Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1994-10-17
2001-01-16
Kunz, Gary L. (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S013800, C514S014800, C530S326000, C530S327000, C530S328000, C530S324000
Reexamination Certificate
active
06174862
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to Activity Dependent Neurotrophic Factor (ADNF). More particularly, the present invention relates to a family of polypeptides derived from ADNF that exhibit neuroprotective
eurotrophic action on neurons originating in the central nervous system and to uses thereof for the treatment of neurological deficiencies and for the prevention of cell death associated with (1) gp120, the envelope protein from HV; (2) N-methyl-D-aspartic acid (excito-toxicity); (3) tetrodotoxin (blockage of electrical activity); and (4) beta-amyloid peptide, a substance related to neuronal degeneration in Alzheimer's disease.
BACKGROUND OF TH INVENTION
Neuronal cell death is characteristic of most developing neural systems in vertebrates. The extent (30-80%) of neuronal death that occurs during development indicates that the regulation of this process is of fundamental importance to the determination of nervous system structure. Although a great deal of descriptive data has been reported concerning the magnitude and ubiquity of this neuronal cell loss, little is known of the mechanism that regulates this process during development. It is clear, however, that electrical activity plays an important role in determining neuronal survival during this regressive phase of development. It has been found, for example, blockage of electrical activity with &agr;-bungarotoxin attenuates the naturally occurring cell death in spinal motoneurons (Pittman and Oppenheim,
Nature
(Lond.) 271, 364-366 (1987)) and in trochlear nucleic in vivo (Creazzo and Sohal,
Exp. Neurol.
66, 135-145 (1979)).
Studies with cultured spinal cord-dorsal root ganglion (SC-DRG) neurons have shown that during development in vivo, neuronal cell death also occurs in a predictable and activity-dependent manner (Brenneman, et al.,
Peptides
6, 35-39 (1985)). Analysis of the effects of activity blockage on neuronal survival in culture has indicated an interaction between conditioning substances and electrical activity. When endogenous conditioning substances were removed before electrical blockade, neuronal cell death was accelerated (Brenneman, et al.,
Dev. Brain Res.
9, 13-27 (1983)). In contrast, when conditioning substances from SC-DRG cultures were supplied during blockage of electrical activity, neuronal cell death was prevented (Brenneman, et al.,
Dev. Brain Res.
15, 211-217 (1984)).
Further studies have indicated that part of the molecular basis of this activity-dependence is the action of vasoactive intestinal peptide (VIP), a neuropeptide which is released during electrical activity (Brenneman, D. E. and Eiden, L. E.,
Proc. Natl. Acad. Sci. U.S.A.
83, 1159-1162 (1986); and Brenneman, et al.,
Peptides
6, 35-39 (1985)). Moreover, studies have indicated that VIP increases the survival of activity-dependent spinal cord neurons by releasing protein growth factors from non-neuronal spinal cord cells (Brenneman, et al.,
J. Cell Biology,
104, 1603-1610 (1987)). More specifically, it has been determined that VIP interacts with its receptors on glial cells (Gozes, et al.,
Soc. Neurosci. Abs.
15, 216 (1989)) to induce the secretion of neuronal survival factor(s) (Brenneman, et al.,
J. Neurosci. Res.
25, 38&394 (1990); and Gozes, I. and Brenneman, D. E.,
Molecular Neurobiology,
3, 201-236 (1989)).
Among the growth factors released from non-neuronal spinal cord cells by VIP is Activity Dependent Neurotrophic Factor (ADNF). This glia-derived, VIP-released growth factor has been isolated from conditioned medium of rat cerebral cortical astroglia stimulated by VIP (Gozes, 1. & Brenneman, D. E.,
Molecular Neurobiology
3, 1-36 (1989); and Brenneman, D. B. & Eiden, L. E.,
Proc. Natl. Acad Sci. U.S.A.
83, 1159-1162 (1986)). Sequential chromatographic separations by ion exchange, gel permeation and hydrophobic interaction have been utilized to obtain about a 1650-fold purification of a single, 14,000 Dalton protein (apparent pI: 8.3±0.25) that increases survival (EC50, 0.075 pg/ml) of electrically blocked spinal cord neurons and, accordingly, this glia-derived, VIP-released growth factor has been named: Activity Dependent Neurotrophic Factor. ADNF has been shown to protect neuronal cells against death. More particularly, ADNF has been shown to increase the growth and survival of developing spinal cord neurons, hippocampal neurons and cerebral cortical neurons. In addition, ADNF has been found to protect neuronal cell viability by preventing neuronal cell death produced by the external envelope protein of the HV virus.
Although ADNF effectively protects against neuronal cell death, it would be advantageous to have polypeptides which are shorter than the full length amino acid sequence of ADNF, but which exhibit the same neuroprotective
eurotrophic action of the intact ADNF growth factor. Quite surprisingly, the present invention provides such polypeptides.
SUMMARY OF TE INVENTION
An active site for Activity Dependent Neurotrophic Factor (ADNF) has now been discovered. Quite surprisingly, this active site, which is only nine amino acids in length, is more potent than intact ADNF and more effective over a greater range of concentrations (i.e., from 0.01 fm to about 1 pM). This is the first time a polypeptide has been identified which mimics the full biological activity of an intact growth factor. It has further been discovered that other ADNF polypeptides containing this active site also posses the neuroprotective
eurotrophic activity of intact ADNF. As such, the present invention provides a family of ADNF polypeptides, consisting essentially of this ADNF active site, which exhibit the neuroprotective
eurotrophic activity of intact ADNF. These smaller ADNF polypeptides are advantages in that they readily cross the brain barrier. Moreover, due to their length, they do not possess any solubility problems associated with the intact ADNF growth factor.
Quite surprisingly, it has further been discovered that such ADNF polypeptide can be used for the treatment of neurological deficiencies and for the prevention of neuronal cell death. Such ADNF polypeptides can be used, for example, to prevent the death of neuronal cells including, but not limited to, spinal cord neurons, hippocampal neurons, cerebral neurons and cholingeric neurons. More particularly, the ADNF polypeptides of the present invention can be used to prevent cell death associated with (1) gp120, the envelope protein from HIV; (2) N-methyl-D-aspartic acid (excito-toxicity); (3) tetrodotoxin (blockage of electrical activity); and (4) beta-amyloid peptide, a substance related to neuronal degeneration in Alzheimer's disease.
As such, the present invention provides methods for preventing neuronal cell death. More particularly, in one aspect, methods are provided for using the ADNF polypeptides of the present invention to prevent gp120-induced neuronal cell death in a patient infected with HIV. In another aspect, methods are provided for using the ADNF polypeptides of the present invention to prevent neuronal cell death associated with excito-toxicity induced by N-methyl-D-aspartate stimulation. In yet another aspect, methods are provided for using the ADNF polypeptides of the present invention to prevent neuronal cell death induced by the beta-amyloid peptide in a patient afflicted or impaired with Alzheimer's disease. In still another aspect, methods are provided for using the ADNF polypeptides of the present invention to alleviate learning impairment produced by cholingeric blockage in a patient impaired or afflicted with Alzheimer's disease. The ADNF polypeptides of the prevent invention can effectively be used to prevent neuronal cell death associated with a number of other neurological diseases and deficiencies.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.
REFERENCES:
patent: 5135956 (1992-08-01), Borg et al.
patent: WO 90/10449 (1990-09-01), None
patent: WO 92/18140 (1992-10-01), N
Gucker Stephen
Kunz Gary L.
Ramot University Authority for Applied Research and Industrial D
Townsend and Townsend / and Crew LLP
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