U.S. patent application number 14/628050 was filed with the patent office on 2015-08-20 for neurotherapeutic nanoparticle compositions and devices.
The applicant listed for this patent is SUSAN MARIE METCALFE, YALE UNIVERSITY. Invention is credited to Tarek Fahmy, Susan Marie Metcalfe.
Application Number | 20150231266 14/628050 |
Document ID | / |
Family ID | 50150386 |
Filed Date | 2015-08-20 |
United States Patent
Application |
20150231266 |
Kind Code |
A1 |
Metcalfe; Susan Marie ; et
al. |
August 20, 2015 |
Neurotherapeutic Nanoparticle Compositions and Devices
Abstract
There are provided compositions and methods for treatment of
neurodegeneative diseases and CNS injury. The compositions a
pharmaceutically acceptable carrier solution; and a plurality of
biodegradable nanoparticles, wherein the nanoparticles comprise a
targeting moiety that is able to bind selectively to the surface of
a neural stem cell and wherein the nanoparticles further comprise
factors such as leukaemia inhibitory factor (LIF); XAV939 and/or
one or more of : brain-derived neurotrophic factor (BDNF) or an
agonist thereof; epidermal growth factor (EGF) or an agonist
thereof; glial cell-derived neurotrophic factor (GDNF) or an
agonist thereof; retinoic acid and derivatives thereof; ciliary
neurotrophic factor (CTNF) or an agonist thereof; and Wnt5A. The
biodegradable nanoparticles may deliver via controlled time
release.
Inventors: |
Metcalfe; Susan Marie;
(Cambridge, GB) ; Fahmy; Tarek; (New Haven,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
METCALFE; SUSAN MARIE
YALE UNIVERSITY |
Cambridge
New Haven |
CT |
GB
US |
|
|
Family ID: |
50150386 |
Appl. No.: |
14/628050 |
Filed: |
February 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2013/056246 |
Aug 22, 2013 |
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14628050 |
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61707723 |
Sep 28, 2012 |
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61692519 |
Aug 23, 2012 |
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Current U.S.
Class: |
424/451 ;
424/85.2 |
Current CPC
Class: |
A61K 38/185 20130101;
A61K 47/42 20130101; A61K 47/6907 20170801; A61K 9/4866 20130101;
A61K 38/1808 20130101; C07K 16/2863 20130101; B82Y 5/00 20130101;
A61P 25/28 20180101; A61K 38/2093 20130101; A61K 38/204 20130101;
A61P 25/16 20180101; A61K 38/185 20130101; A61K 2300/00 20130101;
A61K 38/1808 20130101; A61K 2300/00 20130101; A61K 38/2093
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 47/42 20060101
A61K047/42; A61K 9/48 20060101 A61K009/48; A61K 38/20 20060101
A61K038/20 |
Claims
1. A composition for the treatment of neurodegenerative disease
comprising: a) a pharmaceutically acceptable carrier solution; and
b) a plurality of biodegradable nanoparticles, the nanoparticles
being comprised of a biodegradable polymer matrix that encapsulates
a cargo, the nanoparticles further comprising a targeting moiety
located on the surface of the nanoparticle, the targeting moiety
selected from a monoclonal antibody and an antigen-binding fragment
thereof, the targeting moiety being able to bind selectively to an
antigen on the surface of a neural cell selected from the group
consisting of: a neural stem cell; a neural progenitor cell; a
precursor cell that is commited to a neurectodermal lineage; and a
neuronal cell; and wherein the cargo comprises leukaemia inhibitory
factor (LIF).
2. The composition of claim 1, wherein the polymer comprises
poly(lactic)-co-glycolic acid (PLGA) and/or PLA.
3. The composition of claim 1, wherein the targeting moiety binds
specifically to a Thy-1 antigen present on the surface of the
neural stem cell.
4. The composition of claim 1, wherein the targeting moiety binds
specifically to a NCAM antigen present on the surface of the neural
stem cell.
5. The composition of claim 1, wherein the targeting moiety binds
specifically to a glial cell line derived neurotrophic factor
receptor .alpha.1(GDNFR-.alpha.1) present on the surface of the
neural stem cell.
6. The composition of claim 1, wherein the nanoparticles further
comprise one or more of the following therapeutic compounds:
brain-derived neurotrophic factor (BDNF) or an agonist thereof;
epidermal growth factor (EGF) or an agonist thereof; glial
cell-derived neurotrophic factor (GDNF) or an agonist thereof;
retinoic acid and derivatives thereof; ciliary neurotrophic factor
(CTNF) or an agonist thereof; and Wnt5A.
7. The composition of claim 1, wherein the nanoparticles have a
diameter of at least about 50 nm and at most about 300 nm.
8. The composition of claim 1, wherein the nanoparticles are
capable of degrading of a period of time in order to effect timed
release of the encapsulated LIF.
9. The composition of claim 8, wherein the period of time is
selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12 days; 1, 2, 3, 4, 5 or 6 weeks; and up to six months.
10. A method for treating a patient suffering from a
neurodegenerative disease (NDD) or CNS damage comprising
administering to the subject a pharmaceutical composition of claim
1.
11. A nanoparticle for use in the treatment of neurodegenerative
disease and/or CNS damage comprising: a biodegradable carrier
material, a therapeutic compound, and a targeting moiety; wherein
the carrier material is configured so to encapsulate the
therapeutic compound and wherein the carrier material further
defines a surface, upon and within which surface is located the
targeting moiety, the nanoparticle further characterised in that
the therapeutic compound comprises LIF and the surface located
targeting moiety comprises an antibody, or an antigen binding
fragment of an antibody, that specifically binds to an antigen
present on the cell surface of a stem cell having the capacity to
act as a neural precursor cell.
12. The nanoparticle of claim 11, wherein the biodegradable carrier
material degrades at a rate that allows for controlled release of
the LIF over a pre-determined period of time.
13. The nanoparticle of claim 11, wherein the targeting moiety
binds specifically to a Thy-1 antigen present on the surface of the
stem cell.
14. The nanoparticle of claim 11, wherein the targeting moiety
binds specifically to a NCAM antigen present on the surface of the
cell.
15. The nanoparticle of claim 11, wherein the targeting moiety
binds specifically to a GDNFR-.alpha.1 present on the surface of
the cell.
16. The nanoparticle of claim 11, wherein the nanoparticle has a
diameter of at least about 50 nm and at most about 300 nm.
17. The nanoparticle of claim 16, wherein the nanoparticle has a
diameter of at least about 100 nm and at most about 200 nm.
18. The nanoparticle of claim 11, wherein the neurodegenerative
disease is selected from the group consisting of: Alzheimer's
Disease (AD), Multiple Sclerosis (MS); Parkinson's Disease (PD);
Huntington's Disease (HD); Frontotemporal dementia (FTD); and
Amyotrophic Lateral Sclerosis (ALS).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US2013/056246, which designated the United
States and was filed on Aug. 22, 2013, published in English, which
claims the benefit of U.S. Provisional Application No. 61/692,519,
filed on Aug. 23, 2012 and U.S. Provisional Application No.
61/707,723, filed on Sep. 28, 2012. The entire teachings of the
above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention is in the field of compositions for
neuroprotection, particularly compositions that promote and protect
neural cells in the central nervous system of a mammal such as a
human. Also described are methods for repairing tissues of the
central nervous system of a mammal such as a human.
Neurodegenerative diseases represent the largest area of unmet
clinical need in the Western world. They are characterised by a
progressive loss of the structure or function of neurons in the
nervous system (neurodegeneration) and include Alzheimer's Disease
(AD), Parkinson's Disease (PD) and a host of other rarer conditions
such as Huntington's Disease (HD), Frontotemporal dementia (FTD)
and Amyotrophic Lateral Sclerosis (ALS). The process of
neurodegeneration is not well understood and so the diseases that
stem from it have no effective cures, nor is it possible to slow
down their progression, as yet.
[0003] Chronic neurodegenerative disorders (NDD) of the central
nervous system, which target the aging brain, are set to increase
as the population ages and finding ways to better understand and
treat these conditions is a major challenge given the personal and
economic costs of these conditions. These disorders are defined by
the loss of specific populations of neurons with a characteristic
pathological pattern of protein aggregation--for example in the
case of PD the loss of the nigrostriatal dopaminergic pathway and
the presence of alpha synuclein-containing Lewy bodies.
[0004] While this is a useful starting point by which to define
these diseases, it is though important to realise that these
chronic neurodegenerative disorders: [0005] (i) have a much greater
extent of pathological burden than was once recognised and as such
these diseases target a whole range of different neuronal
populations, rather than just one neuronal network; [0006] (ii)
have pathology that is not confined to the neurons but involves
glial cells and an inflammatory element; [0007] (iii) often display
mixed profiles of pathology typically with a significant vascular
disease burden in the brain of some of those conditions that affect
the more elderly; [0008] (iv) are heterogeneous with a complex
aetiology.
[0009] Taking Parkinson's Disease (PD) as an example, this is a
degenerative disorder of the central nervous system (CNS) that
currently affects approximately 1% of people over 65 years of age
and is likely to become more common as the population ages and
lives longer. It is characterised clinically by the development of
bradykinesia, rigidity and a resting tremor, which has been
attributed in part to the progressive degeneration of the
dopaminergic input from the substantia nigra to the striatum of the
brain. It is increasingly being understood that PD is a disorder
which has widespread pathology from its onset and that, therefore,
the nigral pathology is only part of a much more diffuse
pathological process. However, the core loss of the dopaminergic
nigrostriatal pathway is not disputed.
[0010] The progressive loss of dopamine can be treated with a range
of symptomatic dopaminergic drug therapies, particularly in the
early stages of the disease. However, as symptoms progress with
time and coupled to the long-term use of dopaminergic drug
therapies, a range of problems arise including the development of
drug-induced motor complications such as "on-off" fluctuations and
levodopa-induced dyskinesias (LID). At this stage of the disease,
drug therapies become increasingly disappointing in terms of a
reliable therapeutic benefit. Therefore, other therapeutic
approaches are used including more invasive ways of delivering more
continuous dopaminergic therapy, such as apomorphine pumps and
DuoDopa.RTM. (constant delivery of L-Dopa into the small bowel), as
well as neurosurgical interventions such as deep brain stimulation,
especially of the subthalamic nucleus.
[0011] These latter therapies can be effective, but only ever treat
the symptoms without any attempt to repair the underlying and
progressive disease. Thus, these treatments also start to fail, in
part because of this progressive nature of the non-nigral,
non-motor aspects of Parkinson's Disease and in part because of the
continued loss of nigral dopaminergic neurones. Therefore, whilst a
better understanding of disease pathogenesis may enable better
treatment of all aspects of PD, more restorative approaches to
repairing the dopaminergic nigrostriatal tract, including cell
replacement, neurotrophic support and pharmacological and gene
therapies, may also prove very useful.
[0012] Thus, NDD are characterised by a slow insidious progression
with increasing misery for the patient and their family, and
increasing burden on healthcare systems worldwide. Alzheimer's
Disease (AD) afflicts some 8 million in the Western World; PD
around 120,000 in the UK; 1 million in the USA; and 4 million
worldwide. Huntington's Disease cases number some 6,000 in the UK,
and 30,000 in the USA. Development of strategies to improve
treatment of NDD is a pressing priority. Currently patients with
NDD are managed in general neurology/medical or specialist clinics,
and offered some symptomatic drugs which, whilst helpful in some of
these conditions, are often only useful in the early stages of
disease. Early management is more in the community, but over time
there are increasing co-morbidities that in turn greatly escalate
costs in their management.
[0013] Stopping or slowing down the disease process at the early
stages of NDD conditions would represent a very major therapeutic
advance with far reaching benefits to those afflicted, and within
the health care organisations worldwide.
[0014] At the cellular level attempts to slow down or reverse the
neurodegenerative disease process have produced variable results.
One of the most effective reparative therapies in patients to date
has been with allotransplants of dopamine neuroblasts obtained from
foetal ventral mesencephalic (VM) tissue. Some grafted patients
have responded well and come off anti-PD medication for years,
whilst others have shown no or only modest clinical improvements.
Moreover, a subset of patients also developed severe, off-state
graft-induced dyskinesias, which in a few cases have required
additional neurosurgical intervention. The reasons behind this
heterogeneity of outcomes and the emergence of graft-induced
dyskinesias, in particular, are unknown. There is, therefore, an
urgent need for an optimised and more standardised procedure that
will translate into more consistently efficacious transplants with
minimal side-effects. Current cell harvest procedures typically
incur an 80% cell death rate of an already scare cell resource;
therefore, there is a need to reduce the cell death rate and reduce
the amount of tissue required for allo--or autografting by
optimising procedures for cellular therapy. Thus, in cell therapy
for PD, problems arise from the scarcity and ex vivo fragility of
fetal dopaminergic cells.
[0015] The newly developed capacity to re-programme adult somatic
cells from patients with neurodegenerative diseases has opened up
new possibilities in this area. The technology of inducible stem
cells has been used to better understand these diseases and in
addition provide a potential future resource for cell
transplantation.
[0016] Other experimental treatments aim to repair the core
pathology, for example by delivering soluble growth factors to
rescue the diseased cells from dying, or by immunising against the
protein that lies at the core of the pathology (e.g. amyloid in
Alzheimer's disease). However, such approachs have so-far failed to
deliver substantial clinical benefits. One exception exists where
L-DOPA-synthesising enzymes were delivered via lentivirus to the
substantia nigra. Whilst this exception proves that repair at the
level of neuro-biochemistry is possible, viral-mediated delivery
involves risk of unwanted side-effects due to viral components in
addition to generating an immune response within the patient
against the therapeutic protein itself. Use of soluble growth
factors alone is not simple, and may incur substantial off-target
side-effects including the risk of carcinogenesis. Even targeted
delivery of growth factor using gene therapy into the CNS,
including leukaemia inhibitory factor (LIF) gene therapy, revealed
the issue of increased endogenous inflammatory gene expression
profiles and severe cachexia due to long term high level of LIF
exposure (Prima et al, 2004, Endocrinology). Thus there is an
outstanding need for a means of controlled, transient,
paracrine-type delivery of growth factor to the CNS at
physiological doses where the aim is to stimulate endogenous repair
within the CNS. This need is combined with the need to protect the
therapeutic growth factor from degradation by circulating proteases
in the blood, plus the need to avoid troughs and peaks of exposure
to the growth factor that are associated with bolus delivery.
[0017] In addition to chronic neurodegenerative disorders, damage
to cells within the CNS may arise following traumatic injury,
hypoxic injury as may occur in newborns, and axonal damage occuring
as a result of demeylinating disorders.
[0018] In summary, the need to improve the treatment of NDDs,
injuries of the CNS, hypoxic injury in newborns and trauma arising
from demyelinating disorders by repairing or replacing damaged CNS
neural tissue requires an approach that is simple, transient,
non-invasive and non-inflammatory, with the aim of harnessing
endogenous repair and slowing down, stopping or even reversing
disease progression.
[0019] Nanomedicine is now recognised worldwide as representing new
opportunities in clinical medicine. Currently untreatable illnesses
including NDD present key future targets for nano-therapeutic
intervention. Within the CNS endogenous neural stem and precursor
cells (NSC and NPC) constitute up to 10% of the brain, providing a
potential resource of healthy cells can be exploited to replace
diseased neural tissue by stimulation with neural growth
factors.
[0020] Accordingly, the present invention seeks to overcome or at
least reduce the problems that exist in the treatment of tissue
damage within the CNS including that caused by neurodegenerative
diseases by providing a nanotherapeutic composition for targeted
delivery of factors to expand, and/or to protect and/or to
differentiate neural stem cells, and/or neural progenitor cells
and/or induced pluripotent stem cells. This includes recruiting the
endogenous stem cells that exist in the adult brain and which are
able to replace damaged cells and so maintain good brain function.
The invention enables (i) expansion and protection of healthy brain
cells; (ii) improved cell therapy for NDD; and (iii) development of
neuronal models of clinical disease to identify new therapies
including nanotherapeutics to abrogate the clinical disease
process. By delivering critical neural growth factors direct to
neural progenitor cells either ex vivo, or direct to endogenous
cells within the brain, the invention will stop or even reverse
disease progression.
SUMMARY OF THE INVENTION
[0021] A first aspect of the invention provides a composition for
the treatment of neurodegenerative disease comprising:
[0022] a) a pharmaceutically acceptable carrier solution; and
[0023] b) a plurality of biodegradable nanoparticles, wherein the
nanoparticles comprise a targeting moiety that is able to bind
selectively to the surface of a neural stem cell and wherein the
nanoparticles further comprise leukaemia inhibitory factor
(LIF).
[0024] In a specific embodiment of the invention the targeting
moiety is further able to bind selectively to the surface of one or
more of the group consisting of: a pluripotent stem cell; a
totipotent stem cell; an embryonic stem cell (ESC); an induced
pluripotent stem cell (iPSC); a T lymphocyte; an ectodermal cell; a
precursor cell having commitment to a neurectodermal lineage; a
neural cell; a neuroglial cell, and a neuronal cell.
[0025] In an embodiment of the invention the nanoparticles comprise
a biodegradable polymer layer that encapsulates the LIF.
Optionally, the polymer comprises poly(lactic)-co-glycolic acid
(PLGA) and/or PLA. In an alternative embodiment of the invention
the nanoparticles comprise a lipid layer that encapsulates the LIF
so as to form a liposome nanoparticle, optionally the lipid layer
may comprise a phospholipid bilayer.
[0026] According to a specific embodiment of the invention the
targeting moiety is selected from a monoclonal antibody; a
polyclonal antibody; an antigen-binding antibody fragment; a
ligand; an aptamer and a small molecule. In one embodiment of the
invention the targeting moiety binds specifically to a Thy-1
antigen present on the surface of the neural stem cell and/or the
neural progenitor cell and/or the induced pluripotent stem
cell.
[0027] In a particular embodiment of the invention the
nanoparticles further comprise one or more of the following
therapeutic (compounds) biologics: brain-derived neurotrophic
factor (BDNF) or an agonist thereof; epidermal growth factor (EGF)
or an agonist thereof; glial cell-derived neurotrophic factor
(GDNF) or an agonist thereof; retinoic acid and derivatives
thereof; ciliary neurotrophic factor (CTNF) or an agonist thereof;
Wnt5A.
[0028] According to an embodiment of the invention the
nanoparticles suitably have a diameter of at least about 50 nm and
at most about 300 nm; optionally at least about 100 nm and at most
about 200 nm. Suitably the nanoparticles are capable of degrading
of a period of time in order to effect timed release of the
encapsulated LIF. Optionally the period of time may be selected
from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12
days; 1, 2, 3, 4, 5 or 6 weeks; and up to six months.
[0029] A second aspect of the invention provides a method for
expanding a population of stem cells having the capacity to act as
a neural precursor cell comprising exposing the cells to a
plurality of biodegradable nanoparticles, wherein the nanoparticles
comprise a targeting moiety that is able to bind selectively to the
surface of the stem cells and wherein the nanoparticles further
comprise leukaemia inhibitory factor (LIF).
[0030] In an embodiment of the invention, the stem cells having the
capacity to act as a neural precursor cell are selected from one or
more of the group consisting of: neural stem cells; neural
progenitor cells; pluripotent stem cells; totipotent stem cells;
embryonic stem cells (ESCs); induced pluripotent stem cell (iPSCs);
induced neural cells (iN); induced dopaminergic cells (iDA);
induced oligodendrocytes (iOD); ectodermal cells; precursor cells
having commitment to a neurectodermal lineage; neural cells; and
neuronal cells.
[0031] In an embodiment of the invention the nanoparticles comprise
a biodegradable polymer layer that encapsulates the LIF. Suitably
the polymer comprises poly(lactic)-co-glycolic acid (PLGA) and/or
PLA or a suitable biocompatible equivalent. In an alternative
embodiment of the invention the nanoparticles comprise a lipid
layer that encapsulates the LIF so as to form a liposome
nanoparticle, suitably a phospholipid bilayer.
[0032] In a particular embodiment of the invention the
nanoparticles comprise a targeting moiety that is selected from a
monoclonal antibody; a polyclonal antibody; an antigen-binding
antibody fragment; a ligand; and a small molecule. Suitably the
targeting moiety may bind specifically to a Thy-1 antigen present
on the surface of the stem cell.
[0033] According to a specific embodiment of the invention the
nanoparticles further comprise one or more of the compounds
selected from: brain-derived neurotrophic factor (BDNF) or an
agonist thereof; epidermal growth factor (EGF) or an agonist
thereof; glial cell-derived neurotrophic factor (GDNF) or an
agonist thereof; retinoic acid and derivatives thereof; ciliary
neurotrophic factor (CTNF) or an agonist thereof; Wnt5A.
[0034] In embodiments of the invention the method is carried out in
vitro, ex vivo or in vivo.
[0035] A third aspect of the invention provides a method for
treating a subject suffering from a neurodegenerative disease (NDD)
or CNS damage comprising administering to the subject a
pharmaceutical composition comprising a plurality of biodegradable
nanoparticles, wherein the nanoparticles comprise a targeting
moiety that is able to bind selectively to the surface of a neural
precursor cell and wherein the nanoparticles further comprise
leukaemia inhibitory factor (LIF). Suitably, the neural precursor
cell comprises a neural stem cell and/or a neural progenitor cell.
In an embodiment of the invention the targeting moiety is further
able to bind selectively to the surface of one or more of the group
consisting of: a pluripotent stem cell; a totipotent stem cell; an
embryonic stem cell (ESC); an induced pluripotent stem cell (iPSC);
induced neural cells (iN); induced dopaminergic cells (iDA);
induced oligodendrocytes (iOD); a T lymphocyte; an ectodermal cell;
a precursor cell having commitment to a neurectodermal lineage; a
neural cell; and a neuronal cell.
[0036] According to a specific embodiment of the invention the
subject is an animal, suitably a mammal, optionally selected from
the group consisting of: sheep; cattle; rodents; rabbits; pigs;
cats; dogs; and primates. Where the mammal is a primate the primate
may be a human.
[0037] A fourth aspect of the invention provides for a nanoparticle
device comprising:
[0038] a biodegradable carrier material, a therapeutic compound,
and a targeting moiety;
[0039] wherein the carrier material is configured so as to
encapsulate the therapeutic compound and wherein the carrier
material further defines a surface, upon and within which surface
is located the targeting moiety,
[0040] the nanoparticle device further characterised in that the
therapeutic compound comprises LIF and the surface located
targeting moiety comprises an antibody, or an antigen binding
fragment of an antibody, that specifically binds to an antigen
present on the cell surface of a stem cell having the capacity to
act as a neural precursor cell.
[0041] In a particular embodiment of the invention the
biodegradable carrier material degrades at a rate that allows for
controlled release of the LIF over a pre-determined period of time.
Suitably, the targeting moiety binds specifically to a Thy-1
antigen present on the surface of the stem cell. In a further
embodiment the moiety binds specifically to a NCAM antigen present
on the surface of the cell. In yet a further embodiment the moiety
binds specifically to a GDNF receptor .alpha.1 (GDNFR-.alpha.1)
located on the surface of the cell.
[0042] In an embodiment of the invention the nanoparticle device
further comprises one or more of the following therapeutic
compounds: brain-derived neurotrophic factor (BDNF) or an agonist
thereof; epidermal growth factor (EGF) or an agonist thereof; glial
cell-derived neurotrophic factor (GDNF) or an agonist thereof;
retinoic acid and derivatives thereof; ciliary neurotrophic factor
(CTNF) or an agonist thereof; Wnt5A.
[0043] In a particular embodiment of the invention the nanoparticle
device has a diameter of at least about 50 nm and at most about 300
nm; optionally at least about 100 nm and at most about 200 nm.
[0044] A fifth aspect of the invention provides for a compositions
or nanoparticle devices as described above for use in the treatment
of NDD and CNS damage. According to a specific embodiment of the
invention the compositions or nanoparticle devices are suitable for
use in the treatment of one or more diseases selected from the
group consisting of: Alzheimer's Disease (AD), Parkinson's Disease
(PD); Huntington's Disease (HD); Frontotemporal dementia (FTD); and
Amyotrophic Lateral Sclerosis (ALS).
[0045] A sixth aspect of the invention provides a composition for
the treatment of NDD and CNS repair comprising:
[0046] a) a pharmaceutically acceptable carrier solution; and
[0047] b) a plurality of biodegradable nanoparticles, wherein the
nanoparticles comprise a targeting moiety that is able to bind
selectively to the surface of a neural stem cell and/or a neural
progenitor cell and wherein the nanoparticles further comprise
XAV939.
[0048] A seventh aspect of the invention provides for a
combinatorial composition for the treatment of NDD comprising:
[0049] a) a pharmaceutically acceptable carrier solution;
[0050] b) a first population of biodegradable nanoparticles,
wherein the first nanoparticles comprise a targeting moiety that is
able to bind selectively to the surface of a neural stem cell
and/or a neural progenitor cell and wherein the first nanoparticles
further comprise leukaemia inhibitory factor (LIF); and
[0051] c) a second population of biodegradable nanoparticles,
wherein the second population of nanoparticles comprise a targeting
moiety that is able to bind selectively to the surface of a neural
stem cell and/or a neural progenitor cell and wherein the second
nanoparticles further comprise one or more of the compounds
selected from: brain-derived neurotrophic factor (BDNF) or an
agonist thereof; epidermal growth factor (EGF) or an agonist
thereof; glial cell-derived neurotrophic factor (GDNF) or an
agonist thereof; retinoic acid and derivatives thereof; ciliary
neurotrophic factor (CTNF) or an agonist thereof; Wnt5A; and
XAV939.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0053] FIG. 1A shows a diagram of the LIF receptor consisting of
two proteins: gp130 and gp190; and FIG. 1B shows
Immunocytochemistry of 5 day old E14 VM cultures with antibodies
against tyrosine hydroxylase and gp130 or gp190 demonstrating that
dopaminergic neurons express gp130 and gp190.
[0054] FIG. 2 shows a graph indicating that dissociation of E14 VM
tissue in LIF supplemented medium increases the number of
dopaminergic neurons in subsequent monolayer culture. Isolated
ventral midbrain tissue from E14 rat foetuses was dissociated in
growth medium alone or medium supplemented with 0.1 ng/ml LIF.
[0055] FIG. 3A and FIG. 3B show that supplementing growth medium
with 0.1 ng/ml LIF increases the dopaminergic cell count at 3 and 5
days in vitro. FIG. 3A shows a graph of results demonstrating that
supplementing the medium with 0.1 ng/ml LIF significantly increased
the number of dopaminergic neurons at 3 and 5 days in vitro. FIG.
3B shows exemplary immunocytochemistry images of E14 VM cultures
after 5 days in vitro demonstrates the increased number of tyrosine
hydroxylase positive neurons (highlighted) in cultures grown with
0.1 ng/ml LIF. The scale bar represents 100 .mu.m.
[0056] FIG. 4 shows micrographs indicating that dopaminergic
neurons in E14 VM cultures express the GDNF receptor .alpha.1. The
scale bar represents 25 .mu.m.
[0057] FIG. 5A shows a graph and FIG. 5B shows immunocytochemistry
indicating that treatment of E14 VM cultures with nanoparticles
targeted to the GDNF receptor .alpha.1 increases the dopaminergic
cell count at 3 days in vitro.
[0058] FIG. 6 shows micrographs indicating that for monolayer
cultures derived from E14 VM cells previously expanded as
neurospheres immunocytochemical analysis revealed presence of
immature neurons (.beta.III tubulin) and astrocytes (GFAP). The
scale bars represent 50 .mu.m.
[0059] FIG. 7 shows graphs that indicate that expansion of E14 VM
neural progenitor cells with 0.1 ng/ml LIF has no impact on
subsequent differentiation. Expansion of E14 VM as neurospheres in
medium supplemented with 0.1 ng/ml LIF had no significant effect on
subsequent levels of neural or astroglial differentiation in
monolayer cultures produced from dissociated neurospheres.
[0060] FIG. 8 shows micrographs indicating that a proportion of
dopaminergic neurons in E14 VM cultures undergo apoptosis. E14 VM
cultures were fixed after 2 days in vitro and analysed via
immunocytochemistry.
[0061] FIG. 9 shows graphs of the results of treatment of E14 VM
cultures with 0.1 ng/ml LIF or targeted LIF nanoparticles after 2,
3 and 5 days, demonstrating a significant reduction in the level of
dopaminergic apoptosis at 2 days in vitro.
[0062] FIG. 10 shows micrographs indicating that serotonin neurons
express GDNF receptor .alpha.1 (GDNFR-.alpha.1). Analysis of the
stained culture demonstrated that serotonin neurons from E14 VM
express GDNFR-.alpha.1 both on their soma and neurites. The scale
bar represents 50 .mu.m.
[0063] FIGS. 11A-11H show graphs of results indicating that rat E14
VM cultures respond to Thy-1 targeted nanoparticles. The
nanoparticles were directed to Thy-1 using biotinylated anti-Thy-1
in the NP surface: they carried a cargo of 7,8 dihydroxyflavone
(7,8 DHF), a BDNF agonist that binds TrkB, the BDNF receptor.
[0064] FIG. 12 shows the experimental protocol for transplanting
primary isolates of rat VM cells into the striatum of isogenic
Lewis rats.
[0065] FIG. 13 shows graphs indicating the response of lesioned
recipient Lewis rats following transplantation of isogenic foetal
VM cells treated with either empty nanoparticles, LIF nanoparticles
or BDNF nanoparticles, or untreated cells.
[0066] FIG. 14 shows micrographs demonstrating the expansion of
primary human foetal ventral mesencephalon culture cell numbers to
provide sufficient cells for testing LIF therapeutic nanoparticles.
Primary=primary cultures; Passage 0=first subculture; Passage
1=second subculture.
[0067] FIG. 15 shows micrographs with the amplified cells of FIG.
14 used to test the effect of LIF nanoparticles at increasing
concentrations (dose) on dopaminergic cell maturation and overall
cell survival.
[0068] FIG. 16 shows a graph providing quantification of the
results of FIG. 15.
[0069] FIG. 17 shows the protocol for testing the effect of
LIF-nanoparticle treatment targeted to Thy-1 on human foetal VM
cell grafts in vitro.
[0070] FIG. 18 shows a schematic of a protocol to measure the
effect of nanotherapy in vitro by incubating hfVM cells for 24 h at
37.degree. C. together with Thy-1 targeted particles loaded with
various cargos prior to transplantation into the striatum of a nude
rat. B
[0071] FIG. 19 shows photographs and micrographs of sections of
striatum of a nude rat brain that comprises LIF-nano treated hfVM
cells. A: low power section showing graft stained for HuNu and TH
positive cells, enlarged in B. Further enlargement in C shows large
numbers of HuNu staining cells plus some TH+ cells both within the
graft site and spreading out from this site.
[0072] FIG. 20 shows photographs and micrographs of sections of
striatum of a nude rat brain following transplantation of
XAV939-Nano treated hfVM cells. A: low power section showing
striatum (brown) with grafted hfVM cells (black nuclei) on left
"grafted striatum"--shown in higher power in B. B also shows human
dopaminergic cell within the graft site (DA cell). Ungrafted
striatal tissue (C) acts as endogenous control for specificity of
HuNu staining of hfVM: no stained nuclei are present.
[0073] FIG. 21 shows photographs and micrographs of sections of
striatum of a nude rat brain following transplantation of Retinoic
Acid (RA)-Nano treated hfVM cells. A: low power section showing
striatum with grafted hfVM cells (black nuclei) where the injection
needle tract (solid arrow) is marked by the presence of the HuNu
stained nuclei. B shows a different section of the same recipient
as in A, at higher power, showing surviving cells plus some cell
debris (solid arrow): the dashed arrow indicates human dopaminergic
TH+ cells. C shows a further higher power of the grafted cells in
situ plus cell debris.
[0074] FIG. 22 shows photographs and micrographs of sections of
striatum of a nude rat brain following transplantation of control
Empty-Nano (i.e. nanoparticles targeted to Thy-1 but without any
cargo) treated hfVM cells. A: low power section showing striatum
(brown) with grafted hfVM cells (black nuclei) where the injection
needle tract (solid arrow) is marked by the presence of the HuNu
stained nuclei. B and C show higher magnifications of the grafted
cells, where cell debris (pale clumps) is also visible.
[0075] FIG. 23 is graph showing survival benefit of
nanotherapeutics for TH positive dopaminergic cells according to
the protocol of FIG. 17. EM-NP represents empty nanoparticle
control.
[0076] FIG. 24 is graph showing total cell numbers survival benefit
of nanotherapeutics counting all DAPI positive cells according to
the protocol of FIG. 17.
[0077] FIG. 25 is graph showing preferential survival benefit on
dopaminergic cells expressed as percentage for each treatment
according to the protocol of FIG. 17.
DETAILED DESCRIPTION OF THE INVENTION
[0078] A description of example embodiments of the invention
follows. A number of definitions are provided that will assist in
the understanding of the invention. All references cited herein are
incorporated by reference in their entirety. Unless otherwise
defined, 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.
[0079] As used herein, the term "comprising" means any of the
recited elements are necessarily included and other elements may
optionally be included as well. "Consisting essentially of" means
any recited elements are necessarily included, elements that would
materially affect the basic and novel characteristics of the listed
elements are excluded, and other elements may optionally be
included. "Consisting of" means that all elements other than those
listed are excluded. Embodiments defined by each of these terms are
within the scope of this invention.
[0080] The term "antibody" as used herein denotes a protein that is
produced in response to an antigen that is able to combine with and
bind to the antigen, preferably at a specific site on the antigen,
known as an epitope. The term as used herein includes antibodies of
polyclonal and monoclonal origin, unless stated otherwise. Also
included within the term are antigen binding fragments of naturally
or non-naturally occurring antibodies, for example, the "Fab
fragment", "Fab' fragment" (a Fab with a heavy chain hinge region)
and "F(ab')2 fragment" (a dimer of Fab' fragments joined by a heavy
chain hinge region).
[0081] The term "growth factor" as used herein denotes a naturally
occurring substance capable of stimulating cellular growth,
proliferation and differentiation. Growth factors are important for
regulating a variety of cellular processes and typically act as
signaling molecules between cells. Certain combinations of growth
factors create gradients able to guide cell differentiation in a
temporal and spatial manner.
[0082] The term "induced pluripotent stem cells" (IPS cells) as
used herein denotes a type of pluripotent stem cell artificially
derived from a non-pluripotent cell by inducing the forced
expression of specific genes. Typically, the non-pluripotent cell
is an adult somatic cell. IPS cells can be used to generate
immuno-compatible cell types for cell based therapy, thereby
avoiding the use of immune suppressive treatment.
[0083] The compositions and methods of the invention can be
utilised with any stem cells that exhibit the capacity to act as a
neural precursor cell or to differentiate into a neural stem cell.
Such stem cells may be selected from one or more of the group
consisting of: neural stem cells; neural progenitor cells;
pluripotent stem cells; totipotent stem cells; embryonic stem cells
(ESCs); induced pluripotent stem cell (iPSCs); ectodermal cells;
precursor cells having commitment to a neurectodermal lineage;
neural cells; and neuronal cells. In certain embodiments of the
invention where the stem cells are ESCs, the ESCs may be derived
from sources other than a human embryo.
[0084] The term "neural stem cells" (NSCs) as used herein denotes
self-renewing multipotent cells that are capable of generating the
main phenotypes of the nervous system, including neurons,
astrocytes and oligodendrocytes.
[0085] The term "neural progenitor cells" (NPCs) as used herein
denotes oligopotent cells that are at a further stage of
differentiation compared to NSCs and are destined to differentiate
into specific target cells.
[0086] The terms "induced neuron" (iN) and "induced dopaminergic
cell" (iDA) and "induced oligodendrocyte" (iOD) are used to denote
cells derived by transdifferentiation from differentiated somatic
cell types usually fibroblastic in origin.
[0087] The invention provides nanoparticle-mediated delivery of
compounds, such as growth factors, signalling proteins, cytokines
and small molecules in novel combinations, as a novel means to
repair damaged tissue in the CNS of an animal, such as a human. The
clinical benefit is considerable for patients with
neurodegenerative diseases or other tissue damage within the CNS
including demyelinating injury. Compounds may be delivered
individually or in combinatorial compositions, thereby allowing for
synergistic therapeutic activity to be localised to the point of
need in the recipient.
[0088] LIF is a member of the IL-6 family of cytokines, which are
growth factors. LIF is a secreted signalling factor that binds to
and signals via heterodimers of the LIF-specific receptor subunit,
"gp190" and the signal-transducing receptor subunit "gp130".
Downstream, intracellular signal propagation following LIF/LIF-R
engagement occurs via both (i) the JAK/STAT pathway especially via
the transcription factor STAT-3, and (ii) the MAPKinase pathway.
Within the immune system there is an exquisite ability to
discriminate between "self " and "non-self" that is orchestrated by
antigen-specific T lymphocytes. Genomic plasticity enables
differentiation of naive CD4+ T lymphocytes into either regulatory
cells (Treg) that express the transcription factor Foxp3 and
actively prevent auto-immune self-destruction, or effector cells
(Teff) that attack and destroy their cognate target. Importantly,
LIF supports Treg maturation in contrast to IL-6 which drives
development of the pathogenic Th17 effector phenotype (Gao et al
2009 Cell Cycle). The inventors have previously demonstrated that
nanoparticle-mediated targeted delivery of LIF can be used to guide
tolerogenesis in a patient (see International (PCT) Publication No.
WO 2009/053718, which is incorporated herein by reference).
[0089] Working in the CNS, the inventors made the unexpected
discovery that nanoparticle-mediated targeted delivery of LIF to
neural precursor has a profound protective effect that is markedly
superior to that of soluble LIF. The cells were of the CNS where
there is commitment to a neural cell fate, such as for neural stem
cells, neural, neuronal oligodendrocyte and glial progenitor cells.
This enables these nano-LIF-treated cell populations to be used
therapeutically with unexpectedly high efficacy, such as in the
treatment of NDD and other CNS conditions,
[0090] In the CNS, LIF is thought to act predominantly as an injury
factor, optimising the pool of neural precursors available for
repopulation during repair (Pitman et al 2004, Mol Cell
Neuroscience). LIF promotes neural stem cell self-renewal in the
adult brain, regulating the emergence of more differentiated cell
types, which ultimately leads to an expansion of the neural stem
cell pool (Bauer, S. et al., 2006). LIF also stimulates the
proliferation of parenchymal glial progenitors, in particular
oligodendrocyte progenitor cells, through the activation of gp130
receptor signaling within these cells. This effect of LIF can be
used to enhance the generation of oligodendrocytes and suggests
that LIF has both reparative and protective activities that makes
it a suitable candidate for the treatment of CNS demyelinating
disorders and injuries (Deverman, B. E. et al., 2012). Furthermore,
LIF has been shown to directly prevent oligodendrocyte death in
animal models of multiple sclerosis, which is a disabling
inflammatory demyelinating disease of the CNS, and this effect
complements endogenous LIF receptor signalling, which already
serves to limit oligodendrocyte loss during immune attack
(Butzkueven, H. et al., 2002). LIF has also been shown to
up-regulate the re-expression of NPCs in the brain of a Parkinson's
Disease mouse model (Liu, J. et al., 2009).
[0091] However, when considering LIF as a potential therapeutic, it
is important to note that LIF is tightly regulated in vivo under
physiological conditions and that recombinant LIF (rLIF)
administered systemically in high bolus doses is toxic. Low doses
of rLIF are ineffective due to rapid degradation by serum
proteases--part of the physiological control imposed on endogenous
LIF in vivo.
[0092] In order to harness the immense therapeutic potential of LIF
as a therapeutic within the CNS, the inventors have created a
device that permits (i) specific targeting to sites of need within
the CNS and (ii) provides low dose paracrine-type delivery of cargo
sustained over several days or weeks, followed by complete
degradation and elimination of the degradation products device
including via CSF transit flow. Unexpectedly, by bringing the
LIF-loaded nanoparticles into direct contact with cells surface
receptors via the targeting moiety, the continuous low dose
paracrine-type delivery of LIF achieves profound efficacy in
promoting and protecting the CNS-derived cells as is shown in the
Examples described herein.
[0093] In an embodiment of the invention, LIF-containing
nanoparticles are provided that are capable of being targeted at
neural stem cells and/or neural progenitor cells, in particular at
specific markers located on the surface of these cells. The
nanoparticles can be targeted to stem cells committed to or capable
of following a neural lineage, including neural stem cells and
neural progenitor cells in vitro (for example to test the
nanoparticle efficacy and cytokine release rate, etc.), ex vivo
(for later transplantation of LIF expanded neural cell populations
into a patient) and/or in vivo (i.e. direct administration of
nanoparticles into a patient requiring treatment for a
neurodegenerative disorder).
[0094] The nanoparticle--also referred to as the nanoparticle
device--suitably comprises a biodegradable non-toxic polymer that
encapsulates LIF polypeptide (multiple cytokine polypeptides are
typically encapsulated) either alone or in combination with one or
more other factors. In this way the LIF represents a cargo load
that is delivered by the nanoparticle. Suitably, the polymer
comprises the copolymer poly(lactic)-co-glycolic acid (PLGA), which
is an FDA approved biodegradable and biocompatible copolymer that
allows for the slow release of LIF into the micro-environment of
the target cell(s). PLGA undergoes hydrolysis (biodegrades) in the
body to produce the original monomers, lactic acid and glycolic
acid. It is possible to adjust the polymer degradation time by
altering the ratio of these monomers in the PLGA copolymer.
Hydrolysis of the polymeric matrix releases entrapped bioactive LIF
in a sustained manner. Nanoparticulate devices and compositions are
described in US-2010/0151436, which is incorparated herein by
reference.
[0095] Alternatively, the nanoparticle polymer may comprise a
combination of PLGA and poly(lactic acid) (otherwise known as
polylactide--PLA). PLA is biodegradable thermoplastic aliphatic
polyester derived from renewable resources. The ratios of PLGA and
PLA can be varied to provide optimal delivery of LIF to neural stem
cells and/or neural progenitor cells. The ratios can also be varied
depending on whether the nanoparticles are to be delivered in
vitro, ex vivo or in vivo.
[0096] The above-described polymers have several features that make
them ideal materials for use in the nanoparticles of the present
invention: 1) control over the size range of the nanoparticles, an
important feature for ensuring that the nanoparticles can pass
through biological barriers (such as the blood brain barrier) when
used in active therapy (i.e. in vivo delivery of nanoparticles to
CNS and brain tissue); 2) reproducible biodegradability without the
necessary addition of enzymes or cofactors; 3) capability for
temporal and special control of sustained release of encapsulated,
protected neurally active factors (such as LIF) that may be tuned
in the range of days to months by varying factors such as the PLGA
to PLA copolymer ratios; 4) well-understood fabrication
methodologies that offer flexibility over the range of parameters
that can be used for fabrication, including choices over the
polymer material, solvent, stabiliser, and scale of production; 5)
control over surface properties facilitating the introduction of
modular functionalities into the surface; and 6) the polymers are
impermeable to serum proteases.
[0097] The nanoparticles of the invention are typically sized at
least 50 nm (nanometres), suitably at least approximately 100 nm
and typically at most 200 nm, although suitably up to 300 nm in
diameter. In one embodiment of the invention the nanoparticle
device has a diameter of approximately 100 nm. This is so that they
are below the threshold for reticuloendothelial system (mononuclear
phagocyte system) clearance, i.e. the particle is small enough not
to be destroyed by phagocytic cells as part of the body's defence
mechanism.
[0098] The nanoparticle device of the invention may suitably
deliver the encapsulated cargo over a period of time that may be
controlled by the particular choice or formulation of the
encapsulating biodegradable non-toxic polymer or biocompatible
material. One exemplary temporal release profile comprises a pulse
of LIF release--characterized by release of up to 50% by weight of
the amount of the cargo--associated with the nanoparticulate device
in 1-5 days following the introduction into a subject. Following
the pulse, the residual amount is slowly released over an extended
period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 days or 2,
3, 4, 5 or more weeks) following the pulse period. In another
embodiment of the invention the initial pulse may be reduced to
less than 50% of the amount of the cargo, less than 30% or even
less than 10% by weight of the total cargo. Likewise, the device
may be configured so as to only deliver the cargo over a sustained
period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 days, 2, 3, 4,
5 or more weeks, or up to six months. It will be appreciated that
the release profile will be best optimised to suit the clinical
needs of the patient and the particular NDD that is being
treated.
[0099] Targeting of the nanoparticles to a specified cell surface
marker on the cell of choice, for example a neural stem cell and/or
a neural precursor cell, is typically achieved by locating a
targeting moiety, such as an antibody, on the surface of the
nanoparticle. The targeting moiety is able to bind selectively to
the cell of choice via affinity-targeted ligand interactions,
Cell-specific targeting is achieved by the choice of surface-bound
antibody. Thus, the nanoparticles of the invention further comprise
a surface exposed antibody that specifically binds to the cell of
choice. Suitable targeting moieties include monoclonal antibodies,
polyclonal antibodies, antigen binding antibody fragments, ligands,
and small molecules. Suitable antibody fragments or derivatives
from a variety of sources may include: F.sub.ab, scF.sub.v,
Bis-scF.sub.v, V.sub.H, V.sub.L, V-NAR, VhH or any other
antigen-binding single domain antibody fragment. The specific
binding activity may also be localised within another antibody-like
affinity binding protein including lactoferrins, cathelicidins,
ficolins, collagenous lectins and defensins.
[0100] The nanoparticle polymer can suitably be decorated with
functional avidin or streptavidin groups on the nanoparticle
surface to enable modification of the surface through the robust
attachment of biotinylated ligands such as specific cell-targeting
antibodies.
[0101] The Thy-1 antigen (Reif and Allen, 1964) has been identified
as one suitable target to localise nanoparticles of the invention
to the surface of neural stem cells and neural progenitor cells. It
may be beneficial to target the nanoparticles to the Thy-1 antigen
rather than a cell surface receptor so as to avoid any potential
interference of receptor function of the target cell. Thy-1 (also
known as CD90--Cluster of Differentiation 90) is a 25-37 kDa
heavily N-glycosylated, glycophosphatidylinositol anchored
conserved cell surface protein with a single V-like immunoglobulin
domain. It can be used as a marker for a variety of stem cells,
including neural stem cells, and for the axonal processes of mature
neurons. T lymphocytes also express Thy-1 on their cell surface.
The co-targeting of the nanoparticles of the invention to neural
committed stem cells, neural progenitor cells and additionally T
lymphocytes is of great benefit when using the nanoparticles to
expand and protect a population of neural stem cells and/or neural
progenitor cells ex vivo for transplantation into a subject. T
lymphocytes mature towards T.sub.reg under the influence of LIF so
that, when the time comes for cell transplantation, a population of
the transplanted cells treated with nanoparticles of the invention
will be surrounded by an artificial stroma comprising, for example,
LIF-containing nanoparticles that promote both cell survival and
repress adverse immune reactions to enhance engraftment of
transplanted cells in the CNS. Thus, in one embodiment of the
invention, LIF's neurogenic and tolerogenic dual characteristics
make it an ideal choice of factor for endogenous support of brain
repair and suppression of inappropriate immune activity and a
profound synergistic effect is provided by the LIF encapsulated
nanoparticles.
[0102] The link between IL6, a potent inducer of pathogenic
inflammatory TH17 lymphocytes and neurodegenerative disease
progression is of further relevance, since the inventors have found
that LIF directly suppresses both IL6 activity and TH17 cell
development and instead promotes tolerogenic T.sub.reg cells (Gao
et al 2009; Park et al 2011). This correlates with the recent
finding that T.sub.reg opposes TH17-driven dopaminergic
neurodegeneration in a mouse model of Parkinson's Disease (Reynolds
et al 2010); and that LIF opposes pathogenic TH17 cells in an
experimental allergic encephalitis (EAE) model of multiple
sclerosis, a demyelinating disease of the CNS (Cao et al 2011).
[0103] It will be appreciated by the skilled person that other
alternative cell surface markers may be used for targeting
nanoparticle devices of the invention to neural stem cells and
neural progenitor cells, or other pluripotent cells having the
capacity to differentiate into neural cells. By way of non-limiting
example, one alternative target is the glial cell line derived
neurotrophic factor receptor .alpha.1 (GDNF-R .alpha.1). Hence, in
specific embodiments of the invention if Thy-1 is the target cell
surface marker the nanoparticle may comprise an anti-Thy-1 antibody
in its surface. Likewise, if GDNF-R .alpha.1 is the target cell
surface marker the nanoparticle may comprise an anti-GDNF-R
.alpha.1 antibody on its surface.
[0104] The nanoparticles of the invention enable the sustained
delivery of factors, such as multiple LIF molecules, to ensure a
relatively high concentration of factor precisely within the
microenvironment of the targeted cells to expand and protect the
cells, whilst avoiding toxic systemic exposure of the recipient
subject to the therapeutic cytokine
[0105] In an embodiment of the invention, the nanoparticles are
suspended in a biocompatible solution to form a composition that
can be targeted to a location on a cell, within a tissue or within
the body of a patient or animal (e.g. the composition can be used
in vitro, ex vivo or in vivo). Suitably, the biocompatible solution
may be phosphate buffered saline or any other pharmaceutically
acceptable carrier solution. One or more additional
pharmaceutically acceptable carriers (such as diluents, adjuvants,
excipients or vehicles) may be combined with the nanoparticles of
the invention in a pharmaceutical composition. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Pharmaceutical
formulations and compositions of the invention are formulated to
conform to regulatory standards and can be administered orally,
intravenously, topically, or via other standard routes.
Administration can be systemic or local or intranasal or
intrathecal.
[0106] In further embodiments of the invention, other growth
factors, signalling proteins and small molecules may be
encapsulated within the nanoparticles either in addition to or
instead of LIF to expand, protect and/or differentiate neural stem
cells, neural progenitor cells or other pluripotent cells having
the capacity to differentiate into neural cells. The provision of
other factors and/or molecules in addition to LIF may augment the
efficacy of LIF or the tolerogenic effect of the composition when
used in vivo.
[0107] Other potential neurogenic and/or neuroprotective agents for
encapsulation in nanoparticles include growth factors such as
brain-derived neurotrophic factor (BDNF), the BDNF-agonist 7,8
dihydroxy flavone (7,8-DHF) epidermal growth factor (EGF), glial
cell-derived neurotrophic factor (GDNF), ciliary neurotrophic
factor (CTNF), amongst others, retinoic acid (RA) and derivatives
thereof, and the signalling protein Wnt5A. Derivatives of retinoic
acid may include, but are not limited to, 9-cis RA, 13-cis RA,
N-(4-hydroxyphenyl) retinamide (4-HPR), and all-trans retinoic acid
(ATRA). Agonists of neural growth factors can also be encapsulated
in the nanoparticles. By way of example, the BDNF agonist 7,8
dihydroxyflavone (7,8,DHF) is shown in the present Examples to
increase the yield of TH+ neuronal cells in primary rat E14 VM
tissue treated with nanoparticles that encapsulate the agonist.
Optional additional factors, such as anti-oxidants, or transforming
growth factor beta (TGF-.beta.) that promotes responsiveness to
GDNF, or retinoic acid that plays an important role in
multipotency, may also be included in the nanoparticles. Single or
multiple agents may be combined with LIF in the same nanoparticle,
or may be used individually in one nanoparticle, for nanoparticle
delivery to target cells.
[0108] Taking EGF as an example, this growth factor has a unique
role as a mediator of dopamine-induced precursor cell proliferation
in the sub-ventricular zone of the brain. EGF receptors are reduced
in Parkinson's Disease, therefore targeted paracrine delivery of
nanoparticles containing EGF can increase dopamine-induced
precursor cell proliferation due to the increase in EGF
potency.
[0109] Wnt5a (Wingless-type MMTV integration site family member 5A)
is a signaling protein that in humans is encoded by the WNT5A gene.
Members of the Wnt5a class of proteins activate non-canonical Wnt
pathways, which involve different kinases such as protein kinase C,
calmodulin-dependent protein kinase II and c-Jun N-terminal kinase,
as well as phosphatases and GTPases. Non-canonical Wnt pathways
inhibit the canonical Wnt-.beta.-catenin pathway. Human frizzled-5
(hFz5) is a receptor for the human Wnt5A protein. Wnt5A has been
implicated as a tumour suppressor gene. Importantly, Wnt5A has been
identified for use in the treatment of primary midbrain precursor
cells to induce their differentiation into dopaminergic (DA)
neurons. Therefore, sustained nanoparticle delivery of Wnt5a
(either with or without LIF) to dopaminergic precursor cell
populations will support DA cell differentiation in addition to
increasing dopaminergic precursor cell recovery ex vivo and also
their survival following subsequent transplantation into patients
suffering from Parkinson's Disease.
[0110] In an embodiment of the invention the nanoparticles may also
comprise as the cargo--in addition to or instead of LIF--the small
molecule XAV939 (structure shown below).
##STR00001##
XAV939 is a known inhibitor of the Wnt/.beta.-catenin signalling
pathway that mediates .beta.-catenin degradation by inhibiting the
poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2, which in
turn stabilises axin. Both tankyrase isoforms interact with a
highly conserved domain of axin and stimulate its degradation
through the ubiquitin-proteasome pathway (Huang et al., 2009).
Importantly, XAV939 promotes remyelination of demyelinated nerve
axons by stabilising Axin2. Axin2 itself is regulatory and provides
a therapeutic target in new born brain injury and for
remyelination. Axin2 is expressed in immature oligodendrocyte
precursor cells (OPC), including those residing in active MS
lesions. Axin2 plays a role in feedback regulation of the wnt
signalling pathway: since wnt signalling can act to inhibit OPC
differentiation in both adult remyelination models and
developmental myelination, manipulation of Axin2 levels in OPC can
repress wnt signalling and promote accelerated differentiation of
OPC to oligodendrocytes (OD) capable of remyelinating nerve axons
within the CNS. By inhibiting tankyrase, involved in Axin2
degradation, XAV939 promotes remyelination (Fancy et al. 2011).
Direct injection of XAV939 direct into spinal cord lesions promotes
markedly accelerated OD differentiation after demyelinating injury.
Hence, the nano-XAV939 device of the present invention targeted to
the surface of, for example, demyelinated axons provides a
non-invasive focussed means of simarly promoting remyelination.
[0111] The nanoparticles and compositions of the invention can be
delivered to target cells in vitro, for example to test their
efficacy, and also ex vivo for the transplantation of LIF expanded
and/or protected target cells into the adult brain of patients
suffering from neurodegenerative disease. Cell therapy promotes
brain repair by maintaining or replacing populations of vulnerable
neurons and/or expanding the endogenous neural stem cells and
progenitor cells that populate the brain, providing an enriched
source of healthy precursor cells with the potential to mediate
repair. Cell therapy can provide precursor cells as autografts (for
example, derived from patient skin fibroblasts by
trans-differentiation to a required phenotypic precursor cell--IPS
cell) or allografts (for example, from foetal precursor cells). In
an embodiment of the invention the transplanted cells may be
dopaminergic cells.
[0112] The nanoparticles and compositions of the invention can also
be delivered to target cells in vivo. In vivo use requires that the
nanoparticles of the invention are able to cross the blood brain
barrier so that they can access the target cells within the brain
of the patient. Self-administered intra-nasal delivery of the
nanoparticles and compositions of the invention is one way in which
the nanoparticles can reach the target cells to promote endogenous
repair and replacement of damaged brain tissues, and to protect
healthy brain structure from toxic damage associated with disease
states.
[0113] The nanoparticles and compositions of the invention can be
used in the treatment of various neurodegenerative diseases,
including Alzheimer's Disease, Parkinson's Disease, Amyotrophic
lateral sclerosis and Huntington's Disease, amongst others, and
will provide huge socio-economic benefit to patients suffering from
neurodegenerative diseases and their families. By way of example,
dopaminergic cell replacement therapy is the focus for the
treatment of Parkinson's Disease.
[0114] IPS cells are an alternative source of cells for therapy and
the nanoparticles and compositions of the invention can be targeted
to IPS cells to expand, protect and/or differentiate these cells
for use in cellular therapy in the treatment of NDD and CNS trauma.
Likewise the nanoparticle devices of the invention may be used to
expand or admix with stem cell preparations ex-vivo prior to
introduction into a subject. In such an embodiment of the invention
the stem cells may be adult derived, foetal-derived, derived from
IPS cells, or from any other allogenic
[0115] The invention further provides for combinatorial
compositions that comprise mixtures of populations of nanoparticles
that comprise more than one therapeutic agent per nanoparticle, or
different nanparticles each comprising a different therapeutic
agent, for the treatment of neurodegenerative disease. Such
combinatorial compositions may suitably comprise a pharmaceutically
acceptable carrier solution; at least a first population of
biodegradable nanoparticles, wherein the first nanoparticles
comprise a targeting moiety that is able to bind selectively to the
surface of a neural stem cell and/or a neural progenitor cell and
wherein the first nanoparticles further comprise leukaemia
inhibitory factor (LIF); and at least second population of
biodegradable nanoparticles, wherein the second population of
nanoparticles comprise a targeting moiety that is able to bind
selectively to the surface of a neural stem cell and/or a neural
progenitor cell and wherein the second nanoparticles further
comprise one or more other than LIF. Suitably, the second
nanoparticles may comprise compounds selected from: brain-derived
neurotrophic factor (BDNF); epidermal growth factor (EGF); glial
cell-derived neurotrophic factor (GDNF); ciliary neurotrophic
factor (CTNF); retinoic acid, and derivatives thereof; Wnt5A; and
XAV939.
[0116] The invention is further exemplified in the following
non-limiting examples.
EXAMPLE 1
1.1 Dopaminergic Neurons Derived from E14 Ventral Mesencephalon
(VM) from Rat Foetuses Express the Components of the LIF Receptor
Complex
[0117] The expression of gp130 and gp190, the two components of the
LIF receptor complex (FIG. 1A), on dopaminergic neurons of
embryonic day 14 ('E14') VM was analysed via immunocytochemistry of
E14 VM cultures after 3 days in vitro (DIV) (FIG. 1B). FIG. 1B
shows that both components of the LIF receptor complex are
expressed by dopaminergic neurons in E14 ventral mesencephalon (VM)
cultures. FIG. 1A: The LIF receptor is a heterodimer consisting of
two proteins: gp130 and gp190. FIG. 1B: Immunocytochemistry of 5
day old E14 VM cultures with antibodies against tyrosine
hydroxylase and gp130 or gp190 demonstrated that dopaminergic
neurons express gp130 and gp190. Dopaminergic neurons were
demonstrated to express both gp130 and gp190, suggesting a
potential for responsiveness to LIF treatment.
1.2 LIE Treatment during Tissue Dissociation Increases the
Subsequent Number of Dopaminergic Neurons
[0118] The VM of E14 rat foetuses was dissected and dissociated in
medium with or without 0.1 ng/ml soluble LIF. The tissue was then
plated in monolayer culture and grown for 2, 3 or 5 days prior to
fixing. Dissociated cells were seeded in monolayer cultures and
fixed after 2, 3 or 5 days in vitro (DIV). Culture derived from
cells dissociated in LIF supplemented medium were found via
immunocytochemical analysis to contain significantly more tyrosine
hydroxylase positive neurons after 2 days in vitro but not later
time points. Subsequent immunocytochemistry of fixed culture
demonstrated that cultures derived from tissue dissociated in the
presence of 0.01 ng/ml LIF had significantly more TH+ neurons after
2 days in vitro; this effect was lost at 3 and 5 days in vitro
(FIG. 2).
1.3 Dopaminergic Cell Count in E14 VM Cultures can be Increased by
Supplementing Growth Medium with 0.1 ng/ml Soluble LIE
[0119] The VM of E14 rat foetuses was dissected and dissociated in
standard conditions. Primary E14 VM tissue was dissociated and
grown as monolayer cultures. After plating cells were chronically
treated with soluble LIF in their growth medium ranging from 0.1
ng/ml to 100 ng/ml. Subsequent immunocytochemistry demonstrated
that supplementation of growth medium with 0.01 ng/ml LIF was able
to significantly increase the number of tyrosine hydroxylase
positive neurons after 3 and 5 days in vitro (FIG. 3A and FIG. 3B).
Treatment of E14 VM cultures with all LIF dosages above 0.1 ng/ml
had no significant effect on the number of TH positive neurons.
1.4 Dopaminergic Neurons Express the Glial Cell Line Derived
Neurotrophic Factor Receptor .alpha.1
[0120] Before the effect of nanoparticle treatment on E14 VM
cultures could be investigated it was necessary to identify a cell
surface protein that could be used as a target for antibodies on
the nanoparticle surface. Given the known neurotrophic effect of
glial cell line derived neurotrophic factor (`GDNF`) on
dopaminergic neurons the expression of the GDNF receptor al
(`GDNF-R .alpha.1`) in E14 VM cultures was analysed via
immunocytochemistry with the aim of potentially using this protein
as a nanoparticle target. The monolayer culture was fixed after 5
days in vitro and analysed for expression of GDNFR-.alpha.1 through
immunocytochemistry. Dual staining with tyrosine hydroxylase
demonstrated that individual neurons express both TH and
GDNFR-.alpha.1. Hence, as expected, dopaminergic neurons were found
to express this protein (FIG. 4).
1.5 LIE Nanoparticles Targeted via Antibodies Against GDNF-R
.alpha.1 Increase the Tyrosine Hydroxylase Positive Cell Count in
E14 VM Cultures
[0121] To investigate the effect of LIF nanoparticle treatment on
tyrosine hydroxylase positive cell counts, primary E14 VM was mixed
with LIF nanoparticles (targeted or non-targeted) or empty
nanoparticles (targeted or non-targeted) immediately prior to
plating in monolayer culture. E14 VM tissue was mixed with 100
.mu.l of a 1 mg/ml nanoparticle solution immediately prior to
plating. Nanoparticles were either empty nanoparticles (with or
without surface bound anti-GDNFR-.alpha.1 antibodies) or LIF
nanoparticles (with or without anti-GDNFR-.alpha.1 antibodies).
Immunocytochemical analysis of these cultures after 3 days in vitro
revealed a significant increase in the number of tyrosine
hydroxylase positive neurons in the cultures treated with targeted
LIF nanoparticles. Cultures were fixed after 3 days in vitro and
analysed via immunocytochemistry for tyrosine hydroxylase. Plating
cells with targeted LIF nanoparticles significantly increased the
TH positive cell count at 3 days in vitro Non-targeted LIF
nanoparticles and empty nanoparticles had no effect on the TH+ cell
count (FIG. 5A and FIG. 5B).
1.6 Treatment of E14 VM Derived Neurospheres with 0.1 ng/ml Soluble
LIE has no Effect on Subsequent Differentiation in Monolayer
Culture
[0122] To investigate the effect of LIF treatment on the
differentiation of E14 VM, tissue was grown as neurospheres in
expansion medium with or without 0.1 ng/ml soluble LIF. Primary
ventral midbrain tissue was expanded in medium containing the
mitogens EGF and FGF-2 for 5 days. These neurospheres were then
dissociated into single cells and plated in monolayer culture in
the absence of LIF. After 5 days of growth these cultures were
analysed via immunocytochemistry for neural and astroglial
differentiation (FIG. 6, showing morphology+or -LIF). The presence
of LIF during the expansion of E14 VM had no effect on subsequent
differentiation (FIG. 7 showing results after 5 and 10 days).
1.7 Treatment of E14 VM Monolayer Cultures with Soluble LIF or
Targeted LIF Nanoparticles Reduces Levels of Dopaminergic
Apoptosis
[0123] A subset of tyrosine hydroxylase neurons co-localised with
cleaved caspase-3 and a condensed nucleus, both markers of
apoptotic cells. This indicates that a proportion of dopaminergic
neurons in E14 VM cultures undergo apoptosis during culture (FIG.
8), contributing to the decrease in the number of these neurons as
culture time progresses. Immunocytochemical analysis was performed
to determine whether LIF treatment (soluble or targeted
nanoparticles) decreased the number of apoptotic dopaminergic
neurons in these cultures.
[0124] E14 VM monolayer cultures, treated with soluble LIF or
LIF/empty nanoparticles were fixed after 2, 3 or 5 days.
Immunocytochemical analysis for cells positive for tyrosine
hydroxylase, cleaved caspase-3 (CC-3) and a condensed nucleus
demonstrated a significant reduction in dopaminergic apoptosis. It
was found that LIF treatment resulted in reduced numbers of
apoptotic dopaminergic neurons after 2 days in vitro (FIG. 9). A
trend towards reduced apoptosis in the presence of LIF remained
after 3 days in vitro but did not reach statistical significance.
Together with the finding that LIF does not bias E14 VM towards
neural differentiation, this result suggests the increase in TH+
cells seen with chronic LIF treatment is an effect of increased
dopaminergic cell survival.
1.8 Serotonin Neurons in E14 VM Cultures Express GDNFR-.alpha.1
[0125] Contaminating serotonin neurons in foetal grafts have been
linked to the development of graft-induced dyskinesias (`GIDs`) in
Parkinson's Disease patients. It was therefore of interest to
determine whether LIF treatment had any effect on the number of
serotonin neurons in E14 VM cultures. An E14 VM culture was fixed
after 5 days in vitro and stained with antibodies against
GDNFR-.alpha.1 and serotonin. As a first step, immunocytochemistry
was performed to reveal whether serotonin neurons in these cultures
express GDNFR-.alpha.1, the protein being used to target LIF
nanoparticles. Dual staining for serotonin and GDNFR-.alpha.1
demonstrated that serotonin neurons express GDNFR-.alpha.1 (FIG.
10).
1.9 Anti-Thy-1 Directed Nanotherapy: Either Nanoparticle-Delivered
BDNF, or Nanoparticle-Delivered 7,8 Dihydroxy-Flavone (7,8-DHF)
Improves Yield of TH+ Cells and this is Comparable to Treatment
with Soluble BDNF, or Soluble 7,8-DHF
[0126] To compare the effect of brain-derived neurotrophic factor
(BDNF), or the BDNF agonist 7,8-dihydroxy flavone (7,8-DHF), when
in a nano-particulate formulation targeted to Thy-1, versus free,
primary rat E14 VM tissue was mixed with 100 .mu.l of nanoparticle
solution (0.05mg; 0.1mg; 1.0mg nanoparticles/ml), or with free
growth factor (10nM; 100nM; 1.mu.M; 10.mu.M) immediately prior to
plating. After first confirming presence of Thy-1 antigen on the
surface of TH+ neurons (data not shown), anti-Thy-1 decorated
nanoparticles were prepared as either empty; or BDNF-nanoparticles;
or 7,8 DHF-nanoparticles. Cultures were fixed after 7 days in vitro
and analysed via immunocytochemistry for tyrosine hydroxylase
positive cells. Plating cells with targeted BDNF-, or 7,8
DHF-nanoparticles significantly increased the TH positive cell
count to levels comparable with the effect of free BDNF or 7,8-DHF.
Analysis of cells demonstrated a response to BDNF, and to the
BDNF-agonist 7,8-dihydroxy flavone (7,8-DHF), delivered in
nano-formulation targeted to Thy-1. This is shown for 7,8
DHF-nanoparticles in FIG. 11A and FIG. 11B where the dose-response
curve is similar to that reported by Jang et al (Jang et al, Proc.
Natl. Acad. Sci. USA, 2010), with the exception of the high dose
(10.mu.M) decline observed here.
[0127] The experiment also tested for the effect of BDNF and
7,8-DHF on serotonergic cells versus dopaminergic cells where a
constant ratio was found (FIG. 11C and FIG. 11D). Measurement of
both longest neurite length and number of primary neurites revealed
a significant increase for both parameters following treatment with
BDNF or BDNF-nano (data not shown): unexpectedly, neither soluble
7,8-DHF nor 7,8-DHF-nano altered neurite length or number (FIG.
11E-FIG. 11H).
1.10 Rat fetal VM Grafts Treated Ex Vivo with LIF or BDNF
Nanoparticles Prior to Grafting into the Striatum of Lesioned
Syngeneic Recipients Show no Evidence of Adverse Effects Though do
not Significantly Alter the Response to Amphetamine
[0128] Following transplantation surgery rats in all groups
continued to gain weight. Post-transplantation weight gain was not
affected by nanoparticle supplementation of grafted tissue. Two way
repeated measures ANOVA: significant effect of time
F.sub.1.74,41.75=99.30, p<0.001, no effect of group
F.sub.3,24=1.3, p=0.311, no time x group interaction
F.sub.9,24=0.74, p>0.05. FIG. 13 (upper).
[0129] In the amphetamine-induced rotation assay, there was a
significant reduction in net ipsilateral rotation across all
groups. There was no significant effect of nanoparticle
supplementation on recovery rate in the reduction of amphetamine
induced rotation post-transplant. Two way repeated measures ANOVA:
significant effect of time F .sub.1.7,41.1=18.41, p<0.001, no
effect of group F.sub.3,24=1.89, p=0.158, no time x group
interaction F.sub.9,24=1.21, p>0.05. FIG. 13 (lower).
EXAMPLE 2
2.1 Human: Monolayer and Neurosphere Cultures--Expansion of Cell
Numbers to Provide Sufficient Cells for Testing Therapeutic LIE
Nanoparticles
[0130] 6-8 week old human foetal midbrain was dissected and
cultured as neurospheres in proliferation medium before being
sectioned and stained. Upon passage, parallel cultures as monolayer
were grown in differentiation medium. Dopaminergic cells are
positive for tyrosine hydroxylase (TH). Total cell numbers are
stained with nuclear antigen (Hoechst). See FIG. 12, where:
Primary=primary cultures; Passage 0=first subculture; Passage
1=second subculture.
[0131] Results show (i) maturation of TH+ neurons within the
neurosphere microenvironment; (ii) differentiation of TH+ neurons
grown in monolayer. Passage 2 also contains dopaminergic (DA)
cells. These amplified cells were used to test LIF-nano effects on
DA cell maturation and overall cell survival.
2.2 Human Foetal Ventral Mesencephalon LIF-Nanoparticle Therapy
Enhances Human Dopaminergic Neuron Derivation
[0132] After establishing culture conditions for expansion of
primary human foetal VM cells, these cells were used to test
therapeutic efficacy of the LIF-nano device (see FIG. 14). Cultures
were stained for tyrosine hydroxylase (TH) after 5 days in culture
in the presence or absence of LIF nanoparticles targeted to Thy-1.
Three sets of experiments were completed. A dose of 1/100
LIF-nanoparticles targeted to Thy-1 resulted in both (i) 3-fold
increase in overall cell numbers and (ii) a percentage fold
increase of 2.5% TH+ cells within this overall cell population.
Thus, there was a greater than 5-fold increase of dopaminergic
cells as a result of LIF-nano therapy (see FIGS. 15 and 16).
EXAMPLE 3
3.1 Treatment of Human Foetal Ventral Mesencephalon (hfVM) with
LIF-Nanoparticle Therapy, or XAV939-Nanoparticle Therapy, Enhances
Human Dopaminergic Neuron Derivation and Increases hfVM Cell
Survival both In Vitro and In Vivo
[0133] To measure the effect of nanotherapy in vivo: hfVM cells
were prepared as for the in vitro experiments as outlined in FIG.
17, primary human fetal mesencephalon tissue was stored at
4.degree. C. for upto 4 days in Hibernate E storage medium. The
cells were then seeded on coverslips and cultured 4d in
differentiation medium after which cells were stained by DAPI to
enumerate nuclei and for tyrosine hydroxylase to identify and
enumerate differentiated dopaminergic cells. Pooled tissue was then
prepared for cell transplantation following the clinical TransEuro
Protocol. The protocol summarised in FIG. 18 follows that of the
TransEuro clinical trial assessing hfVM cell grafts as cell therapy
in patients with Parkinson's disease: http://www.transeuro.org.uk.
The harvested cells were divided into four aliquots in
proliferation medium and treated with nanoparticles targeted to
Thy-1 and carrying a cargo of (i) no cargo; (ii) LIF; (iii) XAV939;
or (iv) retinoic acid for upto 24 h. The cells were then
transplanted in to the striatum of nude rats aged between 12 -16
weeks following the protocol in FIG. 22 using standard techniques.
At 3 months the rats were perfused with BrdU according to standard
protocols and then culled when the brain was harvested and
sectioned for immune-cytochemical analysis.
[0134] Human nuclear antigen specific antibody (HuNu) stained
transplanted human cells: tyrosine hydroxylase staining revealed
human dopaminergic (DA) cells within the grafts (FIGS. 19, 20, 21
and 22). Beta III tubulin stained neurons, and BrdU identified any
dividing cells post infusion and pre-cull. Numbers and localisation
of cells were identified following image capture (Imagescope
Aperio). The results show highly significant increased survival and
distribution of transplant-derived neurons and DA cells in the
striatum of rats receiving grafts pretreated with LIF-nano, or with
XAV939-nano, when compared to the empty-nano controls. In
particular the results quite clearly show the surprising and
beneficial effects of nanoparticle devices of the invention (see
FIGS. 19(C) and 20(B)) on cell survival and differentiation in the
brain compared to control (see FIG. 22(C)).
[0135] In vitro experiments paralleled the above in vivo study, but
instead of transplantation, the cells were seeded onto coverslips
in differentiation medium and grown for 4 days, fixed, and stained
for DA cells and total neurons. Here six groups of nanoparticles
were tested namely empty-nano; LIF-nano; XAV939-nano;
LIF+XAV939-nano; BDNF-nano; and GDNF-nano; and the results are
shown in FIGS. 23, 24 and 25. All cargos promoted cell survival
with increased numbers of TH+ cells compared to the empty
nanoparticle control group (Em-NP).
EXAMPLE 5
5.1 Preparation of Surface Targeted Nanoparticles Containing hLIF,
mLIF or XAV939
[0136] Human LIF (Santa Cruz cat. SC-4377), mouse LIF (Santa Cruz
cat. SC-4378), or XAV939 (Sigma Aldrich cat. X3004) was
encapsulated in avidin-coated PLGA nanoparticles using a modified
water/oil/water double emulsion technique.
[0137] Briefly, 50 .mu.g of cytokine was dissolved in 200 .mu.L PBS
or 1 mg of XAV939 dissolved in DMSO at a concentration of 10 mg/ml
(100 .mu.l) was added dropwise with vortexing to 100 mg PGLA (50/50
monomer ratio, Durect Corp. cat. B0610-2) in 2 ml dichloromethane.
The resulting emulsion was added to 4 ml of aqueous surfactant
solution containing 2.5 mg/ml polyvinyl alcohol (PVA)
(Sigma-Aldrich cat. 363138) and 2.5 mg/ml avidin-palmitate
bioconjugate (see 5.2 below), and sonicated to create an emulsion
containing nano-sized droplets of polymer/solvent, encapsulated
cytokine and surfactant. Solvent was removed by magnetic stirring
at room temperature; hardened nanoparticles were then washed
3.times. in DI water and lyophilized for long-term storage.
[0138] Targeted nanoparticles were formed by reacting the
avidin-coated NPs in PBS with 4 biotin-antibody (0.5 mg/ml) per mg
NP for 15 minutes and used immediately. Nanoparticle size and
morphology are analyzed via scanning electron microscopy and
dynamic light scattering in 1.times. PBS (Brookhaven Instruments,
ZetaPALS). Drug or cytokine release was measured by incubating
particles in PBS at 37.degree. C. and measuring cytokine or drug
concentrations in supernatant fractions by ELISA or UV
Spectroscopy. Total encapsulation was approximated as the amount of
LIF or XAV939 released over a seven day period and percent
encapsulation efficiency calculated as total encapsulation divided
by maximum theoretical encapsulation. Capture of biotinylated
ligands was quantified using biotin-R-phycoerythrin as a model
protein. NPs were suspended at 1.0 mg/ml in 1.times. PBS, and 200
.mu.l added to eppendorfs containing varying concentrations of
biotin-R-PE. NPs were reacted for 15-30 minutes at room
temperature, centrifuged for 10 minutes at 12k RPM, and the
remaining biotin-R-PE in the supernatant quantified by fluorescence
at excitation/emission 533/575 nm.
5.2 Preparation of the Avidin Palmitate Bioconjugate for use in
Surface Modification of Biodegradable Nanoparticles
[0139] Stable avidin-lipid conjugates were formed using a
zero-length crosslinking agent to create a covalent bond between
the lipid carboxyl end groups and free amines on the avidin
protein. Fatty acid (Palmitic acid, Sigma) was first reacted in
0.1.times. PBS with 1-ethyl-3 [3-dimethylaminopropyl] carbodiimide
(EDC) and N-hydroxylsulfosuccinimide (sulfo-NHS) (Invitrogen) to
convert the terminal carboxyl group to an amine-reactive sulfo-NHS
ester. Avidin (Sigma) at 5 mg/ml was then reacted with 10-fold
molar excess of the NHS-functionalized fatty acid in 0.1.times. PBS
and the solution was gently mixed at 37.degree. C. for 2 hours.
Reactants were then dialyzed against 1.0.times. PBS at 37oC for 24
hours to remove excess reactants and/or hydrolyzed esters.
[0140] The above protocol may be adapted for encapsulation of the
other compounds described herein.
[0141] Although particular embodiments of the invention have been
disclosed herein in detail, this has been done by way of example
and for the purposes of illustration only. The aforementioned
embodiments are not intended to be limiting with respect to the
scope of the appended claims, which follow. It is contemplated by
the inventors that various substitutions, alterations, and
modifications may be made to the invention without departing from
the spirit and scope of the invention as defined by the claims.
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[0160] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0161] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References