U.S. patent application number 16/657920 was filed with the patent office on 2020-07-09 for igg stimulated remyelination of peripheral nerves.
The applicant listed for this patent is Baxalta Incorporated Baxalta GmbH. Invention is credited to Sebastian Bunk, Hartmut Ehrlich, Hans-Peter Hartung, Corinna Hermann, Patrick Kury, Birgit Maria Reipert, Hans-Peter Schwarz, Nevena Tzekova.
Application Number | 20200216518 16/657920 |
Document ID | / |
Family ID | 47884569 |
Filed Date | 2020-07-09 |
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United States Patent
Application |
20200216518 |
Kind Code |
A1 |
Kury; Patrick ; et
al. |
July 9, 2020 |
IgG STIMULATED REMYELINATION OF PERIPHERAL NERVES
Abstract
The present invention is based on the discovery of polyclonal
IgG's ability to promote Schwann cell maturation, differentiation,
and myelin production. Methods for treating non-idiopathic,
demyelinating peripheral neuropathies in mammals, where the
neuropathy is not immune-mediated or infection-mediated, through
the administration of polyclonal IgG are provided. Types of
demyelinating peripheral neuropathies treatable with the present
invention include peripheral nerve trauma and toxin-induced
peripheral neuropathies. Alternatively, a composition of polyclonal
IgGs can be applied directly to a peripheral nerve cell to induce
maturation, differentiation into a myelinating state, and myelin
expression or promote cell survival.
Inventors: |
Kury; Patrick; (Dusseldorf,
DE) ; Tzekova; Nevena; (Dusseldorf, DE) ;
Hartung; Hans-Peter; (Dusseldorf, DE) ; Hermann;
Corinna; (Vienna, AT) ; Reipert; Birgit Maria;
(Deutsch-Wagram, AT) ; Schwarz; Hans-Peter;
(Vienna, AT) ; Ehrlich; Hartmut; (Vienna, AT)
; Bunk; Sebastian; (Vienna, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta GmbH |
Bannockburn
Zug |
IL |
US
CH |
|
|
Family ID: |
47884569 |
Appl. No.: |
16/657920 |
Filed: |
October 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15798313 |
Oct 30, 2017 |
10494418 |
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16657920 |
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14625542 |
Feb 18, 2015 |
9834593 |
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15798313 |
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13781283 |
Feb 28, 2013 |
8986670 |
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14625542 |
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61605117 |
Feb 29, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 25/02 20180101; C07K 16/065 20130101; A61K 2039/505 20130101;
A61K 45/06 20130101; A61K 39/39516 20130101; C07K 16/06 20130101;
A61P 25/00 20180101; A61P 43/00 20180101; A61P 35/02 20180101; A61K
39/39516 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/06 20060101
C07K016/06; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating i) a demyelinating peripheral neuropathy
comprising administering a therapeutically effective amount of
polyclonal IgG to a mammal diagnosed with said neuropathy, with the
proviso that said neuropathy is not an immune-mediated or
infection-mediated neuropathy and excludes Guillain-Barre syndrome,
chronic demyelinating polyneuropathy and multifocal motor
neuropathy, or ii) a toxin-induced peripheral neuropathy comprising
administering a therapeutically effective amount of polyclonal IgG
to a mammal diagnosed with said neuropathy, wherein said neuropathy
is not infection-mediated.
2. The method of claim 1, wherein the mammal is human.
3. The method of claim 1, wherein the polyclonal IgG is
administered locally.
4. The method of claim 3, wherein the polyclonal IgG is
administered intramuscularly or intradermally.
5. The method of claim 1, wherein the polyclonal IgG is
administered systemically.
6. The method of claim 5, wherein the polyclonal IgG is
administered intranasally, subcutaneously, orally, intra-arterially
or intravenously.
7. The method of claim 1, wherein an anti-inflammatory agent is
co-administered with the polyclonal IgG to the mammal.
8. The method of claim 7, wherein the anti-inflammatory agent is
adrenocorticotropic hormone, a corticosteroid, an interferon,
glatiramer acetate, or a non-steroidal anti-inflammatory drug.
9. The method of claim 1, wherein the demyelinating peripheral
neuropathy is selected from a trauma-induced neuropathy, a
toxin-induced neuropathy, an inherited neuropathy, and a neuropathy
induced by a metabolic disease.
10. The method of claim 9, wherein the peripheral neuropathy is a
trauma-induced neuropathy.
11. The method of claim 9, wherein the peripheral neuropathy is a
toxin-induced neuropathy.
12. The method of claim 9, wherein the peripheral neuropathy is an
inherited neuropathy.
13.-43. (canceled)
44. A method of promoting myelination of a peripheral nerve cell by
a Schwann cell comprising contacting said Schwann cell with an
amount of polyclonal IgG sufficient to promote myelination of said
peripheral nerve cell by the Schwann cell.
45. A method of promoting the differentiation of an immature
Schwann cell into a myelinating state comprising contacting said
Schwann cell with polyclonal IgG in an amount sufficient to induce
the Schwann cell differentiation.
46. A method of promoting the production of myelin by a Schwann
cell comprising contacting said Schwann cell with an amount of
polyclonal IgG sufficient to upregulate MBP genes.
47. A method of culturing mammalian nervous tissue which comprises
axons, said method comprising contacting the tissue in culture with
an effective amount of Schwann cells and an effective amount of
polyclonal IgG, whereby the contacting of Schwann cells with
polyclonal IgG induces upregulation of MBP genes.
48.-49. (canceled)
Description
CROSS REFERENCES TO APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 15/798,313, filed Oct. 30, 2017, which is a
Continuation of U.S. patent application Ser. No. 14/625,542, filed
Feb. 18, 2015, which is a Divisional of U.S. patent application
Ser. No. 13/781,283 (issued as U.S. Pat. No. 8,986,670), filed Feb.
28, 2013, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/605,117 filed Feb. 29, 2012, the
disclosures of which are hereby incorporated herein by reference in
their entireties for all purposes.
BACKGROUND OF THE INVENTION
[0002] Peripheral neuropathy is a manifestation of disorders that
inflict damage to the peripheral nervous system (PNS), a network of
ganglia and neurons that transmit signals between the central
nervous system (CNS), i.e. brain and spinal cord, and every other
part of the body. Neurons of the PNS rely on Schwann cells for,
e.g. myelination, accelerated nerve conduction, nerve development
and regeneration, trophic support, production of nerve
extracellular matrix, and modulation of neuromuscular synaptic
activity. These Schwann cells provide electric insulation by
wrapping a protein and lipid-rich myelin sheath around axons of
motor and sensory neurons. Given myelin's critical role, it is not
surprising that demyelination of peripheral axons is a hallmark of
acute and chronic peripheral neuropathies such as Guillain-Barre
syndrome (GBS), chronic demyelinating polyneuropathy (CIDP) and
multifocal motor neuropathy (MMN) as well as other peripheral nerve
pathologies induced by toxins, drugs or systemic diseases, e.g.
diabetes.
[0003] Peripheral neuropathies can distort signal transmission,
causing symptoms that vary with the origin of the neuropathy and
type or number of nerves affected. For example, symptoms may depend
on whether the disorder affects sensory nerve fibers, which
transmit sensory information from the affected area to the CNS, or
motor nerve fibers, which transmit impulses and coordinate motor
activity from the CNS to a muscle, or both. Peripheral neuropathies
can be classified as mononeuropathies, involving damage to one
nerve, or polyneuropathies, involving damage of multiple nerves;
acute, where symptoms appear suddenly, progress rapidly, and
resolve slowly, or chronic, where symptoms begin subtly, and
progress slowly. Over 100 different types of peripheral neuropathy
have been identified to date. Clinical diagnoses of peripheral
neuropathy can be made based on the clinical history of the
subject, a physical examination, the use of electromyography (EMG)
and nerve conduction studies (NCS), autonomic testing, and nerve
biopsies, etc.
[0004] Current treatments for peripheral neuropathies are directed
at the underlying condition, where possible, and often used in
conjunction with symptomatic treatments, such as anti-inflammatory
agents, pain management, mechanical aids, and/or surgical
intervention, etc. The body also possesses its own regenerative
capacity in response to injury or damage of the PNS. After injury
to the PNS, Wallerian degeneration of distal nerve stumps occur,
followed by Schwann cell degradation of myelin, phagocytosis of
extracellular myelin, and recruitment of macrophages for further
myelin clearance. Schwann cells can further adapt to pathological
situations by its ability to dedifferentiate, proliferate, promote
axonal regeneration and redifferentiate, and produce myelin. See
Bhatheja et al. (2006) Int. J. Biochem. Cell Biol. 38(12):1995-9.
In the course of repair, Schwann cells stimulate, guide axonal
regeneration, and target reinnervation, forming a regeneration tube
of the axon, known as Bunger's band, by proliferating rapidly and
providing the axon with a path to grow along. See Burstyn-Cohen et
al. (1998) J. Neurosci 18(21): 8875-8885. While functional nerve
regeneration in the PNS can generally be observed (in contrast to
CNS which lacks a regenerative mechanism for myelin clearance and
axon regeneration), it is often limited or chronically impaired.
Novel repair promoting approaches for the PNS are therefore
needed.
[0005] Recent studies on the CNS have yielded evidence of IgM's
direct effect on oligodendrocytes, the myelinating glial cells of
the central nervous system. For instance, targeting of
oligodendrocyte-reactive IgM.kappa. antibodies to oligodendrocytes
was found to promote CNS remyelination (Asakura et al., 1998).
Other studies showed that treatment of a non-immune, toxin-induced
model of demyelinating disease with pooled human IgM molecules
results in a significantly enhanced oligodendrocyte differentiation
in the CNS (Bieber et al., 2000; Bieber et al., 2002; Warrington et
al., 2007). The discovery of Fc receptors for IgM on
oligodendrocytes, their precursor cells, and myelin in the CNS,
offers further clues of a possible ligand-receptor interaction
(Nakahara et al., 2003).
[0006] Knowledge gained from these oligodendrocyte--IgM studies,
though meaningful for CNS repair, fails to harness the regenerative
capacity of the PNS (which contains no oligodendrocytes). In more
relevant studies, administration of human IVIG was found to reduce
disease duration in an EAN (autoimmune neuritis) rat model,
simulating the PNS-specific, demyelinating Guillain-Barre syndrome
(GBS) (Lin et al., 2007). The effects were postulated as being
attributable to IVIG's immunomodulatory role and possible
anti-inflammatory and secondary bystander axonal loss reduction
capability. In a separate study of the humoral immune system,
B-cell knockout RID mice exhibited significant delay in macrophage
influx, myelin clearance, and axon regeneration after PNS injury.
Rapid myelin debris clearance was restored through passive transfer
of antibodies from naive WT mice or anti-PNS myelin antibody,
thereby confirming the role of endogenous antibodies in promoting
macrophage entrance and phagocytic activity (Vargas et al., 2010).
Clinical trials with administration of intravenous immunoglobulins
(IVIG) have shown positive effects for GBS, chronic demyelinating
polyneuropathy (CIDP) and multifocal motor neuropathy (MMN), with
the assumption that treatment in each of these autoimmune or
immune-mediated neuropathies was accomplished through IVIG's
immunomodulatory role.
[0007] The effect of polyclonal IgG on Schwann cells, if any, was
heretofore unknown. A question, therefore, remained as to how the
regenerative function of Schwann cells could be harnessed for
therapeutic purposes in demyelinating, peripheral neuropathies. The
present discovery of exogenous polyclonal IgG's ability to induce
Schwann cell maturation, differentiation, and myelin production, is
an important clarification of mechanism that provides novel
approaches to the treatment of all demyelinating peripheral
neuropathies.
SUMMARY OF THE INVENTION
[0008] In one aspect of the invention, there is provided methods of
treating a demyelinating peripheral neuropathy in mammals, wherein
the neuropathy is not immune-mediated or infection-mediated, by
administering a therapeutically effective amount of polyclonal IgG
to a mammal diagnosed with said neuropathy. In some embodiments of
the invention, the demyelinating peripheral neuropathy being
treated is not Guillain-Barre syndrome, chronic demyelinating
polyneuropathy, or multifocal motor neuropathy. In other
embodiments of the invention, the demyelinating peripheral
neuropathy is a non-idiopathic neuropathy. The demyelinating
peripheral neuropathy treatable by the present invention may be
selected from a trauma-induced neuropathy, a toxin-induced
neuropathy, an inherited neuropathy, and a neuropathy induced by a
metabolic disease, e.g. diabetic neuropathy.
[0009] In another aspect of the invention, there is provided
methods of treating peripheral nerve trauma by administering a
therapeutically effective amount of polyclonal IgG to a mammal with
peripheral nerve trauma.
[0010] In yet another aspect of the invention, there is provided
methods of treating toxin-induced peripheral neuropathy, wherein
the neuropathy is not infection-mediated, by administering a
therapeutically effective amount of polyclonal IgG to a mammal
diagnosed with said neuropathy.
[0011] For treatment of a demyelinating peripheral neuropathy
described herein, polyclonal IgG of the invention may be
administered locally or systemically. Local administration of the
polyclonal IgG can occur intramuscularly or intradermally. Systemic
administration of the polyclonal IgG can occur intranasally,
subcutaneously, orally, intra-arterially or intravenously. In some
embodiments of the invention, an anti-inflammatory agent is
co-administered with the polyclonal IgG to the mammal. The
anti-inflammatory agent may be selected from an adrenocorticotropic
hormone, a corticosteroid, an interferon, glatiramer acetate, or a
non-steroidal anti-inflammatory drug.
[0012] The polyclonal IgG of the invention may be administered
weekly, biweekly, or monthly at a dose of about 0.05 to 5 g per kg
of patient body weight or about 0.5 to 2 g per kg of patient body
weight.
[0013] In a further aspect of the invention, there is provided
methods of promoting myelination of a peripheral nerve cell by a
Schwann cell by contacting the Schwann cell with an amount of
polyclonal IgG sufficient to promote myelination of said peripheral
nerve cell by the Schwann cell.
[0014] In another aspect of the invention, there is provided
methods of promoting the differentiation of an immature Schwann
cell into a myelinating state by contacting said Schwann cell with
polyclonal IgG in an amount sufficient to induce the Schwann cell
differentiation.
[0015] In yet another aspect, there is provided methods of
promoting myelin production by a Schwann cell comprising contacting
said Schwann cell with an amount of polyclonal IgG sufficient to
upregulate MBP gene.
[0016] In a further aspect of the invention, there is provided
methods of culturing mammalian nervous tissue which comprises axons
by contacting the tissue in culture with an effective amount of
Schwann cells and an effective amount of polyclonal IgG, whereby
the contacting of Schwann cells with polyclonal IgG induces
upregulation of MBP gene.
[0017] In yet another aspect of the invention, there is provided
methods of treating a peripheral nerve injury in a mammal by:
transplanting nerve cells to a site of the peripheral nerve injury;
and contacting the nerve cells with a composition comprising
Schwann cells and polyclonal IgG.
[0018] In the methods described herein, the polyclonal IgG can be
given through one or more routes of administration, such as
intramuscularly, intradermally, subcutaneously, buccally, orally,
intranasally, or intra-arterially or intravenously to an individual
in need of such therapy. The individual may be a human or
domesticated animal. In some embodiments, the polyclonal IgG is
derived from pooled human serum.
[0019] In some embodiments, the polyclonal IgG Is co-administered
with an anti-inflammatory agent to mammal in need of such therapy.
The anti-inflammatory agent may be selected from an
adrenocorticotropic hormone, a corticosteroid, an interferon,
glatiramer acetate, or a non-steroidal anti-inflammatory drug.
[0020] In yet another aspect of the invention, there is provided
pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and an effective amount of polyclonal IgG for
treating a non-idiopathic, demyelinating peripheral neuropathy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] More particular descriptions of the invention are made by
reference to certain exemplary embodiments thereof which are
illustrated in the appended Figures. These Figures form a part of
the specification. It is to be noted, however, that the appended
Figures illustrate exemplary embodiments of the invention and
therefore are not to be considered limiting in their scope.
[0022] FIG. 1A and FIG. 1B show the relative proliferation rates of
immature Schwann cells that were exposed to nondialysed (FIG. 1A)
and dialysed (FIG. 1B) IVIG/buffer formulations after 2 days as
measured by BrdU incorporation assays. These relative proliferation
rates were generated based on the number of cells positive for of
5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA
during cell proliferation.
[0023] FIG. 2A and FIG. 1B show the relative proliferative rates of
immature Schwann cells that were exposed to nondialysed (FIG. 2A)
and dialysed (FIG. 2B) IVIG/buffer formulations after 2 days as
measured using Ki-67 assays. These relative proliferation rates
were generated based on the number of cells positive for Ki-67
expression during cell proliferation.
[0024] FIG. 3A and FIG. 3B show the levels of P0 (FIG. 3A) and MBP
(FIG. 3B) gene expression in immature Schwann cells that were
exposed to dialysed IVIG/buffer formulations at 1 day and 3 day
time-points.
[0025] FIG. 4A and FIG. 4B show the levels of P0 (FIG. 4A) and MBP
(FIG. 4B) gene expression in p57kip2 suppressed Schwann cells that
were exposed to dialysed IVIG/buffer formulations at the 7 day
time-point (9 days suppression).
[0026] FIG. 5 shows the expression levels of CD64 Fc receptor in
p57kip2 suppressed Schwann cells as compared to control transfected
Schwann cells (without p57kip2 suppression). Neither group of
Schwann cells were exposed to IVIG/buffer formulations.
[0027] FIG. 6A and FIG. 6B show fluorescent images of p57kip2
suppressed Schwann cells (FIG. 6B) and control transfected cells
(FIG. 6A) after stimulation with 20 mg dialyzed IVIG/buffer
formulations. The location and length of cellular processes are
indicated by the arrows superimposed onto the fluorescent
images.
[0028] FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D show a graph of the
cell outgrowth length for p57kip2 suppressed Schwann cells and
control transfected cells (FIG. 7A) after 3 days of stimulation
with dialysed IVIG/buffer formulations (5 days suppression) along
with the respective fluorescent images of the p57kip2 suppressed
Schwann cells stimulated with 20 mg of IVIG (FIG. 7B), p57kip2
suppressed Schwann cells stimulated with buffer (FIG. 7C), control
transfected cells treated with 20 mg IVIG (FIG. 7D), and control
transfected cells treated with buffer (FIG. 7E).
[0029] FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D show a graph of the
cell outgrowth length for p57kip2 suppressed Schwann cells and
control transfected cells (FIG. 8A) after 7 days of stimulation
with dialysed IVIG/buffer formulations (9 days suppression) along
with the respective fluorescent images of the p57kip2 suppressed
Schwann cells stimulated with 20 mg of IVIG (FIG. 8B), p57kip2
suppressed Schwann cells stimulated with buffer (FIG. 8C), control
transfected cells treated with 20 mg IVIG (FIG. 8D), and control
transfected cells treated with buffer (FIG. 8E).
[0030] FIG. 9 is a flow diagram of the process for establishing a
cocultue of PNS neurons (rat dorsal root ganglion) and myelinating
Schwann cells.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The discovery of polyclonal IgG's ability to promote Schwann
cell homeostasis, maturation, differentiation, and myelin
production can be applied for treatment of demyelinating peripheral
neuropathies of varying origins, e.g. toxin-induced neuropathies,
diabetic neuropathy, trauma-induced neuropathy, by promoting the
regenerative capacity of native Schwann cells. Contemplated is the
administration of polyclonal IgG as an adjunct or replacement of
existing therapeutic regimes or symptomatic treatments for
demyelinating peripheral neuropathies. Furthermore, the present
invention can be used in the laboratory setting for effecting
peripheral nerve remyelination. Based on the findings described
herein, polyclonal IgGs can be applied in nerve transplant, cell
culture, e.g. induction of Schwann cell differentiation,
determination of precursor cell fate, myelin gene regulation or
protein expression, and as a pretreatment to or post-operative care
regimen for surgical techniques threatening or involving peripheral
nerves.
I. Definitions
[0032] The term "non-idiopathic" refers to a disorder where the
underlying cause is known.
[0033] The term "peripheral neuropathy," as used herein, refers to
a disorder affecting the peripheral nervous system, which excludes
ganglion and nerves of the brain and the spinal cord. "Peripheral
neuropathy" can manifest as one or a combination of motor, sensory,
sensorimotor, or autonomic neural dysfunction. The variety of
morphologies exhibited by peripheral neuropathies can be attributed
to a number of different causes. For example, peripheral
neuropathies can be genetically acquired, can result from a
systemic disease, or can be induced by a toxic agent. Examples
include but are not limited to diabetic peripheral neuropathy,
distal sensorimotor neuropathy, or autonomic neuropathies such as
reduced motility of the gastrointestinal tract or atony of the
urinary bladder. Examples of peripheral neuropathies associated
with systemic disease include post-polio syndrome or
AIDS-associated neuropathy; examples of hereditary peripheral
neuropathies include Charcot-Marie-Tooth disease,
Abetalipoproteinemia, Tangier disease, Metachromatic
leukodystrophy, Fabry's disease, and Dejerine-Sottas syndrome; and
examples of peripheral neuropathies caused by a toxic agent include
those caused by treatment with a chemotherapeutic agent such as
vincristine, cisplatin, methotrexate, or
3'-azido-3'-deoxythymidine.
[0034] One variety of peripheral neuropathy is "demyelinating
peripheral neuropathy." As used herein, a "demyelinating peripheral
neuropathy" describes a broad class of peripheral neuropathies that
are associated with the destruction or removal of myelin, the
lipid-rich sheath surrounding and insulating nerve fibers, from
nerves. Non-limiting examples of demyelinating peripheral
neuropathy diseases include diabetic peripheral neuropathy, distal
sensorimotor neuropathy, or autonomic neuropathies such as reduced
motility of the gastrointestinal tract or atony of the urinary
bladder. Further examples and descriptions of demyelinating
peripheral neuropathy can be found in Section II of the Detailed
Description.
[0035] An "immune-mediated" disorder, as used herein, refers to a
condition which results from abnormal activity of the body's immune
system. Subsets of "immune-mediated" disorder include, without
limitation, autoimmune disease, wherein the immune system attacks
the body, immune-complex disorders, disorders involving
post-transplant rejection, inflammatory disease, and allergies.
[0036] An "infection-mediated" peripheral neuropathy refers to a
dysfunction of the peripheral nervous system sustained as a result
of viral, bacterial, or fungal infections.
[0037] A "trauma-induced peripheral neuropathy" or "traumatic
peripheral neuropathy" refers to dysfunction of the peripheral
nervous system caused by bodily shock, injury, or "physical
trauma." Physical trauma, e.g. from combat, vehicular accidents,
falls, and sports-related activities, can cause nerves to be
partially or completely severed, crushed, compressed, or stretched,
sometimes so forcefully that they are partially or completely
detached from ganglia or the spinal cord and result in
demyelination. Traum-induced peripheral neuropathies can also be
sustained as a result of, e.g. electric shock, hypothermia,
etc.
[0038] A "toxin" or "chemical induced" peripheral neuropathy refers
to dysfunction of the peripheral nervous system caused by toxins
(e.g., chemical agents). Toxins that produce peripheral neuropathy
can generally be divided into three groups: drugs and medications;
industrial chemicals; and environmental toxins. Non-limiting
examples of toxins that can cause peripheral neuropathy are
described below in Section II of the Detailed Description.
[0039] An "anti-inflammatory agent" as used herein includes any
agent that reduces inflammation of an affected blood vessel and/or
adjacent tissue. Non-limiting examples of anti-inflammatory agents
are steroids (e.g., glucocorticoids and corticosteroids), immune
selective anti-inflammatory derivatives (ImSAIDs), cooling agents,
herbal supplements (e.g., devil's claw, hyssop, ginger, turmeric,
arnica Montana, and willow bark (containing alicylilc acid), and
foods with anti-inflammatory effects (e.g., pomegranate, green tea,
vegetables, foods that contain omega-3 fatty acids), nuts, seeds,
and extra-virgin olive oil). Specifically, prostaglandin 2 (PGE2)
is a pro-inflammatory compound and PGE1 and PGE3 are
anti-inflammatory compounds. Accordingly, agents that decrease PGE2
or increase PGE1 and PGE3 can also act as anti-inflammatory agents.
Additional non-limiting examples of anti-inflammatory agents can be
found in Section VI, "Combination Therapy," below.
[0040] An "immature Schwann cell," as used herein, refers to a
specific stage in the Schwann cell lineage. The first step along
the Schwann cell lineage gives the Schwann cell precursor, a
proliferative cell that becomes associated with many axons and
expresses the nerve growth factor receptor (NGF-R),
growth-associated protein 43 (GAP-32), and the neural cell adhesion
molecules N-CAM and L1. The subsequent "committed" Schwann cell is
known as an immature Schwann cell; it becomes associated with
progressively fewer axons and expresses, in addition to the
previously noted markers, S-100 protein (from this stage onward,
all Schwann cells express S-100). Committed Schwann cells develop
into either nonmyelinating Schwann cells, which remain associated
with several axons and express galactocerebroside (GalC) in
addition to the previous markers, or into myelinating Schwann
cells. Myelinating Schwann cells progress through a proliferative
"premyelinating" stage, characterized by transient expression of
suppressed cAMP-inducible Pou-domain transcription factor (SCIP),
followed by a "promyelinating" GalC-positive stage, becoming
associated with a single axon in the progress. The final
differentiation into a mature myelinating Schwann cell involves
downregulation of NGF-R, GAP-43, N-CAM, and L1 expression, with
upregulation of expression of GalC and myelin proteins, and in
vivo, the synthesis and elaboration of myelin.
[0041] The term "IgG," as used herein, refers to a composition of
IgG immunoglobulins. The IgG class of immunoglobulins, as the name
suggests, is characterized by the presence of a .gamma. (gamma)
heavy chain. An exemplary whole IgG immunoglobulin structure
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms variable light chain (V.sub.L) and variable heavy chain
(V.sub.H) refer to these light and heavy chains respectively.
[0042] An "immunoglobulin" or "antibody" is a polypeptide that is
immunologically reactive with a particular antigen. The term
"immunoglobulin," as used herein, encompasses intact molecules of
various isotypes as well as fragments with antigen-binding
capability, e.g., Fab', F(ab').sub.2, Fab, Fv and rIgG. See, e.g.,
Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,
Rockford, Ill.); Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman
& Co., New York (1998). The term also encompasses recombinant
single chain Fv fragments (scFv). The term further encompasses
bivalent or bispecific molecules, diabodies, triabodies, and
tetrabodies. Bivalent and bispecific molecules are described in,
e.g., Kostelny et al. (1992) J. Immunol. 148:1547, Pack and
Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993,
supra, Gruber et al. (1994) J. Immunol.: 5368, Zhu et al. (1997)
Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et
al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995)
Protein Eng. 8:301.
[0043] The term "polyclonal IgG," as used herein, refers to a
heterogeneous collection of IgG immunoglobulins derived from
multiple B-cells and having different specificities and epitope
affinities. Methods of preparing polyclonal antibodies are known to
the skilled artisan (e.g., Harlow & Lane, 1988, Antibodies: A
Laboratory Manual. (Cold Spring Harbor Press)). The polyclonal IgGs
of the invention can be extracted from plasma pooled from different
mammalian individuals who have been prescreened for pathogenic
disorders. In some embodiments, the polyclonal IgGs of the present
invention are representative of over 100 individuals, over 200
individuals, over 300 individuals, over 400 individuals, over 500
individuals, over 600 individuals, over 700 individuals, over 800
individuals, over 900 individuals, over 1000 individuals, over 1100
individuals, over 1200 individuals, over 1300 individuals, over
1400 individuals, over 1500 individuals, over 1600 individuals,
over 1700 individuals, over 1800 individuals, over 1900
individuals, or over 2000 individuals.
[0044] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein, in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein sequences at least two times the
background and more typically more than 10 to 100 times background.
A ligand (e.g., an antibody) that specifically binds to a protein
generally has an association constant of at least 10.sup.3 M.sup.-1
or 10.sup.4 M.sup.-1, sometimes 10.sup.5 M.sup.-1 or 10.sup.6
M.sup.-1, in other instances 10.sup.6 M.sup.-1 or 10.sup.7
M.sup.-1, preferably 10.sup.8 M.sup.-1 to 10.sup.9 M.sup.-1, and
more preferably, about 10.sup.10 M.sup.-1 to 10.sup.11 M.sup.-1 or
higher. A variety of immunoassay formats can be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0045] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0046] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0047] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0048] "Myelin basic protein" (MBP), as used herein, refers to the
gene as well as the protein encoded thereby, which is a major
protein component of myelin, comprising approximately 30% of the
total protein content of the myelin sheath. MBP has been shown to
be a major target autoantigen in MS, and T cells reactive with MBP
play a key role in its pathogenesis (see, for example, Schwartz, R
S, "Autoimmunity and Autoimmune Diseases" in Paul, Fundamental
Immunology, 3rd Ed. Raven Press, New York, 1993, pp. 1033 1097;
Brown and McFarlin 1981. Lab Invest 45, pp. 278 284; Lehmann et al.
1992. Nature 358, pp. 155 157; Martin et al. 1992. Ann Rev Immunol
10, pp. 153 187; Sprent 1994. Cell 76, pp. 315 322; Su and Sriram.
1991. J of Neuroimmunol 34, pp. 181 190; and Weimbs and Stoffel.
1992. Biochemistry 31, pp. 12289 12296).
[0049] The term "axon" refers to an elongated fiber of a nerve cell
responsible for conducting signals in the body.
[0050] The terms "individual," "subject," and "patient," used
interchangeably herein, refer to a mammal, including, but not
limited to, murines, simians, humans, mammalian farm animals,
mammalian sport animals, and mammalian pets. In preferred
embodiments, the individual is a human.
[0051] The terms "dose" and "dosage" are used interchangeably
herein. A dose refers to the amount of active ingredient given to
an individual at each administration. The dose will vary depending
on a number of factors, including frequency of administration; size
and tolerance of the individual; severity of the condition; risk of
side effects; and the route of administration. One of skill in the
art will recognize that the dose can be modified depending on the
above factors or based on therapeutic progress. The term "dosage
form" refers to the particular format of the pharmaceutical, and
depends on the route of administration. For example, a dosage form
can be in a liquid, e.g., a saline solution for injection.
[0052] A "therapeutically effective" amount or dose or
"sufficient/effective" amount or dose, is a dose that produces
effects for which it is administered. The exact dose will depend on
the purpose of the treatment, and will be ascertainable by one
skilled in the art using known techniques (see, e.g., Lieberman,
Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art,
Science and Technology of Pharmaceutical Compounding (1999);
Pickar, Dosage Calculations (1999); and Remington: The Science and
Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,
Williams & Wilkins).
[0053] The term "treatment" or "therapy" generally means obtaining
a desired physiologic effect. The effect may be prophylactic in
terms of completely or partially preventing a disease or condition
or symptom thereof and/or may be therapeutic in terms of a partial
or complete cure for an injury, disease or condition and/or
amelioration of an adverse effect attributable to the injury,
disease or condition and includes arresting the development or
causing regression of a disease or condition. Treatment can also
include prophylactic use to mitigate the effects of injury, should
it occur. For example, in one aspect, the present invention
includes pre-administration to mitigate damage prior to surgery
involving the peripheral nervous system. Treatment can also refer
to any delay in onset, amelioration of symptoms, improvement in
patient survival, increase in survival time or rate, etc. The
effect of treatment can be compared to an individual or pool of
individuals not receiving the treatment.
[0054] A "control" is used herein, refers to a reference, usually a
known reference, for comparison to an experimental group. One of
skill in the art will understand which controls are valuable in a
given situation and be able to analyze data based on comparisons to
control values. Controls are also valuable for determining the
significance of data. For example, if values for a given parameter
vary widely in controls, variation in test samples will not be
considered as significant.
[0055] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to
particular embodiments described, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0056] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0057] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0058] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0059] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0060] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
II. Demyelinating Peripheral Neuropathies
[0061] The present invention is based on the discovery that
polyclonal IgG can harness Schwann cells' regenerative capacity
through stimulation of Schwann cell maturation, differentiation,
and myelin production. In this manner, the invention targets a
unifying mechanism of demyelinating peripheral neuropathies so as
to provide a broad-spectrum treatment for such disorders. For
example, this invention targets demyelinating peripheral
neuropathies caused by physical trauma, toxic agents, and
diabetes.
[0062] Demyelinating disorders treatable by the polyclonal IgG
composition described herein include, for example, peripheral
neuropathies that are genetically acquired, result from a systemic
disease, or induced by a toxin or by trauma.
[0063] Genetic demyelinating neuropathies (also known as hereditary
neuropathies) are one of the most common inherited neurological
diseases. Genetic demyelinating neuropathies are divided into four
major subcategories: 1) motor and sensory neuropathy, 2) sensory
neuropathy, 3) motor neuropathy, and 4) sensory and autonomic
neuropathy. Specifically, the demyelinating hereditary neuropathies
are often progressive neuropathies with markedly decreased nerve
conduction and velocity and chronic segmental demyelination of the
peripheral nerve. Gabreels-Festen et al., "Hereditary demyelinating
motor and sensory neuropathy," Brain Pathol. 3(2):135-146 (1993).
Examples of general classes of genetic deyelinating neuropathies
include but are not limited to diabetic peripheral neuropathy,
distal sensorimotor neuropathy, or autonomic neuropathies such as
reduced motility of the gastrointestinal tract or atony of the
urinary bladder. Examples of hereditary peripheral neuropathies
include Charcot-Marie-Tooth disease, Abetalipoproteinemia, Tangier
disease, Metachromatic leukodystrophy, Fabry's disease, and
Dejerine-Sottas syndrome.
[0064] Systemic demyelinating peripheral neuropathies arise as side
effects of a systemic illness. Non-limiting examples of peripheral
neuropathies associated with systemic disease include post-polio
syndrome and AIDS-associated neuropathy. Furthermore, the following
non-limiting systemic diseases can have peripheral neuropathy
symptoms: cancer, malnutrition, alcoholism, diabetes, AIDS, Lyme
disease, Rheumatoid arthritis, chronic kidney failure, autoimmune
disorders, hypothyroidism, and viral infections (e.g.,
hepatitis).
[0065] Toxin induced demyelinating peripheral neuropathies are
caused by exposure to neurotoxic agents such as pharmaceutical
agents, biological agents, and chemical exposure. Examples of
toxins that cause peripheral neuropathies include, but are not
limited to, chemotherapeutic agents (e.g., vincristine, paclitaxel,
cisplatin, methotrexate, or 3'-azido-3'-deoxythymidine), lead,
mercury, thallium, organic solvents, pesticides, carbon disulfide,
arsenic, acrylamide, diphtheria toxin, alcohol, anti-HIV
medications (e.g., didanosine and zalcitabine), anti-tuberculosis
medications (e.g., isoniazid and ethamubtol), antimicrobial drugs
(e.g., dapsone, metronidazole, chloroquine, and chloamphenicol),
psychiatric medications (e.g., lithium), radiation, and medications
such as amiodarone, aurothioglucose, phenytoin, thalidomide,
colchicine, cimetidine, disulfiram, hydralazine, and high levels of
vitamin B6. Additional toxic agents that may cause peripheral
neuropathy are listed below.
[0066] Trauma induced demyelinating peripheral neuropathies, as
described above, are caused by bodily shock, injury, or physical
trauma.
[0067] Accordingly, causes of peripheral neuropathy range widely,
e.g. from diabetic complications; trauma; toxins including, without
limitation, drugs and medications, industrial chemicals, and
environmental toxins; autoimmune response; nutritional
deficiencies; to vascular and metabolic disorders. For example,
demyelinating peripheral neuropathies may occur as a result of
osteosclerotic myeloma, monoclonal protein-associated peripheral
neuropathy, hereditary motor and sensory peripheral neuropathies
types 1 and 3, and hereditary susceptibility to pressure
palsies.
[0068] Similarly, symptoms of a demyelinating peripheral neuropathy
also vary, e.g. with the type of nerves affected. For example, a
human patient having a demyelinating disorder can have one or more
symptoms of a demyelinating disorder such as, but not limited to,
impaired vision, numbness, weakness in extremities, tremors or
spasticity, heat intolerance, speech impairment, incontinence,
dizziness, or impaired proprioception (e.g., balance, coordination,
sense of limb position). A human (e.g., a human patient) with a
family history of a demyelinating disorder (e.g., a genetic
predisposition for a demyelinating disorder), or who exhibits mild
or infrequent symptoms of a demyelinating disorder described above
can be, for the purposes of the method, considered at risk of
developing a demyelinating disorder.
[0069] Specifically, sensory nerve damage caused by a demyelinating
peripheral neuropathy can cause a more complex range of symptoms
because sensory nerves have a wider, more highly specialized range
of functions. Larger sensory fibers enclosed in myelin (lipid-rich
membrane folds that are spirally wrapped and insulate many nerves)
register vibration, light touch, and position sense. Damage to
large sensory fibers lessens the ability to feel vibrations and
touch, resulting in a general sense of numbness, especially in the
hands and feet. Many patients cannot recognize by touch alone the
shapes of small objects or distinguish between different shapes.
This damage to sensory fibers may contribute to the loss of
reflexes (as can motor nerve damage). Loss of position sense often
makes individuals unable to coordinate complex movements like
walking or fastening buttons, or to maintain their balance when
their eyes are shut. Neuropathic pain is difficult to control and
can seriously affect emotional well-being and overall quality of
life.
[0070] Smaller sensory fibers without myelin sheaths transmit pain
and temperature sensations. Damage to these fibers can interfere
with the ability to feel pain or changes in temperature.
Individuals may fail to sense that they have been injured from a
cut or that a wound is becoming infected. Others may not detect
pains that warn of impending heart attack or other acute
conditions. (Loss of pain sensation is a particularly serious
problem for individuals with diabetes, contributing to the high
rate of lower limb amputations among this population.) Pain
receptors in the skin can also become oversensitized, so that
severe pain is felt (allodynia) from stimuli that are normally
painless.
[0071] Symptoms of autonomic nerve damage are diverse and depend
upon which organs or glands are affected. Autonomic nerve
dysfunction can become life threatening and may require emergency
medical care in cases when breathing becomes impaired or when the
heart begins beating irregularly. Common symptoms of autonomic
nerve damage include an inability to sweat normally, which may lead
to heat intolerance; a loss of bladder control, which may cause
infection or incontinence; and an inability to control muscles that
expand or contract blood vessels to maintain safe blood pressure
levels. A loss of control over blood pressure can cause dizziness,
lightheadedness, or even fainting when an individual moves suddenly
from a seated to a standing position (a condition known as postural
or orthostatic hypotension).
[0072] Gastrointestinal symptoms frequently accompany autonomic
neuropathy. Nerves controlling intestinal muscle contractions often
malfunction, leading to diarrhea, constipation, or incontinence.
Individuals may also experience difficulty eating or swallowing if
certain autonomic nerves are affected.
[0073] The polyclonal IgG composition of the invention may also be
used to treat demyelinating peripheral neuropathy which developed
as a complication of diabetes, i.e. Type I, Type II. Peripheral
neuropathy is one of the major complications of diabetes. Both a
decrease in nerve conduction velocity and increased resistance to
conduction failure caused by ischemia are among the earliest
changes detected in diabetic patients and animal models of the
disease. Ultrastructural studies have demonstrated changes in both
axons and Schwann Cells (SC) (e.g., decrease in axon caliber and
segmental demyelination) as well as in the microvasculature, all of
which appear to develop independently. Some studies concluded that
the progressive loss of fibers in peripheral nerves observed in
human diabetic neuropathy may be due, at least in part, to delayed
nerve degeneration and impaired nerve regeneration. Metabolic and
microvascular abnormalities, as well as a deficiency in
neurotrophins, have been considered responsible for the
pathogenesis of diabetic neuropathy. The vascular alterations in
diabetes consists mainly of ischemia and endoneurial hypoxia. The
mechanisms underlying these vascular abnormalities include
degenerative changes in the sympathetic nerve endings of vasa
nervorum, with the consequent impairment in neural control of nerve
blood flow and reduced production of prostacyclin and nitric oxide
in nerves.
[0074] Two distinct clinical manifestations of diabetic neuropathy
are those represented by patients suffering from painful
symmetrical polyneuropathy, and by patients with insensitive,
painless feet. The painless neuropathy is the prevalent disorder
and, according to several studies, is likely to reflect the degree
of nerve degeneration. The painful syndrome, on the other hand, is
associated with fewer morphological abnormalities. While it has
also been proposed that the painful syndrome may reflect nerve
regeneration, as opposed to degeneration, several reports suggest
that nerve regeneration is impaired in diabetes. Analysis of
several functional indices in peripheral sensory nerves of diabetic
rodents also suggests depressed, rather than increased, function.
For instance, experimental diabetes induces several nociceptive
responses including early thermal hyperalgesia that with time turns
into hypoalgesia, mechanical hyperalgesia, thermal and tactile
allodynia, increased C fiber activity and reduced sensitivity to
opioids. In this context, mechanical hyperalgesia may result from
increased firing after sustained suprathreshold mechanical
stimulation of C fibers.
[0075] While therapies with antioxidants, vasodilators and
neurotrophins may reverse some functional and metabolic
abnormalities in diabetic nerves, they only result in a partial
amelioration of abnormal pain perception, suggesting that other
pathways are at play. The present invention is able to promote
Schwann cell's healing capacity towards treatment of diabetic
neuropathy.
[0076] The polyclonal IgG composition of the invention may also be
used to treat demyelinating peripheral neuropathy resulting from
trauma. A "trauma-induced" neuropathy refers to damage to the
nervous system from external physical injury. Injury or sudden
trauma, e.g. from warfare, automobile accidents, falls, and
sports-related activities, can cause nerves to be partially or
completely severed, crushed, compressed, or stretched, sometimes so
forcefully that they are partially or completely detached from the
spinal cord and result in demyelination. Less dramatic traumas also
can cause serious nerve damage.
[0077] The polyclonal IgG composition of the invention may also be
used to treat peripheral neuropathy caused by a toxic agent. Toxins
that produce peripheral neuropathy can generally be divided into
three groups: drugs and medications; industrial chemicals; and
environmental toxins. As used herein, the term "toxic agent" is
defined as any substance that, through its chemical action, impairs
the normal function of one or more components of the peripheral
nervous system. The definition includes agents that are airborne,
ingested as a contaminant of food or drugs, or taken deliberately
as part of a therapeutic regime.
[0078] The list of toxic agents that may cause peripheral
neuropathy includes, but is not limited to,
3'-azido-3'-deoxythymidine, acetazolamide, acrylamide, adriamycin,
alcohol, allyl chloride, almitrine, amitriptyline, amiodarone,
amphotericin, arsenic, aurothioglucose, carbamates, carbon
disulfide, carbon monoxide, carboplatin, chloramphenicol,
chloroquine, cholestyramine, cimetidine, cisplatin, cis-platinum,
clioquinol, colestipol, colchicine, colistin, cycloserine,
cytarabine, dapsone, dichlorophenoxyacetic acid, didanosine;
dideoxycytidine, dideoxyinosine, dideoxythymidine,
dimethylaminopropionitrile, disulfiram, docetaxel, doxorubicin,
ethambutol, ethionamide, ethylene oxide, FK506 (tacrolimus),
glutethimide, gold, hexacarbons, hexane, hormonal contraceptives,
hexamethylolmelamine, hydralazine, hydroxychloroquine, imipramine,
indomethacin, inorganic lead, inorganic mercury, isoniazid,
lithium, methylmercury, metformin, methotrexate, methylbromide,
methylhydrazine, metronidazole, misonidazole, methyl N-butyl
ketone, nitrofurantoin, nitrogen mustard, nitrous oxide,
organophosphates, ospolot, paclitaxel, penicillin, perhexiline,
perhexiline maleate, phenytoin, platinum, polychlorinated
biphenyls, primidone, procainamide, procarbazine, pyridoxine,
simvastatin, sodium cyanate, streptomycin, sulphonamides, suramin,
tamoxifen, thalidomide, thallium, toluene, triamterene,
trimethyltin, triorthocresyl phosphate, L-tryptophan, vacor, vinca
alkaloids, vincristine, vindesine, megadoses of vitamin A,
megadoses of vitamin D, zalcitamine, zimeldine; industrial agents,
especially solvents; heavy metals; and sniffing glue or other toxic
compounds.
[0079] The polyclonal IgG composition of the invention may also be
used to treat demyelinating peripheral neuropathy resulting from
the administration of chemotoxins for cancer therapy. Among the
chemotoxins known to cause peripheral neuropathy are vincristine,
vinblastine, cisplatin, paclitaxel, procarbazine, dideoxyinosine,
cytarabine, alpha interferon, and 5-fluorouracil (see Macdonald,
Neurologic Clinics 9: 955-967 (1991)).
III. Diagnosis and Monitoring of Demyelinating Peripheral
Neuropathies
[0080] Diagnosis of demyelinating peripheral neuropathy can be made
by a physician or clinician using one or more methods known in the
art. A neurological examination is typically required and involves
taking a patient history (including the patient's symptoms, work
environment, social habits, exposure to any toxins, history of
alcoholism, risk of HIV or other infectious disease, and family
history of neurological disease), performing tests that may
identify the cause of the neuropathic disorder, and conducting
tests to determine the extent, site, and type of nerve damage.
[0081] A general physical examination and related tests may reveal
the presence of a systemic disease causing nerve damage. Blood
tests can detect diabetes, vitamin deficiencies, liver or kidney
dysfunction, other metabolic disorders, and signs of abnormal
immune system activity. An examination of cerebrospinal fluid that
surrounds the brain and spinal cord can reveal abnormal antibodies
associated with neuropathy. More specialized tests may reveal other
blood or cardiovascular diseases, connective tissue disorders, or
malignancies. Tests of muscle strength, as well as evidence of
cramps or fasciculations, indicate motor fiber involvement.
Evaluation of a patient's ability to register vibration, light
touch, body position, temperature, and pain reveals sensory nerve
damage and may indicate whether small or large sensory nerve fibers
are affected.
[0082] Based on the results of the neurological exam, physical
exam, patient history, and any previous screening or testing,
additional testing may be ordered to help determine the nature and
extent of the neuropathy. Exemplary technologies for aiding in the
diagnosis of peripheral neuropathies include: computed tomography
scan, magnetic resonance imaging, electromyography, nerve
conduction velocity, nerve biopsy, or skin biopsy. Apparatuses
useful in the diagnosis of peripheral neuropathies include, without
limitation, U.S. Pat. No. 7,854,703.
[0083] Computed tomography, or CT scan, is a noninvasive, painless
process used to produce rapid, clear two-dimensional images of
organs, bones, and tissues. X-rays are passed through the body at
various angles and are detected by a computerized scanner. The data
is processed and displayed as cross-sectional images, or "slices,"
of the internal structure of the body or organ. Neurological CT
scans can detect bone and vascular irregularities, certain brain
tumors and cysts, herniated disks, encephalitis, spinal stenosis
(narrowing of the spinal canal), and other disorders.
[0084] Magnetic resonance imaging (MRI) can examine muscle quality
and size, detect any fatty replacement of muscle tissue, and
determine whether a nerve fiber has sustained compression damage.
The MRI equipment creates a strong magnetic field around the body.
Radio waves are then passed through the body to trigger a resonance
signal that can be detected at different angles within the body. A
computer processes this resonance into either a three-dimensional
picture or a two-dimensional "slice" of the scanned area.
[0085] Electromyography (EMG) involves inserting a fine needle into
a muscle to compare the amount of electrical activity present when
muscles are at rest and when they contract. EMG tests can help
differentiate between muscle and nerve disorders.
[0086] Nerve conduction velocity (NCV) tests can precisely measure
the degree of damage in larger nerve fibers, revealing whether
symptoms are being caused by degeneration of the myelin sheath or
the axon. During this test, a probe electrically stimulates a nerve
fiber, which responds by generating its own electrical impulse. An
electrode placed further along the nerve's pathway measures the
speed of impulse transmission along the axon. Slow transmission
rates and impulse blockage tend to indicate damage to the myelin
sheath, while a reduction in the strength of impulses is a sign of
axonal degeneration.
[0087] Nerve biopsy involves removing and examining a sample of
nerve tissue, most often from the lower leg. Although this test can
provide valuable information about the degree of nerve damage, it
is an invasive procedure that is difficult to perform and may
itself cause neuropathic side effects.
[0088] Skin biopsy is a test in which doctors remove a thin skin
sample and examine nerve fiber endings. Unlike NCV, it can reveal
damage present in smaller fibers; in contrast to conventional nerve
biopsy, skin biopsy is less invasive, has fewer side effects, and
is easier to perform.
[0089] Methods of monitoring an individual for demyelination or
remyelination are known in the art. Monitoring a subject (e.g., a
human patient) for remyelination, as defined herein, means
evaluating the subject for a change, e.g., an improvement in one or
more parameters that are indicative of remyelination, e.g., one can
monitor improvement in one or more symptoms of a demyelinating
disorder. Such symptoms include any of the symptoms of a
demyelinating disorder described herein. Remyelination can also be
monitored by methods which include direct determination of the
state of myelin in the subject, e.g., one can measure white matter
mass using magnetic resonance imaging (MRI) or measure the
thickness of myelin fibers using a magnetic resonance spectroscopy
(MRS) brain scan.
[0090] In some embodiments, the evaluation is performed at least 1
hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1
day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9
days, 10 days, 11, days, 12 days, 13 days, 14 days, 15 days, 16
days, 17 days, 18 days, 19 days, or 20 days or more, or at least 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8
weeks, 9 weeks, 10 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks,
16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or more, or any
combination thereof, after an administration, preferably the first
administration, of the polyclonal IgG. The subject can be evaluated
in one or more of the following periods: prior to beginning of
treatment; during the treatment; or after one or more elements of
the treatment have been administered. Evaluating can include
evaluating the need for further treatment, e.g., evaluating whether
a dosage, frequency of administration, or duration of treatment
should be altered. It can also include evaluating the need to add
or drop a selected therapeutic modality, e.g., adding or dropping
any of the treatments for demyelinating disorders described herein.
For example, continued administration of the polyclonal IgG could
be done with one or more additional therapeutic agents where
necessary. In a preferred embodiment, if a preselected outcome of
the evaluation is obtained, an additional step is taken, e.g., the
subject is administered another treatment or another evaluation or
test is performed. The level of remyelination can be used to make a
determination on patient care, e.g., a selection or modification of
a course of treatment or the decision of a third party to reimburse
for the treatment.
[0091] In some embodiments, monitoring a subject (e.g., a human
patient) for remyelination can also include monitoring for a
reduction in the size or number of inflammatory lesions (i.e.,
scleroses) using, e.g., Magnetic Resonance Imaging (MRI) scans,
Positron-Emission Tomography (PET) scans, Diffusion-Weighted
Imaging (DW-I, or DW-MRI), Diffusion Tensor Imaging, Myelography,
Magnetization Transfer. In some embodiments, monitoring a subject
for remyelination can include the detection of, e.g., (i) abnormal
proteins such as tiny fragments of myelin, (ii) elevated levels of
or specific types of lymphocytes, and/or (iii) abnormal levels of
immunoglobulin (IgG) molecules. In other embodiments, monitoring a
subject for remyelination can include assessment of a change in the
subject's neuropsychology (e.g., the status of various abilities
such as memory, arithmetic, attention, judgment and reasoning). In
some embodiments, the monitoring of a subject (e.g., a human
patient) for remyelination can involve testing a patient's urine
for a decrease in levels of myelin basic protein-like material
(MBP-like material), which substance becomes elevated as axonal
damage occurs during disease progression. In some embodiments,
where the demyelinating disorder affects a subject's eyes or
vision, the monitoring of a subject for remyelination can involve
testing for improvements in, e.g., color blindness.
[0092] Provided herein are methods of evaluating a subject, to
determine, e.g., if a subject is responding or not responding to a
treatment for a demyelinating disorder, e.g., a therapy that
increases remyelination in a subject such as administering a
polyclonal IgG. The method includes providing a reference value
(e.g., a pre-administration value) for the level or state of myelin
in the subject, and optionally, administering to the subject a
medicament that increases remyelination (e.g., a polyclonal IgG).
In embodiments where a medicament is administered, the method also
includes providing a post-administration value for the level or
state of myelin in the subject (e.g., the level or state of myelin
following administration of a remyelination therapy) and comparing
the post-administration value with the reference value, thereby
evaluating the subject, e.g., determining if the subject is
responding or not responding to the therapy. The
post-administration value (i.e., the value corresponding to the
state or level of myelin in a subject following a remyelination
therapy) can be determined, e.g., by any of the assessment methods
described herein. The reference value (i.e., the state or level of
myelin in a subject prior to treatment with a remyelination
therapy) can also be determined, e.g., by any of the assessment
methods described herein.
[0093] In some embodiments, a determination that a subject is
responding indicates that a shorter duration of treatment
can/should/will be/is administered to the subject (e.g., shorter
than the treatment which is recommended for a subject who is not
responding to a therapy, or a duration shorter than currently used
with existing therapies for demyelinating disorders, and
optionally, that indication is entered into a record.
[0094] In some embodiments, a determination that a subject is
responding indicates that a shorter duration of treatment is
counter-indicated for the subject (e.g., a duration shorter than
currently used with existing treatments for demyelinating
disorders, e.g., any of the treatments for demyelinating disorders
described herein), and optionally, that indication is entered into
a record.
[0095] In some embodiments, providing a comparison of the
post-administration value with a reference value includes:
providing a determination of a post-administration level of myelin
in a subject at a first time point (e.g., wherein the first time
point is 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days (e.g., 3, 4,
5, 6, 8 or more weeks (e.g., 3, 4, 6, 12 or more months))) after
the commencement of administration of the remyelination therapy
(e.g., polyclonal IgG); providing a determination of a reference
value of the state or level of myelin in the subject at a second
time point that is prior to the first time point (e.g., wherein the
second time point is prior to, or within about 1, 2, 3, 4, or 5
days of the commencement of, administration of a remyelination
therapy (e.g., polyclonal IgG); and providing a comparison of the
post administration level and reference value of a subject's
myelin, wherein increased levels of myelin in a subject (e.g., the
levels differ by no more than about 60%, about 50%, about 40%,
about 30%, about 20%, about 10%, about 5%, about 2%, or about 1%)
between the post-administration level and reference value indicates
that the subject is responding.
[0096] In some embodiments, the determination of whether a patient
is responding to a therapy is made by evaluating the subject for a
change, an improvement, in one or more parameters that are
indicative of remyelination, e.g., one can monitor improvement in
one or more symptoms of a demyelinating disorder. Such symptoms
include any of the symptoms of a demyelinating disorder described
herein. Remyelination can also be monitored by methods which
include direct determination of the state of myelin in the subject,
e.g., one can measure white matter mass using magnetic resonance
imaging (MRI), measure the thickness of myelin fibers using a
magnetic resonance spectroscopy (MRS) brain scan, or any other
direct measures described herein.
[0097] In another embodiment, the determination of whether a
patient is responding to a therapy can also be evaluated by any
other assessment or indicia described herein, including, but not
limited to, monitoring a patient for a reduction in the size or
number of inflammatory lesions (i.e., scleroses) present in the
patient; monitoring a patient's endoneurial fluid for a reduction
in the presence or amount of, e.g., (i) elevated levels of or
specific types of lymphocytes, and/or (ii) abnormal levels of
immunoglobulin (IgG) molecules; monitoring a patient for a positive
change in neuropsychology (e.g., the status of various abilities
such as memory, arithmetic, attention, judgment and reasoning);
and/or monitoring a patient's urine for a decrease in levels of
myelin basic protein-like material (MBP-like material).
[0098] In some embodiments, at least a 5% (e.g., at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 50%, at least 60%, at least 70%) improvement
in one or more symptoms of a demyelinating disorder or other
above-described indicia following a remyelination therapy (e.g., a
therapy that induces remyelination in a subject, e.g., a therapy
such as a polyclonal IgG) is sufficient to classify the patient as
responding to a therapy.
IV. Preparation of Polyclonal IGG
[0099] Immunoglobulin preparations according to the present
invention can be prepared from any suitable starting materials. For
example, immunoglobulin preparations can be prepared from donor
serum or monoclonal or recombinant immunoglobulins. In a typical
example, blood is collected from healthy donors. Usually, the blood
is collected from the same species of animal as the subject to
which the immunoglobulin preparation will be administered
(typically referred to as "homologous" immunoglobulins). The
immunoglobulins are isolated from the blood and purified by one or
more suitable procedures, such as, for example, Cohn fractionation,
ultracentrifugation, electrophoretic preparation, ion exchange
chromatography, affinity chromatography, immunoaffinity
chromatography, polyethylene glycol fractionation, alcohol
fractionation, nanofiltration, ultrafiltration/diafiltration or the
like. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459-75 (1946);
Oncley et al., J. Am. Chem. Soc. 71:541-50 (1949); Barundern et
al., Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102
(1967); Teschner et al. Vox Sang (92):42-55 (2007); Hoppe et al.
Munch Med Wochenschr (34): 1749-1752 (1967), Falksveden (Swedish
Patent No. 348942); Tanaka et al., Braz J Med Biol Res (33)37-30
(2000); Lebing et al., Vox Sang (84):193-201 (2003); U.S. Pat. Nos.
5,122,373 and 5,177,194; PCT/US2010/036470; and PCT/US2011/038247;
the disclosures of which are incorporated by reference herein.)
[0100] To inactivate various viral contaminants present in
plasma-derived products, the clarified PptG filtrate may be
subjected to a solvent detergent (S/D) treatment. Methods for the
detergent treatment of plasma derived fractions are well known in
the art (for review see, Pelletier J P et al., Best Pract Res Clin
Haematol. 2006; 19(1):205-42). Generally, any standard S/D
treatment may be used in conjunction with the methods provided
herein.
[0101] To further purify and concentrate IgG, cation exchange
and/or anion exchange chromatography can be employed. Methods for
purifying and concentrating IgG using ion exchange chromatography
are well known in the art. For example, U.S. Pat. No. 5,886,154
describes a method in which a Fraction II+III precipitate is
extracted at low pH (between about 3.8 and 4.5), followed by
precipitation of IgG using caprylic acid, and finally
implementation of two anion exchange chromatography steps. U.S.
Pat. No. 6,069,236 describes a chromatographic IgG purification
scheme that does not rely on alcohol precipitation at all. PCT
Publication No. WO 2005/073252 describes an IgG purification method
involving the extraction of a Fraction II+III precipitate, caprylic
acid treatment, PEG treatment, and a single anion exchange
chromatography step. U.S. Pat. No. 7,186,410 describes an IgG
purification method involving the extraction of a Fraction I+II+III
or Fraction II precipitate followed by a single anion exchange step
performed at an alkaline pH. U.S. Pat. No. 7,553,938 describes a
method involving the extraction of a Fraction I+II+III or Fraction
II+III precipitate, caprylate treatment, and either one or two
anion exchange chromatography steps. U.S. Pat. No. 6,093,324
describes a purification method comprising the use of a macroporous
anion exchange resin operated at a pH between about 6.0 and about
6.6. U.S. Pat. No. 6,835,379 describes a purification method that
relies on cation exchange chromatography in the absence of alcohol
fractionation. The disclosures of the above publications are hereby
incorporated by reference in their entireties for all purposes
[0102] To reduce the viral load of an IgG composition provided
herein, the composition may be nanofiltered using a suitable
nanofiltration device. In certain embodiments, the nanofiltration
device will have a mean pore size of between about 15 nm and about
200 nm. Examples of nanofilters suitable for this use include,
without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP,
Viresolve NFR (Millipore), Planova 15N, 20N, 35N, and 75N
(Planova). In a specific embodiment, the nanofilter may have a mean
pore size of between about 15 nm and about 72 nm, or between about
19 nm and about 35 nm, or of about 15 nm, 19 nm, 35 nm, or 72 nm.
In a preferred embodiment, the nanofilter will have a mean pore
size of about 35 nm, such as an Asahi PLANOVA 35N filter or
equivalent thereof. In a particular embodiment, the IgG composition
recovered from the anion exchange step is nanofiltered using a
nanofilter having a pore size between 30 nm and 40 nm, preferably
35.+-.2 nm. In another preferred embodiment, the nanofilter will
have a mean pore size of about 19 or 20 nm, such as an Asahi
PLANOVA 20N filter (19.+-.2 nm) or equivalent thereof. In a
particular embodiment, the IgG composition recovered from the anion
exchange step is nanofiltered using a nanofilter having a pore size
between 15 nm and 25 nm, preferably 19.+-.2 nm.
[0103] In certain embodiments, immunoglobulin is prepared from
gamma globulin-containing products produced by the alcohol
fractionation and/or ion exchange and affinity chromatography
methods well known to those skilled in the art. Purified Cohn
Fraction II is commonly used. The starting Cohn Fraction II paste
is typically about 95 percent IgG and is comprised of the four IgG
subtypes. The different subtypes are present in Fraction II in
approximately the same ratio as they are found in the pooled human
plasma from which they are obtained. The Fraction II is further
purified before formulation into an administrable product. For
example, the Fraction II paste can be dissolved in a cold purified
aqueous alcohol solution and impurities removed via precipitation
and filtration. Following the final filtration, the immunoglobulin
suspension can be dialyzed or diafiltered (e.g., using
ultrafiltration membranes having a nominal molecular weight limit
of less than or equal to 100,000 daltons) to remove the alcohol.
The solution can be concentrated or diluted to obtain the desired
protein concentration and can be further purified by techniques
well known to those skilled in the art.
[0104] Preparative steps can be used to enrich a particular isotype
or subtype of immunoglobulin. For example, protein A, protein G or
protein H sepharose chromatography can be used to enrich a mixture
of immunoglobulins for IgG, or for specific IgG subtypes. (See
generally Harlow and Lane, Using Antibodies, Cold Spring Harbor
Laboratory Press (1999); Harlow and Lane, Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1988); U.S. Pat. No.
5,180,810.)
[0105] Commercial sources of polyclonal immunoglobulins can also be
used. Such sources include but are not limited to: Kiovig.RTM. 10%
IVIG (Baxter Healthcare); Gammagard Liquid.RTM. 10% IVIG (Baxter
Healthcare); Gammagard S/D.RTM. (Baxter Healthcare); Gammagard
S/D.RTM. with less than 1 mg/mL of IgA in a 5% solution (Baxter
Healthcare); Gamunex.RTM.-C, 10% (Grifols USA); Flebogamma.RTM., 5%
and 10% DIF (Grifols USA); Privigen.RTM. 10% Solution (CSL
Behring); Carimune NF or Sandoglobulin.RTM. (CSL Behring); and
Hizentra.RTM. 20% Liquid (CSL Behring); Octagam.RTM., 5% and 10%
IVIG (Octapharma AG); Gammanorm.RTM. 16.5% SCIG (Octapharma AG).
The commercial source of immunoglobulin preparation for use in the
methods of the present invention is not critical.
[0106] An alternative approach is to use fragments of antibodies
with antigen-binding capability, e.g., Fab', F(ab')2, Fab, Fv and
rIgG. See, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce
Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3.sup.rd Ed.,
W.H. Freeman & Co., New York (1998). The polyclonal IgG
composition of the invention may include fragments of one
immunoglobulin isotype, i.e. IgG, or can contain a mixture of
immunoglobulin fragments of different isotypes (e.g., IgA, IgD,
IgE, IgG and/or IgM). The Fc preparation also can contain
predominantly (at least 60%, at least 75%, at least 90%, at least
95%, or at least 99%) fragments from the IgG immunoglobulin
isotype, and can contain minor amounts of the other subtypes. For
example, an Fc preparation can contain at least at least about 75%,
at least about 90%, at least about 95%, or at least about 99% IgG
fragments. In addition, the polyclonal IgG preparation can comprise
a single IgG subtype or a mixture of two or more of IgG subtypes.
Suitable IgG subtypes include IgG1, IgG2, IgG3, and IgG4. In a
specific embodiment, the polyclonal IgG preparation comprises IgG1
fragments.
[0107] Immunoglobulins can be cleaved at any suitable time during
preparation to yield Fab, F(ab') and/or F(ab')2 fragments, as
applicable. A suitable enzyme for cleavage is, for example, papain,
pepsin or plasmin. (See, e.g., Harlow and Lane, Using Antibodies,
Cold Spring Harbor Laboratory Press (1999); Plan and Makula, Vox
Sanguinis 28:157-75 (1975).) After cleavage, the Fc portions can be
separated from the Fab, F(ab') and/or F(ab')2 fragments by, for
example, affinity chromatography, ion exchange chromatography, gel
filtration, or the like. In a specific example, immunoglobulins are
digested with papain to separate the Fc fragment from the Fab
fragments. The digestion mixture is then subjected to cationic
exchange chromatography to separate the Fc fragments from the Fab
fragments.
[0108] Immunoglobulin fragments can also be prepared from
hybridomas or other culture system which express monoclonal
antibody. (See, e.g., Kohler and Milstein, Nature 256:495-97
(1975); Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15-19
(1993); Kozbor et al., Immunology Today 4:72 (1983); Cole et al.,
in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
pp. 77-96 (1985).) Human monoclonal antibodies can be obtained, for
example, from human hybridomas (see, e.g., Cote et al., Proc. Natl.
Acad. Sci. USA 80:2026-30 (1983)) or by transforming human B cells
with EBV virus in vitro (see, e.g., Cole et al., supra). Monoclonal
antibodies produced from hybridomas can be purified and the Fc
fragments separated from the Fab, F(ab') and/or F(ab')2 fragments
as described herein or as known to the skilled artisan.
[0109] IgG fragments also can be produced recombinantly, such as
from eukaryotic cell culture systems. For example, a single chain
Fv fragments (scFv) can be recombinantly produced by Chinese
hamster ovary (CHO) cells transfected with a vector containing a
DNA sequence encoding the Fv fragments. Methods for creating such
recombinant mammalian cells are described in, for example, Sambrook
and Russell, Molecular Cloning, A Laboratory Manual, 3rd ed. (Cold
Spring Harbor Laboratory Press (New York) 2001) and Ausubel et al.,
Short Protocols in Molecular Biology, 4th ed. (John Wiley &
Sons, Inc. (New York) 1999) and are known to the skilled artisan.
Recombinant immunoglobulin fragments can also be produced in other
mammalian cell lines, such as baby hamster kidney (BHK) cells.
Methods of culturing recombinant cells to produce recombinant
proteins are also known to the art.
[0110] A variety of other expression systems can be utilized to
express recombinant immunoglobulins IgG fragments. These include,
but are not limited to, insect cell systems and microorganisms such
as yeast or bacteria which have been transfected or transformed
with an expression cassette encoding the desired IgG fragment. In
certain embodiments, the microorganism optionally can be engineered
to reproduce glycosylation patterns of mammalian or human IgG
fragments.
[0111] In certain embodiments, further preparative steps can be
used in order to render an immunoglobulin preparation safe for use
in the methods according to the present invention. Such steps can
include, for example, treatment with solvent/detergent,
pasteurization and sterilization. Additional preparative steps may
be used in order to ensure the safety of a polyclonal IgG
preparation. Such preparative steps can include, for example,
enzymatic hydrolysis, chemical modification via reduction and
alkylation, sulfonation, treatment with B-propiolactone, treatment
at low pH, or the like. Descriptions of suitable methods can also
be found in, for example, U.S. Pat. Nos. 4,608,254; 4,687,664;
4,640,834; 4,814,277; 5,864,016; 5,639,730 and 5,770,199; Romer et
al., Vox Sang. 42:62-73 (1982); Romer et al., Vox Sang. 42:74-80
(1990); and Rutter, J. Neurosurg. Psychiat. 57 (Suppl.):2-5 (1994)
(the disclosures of which are incorporated by reference
herein).
V. Pharmaceutical Compositions and Dosages
[0112] An individual in whom administration of the polyclonal IgG
as set forth herein is an effective therapeutic regimen for
demyelinating peripheral neuropathy, is preferably a human, but can
be any mammal. Thus, as can be readily appreciated by one of
ordinary skill in the art, the methods and pharmaceutical
compositions of the present invention are particularly suited to
administration to any a mammal, and including, but by no means
limited to, domestic animals, such as feline or canine subjects,
farm animals, such as but not limited to bovine, equine, caprine,
ovine, and porcine subjects, wild animals (whether in the wild or
in a zoological garden), research animals, such as mice, rats,
rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., for veterinary
medical use.
[0113] It is contemplated that a pharmaceutical composition
comprising polyclonal IgG of the present invention can be
administered by a variety of methods known in the art. The route
and/or mode of administration vary depending upon the desired
results, but will typically be intravenous, intramuscular,
intranasal, intraperitoneal, intra-arterial, or subcutaneous. The
pharmaceutical composition can include an acceptable carrier
suitable for intravenous, intramuscular, subcutaneous, parenteral,
spinal or epidermal administration (e.g., by injection or
infusion).
[0114] The polyclonal IgG of this invention are useful for local or
systemic administration for prophylactic and/or therapeutic
treatment. Exemplary modes of administration include, without
limitation, transdermal, subcutaneous, intra-arterial, intravenous,
intranasal, intramuscular, rectal, buccal, and oral administration.
The pharmaceutical compositions can be administered in a variety of
unit dosage forms depending upon the method of administration. For
example, unit dosage forms include powder, tablets, pills,
capsules, suppositories, ampoules, and lozenges. It is only
necessary that the active ingredient constitute an effective
amount, i.e., such that a suitable effective dosage will be
consistent with the dosage form employed in single or multiple unit
doses. The exact individual dosages, as well as daily dosages,
will, of course, be determined according to standard medical
principles under the direction of a physician or veterinarian. The
pharmaceutical polyclonal IgG immunoglobin compositions of this
invention, when administered orally, are preferably protected from
digestion. This is typically accomplished either by complexing the
antibodies with a composition to render them resistant to acidic
and enzymatic hydrolysis or by packaging the antibodies in an
appropriately resistant carrier such as a vesicle, in particular a
liposome (see Langer, Science 249:1527-1533 (1990); Treat et al.,
in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365
(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
Means of protecting proteins from digestion are well known in the
art.
[0115] The pharmaceutical compositions of this invention are
particularly useful for parenteral administration, such as
intravenous administration or administration into a body cavity or
lumen of an organ. The compositions for administration will
commonly comprise a composition of polyclonal IgG with a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like.
[0116] Diluents that can be used in pharmaceutical compositions
(e.g., granulates) containing the active compound adapted to be
formed into tablets, dragees, capsules and pills include the
following: (a) fillers and extenders, e.g., starch, sugars,
mannitol and silicic acid; (b) binding agents, e.g., carboxymethyl
cellulose and other cellulose derivatives, alginates, gelatine and
polyvinyl pyrrolidone; (c) moisturizing agents, e.g., glycerol; (d)
disintegrating agents, e.g., agar-agar, calcium carbonate and
sodium bicarbonate; (e) agents for retarding dissolution, e.g.,
paraffin; (f) resorption accelerators, e.g., quaternary ammonium
compounds; (g) surface active agents, e.g., cetyl alcohol, glycerol
monostearate; (g) adsorptive carriers, e.g., kaolin and bentonite;
(i) lubricants, e.g., talc, calcium and magnesium stearate and
solid polyethylene glycols. The diluents to be used in
pharmaceutical compositions adapted to be formed into suppositories
can, for example, be the usual water-soluble diluents, such as
polyethylene glycols and fats (e.g., cocoa oil and high esters,
[e.g., C.sub.14-alcohol with C.sub.16-fatty acid]) or mixtures of
these diluents.
[0117] The pharmaceutical compositions of the invention are sterile
and generally free of undesirable matter. For parental
administration, solutions and suspensions should be sterile, e.g.,
water or arachis oil contained in ampoules and, if appropriate,
blood-isotonic. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of the polyclonal IgG in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, patient body weight and the like in accordance with
the particular mode of administration selected and the patient's
needs.
[0118] Proper fluidity of the composition can be maintained, for
example, by use of coating such as lecithin, by maintenance of
required particle size in the case of dispersion and by use of
surfactants. In some cases, it is preferable to include isotonic
agents, for example, sugars such as sucrose, polyalcohols such as
mannitol or sorbitol, and sodium chloride in the composition.
Stabilizers such as nicotinamide, L-proline, L-glycine, or
L-isoleucine may also be employed. Long-term absorption of the
injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0119] The pharmaceutical compositions which are suspensions can
contain the usual diluents, such as liquid diluents, e.g., water,
ethyl alcohol, propylene glycol, surface active agents (e.g.,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitols and
sorbitan esters), microcrystalline cellulose, aluminum
methahydroxide, bentonite, agar-agar and tragacanth, or mixtures
thereof.
[0120] The pharmaceutical compositions can also contain coloring
agents and preservatives, as well as perfumes and flavoring
additions (e.g., peppermint oil and eucalyptus oil), and sweetening
agents, (e.g., saccharin and aspartame).
[0121] The pharmaceutical compositions will generally contain from
0.5 to 90% of the active ingredient by weight of the total
composition.
[0122] In addition to the monoclonal antibodies, the pharmaceutical
compositions and medicaments can also contain other
pharmaceutically active compounds, e.g. steroids, anti-inflammatory
agents or the like.
[0123] Any diluent in the medicaments of the present invention may
be any of those mentioned above in relation to the pharmaceutical
compositions. Such medicaments may include solvents of molecular
weight less than 200 as the sole diluent.
[0124] Pharmaceutical compositions of the invention can be prepared
in accordance with methods well known and routinely practiced in
the art. See, e.g., Remington: The Science and Practice of
Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions
are preferably manufactured under GMP conditions. Typically, a
therapeutically effective dose or efficacious dose of the
immunoglobulin preparation is employed in the pharmaceutical
compositions of the invention. The pharmaceutical composition can
be formulated into dosage forms by conventional methods known to
those of skill in the art. Dosage regimens are adjusted to provide
the optimum desired response (e.g., a therapeutic response). For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation. It can be advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit contains a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier.
[0125] Actual dosage levels can be varied so as to obtain an amount
of the active ingredient which is effective to achieve the desired
therapeutic response for a particular patient without being toxic
to the patient. A physician can start doses of the pharmaceutical
composition at levels lower than that required to achieve the
desired therapeutic effect and gradually increase the dosage until
the desired effect is achieved. In general, effective doses vary
depending upon many different factors, including the specific
disease or condition to be treated, its severity, physiological
state of the patient, other medications administered, and whether
treatment is prophylactic or therapeutic.
[0126] The polyclonal IgG composition can be administered on
multiple occasions. Intervals between single dosages can be daily,
weekly, biweekly, every 3 weeks, every 4 weeks, monthly or yearly.
Intervals can also be irregular as indicated by measuring
therapeutic progress in the patient. Dosage and frequency can vary
depending on the half-life of the antibodies in the patient.
[0127] Alternatively, the polyclonal IgG can be delivered in a
controlled release system. For example, the polyclonal
immunoglobulins may be administered using intravenous infusion, an
implantable osmotic pump, a transdermal patch, liposomes, or other
modes of administration. In one embodiment, a pump may be used (see
Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);
Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J.
Med. 321:574 (1989)). In another embodiment, polymeric materials
can be used (see Medical Applications of Controlled Release, Langer
and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled
Drug Bioavailability, Drug Product Design and Performance, Smolen
and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et
al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351
(1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another
embodiment, a controlled release system can be placed in proximity
of the therapeutic target, i.e., a site of injury in the peripheral
nervous system, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)). Other controlled release
systems are discussed in the review by Langer (Science
249:1527-1533 (1990)).
[0128] In the case of a polyclonal IgG immunoglobulin preparation,
intravenous immunoglobulin (IVIG) is commonly used. IVIG
formulations are designed for administration by injection. Because
polyclonal IgG preparations have achieved an exceptionally high
immunoglobulin concentration (e.g. 10% w/v in some embodiments, 15%
w/v in other embodiments, 20% w/v in still other embodiments, and
up to 25% w/v in still further embodiments), which significantly
reduces the volume for a therapeutically effective dose, the
composition of the present invention is particularly advantageous
for subcutaneous and/or intramuscular administration to a patient,
as well as intravenous administration.
[0129] The term "effective amount" refers to an amount of
polyclonal IgG preparation that results in an improvement or
remediation of a medical condition being treated in the subject
(e.g., for treating peripheral nerve trauma, for treating
toxin-induced peripheral neuropathy, etc.). An effective amount to
be administered to the subject can be determined by a physician
with consideration of individual differences in age, weight,
disease severity, route of administration (e.g., intravenous v.
subcutaneous) and response to the therapy.
[0130] The dosing schedule may vary, depending on the circulation
half-life, and the formulation used. The compositions are
administered in a manner compatible with the dosage formulation in
the therapeutically effective amount. Precise amounts of active
ingredient required to be administered depend on the judgment of
the practitioner and are peculiar to each individual.
[0131] A suitable dose of polyclonal IgG may be administered to a
patient weekly, biweekly, every 3 weeks, every 4 weeks, or monthly
to a subject, wherein the dose ranges from about 0.050 to 5
g/kilogram of patient body weight, about 0.095 to 4.7 g/kilogram of
patient body weight, about 0.140 to 4.4 g/kilogram of patient body
weight, about 0.185 to 4.1 g/kilogram of patient body weight, about
0.230 to 3.8 g/kilogram of patient body weight, about 0.275 to 3.5
g/kilogram of patient body weight, about 0.320 to 3.2 g/kilogram of
patient body weight, about 0.365 to 2.9 g/kilogram of patient body
weight, about 0.410 to 2.6 g/kilogram of patient body weight, about
0.455 to 2.3 g/kilogram of patient body weight, about 0.500 to 2.0
g/kilogram of patient body weight.
[0132] In alternative embodiments, the polyclonal IgG composition
of the invention is administered weekly, biweekly, every 3 weeks,
every 4 weeks, or monthly to a subject at a dose of about 0.05 to
4.9 g/kilogram of patient body weight, about 0.05 to 4.8 g/kilogram
of patient body weight, about 0.05 to 4.7 g/kilogram of patient
body weight, about 0.05 to 4.6 g/kilogram of patient body weight,
about 0.05 to 4.5 g/kilogram of patient body weight, about 0.05 to
4.4 g/kilogram of patient body weight, about 0.05 to 4.3 g/kilogram
of patient body weight, about 0.05 to 4.2 g/kilogram of patient
body weight, about 0.05 to 4.1 g/kilogram of patient body weight,
about 0.05 to 4.0 g/kilogram of patient body weight, about 0.05 to
3.9 g/kilogram of patient body weight, about 0.05 to 3.8 g/kilogram
of patient body weight, about 0.05 to 3.7 g/kilogram of patient
body weight, about 0.05 to 3.6 g/kilogram of patient body weight,
about 0.05 to 3.5 g/kilogram of patient body weight, about 0.05 to
3.4 g/kilogram of patient body weight, about 0.05 to 3.3 g/kilogram
of patient body weight, about 0.05 to 3.2 g/kilogram of patient
body weight, about 0.05 to 3.1 g/kilogram of patient body weight,
about 0.05 to 3.0 g/kilogram of patient body weight, about 0.05 to
2.9 g/kilogram of patient body weight, about 0.05 to 2.8 g/kilogram
of patient body weight, about 0.05 to 2.7 g/kilogram of patient
body weight, about 0.05 to 2.6 g/kilogram of patient body weight,
about 0.05 to 2.5 g/kilogram of patient body weight, about 0.05 to
2.4 g/kilogram of patient body weight, about 0.05 to 2.3 g/kilogram
of patient body weight, about 0.05 to 2.2 g/kilogram of patient
body weight, about 0.05 to 2.1 g/kilogram of patient body weight,
about 0.05 to 2.0 g/kilogram of patient body weight, about 0.05 to
1.9 g/kilogram of patient body weight, about 0.05 to 1.8 g/kilogram
of patient body weight, about 0.05 to 1.7 g/kilogram of patient
body weight, about 0.05 to 1.6 g/kilogram of patient body weight,
about 0.05 to 1.5 g/kilogram of patient body weight, about 0.05 to
1.4 g/kilogram of patient body weight, about 0.05 to 1.3 g/kilogram
of patient body weight, about 0.05 to 1.2 g/kilogram of patient
body weight, about 0.05 to 1.1 g/kilogram of patient body weight,
about 0.05 to 1.0 g/kilogram of patient body weight. Clinicians
familiar with the diseases treated by IgG preparations can
determine the appropriate dose for a patient according to criteria
known in the art.
[0133] In other embodiments, an IVIG product can be administered to
a subject within the range of about 0.2 g/kilogram of patient body
weight to about 4 g/kilogram patient body weight each time, and the
frequency of administration may range from twice a week, once a
week, twice a month, once a month, or once every other month. One
exemplary dose range of IVIG is between about 0.1 to about 1 or
about 0.2 to about 0.8 g/kg patient body weight, typically
administered at the frequency of twice a month or once a month. For
instance, IVIG is administered to some patients at the dose of 0.2,
0.4, 0.6, or 0.8 g/kg patient body weight according to a
twice-a-month schedule. In other cases, IVIG is administered at the
dose of 0.2, 0.4, 0.6 or 0.8 g/kg patient body weight according to
a once-a-month schedule.
[0134] The duration of IVIG treatment for a demyelinating
peripheral neuropathy can vary: it may be as short as 3 or 6
months, or may be as long as 18 months, 2 years, 5 years, or 10
years. In some cases, the IVIG treatment may last the remainder of
a patient's natural life. Effectiveness of the IVIG treatment may
be assessed during the entire course of administration after a
certain time period, e.g., every 3 months or every 6 months for an
18-month treatment plan. In other cases, effectiveness may be
assessed every 9 or 12 months for a longer treatment course. The
administration schedule (dose and frequency) may be adjusted
accordingly for any subsequent administration.
[0135] For intravenous administration, the polyclonal IgG is
administered at an exemplary initial infusion rate of 0.5 mL/kg/hr
(0.8 mg/kg/min) for 30 minutes whereas the exemplary maintenance
infusion rate would be to increase the rate every 30 minutes if
tolerated up to 5 mL/kg/hr (8 mg/kg/min). Infusion times may vary
depending on the dose, rate of infusion and tolerability.
[0136] For subcutaneous administration to individuals of 40 kg
patient body weight and greater, an exemplary initial infusion rate
is 30 mL/site at 20 mL/hr/site whereas an exemplary maintenance
infusion rate is 30 mL/site at 20-30 mL/hr/site. For subcutaneous
administration to individuals of less than 40 kg patient body
weight, an exemplary initial infusion rate is 20-30 mL/site at 15
mL/hr/site whereas an exemplary maintenance infusion rate is 20
mL/site at 15-20 mL/hr/site. Infusion times may vary depending on
the dose, rate of infusion and tolerability.
[0137] In accordance with the present invention, the time needed to
complete a course of the treatment can be determined by a physician
and may range from as short as one day to more than a month. In
certain embodiments, a course of treatment can be from 1 to 6
months.
[0138] Methods for preparing parenterally administrable
compositions will be known or apparent to those skilled in the art
and are described in more detail in such publications as
Remington's Pharmaceutical Science, 15th ed., Mack Publishing
Company, Easton, Pa. (1980).
VI. Combination Therapy
[0139] In some embodiments, the polyclonal IgG can be administered
to a subject as a combination therapy with another treatment, e.g.,
another treatment for a demyelinating disorder (e.g., any of the
demyelinating disorders described herein. For example, the
combination therapy can include administering to the subject (e.g.,
a human patient) one or more additional agents that provide a
therapeutic benefit to the subject who has, or is at risk of
developing, a demyelinating disorder. In some embodiments, the
polyclonal IgG and the one or more additional agents are
administered at the same time. In other embodiments, the polyclonal
IgG is administered first in time and the one or more additional
agents are administered second in time. In some embodiments, the
one or more additional agents are administered first in time and
the polyclonal IgG is administered second in time. The polyclonal
IgG can replace or augment a previously or currently administered
therapy. For example, upon treating with polyclonal IgG,
administration of the one or more additional agents can cease or
diminish, e.g., be administered at lower levels. In other
embodiments, administration of the previous therapy is maintained.
In some embodiments, a previous therapy will be maintained until
the level of polyclonal IgG reaches a level sufficient to provide a
therapeutic effect. The two therapies can be administered in
combination.
[0140] In some embodiments, the individual receiving a first
therapy for a demyelinating disorder, e.g., Interferon Beta 1a
(Avonex), Interferon Beta 1b (Rebif), glatiramer acetate
(Copaxone), mitoxantrone (Novantrone), azathiprine (Imuran),
cyclophosphamide (Cytoxan or Neosar), cyclosporine (Sandimmune),
methotrexate, Cladribine (Leustatin), methylprednisone (Depo-Medrol
or Solu-Medrol), prednisone (Deltasone), prednisolone
(Delta-Cortef), dexamethasone (Medrol or Decadron),
adreno-corticotrophic hormone (ACTH), or Corticotropin (Acthar),
can also be administered polyclonal IgG. In some embodiments, when
the human is administered polyclonal IgG, the first therapy is
halted. In other embodiments, the human is monitored for a first
pre-selected result, e.g., an improvement in one or more symptoms
of a demyelinating disorder (such as increased remyelination),
e.g., any of the symptoms of demyelinating disorders described
herein. In some embodiments, when the first pre-selected result is
observed, treatment with polyclonal IgG is decreased or halted. In
some embodiments, the human is then monitored for a second
pre-selected result after treatment with polyclonal IgG is halted,
e.g., a worsening of a symptom of a demyelinating disorder. When
the second pre-selected result is observed, administration of the
polyclonal IgG to the human is reinstated or increased, or
administration of the first therapy is reinstated, or the human is
administered both polyclonal IgG, or an increased amount of
polyclonal IgG, and the first therapeutic regimen.
[0141] In one embodiment, a human receiving a first therapy for a
demyelinating disorder, who is then treated with polyclonal IgG,
continues to receive the first therapy at the same or a reduced
amount. In another embodiment, treatment with the first therapy
overlaps for a time with treatment with polyclonal IgG, but
treatment with the first therapy is subsequently halted.
[0142] In some embodiments of the invention, a therapeutically
effective amount of polyclonal IgG is co-administered with an
anti-inflammatory to a patient in need thereof. Anti-inflammatory
agents are a well-known class of pharmaceutical agents which reduce
inflammation by acting on body mechanisms (Stedman's Medical
Dictionary 26 e., Williams and Wilkins, (1995); Physicians Desk
Reference 51 ed, Medical Economics, (1997)).
[0143] Anti-inflammatory agents useful with the methods of the
invention include Non-steroidal Anti-Inflammatory Agents (NSAIDS).
NSAIDS typically inhibit the body's ability to synthesize
prostaglandins. Prostaglandins are a family of hormone-like
chemicals, some of which are made in response to cell injury.
Specific NSAIDS approved for administration to humans include
naproxen sodium, diclofenac, sulindac, oxaprozin, diflunisal,
aspirin, piroxicam, indomethocin, etodolac, ibuprofen, fenoprofen,
ketoprofen, mefenamic acid, nabumetone, tolmetin sodium, and
ketorolac tromethamine.
[0144] Other anti-inflammatory agents useful with the methods of
the invention include salicylates, such as, for example, salicyclic
acid, acetyl salicylic acid, choline salicylate, magnesium
salicylate, sodium salicylate, olsalazine, and salsalate.
[0145] Other anti-inflammatory agents useful with the methods of
the invention include cyclooxygenase (COX) inhibitors. COX
catalyzes the conversion of arachidonate to prostaglandin H2
(PGH2); a COX inhibitor inhibits this reaction. COX is also known
as prostaglandin H synthase, or PGH synthase. Two Cox genes, Cox-1
and Cox-2 have been isolated in several species. COX-2 is tightly
regulated in most tissues and usually only induced in abnormal
conditions, such as inflammation, rheumatic and osteo-arthritis,
kidney disease and osteoporosis. COX-1 is believed to be
constitutively expressed so as to maintain platelet and kidney
function and inter homeostasis. Typical COX inhibitors useful in
the methods of the invention include etodolac, celebrex, meloxicam,
piroxicam, nimesulide, nabumetone, and rofecoxib.
[0146] Preferred anti-inflammatory agents that can be incorporated
into a polymer matrix for administration in the methods of the
invention include: Isonixin, Amtolmetin Guacil, Proglumetacin,
Piketoprofen, Difenamizole, Epirizole, Apazone, Feprazone,
Morazone, Phenylbutazone, Pipebuzone, Propyphenazone, Ramifenazone,
Thiazolinobutazone, Aspirin, Benoiylate, Calcium Acetylsalicylate,
Etersalate, Imidazole Salicylate, Lysine Acetyisalicylate,
Morpholine Salicylate, 1-Naphthyl Salicylate, Phenyl
Acetysalicylate, Ampiroxicam, Droxicam, S-Adenosylmethionine,
Amixetine, Benzydamine, Bucolome, Difenpiramide, Emorfazone,
Guaiazulene, Nabunetone, Nimesulide, Proquazone, Superoxide
Dismutase, and Tenidap.
[0147] Anti-inflammatory agents that can be appended to a polymer
for administration in the methods of the invention include:
Etofenamate, Talniflumate Terofenamate, Acemetacin, Alclofenac,
Bufexamac, Cinmetacin, Clopirac, Felbinac, Penclozic Acid,
Fentiazac, Ibufenac, Indomethacin, Isofezolac, Isoxepac, Lonazolac,
Metiazinic Acid, Mofezolac, Oxametacine, Pirazolac, Sulindac,
Tiaramide, Tolmetin, Tropesin, Zomepirac, Bumadizon, Butibufen,
Fenbufen, Xenbucin Clidanac, Ketorolac, Tinoridine, Benoxaprofen,
Bermoprofen, Bucloxic Acid, Fenoprofen, Flunoxaprofen,
Flurbiprofen, Tbuprofen, Tbuproxam, Indoprofen, Ketoprofen,
Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Pranoprofen, Prodznic
Acid, Suprofen, Tiaprofenic Acid, Zaltoprofen, Benzpiperylon,
Mofebutazone, Oxyphenbutazone, Suxibuzone, Acetaminosalol,
Parsalmide, Phenyl Salicylate, Salacetamide, Salicylsulfuric Acid,
Isoxican, Lomoxicam, Piroxicam, Tenoxicam,
.epsilon.-Acetamidocaproic Acid, Bendazac, .alpha.-Bisabolol,
Paranyline, Perisoxal, and Zileuton.
[0148] Anti-inflammatory agents that can be incorporated into a
polymer backbone for administration in the methods of the invention
include: Enfenamic Acid, Aceclofenac, Glucametacin, Alminoprofen,
Caiprofen, Xinoprofen, Salsalate, 3-Amino-4-hydroxybutyric Acid,
Ditazol, Fepradinol, and Oxaceprol.
[0149] Anti-inflammatory agents that possess suitable ortho
functionality to be incorporated into the backbone of a polymer of
formula (I) as described herein include: Flufenamic Acid,
Meclofenamic Acid, Mefenamic Acid, Niflumic Acid, Tolfenamic Acid,
Amfenac, Bromfenac, Diclofenac Sodium, Etodolac, Bromosaligenin,
Diflunisal, Fendosal, Getitisic Acid, Glycol Salicylate, Salicilic
Acid, Mesalamine, Olsalazine, Salicylamide 0-Acetic Acid,
Sulfasalazine,
[0150] For any anti-inflammatory agent referred to herein by a
trade name it is to be understood that either the trade name
product or the active ingredient possessing anti-inflammatory
activity from the product can be used. Additionally, preferred
agents identified herein for incorporation into a polymer backbone
can also preferably be appended to a polymer or can be incorporated
into a polymer matrix. Preferred agents that can be appended to a
polymer can also preferably be incorporated into a polymer
matrix.
EXAMPLES
[0151] Examples are provided below to illustrate the present
invention. These examples are not meant to constrain the present
invention to any particular application or theory of operation.
Example 1: Investigation of Ivig Effect on Schwann Cells
[0152] The direct effect of human serum-derived polyclonal
immunoglobulins on Schwann cell homeostasis, differentiation, and
maturation as demonstrated through various molecular and cellular
variables was investigated using three models: 1) a primary rat
Schwann cell culture model; 2) a p57kip2 suppressed Schwann cell
model; and 3) a co-culture of PNS neurons and myelinating Schwann
cells.
[0153] 1.1. Preparation of the Rat Schwann Cell Model 1:
[0154] In this model, naive primary Schwann cells (SCs) isolated
from the sciatic nerves of newborn rats were cultured. At this
stage, SCs are immature and have not yet initiated differentiation
processes. In culture, they do not progress along their
differentiation program and remain proliferative but immature, most
likely due to the presence of intrinsic differentiation inhibitors
(Heinen et al., 2008a).
[0155] 1.2. Preparation of p57kip2 Suppressed Schwann Cell Model
2:
[0156] The present inventors have identified the p57kip2 gene as a
novel intrinsic inhibitor of myelinating glial cell
differentiation, maturation and myelination. It has been
demonstrated that long-term shRNA dependent suppression of the
p57kip2 gene uncouples primary SC differentiation from axonal
contact. This was revealed by cell cycle exit, altered SC
morphology as well as induced myelin expression (Kury et al., 2002;
Heinen et al., 2008a; Heinen et al., 2008b). In this second model,
p57kip2 suppressed SC was used for comparison with control
transfected cells, i.e. non-differentiating cells. This culture
system provides the unique opportunity to observe SC
differentiation and maturation in vitro in the absence of axons in
a quantitative way.
[0157] 1.3. Preparation of a Co-Culture of PNS Neurons and
Myelinating Schwann Cells--Model 3:
[0158] In this model, myelinating neuron/SC co-cultures were
generated. Culture preparations were made from embryonic Wistar rat
or C57/BL6 mouse dorsal root ganglia containing both immature
sensory neurons and Schwann cell precursors of the PNS. This
co-culture simulates the in vivo situation and offers the
possibility of studying the final wrapping/myelination process and
whether this complex interaction can be influenced by
immunoglobulin administration. Optimization of the co-culture
conditions and preparations was done according to established
protocols used in the inventors' laboratory or the protocol
published from Paivalainen et al., (2008) with some modifications.
IVIG stimulation was performed in parallel to initiation of the
myelination process with dialysed IGIV/buffer preparations.
IGIV/buffer dialysis was performed against cell culture medium
without supplements. All experiments were performed with one
concentration of IGIV: 20 mg/ml. The duration of stimulation was
determined by analyzing the myelination kinetics (internode
formation) after 3 and 6 days following addition of dialysed
IGIV/buffer.
[0159] 1.4. Cell Morphology:
[0160] Cell morphology was investigated in model 1 (rat SCs in
culture) and model 2 (p57kip2-suppressed SCs) for up to 9 days with
stimulation by 10 mg/ml and 20 mg/ml of IVIG for model 1 (to
observe dose dependency) and up to 7 days stimulation (9 days
transfection) for model 2. Experiments were performed with both
non-dialysed and dialysed IGIV and buffer preparations. IVIG and
buffer dialysis was performed against cell culture medium without
supplements. All model 2 experiments were performed with one
concentration of dialysed IGIV (20 mg/ml). In model 2, the cell
growth and differentiation kinetics was also determined by
measuring the cell protrusion length after 3 and 7 days of
stimulation with dialysed IVIG.
[0161] 1.5. Cell Death/Proliferation:
[0162] Cell death/proliferation was investigated in model 1 after 2
days stimulation with non-dialyzed and dialyzed IVIG/buffer
preparations. IVIG/buffer dialysis was performed against cell
culture medium without supplements. All experiments were performed
with one concentration of IVIG (20 mg/ml). Two assays for measuring
cell proliferation were employed: immunocytochemical staining
against the Ki-67 antigen and imunocytochemical staining against
BrdU. Ki-67 antigen is a nuclear protein which serves as a cellular
marker for proliferation. BrdU (bromodeoxyuridine) is a nucleotide
analogue of thymidine used for labeling of proliferating cells.
Immunocytochemical staining against caspase-3 was employed as an
apoptosis marker. Caspase-3 is a protease activated in apoptotic
cells and therefore used as a cell death marker. Cells were fixed
after two different BrdU-pulse durations of 8 h and 24 h.
[0163] 1.6. Gene Expression:
[0164] Gene expression was analyzed in model 1 (rat SCs in
culture--section 1.1) and model 2 (p57kip2-suppressed SCs--section
1.2) exposed for up to 9 days stimulation for model 1 and 7 days
stimulation (9 days transfection) for model 2 using both
non-dialyzed and dialyzed IVIG/buffer preparations. Dialysed
SYNAGIS preparations were used as a IgG1 control on naive SCs
(model 1). IVIG/buffer/SYNAGIS dialysis was performed against cell
culture medium without supplements. All experiments were performed
with one concentration of IVIG: 20 mg/ml. Transcription of myelin
genes (P.sub.0, MBP) and Fc receptors (CD64, CD32 and CD16) were
measured using real-time RT-PCR.
Example 2: Schwann Cell Responds to Incubation with IVIG
[0165] 2.1. Morphology:
[0166] IVIG treatment was observed to affect Schwann cell
morphology. SCs cultured in the presence of 10 mg/ml IVIG, and to a
larger extent, in the presence of 20 mg/ml IVIG, appeared to have
larger somata and nuclei. It is currently unclear whether this is a
direct impact on SC shape and cytoskeleton or adhesion properties,
a result from different cell densities, or is reflective of
discrete cell surface alterations possibly connected to the IVIG
binding site(s) on the cell surface.
[0167] Significantly accelerated growth of cellular protrusions was
measured upon stimulation with IGIV using model 2 (p57kip2
suppression). This effect was observed only in the early stages of
the differentiation process, indicating an IVIG effect on the
differentiation kinetics of the Schwann cells. To explain, the
growth of cellular protrusions is a maturation parameter which was
found to be dependent on suppressed p57kip2 levels. On the other
hand, no effect on actin filament assembly and structure could be
observed after IVIG stimulation as revealed by TRITC conjugated
phalloidin stainings.
[0168] 2.2. Cell Death/Proliferation (Model 1):
[0169] After stimulation with non-dialyzed IVIG (20 mg/ml)
preparations, the proliferation rate of naive SC was significantly
reduced, as revealed by assays using proliferation markers BrdU and
Ki-67. See FIGS. 1-2. The IVIG-dependent effect on the
proliferation rate was diminished with IVIG dialysis, but remained
statistically significant thereafter. There is currently no
evidence of induction of apoptosis after treatment with IVIG based
on the negative staining for caspase-3.
[0170] 2.3. Gene Expression:
[0171] Stimulation of non-transfected SCs (model 1) with
non-dialyzed and dialyzed IVIG/buffer preparations led to slight
upregulation of P.sub.0 and strong upregulation of the MBP genes
within the first 3 days of treatment, but not after longer
incubation periods. Stimulation of p57kip2 suppressed cells (model
2) with non-dialyzed and dialyzed IVIG/buffer preparations also led
to similar results regarding myelin gene expression. The expression
and upregulation of both myelin genes were significantly stronger
in the p57kip2-suppressed cells than in the control transfected
cells. Observations of the gene regulation of Fc receptors showed
that Schwann cells express CD64 and CD32 and that long term
suppression for p57kip2 leads to significant upregulation of these
genes. There was a detectable level of CD64 Fc receptor expression
in immature SCs. In differentiating Schwann cells (upon suppression
of the intrinsic inhibitor p57kip2), CD64 levels were significantly
increased with IVIG stimulation.
[0172] Importantly, the monoclonal IgG1 controls (Synagis, Avastin
and Herceptin) showed no significant effect on myelin gene
expression. Stimulation of p57kip2 suppressed cells (model 2) with
non-dialysed and dialysed IVIG/buffer preparations induced myelin
gene expression to a similar extent. Again MBP expression was
strongly induced upon IVIG stimulation whereas P0 expression was
mildly induced by the treatment. Note that myelin gene induction
could be observed during a period of seven days of stimulation and
was therefore not limited to early phases. Furthermore, the
expression of the p57kip2 gene was found to encode an intrinsic
inhibitor of Schwann cell differentiation and was significantly
lowered in control transfected (non-differentiating) cells.
[0173] Observations of the gene regulation of all known Fc.gamma.
receptors showed that Schwann cells express the CD64 Fc receptor.
In differentiating Schwann cells (model 2), CD64 levels were
significantly increased in comparison to control transfected
(non-differentiating) cells. Regulation of the CD64 receptor
expression in response to IVIG stimulation could not be observed.
Of note, effects of the non-dialysed buffer control were observed
in all the gene expression experiments performed. This effect was,
however, diminished after dialysis. Further gene expression
analyses were therefore performed with dialysed IVIG preparations
only.
[0174] 2.4. Summary of Findings:
[0175] In the first 18 months of the investigation, it was
discovered that primary SCs respond to IVIG incubation with altered
cell morphology accompanied by an accelerated growth of cellular
protrusions in early stages of the differentiation process.
Incubation with IVIG was also found to reduce Schwann cell
proliferation without affecting cell survival. Furthermore,
expression of two major myelin genes, P.sub.0 and MBP, was induced
in immature as well as differentiating SCs following stimulation
with IVIG. Data shows that primary rat Schwann cells were express
the CD64 Fc receptor and that in differentiating Schwann cells
(upon suppression of the intrinsic inhibitor p57kip2), CD64 levels
were significantly increased with exposure to IVIG. The evidence
also provides strong indications for an upregulation of Fc
receptors (in particular CD64) in differentiating SCs. Furthermore,
a specific binding of the human IVIGs on the Schwann cell surface
was shown.
[0176] These findings support the hypothesis that SCs might exhibit
immune competence. Reduced proliferating rate with no signs of
apoptosis as well as the induction of myelin genes, combined with
accelerated growth of cellular protrusions, suggest a promotion of
the differentiation process in the immature SC by IVIG. These are
the first in vitro results demonstrating that Schwann cells are not
only able to respond to but also to specifically bind
immunoglobulins and that IVIG stimulation can promote Schwann cell
precursor maturation.
Example 3: Gene Expression
[0177] For further examination of the IVIG dependent effects on
differentiating (p57kip2 suppressed cells, model 2) and
non-differentiating (control suppressed cells, model 2) Schwann
cell gene expression we collected 16 RNA samples from 4 independent
experiments for a GeneChip Array analysis (performed by Miltenyi
Biotec, Germany). Sample validation was performed by determination
of expression levels of MBP, P.sub.0, p57kip2 and CD64 genes.
[0178] Statistical and functional analysis was performed. Genes
that were identified as significantly up- or down-regulated upon
treatment with IVIG are provided in Tables 1 and 2. Future aims are
at further gene identification as well as validation of the
obtained results.
TABLE-US-00001 TABLE 1 Comparison of non-differentiating Schwann
cells +/- IVIG Up regulated genes after Down regulated genes after
treatment (gene sequence name) treatment (gene sequence name) Tyrp1
RGD1562551 Tyrp1 Ctnna2 Col24a1 Olr832 Fat3 Phgr1 Tmem72 RGD1566220
Tesc Nedd9 lI18 Slc12a3 Mt1a Arhgef9 Slc40a1 Gckr Asgr1 TC636329
LOC678704 A_64_P023581 TC609365 Ptprr Bcl6b Olr749 A_64_P063062
Nebl Npas2 RGD1562545 Gpx2 Hes5 Matn1 Mpzl2 A_64_P022503 Ezr Fbxo32
Cryab Pls1 Fcgr2b A_64_P094596 A_64_P025678 Olig1 Sox2 Plp1
TABLE-US-00002 TABLE 2 Comparison of differentiating Schwann cells
+/- IVIG Up regulated genes after Down regulated genes after
treatment (gene sequence name) treatment (gene sequence name)
ENSRNOT00000064975 XM_346212 Zfp334 XR _009266 Mmp25 LOC688695
A_64_P117674 Ak3l1 A_64_P151655 A_64_P163956 A_44_P386999 Olig1
Sox10 Hes5
[0179] In order to confirm the observed induction of myelin gene
expression (in particular P.sub.0 and MBP) at protein level, we
performed Western-blot analysis on p57kip2 suppressed versus
control suppressed cells (model 2) after treatment with dialysed
IVIG/buffer. We could demonstrate that in differentiating Schwann
cells protein levels of P.sub.0 and to a lesser extent of MBP were
increased after IVIG treatment.
Example 4: Immune-Related Proteins
[0180] It was important to confirm direct IVIG binding to the
Schwann cell surface. Applying an anti-human Fab-specific F(ab)'2
and anti-human Fc.gamma.-specific F(ab)'2 antibodies, it was shown
that human immunoglobulins in the IVIG specifically bound to the
Schwann cell surface. Live Schwann cells in culture were stimulated
with IVIG, washed, fixed and then separately stained against human
Fab fragments, human Fc.gamma. fragments or against both epitopes
in combination of a double-staining. A specific surface binding
could be localized within the perinuclear region of the cells.
These binding studies were performed with naive Schwann cells
(model 1) using IVIG and IgG1 controls (Avastin and Herceptin) as
well as with differentiating Schwann cells (model 2) using IVIG. In
order to address the question of whether CD64 receptor protein is
also expressed on the Schwann cell surface, staining experiments
with two anti-CD64 antibodies have been initiated.
[0181] In order to determine whether CD64 receptor protein was also
expressed on the Schwann cell surface staining experiments with two
anti-CD64 antibodies were performed. One anti-CD64 antibody
appeared to bind specifically to the rat CD64 receptor on the
Schwann cells and diffuse receptor staining was distributed over
the cell surface of the non-differentiating cells. In comparison,
the receptor staining on differentiating cells was concentrated to
the cell soma above the perinuclear region. The detected CD64
signals did not coincide with the IVIG binding signals (comparison
of immunological stainings).
Example 5: Internode Formation
[0182] In order to improve efficiency and reproducibility of the in
vitro myelination model (model 3), a number of experimental
improvement steps using DRG cultures derived C57/BL6 mouse embryos
were performed and established. To this end, the protocol according
to Paivalainen et al. (2008) was modified and can now be used to
study the effects of IVIG application on axon/Schwann cell
interactions. IVIG stimulation (20 mg/ml) was performed concomitant
to the initiation of the myelination process using dialysed
IGIV/buffer preparations.
[0183] After determination of the optimal time point for the
analysis at 7 days upon initiation of myelinisation, a
statistically significant number of IVIG stimulation experiments
(n=9) were performed. In order to evaluate the ability of
immunoglobulin treatment to modulate the generation of myelin
sheaths (internode formation), the number of internodes
(normalizing to the whole number of nuclei in the co-culture) of
IVIG treated were compared to the number of internodes in control
co-cultures. Although a trend towards slightly increased internode
densities could be observed, no statistically significant
difference in myelin segment formation was detected after
treatment.
Example 6: In Vivo Nerve Repair Paradigm
[0184] 6.1. Summary
[0185] In order to translate in vitro findings based on primary rat
Schwann cell cultures to an in vivo paradigm, chronic peripheral
nerve lesions were induced in adult rats treated with IVIG or
control buffer during a so called "nerve regeneration period".
Sciatic nerves were transected and, by means of suturing religation
of nerve ends, nerve regeneration was prevented for a period of
three months. After this degeneration period, nerves were ligated
to allow regeneration to take place and IVIG or buffer was
administered (i.p. injections). Nerves were allowed to regenerate
for another three months until the animals were sacrificed.
[0186] The above-described surgical approach on Schwann cells was
used to determine whether IVIG stimulation can repair the activity
of injured peripheral nerves. During the three months regeneration
(and IVIG/buffer treatment) period a number of functional tests
were performed on live rats. Afterwards animals were sacrificed and
sciatic nerves were dissected, fixed and embedded for morphological
and immunohistochemical future analyses aiming at the description
of Schwann cell/myelin and axonal reactions. Preliminary results
were acquired from the functional analyses. These preliminary
findings indicate that differences between the two groups (IVIG vs.
buffer treated animals) exist. Specifically, IVIG treated animals
displayed longer and broader footprint areas (contact zones between
foot and floor) as compared to buffer treated animals. These
footprint areas also gradually increased during the treatment
period and this was accompanied with an increased landing pressure
(corresponding to the force that is used by the leg to make a step
or to the pressure the foot exerts to the surface). Overall these
first preliminary data suggest that IVIG treated animals experience
an accelerated normalization of walking behavior and an increased
strength in their leg usage.
[0187] 6.2. Methods
[0188] IVIG dependent effects on Schwann cell survival were
investigated. Specifically, a previously established chronic
peripheral nerve denervation model (Fu and Gordon; J Neurosci 1995)
was used to study the proliferation as well as remyelination and
axonal regeneration in denervated nerve segments in vivo. This in
vivo model features similar nerve conditions to those observed in
many human nerve pathologies. This in vivo model also provides the
advantage of focusing on regenerative events only as degeneration
processes (i.e., immune reactions are temporally excluded).
[0189] For this purpose sciatic nerves of 24 adult Lewis rats were
transected and nerve regeneration was prevented by means of
surturing religation of the nerve ends. This setup results in
chronically injured and denervated nerve segments. Regeneration was
prevented for the period of three months after nerve transaction.
During this period, no functional tests were performed with the
animals.
[0190] After three months of degeneration all 24 rats were exposed
to a delayed sciatic nerve ligation (anastomosis) in that proximal
nerve segments were sutured to the distal nerve segments thereby
allowing nerve regeneration to take place. Note that in this
chronic setup, the overall regeneration capacity was significantly
reduced as compared to acute nerve lesions. During this first three
months period axonal and myelin degeneration process were
completed.
[0191] In a first set of experiments (study 1), the generation of
anti-drug antibodies (ADA) and human IgG plasma levels after IVIG
application was studied in healthy rats (unlesioned nerves) using
ELISA tests. ADA against IVIGs was then monitored in lesioned and
treated animals as secondary readout in study 2 (see below).
[0192] In a second set of experiments (study 2), Lewis Rats with
chronic peripheral nerve lesions was treated with 1 g IVIG/kg body
weight (high-dose treatment) following nerve ligation (regeneration
period of 3 month). IVIG application was done by means of i.p.
injections once every week in the first month and then once every
second week in the last two months of the regeneration phase.
Control rats with nerve lesions received IVIG formulation buffer
injections. Control buffer treated and IVIG treated animal groups
comprised of 12 adult female rats each. During the period of IVIG
treatment, blood samples were collected from the tail vein in order
to monitor ADA and to determine the half-life of human IgG (see
study 1). Blood plasma samples were collected every second week
prior to treatment.
[0193] 6.2. Results
[0194] In order to test the degree of recovery of function of the
target organs after religation of the nerve ends, a weekly set of
functional evaluation tests were conducted. Sensory function was
evaluated by testing the withdrawal response of toe 4 and 5 after
application of a pain stimulus (pinch test with a forceps). Muscle
strength and regeneration of muscle fibers were analyzed using the
leg spread test. These two functional tests as well as monitoring
of the animals' weight (health and wellbeing parameter) were done
on a weekly basis. The animals were further subjected to weekly to
monitoring of footprints and walking tracks (i.e., the "cat walk
analysis") to evaluate functional recovery of the sciatic
nerves.
[0195] At the end of the study, 21 animals were left: 10 animals
that received buffer control injections and 11 animals treated with
IVIG. All rats were sacrificed and the regenerating peripheral
nerve segments, as well as contralateral healthy control nerves
were collected for further analysis. For this purpose animals were
divided in three groups:
[0196] Group I consists of 4 buffer treated and 4 IVIG treated
animals. Sciatic nerves segments (healthy and transected) of these
animals will be processed for electron microscopy analysis (EM).
Apart from determining axonal density (thus measuring regeneration
efficiency) this will also include a g-ratio calculation (axonal
diameter divided by the diameter of the axon and its myelin sheath)
in order to determine remyelination efficiencies. This analysis is
currently ongoing. Functional evaluation data of these animals (cat
walk data, pinch-test and leg spread behavior) were determined and
preliminary results are described below.
[0197] Group II consists of 3 buffer treated and 4 IVIG treated
animals. Sciatic nerves segments (healthy and transected) of these
animals will be used for immunohistochemical stainings (IHC)
against axonal, myelin and glial markers in order to determine the
degree of cellular redifferentiation and regeneration. Nerves are
currently processed and this study is also ongoing. Functional
evaluation data of these animals (cat walk data, pinch-test and leg
spread behavior) were determined and preliminary results are
available are described below.
[0198] Group III consists of 3 buffer treated and 3 IVIG treated
animals. The transected sciatic nerves segments of these animals
displayed no anatomical regeneration signs since the anastomosis
did not take place. The functional evaluation data of these animals
will not be included in the overall analysis.
[0199] A preliminary evaluation of the cat walk data indicates that
differences between the two groups (IVIG vs. buffer treated
animals) exist. IVIG treated animals displayed longer and broader
footprint areas (contact zones between foot and floor) as compared
to buffer treated animals. These footprint areas also gradually
increased during the treatment period and this was accompanied with
an increased landing pressure (corresponding to the force that is
used by the leg to make a step or to the pressure the foot exerts
to the surface). Overall this data suggest that IVIG treated
animals experience an accelerated normalization of walking behavior
and an increased strength in their leg usage.
Example 7: Supplemental Studies to Determine the Underyling
Mechanisms of Ivig Action
[0200] To better understand the underlying mechanisms of IVIG
action and mechanisms by which IVIGs promote cellular maturation,
detailed molecular/cellular investigations on stimulated Schwann
cells will be performed.
[0201] As outlined above (see 5.1), a GeneChip analysis on
non-differentiating and differentiating Schwann cells exposed to
IVIG treatment was performed and analyzed. Based upon the newly
discovered unregulated and downregulated genes (Tables 1 and 2),
further validation experiments will be conducted using quantitative
real-time RT-PCR on selected genes. If necessary and applicable,
additional validations using antibodies (Western-blot,
immunological stainings as well as ELISA) will be performed. This
will be particularly interesting for genes related to immune
competence. Of note, this expression analysis will not only be
analyzed in order to understand what cellular processes are most
IVIG sensitive, it will most likely also serve to define additional
marker genes that can be used to monitor and quantify IVIG
dependent reactions.
[0202] Following establishment of a suitable in vitro myelination
assay (model 3), a statistically significant number of IVIG
stimulation experiments will be performed. The active time windows
and to which extent immunoglobulin treatment can modulate the
generation of myelin sheaths (internode formation) will be
evaluated.
[0203] Using a Cy3 conjugated anti-human Fab antibody, the specific
binding of IVIGs to Schwann cell surfaces can be demonstrated. It
remains to be shown whether this is due to interaction with the
CD64 Fc receptor or whether Schwann cell-specific epitopes are
recognized by Fab-mediated binding. For this purpose, Schwann cells
(model 1) will either be contacted with Fc and F(ab)2 fractions of
papain-digested IVIG or bound IVIGs on Schwann cells will be
digested with papain in situ. Furthermore, the application of a
FITC-conjugated anti-human Fc antibody in combination with Cy3
conjugated anti-human Fab antibody is expected to result in papain
sensitive stainings. Two anti-CD64 antibodies will be applied on
non-differentiating and differentiating (model 2) Schwann cells in
order to determine whether CD64 is also expressed as a receptor
protein on the Schwann cell surface. In case that the IVIG binding
is really mediated via this Fc receptor, it will be expected that
the CD64 signals coincide with the IVIG binding (immunological
stainings). Further to this end, it will be examined whether an
increase in CD64 protein levels can be observed as a consequence of
the differentiation process (Western-blot).
[0204] To provide functional proof for Fc-receptor involvement,
pharmacological inhibitors such as
3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfon-
amide or Ly294002 interfering with spleen tyrosine kinase (Syk) and
phosphatidylinositol-3-kinase (PI3K) will be applied, respectively,
prior to IVIG stimulation of naive Schwann cells (model 1). This
will indicate whether these Fc-dependent signaling components are
involved in MBP induction (or appropriate marker genes identified
in 1.). Furthermore, digested IVIGs will be used to stimulate
cultured Schwann cells (model 1) in order to reveal whether Fc
or/and Fab fractions are responsible for IVIG specific gene
regulations (MBP and other marker genes identified in the gene
expression analysis). Finally, shRNA-mediated suppression of CD64
expression in Schwann cells (model 1) can be used to confirm that
IVIG binding is CD64 dependent as well as responsible for the IVIG
dependent induction of MBP expression (or other marker genes
identified in the gene expression analysis).
[0205] Standard Schwann cell culture (maintenance and
differentiation) conditions feature high fetal calf serum
concentrations (up to 10% of volume). It is therefore conceivable
that immunoglobulins present in the serum are diminishing
IVIG-dependent Schwann cell reactions. To test this, the serum
concentration will be reduced to the lower limit needed in order to
assure cell survival and differentiation, the Schwann cells
stimulated with IVIGs and MBP expression levels (models 1 and 2) as
well as morphological parameters measured (model 2).
[0206] The present inventors' recent investigations revealed that
Schwann cell differentiation is critically dependent of the histone
methyltransferase enhancer of zeste homolog 2 (EZH2; Heinen et al.,
in revision). Upon suppression of EZH2 activity, cultured Schwann
cells show dedifferentiation reactions similar to what is observed
in nerve pathologies. As part of future investigations, such
dedifferentiating Schwann cells will be stimulated with IVIGs to
determine expression of Schwann cell marker and myelin genes. The
latter of which were shown to be downregulated below control
levels. It will be of interest to see whether immunoglobulin
treatment is not only able to promote differentiation/maturation
reactions (as seen with model 2; i.e. upon suppression of the
inhibitory gene p57kip2) but can also interfere with
dedifferentiation processes (such as normalization of myelin gene
expression levels).
[0207] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
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