Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-04-23
2002-07-09
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S014800, C514S015800, C514S214020, C514S365000, C514S424000, C514S570000, C514S634000, C514S648000
Reexamination Certificate
active
06417159
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for enhancing an effect of a neutrophin, preferably, but not limited to enhancing the growth and survival promoting properties of neurotrophins, using an analogue of a portion of p75
NTR
.
BACKGROUND OF THE INVENTION
The neurotrophins are a family of structurally and functionally related neurotrophic factors. The family includes prototypic member nerve growth factor (NGF), as well as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) (Heumann, 1994) and neurotrophin-6 (NT-6) (Gotz et al., 1994). The neurotrophins have similar structural conformations, including three surface &bgr;-hairpin loops, a &bgr;-strand, an internal reverse turn region, and N- and C-termini, and exhibit approximately 50% amino acid sequence identity.
Neurotrophins function to promote growth and survival of certain classes of peripheral and central nervous both during development and following neuronal damage. For example, NGF is involved in the development of neurons in the peripheral nervous system, supports neuronal survival, and enhances and maintains the differentiated state of neurons. Neurotrophins can promote neurite differentiation such as sprouting or process formation, and process growth. Neurotrophins can also modulate cell motility (Anton et al., 1994), for example, both accelerate nerve process growth and decrease general cell motility. Another neurotrophin-mediated activity is induction of particular enzymes.
Furthermore, with respect to functional similarity, each of the neurotrophins can bind to a membrane-bound receptor protein (MW~75 kDa) called the common neurotrophin receptor, or “p75
NTR
”. Each neurotrophin also binds with higher affinity to a second membrane-bound receptor protein of the tyrosine kinase receptor (Trk) family. In particular, NGF binds selectively to the TrkA receptor, and BDNF and NT-4/5 bind selectively to the TrkB receptor. NT-3 is less selective and, though it binds primarily to the TrkC receptor, NT-3 also exhibits some binding to the TrkA and TrkB receptors (banez et al., 1993).
A variety of cell types express either p75
NTR
and/or a member of the Trk family of receptor tyrosine kinases. These include neurons, mast cells, glial cells such as astrocytes, oligodendrocytes and Schwann cells, and dysplasic or malignant cells such as neuroblastoma or melanoma cells. Cells of neuronal lineage that differentiate by extension of neurites in the presence of a neurotrophin express both a member of the Trk receptor family and lower molecular weight receptor protein p75
NTR
.
Neurite growth is the best characterized differentiation response to NGF, and evidence is beginning to emerge that p75
NTR
can modulate this activity. Gene targeting studies resulting in nonfunctional p75
NTR
demonstrate reduced density of sensory and sympathetic innervation in vivo (Lee et al., 1994 a and b), possibly related to a shift to the right of dose response curves for NGF (Davies et al., 1993). The low molecular weight neurotrophin receptor p75
NTR
is a member of a family of receptors designated the NGF receptor superfamily (Krammer and Debatin, 1992; Mallett and Barclay, 1991). In addition to p75
NTR
, this family includes TNFR1, TNRF2, CD30, Fas, Fas/Apo-1, Apo-3, CD40, 4-1BB, CD-27, SFV-T2, and OX-40. While these receptors have been grouped according to structural similarities in transmembrane and extracellular domains, the inventors have provided evidence supporting existence of shared pulative signalling motifs in the cytoplasmic domains (Myers et al., 1994, incorporated herein by reference). Similar to the wasp venom tetradecapeptide mastoparan (MP), p75
NTR
(rat, chick, human), human TNFR-1, and human 4-1BB have been found to have secondary structure domains with putative plasma membrane associating properties that have been implicated in intracellular signalling (Higashijima, 1983, 1990, each incorporated herein by reference).
Fas/Apo-1, TNFR1, Apo-3, and p75
NTR
all activate apoptosis via “death domains” that are rich in sequences predicted to form &agr;-helices (Myers et al., 1994). These &agr;-helices are potentially involved in oligomerization that mediates signalling in ligand-independent or ligand-dependent states (Huang et al., 1996). TNFR1 and p75
NTR
contain within their death domain regions, motifs that confer surface membrane associating properties (Myers et al., 1994) as determined by maximum mean hydrophobic moment (Eisenberg et al., 1984a,b), and both TNFR1 and p75
NTR
signal translocation of transcription factor NFkB (Marsters et al., 1996; Carter et al., 1996). In contrast, Apo-1/Fas, which contains a death domain but no amphiphilic sequence therein (Myers et al., 1994), does not translocate NFkB (Marsters et al., 1996). The inventors reasoned from these observations that the amphiphilic motif of p75
NTR
could be involved in NFkB activation/translocation, and thus conducted a series of experiments to determine whether the amphiphilic motif of p75
NTR
and NFkB interact directly.
Based on knowledge of their biological actions, neurotrophins have been considered for therapeutic use in neurological disorders characterized by loss of neurons and/or loss of connectivity. Such disorders include stroke, cerebral and spinal cord injury, a host of neurodegenerative disorders, the most prevalent of which is Alzheimer's disease, and a variety of peripheral nerve disorders, most notably those associated with diabetes mellitus and cancer chemotherapy.
These are limitations to use of neurotrophins for therapy of neurological disorders. Such limitations relate to the prohibitive costs of production of recombinant human neurotrophins, their stability in vivo, the routes of administration, and penetration of the blood-brain and nerve-brain barriers.
To circumvent such limitations, stable small molecule neurotrophin agonists or stable small molecule modulators of neurotrophin levels or neurotrophin effects would be desirable.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and compositions for enhancing an effect of a neurotrophin, preferably enhancing the growth and survival promoting properties of a neurotrophin.
It is a further object to provide low molecular weight analogues of p75
NTR
367-379 useful as therapeutic agents for in situ treatment of certain neurological disorders.
It is a yet another object to provide low molecular weight analogues of p75
NTR
367-379 useful for ex vivo treatment of cells. Such ex vivo uses include diagnostic applications, assays wherein various factors are screened, and applications wherein cells, e.g., hematopoientic cells, are treated prior to their reintroduction to a patient.
Utilizing peptide analogues of a putative amphiphilic domain of p75
NTR
, the inventors have discovered that these analogues have biophysical and biochemical properties in MP (Sequence ID No. 5) that they are taken up into cells, that the amphiphilic properties and polar group relationships of p75
NTR
367-379 analogues influence signalling pathways involved in neurite growth by NGF-responsive cells of neuronal lineage, and that these analogues depend upon the functional expression of TrKA to exert their influence. The findings suggest that p75
NTR
participates by interaction of its amphiphilic domain with a Trk receptor to modulate neurite growth.
In the broadest aspect, the present invention provides a method for enhancing the effect of a neurotrophin on cells having a plasma membrane and expressing a neurotrophin receptor. The method comprises providing an effective compound having an effective number of charged moieties spaced thereon and, when in close proximity to the plasma membrane, an amphipathic conformation comprising a substantially hydrophobic membrane associating face and a substantially hydrophilic face. The method includes exposing the cells for an effective period of time to an effective amount of the compound in the presence of the neurotrophin for enhancing the effect of the neurotro
Riopelle Richard J.
Ross Gregory M.
Shamovsky Igor L.
Weaver Donald F.
Dowell & Dowell , P.C.
Hayes Robert C
Kunz Gary L.
Miernicki Steeg Carol
Queen's University at Kingston
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