Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-09-18
2001-07-17
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S399000, C530S350000, C530S324000, C435S069400
Reexamination Certificate
active
06262024
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a composition of matter comprising a neuron regulatory factor or a component thereof, for promoting neurite outgrowth and enhancing survival of both neuronal and non-neuronal cells, to a pharmaceutical preparation containing the neuron regulatory factor or component thereof, and to its use in the treatment of damaged neurons or non-neuronal cells.
BACKGROUND OF THE INVENTION
Neurotrophic factors are considered to be vital for normal development of the nervous system. During development, neuronal target structures produce limited amounts of specific neurotrophic factors necessary for both the survival and differentiation of neurons projecting into the structures. The same factors have been found to be involved in the survival and/or maintenance of mature neurons.
A neurotrophic factor is defined as a substance capable of increasing and/or maintaining survival of a neuron population, and possibly affecting outgrowth of neurites (neuron processes) and certain other metabolic activities of a neuron. Neurotrophic factors are generally described as soluble molecules synthesized in the peripheral targets of neurons and transported to their cell bodies, where they exert their effects.
Studies with isolated neurotrophic factors have shown that exogenously added neurotrophic factors can exert their neurotrophic effects upon cultured neurons in vitro, or by administration to damaged or degenerated neurons in vivo. For this reason, various neurotrophic factors have received great attention as potential therapeutic agents for treatment of degenerative diseases of the central nervous system, as well as traumatic damage to the CNS. For example, nerve growth factor (NGF) has been shown to increase the survival, function and regeneration of cholinergic neurons in the basal forebrain. Degeneration of this population of cholinergic neurons has been associated with patients having Alzheimer's disease, and could be the primary neuronal defect responsible for the loss of cognitive function associated with Alzheimer's disease. NGF has been found to be synthesized and released from the target areas of these cholinergic neurons in the hippocampus and neurocortex, both areas of the brain associated with learning and memory. See Springer, J. E., Drug News and Perspectives, 4: 394-99 (1991). As another example, a dopaminergic neurotrophic factor (DNTF) has been purified and characterized, and found to promote survival and neurite outgrowth of dopaminergic neurons of the substantia nigra. DNTF is considered a potentially valuable therapeutic agent for the treatment of Parkinson's disease which involves degeneration of dopaminergic motor neurons of the central nervous system (U.S. Pat. No. 5,215,969 to Springer et al., 1993).
It can be seen from the foregoing examples that neurotrophic factors are a valuable source of therapeutic agents for the treatment of neuron damage and neurodegenerative disease. However, the development of such factors as therapeutic agents can be problematic. For example, it is difficult to determine the specificity of an endogenous neurotrophic agent, i.e., whether different factors exist for different nervous system pathways, and which neuron populations in those pathways are affected by a factor. In fact, many identified neurotrophic agents have been shown to have a wide range of biological functions, acting on both central and peripheral neurons, as well as non-neuronal cells in vitro (e.g., polypeptide growth factors and ciliary neurotrophic factor, CNTF). In the central nervous system, with its complex interconnections and heterogeneous neuron types, it is difficult to determine which neurotrophic factors are effective on a particular neuronal population. This difficulty is further exacerbated by the fact that many of the neurotrophic factors that have been characterized have been found to be closely related to one another. For example, it is now known that NGF possesses amino acid sequence homology to brain-derived neurotrophic factor (BNDF), a protein with similar, but not identical, in vitro properties as NGF (Barde et al., EMBO J., 1: 549-53, 1982; Leibrock et al., Nature, 341: 149-52, 1989). In fact, NGF, BNDF and the neurotrophin (NT) series have been classified as members of a superfamily of neurotrophic factors (NGF superfamily). Because of their similarity in amino acid sequence (and hence nucleotide sequences encoding the factor), it has been difficult to develop nucleic acid or antibody probes that are specific for a particular member of the family. The lack of a specific means for identifying a particular neurotrophic factor has hindered the elucidation of particular neuronal populations affected by a specific factor.
An additional obstacle to developing neurotrophic factors as therapeutic agents for treatment of damaged neurons is that few in vivo models exist to study the survival-promoting activity of these factors in the central nervous system. In order to develop a neurotrophic factor as an effective therapeutic agent for the treatment of neuron degeneration, it is important to be able to determine where in the central nervous system the neurotrophic factor operates, whether the treatment with exogenous neurotrophic factor is effective, and the concentration of neurotrophic factor effective for imparting a therapeutic effect. Such an objective would best be accomplished with a neurotrophic factor that is identifiable and distinct from other factors, that is capable of exerting an effect on many different neuron populations, and for which in vivo models are available to test the efficacy of the neurotrophic factor on a specific neuron population.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, there is provided a purified neuron regulatory factor for supporting survival of neurons. This factor is referred to as “neuron regulatory factor” (“NRF”) because it possesses a variety of neuroprotective activities, including the neuron growth and survival-promoting activities traditionally attributed to neurotrophic factors. NRF comprises a complex of polypeptides of molecular weight between about 2,500 and 250,000 Da as determined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and exhibits a neurotrophic and neuron-regulatory effect on cells of the nervous system in vitro and in vivo.
As described in the Examples set forth below, NRF can be purified from a variety of sources. The complex has been purified from conditioned medium from embryonic primordia comprising the geniculocortical pathway. NRF from conditioned medium is sometimes referred to herein as NRFcm. Following isolation from tissue culture media, the complex is subjected to HPLC. Following this purification step, the protein may be further purified over an immunoaffinity column. Electrophoretic resolution of NRF so purified reveals bands migrating at approximately 55, 110 and 200 kDa. It is possible that the higher molecular weight polypeptide is comprised of multimers of the lower molecular weight polypeptides.
NRF has also been purified from homogenates of rat cerebral cortex tissue. The purification of NRF from cytosolic fractions of cerebral cortex tissue is described in Example 7. Like the NRF purified from conditioned medium, resolution of NRF from the cerebral cortex on polyacrylamide gels reveals a protein migrating at approximately 200 kDa.
Example 9 sets forth methods utilized to purify NRF from human retinoblastoma cells. This protein, sometimes referred to herein as NRFrb, also migrates at approximately 200 kd on polyacrylamide gels.
In another embodiment of the invention, NRF is further purified over an immunoaffinity column specific for cytosolic or secreted actin, followed by acid separation on a superose 12 column. NRF so purified is referred to herein as NRFSCI for neuron regulatory factor subcomponent 1. Resolution of NRFSCI on polyacrylamide gels reveals bands migrating at 3-8, 14-21, 30 and 66-69 kDa. The 3-8 and 14-21 bands have been eluted from the gel and
Cunningham Timothy J.
Eagleson Kathie L.
Haun Forrest
Kennedy Sarah E.
Levitt Pat R.
Dann Dorfman Herrell and Skillman
Hayes Robert C.
Kunz Gary L.
Philadelphia Health and Education Corporation
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