Nerve regeneration

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Reexamination Certificate

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C530S399000

Reexamination Certificate

active

06506727

ABSTRACT:

BACKGROUND OF THE INVENTION
This application relates to nerve regeneration by the administration of growth factors.
Growth factors are polypeptide hormones which stimulate a defined population of target cells. Examples of growth factors are platelet-derived growth factor (PDGF), insulin-like growth factors (IGF's), transforming growth factors beta (TGF-&bgr;), and alpha (TGF-&agr;), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic FGF(bFGF), and nerve growth factor (NGF).
The application of a combination of PDGF and IGF-I or PDGF and IGF-II in wound healing and bone regeneration has been described (Lynch et al, 1987
, Proc. Nat'l. Acad. Sci. USA
. 84:7696-7700; Lynch et al, 1989
, J. Clin. Invest
. 84:640-646; Lynch et al, 1989
, J. Clin. Periodontol
, 16:545-588; Lynch et al, 1991
, J. Periodontol
; 62:458-467. U.S. Pat. Nos. 4,861,757 and 5,019,559, hereby incorporated by reference).
IGF's, or somatomedins, are polypeptides of about 7.5 KD that have a strong homology to human proinsulin (Humbel, 1984 in Hormonal Proteins and Peptides 12:57-79). IGF-I and II share a 62% sequence homology. Their actions are mediated through two distinct receptors., The IGF-I receptor is named type-I receptor (IGF-IR), and the IGF-II receptor is named type-II receptor (IGF-IIR). The IGF-IR is a transmembrane protein structurally related to the insulin receptor (Ullrich et al, 1986
EMBO J
. 5:2503-2512). It contains an extracellular binding domain consisting of two &agr;-subunits and an intracellular tyrosine kinase domain consisting of two &bgr;-subunits. The type-I receptor has a high affinity for IGF-I and a lower affinity for IGF-II and insulin. The type II receptor is distinct from the IGF-I and insulin receptors (Morgan et al, 1987
Nature
329:301-307). It has a high affinity for IGF-II, a low affinity for IGF-I and it does not bind insulin. It is a transmembrane protein with a large extracellular binding domain and it does not seem to process tyrosine kinase activity. Its primary sequence is identical to that of the cation-independent mannose-6-phosphate receptor (Morgan et al, 1987 ibid). In addition to IGF-I and IGF-II, a truncated form of IGF-I has been obtained from brain and was named IGF-III (Sara et al, 1986
; Proc. Nat'l. Acad. Sci.: USA
; 83:4904-4907). IGF-III is lacking the three amino-terminal amino acid residues of IGF-I, but it retains functional properties similar to those of IGF-I. In vitro, IGF's exert diverse metabolic activities and they act as growth factors on a variety of cells including cells of mesenchymal origin (Froesch et al, 1985
Ann. Rev. Physiol
. 47:443-467; Van Wyk, (1984) Hormonal proteins and peptides; 12: 81-125; Daugheday and Rotwein.
Endocrine Rev
. 1989; 10:68-91; Baxter et al (1985)
Comp. Biochem. Physiol
. 91&bgr;:229-235; Baskin et al (1988)
TINS
11:107-111). IGF-I was also shown to be a potent inducer of oligodendrocyte development (McMorris et al,
Proc. Natl. Acad. Sci. USA
, 1986; 83:822-826) and a mitogen for cultured neonatal rat astroglial cells (Han et al,
J. Neurosci
. 1987; 7:501-506).
High levels of expression of IGF-I and IGF-II have been reported in fetal and neonatal tissues including brain (Han et al,
J. Clin. Endocrinol Metab
, 1988; 66:422-426; Schofield and Tate, 1987
; Development
101:793-803; D'Ercole and Underwood,
Pediar. Pulmonol
, 1985; 1:599-606; D'Ercole (1987)
J. Devel. Physiol
. 9:481-495; Bondy et al. (1990),
Mol. Endocrinol
. 4:1386-1398).
IGF's have been suggested to act as neurotrophic factors in vitro (Aizenman et al, Brian Res. 1987; 406:32-42; Bothwell,
J. Neurosci. Res
. 1982; 8:225-231; European Patent Application No. 86850417.6; Recio-Pinto et al,
J. Neurosci
1986; 6:1211-1219; Shemer et al,
J. Biol Chem
. 1987; 262:7693-7699) and in vivo (Hansson et al,
Acta Physiol. Scand
. 1986; 126:609-614; Anderson et al,
Acta Physiol. Scand
. 1988; 132:167-173; Kanje et al,
Brain Res
. 1988; 475:254-258; Sjoberg and Kanje,
Brain Res
. 1989; 485:102-108; Nachemson et al, Growth Factors 1990; 3:309-314) and to affect growth of undifferentiated neurons (Re-cio-Pento et al,
J. Neurosci. Res
. 1988; 19:312-320; Matteson et al.(1986)
J. Cell Biol
. 102:1949-1954). Addition of IGF-I or IGF-II alone or in combination with NGF appears to enhance in vitro the survival of neuronal cells (European Patent Application No. 63196524). Local administration of IGF-I to injured rat sciatic nerve has been reported to promote nerve regeneration (Hansson et al, 1986; Sjoberg and Kenje, 1989; Nachemson et al, 1990). Immunohistochemistry studies with specific anti-IGF-I antisera demonstrated increased amounts of endogenous IGF-I expression in the nerve and within the Schwann cells of injured rat sciatic nerve in vivo (Hansson et al,
Cell Tissue Res
. 1987; 247:241-247; and Hansson-et al,
Acta Physiol. Scand
. 1988; 132:35-41).
No data have been previously reported on the effect of exogenous platelet-derived growth PDGF) alone or in combination with other biologically active agents on nerve regeneration in vivo. In situ hybridization and immunostaining of tissues with antigen-specific antisera has demonstrated high levels of PDGF-A chain mRNA and immunoreactive PDGF-A in the neurons of embryonic and adult mice (Yeh et al,
Cell
1991; 64:209-216). In the same study, significantly weaker signals of the PDGF-A chain were observed in glial cells. In vitro Schwann cells in both short and long term culture possess PDGF receptors and synthesize DNA in response to PDGF. The receptors were found to be mostly of the &bgr; type and PDGF-BB homodimer (i.e. PDGF-2) was a more potent mitogen than PDGF-AA homodimer. It was suggested that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development. (Eccleston et al,
Eur. J. Neurosci
. 1990; 2:985-992.) PDGF-&bgr; type receptors have also been reported on newborn rat brain neurons in vivo and in vitro. In vitro continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival (Smits et al.,
Proc. Natl. Acad. Sci. USA
1991; 88:8159-8163). The mRNA for PDGF-A is found in cultured Type-I astrocytes and in perinatel rat brain (Richardson et al,
Cell
1988; 53:309-319). Type-I astrocytes have been suggested to be a source of PDGF in the nervous system (Pring et al,
EMBO J
. 1988; 18:1049-1056). PDGF has also been implicated as a factor in the proliferation and differentiation of rat optic nerve 0-2A progenitor cells (Raff et al,
Nature
1988; 333:560-562; Noble et al,
Nature
; 1988; 333:560-562). PDGF appears to have a role in the proliferation and development of glial cells in the central nervous system (reviewed in Raff M, Science 1989; 243:1450-1455).
Peripheral Nerve Repair
Injury to peripheral nerves induces profound changes in the nerve cell body, its processes, and its surroundings (reviewed by Seckel, 1990
; Muscle & Nerve
13:785-800). Following injury, the central nerve cell body becomes swollen, the nissl substance is dispersed, and the nucleus is displaced peripherally. The central cell body synthesizes a host of new mRNA'a, lipids, and cytoskeletal proteins (Grafstein B, et al, in
Neuronal Plasticity
, Cotman CW (ed) 1978). In addition other growth associated proteins (GAP's) are synthesized. Although GAP's do not appear to initiate growth, they are an essential component of the regenerative response. Electrophysiologic changes occur in the cell body that indicate differentiation towards a more plastic or embryonic state permitting growth (Foehring et al, (1986)
J. Neurophysiol
55:947-965; Gorden et al, in
Somotic and Autonomic Nerve-Muscle Interactions
, Burnstock et al, (ed's) 1983).
The proximal axonal segment undergoes a variable degree of traumatic degeneration following nerve injury. This degenerative process extends at a minimum back to the next node of Ranvier, or maximally may result is cell death. When cell death is not the sequela, the area of the first node of Ranvier proximal t

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