Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
IMMUNOMODULATORY PEPTIDES FOR TREATMENT OF PROGRESSIVE NEURODEGENERATIVE DISEASES
Document Type and Number:
WIPO Patent Application WO/2013/034982
Kind Code:
A2
Abstract:
Compositions and methods for the prevention or treatment of neurodegenerative disease are provided. The methods provide administering an innate defense regulator (IDR) peptide composition to the subject in an amount effective to reduce or eliminate the symptoms and causes of neurodegenerative disease including neuroinflammation.

Inventors:
HANCOCK, Robert, E., W. (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
CASHMAN, Neil, R. (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
WELLINGTON, Cheryl (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
MALLARD, Carina (Institute of Neuroscience and Physiology, Sahlgrenska AcademyUniversity of Gothenbur, Box 432 Gothenburg, SE-40530, SE)
Application Number:
IB2012/002216
Publication Date:
March 14, 2013
Filing Date:
September 10, 2012
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
THE UNIVERSITY OF BRITISH COLUMBIA (University -Industry Liaison Office, # Agronomy RoadVancouver, BC V6T 1Z3, 103-6190, CA)
HANCOCK, Robert, E., W. (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
CASHMAN, Neil, R. (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
WELLINGTON, Cheryl (University-Industry Liaison Office# Agronomy Roa, Vancouver BC V6T 1Z3, 103-6190, CA)
MALLARD, Carina (Institute of Neuroscience and Physiology, Sahlgrenska AcademyUniversity of Gothenbur, Box 432 Gothenburg, SE-40530, SE)
International Classes:
A61K38/08
Domestic Patent References:
WO2008022444A12008-02-28
WO2010026489A12010-03-11
WO2010043039A12010-04-22
Other References:
STREIT W.J. ET AL.: 'Microglia and Neuroinflammation: a Pathological Perspective' J. NEUROINFLAMMATION., [Online] vol. 1, no. 14, 30 July 2004, pages 1 - 4, XP021010065 Retrieved from the Internet: [retrieved on 2013-03-04]
Download PDF:
Claims:
What is Claimed:

1. An isolated innate defense regulator (IDR) peptide for the treatment of

neurodegenerative disease having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

2. The peptide of claim 1, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%>, or 95% identity thereto.

3. The peptide of claim 1, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95%) identity thereto.

4. The peptide of claim 1, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR- HH2), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

5. An isolated polynucleotide that encodes a peptide of claim 1, 2, 3, or 4.

6. The peptide of claim 1, 2, 3, or 4, wherein the neurogenerative disease is ALS.

7. The peptide of claim 1, 2, 3, or 4, wherein the neurodegenerative disease is

Alzheimer's disease.

8. The peptide of claim 1, 2, 3, or 4, wherein the neurodegenerative disease is selected from the group consisting of MS, viral encephalitis, meningitis, cerebral malaria, and neural inflammatory responses.

9. The peptide of claim 1, 2, 3, or 4, wherein the peptide reduces neuroinflammation.

10. The peptide of claim 1, 2, 3, or 4, wherein the peptide inhibits microglial activation.

11. The peptide of claim 1 , 2, 3, or 4, wherein the peptide results in improved motor function.

12. The peptide of claim 7, wherein the peptide results in improved cognitive function.

13. The peptide of claim 7, wherein the peptide results in reduced levels of Αβ peptides.

14. A method of preventing or treating a neurodegenerative disease, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neurodegenerative disease having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

15. The method of claim 14, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%>, or 95% identity thereto.

16. The method of claim 14, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

17. The method of claim 14, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR-HH2), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

18. The method of claim 14, 15, 16, or 17, wherein the neurodegenerative disease is ALS.

19. The method of claim 14, 15, 16, or 17, wherein the neurodegenerative disease is Alzheimer's disease.

20. The method of claim 14, 15, 16, or 17, wherein the neurodegenerative disease is selected from the group consisting of viral encephalitis, meningitis, cerebral malaria, and neural inflammatory responses.

21. The method of claim 14, 15, 16, or 17, wherein the treating results in reduced neuroinflammation.

22. The method of claim 14, 15, 16, or 17, wherein the treating results in improved motor function.

23. The method of claim 14, 15, 16, or 17, wherein the treating results in suppressed microglial activation.

24. The method of claim 19, wherein the treating results in improved cognitive function.

25. The method of claim 19, wherein the treating results in reduced levels of Αβ peptides.

26. A method of preventing or treating neuroinflammation, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neuroinflammation having 7 to 13 amino acids, having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

27. The method of claim 26, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%>, or 95% identity thereto.

28. The method of claim 26, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

29. The method of claim 26, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR-HH2), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

30. The method of claim 26, 27, 28, or 29, wherein the treating results in improved motor function.

31. The method of claim 26, 27, 28, or 29, wherein the treating results in improved cognitive function.

32. The method of claim 26, 27, 28, or 29, wherein the treating results in suppressed microglial activation.

33. A method for suppressing microglial activation, the method comprising contacting a microglial cell with an effective amount of the peptide of claims 1, 2, 3, or 4, wherein contacting the microglial cell with the peptide inhibits the secretion of neurotoxic molecules.

34. The method of claim 33, wherein the neurotoxic molecule is selected from the group consisting of proinflammatory cytokines, glutamate, free radical species, nitric oxide, and a combination thereof.

35. The method of claim 34, wherein the proinflammatory cytokine is TNF-a, IL-Ιβ, or both.

36. A method of preventing or treating neonatal brain injury, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neurodegenerative disease having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

37. The method of claim 36, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%>, or 95% identity thereto.

38. The method of claim 36, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

39. The method of claim 36, wherein the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR-HH2), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

40. The method claim 36, 37, 38, or 39, wherein the treating results in reduced secretion of TNF-a.

41. The method of claim 36, 37, 38, or 39, wherein the treating results in protection of white and gray cerebral matter in the neonatal brain.

42. The method of claim 36, 37, 38, or 39, wherein the treating results in inhibition of the p53 pathway.

43. The method of claim 36, 37, 38, or 39, wherein the peptide is administered after an initial insult that gives rise to the neonatal brain injury.

Description:
IMMUNOMODULATORY PEPTIDES FOR TREATMENT OF PROGRESSIVE

NEURODEGENERATIVE DISEASES

FIELD

[0001] The present invention relates generally to peptides and more specifically to immunomodulatory peptides useful in the prevention or treatment of inflammatory neurodegenerative or infectious diseases.

BACKGROUND

[0002] Neurodegenerative diseases are characterized by the progressive loss of structure or function of neurons, including neuronal death. Neurodegenerative nerve diseases cause worsening of many bodily functions, including balance, movement, talking, and breathing. Examples of degenerative nerve diseases include: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, and multiple sclerosis (MS). Many of these have a profound inflammatory component that contributes to pathology.

[0003] The underlying pathogenesis of these diseases is varied. Some of these diseases are strictly or partly genetic, but most have sporadic forms for which the etiology is unclear. Toxins, chemicals or viruses might cause still other types.

[0004] For example, amyotrophic lateral sclerosis (ALS/Lou Gehrig's Disease) is a disease in which motor neurons are selectively targeted for degeneration. Missense mutations in the gene encoding the antioxidant enzyme Cu/Zn superoxide dismutase 1 (SOD1) were discovered in subsets of patients with familial ALS.

[0005] In contrast, MS is an autoimmune disease caused by damage to the myelin sheath, the protective covering that surrounds nerve cells. When this nerve covering is damaged, nerve impulses are slowed down or stopped. The nerve damage is caused by inflammation, which occurs when the body's own immune cells attack the nervous system. Repeated episodes of inflammation can occur along any area of the brain, optic nerve, and spinal cord.

[0006] Neuroinflammation has been recognized as a hallmark of many neurodegenerative diseases. Recently, it has been appreciated that activated microglial cells can secret neurotoxic molecules which can contribute to neuroinflammation and increasing severity of neurodegenerative diseases, leading to, for example, motoneuron dysfunction, injury, and loss, in diseases such as ALS. In addition, certain infectious circumstances including cerebral malaria, bacterial meningitis, viral encephalitis and endotoxin mediated brain inflammation can also cause severe damage or death through inflammation mediated processes.

[0007] Given that there is no cure for many neurodegenerative diseases or other neural inflammatory conditions, there remains a need to develop treatment methods that take into account the latest research findings in an effort to improve symptoms, and increase function, if not cure these progressive diseases. The present invention provides solutions to these and other unmet needs.

SUMMARY

[0008] Described herein are compositions and methods for preventing or treating neurodegenerative diseases using effective amounts of innate defence regulator (IDR) peptides, where the peptides have 7 to 13 amino acids and comprise an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof.

[0009] In a first aspect, the present invention provides an isolated innate defense regulator (IDR) peptide for the treatment of neurodegenerative or neuroinflammatory disease having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

[0010] In a second aspect, the present invention provides a method of preventing or treating a neurodegenerative disease, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neurodegenerative disease having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

[0011] In a third aspect, the present invention provides a method of preventing or treating neuroinflammation, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neuroinflammation having 7 to 13 amino acids, having 7 to 13 amino acids, said peptide possessing an overall cationic amphipathic character.

[0012] In various embodiments of the above aspects, the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

[0013] In other embodiments of the above aspects, the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

[0014] In yet other embodiments of the above aspects, the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR-HH2), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

[0015] In another aspect, the present invention provides an isolated polynucleotide that encodes the peptides of the above aspects and embodiments.

[0016] In various embodiments of the above, the neurogenerative disease is ALS. In other embodiments, the neurodegenerative disease is Alzheimer's disease. In further embodiments, the neurodegenerative disease is selected from the group consisting of MS, viral encephalitis, meningitis, cerebral malaria, and neural inflammatory responses.

[0017] In various embodiments, the peptides reduce neuroinflammation. In other embodiments, the peptides inhibit microglial activation. In further embodiments, the peptides result in improved motor function. In yet further embodiments, the peptides result in improved cognitive function. In further embodiments, the peptides result in reduced levels of Αβ peptides.

[0018] In a fourth aspect, the present invention provides a method for suppressing microglial activation, the method comprising contacting a microglial cell with an effective amount of the peptides of the above aspects and embodiments, where contacting the microglial cell with the peptides inhibits the secretion of neurotoxic molecules.

[0019] In various embodiments of this aspect, the neurotoxic molecule can be

proinflammatory cytokines, glutamate, free radical species, nitric oxide, or a combination thereof. Examples of proinflammatory cytokines include TNF-a, IL-Ιβ, or both.

[0020] In a fifth aspect, the present invention provides a method of preventing or treating neonatal brain injury, the method comprising the administrating to a subject in need thereof an effective amount of an isolated innate defense regulator (IDR) peptide for the treatment of neurodegenerative disease having 7 to 13 amino acids, said peptide having an overall cationic amphipathic character. [0021] In various embodiments of the above aspect, the peptide comprises an amino acid sequence of SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%>, or 95% identity thereto.

[0022] In other embodiments of the above aspect, the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1215 (IDR-1003), 1222 (IDR-1010), 1233 (IDR-1011), 1225 (IDR-1013), 1227 (IDR-1015), 1230 (IDR-1018), 1231 (IDR-1019), 1232 (IDR-1020), 1234 (IDR-1022), 1236 (IDR-1024), 1237 (IDR-1025), 1238 (IDR-1026), 1247 (IDR-1035), 1248 (IDR-1036), 1249 (IDR-1037), 2 (IDR-HH2) or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto.

[0023] In yet other embodiments of the above aspect, the peptide comprises an amino acid sequence of SEQ ID NOS: 1214 (IDR-1002), 1222 (IDR-1010), 1230 (IDR-1018), 1232 (IDR-1020), 2 (IDR-HH2) ), or analogs, derivatives, amidated variations and conservative variations thereof, or an amino acid sequence having at least 80%>, 85%>, 90%>, or 95%> identity thereto.

[0024] In an embodiment of the above aspect, the treating results in reduced secretion of TNF-a. In another embodiment of the above aspect, the treating results in protection of white and gray cerebral matter in the neonatal brain. In a further embodiment of the above aspect, the treating results in inhibition of the p53 pathway. In a yet further embodiment, the peptide is administered after an initial insult that gives rise to the neonatal brain injury.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows the effect of IDR peptide (IDR 1018) administration in inhibiting behavioral deterioration in a mouse model of ALS using a rotarod test. Average rotarod deterioration is delayed by treatment with IDR peptide in G93A mice.

[0026] Figure 2 shows the effect of IDR peptide (IDR 1018) administration in inhibiting behavioral deterioration in a mouse model of ALS using a test of hindlimb reflex

deterioration. Average hindlimb reflex deterioration is delayed by treatment with IDR peptide in G93A mice.

[0027] Figure 3 shows IDR-1018 effects in enhancing some chemokines/cytokines and inhibiting others in the ALS model of hSODl G93A mice. Spinal cords and three brain regions from hSODl G93A mice treated with saline or IDR-1018 were used for mRNA isolation and qRT-PCR. Data were analyzed using a comparative Ct method and normalized to β-actin.

[0028] Figure 4 shows the reduction of LPS-induced pro-inflammatory cytokine release from murine microglial (BV2) cells with IDR peptides at 4 hours (similar results were observed at 24 hours). BV-2 cells were treated with 2 ng/ml LPS with or without 20 or 50 μg/ml of IDR-1018 for 4 h, and then supematants were used in an ELISA.

[0029] Figure 5 shows that IDR-1018 and mCRAMP decrease inflammation in mouse BV-2 microglial cells. BV-2 cells were treated with 10 ng/ml LPS alone (i.e. higher LPS concentrations than in Fig. 4) or with the addition of IDR-1018 or mCRAMP. mRNA was isolated from 4 h samples and used for qRT-PCR (n = 3), and data were analyzed using comparative Ct method and normalized to β-actin. Supematants from 24 h samples were used for ELI S As (n = 5), and data were analyzed using one-way ANOVA and Bonferroni's multiple comparisons. *: p < 0.05, **: p < 0.01, p < 0.001.

[0030] Figure 6 shows IDR-1018 effects in improving novel object recognition in Alzheimers model APP/PS1 mice carrying mutated human amyloid precursor protein (APP) and presenilin-1 (PS1) transgenes. Mice underwent a training phase followed by a testing phase with one novel (N) object and one familiar object. Exploration time around each object was recorded and analyzed by a Logitech Quickcam Pro 5000 with ANY-maze software. Data represent mean ± SEM.

[0031] Figure 7 shows that IDR-1018 increases gene expression of the chemokines MCP- 1 and MCP-3 in APP/PS1 mice. Brains from APP/PS1 mice treated with saline or IDR-1018 were used for mRNA isolation and qRT-PCR. Data were analyzed using comparative Ct method and normalized to β-actin, ** p < 0.01.

[0032] Figure 8 shows that IDR-1018 increases ABCA1 and ApoE expression in human monocyte derived macrophages (MDMs). MDMs were generated by culturing human monocytes for 7 days in the presence of 10 ng/ml M-CSF. On day 7 media was changed and replaced with RPMI media containing 2% human serum. Cells were treated with 10 ng/ml lipopolysaccharide (LPS) or 20 μg/ml IDR-1018 for 24 h. Whole cell lysates were collected in NP-40 lysis buffer and stored at -80°C until used for Western blots. Samples were ran on a 7.5% gel for ABCA1 and a 12% gel for ApoE. Densitometry was completed using Image J software and statistics analyzed using GraphPad Prism. Data are from 3 or 4 donors and expressed as mean ± SEM.

[0033] Figure 9 shows that IDR-1018 decreases Αβ levels in vivo. Female Alzheimers model APP/PS1 mice (8 months of age) were treated with IDR-1018 (50 mg/mouse iv 3x/week for 14 days, N=4)) or vehicle control (N=4). Hemibrains were used for biochemical analyses. (A) IDR-1018 does not affect total APP levels. (B) IDR-1018 significantly decreases CTF levels, suggesting that Αβ production is inhibited. (C,D) IDR-1018 significantly decreases deposited Αβ42 levels and shows a trend toward reduced Αβ40 deposition. (E,F) Soluble total Αβ40 and Αβ42 levels are not affected by IDR-1018. (E) IDR- 1018 leads to a clear trend toward reduced levels of 56 kDa Αβ oligomers (F) IDR-1018 significantly elevates MCP-1 and MCP-3 chemokine levels. Data represent mean and SEM. * indicates p<0.05 by Student's t test.

[0034] Figure 10 shows the anti-inflammatory potency of IDRs. Prototypic inflammatory mediators produced by LPS-activated microglial cells in the absence (-) and presence of IDRs 1018, 1002 and HH2 and a negative control peptide, 1006 (x-axis) were measured (pg/ml, y- axis) by 20-plex immunoassay. Data represent the mean concentration of proteins + SEM from 4 independent experiments. Asterisks indicate a statistically significant reduction (p<0.05) in LPS-induced inflammatory mediators by individual IDRs.

[0035] Figure 11 shows the positive effects, in preventing brain injury/damage in response to bacterial lipopolysaccharide (LPS at 0 hr) plus hypoxia ischemia (HI at +14 hr) treatment, of pre-injury administration of IDR-1018 in vivo to neonatal mice. (A) In the pre- injury treatment schema, pups on postnatal day 8 (PND8) received either the vehicle (veh) or IDR-1018, 4 h prior to LPS (0 h) and HI (+14 h). (B) Histological scoring 7 days post-injury (PND16) was used to assess animals treated with either the vehicle (veh, n=13) or IDR-1018 (n=13) (x-axis) that suffered severe (black) or mild (white) injury to the hippocampus (y- axis). (C-D) The distribution of 3 H-IDR-1018 in (C) plasma (y-axis; μg 1018), (D) liver (closed circles), spleen (open triangles) and brain (stars) was monitored over time (x-axis, minutes). Data represent the mean quantity of IDR-1018 ± SEM (n=3 per time point).

[0036] Figure 12 A shows a bioinformatics-driven network analysis of the protective effects of IDR-1018 on LPS-associated gene expression in the neonatal brain in vivo. (A) Demonstration of selected genes ('hubs' representing highly connected proteins thai receive and disburse signals in signalling repsonses; indicated by the named coloured circles) that are central to LPS-responsiveness in brain tissue in the absence (upper image, LPS) and the presence (lower image, LPS+1018) of IDR-1018. IDR-1018 altered the importance ('hub degree'; represented as the relative size of the circles) of various key inflammatory genes, with the larger hubs being the most important. Node colour indicates down- (green) or up- (red) regulation of genes (relative to vehicle control). [0037] Figure 12 B shows a bioinformatics-driven network analysis of the effects of IDR- 1018 on LPS-associated alterations in gene expression in the neonatal brain in vivo. (B) Dysregulated genes in LPS-HI-injured brain tissue pre-treated with IDR-1018 are uniquely associated with p53 -signaling (top) and Ca 2+ -signaling (bottom). Node colour indicates down- (green) or up- (red) regulation of genes relative to uninjured brain tissue.

[0038] Figure 13 shows that therapeutic administration of IDR-1018 protected both white and grey matter in vivo. (A) In the post-injury treatment schema, vehicle (veh) and IDR-1018 were given to PND9 pups within a clinically-relevant therapeutic window, 3 hr after LPS/HI. (B) Histological scoring 7 days post-injury (PND16) was used to assess animals treated with either the vehicle (veh, n=9) or IDR-1018 (n=8) (x-axis) that suffered either severe (black) or mild (white) injury (y-axis) showing that IDR-1018 had a clear protective effect. (C) Total histological injury score ± SEM (y-axis) in the injured brain hemisphere (left), tissue volume loss in grey matter (middle; measured as MAP2-positive tissue loss), and in white matter (right, measured as MBP-positive tissue loss) from animals treated with vehicle (veh, n=9) and IDR-1018 (1018, n=8-l 1). Horizontal lines show the mean injury score or mean percentage loss for the groups and IDR-1018 had a singificant protective effect. (D)

Microscopic images are representative of untreated (veh) and treated (1018) brains stained for acid fuchsin/thionin (left), MAP2 (middle) and MBP (right) demonstrating the beneficial effects of peptide treatment. (E) Mean injury score + SEM (y-axis) in different regions of brain tissue in the absence (veh, n=9) and presence of IDR-1018 (n=l 1) showing the beneficial effects of the peptide.

DETAILED DESCRIPTION

[0039] The case for a primary role for host defense peptides in innate host defences includes data demonstrating altered susceptibility to infection and altered inflammation (a) in specific human diseases, (b) in knockout animals, and (c) upon delivery of excess natural and artificial peptides to animal infection models. These results cannot be explained by direct antimicrobial activity. Indeed, there are a variety of activities of cationic peptides other than in direct killing, in which these peptides act directly on cells of the immune system to modulate both innate and adaptive immunity, thus, impacting on the quality and effectiveness of innate immune responses and suppressing inflammation.

[0040] A wide variety of innate defence regulator (IDR) peptides have been designed. These peptides have been shown to protect against infections when given as early as 48 hours prior to or 6 hours after initiation of an invasive S. aureus infection by selectively modulating innate immunity (i.e., upregulating elements of protective immunity, particularly chemokines, and suppressing inflammatory responses). The in vivo and in vitro data has implicated chemokines and cell recruitment in IDR anti-infective activity, with the principal cell type being monocytes/macrophages, since protection was lost in liposomal clodronate-treated mice.

[0041] IDR peptides also demonstrate protection in animal models against invasive S. aureus infections (via IV, IP and SC), local thigh S. aureus infections (via IM), MRSA, VRE, Escherichia coli and Salmonella. The major in vitro elements pertaining to the

immunomodulatory activities, namely upregulation of chemokines (e.g. MCP-1, MCP-3 and Gro-a), suppression of pro-inflammatory cytokines (TNF-a, IL-6) and enhanced immune cell recruitment, were demonstrated in the context of protection against infections. In more recent unpublished studies, we also have obtained evidence for protection in mice against

Pseudomonas aeruginosa, Citrobacter rodentium, Pox virus, tuberculosis, and inflammatory bowel disease. We also observed the effective stimulation of adaptive immunity with an adjuvant formulation combining IDR peptides plus CpG and polyphosphazene, which induced protective titers against pertussis toxoid of 40,000 in a mixed Thl/Th2 response in a single dose in mice, cattle and pigs. However, none of this data indicated that there would be protection in models of degenerative brain disease or other inflammatory diseases of the brain.

[0042] Microglia are the resident innate immune cells of the CNS. They continuously evaluate the extracellular spaces in the CNS for signs of cell stress and foreign material. Microglia respond to a variety of immunological alarm signals, and become activated. The activated microglia will exert either a toxic or protective effect on neurons depending on the physiological conditions. In a neuroprotective role, the activated microglial cells can clear toxic material (apoptotic neurons, protein aggregates), secrete neurotrophic factors (e.g.

BDNF) and protective factors (e.g. glutathione) and increase clearance of excitotoxic glutamate by astrocytes. However, activated microglial cells can be detrimental to nearby neurons by secreting neurotoxic molecules such as proinflammatory cytokines (TNF-a, IL- 1β), glutamate, free radical species and nitric oxide.

[0043] Neuroinflammation is a hallmark of ALS, highlighted by the presence of activated microglia and infiltrating lymphocytes at sites of motoneuron injury. Microglial activation in human postmortem brain and spinal cord tissue of ALS cases indicate a role of

proinflammatory cascades in ALS pathology. PET imaging coupled to [11C](R)-PK11195, a ligand for the peripheral benzodiazepine binding site, which is expressed by activated microglial cells, has shown microglia activation in ALS patients but not in healthy controls. It also shows that there is an increase in microglia activation correlating with increasing severity of the disease. Microglia activation likely contributes to motoneuron dysfunction, injury and loss, and hence, to disease progression.

[0044] The importance of microglia activation in the pathogenesis of ALS has been amply demonstrated using ALS mouse models harboring a mutant human SOD1 transgene. Using chimeric mice, motoneurons expressing mutant SOD1 survive when surrounded by wild-type glia cells, while wild-type motoneurons surrounded by mutant SOD1 expressing glia cells are affected, consistent with a "non-cell autonomous" toxic process at least partly dependent on microglia. In G37R SOD1 mice, a Cre-loxP system was employed to create mice with reduced expression of the mutant SOD1 transgene in specific cell types. When the expression is reduced in motor neurons, there is little effect on disease onset, but a delay in early disease progression, extending the lifespan by -22% (64 days). When the expression is reduced in microglial cells, there is no effect on onset or the early disease progression, but there is a large protective effect in late-stage disease progression, resulting in an extension of lifespan by 99 days (mean). Other studies aimed at finding the cell types responsible for disease looked at hemizygous expression of mutant SOD 1 in motoneurons, astrocytes, or microglia. These studies indicate that mutant SOD1 expression in a single cell type does not cause the same motoneuron disease phenotype as when the mutant SOD1 is ubiquitously expressed. Cultured media preconditioned by glial cells expressing mutant SOD1 is toxic to cultured wild-type motor neurons, but is not toxic to other neuronal cell types (e.g.

dopaminergic neurons), suggesting the presence of a soluble motor neuron-specific toxic factor. A different experiment involving cultured spinal cord cells showed that addition of mutant SOD1 was toxic for motor neurons in contrast to wild-type SOD1, and that this toxicity was dependent on the presence of microglial cells. These observations offer a possible pathological mechanism where mutant SOD 1 secreted by motor neurons would activate microglial cells that would in turn secrete neurotoxic factors and affect motor neurons. As disclosed herein, we have used IDR peptides to, among other things, modulate the activities of microglial cells in the treatment of neurodegenerative diseases.

[0045] Accordingly, the present invention provides compositions and methods for treating neurodegenerative diseases with IDR peptides. DEFINITIONS

[0046] It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise.

[0047] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0049] As used herein, a neurodegenerative disease refers generally to diseases or conditions that result in the progressive loss of structure or function of neurons, including death of neurons. As used herein, examples of such diseases can include, but are not limited to, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Friedreich's ataxia , Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, multiple sclerosis (MS), viral encephalitis, meningitis, cerebral malaria, endotoxin inflammatory responses, and neonatal brain injury. Many of these have a profound inflammatory component that contributes to pathology.

[0050] "Treating" or "treatment" refers to either (i) the prevention of a disease of interest, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g., therapy. "Treating" or "treatment" can refer to the administration of a composition comprising a peptide of interest, e.g., SEQ ID NOS: 1-969, 973-1264, or analogs, derivatives, amidated variations and conservative variations thereof. Treating a subject with the composition can prevent, reduce or slow the course of a neurodegenerative disease. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the

manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. [0051] "Preventing" or "prevention" refers to prophylactic administration of the compositions of the invention.

[0052] "Therapeutically-effective amount" or "an amount effective" refers to an amount of peptide that is sufficient to prevent or treat at least one of the symptoms associated with a disease of interest, e.g., a neurodegenerative disease. It is not necessary that the

administration of the composition eliminate the symptoms of a disease, as long as the benefits of administration of composition outweigh the detriments. Likewise, the terms "treat" and "treating" in reference to neurodegenerative diseases, as used herein, are not intended to mean that the subject is necessarily cured of the disease or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.

[0053] An "innate defence regulator" (IDR) peptide refers to a peptide that has immunomodulatory activity by acting directly on cells to effect an innate immune response, as described in more detail below. It is understood by one of skill in the art that these peptides are related to natural host defence peptides and natural and synthetic antimicrobial peptides and that many of these peptides are anticipated to have similar properties in affecting neural inflammation.

[0054] A "peptide possessing an overall cationic amphipathic character" refers generally to short cationic peptides of, for example, about 9 or less to about 50 amino acid residues, with a net positive charge of, for example +2 to +9, and a high proportion of hydrophobic residues. See, e.g., Wieczorek et al, Chemistry and Biology, 17: 970-980 (2010). These peptides generally fold in membrane-like environments such that the hydrophobic and polar (including cationic) residues form distinct domains in 3-dimensional space. The term

"amphipathic" is synonymous with the term amphiphilic, and is a term used in describing a chemical compound possessing both hydrophilic (water-loving, polar) and lipophilic

(lipid/membrane-loving, hydrophobic) properties.

PEPTIDES

[0055] The invention provides an isolated peptide with immunomodulatory activity, in particular, neural anti-inflammatory activity. Exemplary peptides of the invention have an amino acid sequence including those listed in Table 1, and analogs, derivatives, amidated variations and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NOS: 1-969 and 973-1264, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof.

[0056] "Isolated" when used in reference to a peptide, refers to a peptide substantially free of proteins, lipids, nucleic acids, for example, with which it might be naturally associated. Those of skill in the art can make similar substitutions to achieve peptides with greater antimicrobial activity and a broader host range. For example, the invention includes the peptides depicted in SEQ ID NOS: 1-969 and 973-1264, as well as analogs or derivatives thereof, as long as the bioactivity (e.g., anti-inflammatory activity or ability to suppress an inflammatory response after stimulation with a Toll-like receptor ligand) of the peptide remains. Minor modifications of the primary amino acid sequence of the peptides of the invention, and especially conservative substitutions, may result in peptides that have substantially equivalent activity as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis or addition of different amino acids during synthesis, or may be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the original peptide still exists.

[0057] Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule that would also have utility. For example, amino or carboxy terminal amino acids that may not be required for biological activity of the particular peptide can be removed. Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein remains. All peptides were synthesized using L amino acids, however, all D forms of the peptides can be synthetically produced. In addition, C-terminal derivatives can be produced, such as C-terminal methyl esters and C-terminal amidates, in order to increase the biological activity of a peptide of the invention. The peptide can be synthesized such that the sequence is reversed whereby the last amino acid in the sequence becomes the first amino acid, and the penultimate amino acid becomes the second amino acid, and so on. It is well known that such reversed peptides, especially when synthesized using D-amino acids, usually have similar biological activities to the original sequence.

[0058] In certain aspects, the peptides of the invention include peptide analogs and peptide mimetics. Indeed, the peptides of the invention include peptides having any of a variety of different modifications, including those described herein. [0059] Peptide analogs of the invention are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the following sequences disclosed in the tables. The present invention clearly establishes that these peptides in their entirety and derivatives created by modifying any side chains of the constituent amino acids have the ability to suppress excessive inflammation in neuronal cells. The present invention further encompasses polypeptides up to about 50 amino acids in length that include the amino acid sequences and functional variants or peptide mimetics of the sequences described herein.

[0060] In another aspect, a peptide of the present invention is a pseudopeptide.

Pseudopeptides or amide bond surrogates refers to peptides containing chemical

modifications of some (or all) of the peptide bonds. The introduction of amide bond surrogates not only decreases peptide degradation but also may significantly modify some of the biochemical properties of the peptides, particularly the conformational flexibility and hydrophobicity.

[0061] To improve or alter the characteristics of polypeptides of the present invention, protein engineering can be employed. Peptide synthesis or recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can show, e.g., increased/decreased biological activity or increased/decreased stability. In addition, they can be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Further, the polypeptides of the present invention can be produced as multimers including dimers, trimers and tetramers. Multimerization can be facilitated by linkers, introduction of cysteines to permit creation of interchain disulphide bonds, or recombinantly though addition of heterologous polypeptides such as Fc regions.

[0062] It is known in the art that one or more amino acids can be deleted from the N- terminus or C-terminus without substantial loss of biological function. See, e.g., Ron, et al, Biol Chem., 268: 2984-2988, 1993. Accordingly, the present invention provides polypeptides having one or more residues deleted from the amino terminus. Similarly, many examples of biologically functional C-terminal deletion mutants are known (see, e.g., Dobeli, et al, 1988). Accordingly, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini as described below. [0063] Other mutants in addition to N- and C-terminal deletion forms of the peptides discussed above are included in the present invention. Thus, the invention further includes variations of the peptides that show substantial peptide activity. Such mutants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity (see, e.g. Hilpert, et al. 2005. Nature Biotech. 23: 1008-1012).

[0064] There are two main approaches for studying the tolerance of an amino acid sequence to change, see, Bowie, et al, Science, 247: 1306-1310, 1994. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene or peptide chemistry means to introduce amino acid changes at specific positions of the chemically synthesized sequence, followed by selections or screens to identify sequences that maintain functionality. These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions.

[0065] Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, He and Met; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr, and Trp. Thus, the polypeptide of the present invention can be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue can or cannot be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which additional amino acids are fused to the above form of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a pro-protein sequence.

[0066] Thus, the polypeptides of the present invention can include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human

manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein. The following groups of amino acids represent equivalent changes with respect to folding: (1) Ala, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

[0067] Furthermore, polypeptides of the present invention can include one or more amino acid substitutions that mimic modified amino acids. An example of this type of substitution includes replacing amino acids that are capable of being phosphorylated (e.g., serine, threonine, or tyrosine) with a negatively charged amino acid that resembles the negative charge of the phosphorylated amino acid (e.g., aspartic acid or glutamic acid). Also included is substitution of amino acids that are capable of being modified by hydrophobic groups (e.g., arginine) with amino acids carrying bulky hydrophobic side chains, such as tryptophan or phenylalanine. Therefore, a specific aspect of the invention includes polypeptides that include one or more amino acid substitutions that mimic modified amino acids at positions where amino acids that are capable of being modified are normally positioned. Further included are polypeptides where any subset of modifiable amino acids is substituted. For example, a polypeptide that includes three arginine residues can be substituted at any one, any two, or all three of said arginines. Furthermore, any polypeptide amino acid capable of being modified can be excluded from substitution with a modification-mimicking amino acid.

[0068] The present invention is further directed to fragments of the peptides of the present invention. More specifically, the present invention embodies purified, isolated, and

recombinant peptides comprising at least any one integer between 6 and 12 of consecutive amino acid residues.

[0069] The present invention also provides for the exclusion of any species of polypeptide fragments of the present invention specified by 5' and 3' positions or sub-genuses of polypeptides specified by size in amino acids as described above. Any number of fragments specified by 5' and 3' positions or by size in amino acids, as described above, can be excluded.

[0070] In addition, it should be understood that in certain aspects, the peptides of the present invention include two or more modifications, including, but not limited to those described herein. By taking into the account the features of the peptide drugs on the market or under current development, it is clear that most of the peptides successfully stabilized against proteolysis consist of a mixture of several types of the above described modifications. This conclusion is understood in the light of the knowledge that many different enzymes are implicated in peptide degradation. PEPTIDES, PEPTIDE VARIANTS, AND PEPTIDE MIMETICS

[0071] "Polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L-phenylglycine; D-or L- 2 thieneylalanine; D- or L-I, -2,3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro- phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso- pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings (See also, table entitled "Non-Natural Amino Acids" in Appendix below).

[0072] "Peptide" as used herein includes peptides that are conservative variations of those peptides specifically exemplified herein. "Conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include, but are not limited to, the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides of the invention. "Cationic" as is used to refer to any peptide that possesses sufficient positively charged amino acids to have a pi (isoelectric point) greater than about 9.0.

[0073] The biological activity of the peptides can be determined by standard methods known to those of skill in the art, for example, in vitro or in vivo tests as described in the present examples.

[0074] The peptides and polypeptides of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity. As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, e.g., a polypeptide fragment of an immunomodulatory protein having neural anti-inflammatory activity.

[0075] Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N- hydroxysuccinimide esters, bifunctional maleimides, Ν,Ν'-dicyclohexylcarbodiimide (DCC) or Ν,Ν'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., ~C(=0) — CH 2 — for—

C(=0)— NH-), aminomethylene (CH 2 -NH), ethylene, olefin (CH=CH), ether (CH 2 -0), thioether (CH 2 — S), tetrazole (CN 4 -), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0076] Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono) alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'— N— C— N— R') such as, e.g., l-cyclohexyl-3(2- morpholin-yl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0077] Mimetics of basic amino acids can be generated by substitution with, e.g. , (in addition to lysine and arginine) the amino acids ornithine, or citrulline. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.

[0078] Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or

carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;

methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.

[0079] A component of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.

[0080] The invention also provides polypeptides that are "substantially identical" to an exemplary polypeptide of the invention. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non- conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or

methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for

asparagine). One or more amino acids can be deleted, for example, from an IDR polypeptide of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.

[0081] The skilled artisan will recognize that individual synthetic residues and

polypeptides incorporating these mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Peptides and peptide mimetics of the invention can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al- Obeidi, MoT

Biotechnol. 9: 205-223, 1998; Hruby, Curr. Opin. Chem. Biol. 1 : 114-119, 1997; Ostergaard,

MoT Divers. 3: 17-27, 1997; Ostresh, Methods Enzymol. 267: 220-234, 1996. Modified peptides of the invention can be further produced by chemical modification methods, see, e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994.

[0082] Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers, Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, Therapeutic Peptides and Proteins,

Formulation, Processing and Delivery Systems Technomic Publishing Co., Lancaster, PA, 1995. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield, Methods Enzymol. 289: 3-13, 1997) and automated synthesis can be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0083] Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan, et ai, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).

[0084] Peptides of the invention can also be synthesized by the well known solid phase or solution phase peptide synthesis methods described in Merrifield, J. Am. Chem. Soc, 85:2149, (1962), and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969, pp.27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF- 10% anisole for about 1/4-1 hours at 0°C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G- 15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.

[0085] Analogs, polypeptide fragment of antimicrobial protein having antimicrobial activity, are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the sequences including SEQ ID NOS: 1-969 and 973-1264.

[0086] The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same

(i.e., about 60% identity, preferably 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region

(e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical."

This term also refers to, or can be applied to, the compliment of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have

substitutions. As described below, the preferred algorithms can account for gaps and the like.

Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0087] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0088] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to

600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoT Biol. 48: 443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444,

1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al, eds. 1995 supplement).

[0089] Programs for searching for alignments are well known in the art, e.g., BLAST and the like. For example, if the target species is human, a source of such amino acid sequences or gene sequences (germline or rearranged antibody sequences) can be found in any suitable reference database such as Genbank, the NCBI protein databank

(http://ncbi.nlm.nih.gov/BLAST/), VBASE, a database of human antibody genes

(http://www.mrc-cpe.cam.ac.uk/imt-doc), and the Kabat database of immunoglobulins 11 (http://www.immuno.bme.nwu.edu) or translated products thereof. If the alignments are done based on the nucleotide sequences, then the selected genes should be analyzed to determine which genes of that subset have the closest amino acid homology to the originating species antibody. It is contemplated that amino acid sequences or gene sequences which approach a higher degree homology as compared to other sequences in the database can be utilized and manipulated in accordance with the procedures described herein. Moreover, amino acid sequences or genes which have lesser homology can be utilized when they encode products which, when manipulated and selected in accordance with the procedures described herein, exhibit specificity for the predetermined target antigen. In certain aspects, an acceptable range of homology is greater than about 50%. It should be understood that target species can be other than human.

[0090] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25: 3389-3402, 1977 and Altschul et al, J. MoT Biol. 215: 403-410, 1990, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length (W) in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:

10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

POLYPEPTIDES AND FUNCTIONAL VARIANTS THEREOF

[0091] "Polypeptide" includes proteins, fusion proteins, oligopeptides and polypeptide derivatives, with the exception that peptidomimetics are considered to be small molecules herein.

[0092] A "protein" is a molecule having a sequence of amino acids that are linked to each other in a linear molecule by peptide bonds. The term protein refers to a polypeptide that is isolated from a natural source, or produced from an isolated cDNA using recombinant DNA technology; and has a sequence of amino acids having a length of at least about 200 amino acids.

[0093] A "fusion protein" is a type of protein that has an amino acid sequence that results from the linkage of the amino acid sequences of two or more normally separate polypeptides.

[0094] A "protein fragment" is a proteolytic fragment of a larger polypeptide, which may be a protein or a fusion protein. A proteolytic fragment may be prepared by in vivo or in vitro proteolytic cleavage of a larger polypeptide, and is generally too large to be prepared by chemical synthesis. Proteolytic fragments have amino acid sequences having a length from about 200 to about 1,000 amino acids.

[0095] An "oligopeptide" or "peptide" is a polypeptide having a short amino acid sequence (i.e., 2 to about 200 amino acids). An oligopeptide is generally prepared by chemical synthesis. [0096] Although oligopeptides and protein fragments may be otherwise prepared, it is possible to use recombinant DNA technology and/or in vitro biochemical manipulations. For example, a nucleic acid encoding an amino acid sequence may be prepared and used as a template for in vitro transcription/translation reactions. In such reactions, an exogenous nucleic acid encoding a preselected polypeptide is introduced into a mixture that is essentially depleted of exogenous nucleic acids that contains all of the cellular components required for transcription and translation. One or more radiolabeled amino acids are added before or with the exogenous DNA, and transcription and translation are allowed to proceed. Because the only nucleic acid present in the reaction mix is the exogenous nucleic acid added to the reaction, only polypeptides encoded thereby are produced, and incorporate the radiolabeled amino acid(s). In this manner, polypeptides encoded by a preselected exogenous nucleic acid are radiolabeled. Although other proteins are present in the reaction mix, the preselected polypeptide is the only one that is produced in the presence of the radiolabeled amino acids and is thus uniquely labeled.

[0097] As is explained in detail below, "polypeptide derivatives" include without limitation mutant polypeptides, chemically modified polypeptides, and peptidomimetics.

[0098] The polypeptides of this invention, including the analogs and other modified variants, may generally be prepared following known techniques. Preferably, synthetic production of the polypeptide of the invention may be according to the solid phase synthetic method. For example, the solid phase synthesis is well understood and is a common method for preparation of polypeptides, as are a variety of modifications of that technique. Merrifield, J. Am. Chem. Soc, 85: 2149, 1964; Stewart and Young, Solid Phase polypeptide Synthesis, Pierce Chemical Company, Rockford, 111., 1984; Bodanszky and Bodanszky, The Practice of polypeptide Synthesis, Springer- Verlag, New York, 1984; Atherton and Sheppard, Solid Phase polypeptide Synthesis: A Practical Approach, IRL Press, New York, 1989. See, also, the specific method described in Example 1 below.

[0099] Alternatively, polypeptides of this invention may be prepared in recombinant systems using polynucleotide sequences encoding the polypeptides.

[00100] A "variant" or "functional variant" of a polypeptide is a compound that is not, by definition, a polypeptide, i.e., it contains at least one chemical linkage that is not a peptide bond. Thus, polypeptide derivatives include without limitation proteins that naturally undergo post-translational modifications such as, e.g., glycosylation. It is understood that a

polypeptide of the invention may contain more than one of the following modifications within the same polypeptide. Preferred polypeptide derivatives retain a desirable attribute, which may be biological activity; more preferably, a polypeptide derivative is enhanced with regard to one or more desirable attributes, or has one or more desirable attributes not found in the parent polypeptide. Although they are described in this section, peptidomimetics are taken as small molecules in the present disclosure.

[00101] A polypeptide having an amino acid sequence identical to that found in a protein prepared from a natural source is a "wildtype" polypeptide. Functional variants of

polypeptides can be prepared by chemical synthesis, including without limitation

combinatorial synthesis.

[00102] Functional variants of polypeptides larger than oligopeptides can be prepared using recombinant DNA technology by altering the nucleotide sequence of a nucleic acid encoding a polypeptide. Although some alterations in the nucleotide sequence will not alter the amino acid sequence of the polypeptide encoded thereby ("silent" mutations), many will result in a polypeptide having an altered amino acid sequence that is altered relative to the parent sequence. Such altered amino acid sequences may comprise substitutions, deletions and 95 additions of amino acids, with the proviso that such amino acids are naturally occurring amino acids.

[00103] Thus, subjecting a nucleic acid that encodes a polypeptide to mutagenesis is one technique that can be used to prepare Functional variants of polypeptides, particularly ones having substitutions of amino acids but no deletions or insertions thereof. A variety of mutagenic techniques are known that can be used in vitro or in vivo including without limitation chemical mutagenesis and PCR-mediated mutagenesis. Such mutagenesis may be randomly targeted (i.e., mutations may occur anywhere within the nucleic acid) or directed to a section of the nucleic acid that encodes a stretch of amino acids of particular interest. Using such techniques, it is possible to prepare randomized, combinatorial or focused compound libraries, pools and mixtures.

[00104] Polypeptides having deletions or insertions of naturally occurring amino acids may be synthetic oligopeptides that result from the chemical synthesis of amino acid sequences that are based on the amino acid sequence of a parent polypeptide but which have one or more amino acids inserted or deleted relative to the sequence of the parent polypeptide. Insertions and deletions of amino acid residues in polypeptides having longer amino acid sequences may be prepared by directed mutagenesis.

[00105] As contemplated by this invention, "polypeptide" includes those having one or more chemical modification relative to another polypeptide, i.e., chemically modified polypeptides. The polypeptide from which a chemically modified polypeptide is derived may be a wildtype protein, a functional variant protein or a functional variant polypeptide, or polypeptide fragments thereof; an antibody or other polypeptide ligand according to the invention including without limitation single-chain antibodies, crystalline proteins and polypeptide derivatives thereof; or polypeptide ligands prepared according to the disclosure. Preferably, the chemical modification(s) confer(s) or improve(s) desirable attributes of the polypeptide but does not substantially alter or compromise the biological activity thereof. Desirable attributes include but are limited to increased shelf-life; enhanced serum or other in vivo stability; resistance to proteases; and the like. Such modifications include by way of non- limiting example N-terminal acetylation, glycosylation, and biotinylation.

[00106] An effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a polypeptide is to add chemical groups at the polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the polypeptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum (Powell et al, Pharma. Res. 10: 1268-1273, 1993). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group.

[00107] The presence of an N-terminal D-amino acid increases the serum stability of a polypeptide that otherwise contains L-amino acids, because exopeptidases acting on the N- terminal residue cannot utilize a D-amino acid as a substrate. Similarly, the presence of a C- terminal D-amino acid also stabilizes a polypeptide, because serum exopeptidases acting on the C-terminal residue cannot utilize a D-amino acid as a substrate. With the exception of these terminal modifications, the amino acid sequences of polypeptides with N-terminal and/or C-terminal D-amino acids are usually identical to the sequences of the parent L-amino acid polypeptide.

[00108] Substitution of unnatural amino acids for natural amino acids in a subsequence of a polypeptide can confer or enhance desirable attributes including biological activity. Such a substitution can, for example, confer resistance to proteolysis by exopeptidases acting on the N-terminus. The synthesis of polypeptides with unnatural amino acids is routine and known in the art (see, for example, Coller, et al. 1993, cited above).

[00109] Different host cells will contain different post-translational modification mechanisms that may provide particular types of post-translational modification of a fusion protein if the amino acid sequence required for such modifications is present in the fusion protein. A large number (about 100) of post-translational modifications have been described, a few of which are discussed herein. One skilled in the art will be able to choose appropriate host cells, and design chimeric genes that encode protein members comprising the amino acid sequence needed for a particular type of modification.

[00110] Glycosylation is one type of post-translational chemical modification that occurs in many eukaryotic systems, and may influence the activity, stability, pharmacogenetics, immunogenicity and/or antigenicity of proteins. However, specific amino acids must be present at such sites to recruit the appropriate glycosylation machinery, and not all host cells have the appropriate molecular machinery. Saccharomyces cerevisieae and Pichia pastoris provide for the production of glycosylated proteins, as do expression systems that utilize insect cells, although the pattern of glyscoylation may vary depending on which host cells are used to produce the fusion protein.

[00111] Another type of post-translation modification is the phosphorylation of a free hydroxyl group of the side chain of one or more Ser, Thr or Tyr residues, Protein kinases catalyze such reactions. Phosphorylation is often reversible due to the action of a protein phosphatase, an enzyme that catalyzes the dephosphorylation of amino acid residues.

[00112] Differences in the chemical structure of amino terminal residues result from different host cells, each of which may have a different chemical version of the methionine residue encoded by a start codon, and these will result in amino termini with different chemical modifications.

[00113] For example, many or most bacterial proteins are synthesized with an amino terminal amino acid that is a modified form of methionine, i.e., N-formyl-methionine (fMet). Although the statement is often made that all bacterial proteins are synthesized with an fMet initiator amino acid; although this may be true for E. coli, recent studies have shown that it is not true in the case of other bacteria such as Pseudomonas aeruginosa (Newton et al, Biol. Chem. 274: 22143-22146, 1999). In any event, in E. coli, the formyl group of fMet is usually enzymatically removed after translation to yield an amino terminal methionine residue, although the entire fMet residue is sometimes removed (see Hershey, Chapter 40, "Protein Synthesis" in: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Neidhardt, Frederick C, Editor in Chief, American Society for Microbiology, Washington, D.C., 1987, Volume 1, pages 613-647, and references cited therein.). E. coli mutants that lack the enzymes (such as, e.g., formylase) that catalyze such post-translational modifications will produce proteins having an amino terminal fMet residue (Guillon et ah, J. Bacteriol. 174: 4294-4301, 1992).

[00114] In eukaryotes, acetylation of the initiator methionine residue, or the penultimate residue if the initiator methionine has been removed, typically occurs co- or post- translationally. The acetylation reactions are catalyzed by N-terminal acetyltransferases (NATs, a.k.a. N-alpha-acetyltransferases), whereas removal of the initiator methionine residue is catalyzed by methionine aminopeptidases (for reviews, see Bradshaw et al, Trends Biochem. Sci. 23: 263-267, 1998; and Driessen et al, CRC Crit. Rev. Biochem. 18: 281-325, 1985). Amino terminally acetylated proteins are said to be "N-acetylated," "N alpha acetylated" or simply "acetylated."

[00115] Another post-translational process that occurs in eukaryotes is the alpha- amidation of the carboxy terminus. For reviews, see Eipper et al. Annu. Rev. Physiol. 50: 333-344, 1988, and Bradbury et al. Lung Cancer 14: 239-251, 1996. About 50% of known endocrine and neuroendocrine peptide hormones are alpha-amidated (Treston et al, Cell Growth Differ. 4: 911-920, 1993). In most cases, carboxy alpha-amidation is required to activate these peptide hormones.

POLYPEPTIDE MIMETIC

[00116] In general, a polypeptide mimetic ("peptidomimetic") is a molecule that mimics the biological activity of a polypeptide but is no longer peptidic in chemical nature. By strict definition, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids). However, the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Examples of some peptidomimetics by the broader definition (where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the polypeptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems that are similar to the biological activity of the polypeptide.

[00117] There are several potential advantages for using a mimetic of a given polypeptide rather than the polypeptide itself. For example, polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action. Peptidomimetics are often small enough to be both orally active and to have a long duration of action. There are also problems associated with stability, storage and immunoreactivity for polypeptides that are not experienced with peptidomimetics.

[00118] Candidate, lead and other polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities. Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original polypeptide (Dean, BioEssays, 16: 683-687, 1994; Cohen and Shatzmiller, J. MoT Graph., 1 1 : 166-173, 1993; Wiley and Rich, Med. Res. Rev., 13: 327-384, 1993; Moore, Trends Pharmacol. Sci., 15: 124-129, 1994; Hruby, Biopolymers, 33: 1073-1082, 1993; Bugg et al, Sci. Am., 269: 92-98, 1993, all incorporated herein by reference).

[00119] Thus, through use of the methods described above, the present invention provides compounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above. The peptidomimetic compounds obtained by the above methods, having the biological activity of the above named polypeptides and similar three-dimensional structure, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified polypeptides described in the previous section or from a polypeptide bearing more than one of the modifications described from the previous section. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.

[00120] Specific examples of peptidomimetics derived from the polypeptides described in the previous section are presented below. These examples are illustrative and not limiting in terms of the other or additional modifications.

[00121] Proteases act on peptide bonds. It therefore follows that substitution of peptide bonds by pseudopeptide bonds confers resistance to proteolysis. A number of pseudopeptide bonds have been described that in general do not affect polypeptide structure and biological activity. The reduced isostere pseudopeptide bond is a suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity (Couder, et al, hit. J. Polypeptide Protein Res. 41 : 181-184, 1993, incorporated herein by reference). Thus, the amino acid sequences of these compounds may be identical to the sequences of their parent L- amino acid polypeptides, except that one or more of the peptide bonds are replaced by an isosteric pseudopeptide bond. Preferably the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.

[00122] To confer resistance to proteolysis, peptide bonds may also be substituted by retro- inverso pseudopeptide bonds (Dalpozzo, et al, Int. J. Polypeptide Protein Res. 41 : 561-566, incorporated herein by reference). According to this modification, the amino acid sequences of the compounds may be identical to the sequences of their L-amino acid parent

polypeptides, except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond. Preferably the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.

[00123] Peptoid derivatives of polypeptides represent another form of modified

polypeptides that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon, et al, Proc. Natl. Acad. Sci. USA, 89: 9367-9371, 1992, and incorporated herein by reference). Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid.

POLYNUCLEOTIDES

[00124] The invention includes polynucleotides encoding peptides of the invention.

Exemplary polynucleotides encode peptides including those listed in Table 1, and analogs, derivatives, amidated variations and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NOS: 1-969 and 973-1264, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof.

[00125] "Isolated" when used in reference to a polynucleotide, refers to a polynucleotide substantially free of proteins, lipids, nucleic acids, for example, with which it is naturally associated. As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct. DNA encoding a peptide of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit. Polynucleotide sequences of the invention include DNA, R A and cDNA sequences. A polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code. Such polynucleotides are useful for the recombinant production of large quantities of a peptide of interest, such as the peptide of SEQ ID NOS: 1-969 and 973-1264.

[00126] In the present invention, the polynucleotides encoding the peptides of the invention may be inserted into a recombinant "expression vector". The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of genetic sequences. Such expression vectors of the invention are preferably plasmids that contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence in the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. For example, the expression of the peptides of the invention can be placed under control of E. coli chromosomal DNA comprising a lactose or lac operon which mediates lactose utilization by elaborating the enzyme beta-galactosidase. The lac control system can be induced by IPTG. A plasmid can be constructed to contain the lac Iq repressor gene, permitting repression of the lac promoter until IPTG is added. Other promoter systems known in the art include beta-lactamase, lambda promoters, the protein A promoter, and the tryptophan promoter systems. While these are the most commonly used, other microbial promoters, both inducible and constitutive, can be utilized as well. The vector contains a replicon site and control sequences which are derived from species compatible with the host cell. In addition, the vector may carry specific gene(s) which are capable of providing phenotypic selection in transformed cells. For example, the beta-lactamase gene confers ampicillin resistance to those transformed cells containing the vector with the beta-lactamase gene. An exemplary expression system for production of the peptides of the invention is described in U.S. Pat. No. 5,707,855.

[00127] Transformation of a host cell with the polynucleotide may be carried out by conventional techniques known to those skilled in the art. For example, where the host is prokaryotic, such as E. coli, competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl 2 method using procedures known in the art. Alternatively, MgCl 2 or RbCl could be used.

[00128] In addition to conventional chemical methods of transformation, the plasmid vectors of the invention may be introduced into a host cell by physical means, such as by electroporation or microinjection. Electroporation allows transfer of the vector by high voltage electric impulse, which creates pores in the plasma membrane of the host and is performed according to methods known in the art. Additionally, cloned DNA can be introduced into host cells by protoplast fusion, using methods known in the art.

[00129] DNA sequences encoding the peptides can be expressed in vivo by DNA transfer into a suitable host cell. "Host cells" of the invention are those in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that not all progeny are identical to the parental cell, since there may be mutations that occur during replication. However, such progeny are included when the terms above are used. Preferred host cells of the invention include E. coli, S. aureus and P.

aeruginosa, although other Gram negative and Gram positive organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.

[00130] The polynucleotide sequence encoding the peptide used according to the method of the invention can be isolated from an organism or synthesized in the laboratory. Specific DNA sequences encoding the peptide of interest can be obtained by: 1) isolation of a double- stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the peptide of interest; and 3) in vitro synthesis of a double- stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed that is generally referred to as cDNA.

[00131] The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known. In the present invention, the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons that are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation. In addition, virtually any peptide can be synthesized, including those encoding natural peptides, variants of the same, or synthetic peptides.

[00132] When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries that are derived from reverse transcription of mRNA that is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the peptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay et al, Nuc. Acid Res., 11 :2325, 1983).

METHODS OF USE: IMMUNOMODULATORY

[00133] The present invention provides novel cationic peptides, characterized by a group of generic formulas which have ability to modulate (e.g., up- and/or down regulate) polypeptide expression, thereby regulating inflammatory responses and/or innate immunity.

[00134] "Innate immunity" as used herein refers to the natural ability of an organism to defend itself. Innate immunity is contrasted with acquired/adaptive immunity in which the organism develops a defensive mechanism based substantially on antibodies and/or immune lymphocytes that is characterized by specificity, amplifiability and self vs. non-self discrimination. With innate immunity, broad, nonspecific immunity is provided and there is no immunologic memory of prior exposure. The hallmarks of innate immunity are

effectiveness against a broad variety of potential pathogens, independence of prior exposure to a pathogen, and immediate effectiveness (in contrast to the specific immune response which takes days to weeks to be elicited). In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like. Some aspects of innate immunity are captured as the inflammatory response. In general, inflammation has good aspects whereby protective immunity against pathogens is triggered. However, the excessive or prolonged activation of inflammation can lead to pathological conditions such as sepsis or chronic inflammation. Cationic host defence peptides and IDR peptides are known to stimulate protective inflammatory/innate immune responses while dampening potentially harmful inflammation. However, prior to the current invention it was not known that they could suppress neural inflammation, and the poor access of drugs into the brain across the blood brain barrier meant that an anti-inflammatory action in this contex could not be predicted.

[00135] In innate immunity, the immune response is not dependent upon antigens. The innate immunity process may include the production of secretory molecules and cellular components and the modulation of the expression of many specific genes as set forth herein.

[00136] In one aspect, the present invention provides the use of compounds including peptides of the invention to reduce inflammatory responses by acting directly on host cells, thereby preventing or treating neurodegenerative diseases. In this aspect, a method of identification of a polynucleotide or polynucleotides that are regulated by one or more inflammatory inducing agents is provided, where the regulation is altered by a cationic peptide. Such inflammatory inducing agents include, but are not limited to endotoxic lipopolysaccharide (LPS), lipoteichoic acid (LTA) and/or CpG DNA or intact bacteria or other bacterial components or triggering agents that are associated with each neural inflammatory disease, such as β-amyloid, although the specific nature of said agents may not be currently known. The identification is performed by contacting the host cell with the inflammatory inducing agents and further contacting with a cationic peptide either simultaneously or immediately after. The expression of the polynucleotide or polypeptide in the presence and absence of the cationic peptide is observed and a change in expression is indicative of a polynucleotide or polypeptide or pattern of polynucleotides or polypeptides that is regulated by a sepsis or inflammatory inducing agent and inhibited by a cationic peptide. In another aspect, the invention provides a polynucleotide identified by the method.

[00137] Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs. Candidate agents are also found among biomolecules including, but not limited to: peptides, peptidiomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives, structural analogs or combinations thereof.

[00138] In another aspect of the methods of the invention, a cationic peptide is utilized to detect and locate a polynucleotide or polypeptide that is essential in the process of inflammation. Once identified, a pattern of polynucleotide or polypeptide expression may be obtained by observing the expression in the presence and absence of the cationic peptide. The pattern obtained in the presence of the cationic peptide is then useful in identifying additional compounds that can inhibit expression of the polynucleotide and therefore block inflammation, for instance in neurodegenerative diseases. It is well known to one of skill in the art that non-peptidic chemicals and peptidomimetics can mimic the ability of peptides to bind to receptors and enzyme binding sites and thus can be used to block or stimulate biological reactions. Where an additional compound of interest provides a pattern of polynucleotide or polypeptide expression similar to that of the expression in the presence of a cationic peptide, that compound is also useful in the modulation of an innate immune response. In this manner, the cationic peptides of the invention are also useful as tools in the identification of additional compounds that inhibit neural inflammation.

[00139] As can be seen in the Examples below, peptides of the invention have an ability to reduce the expression of polynucleotides or polypeptides regulated by LPS. See, e.g., Example 3.

[00140] In another aspect, the invention identifies agents that enhance innate immunity. Human cells that contain a polynucleotide or polynucleotides that encode a polypeptide or polypeptides involved in innate immunity are contacted with an agent of interest. Expression of the polynucleotide is determined, both in the presence and absence of the agent. The expression is compared and of the specific modulation of expression was indicative of an enhancement of innate immunity. In still another aspect the agent reduces or blocks the inflammatory response.

[00141] In another aspect, the invention provides methods of direct polynucleotide or polypeptide regulation by cationic peptides and the use of compounds including cationic peptides to stimulate elements of innate immunity. In this aspect, the invention provides a method of identification of a pattern of polynucleotide or polypeptide expression for identification of a compound that enhances innate immunity. In the method of the invention, an initial detection of a pattern of polypeptide expression for cells contacted in the presence and absence of a cationic peptide is made. The pattern resulting from polypeptide expression in the presence of the peptide represents stimulation of innate immunity. A pattern of polypeptide expression is then detected in the presence of a test compound, where a resulting pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide is indicative of a compound that enhances innate immunity. In another aspect, the invention provides compounds that are identified in the above methods.

TREATMENT REGIMES

[00142] The invention provides pharmaceutical compositions comprising one or a combination of immunomodulatory compounds, particularly IDR peptides, for example, formulated together with a pharmaceutically acceptable carrier. Some compositions include a combination of multiple (e.g., two or more) peptides of the invention.

[00143] As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one aspect, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for

intravenous, intraperitoneal, intranasal, subcutaneous, or intramuscular administration. In another aspect, the carrier is suitable for oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the

extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is compatible with the active compound, use thereof in the pharmaceutical compositions is contemplated. Supplementary active

compounds can also be incorporated into the compositions.

[00144] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (See, e.g., Berge, et al, J. Pharm. Sci,. 66: 1-19, 1977). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[00145] In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition {i.e., as a result of bacteria, fungi, viruses, parasites or the like) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease or condition in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease or condition (e.g., biochemical and/or histologic), including its complications and intermediate pathological phenotypes in development of the disease or condition. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.

[00146] The pharmaceutical composition of the present invention should be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), a detergent or surfactant, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the

composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[00147] When the active compound is suitably protected, as described above, the compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.

[00148] Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, in treatment of bacteria, the combination therapy can include a composition of the present invention with at least one agent or other conventional therapy.

ROUTES OF ADMINISTRATION

[00149] A composition of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. The phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,

subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intranasal, intraspinal, epidural and intrasternal injection and infusion. The peptide of the invention can be administered parenterally by injection or by gradual infusion over time. The peptide can also be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Further methods for delivery of the peptide include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by

iontophoresis or transdermal electroporation. To administer a peptide of the invention by certain routes of administration, it can be necessary to coat the compound with, or coadminister the compound with, a material to prevent its inactivation. The method of the invention also includes delivery systems such as microencapsulation of peptides into liposomes or a diluent. Microencapsulation also allows co-entrapment of antimicrobial molecules along with the antigens, so that these molecules, such as antibiotics, may be delivered to a site in need of such treatment in conjunction with the peptides of the invention. Liposomes in the blood stream are generally taken up by the liver and spleen.

Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al, J. NeuroimmunoL, 7: 27, 1984).Thus, the method of the invention is particularly useful for delivering antimicrobial peptides to such organs. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J.R.

Robinson, Ed., 1978, Marcel Dekker, Inc., New York. Other methods of administration will be known to those skilled in the art.

[00150] Preparations for parenteral administration of a peptide of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, PEGylated lipids, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. [00151] Therapeutic compositions typically must be sterile, substantially isotonic, and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00152] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Therapeutic compositions can also be administered with medical devices known in the art. For example, in a preferred aspect, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851,

5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent

No. 4. ,486, 194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and

U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known. [0166] When the peptides of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (or 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

EFFECTIVE DOSAGES

[00153] "Therapeutically effective amount" as used herein for treatment of

neurodegenerative diseases and conditions refers to the amount of peptide used that is of sufficient quantity to reduce or ameliorate any of the symptoms of a neurodegenerative disease in a subject. The dosage ranges for the administration of peptides are those large enough to produce the desired effect. The amount of peptide adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and

concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest

Remington's (Remington's Pharmaceutical Science, Mack Publishing Company, Easton, PA); Egleton, Peptides 18: 1431-1439, 1997; Langer Science 249: 1527-1533, 1990. The dosage regimen can be adjusted by the individual physician in the event of any contraindications.

[00154] Dosage regimens of the pharmaceutical compositions of the present invention are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. [00155] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

[00156] A physician or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or

administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[00157] An effective dose of each of the peptides disclosed herein as potential therapeutics for use in treating neurodegenerative diseases and conditions is from about 1 μg to 500 mg/kg body weight, per single administration, which can readily be determined by one skilled in the art. As discussed above, the dosage depends upon the age, sex, health, and weight of the recipient, kind of concurrent therapy, if any, and frequency of treatment. Other effective dosage range upper limits are 100 mg/kg body weight, 50 mg/kg body weight, 25 mg/kg body weight, and 10 mg/kg body weight.

[00158] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a

prophylactic regime.

[00159] Some compounds of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, See, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (See, e.g., Ranade, J. Clin. Pharmacol, 29: 685, 1989). Exemplary targeting moieties include folate or biotin (See, e.g., U.S. Patent 5,416,016 to Low, et al); mannosides (Umezawa, et al, Biochem. Biophys. Res. Commun., 153: 1038, 1988); antibodies (Bloeman, et al, FEBS Lett., 357: 140, 1995; Owais, et al, Antimicrob. Agents Chemother., 39: 180, 1995); surfactant protein A receptor (Briscoe, et al, Am. J. Physiol, 1233: 134, 1995), different species of which can comprise the formulations of the inventions, as well as components of the invented molecules; pl20 (Schreier, et al, J. Biol. Chem., 269: 9090, 1994). See also Keinanen, et al, FEBS Lett., 346: 123, 1994; KiUion, et al, Immunomethods, 4: 273, 1994. In some methods, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred aspect, the liposomes include a targeting moiety. In some methods, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition should be fluid to the extent that easy AQ syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

FORMULATION

[00160] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. [00161] In some formulations, compositions may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), and solubilizing agents (e.g. glycerol, polyethylene glycerol).

[00162] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[00163] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%>-95%> of active ingredient, preferably 25%-70%.

[00164] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al, Nature 391 : 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.

[0186] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem.

Biophys. Acta 1368: 201-15, 1998.

[00165] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[00166] From the foregoing description, various modifications and changes in the compositions and methods will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein. Each recited range includes all combinations and sub-combinations of ranges, as well as specific numerals contained therein. EXAMPLES

Example 1: Materials, methods, and peptides

[00167] Peptide Synthesis - Peptides were synthesized by suitable manufacturers such as GenScript (Piscataway, NJ) using solid phase Fmoc chemistry and purified (>95% purity) using reversed phase HPLC. The correct peptide mass was confirmed by mass spectrometry. Table 1: Peptides utilized in these studies

Name Sequence ID

HH1 QRLRIRVAVIRA SEQ ID NO 1

HH2 VQLRIRVAVIRA SEQ ID NO 2

HH3 VRFRIRVAVIRA SEQ ID NO 3

HH4 VRWRIRVAVIRA SEQ ID NO 4

HH5 VRLWIRVAVIRA SEQ ID NO 5

HH6 VRLRIRVWVIRA SEQ ID NO 6

HH7 VRLRIRVAVRRA SEQ ID NO 7

HH8 VRLRIRVAVIRK SEQ ID NO 8

HH9 VQLRIRVRVIRK SEQ ID NO 9

HH10 KRFRIRVAVRRA SEQ ID NO 10

HH11 VRLRIRVRVIRK SEQ ID NO 11

HH12 KQFRIRVRVIRK SEQ ID NO 12

HH13 HQFRFRFRVRRK SEQ ID NO 13

HH14 HQWRIRVAVRRH SEQ ID NO 14

HH15 KRFRIRVRVIRK SEQ ID NO 15

HH16 KRWRIRVRVIRK SEQ ID NO 16

HH17 KIWVRWK SEQ ID NO 17

HH18 IWVIWRR SEQ ID NO 18

HH19 ALPWKWPWWPWRR SEQ ID NO 19

HH20 IAPWKWPWWPWRR SEQ ID NO 20

HH21 ILAWKWPWWPWRR SEQ ID NO 21

HH22 ILPAKWPWWPWRR SEQ ID NO 22

HH23 ILPWAWPWWPWRR SEQ ID NO 23

HH24 ILPWKAPWWPWRR SEQ ID NO 24

HH25 ILPWKWAWWPWRR SEQ ID NO 25

HH26 ILPWKWPAWPWRR SEQ ID NO 26

HH27 ILPWKWPWAPWRR SEQ ID NO 27

HH28 ILPWKWPWWAWRR SEQ ID NO 28

HH29 ILPWKWPWWPARR SEQ ID NO 29

HH30 ILPWKWPWWPWAR SEQ ID NO 30

HH31 ILPWKWPWWPWRA SEQ ID NO 31

HH32 DLPWKWPWWPWRR SEQ ID NO 32

HH33 IDPWKWPWWPWRR SEQ ID NO 33

HH34 ILDWKWPWWPWRR SEQ ID NO 34

HH35 ILPDKWPWWPWRR SEQ ID NO 35

HH36 ILPWDWPWWPWRR SEQ ID NO 36

HH37 ILPWKDPWWPWRR SEQ ID NO 37 HH38 ILPWKWDWWPWRR SEQ ID NO 38

HH39 ILPWKWPDWPWRR SEQ ID NO 39

HH40 ILPWKWPWDPWRR SEQ ID NO 40

HH41 ILPWKWPWWDWRR SEQ ID NO 41

HH42 ILPWKWPWWPDRR SEQ ID NO 42

HH43 ILPWKWPWWPWDR SEQ ID NO 43

HH44 ILPWKWPWWPWRD SEQ ID NO 44

HH45 ELPWKWPWWPWRR SEQ ID NO 45

HH46 IEPWKWPWWPWRR SEQ ID NO 46

HH47 ILEWKWPWWPWRR SEQ ID NO 47

HH48 ILPEKWPWWPWRR SEQ ID NO 48

HH49 ILPWEWPWWPWRR SEQ ID NO 49

HH50 ILPWKEPWWPWRR SEQ ID NO 50

HH51 ILPWKWEWWPWRR SEQ ID NO 51

HH52 ILPWKWPEWPWRR SEQ ID NO 52

HH53 ILPWKWPWEPWRR SEQ ID NO 53

HH54 ILPWKWPWWEWRR SEQ ID NO 54

HH55 ILPWKWPWWPERR SEQ ID NO 55

HH56 ILPWKWPWWPWER SEQ ID NO 56

HH57 ILPWKWPWWPWRE SEQ ID NO 57

HH58 FLPWKWPWWPWRR SEQ ID NO 58

HH59 IFPWKWPWWPWRR SEQ ID NO 59

HH60 ILFWKWPWWPWRR SEQ ID NO 60

HH61 ILPFKWPWWPWRR SEQ ID NO 61

HH62 ILPWFWPWWPWRR SEQ ID NO 62

HH63 ILPWKFPWWPWRR SEQ ID NO 63

HH64 ILPWKWFWWPWRR SEQ ID NO 64

HH65 ILPWKWPFWPWRR SEQ ID NO 65

HH66 ILPWKWPWFPWRR SEQ ID NO 66

HH67 ILPWKWPWWFWRR SEQ ID NO 67

HH68 ILPWKWPWWPFRR SEQ ID NO 68

HH69 ILPWKWPWWPWFR SEQ ID NO 69

HH70 ILPWKWPWWPWRF SEQ ID NO 70

HH71 GLPWKWPWWPWRR SEQ ID NO 71

HH72 IGPWKWPWWPWRR SEQ ID NO 72

HH73 ILGWKWPWWPWRR SEQ ID NO 73

HH74 ILPGKWPWWPWRR SEQ ID NO 74

HH75 ILPWGWPWWPWRR SEQ ID NO 75

HH76 ILPWKGPWWPWRR SEQ ID NO 76

HH77 ILPWKWGWWPWRR SEQ ID NO 77

HH78 ILPWKWPGWPWRR SEQ ID NO 78

HH79 ILPWKWPWGPWRR SEQ ID NO 79

HH80 ILPWKWPWWGWRR SEQ ID NO 80

HH81 ILPWKWPWWPGRR SEQ ID NO 81

HH82 ILPWKWPWWPWGR SEQ ID NO 82

HH83 ILPWKWPWWPWRG SEQ ID NO 83

HH84 HLPWKWPWWPWRR SEQ ID NO 84

HH85 IHPWKWPWWPWRR SEQ ID NO 85 HH86 ILHWKWPWWPWRR SEQ ID NO 86

HH87 ILPHKWPWWPWRR SEQ ID NO 87

HH88 ILPWHWPWWPWRR SEQ ID NO 88

HH89 ILPWKHPWWPWRR SEQ ID NO 89

HH90 ILPWKWHWWPWRR SEQ ID NO 90

HH91 ILPWKWPHWPWRR SEQ ID NO 91

HH92 ILPWKWPWHPWRR SEQ ID NO 92

HH93 ILPWKWPWWHWRR SEQ ID NO 93

HH94 ILPWKWPWWPHRR SEQ ID NO 94

HH95 ILPWKWPWWPWHR SEQ ID NO 95

HH96 ILPWKWPWWPWRH SEQ ID NO 96

HH97 IIPWKWPWWPWRR SEQ ID NO 97

HH98 ILIWKWPWWPWRR SEQ ID NO 98

HH99 ILPIKWPWWPWRR SEQ ID NO 99

HH100 ILPWIWPWWPWRR SEQ ID NO 100

HH101 ILPWKIPWWPWRR SEQ ID NO 101

HH102 ILPWKWIWWPWRR SEQ ID NO 102

HH103 ILPWKWPIWPWRR SEQ ID NO 103

HH104 ILPWKWPWIPWRR SEQ ID NO 104

HH105 ILPWKWPWWIWRR SEQ ID NO 105

HH106 ILPWKWPWWPIRR SEQ ID NO 106

HH107 ILPWKWPWWPWIR SEQ ID NO 107

HH108 ILPWKWPWWPWRI SEQ ID NO 108

HH109 KLPWKWPWWPWRR SEQ ID NO 109

HH110 IKPWKWPWWPWRR SEQ ID NO 110

HH111 ILKWKWPWWPWRR SEQ ID NO 111

HH112 ILPKKWPWWPWRR SEQ ID NO 112

HH113 ILPWKKPWWPWRR SEQ ID NO 113

HH114 ILPWKWKWWPWRR SEQ ID NO 114

HH115 ILPWKWPKWPWRR SEQ ID NO 115

HH116 ILPWKWPWKPWRR SEQ ID NO 116

HH117 ILPWKWPWWKWRR SEQ ID NO 117

HH118 ILPWKWPWWPKRR SEQ ID NO 118

HH119 ILPWKWPWWPWKR SEQ ID NO 119

HH120 ILPWKWPWWPWRK SEQ ID NO 120

HH121 LLPWKWPWWPWRR SEQ ID NO 121

HH122 ILLWKWPWWPWRR SEQ ID NO 122

HH123 ILPLKWPWWPWRR SEQ ID NO 123

HH124 ILPWLWPWWPWRR SEQ ID NO 124

HH125 ILPWKLPWWPWRR SEQ ID NO 125

HH126 ILPWKWLWWPWRR SEQ ID NO 126

HH127 ILPWKWPLWPWRR SEQ ID NO 127

HH128 ILPWKWPWLPWRR SEQ ID NO 128

HH129 ILPWKWPWWLWRR SEQ ID NO 129

HH130 ILPWKWPWWPLRR SEQ ID NO 130

HH131 ILPWKWPWWPWLR SEQ ID NO 131

HH132 ILPWKWPWWPWRL SEQ ID NO 132

HH133 MLPWKWPWWPWRR SEQ ID NO 133 HH134 IMPWKWPWWPWRR SEQ ID NO 134

HH135 ILMWKWPWWPWRR SEQ ID NO 135

HH136 ILPMKWPWWPWRR SEQ ID NO 136

HH137 ILPWMWPWWPWRR SEQ ID NO 137

HH138 ILPWKMPWWPWRR SEQ ID NO 138

HH139 ILPWKWMWWPWRR SEQ ID NO 139

HH140 ILPWKWPMWPWRR SEQ ID NO 140

HH141 ILPWKWPWMPWRR SEQ ID NO 141

HH142 ILPWKWPWWMWRR SEQ ID NO 142

HH143 ILPWKWPWWPMRR SEQ ID NO 143

HH144 ILPWKWPWWPWMR SEQ ID NO 144

HH145 ILPWKWPWWPWRM SEQ ID NO 145

HH146 NLPWKWPWWPWRR SEQ ID NO 146

HH147 INPWKWPWWPWRR SEQ ID NO 147

HH148 ILNWKWPWWPWRR SEQ ID NO 148

HH149 ILPNKWPWWPWRR SEQ ID NO 149

HH150 ILPWNWPWWPWRR SEQ ID NO 150

HH151 ILPWKNPWWPWRR SEQ ID NO 151

HH152 ILPWKWNWWPWRR SEQ ID NO 152

HH153 ILPWKWPNWPWRR SEQ ID NO 153

HH154 ILPWKWPWNPWRR SEQ ID NO 154

HH155 ILPWKWPWWNWRR SEQ ID NO 155

HH156 ILPWKWPWWPNRR SEQ ID NO 156

HH157 ILPWKWPWWPWNR SEQ ID NO 157

HH158 ILPWKWPWWPWRN SEQ ID NO 158

HH159 PLPWKWPWWPWRR SEQ ID NO 159

HH160 IPPWKWPWWPWRR SEQ ID NO 160

HH161 ILPPKWPWWPWRR SEQ ID NO 161

HH162 ILPWPWPWWPWRR SEQ ID NO 162

HH163 ILPWKPPWWPWRR SEQ ID NO 163

HH164 ILPWKWPPWPWRR SEQ ID NO 164

HH165 ILPWKWPWPPWRR SEQ ID NO 165

HH166 ILPWKWPWWPPRR SEQ ID NO 166

HH167 ILPWKWPWWPWPR SEQ ID NO 167

HH168 ILPWKWPWWPWRP SEQ ID NO 168

HH169 QLPWKWPWWPWRR SEQ ID NO 169

HH170 IQPWKWPWWPWRR SEQ ID NO 170

HH171 ILQWKWPWWPWRR SEQ ID NO 171

HH172 ILPQKWPWWPWRR SEQ ID NO 172

HH173 ILPWQWPWWPWRR SEQ ID NO 173

HH174 ILPWKQPWWPWRR SEQ ID NO 174

HH175 ILPWKWQWWPWRR SEQ ID NO 175

HH176 ILPWKWPQWPWRR SEQ ID NO 176

HH177 ILPWKWPWQPWRR SEQ ID NO 177

HH178 ILPWKWPWWQWRR SEQ ID NO 178

HH179 ILPWKWPWWPQRR SEQ ID NO 179

HH180 ILPWKWPWWPWQR SEQ ID NO 180

HH181 ILPWKWPWWPWRQ SEQ ID NO 181 HH182 RLPWKWPWWPWRR SEQ ID NO 182

HH183 IRPWKWPWWPWRR SEQ ID NO 183

HH184 ILRWKWPWWPWRR SEQ ID NO 184

HH185 ILPRKWPWWPWRR SEQ ID NO 185

HH186 ILPWRWPWWPWRR SEQ ID NO 186

HH187 ILPWKRPWWPWRR SEQ ID NO 187

HH188 ILPWKWRWWPWRR SEQ ID NO 188

HH189 ILPWKWPRWPWRR SEQ ID NO 189

HH190 ILPWKWPWRPWRR SEQ ID NO 190

HH191 ILPWKWPWWRWRR SEQ ID NO 191

HH192 ILPWKWPWWPRRR SEQ ID NO 192

HH193 SLPWKWPWWPWRR SEQ ID NO 193

HH194 ISPWKWPWWPWRR SEQ ID NO 194

HH195 ILSWKWPWWPWRR SEQ ID NO 195

HH196 ILPSKWPWWPWRR SEQ ID NO 196

HH197 ILPWSWPWWPWRR SEQ ID NO 197

HH198 ILPWKSPWWPWRR SEQ ID NO 198

HH199 ILPWKWSWWPWRR SEQ ID NO 199

HH200 ILPWKWPSWPWRR SEQ ID NO 200

HH201 ILPWKWPWSPWRR SEQ ID NO 201

HH202 ILPWKWPWWSWRR SEQ ID NO 202

HH203 ILPWKWPWWPSRR SEQ ID NO 203

HH204 ILPWKWPWWPWSR SEQ ID NO 204

HH205 ILPWKWPWWPWRS SEQ ID NO 205

HH206 TLPWKWPWWPWRR SEQ ID NO 206

HH207 ITPWKWPWWPWRR SEQ ID NO 207

HH208 ILTWKWPWWPWRR SEQ ID NO 208

HH209 ILPTKWPWWPWRR SEQ ID NO 209

HH210 ILPWTWPWWPWRR SEQ ID NO 210

HH211 ILPWKTPWWPWRR SEQ ID NO 211

HH212 ILPWKWTWWPWRR SEQ ID NO 212

HH213 ILPWKWPTWPWRR SEQ ID NO 213

HH214 ILPWKWPWTPWRR SEQ ID NO 214

HH215 ILPWKWPWWTWRR SEQ ID NO 215

HH216 ILPWKWPWWPTRR SEQ ID NO 216

HH217 ILPWKWPWWPWTR SEQ ID NO 217

HH218 ILPWKWPWWPWRT SEQ ID NO 218

HH219 VLPWKWPWWPWRR SEQ ID NO 219

HH220 IVPWKWPWWPWRR SEQ ID NO 220

HH221 ILVWKWPWWPWRR SEQ ID NO 221

HH222 ILPVKWPWWPWRR SEQ ID NO 222

HH223 ILPWVWPWWPWRR SEQ ID NO 223

HH224 ILPWKVPWWPWRR SEQ ID NO 224

HH225 ILPWKWVWWPWRR SEQ ID NO 225

HH226 ILPWKWPVWPWRR SEQ ID NO 226

HH227 ILPWKWPWVPWRR SEQ ID NO 227

HH228 ILPWKWPWWVWRR SEQ ID NO 228

HH229 ILPWKWPWWPVRR SEQ ID NO 229 HH230 ILPWKWPWWPWVR SEQ ID NO 230

HH231 ILPWKWPWWPWRV SEQ ID NO 231

HH232 WLPWKWPWWPWRR SEQ ID NO 232

HH233 IWPWKWPWWPWRR SEQ ID NO 233

HH234 ILWWKWPWWPWRR SEQ ID NO 234

HH235 ILPWWWPWWPWRR SEQ ID NO 235

HH236 ILPWKWWWWPWRR SEQ ID NO 236

HH237 ILPWKWPWWWWRR SEQ ID NO 237

HH238 ILPWKWPWWPWWR SEQ ID NO 238

HH239 ILPWKWPWWPWRW SEQ ID NO 239

HH240 YLPWKWPWWPWRR SEQ ID NO 240

HH241 IYPWKWPWWPWRR SEQ ID NO 241

HH242 ILYWKWPWWPWRR SEQ ID NO 242

HH243 ILPYKWPWWPWRR SEQ ID NO 243

HH244 ILPWYWPWWPWRR SEQ ID NO 244

HH245 ILPWKYPWWPWRR SEQ ID NO 245

HH246 ILPWKWYWWPWRR SEQ ID NO 246

HH247 ILPWKWPYWPWRR SEQ ID NO 247

HH248 ILPWKWPWYPWRR SEQ ID NO 248

HH249 ILPWKWPWWYWRR SEQ ID NO 249

HH250 ILPWKWPWWPYRR SEQ ID NO 250

HH251 ILPWKWPWWPWYR SEQ ID NO 251

HH252 ILPWKWPWWPWRY SEQ ID NO 252

HH253 ARLRIRVAVIRA SEQ ID NO 253

HH254 DRLRIRVAVIRA SEQ ID NO 254

HH255 ERLRIRVAVIRA SEQ ID NO 255

HH256 FRLRIRVAVIRA SEQ ID NO 256

HH257 GRLRIRVAVIRA SEQ ID NO 257

HH258 HRLRIRVAVIRA SEQ ID NO 258

HH259 IRLRIRVAVIRA SEQ ID NO 259

HH260 KRLRIRVAVIRA SEQ ID NO 260

HH261 LRLRIRVAVIRA SEQ ID NO 261

HH262 MRLRIRVAVIRA SEQ ID NO 262

HH263 NRLRIRVAVIRA SEQ ID NO 263

HH264 PRLRIRVAVIRA SEQ ID NO 264

HH265 QRLRIRVAVIRA SEQ ID NO 265

HH266 RRLRIRVAVIRA SEQ ID NO 266

HH267 SRLRIRVAVIRA SEQ ID NO 267

HH268 TRLRIRVAVIRA SEQ ID NO 268

HH269 WRLRIRVAVIRA SEQ ID NO 269

HH270 YRLRIRVAVIRA SEQ ID NO 270

HH271 VALRIRVAVIRA SEQ ID NO 271

HH272 VDLRIRVAVIRA SEQ ID NO 272

HH273 VELRIRVAVIRA SEQ ID NO 273

HH274 VFLRIRVAVIRA SEQ ID NO 274

HH275 VGLRIRVAVIRA SEQ ID NO 275

HH276 VHLRIRVAVIRA SEQ ID NO 276

HH277 VILRIRVAVIRA SEQ ID NO 277 HH278 VKLRIRVAVIRA SEQ ID NO 278

HH279 VLLRIRVAVIRA SEQ ID NO 279

HH280 VMLRIRVAVIRA SEQ ID NO 280

HH281 VNLRIRVAVIRA SEQ ID NO 281

HH282 VPLRIRVAVIRA SEQ ID NO 282

HH283 VQLRIRVAVIRA SEQ ID NO 283

HH284 VSLRIRVAVIRA SEQ ID NO 284

HH285 VTLRIRVAVIRA SEQ ID NO 285

HH286 WLRIRVAVIRA SEQ ID NO 286

HH287 VWLRIRVAVIRA SEQ ID NO 287

HH288 VYLRIRVAVIRA SEQ ID NO 288

HH289 VRARIRVAVIRA SEQ ID NO 289

HH290 VRDRIRVAVIRA SEQ ID NO 290

HH291 VRERIRVAVIRA SEQ ID NO 291

HH292 VRFRIRVAVIRA SEQ ID NO 292

HH293 VRGRIRVAVIRA SEQ ID NO 293

HH294 VRHRIRVAVIRA SEQ ID NO 294

HH295 VRIRIRVAVIRA SEQ ID NO 295

HH296 VRKRIRVAVIRA SEQ ID NO 296

HH297 VRMRIRVAVIRA SEQ ID NO 297

HH298 VRNRIRVAVIRA SEQ ID NO 298

HH299 VRPRIRVAVIRA SEQ ID NO 299

HH300 VRQRIRVAVIRA SEQ ID NO 300

HH301 VRRRIRVAVIRA SEQ ID NO 301

HH302 VRSRIRVAVIRA SEQ ID NO 302

HH303 VRTRIRVAVIRA SEQ ID NO 303

HH304 VRVRIRVAVIRA SEQ ID NO 304

HH305 VRWRIRVAVIRA SEQ ID NO 305

HH306 VRYRIRVAVIRA SEQ ID NO 306

HH307 VRLAIRVAVIRA SEQ ID NO 307

HH308 VRLDIRVAVIRA SEQ ID NO 308

HH309 VRLEIRVAVIRA SEQ ID NO 309

HH310 VRLFIRVAVIRA SEQ ID NO 310

HH311 VRLGIRVAVIRA SEQ ID NO 311

HH312 VRLHIRVAVIRA SEQ ID NO 312

HH313 VRLIIRVAVIRA SEQ ID NO 313

HH314 VRLKIRVAVIRA SEQ ID NO 314

HH315 VRLLIRVAVIRA SEQ ID NO 315

HH316 VRLMIRVAVIRA SEQ ID NO 316

HH317 VRLNIRVAVIRA SEQ ID NO 317

HH318 VRLPIRVAVIRA SEQ ID NO 318

HH319 VRLQIRVAVIRA SEQ ID NO 319

HH320 VRLSIRVAVIRA SEQ ID NO 320

HH321 VRL IRVAVIRA SEQ ID NO 321

HH322 VRLVIRVAVIRA SEQ ID NO 322

HH323 VRLWIRVAVIRA SEQ ID NO 323

HH324 VRLYIRVAVIRA SEQ ID NO 324

HH325 VRLRARVAVIRA SEQ ID NO 325 HH326 VRLRDRVAVIRA SEQ ID NO 326

HH327 VRLRERVAVIRA SEQ ID NO 327

HH328 VRLRFRVAVIRA SEQ ID NO 328

HH329 VRLRGRVAVIRA SEQ ID NO 329

HH330 VRLRHRVAVIRA SEQ ID NO 330

HH331 VRLRKRVAVIRA SEQ ID NO 331

HH332 VRLRLRVAVIRA SEQ ID NO 332

HH333 VRLRMRVAVIRA SEQ ID NO 333

HH334 VRLRNRVAVIRA SEQ ID NO 334

HH335 VRLRPRVAVIRA SEQ ID NO 335

HH336 VRLRQRVAVIRA SEQ ID NO 336

HH337 VRLRRRVAVIRA SEQ ID NO 337

HH338 VRLRSRVAVIRA SEQ ID NO 338

HH339 VRLRTRVAVIRA SEQ ID NO 339

HH340 VRLRVRVAVIRA SEQ ID NO 340

HH341 VRLRWRVAVIRA SEQ ID NO 341

HH342 VRLRYRVAVIRA SEQ ID NO 342

HH343 VRLRIAVAVIRA SEQ ID NO 343

HH344 VRLRIDVAVIRA SEQ ID NO 344

HH345 VRLRIEVAVIRA SEQ ID NO 345

HH346 VRLRIFVAVIRA SEQ ID NO 346

HH347 VRLRIGVAVIRA SEQ ID NO 347

HH348 VRLRIHVAVIRA SEQ ID NO 348

HH349 VRLRIIVAVIRA SEQ ID NO 349

HH350 VRLRIKVAVIRA SEQ ID NO 350

HH351 VRLRILVAVIRA SEQ ID NO 351

HH352 VRLRIMVAVIRA SEQ ID NO 352

HH353 VRLRI VAVIRA SEQ ID NO 353

HH354 VRLRIPVAVIRA SEQ ID NO 354

HH355 VRLRIQVAVIRA SEQ ID NO 355

HH356 VRLRISVAVIRA SEQ ID NO 356

HH357 VRLRI VAVIRA SEQ ID NO 357

HH358 VRLRIWAVIRA SEQ ID NO 358

HH359 VRLRIWVAVIRA SEQ ID NO 359

HH360 VRLRIYVAVIRA SEQ ID NO 360

HH361 VRLRIRAAVIRA SEQ ID NO 361

HH362 VRLRIRDAVIRA SEQ ID NO 362

HH363 VRLRIREAVIRA SEQ ID NO 363

HH364 VRLRIRFAVIRA SEQ ID NO 364

HH365 VRLRIRGAVIRA SEQ ID NO 365

HH366 VRLRIRHAVIRA SEQ ID NO 366

HH367 VRLRIRIAVIRA SEQ ID NO 367

HH368 VRLRIRKAVIRA SEQ ID NO 368

HH369 VRLRIRLAVIRA SEQ ID NO 369

HH370 VRLRIRMAVIRA SEQ ID NO 370

HH371 VRLRIRNAVIRA SEQ ID NO 371

HH372 VRLRIRPAVIRA SEQ ID NO 372

HH373 VRLRIRQAVIRA SEQ ID NO 373 HH374 VRLRIRRAVIRA SEQ ID NO 374

HH375 VRLRIRSAVIRA SEQ ID NO 375

HH376 VRLRIR AVIRA SEQ ID NO 376

HH377 VRLRIRWAVIRA SEQ ID NO 377

HH378 VRLRIRYAVIRA SEQ ID NO 378

HH379 VRLRIRVDVIRA SEQ ID NO 379

HH380 VRLRIRVEVIRA SEQ ID NO 380

HH381 VRLRIRVFVIRA SEQ ID NO 381

HH382 VRLRIRVGVIRA SEQ ID NO 382

HH383 VRLRIRVHVIRA SEQ ID NO 383

HH384 VRLRIRVIVIRA SEQ ID NO 384

HH385 VRLRIRVKVIRA SEQ ID NO 385

HH386 VRLRIRVLVIRA SEQ ID NO 386

HH387 VRLRIRVMVIRA SEQ ID NO 387

HH388 VRLRIRV VIRA SEQ ID NO 388

HH389 VRLRIRVPVIRA SEQ ID NO 389

HH390 VRLRIRVQVIRA SEQ ID NO 390

HH391 VRLRIRVRVIRA SEQ ID NO 391

HH392 VRLRIRVSVIRA SEQ ID NO 392

HH393 VRLRIRVTVIRA SEQ ID NO 393

HH394 VRLRIRVWIRA SEQ ID NO 394

HH395 VRLRIRVWVIRA SEQ ID NO 395

HH396 VRLRIRVYVIRA SEQ ID NO 396

HH397 VRLRIRVAAIRA SEQ ID NO 397

HH398 VRLRIRVADIRA SEQ ID NO 398

HH399 VRLRIRVAEIRA SEQ ID NO 399

HH400 VRLRIRVAFIRA SEQ ID NO 400

HH401 VRLRIRVAGIRA SEQ ID NO 401

HH402 VRLRIRVAHIRA SEQ ID NO 402

HH403 VRLRIRVAIIRA SEQ ID NO 403

HH404 VRLRIRVAKIRA SEQ ID NO 404

HH405 VRLRIRVALIRA SEQ ID NO 405

HH406 VRLRIRVAMIRA SEQ ID NO 406

HH407 VRLRIRVANIRA SEQ ID NO 407

HH408 VRLRIRVAPIRA SEQ ID NO 408

HH409 VRLRIRVAQIRA SEQ ID NO 409

HH410 VRLRIRVARIRA SEQ ID NO 410

HH411 VRLRIRVASIRA SEQ ID NO 411

HH412 VRLRIRVA IRA SEQ ID NO 412

HH413 VRLRIRVAWIRA SEQ ID NO 413

HH414 VRLRIRVAYIRA SEQ ID NO 414

HH415 VRLRIRVAVARA SEQ ID NO 415

HH416 VRLRIRVAVDRA SEQ ID NO 416

HH417 VRLRIRVAVERA SEQ ID NO 417

HH418 VRLRIRVAVFRA SEQ ID NO 418

HH419 VRLRIRVAVGRA SEQ ID NO 419

HH420 VRLRIRVAVHRA SEQ ID NO 420

HH421 VRLRIRVAVKRA SEQ ID NO 421 HH422 VRLRIRVAVLRA SEQ ID NO 422

HH423 VRLRIRVAV RA SEQ ID NO 423

HH424 VRLRIRVAWRA SEQ ID NO 424

HH425 VRLRIRVAVPRA SEQ ID NO 425

HH426 VRLRIRVAVQRA SEQ ID NO 426

HH427 VRLRIRVAVRRA SEQ ID NO 427

HH428 VRLRIRVAVSRA SEQ ID NO 428

HH429 VRLRIRVAVTRA SEQ ID NO 429

HH430 VRLRIRVAWRA SEQ ID NO 430

HH431 VRLRIRVAWRA SEQ ID NO 431

HH432 VRLRIRVAVYRA SEQ ID NO 432

HH433 VRLRIRVAVIAA SEQ ID NO 433

HH434 VRLRIRVAVIDA SEQ ID NO 434

HH435 VRLRIRVAVIEA SEQ ID NO 435

HH436 VRLRIRVAVIFA SEQ ID NO 436

HH437 VRLRIRVAVIGA SEQ ID NO 437

HH438 VRLRIRVAVIHA SEQ ID NO 438

HH439 VRLRIRVAVIIA SEQ ID NO 439

HH440 VRLRIRVAVIKA SEQ ID NO 440

HH441 VRLRIRVAVILA SEQ ID NO 441

HH442 VRLRIRVAVIMA SEQ ID NO 442

HH443 VRLRIRVAVINA SEQ ID NO 443

HH444 VRLRIRVAVIPA SEQ ID NO 444

HH445 VRLRIRVAVIQA SEQ ID NO 445

HH446 VRLRIRVAVISA SEQ ID NO 446

HH447 VRLRIRVAVITA SEQ ID NO 447

HH448 VRLRIRVAVIVA SEQ ID NO 448

HH449 VRLRIRVAVIWA SEQ ID NO 449

HH450 VRLRIRVAVIYA SEQ ID NO 450

HH451 VRLRIRVAVIRD SEQ ID NO 451

HH452 VRLRIRVAVIRE SEQ ID NO 452

HH453 VRLRIRVAVIRF SEQ ID NO 453

HH454 VRLRIRVAVIRG SEQ ID NO 454

HH455 VRLRIRVAVIRH SEQ ID NO 455

HH456 VRLRIRVAVIRI SEQ ID NO 456

HH457 VRLRIRVAVIRK SEQ ID NO 457

HH458 VRLRIRVAVIRL SEQ ID NO 458

HH459 VRLRIRVAVIRM SEQ ID NO 459

HH460 VRLRIRVAVIRN SEQ ID NO 460

HH461 VRLRIRVAVIRP SEQ ID NO 461

HH462 VRLRIRVAVIRQ SEQ ID NO 462

HH463 VRLRIRVAVIRR SEQ ID NO 463

HH464 VRLRIRVAVIRS SEQ ID NO 464

HH465 VRLRIRVAVIRT SEQ ID NO 465

HH466 VRLRIRVAVIRV SEQ ID NO 466

HH467 VRLRIRVAVIRW SEQ ID NO 467

HH468 VRLRIRVAVIRY SEQ ID NO 468

HH469 RRRRVKWWR SEQ ID NO 469 HH470 WLRKKQGRL SEQ ID NO 470

HH471 KWVRVYLRW SEQ ID NO 471

HH472 GKVMISIVR SEQ ID NO 472

HH473 IKWRWRWR SEQ ID NO 473

HH474 RRRRRWVRR SEQ ID NO 474

HH475 HM RFRTVY SEQ ID NO 475

HH476 VRKRGSWRM SEQ ID NO 476

HH477 RIIRTYKRG SEQ ID NO 477

HH478 WWRWRLRLI SEQ ID NO 478

HH479 WLNRLYIRL SEQ ID NO 479

HH480 IWRWTKWFW SEQ ID NO 480

HH481 RFKGSWKYR SEQ ID NO 481

HH482 VWVIRKKKW SEQ ID NO 482

HH483 RGRRVWRLF SEQ ID NO 483

HH484 WRWRKVKQW SEQ ID NO 484

HH485 WWKYWRKVI SEQ ID NO 485

HH486 WLVRIRKRI SEQ ID NO 486

HH487 WWRWWQRRW SEQ ID NO 487

HH488 RKKWWWKIR SEQ ID NO 488

HH489 WVRKKIRRR SEQ ID NO 489

HH490 RYRRRWYIR SEQ ID NO 490

HH491 LYRWVWKVG SEQ ID NO 491

HH492 VRRRWFKWL SEQ ID NO 492

HH493 RRLWWWKWL SEQ ID NO 493

HH494 WRFKWTRRG SEQ ID NO 494

HH495 KWWRHRRMW SEQ ID NO 495

HH496 RRKRWWWRT SEQ ID NO 496

HH497 WRRKIVRVW SEQ ID NO 497

HH498 KLRRGSLWR SEQ ID NO 498

HH499 RVIWWWRRK SEQ ID NO 499

HH500 TWRVWKVRW SEQ ID NO 500

HH501 QRGIVIWRK SEQ ID NO 501

HH502 GKWWKWGIW SEQ ID NO 502

HH503 RVRRWWFVR SEQ ID NO 503

HH504 FWRRRVKWR SEQ ID NO 504

HH505 FRRYQNIVR SEQ ID NO 505

HH506 RFWRWIFKW SEQ ID NO 506

HH507 KR VKR WK SEQ ID NO 507

HH508 WYSLIIFKR SEQ ID NO 508

HH509 RKNRRIRW SEQ ID NO 509

HH510 FFRKRRWRI SEQ ID NO 510

HH511 WKIRKVIKW SEQ ID NO 511

HH512 IKWYWRKKK SEQ ID NO 512

HH513 KRGWRKRWW SEQ ID NO 513

HH514 RKWMGRRIR SEQ ID NO 514

HH515 WKGKKRRVI SEQ ID NO 515

HH516 KVIRYKVYI SEQ ID NO 516

HH517 RRTRKWILR SEQ ID NO 517 HH518 Y W WLRRW SEQ ID NO 518

HH519 KWKHWRWQW SEQ ID NO 519

HH520 RKIWKVRV SEQ ID NO 520

HH521 QYLGWRFKW SEQ ID NO 521

HH522 KIKTRKVKY SEQ ID NO 522

HH523 VWIRWRRRW SEQ ID NO 523

HH524 WGVRVRRLI SEQ ID NO 524

HH525 WWKRVWKFI SEQ ID NO 525

HH526 YWIYSRLRR SEQ ID NO 526

HH527 RRYWKFKRR SEQ ID NO 527

HH528 IVRRVIIRV SEQ ID NO 528

HH529 ARRRGLKVW SEQ ID NO 529

HH530 RRWVRRWWR SEQ ID NO 530

HH531 WKWKWKWQS SEQ ID NO 531

HH532 RWKVKQRRR SEQ ID NO 532

HH533 YWTKFRLRI SEQ ID NO 533

HH534 WVIKVRIRW SEQ ID NO 534

HH535 ARVQVYKYR SEQ ID NO 535

HH536 KWRWHWVYV SEQ ID NO 536

HH537 KVKYKFRRW SEQ ID NO 537

HH538 RFRKRKNRI SEQ ID NO 538

HH539 MFRRRFIWK SEQ ID NO 539

HH540 WRLRRFRLW SEQ ID NO 540

HH541 WIQRIRIWV SEQ ID NO 541

HH542 RRYHWRIYI SEQ ID NO 542

HH543 SRFWRRWRK SEQ ID NO 543

HH544 YRVWIIRRK SEQ ID NO 544

HH545 WRVSWLIWR SEQ ID NO 545

HH546 RFVKRKIVW SEQ ID NO 546

HH547 RIYKIRWII SEQ ID NO 547

HH548 RKFWHRG I SEQ ID NO 548

HH549 AWWWRKRW SEQ ID NO 549

HH550 WVWGKVRWG SEQ ID NO 550

HH551 FGIRFRRMV SEQ ID NO 551

HH552 FWIRKVFRI SEQ ID NO 552

HH553 KRWKVRWW SEQ ID NO 553

HH554 KIRIWRIWV SEQ ID NO 554

HH555 RGRWKRIKK SEQ ID NO 555

HH556 RLWFLVLRR SEQ ID NO 556

HH557 IIRVTRWTK SEQ ID NO 557

HH558 AMWRWKWRK SEQ ID NO 558

HH559 TRKYFGRFV SEQ ID NO 559

HH560 ARRVKKKRR SEQ ID NO 560

HH561 RWWKIWKRR SEQ ID NO 561

HH562 RWRYKIQKW SEQ ID NO 562

HH563 RVGIKIKMK SEQ ID NO 563

HH564 WVLKLRYKW SEQ ID NO 564

HH565 FRRKWIFKK SEQ ID NO 565 HH566 WIQKLWRQR SEQ ID NO 566

HH567 RIVRLHVRK SEQ ID NO 567

HH568 VRIGWRRVK SEQ ID NO 568

HH569 RRRIGIKRF SEQ ID NO 569

HH570 RRRRKKVRI SEQ ID NO 570

HH571 KLWRYKRWR SEQ ID NO 571

HH572 RIRRFIKKW SEQ ID NO 572

HH573 LWHKKKKIW SEQ ID NO 573

HH574 LTRRFWLRR SEQ ID NO 574

HH575 RRRYVIRRR SEQ ID NO 575

HH576 WGWRWIWIK SEQ ID NO 576

HH577 RWRWQRGRF SEQ ID NO 577

HH578 RRKKWKVRI SEQ ID NO 578

HH579 KMKLYKGSM SEQ ID NO 579

HH580 GTIRWWRRR SEQ ID NO 580

HH581 SLRRYIWRF SEQ ID NO 581

HH582 GRYWKKWRR SEQ ID NO 582

HH583 WIRQFRWKK SEQ ID NO 583

HH584 AKVRRIKHW SEQ ID NO 584

HH585 YSRRKTWWI SEQ ID NO 585

HH586 RGRWWIRRQ SEQ ID NO 586

HH587 WVFRWVWWR SEQ ID NO 587

HH588 VYRVWWLKW SEQ ID NO 588

HH589 WWVRRRVGW SEQ ID NO 589

HH590 WFKIKRLYL SEQ ID NO 590

HH591 WKMWKRGWT SEQ ID NO 591

HH592 RWWRKSRRL SEQ ID NO 592

HH593 FWRIRWWRW SEQ ID NO 593

HH594 VWWFGKRTT SEQ ID NO 594

HH595 VRIIWWIWR SEQ ID NO 595

HH596 WWVRIWRWM SEQ ID NO 596

HH597 RKWKKWFHR SEQ ID NO 597

HH598 RKWKFWGYK SEQ ID NO 598

HH599 FWYIWSKRV SEQ ID NO 599

HH600 YWRQFRRKQ SEQ ID NO 600

HH601 WWWKVKSRR SEQ ID NO 601

HH602 WRLWIWWIR SEQ ID NO 602

HH603 QFRV RRKY SEQ ID NO 603

HH604 RYRFWWVRR SEQ ID NO 604

HH605 THIWLRRRR SEQ ID NO 605

HH606 RRRFRKRRM SEQ ID NO 606

HH607 LYTRVRRYS SEQ ID NO 607

HH608 WSIRRLWWL SEQ ID NO 608

HH609 YKIKRRRYG SEQ ID NO 609

HH610 WKRIQFRRK SEQ ID NO 610

HH611 HKKRRIWRK SEQ ID NO 611

HH612 WRLIRWWIR SEQ ID NO 612

HH613 LRK WWWRR SEQ ID NO 613 HH614 VKRIRIWML SEQ ID NO 614

HH615 IRYR WKWL SEQ ID NO 615

HH616 GRILSRRWK SEQ ID NO 616

HH617 KHWKIHVRW SEQ ID NO 617

HH618 WIYWKVWRR SEQ ID NO 618

HH619 KLWKVRNRR SEQ ID NO 619

HH620 RRVYYYKWV SEQ ID NO 620

HH621 WRWGVFRLR SEQ ID NO 621

HH622 IWRVLKKRV SEQ ID NO 622

HH623 AKKFWR WI SEQ ID NO 623

HH624 RQWRKWKK SEQ ID NO 624

HH625 GWKRWWVML SEQ ID NO 625

HH626 KWRRTRRRK SEQ ID NO 626

HH627 FRRMKRFLR SEQ ID NO 627

HH628 RSW WWWIR SEQ ID NO 628

HH629 WRRRIWINR SEQ ID NO 629

HH630 RWKWFYLKR SEQ ID NO 630

HH631 RKR IWRII SEQ ID NO 631

HH632 RRRVWWRRR SEQ ID NO 632

HH633 KWRFKWWKR SEQ ID NO 633

HH634 KWIWGWRRW SEQ ID NO 634

HH635 WIKRKWKMR SEQ ID NO 635

HH636 MWKKVLRRV SEQ ID NO 636

HH637 WRWRIFHWL SEQ ID NO 637

HH638 KIQRWKGKR SEQ ID NO 638

HH639 LWYKYWRWR SEQ ID NO 639

HH640 YVRRIWKIT SEQ ID NO 640

HH641 RWRQYRSRW SEQ ID NO 641

HH642 VGRWKRRRW SEQ ID NO 642

HH643 KSSRIYILF SEQ ID NO 643

HH644 AKWWWYRKI SEQ ID NO 644

HH645 FYWWRWFRV SEQ ID NO 645

HH646 RTRWLRYRR SEQ ID NO 646

HH647 W IIWWIRR SEQ ID NO 647

HH648 KRGFWWWRI SEQ ID NO 648

HH649 RRRKKYIIR SEQ ID NO 649

HH650 VWKVGWYYR SEQ ID NO 650

HH651 LKFSTGRVR SEQ ID NO 651

HH652 RRVWVRRKR SEQ ID NO 652

HH653 RFWYMWKYV SEQ ID NO 653

HH654 WYVRWMGRR SEQ ID NO 654

HH655 WKRRMRRRK SEQ ID NO 655

HH656 RVLRRVSWV SEQ ID NO 656

HH657 RRLRKKWGW SEQ ID NO 657

HH658 WYKKIRLII SEQ ID NO 658

HH659 IYIIIWRTK SEQ ID NO 659

HH660 TWRMRVKVS SEQ ID NO 660

HH661 AWWKIRWRI SEQ ID NO 661 HH662 RVRRYRWSW SEQ ID NO 662

HH663 IWRIRRFRI SEQ ID NO 663

HH664 KIRRKWWWF SEQ ID NO 664

HH665 RRFWWIKIR SEQ ID NO 665

HH666 WYWWRVRRV SEQ ID NO 666

HH667 WYKLWRRKV SEQ ID NO 667

HH668 WWFSWRWRV SEQ ID NO 668

HH669 RFKTRRGWR SEQ ID NO 669

HH670 WIWIVRRRV SEQ ID NO 670

HH671 RRFKKWMYW SEQ ID NO 671

HH672 RWYRVIRWK SEQ ID NO 672

HH673 YRWMVRWVR SEQ ID NO 673

HH674 KVRRYNRRR SEQ ID NO 674

HH675 WFVW RRW SEQ ID NO 675

HH676 RWKWRWRWY SEQ ID NO 676

HH677 ARWRVRKWW SEQ ID NO 677

HH678 KIKFWIIRR SEQ ID NO 678

HH679 WYWRVRLQW SEQ ID NO 679

HH680 YWWWKRRRR SEQ ID NO 680

HH681 FIKRVRRRW SEQ ID NO 681

HH682 VSWFRRRY SEQ ID NO 682

HH683 KFRV VRVL SEQ ID NO 683

HH684 WMYYKRRRR SEQ ID NO 684

HH685 IWIWWRWRW SEQ ID NO 685

HH686 WKKKKIIRV SEQ ID NO 686

HH687 RRGWRRRRR SEQ ID NO 687

HH688 WRWRKIWKW SEQ ID NO 688

HH689 WWRWKRRII SEQ ID NO 689

HH690 WKVRWKIRR SEQ ID NO 690

HH691 RFWVRGRRS SEQ ID NO 691

HH692 RRWVLWRRR SEQ ID NO 692

HH693 KYIWKKRRY SEQ ID NO 693

HH694 KWQWIRKIR SEQ ID NO 694

HH695 YWIRRRWRL SEQ ID NO 695

HH696 RVKWIKWLH SEQ ID NO 696

HH697 YVRQWKKRR SEQ ID NO 697

HH698 WKIVGVFRV SEQ ID NO 698

HH699 VIKYVRMWW SEQ ID NO 699

HH700 RRRRVWRVR SEQ ID NO 700

HH701 RRRKIRVYR SEQ ID NO 701

HH702 RRNRWRRIR SEQ ID NO 702

HH703 IRKWIWRRV SEQ ID NO 703

HH704 QRWRVRRRY SEQ ID NO 704

HH705 WWMIIKIRN SEQ ID NO 705

HH706 ARRRGRRVM SEQ ID NO 706

HH707 RRWHWRKRK SEQ ID NO 707

HH708 KRFLRKRRF SEQ ID NO 708

HH709 RWKGWYLRT SEQ ID NO 709 HH710 WSWRGRRKF SEQ ID NO 710

HH711 KIIMKRRRW SEQ ID NO 711

HH712 VWKRFLHWR SEQ ID NO 712

HH713 RLKRRKKWR SEQ ID NO 713

HH714 AVRKFRRVT SEQ ID NO 714

HH715 IKQRFWWRT SEQ ID NO 715

HH716 WKIWWIIK SEQ ID NO 716

HH717 LYRWIVWKR SEQ ID NO 717

HH718 WWWRWRIRK SEQ ID NO 718

HH719 RLWRKWQW SEQ ID NO 719

HH720 RVKLRWGWR SEQ ID NO 720

HH721 AWRYKRRIF SEQ ID NO 721

HH722 KRWQIRGIT SEQ ID NO 722

HH723 KRWRWRWRW SEQ ID NO 723

HH724 KRWVYKYRV SEQ ID NO 724

HH725 VHWRWRFWK SEQ ID NO 725

HH726 FVGKTKRKR SEQ ID NO 726

HH727 RLRFGWFLF SEQ ID NO 727

HH728 AKRWIWIQV SEQ ID NO 728

HH729 RKYVRRWVY SEQ ID NO 729

HH730 YRVYWWWWR SEQ ID NO 730

HH731 KRRKKRRVR SEQ ID NO 731

HH732 KKVRF I W SEQ ID NO 732

HH733 KLWYWKKW SEQ ID NO 733

HH734 WRWGLRWWQ SEQ ID NO 734

HH735 AFFYRWWIR SEQ ID NO 735

HH736 WYWRRRRLK SEQ ID NO 736

HH737 YKFRWRIYI SEQ ID NO 737

HH738 WLRKVW WR SEQ ID NO 738

HH739 RVRFKVYRV SEQ ID NO 739

HH740 RWLSKIWKV SEQ ID NO 740

HH741 RRRLGWRRG SEQ ID NO 741

HH742 KKWGGGLVK SEQ ID NO 742

HH743 YWWLWRKKR SEQ ID NO 743

HH744 WIRLWVKWR SEQ ID NO 744

HH745 GRRSTHWRI SEQ ID NO 745

HH746 KKKLFINTW SEQ ID NO 746

HH747 VYRRRRVKG SEQ ID NO 747

HH748 KGWIIWKIV SEQ ID NO 748

HH749 VFHRIRRIK SEQ ID NO 749

HH750 RLRLWKSKR SEQ ID NO 750

HH751 RRKVFKLRR SEQ ID NO 751

HH752 VWLKVYWFK SEQ ID NO 752

HH753 VRWGRRRWV SEQ ID NO 753

HH754 RY WVRRKK SEQ ID NO 754

HH755 KIRWRKYHL SEQ ID NO 755

HH756 VIWRWRKFY SEQ ID NO 756

HH757 RRWWKWWWR SEQ ID NO 757 HH758 WRVKGKRSK SEQ ID NO 758

HH759 RWRTRR IV SEQ ID NO 759

HH760 WWFSIRLWR SEQ ID NO 760

HH761 YTWYIKKKR SEQ ID NO 761

HH762 VWRRKKYWR SEQ ID NO 762

HH763 YLTRFVKYF SEQ ID NO 763

HH764 KRWKHIRRI SEQ ID NO 764

HH765 WIVWIRKRI SEQ ID NO 765

HH766 RRWVIRIYK SEQ ID NO 766

HH767 WFWRRKMIR SEQ ID NO 767

HH768 RYRRWVRKR SEQ ID NO 768

HH769 RKWWWKWRR SEQ ID NO 769

HH770 RIWMFKIFR SEQ ID NO 770

HH771 IVRVGIFRL SEQ ID NO 771

HH772 IIRLIKWWR SEQ ID NO 772

HH773 WVRRYQMRR SEQ ID NO 773

HH774 WQWMRYRR SEQ ID NO 774

HH775 KKWKVWRFG SEQ ID NO 775

HH776 WRYWWTRRI SEQ ID NO 776

HH777 RIRKGWKWG SEQ ID NO 777

HH778 KKRRGNRVR SEQ ID NO 778

HH779 V RKLRRRW SEQ ID NO 779

HH780 RNRTHWWRK SEQ ID NO 780

HH781 RFTWWWRKF SEQ ID NO 781

HH782 KRIRYKRWH SEQ ID NO 782

HH783 RWRRYGRVY SEQ ID NO 783

HH784 TWKKRVKK SEQ ID NO 784

HH785 RKYRRRYRR SEQ ID NO 785

HH786 YFRWWKRWI SEQ ID NO 786

HH787 WWQWIVWRK SEQ ID NO 787

HH788 RKRLYRWIK SEQ ID NO 788

HH789 GWWK WRWW SEQ ID NO 789

HH790 KWWWYWYRR SEQ ID NO 790

HH791 RFKWFIRRF SEQ ID NO 791

HH792 RIRRLW IV SEQ ID NO 792

HH793 ARWMWRRWR SEQ ID NO 793

HH794 LVRWVWGKR SEQ ID NO 794

HH795 KRWLKWWRV SEQ ID NO 795

HH796 FVYRGWRRK SEQ ID NO 796

HH797 RRRWKIYKW SEQ ID NO 797

HH798 KRWWQWRWF SEQ ID NO 798

HH799 KRVKVRWVT SEQ ID NO 799

HH800 RFKYWRWWQ SEQ ID NO 800

HH801 KRQWWRVFK SEQ ID NO 801

HH802 FKIVWWRRR SEQ ID NO 802

HH803 QWWWKYRWK SEQ ID NO 803

HH804 RWLRIRKVY SEQ ID NO 804

HH805 RYKRWYRH SEQ ID NO 805 HH806 KVRWKWWGW SEQ ID NO 806

HH807 IWKVRIFKR SEQ ID NO 807

HH808 AIWHKTRRL SEQ ID NO 808

HH809 IRQRVRWRW SEQ ID NO 809

HH810 MKVWIRWRI SEQ ID NO 810

HH811 QRRWWGRFK SEQ ID NO 811

HH812 NKRVWFIYR SEQ ID NO 812

HH813 RW WKGGL SEQ ID NO 813

HH814 RYRRFRVRW SEQ ID NO 814

HH815 KKVRRVIWW SEQ ID NO 815

HH816 WFTRWKWRW SEQ ID NO 816

HH817 KWVWFRWRK SEQ ID NO 817

HH818 KYLRSVIFY SEQ ID NO 818

HH819 FKRSWVQIV SEQ ID NO 819

HH820 RWWFIRKWW SEQ ID NO 820

HH821 IRRWKRVWW SEQ ID NO 821

HH822 QKWYRQRRN SEQ ID NO 822

HH823 VWRKWYRVK SEQ ID NO 823

HH824 KKKLWRKFR SEQ ID NO 824

HH825 RRWWWWRFN SEQ ID NO 825

HH826 WFFKSKVYW SEQ ID NO 826

HH827 RW L WRW SEQ ID NO 827

HH828 RWRR WMTK SEQ ID NO 828

HH829 WKIWKIRWF SEQ ID NO 829

HH830 WWFWVIRKY SEQ ID NO 830

HH831 RYVKIRWVR SEQ ID NO 831

HH832 RIWILSWRW SEQ ID NO 832

HH833 KSWRKLFIW SEQ ID NO 833

HH834 VWVRWKIWY SEQ ID NO 834

HH835 KKRRFKRRY SEQ ID NO 835

HH836 RFWKKIRRH SEQ ID NO 836

HH837 RKVWWRVFY SEQ ID NO 837

HH838 YWRRKWRRK SEQ ID NO 838

HH839 KRIRRWKWW SEQ ID NO 839

HH840 YWRYLWIRF SEQ ID NO 840

HH841 IIYKWRWYW SEQ ID NO 841

HH842 QTVYLIFRR SEQ ID NO 842

HH843 AKKIKWLVW SEQ ID NO 843

HH844 YRFVRRWIV SEQ ID NO 844

HH845 VWRRYWWYR SEQ ID NO 845

HH846 ARKWKYWRF SEQ ID NO 846

HH847 RKRVIKRWR SEQ ID NO 847

HH848 RSFWWMWFK SEQ ID NO 848

HH849 WRINIFKRI SEQ ID NO 849

HH850 RWRVLKRRK SEQ ID NO 850

HH851 RWWVIWWWK SEQ ID NO 851

HH852 KLIRIWWWW SEQ ID NO 852

HH853 FKRKRWWGI SEQ ID NO 853 HH854 VWHWWRWRW SEQ ID NO 854

HH855 WKRWLIIGR SEQ ID NO 855

HH856 AYRWWTRFK SEQ ID NO 856

HH857 SWWWIWLKK SEQ ID NO 857

HH858 FVIWKYIRV SEQ ID NO 858

HH859 RWVRTRRRR SEQ ID NO 859

HH860 RRSWWYKRR SEQ ID NO 860

HH861 RKYVWWKSI SEQ ID NO 861

HH862 WWKRYIVKK SEQ ID NO 862

HH863 WFIRVWRYR SEQ ID NO 863

HH864 WKMWLRKHW SEQ ID NO 864

HH865 RRFFWKKGI SEQ ID NO 865

HH866 KRWTFWSRR SEQ ID NO 866

HH867 AVQRWRWW SEQ ID NO 867

HH868 IWKYGWRYK SEQ ID NO 868

HH869 IIKWWRRWR SEQ ID NO 869

HH870 AFRKVKRWG SEQ ID NO 870

HH871 MGFTRKWQF SEQ ID NO 871

HH872 WIRWRKWR SEQ ID NO 872

HH873 RIGRKLRIR SEQ ID NO 873

HH874 RWWRWRHVI SEQ ID NO 874

HH875 RLVSKRRRK SEQ ID NO 875

HH876 RRKYWKKYR SEQ ID NO 876

HH877 IILWWYRRK SEQ ID NO 877

HH878 IYFWWWRIR SEQ ID NO 878

HH879 HKRKWWRFR SEQ ID NO 879

HH880 IGRFWRRWL SEQ ID NO 880

HH881 RIRRVLVYV SEQ ID NO 881

HH882 WWLRGRRWL SEQ ID NO 882

HH883 VRIRKRRWR SEQ ID NO 883

HH884 WWRRKWWRR SEQ ID NO 884

HH885 WWWRSFRKR SEQ ID NO 885

HH886 VGQKWRKRT SEQ ID NO 886

HH887 FRRRYRVYR SEQ ID NO 887

HH888 RIRRKRKGR SEQ ID NO 888

HH889 WKWVTRMYI SEQ ID NO 889

HH890 KWRKKRLR SEQ ID NO 890

HH891 RKRRKHWRY SEQ ID NO 891

HH892 RVTRTWQRW SEQ ID NO 892

HH893 RRRITRKRI SEQ ID NO 893

HH894 RLILIKKKW SEQ ID NO 894

HH895 WKRRWSRSR SEQ ID NO 895

HH896 MWWWFLWRR SEQ ID NO 896

HH897 RWVRIWKKK SEQ ID NO 897

HH898 KRRVWRMWR SEQ ID NO 898

HH899 WHWWIRWWR SEQ ID NO 899

HH900 WWRRLRWLV SEQ ID NO 900

HH901 KWWIWKRRR SEQ ID NO 901 HH902 RYGRKWMIW SEQ ID NO 902

HH903 RVKKIKLFI SEQ ID NO 903

HH904 RIRYIQRVW SEQ ID NO 904

HH905 RLIRWWRKR SEQ ID NO 905

HH906 QRGRWLRRG SEQ ID NO 906

HH907 RRRRWIRKK SEQ ID NO 907

HH908 LGRRWRYRR SEQ ID NO 908

HH909 FKIVHVKVR SEQ ID NO 909

HH910 FRKKYRVRR SEQ ID NO 910

HH911 WKYKYRIRL SEQ ID NO 911

HH912 HVRRWWRII SEQ ID NO 912

HH913 RFKWWRRYW SEQ ID NO 913

HH914 RRRRMRKKI SEQ ID NO 914

HH915 RRIRGRVGR SEQ ID NO 915

HH916 AFWRWIRFK SEQ ID NO 916

HH917 VKKRKIVIY SEQ ID NO 917

HH918 KRVKWTWRK SEQ ID NO 918

HH919 TGVGRGYRI SEQ ID NO 919

HH920 LSWKWWRRV SEQ ID NO 920

HH921 IKTFIKRWR SEQ ID NO 921

HH922 KMRLKWKRR SEQ ID NO 922

HH923 WRWYVTRRK SEQ ID NO 923

HH924 IYRRRRKLR SEQ ID NO 924

HH925 VWWKWWRWW SEQ ID NO 925

HH926 KYKKGWRW SEQ ID NO 926

HH927 KWRRWYYWR SEQ ID NO 927

HH928 RRWVFGRRY SEQ ID NO 928

HH929 GFTWKKKRR SEQ ID NO 929

HH930 YKKIRIKRR SEQ ID NO 930

HH931 VWIRRIKRR SEQ ID NO 931

HH932 WWKWIRKIV SEQ ID NO 932

HH933 WRRKWWSRW SEQ ID NO 933

HH934 VTRRRTRIK SEQ ID NO 934

HH935 RKRWFVYIW SEQ ID NO 935

HH936 IIKWKRIMI SEQ ID NO 936

HH937 FNRWWWKKI SEQ ID NO 937

HH938 RYKSRRVRR SEQ ID NO 938

HH939 VKVIKKFVR SEQ ID NO 939

HH940 KWKWLQGRR SEQ ID NO 940

HH941 KVRWWYNIK SEQ ID NO 941

HH942 FWFRIRKLK SEQ ID NO 942

HH943 KRRKQRKYR SEQ ID NO 943

HH944 AKNSKRRLW SEQ ID NO 944

HH945 RNRRIFRYS SEQ ID NO 945

HH946 RWTKWFLVR SEQ ID NO 946

HH947 RIRRTRRTR SEQ ID NO 947

HH948 KIRWWRISI SEQ ID NO 948

HH949 YKGRWGRRW SEQ ID NO 949 HH950 MYYRIKQKW SEQ ID NO 950

HH951 WRIQRWRWQ SEQ ID NO 951

HH952 IRRWSYRRW SEQ ID NO 952

HH953 VRIWKIIWW SEQ ID NO 953

HH954 RWRWWWLWK SEQ ID NO 954

HH955 TKRRWIWIT SEQ ID NO 955

HH956 RRWHYWKGW SEQ ID NO 956

HH957 WRIRKWWMR SEQ ID NO 957

HH958 KRRTRWWVR SEQ ID NO 958

HH959 RKWRVWKRR SEQ ID NO 959

HH960 WRVWKIRVR SEQ ID NO 960

HH961 KYWGIGGWR SEQ ID NO 961

HH962 RLISRRRKK SEQ ID NO 962

HH963 VSRRIVRRM SEQ ID NO 963

HH964 I KWWRKRR SEQ ID NO 964

HH965 KWKIQLWKI SEQ ID NO 965

HH966 KKWTWWYVI SEQ ID NO 966

HH967 SWKKNRKIW SEQ ID NO 967

HH968 HKRQYRKWF SEQ ID NO 968

HH969 IFKWFYRRK SEQ ID NO 969

Bac2A RLARIWIRVAR SEQ ID NO 970

Indolicidin ILPWKWPWWPWRR SEQ ID NO 971

Scrambled VRLRIRVAVIRA SEQ ID NO 972

HH970 ILKWKWPWWKWRR SEQ ID NO 973

HH971 ILPWKWRWWKWRR SEQ ID NO 974

HH972 FLPKKFRWWKYRK SEQ ID NO 975

HH973 FIKWKFRWWKWRK SEQ ID NO 976

HH974 KWPWWPWRR SEQ ID NO 977

HH975 KWPWWPWRK SEQ ID NO 978

HH976 KFPWWPWRR SEQ ID NO 979

HH977 KKPWWPWRR SEQ ID NO 980

HH978 KWRWWPWRR SEQ ID NO 981

HH979 KWPKWPWRR SEQ ID NO 982

HH980 KWPWKPWRR SEQ ID NO 983

HH981 KWPWWKWRR SEQ ID NO 984

HH982 KWPWWPKRR SEQ ID NO 985

HH983 KWPWWPWRR SEQ ID NO 986

HH984 KFRWWPWRR SEQ ID NO 987

HH985 KFRWWKWRR SEQ ID NO 988

HH986 KWRWWKKRR SEQ ID NO 989

HH987 KKKWWKWRR SEQ ID NO 990

HH988 KFHWWIWRK SEQ ID NO 991

HH989 KFHWWKWRK SEQ ID NO 992

HH990 KFKWWKYRK SEQ ID NO 993

HH991 KFKFFKYRK SEQ ID NO 994

HH992 KFKFFKFRK SEQ ID NO 995

HH993 PWWPWRR SEQ ID NO 996

HH994 KWWPWRR SEQ ID NO 997 HH995 PWWKWRR SEQ ID NO 998

HH996 RWWPWRR SEQ ID NO 999

HH997 PKWPWRR SEQ ID NO 1000

HH998 PWKPWRR SEQ ID NO 1001

HH999 PWWKWRR SEQ ID NO 1002

HH1000 PWWPKRR SEQ ID NO 1003

HH1001 PWWPWRK SEQ ID NO 1004

HH1002 RWWKWRR SEQ ID NO 1005

HH1003 RWWKWRK SEQ ID NO 1006

HH1004 RFWKWRR SEQ ID NO 1007

HH1005 RWWIKRR SEQ ID NO 1008

HH1006 RWWIYRR SEQ ID NO 1009

HH1007 RFFKFRR SEQ ID NO 1010

HH1008 KWWKWKK SEQ ID NO 1011

HH1009 KFFKFKK SEQ ID NO 1012

HHC1 RWRWKRWWW SEQ ID NO 1013

HHC2 RWRRWKWWW SEQ ID NO 1014

HHC3 RWWRWRKWW SEQ ID NO 1015

HHC4 RWRRKWWWW SEQ ID NO 1016

HHC5 RWRWWKRWY SEQ ID NO 1017

HHC6 RRKRWWWWW SEQ ID NO 1018

HHC7 RWRIKRWWW SEQ ID NO 1019

HHC8 KIWWWWRKR SEQ ID NO 1020

HHC9 RWRRWKWWL SEQ ID NO 1021

HHC10 KRWWKWIRW SEQ ID NO 1022

HHC11 KRWWWWWKR SEQ ID NO 1023

HHC12 IRWWKRWWR SEQ ID NO 1024

HHC13 IKRWWRWWR SEQ ID NO 1025

HHC14 RRKWWWRWW SEQ ID NO 1026

HHC15 RKWWRWWRW SEQ ID NO 1027

HHC16 KRWWWWRFR SEQ ID NO 1028

HHC17 IKRWWWRRW SEQ ID NO 1029

HHC18 KRWWWVWKR SEQ ID NO 1030

HHC19 KWRRWKRWW SEQ ID NO 1031

HHC20 WRWWKIWKR SEQ ID NO 1032

HHC21 WRWRWWKRW SEQ ID NO 1033

HHC22 WKRWKWWKR SEQ ID NO 1034

HHC23 RIKRWWWWR SEQ ID NO 1035

HHC24 IWKRWWRRW SEQ ID NO 1036

HHC25 KWWKIWWKR SEQ ID NO 1037

HHC26 RKRWLWRWW SEQ ID NO 1038

HHC27 KRWRWWRWW SEQ ID NO 1039

HHC28 KKRWLWWWR SEQ ID NO 1040

HHC29 RWWRKWWIR SEQ ID NO 1041

HHC30 KWWRWWRKW SEQ ID NO 1042

HHC31 KRWWIRWWR SEQ ID NO 1043

HHC32 KIWWWWRRR SEQ ID NO 1044

HHC33 RRRKWWIWW SEQ ID NO 1045 HHC34 RRRWWWWWW SEQ ID NO 1046

HHC35 RWWIRKWWR SEQ ID NO 1047

HHC36 KRWWKWWRR SEQ ID NO 1048

HHC37 KRWWRKWWR SEQ ID NO 1049

HHC38 RRIWRWWWW SEQ ID NO 1050

HHC39 IRRRKWWWW SEQ ID NO 1051

HHC40 KRKIWWWIR SEQ ID NO 1052

HHC41 RKIWWWRIR SEQ ID NO 1053

HHC42 KRWWIWRIR SEQ ID NO 1054

HHC43 RWFRWWKRW SEQ ID NO 1055

HHC44 WRWWWKKWR SEQ ID NO 1056

HHC45 WKRWWKKWR SEQ ID NO 1057

HHC46 WKRWRWIRW SEQ ID NO 1058

HHC47 WRWWKWWRR SEQ ID NO 1059

HHC48 WKKWWKRRW SEQ ID NO 1060

HHC49 WRWYWWKKR SEQ ID NO 1061

HHC50 WRRWWKWWR SEQ ID NO 1062

HHC51 IRMWVKRWR SEQ ID NO 1063

HHC52 RIWYWYKRW SEQ ID NO 1064

HHC53 FRRWWKWFK SEQ ID NO 1065

HHC54 RVRWWKKRW SEQ ID NO 1066

HHC55 RLKKVRWWW SEQ ID NO 1067

HHC56 RWWLKIRKW SEQ ID NO 1068

HHC57 LRWWWIKRI SEQ ID NO 1069

HHC58 TRKVWWWRW SEQ ID NO 1070

HHC59 KRFWIWFWR SEQ ID NO 1071

HHC60 KKRWVWVIR SEQ ID NO 1072

HHC61 KRWVWYRYW SEQ ID NO 1073

HHC62 IRKWRRWWK SEQ ID NO 1074

HHC63 RHWKTWWKR SEQ ID NO 1075

HHC64 RRFKKWYWY SEQ ID NO 1076

HHC65 RIKVIWWWR SEQ ID NO 1077

HHC66 RKRLKWWIY SEQ ID NO 1078

HHC67 LVFRKYWKR SEQ ID NO 1079

HHC68 RRRWWWIIV SEQ ID NO 1080

HHC69 KKRWVWIRY SEQ ID NO 1081

HHC70 RWRIKFKRW SEQ ID NO 1082

HHC71 KWKIFRRWW SEQ ID NO 1083

HHC72 IWKRWRKRL SEQ ID NO 1084

HHC73 RRRKWWIWG SEQ ID NO 1085

HHC74 RWLVLRKRW SEQ ID NO 1086

HHC75 RKWIWRWFL SEQ ID NO 1087

HHC76 KRRRIWWWK SEQ ID NO 1088

HHC77 IWWKWRRWV SEQ ID NO 1089

HHC78 LRWRWWKIK SEQ ID NO 1090

HHC79 RWKMWWRWV SEQ ID NO 1091

HHC80 VKRYYWRWR SEQ ID NO 1092

HHC81 RWYRKRWSW SEQ ID NO 1093 HHC82 KRKLIRWWW SEQ ID NO 1094

HHC83 RWRWWIKII SEQ ID NO 1095

HHC84 KFRKRVWWW SEQ ID NO 1096

HHC85 IWIWRKLRW SEQ ID NO 1097

HHC86 LRFILWWKR SEQ ID NO 1098

HHC87 RVWFKRRWW SEQ ID NO 1099

HHC88 RRWFVKWWY SEQ ID NO 1100

HHC89 KWWLVWKRK SEQ ID NO 1101

HHC90 RWILWWWRI SEQ ID NO 1102

HHC91 KRWLTWRFR SEQ ID NO 1103

HHC92 RKWRWRWLK SEQ ID NO 1104

HHC93 IRRRWWWIV SEQ ID NO 1105

HHC94 IKWWWRMRI SEQ ID NO 1106

HHC95 RWKIFIRWW SEQ ID NO 1107

HHC96 IRQWWRRWW SEQ ID NO 1108

HHC97 RRRKTWYWW SEQ ID NO 1109

HHC98 RRWWHLWRK SEQ ID NO 1110

HHC99 RRWWMRWWV SEQ ID NO 1111

HHCIOO RRFKFIRWW SEQ ID NO 1112

HHC101 INRKRRLRW SEQ ID NO 1113

HHC102 RRMKKLRRK SEQ ID NO 1114

HHC103 RKVRWKIRV SEQ ID NO 1115

HHC104 VRIVRVRIR SEQ ID NO 1116

HHC105 IKRVKRRKR SEQ ID NO 1117

HHC106 RVKTWRVRT SEQ ID NO 1118

HHC107 RVFVKIRMK SEQ ID NO 1119

HHC108 IRGRIIFWV SEQ ID NO 1120

HHC109 ATWIWVFRR SEQ ID NO 1121

HHC110 KKSKQLWKR SEQ ID NO 1122

HHC111 MINRVRLRW SEQ ID NO 1123

HHC112 GGIRRLRWY SEQ ID NO 1124

HHC113 RLVHWIRRV SEQ ID NO 1125

HHC114 AWKIKKGRI SEQ ID NO 1126

HHC115 FW KRIVW SEQ ID NO 1127

HHC116 GIKWRSRRW SEQ ID NO 1128

HHC117 RWMVSKIWY SEQ ID NO 1129

HHC118 IWRVWWR SEQ ID NO 1130

HHC119 RWIGVIIKY SEQ ID NO 1131

HHC120 WIRKRSRIF SEQ ID NO 1132

HHC121 GWKILRKRK SEQ ID NO 1133

HHC122 YQRLFVRIR SEQ ID NO 1134

HHC123 AVWKFVKRV SEQ ID NO 1135

HHC124 IRKKRRRWT SEQ ID NO 1136

HHC125 ILRVISKRR SEQ ID NO 1137

HHC126 AWRFKNIRK SEQ ID NO 1138

HHC127 HYKFQRWIK SEQ ID NO 1139

HHC128 RRIRRVRWG SEQ ID NO 1140

HHC129 VLVKKRRRR SEQ ID NO 1141 HHC130 RWRGIVHIR SEQ ID NO 1142

HHC131 WRNRKWWR SEQ ID NO 1143

HHC132 KFWWW YLK SEQ ID NO 1144

HHC133 KRIMKLKMR SEQ ID NO 1145

HHC134 IRRRKKRIK SEQ ID NO 1146

HHC135 RKWMGRFLM SEQ ID NO 1147

HHC136 RRVQRGKWW SEQ ID NO 1148

HHC137 WHGVRWWKW SEQ ID NO 1149

HHC138 WVRFVYRYW SEQ ID NO 1150

HHC139 RKRTKVTWI SEQ ID NO 1151

HHC140 IRRIVRRKI SEQ ID NO 1152

HHC141 KIRRKVRWG SEQ ID NO 1153

HHC142 AIRRWRIRK SEQ ID NO 1154

HHC143 WRFKVLRQR SEQ ID NO 1155

HHC144 RSGKKRWRR SEQ ID NO 1156

HHC145 FMWVYRYKK SEQ ID NO 1157

HHC146 RGKYIRWRK SEQ ID NO 1158

HHC147 WVKVWKYTW SEQ ID NO 1159

HHC148 WLKIVRRF SEQ ID NO 1160

HHC149 GKFYKVWVR SEQ ID NO 1161

HHC150 SWYRTRKRV SEQ ID NO 1162

HHC151 KNRGRWFSH SEQ ID NO 1163

HHC152 AFRGSRHRM SEQ ID NO 1164

HHC153 GRNGWYRIN SEQ ID NO 1165

HHC154 AGGMRKRTR SEQ ID NO 1166

HHC155 ATRKGYSKF SEQ ID NO 1167

HHC156 SSGVRWSWR SEQ ID NO 1168

HHC157 RVWRNGYSR SEQ ID NO 1169

HHC158 WGRTRWSSR SEQ ID NO 1170

HHC159 GKRVWGRGR SEQ ID NO 1171

HHC160 SF WKRSGK SEQ ID NO 1172

HHC161 WGRGGWTNR SEQ ID NO 1173

HHC162 ANRWGRGIR SEQ ID NO 1174

HHC163 WGGHKRRGW SEQ ID NO 1175

HHC164 WHGGQKWRK SEQ ID NO 1176

HHC165 FVWQKGTNR SEQ ID NO 1177

HHC166 HGVWGNRKR SEQ ID NO 1178

HHC167 TRGWSLGTR SEQ ID NO 1179

HHC168 GRRVM QKR SEQ ID NO 1180

HHC169 RNKFGG WR SEQ ID NO 1181

HHC170 GVRVQRNSK SEQ ID NO 1182

HHC171 NQKWSGRRR SEQ ID NO 1183

HHC172 RQNGVWRVF SEQ ID NO 1184

HHC173 GRMRLW GR SEQ ID NO 1185

HHC174 WHYRSQVGR SEQ ID NO 1186

HHC175 GW TMGRRW SEQ ID NO 1187

HHC176 RRMGNGGFR SEQ ID NO 1188

HHC177 SK VRTWRQ SEQ ID NO 1189 HHC178 ARGRWINGR SEQ ID NO 1190

HHC179 GSRRSVWVF SEQ ID NO 1191

HHC180 WSQ VRTRI SEQ ID NO 1192

HHC181 GMRRWRGKN SEQ ID NO 1193

HHC182 RGRTS WKM SEQ ID NO 1194

HHC183 GRRWGMGVR SEQ ID NO 1195

HHC184 WGKRRGW T SEQ ID NO 1196

HHC185 AMLGGRQWR SEQ ID NO 1197

HHC186 QRNKGLRHH SEQ ID NO 1198

HHC187 ARGKSIKNR SEQ ID NO 1199

HHC188 NRRNGQMRR SEQ ID NO 1200

HHC189 RGRRQIGKF SEQ ID NO 1201

HHC190 ASKRVGVRN SEQ ID NO 1202

HHC191 GRIGGK VR SEQ ID NO 1203

HHC192 NKTGYRWRN SEQ ID NO 1204

HHC193 VSG WRGSR SEQ ID NO 1205

HHC194 GWGGKRRNF SEQ ID NO 1206

HHC195 K RRWQGR SEQ ID NO 1207

HHC196 GRTMGNGRW SEQ ID NO 1208

HHC197 GRQISWGRT SEQ ID NO 1209

HHC198 GGRGTRWHG SEQ ID NO 1210

HHC199 GVRSWSQRT SEQ ID NO 1211

HHC200 GSRRFGW R SEQ ID NO 1212

1001 LVRAIQVRAVIR SEQ ID NO 1213

1002 VQRWLIVWRIRK SEQ ID NO 1214

1003 IVWKIKRWWVGR SEQ ID NO 1215

1004 RFWKVRVKYIRF SEQ ID NO 1216

1005 VQLRIRVAV SEQ ID NO 1217

1006 VQLRIWVRR SEQ ID NO 1218

1007 WNRVKWIRR SEQ ID NO 1219

1008 RIKWIVRFR SEQ ID NO 1220

1009 AIRWRARLVRR SEQ ID NO 1221

1010 IRWRIRVWVRRI SEQ ID NO 1222

1011 RRWWWRIVQRR SEQ ID NO 1223

1012 IFWRRIVIVKKF SEQ ID NO 1224

1013 VRLRIRVAV SEQ ID NO 1225

1014 RQVIVRRW SEQ ID NO 1226

1015 VLIRWNGKK SEQ ID NO 1227

1016 LRIRWIFKR SEQ ID NO 1228

1017 KRIVRRLVARIV SEQ ID NO 1229

1018 VRLIVAVRIWRR SEQ ID NO 1230

1019 IWWRRQLVKNK SEQ ID NO 1231

1020 VRLRIRWWVLRK SEQ ID NO 1232

1021 VRLRIRVAV SEQ ID NO 1233

1022 LRIRVIVWR SEQ ID NO 1234

1023 IRVWVLRQR SEQ ID NO 1235

1024 RIRVIVLKK SEQ ID NO 1236

1025 RRIVKKFQIVRR SEQ ID NO 1237 1026 VQWRIRVRVIKK SEQ ID NO 1238

1027 KKQVSRVKVWRK SEQ ID NO 1239

1028 LIQRIRVRNIVK SEQ ID NO 1240

1029 KQFRIRVRV SEQ ID NO 1241

1030 FRIRVRVIR SEQ ID NO 1242

1031 WRWRVRVWR SEQ ID NO 1243

1032 IRVRVIWRK SEQ ID NO 1244

1033 RRVIVKKFRIRR SEQ ID NO 1245

1034 KQFRNRLRIVKK SEQ ID NO 1246

1035 KRWRWIVRNIRR SEQ ID NO 1247

1036 VQFRIRVIVIRK SEQ ID NO 1248

1037 KRFRIRVRV SEQ ID NO 1249

1038 IWRRVIRK SEQ ID NO 1250

1039 IWVIRRVWR SEQ ID NO 1251

1040 FQWKIKVR SEQ ID NO 1252

1041 VIWIRWR SEQ ID NO 1253

1042 IVWIWRR SEQ ID NO 1254

1043 WIVIWRR SEQ ID NO 1255

1044 RRWIVWI SEQ ID NO 1256

1045 RWWRIVI SEQ ID NO 1257

1046 WIRVIRW SEQ ID NO 1258

1047 IIRRWWV SEQ ID NO 1259

1048 IRWVIRW SEQ ID NO 1260

HH1010 ILRWKWRWWRWRR SEQ ID NO 1261

HH1011 RWRWWRWRR SEQ ID NO 1262

HH1012 KWKWWKWKK SEQ ID NO 1263

HH1013 RWWRWRR SEQ ID NO 1264

Example 2: Effect of IDR peptide administration on behavioral deterioration in a mouse model of ALS using a rotarod test

[00168] We performed an experiment in G93A mice, an aggressive model of ALS due to transgenic overexpression of G93A human mutant SOD1, resulting in progressive paralysis and death by about 5 months of age. We treated G93A mice with 200 μg/mouse dose of IDR peptide (IDR-1018; "TXl") delivered IV 3 times per week. To model human ALS as closely as possible, in which treatment can only be initiated upon disease onset, we treated mouse cohorts of ~10 animals that had begun to display motor neuron function symptoms, as indicated by evidence of hind limb reflex abnormalities (i.e., transition for normal HLR4 to HLR3). Statistically significant slowing of behavioral deterioration was noted in IDR peptide- administered animals compared to saline injected controls in average rotarod function (Figure 1). No significant effects were observed on survival by Kaplan- Meier analysis, possibly due to the small sample, and the aggressive nature of the G93A disease (e.g., treatment effects may be observed in more indolent models such as G37R). Example 3: Effect of IDR peptide administration on behavioral deterioration in a mouse model of ALS using a test of hindlimb reflex deterioration

[00169] We performed an experiment in G93A mice, an aggressive model of ALS due to transgenic overexpression of G93A human mutant SOD1, resulting in progressive paralysis and death by about 5 months of age. We treated G93A mice with 200 μg/mouse dose of IDR peptide (IDR-1018; "TXl") delivered IV 3 times per week. To model human ALS as closely as possible, in which treatment can only be initiated upon disease onset, we treated mouse cohorts of ~10 animals that had begun to display motor neuron function symptoms, as indicated by evidence of hind limb reflex abnormalities (i.e., transition for normal HLR4 to HLR3). Statistically significant slowing of behavioral deterioration was noted in IDR peptide- administered animals compared to saline injected controls in HLR score (Figure 2). No significant effects were observed on survival by Kaplan- Meier analysis, possibly due to the small sample, and the aggressive nature of the G93A disease (e.g., treatment effects might be observed in more indolent models such as G37R).

Example 4: Use of IDR-1018 in a mouse model of amyotrophic lateral sclerosis and determination of its effects on motor function

[00170] Another study was performed to test IDR- 1018 in the transgenic hSOD 1 G93 A model of amyotrophic lateral sclerosis (ALS). The study showed improved motor abilities in the IDR-1018 group as measured by hind limb reflex and rotarod (Figures 1 and 2). Brains and spinal cords were used for qRT-PCR to examine chemokines and several genes often found dysregulated in ALS (Figure 3). While the limited number of mice prevented the achievement of significance, notable trends in the IDR-1018 group include increased TGF-βΙ (p = 0.058) in the forebrain and the greatly increased expression of several genes in the olfactory bulb except for TGF-βΙ .

Example 5: Reduction of LPS-induced cytokine release from microglial cells with IDR peptides

[00171] In this example, we examined how IDR peptides influenced the inflammatory response in LPS-stimulated microglial cells in vitro.

[00172] The murine microglial cell line BV-2 was cultured in vitro. Isolated microglial cells (100,000 cells/well) were exposed to LPS (100 ng/well) with or without IDR peptides. The peptides IDR-1018 and IDR- 1002 were tested. The cell supematants were collected after 4 and 24 hours of stimulation with or without LPS ± IDR peptide treatment (n=8) and quantification of inflammatory mediators was carried out using multiplex ELISA analyses (Cytokine Mouse 20-Plex Panel, Invitrogen).

[00173] As shown in Figure 4, IDR peptides by themselves in vehicle treated murine microglial cells had no effect on induction of the pro-inflammatory cytokine TNFa. In contrast LPS was able to significantly induce TNFa production. In contrast at both 4 and 24 hours IDR- 1002 and IDR-1018 significantly, and in certain situations totally, decreased the expression of proinflammatory cytokine TNFa.

[00174] We have also examined the ability of IDR peptides to suppress LPS-induced proinflammatory cytokine expression in human peripheral blood monuclear cells (PBMC). The results of this study are shown in Table 2.

Table 2: Percent suppression of TNF-a production in human PBMC treated with 10 ng/ml of LPS without (control) or with 20 μg/ml peptide. Data are expressed relative to the positive control (LPS treated but no peptide = 100%) and the negative control (no LPS or peptide (0%).

1024 RIRVIVLK 86

1025 RRIVKKFQIVRR 31

1026 VQWRIRVRVIK 46

1027 K QVSRVKVWRK 5

1028 LIQRIRVRNIVK 0

1029 KQFRIRVRV 0

1030 FRIRVRVIR 0

1031 WRWRVRVWR 4

1032 IRVRVIWRK 0

1033 RRVIVKKFRIRR 0

1034 KQFRNRLRIVK 0

1035 KRWRWIVRNIRR 94

1036 VQFRIRVIVIRK 60

1037 KRFRIRVRV 91

1038 IVVRRVIRK 2

1039 IWVIRRVWR 0

1040 FQVVKIKVR 5

[00175] Among these immunomodulatory peptides, a clear pattern of related peptides were found that obviously represented minor substitutions, deletions or additions to a base sequence represented by SEQ ID NO: 2. Thus, these peptides have clear unitary relationship.

[00176] Among twelve amino acid immunomodulatory peptides, a clear pattern of related peptides were found that obviously represented minor substitutions, deletions or additions to a base sequence represented by SEQ ID NO: 1230, as shown by the alignments of subsets of peptides below.

[00177] Alignment of nine sequences

HH2 VQLR-IRV-AVIRA SEQ ID NO 2

1001 LV--RAIQVRAVIR SEQ ID NO 1213

1002 VQRWLI-VWR-IRK SEQ ID NO 1214

1010 I-RWRIRVW-VRRI SEQ ID NO 1222

1012 IFWRRI-VI-VKKF SEQ ID NO 1224

1018 V--RLIVAVRIWRR SEQ ID NO 1230

1020 VRLR-IRWW-VLRK SEQ ID NO 1232

HH12 KQFR-IRV-RVIRK SEQ ID NO: 12

1026 VQWR-IRV-RVIKK SEQ ID NO 1238

[00178] Alignment of five sequences

1020 V-RLRIRWW-VLRK SEQ ID NO: 1232

1018 V-RLIVAV-RIWRR SEQ ID NO: 1230

HH2 VQ-LRIRV-AVIRA SEQ ID NO: 2

1002 VQRWLI-V RI-RK SEQ ID NO: 1214

1010 I-RWRIRVW-VRRI SEQ ID NO: 122 [00179] Alignment of three sequences

1018 VRLIVAVRIWRR SEQ ID NO: 1230

HH2 VQLRIRVAVIRA SEQ ID NO: 2

1002 VQRWLIVWRIRK SEQ ID NO: 1214

[00180] Thus, these peptides have a clear unitary relationship.

Example 6: Ability of IDR-1018 and mCRAMP to reduce endotoxin-induced

inflammation in cultured microglial cells

[00181] Neurodegenerative diseases affect tens of millions worldwide, with the numbers expected to increase to 100 million over the next thirty years (Farooqui, A. A. 2010.

Neurochemical Aspects ofNeurotraumatic and Neurodegenerative Diseases. Springer). Three of the most common diseases, Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) have no cures and limited treatment options. Generating new therapies is critical, but is hampered by complex or unidentified disease etiologies, the lack of known therapeutic targets, and the difficulty of transporting drugs across the blood- brain barrier (BBB).

[00182] Two common features in neurodegenerative diseases are neuronal loss and neuroinflammation, which both depend on the interactions of the two cell populations in the brain, neurons and neuroglia Perry VH, et al. 2010. Nat. Rev. Neurol. 6: 193-201.; Heneka, MT et al. 2010. Brain Res. Rev. 63: 189-211). The release of proinflammatory cytokines and reactive nitrogen or oxygen species by neuroglia, particularly activated microglia, leads to neuroinflammation, which in turn can cause the death of neurons through activation of necrotic, apoptotic, or autophagic pathways (Czirr and Wyss-Coray. 2012. J. Clin. Invest. 122: 1156-1163). However, neuroinflammation also seems to have a protective role in some conditions (Wee Yong, V. 2010. Neuroscientist 16:408-420). Therefore, treating

neurodegeneration by targeting inflammation requires modulating the immune response to prevent harmful inflammation while still allowing a restricted inflammatory response.

Host defence peptides (HDPs) are one possible therapy for neurodegeneration. HDPs are small peptides 10-50 amino acids in length that have been shown to selectively activate the immune response and have a key role in host immunity (Hancock, REW et al. 2012. Nature Rev. Microbiol. 10:243-254; Mookherjee, N., and R. E. W. Hancock. 2007. Cell. Molec. Life Sci. 64:922-933). In the central nervous system (CNS), naturally occurring HDPs, including LL-37 and its murine homolog mCRAMP, have not been studied extensively, but the HDP β- defensin-2 is expressed in cultured primary astrocytes and rat CRAMP (rCRAMP) activates primary neuroglia in vitro and is upregulated during meningitis in a rat model, but these peptides are not feasible as therapies due to cytotoxicity (Brandenburg, LO et al. 2010.

Molec. Immunol. 47: 1905-1913; Su, Y., et al. 2010. Arch. Immunol. Ther. Exp. 58:365-377; Brandenburg, LO, et al. 2008. J. Neuropathol. Exp. Neurol. 67: 1041-1054. Hao, HN, et al. 2001. J. Neurochem. 77: 1027-1035). None of these papers however revealed that these peptides might be useful in therapy for treating neural inflammation. Innate defence regulators (IDRs) are synthetic versions of HDPs that have been modified to exhibit stronger immunomodulatory effects but with reduced cytotoxicity. IDR-1018 has demonstrated the ability to manipulate inflammation in various leukocytes and epithelial cells as well as in vivo models by affecting the production of cytokines and chemokines, and preliminary data also indicate that IDR-1018 affects autophagy (Hancock, REW et al. 2012. Nature Rev. Microbiol. 10:243-254). Critically, in additon to the effects on ALS described above, IDR-1018 was demonstrated, after the initial submission of this patent application, to be effective in a mouse model cerebral malaria (Achtman AH, et al. 2012. SciTrans Med 4: 135ral64.), indicating it is capable of exerting its effects in the CNS ( Achtman, A. H., et al., . 2012. Effective adjunctive therapy by an innate defense regulatory peptide in a preclinical model of severe malaria. Sci. Transl. Med. 4: 135ral64.). These studies and others described here provide evidence that IDR-1018 may be effective in treating neurodegenerative and neural injury diseases. The BV-2 mouse microglial cell line was used as a model to determine the effects of IDR-1018 and mCRAMP under inflammatory conditions in vitro.

[00183] Microglia are the resident innate immune cells of the central nervous system (CNS). Microglia become activated in response to a pathological stimulus and exert either a toxic or protective effect. In a neuroprotective role, activated microglial clear toxic material (protein aggregates such as Αβ, neuronal debris), secrete neurotrophic factors (e.g. brain derived neurotrophic factor and other protective factors (e.g. glutathione) and increase clearance of excitotoxic glutamate by astrocytes. However, activated microglia can also secrete neurotoxic molecules such as pro-inflammatory cytokines (TNF-a, IL-Ιβ), glutamate, free radicals and nitric oxide (Rogers J, et al. 2002. Glia 40, 260; Rogers J. and LF Lue. 2001. Neurochem Int 39, 333). Although not typically considered to be part of the innate immunity network, astrocytes also have the potential to mount an inflammasome reaction upon recognition of danger signals such as Αβ (mimimcked here with LPS) (Yazdi AS, et al. 2010. J Clin Immunol 30, 623). We hypothesized that IDR-1018 will attenuate LPS-mediated inflammation in glial cultures. As shown in Figure 4, IDR-1018 significantly reduces TNFa levels after 2 ng/ml LPS stimulation in BV2 microglia, supporting our hypothesis that IDR- 1018 attenuates inflammation in relevant cells.

[00184] Microglia are the resident macrophages of the brain (and spinal cord), and thus act as the first and main form of active immune defenses but also contribute markedly to the production of brain inflammatory responses. BV-2 cells were treated with the inflammatory stimulus lipopolysaccharide (LPS at a higher dose of 10 ng/ml) and either saline or IDR- 1018 or mCRAMP, and then cell lysates and supematants were collected at 4 and 24 h. RNA was isolated from the 4 h lysates and used for qRT-PCR. Supematants from both timepoints were used in ELI S As.

[00185] Both IDR- 1018 and mCRAMP produce significant reductions in the LPS-induced release of the pro-inflammatory cytokines TNF and IL-6 and the chemokine MCP-1 (Figure 4, 5). Changes were seen at both the protein and mRNA levels. The results show that both peptides are effective in reducing inflammation in microglia in vitro.

Example 7: Use of IDR-1018 in a mouse model of Alzheimer's disease and

determination of its effects on cognitive function

[00186] APP/PSl mice have mutations in both amyloid precursor protein (APP) and presenilin-1 (PS1), two known genetic risk factors for Alzheimer's disease (AD), and develop Alzheimer's disease at approximately ten months. APP/PSl mice were injected intravenously three times a week for two weeks with 4 mg/kg of IDR-1018 diluted in 0.9% saline and 0.01% Tween 80. After the injection period ended, mice were tested for memory loss in an open-field novel object recognition trial. The IDR-1018-treated mice demonstrated a very strong trend towards improved cognitive function (Figure 6; p = 0.054). In the brains, the IDR-1018 group also showed significant reduction in APP-C-terminal fragment (APP-CTF) in both male and female mice, but no significant changes in APP or β-amyloid were observed. The transcriptional expression of several chemokines and cytokines were examined in qRT- PCR, and the chemokines MCP-1 and MCP-3 showed very significant increases in the IDR- 1018-treated mice (Figure 7). Additionally, BV-2 cells were treated with combinations of Αβ 42 , LPS, and IDR-1018, but no significant changes in TNF or MCP-1 production were seen. Overall, the results indicated that mice treated with IDR-1018 strongly trended towards cognitive improvement. Example 8: Ability of IDR-1018 to stimulate Αβ clearance and inhibit A3 production

[00187] As professional phagocytes, microglia engulf and degrade Αβ peptides. This process is facilitated by Liver-X-Receptor (LXR) agonists, which stimulate genes that suppress inflammation and maintain lipid homeostasis (Joseph SB, et al. 2003. Nat Med 9, 213). Key LXR targets for microglial Αβ clearance are ABCA1 and apoE (Donkin JJ, et al.2010. J Biol Chem 285:34144). ABCA1 effluxes cellular cholesterol and phospholipids onto apoE. ABCA1 -deficient microglia are unable to degrade exogenous Αβ, and this defect is rescued by the addition of exogenous lipidated apoE (24). We hypothesized that IDR-1018 will promote Αβ degradation by microglia via upregulation of ABCA1 and/or apoE expression. Beta-site APP-cleaving enzyme 1 (BACE1), which generates the N-terminus of Αβ, is regulated in part through NF-κΒ signaling (Jiang Q. et al. 2008 Neuron 58:681) and may exhibit distinct responses to Αβ- or TNFa-induced NF-κΒ activation in neurons and glia (Chen CH, et al.2011. Int J Neuropsycopharmacol 8: 1). Because IDR-1018 suppresses TNFa production from BV2 microglia (Figures 4,5), we propose that IDR-1018 may inhibit NF-KB- mediated BACE1 expression and reduce Αβ production.

[00188] As shown in Figure 8, IDR-1018 but not LPS potently elevates ABCA1 and apoE expression in human monocyte-derived macrophages, supporting our hypothesis that IDR- 1018 will promote Αβ clearance through the ABCAl-apoE pathway.

Example 9: The ability of IDR-1018 to preserve cognitive function, reduce

neuropathology and attenuate inflammation in APP/PS1 mice

[00189] As described above, IDR peptides suppress harmful inflammation and maintain protective mechanisms without leading to negative immune pathologies in infection, cerebral malaria and ALS models. Given the major role for inflammation in AD (Bales KR, et al. 2000. Neurobiol Aging 21 :427) and the ability of IDR-1018 to attenuate TNFa production and elevate the ABCAl-apoE pathway in BV2 cells (Figures 4,8), we hypothesize that IDR- 1018 in preserving cognitive function (Figure 6), will reduce neuropathology and attenuate inflammation in APP/PS1 mice.

[00190] Female APP/PS1 mice aged 32 weeks received 50 μg IDR-1018 (N=4) or vehicle control (N=4) intravenously 3x/week for 2 weeks. APP and CTFs levels were determined by immunoblot, soluble and insoluble Αβ levels were determined by ELISA, and Αβ

conformational status was determined by immunoblot. IDR-1018 did not alter APP levels but significantly reduced CTFa/β levels (Figure 9 A, B). Insoluble Αβ40 and Αβ42 levels showed a clear trend toward reduction, whereas carbonate-soluble Αβ40 and Αβ42 levels remained steady state (Figure 9 C,D). Oligomeric Αβ, immunodetected with 5E3, also showed a clear trend toward reduced levels in treated mice when expressed as total oligomeric Αβ or a function of total APP (Figure 9, E). Forebrain levels of MCP-1 and MCP-3 were significantly elevated in treated mice (Figure 7, 9, F,). Together, these results support our hypothesis that IDR-1018 will protect from AD phenotypes in APP/PS 1 mice by promoting beneficial microglial recruitment and clearance of Αβ oligomers and also by reducing Αβ production.

Example 10: Use of IDR peptides in neonatal brain injury

[00191] Perinatal brain injury is a major clinical problem associated with high neonatal mortality and morbidity and an increased risk of life-long chronic disabilities. Despite improved survival rates, the absolute numbers of neurological handicaps of perinatal origin have not decreased (Kurinczuk JJ, et al. 2010. Early Hum Dev 86:329-338; Marlow N, et al. 2005. N Engl J Med 352:9-19). There is currently no pharmacological treatment that provides neuroprotection in neonates. Activation of an inflammatory response is a key contributing factor to brain injury in both term and preterm infants (Dammann O & Leviton A. 1997. Pediatr Res 42: 1-8; Dean, J.M., 2011. Annals Neurology 70:846-856;). Therefore, the therapeutic potential of novel immunomodulatory innate defence regulator peptides (IDRs) in perinatal brain injury was investigated here since IDRs have been shown to suppress proinflammatory cytokines while maintaining protective responses. IDR-1018 significantly reduced the production of inflammatory mediators by LPS -stimulated microglia cells in vitro. Consistent with this, IDR-1018 modulated neuroinflammation and was neuroprotective in an animal model of neonatal brain injury, exerting effects on regulatory molecules of TLR-, Ca 2+ - and p53-signaling. Of utmost importance, IDR-1018 markedly protected both white and grey brain matter when administered after the injury, thus demonstrating tremendous applicability to the clinical setting. Thus we have shown for the first time that peripheral administration of a selectively immunomodulatory peptide is neuroprotective in vivo in a clinically relevant model of perinatal brain damage.

[00192] Susceptibility and progression of CNS injury in the newborn is closely associated with an exacerbated innate immune response (Dammann O & Leviton A. 1997. Pediatr Res 42: 1-8; Dean, J.M., 2011. Annals Neurology 70:846-856; Favrais, G., et al. 2011. Annals Neurology 70:550-65). Neuroinflammation can be elicited by infectious challenge as well as tissue damage, and perinatal brain injury is commonly associated with a combination of both sterile and infectious inflammation (Volpe, J.J. 2011. Pediatr Res 50, 553-562). Experimental studies that use animal models of neonatal brain injury suggest that the inhibition of microglia-associated pro-inflammatory processes is neuroprotective (Hedtjarn, M., et al. 2002. J Neurosci 22:5910-9; Wang, X, et al. 2009. J Immunol 183:7471-7; Doverhag C, et al. 2010. Neurobiol Dis 38:36-46). Results are, however, contradictory(Fox C, et al. 2005. J Cerebral Blood Flow Metab 25: 1138-1149) and recent evidence from adult models suggests that activated microglial cells may also have protective properties (Lalancette-Hebert M, et al. 2007. J Neurosci 27:2596-2605). Thus, neuroprotective treatments that aim to modulate, rather than eliminate, particular immune cells and processes are particularly attractive for the treatment of perinatal brain injury. Novel immunomodulatory innate defence regulator peptides (IDRs) are synthetic derivatives of endogenous cationic host defence peptides (HDP), which have enhanced effects in suppressing inflammation that is induced in vitro and in vivo by a wide range of infectious agents, while augmenting, rather than compromising, protective immunity to the pathogen. Thus both natural HDP and enhanced synthetic IDR peptides modulate inflammatory responses to augment the recruitment of immune cells to sites of infection and inflammation, and increasing their anti-infective and wound healing properties while suppressing cellular hyperactivation and production of proinflammatory cytokines. Intriguingly such peptides also promote the transition to more effective adaptive responses (Kindrachuk J, et al. 2009. Vaccine 27:4662-4671).

[00193] Three biologically-active IDRs (IDR-HH2, IDR-1002 and IDR-1018) and one inactive, control peptide (P-1006) were selected for this study. Active IDRs possessed anti- endotoxin effects on human PBMC in the absence of direct antimicrobial activity or immune cell activation, but their effects on microglia, the resident immune cell in the brain, were not known prior to the work reported here. To determine if IDRs could modulate innate immune cells of the brain, thus showing neuroprotective potential, the effect of IDRs on inflammatory mediators produced by LPS-activated murine microglia were measured. Neither LPS nor the IDRs had any effect on microglia viability (data not shown). In addition, none of the IDRs stimulated microglia, with the exception of IDR-1002, which induced a modest IP- 10 release (1-way ANOVA, p<0.05). As expected, the control peptide P-1006 did not affect LPS- induced inflammation (Figure 10). IDR-HH2 reduced LPS-induced TNF-a production but not the production of other inflammatory mediators. In contrast, IDR-1002 and IDR-1018 substantially reduced the release of GM-CSF, IL-4, IL-10, TNF-a, IL-17 and KC (1-way ANOVA, Figure 10). In addition, IDR-1018 reduced IFN-γ, IL-Ιβ and IL-6 (1-way ANOVA, Figure 10). Thus, IDR-1018, and to a somewhat lesser extent IDR-1002, exerted broad antiinflammatory effects in suppressing LPS-induced inflammatory mediators produced in vitro by primary murine microglia.

[00194] Next we assessed the ability of IDR- 1002 and IDR- 1018 to confer neuroprotection in vivo in a clinically relevant model of neonatal brain injury that combines infectious challenge (LPS) with hypoxia-ischemia (HI) (Wang, X, et al. 2009. J Immunol 183:7471-7;

Eklind S, et al. 2001. Europ J Neurosci 13: 1101-1106). Injury was evaluated blinded by regional as well as overall neuropathological scoring by two independent researchers. IDR-

1018 and IDR-1002 were given systemically 4 h before LPS/HI (Figure 11A). A single prophylactic dose of IDR-1018, but not IDR-1002 (data not shown), caused a significant reduction in the number of animals that suffered severe insult to the hippocampus (Chi- square, p=0.0271, Figure 1 IB). To determine if this protection was associated with the presence of IDR-1018 in the brain, IDR-1018 was isotope-labelled and its distribution tracked in the serum, liver, spleen and brain. IDR-1018 was cleared from circulation with a half-life of about 1 minute (Figure 11C). The labelled peptide appeared predominantly in the liver and kidneys at -10 μg/g of tissue within 2 min of delivery, decreasing to -3.5 μg/g of tissue after

4 h. In contrast to decreasing peptide concentrations in the liver and spleen, IDR-1018 accumulated in cerebral tissue over time, starting at 0.3 μg/g after 2 min and increasing to 1.5 μg/g over 4 h (Figure 1 ID). These data suggest that in vivo IDR-1018 delivered to the periphery can transit to the brain and reside there for an extended period. Thus IDR-1018 might exert neuroprotective effects directly on brain immune cells.

[00195] To gain insight into the mechanisms of IDR-1018 actions in the brain, global cerebral gene expression patterns were analyzed in neonatal pups that were pre-treated with

IDR-1018 4 h before inflammatory challenge (LPS only) or injury (LPS+HI), and compared to peptide -untreated pups. The effect of IDR-1018 on gene expression associated with neuroinflammation was evaluated 6 h after LPS injection and the effect of IDR-1018 on injury-related gene expression 6 h after the completion of LPS+HI (Figure 11A scheme).

Administration of IDR-1018 alone (without LPS or LPS/HI) altered the expression of less than 200 genes compared to vehicle treated animals. Those changes were not notably different from those seen after administration of vehicle alone, consistent with other observations that in the absence of an inflammatory or infectious stimulus, the peptides alone have very mild effects on host gene expression. There were hundreds of changes in gene expression in response to LPS alone that through network analysis were shown to involve 44 key 'hub' genes (hubs are key proteins involved in trafficking of information) (Figure 12A). Pre -treatment with IDR-1018 increased the importance of one LPS-specific hub (STAT3) and diminished the relative role of 24 others including those involving the TLR-signalling adapter MyD88, transcription factor NFKB p65/RelA, TNF -related signalling intermediate genes TRAF5 and TNFRSF 1A, and the IL 1 receptor IL1R1 (Figure 12A). IDR-1018 treatment in association with LPS evoked the participation of 26 novel hubs, including negative regulators of NFKB (NFKBIA, PIk3rl, HDAC1 and SALL1), heat shock protein HSPA8, cell cycle regulator CDK4, Ca 2+ -ion exchanger SLC8A1, and the transcription factor IRF3 (Figure 12A). These data provide evidence that systemic administration of IDR-1018 can disengage genes/proteins that drive LPS-mediated inflammatory responses in the brain.

[00196] Consistent with these results, specific pathways were altered in LPS/HI-injured brain hemispheres pre-treated with IDR-1018. Most notably, the presence of IDR-1018 was associated with the down regulation of Ca 2+ -signalling (p=0.0027) and p53-signaling

(p=0.039) (Figure 12B). Interestingly, molecules that promote Ca 2+ -signalling were suppressed while negative regulators of p53 -signalling were enhanced. Increased intracellular Ca 2+ concentration is a key initiating factor in perinatal brain injury and results in the activation of cell degrading enzymes, production of oxygen free radicals with subsequent mitochondrial dysfunction and increased apoptosis (Blomgren K & Hagberg H. 2006. Free Radic Biol Med 40:388-397). Transcription factor p53 is involved in the expression of pro- apoptotic proteins (Nijboer CH et al. 2008. Stroke 39:2578-2586) but may also induce mitochondrial dysfunction and promote apoptosis in a non transcription-dependent manner after neonatal HI and inhibition of p53 is protective in animal models for perinatal brain injury (Nijboer CH, et al. 2011. Annals Neurology 70:255-264). Together these data provided evidence that systemic administration of IDR-1018 can modulate key molecules and pathways associated with neuroinflammation and injurious responses in the neonatal brain.

[00197] Encouraged by these results, the neuroprotective properties of IDR-1018 were tested when given after LPS/HI, as this would mimic the clinical situation where treatment could only be initiated after the primary insult. Experimental studies indicate a therapeutic window following perinatal insults of up to 6 h 27 which has been confirmed in clinical studies of post-HI hypothermia (Shankaran, S., et al. 2008. Pediatrics 122:e791-798). Thus IDR-1018 was administered 3 h after LPS/HI (Figure 13 A). Under these conditions, IDR-1018 reduced the overall injury score (Figure 13B), confirming that IDR-1018 was able to demonstrate therapeutic neuroprotection against neonatal brain injury. As the pattern of perinatal brain injury can vary with insult and brain maturity, the effect of IDR-1018 on cerebral white matter and grey matter was separately determined by immunohistochemistry for myelin basic protein (MBP) and microtubule-associated protein-2 (MAP-2), respectively. IDR-1018 strongly reduced the white matter tissue loss as well as injury in the grey matter (Figure 13C). Neuroprotection was evident in all brain regions examined, including cerebral cortex, hippocampus, thalamus and striatum (Figure 14D) and was most dramatic in the thalamus and striatum, the areas most commonly affected in infants with severe sequelae following birth asphyxia.

[00198] This study provides evidence for a new approach to treat neonatal brain injury using a selectively immunomodulatory peptide IDR-1018. Our findings demonstrate that IDR-1018 has potential clinical applicability, since a single, peripheral dose of IDR-1018 given after the initial insult was sufficient to confer neuroprotection. Furthermore, IDR-1018 in the absence of LPS or LPS/HI caused only minor changes in gene expression in cerebral tissue, thus, unlike non-steroidal anti-inflammatory drugs, IDR-1018 is not expected to non- specifically suppress inflammatory responses and compromise immunity. These results also indicate that IDR-1018 may have limited effects on healthy neonatal brains, which is clinically significant as the identification of 'risk infants' in need of treatment inadvertently will include some infants that would otherwise develop normally. The action of IDR-1018 on key inflammatory pathways evoked by both LPS and LPS/HI is consistent with the conclusion that IDR-1018 can protect against neuroinflammation and subsequent injury elicited by both sterile and infectious inflammation.

Materials and methods

Chemicals and reagents

[00199] Ultra pure lipopolysaccharide (LPS) from E. coli 055 :B5 was purchased from List Biological Laboratories Inc., USA. Synthetic peptides IDR-HH2 (VQLRIRVAVIRA-NH 2 ), IDR-1018 (VRLIVAVRIWRR-NH 2 ), PI 006 (VQLRIWVRR-NH 2 ) and IDR-1002

(VQRWLIVWRIR -NH 2 ) were synthesized using F-moc chemistry at the Nucleic

Acid/Protein Synthesis Unit (University of British Columbia, Vancouver, British Columbia, Canada). All peptides were selected from a library of more than 100 peptides based loosely on the weakly active bovine peptide Bac2A, with the same size and similar overall amino acid compositions. Peptides HH2, 1002 and 1018 were designated as immunomodulatory based on their superior ability to induce chemokines and suppress LPS-induced inflammatory responses in PBMC, while PI 006 was inactive in this assay. All other reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Stockholm, Sweden). Animals

[00200] For brain injury models, C57/B16 male and female mice were purchased from B&K Universal, Stockholm, Sweden and pups were bred at the Experimental Biomedicine animal facility (University of Gothenburg, Gothenburg; Sweden). All experiments were approved by the local Ethical Committee at University of Gothenburg (No. 277-2007 and 374-2009) and performed according to the Guidelines for the Care and Use of Laboratory Animals. For tracking experiments, female CD1 mice were purchased from Harlan

Laboratories and housed at the Experimental Therapeutics and the Animal Resource Centre, Vancouver. Mice were used in accordance with the Canadian Council on Animal Care guidelines.

In vitro study

[00201] Microglia were isolated from 1- to 2- day-old mice as previously described 29 . Microglia from 4-6 mice were pooled, seeded in 24-well plates (100,000 cells/well) for 24 h (37°C, 5% C0 2 , 95% relative humidity), then activated with 10 ng/mL LPS ± IDRs (20 μg/mL) for a subsequent 24 h. Microglia-conditioned medium (MCM) was collected and stored at -80°C until analysis. The lactate dehydrogenase (LDH) assay was used according to the manufacturer's instructions (Roche, Stockholm, Sweden) to verify that IDRs did not compromise microglia viability. Inflammatory mediators in MCM were measured using the Cytokine Mouse 20-Plex Panel (Invitrogen) and a Luminex 100 analyzer (Luminex

Corporation, Austin, Tex., USA), according to the manufacturer's instructions. The multiplex assay included FGF-basic, GM-CSF, IFN-γ, IL-l , IL-Ιβ, IL-2, IL-4, IL-5, IL-6, IL-10, IL- 12, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-la, TNF-a and VEGF. Cytokine

concentrations were calculated using Bio-Plex Manager 3.0 software with a five parameter curve-fitting algorithm applied for standard curve calculations. Analyses were performed in duplicates from four independent repeats, using 4-6 animals/repeat.

In vivo model

[00202] Neonatal LPS-sensitized hypoxic-ischemic (LPS/HI) damage was induced in

C57/B16 male and female mice at postnatal day 9 (PND9) as a clinically relevant animal model as previously described (Wang, X, et al. 2009. J Immunol 183:7471-7 ). In this model,

LPS (0.3 mg/kg) is administered 14 hr prior to unilateral hypoxia-ischemia induced by carotid ligation followed by 20 min hypoxia in 10% oxygen. IDR-1002 and IDR-1018 (8 μg/g, i.p.) or vehicle (NaCl, 0.9%) were administered 4 h prior to LPS/HI or 3 h after LPS/HI. On

PND16, regional brain injury (striatum, hippocampus, thalamus and cerebral cortex) was assessed blindly by two independent researchers in acid fuchsin/thionin stamed sections using a semi-quantitative neuropathological scoring system (Hedtjarn, M., et al. 2002. J Neurosci 22:5910-9). Damage to the hippocampus, striatum and thalamus was assessed by grading both the degree of atrophy (scale from 0-3) and the observable cell injury/infarction (0-3), yielding a neuropathological score (0-6) for each of these three regions. Injury to the cerebral cortex was graded on a scale from 0-4. Scores from all four regions of the brain (the cerbral cortex, hippocampus, striatum and thalamus) were combined for a total injury score in the range from 0-22. Scores in the upper 50% of the range (total and for each brain region) were defined as severe injury while lower scores (<50%) are representative of mild injury. To examine grey and white matter damage, adjacent sections were stained with microtubule- associated protein-2 (MAP-2; 1 :2000, mouse-anti-MAP-2, Sigma- Aldrich), a marker of neurons and dendrites, and myelin basic protein (MBP, 1 : 10000, SMI-94R, BioSite), respectively. Every 50th brain section throughout the forebrain was analyzed and tissue volume loss was calculated as previously described (Doverhag C, et al. 2010. Neurobiol Dis 38:36-46).

Microarray

[00203] PND8 mice were pre-treated with IDR-1018 4 h before LPS as described (in vivo model). Six hours after LPS alone or 6 h after mice were subject to both LPS+HI, brains were removed, rapidly frozen on dry ice and total RNA extracted (RNeasy® Lipid Tissue Mini Kit, including DNase treatment, Qiagen, UK). Total brain RNA (n=5/group) from animals treated with LPS ± IDR-1018 as well as RNA from both injured (left) and uninjured (right) hemispheres from animals treated with LPS/HI ± IDR-1018 were analyzed separately on Affymetrix GeneChip Mouse Gene LOST Array (Swegene, Lund, Sweden), Gene expression data was submitted to NCBI GEO (accession number GSE36215).

Network Analysis

[00204] Gene expression data were analyzed with MetaGEX analysis software

(http://marray.cmdr.ubc.ca/metagex/). Gene expression patterns were first compared to vehicle control (LPS vs. veh, IDR-1018 vs. veh) or to the uninjured brain hemisphere

(LPS/HI injured vs. uninjured, LPS/HI/IDR- 1018 injured vs. uninjured), then contrasted with to one another (MetaGEX meta-analysis). Network analysis using minimum-spanning tree analysis (BioNet package [ Beisser, D.; Kiau, G. W.; Dandekar, T.; Mueller, T. and Dittrich,

M. (2009). Bioinformatics 26: 1129-1130.] from Bioconductor) identified IDR-1018 responsive genes (p<0.05) that encoded protein products, termed 'hubs', that had the greatest number of interactions with other differentially expressed genes/proteins; thus these key hub genes represent key proteins involved in the trafficking of signals and gene expression responses. Over-representation analysis (ORA) (hypergeometric distribution for differentially expressed genes p<0.05) was used to identify KEGG pathways (adjusted p<0.05) that had a disproportionately high number of differentially expressed genes, thereby indicating dysregttlation of particular biological pathways. Results from the network and ORA analysis were visualized using InnateDB that employs the Cerebral plugin of Cytoscape (Lynn, DJ, et al. 2008. Molec Syst Biol 4:218).

Tracking

[00205] 3 H-radiolabeled IDR-1018, tritiated at alanine-6 during synthesis, was synthesized at the Centre for Drug Research and Development (specific activity of 0.329 .αΐ peptide; purity by HPLC >95%). The peptide was formulated in physiological saline solution containing 0.01% (v/v) Tween 80. Female 5-week old CD-I mice (3 per time point) were injected intravenously with the appropriate volume of test article, by weight (10 μΤ/kg), to achieve a target dose of 2 mg/kg IDR-1018 (40 μα/kg 3 H). Blood and organs (liver, spleen, lung, kidney, heart and brain) were collected 2, 5, 15, 30, 60 and 240 min post-administration. Blood/plasma and tissue samples were analyzed for peptide content by scintillation counting after digestion with Solvable tissue solubilizer and decolorization with hydrogen peroxide; tissues were homogenized prior to tissue solubilization. Pharmacokinetic parameters were calculated using PK Solutions software. Mice were observed post-administration and throughout the study period for morbidity and mortality.

Statistics

[00206] Differences between groups were analyzed by t-test or one-way ANOVA followed by Dunnet ' s correction, and differences in frequency distribution between groups were analyzed by the Chi-Square test. A p-value below 0.05 was considered statistically

significant, where * indicate p-values <0.05 (*), <0.005 (**), <0.0001 (***). Differential expression of microarray values was assessed with the limma package [Smyth, G. K. (2005). Limma: linear models for microarray data. In: 'Bioinformatics and Computational Biology Solutions using R and Bioconductor'. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds), Springer, New York, pages 397—420] from the Bioconductor project, and reported p-values were adjusted for multiple comparison with the Benjamini-Hochberg method.

[00207] While specific aspects of the invention have been described and illustrated, such aspects should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims. [00208] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[00209] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.