Note: Descriptions are shown in the official language in which they were submitted.
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PERINATAL HYPOXIC-ISCHEMIC BRAIN DAMAGE TREATMENT
This application claims the benefit, under 35 U.S.C. ~ 119(e) of United
States Provisional Application Serial No. 60/527,056, filed December 3, 2003
and United
States Provisional Application Serial No. 60/489,198, filed July 21, 2003,
which are
herein both incorporated by reference in their entirety.
Field of the invention
This invention relates to methods of treatment and prevention of perinatal
hypoxic-ischemic brain damage.
BACKGROUND OF THE INVENTION
Perinatal hypoxic-ischemic brain damage is a major cause of acute
mortality and chronic neurological morbidity in infants and children. The
condition is
particularly prevalent in low birth weight (premature) newborns. The basic
cause is a
reduction or interruption in the supply of oxygen to the brain of the fetus or
newborn,
commonly as a result of diminished placental perfusion, uteroplacental
insufficiency or
umbilical cord compression, and complications of delivery in the newborn,
leading to
asphyxiation. Pre-exposure to an inflammatory insult such as infection makes
the infant
more susceptible to hypoxic-ischemic brain injury. Between 20% and 50% of
asphyxiated
newborn infants who exhibit hypoxic-ischemic encephalopathy expire during the
newborn period. Of the survivors, up to 25% exhibit permanent
neuropsychological
handicaps in the form of mental retardation, cerebral palsy, learning
disability, or
epilepsy. A fetus and newborn at risk for perinatal hypoxic-ischemic brain
damage is
readily recognized from experiences and events during late pregnancy and
during
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delivery. Moreover, the atrophy of the brain resulting therefrom continues
over several
days if not weeks. The brains of neonates that have experienced perinatal
hypoxic-
ischemic brain damage do not exhibit normal growth in subsequent years.
Infants born preterm are most susceptible to brain injury. They are prone
to periventricular hemorrhage and periventricular leukomalacia. Both are
caused by
vascular dysfunction with impairment of blood flow to the injured region as an
important
part of the pathogenesis. The most common reason for premature onset of labor
and
delivery of a preterm infant is an inflammatory reaction of the placenta
(chorioamnionitis)
which in most cases is caused by infection. The inflammatory response in the
placenta
induces early labor. The infection of the placenta is also associated with
elevated
inflammatory cytokines in the amniotic fluid and the fetal brain. This
systemic response
to infection is referred to as the fetal inflammatory response and is
associated with
increased damage to the areas of the brain with lowest blood flow as well as
an increase
in the incidence of bronchopulmonary dysplasia , a chronic inflammatory
process in the
premature infants lung requiring prolonged hospitalization for mechanical
ventilation and
supplemental oxygen delivery. Full term infants who develop cerebral palsy
have been
shown to have elevated inflammatory cytokines when their umbilical cord blood
samples
are analyzed, supporting the notion that inflammation contributes to neonatal
brain injury.
The mechanisms whereby inflammation can contribute to injury is by activating
endothelial neutrophil interactions, including the production of free radical
species, and
activating the coagulation pathway in blood vessels thereby impairing micro
vascular
function, red cell trafficking and tissue oxygen deliver. In addition
peripheral
macrophages (monocytes) are activated to produce inflammatory chemokines and
cytokines that recruit inflammatory cells and induce them to release cytotoxic
agents.
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On occasion, it is necessary to conduct cardiac surgery on a neonate, for
example surgical repair to the heart. This requires that the heart be bypassed
and stopped,
for up to thirty minutes, while surgical repairs are conducted on the heart.
Hypoxic-
ischemic brain damage in the neonate is a likely consequence of such a
procedure. It can
similarly arise from complications where the umbilical cord becomes entangled
with the
neonate's circulatory system during delivery, to cut off blood flow to the
brain.
In short, situations where there is significant risk of development of
hypoxic-ischemic brain damage in the neonate are readily recognizable, and not
infrequent.
An animal model of perinatal hypoxic-ischemic brain damage has been
developed and extensively studied, to gain insight into the underlying
mechanisms of
perinatal hypoxic-ischemic brain damage, and how tissue injury can be
prevented or
minimized through therapeutic intervention. The 7-day postnatal rat has a
brain which is
histologically similar to that of a 32-34 week gestation human fetus or
newborn infant, in
that the cerebral cortical neuronal layering is complete, the germinal matrix
is involuting,
and white matter as yet has undergone little myelination. In addition, the
brain of the 12-
13 day postnatal rat is roughly equivalent to that of the full-term newborn
human infant
(R.C. Vannucci, et al., "Rat model of perinatal hypoxic-ischemic brain
damage", Journal
of Neuroscience Research, 55:158-163 (1999).
Currently there is no intervention based on controlled, randomized clinical
trial evidence that can be recommended for the treatment of newborns with
perinatal
hypoxic -ischemic brain injury. In neonatal animal models various modalities
have
demonstrated that injury can be reduced if treatment is administered either
before or after
the insult. This shows that there is a therapeutic window for the appropriate
agent.
Strategies that impair the inflammatory component that accompanies and follows
the
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insult are protective in animal models. To date there have been limited
clinical trials of
potential pharmacologic agents because of the potential for toxicity
especially in the
newborn.
Arvin et. al., (Annals of Neurology Vol. 52, No. 1, July 2002, incorporated
by reference in its entirety) report studies of the effectiveness of the
antibiotic
minocycline given as a single dose intraperitoneally, either immediately
before or
immediately after the hypoxic-ischemic injury in rats. They report a
significant
neuroprotective effect from minocycline and postulate that it is acting early
in the
neuronal death-promoting cascades to block necrosis and apoptosis.
Palmer, C. and R.L. Roberts, (Pedatr. Res., 1997. 41: 294A, incorporated
by reference in its entirety) report promising results with delayed
administration of
allopurinol after cerebral hypoxia-ischemia brain injury in neonatal rats.
There remains a need for a safe, systemic neuroprotective therapy that can
easily be used immediately on recognition that a hypoxic-ischemic injury in a
perinatal
patient is occurring or has occurred.
SUlVIMAIZY ~F TIIE INVENTI~N.
The present invention provides treatments and compositions beneficially
affecting perinatal hypoxic-ischemic brain damage.
The present invention is based upon the discovery that pharmaceutically
acceptable bodies, such as liposomes, beads or similar particles, with
presented
phosphate-glycerol head groups may be used to treat perinatal hypoxic-ischemic
brain
damage.
In a preferred embodiment, the invention is directed to a method for
inhibiting and/or reducing symptoms associated with perinatal hypoxic-ischemic
brain
damage in a perinatal patient comprising administering to the perinatal
patient an
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effective amount of phosphatidylglycerol (PG)-carrying bodies. Preferably, the
bodies
are of a size from about 20 nanometers (nm) to about 500 micrometers (~.m) (as
measured
on its longest axis), more preferably from about 50 nanometers to about 1000
nanometers,
and still more preferably from about 80 nanometers to about 120 nanometers.
Preferably, the bodies are essentially free of pharmaceutically active
entities other than the phosphate-containing groups. Thus the bodies are not
being used as
carriers for pharmaceutical entities, but are active themselves, through the
phosphate-
glycerol groups. Preferably the phosphate-glycerol groups constitute 60% -
100% of the
available phosphate-containing groups on the bodies, the balance thereof being
inactive or
active through a different mechanism. Thus the bodies described above may
additionally
comprise an inactive constituent surface group (such as phosphate-choline
groups), and/or
a constituent surface group which is active through another mechanism, (such
as
phosphate-serine groups from phosphatidylserine as described in Fadok et al.,
International Publication WO 01/66785).
Such constituent surface groups, if present, should not constitute more than
about 40% of the total functional surface groups, balance phosphate glycerol.
In another aspect this invention provides the use of phosphatidylglycerol
(PG)-carrying bodies in the preparation of a medicament to inhibit and/or
reduce the
progression of perinatal hypoxic ischemic brain damage in a mammalian patient.
It is contemplated that the methods may be accompanied with one or more
other modalities, such as, but not limited to, those described in Arvin, et
al. and Palmer, et
al. Administration in combination includes, for example, administration of the
compositions described herein, prior to, during or after administration of the
one or more
other modalities. One of skill in the art will be able to determine the
administration
schedule and dosage.
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BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a diagrammatic illustration of a brain of a neonatal rat, indicating
how measurements were taken in the experimental Example 2 described below.
DESCRIPTION OF PREFERRED EMBODIMENTS
1. Definitions
This section sets forth certain defined terms; other terms used herein are
defined in context and/or have the meanings generally attributable to them in
standard
usage by those skilled in the art.
The term "biocompatible" refers to substances that, in the amount
employed, are either non-toxic or have acceptable toxicity profiles such that
their use in
vivo is acceptable.
The terms "liposomes" and "lipid vesicles" refer to sealed membrane sacs,
having diameters in the micron or sub-micron range, the walls of which consist
of layers,
typically bilayers, of suitable, membrane-forming amphiphiles. They normally
contain an
aqueous medium.
The term "pharmaceutically acceptable" has a meaning that is similar to
the meaning of the term "biocompatible." As used in relation to
"pharmaceutically
acceptable bodies" herein, it refers to bodies of the invention comprised of
one or more
materials which are suitable for administration to a mammal, preferably a
human, i~c vivo,
according to the method of administration specified (e.g., intramuscular,
intravenous,
subcutaneous, topical, oral, and the like).
The term "phosphate choline" refers to the group -O-P(=O)(OH)-O-CH2-
CH2-N+-(CH3)3, which can attached to lipids to form "phosphatidylcholine" (PC)
as
shown in the following structure:
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RZ-CO-O- ~ H~
R3-CO-O-CH O
H2C-O- ~ -O-CHz-N(CH3)s
O'
and salts thereof, wherein R2 and R3 are independently selected from C1-C24
hydrocarbon
chains, saturated or unsaturated, straight chain or containing a limited
amount of
branching wherein at least one chain has from 10-24 carbon atoms.
The teen "phosphate-glycerol-carrying bodies" refers to biocompatible,
pharmaceutically-acceptable, three-dimensional bodies having on their surfaces
phosphate-glycerol groups or groups that call be converted to phosphate-
glycerol groups,
as described herein.
A "phosphate-glycerol group" is a group having the general structure: O-
P(=O)(OH)-O-CH2CH(OH)CHZOH, and derivatives thereof, including, but not
limited to
groups in which the negatively charged oxygen of the phosphate group of the
phosphate-
glycerol group is conveuted to a phosphate ester group (e.g., L-
OP(O)(OR')(OR"), where
L is the remainder of the phosphate-glycerol group, R' is -CH2CH(OH)CHZOH and
R" is
alkyl of from 1 to 4 carbon atoms, or a hydroxyl substituted alkyl of from 2
to 4 carbon
atoms, and 1 to 3 hydroxyl groups provided that R" is more readily hydrolyzed
in vivo
than the R' group; to a diphosphate group including diphosphate esters (e.g.,
L-
OP(O)(OR')OP(O)(OR")Z wherein L and R' are as defined above and each R" is
independently hydrogen, alkyl of from 1 to 4 carbon atoms, or a hydroxyl
substituted
allcyl of from 2 to 4 carbon atoms and 1 to 3 hydroxyl groups, provided that
the second
phosphate [-P(O)(OR")2] is more readily hydrolyzed i~ vivo than the R' group;
or to a
triphosphate group including triphosphate esters (e.g., L-
OP(O)(OR')OP(O)(OR")OP(O)(OR")2 wherein L and R' are defined as above and each
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R" is independently hydrogen, alkyl of from 1 to 4 carbon atoms, or a hydroxyl
substituted alkyl of from 2 to 4 carbon atoms and 1 to 3 hydroxyl groups
provided that the
second and third phosphate groups are more readily hydrolyzed irz vivo than
the R' group;
and the like. Such synthetically altered phosphate-glycerol groups are capable
of
expressing phosphate-glycerol in vivo and , accordingly, such altered groups
are
phosphate-glycerol convertible groups within the scope of the invention. A
specific
example of a phosphate-glycerol group is the compound phosphatidylglycerol
(PG),
further defined herein.
"Phosphatidylglycerol" is also abbreviated herein as "PG." This term is
intended to cover phospholipids carrying a phosphate-glycerol group with a
wide range of
at least one fatty acid chain provided that the resulting PG entity can
participate as a
structural component of a liposome. Chemically, PG has a phosphate-glycerol
group and
a pair of similar, but different fatty acid side chains. Preferably, such PG
compounds can
be represented by the Formula I:
R-CO-O- ~ Ha
R~-CO-O-CH O
CHZ-O-~ O-CHzCH(OH)CHaOH
0'
where R and RI are independently selected from C1 -C24 hydrocarbon chains,
saturated or
unsaturated, straight chain or containing a limited amount of branching
wherein at least
one chain has from 10 to 24 carbon atoms. R and Rl can be varied to include
two or one
lipid chain(s), which can be the same or different, provided they fulfill the
structural
function. As mentioned above, the fatty acid side chains may be from about 10
to about
24 carbon atoms in length, saturated, mono-unsaturated or polyunsaturated,
straight-chain
or with a limited amount of branching. Laurate (C 12), myristate (C 14),
palmitate (C 16),
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stearate (C18), arachidate (C20), behenate (C22) and lignocerate (C24) are
examples of
useful saturated fatty acid side chains for the PG for use in the present
invention.
Palmitoleate (C15), oleate (C18) are examples of suitable mono-unsaturated
fatty acid
side chains. Linoleate (C18), linolenate (C18) and arachidonate (C20) are
examples of
suitable poly-unsaturated fatty acid side chains for use in PG in the
compositions of the
present invention. Phospholipids with a single such fatty acid side chain,
also useful in the
present invention, are known as lysophospholipids.
The term PG also includes dimeric forms of PG, namely cardiolipin, but
other dimers of Formula I are also suitable. Preferably, such dimers are not
synthetically
cross-linked with a synthetic cross-linking agent, such as maleimide but
rather are cross-
linked by removal of a glycerol unit as described by Lehninger, Biochemistry
and
depicted in the reaction below:
R-CO-O- ~ Hz
R~-CO-O-CH O
Hz-O ~~-O-CHzCH(OH)CHzOH
O'
PG
R-CO-O- ~ Hz ~ Hz-O-CO-R
R'-CO-O-CH O O CH-0-CO-Ri
Hz-O ~~-O~CHZCH(OH)CH20 ~~-O-~ Nz
O' O'
HOCHzCH(OH)CHZOH
cardiolipin
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Purified forms of phosphatidylglycerol are commercially available, for
example, from Sigma-Aldrich (St. Louis, MO). Alternatively, PG can be
produced, for
example, by treating the naturally occurring dimeric form of
phosphatidylglycerol,
cardiolipin, with phospholipase D. It can also be prepared by enzymatic
synthesis from
pho,sphatidyl choline using phospholipase D. See, for example, U.S. Patent
5,188,951
(Tremblay et al.), incorporated herein by reference.
"PG-carrying bodies" are three-dimensional bodies, as described above,
that have surface PG molecules. By way of example, PG can form the membrane of
a
liposome, either as the sole constituent of the membrane or as a major or
minor
component thereof, with other phospholipids and/or membrane forming materials.
The term "phosphatidylserine" or "PS" is intended to cover phosphatidyl
serine and analogs/derivatives thereof.
In the context of the present invention, "three-dimensional bodies" refer to
biocompatible synthetic or semi-synthetic entities, including but not limited
to liposomes,
solid beads, hollow beads, filled beads, particles, granules and microspheres
of
biocompatible materials, natural or synthetic, as commonly used in the
pharmaceutical
industry. Liposomes may be formed of lipids, including phosphatidylglycerol
(PG).
Beads may be solid or hollow, or filled with a biocompatible material. Such
bodies have
shapes that are typically, but not exclusively spheroidal, cylindrical,
ellipsoidal, including
oblate and prolate spheroidal, serpentine, reniform and the like, and have
sizes ranging
from 200 nm to 500 Vim, preferably measured along the longest axis.
"Treatment" includes, for example, a reduction in the number of
symptoms, a decrease in the severity of at least one symptom of the particular
disease or a
delay in the further progression of at least one symptom of the particular
disease.
2. Phosphate-Glycerol-Carrying Bodies
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This section describes various embodiments of phosphate-glycerol-
carrying bodies contemplated by the present invention, including specific
embodiments
thereof. With the guidance provided herein, persons having requisite skill in
the art will
readily understand how to make and use phosphate-glycerol-carrying bodies in
accordance with the present invention.
In the context of the present invention, phosphate-glycerol-carrying bodies
refer to biocompatible, pharmaceutically-acceptable, three-dimensional bodies
having on
their surfaces phosphate-glycerol groups or groups that can be conveuted to
phosphate-
glycerol groups, as described herein.
a. Phosphate-Glycerol Groups
According to a general feature of the invention, phosphate-glycerol groups
useful in the present invention have the general structure:
O-P(=O)(OH)-O-CH2CH(OH)CH2OH
Such phosphate-glycerol groups include synthetically altered versions of
the phosphate-glycerol group shown above, and may include all, part of or a
modified
version of the original phosphate-glycerol group.
Preferably the fatty acid side chains of the chosen PG will be suitable for
formation of liposomes, and incorporation into the lipid membranes) forming
such
liposomes, as described in more detail below.
More generally, without being limited to any particular theory, it is
believed that phosphate-glycerol groups according to the present invention are
capable of
interacting with one or more receptors present in relevant brain tissue, such
as the
hippocampus. A specific example of a phosphate-glycerol group is the compound
phosphatidylglycerol (PG), described above.
PG groups of the present invention, including dimers thereof, are believed
to act as ligands, binding to specific sites on a protein or other molecule
("PG receptor")
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and, accordingly, PG (or derivatives or dimeric forms thereof) are sometimes
referred to
herein as a "ligand" or a "binding group." Such binding is believed to talee
place through
the phosphate-glycerol group -O-P(=O)(OH)-O-CH2CH(OH)CH2OH, which is
sometimes referred to herein as the "head group," "active group," or "binding
group,"
while the fatty acid side chains) are believed to stabilize the group and/or,
in the case of
liposomal preparations, form the outer lipid layer or bilayer of the liposome.
More
generally, again without being limited to any particular theory, it is
believed that
phosphate-glycerol groups, including PG are capable of interacting with one or
more
receptors in the brain and that such interactions may provide positive effects
on synaptic
transmission, and, by extension, memory, as described herein.
As noted above, analogues of phosphatidylglycerol with modified active
groups, which also interact with PG receptors on the antigen presenting cells,
through the
same receptor pathway as PG or otherwise resulting in an anti-inflammatory
reaction in
the recipient body are contemplated within the scope of the term
phosphatidylglycerol.
This includes, without limitation, compounds in which one or more of the
hydroxyl
groups and/or the phosphate group is derivatized, or in the form of a salt.
Many such
compounds form free hydroxyl groups in vivo, upon or subsequent to
administration and,
accordingly, comprise convertible PG groups.
b. Formation of Phosphate-Glycerol Carrying Bodies
Phosphate-glycerol carrying bodies are three-dimensional bodies that have
surface phosphate-glycerol molecules. This section will describe general and
exemplary
phosphate-glycerol carrying bodies suitable for use in the present invention.
Generally, phosphate-glycerol carrying bodies of the present invention
carry phosphate-glycerol molecules on their exterior surfaces to facilitate in
vivo
interaction of the binding groups.
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Three-dimensional bodies are preferably formed to be of a size or sizes
suitable for administration to a living subject, preferably by injection;
hence such bodies
will preferably be in the range of 20 to 1000 nm (0.02-1 micron), more
preferably 20 to
500 run (0.02-0.5 micron), and still more preferably 20-200 nm in diameter,
where the
diameter of the body is determined on its longest axis, in the case of non-
spherical bodies.
Suitable sizes are generally in accordance with blood cell sizes. While bodies
of the
invention have shapes that are typically, but not exclusively spheroidal, they
can
alternatively be cylindrical, ellipsoidal, including oblate and prolate
spheroidal,
serpentine, reniform in shape, or the like. '
Suitable forms of bodies for use in the compositions of the present
invention include, without limitation, particles, granules, microspheres or
beads of
biocompatible materials, natural or synthetic, such as polyethylene glycol,
polyvinylpyrrolidone, polystyrene, and the like; polysaccharides such as
hydroxethyl
starch, hydroxyethylcellulose, agarose and the like; as are commonly used in
the
pharmaceutical industry. Preferably, such materials will have side-chains or
moieties
suitable for derivatization, so that a phosphate-glycerol group, such as PG,
may be
attached thereto, preferably by covalent bonding. Bodies of the invention may
be solid or
hollow, or filled with biocompatible material. They are modified as required
so that they
carry phosphate-glycerol molecules, such as PG on their surfaces. Methods for
attaching
phosphate-glycerol in general, and PG in particular, to a variety of
substrates are known
in the art.
In addition to the various bodies listed above, the liposome is a particularly
useful form of body for use in the present invention. Liposomes are
microscopic vesicles
composed of amphiphilic molecules forming a monolayer or bilayer surrounding a
central
chamber, which may be fluid-filled. Amphophlilic molecules (also referred to
as
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"amphiphiles"), are molecules that have a polar water-soluble group attached
to a water-
insoluble (lipophilic) hydrocarbon chain, such that a matrix of such molecules
will
typically form defined polar and apolar regions. Amphiphiles include naturally
occurring
lipids such as PG, phosphatidylserine, phosphatidylethanolamine,
phosphatidylinositol,
phosphatidylcholine, cholesterol, cardiolipin, ceramides and sphingomyelin,
used alone or
in admixture with one another. They can also be synthetic compounds such as
polyoxyethylene allcyl ethers, polyoxyethylene alkyl esters and
saccharosediesters.
Preferably, for use in forming liposomes, the amphiphilic molecules will
include one or more forms of phospholipids of different head groups (e.g~.,
phosphatidylglycerol, phosphatidylserine, phosphatidylcholine) and having a
variety of
fatty acid side chains, as described above, as well as other lipophilic
molecules, such as
cholesterol, sphingolipids and sterols.
In accordance with the present invention, phosphatidylglycerol (PG) will
constitute the major portion or the entire portion of the liposome layers) or
wall(s),
oriented so that the phosphate-glycerol group portion thereof is presented
exteriorly, as
described above, while the fatty acid side chains form the structural wall.
~Jhen, as in the
present invention, the bilayer includes phospholipids, the resulting membrane
is usually
referred to as a "phospholipid bilayer," regardless of the presence of non-
phospholipid
components therein.
Liposomes of the invention are typically formed from phospholipid
bilayers or a plurality of concentric phospholipid bilayers which enclose
aqueous phases.
In some cases, the walls of the liposomes may be single layered; however, such
liposomes
(termed "single unilamellar vesicles" or "SUVs") are generally much smaller
(diameters
less than about 70 nm) than those formed of bilayers, as described below.
Liposomes
formed in accordance with the present invention are designed to be
biocompatible,
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WO 2005/009540 PCT/US2004/023509
biodegradable and non-toxic. Liposomes of this type are used in a number of
pharmaceutical preparations currently on the market, typically carrying active
drug
molecules in their aqueous inner core regions. In the present invention,
however, the
liposomes are not filled with pharmaceutical preparation. The liposomes are
active
themselves, not acting as drug carrier.
Preferred PG-carrying liposomes of the present invention are constituted to
the extent of 50% -100% by weight of phosphatidyl glycerol, the balance being
phosphatidylcholine (PC) or other such biologically acceptable
phospholipid(s). More
preferred are liposomes constituted by PG to the extent of 65% -90% by weight,
most
preferably 70°/~ -~0% by weight, with the single most preferred
embodiment, on the basis
of current experimental experience, being PG 75% by weight, the balance being
other
phospholipids such as PC. Such liposomes are prepared from mixtures of the
appropriate
amounts of phospholipids as starting materials, by known methods. According to
an
important feature of the invention, PG-carrying bodies comprise less than 50%,
preferably less than 40%, still preferably less than 25% and even still
preferably less than
10% phosphatidyl serine.
The present invention contemplates the use, as PG-carrying bodies, not
only of those liposomes having PG as a membrane constituent, but also
liposomes having
non-PG membrane substituents that carry on their external surface molecules of
phosphate-glycerol, either as monomers or oligomers (as distinguished from
phosphatidylglycerol), e.g., chemically attached by chemical modification of
the
liposome surface of the body, such as the surface of the liposome, making the
phosphate-
glycerol groups available for subsequent interaction. Because of the inclusion
of
phosphate-glycerol on the surface of such molecules, they are included within
the
definition of PG-carrying bodies.
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Liposomes may be prepared by a variety of techniques known in the art,
such as those detailed in Szoka et al. (Ann. Rev. Biophys. Bioeng. 9:467
(1980)).
Depending on the method used for forming the liposomes, as well as any after-
formation
processing, liposomes may be formed in a variety of sizes and configurations.
Methods of
preparing liposomes of the appropriate size are known in the art and do not
form part of
this invention. Reference may be made to various textbooks and literature
articles on the
subject, for example, the review at-ticle by Yechezkel Barenholz and Daan J.
A.
Chromeline, and literature cited therein, for example New, R. C. (1990), and
Nassander,
U. IC., et al. (1990), and Barenholz, Y and Lichtenberg, D., Liposomes:
py~epa~atiojz,
charactef°ization, ahd prese~vatio~c. Methods Biochem Aszal. (1988)
33:337-462.
Multilamellar vesicles (MLV's) can be formed by simple lipid-film
hydration techniques according to methods known in the art. In this procedure,
a mixture
of liposome-forming lipids is dissolved in a suitable organic solvent. The
mixture is
evaporated in a vessel to form a thin film on the inner surface of the vessel,
to which an
aqueous medium is then added. The lipid film hydrates to form MLV's, typically
with
sizes between about 100-1000 nm (0.1 to 10 microns) in diameter.
A related, reverse evaporation phase (REV) technique can also be used to
form unilamellar liposomes in the micron diameter size range. The REV
technique
involves dissolving the selected lipid components, in an organic solvent, such
as diethyl
ether, in a glass boiling tube and rapidly inj ecting an aqueous solution,
into the tube,
through a small gauge passage, such as a 23-gauge hypodermic needle. The tube
is then
sealed and sonicated in a bath sonicator. The contents of the tube are
alternately
evaporated under vacuum and vigorously mixed, to form a final liposomal
suspension.
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By way of example, but not limitation, Example 1 provides a detailed
description of a method of preparing a PG-liposomal preparation for use in the
present
invention.
The diameters of the PG-carrying liposomes of the preferred embodiment
of this invention range from about 20 mn to about 1000 nm, more preferably
from about
20 nm to about 500 nm, and most preferably from about 20 nm to about 200 nm.
Such
preferred diameters will correspond to the diameters of mammalian apoptotic
bodies,
such as may be apprised from the art.
One effective sizing method for REV's and MLV's involves extruding an
aqueous suspension of the liposomes through a series of polycarbonate
membranes
having a selected uniform pore size in the range of 0.03 to 0.2 micron,
typically 0.05,
0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly
to the
largest sizes of liposomes produced by extrusion through that membrane,
particularly
where the preparation is extruded two or more times through the same membrane.
This
method of liposome sizing is used in preparing homogeneous-size REV and MLV
compositions. U.S. Patents 4,737,323 and 4,927,637, incorporated herein by
reference,
describe methods for producing a suspension of liposomes having uniform sizes
in the
range of 0.1-0.4 ~m (100-400 nm) using as a starting material liposomes having
diameters
in the range of 1 ~,m. Homogenization methods are also useful for down-sizing
liposomes
to sizes of 100 nm or less (Martin, F. J. (1990) In: Specialized Drug Delivery
Systems--
Manufacturing and Production Technology, P. Tyle (ed.) Marcel Dekker, New
York, pp.
267-316.). Another way to reduce liposomal size is by application of high
pressures to the
liposomal preparation, as in a French Press.
Liposomes can be prepared to have substantially homogeneous sizes of
single, bi-layer vesicles in a selected size range between about 0.07 and 0.2
microns (70-
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200 nm) in diameter, according to methods known in the art. In particular,
liposomes in
this size range are readily able to extravasate through blood vessel
epithelial cells into
surrounding tissues. A further advantage is that they can be sterilized by
simple filtration
methods lenown in the art.
Whilst a preferred embodiment of PG-carrying bodies for use in the
present invention is liposomes with PG presented on the external surface
thereof, it is
understood that the PG-carrying body is not limited to a liposomal structure,
as mentioned
above.
3. Dosages, Modes of Administration and Utility
The phosphate-glycerol-carrying bodies of the invention may be
administered to the patient by any suitable route of administration, including
oral, nasal,
topical, rectal, intravenous, subcutaneous and intramuscularly. At present,
intramuscular
administration is preferred, especially in conjunction with PG-liposomes.
The PG-carrying bodies may be suspended in a pharmaceutically
acceptable carrier, such as physiological sterile saline, sterile water,
pyrogen-free water,
isotonic saline, and phosphate buffer solutions, as well as other non-toxic
compatible
substances used in pharmaceutical formulations. Preferably, PG-carrying bodies
are
constituted into a liquid suspension in a biocompatible liquid such as
physiological saline
and administered to the patient in any appropriate route which introduces it
to the immune
system, such as intra-arterially, intravenously, intra-arterially or most
preferably
intramuscularly or subcutaneously.
The quantities of PG-carrying bodies to be administered will vary
depending on the identity and characteristics of the patient. It is important
that the
effective amount of PG-bodies is non-toxic to the patient. The most effective
amounts are
unexpectedly small. When using intra-arterial, intravenous, subcutaneous or
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intramuscular administration of a liquid suspension of PG-carrying bodies, it
is preferred
to administer, for each dose, from about 0.1-50 ml of liquid, containing an
amount of PG-
carrying bodies generally equivalent to 10% -1000% of the number of leukocytes
normally found in an equivalent volume of whole blood or the number of
apoptotic
bodies that can be generated from them. Generally, the number of PG-carrying
bodies
administered per delivery to a patient is in the range from about 500 to about
2.5 x 1012
(about 260 nanograms by weight), preferably from about 5,000 to about
500,000,000,
more preferably from about 10,000 to about 10,000,000, and most preferably
from about
200,000 to about 2,000,00
According to one feature of the invention, the number of such bodies
administered to an injection site for each administration is believed to be a
more
meaningful quantization than the number or weight of PG-carrying bodies per
unit of
patient body weight. Thus, it is contemplated that effective amounts or
numbers of PG-
carrying bodies for small animal use may not directly translate into effective
amounts for
larger mammals on a weight ratio basis.
It is contemplated that the PG-carrying bodies may be freeze-dried or
lyophilized to a form which may be later resuspended for administration. This
invention
therefore also includes a kit of parts comprising lyophilized or freeze-dried
PG- carrying
bodies and a pharmaceutically acceptable carrier, such as physiological
sterile saline,
sterile water, pyrogen-free water, isotonic saline, and phosphate buffer
solutions, as well
as other non-toxic compatible substances used in pharmaceutical formulations.
Such a kit
may optionally provide injection or administration means for administering the
composition to a subject.
It is contemplated that the methods of the invention will be useful for
treating perinatal patients. As used herein, the term "perinatal patient"
refers to infants
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who are at risk of or showing signs of development of perinatal hypoxic-
ischemic brain
damage. These perinatal patients may be treated prior to or post birth.
A preferred manner of administering the pharmaceutically acceptable
bodies to the patient is by injection, administered intramuscularly or
subcutaneously to
the newborn at risk of or showing signs of development of perinatal hypoxic-
ischemic
brain damage. One or more injections may be administered, on a daily or twice
daily
basis, for the first several days of life, or on a different schedule
according to the
observations of the attending clinician. Since the brain atrophy resulting
from the
hypoxic-ischemic insult can continue for an extended period, the treatment
according to
the preferred aspects of the invention may beneficially be continued for
several months.
A pre-term infant is at rislc of developing brain injury for several weeks
after birth, depending on the level of prematurity, so that prophylactic
administration for
weeks may be necessary for this population.
It is also within the scope of the present invention to administer the
pharmaceutically acceptable bodies described above to the mother, prior to
delivery and
birth of the neonate or neonatal infant. Typically a neonatal infant is an
infant born
prematurely or otherwise, under 4 weeks old. As noted above, mothers at risk
of giving
birth to a premature neonate or a neonate likely to experience hypoxic-
ischemic brain
damage, are often recognizable. Any method of administration of the
compositions of the
invention that ensures that an effective amount of the composition reaches the
placenta
and crosses the placental barrier can be used on the mother. Intramuscular
injection is
preferred. Any mother giving birth to a pre-term baby is a candidate for
treatment
according to this invention.
It is postulated that, in many embodiments of the present invention,
pharmaceutically acceptable bodies comprising the phosphate-glycerol head
groups as
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binding groups on their surface are acting as modifiers of the patient's
immune system, in
a manner similar to that of a vaccine. Accordingly they are used in quantities
and by
administration methods to provide a sufficient localized concentration of the
bodies at the
site of introduction. Quantities of such bodies appropriate for immune system
modification may not be directly correlated with body size of a recipient and
can,
therefore, be clearly distinguished from drug dosages, which are designed to
provide
therapeutic levels of active substances in a patient's bloodstream and
tissues. Drug
dosages are accordingly likely to be much larger than immune system modifying
dosages.
The correlation between weights of liposomes and numbers of liposomes
is derivable from the knowledge, accepted by persons skilled in the art of
liposomal
formulations, that a 100 mn diameter bilayer vesicle has 81,230 lipid
molecules per
vesicle, distributed approximately 50:50 between the layers (see Richard
Harrigan -1992
University of British Columbia PhI~ Thesis "Transmembrane pH gradients in
liposomes
(microform): drug-vesicle interactions and proton flux", published by National
Library of
Canada, ~ttawa, Canada (1993); University Microfilms order no. UMI00406756;
Canadians no. 942042220, ISBN 0315796936). From this one can calculate, for
example, that a dose of 5 x 108 vesicles, of the order of the dose used in the
specific i~
vivo examples below, is equivalent to 4.06 x 1013 lipid molecules. Using
Avogadro's
number for the number of molecules of lipid in a gram molecule (mole), 6.023 x
1023, one
determines that this represents 6.74 x 1011 moles which, at a molecular weight
of 729 for
PG is approximately 4.92 x 10-8 gm, or 49.2 nanograms of PG for such dosage.
For a
dose of 6 x 1 OS vesicles , of the order of the dose used in the specific in
vivo examples
below, using animals of average weight 15 grams, the corresponding calculation
gives a
weight of 5.89 x 10-1 gm, or 0.059 nanograms.
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The quantities of the pharmaceutically acceptable bodies to be
administered are very small. Preferably, the effective amount of
pharmaceutically
acceptable bodies is non-toxic to the patient, and is not so large as to
overwhelm the
immune system. When using intra-arterial, intravenous, subcutaneous or
intramusculax
administration of a sterile aqueous suspension of pharmaceutically acceptable
bodies, it is
preferred to administer, for each dose, from about 0.1-50 ml of liquid,
containing a
number of bodies in the range from about 500 to about 2.5 x 109 (<250 ng of
bodies, in
the case of liposomes, pro-rated for density differences for other embodiments
of bodies),
more preferably from about 1,000 to about 1,500,000,000, even more preferably
10,000
to about 100,000,000, and most preferably from about 200,000 to about
2,000,000.
Since the pharmaceutically acceptable bodies axe believed to be acting, in
the process of the invention, as immune system modifiers, in the nature of a
vaccine, the
number of such bodies administered to an injection site for each
administration may be a
more meaningful quantitation than the number or weight of bodies per unit of
patient
body weight. For the same reason, it is now contemplated that effective
amounts or
numbers of bodies for small animal use may not directly translate into
effective amounts
for human newborns on a weight ratio basis.
EXAMPLES
The following examples are intended to illustrate methods for preparing
therapeutic compositions of the present invention and exemplary treatment
results. The
examples are in no way intended to limit the scope of the invention.
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Example 1
Preparation of Liposomes
A dry mixture ("Lipid Premix") was prepared, consisting of semi-synthetic
POPG (1-palmitoyl-2-oleoly-sn-glycero-3-phosphoglycerol sodium salt), 3 parts
by mass,
and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), 1 part by mass.
The POPC ingredient was prepared from DPPC (dipalmitoyl-sn-glycero-3-
phosphocholine) which was purified from soybean and enzymatically hydrolyzed
with
porcine pancreas phospholipase A2 (E.C. 3.1.1.4) to generate monopalmitoyl
phosphatidylcholine (MPPC). The MPPC was acylated with oleic acid to generate
POPC.
The POPC was recovered and further purified by liquid phase chromatography to
a purity
of not less than 9~%. The purified material was dried, dissolved in
appropriate solvent
(ethanol, t-butanol or chloroform), filtered through 0.22 micron filter and
subsequently
dried in a clean room.
The POPG ingredient was prepared from POPC. The POPC was dissolved
in a suitable solvent (ethanol, t-butanol or chloroform) and incubated with
excess glycerol
in the presence of recombinant phospholipase D (E.C. 3.1.4.4). POPG was
recovered and
purified by liquid phase chromatography to a purity of not less than 9~%. The
material
was dried, dissolved in appropriate solvent (ethanol, t-butanol or
chloroform), filtered
through 0.22 micron filter and subsequently dried in a clean room.
POPG and POPC were dissolved at a ratio of 3:1 by mass in t-butanol,
followed by filtration (0.22 micron) and drying in a clean room, to form the
Lipid Premix.
These steps were performed for the Applicants by Lipoid GmbH, Frigensr 4,
Ludwigshafen.
The Lipid Premix was hydrated with phosphate buffered saline (PBS, pH
7.0, sterilized by filtration through a 0.22 micron sterilizing filter). A
suspension of
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multilamellar vesicles (MLV's) formed. The suspension was passed through
polycarbonate filter (100 nm pore size) under pressure, generating unilamellar
vesicles of
about 100 nm in diameter. Vesicle size was verified, in-process, using a Quasi-
Elastic
Light Scattering (QELS) analysis. The suspension of unilamellar vesicles
(liposomes)
was immediately removed to a class 1,000 clean room, where it was redundantly
filtered
(0.22 micron) and filled into vials (1 mL per 2 mL amber vial) in a class 100
laminar flow
hood. The vials were baclcfilled with nitrogen and sealed with butyl rubber
stopper and
aluminium crimp seals.
Example 2
Animal Model Testing
The Rice-Vannucci model of hypoxic ischemic brain injury in the neonatal
rat is used for testing the formulations in the present invention - see Rice,
J.E., R.C.
Vannucci and J.B. Brierley, "The influence of immaturity on hypoxia-ischemic
brain
damage in the rat", Ann. Neurol., 1981.9: p.l 13-141. There is an evolving
inflammatory
reaction in the injured brain following hypoxia-ischemia in the immature rat
pup model.
Inflammatory cytolcines such as IL-1 ~i increase in the first 24 hours of
recovery (1). The
inflammatory reaction is characterized by an increase in activated microglial
cells,
expression of inflammatory cytolcines and chemolcines, and even the influx of
CD4
lymphocytes after a few days. Prior exposure to endotoxin (LPS) increases the
rats'
susceptibility to brain damage following hypoxic-ischemia (2). Also, there is
a delayed
component of brain cell death (mainly by apoptosis) that continues for at
least a week
after the initial insult (3, 4). There is evidence of foci of injury
surrounded by microglial
for over 6 months in the immature rat pup model used here (5).
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In order to demonstrate modulation of the inflammatory response
following the insult in an attempt to reduce injury in accordance with a
preferred
embodiment of the invention, the following experiments are conducted.
Timed pregnant rats (2 per weelc) were allowed to deliver. On day one of
life, 88 rat pups were numbered sequentially and assigned to one of two
groups. Also on
day 1 of life, one group was injected subcutaneously, at a fold of skin at the
back of the
neck, with 75% PG/ 25% PC liposomes, and the other group similarly injected,
on the
same schedule, with saline as control. Each injection consisted of a volume of
0.01 ml,
per gram of body weight of the pup. The concentration of the liposomes in the
suspension
was 3 x 106 liposomes per ml, so that each pup received 3x104 vesicles per
gram body
weight in each injection. The treatments (injections) were repeated on day 2
of life, day 6
of life and day ~ of life.
On day 7 of life, the rats were subjected to hypoxic-ischemic insult. For
this purpose, the rats were anesthetized with Halothane/nitrous oxide and the
right
common carotid artery was permanently ligated with silk. After three hours
recovery, the
pups were placed in glass jars resting in a temperature controlled incubator
and exposed
to a hypoxic gas mixture (8% oxygen, balance nitrogen) for 2.25 hrs. Then the
pups were
returned to room air. This insult produces atrophy to the right hemisphere of
the brain,
while leaving the left hemisphere virtually intact. Following the hypoxic
interval, the jars
were opened to room air, and the pups were returned to their dams and returned
to the
animal care facility to recover for 21 days. Then they were sacrificed with a
lethal dose of
pentobarbital and their brains carefully removed intact and immersed in
fixative.
Gross Assessment
After a weelc in fixative, the brains were removed from fixative and
examined intact, to arrive at a gross assessment. The brains were examined by
two
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experienced investigators who were unaware of the identity of the treatment
group. They
allocated the brains an injury score (0-4) based on the difference in size of
the ipsilateral
(right) vs. contralateral (left) hemisphere. The examiners came to a consensus
and agreed
on each score, using the following criteria:
0 = no injury; no difference in size between hemispheres;
1 = mild injury; about 15% reduction in R hemisphere size;
2 = mild injury, about 15 - 30% atrophy, no cavitating injury;
3 = moderate injury, >30% atrophy, cavitating lesion with some preservation of
R
hemisphere;
4 = severe injury, almost complete destruction of the R hemisphere.
There were 42 rat brains from liposome-treated animals and 37 rat brains
from saline treated animals, that had survived the hypoxia and the 3 weelcs
recovery. The
brains from liposome treated rats had lower categories of injury that the
brains from
saline treated rats (P=0.0002, Mantzel-Hantzel test). ~f the brains from
saline treated
rats, there were four with category 4 injury and eight with category 3 injury.
None of the
brains from liposome treated animals showed category 4 injury, and only three
showed a
category 3 injury, despite the fact that this was a larger group than the
saline treated
animals.
Morphometric analysis of brain atrophy
The fixed brains were cut with a blade into three 2 mm thick coronal slices
from the mid section of the brain region that represented the area of maximum
injury, as
indicated on the accompanying Figure. This shows diagrammatically the rat pup
brain 10
with its left hemisphere 12 and its right hemisphere 14. The area of hypoxic-
ischemic
damage 16 is in the right hemisphere 14. The slices were made by cutting
coronal to the
base of the brain, to obtain slice 2, slice 3 and slice 4.
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WO 2005/009540 PCT/US2004/023509
A digital image was made of the three sections. The area of each
hemisphere was measured using NIH image software. The percentage atrophy of
the right
hemisphere was calculated using the formula:
Area L - Area R x 100
Area L
The atrophy in each slice 2, 3 and 4 was calculated and the results
averaged to show the average atrophy. The results from the treatment group
were
compared with those from the saline, control group.
Brains from the saline treated rats had approximately double the amount of
atrophy compared with those from the liposome treated rats. This was
consistent for each
slice. For all the slices, the average amount of atrophy for the saline
treated group was
33.6 ~ 21.7% (mean ~ SIB), while the liposome treated group had only 16.8
X14.4%, a
statistically significant difference (p< 0.001, Mann Whitney U. test). Thus
liposome
treatment according to this embodiment of the invention reduced atrophy by
approximately 50%. The results are given in the Table below.
Table - Percentage Right Hemisphere Atrophy Comparisons.
Saline, n=37 Liposome, n=42 Mann-Whitney
U
Slice 2 27.818.8 14.214.8 0.001
Slice 3 35.4 X24.0 17.1413.9 <0.001
Slice 4 37.523.6 19.0115.37 <0.001
Average 33.621.7 16.814.4 <0.001
This indicated a clinically significant reduction in injury consequent upon
the hypoxia-ischemia insult in this model, and highlights the potential for
the use of the
invention in the treatment of perinatal hypoxic-ischemic brain damage.
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References
1. Bona , E., et al., Chemokine and inflammatory cell response to hypoxia-
ischemia in immature rats. Pediatr Res, 1999. 45(4 Pt 1): p. 500-509.
2. Elclind, S., et al., Bacterial endotoxin sensitizes the immature brain to
hypoxic-ischaemic injury. Eur J Neurosci, 2001. 13(6): p.1931-8.
3. Northington, F.J., et al., Delayed neurodegeneration in neonatal rat
thalamus after hypoxia-ischemia is apoptosis. J Neurosci, 2001. 21 (6): p.
1931-8.
4. Nakajima, W., et al., Apoptosis has a prolonged role in the
neurodegeneration after hypoxic ischemia in the newborn rat. J. Neurosci,
2000 20(21): p. 7994-8004.
5. Palmer, C., et al., Iron containing "plaques" developing the brains of rats
months after neonatal hypoxic-ischemic brain injury. Pediatr Res, 1998.
43: p. 322A.
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