Note: Descriptions are shown in the official language in which they were submitted.
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Compositions and methods for protection and/or repair
of the nervous system
Field of the Invention
[0001] The present application claims priority from Australian Patent
Application No.
2015904055, filed on 6 October 2015.
[0002] The present invention relates to compositions and methods for
protecting
and/or promoting repair of the nervous system. In particular, the present
invention
relates to the use of muramyl dipeptide crosslinked to form a microparticle
for protecting
and/or promoting repair of the nervous system.
Background of the Invention
[0003] Any discussion of the prior art throughout the specification should
in no way
be considered as an admission that such prior art is widely known or forms
part of
common general knowledge in the field.
[0004] The nervous system consists of two main parts, the central nervous
system
(CNS) and the peripheral nervous system (PNS). The CNS contains the brain and
spinal cord, while the PNS consists mainly of nerves, which connect the CNS to
every
other part of the body.
[0005] At the cellular level, the nervous system contains neurons and glial
cells.
Neurons have special structures that allow them to send signals rapidly and
precisely to
other cells. The signals are in the form of electrochemical waves that travel
from the
body of the neuron along an axon to other neurons. Glial cells provide
structural and
metabolic support for neurons. Glial cells include oligodendrocytes, which
insulate
axons by wrapping around them and forming a myelin sheath, and astrocytes,
which
provide structural and metabolic support.
[0006] Damage or injury to the nervous system leads to abnormal
neurological
function, including inability to speak, decreased touch sensation, loss of
balance,
weakness, mental function problems, visual changes, abnormal reflexes, and
walking
problems.
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[0007] Damage or injury to the nervous system may be the result of a
variety of
traumas or diseases, including genetic defects (e.g., Huntington's disease and
muscular
dystrophy), developmental problems (e.g., spina bifida), physical trauma,
degeneration
(e.g., Parkinson's disease and Alzheimer's disease), loss of blood supply
(e.g., stoke),
infection (e.g., meningitis), seizures (e.g., epilepsy) and cancer (e.g.,
brain tumours).
[0100] Damage or injury to the nervous system results in disruption of the
precisely
controlled microenvironment, resulting in tissue damage and often leading to a
lack of
significant functional repair and nerve regeneration. When the CNS is injured
by
trauma or disease, a cascade of secondary damage ensues. Vascular, cellular
and
chemical responses to the injury include tissue inflammation, reduced blood
flow and
scar formation. Demyelination occurs on injured axons, slowing the conduction
of nerve
impulses and stripping axons of protection against further damage.
[0101] There is a need for agents that protect the nervous system from
trauma or
disease. There is also a need for agents that promote the repair of the
nervous system
following trauma or disease.
[0008] It is an object of the present invention to overcome or ameliorate
at least one
of the disadvantages of the prior art, or to provide a useful alternative.
Summary of the Invention
[0009] It has been surprisingly found that muramyl dipeptide crosslinked to
form a
microparticle (MDP microparticle) enhances motor function following
demyelination
and/or spinal cord injury, increases the levels of neuroprotective/repair
factors and
decreases macrophage activation.
[0010] According to another aspect, the present invention provides use of
muramyl
dipeptide crosslinked to form a microparticle (MDP microparticle) for the
manufacture of
a medicament for the prophylactic and/or therapeutic treatment of neural
injury in a
subject.
[0011] According to a further aspect, the present invention provides a
composition
comprising muramyl dipeptide crosslinked to form a microparticle (MDP
microparticle)
for use in the prophylactic and/or therapeutic treatment of neural injury in a
subject.
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[0012] According to one aspect, the present invention provides a method for
the
prophylactic and/or therapeutic treatment of neural injury in a subject,
comprising
administering to the subject a composition comprising murannyl dipeptide
crosslinked to
form a microparticle (MDP microparticle).
[0013] In one embodiment, the MDP microparticle comprises DNA fragments.
[0014] In another embodiment, the MDP microparticle is isolated from
macrophages.
[0015] In another embodiment, the neural injury is spinal cord injury.
[0016] In another embodiment, the neural injury is demyelination.
[0017] In another embodiment, the composition or medicament increases the
levels
of one or more neuroprotective/repair factors in the subject.
[0018] In another embodiment, the neuroprotective/repair factors are
selected from
vascular endothelial growth factor (VEGF), insulin growth factor-1 (IGF-1),
hepatocyte
growth factor (HOE) and erythropoietin (EPO).
[0019] In another embodiment, the composition or medicament reduces
macrophage
activation.
[0020] In another embodiment, the composition or medicament reduces the
number
of macrophages expressing MHC class II.
[0021] In another embodiment, the composition or medicament decreases the
level
of expression of MHC class II on macrophages.
[0022] In another embodiment, the composition or medicament comprises one
or
more pharmaceutically-acceptable excipients, carriers, vehicles or diluents.
[0023] In another embodiment, the MDP microparticle is not encapsulated in
a
liposome.
[0024] In another embodiment, the composition or medicament is administered
or
administrable to the subject parenterally.
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[0025] In another embodiment, the composition or medicament is
administered or
administrable to the subject intravenously.
[0026] In another embodiment, the composition or medicament is
administered or
administrable to the subject orally.
[0027] In another embodiment, the composition or medicament is
administered or
administrable to the subject at a dosage of about lpg to about 100pg of MDP
microparticles.
[0028] In another embodiment, the composition or medicament is
administered or
administrable to the subject at a dosage of about 100pg to about 1000pg of MDP
microparticles.
[0029] In another embodiment, the composition or medicament is
administered to the
subject at a dosage of about 100pg to about 700pg of MDP microparticles.
[0030] In another embodiment, the composition or medicament is
administered or
administrable to the subject at a dosage of about 300pg to about 700pg of MDP
microparticles.
[0031] In another embodiment, the composition or medicament is
administered or
administrable to the subject at a dosage of about 500pg to about 700pg of MDP
microparticles.
[0032] In another embodiment, the composition or medicament is
administered or
administrable to the subject at a dosage of about 1, 25, 50, 75, 100, 125,
150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,
600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000
pg of
MDP microparticles.
[0033] In another embodiment, the composition or medicament is
administered or
administrable to the subject once a day.
[0034] In another embodiment, the composition or medicament is
administered or
administrable to the subject once a week.
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[0035] In another embodiment, the composition or medicament is administered
or
administrable to the subject once a fortnight.
[0036] In another embodiment, the composition or medicament is administered
or
administrable to the subject once a month.
[0037] The appropriate dosage of MDP microparticles, total amount
administered
and duration of administration can be easily determined by a medical
practitioner based
on guidance provided herein, the nature and severity of the disease to be
treated, and
the response by the subject to the treatment. As an example, useful individual
dosages
may be selected from the range 1pg to 1000pg of MDP microparticles, and may be
administered once a day, once a week, once a fortnight or once month depending
on
the subject's condition, symptoms, tolerance and response to treatment. Doses
in a
higher range can also be used depending on the requirements, for examples
doses in
the range of 1000pg to 1500pg of MDP microparticles. Dosages at other
frequencies
may also be employed. An example of a suitable dosage regimen could be to
start with
an initial dose of 100 pg followed by escalated doses until appropriate
beneficial
therapeutic effects are observed in the subject, without significant side-
effects. The
dosage may be given as single bolus dose or infused overtime, or given in
divided
doses. The total amount of MDP microparticles administered will depend on
subject
response and tolerance to treatment. The composition may be administered once
a
day, once a week, once a fortnight or once a month for a total period that
depends on
the subject's response.
[0038] MDP microparticle-containing compositions or medicaments may be
suitable
for oral, nasal, topical (including buccal and sublingual), rectal, vaginal,
aerosol and/or
parenteral administration. The compositions may conveniently be presented in
unit
dosage form and may be prepared by any methods well known in the art of
pharmacy.
The amount of MDP microparticle-containing composition which may be combined
with
a carrier material to produce a single dose may vary depending upon the
subject being
treated, and the particular mode of administration.
[0039] The MDP microparticle-containing compositions or medicaments may be
administered alone or in combination with pharmaceutically acceptable
excipients,
carriers, vehicles or diluents, in either single or multiple doses. Suitable
pharmaceutical
acceptable excipients, carriers, vehicles and diluents include inert solid
diluents or
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fillers, sterile aqueous solutions and various organic solvents. The
compositions formed
by combining the MDP microparticle-containing compositions and the
pharmaceutically
acceptable excipients, carriers, vehicles or diluents are then readily
administered in a
variety of dosage forms such as tablets, powders, lozenges, syrups, injectable
solutions
and the like. These pharmaceutical compositions can, if desired, contain
additional
ingredients such as flavourings, binders, excipients and the like. Thus, for
purposes of
oral administration, tablets containing various excipients such as L-arginine,
sodium
citrate, calcium carbonate and calcium phosphate may be employed along with
various
disintegrates such as starch, alginic acid and certain complex silicates,
together with
binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
Additionally,
lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc
are
often useful for tabletting purposes. Solid composition of a similar type may
also be
employed as fillers in soft and hard filled gelatin capsules. Appropriate
materials for this
include lactose or milk sugar and high molecular weight polyethylene glycols.
When
aqueous suspensions or elixirs are desired for oral administration, the
essential active
ingredient therein may be combined with various sweetening or flavouring
agents,
colouring matter or dyes and, if desired, emulsifying or suspending agents,
together
with diluents such as water, ethanol, propylene glycol, glycerin and
combinations
thereof. The MDP microparticle-containing compositions may also comprise
enterically
coated dosage forms.
[0040] Suitable formulation protocols and suitable excipients, carriers,
vehicles and
diluents can be found in standard texts such as Remington: The Science and
Practice
of Pharmacy, 19th Ed, 1995 (Mack Publishing Co. Pennsylvania, USA), British
Pharmacopoeia, 2000, and the like.
Definitions
[0041] In the context of the present invention, the terms "muramyl
dipeptide
crosslinked to form a microparticle" and "MDP microparticle" refer to a
microparticle
formed by crosslinked repeats of muramyl dipeptide (MDP), wherein the MDP
repeats
are crosslinked to each other. The MDP microparticle may also contain DNA
fragments
and/or other agents that stimulate and/or regulate the immune system.
[0042] In the context of the present invention, the words "comprise",
"comprising"
and the like are to be construed in their inclusive, as opposed to their
exclusive, sense,
that is in the sense of "including, but not limited to".
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[0043] In the context of the present invention, the terms
"treatment" or "treating"
include preventing a disease, disorder or condition from occurring in an
animal which
may be predisposed to the disease, disorder and/or condition but has not yet
been
diagnosed as having it; inhibiting the disease, disorder or condition, e.g.,
impeding its
progress; and relieving the disease, disorder, or condition, e.g., causing
regression of
the disease, disorder and/or condition. Treating the disease or condition
includes
ameliorating at least one symptom of the particular disease or condition, even
if the
underlying pathophysiology is not affected.
[0044] In the context of the present invention, the phrase
"therapeutically effective
amount" refers to an amount of MDP microparticles that produces some desired
effect
at a reasonable benefit/risk ratio applicable to any medical treatment. In
certain
embodiments, the term refers to that amount necessary or sufficient to
eliminate or
reduce medical symptoms for a period of time. The effective amount may vary
depending on such factors as the disease or condition being treated, the
particular
targeted constructs being administered, the size of the subject, or the
severity of the
disease or condition. One of ordinary skill in the art may empirically
determine the
effective amount of a particular composition without necessitating undue
experimentation. In certain embodiments, compositions are formulated in a
manner
such that they will be delivered to a patient in a therapeutically effective
amount, as part
of a prophylactic or therapeutic treatment. The desired amount of the
composition to the
administered to a patient will depend on absorption, inactivation and
excretion rates of
the MDP microparticles, as well as the delivery rate of the MDP
microparticles.
[0045] In the context of the present invention, the phrase
"parenteral administration"
as used herein refer to modes of administration other than enteral and topical
administration, such as injections, and include without limitation
intravenous,
intramuscular, intrapleural, intravascular, intrapericardial, intraarterial,
intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal and
intrasternal injection and infusion.
[0046] In the context of the present invention, the phrase
"pharmaceutically
acceptable" is art-recognized. In certain embodiments, the term includes
compositions,
polymers and other materials and/or dosage forms which are, within the scope
of sound
medical judgment, suitable for use in contact with the tissues of mammals,
human
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beings and animals without excessive toxicity, irritation, allergic response,
or other
problem or complication, commensurate with a reasonable benefit/risk ratio.
[0047] In the context of the present invention, the phrase "neural injury"
means injury
to the tissue or cells of the nervous system.
[0048] In the context of the present invention, the phrase "nervous system"
encompasses the central nervous system and the peripheral nervous system.
Brief Description of the Figures
[0049] Figure 1: MIS416 treatment enhances motor function recovery in the
cuprizone dennyelination model when administered in the recovery/repair phase.
[0050] Figure 2: MIS416 treatment increases the levels of vascular
endothelial
growth factor (VEGF), Insulin growth factor-1 (IGF-1), hepatocyte growth
factor (HGF)
and erythropoietin (EPO) in human peripheral blood plasma samples.
[0051] Figure 3: Basso Mouse Scale measure in mice with spinal cord injury
(SCI) on
day 1, 3 and 7 following injury. MIS416 (3 mg/Kg or 6 mg/Kg) or saline was
administered systemically on day 1 post injury. In the saline treated SCI
group there is
minimal recovery by day 7 post SCI. In contrast, both MIS416 treated groups
show
locomotive recovery by day 7, with statistical significance reached in the 6
mg/kg
MIS416 treated group compared to saline control.
[0052] Figure 4: Flow cytometric analysis of the spinal cords from mice
harvested at
day 7 post SCI. Figure 4A shows that macrophage activation is clearly evident
in saline
treated SCI mice compared to healthy controls, based on the % of total
macrophages
expressing MHC class II. Both MIS416 treated groups show a clear reduction in
the
proportion of activated macrophages relative to the saline/SCI treated group.
Figure 4B
shows lower expression levels of MHC class II on MHC II class + macrophages
compared to the saline/SCI treated group.
Preferred Embodiment of the Invention
[0053] Although the invention has been described with reference to certain
embodiments detailed herein, other embodiments can achieve the same or similar
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results. Variations and modifications of the invention will be obvious to
those skilled in
the art and the invention is intended to cover all such modifications and
equivalents.
[0054] The present invention is further described by the following non-
limiting
examples.
Examples
Example 1: Preparation of MIS416
[0055] Prop/on/bacterium acnes was grown to a mid-stationary growth phase and
washed to remove contaminants of bacterial culture origin by employing
techniques well
known to those in the art. Hydrophobic components contained in the cell walls
and
cytoplasm were sequentially extracted by successive washes with increasing
concentrations of ethanol/isopropanol/water (10%:10%:80%, 25%:25%:50% and
40%:40%:20%) at elevated temperatures. The isopropanol was then removed with
successive washes with decreasing concentrations (80%, 50%, 40% and 20%) of
ethanol at elevated temperatures. The resultant microparticles (MIS416) were
then
suspended in 6M guanidine-HCI and then washed in water for irrigation and its
concentration measured by relating its absorbance at 565 nm to the absorbance
of
turbidity standards.
[0056] MIS416 contains extensively crosslinked MDP, amino-linked L-alanine-
D-
isoglutamine dipeptides and bacterial DNA fragments. The MIS416 generated by
the
present methods can have a broad range of sizes (for example, 0.01 to 30
microns) but
the most common size range is from 1 to 7 microns. The preferred size is in
the range
of 0.5 to 3 microns.
[0057] MIS416 can be isolated from natural sources, as described above, or
synthesized using well-known synthetic procedures (see, e.g., Liu et al.,
Bioorganic and
Medicinal Chemistry Letters, 10 (12), 2000, pp. 1361-1363(3); Schwartzman &
Ribi,
Prep Biochem. 1980; 10(3): 255-67; Ohya et al. Journal of Bioactive and
Compatible
Polymers, 1993; 8: 351-364).
[0058] The concentration of MIS416 was adjusted to 0.2 mg/mL in sodium
chloride
for intravenous administration.
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Example 2: Effect of MIS416 on early remyelination
[0059] The effect of MIS416 therapy on early remyelination was examined in
the
Cuprizone demyelination model. C57/BI6 mice (n=7 or 8 per group) were fed
continuously ad libitum a diet of 0.3 A (w/w) cuprizone [oxalic bis-
(cyclohexylidenehydrazide); Sigma¨Aldrich] mixed into chow pellets for 6
weeks. After
6 weeks of cuprizone feeding, the mice were fed normal mouse chow to allow
spontaneous remyelination to progress (recovery phase) for 2 weeks. To study
the
effect of MIS416 treatment on the recovery phase, 100 pg/mouse or 200 pg/mouse
was
administered intravenously on a weekly dosing cycle at weeks 5, 6, 7 and 8
weeks, the
first dose overlapping with the last week of cuprizone feeding.
[0060] Cuprizone specifically causes the death oligodendrocytes, which are
the
myelinating cells within the central nervous system. Their main function is to
provide
support to axons and also to produce the myelin sheath, which serves to
insulate the
axons. Loss of oligodendrocytes as a result of cuprizone exposure, leads to
progressive loss of motor function.
[0061] The effect of cuprizone demyelination on neurological function was
measured
weekly, using the rotarod test that measures motor control, coordination and
balance.
All rotarod assessments were performed blinded.
[0062] The results show that MIS416 treated animals demonstrated a steady
increase in motor function during the recovery phase (week 6 onwards). This is
in
contrast to untreated animals, where there was little evidence of spontaneous
recovery
at the early stage of remyelination (Figure 1).
Example 3: Effect of MIS416 on levels of neuroprotective/repair factors
[0063] The effect of MIS416 treatment on the levels of
neuroprotective/repair factors
was investigated in peripheral blood plasma samples from patients
participating in a
phase 1b/2a clinical trial for progressive MS. Human trial patient blood
plasma was
collected at baseline and at 24 hr post MIS46 dosing (500 pg/dose) and stored
at -80 C
until analysis for the levels of neuroprotective factors. Levels of vascular
endothelial
growth factor (VEGF), Insulin growth factor-1 (IGF-1) and hepatocyte growth
factor
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(HGF) were determined using flow cytometry cytokine bead arrays (Antigenix),
and
erythropoietin (EPO) was quantified using standard ELISA (Cusbio Asia).
[0064] At 24 hr post MIS416 dosing, there were elevated plasma levels of
VEGF,
EPO, IGF-1 and HGF compared to pre-treatment levels (Figure 2). VEGF has been
shown to have neurotrophic and neuroprotective effects on neurons and glial
cells
(Zachary 2005, Neuro-Signals, 14(5): 207-21; Nicoletti et al 2008,
Neuroscience,
151(1): 232-41), as well as promoting angiogenesis in demyelinated lesions
(Girolamo
et al 2014, Acta Neuropathologica Communications, 2: 84). IGH-1 has been shown
to
promote the survival of neurons (Heck et al 1999, Journal of Biological
Chemistry,
274(14): 9828-35) and its levels are increased in repair responses (Mangolia
et al 2014,
BioMed Research International, Article ID 736104). HCF has mitogenic,
motogenic,
morphologic, anti-apoptotic activities and neurotrophic activities, and has
been shown to
prevent cell death (Kadoyama et al 2011, Current Drug Therapy, 6(3): 197-206).
EPO
decreases the development of pro-inflammatory cytokines and provides trophic
support
to enable tissue regeneration (Brines & Cerami 2008, Journal of Internal
Medicine,
264(5): 405-32). EPO has been shown to enhance nerve recovery after spinal
trauma
(Celik et al 2002, Proceedings of the National Academy of Sciences of the
United
States of America, 99(4): 2258-63) and to have a role in neurogenesis and post-
stroke
recovery (Tsai et al 2006, Journal of Neuroscience, 26(4): 1269-74).
Accordingly,
these results demonstrate that administration of MIS416 results in the
elevation of
neuroprotective/repair factors.
Example 4: MIS416 toxicology studies
[0065] It has been well established that free/soluble MDP has significant
toxicity in
vivo. Attempts to reduce MDP toxicity have employed procedures to delay
release,
such as MDP incorporation into liposomes or other related compounds, or
modification
of terminal groups. Chemical modification has resulted in marked reduction in
activity,
and designs which change delivery rate have been difficult to control.
[0066] In vivo toxicology studies for MIS416 were performed as summarized
in
Table 1.
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Study ID. Study Title Method Quantity of Outcome
MIS416
G6121 MIS416: MIS416 was administered 10,000, 15,000 Maximum
tolerated
Acute as single escalating 30,000 and 45,000 dose (MTD)
Toxicology doses. Animals were mcg/kg body weight established as
Study by IV monitored for toxic signs 15,000 mcg/kg body
route in and mortality up to day 15 weight
Swiss Albino and subjected to detailed
Mice necropsy at terminal
sacrifice on day 15
G6122 MIS416: MIS416 was administered 50, 200, 800 and MTD
established as
Acute as single escalating 3200 mcg/kg body 3,200 p.g/kg body
Toxicology doses. Animals were weight (rabbits) weight
Study by IV monitored for toxic signs
route in New and mortality up to day 15
Zealand and subjected to detailed
White necropsy at terminal
Rabbits sacrifice on day 15
G6123 MIS416: MIS416 doses selected 1,000 3,000 and The no-
observed-
Four Week from acute toxicology 10,000 mcg/kg adverse-effect-
level
Repeated study were administered body weight (mice) (NOAEL) was
Dose twice weekly for 4 weeks. considered to be
Toxicology Animals were subjected to 1,000 mg/kg (or a
Study by IV detailed necropsy at total weekly dose of
Route in terminal sacrifice. 2,000 mcg/kg/week)
Swiss Albino
Mice
G6124 MIS416: MIS416 doses selected 50, 500 and 5,000 The NOAEL was
Four Week from acute toxicology mcg/kg body weight considered to
be 50
Repeated study were administered mcg/kg injected twice
Dose twice weekly for 4 weeks. weekly (100
Toxicology Animals were subjected to mcg/kg/week)
Study by IV detailed necropsy at
Route in New terminal sacrifice.
Zealand
White
Rabbits
1370-002 MIS416: A MIS416 was administered 20, 200, 1000 NOAEL in
this study
26-Week IV once weekly for 26 mcg/kg body weight could be
considered
Toxicity weeks. Animals were as being close to 20
Study In subjected to detailed mcg/kg for the
Rabbits necropsy at terminal purpose of
sacrifice. A one month estimation of human
recovery arm comprised 2 safety margins
animals/sex/group.
Table 1 Summary of toxicology studies
[0067] The toxicity studies, conducted in mice and rabbits for up to 26
weeks
duration, provide adequate safety margins to support long term clinical
studies at
dosage levels in patients up to 20 mcg/kg/week.
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[0068] The toxicity studies show that MI5416 has significantly lower
toxicity than
free/soluble MDP.
Example 5: Determination of the ability of MIS416 to enhance functional
recovery
following spinal cord injury
[0069] Mice were anesthetized by IP injection of 50/50 mixture of xylazine
(10 mg/kg,
Troy laboratories Pty Ltd, Australia) and zoletil (Tiletamine/Zolazepam) (50
mg/kg,
Virbac Australia Pty Ltd, Australia). The hair of the dorsal thoracic
vertebrae was
shaved and bare skin was wiped with saline and sterilized with chlorhexidine
and 70%
alcohol. The animal was located in prone position on the fixation plate of a
IH-0400
impactor (Precision Systems and Instrumentation (PSI), LLC, USA). Aseptic
procedure
was maintained during surgery. A vertical incision (-2 cm) was performed over
the
laminecomy site extending from about thoracic vertebrae T6 to T11 using a type
#11
scalpel blade. The fat tissue was located towards the upper vertebrae (around
T7
upwards) and was carefully separated on both sides to locate a large vessel
(superior
vena cava) in that vicinity to prevent potentially fatal bleeding. The
paravertebral
muscles on both right and left sides of the vertebrate column were dissected
with #5
forceps from T10 towards T8. All the muscle flesh in the middle was cut away
and the
spinous processes of the vertebrae were exposed. T8 and T10 vertebrate were
localized as T7, 8 & 9's spinous processes are in caudal direction, T10's is
almost in
posterior direction, and there is a bigger gap between T10 and T11's spinous
processes
compared to T10 and T9. T9 lanninectomy was performed by first holding T9's
spinous
process with Adson forceps and removing the lamina bone on both sides with #2
forceps, then holding T8's spinous process with Adson forceps and removing
T9's
spinous process.
[0070] After laminectomy, the vertebral column was stabilized by rostral
and caudal
clamping of T8 and T10's transverse process with Adson microforceps on the
impactor's fixation plate. The mice were placed under the impactor and the tip
was
adjusted by first lowering the tip as close as possible to the spinal cord,
then bringing
the tip upwards by three counter-clockwise turns of the position sensor. Using
the IH
impactor software, a 70 kilodyne-controlled force was applied to the spinal
cord by IH-
0400 impactor's 1.3 mm diameter probe. Animals were moved away from the
impactor,
the paravertebral muscles and superficial fascia on both sides of the incision
were
sutured to cover the injury site, the wound was cleaned with saline, and the
skin was
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clipped. There was no significant difference in delivered force and tissue
displacement
between experimental groups.
[0071] Mice were given Hartman's solution (25 pL/g) supplemented with
gentamycin
(1 mg/kg, Troy laboratories Pty limited, Australia) subcutaneous prior to
surgery as
premedication for possible dehydration during surgery. Animals were visually
monitored
every 10-15 min after surgery till the reflexes were returned and every 15-30
min till
they were fully recovered. Mice were left in recovery room on the heating pad
(half on,
half off) overnight and moved to the animal holding room the next day. For the
first
week post surgery mice were weighed and given a dose of subcutaneous
gentamycin
(1 mg/kg) on daily basis. The bladder was manually expressed twice daily.
Impaired
mice were not picked/handled by tail for the duration of the experiment and
were
supplied with unlimited access to food and water/gel.
[0072] MIS416 at 3 mg/kg or 6 mg/kg was administered systemically via retro-
orbital
injection, following isoflurane anesthetization at 24 hr following SCI
procedure.
[0073] The Basso Mouse Scale (BMS) for locomotion scoring system, a well-
accepted clinical measure of locomotion (Basso et al 2006, Journal of
Neurotrauma
23(5):635-59) was used to assess the locomotor recovery post spinal cord
injury. To
assess the effects of injury, two blinded assessors scored mice independently
at days
1,3, and 7 post injury.
[0074] Mice were sacrificed at day 7 post injury. The mice were perfused
with PBS
transcardially and the whole spinal cord was isolated and mononuclear cells
were
extracted for flow cytometry analysis. Briefly, the spinal cord tissue was cut
in small
pieces and digested with DNAase and Collagenase at 37 C for 45 min. The tissue
then
was homogenized by passing through 70 pm sieves and mononuclear cells were
separated 33% percoll gradient and were collected at the bottom of the tube.
The cells
were labelled with the following antibody-fluorophore conjugates (Biolegend,
USA):
CD45-BV510, CD115-AF488, CD11b-BV421, CD11c-PE/Dazzle 594, MHC class 11 (IA-
IE)-APC/Cy7, CX3CR1-AF647, CCR2-PE, GR1-PerCp/Cy5.5, and acquired using the
Fortessa X20 flow cytometer (BD Biosciences, USA). The data was analysed with
FlowJo software (Treestar, USA). Peripheral blood-derived macrophages in the
spinal
cord were distinguished from other peripheral blood-derived myeloid cells and
from
microglia based on the following phenotype: CD45HICD1Ib+CCR2L0CX3CR1+Gri
CA 02926507 2016-04-06
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CD11e. The activation status of these cells was determined by the extent of
MHC
class ll co-expression.
[0075] Figure 3 shows the Basso Mouse Scale measure in mice with spinal
cord
injury (SCI) on day 1, 3 and 7 following injury. MIS416 (3 mg/kg or 6 mg/kg)
or saline
was administered systemically on day 1 post injury. In the saline treated SCI
group
there is minimal recovery by day 7 post SCI. In contrast, both MIS416 treated
groups
show locomotive recovery by day 7, with statistical significance reached in
the 6 mg/kg
MIS416 treated group compared to saline control.
[0076] Figure 4 shows the results from flow cytometric analysis of the
spinal cords
from mice harvested at day 7 post SCI to quantify activated peripheral-blood
derived
macrophages in response to injury. Macrophage activation in the spinal cord
parenchyma is a direct response to the inflamed spinal cord microenvironment.
Activated macrophages are sources of pro-inflammatory cytokines and
neurotoxins.
Macrophages that are less inflammatory are more able to support repair
pathways.
Macrophage activation is clearly evident in saline treated SCI mice compared
to healthy
controls, based on the % of total macrophages expressing MHC class ll (Figure
4A).
Both MIS416 treated groups show a clear reduction in the proportion of
activated
macrophages relative to the saline/SCI treated group. This finding is
supported the
lower expression level of MHC class II on MHC class II+ macrophages compared
to the
saline/SCI treated group (Figure 4B)
