WO2012015910A2 - Use of adnf polypeptides for treating neurodegenerative diseases - Google Patents

Use of adnf polypeptides for treating neurodegenerative diseases Download PDF

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WO2012015910A2
WO2012015910A2 PCT/US2011/045522 US2011045522W WO2012015910A2 WO 2012015910 A2 WO2012015910 A2 WO 2012015910A2 US 2011045522 W US2011045522 W US 2011045522W WO 2012015910 A2 WO2012015910 A2 WO 2012015910A2
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adnf
polypeptide
subject
ser
leu
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PCT/US2011/045522
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French (fr)
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WO2012015910A3 (en
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Alistair James Stewart
Bruce Hisashi Morimoto
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Allon Therapeutics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • This invention relates to the use of ADNF polypeptides in the treatment of neurodegenerative diseases.
  • the present invention also relates to the manufacture of medicaments, methods of formulation and uses thereof.
  • specific therapeutically effective pharmacokinetic parameters, dosing levels and formulations of the ADNF polypeptides useful in treating or preventing these diseases are described.
  • NAP an 8-amino-acid peptide
  • NAPVSIPQ Asn-Ala-Pro-Val-Ser-Ile- Pro-Gin
  • ADNP (U.S. Pat. No. 6,613,740; Bassan et al., J. Neurochem. 72:1283-1293 (1999)).
  • the NAP sequence within the ADNP gene is identical in rodents and humans (U.S. Pat.
  • NAP is a potent neuroprotectant in a range of in vitro models (Gozes et al, CNS Drug Rev. 11(4):353- 68 (2005)) against a number of toxic insults including several relevant to neurodegenerative diseases such as amyloid beta peptides (Bassan et al., J. Neurochem.
  • NAP neurotrophic factor
  • NAP has been shown to be active in a number of transgenic mouse models of dementia including Alzheimer's disease.
  • the triple transgenic mouse model of Alzheimer's disease expressing mutant APP (Swedish), tau (P301L), and presenilin-1 (Ml 46V) develops both neurofibrillary tangles and amyloid beta plaques in a progressive fashion (Oddo et al, Neuron 39(3):409-21 (2003)).
  • NAP has been shown to reduce neurofibrillary tangles and tau hyperphosphorylation in a transgenic mouse model carrying double human tau mutations (P301S; K257T) (Shiryaev et al., Neurobiol Dis. 34(2):381-8 (2009)).
  • NAP neuroprotective effects of NAP have also been demonstrated in animal model expressing half the gene dosage of the parent protein of NAP, ADNP.
  • ADNP heterozygous mice exhibit severe learning deficiencies which are ameliorated, in part, by intranasal NAP treatment.
  • Tau hyperphosphorylation occurs in these ADNP deficient mice and is reduced with NAP treatment (Vulih-Shultzman et al.,
  • ApoE homozygous knock out mice are born with reduced cognitive abilities.
  • Daily subcutaneous NAP injections to ApoE knockout mice results in accelerated acquisition of developmental reflexes as well as prevention of cholinergic deficits and short-term memory impairment compared to placebo groups (Bassan et al.,
  • AF64A ethylcholine aziridium
  • NAP treatment also reduced infarct volume and motor function deficits after ischemic injury in the spontaneous hypertensive rat model (animal model for stroke) (Leker et al., Stroke 33:1085-1092 (2002)) and reduced damage and inflammation in the closed head injury in mice (Beni Adani et al., J. Pharmacol. Exp. Ther. 296:57-63 (2001); Romano et al., J Mol. Neurosci. 18:3745 (2002); Zaltzman et al, NeuroReport 14:481-484 (2003)).
  • fetal alcohol syndrome fetal death after intraperitoneal injection of alcohol was inhibited by NAP treatment (Spong et al., J Pharmacol. Exp. Ther. 297:774-779 (2001); see also WO 00/53217).
  • NAP can cross the blood-brain barrier and can be detected in rodents' brains either after intranasal treatment (Gozes et al., J Pharmacol Exp Ther. 293(3): 1091 -8 (2000)) or intravenous injection (Leker et al., Stroke 33:1085-1092 (2002)) or intraperitoneal administration (Spong et al., 2001).
  • SAL has been shown in in-vitro assays and in vivo disease models to keep neurons of the central nervous system alive in response to various insults (Gozes et al., J Pharmacol Exp Ther. 293(3):1091-8 (2000); Brenneman et al., J Pharmacol. Exp. Ther. 285:619-627 (1998)).
  • D-SAL is an all D- amino acid derivative of SAL that is stable and orally available (Brenneman et al., J Pharmacol Exp Ther. 309:1190-7 (2004)) and surprisingly exhibits similar biological activity (potency and efficacy) to SAL in the systems tested.
  • ADNF-1 complexes are described in International PCT Application No, PCT7US02/29146, filed September 12, 2002 (published as WO 03/022226).
  • ADNF polypeptides including NAP and SAL, and uses thereof in neuroprotection against disorders of the central nervous system, are the subject of numerous patents and patent applications including International PCT Publication No. WO 01/92333; U.S. Application No. 07/871,973 filed April 22, 1992, now U.S. Patent No. 5,767,240; U.S. Application No. 08/342,297 filed October 17, 1994 (published as WO 96/11948), now U.S. Patent No. 6,174,862; U.S. Application No. 60/037,404 filed February 7, 1997 (published as WO 98/35042); U.S. Application No. 09/187,330 filed November 11, 1998 (published as WO 00/27875); U.S. Application No.
  • the present invention is directed to various therapeutically effective pharmacokinetic parameters, dosing levels and formulations of ADNF polypeptides useful in treating or preventing neurodegenerative diseases.
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10 "11 to about 10 " M for a period of time.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10 " to about 10 " M for a period of time.
  • the period of time is at least about 24 hours.
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (C max ) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (C max ) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
  • the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL. In other more specific embodiments, the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL. In other more specific embodiments, the total maximum concentration (C ma ) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr.
  • the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo- ⁇ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo- ⁇ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUC 0- T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/n L. In other more specific embodiments, the AUCo- ⁇ of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL. In other more specific embodiments, the AUC 0- x of the ADNF polypeptide is equal to or greater than about
  • a method for administration of an ADNF polypeptide to a subject comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-oo of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo - ⁇ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUC 0- ⁇ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL. In other more specific embodiments, the AUC 0-0 o of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL. In further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses (e.g., sprays) to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • doses e.g., sprays
  • the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10 "11 to about 10 "12 M for a period of time. In more specific embodiments, the period of time is at least about 24 hours.
  • the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (C max ) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL. In more specific embodiments, the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL. In other more specific embodiments, the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL. In other more specific embodiments, the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
  • the ADNF polypeptide is administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In more specific embodiments, the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
  • the ADNF polypeptide is administered to the subject in one or more doses to produce a AUC 0- T of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUC 0- x of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL.
  • the AUCO-T of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL.
  • the AUCo- ⁇ of the ADNF polypeptide is equal to or greater than about 5.8 h*ng/mL.
  • the ADNF polypeptide is administered to the subject in one or more doses to produce a AUC 0 .
  • ⁇ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUC 0- ⁇ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL.
  • the AUC 0- oo of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL.
  • the method comprises intranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
  • the composition has an iso-osmolarity of about 250 to about 375 mOsm.
  • a liquid composition for intranasal administration of an ADNF polypeptide comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
  • a method for intranasal administration of an ADNF polypeptide to a subject comprising intranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
  • a method of treating or preventing a neurodegenerative disease in a subject comprising the step of instranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
  • the composition is intranasally administered to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10 "11 to about 10 "12 M for a period of time. In more specific embodiments, the period of time is at least about 24 hours.
  • the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (C max ) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
  • the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL.
  • the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL.
  • the total maximum concentration (C max ) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
  • the composition is intranasally administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In more specific embodiments, the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
  • the composition is intranasally administered to the subject in one or more doses to produce a AUCo- ⁇ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUCo- ⁇ of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL.
  • the AUCQ-T of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL.
  • the AUC 0- x of the ADNF polypeptide is equal to or greater than about 5.8 h*ng/mL.
  • the composition is intranasally administered to the subject in one or more doses to produce a AUC 0 .oo of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
  • the AUCo- ⁇ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL.
  • the AUC 0- ⁇ of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL.
  • the composition has an iso-osmolarity of about 250 to about 375 mOsm.
  • ADNF polypeptide may be selected as follows.
  • the ADNF polypeptide is a member selected from the group consisting of: (a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence: Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l); (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2); and (c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b).
  • the ADNF polypeptide is a member selected from the group consisting of a full length ADNF I polypeptide, a full length ADNF III polypeptide (ADNP), and a mixture of a full length ADNF I polypeptide and a full length ADNF III polypeptide.
  • the ADNF polypeptide is an ADNF I polypeptide. In another embodiment, the ADNF polypeptide is a full length ADNF I polypeptide. In another embodiment, the ADNF polypeptide has the formula (R ! ) x - Ser-Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala -(R ) y (SEQ ID NO: 14) in which: R is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R 2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one.
  • the ADNF I polypeptide is Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l).
  • the active core site of the ADNF I polypeptide comprises at least one D-amino acid.
  • the active core site of the ADNF I polypeptide comprises all D- amino acids.
  • the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site.
  • the ADNF I polypeptide is selected from the group consisting of:
  • Gly-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala SEQ ID NO:6
  • Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala SEQ ID NO:7;
  • the ADNF polypeptide is an ADNF III polypeptide. In another embodiment, the ADNF polypeptide is a full length ADNF III polypeptide. In another embodiment, the the ADNF polypeptide has the formula (R ⁇ x-Asn-Ala-Pro- Val-Ser-Ile-Pro-Gln-(R 2 )y (SEQ ID NO: 13) in which: R 1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R 2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one.
  • the ADNF III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
  • the active core site of the ADNF III polypeptide comprises at least one D- amino acid.
  • the active core site of the ADNF III polypeptide comprises all D-amino acids.
  • the ADNF III polypeptide comprises up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site.
  • the ADNF III polypeptide is a member selected from the group consisting of:
  • At least one of the ADNF polypeptides is encoded by a nucleic acid that is administered to the subject.
  • an ADNF I polypeptide and an ADNF III polypeptide are administered to the subject.
  • either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise at least one D-amino acid.
  • either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise all D-amino acids.
  • the ADNF I polypeptide is Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l)
  • the ADNF III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
  • the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF I polypeptide
  • the ADNF III polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF III polypeptide.
  • the ADNF I polypeptide is a member selected from the group consisting of: Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID
  • Gly-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala SEQ ID NO:6
  • Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala SEQ ID NO:7;
  • ADNF III polypeptide is selected from the group consisting of:
  • the subject suffers from a neurodegenerative disease.
  • the neurodegenerative disease is Alzheimer's disease, corticobasal ganglionic degeneration, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, amyotrophic lateral sclerosis, Pick's atrophy, diffuse Lewy body disease, a neurodegenerative pathology associated with aging, a pathological change resulting from a focal trauma, peripheral neuropathy, retinal neuronal degeneration, or dopamine toxicity.
  • the neurodegenerative disease is neurodegeneration associated with schizophrenia.
  • Figure 1 shows a process flow chart for clinical trial manufacture for AL-108 Nasal Spray, 25 mg/mL.
  • Figure 2 shows a process flow chart for clinical trial manufacture for
  • Figures 3 A and 3B show a comparison of PK concentrations for QD and BID administration of 15 mg.
  • the line shows concentrations based on the C max and half-life generated for the 15 mg dose of AL-108 in study AL-108-121.
  • PK modeling using these parameters was carried out on a single daily dose ( Figure 3 A) and two doses separated by 12 hours ( Figure 3B). Data is shown for a continuous 48 hour period.
  • Figure 4 shows a PK model comparison of AL-108 concentrations based on Cmax and half-lives obtained in studies AL-208-110 and AL-108-121.
  • the triangles show concentrations modeled from the C max and half-life generated for the 30 mg dose in study AL-108-121.
  • the squares represent concentrations predicted for a 30 mg dose based on the C max and half- life obtained from the AL-208-110 study in which healthy volunteers were given 15 mg. For this predicted curve, the C max from study AL-108- 110 was doubled and the actual half-life of 0.8 h used.
  • Figure 5 shows PK parameters corrected for drug dose.
  • ADNF polypeptide refers to one or more activity dependent neurotrophic factors (ADNF) that have an active core site comprising the amino acid sequence of SALLRSIPA (referred to as “SAL”) or NAPVSIPQ (referred to as “NAP” or “AL-108” or “davunetide”), or conservatively modified variants thereof that have neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., Brain Res. 603:222-233 (1993); Brenneman & Gozes, J. Clin. Invest 97:2299-2307 (1996), Forsythe & Westbrook, J. Physiol. Land.
  • An ADNF polypeptide can be an ADNF I polypeptide, an ADNF III polypeptide, their alleles, polymorphic variants, analogs, interspecies homolog, any subsequences thereof (e.g., SALLRSIPA or NAPVSIPQ) or lipophilic variants that exhibit neuroprotective/neurotrophic action on, e.g., neurons originating in the central nervous system either in vitro or in vivo.
  • An "ADNF polypeptide” can also refer to a mixture of an ADNF I polypeptide and an ADNF III polypeptide.
  • ADNF I refers to an activity dependent neurotrophic factor polypeptide having a molecular weight of about 14,000 Daltons with a pi of 8.3 ⁇ 0.25.
  • ADNF I polypeptides have an active site comprising an amino acid sequence of Ser- Ala-Leu-Leu- Arg-Ser-IlePro-Ala (also referred to as "SALLRSIPA” or "SAL” of "ADNF-9”).
  • SALLRSIPA Ser- Ala-Leu-Leu- Arg-Ser-IlePro-Ala
  • SAL refers to a peptide having an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala, not a peptide having an amino acid sequence of Ser-Ala-Leu.
  • a full length amino acid sequence of ADNF I can be found in WO 96/11948 herein incorporated by reference in its entirety.
  • ADNF III polypeptide or "ADNF ⁇ ” also called activity- dependent neuroprotective protein (ADNP) refers to one or more activity dependent neurotrophic factors (ADNF) that have an active core site comprising the amino acid sequence of NAPVSIPQ (referred to as "NAP” or “AL-108” or “davunetide”), or conservatively modified variants thereof have neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., Brain Res. 603:222-233 (1993); Gozes et al, Proc. Natl. Acad. Set USA 93:427-432 (1996).
  • ADNF activity dependent neurotrophic factors
  • An ADNF polypeptide can be an ADNF III polypeptide, allelic or polymorphic variant, analog, interspecies homolog, or any subsequences thereof (e.g., NAPVSIPQ) that exhibit neuroprotective/neurotrophic action on, e.g., neurons originating in the central nervous system either in vitro or in vivo.
  • ADNF III polypeptides can range from about eight amino acids and can have, e.g., between 8-20, 8-50, 10-100 or about 1000 or more amino acids.
  • Full length human ADNF III has a predicted molecular weight of
  • ADNF III polypeptides haven active site comprising an amino acid sequence of Asn- Ala-Pro-Val-Ser-Ile-Pro-Gln (also referred to as "NAPVSIPQ” or “NAP” or “AL-108” or “davunetide”). See Zamostiano et al., J. Biol. Chem. 476:708-714 (2001) and Bassan et al., J Neurochem. 72: 1283-1293 (1999), each of which is incorporated herein by reference.
  • NAP refers to a peptide having an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln, not a peptide having an amino acid sequence of Asn- Ala-Pro.
  • Full-length amino acid and nucleic acid sequences of ADNF III can be found in WO 98/135042, WO 00/27875, U.S. Pat. Nos. 6,613,740 and 6,649,411. The Accession number for the human sequence is NP 852107, see also Zamostiano et al., supra.
  • subject refers to any mammal, in particular human, at any stage of life.
  • ADNF polypeptides or nucleic acids encoding them of the present invention can be "administered" by any conventional method such as, for example, parenteral, oral, topical, and inhalation routes. In some embodiments, parenteral and nasal inhalation routes are employed.
  • a “neurodegenerative disease” refers to a condition (disease and/or insult) associated with neuronal cell death and/or sub-lethal neuronal pathology, including, but not limited to, thos arising from a disease stat and/or having an excitotoxic/ischemic mechanism. These diseases include, but are not limited to:
  • diseases of central motor systems including degenerative conditions affecting the basal ganglia (Huntington's disease, Wilson's disease, striatonigral degeneration, corticobasal ganglionic degeneration), Tourette's syndrome, Parkinson's disease, progressive supranuclear palsy (PSP), progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration, and dopamine toxicity;
  • Basal ganglia Heuntington's disease, Wilson's disease, striatonigral degeneration, corticobasal ganglionic degeneration
  • Tourette's syndrome Parkinson's disease, progressive supranuclear palsy (PSP), progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, oli
  • diseases of limbic and cortical systems such as cerebral amyloidosis, Pick's atrophy, Retts syndrome;
  • neurodegenerative pathologies involving neuronal systems and/or brainstem including Alzheimer's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, epilepsy, multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, late-degenerative stages of Down's syndrome, Alper's disease, vertigo as result of CNS degeneration;
  • pathologies associated with developmental retardation and learning impairments, Down's syndrome, and oxidative stress induced neuronal death pathologies associated with developmental retardation and learning impairments, Down's syndrome, and oxidative stress induced neuronal death; pathologies arising with aging and chronic alcohol or drug abuse including, for example, with alcoholism the degeneration of neuron in locus coeruleus, cerebellum, cholinergic basal forebrain; with aging degeneration of cerebellar neurons and cortical neurons leading to cognitive and motor impairments; and with chronic amphetamine abuse degeneration of basal ganglia neurons leading to motor impairments;
  • focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or direct trauma;
  • pathologies arising as a negative side-effect of therapeutic drugs and treatments e.g., degeneration of cingulate and entorhinal cortex neurons in response to anticonvulsant doses of antagonists of the NMDA class of glutamate receptor;
  • peripheral neurotoxicity including, but not limited to, peripheral neurotoxicity resulting from treatment with one or more chemical agents (e.g., chemical agents for cancer, multiple sclerosis, gout, arthritis, Behcet's disease, psychiatric disorder, immunosuppression and infectious disease, such as vinca alkaloids, platinum drugs, L-asparaginase, taxanes, thalidomide, methotrexate, colchicines, and anti- infective agents); and
  • chemical agents e.g., chemical agents for cancer, multiple sclerosis, gout, arthritis, Behcet's disease, psychiatric disorder, immunosuppression and infectious disease, such as vinca alkaloids, platinum drugs, L-asparaginase, taxanes, thalidomide, methotrexate, colchicines, and anti- infective agents
  • a “mental disorder” or “mental illness” or “mental disease” or “psychiatric or neuropsychiatric disease or illness or disorder” refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder and attention deficit disorders) as well as other mental disorders such as substance-related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV). Typically, such disorders have a complex genetic and/or a biochemical component.
  • a "mood disorder” refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time.
  • Mood disorders include major depression disorder (i.e., unipolar disorder), ma, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
  • Major depression disorder refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or “empty” mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being “slowed down”; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability, or persistent physical symptoms that do not respond to treatment, such as headaches digestive disorders, and chronic pain.
  • DSM depressive disorder
  • Bipolar disorder is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing try being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
  • Anxiety refers to psychiatric syndromes characterized by a subjective sense of unease, dread, or foreboding, e.g., panic disorder, generalized anxiety disorder, attention deficit disorder, attention deficit hyperactive disorder, obsessive-compulsive disorder, and stress disorders, e.g., acute and post-traumatic. Diagnostic criteria for these disorders are well known to those of skill in the art (see, e.g., Harrison's Principles of Internal Medicine, pp. 2486-2490 (Wilson et al., eds., 12th ed. 1991) and DSM IV).
  • Neurotoxicity as used herein is defined as adverse effects on the structure or functioning of the cells of the nervous system that result from exposure to chemical substances or to disease processes.
  • neurotoxicants can cause morphological changes that lead to generalized damage to nerve cells (neuronopathy), injury to axons (axonopathy), or destruction of the myelin sheath (myelinopathy).
  • axonopathy injury to axons
  • myelinopathy myelin sheath
  • Symptoms of neurotoxicity include muscle weakness, loss of sensation and motor control, tremors, alterations in cognition, and impaired functioning of the autonomic nervous system.
  • Neurotoxicological assessments use a battery of functional and observational tests. Neurotoxicity in humans is most commonly measured by neurological tests that assess cognitive, sensory, and motor function.
  • PNS neurotoxicity refers to neurotoxicity of the peripheral nervous system (PNS).
  • PNS peripheral nervous system
  • DRG dorsal root ganglia
  • DRG dorsal root ganglia
  • Central nervous system or “CNS” means the brain and spinal cord.
  • Laser-induced retinal damage includes both intentional and accidental retinal damage resulting from laser exposure.
  • Laser photocoagulation refers to a surgical procedure wherein a laser is used to seal leaking blood vessels within the retina and reduce the future growth of abnormal blood vessels. By sealing leaking blood vessels, laser photocoagulation slows down, e.g., the buildup of fluid under the retina that distors the shape and position of the macula, the growth of scar tissue and the abnormal membrane under the retina that damage the cells in the macula, and central vision loss.
  • Constants involving retinal degeneration include, but are not limited to, laser-induced retinal damage and ophthalmic diseases, such as glaucoma, Retinitis pigmentosa, Usher syndrome, artery or vein occlusion, diabetic retinopathy, retrlental fibroplasias or retinopathy of prematurity (R.L.F./R.O.P), retinoschisis, lattic degeneration, macular degeneration and ischemic optic neuropathy.
  • laser-induced retinal damage and ophthalmic diseases such as glaucoma, Retinitis pigmentosa, Usher syndrome, artery or vein occlusion, diabetic retinopathy, retrlental fibroplasias or retinopathy of prematurity (R.L.F./R.O.P), retinoschisis, lattic degeneration, macular degeneration and ischemic optic neuropathy.
  • polypeptide refers to a polymer of amino acid residues.
  • peptide refers to a short polypeptide.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereofm either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 20-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may include those having non-naturally occurring D- chirality, as disclosed in WO 01/12654, incorporated herein by reference, which may improve oral availability and other drug like characteristics of the compound.
  • one or more, and potentially all of the amino acids of NAP or the ADNF- polypeptide will have D-chirality.
  • the therapeutic use of peptides can be enhanced by using D-amino acids to provide longer half life and duration of action.
  • NAP and ADNF polypeptides also retain activity in the D-amino acid form (Brenneman et al, J Pharmacol. Exp. Ther. 309(30):1190-1197 (2004)).
  • Amino acids may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The amino acids referred to herein are described by shorthand designations as follows: TABLE 1
  • Constantly modified variants applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent a variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar-amino acid. Conservative substitution-tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following groups each contain amino acids that are conservative substitutions for one another:
  • nucleic acid and polypeptide sequences yield functionally identical products.
  • silent substitutions i.e., substitutions of a nucleic acid sequence that do not result in an alteration in an encoded polypeptide
  • conservative amino acid substitutions in one or a few amino acids in an amino acid-sequence are substituted with different amino acids with highly similar properties (see the definitions section, supra), are. also readily identified as being highly similar to a disclosed no acid sequence, or to a disclosed nucleic acid sequence that encodes an amino acid.
  • conservatively substituted variations of each explicitly listed nucleic acid and amino acid sequences are a feature of the present invention.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated ADNF nucleic acid is separated from open reading frames that flank the ADNF gene and encode proteins other than ADNF.
  • purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • an amount sufficient” or “an efficient amount” or a “therapeutically effective amount” is that amount of a given ADNF polypeptide that exhibits the activity of interest or which provides either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • the NAP or ADNF polypeptides of the invention are administered to a patient in an amount sufficient to reduce or eliminate symptoms of a neurodegenerative disease. An amount adequate to accomplish this is defined as the "therapeutically effective dose.”
  • the dosing range varies with the NAP or ADNF polypeptide, as further set out below, and in CA patent 2202496, U.S. Pat. No. 6,174,862 and U.S. Pat. No. 6,613,740, herein incorporated by reference in their entirety.
  • Inhibitors are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonist and their homologs and mimetics.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactive, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of polypeptide or polynucleotide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity.
  • Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of the effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%.
  • Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • the ADNF polypeptides of the present invention comprise the following amino acid sequence: (R ⁇ x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln- (R 2 ) y (SEQ ID NO: 13) and conservatively modified variations thereof.
  • R 1 denotes the orientation of the amino terminal (NH 2 or N- terminal) end and R 2 represents the orientation of the carboxyl terminal (COOH or C- terminal) end.
  • R 1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino, acids and amino acid analogs.
  • the term "independently selected” is used herein to indicate that the amino acids making up the amino acid sequence R 1 may be identical or different (e.g., all of the amino acids making up the amino sequence may be threonine, etc.).
  • the amino acids making up the amino acid sequence R 1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs).
  • Suitable amino acids that can be used to form the amino acids sequence R 1 include, but are not limited to, those listed in Table 1, infra.
  • the indexes "x" and “y” are independently selected and can be equal to one or zero.
  • R 1 , R 2 in the above formula is an amino acid sequence comprising from 1 to 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid
  • amino acids making up the amino acid sequence R may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs).
  • Suitable amino acids that can be used to form R 2 include, but are not limited to, those listed in Table 1 , infra.
  • NAP or “NAP peptide” refers to the formula above where x and y both equal 0.
  • NAP related peptide refers to any of the other variants of NAP which are described the formula.
  • R and R are independently selected. If R and R are the same, they are identical in terms of both chain length and amino acid composition.
  • both R 1 and R 2 may be Val-Leu-Gly-Gly-Gly- (SEQ ID NO: 15).
  • R 1 and R 2 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences.
  • R 1 may be Val-Leu-Gly-Gly-Gly- (SEQ ID NO: 16)
  • R 2 may be Val-Leu-Gly-Gly- (SEQ ID NO: 17).
  • R 1 may be Val-Leu-Gly-Gly- (SEQ ID NO: 18).
  • NAP and NAP related polypeptide are also preferred, namely those in which x and y are both zero (i.e. NAP).
  • NAP and NAP related polypeptides in which x is one; R 1 is Gly- Gly-; and y is zero (SEQ ID NO: 19).
  • NAP and NAP related polypeptides in which x is one; R 1 is Leu-Gly-Gly-; y is one; and R 2 is -Gln-Ser (SEQ ID NO:20).
  • NAP and NAP related polypeptides in which x is one; R 1 is Leu-Gly-Leu-Gly-Gly- (SEQ ID NO: 21); y is one; and R 2 is -Gln-Ser (SEQ ID NO:22). Also equally preferred are NAP and NAP related polypeptides in which x is one; R 1 is Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly- (SEQ ID NO:23); y is one; and R 2 is - Gln-Ser (SEQ ID NO:24). Additional amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity.
  • the present invention provides pharmaceutical compositions comprising one of the previously described NAP and NAP related polypeptides in an amount sufficient to exhibit the desired activity, in a pharmaceutically acceptable diluent, carrier or excipient.
  • the NAP or NAP related peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 9-12, and conservatively modified variations thereof.
  • the ADNF polypeptide comprises the following amino acid sequence: (R 1 ) x -Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R 2 ) y (SEQ ID NO: 14) and conservatively modified variations thereof.
  • R 1 denotes the orientation of the amino terminal (NH 2 or N-terminal) end and R 2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.
  • R 1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs.
  • the term "independently selected” is used herein to indicate that the amino acids making up the amino acid sequence R 1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc).
  • the amino acid making up the amino acid sequences R 1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to naturally occurring amino acids (i.e., amino acid mimetics and analogs).
  • Suitable amino acids that can be used to form the amino acid sequence R 1 include, but are not limited to, those listed in Table 1, infra.
  • the indexes "x" and "y” are independently selected and can be equal to one or zero.
  • R 2 in the above formula is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected form the groups consisting of naturally occurring amino acids and amino acid analogs.
  • the amino acids making up the amino acid sequence R 2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs).
  • Suitable amino acids that can be used to form R 2 include, but are not limited to those listed in Table 1, infra.
  • SAL SAL or SAL peptide refers to the formula above where x and y both equal 0.
  • SAL related peptide refers to any of the other variants of SAL which are described the formula.
  • R 1 and R 2 are independently selected. If
  • R and R z are the same, they are identical in terms of both chain length and amino acid composition. Additional amino acids can be added to both the N-terminus and the C- terminus of the active peptide without loss of biological activity.
  • the present invention provides pharmaceutical compositions comprising one of the previously described SAL and SAL-related polypeptides in an amount sufficient to exhibit the desired activity, in a pharmaceutically acceptable diluent, carrier or excipient.
  • the SAL or SAL related peptide has an amino acid sequence selected form the group consisting of SEQ ID NOS: 1 and 3-8, and conservatively modified variations thereof.
  • Polypeptides and peptides comprising the core NAPVSIPQ or SALLRSIPA active site can be easily made, e.g., by the synthetic chemical process of systematically adding one amino acid at a time and screening the resulting peptide for biological activity, as described herein.
  • the contributions made by the side chains of various amino acids residues in such peptides can be probed via a systematic scan with a specified amino acid, e.g., Ala.
  • alterations in a given nucleic acid sequence include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells continuing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see Giliman & Smith, Gene 8:81-97 (1979); Roberts & ⁇ ., Nature 1328:731-734 (1987)).
  • polypeptide sequences are altered by changing the corresponding nucleic acid sequence and expressing the polypeptide.
  • polypeptide sequences are also optionally generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide (see Merrifield, Am. Chem. Soc. 85:2149-2154 (1963); Stewart & Young, Solid Phase Peptide Synthesis (2nd ed. 1984)).
  • nucleic acid or polypeptide of the invention can select a desired nucleic acid or polypeptide of the invention based upon the sequences provided and upon knowledge in the art regarding proteins generally. Knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids and polypeptides disclosed herein. The definitions section, supra, describes exemplar conservative amino acid substitutions.
  • Modifications to the ADNF polypeptides are evaluated by routine techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of a polypeptide can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a target nucleic acid, redox or thermal stability of a protein, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
  • ADNF polypeptides in accordance with the teachings of the present invention and, in turn, screen them using the foregoing animal models to find ADNF polypeptides, in addition to those set forth herein, which possess the desired activity.
  • NAP peptide i.e., Asn- Ala-Pro- Val-Ser-Ile-Pro-Gln (SEQ ID NO:2)
  • SAL peptide i.e., Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l)
  • Gly-, Gly-Gly-, Leu-Gly-Gly- to the N-terminus of the peptide and, in turn, screen each of these NAP or ADNP polypeptides in the foregoing assay to determine whether the desired activity.
  • the peptides of the invention may be prepared via a wide variety of well-known techniques. Peptides of relatively short size are typically synthesized on a solid support or in solution in accordance with conventional techniques (see, e.g., Merrifield et ah, Am. Chem. Soc. 85:2149-2154 (1963)). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis (2nd 1984)). Solid phase synthesis in which the C-terminal amino acid of die sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the peptides of this invention.
  • the peptides for use in the invention may be prepared by recombinant DNA methodology. Generally, this involves creating a nucleic acid sequence that encodes the protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, and expressing the protein in a host cell.
  • Recombinantly engineered cells known to those of skill in the art include, but are not limited of bacteria, yeast, plant, filamentous fungi, insect (especially employing baculoviral vectors) of mammalian cells.
  • control sequences include the T7, tip, or lambda promoters, a ribosome binding site and, preferably, a transcription termination signal.
  • control sequences typically include a promoter and, preferably, an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • the plasmids of the invention can be transferred into the chosen host cell by well-known methods. Such methods include, for example, the calcium chloride transformation method for E. coli and the calcium phosphate treatment or electroporation methods for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo, and hyg genes.
  • the recombinant peptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Polypeptide Purification (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Polypeptide Purification (1990)).
  • the NAP and ADNF polypeptides may then be used, e.g., to prevent neuronal cell death or as immunogens for antibody production.
  • Optional additional steps include isolating the expressed protein to a higher degree, and, if required, cleaving or otherwise modifying the peptide, including optionally generating the protein.
  • the peptide(s) may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is helpful to denature and reduce the peptide and then to cause the peptide to re-fold into the preferred conformation.
  • Methods of reducing and denaturing peptides and inducing re-folding are well known to those of skill in the art (see Debinski et al, Biol Chem. 268:14065- 14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:585 (1993); and Buchner et al, Anal Biochem. 205:263-270 (1992)).
  • Debinski et al. describe the denaturation and reduction of inclusion body peptides in guanidine-DTE.
  • the peptide is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
  • modifications can be made to the peptides without diminishing their biological activity. Some modifications' may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion peptide. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
  • the present invention provides a method for administration of an ADNF polypeptide to a subject, wherein the method comprises administering the ADNF polypeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • the present invention provides a method of treating or preventing a neurodegenerative disease in a subject, wherein the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
  • this dosing level (i.e., 30 mg BID) has been found to produce certain surprising effects on various pharmacokinetic parameters, including, the total maximum concentration (C max ), half-life, AUCo- ⁇ and AUCo-oo, of an ADNF polypeptide in the blood plasma of a subject.
  • the present invention also provides methods for administration of an ADNF polypeptide to a subject and methods of treating or preventing a neurodegenerative disease in a subject, wherein the methods comprise administering the ADNF polpeptide to the subject in one or more doses to produce one or more of these pharmacokinetic effects. More specifically, the present invention provides methods wherein the methods comprise administering the ADNF polypeptide to the subject in one or more doses to:
  • ADNF polypeptide equal to or greater than about 10 " to about 10 " M for a period of time (including methods wherein the period of time is at least about 24 hours);
  • the ADNF polypeptides of the invention may be administered to a patient in any amount sufficient to produce one or more of these pharmacokinetic effects. Amounts effective for this use will depend on, for example, the particular ADNF polypeptide employed, the manner of administration, the weight and general state of health ofthe patient, and the judgement of the prescribing physician. For example, an amount of polypeptide falling within the range of a 100 ng to 30 mg dose given intranasally once a day (e.g., in the evening) or twice daily would be an effective amount. Alternatively, dosages may be outside of this range, or on a different schedule.
  • dosages may range from 0.0001 mg/kg to 10,000 mg/kg, and will preferably be about 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 50 mg/kg or 500 mg/kg per dose.
  • Doses may be administered hourly, every 4, 6 or 12 hours, with meals, daily, every 2, 3, 4, 5, 6, for 7 days, weekly, every 2, 3, 4 weeks, monthly or every 2, 3 or 4 months, or any combination thereof.
  • the duration of dosing may be single (acute) dosing, or over the course of days, weeks, months, or years, depending on the condition to be treated. Those skilled in the art can determine the suitable dosage, and may rely on preliminary data reported in Gozes et al., J Pharmacol Exp Ther.
  • the present invention provdes a liquid composition for intranasal administration of an ADNF polypeptide, wherein the composition comprises: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
  • such composition has an iso-osmolarity of about 250 to about 375 mOsm.
  • the ADNF polypeptides and pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems.
  • Peptides that have the ability to cross the blood brain can be administered, e.g., systemically, nasally, etc., using methods known to those of skill in the art.
  • Larger peptides that do not have the ability to cross the blood brain barrier can be administered to the mammalian brain via intracerebro ventricular (ICV) injection or via a cannula using techniques well known to those of skill in the art (see, e.g., Motta & Martini, Proc. Soc. Exp. Biol Med. 168:62-64 (1981); Peterson et al., Biochem. Pharmacol.
  • ICV intracerebro ventricular
  • the present invention provides for therapeutic compositions or medicaments comprising one or more of the ADNF polypeptides described hereinabove in combination with a pharmaceutically acceptable excipient, wherein the amount of the ADNF polypeptide is sufficient to provide a therapeutic effect.
  • the ADNF polypeptides of the present invention are embodied in pharmaceutical compositions intended for administration by any effective means, including parenteral, topical, oral, pulmonary (e.g. by inhalation) or local administration.
  • the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, sublingually, buccally or intramuscularly, or intranasally.
  • compositions for parenteral administration that comprise a solution of an ADNF polypeptide, as described above, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used including, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques or, they may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as, for example, sodium acetate, sodium lactate, sodium chloride potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • conventional nontoxic solid carriers may be used that include, for, example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.
  • the ADNF polypeptides are preferably supplied in finely divided from along with a surfactant and propellant.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • An example includes a solution in which each milliliter included 7.5 mg NaCl, 1.7 mg citric acid monohydrate, 3 mg disodium phosphate dihydrate and 0.2 mg benzalkonium chloride solution (50%) (Gozes et al., J Mol Neurosci. 19(1-2): 167-70 (2002)).
  • the 25 mg/mL nasal spray was a solution of AL-108 in 129 mM sodium chloride, 8 mM citric acid, 17 mM sodium phosphate, and 0.005% benzalkonium chloride, had pH 5, and was packaged in the Pfeiffer Advanced Preservative Free (APF) System, a mechanical multidose device designed for the application of solutions via the nasal route.
  • APF Pfeiffer Advanced Preservative Free
  • Each spray delivered 0.1 mL containing 2.5 mg of AL-108.
  • Each nasal spray device contained 10 mL of product and could accurately dispense 75 sprays.
  • the 75 mg/mL nasal spray was a solution of AL-108 in 8 mM citric acid, 17 mM sodium phosphate, and 0.005% benzalkonium chloride, had pH 5, and was packaged (9 mL per device) in the Valois VP7 pump, a mechanical multidose device designed for the application of solutions via the nasal route.
  • Each spray delivered 0.1 mL containing 7.5 mg of AL-108.
  • Sodium chloride was removed from the 75 mg/mL formulation to maintain the target iso-osmolarity of 250-375 mOsm.
  • Nasal drug absorption is affected by molecular weight, size, formulation pH, pKa of molecule, and delivery volume among other formulation characteristics. Specifically, the delivery volume is limited by the size of the nasal cavity. A volume of 0.3 to 0.5 mL/nostril, to a maximum volume of 1 mL has been suggested. More will run or drip out of the nose (see Romeo et al., Adv Drug Delivery Rev. 29:89-116 (1998)). Ideally, one would limit the volume of nasal administration to ensure delivery of the entire dosage of drug. Therefore, in order to administer doses higher than 15 mg BID of AL-108 intranasally in an acceptable spray volume, a higher dosage strength formulation was needed.
  • the initial test batch of 75 mg/mL dosage strength was manufactured with identical excipient components as the 25 mg/mL dosage strength.
  • osmolarity measurement of the 75 mg/mL test batch revealed that the formulation resulted in a hyperosmotic solution (-440 mOsm).
  • a hyperosmotic solution when delivered intranasally could potentially result in nasal irritation. Therefore, there was a need to develop a nasal formulation with an osmolarity within normal physiological range of 250-375 mOsm.
  • a sodium chloride free formulation for AL-108 peptide overcame the hyperosmotic issue associated with the initial test formulation.
  • the osmolarity measurement of the new sodium free formulation of intranasal AL-108 fell with the physiologically acceptable range of 250-375 mOsm.
  • the process flow chart for manufacturing the 25 mg/mL batch of AL- 108 is provided in Figure 1.
  • the benzolkonium chloride was the last excipient to be added, prior to the active substance (AL-108), and the container was thoroughly rinsed out at least three times with portions of the purified water, to ensure that preservative was not lost in the container.
  • the mixer settings were at 100 ⁇ 20 rpm during mixing of excipients, and then reduced to 50 ⁇ 10 rpm during and after addition of active substance, so as not to disrupt the peptide.
  • the solution was mixed for at least 10 minutes each time. Approximately 500 g of placebo solution was passed through the membrane filter prior to filtration of the active solution. This was used as a filter conditioning solution, to saturate the membrane filter with preservative, so minimizing loss of preservative from the active solution, through adsorption onto the membrane.
  • the process flow chart for manufacturing the 75 mg/niL batch of AL- 108 is provided in Figure 2.
  • 80.0 kg of purified water was dispensed into the vessel.
  • Citrate acid monohydrate and sodium phosphate dibasic dihydrate were added to purified water and the solution was mixed until completely dissolved.
  • Benzolkonium chloride was diluted with 500 g of purified water and added into the vessel.
  • the original container was rinsed at least three times with 250.0 g of purified water and the rinses were added to the vessel.
  • the active agent (AL-108) was then added to the vessel.
  • the containers were rinsed with approximately 2.0 kg of purified water, if required.
  • the pH was adjusted with sodium hydroxide to reach an acceptable range of pH 4.8-5.2.
  • a 30 mg dose of AL-108 of Example 1 was tested in a phase 1 study in order to assess safety and PK.
  • the expected result from the study (AL-108-121) was that a 30 mg dose would be well- tolerated and provide exposure double that seen with a 15 mg dose and three-times that seen with a 10 mg dose.
  • the results showed that a 30 mg dose provided a half-life and systemic exposure substantially greater than would have been expected from linear pharmacokinetics and consistent half-life based on earlier clinical studies. This data, coupled with predictions of increased efficacy based on PK modeling, indicates that this unexpected increase in exposure will translate to a similarly improved efficacy.
  • Phase I study AL-108-121 generated data showing that 30 mg gave unexpectedly greater drug exposure than predicted. C max concentrations were higher, the exposure (measured by AUC) was greater and the half-life was longer. Based on the 15 mg dose pharmacokinetics, a 30 mg dose was expected to maintain plasma concentrations above a threshold level between 10 "13 to 10 "14 M. Suprisingly, the 30 mg dose data from the AL-108-121 study showed sustained exposure of AL-108 at concentrations greater than 10 "11 to 10 "12 M, as shown in Figure 4. Further, the half-life was extended to 1.46 hours, rather than the 0.8 hours expected from previous studies. Using the C max from this data (3.44 ng/ml) and the observed half- life of 1.46 hours, the PK modeling exposure graph in Figure 4 shows a very dramatic effect on exposure and duration of concentrations that are predicted to be more active.
  • This PK modeling data based on actual PK parameters and the knowledge that 15 mg BID is an active dose, predicts that the 30 mg dose will be active and more active than would have been predicted using the PK parameters available before this new data was generated in the AL-108-121 study.
  • PK modeling data indicating novel and unexpected findings is supported by comparison of data generated from the AL-108-121 study and previous pharmacokinetic (PK) studies carried out in normal healthy volunteers and AD patients where AL-108 concentrations were measured in plasma at a series of time points.
  • the calculated PK parameters provide a measure of drug residence time and drug exposure in the different studies. As it pertains to assessing the effect of different drug products on drug exposure, the most relevant parameters are half-life (T ⁇ ), C max ,
  • Table 1 shows a dramatically longer half-life at 30 mg than seen at 10 or 15 mg, a parameter which was not expected to change at all when increasing the dose.
  • the half-life at 10 mg and 15 mg is consistent at 0.61 and 0.62 hours, respectively.
  • administration of 30 mg produced a half-life of 1.46 hours. While exposure parameters were expected to increase in a linear manner over this range, and indeed did so between 10 and 15 mg, linearity was lost between 15 and 30 mg.
  • the data in Figure 5 shows this in a graphical format: by dividing the PK parameters (C MAX , AUC 0- T, and AUC 0- ⁇ ) by the dose administered. If there was a linear dose-to-PK relationship, this would present itself as no change in the y-axis as the dose (x-axis) changes. Indeed, there is essentially no change with PK parameters corrected for dose between 10 and 15 mg. However, this can be contrasted with the large shift in each of the exposure parameters when the dose was increased to 30 mg.
  • Table 2 Further data analysis in Table 2 was carried out to take into account changes in the strength of the drug formulation.
  • the formulation change was made to allow smaller volumes to be administered to patients, increasing the drug strength from 25 mg/mL to 75 mg/n L.
  • the closest comparison of PK generated for the 30 mg dose must be made for similar drug strengths meaning that the 15 mg and 30 mg doses should be compared in data from study AL-108-121 where the same drug strength was used for the two doses. This provides a direct comparison of samples taken from the same study, analysed at the same time and in the same patients since this was a crossover design.
  • Table 2 presents the parameters from study AL-108- 121, again using data only from day 1 since the 15 mg dose was given once only.
  • Table 3 presents the data from three Phase 1 clinical trials where intranasal AL-108 plasma PK was assessed (studies AL-108- 102, AL-208-110 and AL-108-121).
  • Table 3 shows a dramatically longer half-life at 30 mg than seen at 10 or 15 mg.
  • the half-life at 10 mg and 15 mg is consistent at 0.59 hours.
  • administration of 30 mg produced a half-life of 2.09 hours, approximately 3.5- fold longer than expected.
  • the half-life of a drug is constant at different doses, so to see a 3.5-fold increase was quite unexpected.
  • the primary objectives of this study are to evaluate AL-108 30 mg BID relative to placebo, when both are administered intranasally (IN) for 52 weeks to subjects with PSP, with respect to:
  • Brain atrophy as measured by change from baseline of ventricular volumes measured by volumetric brain MRI at 52 weeks.
  • ⁇ Efficacy as measured by the change from baseline of cognition by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) at 52 weeks.
  • Cerebrospinal fluid (CSF) biomarkers including total tau, phosphorylated tau, amyloid beta peptide (1-42), neurofilament protein light chain (NFL), and phosphorylated neurofilament heavy chain (pNFH) as measured by the change from baseline at 52 weeks in the subgroup of subjects consenting to lumbar puncture.
  • CSF Cerebrospinal fluid
  • Plasma phosphorylated neurofilament protein heavy chain (pNFH), as measured by the change from baseline at 6, 13, and 52 weeks.
  • the AL-108 dose of 30 mg intranasal (IN) BID was chosen because AL- 108 15 mg IN BID demonstrated efficacy in a prior study of memory function in subjects with aMCI and the 30 mg dose is the maximum feasible dose delivered IN with 2 sprays to each nostril while maintaining iso-osmolarity.
  • the 30 mg IN BID dose was well-tolerated and provided four- to five-fold greater exposure than the 15 mg IN BID dose in Phase 1 evaluation of age-matched healthy volunteers (45 to 85 years).
  • the study duration of twelve months is appropriate for a Phase 2/3 study in PSP to assess the magnitude of motor changes and brain atrophy.
  • AL-108 demonstrated cognitive improvement in twelve weeks in subjects with aMCI. In that study, the treatment effect may not have reached a maximum level.
  • a study duration of twelve months will allow a better assessment of the potential treatment effect and time course of that effect.
  • the twelve-month duration will also allow an assessment as to whether the rate of decline has been affected by treatment.
  • the endpoints are appropriate measures of treatment effect in this study.
  • the PSPRS is a robust measure of PSP disease activity and progression.
  • the instrument measures 6 relevant categories of PSP symptoms: daily activities, behavior, bulbar, oculomotor, limb motor, and gait/midline.
  • the instrument has been validated and has been shown to correlate well with survival and retention of gait function (Golbe and Ohman-Strickland, Brain, 130(Pt 6): 1552-65, 2007 ( Epub 2007 Apr 2)).
  • the SEADL (Schwab RS, Trans Am Neurol Assoc, 94:363-4, 1969) is being used in this study to assess subject's functional capacity and potential changes with treatment. Additionally, the SEADL has been found to be one of the most sensitive measures of disease progression (Bensimon et al., Brain, 132(Pt 1): 156-71, 2009 (Epub 2008 Nov 23)). The CGI has been used in studies of therapies for PSP and other movement disorders (Bensimon et al., Brain, 132(Pt 1):156-71, 2009 (Epub 2008 Nov 23)).
  • the RBANS and Executive Function tests are appropriate measures of cognition in this population.
  • the RBANS is a validated instrument that assesses 5 cognitive domains: Immediate Memory, Delayed Memory, Attention, Language, and Visuospatial/Constructional (Randolph et al., J Clin Exp Neuropsychol, 20(3):310-9, 1998).
  • the tests used to assess executive function are phonemic fluency, the colors trail test and letter- number sequencing.
  • Executive function is typically compromised in subjects with PSP, but is not specifically assessed by the RBANS. Therefore, it is appropriate to include both sets of tests in this study.
  • Structural MR! and plasma and CSF biomarkers are appropriate for this study because AL-108 has the potential to modify the course of PSP. Disease progression as measured by ventricular volume enlargement on MRI is expected to be about 9% in this study population over twelve months (Paviour et al., Mov Disord, 21(7):989-96, 2006; Whitwell et al., Brain, 130(Pt 4):1148-58, 2007 (Epub 2007 Mar 8)). Therefore, decreases in the rate of brain atrophy may be detectable with AL-108 treatment that would suggest disease modification.
  • the elevated neurofilament levels are believed to represent neuronal-axonal degeneration (Holmberg et al., Mov Disord, 13(l):70-7, 1998; Holmberg et al., Parkinsonism Relat Disord, 8(1):23-31, 2001 ; Brettschneider et al., Dement Geriatr Cogn Disord, 21(5-6):291-5, 2006 (Epub 2006 Feb 10); Constantinescu et al., Parkinsonism Relat Disord, 16(2):142-5, 2010 (Epub 2009 Jul 31); Perrot and Eyer, Brain Res Bull, 80(4-5):282-95, 2009 (Epub 2009 Jun 17)). Therefore, decreased levels of CSF or plasma neurofilament chains would suggest that AL-108 decreased the rate of neuronal degeneration.
  • Mood status of the subjects will be assessed by the GDS. This scale has been used extensively in clinical trials.
  • Treatments evaluated in this study will be placebo and AL-108 30 mg BID. All treatments will be blinded and administered intranasally. Each dose of study drug will consist of 2 sprays in each nostril for a total of 4 sprays. Therefore, the total daily dose will be eight (8) sprays daily (4 sprays, twice daily, 8 to 12 hours apart). Each spray will deliver 0.1 mL of study drug solution.
  • the AL-108 dose for this study was selected on the basis of prior preclinical and clinical data.
  • Preclinical toxicology evaluation has shown AL-108 to be safe and well- tolerated at doses of 80 mg/kg/day in rats and 20 mg/kg/day in dogs. No evidence of harm was observed in safety pharmacology evaluation of cardiovascular, respiratory and neurological function in rats and dogs at doses up to 100 mg/kg IV.
  • Preclinical efficacy studies have demonstrated improvement in cognitive function and decreases in both amyloid beta (1-42) and phosphorylated tau in transgenic murine models of tauopathies at doses of approximately 20 to 1200 ug/kg/day.
  • the effect of AL-108 on cognitive function was evaluated in a study of subjects with aMCI.
  • AL-108 was safe and well-tolerated when administered at doses of 5 mg QD and 15 mg BID for 12 weeks.
  • Statistically significant improvement was observed at the 15 mg BID dose in cognitive tests that primarily assessed working memory. Improvement was not observed in cognitive tests that more prominently evaluated other cognitive functions. This finding is believed to reflect the clinically normal functioning of the study subjects in non-memory clinical domains. No improvement in cognition was observed at the 5 mg QD dose.
  • AL-108 was also safe and well-tolerated when administered as a single 300-mg IV dose to subjects undergoing coronary artery bypass graft surgery.
  • the AL-108 dose of 30 mg BID was chosen because:
  • 30 mg is the highest feasible dose that can be delivered at any single administration time point while maintaining iso-osmolarity of the formulation
  • Group 1 15 mg AL- 108 (healthy adults)
  • the nasal respiratory mucosa is considered the most important area for delivering drugs systemically (Pires et al., J. Pharm Pharmaceutical Sci 12(3):288-311, (2009)).
  • the mucosa is made up of the epithelium, basement membrane and lamina basement.
  • the lamina basement membrane is located beneath the basal lamina and contains many blood vessels, nerves and glands.
  • the intense blood flow in the arteriovenous anastomosis and the large surface of the respiratory epithelium favors transmucosal nasal drugs absorption. Indeed, the pharmacokinetic parameters obtained after intranasal and oral administration of certain drugs showed comparable systemic exposure independent of the route of administration (Ding et al., Drug Delivery 14:101- 104 (2007)).
  • Alzheimer's disease patients olfactory impairment and neuroanatomical changes in the central portions of the olfactory system occur early in the disease. AD patients often report loss of olfactory sensory function. Further, histological examination of the Alzheimer's patients' epithelial layer showed dystrophic cytoskeletal changes (Hock et al., Eur Neurol, 40(l):31-6 (1998)) and increased oxidative damage to the cell (Ghanbari et al, Aging Cell 3(l):41-44 (2004), Perry et al., Acta Neuropathol 106(6):552-556 (2003)). The compromised epithelial lining may result in increased drug absorption in Alzheimer's patients compared to healthy subjects.
  • the pharmacokinetic parameters for the 15 mg dose strength in AL-108-110 study demonstrated increased systemic exposure of AL-108 in Alzheimer's disease patients compared to healthy subjects while the terminal half-life was unchanged.
  • the pharmacokinetic parameters achieved when administering the 30 mg dose strength to patients with compromised olfactory epithelial lining resulting from their neurodegenerative disease will be substantially higher than that obtained with the 15 mg dose seen in the AL-108-121 study.
  • this effect will also be seen in patients with neurodegenerative conditions and that the following calculated parameters will be seen.
  • the ratio between PK parameters for AD patients and healthy subjects (both from study AL- 108-110) is derived and multiplied by the PK parameters obtained with 30 mg in AD subjects from study AL-108-110.
  • the ratio between PK parameters for 30 mg versus 15 mg healthy subjects (both from study AL-108-121) is derived and multiplied by the PK parameters obtained for 15 mg in healthy subjects from study AL- 108- 121.

Abstract

This invention relates to the use of ADNF polypeptides in the treatment of neurodegenerative diseases based upon the identification of certain therapeutically effective pharmacokinetic parameters, dosing levels and formulations. The ADNF polypeptides include ADNF I and ADNF III (also referred to as ADNP) polypeptides, analogs, subsequences such as NAP and SAL, and D-amino acid versions (either wholly D-amino acid peptides or mixed D- and L-amino acid peptides), and combinations thereof which contain their respective active core sites.

Description

USE OF ADNF POLYPEPTIDES FOR TREATING NEURODEGENERATIVE
DISEASES
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit under 35 U.S.C. §119(e) of U.S.
Provisional Patent Application No. 61/368,489 filed July 28, 2010. The foregoing application is incorporated herein by reference in its entirety.
BACKGROUND
Field
This invention relates to the use of ADNF polypeptides in the treatment of neurodegenerative diseases. The present invention also relates to the manufacture of medicaments, methods of formulation and uses thereof. In particular, specific therapeutically effective pharmacokinetic parameters, dosing levels and formulations of the ADNF polypeptides useful in treating or preventing these diseases are described.
Description of the Related Art
NAP, an 8-amino-acid peptide (NAPVSIPQ = Asn-Ala-Pro-Val-Ser-Ile- Pro-Gin), is derived from a novel protein, activity-dependent neuroprotective protein,
ADNP (U.S. Pat. No. 6,613,740; Bassan et al., J. Neurochem. 72:1283-1293 (1999)).
The NAP sequence within the ADNP gene is identical in rodents and humans (U.S. Pat.
No. 6,613,740, Zamostiano et al., J. Biol. Chem. 276:708-714 (2001)). NAP is a potent neuroprotectant in a range of in vitro models (Gozes et al, CNS Drug Rev. 11(4):353- 68 (2005)) against a number of toxic insults including several relevant to neurodegenerative diseases such as amyloid beta peptides (Bassan et al., J. Neurochem.
72:1283-1293 (1999); Gozes et al., BMC Neurosci. (2008)), excitotoxicity (Bassan et al, J. Neurochem. 72:1283-1293 (1999)), and oxidative stress (Steingart et al., J. Mol.
Neurosci. 15(3): 137-45 (2000)). Further experiments identified NAP as a neurotrophic factor, stimulating neurite elongation and synapse formation (Smith-Swintosky et al., J.
Mol. Neurosci. 25(3):225-38 (2005)). NAP has been shown to be active in a number of transgenic mouse models of dementia including Alzheimer's disease. For instance, the triple transgenic mouse model of Alzheimer's disease expressing mutant APP (Swedish), tau (P301L), and presenilin-1 (Ml 46V) develops both neurofibrillary tangles and amyloid beta plaques in a progressive fashion (Oddo et al, Neuron 39(3):409-21 (2003)). Treatment of 12-month-old animals with an intranasal dose of 2 μg/day (-0.07 mg/kg/day) for 3 months resulted in a 70% decrease in phosphorylated tau at Ser202/Thr205, Thr231, and Ser202 residues (Matsuoka et al, J. Pharmacol Exp Ther. 325(l):146-53 (2008)).
Histological examination of the hippocampal CA1 region confirmed that NAP treatment resulted in a reduction of phosphorylated tau. Treatment of 9-month-old animals with an intranasal dose of 0.5 μg/day (-0.017 mg/kg/day) for 3 months resulted in a 30% to 40% decrease in phosphorylated tau (Matsuoka et al, J Mol Neurosci.
31(2): 165-70 (2007)).
In addition, NAP has been shown to reduce neurofibrillary tangles and tau hyperphosphorylation in a transgenic mouse model carrying double human tau mutations (P301S; K257T) (Shiryaev et al., Neurobiol Dis. 34(2):381-8 (2009)).
The neuroprotective effects of NAP have also been demonstrated in animal model expressing half the gene dosage of the parent protein of NAP, ADNP.
These ADNP heterozygous mice exhibit severe learning deficiencies which are ameliorated, in part, by intranasal NAP treatment. Tau hyperphosphorylation occurs in these ADNP deficient mice and is reduced with NAP treatment (Vulih-Shultzman et al.,
J. Pharmacol Exp Ther. 323(2):438-49 (2007)).
ApoE homozygous knock out mice are born with reduced cognitive abilities. Daily subcutaneous NAP injections to ApoE knockout mice results in accelerated acquisition of developmental reflexes as well as prevention of cholinergic deficits and short-term memory impairment compared to placebo groups (Bassan et al.,
J. Neurochem. 72:1283-1293 (1999)).
In the AF64A rat model, the acetylcholine producing neurons are selectively killed by injection of a toxin called AF64A (ethylcholine aziridium). This produces characteristic Alzheimer's disease-like cognitive impairment in the rats. Following AF64A toxin administration, intranasal administration of NAP provided significant improvements in short-term spatial memory measured in the Morris water maze (Gozes et al., J Pharmacol Exp Ther. 293(3):1091-1098 (2000)).
Furthermore, NAP treatment also reduced infarct volume and motor function deficits after ischemic injury in the spontaneous hypertensive rat model (animal model for stroke) (Leker et al., Stroke 33:1085-1092 (2002)) and reduced damage and inflammation in the closed head injury in mice (Beni Adani et al., J. Pharmacol. Exp. Ther. 296:57-63 (2001); Romano et al., J Mol. Neurosci. 18:3745 (2002); Zaltzman et al, NeuroReport 14:481-484 (2003)). In a model of fetal alcohol syndrome, fetal death after intraperitoneal injection of alcohol was inhibited by NAP treatment (Spong et al., J Pharmacol. Exp. Ther. 297:774-779 (2001); see also WO 00/53217).
Utilizing radiolabeled peptides these studies showed that NAP can cross the blood-brain barrier and can be detected in rodents' brains either after intranasal treatment (Gozes et al., J Pharmacol Exp Ther. 293(3): 1091 -8 (2000)) or intravenous injection (Leker et al., Stroke 33:1085-1092 (2002)) or intraperitoneal administration (Spong et al., 2001).
SAL, a 9-amino acid peptide (SALLRSIPA = Ser-Ala-Leu-Leu-Arg-Ser- lle-Pro-Ala), also known as ADNF-9 or ADNF-1, was identified as the shortest active form of ADNF (U.S. Patent No. 6,174,862). SAL has been shown in in-vitro assays and in vivo disease models to keep neurons of the central nervous system alive in response to various insults (Gozes et al., J Pharmacol Exp Ther. 293(3):1091-8 (2000); Brenneman et al., J Pharmacol. Exp. Ther. 285:619-627 (1998)). D-SAL is an all D- amino acid derivative of SAL that is stable and orally available (Brenneman et al., J Pharmacol Exp Ther. 309:1190-7 (2004)) and surprisingly exhibits similar biological activity (potency and efficacy) to SAL in the systems tested. ADNF-1 complexes are described in International PCT Application No, PCT7US02/29146, filed September 12, 2002 (published as WO 03/022226).
ADNF polypeptides, including NAP and SAL, and uses thereof in neuroprotection against disorders of the central nervous system, are the subject of numerous patents and patent applications including International PCT Publication No. WO 01/92333; U.S. Application No. 07/871,973 filed April 22, 1992, now U.S. Patent No. 5,767,240; U.S. Application No. 08/342,297 filed October 17, 1994 (published as WO 96/11948), now U.S. Patent No. 6,174,862; U.S. Application No. 60/037,404 filed February 7, 1997 (published as WO 98/35042); U.S. Application No. 09/187,330 filed November 11, 1998 (published as WO 00/27875); U.S. Application No. 09/267,511 filed March 12, 1999 (published as WO 00/53217); U.S. Patent No. 6,613,740; U.S. Application No, 60/149,956 filed August 18, 1999 (published as WO 01/12654); U.S. Application No, 60/208,944 filed May 31, 2000; U.S. Application No. 60/267,805 filed February 8, 2001; International PCT Application No. PCT/IL2004/000232 filed March 11, 2004 (published as WO 2004/080957); International PCT Application No. PCT/US02/29146 filed September 12, 2002 (published as WO 2003/022226); and U.S. Patent Application No. 61/141,588 filed December 30, 2008, each of which are incorporated by reference in their entirety.
Accordingly, while progress has been made in this field, there remains a need for new chemical entities for the treatment of neurodegenerative diseases, and for improved therapeutic treatments using known chemical entities. In particular, improved therapeutic treatments based upon therapeutically effective pharmacokinetic parameters, dosing levels and formulations. The present disclosure fulfils these needs and provides further related advantages.
BRIEF SUMMARY
In brief, the present invention is directed to various therapeutically effective pharmacokinetic parameters, dosing levels and formulations of ADNF polypeptides useful in treating or preventing neurodegenerative diseases.
In one aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
In another aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10" M for a period of time. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10" to about 10" M for a period of time. In more specific embodiments, the period of time is at least about 24 hours.
In another aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL. In more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL. In other more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL. In other more specific embodiments, the total maximum concentration (Cma ) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
In another aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In more specific embodiments, the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
In another aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-τ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-τ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUC0-T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/n L. In other more specific embodiments, the AUCo-τ of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL. In other more specific embodiments, the AUC0-x of the ADNF polypeptide is equal to or greater than about
Figure imgf000008_0001
In another aspect, a method for administration of an ADNF polypeptide to a subject is disclosed, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-oo of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUC0-∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL. In other more specific embodiments, the AUC0-0o of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL. In further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses (e.g., sprays) to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
In other further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10"12 M for a period of time. In more specific embodiments, the period of time is at least about 24 hours.
In other further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL. In more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL. In other more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL. In other more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
In other further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In more specific embodiments, the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
In other further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses to produce a AUC0-T of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUC0-x of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL. In other more specific embodiments, the AUCO-T of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL. In other more specific embodiments, the AUCo-τ of the ADNF polypeptide is equal to or greater than about 5.8 h*ng/mL. In other further embodiments of the above aspects, the ADNF polypeptide is administered to the subject in one or more doses to produce a AUC0.∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUC0-∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL. In other more specific embodiments, the AUC0-oo of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL.
In other further embodiments of the above aspects, the method comprises intranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride. In further embodiments, the composition has an iso-osmolarity of about 250 to about 375 mOsm.
In another aspect, a liquid composition for intranasal administration of an ADNF polypeptide is disclosed, the composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
In another aspect, a method for intranasal administration of an ADNF polypeptide to a subject is disclosed, the method comprising intranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride. In a related aspect, a method of treating or preventing a neurodegenerative disease in a subject is disclosed, the method comprising the step of instranasally administering to the subject in one or more doses a liquid composition comprising: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride.
In further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
In other further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10"12 M for a period of time. In more specific embodiments, the period of time is at least about 24 hours.
In other further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL. In more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL. In other more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 6.8 ng/mL. In more specific embodiments, the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 8.2 ng/mL.
In other further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr. In more specific embodiments, the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
In other further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to produce a AUCo-τ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUCo-τ of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL. In other more specific embodiments, the AUCQ-T of the ADNF polypeptide is equal to or greater than about 4.6 h*ng/mL. In other more specific embodiments, the AUC0-x of the ADNF polypeptide is equal to or greater than about 5.8 h*ng/mL.
In other further embodiments of the above aspects, the composition is intranasally administered to the subject in one or more doses to produce a AUC0.oo of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL. In more specific embodiments, the AUCo-∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL. In other more specific embodiments, the AUC0-∞ of the ADNF polypeptide is equal to or greater than about 5.3 h*ng/mL.
In other further embodiments of the above aspects, the composition has an iso-osmolarity of about 250 to about 375 mOsm.
In other further embodiments of all of the above aspects, the ADNF polypeptide may be selected as follows.
In one embodiment, the ADNF polypeptide is a member selected from the group consisting of: (a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence: Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l); (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2); and (c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b).
In one embodiment, the ADNF polypeptide is a member selected from the group consisting of a full length ADNF I polypeptide, a full length ADNF III polypeptide (ADNP), and a mixture of a full length ADNF I polypeptide and a full length ADNF III polypeptide.
In one embodiment, the ADNF polypeptide is an ADNF I polypeptide. In another embodiment, the ADNF polypeptide is a full length ADNF I polypeptide. In another embodiment, the ADNF polypeptide has the formula (R!)x- Ser-Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala -(R )y (SEQ ID NO: 14) in which: R is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one. In another embodiment, the ADNF I polypeptide is Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l). In another embodiment, the active core site of the ADNF I polypeptide comprises at least one D-amino acid. In another embodiment, the active core site of the ADNF I polypeptide comprises all D- amino acids. In another embodiment, the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site. In another embodiment the ADNF I polypeptide is selected from the group consisting of:
Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID
NO:3);
Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser- Ile-Pro-Ala (SEQ ID NO:4);
Leu-Gly-Gly-Gly-Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID
NO:5);
Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:6); Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:7);
Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:8); and
Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1).
In one embodiment, the ADNF polypeptide is an ADNF III polypeptide. In another embodiment, the ADNF polypeptide is a full length ADNF III polypeptide. In another embodiment, the the ADNF polypeptide has the formula (R^x-Asn-Ala-Pro- Val-Ser-Ile-Pro-Gln-(R2)y (SEQ ID NO: 13) in which: R1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one. In another embodiment, the ADNF III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2). In another embodiment, the active core site of the ADNF III polypeptide comprises at least one D- amino acid. In another embodiment, the active core site of the ADNF III polypeptide comprises all D-amino acids. In another embodiment, the ADNF III polypeptide comprises up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site. In another embodiment, the ADNF III polypeptide is a member selected from the group consisting of:
Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:9);
Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO:10);
Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ
ID O:l l);
Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln- Gln-Ser (SEQ ID NO: 12); and
Asn- Ala-Pro- Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
In one embodiment, at least one of the ADNF polypeptides is encoded by a nucleic acid that is administered to the subject.
In one embodiment, an ADNF I polypeptide and an ADNF III polypeptide are administered to the subject. In another embodiment, either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise at least one D-amino acid. In another embodiment, either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise all D-amino acids. In another embodiment, the ADNF I polypeptide is Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l), and the ADNF III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2). In another embodiment, the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF I polypeptide, and wherein the ADNF III polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF III polypeptide. In another embodiment, the ADNF I polypeptide is a member selected from the group consisting of: Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID
NO:3);
Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser- Ile-Pro-Ala (SEQ ID NO:4);
Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID
NO:5);
Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:6); Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:7);
Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 8); and
Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1);
and the ADNF III polypeptide is selected from the group consisting of:
Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:9); Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO:
10);
Leu-Gly-Leu-Gly-Gly- Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ
ID NO: 11);
Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln- Gln-Ser (SEQ ID NO: 12); and
Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
In other further embodiments of all of the above aspects, the subject suffers from a neurodegenerative disease. In a further embodiment, the neurodegenerative disease is Alzheimer's disease, corticobasal ganglionic degeneration, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, amyotrophic lateral sclerosis, Pick's atrophy, diffuse Lewy body disease, a neurodegenerative pathology associated with aging, a pathological change resulting from a focal trauma, peripheral neuropathy, retinal neuronal degeneration, or dopamine toxicity. In another further embodiment, the neurodegenerative disease is neurodegeneration associated with schizophrenia.
These and other aspects of the invention will be apparent upon reference to the following detailed description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a process flow chart for clinical trial manufacture for AL-108 Nasal Spray, 25 mg/mL.
Figure 2 shows a process flow chart for clinical trial manufacture for
AL-108 Nasal Spray, 75 mg/mL.
Figures 3 A and 3B show a comparison of PK concentrations for QD and BID administration of 15 mg. The line shows concentrations based on the Cmax and half-life generated for the 15 mg dose of AL-108 in study AL-108-121. PK modeling using these parameters was carried out on a single daily dose (Figure 3 A) and two doses separated by 12 hours (Figure 3B). Data is shown for a continuous 48 hour period.
Figure 4 shows a PK model comparison of AL-108 concentrations based on Cmax and half-lives obtained in studies AL-208-110 and AL-108-121. The triangles show concentrations modeled from the Cmax and half-life generated for the 30 mg dose in study AL-108-121. The squares represent concentrations predicted for a 30 mg dose based on the Cmax and half- life obtained from the AL-208-110 study in which healthy volunteers were given 15 mg. For this predicted curve, the Cmax from study AL-108- 110 was doubled and the actual half-life of 0.8 h used.
Figure 5 shows PK parameters corrected for drug dose.
DETAILED DESCRIPTION
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention may be practiced without these details.
Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is as "including, but not limited to". Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Definitions
As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated.
The phrase "ADNF polypeptide" refers to one or more activity dependent neurotrophic factors (ADNF) that have an active core site comprising the amino acid sequence of SALLRSIPA (referred to as "SAL") or NAPVSIPQ (referred to as "NAP" or "AL-108" or "davunetide"), or conservatively modified variants thereof that have neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., Brain Res. 603:222-233 (1993); Brenneman & Gozes, J. Clin. Invest 97:2299-2307 (1996), Forsythe & Westbrook, J. Physiol. Land. 396:515 (1988). An ADNF polypeptide can be an ADNF I polypeptide, an ADNF III polypeptide, their alleles, polymorphic variants, analogs, interspecies homolog, any subsequences thereof (e.g., SALLRSIPA or NAPVSIPQ) or lipophilic variants that exhibit neuroprotective/neurotrophic action on, e.g., neurons originating in the central nervous system either in vitro or in vivo. An "ADNF polypeptide" can also refer to a mixture of an ADNF I polypeptide and an ADNF III polypeptide.
The term "ADNF I" refers to an activity dependent neurotrophic factor polypeptide having a molecular weight of about 14,000 Daltons with a pi of 8.3±0.25. As described above, ADNF I polypeptides have an active site comprising an amino acid sequence of Ser- Ala-Leu-Leu- Arg-Ser-IlePro-Ala (also referred to as "SALLRSIPA" or "SAL" of "ADNF-9"). See Brenneman & Gozes, J Clin. Invest. 97:2299-2307 (1996), Glazner et al., Anat. Embryol. (Berl), 200; 65-71 (1999), Brenneman et al., J Pharm. Exp. Ther. 285:619-27 (1998), Gozes & Brenneman, J. Mol. Neurosci. 7:235-244 (1996), and Gozes et al., Dev. Brain Res. 99: 167175 (1997), all of which are herein incorporated by reference. Unless indicated as otherwise, "SAL" refers to a peptide having an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala, not a peptide having an amino acid sequence of Ser-Ala-Leu. A full length amino acid sequence of ADNF I can be found in WO 96/11948 herein incorporated by reference in its entirety.
The phrase "ADNF III polypeptide" or "ADNF ΙΠ" also called activity- dependent neuroprotective protein (ADNP) refers to one or more activity dependent neurotrophic factors (ADNF) that have an active core site comprising the amino acid sequence of NAPVSIPQ (referred to as "NAP" or "AL-108" or "davunetide"), or conservatively modified variants thereof have neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., Brain Res. 603:222-233 (1993); Gozes et al, Proc. Natl. Acad. Set USA 93:427-432 (1996). An ADNF polypeptide can be an ADNF III polypeptide, allelic or polymorphic variant, analog, interspecies homolog, or any subsequences thereof (e.g., NAPVSIPQ) that exhibit neuroprotective/neurotrophic action on, e.g., neurons originating in the central nervous system either in vitro or in vivo. ADNF III polypeptides can range from about eight amino acids and can have, e.g., between 8-20, 8-50, 10-100 or about 1000 or more amino acids.
Full length human ADNF III has a predicted molecular weight of
123,562.8 Da (>1000 amino acid residues) and a pi of about 6.97. As described above, ADNF III polypeptides haven active site comprising an amino acid sequence of Asn- Ala-Pro-Val-Ser-Ile-Pro-Gln (also referred to as "NAPVSIPQ" or "NAP" or "AL-108" or "davunetide"). See Zamostiano et al., J. Biol. Chem. 476:708-714 (2001) and Bassan et al., J Neurochem. 72: 1283-1293 (1999), each of which is incorporated herein by reference. Unless indicated as otherwise, "NAP" refers to a peptide having an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln, not a peptide having an amino acid sequence of Asn- Ala-Pro. Full-length amino acid and nucleic acid sequences of ADNF III can be found in WO 98/135042, WO 00/27875, U.S. Pat. Nos. 6,613,740 and 6,649,411. The Accession number for the human sequence is NP 852107, see also Zamostiano et al., supra.
The term "subject" refers to any mammal, in particular human, at any stage of life.
The ADNF polypeptides or nucleic acids encoding them of the present invention can be "administered" by any conventional method such as, for example, parenteral, oral, topical, and inhalation routes. In some embodiments, parenteral and nasal inhalation routes are employed.
A "neurodegenerative disease" refers to a condition (disease and/or insult) associated with neuronal cell death and/or sub-lethal neuronal pathology, including, but not limited to, thos arising from a disease stat and/or having an excitotoxic/ischemic mechanism. These diseases include, but are not limited to:
diseases of central motor systems including degenerative conditions affecting the basal ganglia (Huntington's disease, Wilson's disease, striatonigral degeneration, corticobasal ganglionic degeneration), Tourette's syndrome, Parkinson's disease, progressive supranuclear palsy (PSP), progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration, and dopamine toxicity;
diseases affecting sensory neurons such as Friedreich's ataxia, diabetes, peripheral neuropathy, retinal neuronal degeneration;
diseases of limbic and cortical systems such as cerebral amyloidosis, Pick's atrophy, Retts syndrome;
neurodegenerative pathologies involving neuronal systems and/or brainstem including Alzheimer's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, epilepsy, multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, late-degenerative stages of Down's syndrome, Alper's disease, vertigo as result of CNS degeneration;
pathologies associated with developmental retardation and learning impairments, Down's syndrome, and oxidative stress induced neuronal death; pathologies arising with aging and chronic alcohol or drug abuse including, for example, with alcoholism the degeneration of neuron in locus coeruleus, cerebellum, cholinergic basal forebrain; with aging degeneration of cerebellar neurons and cortical neurons leading to cognitive and motor impairments; and with chronic amphetamine abuse degeneration of basal ganglia neurons leading to motor impairments;
pathological changes resulting from focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or direct trauma;
pathologies arising as a negative side-effect of therapeutic drugs and treatments (e.g., degeneration of cingulate and entorhinal cortex neurons in response to anticonvulsant doses of antagonists of the NMDA class of glutamate receptor);
neurodegeneration associated with a mental disease;
peripheral neurotoxicity, including, but not limited to, peripheral neurotoxicity resulting from treatment with one or more chemical agents (e.g., chemical agents for cancer, multiple sclerosis, gout, arthritis, Behcet's disease, psychiatric disorder, immunosuppression and infectious disease, such as vinca alkaloids, platinum drugs, L-asparaginase, taxanes, thalidomide, methotrexate, colchicines, and anti- infective agents); and
laser-induced retinal damage, laser photocoagulation or conditions involving retinal degeneration.
A "mental disorder" or "mental illness" or "mental disease" or "psychiatric or neuropsychiatric disease or illness or disorder" refers to mood disorders (e.g., major depression, mania, and bipolar disorders), psychotic disorders (e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, delusional disorder, brief psychotic disorder, and shared psychotic disorder), personality disorders, anxiety disorders (e.g., obsessive-compulsive disorder and attention deficit disorders) as well as other mental disorders such as substance-related disorders, childhood disorders, dementia, autistic disorder, adjustment disorder, delirium, multi-infarct dementia, and Tourette's disorder as described in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV). Typically, such disorders have a complex genetic and/or a biochemical component.
A "mood disorder" refers to disruption of feeling tone or emotional state experienced by an individual for an extensive period of time. Mood disorders include major depression disorder (i.e., unipolar disorder), ma, dysphoria, bipolar disorder, dysthymia, cyclothymia and many others. See, e.g., Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
"Major depression disorder," "major depressive disorder," or "unipolar disorder" refers to a mood disorder involving any of the following symptoms: persistent sad, anxious, or "empty" mood; feelings of hopelessness or pessimism; feelings of guilt, worthlessness, or helplessness; loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex; decreased energy, fatigue, being "slowed down"; difficulty concentrating, remembering, or making decisions; insomnia, early-morning awakening, or oversleeping; appetite and/or weight loss or overeating and weight gain; thoughts of death or suicide or suicide attempts; restlessness or irritability, or persistent physical symptoms that do not respond to treatment, such as headaches digestive disorders, and chronic pain. Various subtypes of depression are described in, e.g., DSM
IV.
"Bipolar disorder" is a mood disorder characterized by alternating periods of extreme moods. A person with bipolar disorder experiences cycling of moods that usually swing try being overly elated or irritable (mania) to sad and hopeless (depression) and then back again, with periods of normal mood in between. Diagnosis of bipolar disorder is described in, e.g., DSM IV. Bipolar disorders include bipolar disorder I (mania with or without major depression) and bipolar disorder II (hypomania with major depression), see, e.g., DSM IV.
"Anxiety," "anxiety disorder," and "anxiety-related disorder refer to psychiatric syndromes characterized by a subjective sense of unease, dread, or foreboding, e.g., panic disorder, generalized anxiety disorder, attention deficit disorder, attention deficit hyperactive disorder, obsessive-compulsive disorder, and stress disorders, e.g., acute and post-traumatic. Diagnostic criteria for these disorders are well known to those of skill in the art (see, e.g., Harrison's Principles of Internal Medicine, pp. 2486-2490 (Wilson et al., eds., 12th ed. 1991) and DSM IV).
"Neurotoxicity" as used herein is defined as adverse effects on the structure or functioning of the cells of the nervous system that result from exposure to chemical substances or to disease processes. Among other things, neurotoxicants can cause morphological changes that lead to generalized damage to nerve cells (neuronopathy), injury to axons (axonopathy), or destruction of the myelin sheath (myelinopathy). It is well established that exposure to certain chemotherapeutic agents, agricultural and industrial chemicals can damage the nervous system, resulting in neurological and behavioral dysfunction. Symptoms of neurotoxicity include muscle weakness, loss of sensation and motor control, tremors, alterations in cognition, and impaired functioning of the autonomic nervous system. Neurotoxicological assessments use a battery of functional and observational tests. Neurotoxicity in humans is most commonly measured by neurological tests that assess cognitive, sensory, and motor function.
"Peripheral neurotoxicity" refers to neurotoxicity of the peripheral nervous system (PNS). The PNS includes all the nerves not in the brain or spinal cord, and includes the dorsal root ganglia (DRG). These nerves carry sensory information and motor impulses. Damage to the nerve fibers of the PNS can disrupt communication between the CNS and the rest of the body. Peripheral neurotoxicity is also sometimes referred to in the literature as peripheral neuropathy, and can include hundreds of identifiable conditions, as further described below. "Peripheral neuropathy" encompasses a wide range of conditions in which the nerves outside of the brain and spinal cord have been damaged, and may include crush injury and section.
"Central nervous system" or "CNS" means the brain and spinal cord.
"Laser-induced retinal damage" includes both intentional and accidental retinal damage resulting from laser exposure.
"Laser photocoagulation" refers to a surgical procedure wherein a laser is used to seal leaking blood vessels within the retina and reduce the future growth of abnormal blood vessels. By sealing leaking blood vessels, laser photocoagulation slows down, e.g., the buildup of fluid under the retina that distors the shape and position of the macula, the growth of scar tissue and the abnormal membrane under the retina that damage the cells in the macula, and central vision loss.
"Conditions involving retinal degeneration" include, but are not limited to, laser-induced retinal damage and ophthalmic diseases, such as glaucoma, Retinitis pigmentosa, Usher syndrome, artery or vein occlusion, diabetic retinopathy, retrlental fibroplasias or retinopathy of prematurity (R.L.F./R.O.P), retinoschisis, lattic degeneration, macular degeneration and ischemic optic neuropathy.
The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Generally, a peptide refers to a short polypeptide. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereofm either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 20-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. For the purposes of this application, amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. For the purposes of this application, amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may include those having non-naturally occurring D- chirality, as disclosed in WO 01/12654, incorporated herein by reference, which may improve oral availability and other drug like characteristics of the compound. In such embodiments, one or more, and potentially all of the amino acids of NAP or the ADNF- polypeptide will have D-chirality. The therapeutic use of peptides can be enhanced by using D-amino acids to provide longer half life and duration of action. However, many receptors exhibit a strong preference for L-amino acids, but examples of D-peptides have been reported that have equivalent activity to the naturally occurring L-peptides, for example, pore-forming antibiotic peptides, beta amyloid peptide (no change in toxicity), and endogenous ligands for the CXCR4 receptor. In this regard, NAP and ADNF polypeptides also retain activity in the D-amino acid form (Brenneman et al, J Pharmacol. Exp. Ther. 309(30):1190-1197 (2004)).
Amino acids may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The amino acids referred to herein are described by shorthand designations as follows: TABLE 1
Figure imgf000025_0001
"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al, Mol. Cell Probes 8:19-98 (1994)). Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent a variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar-amino acid. Conservative substitution-tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following groups each contain amino acids that are conservative substitutions for one another:
1 ) Alanine (A), Glycine (G);
2) Serine (S), Threonine (T);
3) Aspartic acid (D), Glutamic acid (E);
4) Asparagine (N), Glutamine (Q);
5) Cysteine (C), Methionine (M);
6) Arginine (R), Lysine (K), Histidine (1);
7) Isoleucine (I), Leucine (L), Valine (V); and
8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,
Creighton, Proteins (1984)).
One of ordinary skill in the art will appreciate that many conservative variations of the nucleic acid and polypeptide sequences provided herein yield functionally identical products. For example, due to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions of a nucleic acid sequence that do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence that encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid-sequence are substituted with different amino acids with highly similar properties (see the definitions section, supra), are. also readily identified as being highly similar to a disclosed no acid sequence, or to a disclosed nucleic acid sequence that encodes an amino acid. Such conservatively substituted variations of each explicitly listed nucleic acid and amino acid sequences are a feature of the present invention.
The terms "isolated," "purified" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated ADNF nucleic acid is separated from open reading frames that flank the ADNF gene and encode proteins other than ADNF. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
"An amount sufficient" or "an efficient amount" or a "therapeutically effective amount" is that amount of a given ADNF polypeptide that exhibits the activity of interest or which provides either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In therapeutic applications, the NAP or ADNF polypeptides of the invention are administered to a patient in an amount sufficient to reduce or eliminate symptoms of a neurodegenerative disease. An amount adequate to accomplish this is defined as the "therapeutically effective dose." The dosing range varies with the NAP or ADNF polypeptide, as further set out below, and in CA patent 2202496, U.S. Pat. No. 6,174,862 and U.S. Pat. No. 6,613,740, herein incorporated by reference in their entirety.
"Inhibitors," "activators," and "modulators" of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonist and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactive, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of polypeptide or polynucleotide of the invention, e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity. Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of the effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
ADNF Polypeptides
In one embodiment the ADNF polypeptides of the present invention comprise the following amino acid sequence: (R^x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln- (R2)y (SEQ ID NO: 13) and conservatively modified variations thereof. In the foregoing formula, R1 denotes the orientation of the amino terminal (NH2 or N- terminal) end and R2 represents the orientation of the carboxyl terminal (COOH or C- terminal) end.
In the above formula, R1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino, acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R1 may be identical or different (e.g., all of the amino acids making up the amino sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acids sequence R1 include, but are not limited to, those listed in Table 1, infra. The indexes "x" and "y" are independently selected and can be equal to one or zero. As with R1, R2 in the above formula, is an amino acid sequence comprising from 1 to 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid
1 9 analogs. Moreover, as with R , the amino acids making up the amino acid sequence R may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R2 include, but are not limited to, those listed in Table 1 , infra.
As used herein, "NAP" or "NAP peptide" refers to the formula above where x and y both equal 0. "NAP related peptide" refers to any of the other variants of NAP which are described the formula.
1 9 1 9
R and R are independently selected. If R and R are the same, they are identical in terms of both chain length and amino acid composition. For example, both R1 and R2 may be Val-Leu-Gly-Gly-Gly- (SEQ ID NO: 15). If R1 and R2 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences. For example, R1 may be Val-Leu-Gly-Gly-Gly- (SEQ ID NO: 16), whereas R2 may be Val-Leu-Gly-Gly- (SEQ ID NO: 17). Alternatively, R1 may be Val-Leu-Gly-Gly- (SEQ ID NO: 18).
Within the scope of the above formula, certain NAP and NAP related polypeptide are also preferred, namely those in which x and y are both zero (i.e. NAP). Equally preferred are NAP and NAP related polypeptides in which x is one; R1 is Gly- Gly-; and y is zero (SEQ ID NO: 19). Also equally preferred are NAP and NAP related polypeptides in which x is one; R1 is Leu-Gly-Gly-; y is one; and R2 is -Gln-Ser (SEQ ID NO:20). Also equally preferred are NAP and NAP related polypeptides in which x is one; R1 is Leu-Gly-Leu-Gly-Gly- (SEQ ID NO: 21); y is one; and R2 is -Gln-Ser (SEQ ID NO:22). Also equally preferred are NAP and NAP related polypeptides in which x is one; R1 is Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly- (SEQ ID NO:23); y is one; and R2 is - Gln-Ser (SEQ ID NO:24). Additional amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity. In another aspect, the present invention provides pharmaceutical compositions comprising one of the previously described NAP and NAP related polypeptides in an amount sufficient to exhibit the desired activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the NAP or NAP related peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 9-12, and conservatively modified variations thereof.
In another embodiment, the ADNF polypeptide comprises the following amino acid sequence: (R 1 )x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R 2 )y (SEQ ID NO: 14) and conservatively modified variations thereof. In this formula, R1 denotes the orientation of the amino terminal (NH2 or N-terminal) end and R2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.
In the above formula, R1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc). Moreover, as previously explained, the amino acid making up the amino acid sequences R1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acid sequence R1 include, but are not limited to, those listed in Table 1, infra. The indexes "x" and "y" are independently selected and can be equal to one or zero.
As with R1, R2 in the above formula, is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected form the groups consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R1, the amino acids making up the amino acid sequence R2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R2 include, but are not limited to those listed in Table 1, infra.
As used herein, "SAL" or "SAL peptide" refers to the formula above where x and y both equal 0. "SAL related peptide"refers to any of the other variants of SAL which are described the formula.
As in the above embodiments wherein the ADNF poypeptides comprise
1 ^ the following amino acid sequence: (R )x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R )y (SEQ ID NO: 14), in the present embodiments, R1 and R2 are independently selected. If
1
R and Rz are the same, they are identical in terms of both chain length and amino acid composition. Additional amino acids can be added to both the N-terminus and the C- terminus of the active peptide without loss of biological activity.
In an another aspect, the present invention provides pharmaceutical compositions comprising one of the previously described SAL and SAL-related polypeptides in an amount sufficient to exhibit the desired activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the SAL or SAL related peptide has an amino acid sequence selected form the group consisting of SEQ ID NOS: 1 and 3-8, and conservatively modified variations thereof.
Design and Synthesis of ADNF Polypeptides
Polypeptides and peptides comprising the core NAPVSIPQ or SALLRSIPA active site can be easily made, e.g., by the synthetic chemical process of systematically adding one amino acid at a time and screening the resulting peptide for biological activity, as described herein. In addition, the contributions made by the side chains of various amino acids residues in such peptides can be probed via a systematic scan with a specified amino acid, e.g., Ala.
One of skill will recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells continuing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see Giliman & Smith, Gene 8:81-97 (1979); Roberts &Χ ., Nature 1328:731-734 (1987)).
Most commonly, polypeptide sequences are altered by changing the corresponding nucleic acid sequence and expressing the polypeptide. However, polypeptide sequences are also optionally generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide (see Merrifield, Am. Chem. Soc. 85:2149-2154 (1963); Stewart & Young, Solid Phase Peptide Synthesis (2nd ed. 1984)).
One of skill can select a desired nucleic acid or polypeptide of the invention based upon the sequences provided and upon knowledge in the art regarding proteins generally. Knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids and polypeptides disclosed herein. The definitions section, supra, describes exemplar conservative amino acid substitutions.
Modifications to the ADNF polypeptides are evaluated by routine techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of a polypeptide can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a target nucleic acid, redox or thermal stability of a protein, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
Using these assays and models, one of ordinary skill in the art can readily prepare a large number of ADNF polypeptides in accordance with the teachings of the present invention and, in turn, screen them using the foregoing animal models to find ADNF polypeptides, in addition to those set forth herein, which possess the desired activity. For instance, using the NAP peptide (i.e., Asn- Ala-Pro- Val-Ser-Ile-Pro-Gln (SEQ ID NO:2)) or SAL peptide (i.e., Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l)) as a starting point, one can systematically add, for example, Gly-, Gly-Gly-, Leu-Gly-Gly- to the N-terminus of the peptide and, in turn, screen each of these NAP or ADNP polypeptides in the foregoing assay to determine whether the desired activity. In doing so, it will be found that additional amino acids can be added to both the N- terminus and the C-terminus of the active site, i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2) or Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l), without loss of biological activity.
The peptides of the invention may be prepared via a wide variety of well-known techniques. Peptides of relatively short size are typically synthesized on a solid support or in solution in accordance with conventional techniques (see, e.g., Merrifield et ah, Am. Chem. Soc. 85:2149-2154 (1963)). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis (2nd 1984)). Solid phase synthesis in which the C-terminal amino acid of die sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the peptides of this invention. Techniques for solid phase synthesis are described by Barany & Merrifield, Solid-Phase Peptid Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al., 1963; Stewart et al., 1984). NAP and related peptides are synthesized using standard Fmoc protocols (Wellings & Atherton, Methods Enzymol. 289:44-67 (1997)).
In addition to the foregoing techniques, the peptides for use in the invention may be prepared by recombinant DNA methodology. Generally, this involves creating a nucleic acid sequence that encodes the protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, and expressing the protein in a host cell. Recombinantly engineered cells known to those of skill in the art include, but are not limited of bacteria, yeast, plant, filamentous fungi, insect (especially employing baculoviral vectors) of mammalian cells.
The recombinant nucleic acids are operably linked to appropriate control sequences for expression in the selected host. For E. coli, example control sequences include the T7, tip, or lambda promoters, a ribosome binding site and, preferably, a transcription termination signal. For eukaryotic cells, the control sequences typically include a promoter and, preferably, an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
The plasmids of the invention can be transferred into the chosen host cell by well-known methods. Such methods include, for example, the calcium chloride transformation method for E. coli and the calcium phosphate treatment or electroporation methods for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo, and hyg genes.
Once expressed, the recombinant peptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Polypeptide Purification (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Polypeptide Purification (1990)). Once purified, partially or to homogeneity as desired, the NAP and ADNF polypeptides may then be used, e.g., to prevent neuronal cell death or as immunogens for antibody production. Optional additional steps include isolating the expressed protein to a higher degree, and, if required, cleaving or otherwise modifying the peptide, including optionally generating the protein.
After chemical synthesis, biological expression or purification, the peptide(s) may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is helpful to denature and reduce the peptide and then to cause the peptide to re-fold into the preferred conformation. Methods of reducing and denaturing peptides and inducing re-folding are well known to those of skill in the art (see Debinski et al, Biol Chem. 268:14065- 14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:585 (1993); and Buchner et al, Anal Biochem. 205:263-270 (1992)). Debinski et al., for example, describe the denaturation and reduction of inclusion body peptides in guanidine-DTE. The peptide is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
One of skill will recognize that modifications can be made to the peptides without diminishing their biological activity. Some modifications' may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion peptide. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
Pharmacokinetic Parameters and Dosing Levels
As noted above, the present invention provides a method for administration of an ADNF polypeptide to a subject, wherein the method comprises administering the ADNF polypeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period. In a related aspect, the present invention provides a method of treating or preventing a neurodegenerative disease in a subject, wherein the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
As shown in the Examples below, this dosing level (i.e., 30 mg BID) has been found to produce certain surprising effects on various pharmacokinetic parameters, including, the total maximum concentration (Cmax), half-life, AUCo-τ and AUCo-oo, of an ADNF polypeptide in the blood plasma of a subject.
Accordingly, as also noted above, the present invention also provides methods for administration of an ADNF polypeptide to a subject and methods of treating or preventing a neurodegenerative disease in a subject, wherein the methods comprise administering the ADNF polpeptide to the subject in one or more doses to produce one or more of these pharmacokinetic effects. More specifically, the present invention provides methods wherein the methods comprise administering the ADNF polypeptide to the subject in one or more doses to:
(i) produce in the blood plasma of the subject a total concentration
1 1 12
of the ADNF polypeptide equal to or greater than about 10" to about 10" M for a period of time (including methods wherein the period of time is at least about 24 hours);
(ii) produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL (including methods wherein the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL, equal to or greater than about 6.8 ng/mL, or equal to or greater than about 8.2 ng/mL);
(iii) produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr (including methods wherein the half- life of the ADNF polypeptide is equal to or greater than about 1.4 hr);
(iv) produce a AUCo-τ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL (including methods wherein the AUCO-T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL, equal to or greater than about 4.6 h*ng/mL, or equal to or greater than about 5.8 h*ng/mL); and/or
(v) produce a AUCo-oo of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL (including methods wherein the AUCo-∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL, or equal to or greater than about 5.3 h*ng/mL).
In these and other therapeutic applications, the ADNF polypeptides of the invention may be administered to a patient in any amount sufficient to produce one or more of these pharmacokinetic effects. Amounts effective for this use will depend on, for example, the particular ADNF polypeptide employed, the manner of administration, the weight and general state of health ofthe patient, and the judgement of the prescribing physician. For example, an amount of polypeptide falling within the range of a 100 ng to 30 mg dose given intranasally once a day (e.g., in the evening) or twice daily would be an effective amount. Alternatively, dosages may be outside of this range, or on a different schedule. For example, dosages may range from 0.0001 mg/kg to 10,000 mg/kg, and will preferably be about 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 50 mg/kg or 500 mg/kg per dose. Doses may be administered hourly, every 4, 6 or 12 hours, with meals, daily, every 2, 3, 4, 5, 6, for 7 days, weekly, every 2, 3, 4 weeks, monthly or every 2, 3 or 4 months, or any combination thereof. The duration of dosing may be single (acute) dosing, or over the course of days, weeks, months, or years, depending on the condition to be treated. Those skilled in the art can determine the suitable dosage, and may rely on preliminary data reported in Gozes et al., J Pharmacol Exp Ther. 293(3):1091-8 (2000), Gozes et al., J Mol Neurosci. 19(1-2): 167- 70 (2002), Bassan et al., J Neurochem. 72:1283-1293 (1999),, Zemlyak et al., Regul. Pept. 96:39-43 (2000); Brenneman et al, Biochem, Soc. Trans. 28:452-455 (2000); Erratum Biochem Soc. Trans. 28:983; Wilkemeyer et al., Proc Natl. Acad, Sci. USA 100:3543-8548 (2003).
Pharmaceutical Formulations
As discussed above, the present invention provdes a liquid composition for intranasal administration of an ADNF polypeptide, wherein the composition comprises: (a) about 75 mg/mL of the ADNF polypeptide; (b) about 0.17% (w/w) citric acid monohydrate; (c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and (d) about 0.005% benzalkonium chloride, wherein the composition comprises essentially no sodium chloride. In various embodiments, such composition has an iso-osmolarity of about 250 to about 375 mOsm.
In other embodiments, the ADNF polypeptides and pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Peptides that have the ability to cross the blood brain can be administered, e.g., systemically, nasally, etc., using methods known to those of skill in the art. Larger peptides that do not have the ability to cross the blood brain barrier can be administered to the mammalian brain via intracerebro ventricular (ICV) injection or via a cannula using techniques well known to those of skill in the art (see, e.g., Motta & Martini, Proc. Soc. Exp. Biol Med. 168:62-64 (1981); Peterson et al., Biochem. Pharmacol. 31 :2807-2810 (1982); Rzepczynski et al, Metab. Brain Dis. 3:211-216 (1939); Leibowitz et al., Brain Res. Bull. 21 :905-912 (1988); Sramka et al., Stereotact. Funct. Neurosurg. 58:79-83 (1992); Peng et al., Brain Res. 632:57-67 (1993); Chern et al., Exp. Neurol. 125:72-81 (1994); Nikkhah et al., Neuroscience 63:57-72 (1994); Anderson et al., J Compo Neurol. 357:196-317 (1995); and Brecknell & Fawcett, Exp. Neurol. 138:338-344 (1996)). Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (17th ed. 1985), which is incorporated herein by reference. In addition, for a brief review of methods for drug delivery, see Langer et al., Science 249:1527-1533 (1990), which is incorporated herein by reference. Suitable dose ranges are described in the examples provided herein, as well as in WO 96/11948, herein incorporated by reference in its entirety.
As such, the present invention provides for therapeutic compositions or medicaments comprising one or more of the ADNF polypeptides described hereinabove in combination with a pharmaceutically acceptable excipient, wherein the amount of the ADNF polypeptide is sufficient to provide a therapeutic effect.
In a therapeutic application, the ADNF polypeptides of the present invention are embodied in pharmaceutical compositions intended for administration by any effective means, including parenteral, topical, oral, pulmonary (e.g. by inhalation) or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, sublingually, buccally or intramuscularly, or intranasally.
Thus, the invention provides compositions for parenteral administration that comprise a solution of an ADNF polypeptide, as described above, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used including, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques or, they may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional nontoxic solid carriers may be used that include, for, example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.
For aerosol administration, the ADNF polypeptides are preferably supplied in finely divided from along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery. An example includes a solution in which each milliliter included 7.5 mg NaCl, 1.7 mg citric acid monohydrate, 3 mg disodium phosphate dihydrate and 0.2 mg benzalkonium chloride solution (50%) (Gozes et al., J Mol Neurosci. 19(1-2): 167-70 (2002)).
EXAMPLES
Example 1
Formulation of 75 mg/mL Dose Strength As described herein, aqueous formulations of AL-108 packaged in a multi-dispensing, metered nasal spray pump device at two dose strengths (25 and 75 mg/mL) were tested.
The 25 mg/mL nasal spray was a solution of AL-108 in 129 mM sodium chloride, 8 mM citric acid, 17 mM sodium phosphate, and 0.005% benzalkonium chloride, had pH 5, and was packaged in the Pfeiffer Advanced Preservative Free (APF) System, a mechanical multidose device designed for the application of solutions via the nasal route. Each spray delivered 0.1 mL containing 2.5 mg of AL-108. Each nasal spray device contained 10 mL of product and could accurately dispense 75 sprays.
The 75 mg/mL nasal spray was a solution of AL-108 in 8 mM citric acid, 17 mM sodium phosphate, and 0.005% benzalkonium chloride, had pH 5, and was packaged (9 mL per device) in the Valois VP7 pump, a mechanical multidose device designed for the application of solutions via the nasal route. Each spray delivered 0.1 mL containing 7.5 mg of AL-108. Sodium chloride was removed from the 75 mg/mL formulation to maintain the target iso-osmolarity of 250-375 mOsm.
75 mg/mL Dose Strength Selection
Nasal drug absorption is affected by molecular weight, size, formulation pH, pKa of molecule, and delivery volume among other formulation characteristics. Specifically, the delivery volume is limited by the size of the nasal cavity. A volume of 0.3 to 0.5 mL/nostril, to a maximum volume of 1 mL has been suggested. More will run or drip out of the nose (see Romeo et al., Adv Drug Delivery Rev. 29:89-116 (1998)). Ideally, one would limit the volume of nasal administration to ensure delivery of the entire dosage of drug. Therefore, in order to administer doses higher than 15 mg BID of AL-108 intranasally in an acceptable spray volume, a higher dosage strength formulation was needed.
Osmolarity and Sodium Chloride Free Formulation of 75 mg/mL Dose Strength
The initial test batch of 75 mg/mL dosage strength was manufactured with identical excipient components as the 25 mg/mL dosage strength. However, osmolarity measurement of the 75 mg/mL test batch revealed that the formulation resulted in a hyperosmotic solution (-440 mOsm). A hyperosmotic solution when delivered intranasally could potentially result in nasal irritation. Therefore, there was a need to develop a nasal formulation with an osmolarity within normal physiological range of 250-375 mOsm. It was found that a sodium chloride free formulation for AL-108 peptide overcame the hyperosmotic issue associated with the initial test formulation. The osmolarity measurement of the new sodium free formulation of intranasal AL-108 fell with the physiologically acceptable range of 250-375 mOsm.
Manufacturing of 25 mg/itiL and 75 mg/mL Dose Strengths
The following shows the composition of the 25 mg/mL and 75 mg/mL formulations:
Figure imgf000042_0001
* Use as 6% w/w solution for pH adjustment, 6.99 g of sodium hydroxide was used.
The process flow chart for manufacturing the 25 mg/mL batch of AL- 108 is provided in Figure 1. The benzolkonium chloride was the last excipient to be added, prior to the active substance (AL-108), and the container was thoroughly rinsed out at least three times with portions of the purified water, to ensure that preservative was not lost in the container. The mixer settings were at 100 ± 20 rpm during mixing of excipients, and then reduced to 50 ± 10 rpm during and after addition of active substance, so as not to disrupt the peptide. In addition, the solution was mixed for at least 10 minutes each time. Approximately 500 g of placebo solution was passed through the membrane filter prior to filtration of the active solution. This was used as a filter conditioning solution, to saturate the membrane filter with preservative, so minimizing loss of preservative from the active solution, through adsorption onto the membrane.
The process flow chart for manufacturing the 75 mg/niL batch of AL- 108 is provided in Figure 2. 80.0 kg of purified water was dispensed into the vessel. Citrate acid monohydrate and sodium phosphate dibasic dihydrate were added to purified water and the solution was mixed until completely dissolved. Benzolkonium chloride was diluted with 500 g of purified water and added into the vessel. The original container was rinsed at least three times with 250.0 g of purified water and the rinses were added to the vessel. The active agent (AL-108) was then added to the vessel. The containers were rinsed with approximately 2.0 kg of purified water, if required. The pH was adjusted with sodium hydroxide to reach an acceptable range of pH 4.8-5.2.
Example 2
Pharmacokinetic Profile of 30 mg BID Dose
Rationale
A 30 mg dose of AL-108 of Example 1 (75 mg/mL dose strength, 2 sprays in each nostril) was tested in a phase 1 study in order to assess safety and PK. The expected result from the study (AL-108-121) was that a 30 mg dose would be well- tolerated and provide exposure double that seen with a 15 mg dose and three-times that seen with a 10 mg dose. Unexpectedly, the results showed that a 30 mg dose provided a half-life and systemic exposure substantially greater than would have been expected from linear pharmacokinetics and consistent half-life based on earlier clinical studies. This data, coupled with predictions of increased efficacy based on PK modeling, indicates that this unexpected increase in exposure will translate to a similarly improved efficacy.
Background
In a Phase 2a trial in amnestic mild cognitive impairment (aMCI) two doses of AL-108 were tested, 5 mg daily (QD) and 15 mg twice daily (BID). Efficacy data showed that the 15 mg BID regimen was active and that BID dosing was effective. PK modeling showed BID dosing was necessary to keep the plasma concentrations above a threshold of approximately 10"13 to 10"14 M. As shown in Figure 3, a single daily 15 mg dose reached a Cmax of 0.9 ng/mL (1.2 nM) (study AL-208-110) which decayed rapidly based on the half-life of 0.8 hours. This meant that the concentration dropped below the anticipated activity threshold by approximately 9 hours. By administering a 15 mg BID dose, the second dose was given as the concentrations were dropping below the predicted optimal range and this prolonged the effective drug exposure.
Based on this data, it was decided to move forward into additional efficacy trials with a dose of 30 mg based on PK modeling using data from prior studies (AL-108-102 and AL-208-110) which indicated that, assuming a 30 mg dose would give twice the exposure of 15 mg, 30 mg would maintain concentrations in the active range for at least 12 hours and would therefore provide greater efficacy than 15 mg.
New Data
Phase I study AL-108-121 generated data showing that 30 mg gave unexpectedly greater drug exposure than predicted. Cmax concentrations were higher, the exposure (measured by AUC) was greater and the half-life was longer. Based on the 15 mg dose pharmacokinetics, a 30 mg dose was expected to maintain plasma concentrations above a threshold level between 10"13 to 10"14 M. Suprisingly, the 30 mg dose data from the AL-108-121 study showed sustained exposure of AL-108 at concentrations greater than 10"11 to 10"12 M, as shown in Figure 4. Further, the half-life was extended to 1.46 hours, rather than the 0.8 hours expected from previous studies. Using the Cmax from this data (3.44 ng/ml) and the observed half- life of 1.46 hours, the PK modeling exposure graph in Figure 4 shows a very dramatic effect on exposure and duration of concentrations that are predicted to be more active.
This PK modeling data, based on actual PK parameters and the knowledge that 15 mg BID is an active dose, predicts that the 30 mg dose will be active and more active than would have been predicted using the PK parameters available before this new data was generated in the AL-108-121 study.
Furthermore, this PK modeling data indicating novel and unexpected findings is supported by comparison of data generated from the AL-108-121 study and previous pharmacokinetic (PK) studies carried out in normal healthy volunteers and AD patients where AL-108 concentrations were measured in plasma at a series of time points. The calculated PK parameters provide a measure of drug residence time and drug exposure in the different studies. As it pertains to assessing the effect of different drug products on drug exposure, the most relevant parameters are half-life (T^), Cmax,
Figure imgf000045_0001
For direct comparison of PK data gathered that allows assessment of dose-linearity, the most relevant data comparison is that for samples collected only on day 1 of each clinical study. While some of the studies include sample collection on the first and seventh day of dosing, variability between day 1 and 7 could add unexpected variability and uncertainty to the PK comparisons since not all studies were carried out over 7 days. Notably study AL-208-110 did not capture data after 7 days of dosing and study AL-108-121 captured 7 days of dosing only for the 30 mg BID group, not the 15 mg BID group. In order to reduce the potential of this confound introduced by variability over 7 days, Table 1 presents data only for PK parameters calculated from plasma concentrations taken on the first day of dosing. The data in Table 1 shows that there were greater than expected increases with exposure to 30 mg in all four parameters, most notably Cmax and AUC0-T which increased 3.3 and 3.5 fold relative to the 15 mg dose, respectively. In addition, the half-life increased approximately 2.35 fold relative to previous studies. Increases in exposure relative to the 10 mg dose were higher still, up to 6.3 fold in the case of AUC0-T. Table 1
PK parameters averaged from Day 1 observations in studies AL- 108- 102, AL-208-110 and AL-108-121
10 mg 15 mg 30 mg 30mg/10mg 30mg/15mg
Cmax (ng/mL) 0.76 1.05 3.44 4.55 3.29
Tl/2 h 0.61 0.62 1.46 2.39 2.35
AUCO-t (h*ng/niL) 0.39 0.70 2.47 6.34 3.51
AUC0-∞ (h*ng/mL) 0.63 0.97 2.59 4.12 2.67
The data in Table 1 shows a dramatically longer half-life at 30 mg than seen at 10 or 15 mg, a parameter which was not expected to change at all when increasing the dose. The half-life at 10 mg and 15 mg is consistent at 0.61 and 0.62 hours, respectively. By contrast, administration of 30 mg produced a half-life of 1.46 hours. While exposure parameters were expected to increase in a linear manner over this range, and indeed did so between 10 and 15 mg, linearity was lost between 15 and 30 mg.
The data in Figure 5 shows this in a graphical format: by dividing the PK parameters (CMAX, AUC0-T, and AUC0-∞) by the dose administered. If there was a linear dose-to-PK relationship, this would present itself as no change in the y-axis as the dose (x-axis) changes. Indeed, there is essentially no change with PK parameters corrected for dose between 10 and 15 mg. However, this can be contrasted with the large shift in each of the exposure parameters when the dose was increased to 30 mg.
Further data analysis in Table 2 was carried out to take into account changes in the strength of the drug formulation. The formulation change was made to allow smaller volumes to be administered to patients, increasing the drug strength from 25 mg/mL to 75 mg/n L. The closest comparison of PK generated for the 30 mg dose must be made for similar drug strengths meaning that the 15 mg and 30 mg doses should be compared in data from study AL-108-121 where the same drug strength was used for the two doses. This provides a direct comparison of samples taken from the same study, analysed at the same time and in the same patients since this was a crossover design. Table 2 presents the parameters from study AL-108- 121, again using data only from day 1 since the 15 mg dose was given once only.
Table 2
PK Parameters from Study AL-108-121
15 mg 30 mg Fold-diff.
Cmax (ng/mL) 0.51 3.44 6.72
Tl/2 h 0.77 ' 1.46 1.90
AUCO-t (h*ng/niL) 0.46 2.47 5.33
AUC0-inf (h*ng/mL) 0.50 2.59 5.21 Again, this data analysis shows the unexpected increase in the half-life of AL-108 changing by 1.9-fold in going from 15 to 30 mg. This change in half-life was accompanied by dramatically greater drug exposure than would be expected when increasing the dose by 2-fold. Each parameter increases well in excess of 2-fold in changing the dose from 15 mg to 30 mg, with Cmax increasing by 6.7-fold and AUCO-T by 5.3-fold and AUC0-∞ by 5.2-fold.
Putting aside the above caveats about comparing data from different studies or after several days of treatment, Table 3 presents the data from three Phase 1 clinical trials where intranasal AL-108 plasma PK was assessed (studies AL-108- 102, AL-208-110 and AL-108-121).
Table 3
PK Parameters Averaged from Studies AL-108-102, AL-208-1 10 and AL-108-121
10 mg 15 mg 30 mg 30mg/10mg 30mg/15mg
Cmax (ng/mL) 0.63 1.28 2.89 4.55 2.26
Tl/2 h 0.59 0.59 2.09 3.51 3.55 AUCO-t (h*ng/mL) 0.34 0.88 2.13 6.22 2.43 AUC0-inf (h*ng/mL) 0.55 1.12 2.28 4.15 2.03 Once again, Table 3 shows a dramatically longer half-life at 30 mg than seen at 10 or 15 mg. The half-life at 10 mg and 15 mg is consistent at 0.59 hours. By contrast, administration of 30 mg produced a half-life of 2.09 hours, approximately 3.5- fold longer than expected. Typically, the half-life of a drug is constant at different doses, so to see a 3.5-fold increase was quite unexpected. While exposure parameters were expected to increase in a linear manner over this range, and indeed did so between 10 and 15 mg, linearity was lost between 15 and 30 mg. The data in Table 3 shows that greater plasma exposure was seen with 30 mg than 10 mg by 4.55-, 6.22-, and 4.15-fold for Cmax, AUC0-T, and AUCo-∞, respectively; and by 2.26-, 2.43-, and 2.03-fold for Cmax, AUCO-T, and AUC0-0O, respectively, relative to 15 mg. This loss of linearity and increase in half-life was not expected.
Example 3
Clinical Trial with 30 mg BID Dosing of AL-108 in Tauopathy Patients
This is a randomized, double-blind, placebo-controlled, parallel group study that will evaluate the efficacy and safety of AL-108 30 mg BID compared to placebo administered to subjects with progressive supranuclear palsy (PSP) for 52 weeks. The study will include 4 visits: screening (Visit 1), randomization (Visit 2), treatment (Visits 3-4).
The primary objectives of this study are to evaluate AL-108 30 mg BID relative to placebo, when both are administered intranasally (IN) for 52 weeks to subjects with PSP, with respect to:
• Efficacy, as measured by change from baseline scores of the Progressive Supranuclear Palsy Rating Scale (PSPRS) at 52 weeks.
• Efficacy, as measured by the change from baseline of the Schwab and England Activities of Daily Living Scale (SEADL) at 52 weeks.
• Safety, as measured by reported AEs, electrocardiograms (ECG), nasal examinations and clinical laboratory measures. The secondary objectives of this study are to evaluate AL-108 30 mg BID relative to placebo, when both are administered IN to subjects with PSP, with respect to:
• Efficacy, as measured by the Clinical Global Impression of Change (CGI-C) at 52 weeks.
• Brain atrophy, as measured by change from baseline of ventricular volumes measured by volumetric brain MRI at 52 weeks.
This study will also explore the effect of AL-108 compared with placebo on the following measures:
· Efficacy, as measured by the change from baseline of cognition by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) at 52 weeks.
• Efficacy, as measured by the change from baseline of the Executive Function Tests at 52 weeks.
· Mood, as measured by the change from baseline of the Geriatric
Depression Scale (GDS) at 52 weeks.
• Cerebrospinal fluid (CSF) biomarkers, including total tau, phosphorylated tau, amyloid beta peptide (1-42), neurofilament protein light chain (NFL), and phosphorylated neurofilament heavy chain (pNFH) as measured by the change from baseline at 52 weeks in the subgroup of subjects consenting to lumbar puncture.
• Plasma phosphorylated neurofilament protein heavy chain (pNFH), as measured by the change from baseline at 6, 13, and 52 weeks.
There are currently no FDA-approved medications indicated for the treatment of PSP. However, a number of agents are commonly used off-label for symptomatic management. Most commonly, these include dopaminergic agonists (e.g., levodopa/carbidopa), medications for mood and behavior (e.g., selective serotonin reuptake inhibitor drugs (SSRIs)), atypical antipsychotic agents, and dietary supplements (e.g., coenzyme Q10 [CoQIO]). Lithium is currently under investigation as a treatment for PSP. Cognitive enhancers, such as donepezil, have been studied in PSP and found to be ineffective (Litvan et al., Neurology. 57(3):467-73 (2001)). Of the more commonly used agents, only CoQIO has demonstrated evidence of efficacy in ameliorating PSP-related symptoms (Stamelou et al, Mov Disord. 23(7):942-9 (2008)). Since lithium and other dietary supplements have an unknown potential to affect disease progression in PSP, their use will be prohibited during this study. Because of known negative effects on function in subjects with PSP, use of cognitive enhancers, such as donepezil and memantine, as well as antipsychotic agents and long-acting benzodiazepines will also be prohibited. Other concomitant medications will be allowed as long as their dose has been stable prior to screening and remains stable throughout the study.
Use of the NINDS-SPSP criteria to define PSP is well-established and accepted for the diagnosis of PSP and has been used in prior clinical studies. These criteria have also been demonstrated to provide high diagnostic accuracy in a multicenter study with clinicopathological correlation (Bensimon et al, Brain, 132(Pt 1): 156-71, 2009 (Epub 2008 Nov 23)).
The AL-108 dose of 30 mg intranasal (IN) BID was chosen because AL- 108 15 mg IN BID demonstrated efficacy in a prior study of memory function in subjects with aMCI and the 30 mg dose is the maximum feasible dose delivered IN with 2 sprays to each nostril while maintaining iso-osmolarity. The 30 mg IN BID dose was well-tolerated and provided four- to five-fold greater exposure than the 15 mg IN BID dose in Phase 1 evaluation of age-matched healthy volunteers (45 to 85 years).
The study duration of twelve months is appropriate for a Phase 2/3 study in PSP to assess the magnitude of motor changes and brain atrophy. AL-108 demonstrated cognitive improvement in twelve weeks in subjects with aMCI. In that study, the treatment effect may not have reached a maximum level. A study duration of twelve months will allow a better assessment of the potential treatment effect and time course of that effect. The twelve-month duration will also allow an assessment as to whether the rate of decline has been affected by treatment.
The endpoints are appropriate measures of treatment effect in this study. The PSPRS is a robust measure of PSP disease activity and progression. The instrument measures 6 relevant categories of PSP symptoms: daily activities, behavior, bulbar, oculomotor, limb motor, and gait/midline. The instrument has been validated and has been shown to correlate well with survival and retention of gait function (Golbe and Ohman-Strickland, Brain, 130(Pt 6): 1552-65, 2007 ( Epub 2007 Apr 2)).
Both global assessments and activities of daily living will be assessed.
These measures complement the PSPRS to provide a more comprehensive view of patient status and treatment effects. The SEADL (Schwab RS, Trans Am Neurol Assoc, 94:363-4, 1969) is being used in this study to assess subject's functional capacity and potential changes with treatment. Additionally, the SEADL has been found to be one of the most sensitive measures of disease progression (Bensimon et al., Brain, 132(Pt 1): 156-71, 2009 (Epub 2008 Nov 23)). The CGI has been used in studies of therapies for PSP and other movement disorders (Bensimon et al., Brain, 132(Pt 1):156-71, 2009 (Epub 2008 Nov 23)).
The RBANS and Executive Function tests are appropriate measures of cognition in this population. The RBANS is a validated instrument that assesses 5 cognitive domains: Immediate Memory, Delayed Memory, Attention, Language, and Visuospatial/Constructional (Randolph et al., J Clin Exp Neuropsychol, 20(3):310-9, 1998). The tests used to assess executive function are phonemic fluency, the colors trail test and letter- number sequencing. Executive function is typically compromised in subjects with PSP, but is not specifically assessed by the RBANS. Therefore, it is appropriate to include both sets of tests in this study.
Structural MR! and plasma and CSF biomarkers are appropriate for this study because AL-108 has the potential to modify the course of PSP. Disease progression as measured by ventricular volume enlargement on MRI is expected to be about 9% in this study population over twelve months (Paviour et al., Mov Disord, 21(7):989-96, 2006; Whitwell et al., Brain, 130(Pt 4):1148-58, 2007 (Epub 2007 Mar 8)). Therefore, decreases in the rate of brain atrophy may be detectable with AL-108 treatment that would suggest disease modification. Also, because AL-108 affects tau phosphorylation in preclinical tauopathy models, changes in CSF biomarkers may also be observed during the study, which would suggest AL-108 may modify the course of disease. Finally, neurofilament protein light and heavy chains are elevated in neurodegenerative conditions, including PSP. The elevated neurofilament levels are believed to represent neuronal-axonal degeneration (Holmberg et al., Mov Disord, 13(l):70-7, 1998; Holmberg et al., Parkinsonism Relat Disord, 8(1):23-31, 2001 ; Brettschneider et al., Dement Geriatr Cogn Disord, 21(5-6):291-5, 2006 (Epub 2006 Feb 10); Constantinescu et al., Parkinsonism Relat Disord, 16(2):142-5, 2010 (Epub 2009 Jul 31); Perrot and Eyer, Brain Res Bull, 80(4-5):282-95, 2009 (Epub 2009 Jun 17)). Therefore, decreased levels of CSF or plasma neurofilament chains would suggest that AL-108 decreased the rate of neuronal degeneration.
Mood status of the subjects will be assessed by the GDS. This scale has been used extensively in clinical trials.
Safety will be assessed by standard measures and investigation of potential anti-AL-108 antibodies. Treatments Administered
Treatments evaluated in this study will be placebo and AL-108 30 mg BID. All treatments will be blinded and administered intranasally. Each dose of study drug will consist of 2 sprays in each nostril for a total of 4 sprays. Therefore, the total daily dose will be eight (8) sprays daily (4 sprays, twice daily, 8 to 12 hours apart). Each spray will deliver 0.1 mL of study drug solution.
Rationale for Selection of Dose
The AL-108 dose for this study was selected on the basis of prior preclinical and clinical data.
Preclinical toxicology evaluation has shown AL-108 to be safe and well- tolerated at doses of 80 mg/kg/day in rats and 20 mg/kg/day in dogs. No evidence of harm was observed in safety pharmacology evaluation of cardiovascular, respiratory and neurological function in rats and dogs at doses up to 100 mg/kg IV. Preclinical efficacy studies have demonstrated improvement in cognitive function and decreases in both amyloid beta (1-42) and phosphorylated tau in transgenic murine models of tauopathies at doses of approximately 20 to 1200 ug/kg/day.
The effect of AL-108 on cognitive function was evaluated in a study of subjects with aMCI. In this study, AL-108 was safe and well-tolerated when administered at doses of 5 mg QD and 15 mg BID for 12 weeks. Statistically significant improvement was observed at the 15 mg BID dose in cognitive tests that primarily assessed working memory. Improvement was not observed in cognitive tests that more prominently evaluated other cognitive functions. This finding is believed to reflect the clinically normal functioning of the study subjects in non-memory clinical domains. No improvement in cognition was observed at the 5 mg QD dose.
An additional Phase 1 study demonstrated that 7-day repeat dosing of 30 mg IN BID was safe and well-tolerated in healthy volunteers (45-65 years). In that study, the 30 mg AL-108 exposure was 4-5 times higher (AUCiast = 1.8 to 2.5 ng*h/mL) than the 15 mg dose (AUCiast = 0.5 ng*hr/mL), but within the nonclinical safety margin established in the chronic canine toxicology study. Exposure in the 39-week chronic canine toxicology study was AUCiast = 9.1 to 21.6 ng*hr/mL; therefore exposure at the highest, maximal feasible dose (which was also the NOAEL) in the dog was 3.6 to 12 times greater when compared to the 30 mg human exposure. The exposure in the rat 26-week chronic toxicology study (AUCiast = 2.45 ng*hr/mL) was similar or slightly lower than the 30 mg human exposure, but the rat was administered the maximum feasible dose (no further exposure could be achieved) and there were no toxicological findings.
AL-108 was also safe and well-tolerated when administered as a single 300-mg IV dose to subjects undergoing coronary artery bypass graft surgery.
The AL-108 dose of 30 mg BID was chosen because:
• higher doses on a mg/kg basis have been safe in preclinical safety assessment, single intravenous doses as high as 300 mg and multiple intranasal doses of 15 mg and 30 mg BID have been safe and well-tolerated in prior clinical studies,
30 mg is the highest feasible dose that can be delivered at any single administration time point while maintaining iso-osmolarity of the formulation,
30 mg BID for seven days was well tolerated, and the 30 mg dose exposure exceeds that of the 15 mg BID dose that was demonstrated to improve measures of memory in subjects with MCI. This provides the potential for greater efficacy with the higher dose and the possibility that more compromised subjects will require a higher dose.
Example 4
Pharmacokinetic Profile of 15 mg Dose in Healthy Elderly Patients and Alzheimer's
Disease Patients
The pharmacokinetics of 15 mg intranasal administration (IN) of AL- 108 has been studied in healthy adults (18-65 yrs) and in mild- to moderate- AD patients in a Phase 1, Open Label, Single Dose Study (study AL-108- 110). Enrolment was planned for 2 groups of ~6 healthy male and/or female subjects and ~6 mild-to moderate AD patients. The study treatment groups were as follows:
Group 1 : 15 mg AL- 108 (healthy adults)
Group 2: 15 mg AL-108 (mild-to-moderate AD patients) AL-108 was administered as 3 x 0.1 mL sprays into each nostril. The pharmacokinetics parameters after a single 15 mg dose is presented in Table 4. Table 4
Figure imgf000055_0001
The mean plasma levels following intranasal administration peaked rapidly in both healthy adult and AD patients (Tmax ~ 0.14 to 0.20 hr). The elimination half-life (T1/2) of AL-108 was similar in both groups (0.78-0.61 hr). However, a greater Cmax was observed in Alzheimer's disease patients. A similar greater AUCo-τ was also seen in Alzheimer's patients compared to healthy subject. While the AUC0-∞ did not show a similar increase in Alzheimer's disease patients, it is thought that this data may be less accurate due to the extrapolation from a somewhat limited data set. As such, AUCO-T may have more relevance in this study.
The nasal respiratory mucosa is considered the most important area for delivering drugs systemically (Pires et al., J. Pharm Pharmaceutical Sci 12(3):288-311, (2009)). The mucosa is made up of the epithelium, basement membrane and lamina propria. The lamina propria is located beneath the basal lamina and contains many blood vessels, nerves and glands. The intense blood flow in the arteriovenous anastomosis and the large surface of the respiratory epithelium favors transmucosal nasal drugs absorption. Indeed, the pharmacokinetic parameters obtained after intranasal and oral administration of certain drugs showed comparable systemic exposure independent of the route of administration (Ding et al., Drug Delivery 14:101- 104 (2007)). In Alzheimer's disease patients, olfactory impairment and neuroanatomical changes in the central portions of the olfactory system occur early in the disease. AD patients often report loss of olfactory sensory function. Further, histological examination of the Alzheimer's patients' epithelial layer showed dystrophic cytoskeletal changes (Hock et al., Eur Neurol, 40(l):31-6 (1998)) and increased oxidative damage to the cell (Ghanbari et al, Aging Cell 3(l):41-44 (2004), Perry et al., Acta Neuropathol 106(6):552-556 (2003)). The compromised epithelial lining may result in increased drug absorption in Alzheimer's patients compared to healthy subjects.
As shown above in Table 4, the pharmacokinetic parameters for the 15 mg dose strength in AL-108-110 study demonstrated increased systemic exposure of AL-108 in Alzheimer's disease patients compared to healthy subjects while the terminal half-life was unchanged. We predict that the pharmacokinetic parameters achieved when administering the 30 mg dose strength to patients with compromised olfactory epithelial lining resulting from their neurodegenerative disease, will be substantially higher than that obtained with the 15 mg dose seen in the AL-108-121 study. Further, based on the unexpectedly high exposure seen with the 30 mg dose in healthy volunteers, we can predict that this effect will also be seen in patients with neurodegenerative conditions and that the following calculated parameters will be seen.
Prediction of PK Parameters in Neurodegenerative Disease with 30 mg
Calculation Method 1 :
The ratio between PK parameters for AD patients and healthy subjects (both from study AL- 108-110) is derived and multiplied by the PK parameters obtained with 30 mg in AD subjects from study AL-108-110.
Cmax- 6.82
Figure imgf000056_0001
AUC0-∞: 2.08
T 2: unchanged. Calculation Method 2:
The ratio between PK parameters for 30 mg versus 15 mg healthy subjects (both from study AL-108-121) is derived and multiplied by the PK parameters obtained for 15 mg in healthy subjects from study AL- 108- 121.
Cmax: 8.26
AUC0-T: 4.69
Figure imgf000057_0001
Ύ ΐ2'. unchanged.
In both cases the Tj/2 is predicted to remain the same as seen with 30 mg in healthy subjects from study AL-108-121. It is notable that in study AL-108-110, the plasma exposure was higher for AD patients than healthy subjects but the elimination half-life remained the same. This argues that the mechanism by which the 30 mg formulation increases exposure is not the same as that responsible for the differences between AD and healthy subjects. As such, we would predict that the elimination half- life for 30 mg administration in neurodegenerative diseases would be similar to healthy subject, i.e. , 1.46 h. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS What is claimed is:
1. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
2. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
3. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10"12 M for a period of time.
4. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the
1 1 1
ADNF polypeptide equal to or greater than about 10" to about 10" M for a period of time.
5. The method of claim 3 or 4, wherein the period of time is at least about
24 hours.
6. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
7. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
8. The method of claim 6 or 7, wherein the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL.
9. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr.
10. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr.
11. The method of claim 9 or 10, wherein the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
12. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-τ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
13. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUC0-T of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
14. The method of claim 12 or 13, wherein the AUC0-T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL.
15. A method for administration of an ADNF polypeptide to a subject, the method comprising administering the ADNF polpeptide to the subject in one or more doses to produce a AUC0-∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
16. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of administering the ADNF polpeptide to the subject in one or more doses to produce a AUCo-∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
17. The method of claim 15 or 16, wherein the AUC0.∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL.
18. The method of any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
19. The method of any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10"12 M for a period of time.
20. The method of claim 19, wherein the period of time is at least about 24 hours.
21. The method of any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
22. The method of claim 21, wherein the total maximum concentration (Cmax) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL.
23. The method of any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hr.
24. The method of claim 23, wherein the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
25. The method of claim any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to produce a AUC0-T of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
26. The method of claim 25, wherein the AUC0-T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL.
27. The method of claim any one of claims 1-17, wherein the ADNF polypeptide is administered to the subject in one or more doses to produce a AUCo-∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
28. The method of claim 27, wherein the AUC0-∞ of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL.
29. A method of any one of claims 1-28, the method comprising intranasally administering to the subject in one or more doses a liquid composition comprising:
(a) about 75 mg/mL of the ADNF polypeptide;
(b) about 0.17% (w/w) citric acid monohydrate;
(c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and
(d) about 0.005% benzalkonium chloride,
wherein the composition comprises essentially no sodium chloride.
30. The method of claim 29, wherein the composition has an iso- osmolarity of about 250 to about 375 mOsm.
31. A liquid composition for intranasal administration of an ADNF polypeptide, the composition comprising:
(a) about 75 mg/mL of the ADNF polypeptide;
(b) about 0.17% (w/w) citric acid monohydrate;
(c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and
(d) about 0.005%> benzalkonium chloride,
wherein the composition comprises essentially no sodium chloride.
32. A method for intranasal administration of an ADNF polypeptide to a subject, the method comprising intranasally administering to the subject in one or more doses a liquid composition comprising:
(a) about 75 mg/mL of the ADNF polypeptide;
(b) about 0.17%> (w/w) citric acid monohydrate;
(c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and
(d) about 0.005%) benzalkonium chloride,
wherein the composition comprises essentially no sodium chloride.
33. A method of treating or preventing a neurodegenerative disease in a subject, the method comprising the step of instranasally administering to the subject in one or more doses a liquid composition comprising:
(a) about 75 mg/mL of the ADNF polypeptide;
(b) about 0.17% (w/w) citric acid monohydrate;
(c) about 0.3% (w/w) sodium phosphate dibasic dehydrate; and
(d) about 0.005% benzalkonium chloride,
wherein the composition comprises essentially no sodium chloride.
34. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to deliver a total of 30 mg of the ADNF polypeptide to the subject two times in a 24 hour period.
35. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total concentration of the ADNF polypeptide equal to or greater than about 10"11 to about 10" M for a period of time.
36. The method of claim 35, wherein the period of time is at least about 24 hours.
37. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to produce in the blood plasma of the subject a total maximum concentration (Cmax) of the ADNF polypeptide equal to or greater than about 3.0 ng/mL.
38. The method of claim 37, wherein the total maximum concentration (Cma ) of the ADNF polypeptide is equal to or greater than about 3.4 ng/mL.
39. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to produce a half-life of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 1.0 hf .
40. The method of claim 39, wherein the half-life of the ADNF polypeptide is equal to or greater than about 1.4 hr.
41. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to produce a AUC0-T of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
42. The method of claim 41, wherein the AUC0-T of the ADNF polypeptide is equal to or greater than about 2.4 h*ng/mL.
43. The method of claim 32 or 33, wherein the composition is intranasally administered to the subject in one or more doses to produce a AUCo.∞ of the ADNF polypeptide in the blood plasma of the subject equal to or greater than about 2.0 h*ng/mL.
44. The method of claim 43, wherein the AUCo.«, of the ADNF polypeptide is equal to or greater than about 2.5 h*ng/mL.
45. The composition of claim 31 or the method of claim 32 or 33, wherein the composition has an iso-osmolarity of about 250 to about 375 mOsm.
46. The composition of claim 31 or 45, or the method of any one of claims 1-30 or 32-45, wherein the ADNF polypeptide is a member selected from the group consisting of:
(a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence:
Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l); (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence:
Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2); and
(c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b).
47. The method of claim 46, wherein the ADNF polypeptide is a member selected from the group consisting of a full length ADNF I polypeptide, a full length ADNF III polypeptide, and a mixture of a full length ADNF I polypeptide and a full length ADNF III polypeptide.
48. The method of claim 46, wherein the ADNF polypeptide is an ADNF I polypeptide.
49. The method of claim 48, wherein the ADNF polypeptide is a full length ADNF I polypeptide.
50. The method of claim 48, wherein the ADNF I polypeptide has the formula (R^-Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala-(R2)y (SEQ ID NO: 14) in which
R1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs;
R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and
x and y are independently selected and are equal to zero or one.
51. The method of claim 48, wherein the ADNF I polypeptide is Ser-Ala- Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l).
52. The method of claim 48, wherein the active core site of the ADNF I polypeptide comprises at least one D-amino acid.
53. The method of claim 48, wherein the active core site of the ADNF I polypeptide comprises all D-amino acids.
54. The method of claim 48, wherein the ADNF I polypeptide is selected from the group consisting of:
Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:3);
Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile- Pro-Ala (SEQ ID NO:4);
Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:5);
Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:6);
Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:7);
Gly-Ser- Ala-Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO : 8); and
Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l).
55. The method of claim 48, wherein the ADNF I polypeptide comprises up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site.
56. The method of claim 46, wherein the ADNF polypeptide is an ADNF III polypeptide.
57. The method of claim 56, wherein the ADNF polypeptide is a full length ADNF III polypeptide.
58. The method of claim 56, wherein the ADNF polypeptide has the formula (R^-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln- R2^ (SEQ ID NO: 13) in which R1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs;
R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and
x and y are independently selected and are equal to zero or one.
59. The method of claim 56, wherein the ADNF III polypeptide is Asn- Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
60. The method of claim 56, wherein the active core site of the ADNF III polypeptide comprises at least one D-amino acid.
61. The method of claim 56, wherein the active core site of the ADNF III polypeptide comprises all D-amino acids.
62. The method of claim 56, wherein the ADNF III polypeptide is a member selected from the group consisting of:
Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:9);
Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO: 10); Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID
NO: 11);
Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO: 12); and
Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
63. The method of claim 56, wherein the ADNF III polypeptide comprises up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site.
64. The method of claim 46, wherein at least one of the ADNF polypeptides is encoded by a nucleic acid that is administered to the subject.
65. The method of claim 46, wherein an ADNF I polypeptide of part (a) and an ADNF III polypeptide of part (b) are administered to the subject.
66. The method of clam 65, wherein either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise at least one D-amino acid.
67. The method of claim 65, wherein either or both active core sites of the ADNF I polypeptide and the ADNF III polypeptide comprise all D-amino acids.
68. The method of claim 65, wherein the ADNF I polypeptide is Ser- Ala- Leu-Leu- Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l), and wherein the ADNF III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
69. The method of claim 65, wherein the ADNF I polypeptide is a member selected from the group consisting of:
Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:3);
Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile- Pro-Ala (SEQ ID NO:4);
Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:5);
Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:6);
Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:7);
Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:8); and
Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:l), and
wherein the ADNF III polypeptide is selected from the group consisting of:
Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:9);
Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO: 10);
Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID
NO: 11); Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln- Ser (SEQ ID NO: 12); and
Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2).
70. The method of claim 65, wherein the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF I polypeptide, and wherein the ADNF III polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site of the ADNF III polypeptide.
71. The method of any one of claims 1 -30 or 32-70, wherein the subject suffers from a neurodegenerative disease.
72. The method of any one of claims 1-30 or 32-71 wherein the neurodegenerative disease is Alzheimer's disease, corticobasal ganglionic degeneration, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, amyotrophic lateral sclerosis, Pick's atrophy, diffuse Lewy body disease, a neurodegenerative pathology associated with aging, a pathological change resulting from a focal trauma, peripheral neuropathy, retinal neuronal degeneration, or dopamine toxicity.
73. The method of any one of claims 1-30 or 32-71 wherein the neurodegenerative disease is neurodegeneration associated with schizophrenia.
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