release preparation is driven by osmotic pressure. Several osmotic delivery devices or osmotic pumps and components thereof have been described. For example, the DUROS® delivery device or similar devices (e.g., U.S. Pat. Nos. 5,609,885, 5,728,396, 5,985,305, 5,997,527, 6,113,938, 6,132,420, 6,156,331, 6,217,906, 6,261,584, 6,270,787, 6,287,295, 6,375,978, 6,395,292, 6,508,808, etc.) may be used. , 6,544,252, 6,635,268, 6,682,522, 6,923,800, 6,939,556, 6,976,981, 6,997,922, 7,014,636, 7,207,982, 7,112,335, and 7,163,688, and U.S. Patent Application Nos. 2005-0175701, 2007-0281024, and 2008-0091176, which are incorporated by reference in their entireties.

[0090] The DUROS® delivery device typically consists of a cylindrical container that contains an osmotic engine, a piston, and a drug formulation. The container is sealed at one end with a rate-controlling water-permeable membrane and at the other end with a diffusion regulator through which the drug formulation is released from the drug container. The piston separates the drug formulation from the osmotic engine. The piston contains a sealant to prevent water in the osmotic engine compartment from flowing into the drug container. The diffusion regulator is designed to prevent bodily fluids from flowing into the drug container through the holes along with the drug preparation (e.g., neuroprotective polypeptide(s) or controlled release formulation).

[0091] The DUROS® device releases therapeutic agents at a predetermined rate based on the principle of osmosis. Extracellular fluid flows directly into the salt engine in the DUROS® device through a semi-permeable membrane, which expands and propels a piston at a slow, constant delivery rate. The movement of the piston releases the drug preparation (e.g., neuroprotective polypeptide(s) or controlled release preparation) through a hole or exit hole at a predetermined shear rate. In any aspect or embodiment of the present invention, a neuroprotective polypeptide(s) or controlled release preparation is introduced into the container of the DUROS® device, which can deliver the neuroprotective polypeptide(s) or controlled release preparation to a subject over an extended period of time (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months) at a predetermined, therapeutically effective delivery rate.

[0092] Any implantable device may be used in the practice of the present disclosure, which may include a regulated implantable pump capable of providing compounds at a constant, adjustable, or programmable flow rate.

[0093] The amount of neuroprotective polypeptide(s) or controlled release preparation used in the delivery device of the present disclosure is the amount required to deliver a therapeutically effective amount of the agent to achieve the desired therapeutic outcome. In practice, this amount may vary depending on variables such as, for example, the type of agent, the delivery site, the severity of the condition, and the desired therapeutic effect. Typically, the volume of the beneficial agent chamber for containing the neuroprotective polypeptide(s) or controlled release preparation of the osmotic delivery device is about 100 μl to about 1000 μl (e.g., about 120 μl and about 500 μl, or about 150 μl and about 200 μl).

[0094] In any aspect or embodiment described herein, the osmotic delivery device is implanted in the subject's body, for example subcutaneously. The device may be inserted in one or both arms (e.g., inside, outside, or back of the upper arm) or in the abdomen. For example, the device may be implanted under the skin of the abdomen in an area below the ribs and above the belt line. In order to provide multiple locations for inserting one or more osmotic delivery devices into the abdomen, the abdominal wall may be divided into four quadrants as follows: a right upper quadrant extending 5-8 centimeters below the right ribs and approximately 5-8 centimeters to the right of the midline; a right lower quadrant extending 5-8 centimeters above the belt line and approximately 5-8 centimeters to the right of the midline; a left upper quadrant extending 5-8 centimeters below the left ribs and approximately 5-8 centimeters to the left of the midline; and a left lower quadrant extending 5-8 centimeters above the belt line and approximately 5-8 centimeters to the left of the midline. This provides multiple locations that can be used to implant one or more devices in one or more instances.

[0095] In other embodiments, administration of a controlled release neuroprotective preparation or providing sustained delivery of a neuroprotective polypeptide alleviates at least one symptom of at least one CNS-related condition selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof in a subject.

[0096] In any aspect or embodiment described herein, administration of a controlled release preparation or providing sustained release of the neuroprotective polypeptide results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0097] In any aspect or embodiment described herein, administration of a controlled release preparation or providing sustained release of a neuroprotective polypeptide cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0098] Neuroprotective Polypeptides The neuroprotective polypeptide may have an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55. The neuroprotective polypeptide may comprise an amino acid sequence selected from SEQ ID NOs:1-55. Furthermore, the neuroprotective polypeptide may consist of an amino acid sequence selected from SEQ ID NOs: 1-55.

[0099] "Analogue of GLP-1 or (or) exendin-4" refers to modified GLP-1 and / or exendin amino acid sequences that exhibit agonist properties (i.e., exhibit one or more biological activities of GLP-1 or exendin-4). Such modifications include chimeric polypeptides that contain one or more amino acid residues present in GLP-1 and one or more amino acid residues present in exendin-4. The above modifications also include truncations of either GLP-1 or exendin-4 or the chimeric polypeptides thereof. For example, a truncated chimeric polypeptide is one in which G at position 36 in exendin-4 7-36 is replaced with R at position 36 in GLP-1. The polypeptides of the present disclosure include one or more additions (i.e., insertions or additions), deletions, or substitutions of amino acids in the amino acid sequence of GLP-1 or exendin-4 to an extent that does not result in a significant decrease in functional activity compared to GLP-1 or exendin-4. For example, the deletion may be of an amino acid that is not essential for conferring the differentiation-inducing activity defined in the present invention, and the substitution may be conservative (i.e., replacing a basic, hydrophilic, or hydrophobic amino acid with an amino acid having the same properties) or non-conservative. A conservative substitution is one in which the structural or chemical properties of the substituted amino acid are similar to those of the corresponding amino acid in the reference sequence.For example, conservative amino acid substitution involves replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic or hydrophobic amino acid, replacing one hydroxyl-containing amino acid, such as serine and threonine, with another hydroxyl-containing amino acid, replacing one acidic residue, such as glutamic acid or aspartic acid, with another acidic residue, replacing one amide-containing residue, such as asparagine and glutamine, replacing one aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue, replacing one basic residue, such as lysine, arginine, and histidine, with another basic residue, and replacing one small amino acid, such as alanine, serine, threonine, methionine, and glycine, with another small amino acid.Therefore, it is understood that the amino acid sequences of GLP-1 and exendin-4 may be modified and changed if desired, and still obtain proteins with similar characteristics. Various modifications may be made to the amino acid sequence of GLP-1 or exendin-4 (or the nucleic acid sequence from which it is derived) without significantly decreasing the biological utility or activity, and preferably, enhancing such utility or activity.

[0100] As used herein, the term "fragment" or "truncation" in reference to GLP-1, or exendin-4, or a polypeptide having an amino acid sequence substantially homologous thereto, refers to a polypeptide sequence consisting of at least 5 consecutive amino acids of either GLP-1, exendin-4, or a polypeptide having an amino acid sequence substantially homologous thereto, which polypeptide sequence has insulin secretion function.

[0101] Other modifications include D-enantiomers, in which at least one naturally occurring L-configuration of an amino acid residue is replaced with the D-configuration of that amino acid residue.

[0102] The present disclosure contemplates the use of spacers, such as lateral spacers. The term "lateral spacer" is defined as a compound incorporated into an amino acid sequence by chemical bond. The compound increases the distance between two or more amino acid residues, thereby making the amino acid sequence less susceptible or non-responsive to cleavage (e.g., cleavage by DPP 1V) at or near the position where the compound is incorporated. For example, a sequence AXB having amino acid residues A and B and a lateral spacer X is less susceptible or non-responsive to cleavage by an enzyme compared to a sequence (AB) without a lateral spacer. For example, one to four compounds may be incorporated into an amino acid sequence as lateral spacers. Thus, in various embodiments, one, two, three, or four compounds are inserted.

[0103] In general, the lateral spacer is any compound capable of forming a peptide bond with an amino acid, i.e., containing at least one amino group and at least one carboxy group (CO2), which may be a carboxylic acid or an ester or salt thereof. In one embodiment, the lateral spacer has the formula H2N-R 1 -COH(I), where R 1 comprises a substituted or unsubstituted, branched or straight chain, alkyl, alkenyl, or alkynyl group having 1 to 20 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 8 carbon atoms, a substituted or unsubstituted aryl group having 6 to 20 carbon atoms, or a substituted or unsubstituted heteroaryl group having 4 to 20 carbon atoms. 1 is the formula (CH2) n where n is 1 to 10. In one embodiment, R 1 is (CH2)3 (3-aminopropionic acid) or (CH2)5 (6-aminohexanoic acid).

[0104] The disclosure provides methods that include administration of a controlled release formulation that includes at least one neuroprotective polypeptide. The polypeptide may be a modified GLP-1 or exendin (e.g., exendin-4) sequence, or an analog or derivative thereof, in which a spacer is placed between the amino acid residues corresponding to the 7th and 8th residues of GLP-1 (e.g., "GLP-1 Aha" for GLP-1 with an Aha spacer). 8 "), or with a spacer between the amino acid residues corresponding to the 8th and 9th residues of GLP-1 (e.g., GLP-1 with an Aha spacer is referred to as "GLP-1Aha"). 9 The lateral spacer, in one embodiment, is one or more aminoproprionic acid residues. In one embodiment, the spacer is a 6-aminohexanoic acid spacer that contains fewer than four 6-aminohexanoic acid residues. A polypeptide may be, for example, one or more 6-aminohexanoic acid residues between the seventh and eighth residues of GLP-1 7-36 (i.e., GLP-1 Aha 8 ), or one or more 6-aminohexanoic acid residues between the 8th and 9th residues of GLP-1 7-36. The polypeptide may include two or more 6-aminohexanoic acid residues between the 7th and 8th residues of GLP-1 7-36 (i.e., GLP-1 Aha 8 ), or two or more 6-aminohexanoic acid residues between the 8th and 9th residues of GLP-1 7-36. The polypeptide may, for example, include three or more 6-aminohexanoic acid residues between the 7th and 8th residues of GLP-1 7-36 (i.e., GLP-1 Aha 8 ), or may include three or more 6-aminohexanoic acid residues between the 8th and 9th residues of GLP-1 7-36.

[0105] In another embodiment, the polypeptide according to the present disclosure has an insulinotropic effect equivalent to an equimolar amount of GLP-1, or in one embodiment, an insulinotropic effect equivalent to an equimolar amount of exendin-4. "Equivalent effect" means about 10-15% of the effect of GLP-1 or exendin-4. In another embodiment, the polypeptide has an insulinotropic effect that exceeds the insulinotropic effect of either GLP-1 or exendin-4. "Exceeding the effect" of GLP-1 or exendin-4 means that the insulinotropic effect is increased compared to GLP-1 or exendin-4, for example, the extent of the increase is greater than about 10% of the effect of GLP-1 or exendin-4. Thus, in one embodiment, the polypeptide according to the present disclosure has the same degree of efficacy as GLP-1 or exendin-4, and in another embodiment, the polypeptide according to the present disclosure has a higher efficacy than GLP-1 and, optionally, a higher efficacy than exendin-4. In another embodiment, the polypeptide according to the present disclosure has a longer acting time than GLP-1. In a further embodiment, the polypeptide has a longer acting time at least as long as exendin-4. In another embodiment, the polypeptide has a longer acting time than exendin-4. "More sustained acting than" means that the polypeptide is more resistant to at least one degradative enzyme than GLP-1 or exendin-4. For example, the polypeptide according to the present disclosure is more resistant to degradation by the enzyme dipeptidyl peptidase-4 (DPPIV) than GLP-1 and, optionally, exendin-4. Such resistance to one or more degradative enzymes can be directly determined by detecting the amount of degradation products (e.g., the amount of N-terminal degradation products) or the amount of uncleaved polypeptide. Alternatively, such resistance to one or more degradative enzymes can be indirectly detected by determining the decrease in insulin secretory effect over time after administration of the polypeptide according to the present disclosure. For example, plasma insulin levels will decrease after a single administration as the degradative enzyme cleaves the polypeptide according to the present disclosure.In additional embodiments, the rate of this decline may be slower than that of GLP-1, and / or possibly even slower than that of exendin-4.

[0106] The polypeptides of the present disclosure may be prepared by any of several chemical polypeptide synthesis techniques, including solution and solid-phase techniques, which are well known to those of ordinary skill in the art. Solid-phase synthesis involves the attachment of the C-terminal amino acid of a polypeptide sequence to an insoluble support, followed by the sequential addition of the remaining amino acids of the sequence, and is one of the synthetic methods for preparing polypeptides. The solid-phase synthesis technique is described by Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963). Many automated systems for performing solid-phase peptide synthesis are commercially available.

[0107] Solid phase synthesis is carried out starting from the carboxy terminus (i.e., C-terminus) of the polypeptide by coupling a protected amino acid through its carboxy group to a suitable solid support. The solid support used is of no critical nature, so long as it is capable of binding to the carboxy group and is substantially inert to the reagents used in the peptide synthesis procedure. For example, the starting material may be prepared by attaching an amino-protected amino acid to a chloromethylated or hydroxymethyl resin via a benzyl ester bond, or to a benzhydrylamine (BHA) or p-methylbenzhydrylamine (MBHA) resin via an amide bond. Materials suitable for use as solid supports are widely known to those skilled in the art and include, but are not limited to, halomethyl resins such as chloromethyl or bromomethyl resins, hydroxymethyl resins, phenolic resins such as 4-(a-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin, and tert-alkyloxycarbonylhydrazide resins. Such resins are commercially available and methods for their preparation are well known to those of ordinary skill in the art.

[0108] The acid form of the peptide may be prepared by a solid phase peptide synthesis procedure using a benzyl ester resin as the solid support. The corresponding amide may be generated by using a benzhydrylamine or methylbenzhydrylamine resin as the solid support. Those skilled in the art will recognize that when BHA or MBHA resins are used, cleavage of the peptide from the solid support is accomplished by treatment with anhydrous hydrofluoric acid to generate a peptide with a terminal amide group.

[0109] The α-amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive α-amino functionality. Amino acids containing additional reactive side chain functional groups (e.g., sulfhydryl, amino, carboxy, hydroxy, etc.) must be protected with an appropriate protecting group to prevent chemical reactions at those sites during peptide synthesis. Protecting groups are well known to those skilled in the art. See, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, NY (1981)).

[0110] An appropriately selected α-amino protecting group should be able to inactivate the α-amino functionality during the coupling reaction, be easily removed after coupling under conditions that do not result in removal of the side chain protecting group, preserve the structure of the peptide fragment unchanged, and prevent racemization due to activation immediately prior to coupling. Similarly, the side chain protecting group must be selected to inactivate the side chain functionality during synthesis, be stable under the conditions used for removal of the α-amino protecting group, and be removable after peptide synthesis under conditions that do not alter the structure of the peptide.

[0111] Coupling of amino acids may be carried out by various techniques well known to those skilled in the art. A typical method involves derivatizing the amino acid to make it more amenable to reaction of its carboxy group with the free N-terminal amino group of the peptide fragment, or using a suitable coupling agent, such as N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIPCDI). Hydroxybenzotriazole (HOBt) is often used as a catalyst for such coupling reactions.

[0112] Generally, peptide synthesis begins by coupling the C-terminal amino acid, whose N-amino position is protected with a protecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solid support. Before coupling Fmoc-Asn, the Fmoc residue must be cleaved from the polymer. For example, Fmoc-Asn can be coupled to 4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin using N,N'-dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt) at about 25°C for about 2 hours with stirring. After coupling of the Fmoc-protected amino acid to the resin support, the α-amino protecting group is removed using 20% ​​piperidine in DMF at room temperature.

[0113] After removal of the α-amino protecting group, the remaining Fmoc-protected amino acids are coupled stepwise in the desired order. Suitably protected amino acids are commercially available from several sources (e.g., Novartis, Switzerland or Bachem, Torrance, Calif.). Instead of adding amino acids stepwise, suitably protected peptide fragments consisting of more than one amino acid may be coupled to the "growing" peptide. Selection of appropriate coupling reagents is well within the knowledge of one skilled in the art, as described above.

[0114] Each of the protected amino acids or amino acid sequences is introduced in excess into a solid-phase reactor, and coupling is carried out in dimethylformamide (DMF), methylene chloride (CH2Cl2), or a mixture thereof. If the coupling is incomplete, the coupling reaction may be repeated until deprotection of the N-amino group and addition of the next amino acid. Monitoring of the coupling efficiency may be carried out by several means well known to those skilled in the art. The method for monitoring the coupling efficiency may be the ninhydrin reaction. The peptide synthesis reaction may be carried out automatically using several commercially available peptide synthesizers, such as the Biosearch 9500™ synthesizer (Biosearch, San Raphael, Calif.).

[0115] The peptide may be cleaved and the protecting groups removed by stirring the insoluble or solid support in anhydrous liquid hydrogen fluoride (HF) in the presence of anisole and dimethyl sulfide at about 0° C. for about 20 to about 90 minutes, for example 60 minutes, or by continuously bubbling hydrogen bromide (HBr) through a suspension of 1 mg / 10 mL of resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting group selected, or by incubating the solid support with 90% trifluoroacetic acid, 5% water, and 5% triethylsilane for about 30 to about 60 minutes in the reaction column used for the solid phase synthesis. Other deprotection methods well known to those skilled in the art may also be used.

[0116] The peptides may be isolated and purified from the reaction mixture by peptide purification means well known to those skilled in the art, for example, the peptides may be purified by well-known chromatographic methods such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent partition.

[0117] The preparation of neuroprotective polypeptides may also be carried out by other means, such as recombinant techniques. For examples of suitable cloning and sequencing techniques and sufficient explanations for those skilled in the art to carry out many cloning exercises, see Sambrook et al. (1989) Molecular Cloning-A Laboratory Manual (2nd ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook).

[0118] For example, the exendin derivative(s) used in the controlled release formulation can be a compound represented by Formula I, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa27 Xaa28 -Z1 (Chemical formula I) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg; Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, or a linear or branched alkanoyl having 1 to 10 carbon atoms; Xaa22 is Ala, Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn, and Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; Xaa39 is Ser, or Tyr (e.g., Ser); and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27, and Xaa28 are Ala; and When Xaa1 is His, Arg, or Tyr, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0119] In some embodiments, the N-alkyl group of N-alkylglycine, N-alkylpentylglycine, and N-alkylalanine can include a lower alkyl group, e.g., having 1 to about 6 carbon atoms or 1 to 4 carbon atoms. Compounds of Formula I can include the compounds set forth in Examples 1-89 (Compounds 1-89, respectively) of PCT Application No. PCT / US98 / 24273, filed November 13, 1998, entitled "Novel Exendin Agonist Compounds," which is incorporated by reference in its entirety for all purposes, as well as the corresponding compounds set forth in Examples 104 and 105 thereof.

[0120] In a specific embodiment, exendin derivatives of formula I may include those in which Xaa1 of formula I is His, Ala, Norval, or 4-imidazopropionyl. In another embodiment, Xaa1 of formula I is His, Ala, or 4-imidazopropionyl. In an additional embodiment, Xaa1 of formula I is His or 4-imidazopropionyl.

[0121] The exendin derivative of formula I can be one where Xaa2 is Gly. The exendin derivative of formula I can be one where Xaa3 is Ala.

[0122] The exendin derivative of formula I can be one where Xaa4 is Ala. The exendin derivative of formula I can be one where Xaa9 is Ala.

[0123] Exendin derivatives of Formula I can be those where Xaa14 is Leu, pentylglycine, or Met.

[0124] The exendin derivative of formula I can be one in which Xaa21 is Lys-NHε-R, where R is Lys, Arg, or a straight or branched alkanoyl having 1 to 10 carbon atoms.

[0125] In one embodiment, the exendin derivative of formula I can be one where Xaa25 is Trp or Phe.

[0126] In another embodiment, the exendin derivative of formula I can be one in which Xaa6 is Ala, Phe, or naphthylalanine, Xaa22 is Phe or naphthylalanine, and Xaa23 is Ile or Val. Additionally, the exendin derivative of formula I can be one in which Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, thioproline, and N-alkylalanine, Z1 can be -NH2, and Z2 can be -NH2.

[0127] In other embodiments, the exendin derivative of formula I may be one in which Xaa1 is Ala, His, or Tyr (e.g., Ala or His), Xaa2 is Ala or Gly, Xaa6 is Phe or naphthylalanine, Xaa14 is Ala, Leu, pentylglycine, or Met, Xaa22 is Phe or naphthylalanine, Xaa23 is Ile or Val, Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, thioproline, and N-alkylalanine, Xaa39 is Ser or Tyr (e.g., Ser), and Z1 may be -NH2.

[0128] According to one embodiment, the exendin derivative of formula I is selected from the group consisting of Xaa1, His or Ala, Xaa2, Gly or Ala, Xaa3, Ala, Asp or Glu, Xaa4, Ala or Gly, Xaa5, Ala or Thr, Xaa6, Phe or naphthylalanine, Xaa7, Thr or Ser, Xaa8, Ala, Ser or Thr, Xaa9, Ala, Asp or Glu, Xaa10, Ala, Leu or pentylglycine, Xaa11, Ala or Ser, Xaa12, Ala or Lys, Xaa13, Ala or Gln, and Xaa14, Ala, Leu, Met, or Glu. or pentylglycine, Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg, Xaa21 is Ala or Leu, Xaa22 is Phe or naphthylalanine, Xaa23 is Ile, Val, or tert-butylglycine, Xaa24 is Ala, Glu, or Asp, Xaa25 is Ala, Trp, or Phe, Xaa26 is Ala or Leu, Xaa27 is Ala or Lys, Xaa28 is Ala or Asn, and Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37Xaa39-Z2, Xaa31, Xaa36, Xaa37, and Xaa38 are independently Pro, homoproline, thioproline, or N-methylalanine, Xaa39 is Ser or Tyr (e.g., Ser), and Z2 is -OH or -NH2, with the proviso that Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Of Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27, and Xaa28, not more than three are Ala, and when Xaa1 is His, Arg, or Tyr, at least one of Xaa3, Xaa4, and Xaa9 may be Ala.

[0129] In further embodiments, compounds of Formula I may include those having the amino acid sequences of SEQ ID NOs: 5-93 as set forth in PCT Application No. PCT / US98 / 25728, or those as set forth in U.S. Provisional Application No. 60 / 066,029, which are incorporated herein by reference.

[0130] In one embodiment, compounds are provided in which Xaa14 is Leu, Ile, Val, or pentylglycine (e.g., Leu or pentylglycine) and Xaa25 is Ala, Phe, Tyr, or naphthylalanine (e.g., Phe or naphthylalanine). These compounds will be less susceptible to oxidative degradation both in vitro and in vivo, and during the synthesis of the compounds.

[0131] In another embodiment, the exendin derivative may include a compound represented by formula II, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala X1-Z1 (Chemical formula II) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg, Xaa21 is Ala, Leu, or Lys-NHε-R (wherein R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloarylene-alkanoyl); Xaa22 is Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; X1 is Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloalkylalkanoyl; Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, and N-alkylalanine; Xaa39 is Ser or Tyr; and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, and Xaa26 are Ala; and When Xaa1 is His, Arg, Tyr, or 4-imidazopropionyl, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0132] In certain embodiments, exendin derivatives of formula II may include those in which Xaa1 is His, Ala, Norval, or 4-imidazopropionyl (e.g., those in which Xaa1 includes His, 4-imidazopropionyl, or Ala, or those in which Xaa1 includes His or 4-imidazopropionyl).

[0133] The exendin derivative of formula II can be one where Xaa2 is Gly. The exendin derivative of formula II can be one where Xaa4 is Ala.

[0134] The exendin derivative of formula II can be one where Xaa9 is Ala. The exendin derivatives of formula II can be those in which Xaa14 is Leu, pentylglycine, or Met.

[0135] The exendin derivatives of formula II can be those in which Xaa25 is Trp or Phe.

[0136] Exendin derivatives of formula II can be those in which Xaa6 is Ala, Phe, or naphthylalanine, Xaa22 is Phe or naphthylalanine, and Xaa23 is Ile or Val.

[0137] The exendin derivative of formula II can be one in which Z1 is -NH2. The exendin derivatives of formula II can be those in which Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, thioproline, and N-alkylalanine.

[0138] The exendin derivatives of formula II can be those in which Xaa39 is Ser or Tyr (eg, Ser).The exendin derivatives of formula II can be those in which Z2 is -NH2.

[0139] The exendin derivative of formula II can be one in which Z1 is -NH2. The exendin derivative of formula II may be one in which Xaa21 is Lys-NHε-R, where R is Lys, Arg, or a straight or branched alkanoyl having 1 to 10 carbon atoms.

[0140] The exendin derivative of formula II may be one in which X1 is Lys Asn, Lys-NHε-R Asn, or Lys-NHε-R Ala, where R is Lys, Arg, or a straight or branched alkanoyl having 1 to 10 carbon atoms.

[0141] In further embodiments, the exendin derivatives of formula II may include compounds having the amino acid sequences shown in SEQ ID NOS: 95-110 in WO 99 / 025728. The exendin derivatives of formula II may include compounds having the amino acid sequences shown in SEQ ID NOS: 5-93 in PCT application PCT / US98 / 24210, filed November 13, 1998, entitled "Novel Exendin Agonist Compounds." In another aspect, the exendin derivatives of formula II may include compounds having the amino acid sequences shown in SEQ ID NOS: 37-40 in WO 99 / 007404. The above documents are incorporated herein by reference.

[0142] The abbreviations used in Formulas I and II have the following meanings: "ACN" and "CH3CN" refer to acetonitrile.

[0143] "Boc", "tBoc", and "Tboc" refer to t-butoxycarbonyl. "DCC" refers to N,N'-dicyclohexylcarbodiimide.

[0144] "Fmoc" refers to fluorenylmethoxycarbonyl. "HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate.

[0145] "HOBt" refers to 1-hydroxybenzotriazole monohydrate. "homoP" and "hPro" refer to homoproline.

[0146] "MeAla" and "Nme" refer to N-methylalanine. "Naph" refers to naphthylalanine.

[0147] "pG" and "pGly" refer to pentylglycine. "tBuG" refers to tert-butylglycine.

[0148] "ThioP" and "tPro" refer to thioproline. "3Hyp" refers to 3-hydroxyproline.

[0149] "4Hyp" refers to 4-hydroxyproline. "NAG" refers to N-alkylglycine.

[0150] "NAPG" refers to N-alkylpentylglycine. "Norval" refers to norvaline.

[0151] In one embodiment, the exendin fragment or derivative may be C-terminally substituted or unsubstituted with an amide group and is selected from the group consisting of exendin-4(1-28) (SEQ ID NO: 15), exendin-4(1-28)amide, exendin-4(1-30) (SEQ ID NO: 7), exendin-4(1-30)amide, exendin-4(1-31) (SEQ ID NO: 54), exendin-4(1-31)amide, 14 Leu 25 PheExendin-4 (SEQ ID NO:55), 14 Leu 25 Phe Exendin-4 Amide, and pharma- ceutically acceptable salts thereof.

[0152] Controlled Release Preparations According to other embodiments, the controlled release composition or microspheres may contain as an active ingredient an exendin, GLP-1, or a therapeutically effective GLP-1 or exendin analog in an amount of about 0.1 to about 10 parts by weight (e.g., about 0.8 to about 6 parts by weight) based on 100 parts by weight of the composition or microspheres comprising an exendin (such as exendin-4), GLP-1, or a therapeutically effective GLP-1 or exendin analog (e.g., an exendin-4 analog), a biodegradable polymer, and a coating material. For example, the controlled release composition or microspheres may contain exendin or a therapeutically effective analog thereof and / or GLP-1 or a therapeutically effective analog thereof, based on 100 parts by weight of the composition or microspheres comprising exendin, GLP-1, or a therapeutically effective GLP-1 or exendin analog, a biodegradable polymer, and a coating material, in an amount of about 0.1 to about 10 parts by weight, about 0.1 to about 9 parts by weight, about 0.1 to about 8 parts by weight, about 0.1 to about 7 parts by weight, about 0.1 to about 6 parts by weight, about 0.1 to about 6 parts by weight, about 0.1 to about 5 parts by weight, about 0.1 to about 4 parts by weight, about 0.1 to about 3 parts by weight, about 0.5 to about 10 parts by weight, about 0.5 to about 9 parts by weight, about 0.5 to about 8 parts by weight, about 0.5 to about 7 parts by weight, about 0.5 to about 6 ... ~about 5 parts by weight, about 0.5 to about 4 parts by weight, about 0.5 to about 3 parts by weight, about 1 to about 10 parts by weight, about 1 to about 9 parts by weight, about 1 to about 8 parts by weight, about 1 to about 7 parts by weight, about 1 to about 6 parts by weight, about 1 to about 6 parts by weight , about 1 to about 5 parts by weight, about 1 to about 4 parts by weight, about 1 to about 3 parts by weight, about 2 to about 10 parts by weight, about 2 to about 9 parts by weight, about 2 to about 8 parts by weight, about 2 to about 7 parts by weight, about 2 to about 6 parts by weight, about 2 to about 6 parts by weight, It may comprise about 2 to about 5 parts by weight, about 2 to about 4 parts by weight, about 3 to about 10 parts by weight, about 3 to about 9 parts by weight, about 3 to about 8 parts by weight, about 3 to about 7 parts by weight, about 3 to about 6 parts by weight, about 4 to about 10 parts by weight, about 4 to about 9 parts by weight, about 4 to about 8 parts by weight, about 4 to about 7 parts by weight, about 5 to about 10 parts by weight, about 5 to about 9 parts by weight, about 5 to about 8 parts by weight, about 6 to about 10 parts by weight, about 6 to about 9 parts by weight, or about 7 to about 10 parts by weight.If the amount of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogue contained in the composition or microsphere of the present disclosure is less than the above range, the high effect of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogue cannot be obtained. Also, if the amount of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogue is more than the above range, the initial burst of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogue increases, which causes side effects caused by the initial burst. Therefore, it is preferable that the amount of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogue is within the above range.

[0153] Biodegradable polymers refer to any polymer that is not harmful to humans, because when administered into the body, biodegradable polymers can be slowly degraded and excreted. Biodegradable polymers may include one or more (e.g., 1, 2, 3, 4, 5, 6, or more) selected from the group consisting of polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkylcarbonate, and copolymers of one or more polymers with polyethylene glycol (PEG). The one or more polymers may be in the form of a copolymer or a simple mixture.

[0154] For example, biodegradable polymers include poly(lactide-co-glycolide)s (PLGAs) such as RG502H (IV=0.16-0.24 dL / g), RG503H (IV=0.32-0.44 dL / g), and RG504H (IV=0.45-0.60 dL / g) with a lactide:glycolide ratio of 1:1, RG752H (IV=0.14-0.22 dL / g) with a lactide:glycolide ratio of 75:25, polylactides (PLAs) such as R202H (IV=0.16-0.24 dL / g) and R203H (IV=0.25-0.35 dL / g) (all from Evnik, Germany), 5050DL (IV=0.16-0.24 dL / g) with a lactide:glycolide ratio of 1:1, and 5050DL (IV=0.16-0.24 dL / g) with a lactide:glycolide ratio of 1:1. The polymer may be one or more selected from the group consisting of poly(lactide-co-glycolides) (copolymers available from Evonik, Parsippany, NJ, USA), such as 5050DL 2A (IV=0.15-0.25 dL / g), 5050DL 3A (IV=0.25-0.43 dL / g), and 5050DL 4A (IV=0.38-0.48 dL / g), although equivalent polymers may be provided and obtained from any suitable source.

[0155] In a further embodiment, the biodegradable polymer may be a polymer-sugar conjugate comprising a sugar coupled to (1) a polymer selected from the group consisting of polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyorthoesters, polyanhydrides, polyhydroxybutyrate, polycaprolactone, and polyalkylcarbonates, (2) a copolymer of at least two of the above polymers, or (3) a copolymer of polyethylene glycol (PEG) and one of the above polymers.

[0156] In other embodiments of the present disclosure, a polymer-sugar conjugate may refer to a conjugate in which the hydroxyl groups of the sugar are replaced with a polymer. The sugar may include monosaccharides and polysaccharides having 1-8 sugar units (each sugar unit having 3-6 hydroxyl groups) and linear sugar alcohols having 3-6 hydroxyl groups and a molecular weight of 20,000 or less. The sugar alcohols may include mannitol, pentaerythritol, sorbitol, ribitol, and xylitol. The polymer and the sugar are coupled at three or more hydroxyl groups of the sugar.

[0157] The polymer-sugar conjugates according to the above embodiments may be suitable as biodegradable polymers, since they have in vivo properties similar to those of the polymers coupled to the sugars, and have different degradation rates depending on the type of polymer used, and degrade in the body into harmless polymers and sugars. In one embodiment, the polymer-sugar conjugate may be a PLA-glucose conjugate, a PGA-glucose conjugate, or a PLGA-glucose conjugate, which may have the following structure:

[0158] [ka]

[0159] In the controlled release microspheres according to the present disclosure, the coating layer formed on the surface thereof can effectively control the initial burst of exendin or GLP-1 or therapeutically effective GLP-1 or exendin analogues, thereby preventing side effects caused by excessive initial burst. Biodegradable polymers may be used without viscosity restrictions.

[0160] In the controlled release composition / preparation according to the present disclosure, the biodegradable polymer serves as a matrix to protect the active ingredient (exendin, GLP-1, or therapeutically effective GLP-1 or exendin analog). Therefore, if the viscosity of the polymer is not low enough, the active ingredient cannot be effectively protected, resulting in an increased initial burst. If the viscosity of the polymer is too high, the total amount of the active ingredient released is reduced, resulting in a reduced bioavailability. In the present disclosure, not only the biodegradable polymer but also the coating material contained in the composition plays a role in controlling drug release, so that a biodegradable polymer with a relatively low viscosity can be used. Therefore, in order to effectively control the initial burst of the drug and improve bioavailability, the intrinsic viscosity (IV) of the biodegradable polymer may be about 0.1 to about 0.6 dL / g (e.g., about 0.15 to about 0.31 dL / g or about 0.16 to about 0.24 dL / g). The intrinsic viscosity is measured by dissolving the biodegradable polymer in chloroform at a concentration of 1% (W / V) using an Ubbelohde viscometer at 25° C.±0.1° C.

[0161] In the compositions, preparations, or microspheres according to the present disclosure, the biodegradable polymer serves as a matrix that protects the active ingredient during release and controls the release rate, and its content in the composition or microspheres may be about 85 to about 99.89 parts by weight (e.g., about 91 to about 99 parts by weight) based on 100 parts by weight of the composition / preparation or microspheres comprising the active ingredient (exendin, GLP-1, or a therapeutically effective GLP-1 or exendin analog), the biodegradable polymer, and the coating material.

[0162] The coating material is used to prevent excessive initial burst and increase the bioavailability of the active ingredient, and in the microspheres according to the present disclosure, the coating material may be in the form of a coating layer formed on the surface of the microsphere. The coating material may be one or more (e.g., 1, 2, or 3) selected from basic amino acids, polypeptides, and organic nitrogen compounds. The basic amino acids may include arginine, lysine, histidine, and derivatives thereof. The polypeptide may include 2 to 10 amino acids (e.g., 2 to 5 amino acids) including one or more (e.g., 1, 2, or 3) selected from arginine, lysine, and histidine. The polypeptide may include more basic amino acids than acidic amino acids and thus may exhibit basicity. For example, the polypeptide may be L-Ala-His-Lys, L-Arg-Phe, Gly-His, Gly-His-Gly, Gly-His-Lys, L-His-Gly, L-His-Leu, L-Lys-L-Tyr-L-Lys, L-His-L-Val, L-Lys-Lys, L-Lys-Lys-L-Lys, and L-Lys-L-Thr-L-Thr-L-Lys-L-Ser, etc. Furthermore, the organic nitrogen compound may be creatine, creatinine, urea, etc.

[0163] The content of the coating material contained in the composition according to the present disclosure or coating the microspheres is about 0.01 to about 5 parts by weight (e.g., about 0.015 to about 3 parts by weight, about 0.01 to about 4, about 0.01 to about 3, about 0.01 to about 2, about 0.01 to about 4, about 0.01 to about 2, about 0.015 to about 10 parts by weight) based on 100 parts by weight of the composition or microspheres comprising exendin, GLP-1, or a therapeutically effective GLP-1 or exendin analog, a biodegradable polymer, and a coating material. 5, about 0.015 to about 4, about 0.015 to about 2, about 0.05 to about 5, about 0.05 to about 4, about 0.05 to about 3, about 0.05 to about 2, about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.5 to about 5, about 0.05 to about 4, about 0.05 to about 3, about 0.05 to about 2, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 3 to about 5, about 3 to about 4, or about 4 to about 5). If the content of the coating material is less than the above range, the drug release cannot be effectively controlled, and if the content of the coating material is greater than the above range, further increase in the initial burst control effect cannot be obtained.

[0164] The controlled release microspheres according to the present disclosure may each have a smooth surface coated with a coating material and may have an average size of about 1 to about 50 μm (e.g., about 5 to about 30 μm, about 1 to about 40 μm, about 1 to about 30 μm, about 1 to about 20 μm, about 1 to about 10 μm, about 5 to about 50 μm, about 5 to about 40 μm, about 5 to about 30 μm, about 5 to about 20 μm, about 5 to about 10 μm, about 10 to about 50 μm, about 10 to about 40 μm, about 10 to about 30 μm, about 10 to about 20 μm, about 20 to about 50 μm, about 20 to about 40 μm, about 20 to about 30 μm, about 30 to about 50 μm, about 30 to about 40 μm, or about 40 to about 50 μm). The smooth surface of the microspheres effectively controls the initial burst and provides excellent bioavailability.

[0165] Unlike conventional forms, the controlled release microspheres or microspheres prepared from the compositions / preparations of the present disclosure are coated with a coating material, which prevents excessive / harmful initial burst and increases bioavailability, which cannot be achieved with conventional microspheres containing exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogs. In particular, excessive / harmful initial burst of exendin, GLP-1, or therapeutically effective GLP-1 or exendin analogs causes various side effects such as vomiting, nausea, and headache, so it is very important to reduce the initial burst amount to 5% or less. The controlled release microspheres or microspheres prepared from the compositions (or preparations) of the present disclosure reduce the release amount in the first 24 hours to 5% or less. To reduce side effects resulting from administration of controlled release microspheres comprising exendin, GLP-1, or a therapeutically active GLP-1 or exendin analog, the initial burst amount during the first hour may be about 5% or less (e.g., about 4% or less, about 3% or less, about 2% or less, or about 1% or less). Because the microspheres according to the present disclosure include a coating layer of coating material on their surface, the initial burst is effectively controlled to avoid excessive / harmful initial burst-induced side effects, and the drug is released sustainably and sufficiently to provide excellent bioavailability.

[0166] In one embodiment of the present disclosure, the preparation or microspheres may further comprise excipients such as protective colloids and / or stabilizers.

[0167] The composition or microspheres may further contain one or more protective colloids selected from polyvinyl alcohol, albumin, polyvinylpyrrolidone, gelatin, etc. The protective colloids do not have a special effect of preventing excessive / harmful initial burst of the active ingredient contained in the microspheres, but they play a role in preventing aggregation of the microspheres and improving dispersibility. Considering such a role, the content of the protective colloid is about 0.02% (W / W) to about 1.0% (W / W) (e.g., about 0.02% to about 0.8%, about 0.02% to about 0.6%, about 0.02% to about 0.4%, about 0.05% to about 1.0%, about 0.04% to about 0.8%, about 0.05% to about 0.5%, about 0.05% to about 0.05%, about 0.05% to about 0.05%, or about 0.05% to about 0.05%, based on the weight of the composition, preparation, or microspheres containing exendin, GLP-1, or a therapeutically effective GLP-1 or exendin analog, a biodegradable polymer, and a coating material. 0.4%, about 0.05% to about 0.3%, about 0.05% to about 0.2%, about 0.1% to about 1.0%, about 0.1% to about 0.8%, about 0.1% to about 0.6%, about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 1.0%, about 0.2% to about 0.8%, about 0.2% to about 0.6%, about 0.2% to about 0.4%, about 0.4% to about 1.0%, about 0.4% to about 0.8%, about 0.4% to about 0.6%, about 0.6% to about 1.0%, about 0.6% to about 0.8%, or about 0.8% to about 1.0%).

[0168] Also, to improve the stability of the microspheres during lyophilization, the compositions / preparations or microspheres according to the present disclosure may further comprise an excipient selected from mannitol, trehalose, sucrose, sodium carboxymethylcellulose, and the like, in an amount of about 5% (W / W) to about 30% (W / W), for example, about 10% (W / W) to about 20% (W / W), based on the weight of the composition or microsphere comprising the exendin, GLP-1, or therapeutically active GLP-1 or exendin analog, the biodegradable polymer, and the coating material.

[0169] Additionally, the compositions, preparations, or microspheres of the present disclosure may further comprise any additives and excipients conventionally used in pharmaceutical preparations, the type and content of which can be readily determined by one skilled in the relevant art.

[0170] The controlled release microspheres containing exendin, GLP-1, or therapeutically active GLP-1 or exendin analogs for use in the methods of the present disclosure may be prepared by various methods for preparing controlled release microspheres, such as, for example, a method in which the surface of the microspheres is coated by suspending the microspheres in a coating material solution during or after the preparation of the microspheres. The method for preparing the microspheres may be carried out by a double emulsion method (W / O / W method), a single emulsion method (O / W method), a phase separation method, a spray drying method, etc.

[0171] Specifically, a method for preparing controlled release microspheres containing exendin, GLP-1, or a therapeutically active GLP-1 or exendin analog may include mixing the active agent and a biodegradable polymer to prepare a W / O emulsion or homogeneous mixture, and emulsifying the emulsion or homogeneous mixture by adding it to an aqueous solution of a coating material to form a coating layer.

[0172] More specifically, when the double emulsion method is used, the method may include the steps of mixing and emulsifying an aqueous solution of the active ingredient with a solution of a biodegradable polymer dissolved in an organic solvent to form a primary emulsion (W / O type), suspending this emulsion in an aqueous solution of a coating material to form a W / O / W type emulsion, heating the W / O / W type emulsion to remove the solvent and harden the resulting microspheres, recovering and washing the hardened microspheres, and freeze-drying the resulting microspheres. The organic solvent may be any organic solvent that can form an emulsion by dissolving the biodegradable polymer and then mixing with an aqueous solution, such as one or more selected from the group consisting of chloroform, ethyl acetate, methylene chloride, and methyl ethyl ketone (e.g., methylene chloride). In this case, the coating material is contained in the second aqueous phase (the outer aqueous phase of the W / O / W emulsion) so that after removal of the organic solvent, a coating layer is formed on the outside of the microspheres comprising at least one active ingredient (e.g., exendin, GLP-1, or a therapeutically active GLP-1 analog or exendin-4 analog, or a combination thereof) and a biodegradable polymer.

[0173] Alternatively, when using a single emulsion method, the method may include dissolving the active ingredient and the biodegradable polymer in an organic solvent to form a homogeneous mixture, adding an aqueous solution containing a coating material to the resulting mixture to form an emulsion, heating the emulsion to remove the solvent and harden the resulting microspheres, recovering and washing the hardened microspheres, and freeze-drying the resulting microspheres. The organic solvent may be any organic solvent that can thoroughly mix the active ingredient and the biodegradable polymer to form a homogeneous mixture and can be mixed with an aqueous solution to form an emulsion. For example, the organic solvent may be a mixed solvent of one or more selected from the group consisting of alcohols having 1 to 5 carbon atoms, glacial acetic acid, formic acid, dimethyl sulfoxide, and n-methylpyrrolidone, and one or more selected from the group consisting of chloroform, ethyl acetate, methyl ethyl ketone, and methylene chloride, as well as a mixed solvent of, for example, methanol and methylene chloride. In this case, a coating layer is formed on the surface of the final microspheres by emulsifying a homogeneous mixture of the biodegradable polymer and the active ingredient, adding the coating material to the aqueous solution and removing the organic solvent.

[0174] A method for preparing controlled release microspheres may include the steps of mixing an active ingredient with a biodegradable polymer to form an emulsion or homogeneous mixture, solidifying the resulting emulsion or homogeneous mixture to prepare primary microspheres, and suspending the resulting primary microspheres in an aqueous solution of a coating material to form a coating layer on each microsphere.

[0175] The solidification method is not limited, and may be any solidification method conventionally used in the related art, such as phase separation method or spray drying method. More specifically, when the phase separation method is used in the solidification step, the method may include the steps of mixing an aqueous solution of an active ingredient with a solution in which a biodegradable polymer is dissolved in an organic solvent to form an emulsion, or mixing an active ingredient and a biodegradable polymer in a mixed solvent to form a homogeneous mixture solution, adding an oil such as silicone oil to the obtained emulsion or solution to prepare primary microspheres, removing the organic solvent from the microspheres and hardening the microspheres by adding a non-solvent for the biodegradable polymer, such as a mixed solvent of an alcohol having 1-5 carbon atoms and an alkane having 1-12 carbon atoms, such as a mixed solvent of ethanol and heptane, suspending the obtained microspheres in an aqueous solution of a coating material to form a coating layer on each microsphere, and collecting, washing, and freeze-drying the microspheres on which the coating layer is formed.

[0176] The organic solvent may be one or more (e.g., 1, 2, 3, or 4) selected from the group consisting of chloroform, ethyl acetate, methylene chloride, and methyl ethyl ketone (e.g., methylene chloride). The mixed solvent may be a mixture of one or more (e.g., 1, 2, 3, 4, 5, or more) selected from the group consisting of at least one alcohol having 1 to 5 carbon atoms, glacial acetic acid, formic acid, dimethyl sulfoxide, and n-methylpyrrolidone, and one or more (e.g., 1, 2, 3, or 4) selected from the group consisting of chloroform, ethyl acetate, methyl ethyl ketone, and methylene chloride (e.g., a mixed solvent of methanol and methylene chloride).

[0177] Alternatively, when a spray drying method is used, the method may include the steps of mixing an aqueous solution of the active ingredient with a biodegradable polymer dissolved in an organic solvent to form an emulsion, or mixing the active ingredient and the biodegradable polymer in a single solvent or a mixture of solvents to form a homogeneous mixture solution, spray drying the resulting emulsion or solution to prepare primary microspheres, suspending the resulting primary microspheres in an aqueous solution of a coating material to form a coating layer on each microsphere, and washing and freeze-drying the microspheres with the coated layer formed thereon.

[0178] The organic solvent may be one or more (e.g., 1, 2, 3, or 4) selected from the group consisting of chloroform, ethyl acetate, methylene chloride, and methyl ethyl ketone (e.g., methylene chloride). The single solvent may be one or more (e.g., 1, 2, 3, 4, or 5) selected from the group consisting of glacial acetic acid and formic acid, and the mixed solvent may be a mixture of one or more (e.g., 1, 2, 3, 4, or more) selected from the group consisting of at least one alcohol having 1 to 5 carbon atoms, glacial acetic acid, formic acid, dimethyl sulfoxide, and n-methylpyrrolidone, and one or more selected from the group consisting of chloroform, ethyl acetate, methyl ethyl ketone, and methylene chloride (e.g., a mixed solvent of methanol and methylene chloride).

[0179] The method may further include the step of adding a protective colloid material by any conventional method, for example, the protective colloid material may be added during the step of coating the microspheres with the coating material.

[0180] The concentration of the coating material dissolved in the aqueous phase or solution may be about 0.01 M to about 1 M (e.g., about 0.1 M to about 0.5 M, about 0.01 M to about 0.8 M, about, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4 M, about 0.1 to about 1 M, about 0.1 M to about 0.8 M, about, about 0.1 M to about 0.6 M, about 0.1 M to about 0.4 M, about 0.2 to about 1 M, about 0.2 M to about 0.8 M, about, about 0.2 M to about 0.6 M, about 0.2 M to about 0.4 M, about 0.4 M to about 1 M, about 0.4 M to about 0.8 M, about, about 0.4 M to about 0.6 M, about 0.6 to about 1 M, about 0.6 M to about 0.8 M, or about 0.8 M to about 1 M). If the concentration of the coating material is lower than the above range, the surface of the microspheres cannot be completely coated with the coating material, and if the concentration of the coating material is higher than the above range, the coating material solution becomes supersaturated, so the effect of controlling the initial burst cannot be improved. Therefore, the concentration of the coating material may be within the above range.

[0181] Administration The controlled release composition according to the present disclosure may be administered by oral or parenteral routes (e.g., parenteral routes), such as intravenous, subcutaneous, intramuscular, and intraperitoneal routes. Thus, in one embodiment of the present disclosure, the controlled release composition or preparation may be administered in the form of a dispersion solution, as an injection solution. The effective amount of the composition may be appropriately adjusted according to the age of the subject, the type and severity of the disease, and the condition of the subject, and the amount of the active ingredient in the composition may be about 0.01 to about 100 μg / kg / day (e.g., about 0.1 to about 10 μg / kg / day), and this amount may be administered once or in multiple doses. The exact amount required may vary depending on the type of polypeptide and on the individual subject, and may vary depending on the species, age, general condition, severity of the disease to be treated, the specific polypeptide used, and its mode of administration, etc. Therefore, it is not possible to specify the exact "insulin secretion amount" or the amount useful for treating neurological diseases or injuries. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation.

[0182] Those skilled in the art will know how to monitor the effectiveness of treatment and how to adjust the treatment accordingly.For example, blood glucose level can be monitored, and if normal blood glucose is observed, the treatment is considered to be optimal.If blood glucose level is higher than the preferred level, then the dosage of the polypeptide should be increased, and if blood glucose level is lower than the preferred level, then the dosage of the polypeptide should be decreased.

[0183] The compound may be administered orally, intravenously, intramuscularly, intraperitoneally, topically, transdermally, locally, systemically, intravenously, intracerebrally, subdurally, or intrathecally. Those skilled in the art will know how to vary the mode of administration, pharmacologic carrier, or other parameters to optimize the insulin secretory effect. The amount of the active compound administered will, of course, depend on the subject being treated, the subject's weight, the manner of administration, and the judgment of the prescribing physician.

[0184] Depending on the intended mode of administration, the pharmaceutical composition may be in a solid, semi-solid, or liquid form, such as, for example, a tablet, suppository, pill, capsule, powder, liquid, suspension, lotion, cream, or gel, and may be in a unit dosage form suitable for single administration of a precise dose. As described above, the composition includes an effective amount of the selected agent in combination with a pharma- ceutically acceptable carrier, and further, the composition may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and the like. See, for example, Remington's Pharmaceutical Sciences, latest edition, by EW Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional pharmaceutical composition preparation methods that may be used in conjunction with the preparation of polypeptide preparations, and is incorporated herein by reference. For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate. Liquid pharma-ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., the active compound as described herein and any pharmaceutical excipients in an excipient, such as, for example, water, saline, aqueous dextrose, glycerin, and ethanol, to obtain a solution or suspension. If desired, the pharmaceutical composition to be administered may further contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents and pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. The actual methods of preparing such dosage forms will be well known or apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, referenced above.

[0185] For oral administration, the fine powder or granules may contain diluents, dispersants, and / or surfactants, or may be in water or syrup, or dried in capsules or sachets, or in non-aqueous solutions, or in non-aqueous suspensions that may contain suspending agents, or in tablets that may contain binders and lubricants, or in suspensions in water or syrup. Flavoring agents, protective agents, suspending agents, thickening agents, or emulsifying agents may be included if desired or necessary. In certain embodiments, the oral dosage form is a tablet or granule, which may be coated. When parental administration is performed, administration is generally characterized by injection.

[0186] Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Recently modified methods for parenteral administration maintain the dosage at a constant level by using a slow-release or sustained-release system. See, for example, U.S. Patent No. 3,710,795, which is incorporated herein by reference.

[0187] For topical administration, solutions, suspensions, lotions, creams, gels, or the like may be used, provided they are capable of delivering the active compound to the skin surface.

[0188] Working Example The following examples are presented for the purpose of fully disclosing and illustrating to one of ordinary skill in the art how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended purely to illustrate the disclosure and are not intended to limit the scope of the disclosure as perceived by the inventors. Efforts have been made to ensure accuracy of numerical values ​​(e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for.

[0189] Statistics: Values ​​are expressed as means ± SEM. Normality of distribution was determined by the Kolmogorov-Smirnov test. Statistical analyses as indicated in the Results section were performed by Student's t test, Mann-Whitney test, Fisher's exact test, or one-way and two-way ANOVA. In cases where the assumption of normality was violated, ANOVA on ranks was performed. All multiple pairwise comparisons were performed by post-hoc Newman-Keuls or Dunn tests, and statistical significance was defined as p<0.05.

[0190] Example 1. Pharmacokinetics and sustained release of exendin-4 in plasma. PT302 is a sustained release preparation of exendin-4, containing a mixture of polymer (98%) and exendin-4 (2%). Plasma levels of exenatide were analyzed after injection of the sustained release preparation of exendin-4 (PT302). In particular, the pharmacokinetics of a single dose of PT302 was investigated in six adult (9-week-old) male Sprague-Dawley rats. PT302 was freshly dissolved in diluent and administered subcutaneously at 2 mg / kg. Blood was collected at 0, 0.5, and 1 hour after injection, and at 1, 3, 5, 7, 9, 11, 14, 18, 21, and 26 days after injection. Plasma levels of exendin-4 were quantified using a Peptron Exenatide EIA Kit (Peptron, Daejeon, Korea). The data are shown in Figure 1A. In the figure, Cmax is 1.85ng / ml, Tmax is 12.5 days, and AUC is 18.55ng. * d / ml (values ​​were calculated from individual data for all 6 animals). As shown in Figure 1A, a single subcutaneous injection of the sustained release formulation PT302 achieves acceptable long-term exendin-4 plasma levels over a 1-2 week dosing regimen.

[0191] The pharmacokinetics of single doses of PT302 at various doses (2.4 mg / kg, 4.8 mg / kg, and 9.6 mg / kg) were investigated using adult (9-week-old) male Sprague-Dawley rats, six per group. PT302 was freshly dissolved in diluent and administered subcutaneously as described above. Blood was collected at 0, 0.5, and 1 hour post-injection, and at 1, 3, 7, 10, 14, 17, 21, 24, 28, and 31 days post-injection. Plasma levels of exendin-4 were quantified as described above. The data are shown in Figure 1B, where the Cmax was 2.23 ng / ml, the Tmax was 14.83 days, and the AUC was 21.13 ng / ml at the 2.4 mg / kg dose. * d / ml, and at 4.8 mg / kg, the Cmax was 5.21 ng / ml, the Tmax was 16.17 days, and the AUC was 49.46 ng * d / ml, and at 9.6 mg / kg, the Cmax was 9.42 ng / ml, the Tmax was 17.17 days, and the AUC was 87.14 ng * d / ml. As shown in Figure 1B, a single subcutaneous injection of the sustained release formulation PT302 achieves long-term exendin-4 plasma levels that are directly related to the dose level of PT302 administered.

[0192] PT304 is a sustained release formulation of exendin-4, containing a mixture of polymer (96%) and exendin-4 (4%). Six adult (9-week-old) male Sprague-Dawley rats were injected with the sustained release formulation of exendin-4 (PT304) and then analyzed for exenatide plasma levels. PT304 was freshly dissolved in diluent and administered subcutaneously at 4 mg / kg. Blood was collected at 1 and 3 hours after injection, and at 1, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39, and 42 days after injection. Plasma levels of exendin-4 were quantified using a Peptron Exenatide EIA Kit. The data are shown in Figure 2, where Cmax was 3.82 ng / ml, Tmax was 14.17 days, and AUC was 48.34 ng. *d / ml (values ​​were calculated from individual data for all 6 animals). As shown in Figure 2, a single subcutaneous injection of the sustained release formulation PT304 achieves acceptable long-term exendin-4 plasma levels over a 2-4 week dosing regimen.

[0193] The pharmacokinetics of PT302 was studied over a 10-week period in adult (9-week-old) Sprague-Dawley rats, 10 per group. Rats were injected subcutaneously with PT302 at 2 mg / kg once weekly or PT304 at 4 mg / kg every other week. PT302 and PT304 were prepared as described above, and blood was collected 1 and 3 hours after the first injection, and at 1, 3, 5, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 days after the first injection. Serum levels of exendin-4 were quantified as described above and are shown in Figure 3. Rats injected weekly had a Cmax of 5.27 ng / ml, a Tmax of 51.60 days, and an AUC of 236.42 ng / ml. * d / mL, and rats injected every other week had a Cmax of 5.08 ng / mL, a Tmax of 47.89 days, and an AUC of 211.51 ng * d / mL.

[0194] Example 2. Meth-mediated rotational behavior in a 6-OHDA Parkinson's disease rat model is reduced by pretreatment with PT302.

[0195] Animals were treated with vehicle (9 rats), PT302 at 0.4 mg / kg (low dose, 9 rats), or PT302 at 2 mg / kg (high dose, 10 rats) on the days indicated in Figure 4A (i.e., 16 and 2 days before unilateral 6-OHDA lesion of the medial forebrain bundle and 12, 26, and 40 days after lesion). Rats were examined for meth-mediated rotational behavior at 20, 30, and 45 days after lesion, and euthanized at 47 days, as shown in Figures 4A and 4B. Lesions were administered as follows: Rats were anesthetized with chloral hydrate (400 mg / kg, i.p.) and placed in a stereotaxic apparatus. 6-OHDA (2.76 μg / μl × 5 μl in 0.9% NaCl containing 0.2 mg / ml ascorbic acid) was injected unilaterally into the medial forebrain bundle (-4.4 mm AP, 1.2 mm ML from bregma, 8.4 mm subcranial) over 4 min using a Hamilton microsyringe held in a stereotaxic arm. A micromanipulator attached to the stereotaxic apparatus was used to lower the microsyringe into the desired target site in the brain. A syringe pump (Micro 4, WPI, Sarasota, FL) was used to control the injection rate (0.5 μl / min). Five minutes after injection, the needle was removed. A piece of bone wax was applied to the skull burr hole to prevent fluid leakage. The wound was sutured or clipped. Body temperature was monitored by a thermistor probe, and body temperature was maintained at 37°C during anesthesia using a heating pad. After recovery from anesthesia, body temperature was maintained at 37° C. for an additional 3 hours using a temperature-controlled incubator.

[0196] Meth-induced rotational behavior [Liu DM, Lin SZ, Wang SD, Wu MI, Wang Y. Xenografting human T2 sympathetic ganglion from hyperhidrotic patients partially restores catecholaminergic functions in hemi-Parkinsonian athymic rats. Cell Transplant 1999;8:563-91; and Luo Y, Hoffer BJ, Wang Y. Rotation, Drug-induced. In: Kompoliti K, Verhagen Metman L, editors. Encyclopedia of Movement Disorders. Oxford: Academic Press, 2010. p 49-51] was assessed using an 8-channel rotometer system (RotoMax, AccuScan Instruments). As previously described [Yin LH, Shen H, Diaz-Ruiz O, Backman CM, Bae E, Yu SJ, Wang Y. Early post-treatment with 9-cis retinoic acid reduces neurodegeneration of dopaminergic neurons in a rat model of Parkinson's disease. BMC Neurosci 2012;13:120], animals were administered methamphetamine (2.5 mg / kg) 6 days after 6-OHDA lesion. Methamphetamine is an indirect agonist that induces dopamine release in the brain. Rats with unilateral injection of 6-OHDA into the nigrostriatal dopaminergic system are used as PD models. Animals treated with indirect dopamine agonists (e.g., methamphetamine) show ipsilateral rotation behavior after administration, whereas animals treated with direct dopamine agonists (e.g., apomorphine) show contralateral rotation behavior after administration. These behaviors are associated with unilateral changes in the expression of dopaminergic markers in the striatum.

[0197] As shown in Figure 4B, PT302 treatment at both the high and low doses significantly reduced rotational behavior in PT302-treated groups compared to vehicle (p=0.018, F2,87=4.309, two-way ANOVA for low dose group). Post-hoc Newman-Keuls tests showed that the high dose of PT302 significantly attenuated meth-mediated rotational behavior (p=0.037). Thus, sustained, steady-state administration of exendin-4 as PT302 significantly inhibited the behavioral effects of unilateral 6-OHDA lesion of the medial forebrain bundle in rats (a well-characterized animal model of PD) and provided neuroprotective activity.

[0198] Exendin-4 plasma levels were quantified as described above. As shown in Figure 4C, the mean exendin-4 plasma levels were 30.845 pg / ml (n=10) in high-dose rats and 8,990 pg / ml (n=9) in low-dose rats. However, excluding outlier rats and measurements (rats 1, 10, and 12), the mean exendin-4 plasma levels were 5,596 pg / ml (n=8) in high-dose rats and 574 pg / ml (n=8) in low-dose rats.

[0199] Example 3. Meth-induced rotational behavior was reduced by post-treatment with PT302 in 6-OHDA-lesioned rats. The 6-OHDA unilateral lesion of the medial forebrain bundle in the PD rodent model described in Example 2 and post-treatment with a highly potent drug can be combined as shown in the scheme of Figure 5A. In this case, rodents are unilaterally lesioned with 6-OHDA (day 0) and treatment begins 6 days later. This PD rodent model is more difficult to treat because dopaminergic cell death is already initiated and ongoing before treatment. Nineteen rats were lesioned as described above. Meth-induced rotational behavior was examined as described above, and animals with more than 300 rotations / hour were divided into two groups, so that the number of rotations in the vehicle group and the PT302 group before the start of treatment was the same. Meth-induced rotational behavior was examined 6, 20, 30, and 45 days after injury, as outlined in Figure 5A. Animals (adult male Sprague-Dawley rats, 2 months old at time of receipt) were treated with vehicle (subcutaneous, n=11) or PT302 (100 mg / kg, containing 2.0 mg / kg exendin-4, subcutaneous, n=8) on days 6, 20, and 34 after 6-OHDA injury. PT302 was prepared as described above. On day 47 after injury, animals were euthanized. Blood and brain samples were collected, and plasma was separated and stored (-80°C). Exendin-4 was measured in collected plasma.

[0200] As shown in FIG. 5B, PT302 significantly reduced meth-induced rotational behavior in unilateral 6-OHDA-lesioned rats compared to vehicle controls (p=0.032, two-way ANOVA).

[0201] TH was examined by immunohistochemistry. Specifically, serial cryostat sections 25 μm thick were cut from whole brains. One in six sections was stained for TH examination. To control for staining variability, each batch contained specimens from all experimental groups and reacted together in one netwell tray under the same conditions. Sections were rinsed in 0.1 M phosphate buffer (PB) and blocked with 4% bovine serum albumin (BSA) and 0.3% Triton x-100 in 0.1 M PB. Sections were then incubated in primary antibody (mouse anti-TH monoclonal antibody, diluted in 4% BSA and 0.3% Triton x-100 in 0.1 M PB, concentration 1:100; Chemicon, Temecula, CA) for 17–19 h at 4°C. Sections were then rinsed in 0.1M PB and incubated in secondary antibody for 1 hour, followed by incubation with avidin-biotin-horseradish peroxidase complex for 1 hour. Sections were mounted on glass slides and covered with a coverslip. Control sections were incubated without primary antibody.

[0202] TH immunoreactivity in the striatum was measured using ImageJ and averaged over three brain sections with visible anterior commissure. TH immunoreactivity in the substantia nigra was measured every 360 μm throughout the midbrain (-4.2 mm to -6.0 mm from bregma). A total of five brain sections were used per animal. Substantia nigra volume was analyzed by Cavalieri's method.

[0203] 6-OHDA-mediated dopaminergic neurodegeneration in the striatum was reduced by PT302. Representative striatal TH immunostaining results and plasma exendin-4 levels for three vehicle-treated rats (#866, 883, 886) and three PT302-treated rats (#881, 875, 882) are shown in FIG. 5C (quantification of plasma concentrations of exendin-4 in the same animals is depicted in FIG. 5C). As shown in FIG. 5D, striatal TH immunoreactivity in vehicle-treated animals was significantly reduced by 6-OHDA injection, while TH immunoreactivity in the lesioned striatum was significantly increased by PT302 ( * p<0.001, two-way ANOVA). L=lesioned side, non-L-nonlesioned side, veh=vehicle treated animals, PT=PT302.

[0204] TH immunoreactivity in the substantia nigra was measured by ImageJ and averaged from three selected brain sections with visible anterior commissure. Representative TH immunostaining results are shown in Figure 5E for vehicle-treated animals (rats #866, #883, #886) or PT302-treated animals (rats #881, #875, #882) (plasma levels of exendin-4 in the same animals are also shown). Quantification of TH immunoreactivity in the substantia nigra every 360 um from -4.2 mm to -6 mm from bregma is shown in Figure 5F. 6-OHDA injection significantly reduced TH immunoreactivity in the substantia nigra in vehicle-treated animals, whereas PT302 significantly inhibited the loss of TH immunoreactivity in the lesioned substantia nigra ( * p<0.001, two-way ANOVA). L=lesioned side, non-L-nonlesioned side, veh=vehicle-treated animals, PT=PT302 PT302 post-treatment protects TH+ neurons in the injured substantia nigra. Brains were sectioned and immunohistochemistry was performed as described above. TH+ neurons were found in the non-injured side of the brain (Figure 5G and Figure 5H). Few TH+ neurons or fibers were found in the substantia nigra on the injured side of the brain (rat #886, Figure 5I). PT302 treatment protected some of the TH+ neurons (Figure 5J, Figure 5K, and Figure 5L).

[0205] Exendin-4-mediated protection in TH neurons correlates with plasma exendin-4 levels. Figure 5M shows a significant correlation between normalized striatal TH (i.e., lesioned / non-lesioned) immunoreactivity and plasma exendin-4 levels in the same animals (p=0.002, R=0.663). Figure 5L shows a significant correlation between plasma exendin-4 levels and TH immunoreactivity in the substantia nigra of the lesioned side (p<0.001, R=0.842).

[0206] Example 4. Meth-mediated rotational behavior in 6-OHDA PD rat model is reduced by pretreatment with PT302. As shown in Figure 6A, animals were subcutaneously treated with Ex-4 (5 μg / kg, BID) or PT302 (0.4 mg / kg) 7 days before and 7 days after 6-OHDA injury, as described above. The exendin-4 dose used in this experiment, 5 μg / kg BID, is higher than the dose that can be used in humans (see Table 1 below), and the lower dose of about 1 μg / kg BID is equivalent to both the dose for humans and the PT302 dose used in this experiment. Both Ex-4 (5 μg / kg, BID) and PT302 (0.4 mg / kg) treatment significantly reduced rotational behavior in 6-OHDA injury rats (p=0.005 for Ex-4 group, p=0.002 for PT302 group) (Figure 6B).

[0207] Pretreatment with either exendin-4 or PT302 provided protection against 6-OHDA-mediated dopaminergic neurodegeneration in the substantia nigra. Representative TH immunoreactivity results for sham and 6-OHDA-lesioned animals administered vehicle, Ex-4 (5 μg / kg, BID), or PT302 (0.4 mg / kg) are shown in FIG. 6C. TH immunoreactivity on the lesioned side was significantly reduced by 6-OHDA injection (shown as ipsi in FIG. 6C). Representative TH immunoreactivity quantification in the substantia nigra is shown in FIG. 6D. Loss of TH immunoreactivity on the 6-OHDA-lesioned side was significantly reduced by treatment with PT302 and Ex-4 (assessed by expressing TH immunoreactivity as a percentage of the immunoreactivity on the lesioned side (ipsi) of the same animal relative to the immunoreactivity on the control non-lesioned side (contra)).

[0208] Example 5. Meth-mediated rotation behavior in 6-OHDA Parkinson's disease rat model is reduced by post-treatment with PT302. As described above, after 6-OHDA injury, animals were treated with Ex-4 (1 μg / kg, BID from day 2) or PT302 (0.4 mg / kg, once on day 2), and meth-mediated rotation behavior was examined on day 9 after injury (Figure 7A). As shown in Figure 7B, rotation behavior in unilateral 6-OHDA-lesioned rats was significantly reduced by PT302 (p=0.0075). In contrast, exendin-4 did not reduce rotation behavior in unilateral 6-OHDA-lesioned rats. Thus, in the 6-OHDA rat PD model, exendin-4 at a human equivalent dose (1 μg / kg BID for rats) was ineffective in suppressing meth-induced rotational behavior, whereas PT302 at a human equivalent dose proved effective. When the dose of exendin-4 was increased to a dose equivalent to a higher dose than can be used in humans (5 μg / kg BID, as shown in Example 4), exendin-4 was also able to suppress 6-OHDA-induced abnormalities. In contrast, clinically equivalent doses of PT302 exhibited beneficial effects in both Examples 4 and 5.

[0209] Example 6. Toxin-induced behavioral defects in MPTP PD mouse model were improved by post-treatment with PT302. As described above, after induction of parkinsonism by MPTP, animals were treated with Ex-4 (16.7ug / kg, BID from day 6) or PT302 (0.6mg / kg; days 6, 20, and 34) (Figure 8A). Specifically, parkinsonism was induced by systemic administration of MPTP at 30mg / kg once a day for 5 days. See, for example, Filichia E, Hoffer B, Qi X, Luo Y. Inhibition of Drp1 mitochondrial translocation provides neural protection in dopaminergic system in a Parkinson's disease model induced by MPTP. Sci Rep. 2016;6:32656. Treatment with either Ex-4 or PT302 was started 1 day after the first MPTP administration and continued for 18 days. The doses selected, Ex-4 (16.7ug / kg, BID, subcutaneous) and PT302 (0.4mg / kg, single subcutaneous dose), were comparable in that the amount of Ex-4 delivered and provided during the 18 days of treatment was 601.2ug / kg and less than 600ug / kg, respectively. On day 18, motor coordination was assessed in all groups of animals by wire grip test. The wire grip test (also known as paw grip endurance (PaGE) method and grip force-recording test) is often used as an index of motor strength. PaGE is designed to assess grip strength in mice, which may indicate impaired motor performance.Herein, PaGE was performed as described by Jessica AL et al. (Jessica AL Hutter-Saunders, Howard E. Gendelman, R. Lee Mosley. Murine Motor and Behavior Functional Evaluations for Acute 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Intoxication. J Neuroimmune Pharmacol. 2012;7(1):279-288). Briefly, each mouse was removed from a conventional rodent housing cage, placed on a wire lid, the lid was gently shaken to allow the mouse to grasp, and the lid was inverted (180 degrees). The latency of the mouse to release both hind paws was measured in seconds. Each mouse was randomly given three trials for up to 240 seconds, and the longest latency to fall or remove both hind paws was recorded (Jessica, et al. 2012).

[0210] As shown in Figure 8B, administration of the dopaminergic cytotoxin MPTP significantly decreased the latency in the grip test (MPTP 94.5 s vs. aggregate 148.3 s for no MPTP controls, p=0.02). PT302 significantly increased the latency of MPTP-treated mice to fall off the wire (118.3 s, not statistically different from aggregate control), but exendin-4 (administered twice daily in a manner similar to human dosing of Byetta, i.e., immediate release Ex-4) did not prevent the toxin-induced behavioral deficits in MPTP-treated mice (72.9 s, p+0.01 vs. aggregate control) (Figure 8B: 7-10 mice per group).

[0211] Example 7. Delivery of Ex-4 to the central nervous system using sustained release exendin-4 is effective in achieving therapeutically effective amounts of exendin-4 in the cerebrospinal fluid. Exendin-4 (injection and mini-pump) and sustained release formulation of exendin-4 (PT302) were administered to adult male Sprague-Dawley rats (9 weeks old) used in this study as described in Table 1. 14 days after the first exendin-4 injection, blood and cerebrospinal fluid (CSF) were collected for exendin-4 level measurement. Quantification of plasma and CSF levels of exendin-4 was performed as described above. As shown in Table 2, exendin-4 was detected in plasma and CSF in the sustained release formulation group and the mini-pump-administered exendin-4 group, while exendin-4 in CSF was below the detection limit in the twice-daily (BID, immediate release) exendin-4 group. In contrast to these results, high levels of exendin-4 were detected in the plasma of the same exendin-4 groups. In the pump-administered exendin-4 groups receiving doses of 3.5 to 15 pM / kg / min, CSF / plasma ratios ranging from 0.0081 (or 0.81%) to about 0.0412 (or 4.12%), with a mean of 0.018 (or 1.8%), were observed. Similarly, in animals receiving PT302 at doses of 0.46 to 2.0 mg / kg / 14 days, CSF / plasma ratios ranging from 0.0117 (or 1.17%) to about 0.016 (or 1.6%). This data indicates that sustained release of exendin-4 provides even higher levels of exendin-4 in the CNS.

[0212] [Table 1]

[0213] [Table 2]

[0214] Example 8. Treating mild traumatic brain injury (mTBI) mice with sustained release exenatide improves novel object recognition. Head injury was inflicted using a drop-weight impact device as previously described (Tweedie et al. 2007). The device consisted of a metal tube (90 cm long, 1.3 cm inner diameter) and a sponge placed under the tube to support the mouse head. Mice were lightly anesthetized with isoflurane inhalation and placed under the device. A metal weight (30 grams) was dropped from the top of the tube and struck the temporal side of the mouse head between the corner of the eye and the ear. Immediately after injury, the mice were returned to their cages and allowed to recover. Mice tolerate this head injury procedure well, which leads to widespread neuronal loss in the hippocampus and cerebral cortex on the injured side, accompanied by cognitive impairment in visual and spatial memory tests (Deselms H, Maggio N, Rubovitch V, Chapman J, Schreiber S, Tweedie D, Kim DS, Greig NH, Pick CG. Novel pharmaceutical treatments for minimal traumatic brain injury and evaluation of animal models and methodologies supporting their development. J Neurosci Methods. 2016;272:69-76).

[0215] One hour after head injury, mice were administered sustained release PT302 (0.6 mg / kg, subcutaneous) as described above (Figure 9A). Seven days after injury, mice were subjected to a novel object recognition paradigm test, a test routinely used to assess recognition memory in rodents. Uninjured rodents have an innate tendency to explore novel objects in their vicinity. This behavioral characteristic allows for the assessment of visual recognition memory function. Perhaps more importantly, it allows for the assessment of the effects of different stimuli on this innate behavior. In this test, two experiments are performed. In the first experiment, the animals are allowed to explore two objects for a defined period of time (5 min). In the second experiment, performed 24 hours after the first experiment, the animals are presented with two objects, the same object presented in the first experiment and one new to the animal. In the second experiment, the mice are allowed to explore the two objects for 5 minutes. A discrimination preference index is calculated and used to evaluate the recognition memory of the animals. The index is calculated as follows: the time the animal spends near the novel object is subtracted from the time the animal spends near the familiar object, and then divided by the sum of the time the animal spends near the novel object and the time the animal spends near the familiar object (Dix SL, Aggleton JP. Extending the spontaneous preference test of recognition: evidence of object-location and object-context recognition. Behav. Brain Res. 1999; 99: 191-200). As shown in Figure 9B, exenatide administration significantly increased novel object recognition in mTBI mice (n = 5) compared to untreated mTBI mice (n = 7), indicating that mTBI-induced visual recognition abnormalities were suppressed in PT302-treated mice.

[0216] Example 9. Examination of plasma exendin-4 levels in normal ICR mice. Exendin-4 plasma concentrations were measured 7 days after subcutaneous administration of PT302 (0.1, 0.3, 0.6, 1.0, and 2.0 mg / kg) in normal ICR mice. Exendin-4 plasma levels were quantified as described above. Plasma exendin-4 levels persisted and accumulated to approximately 4000 pg / ml in a dose-dependent manner until 7 days after injection (Figure 10A).

[0217] Example 10. Examination of plasma exendin-4 levels in normal mice and traumatic brain injury mice. 7 days after subcutaneous administration of PT302 (0.6mg / kg) in normal mice and TBI model mice, the exendin-4 plasma levels were measured. The plasma levels of exendin-4 were quantified as described above. No difference in the exendin-4 plasma levels was observed between normal mice and TBI-induced mice (Figure 10B).

[0218] Example 11. Sustained release exenatide maintains exendin-4 plasma levels for a long period of time. Normal ICR mice were given a single subcutaneous injection of PT302 at three different doses (0.024mg / kg, 0.12mg / kg, and 0.6mg / kg). Blood was collected for plasma measurement at 0 hours, 0.5 hours, and 1 hour after the first injection, and at days 1, 3, 7, 14, and 21. Plasma concentrations of exendin-4 were quantified as described above. As shown in Figure 10C, a single dose of PT302 maintained exendin-4 plasma levels for more than 20 days.

[0219] Example 12. Sustained release exenatide significantly increases the recognition of novel objects and new arms of the maze 7 days after traumatic brain injury. Head injury was administered as described above, and mice were quickly returned to their original cages to recover. Mice were administered PT302 (0.024 mg / kg, 0.12 mg / kg, and 0.6 mg / kg) subcutaneously by single injection 1 hour after TBI induction. Behavior was evaluated 7 days after mTBI (novel object recognition and Y-maze test). The Y-maze paradigm is commonly used to evaluate spontaneous exploration and response to novel environments and spatial working memory function (Deselms, et al., 2016). The test apparatus consisted of multiple identical black plexiglass arms (8 x 30 x 15 cm) that extended from a central point at an angle of 120 degrees from the center. In order to provide the mouse with a visual memory anchor, each arm is provided with a different spatial cue. Typically, the two experiments are performed 2-3 minutes apart, but in this example, these experiments were performed 5 minutes apart. In the first experiment, the starting arm is selected randomly for each experiment. One animal is placed in the center of the Y-maze environment, and one of the remaining two arms is randomly closed during the 5 minutes of the first experiment, and all three arms are opened and explored during the 2 minutes of the second experiment. The total time that the mouse explored in each arm during the second experiment is recorded. The T-maze is thoroughly cleaned between experiments to prevent confusion. The time spent in a previously explored, novel arm versus a previously explored, known arm, i.e., (time in new arms - time in known arms) / (time in new arms + time in known arms), is used to assess whether there are behavioral differences between animal treatment groups.

[0220] Vehicle-treated mTBI mice exhibited visual memory deficits and spent less time near the novel object compared to control mice (p<0.001) (Figure 11A). One hour after mTBI induction, mice treated with a single subcutaneous injection of PT302 at doses of 0.12 mg / kg (p<0.05) and 0.6 mg / kg (p<0.01) were observed to have a higher preference for the novel object compared to untreated mTBI mice (Figure 11A).

[0221] mTBI-treated mice exhibited significant spatial memory abnormalities, spending less time in the novel arm of the Y-maze compared to sham (control) animals (p<0.001) (Figure 11B). A single subcutaneous injection of PT302 at doses of 0.12 mg / kg and 0.6 mg / kg, administered 1 hour after mTBI induction, ameliorated the mTBI spatial memory deficits compared to untreated mTBI mice (p<0.01) (Figure 11B). [F(4,50)=4.83, p=0.002, Fisher's least significant difference, post hoc].

[0222] All groups spent approximately the same amount of time in the open arms of the elevated plus maze, making it impossible to distinguish between groups in terms of anxiety-like behavior (FIG. 11C). Importantly, this indicated that anxiety-like behavior was not a confounding factor in the previously performed novel object recognition and Y-maze paradigms (p>0.05) [F(4,36)=0.28, p=0.89].

[0223] Example 13. Sustained release exenatide significantly increases the recognition of novel objects and new arms of the maze 30 days after traumatic brain injury. Head injury is administered as described above, and mice are quickly returned to their original cages to recover. One hour after TBI induction, mice are subcutaneously administered PT302 (0.6 mg / kg) by a single injection. 30 days after mTBI, behavior is evaluated (novel object recognition and Y-maze test).

[0224] Vehicle-treated mTBI mice exhibited visual memory deficits and spent less time near the novel object compared to control mice (p<0.001, FIG. 12A). One hour after mTBI induction, mice treated with a single subcutaneous injection of PT302 at doses of 0.12 mg / kg or 0.6 mg / kg were observed to have a higher preference for the novel object compared to mTBI mice (p<0.01 and p<0.001, respectively, FIG. 12A).

[0225] Mice treated with mTBI exhibited significant spatial memory abnormalities and spent less time in the novel arms of the maze compared to sham / control animals (p<0.001, FIG. 12B). A single subcutaneous injection of PT302 at a dose of 0.6 mg / kg, administered 1 hour after mTBI induction, ameliorated the mTBI spatial memory deficits compared to mTBI alone (p<0.01, FIG. 12B).

[0226] All groups spent approximately the same amount of time in the open arms of the maze, and it was not possible to distinguish between groups in terms of anxiety-like behavior (FIG. 12C), indicating that anxiety-like behavior was not a factor in the previously performed novel object recognition and Y-maze paradigms (p>0.05).

[0227] Example 14. PT302 administration prevented the decrease in NeuN immunoreactivity in the temporal cortex and hippocampus regions of mice after traumatic brain injury. NeuN is a mature neuron marker and can be used to investigate the neuronal loss caused by traumatic brain injury. To evaluate NeuN cells in defined brain regions, mice were anesthetized with an overdose of ketamine + xylazine and rapidly perfused transcardially with physiological buffered saline followed by 4% paraformaldehyde (PFA, 0.1M phosphate buffer, pH 7.4). Brains were removed and fixed overnight (4% PFA, pH 7.4, 0.1M phosphate buffer), then placed in 30% sucrose for 48 hours. Coronal sections (30 μm) were made with a cryostat, placed in cryoprotectant, and stored at -20°C until use. Five cortical and five hippocampal sections were then blocked by incubation with 0.1% Triton X-100 and 10% normal horse serum in phosphate-buffered saline (PBST) for 1 h at 25° C. The primary antibody, mouse anti-neuronal nuclei antibody (NeuN; 1:50, Millipore, Danvers, MA, USA, Cat#MAB3377), was then dissolved in PBST and 2% normal horse serum and incubated with the sections for 48 h at 4° C. After rinsing in PBST, the sections were incubated with DyLight™ 594-labeled AffinityPure donkey anti-rabbit IgG antibody and DyLight™ 488-labeled AffinityPure donkey anti-mouse IgG antibody (1:300; Jackson Laboratories, Bar Harbor, ME, USA) for 1 h at 25° C. After rinsing in PBST, sections were mounted on dry gelatin-coated slides and fluorescence was evaluated under a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) equipped with ×20 and ×63 lenses. Three to five sections were used per brain, with a defined field of view of 140 mm in the hippocampus and temporal cortex. 2 or 440 2The mean cell number in μM was calculated. Immunofluorescence of immunohistochemistry slides was evaluated in a blinded manner, and primary antibodies were routinely omitted when negative control sections were prepared. Analysis of color quantification was performed with the Imaris program (Bitplane AG, Zurich, Switzerland).

[0228] FIG. 13A shows representative images of NeuN (red, as assessed) positive neurons in the cortex, CA3, and dentate gyrus of control, untreated mTBI, and PT302-treated mTBI mice (0.6 mg / kg 1 hour after mTBI) 30 days after mTBI. Bar graphs in FIG. 13B, FIG. 13C, and FIG. 13D show quantification of surviving neurons in the cortex, CA3, and dentate gyrus, respectively. Quantification was performed using the number of anti-NeuN positively stained neurons in the sham control, mTBI, and mTBI+PT302 0.6 mg / kg groups ( ** p<0.01, *** p<0.001). Values ​​are means ± SEM. Administration of exendin-4 via sustained release as PT302 prevented the neuronal loss observed following traumatic brain injury.

[0229] Example 15. PT302 administration reduced the number of degenerated neurons in mTBI mice. Fluoro-Jade® C is a fluorescent stain that labels damaged degenerated brain neurons and can be used to study neuronal loss due to traumatic brain injury. Ionized calcium-binding adaptor molecule 1 (IBA1) is specifically expressed in microglia and upregulated in activated microglia. It can be used as a marker of neuroinflammation. Mice were euthanized and their brains were prepared for immunohistochemical analysis as described above. Coronal sections (40 μm) of the hippocampus and parietal cortex were cryostat-cut and collected in cryoprotectant solution. In addition to the parietal cortex, analysis was also performed on CA1, CA3, and dentate gyrus from the hippocampus.

[0230] To perform FluoroJade C staining, brain sections were first immersed in 1% NaOH in 80% ethanol for 5 min. After rinsing in 70% ethanol for 2 min, they were washed in distilled water and then incubated in 0.06% potassium permanganate solution for 10 min. After washing with water, slides were incubated in FJC staining solution (obtained by adding 4 ml of FJC 0.01% stock solution in distilled water to 96 ml of 0.1% acetic acid) and stained for 10 min. After washing three times with distilled water, slides were thoroughly air-dried on a slide warmer, cleared in xylene, and cover-slipped with DPX. Fluorojade C-positive cells were counted in each region of both hemispheres using an FV 1000MPE Olympus confocal microscope.

[0231] To perform double labeling of IBA1 / TNF-α, sections were incubated with IBA1 antibody (goat anti-IBA1 polyclonal antibody 1:200, Abcam, USA) and TNF-α antibody (TNF-α rabbit anti-TNF-α polyclonal antibody 1:800, Abbiotec, USA) for 48 h. After PBS washing, sections were incubated with secondary antibody for IBA1, and three-step detection with biotin-labeled IgG (IgG(H+L)biotin-goat anti-rabbit 1:500, Invitrogen, USA) and streptavidin-fluorescein (1:200, Vector, UK) was performed to increase the TNF-α signal.

[0232] Qualitative and quantitative analysis of IBA1 and TNFα was performed using an Olympus confocal laser scanning microscope FV 1000MPE. Z-series images were processed with ImageJ 1.47v, and the volume of colocalized elements was measured with Imaris 7.4.2 as follows. For each data set, the software automatically constructed a colocalization channel. In the final layer, target regions were selected for each brain region (CA1, CA3, DG, and cortex) in each hemisphere of each animal, and the volumes of target elements were calculated, summed, and expressed as volume / μm3.

[0233] As shown in FIG. 14A, after mTBI injury, a significant increase in the number of Fluoro-Jade positive neurons was observed in all investigated regions (CA1, CA3, DG, and lateral cortex for hippocampus) compared to controls (p<0.05 for CA1; p<0.001 for CA3, DG, and CTX; FIG. 14A, and FIG. 14B, FIG. 14C, and FIG. 14D, and FIG. 14E, respectively). PT302 treatment at a dose of 0.6 mg / kg counteracted the effects of mTBI-induced neurodegeneration in all investigated regions (p<0.01 for CA3 and DG, FIG. 14C, and FIG. 14D; p<0.05 for CTX and CA1, FIG. 14E, and FIG. 14B), with a significant effect in the CA3 region seen with a dose of 0.12 mg / kg PT302 (p<0.05).

[0234] As shown in FIG. 15A, after mTBI injury, IBA1 immunoreactivity was increased in all analyzed regions compared to vehicle controls (p<0.05 for CA1; p<0.001 for CA3, DG, and CTX). In the control group, microglial cells had smaller cell bodies and longer, thinner processes, exhibiting a resting morphology (FIG. 15A). After mTBI injury, microglia exhibited an activated morphology characterized by larger cell bodies and shorter, thicker processes (FIG. 15A). PT302 inhibited microglial activation at all doses, with PT302 0.6 mg / kg being effective in all brain regions (p<0.001 for CTX, p<0.001 for CA1 and CA3, p<0.05 for DG).

[0235] Immunoreactivity for the proinflammatory cytokine TNF-α was increased in all analyzed regions of IBA1+ cells in the mTBI injury group (p<0.05 for DG; p<0.01 for CA1; p<0.001 for CA3 and cortex; Figures 15D, 15B, 15C, and 15E, respectively). PT302 administration at doses of 0.6 and 0.12 mg / kg reduced the levels of IBA1 / TNF-α IR colocalization volume in both the hippocampus and cortex.

[0236] Specific embodiments One aspect of the present disclosure provides a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject, comprising administering a therapeutically effective amount of a neuroprotective polypeptide to the systemic circulatory bloodstream of the subject using a controlled release preparation or device that provides sustained release or delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprising at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation or sustained release of the neuroprotective polypeptide increases the delivery and / or uptake of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​of the subject to at least a portion of the central nervous system (CNS) compared to a fast release preparation of the neuroprotective peptide.

[0237] Another aspect of the present disclosure provides a method for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method includes administering a therapeutically effective amount of a neuroprotective polypeptide to the subject's systemic circulatory bloodstream using a controlled release preparation or device that provides sustained release or sustained delivery of the neuroprotective polypeptide, the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog, the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation or device increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to a fast release preparation of the neuroprotective polypeptide.

[0238] One aspect of the present disclosure provides a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject, comprising administering a controlled release preparation comprising at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog into the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery and / or uptake of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​to at least a portion of the central nervous system (CNS) of the subject compared to an immediate release preparation of the neuroprotective polypeptide.

[0239] In any aspect or embodiment described herein, the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide.

[0240] In any aspect or embodiment described herein, the controlled release preparation further comprises a biodegradable polymer having a particular viscosity and a coating material to achieve bioavailability and sustained release of an effective amount of the neuroprotective polypeptide over a sufficient period of time (e.g., without an initial, such as a harmful, initial burst of active ingredient).

[0241] In any aspect or embodiment described herein, the controlled release neuroprotective preparation comprises a neuroprotective polypeptide. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0242] In any aspect or embodiment described herein, the sustained release preparation comprises a depot preparation for sustained release of the neuroprotective polypeptide.

[0243] In any aspect or embodiment described herein, the sustained release preparation comprises a composition for sustained release of the neuroprotective polypeptide.

[0244] In any aspect or embodiment described herein, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in a subject.

[0245] In any aspect or embodiment described herein, the CNS-related condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0246] In any aspect or embodiment described herein, administering the controlled release neuroprotective preparation comprises injection of the controlled release neuroprotective preparation.

[0247] In any aspect or embodiment described herein, the injection of the controlled release neuroprotective preparation is a subcutaneous injection.

[0248] In any aspect or embodiment described herein, administration of the controlled release neuroprotective preparation results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0249] In any aspect or embodiment described herein, administration of the controlled release neuroprotective preparation cumulatively increases the concentration of the neuroprotective polypeptide in the subject's cerebrospinal fluid (CSF), brain, or a combination thereof.

[0250] In any aspect or embodiment described herein, the concentration of the neuroprotective polypeptide in the CSF is in the range of about 5 to about 400 pg / mL.

[0251] A further aspect of the present disclosure provides a method for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method comprises administering a therapeutically effective amount of a controlled release neuroprotective preparation comprising at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue to the subject's systemic circulatory bloodstream, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of the subject through the blood-brain barrier (BBB) ​​compared to an immediate release preparation of the neuroprotective polypeptide.

[0252] In any aspect or embodiment described herein, the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide.

[0253] In any aspect or embodiment described herein, the controlled release preparation further comprises a biodegradable polymer having a particular viscosity and a coating material, such that the bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time (e.g., without an initial burst, such as a harmful initial burst, of the active ingredient).

[0254] In any aspect or embodiment described herein, the controlled release neuroprotective preparation comprises a neuroprotective polypeptide. Do and and a biodegradable polymer. Including the core, It comprises a controlled release microsphere and a coating layer coating the core.

[0255] In any aspect or embodiment described herein, the sustained release preparation comprises a depot preparation for sustained release of the neuroprotective polypeptide.

[0256] In any aspect or embodiment described herein, the sustained release preparation comprises a composition for sustained release of the neuroprotective polypeptide.

[0257] In any aspect or embodiment described herein, administration of the controlled release neuroprotective preparation alleviates at least one symptom of at least one CNS-related condition in a subject.

[0258] In any aspect or embodiment described herein, the CNS-related condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy, motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

[0259] In any aspect or embodiment described herein, administering the controlled release neuroprotective preparation comprises injecting the controlled release neuroprotective preparation into the subject.

[0260] In any aspect or embodiment described herein, the injection of the controlled release neuroprotective preparation into the subject is a subcutaneous injection.

[0261] In any aspect or embodiment described herein, administration of the controlled release preparation results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0262] In any aspect or embodiment described herein, administration of the controlled release preparation cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0263] In any aspect or embodiment described herein, the preparation is administered once every 7 to 21 days (e.g., once every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days).

[0264] In any aspect or embodiment described herein, the administration of the preparation is performed a second time about 7 to about 21 days after the previous administration (e.g., once every about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 21 days).

[0265] In any aspect or embodiment described herein, when the preparation is readministered within about 28 days (e.g., within about 21 or about 14 days) of the preceding preparation administration, the percent change in the concentration of the neuroprotective polypeptide in plasma is about 30% or less.

[0266] An additional aspect of the present disclosure provides a method for delivering a neuroprotective polypeptide to at least a portion of the central nervous system (CNS) of a subject, comprising providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases delivery and / or uptake of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​to at least a portion of the central nervous system (CNS) of the subject compared to an immediate release preparation of the neuroprotective peptide.

[0267] A further aspect of the present disclosure provides a method for treating a subject with a central nervous system (CNS)-related disease or reducing at least one symptom of a CNS-related disease in a subject in need of such reduction.The method includes providing sustained delivery of at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analog to the systemic circulatory bloodstream of the subject, wherein the neuroprotective polypeptide binds to and activates a receptor that binds to at least one of GLP-1, exendin-4, or a combination thereof, and the controlled release neuroprotective preparation increases the delivery of the neuroprotective polypeptide through the blood-brain barrier (BBB) ​​of the subject to at least a portion of the central nervous system (CNS) compared to a fast release preparation of the neuroprotective polypeptide.

[0268] In any aspect or embodiment described herein, providing sustained release of the neuroprotective polypeptide(s) includes administering the neuroprotective polypeptide(s) via a device (e.g., a pump, a mini-pump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini-infusion pump, etc.).

[0269] In any aspect or embodiment described herein, when the preparation is administered (e.g., once every 7-28 days, or once every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days), the percent change in plasma neuroprotective polypeptide concentration at steady state after reaching steady state is about 80% or less (e.g., about 50% or less, or about 40% or less).

[0270] In any aspect or embodiment described herein, the neuroprotective polypeptide(s) is administered by a device (e.g., a pump, a mini-pump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini-infusion pump, etc.).

[0271] In any aspect or embodiment described herein, the neuroprotective polypeptide(s) is administered at a rate of about 1 pM / kg / min to about 30 pM / kg / min (e.g., about 3 pM / kg / min to about 17.5 pM / kg / min).

[0272] In any aspect or embodiment described herein, administering a controlled release neuroprotective preparation or providing sustained delivery of a neuroprotective polypeptide alleviates at least one symptom of at least one CNS-related condition in a subject.

[0273] In any aspect or embodiment described herein, administration of a controlled release preparation or providing sustained release of the neuroprotective polypeptide results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

[0274] In any aspect or embodiment described herein, administration of a controlled release preparation or providing sustained release of a neuroprotective polypeptide cumulatively increases the concentration of the neuroprotective polypeptide in at least one of the cerebrospinal fluid (CSF), the brain, or a combination thereof.

[0275] In any aspect or embodiment described herein, the concentration of the neuroprotective polypeptide in the CSF is in the range of about 10 to about 400 pg / mL.

[0276] In any aspect or embodiment described herein, the ratio of the concentration of the polypeptide in the CFS at steady state to the concentration of the polypeptide in plasma at steady state is within the range of about 0.1% to about 5%.

[0277] In any aspect or embodiment described herein, the exendin-4 analog is represented by formula I, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa27 Xaa28 -Z1 (Chemical formula I) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg; Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, or a linear or branched alkanoyl having 1 to 10 carbon atoms; Xaa22 is Ala, Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn, and Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; Xaa39 is Ser, or Tyr (e.g., Ser); and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27, and Xaa28 are Ala; and When Xaa1 is His, Arg, or Tyr, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0278] In any aspect or embodiment described herein, the exendin-4 analog is represented by formula II, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala X1-Z1 (Chemical formula II) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln, Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu, Xaa16 is Ala or Glu, Xaa17 is Ala or Glu, Xaa19 is Ala or Val, Xaa20 is Ala or Arg, Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloarylene-alkanoyl; Xaa22 is Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; X1 is Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloalkylalkanoyl; Z1 is -OH, -NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2, or Gly Gly Xaa31 Ser Ser Gly Ala Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, and N-alkylalanine; Xaa39 is Ser or Tyr; and Z2 is -OH or -NH2; however, Not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, and Xaa26 are Ala; and When Xaa1 is His, Arg, Tyr, or 4-imidazopropionyl, then at least one of Xaa3, Xaa4, and Xaa9 is Ala).

[0279] In any aspect or embodiment described herein, the neuroprotective polypeptide is selected from the group consisting of SEQ ID NOs: 1-55.

[0280] Each document cited in this specification is incorporated herein by reference. Except in the examples and where otherwise specified, all numerical values ​​specifying the amount of a substance or the like in this description are understood to be modified by the word "about". It is understood that the upper limit, lower limit, range limit, and ratio limit described herein may be independently combined. Similarly, the ranges and amounts of each element according to this disclosure can be used together with the ranges or amounts of any other element.

[0281] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein which are intended to be encompassed by the scope of the following claims.

[0282] It is understood that the detailed examples and embodiments described herein are provided by way of example for illustrative purposes only, and are not to be considered as limiting the present disclosure. Various modifications or changes in this regard will be suggested to those skilled in the art, and such modifications or changes are considered to be within the spirit and scope of the present application and within the scope of the appended claims. For example, the relative amounts of ingredients may be changed, additional ingredients may be added, and / or one or more of the ingredients described may be replaced with similar ingredients in order to optimize the desired effect. Further advantageous features and functionalities associated with the systems, methods, and processes according to the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize that there are many equivalents to the specific embodiments of the present disclosure described herein, or such equivalents will be ascertainable using no more than routine experimentation. Such equivalents are intended to be encompassed within the scope of the following claims.

[0283] References

[0284]

Table 3

Claims

1. 1. A controlled release preparation or device for use in a method of treating a subject having a central nervous system (CNS)-related disorder or reducing at least one symptom of a CNS-related disorder in a subject in need thereof, comprising: The method includes administering a therapeutically effective amount of a neuroprotective polypeptide into the systemic circulatory bloodstream of the subject using the controlled release preparation or device that provides sustained release of the neuroprotective polypeptide, wherein the neuroprotective polypeptide comprises at least one neuroprotective polypeptide selected from the group consisting of GLP-1, exendin-4, or a therapeutically effective exendin-4 analog; the neuroprotective polypeptide binds to and activates a receptor that binds at least one of GLP-1, exendin-4, or a combination thereof; The controlled release preparation or device further comprises a coating material and a biodegradable polymer; and The controlled release preparation or device comprises: a controlled release microsphere comprising a core comprising said neuroprotective polypeptide and a biodegradable polymer; a coating layer coating the controlled release microspheres; Including, the biodegradable polymer has a specific viscosity to provide bioavailability and sustained release of an effective amount of the neuroprotective polypeptide over a sufficient period of time, the specific viscosity being between 0.1 and 0.6 dL / g; The biodegradable polymer is selected from the group consisting of polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyorthoester, polyanhydride, polyhydroxybutyric acid, polycaprolactone, and polyalkylcarbonate, or a copolymer or simple mixture of two or more polymers selected from the group of polymers, or a copolymer of the polymer and polyethylene glycol (PEG), or a polymer-sugar complex in which a sugar is coupled to the polymer or the copolymer; the coating material is one or more selected from basic amino acids, The basic amino acid is one or more selected from the group consisting of arginine, lysine, and histidine; and The sustained release of the neuroprotective polypeptide increases delivery of the neuroprotective polypeptide across the blood-brain barrier (BBB) ​​to at least a portion of the central nervous system (CNS) of the subject, as compared to an immediate release preparation of the neuroprotective polypeptide. Said controlled release preparation or device.

2. The exendin-4 analog is represented by formula I, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 -Z 1 (Chemical formula I) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val, Xaa20 is Ala or Arg; Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, or a linear or branched alkanoyl having 1 to 10 carbon atoms; Xaa22 is Ala, Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn, and Z 1 is -OH, -NH 2 , Gly-Z 2 , Gly Gly-Z 2 , Gly Gly Xaa31-Z 2 , Gly Gly Xaa31 Ser-Z 2 , Gly Gly Xaa31 Ser Ser-Z 2, Gly Gly Xaa31 Ser Ser Gly-Z 2, Gly Gly Xaa31 Ser Ser Gly Ala-Z 2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z 2 , Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z 2 , Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z 2 , or Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z 2 , Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, or N-alkylalanine; Xaa39 is Ser, or Tyr (e.g., Ser); and Z 2 is —OH or —NH 2 ; however, not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27, and Xaa28 are Ala; and When Xaa1 is His, Arg, or Tyr, at least one of Xaa3, Xaa4, and Xaa9 is Ala; Alternatively, it is represented by formula II, or a pharma- ceutically acceptable salt thereof: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 X 1 -Z 1 (Chemical formula II) (In the formula, Xaa1 is His, Arg, Tyr, Ala, Norval, Val, Norleu, or 4-imidazopropionyl; Xaa2 is Ser, Gly, Ala, or Thr; Xaa3 is Ala, Asp, or Glu; Xaa4 is Ala, Norval, Val, Norleu, or Gly; Xaa5 is Ala or Thr; Xaa6 is Ala, Phe, Tyr, or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser, or Thr; Xaa9 is Ala, Norval, Val, Norleu, Asp, or Glu; Xaa10 is Ala, Leu, Ile, Val, pentylglycine, or Met; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu, Ile, pentylglycine, Val, or Met; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val, Xaa20 is Ala or Arg; Xaa21 is Ala, Leu, or Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloarylene-alkanoyl; Xaa22 is Phe, Tyr, or naphthylalanine; Xaa23 is Ile, Val, Leu, pentylglycine, tert-butylglycine, or Met; Xaa24 is Ala, Glu, or Asp; Xaa25 is Ala, Trp, Phe, Tyr, or naphthylalanine; Xaa26 is Ala or Leu; X 1 is Lys Asn, Asn Lys, Lys-NHε-R Asn, Asn Lys-NHε-R, Lys-NHε-R Ala, Ala Lys-NHε-R, where R is Lys, Arg, linear or branched alkanoyl having 1 to 10 carbon atoms, or cycloalkylalkanoyl; Z 1 is -OH, -NH 2 , Gly-Z 2 , Gly Gly-Z 2 , Gly Gly Xaa31-Z 2 , Gly Gly Xaa31 Ser-Z 2 , Gly Gly Xaa31 Ser Ser-Z 2, Gly Gly Xaa31 Ser Ser Gly-Z 2, Gly Gly Xaa31 Ser Ser Gly Ala-Z 2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z 2 , Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z 2 , Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z 2 , or Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z 2 , Xaa31, Xaa36, Xaa37, and Xaa38 are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine, and N-alkylalanine; Xaa39 is Ser or Tyr; and Z 2 is —OH or —NH 2 ; however, not more than three of Xaa3, Xaa4, Xaa5, Xaa6, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, and Xaa26 are Ala; and When Xaa1 is His, Arg, Tyr, or 4-imidazopropionyl, at least one of Xaa3, Xaa4, and Xaa9 is Ala.

2. The controlled release preparation or device of claim 1.

3. The controlled release preparation or device of claim 1 or 2, wherein the controlled release preparation is a sustained release preparation of the neuroprotective polypeptide.

4. The controlled release preparation or device of any one of claims 1 to 3, further comprising a biodegradable polymer having a particular viscosity and a coating material, wherein the bioavailability and sustained release of the neuroprotective polypeptide at effective concentrations continues over a period of time.

5. The controlled release preparation or device of claim 3 , wherein the sustained release preparation comprises a depot preparation for sustained release of the neuroprotective polypeptide.

6. The controlled release preparation or device of claim 3 , wherein the sustained release preparation comprises a composition for sustained release of the neuroprotective polypeptide.

7. The controlled release preparation or device of any one of claims 1 to 6, wherein administering the controlled release neuroprotective preparation comprises injecting the controlled release neuroprotective preparation into the subject.

8. 8. The controlled release preparation or device of claim 7, wherein the injection of the controlled release preparation into the subject is a subcutaneous injection.

9. 9. The controlled release preparation or device of any one of claims 1 to 8, wherein administration of the controlled release preparation is performed about 7 to about 21 days (e.g., once every about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or about 21 days) after the preceding administration.

10. The controlled release preparation or device of any one of claims 1 to 3, wherein the device provides a sustained release of the controlled release preparation.

11. 11. The controlled release preparation or device of claim 3 or 10, wherein the device is a pump (e.g., a pump, a minipump, an osmotic pump, an osmotic delivery device, an infusion pump, an intravenous administration device, a peristaltic pump, or a mini infusion pump, etc.).

12. The controlled release preparation or device of any one of claims 3, 10 and 11, wherein the neuroprotective polypeptide is administered using the device at a rate of about 1 pM / kg / min to about 30 pM / kg / min (e.g., about 3 pM / kg / min to about 17.5 pM / kg / min).

13. The controlled release preparation or device of any one of claims 1 to 12, wherein administration of said neuroprotective polypeptide into the systemic circulatory bloodstream alleviates at least one symptom of at least one CNS-related condition in said subject.

14. 14. The controlled release preparation or device of claim 13, wherein the CNS-related condition is selected from the group consisting of Parkinson's disease (PD), traumatic brain injury (TBI), multiple sclerosis, drug addiction, alcohol addiction, neurodegenerative conditions, brain inflammation, Alzheimer's disease (AD), multiple system atrophy, Huntington's disease, chronic traumatic encephalopathy (CTE), motor neuron diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury, spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA)), vascular dementia, dementia with Lewy bodies (DLB), mixed dementia, frontotemporal dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, or a combination thereof.

15. The controlled release preparation or device of any one of claims 1 to 14, wherein administration of the controlled release preparation or providing sustained release of the neuroprotective polypeptide using the device results in a steady state concentration of the neuroprotective polypeptide in plasma within the range of about 50 to about 4500 pg / mL.

16. The controlled release preparation or device of any one of claims 1 to 15, wherein administration of the controlled release preparation or providing sustained release of the neuroprotective polypeptide using the device cumulatively increases the concentration of the neuroprotective polypeptide in at least one of cerebrospinal fluid (CSF), brain, or a combination thereof.

17. The controlled release preparation or device of any one of claims 1 to 16, wherein the concentration of said neuroprotective polypeptide in CSF is in the range of about 10 to about 400 pg / mL.

18. The controlled release preparation or device of any one of claims 1 to 17, wherein the ratio of the concentration of said polypeptide in CFS at steady state to the concentration of said polypeptide in plasma at steady state is within the range of about 0.1% to about 5%.

19. The controlled release preparation or device of any one of claims 1 to 18, wherein the neuroprotective polypeptide is selected from the group consisting of SEQ ID NOs: 1-55.

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