WO2017142961A1 - Treatment of traumatic brain injury or stroke - Google Patents

Abstract

The present invention provides a method for treating neurodegenerative disease, traumatic brain injury, or stroke. In particular, the present invention provides a method for using a glycopeptide PACAP/VIP analogues with enhanced CNS penetration for treatment of neurodegenerative disease, traumatic brain injury, or stroke.

Description

TREATMENT OF TRAUMATIC BRAIN INJURY OR STROKE

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0001] This invention was made with government support under Grant Nos. R01

NS091238 and R01 NS052727, awarded by the National Institutes of Health and

CHE9526909 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for treating traumatic brain injury or stroke. In particular, the present invention relates to using a glycopeptide PACAP/VIP analogues with enhanced CNS penetration for treatment of traumatic brain injury or stroke.

BACKGROUND OF THE INVENTION

[0003] Endogenous opioid peptides, lumped together under the generic term endorphins, have been the subject of intense study since their discovery in the mid 1970's. Some neuropeptides have the potential for selective pharmacological intervention with fewer off-target side effects. If these naturally occurring opioid peptides and their derivatives could be rendered permeable to the blood-brain barrier (BBB), then a new vista of

psychopharmacology would be opened to exploration and exploitation. After three decades of research, many potent and selective opioid agonists have been developed, and stability problems have been largely overcome. The remaining problem that prevents the use of opioid peptides as drugs is poor bioavailability. Without being bound by any theory, poor bioavailability is believed to be primarily due to poor penetration of the BBB.

[0004] The BBB is composed of endothelial cells in the cerebrovascular capillary beds. The BBB acts as a lipophilic barrier to chemical substances, but admits vital nutrients through selective transport proteins for proper function of the CNS. The flow is bidirectional, allowing for export of materials from the CNS and the import of materials from the blood. The BBB represents not only a physical obstacle, but a metabolic one as well, possessing both oxidative enzymes and peptidases such as aminopeptidase, arylamidase, and enkephalinase. Thus, metabolically unstable substances (e.g. peptides) are generally degraded before they reach the CNS. It should also be noted that entry to the CNS does not guarantee that a drug will accumulate in useful concentrations, as many peptides are rapidly exported back to the bloodstream. Several strategies have been reported to overcome the BBB penetration problem, including substitution of unnatural amino acids, the use of conformational constraints, and the addition of lipophilic side chains or other transport vectors.

[0005] Glycosylation has proven to be a successful methodology to improve both the stability and bioavailability of short peptide "messages" by incorporation of the peptide pharmacophore into a glycopeptide. Some BBB penetration studies with opioid glycopeptide agonists based on enkephalins have shown up to 3-fold increases in the rate of brain delivery of these compounds compared with the unglycosylated parent peptides. Recent studies with glycopeptides in artificial membrane systems indicate that amphipathicity of the

glycopeptides is an important factor in BBB penetration. In addition, there is evidence that suggests that the type of glycosylation can alter tissue distribution patterns, BBB penetration and peptide/receptor interactions.

[0006] The endogenous neuropeptide β-endorphin is a 31 residue naturally occurring opioid peptide agonist that binds to μ and δ opioid receptors. Its N-terminal 5 residues are identical to the Met-Enkephalin sequence, and may be considered to be the pharmacophore or "opioid message." It was shown that the C-terminal region of β-endorphin has an

amphipathic a-helical structure that plays a role in receptor binding and opioid agonism, and may impart resistance to proteolysis. It is believed that the N-terminal sequence is the essential "message," and the C-terminal helical region is the "address" that limits delivery of the message to a subset of otherwise available opioid receptors. Some have proposed that B- endorphin consists of the Met-enkephalin peptide sequence at the N-terminus, a hydrophilic linker region from residues 6 through 12, and an amphiphilic helical region between the helix breaker residues Pro(13) and Gly(30). This was later proven by synthesizing a number of B- endorphin mimics with artificial C-terminal helical regions with amphipathic character. These de novo amphipathic helices were not homologous with the B-endorphin C-terminal region, and they were shown to be largely a-helical by circular dichroism (CD)

measurements. These hybrid structures showed good opioid agonism in vitro when compared to B-endorphin. These studies suggested that the overall amphipathicity of the C-terminal helix plays a key role in the selectivity of these compounds, rather than the identity of specific amino acid residues present in the C-terminal.

[0007] Dynorphin A (1-17) is also an endogenous opioid peptide, but it binds preferentially to the κ opioid receptor and has an N-terminal message segment identical to Leu-Enkephalin. It has been suggested that an address sequence in the C-terminal region imparts selectivity for κ receptors. Dynorphin A displayed an extended and/or random coil structure when subjected to structural analysis by various spectroscopic measurements. A 2D (1) H- MR study in DPC micelle shows that Dynorphin A(l-17) contains a less ordered N- terminal segment, a well-defined a-helix segment spanning between Phe(4) and Pro(10) or Lys(l 1), and a B-turn from Trp(14) to Gln(17). Based on NMR results, it is believed that both the a-helix and the C-terminal B-turn are due to dynorphin-micelle interactions, and may be important structural features of the full-length peptide when bound to the cell membrane in vivo.

[0008] The biological importance of helical C-terminal address segments in larger opioid peptides has been well documented. Studies have shown several potent nociceptin (NC) peptide analogs exploiting the a-helix-promoting residues a-aminoisobutyric acid (Aib) and N-methyl alanine (MeAla) at the C-terminus of NC. Nociceptin is the endogenous ligand for the recently identified opioid receptor-like 1 receptor (ORL-1). Thus, it seems logical to approach the design of opioid agonist B-endorphin or dynorphin peptide analogs by combining C-terminal amphipathic helical address segments that can also promote BBB penetration by virtue of glycosylation.

SUMMARY OF THE INVENTION

[0009] One aspect of the present invention provides glycopeptides that are capable of penetrating the blood-brain-barriers (BBB) for treating traumatic brain injury or stroke. In particular, the present inventors have discovered that a variation of glycosylated pleiotropic peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) or vasoactive intestinal peptide (VIP), which can both activate PAC_1; Vn & VIP₂ receptors, can cause

neuroprotective effects. Also, in their N-terminal truncated forms, these peptides can antagonize these receptors, having anti-inflammatory effects in several models of acute neuronal damage and neurodegenerative diseases, including ALS, PD, AD, HD, migraines, traumatic brain injury, stroke and certain forms of dementia.

[0010] The present invention provides a method of relieving symptoms of ALS, PD,

AD, HD, migraines, traumatic brain injury, stroke and certain forms of dementia. Such method generally comprises administering to a subject in need thereof an effective amount of a glycosylated PACAP or VIP analogue.

[0011] One particular aspect of the invention provides a method for treating traumatic brain injury or stroke in a subject. The method comprises administering a therapeutically effective amount of a glycosolated pleiotropic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) or a derivative thereof. In some embodiments, said PACAP comprises PACAP-27. Yet in other embodiments, said PACAP comprises PACAP-38. The therapeutically effective amount can be a unit dose amount of between 0.1 and 10 milligrams per kilo. Such a dose can be administered once a day or twice a day.

[0012] Yet in other embodiments, said traumatic brain injury comprise central nervous system injury. Still in other embodiments, said traumatic brain injury comprises neurotrauma. In some embodiments, said traumatic brain injury is caused by violence, transportation accidents, construction accidents, sports, war, or any other damage to the brain resulting from external mechanical force due to rapid acceleration or deceleration, impact, blast waves, or penetration by a projectile.

[0013] Still in other embodiments, said glycosylated PACAP or a derivative thereof comprises SEQ ID NO:4, 7, 8, 10 or 11 or a mixture thereof. Yet in other embodiments, said glycosylated PACAP or a derivative thereof comprises a mono- or disaccharide. In some instances, said mono- or disaccharide is O-linked to said glycosylated PACAP or a derivative thereof. In other embodiments, said glycosylated PACAP or a derivative thereof comprises a serine on the C-terminus, and wherein said glycosylate is attached to a hydroxyl group of said serine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 shows increased stability of the Serine glucoside in mouse serum compared to native peptide PACAPi_-27 and the corresponding lactoside; and

[0015] Fig. 2 shows PACi - CHO calcium flux of PACAPi_-27 and various truncated derivatives, demonstrating that PACAP_I-27SG is potent and efficacious at the PACi receptor.

[0016] Figs. 3A-3D show PC 12 cell morphology after vehicle versus various PACAP analog treatments.

DETAILED DESCRIPTION OF THE INVENTION

[0017] It must be noted that, as used herein, and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the preferred methods are now described. All publications and references mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0018] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. [0019] "Administering" when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted.

"Administering" a composition may be accomplished by oral or rectal administration, injection, infusion, inhalation, absorption or by any method in combination with other known techniques. Such combination techniques include heating, radiation and ultrasound.

[0020] The term "improves" is used to convey that the present disclosure changes the appearance, form, characteristics and/or physical attributes of the tissue to which it is being provided, applied or administered. "Improves" may also refer to the overall physical state of an individual to whom an active agent has been administered. For example, the overall physical state of an individual may "improve" if one or more symptoms of a

neurodegenerative disorder are alleviated by administration of an active agent, and is not limited to increased stability or BBB penetration.

[0021] As used herein, the term "therapeutic" means an agent utilized to treat, combat, ameliorate or prevent an unwanted condition or disease of a patient.

[0022] The terms "therapeutically effective amount" or "therapeutic dose" as used herein are interchangeable and may refer to the amount of an active agent or pharmaceutical compound or composition that elicits a biological or medicinal response in a tissue, system, animal, individual, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. A biological or medicinal response may include, for example, one or more of the following: (1) preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display pathology or symptoms of the disease, condition or disorder, (2) inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptoms of the disease, condition or disorder or arresting further development of the pathology and/or symptoms of the disease, condition or disorder, and (3) ameliorating a disease, condition or disorder in an individual that is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder or reversing the pathology and/or symptoms experienced or exhibited by the individual.

[0023] The term "treating" may be taken to mean prophylaxis of a specific disorder, disease or condition, alleviation of the symptoms associated with a specific disorder, disease or condition and/or prevention of the symptoms associated with a specific disorder, disease or condition. In some embodiments, the term refers to slowing the progression of the disorder, disease or condition or alleviating the symptoms associated with the specific disorder, disease or condition. In some embodiments, the term refers to slowing the progression of the disorder, disease or condition. In some embodiments, the term refers to alleviating the symptoms associated with the specific disorder, disease or condition. In some embodiments, the term refers to restoring function, which was impaired or lost due to a specific disorder, disease or condition.

[0024] The term "patient" generally refers to any living organism to which the compounds described herein, are administered and may include, but is not limited to, any non-human mammal, primate or human. Such "patients" may or may not be exhibiting the signs, symptoms or pathology of the particular diseased state.

[0025] For the purposes of this disclosure, a "salt" is any acid addition salt, preferably a pharmaceutically acceptable acid addition salt, including but not limited to, halogenic acid salts such as hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acid salt such as, for example, nitric, perchloric, sulfuric and phosphoric acid salt; an organic acid salt such as, for example, sulfonic acid salts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or /?-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, palmoic, pantothenic, oxalic and maleic acid salts; and an amino acid salt such as aspartic or glutamic acid salt. The acid addition salt may be a mono- or di-acid addition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di- organic acid salt. In all cases, the acid addition salt is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or

precipitation of a specific optical isomer of the products of this disclosure.

[0026] "Pharmaceutically acceptable salt" is meant to indicate those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response and the like, and are

commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. (1977) J. Pharm. Sciences, Vol 6. 1-19, which is hereby incorporated by reference in its entirety, describes pharmaceutically acceptable salts in detail.

[0027] As used herein, the term "daily dose amount" refers to the amount of pharmaceutically active compound per day that is administered or prescribed to a patient. This amount can be administered in multiple unit doses or in a single unit dose, in a single time during the day or at multiple times during the day. [0028] A "dose amount" as used herein, is generally equal to the dosage of the active ingredient, which may be administered per day. For example, an effective dose amount may be between about 0.1 and 10 milligrams per kilo, administered 1-2 times a day.

[0029] The term "unit dose" as used herein may be taken to indicate a discrete amount of the therapeutic composition that contains a predetermined amount of the active compound. The amount of the active compound is generally equal to the dosage of the active ingredient, which may be administered one or more times per day. For example, the unit dose may be a fraction of the desired daily dose which may be given in fractional increments, such as, for example, one-half or one-third the dosage.

[0030] The present invention is based in part on the recent discovery that penetration of the blood-brain barrier (BBB) by peptides as well as their stability in vivo is significantly enhanced by glycosylation. As used herein, the terms "glycosylation" and "glycosylated" means that an amino acid residue is functionalized with a glycosyl group. A glycosyl group is composed of saccharide units. These terms are well-known in the field of peptide and protein chemistry and have such meanings as used herein. In some embodiments, the glycosyl group has at most 8 saccharide units. In other embodiments, the glycosyl group has at most 4 saccharide units. Still in another embodiments, the glycosyl group is at most a disaccharide, i.e., the glycosyl group has at most 2 saccharide units. Thus, the total number of saccharide units can range from 1 to 8, inclusive of all specific values and ranges between. Examples of glycosyl groups include B-D-glucose, B-maltose, B-lactose, B-melibiose and B-maltotriose. Other examples include sucrose, trehalose, saccharose, maltose, cellobiose, gentibiose, isomaltose and primeveose. Other glycosyl groups include galactose, xylose, mannose, manosaminic acid, fucose, GalNAc, GlcNAc, idose, iduronic acid, glucuronic acid and sialic acid.

[0031] The present inventors have discovered that a glycosylated pleiotropic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) of the invention provides neuroprotective and anti-inflammatory properties. Vasoactive intestinal peptide (VIP) is a closely related secretin-class peptide. Thus, glycosylated peptides disclosed herein are useful in treating traumatic brain injury and stroke. Exemplary traumatic brain injuries (TBI) that can be treated using the compounds of the invention include, but are not limited to, those caused by violence, transportation accidents, construction accidents, sports, war and any other damage to the brain resulting from external mechanical force, such as rapid acceleration or deceleration, impact, blast waves, or penetration by a projectile. Traumatic brain injuries can be categorized as either central nervous system injuries or neurotrauma. Methods of the invention are suitable for treating both of these types of traumatic brain injuries. In one particular embodiment, the TBI is central nervous system injuries. Yet in another embodiment, the method of the invention is used to treat neurotrauma.

[0032] PACAP (SEQ ID NO: 1) is a neuropeptide. Two forms of PACAP have been identified: PACAP-38 and PACAP-27 consisting of 38 amino acids (SEQ ID NO:2) and 27 amino acids (SEQ ID NO:3), which is shortened at the C-terminus. As can be seen SEQ ID NO: 1 and SEQ ID NO: 2 differs only in that the -OH group on the C-terminus of SEQ ID NO:2 is replaced with -NH₂ group.

[0033] PACAPi.27 has a 68% homology to VIP. PACAP was first isolated from ovine hypothalamus, and is known to regulate the development, maintenance, function, and plasticity of the nervous system, providing neuroprotective and neurotrophic support.

PACAP has been shown to activate 3 closely-related G protein coupled receptors: PACi (which has higher affinity for PACAP), VPACi, and VPAC₂. These receptors bind to both PACAP and VIP and are expressed on neurons, microglia, and also by many other cell types. Constitutive expression of PACAP and its receptor PACi is believed to confer

neuroprotection to central visceromotor neurons via the PACi receptor. PACAP also promotes cytodestructive functions of microglia (Ml amoeboid→ M2 hypertrophic phenotype), thought to drive ALS disease progression via the VPACi receptor. Thus, the ideal drugs for neuroprotection would be PACi agonists at motor neurons to promote neuroprotection in case of ALS, or dopaminergic neurons in case of PD, or hippocampal neurons in case of AD, and in each case VPACi antagonists at microglia to reduce inflammation by maintaining the Ml ('alternatively activated'/resolving anti-inflammatory cells) phenotype vs. the M2 (the classical, proinflammatory macrophages) microglia phenotype or Tau-opathies.

[0034] Glycopeptide analogs of PACAP were prepared with different binding properties to either be only a PACi agonist or only a VPACi antagonist. The glycopeptides of the present invention were produced using Fmoc-based solid-phase peptide methods, and purified by HPLC. Typically, the glycosyl group was linked to the amino acid sequence by an O-linkage to a side chain in the address segment of the sequence. See, for example,

Tetrahedron Asymmetry, 2005, 16, 65-75 and U.S. Pat. No. 5,727,254, all of which are incorporated herein by reference in their entirety. Briefly, the C-terminal amino acids were loaded onto Fmoc-Rink resin (Advanced ChemTech, Louisville, KY, USA) at 0.1 mmol/g resin loading in 25 mL fritted syringes. Initially, the resin was swelled using dimethylformamide (DMF, ~5 mL solvent per gram resin), agitating at room temperature ("RT") for two minutes (x2). A solution of 2% DBU and 3% piped dine in DMF (v:v) was introduced and agitated for 5 minutes, refreshed, and agitated for an additional 10 minutes. The resin was washed with DMF (x5), and finally with N-methylpyrrolidine ( MP). In a separate vial, Fmoc-P-OGlc(OAc)4-Ser-OH (0.12 mmol, 1.2 eq) was dissolved in 5 mL NMP, and HOBt^»H₂0 (0.13 mmol, 1.3 eq) was added and allowed to mix for 5 minutes. Condensing agent DIC (0.26 mmol, 2.6 eq) was then added, and mixed for 5 minutes. This solution was added to the resin and agitated for 10 minutes. Next, the syringe was placed in a microwave (Emerson 900W Microwave - MW9338SB) set to power level 1 and irradiated for 10 minutes, stopping to shake the syringe every 90 seconds. The syringe was then agitated at RT for an additional 30 minutes. The resin was washed with NMP (xl), DMF (x5), and CH₂C1₂ (x5), and dried in vacuo overnight.

[0035] Peptides and glycopeptides also were assembled on a Prelude^® Peptide

Synthesizer (Protein Technologies, Inc., Tucson, AZ, USA) using the following procedure. Rink resin (100 mg) was placed into the fritted reaction vessels (RVs). Amino acids were dissolved in DMF at 250 mM concentration, HATU at 375 mM, and TMP at 3M. The following steps were performed for coupling: DMF Top Wash (1.5 mL, 2 min mix and drain; x6), Deprotection (2% DBU/3% piperidine in DMF; 1.5 mL, 4 min mix and drain; 8 min mix and drain), DMF Top Wash (1.5 ml, 2 min mix and drain; x5), Amino Acid Building Block (0.950 mL, 30 sec mix), Activator 1 (HATU, 0.650 mL, 30 sec mix), Base (TMP, 0.300 mL, 35 min mix and drain), DMF Top Wash (1.5 mL, 2 min mix and drain; x2). After coupling aspartic acid D7, the deprotection solution was changed to 0.1 M HOBt^»H₂0/5% piperazine in DMF to minimize aspartimide formation.

[0036] Cleavage of the peptides and glycopeptides from the resin was accomplished with a mixture of F₃CCOOH:Et₃SiH:H₂0:CH₂Cl₂:Ph-OCH₃ (by volume, 9:0.3 :0.2: 1 :0.05), agitating at RT for 2 hours. The resulting solutions were expelled into 15 mL centrifuge tubes, evaporated under argon, precipitated in ice-cold Et₂0, decanted, and rewashed with Et₂0, then dissolved in H₂0 and lyophilized to generate the crude material as fluffy white solids.

[0037] Purification of the crude glycopeptides was accomplished by Reversed Phase

HPLC (RP-HPLC) with a preparative RP (C-18) Phenomenex (250 X 22 mm) column using a CH₃CN-H₂0 gradient solvent system containing 0.1% F₃CCOOH. Homogeneity of the purified glycopeptides was confirmed by analytical RP-HPLC and high resolution mass spectrometry.

[0038] Neuroprotective effects of the PACi agonist and the anti-inflammatory effects of the VPACi antagonist were tested in cell culture models. In addition, stability and BBB penetration in vivo were also tested. Flow-injection tandem mass spectrometry (FI-MSⁿ) was used to observe the degradation of the peptides and glycopeptides with a Thermo LCQ with electrospray ionization (ESI). The technique involved injection of a sample bolus of material in mouse serum via a six port valve with fluid flow delivered via a syringe pump, and subsequent ESI followed by mass spectral analysis. Samples were diluted to a concentration of ~5 μΜ of each PACAP analogue, and were incubated at 37 °C for times varying from 1 to 60 minutes. After samples had been incubated for the prescribed amount of time they were prepared for mass spectrometry analysis by withdrawing 10 microliters of solution and spiking with 1 microliter of a 10 μΜ solution of peptide internal standard (angiotensin II) in 50% acetic acid and subjecting them to a standard CI 8 zip tip desalting. These solutions, once eluted from the zip tip were diluted to 100 μΙ_, in 50:50 acetonitrile/water with 0.1% formic acid. Tandem mass spectrometry analysis (MS³) was conducted to yield specific, quantitative signals proportional to the amount of PACAP analogue at each time point. This technique was also used with microdialysate samples from a mouse after i.p. administration of PACAPi__27-SG.

[0039] A custom DNA clone of the human PACi gene with 3 hemagglutinin (HA) tags inserted 3' to the signal peptide sequence was obtained from Genecopoeia (Rockville, MD). The construct was electroporated into Chinese Hamster Ovary (CHO) cells, and selected for with 500 μg/mL of G418. The resulting population was screened for high expressing clones, and one such clone selected for further analysis. The clonal cell line (PACi-CHO) displayed high receptor expression by immunocytochemistry and Western blot, and showed selective activation of signaling in response to PACAPi_₂₇. This cell line was used for all molecular pharmacology experiments. The cells were maintained in DMEM/F12 with 10%) heat-inactivated FBS, IX penicillin/streptomycin, and 500 μg/mL G418, at 37°C and 5% C0₂.

[0040] All molecular pharmacology experiments were carried out using a FLIPR

Tetra from Molecular Devices (Sunnyvale, CA) set to image calcium flux using the manufacturer's recommended settings and protocols. The day before an experiment, the PACi-CHO cells were split into 384 well black walled, clear bottom microplates, 10,000 cells per well. The cells were recovered overnight in growth medium (as above). The next day, the growth medium was replaced with Calcium 6 dye (Molecular Devices) using the manufacturer recommended buffer with 2.5 mM probenecid. The cells were incubated for 2 hours in the culture incubator, and removed during the last 15 minutes to allow equilibration to room temperature. Compound was added to the cells using a 384 tip block, with real time monitoring before, during, and 15 minutes after compound addition. The resulting calcium flux was recorded, and the maximum-minimum response over the entire observation time calculated and reported as the mean ± SEM (4 wells per point, Figure 2).

[0041] For agonist mode experiments, compound was added in an 11 point concentration curve, with a vehicle control (buffer). The resulting response was normalized to the stimulation caused by PACAPi_-27 (100%) and vehicle (0%). The response was analyzed using a 3 variable non-linear curve fit, and the EC50 (nM) and E_Max (%) calculated and reported (Prism, GraphPad, La Jolla, CA).

[0042] Activation of PAC 1R, VP AC 1R and VPAC2R result in a wide variety of effects throughout the body, both peripherally and centrally. The neuroprotective effects are associated with each of these receptors, but especially with the PAC1R activation. PAC1 receptors are found extensively in the CNS; peripherally they are highly expressed in the adrenal medulla. The rat pheochromocytoma (PC 12) cell line was used as a model for PAC1R activation based on treated cells undergoing differentiation resulting in increased cell-body volume, extension of neurite-like cell processes, along with an antiproliferative action to decrease cell numbers. This cell type also responds in a similar manner to nerve growth factor (NGF) through the tyrosine receptor kinase A (TrkA) receptor, the pathways for the two receptors being complementary, working through different transduction pathways to act synergistically to promote neurite-like process outgrowth.

[0043] As an initial screening tool for the PACAP glycopeptide analogs, PC12 cells were used for assessing the ability of the analogs to initiate a morphological change. The PC12 cells were cultured in RPMI containing 5% heat inactivated fetal bovine serum and 10% horse serum in the presence of 100 units/mL penicillin and 100 microgram/mL streptomycin. The cells were plated on poly-D-Lysine coated 6-well tissue culture plates at a density of 150,000 cells per well in 2 mL media. After 48 hours at 37 °C in 5% C0₂ atmosphere, media exchange was performed and plates were dosed, using the peptide diluent (water) for the control samples. PACAPi_₂₇, PACAP _I__27-S-G, and PAC AP_I__27-S-_L were used to screen for PACi receptor activation. Four groups of cells were used; one control group (diluent treated) and three treatment groups, each treatment group was exposed to 100 nM concentrations of PACAPi_₂₇, PACAP _I__27-S-G, or PACAP _I__27-S-_L All groups were run in triplicate. Cells were treated daily for 5 days, then viewed at 400X magnification (Nikon Eclipse TE2000-U). Cell images of each treatment group were captured and compared to the control cells to screen for differentiation and cell body volume increases. Cells having neurite-like process outgrowth were noted and photographed. The neurite-like outgrowth was deemed positive if its length was at least two times the width of the cell body.

[0044] Cell number and cell body volume were also noted to evaluate

antiproliferative and differentiation effects, both indications of PAC1R activation in PC12 cells. The cell body size of the control cells remained relatively constant, without showing signs of increased cell volume when compared to the treated cells. Since there was limited cell adherence to the culture dishes and significant cell number loss upon media exchange, cell counts were not performed during this initial screen. With adherent cells, in order to quantify the antiproliferative action of the PACAP analogs the cells may be detached using AccutaseTM (Innovative Cell Technologies, Inc.) and cell size and number can be measured using the Beckman Z2 Coulter Counter, with the lower and upper cell size limits set to 10 and 17 micrometers respectively, and the results expressed as % compared to control.

[0045] As shown in Figures 3 A-3D, the control cells (Fig. 3 A) grew in clumps and did not exhibit detectible process extension or differentiation. Neurite-like process outgrowth and cell body volume expansion was evident in the cells treated daily for 5 days with 100 nM PACAP27 (Fig. 3B), PACAP27S-glucoside (Fig. 3C) and PACAP27S-lactoside (Fig. 3D).

[0046] RESULTS: PACAP derivatives and glycosides were synthesized using solid- phase methods. Some of the representative PACAP derivatives and glycoside analogs of the invention are shown in Table 1. One analog of PACAPi_₂7 was produced by replacing the terminal Leucine 27 amide with a Serine lactoside amide (Ser-O- -D-Glc- -D-Gal)

("PACAP I-27-S-L") Modeling of this compound suggested that the PACAP glycosides can adopt a largely helical conformation.

[0047] In Table 1, the truncated peptides and glycopeptides are missing five N- terminal amino acids responsible for binding to the transmembrane portion of the GPCR receptors. These truncated compounds are expected to be antagonists. For the glycosides, Leucine 27 was replaced by a Serine glycoside bearing glucose (-β-D-Glc, indicated by the "S-G" subscript) or lactose (- -D-Glc- -D-Gal as indicated by the "S-L" subscript). The final 3 alternate compounds were modified by replacing methionine 17 with Leucine, and Lysines 15, 20 and 21 with Arginines to enhance stability in vivo. These compounds are expected to be antagonists at PACi. Table 1: Re resentative analo s of PACAP

[0048] Chemical stability of the glycopeptides in vivo clearly plays an important role in the deliverability of the drugs to the site(s) of action within the brain. Tandem mass spectroscopy (MSⁿ) was used to determine both the stability of the PACAP compounds in mouse serum, and to identify specific cleavage products, which can stem from inherent chemical instability, or from enzymatic hydrolysis. Collision induced fragmentation of the 2^" ion at 1642.7 m/z from mouse brain dialysate was carried out using MS/MS. This spectrum showed a clear pattern from the N-terminus in which fragmentation occurs at the aspartic acid residues resulting y²⁺ ions. The glycosylated yig²⁺ and y₂₄ ²⁺ at 1206.0 and 1472.7 m/z respectively, were the predominant ions in the spectrum (NH₃ loss omitted for clarity). β-Glc cleavage gave 1560.9 m/z, matching the calculated mass for PACAPi_-27 with a C-terminal amide. Loss of the glucoside along with peptide backbone cleavage resulted in yi₉ ²⁺ and y₂₄ ²⁺ ions at 1124.9 and m/z 1391.6.

[0049] Stability of PACAPi_-27, PACAP_I-27-S-G and PACAP _I-27-S-_L was tested.

PACAPi_₂₇ and its glycosylated analogues degraded over 30 min in mouse serum at 37 °C. Data were fitted using a single exponential decay model (R² > 0.71, in all cases). Serine glucoside (PACAP _I__27-S-G_, Glc) showed a significant increase in mouse serum tm in vitro compared to the native peptide PACAPi_₂₇ and the corresponding lactoside (PACAP _I__27-S-_L, Lac) when compared using a 1-way analysis of variance (F2 = 12.91, p = 0.0067, Tukey's multiple comparison Native vs Glc, q = 5.760 p < 0.05, Lac vs Glc, q = 6.602 p < 0.5). See Figure 1. [0050] Using CHO cells that express human PACi receptors, the following compounds were tested for agonist activity using FLIPR: PACAPi_₂₇, the glucoside

PACAP27-S-G, and the truncated putative antagonist PACAP_6-27 and its derivatives. It was found that PACAPi_₂7 and PACAP_I_₂7-S-G, the serine glucoside, activated PACi with high potency (0.95 ± 0.4 nM and 5.68 ± 2.3 nM respectively, Figure 2 and Table 2). These values are similar to what has been reported for PACAPi_₂₇. In addition, the normalized efficacy of the PACAPi__27-s-G glucoside at 101.9 ± 1.6 % was nearly identical to the native PACAPi_₂₇ peptide. These findings strongly suggest that glycosylation of PACAPi_₂₇ does not significantly (i.e., within about ± 10%, typically within about ± 5%) alter binding and activation of the PACi receptor, supporting the use of such a glycopeptide for therapeutic purposes. As expected, none of the PACAP₆_₂₇ derivatives showed any significant agonist activity at concentrations up to 1 μΜ (Figure 2 and Table 2).

Table 2

[0051] PC12 cells are non-adherent cells, and in spite of using the poly-D-Lysine coated plates, the majority of the cells remained suspended. During the media exchange many of the cells were removed with the spent media. The remaining cells could be visually evaluated for qualitative morphological changes at the end of the treatment period, but meaningful cell quantification could not be done reliably using this approach. It was found that glucoside and lactoside PACAPi_-27 derivative treatment produced neurite outgrowth and arborization when compared to vehicle treated cells. See Figures 3 A-D. Qualitatively, it appeared that the arborization caused by PACAPi_-27 may be more extensive than that caused by the glucoside and lactoside derivatives, but this could not be quantified. In any case, both PACAPi_-27 and the derivatives induced neurite outgrowth, suggesting native PACi agonist activity. In all cases the process outgrowths on the treated cells were greater than 2X the cell body width. [0052] Figures 3A-3D show PC12 cell morphology after Vehicle vs PACAP treatment (100 nM). Fig. 3A: diluent only. Fig. 3B: PACAPi_₂₇, Fig. 3C: PACAP _I_₂₇-S-G, Fig. 3D: PACAP_I__27-S-_L- The cell body volumes all showed increase when treated with each of the PACAP derivatives in Table 1. In all cases the process outgrowths on the treated cells were greater than 2X the cell body width.

[0053] Endogenous PACAP peptides occur as C-terminal peptide amides that have either 27 or 38 amino acid residues, and are typically regarded as PACi agonists in assays using intact tissue or in cell culture. Use of solid-phase peptide synthesis has allowed replacement of the terminal Leucine amide with glycosides of Serine amide bearing the simple sugars glucose or lactose. These O-linked glycopeptides not only retained their agonist activity on PC12 cell cultures and in the quantitative CHO cell assay, but also showed extended lifetimes in mouse serum, and provided evidence via microdialysis studies that the glycopeptides can cross the blood brain barrier in mice. In use, an effective amount of the PACAP-VIP glycopeptides of the present invention can be administered to a patient in need of treatment in a therapeutically effective unit dose delivery amount of between about 0.1 and 10 milligrams per kilo, typically 1 - 2 doses per day, or even less frequently. The PACAP- VIP glycopeptides can be delivered in a pharmaceutically acceptable carrier.

[0054] Pharmaceutical formulations and pharmaceutical compositions are well known in the art, and can be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., USA, which is hereby incorporated by reference in its entirety. Any formulations described therein or otherwise known in the art are embraced by embodiments of the disclosure.

[0055] Pharmaceutical excipients are well known in the art and include, but are not limited to, saccharides such as, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations, calcium phosphates such as tricalcium phosphate or calcium hydrogen phosphate, as well as binders, such as, starch paste, which includes maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose,

hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone or combinations thereof.

[0056] Pharmaceutical formulations may include the active compound described and embodied above, a pharmaceutically acceptable carrier or excipient and any number of additional or auxiliary components known in the pharmaceutical arts such as, for example, binders, fillers, disintegrating agents, sweeteners, wetting agents, colorants, sustained release agents, and the like, and in certain embodiments, the pharmaceutical composition may include one or more secondary active agents. Disintegrating agents, such as starches as described above, carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate and combinations thereof. Auxiliary agents may include, for example, flow-regulating agents and lubricants, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, polyethylene glycol and combinations thereof. In certain embodiments, dragee cores may be prepared with suitable coatings that are resistant to gastric juices, such as concentrated saccharide solutions, which may contain, for example, gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures and combinations thereof. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethyl- cellulose phthalate may also be used. In still other embodiments, dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

[0057] Pharmaceutical compositions of the disclosure can be administered to any animal, and in particular, any mammal, that may experience a beneficial effect as a result of being administered a compound of the disclosure including, but not limited to, humans, canines, felines, livestock, horses, cattle, sheep, and the like. The dosage or amount of at least one compound according to the disclosure provided pharmaceutical compositions of embodiments may vary and may depend, for example, on the use of the pharmaceutical composition, the mode of administration or delivery of the pharmaceutical composition, the disease indication being treated, the age, health, weight, etc. of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired and so on.

Various embodiments of the disclosure include pharmaceutical compositions that include one or more compounds of the disclosure in an amount sufficient to treat or prevent diseases such as, for example, cancer. An effective amount of the one or more compounds may vary and may be, for example, from about 0.1 to 10 milligrams per kilo, typically 1-2 doses per day.

[0058] The pharmaceutical compositions of the disclosure can be administered by any means that achieve their intended purpose. For example, routes of administration

encompassed by the disclosure include, but are not limited to, subcutaneous, intravenous, intramuscular, intraperitoneal, buccal, or ocular routes, rectally, parenterally,

intrasystemically, intravaginally, topically (as by powders, ointments, drops or transdermal patch), oral or nasal spray are contemplated in combination with the above described compositions. [0059] Embodiments of the disclosure also include methods for preparing pharmaceutical compositions as described above by, for example, conventional mixing, granulating, dragee-making, dissolving, lyophilizing processes and the like. For example, pharmaceutical compositions for oral use can be obtained by combining the one or more active compounds with one or more solid excipients and, optionally, grinding the mixture.

[0060] Suitable auxiliaries may then be added and the mixture may be processed to form granules which may be used to form tablets or dragee cores. Other pharmaceutical solid preparations include push- fit capsules containing granules of one or more compound of the disclosure that can, in some embodiments, be mixed, for example, with fillers, binders, lubricants, stearate, stabilizers or combinations thereof. Push-fit capsules are well known and may be made of gelatin alone or gelatin in combination with one or more plasticizer such as glycerol or sorbitol to form a soft capsule. In embodiments in which soft capsules are utilized, compounds of the disclosure may be dissolved or suspended in one or more suitable liquids, such as, fatty oils or liquid paraffin and, in some cases, one or more stabilizers.

[0061] Liquid dosage formulations suitable for oral administration are also

encompassed by embodiments of the disclosure. Such embodiments, may include one or more compounds of the disclosure in pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs that may contain, for example, one or more inert diluents commonly used in the art such as, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethyl formamide, oils (for example, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, fatty acid derivatives of glycerol (for example, labrasol), tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions may further contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum

metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0062] Formulations for parenteral administration may include one or more compounds of the disclosure in water-soluble form, for example, water-soluble salts, alkaline solutions, and cyclodextrin inclusion complexes in a physiologically acceptable diluent which may be administered by injection. Physiologically acceptable diluent of such embodiments, may include, for example, sterile liquids such as water, saline, aqueous dextrose, other pharmaceutically acceptable sugar solutions; alcohols such as ethanol, isopropanol or hexadecyl alcohol; glycols such as propylene glycol or polyethylene glycol; glycerol ketals such as 2,2-dimethyl-l,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol)400; pharmaceutically acceptable oils such as fatty acid, fatty acid ester or glyceride, or an acetylated fatty acid glyceride. In some embodiments, formulations suitable for parenteral administration may additionally include one or more pharmaceutically acceptable surfactants, such as a soap or detergent; suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose; an emulsifying agent;

pharmaceutically acceptable adjuvants or combinations thereof. Additional pharmaceutically acceptable oils which may be useful in such formulations include those of petroleum, animal, vegetable or synthetic origin including, but not limited to, peanut oil, soybean oil, sesame oil, cottonseed oil, olive oil, sunflower oil, petrolatum, and mineral oil; fatty acids such as oleic acid, stearic acid, and isostearic acid; and fatty acid esters such as ethyl oleate and isopropyl myristate. Additional suitable detergents include, for example, fatty acid alkali metal, ammonium, and triethanolamine salts; cationic detergents such as dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; and anionic detergents, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates. In some embodiments, non-ionic detergents including, but not limited to, fatty amine oxides, fatty acid alkanolamides and polyoxyethylenepolypropylene copolymers or amphoteric detergents such as alkyl-P-aminopropionates and 2- alkylimidazoline quaternary salts, and mixtures thereof may be useful in parenteral formulations of the disclosure.

[0063] Pharmaceutical compositions for parenteral administration may contain from about 0.5 to about 25% by weight of one or more of the compounds of the disclosure and from about 0.05% to about 5% suspending agent in an isotonic medium. In various embodiments, the injectable solution should be sterile and should be fluid to the extent that it can be easily loaded into a syringe. In addition, injectable pharmaceutical compositions may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0064] Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye. Compositions for topical administration, may be prepared as a dry powder which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredients in admixture are prepared as a finely divided powder. In such embodiments, at least 95% by weight of the particles of the admixture may have an effective particle size in the range of 0.01 to 10 micrometers. In some embodiments, the finely divided admixture powder may be additionally mixed with an inert carrier such as a sugar having a larger particle size, for example, of up to 100 micrometers in diameter.

Alternatively, the composition may be pressurized using a compressed gas, such as nitrogen or a liquefied gas propellant. In embodiments, in which a liquefied propellant medium is used, the propellant may be chosen such that the compound and/or an admixture including the compound do not dissolve in the propellant to any substantial extent. In some

embodiments, a pressurized form of the composition may also contain a surface-active agent. The surface-active agent may be a liquid or solid non-ionic surface-active agent or may be a solid anionic surface-active agent, which in certain embodiments, may be in the form of a sodium salt.

[0065] Compositions for rectal administration may be prepared by mixing the compounds or compositions of the disclosure with suitable non-irritating excipients or carriers such as for example, cocoa butter, polyethylene glycol or a suppository wax. Such carriers may be solid at room temperature but liquid at body temperature and therefore melt in the rectum and release the drugs.

[0066] In still other embodiments, the compounds or compositions of the disclosure can be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances that form mono- or multi-lamellar hydrated liquid crystals when dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used, and in particular embodiments, the lipids utilized may be natural and/or synthetic phospholipids and phosphatidyl cholines (lecithins). Methods to form liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell Biol. 14:33 (1976), which is hereby incorporated by reference in its entirety). Compositions including one or more compounds of the disclosure in liposome form can contain, for example, stabilizers, preservatives, excipients and the like.

[0067] In general, methods of embodiments of the disclosure may include the step of administering or providing an "effective amount" or a "therapeutically effective amount" of a compound or composition of the disclosure to an individual. In such embodiments, an effective amount of the compounds of the disclosure may be any amount that produces the desired effect. As described above, this amount may vary depending on, for example, the circumstances under which the compound or composition is administered (e.g., to incite treatment or prophylactically), the type of individual, the size, health, etc. of the individual and so on. The dosage may further vary based on the severity of the condition. For example, a higher dose may be administered to treat an individual with a well-developed

neurodegenerative condition, compared to the amount used to prevent a subject from developing the neurodegenerative condition. Those skilled in the art can discern the proper dosage based on such factors. For example, in some embodiments, the dosage may be within the range of about 0.01 mg/kg body weight to about 10 mg/kg body weight.

[0068] The administration schedule may also vary. For example, in some

embodiments, the compounds or compositions of the disclosure may be administered in a single dose once per day or once per week. In other embodiments, the compounds or compositions of the disclosure may be administered in one or two or more doses per day. For example, in one embodiment, an effective amount for a single day may be divided into separate dosages that may contain the same or a different amount of the compound or composition and may be administered several times throughout a single day. The dosage per administration and frequency of administration may depend, for example, on the specific compound or composition used, the condition being treated, the severity of the condition being treated, and the age, weight, and general physical condition of the individual to which the compound or composition is administered and other medications which the individual may be taking. In another exemplary embodiment, treatment may be initiated with smaller dosages that are less than the optimum dose of the compound, and the dosage may be increased incrementally until a more optimum dosage is achieved.

[0069] In each of the embodiments above, the compound administered can be provided as a pharmaceutical composition including compound as described above and a pharmaceutically acceptable excipient or a pure form of the compound may be administered.

[0070] In additional embodiments, the compound or composition of the disclosure may be used alone or in combination with one or more additional agents. For example, in some embodiments, a compound or composition of disclosure may be formulated with one or more additional neuroprotective agents or combinations thereof such that the pharmaceutical composition obtained including the compound or composition of the disclosure and the one or more additional agents can be delivered to an individual in a single dose. In other embodiments, the compound or composition of the disclosure may be formulated as a separate pharmaceutical composition that is delivered in a separate dose from pharmaceutical compositions including the one or more additional agents. In such embodiments, two or more pharmaceutical compositions may be administered to deliver effective amounts of a compound or composition of the disclosure and the one or more additional agents.

[0071] The results of studies by the present inventors strongly support the notion that glycopeptides related to PACAP produce PACi agonism, or VPACi agonism or VPAC₂ agonism, all of which may be useful feature for the treatment of neurodegeneration, particularly for PD or treatment of traumatic brain injury or stroke. In addition, VPACi antagonism or VPAC₂ antagonism combined with PACi agonism may be particularly effective.

[0072] Results also show that glycosylation of PACAP peptides is a good strategy for increasing the in vivo stability and CNS penetration of peptide drugs, which may be useful as a strategy for the treatment of diseases like PD migraine, traumatic brain injury and stroke.

[0073] The PACAP- VIP glycopeptides of the present invention have several significant advantages. For example, by binding selectively to PACi and VP AC receptors, the protein has a very narrow range of influence. Side effects are therefore minimized.

Another advantage is that glycosylation allows the protein to cross the blood-brain barrier allowing the protein to carry out its function. Glycopeptides of the invention can be used to treat one of the causes of ALS, PD, AD, HD migraines, traumatic brain injury, stroke and forms of dementia rather than just treating symptoms. Thus, the protein has stronger effects than symptom -treating drugs.

[0074] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

Claims

What is Claimed is:

1. A method for treating traumatic brain injury or stroke in a subject, said method comprising administering a therapeutically effective amount of a glycosolated pleiotropic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) or a derivative thereof.

2. The method of Claim 1, wherein said PACAP comprises PACAP-27.

3. The method of Claim 1, wherein said PACAP comprises PACAP-38.

4. The method of Claim 1, wherein said therapeutically effective amount comprises a unit dose amount of between 0.1 and 10 milligrams per kilo.

5. The method of Claim 4, wherein the dose is administered once a day.

6. The method of Claim 4, wherein the dose is administered twice a day.

7. The method of Claim 2, wherein said therapeutically effective amount comprises a unit dose amount of between 0.1 and 10 milligrams per kilo.

8. The method of Claim 7, wherein the dose is administered once a day.

9. The method of Claim 7, wherein the dose is administered twice a day.

10. The method of Claim 3, wherein the therapeutically effective amount comprises a unit dose amount of between 0.1 and 10 milligrams per kilo.

11. The method of Claim 10, wherein the dose is administered once a day.

12. The method of Claim 10, wherein the dose is administered twice a day.

13. The method of Claim 1, wherein said traumatic brain injury comprise central nervous system injury.

14. The method of Claim 1, wherein said traumatic brain injury comprises neurotrauma.

15. The method of Claim 1, wherein said traumatic brain injury is caused by violence, transportation accidents, construction accidents, sports, war or any other damage to the brain resulting from external mechanical force due to rapid acceleration or deceleration, impact, blast waves, or penetration by a projectile.

16. The method of Claim 1, wherein said glycosolated PACAP or a derivative thereof comprises SEQ ID NO:4, 7, 8, 10 or 11 or a mixture thereof.

17. The method of Claim 1, wherein said glycosolated PACAP or a derivative thereof comprises a mono- or disaccharide.

18. The method of Claim 17, wherein said mono- or disaccharide is O-linked to said glycosolated PACAP or a derivative thereof.

19. The method of Claim 1, wherein said glycosolated PACAP or a derivative thereof comprises a serine on the C-terminus, and wherein said glycosylate is attached to said a hydroxyl group of said serine.