Note: Descriptions are shown in the official language in which they were submitted.
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Apolipoprotein E/Growth Factor Complexes
to and Methods of Use
Related Application Information
This application claims the benefit of United States Provisional Application
No. 601060,533 filed September, 30, 1997, which is incorporated by reference
herein in its entirety.
Statement of Federal Support
This invention was made with Government support under grant number RO1
AG12532-O1 from the National Institutes of Health. The government has certain
rights in this invention.
Field of the Invention
The present invention relates to compositions comprising complexes
containing apolipoprotein E and nerve growth factor, neurotrophin 4, or y-
interferon
and methods of administering the same.
Background of the Invention
Apolipoprotein E (protein: apoE; allele: APOE) is the principal
apolipoprotein in the brain (for review, see Mahley, (,1988) Science 240, 622)
and
cerebrospinal fluid (CSF) (Pitas et al. , (1987) J. Biol. Cnem. 262, I4352).
Several
observations have implicated a role for apoE in the injured nervous system.
Expression of apoE mRNA by astrocytes in the hippocampus increases following
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entorhinal cortex lesion (Poirier et al. , {199/) Mol. Brain Res. 11, 97).
Oligodendrocytes and macrophages increase expression of apoE Following optic
and
sciatic nerve injury, respectively (optic: Stoll et al. , (1989) GLIA 2, 170;
sciatic:
Skene and Shooter, (1983) Proc. Nat. Acad. Sci. USA 80, 4169; Stoll and
Mueller,
(1986) Neurosci. Len. 72, 233), and apoE protein accumulates to 5% of total
extracellular protein following peripheral nervous system (PNS) injury (Skene
and
Shooter, (1983) Proc. Nat. Acad. Sci. USA 80, 4169). APOE is a susceptibility
gene for familial and late-onset Alzheimer's disease (AD: Strittmatter et al.,
(1993)
Proc. Nat. Acad. Sci. USA 90, 1977; for review see Strittmatter and Roses,
(1995)
Proc. Nat. Acad. Sci. USA 92, 4725). The gene dose of APOE4, one of the three
major alleles of APOE in humans, is correlated with increased iiak and-
decreased
average age of onset of AD. These observations suggest a role for apoE in the
injured or diseased nervous system.
Three major isoforms of apoE in humans -- apoE2, apoE3 and apoE4 -- are
distinguished by cysteine-arginine substitutions at positions l I2 and 158.
The most
common isoform, apoE3, is secreted as a 299 amino acid protein with a single
cysteine at position 112 and an arginine at position 158: apoE2 contains a
cysteine at
position 158 and apoE4 contains an arginine at position 112. AgoE contains two
distinct structural and functional domains, a hydrophobic domain and a
hydrophilic
receptor binding domain (Weisgraber, (1994) Adv. Prot. Chem. 4~, 249). The
crystal structure of the hydrophilic domain of apoE is homologous to the
family of
four-helix bundle growth factors, including ciliary neurotrophic factor,
although the
sequences of these proteins diverge greatly. CNTF; reviewed by Bazan, (1991)
Neuron 7, 197; Mott and Campbell, (1995) Curr. Opin. Struc. Biol. ~, 114; apoE
crystal structure by Wilson et al., (1991) Science 252, 1817; CNTF crystal
structure
by McDonald et al., (1995) EMBO J. 14, 2689.
Nerve growth factor (NGF) belongs to a family of neurotrophins that
includes brain-derived neurotrophic factor {BDNF), neurotrophin 3 (NT3),
neurotrophin 415 (NT415), and neurotrophin 6 (NT6). All of these proteins bind
to
the low affinity NGF receptor p75 and a specific member of the trk family of
tyrosine kinase receptors. For NGF, this is the trkA receptor. Binding of the
26
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kDa homodimer of NGF to trkA induces an intracellular signaling cascade that _
begins with autophosphorylation of trkA. Ultimately, activation of trkA ends
with
changes in gene expression that effect neuronal survival, outgrowth,
excitability,
and differentiation.
It is well-established that NGF promotes survival and neurite outgrowth of
sympathetic and neural-crest derived sensory cells during development (for
review,
see Snider and Johnson, (1989) Annual Neurology 2b, 489). This is evidenced by
the fact that mice that are homozygote mutants for either NGF or trkA have
severe
loss of cells in the superior cervical ganglion (SCG; >90%) and in dorsal root
ganglia (DRG, 70-80%a) (Crowley et al., (1994) Cell 78, 1001; Smeyne et al.,
(1994) Nature 368, 246) and die within 3 weeks of birth. The ability-of NGF to
promote neurite outgrowth is one of the most dramatic activities of NGF.
Indeed,
neurite outgrowth from explanted embryonic chick DRGs is the standard bioassay
to
test for NGF activity. Dissociated cultures of DRGs and SCGs also respond to
NGF with increased neuritic growth. The PC12 cell Line, derived from rat
adrenal
medulla, has provided an excellent system in which to study the neurite
outgrowth
and survival effects of NGF. If PC12 cells are cultured in medium supplemented
with fetal bovine and horse serum, they proliferate and appear as small, round
phase-bright cells. Upon addition of NGF to the culture medium, these cells
stop
dividing and differentiate into a sympathetic-like neuronal phenotype with
multiple
neuritic processes and large cell bodies. NGF is also required for survival of
PC12
cells in a serum-free environment. Because of the ease of this paradigm, much
of
the work elucidating the mechanism of NGF action has used PC12 cells.
As DRG neurons mature, the time course and sensitivity of their dependence
2~ on NGF for survival changes. To illustrate this, 24 hours following
transection of
the sciatic nerve in a newborn rat there is loss of 45 % of the cells in the
corresponding DRG. Yip et al., (1984) J. Neurosci. 4, 2986. Systemic
administration of NGF reduces cell loss to 18 % . Id. In contrast, in the
adult rat,
' transection without regeneration results in a 22 %a loss of axotomized cells
in the
DRG at 3 weeks post axotomy. Application of NGF directly to the proximal
stump,
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prevenu all of the axotomy induced cell death. Rich et al., (1987) J. Neuro.
Cytol.
16, 569.
Exogenous NGF can promote the survival and regeneration of transected
sciatic nerves. Multiple studies have confirmed that silastic tube implants
filled with
NGF enhance the rate and degree of functional sensory recovery. Rich et al. ,
(1987) J. Neuro. Cytol. I6, 261; Rich et al., (1989) J. Neuro. Cytol. 18, 569;
Derby et al., (I993) Exp. Neurol. 119, 176.
Neurotrophin 4 (NT4) is another member of the neurotrophin family. NT4
has been reported to promote survival of corticospinal motor neurons from
neonatal
rats (Iunger and Varon, (1997) Brain Res. 762, 56), increase survival of
cultured rat
septal neurons under normal and stress conditions (Nonner et al, (1996) J.
Neurosci.
16, 6665), promote survival and morphological and biochemical differentiation
of
embryonic rat striatal neurons in culture (Ventimiglia et al. (1995), Eur. J.
Neurosci. 7, 213), and enhance survival of cultured rat vestibular ganglion
neurons
and protect these cells against neurotoxic agents (Zheng et al., (1995) J.
Neurobiol.
28, 330).
Interferons (IFN) are proteins secreted by eukaryotic cells after virus
infections, which in turn ~ protect against virus infections. Three classes of
interferons are known at present: they are referred to as IFNa., IFN~i and
IFNy,
which differ in both their structure and biological effects. All of the IFNy
identified
thus far are glycosylated, although glycosylation does not appear to influence
biological activity. Keller et al., (1983) J. Biol. Chem. 258, 8010. Gamma-
interferon is regarded as a Iymphokine since it is produced by lymphoctyes
after
either specific or non-specific stimulation by antigens. Gamma-interferon is
well-
known as an anti-viral and anti-tumor agent. See U.S. Patent No. 5,602,010 to
Hauptmann et al. Other investigators have found that IFNy, increases
intravascular
C1 inhibitor concentrations in patients exhibiting or at risk for C1 inhibitor
deficiencies. U.S. Patent No. 5,271,931 to Lotz et al. C1 inhibitor is a
serine
protease inhibitor that is involved in the regulation of several protedlytic
systems
including the complement, contact, coagulation, and fibrinolytic systems.
Davis et
al. (I988) Ann. Rev. Immunol. 6, 595. Other activities of IFNy include
.promotion
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of polynuclear giant cell formation and activation of macrophages (Weinberg et
al. ,
{1984) Proc. Natl. Acad. Sci. USA 81, 4554), inhibition of bone resorption
{U.S.
Patent No. 4,921,697 to Peterlik et al.), and prevention of type I diabetes
mellitus
{U.S. Patent No. 5,624,895 to Sobel).
Thus, NGF, NT4 and IFNy possess biologically significant activities.
Accordingly, there is a need in the art for strategies of potentiating the
activity of
these factors. In particular, it is an object of this invention to provide
means of
enhancing the activity of the neurotrophic growth factors, NGF and NT4, to
slow
the progression of neurodegenerative diseases, to protect against neural
degeneration
after injury, and to facilitate nerve regeneration.
Summary of the Invention
Disclosed herein is the novel discovery that apoE binds to and potentiates the
biological action of the neurotrophic factor NGF. By "potentiate" it is meant
that
apoE enhances at least one biological action of NGF, by increasing the maximal
response achieved andlor increasing the potency of the growth factor (i. e. ,
shifting
the dose-response curve to the left). Alternately, the term "potentiate"
indicates that
. apoE stabilizes the NGF molecule and decreases its degradation. Further
disclosed
is the finding that apoE advantageously binds to NT4 and IFNy.
As a first aspect, the present invention provides a composition comprising a
complex of apoE and NGF. Also disclosed are compositions comprising a complex
of apoE and NT4. Further disclosed are compositions comprising a complex of
apoE and IFNy. The apoE component can be apoE2, apoE3, or apoE4, and can be
in the native lipid-bound or a delipidated state. Furthermore, the complex can
be
formed by either covalent or noncovalent interactions between the apoE and
NGF,
NT4 or IFNy molecules.
As a second aspect, the present invention provides a method of enhancing the
survival of neural cells, comprising administering to the neural cells a
survival-
enhancing amount of a composition comprising a complex of apoE and NGF or
NT4.
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As a third aspect, the present invention provides a method of administering a
_
composition comprising a complex of apoE and NGF or NT4 in a therapeutically-
effective amount to a subject in need thereof.
As a fourth aspect, the present invention provides a method of protecting
cells against virus infection, comprising administering to the cells an anti-
virus
effective amount of a composition comprising a complex of apoE and IFNy.
Further provided is a method of administering IFNy in an anti-viral effective
amount
to a subject in need thereof.
As a further aspect, the present invention provides a method of administering
IO IFNy in an anti-tumor effective amount to a subject in need thereof.
These and other aspects of the invention are set forth in more detail in the
description of the invention below.
_ ___ _ _ _. __ ___ __ __ ___ _ _ ___ _ _ _ __ g~ie-f-DesEription-of the-
Drawings- _ . _ _ __ ___ __ __ _ ___ __ . _
Figure 1 demonstrates that apoE3 potentiates the survival promoting activity
of NGF in PC12 cells. The data are presented as the mean percent survival ~
standard deviation as compared to the control treatment of 1000 ng/ml NGF and
no
apoE. Using the Student's t test for the difference between the means, p <
0.001 at
100 and 1000 nglml NGF. Open bars: substrate-bound apoE3 (50 nglml)
preincubated with 0, 100 and /000 nglml NGF. Black bars: soluble NGF alone
added at 0, 100 and 1000 nglml.
Figure 2 demonstrates that apoE3, but not apoE4, enhances neurite
outgrowth from PC12 cells in response to NGF. Neurite outgrowth was measured
2~ after 3 days in culture with serum-free medium containing 100 ng/ml NGF and
1
p.g/ml apoE3, apoE4 or bovine serum albumin (BSA) as a control protein. Phase
images of cells were digitally collected on random fields using IMAGE lT"
analysis
software (Universal Imaging Corp.; West Chester, PA). Neurite length is
displayed
as percent of outgrowth of the control treatment (NGF+BSA) t SEM. Using a
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WO 99/16460 PCT7US98120591
paired Student's t-test, p < 0.00 for apoE3 vs. BSA and p < 0.01 for apoE3 vs.
_
apoE4.
Figure 3 demonstrates that apoE knockout (KO) mice have a reduced
number of unmyelinated fibers as compared with wildtype (WT) mice. The total
area covered by high-powered electron micrographs from 3 wildtype and 3
knockout
animals was approximately 18,000 pm'- for each group (17,900 for wildtype and
18,200 for knockout). The total number of myelinated and unmyelinated axons in
this area were counted. It was found that knockout nerves had 50 % fewer
unmyelinated fibers, but an equal number of myelinated fibers, as compared
with
nerves from wildtype animals.
Figure 4 demonstrates that apoE knockout mice have a delayed response to
noxious thermal stimuli. For this experiment, the withdrawal latency in
seconds for
mice to remove their hind feet from a 55°C water bath was measured. The
data
presented are the means ~ SEM for 3 mice in each group and 3 measurements/per
hind foot for each mouse. Knockout mice have over a 50% increase in their
response latency as compared with wildtype mice. The p value for the
difference
between the two means is p < 0.001 using a paired Student's t test.
Detailed Description of the Invention
A. Neurotro~hic Factors - NGF and NT4.
The nervous system responds to acute injury and to chronic
neurodegenerative diseases by the coordinated expression of many proteins,
including growth and survival factors, cell surface receptors, and secreted
extracellular proteins. Many of these proteins, most notably the neurotrophic
factors, play a role in recovery from injury and protection from
neurode~enerative
disease. Within the complex milieu of the traumatized nervous system it is
extremely unlikely that any one growth factor functions alone. Cooperative
interactions of growth factors with other proteins are likely to play
important roles
in the stability, localization or presentation of jrowth factors. Optimal
functioning
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of growth and survival factors released at sites of injury is critical for
recovery from _
neural trauma. In both the peripheral and central nervous systems the
expression of
apoE is increased in acute and chronic stress.
Disclosed herein is the discovery that apoE binds to the neurotrophic factors
NGF and NT4. Further disclosed is the discovery that apoE potentiates the
biological activity of NGF. As described above, by "potentiate" it is meant
that
apoE enhances at least one biological action of NGF, by increasing the maximal
response achieved andlor increasing the potency of the growth factor (i. e. ,
shifting
the dose-response curve to the left). Alternately, the term "potentiate"
indicates that
apoE stabilizes the NGF molecule and decreases its degradation.
While not wishing to be held to any particular theory of the invention, the
finding that apoE binds to NGF and NT4 suggests that apoE may act as an
accessory protein for these growth factors, regulating their metabolism or
biological
activities. One mechanism may entail the localization of the growth factors to
extracellular matrix by tethering them through an apoE molecule. It has
previously
been shown that apoE binds to the extracellular matrix protein laminin, and
thereby
increases neuron adhesion, and alters growth cone spreading (Huang et al.,
(1995)
Exp. Neurology 136, 251). ApoE may additionally alter the biological activity
of the
bound growth factor by other mechanisms, such as inhibiting its proteolytic
inactivation, or by altering its ability to interact with its cell-surface
receptor.
Interactions between apoE and NGF and/or NT4 may be important in modulating
the role of these growth factors in neural regeneration responses, both in
acute and
in chronic disorders of the nervous system.
Disclosed herein are compositions containing apoE:NGF and apoE:NT4
complexes. The claimed complexes can be formed by simply mixing apoE and the
neurotrophic factors) (typically aqueous solutions) together, or by any other
suitable method known in the art. The compositions can contain both complexes
of
apoE with NGF and complexes of apoE with NT4. Also encompassed by the
present invention are compositions containing apoE:NGF andlor apoE:NT4
complexes andlor complexes of apoE with other neurotrophic factors, such as
ciliary neurotrophic factor.
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Those skilled in the art will appreciate that compositions containing the _
claimed apoE complexes produced in this manner will generally contain apoE,
NGF
and/or NT4 monomers, homodimers and multimers. In general, as low as 35%,
25 % , 20 % , 15 %a , or lower, or as high as 40 % , 50 % , 70 %a , 85 % , 90
% , 95 % , 99 %
or higher of the NGF or NT4 molecules in the composition will be complexed
with
apoE. The terms "apoE:NGF" and "apoE:NT4", as used herein, encompass
apoE:NGF and apoE:NT4 complexes containing monomers, dimers, trimers and
larger multimers of NGF or NT4, respectively. Alternately, apoE monomers,
homodimers, homotrimers, etc. may associate with NGF or NT4 monomers,
homodimers, homotrimers, erc. Thus, the present invention encompasses
complexes between one or more apoE molecules with one or more NGF or NT4
molecules.
While not wishing to held to any particular theory of the invention, the
active form of NGF is a homodimer, and it is believed that the NGF homodimer
complexes with an apoE monomer. Heterodimeric apoE:NGF and heterotrimeric
apoE:(NGF), complexes are preferred. Likewise, heterodimers of apoE and NT4
are preferred. Complexes between one or more apoE molecules with one or more
NT4 molecules are also aspects of the present invention. Those skilled in the
art
will appreciate that apoE:NGF and apoE:NT4 complexes may also be more loosely
associated with additional molecules.
Alternatively, apoE may first be bound to a substrate, such as a polymeric
surface (i. e. , tissue culture plate, test tube), prior to being exposed to
and forming a
complex with NGF or NT4. Such apoE-bound substrates are useful for collecting
NGF or NT4 from a solution. As a further alternative, the apoE:NGF or apoE:NT4
complex can be bound to a substrate after complex formation. Such substrates
are
useful for culturing cells in vitro.
The strength of the binding interaction between apoE and NGF or NT4 is
high; with a dissociation constant of the apoE complexes of at least 10-',
preferably
at least 108, more preferably at least 10'9. Complexes can be formed by
covalent or
noncovalent interactions, with covalent complexes of apoE and NGF or NT4 being
preferred. Typically, apoE:NGF and apoE:NT4 complexes are stable in a solution
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containing at least 1 % SDS. Also preferred are complexes formed between apoE3
-
and NGF or NT4.
The apoE component of the claimed complexes can be apoE2, apoE3, apoE4
or a combination thereof. Preferably, the complexes contain apoE3. ApoE
variants
and fragments that bind to the apoE receptor and/or complex with and
potentiate the
biological actions of NGF andlor NT4 are also encompassed by the present
invention. ApoE can be from any species of origin, preferably of mammalian
origin, more preferably human origin. The apoE molecules can be in the native
lipid-bound state or a delipidated state, with delipidated being preferred.
ApoE can be purified from natural sources (i. e. , blood, serum or peritoneal
fluid). United States Patent No. 5,672,685 describes the isolation of native
apoE
from peritoneal fluid, the disclosure of which is incorporated herein in its
entirety
by reference. The majority of apoE from sera is associated with lipoprotein
particles. Purification of apoE from sera requires delipidation with organic
solvents
or detergents, which causes significant protein denaturation. Lipoprotein
isolation
by ultracentrifugation, with subsequent lyophilization , and delipidation of
lipoproteins, and chromatographic isolation of apoE, is described in Rall et
al. ,
(1986) E. Methods Enzymol. 128, 273. An alternative method for isolation of
apoE
from a mixture of apolipoproteins utilizes gel electrophoresis. Purification
of apoE
isoforms may be accomplished using isoelectric focusing techniques (Rail et
al. ,
(1986) E. Methods Enrymol. 128, 273).
ApoE may also be separated from contaminating proteins using heparin-
sepharose chromatography, which utilizes the heparin-binding property of apoE.
Rall et al. , ( 1986) E. Methods Enryrnol. 128, 273. ApoE may be isolated
and/or
purified, optionally to homogeneity, by conventional techniques such as
affinity
chromatography, size-exclusion chromatography, gas chromatography, HPLC, and
combinations thereof. Separation of the non-cysteine containing E4 isoform of
apoE
from contaminating cysteine-containing proteins may be accomplished using
thiopropyl chromatography on thiopropyl Sepharose (Weisgraber et al. (1983),
J.
Biol. Chem. 258, 2508).
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Recombinant apoE can be produced using methods known in the art, and -
human recombinant apoE is commercially available. However, recombinant protein
is not in the native glycosylated form and is subject to denaturation and
oxidation
during purification.
The NGF component of the complex can also be from a native or
recombinant source, and can be produced by any means known in the art. Native:
U.S. Patent Nos. 5,210,185 and 5,057,223 to Della Valle et al.; U.S. Patent
No.
4,407,744 to Young. Recombinant: U.S. Patent No. 5,272,063 to Chan et al.;
U.S.
Patent No. 5,288,622 to Gray et al. ; U.S. Patent No. 5,082,774 to Heinrich.
Moreover, the NGF component of the claimed apoE:NGF complexes can be from
any species of origin, preferably of mammalian origin, more preferably of
human
origin. The term "NGF" encompasses NGF variants, analogs and derivatives. For
example, U.S. Patent No. 5,349,055 to Persson et al. discloses NGF analogs
with
significantly reduced binding to the low affinity p75 receptor and essentially
no
alteration in binding to the trk receptor. Further disclosed by this reference
are NGF
analogs having increased stability. Also known in the art are chimeric NGF
molecules in which regions of the NGF peptide are replaced by the
corresponding
residues of brain derived growth factor (BDGF). ibanez et al., (/991) EMBD J.
10,
2105. Finally, the term "NGF" also includes NGF fragments that bind to NGF
receptors andlor elicit the neurotrophic actions of NGF. For example, U.S.
Patent
No. 5,134,121 to Mobley et al. discloses NGF peptides and analogs thereof that
induce NGF-associated biological response.
Likewise, the NT4 component of the disclosed apoE:NT4 complexes can be
from a native or recombinant source, and can be produced by any means known in
the art. Recombinant: U.S. Patent No. 5,364,769 to Rosenthal. Moreover, the
NT4 component of the claimed apoE:NT4 complexes can be from any species of
origin, preferably of mammalian origin, more preferably of human origin. The
term "NT4" also encompasses NGF variants, analogs and derivatives, such as
those
disclosed in U.S. Patent No. 5,364,769 to Rosenthal and U.S. Patent No.
5,349,055
to Persson. The term "NT4" also includes NT4 fragments that bind to NGF
receptors and/or elicit the neurotrophic actions of NT4.
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Methods of enhancing the survival of neural cells by administration of an
apoE:NGF and/or apoE:NT4 complex are also an aspect of the- present invention.
The term "enhancing the survival" of neural cells is intended to be construed
broadly, and includes neurotrophic and neuro-regenerative actions of the
claimed
compositions. Alternatively, the expression "enhancing the survival" of neural
cells
refers to the action of the claimed compositions in protecting neural cells
from
injury and/or improving recovery from neural injury. By "enhancing the
survival"
it is meant that the inventive complexes provide some improvement to neural
cell
survival, as defined above. The improvement in neural cell survival can be 5 %
,
10 % , 25 % , 50 % , 75 % , 100 % or more. "Neural cells" includes the cells
and
tissues of the central nervous system and the peripheral nervous system, both
in
vitro and in vivo. Compositions comprising apoE:NGF and/or apoE:NT4
complexes for use in the claimed methods are as described in more detail
hereinabove.
The claimed apoE complexes can be administered to neural cells in vitro.
Typically, in vitro administration will simply require adding a solution
(i.e.,
aqueous) containing the apoE:NGF andlor apoE:NT4 complexes to the culture
medium. Alternatively, the apoE and NGF or NT4 components can be added
individually, either concurrently or sequentially, to the culture medium. As a
further alternative, the apoE molecule or the apoE:growth factor complex can
be
bound to a substrate (i. e. , a tissue culture dish or petri dish), preferably
prior to
culturing cells on the substrate. It is well-known in the art that apoE binds
to
polymeric or coated (i. e. , with extracellular matrix proteins such as
laminin)
surfaces of tissue culture plates.
Complexes of apoE and NGF or NT4, which potentiate the activity of these
neurotrophic factors, are useful in vitro for culturing neural cells, such as
immortalized PCI2 cells (see, e.g., U.S. Patent No. 5,349,055 to Persson et
al.)
and primary cuI~~:~s of neurons (see, e.g., Varon, (1997) Brain Res. 7b2, 56;
Barren and Barr=n, (1996) J. Neurosci. 16, 6665; Abiru et al., (1996) Brain
Res.
Dev. Brain Res. 91, 260; Snider and Johnson, (1989) Annual Neurology 26, 489;
Yip et al., (1984) J. Neurosci. 4, 2986).
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As provided by U.S. Patent No. 5,134,121 to Mobley, NGF additionally _
finds use as a component of culture medium to enhance the survival of neurons.
Also disclosed is the use of NGF to provide a therapeutic treatment for
Alzheimer's
disease, Huntington's disease, and other neurodegenerative disorders. As
established by U.S. Patent No. 5,604,202 to Kessler et al., NGF also finds use
in
methods of treating drug-induced neuropathy. Thus, the presently claimed NGF
complexes possessing enhanced neurotrophic activity find similar uses as
previously
known NGF compositions.
As established by U.S. Patent No. 5,364,769 to Rosenthal, NT4 finds use as
a component of culture media to enhance the survival or induce the outgrowth
of
nerve cells in vitro. Also disclosed is use of NT4 in the therapy of
neurodegenerative diseases. Thus, the presently claimed NT4 complexes
possessing
enhanced neurotrophic activity find similar uses as previously known NT4
compositions.
The claimed apoE:NGF and apoE:NT4 complexes also find use in tissue
culture, because the complexed growth factors are protected against
proteolytic
degradation.
Finally, the claimed complexes find use in methods of quantifying or
purifying NGF or NT4 receptors or other proteins that bind to these
neurotrophic
factors. ApoE is readily bound to surfaces, such as test tubes and microtiter
plates.
Thus, NGF or NT4 can be tethered to surfaces for use in sandwich assays or
affinity
purification techniques, which are well-known to those skilled in the art.
The claimed apoE:NGF or apoE:NT4 complexes can also be administered to
a subject in vivo. Methods of administration and gharmaceutical formulations
of the
claimed compositions are described in more detail hereinbelow. The methods of
the
invention are useful for treating a subject afflicted with a neurodegenerative
disease
and subjects who have experienced injury or trauma to neural tissue. Such
subjects
include but are not limited to those afflicted with Alzheimer's Disease,
Parkinson's
Disease, peripheral nerve injury, peripheral neuropathy (in particular,
diabetes-
induced peripheral neuropathy), amyotrophic lateral sclerosis, head injury,
and
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stroke. The present invention is particularly useful in treating Alzheimer's
Disease -
and diabetes-induced peripheral neuropathy.
Subjects suitable for carrying out the present invention are, in general,
mammals and avians, including but not limited to humans, monkeys, equines,
caprines, bovines, ovines, porcines, dogs, cats, rabbits, rats, hamsters,
mice, quail,
chickens and turkeys. Human subjects are presently preferred for in vivo
administration. Similarly, cultured neural tissues/cells for use with the
inventive
methods include mammalian and avian tissues and cells, including but not
limited to
neural tissues and cells derived from humans, monkeys, equines, caprines,
bovines,
ovines, porcines, dogs, cats, rabbits, rats, hamsters, mice, quail, chickens
and
turkeys.
The disclosed compositions may be contained in a physiologically acceptable
carrier (preferably sterile), which is a carrier that is not unduly
detrimental to cells and
includes pharmaceutically acceptable carriers.
In the manufacture of a medicament according to the present invention,
hereinafter referred to as a pharmaceutical formulation, the inventive
compositions are
typically admixed with a pharmaceutically acceptable carrier. For injection,
the
carrier will typically be a liquid. For other methods of administration, the
carrier may
be either solid or liquid, such as sterile, pyrogen-free water or sterile
pyrogen-free
phosphate-buffered saline solution. Alternately, one may incorporate or
encapsulate
the claimed complexes in a suitable polymer matrix, Iiposome or membrane, thus
providing a sustained-release delivery device suitable for implantation near
the site to
be treated locally.
For the preparation of these compositions for administration to a subject, use
2~ can be made of pharmaceutical carriers adapted for conventional forms of
administration, for example, injection solutions, tablets, capsules, dragees,
syrups,
solutions, suspension and the like. As an injection medium, it is preferred to
use water
which contains the additives usual in the case of injection solutions, such as
stabilizing
agents, salts or saline; andlor buffers. The active agent or its
pharmaceutical
formulation may be contained within a nutritional medium, e. g. , in
nutritional
14
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supplements. Oral formulations may be slow release preparations or enteric
coated _
preparations to facilitate delivery of the peptide to the small intestine.
When administering the claimed apoE complexes to a subject in need thereof,
any suitable route of administration may be employed, including administration
by
parenteral injection (e.g., subcutaneous, intramuscular, or intradermal), or
by oral,
rectal, topical, nasal, ophthalmic, intrathecal, and intracerebral
administration.
The apoE:NGF and apoE:NT4 complexes are included in an amount effective
to accomplish the intended treatment. In general, the claimed apoE complexes
are
present in an amount effective to enhance survival of neural cells. ApoENGF
and
apoE:NT4 complexes may be administered concurrently or in combination with
other
agents. In particular, apoE:NGF andlor apoE:NT4 complexes can be administered
with other neurotrophic factors, such as CNTF.
The precise amount of an apoE:NGF or apoE:NT4 complex to be administered
is determined in a routine manner, and will vary depending on the age and
species of
subject, the desired effect, the apoE isoform, and the route of
administration.
Preferred dosages may be determined by simply administering a composition
containing a known amount of apoE:NGF or apoE:NT4 complexes in vitro or in
vivo
to a subject, and monitoring the cells, tissue or subject for the desired
effect, as would
be known by one skilled in the art.
There are no particular upper or lower limits to the dosage of apoE:NGF or
apoE:NT4 complexes to be administered according to the present invention. For
in
vivo administration, dosages can be as low as 10, 3, l, 0.5 or 0.1 ~glkg body
weight,
or less. In vivo dosages can be as high as 10, 30, 50 or 100 ~g/k? body
weight, or
more. In general, the in vivo dosage of apoE:NGF or apoE:NT4 complexes
administered will be sufficient to result in peak plasma concentration of the
complex of
from about 1x10'', 1 x 10° or 1 x 101 picomole per Liter to about 1 x
10'-, 1 x lOj or
even 1 x 10~ picomole per Liter or more.
Similarly, there are no particular lower or upper limits to the dosages of the
claimed complexes to be administered in vitro. For in vitro administration
dosages can
be as low as 10, 5, 1, 0.5, or 0.1 nglml of medium, or less. In vitro dosages
can be as
high as 10, 50, 100, 500, 1000, or 1500 nglml of medium, or more.
IS
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B. Gamma-Interferon.
Another aspect of the present invention is apoE:IFNy complexes. In
preferred embodiments, the biological activity of IFNy is potentiated (as
defined
above with respect to NGF and NT4) by complexing with apoE. ApoE:IFNy
complexes can be formed as described above for apoE:NGF and apoE:NT4
complexes.
Those skilled in the art will appreciate that compositions containing the
claimed apoE:IFNy complexes will generally contain apoE and IFNy monomers,
homodimers and multimers as well. In general, as low as 35 %a , 25 % , 20 %a ,
15 %a ,
or lower, or as high as 40 % , 50 % , 70 % , 85 % , 90 % , 95 % , 99 % or
higher of the
IFNy molecules in the composition are complexed with apoE. The term
"apoE:IFNy" as used herein encompasses apoE:IFNy complexes containing
monomers, dimers, trimers and larger riiultimers of IFNy. Alternately, apoE
monomer, homodimers, homotrimers, etc. may associate with IFNy monomers,
homodimers, homotrimers, etc. Thus, the present invention encompasses
complexes between one or more apoE molecules with one or more IFNy molecules.
While not wishing to be held to any particular theory of the invention, the
active
form of IFNy is a homodimer, and it is believed that the IFNy homodimer
complexes with an apoE monomer. Such heterotrimeric complexes of apoE:IFNy
are preferred. Those skilled in the art will appreciate that apoE:IFNy
complexes
may also be more loosely associated with additional molecules.
Alternatively, apoE may first be bound to a substrate prior to being exposed
to and forming a complex with IFNy. As a further alternative, the apoE:IFNy
complex can be bound to a substrate after complex formation.
The strength of the binding interaction between apoE and IFNy is as
described above for apoE:NGF and apoE:NT4 complexes.
The apoE component of the disclosed complexes is as described above in
connection with apoE:NGF and apoE:NT4 complexes. The IFNy component of the
complex can be from a native or recombinant source, and can be produced by any
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WO 99/16460 PCT/US98120591
means known in the art. Native: U.S. patent No. 5,518,899 to Kurimoto; U.S.
Patent No. 4,723,000 to Georgiades et al.; U.S. Patent No. 5,132,110 to
Fleischmann et al. Recombinant: U.S. Patent No. 5,602,010 to Hauptmann et al.;
U.S. Patent No. 4,970,161 to Kakutatu et al.; U.S. Patent No. 4,889,803 to
Revel
et al. Moreover, the IFNy component of the claimed apoE:IFNy complexes can be
from any species of origin, preferably of mammalian origin, more preferably of
human origin. The term "IFNy" also encompasses IFNy variants, analogs and
derivatives, where the variants, analogs and derivatives retain IFNy
biological
activity. Exemplary are IFNy variants, analogs and derivatives disclosed in
U.S.
Patent No. 4,845,196 to Cowling; PCT application No. 83104053; U.S. Patent
Nos.
4,898,931 and 4,758,656 to Itoh et al.; Franke et al., (1982) DNA 1, 223; King
et
al. , (1983) J. Gen. Virol. 64, 1815. The term "IFNy" also includes fragments
of
the IFNy molecule that bind to IFNy receptors andlor elicit the biological
actions of
IFNy.
A further aspect of the present invention is methods of administering
compositions containing apoE:IFNy complexes to cells in a biologically
effective
amount. Compositions containing apoE:IFN ~ complexes find use in methods of
protecting cells against virus infection, such methods comprise administering
to the
cells an anti-virus effective amount of a composition comprising a complex of
apolipoprotein E and IFNy. See, e.g., Wheelock (1965) Science 149, 310. By
"protecting" cells against viral infection, it is meant that virus infection
rates are
reduced or eliminated or that an already-existing infection is reduced or
eliminated
by the disclosed methods. Such methods can be used to protect cultured cells
in
vitro or a subject in vivo.
Another aspect of the invention is methods of administering an anti-tumor
effective amount of a composition containing apoE:IFNy complexes. An "anti-
tumor effective amount" of the claimed complexes is a dosage that is effective
in
reducing the incidence of tumor formation or in decreasing the size, growth or
metastasis of a tumor. Likewise, the present invention can be employed to
administer the inventive apoE:IFNy complexes to treat a subject afflicted with
a
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tumor (e.g., by decreasing the size, growth or metastasis of a tumor) or to
reduce
the incidence of tumor formation in a subject at risk of developing tumors.
The claimed apoE complexes can be administered to cells in vitro.
Typically, in vitro administration will simply require adding a solution (i.
e. ,
aqueous) containing the apoE:IFNy complexes to the culture medium.
Alternatively, the apoE and IFNy components can be added individually, either
concurrently or sequentially, to the culture medium. As a further alternative,
the
apoE molecule or the apoE complex can be bound to a substrate (i. e. , a
tissue
culture dish or petri dish), preferably prior to culturing cells on the
substrate. It is
well-known in the art that apoE binds to polymeric or coated (i. e. , with
extracellular
matrix proteins such as laminin) surfaces of tissue culture plates.
The disclosed apoE:IFNy complexes can also be administered to a subject in
vivo. Suitable subjects, methods of administration, pharmaceutical
formulations of
the compositions containing apoE:IFNy, and suitable dosages thereof are as
1~ described above in connection with apoE:NGF and apoE:NT4 complexes. The
disclosed ApoE:IFNY complexes can be administered alone or in combination with
other therapeutic agents.
Compositions containing apoE:IFNy complexes can be administered to a
subject in vivo to provide anti-viral and anti-tumor treatments to a subject
in need
thereof. The inventive complexes can also be administered to treat bone
degradation and resorption, for example in the elderly, post-menopausal women,
and women afflicted with or at risk for developing osteoporosis. Finally, IFNy
can
be administered to facilitate blood coagulation, by stimulating blood C 1
inhibitor
levels, and to treat type I diabetes mellitus.
2~ Complexes of apoE and IFNy, which potentiate the activity of the IFNy, are
useful in vitro for culturing cells that are responsive to IFNy, including but
not
limited to bone cells (see U.S. Patent No. 4,921,697 to Peterlik et al.).
As established by U. S. Patent No. 6,268,169 to Brandely et al. , IFNy is
useful in methods of treating ovarian cancer. Also known is use of IFNy to
treat
type I diabetes mellitus. U.S. Patent No. 5,624,895 to Sobel. In addition, it
has
I8
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WO 99/16460 PCT/US98/20591
been established that IFNy is useful in methods of treating blood trauma (U.S.
_
Patent No. 5,271,931 to Lotz et al.) and treating bone degradation-(U.S.
Patent No.
4,921,697 to Peterlik et al.). Thus, the presently claimed IFNy complexes
possessing enhanced biological activity find similar uses as previously known
IFNy
compositions.
The claimed apoE:IFN~r complexes also find use in tissue culture, because
the complexed IFNy molecule is protected against proteolytic degradation.
Finally, the apoE:IFNy complexes find use in methods of quantifying or
purifying IFNy receptors or other proteins that bind to IFNy. ApoE is readily
bound
to plastic surfaces, such as test tubes and microtiter plates. Thus, IFNy can
be
tethered to plastic surfaces for use in sandwich assays or affinity
purification
techniques, which are well-known to those skilled in the art.
The following Examples are provided to illustrate the present invention, and
IS should not be construed as limiting thereof.
25
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E~cample 1
Gel-Shift Assavs
Delipidated apoE was purified from individuals homozygous for apoE3 or
apoE4, as previously described (Rall et al., (1986) Methods Enzymol. I28,
273).
ApoE3 or apoE4 were incubated with growth factors in Tris-buffered saline
(TBS)
for up to 4 hours at 37°C. Incubations were terminated by adding 4X SDS
Laemmli buffer without reducing agents. Proteins were electrophoretically
separated by SDS-PAGE, and transferred to PVDF membrane (Immobilon P,
Millipore, Bedford, MA). The membranes were blocked in Blotto (5 % dried milk
in TBS, pH 7.6, with 0.05 % Tween [Surfact Amps-20, Pierce, Rockford, IL]) for
one hour, then incubated in primary antibody for one hour. The anti-apoE
antibody
was a polyclonal goat anti-human apoE (Calbiochem, San Diego, CA) diluted
1:2000 in Blotto. Membranes were washed in Blotto three times, ten minutes
each
wash, then incubated with the secondary antibody for one hour. Ali incubations
and
washes were done at 25°C.
For detecting the anti-apoE antibody, the secondary antibody was a porcine
anti-goat IgG conjugated fo horseradish peroxidase (HRP; Boehringer Mannheim
Biochemicais, Indianapolis, IN) diluted 1:3000 in Blotto. The enzyme-
conjugated
antibodies were visualized by addition of ECL chemiluminescent substrate
{Amersham, Arlington Heights, IL) and exposure to Hyperfilm (Amersham) as
previously described (Strittmatter et al. , (1993) Proc. Nat. Acad. Sci. USA
90,
1977) .
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Example 2 -
AgoE3 forms an SDS-Stable Complex with NGF, IFNv and NT4
Gel shift assays, as described in Example 1, were used to screen for growth
factors that bind apoE. Fifty ng of apoE3 or apoE4 and 50 ng of growth factor
were incubated together for up to four hours. The growth factors evaluated
were:
recombinant human ciliary neurocrophic factor (CNTF), IFNy, neurotrophin-3
(NT3), neurotrophin-4 (NT4), NGF, the fibroblast growth factor bFGF, and LIF.
CNTF, IFNy and LIF are members of the four-helix bundle family of growth
factors. Sources of growth factors are as follows: CNTF -- generously provided
by
Regeneron Pharmaceuticals (Tarrytown, NY); IFN,I -- Human, recombinant
#40044, Collaborative Biomedical Products (Bedford, MA); NT3 -- Regeneron
Pharmaceuticals; NT4 -- human, recombinant #G1511, Promega (Madison, WI);
NGF -- mouse, natural #40005, Collaborative Biomedical Products (Bedford, MA);
bFGF -- Collaborative Biomedical Products (Bedford, MA); and LIF -- R&D
IS Systems (Minneapolis, MN).
Samples were boiled in non-reducing SDS sample buffer, separated by SDS-
PAGE, transferred to PVDF paper and probed with anti-apoE and peroxidase
conjugated secondary antibody. Immunoreactiviry was detected using the
Amersham chemiluminescent detection reagent.
ApoE3 forms a SDS-stable complex, in the absence of reducing agents, with
CNTF, IFNy, NGF and NT4. Complexes were not observed between ApoE3 and
IL-6, LIF, NT3, bFGF or BDNF. ApoE4 does not form a SDS-stable complex
with any of these growth factors. The gel mobility of each apoE3lgrowth factor
complex is consistent with a bimolecular complex between apoE3 and the growth
factor (the active forms of NGF and IFNy are dimers, so the apoE3lNGF complex
is likely a trimer).
In further studies it was found that the apoE3 complexes with NGF and
CNTF are detected within 30 minutes of incubation and reach equilibrium by 3-6
hours. The SDS-stable apoE3/CNTF complex reducible with ~i-merGaptoethanol,
suggesting disulfide bond formation. Rigorous proof that one of these bands is
a
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WO 99/16460 PCTIUS98/20591
true molecular complex has been obtained by showing that the CNTFIapoE complex
-
is immunoreactive with both CNTF and apoE antibodies.
Example 3
AnoE Promotes NGF Activity in PCI2 Cells
PC12 cells require NGF to survive in serum-free medium. Experiments
were carried out to assess PC12 cell survival in the presence of serum-free
medium
containing NGF, apoE3 or NGF+apoE3.
PC12 cells were plated in 96-well dishes that had been pre-coated with poly-
I-lysine and then treated with a solution containing serial dilutions of NGF
and
apoE3 that had been preincubated together. It is known that apoE3 will adsorb
to
tissue culture dishes under these conditions. For the preincubation, serial
dilutions
of NGF were added to a constant amount of apoE3 (50 ng/ml), incubated for 3
hours at 37°C, and then added to each well. These proteins were allowed
to bind to
the substrate for an additional 3 hours at 37°C. The final treatments
were 50 ng/ml
apoE3 + 0, I00 and 1000 nglml NGF. All the wells were washed three times with
PBS to remove any soluble NGF and apoE. Another set of wells received soluble
NGF at the same concentrations that were adsorbed to the wells (0, 100 and
1000
ng/ml) .
The PC12 cells were incubated for 72 hours in serum-free medium alone or
with substrate-bound apoE3 +NGF, or soluble NGF as described above. At 72
hours, cell viability was assessed using the Promega CellTiter Aqueous MTS
Assay
as described in Example 4. Each condition was done in triplicate and non-
specific
back?round was subtracted.
The substrate-bound complex of apoE3 and NGF promotes the survival of
serum-deprived PCI2 cells to a greater degree than does substrate-bound NGF or
soluble NGF alone (Figure 1). In fact, 50 nglml of apoE3 mixed with 100 ng/ml
NGF and bound to the substrate enhanced survival of PC 12 cells by more than 2-
fold as compared with 100 ng/ml soluble NGF alone (p < 0.001). The differences
at
1000 ng/ml NGF were also significant at the p < 0.001 level. ApoE alone has no
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WO 99/16460 PCT/US98/20591
survival promoting activity toward PCI2 cells (even at 1 pg/ml), regardless of
_
whether the apoE is absorbed to the substrate or included in the culture
medium.
The best interpretation of the survival promotinJ activity of the apoE/NGF
complex is a facilitation of NGF activity. Thus, apoE potentiates the activity
of
NGF in the maintenance of NGF-responsive peripheral nerve cells.
Example 4
1~ITS Survival Assay
Viability of PCI2 cells was assayed using the Promega viability assay
(CeIITiter 96 Aqueous, Promega, Madison, WI). See Ip and Yancopoulos, (1992)
Progress in Growth Factor Res. 4, 1; Riddle et al. , (1995) Nature 378, 189.
This is
a colometric assay in which the tetrazolium compound {3-(4,5-dimethylthiazol-2-
yI)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-trazolium (MTS) is reduced by
viable cells into a soluble formazan product. The absorbance of the formazan
is
measured directly from the 96-well plates. The quantity of formazan product as
measured by absorbance at 490nm is proportional to the number of viable cells.
For
performing this assay, 100 ~.I of media were removed from each well before
adding
~,1 of MTS solution to each well. Plates were returned to the incubator and
absorbance read by a Dynatech MR5000 microplate reader (Dynatech Laboratories)
20 after a four-hour incubation. The data were collected and calculated as the
percent
difference between each treatment group and the matched control (no apoElno
NGF
condition) for each experiment. The value for the no apoElno NGF condition was
set at 100 % . The percent differences for the separate experiments were ~
then
averaged.
Example 5
AooE Potentiates NGF Activity on Neurite Outgrowth
Another well-described activity of NGF is the ability of NGF to promote
neurite outgrowth. Studies were carried out in PC12 cells to evaluate whether
apoE
would promote NGF activity in this regard. PC12 cells were first primed for
neurite outgrowth by growing cells in 500 nglml NGF and serum for 7 days. At
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WO 99/16460 PCTIUS98/20591
this time, alI cells possessed neurites greater than 1 cell body in diameter.
These _
neurites were mechanically removed as the cells were replated in 96-well
plates.
This NGF priming allows far rapid growth of neurites in the experimental
condition.
After 3 days in culture in serum-free medium with 100 nglml NGF and 1
~g/ml apoE3, apoE4 or BSA (as a control), phase images of cells were digitally
collected. Measurements of neurites were made on random fields using IMAGE 1~"
analysis software (Universal Imaging Corp.; West Chester, PA). ApoE3, but not
apoE4, potentiates neurite outgrowth from primed PC12 cells (Figure 2). The
average neurite outgrowth of cells grown in apoE3 plus NGF was 1.5-fold
greater
than in cells grown in BSA or apoE4.
Example 6
ApoE "Knockout" Mice Exhibit Peripheral Neuropathv
In three separate experiments, we analyzed the ultrastructure of the sciatic
nerves from apoE knockout mice and control mice. ApoE knockout mice were
generated using gene targeting in embryonic stem cells. Piedrahita et al.,
(1992)
Proc. Natl. Acad. Sci. USA 89, 4471. Sciatic nerves from 12-week-old wildtype
and knockout mice were carefully dissected, fixed by immersion in 2.5
glutaraldehyde and post-fixed in 1 % osmium tetroxide. Samples were rinsed,
then
dehydrated, in an ethanol gradient with final rinses in propylene oxide. The
nerves
were embedded in epoxy resin which was cured at 60°C for 24 hours.
Eighty
manometer thin sections were cut on a diamond knife and picked up on formvar
covered slot grids which made it possible to view the entire cross-section.
Photomicrographs were made on a Phillips 410 electron microscope.
In all three experiments, a striking loss of unmyelinated fibers was observed,
particularly around the perimeter of the nerve. Although the number of
myelinated
fibers between knockout and wildtype mice was essentially equal, the number of
unmyelinated fibers is reduced by 50% in the apoE knockout mice (Figure 3).
Furthermore, the morphology of the remaining unmyelinated fibers is highly
abnormal. While the unmyelinated fibers in the wildtype are circular and are
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WO 99/16460 PCT/US98/20591
clearly separated from each other by Schwann cell cytoplasm, the knockout
fibers _
are irregularly shaped and are surround by very little Schwanri cell
cytoplasm.
There is also a loss of the sharp distinction between the axons and Schwann
cell
cytoplasm in the knockout.
These abnormalities in apoE knockout mice are consistent with a role for
apoE in potentiating NGF activity in vivo. The unmyelinated fibers within the
sciatic nerve express trkA NGF receptors and are responsive to NGF. These
fibers
are lost from animals treated with anti-NGF antibodies and, following nerve
transection, these neurons can be rescued by NGF. These results are also
relevant
to diabetic neuropathy, in which NGF-sensitive neurons are compromised.
Example 7
Neurite outgrowth and Schwann Cell Migration from Neonatal Rat
DRGs are Impaired on NGF Treated Distal Stumps from ApoE Knockout Mice
Neurite outgrowth and Schwann cell migration from neonatal rat dorsal root
ganglia (DRGs) were measured on NGF-treated distal stumps from apoE knockout
and wildtype mice. For these experiments, neonatal rat DRGs were explanted
onto
cryostat sections of distal stump from either apoE knockout or wildtype
animals.
Five days following transection, distal stumps were removed, fresh frozen in
O.C.T. compound and cut into 20 lzm thick longitudinal sections. Sections were
incubated in 100 pl of 100 nglml NGF for 3 hours at 37°C. Nerves were
then
washed to remove unbound NGF and neonatal DRG explants were placed on the
nerves and cultured in serum-free medium. Images of neurite outgrowth and
Schwann cell migration were obtained after 72 hours using the vital dye
carboxyfluorescein and fluorescence microscopy.
Both neurite outgrowth and Schwann cell migration are impaired on NGF-
treated distal stumps of transected sciatic nerve prepared from apoE knockout
mice.
It has previously been established that NGF promotes both neurite outgrowth
(see
Example 3) and Schwann cell migration. Thus, these studies provide further
evidence of the role of apoE in enhancing NGF action in vivo.
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Example 8 _
ApoE Knockout ylice have a Delayed Response to Noxious Thermal Stimuli
Unmyelinated fibers are the fibers that relay pain and temperature
sensitivity. Thus, the finding that apoE knockout animals have a substantial
decrease in unmylenated fibers (Example 6) suggests that these animals might
have
a reduced response to noxious thermal stimuli. To test this, the withdrawal
latency
for wildtype and apoE knockout mice from a hot waterbath was measured.
Withdrawal latency was determined for wildtype and knockout mice to
remove their hind feet from a 55°C water bath. As shown in Figure 4,
apoE
I0 knockout mice exhibit over a 50% increase in their response latency (p <
0.001). A
similar effect was observed with a tail withdrawal paradigm (data not shown).
These data further support a role for apoE in promoting the health and
survival of unmyelinated fibers through interaction with NGF.
Example 9
Summary of Primary Findings
Suonorting the Hypothesis that ApoE Potentiates NGF Activity
The data presented in the Examples above strongly suggest that apoE
potentiates NGF activity. This evidence is summarized below:
~ ApoE3, but not ApoE4, forms an SDS-stable complex with NGF
~ Substrate-bound complexes of apoE3 and NGF potentiate the survival-
promoting activity of NGF
~ ApoE3, but not apoE4, potentiates the neurite-outgrowth activity of NGF
~ NGF-dependent neurite outgrowth and Schwann cell migration are
impaired on substrates from apoE knockout animals
~ ApoE knockout animals have a 50 % loss of unmyelinated, NGF-
sensitive, sensory fibers
~ ApoE knockout mice have a delayed response to noxious thermal stimuli
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WO 99116460 PCT/US98/20591
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity and undersiandin~, it will
be
obvious that certain changes and modifications may be practiced within the
scope of
the appended claims.
27
