NZ624736B2 - Apoaequorin for reducing neuronal injury due to ischemia - Google Patents
Apoaequorin for reducing neuronal injury due to ischemia Download PDFInfo
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- NZ624736B2 NZ624736B2 NZ624736A NZ62473612A NZ624736B2 NZ 624736 B2 NZ624736 B2 NZ 624736B2 NZ 624736 A NZ624736 A NZ 624736A NZ 62473612 A NZ62473612 A NZ 62473612A NZ 624736 B2 NZ624736 B2 NZ 624736B2
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- apoaequorin
- injection
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- ischemia
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
Disclosed is the use of apoaequorin in the manufacture of a medicament for preconditioning neurons to reduce neuronal injury due to brain ischemia in a subject by administering the apoaequorin to the subject 48 hours before the brain ischemia occurs.
Description
WO 74798
APOAEQUORIN FOR NG NEURONAL INJURY DUE TO ISCHEMIA
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of US. ation No. 61/559,816, filed
November 15, 2011, which is incorporated herein by nce in its entirety for all
purposes.
FIELD OF THE ION
This invention relates to compositions and methods for reducing neuronal
injury due to ischemia. In particular, this invention is directed to apoaequorin and its use
in reducing neuronal injury due to brain ischemia in a subject.
BACKGROUND OF INVENTION
According to the American Heart Association, every 40 s, someone in
the United States suffers from a stroke. Currently, there is only one Food and Drug
Administration approved treatment for stroke, recombinant tissue nogen activator
(rTPA). Although rTPA helps many people, it can also have deleterious side effects, such
as hemorrhage.
During ischemia, neurons are subjected to an excess of intracellular calcium.
Although intracellular calcium is necessary for normal neuronal functions, too much
calcium can trigger cascades of events, leading up to, and including cell death. Several
mechanisms enable neurons to limit or control cytosolic calcium levels, including
calcium binding proteins (CaBPs). It has been shown that treatment with the CaBP
calbindin D—28k reduces deleterious effects from ischemia. Unfortunately, there is a lack
of alternative therapeutics for preventing neuron death due to ischemia and, accordingly,
there exists a need for novel therapeutics useful in treating the rious effects of
ia on neurons.
SUMMARY OF THE INVENTION
Here, the ors demonstrate the use of apoaequorin for providing ischemic
protection to neurons. Accordingly, the invention encompasses in a first aspect a method
of preconditioning s in a subject, preferably human, to reduce neuronal injury due
to brain ischemia. Such a method includes the step of administering apoaequorin to
neurons in a subject, wherein the apoaequorin initiates a change in cytokine expression
levels in the neurons that results in a ion in al injury due to brain ischemia.
A preferred route of administration is by ion to the subject, preferably
intra-hippocampal injection.
In n embodiments, the reduction in neuronal injury effect lasts at least 24
hours from the time of apoaequorin administration. In other embodiments, the reduction
in neuronal injury effect lasts at least 48 hours from the time of apoaequorin
administration.
In other aspects, the invention is directed to the use of apoaequorin for the
cture of a medicament for preconditioning neurons in a subject to reduce
neuronal injury due to brain ischemia.
The invention further encompasses compositions for preconditioning neurons
in a subject to reduce neuronal injury due to brain ischemia. Such compositions include:
(a) a therapeutically effective dosage of apoaequorin to precondition s in a subject
to reduce neuronal injury due to brain ischemia; and (b) a pharmaceutically-acceptable
carrier. Preferred compositions are formulated as inj ectable dosages.
In another aspect, the invention is directed to a kit for preconditioning neurons
in a subject to reduce neuronal injury due to brain ischemia. Such a kit includes: (a) a
therapeutically effective dosage of apoaequorin to precondition neurons in a subject to
reduce neuronal injury due to brain ischemia; and (b) a delivery device configured to
administer the amount of apoaequorin to the t.
Other objects, features and advantages of the t invention will become
nt after review of the specification, claims and gs.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a time-dependent increase in IL—lO mRNA after apoaequorin
infusion (A) as a percent of vehicle control. No change in B—actin mRNA (B) was
ed. (Apoaequorin is abbreviated as "AQ").
Figure 2 shows (A) the dose-dependent effect ofAQ on cell rescue; (B) 4%
AQ administered 2 days prior to ischemia is rotective (note the reduction in trypan
blue labeled neurons following AQ); (C) the Window of neuroprotection provided by AQ;
(D) AQ availability after injection into the hippocampus.
Figure 3 s (A) IL-lO mRNA expression; (B) B-actin mRNA expression;
and (C) PCR arrays displaying changes in multiple cytokines and chemokines ing
AQ administration.
Figure 4 depicts a graph showing how cytokines are significantly d with
AQ injection.
Figure 5 depicts a schematic showing the interplay of immune response genes
examined by the inventors.
Figure 6 depicts graphs showing (A) change in acute inflammatory response
genes following AQ injection; (B) change in T/B cell activators following AQ ion;
(C) change in angiogenesis/cell proliferator genes following AQ injection; (D) pattern of
change in macrophage ting genes following AQ ion; (E) change in calcium
mediator genes ing AQ injection; and (F) genes (grouped by function) that change
with AQ injection.
DETAILED DESCRIPTION OF THE INVENTION
1. IN GENERAL
Before the present materials and methods are described, it is understood that
this invention is not limited to the particular methodology, protocols, materials, and
reagents described, as these may vary. It is also to be tood that the terminology
used herein is for the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention, which will be limited only by any
later-filed nonprovisional applications.
It must be noted that as used herein and in the appended claims, the singular
forms 66 ,9 ‘6
, an”, and “the” e plural reference unless the context clearly dictates
2012/065291
otherwise. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used
interchangeably herein. It is also to be noted that the terms “comprising”, “including”,
and g” can be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings as ly understood by one of ordinary skill in the art to which
this invention belongs. gh any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of the present invention, the
preferred methods and materials are now described. All publications and patents
specifically mentioned herein are incorporated by reference for all purposes including
describing and disclosing the chemicals, instruments, statistical analysis and
methodologies which are reported in the publications which might be used in connection
with the ion. All references cited in this cation are to be taken as indicative
of the level of skill in the art. Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such sure by virtue of prior invention.
II. THE INVENTION
As used herein, "subj ect" means mammals and non—mammals. “Mammals”
means any member of the class Mammalia including, but not limited to, humans, non-
human primates such as chimpanzees and other apes and monkey species; farm animals
such as cattle, , sheep, goats, and swine; domestic animals such as s, dogs,
and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and
the like. Examples ofnon—mammals include, but are not limited to, birds, and the like.
The term "subject" does not denote a particular age or sex.
As used , “administering” or “administration” includes any means for
introducing apoaequorin into the body, preferably into the brain of the subject, more
preferably into the hippocampus of the subject. Examples of administration include but
are not limited to oral, nasal, otic, ophthalmic, buccal, sublingual, pulmonary,
transdermal, transmucosal, as well as subcutaneous, intraperitoneal, intravenous, epidural
and uscular injection.
A "therapeutically effective amount" means an amount of apoaequorin that,
when stered to a subject for treating a disorder, condition, or disease, is sufficient
to effect such treatment for the disorder, or ion, or disease. The "therapeutically
effective amount" will vary depending on the disorder, or condition, or disease state
being treated, the severity or the disorder, or condition, or disease d, the age and
relative health of the subject, the route and form of administration, the judgment of the
attending medical or veterinary practitioner, and other factors.
For es ofthe present invention, “treating” or “treatment” bes the
management and care of a patient for the purpose of combating the e, ion, or
disorder. The terms e both preventative, i.e., prophylactic, and palliative
treatments. Treating includes the administration of apoaequorin to prevent the onset of
the symptoms or complications, alleviating the symptoms or complications, or
eliminating the disease, condition, or disorder.
The term quorin" refers to the apoprotein portion of the calcium
binding n aequorin. Aequorin is composed oftwo distinct units, the respective
apoprotein, apoaequorin, with an approximate molecular weight of 22 kDa, and the
prosthetic group coelenterazine, a luciferin. Aequorin is a photoprotein isolated from
luminescent jellyfish (such as ea species, e.g., Aequorea Victoria) and a variety of
other marine organisms. It was originally isolated from the coelenterate by Osamu
Shimomura. Apoaequorin usefiil in the present invention is available from its natural
source, Via previously known isolation and purification schemes, or publicly known
recombinant methods utilizing expression systems which make use of, e.g., recombinant
DNA constructs and genetically-modified host cells useful for logous expression of
apoaequorin.
As used herein, the term ine" shall refer to small cell—signaling protein
molecules that are secreted by the glial cells of the nervous system and by numerous cells
of the immune system and are a ry of signaling molecules used extensively in
intercellular communication. Cytokines can be classified as proteins, peptides, or
glycoproteins; accordingly, the term "cytokine" encompasses a large and diverse family
of regulators ed throughout the body by cells of diverse embryological origin. The
term "cytokine" shall encompass immunomodulating agents, such as interleukins and
interferons.
As used herein, the term "preconditioning" refers to a technique for producing
resistance to the loss ofblood , and thus oxygen, to tissues of the body. In the
present case, preconditioning refers to producing resistance to the loss of blood supply in
neurons, as measured by, e.g., assaying t or number of cells d following
apoaequorin administration as compared to a control.
The inventors have shown that, when injected directly into dorsal
hippocampus (dhpc), the 22 kD CaBP apoaequorin (AQ) derived from the coelenterate
Aequoria Victoria significantly decreases cell death when subjected to an in vitro
ischemic-type insult (Detert et al., 2009; 2011). This decrease in cell death was present at
24 and 48 hours, but not 1 hour post AQ injection. Interestingly, the AQ protein was
present at 1 hour, with cantly less at 24 hours, and by 48 hours post-injection, was
barely present. The inverse pattern n AQ’s effect and its presence made the
ors question r or not this change was due to a neuroimmunomodulatory
response. Some forms of ischemic preconditioning, such as small ischemic s, reduce
infarcts when given prior to larger, global s (Ara et al., 2010; Jones & on,
2001). Ischemic preconditioning’s protective effects have been associated with immune
system activation (Rehni & Singh, 2012; .Wei et al., 2012). While no one mechanism of
action is adopted herein, it is possible that ifAQ is activating cytokines, cell death
decreases by a sort of ischemic preconditioning where the neuroprotective response seen
from apoaequorin injections may be due to, in part, a neuroimmunomodulatory response.
Apoaequorin is administered to a patient in a therapeutically effective amount.
Apoaequrorin can be administered alone or as part of a pharmaceutically acceptable
composition. In addition, apoaequorin or a composition can be administered all at once,
as for example, by a bolus ion, multiple times, such as by a series of tablets, or
delivered substantially uniformly over a period of time, as for e, using transdermal
delivery. Further, the dose of the active agent can be varied over time. Apoaequorin can
be administered using an immediate e formulation, a controlled release formulation,
or combinations thereof. The term "controlled release" includes sustained release,
delayed release, and combinations thereof.
A ition of the invention can be prepared, packaged, or sold in bulk, as
a single unit dose, or as a ity of single unit doses. As used herein, a "unit dose" is
discrete amount of the composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal to the dosage of the
active ingredient that would be administered to a patient or a convenient fraction of such
a dosage such as, for example, one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically acceptable
r, and any additional ingredients in a composition of the invention will vary,
depending upon the identity, size, and condition of the human treated and further
depending upon the route by which the composition is to be stered.
Another aspect of the invention relates to a kit comprising a composition of
the invention and instructional al. Instructional material includes a publication, a
recording, a diagram, or any other medium of expression which is used to icate
the usefulness of the composition of the ion for one of the es set forth herein
in a human. The instructional material can also, for example, describe an appropriate
dose ofthe pharmaceutical composition ofthe invention. The instructional material of
the kit of the invention can, for e, be affixed to a ner which contains a
pharmaceutical composition of the invention or be shipped together with a container
which contains the pharmaceutical composition. Alternatively, the instructional al
can be shipped separately from the container with the intention that the instructional
material and the pharmaceutical composition be used cooperatively by the recipient.
The invention also includes a kit comprising a composition of the invention
and a delivery device for delivering the composition to a human. By way of example, the
delivery device can be a syringe, a needle, or a dosage- measuring container. The kit can
further se an instructional material as described herein. The kit also comprises a
container for the separate compositions, such as a divided bottle or a divided foil packet.
Additional examples of containers include syringes, boxes, bags, and the like. Typically,
a kit comprises directions for the administration of the separate components. The kit
form is ularly advantageous when the separate components are preferably
administered in different dosage forms (e.g., oral and parenteral), are administered at
different dosage intervals, or when titration of the individual components of the
combination is desired by the prescribing physician.
Parenteral administration of a pharmaceutical composition includes any route
of administration characterized by physical breaching of a tissue of a human and
administration of the ceutical composition through the breach in the tissue.
Parenteral administration thus includes administration of a pharmaceutical composition
by injection of the composition, by ation of the composition through a al
incision, by application of the composition through a tissue-penetrating non—surgical
wound, and the like. In particular, parenteral administration includes subcutaneous,
intraperitoneal, intravenous, intraarterial, intramuscular, or intrastemal injection and
intravenous, intraarterial, or kidney dialytic infusion techniques. For example, the
compositions of the present invention can be administered to a subject by brain (via
VPAG) injections, intrathecal injections, intraperitoneal injections, or blood injections.
Compositions suitable for eral ion comprise the active ingredient
combined with a pharmaceutically acceptable carrier such as physiologically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or may
comprise sterile powders for reconstitution into sterile injectable ons or dispersions.
Examples of le aqueous and nonaqueous carriers, diluents, solvents, or vehicles
include water, isotonic saline, ethanol, polyols (propylene glycol, polyethylene glycol,
glycerol, and the like), suitable mixtures thereof, triglycerides, including vegetable oils
such as olive oil, or injectable organic esters such as ethyl oleate. Proper fluidity can be
ined, for example, by the use of a coating such as lecithin, by the maintenance of
the required particle size in the case of dispersions, and/or by the use of surfactants. Such
formulations can be ed, packaged, or sold in a form suitable for bolus
administration or for continuous administration. Injectable ations can be prepared,
packaged, or sold in unit dosage form, such as in arnpoules, in multi-dose containers
ning a preservative, or in single-use devices for auto-inj ection or injection by a
medical practitioner.
Formulations for parenteral administration include sions, solutions,
emulsions in oily or aqueous es, pastes, and implantable sustained-release or
biodegradable ations. Such formulations can further comprise one or more
onal ients including suspending, stabilizing, or dispersing agents. In one
embodiment of a formulation for parenteral administration, the active ingredient is
provided in dry (i.e. powder or granular) form for titution with a suitable e
(e.g. sterile pyrogen-free water) prior to parenteral administration of the tituted
composition.
The compositions can be prepared, packaged, or sold in the form of a sterile
injectable aqueous or oily (emulsion) suspension or solution. This suspension or solution
can be formulated according to the known art, and can comprise, in addition to the active
ingredient, additional ingredients such as the dispersing agents, wetting agents, or
suspending agents described herein. Such sterile inj ectable formulations can be prepared
using a non-toxic parenterally—acceptable diluent or solvent, such as water or 1,3—
butanediol, for example. Other acceptable diluents and solvents include Ringer's
solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-
glycerides. Other parentally-administrable formulations which are useful include those
which comprise the active ingredient in microcrystalline form, in a liposomal preparation,
or as a component ofbiodegradable polymer systems. Compositions for sustained
release or implantation can comprise pharmaceutically acceptable polymeric or
hydrophobic materials such as an emulsion, an ion exchange resin, a gly soluble
polymer, or a sparingly soluble salt.
Compositions ofthe ion may also contain adjuvants such as preserving,
g, emulsifying, and/or sing agents, ing, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include
isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption
of injectable pharmaceutical compositions can be brought about by the use of agents
capable of delaying absorption, for example, um monostearate and/or gelatin. In
particular, mes, mysomes and emulsifiers can be used in to make the present
compounds more soluble for delivery.
Dosage forms can e solid or inj ectable implants or depots. In preferred
embodiments, the implant comprises an effective amount of an active agent and a
biodegradable polymer. In preferred embodiments, a suitable biodegradable polymer can
be selected from the group consisting of a polyaspartate, polyglutamate, poly(L—lactide),
a poly(D,L-lactide), a poly(lactide-co-glycolide), a poly(s-caprolactone), a
polyanhydride, a eta-hydroxy-butyrate), a poly(orth0—ester) and a
polyphosphazene. In other embodiments, the implant comprises an effective amount of
active agent and a silastic polymer. The implant provides the release of an effective
amount of
Liquid solutions of the active ingredient in aqueous or oily solvents can be
prepared in substantially the same manner as liquid suspensions, the primary difference
being that the active ingredient is ved, rather than suspended in the solvent. Liquid
solutions of the composition of the invention can comprise each of the components
described with regard to liquid suspensions, it being understood that ding agents
will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous
solvents include, for example, water and isotonic . Oily solvents include, for
example, almond oil, oily esters, ethyl l, vegetable oils such as arachis, olive,
sesame, or t oil, fractionated vegetable oils, and mineral oils such as liquid
paraffin.
Compositions ofthe t invention may further include a cyclodextrin
component in order to, e.g., improve water solubility of an active ceutical
ingredient, prolong drug release, and improve tableting teristics. In general, cyclic
structure oligomers of glucose odextrin") are obtained from the starch digests of
certain bacteria. The most abundant cyclodextrins are alpha, beta and gamma
cyclodextrin which have 6,7 and 8 glucose units, respectively. The interior cavity of a
cyclodextrin is hydrophobic and the exposed surface of the molecule is hydrophilic.
Cyclodextrins are known to enhance active pharmaceutical ingredient stability, aqueous
solubility, and reduce volatility. Some examples of commercially available cyclodextrin,
or tives thereof, are as follows: alpha-Cyclodextrin (CAS #: 100163); (2-
Hydroxypropyl)-alpha-cyclodextrin (CAS #: 1284463); beta-Cyclodextrin (CAS #:
75859); 6-O-alpha-D-Glucosyl-beta—cyclodextrin (CAS #: 92517-02—7); gamma-
Cyclodextrin (CAS #: 174650); and (2-Hydroxypropyl)gamma-cyclodextrin (CAS
#: 128446—34-4). Cyclodextrins particularly useful in formulating a delivery vehicle to
ster the present active agents include: the sulfobutyl ether beta-cyclodextrin (SBE-
D) available from Cydex Pharmaceuticals, Inc. under the tradename CAPTISOL;
and the cyclodextrin and hydroxypropyl betacyclodextrins ble from Roquette
Pharma under the tradename KLEPTOSE.
For parenteral administration in non-human animals, apoaequorin may be
prepared in the form of a paste or a pellet and administered as an implant. Paste
formulations can be prepared by dispersing apoaequorin in pharrnaceutically able
oil such as peanut oil, sesame oil, corn oil or the like. Pellets containing a therapeutically
effective amount can be prepared by admixing with a diluent such as a carbowax,
camauba wax, and the like, and a lubricant, such as magnesium or calcium stearate, can
be added to improve the pelleting process. It is, of course, recognized that more than one
pellet may be administered to an animal to achieve the desired dose level. Moreover, it
has been found that such implants may also be administered periodically during the
animal ent period in order to maintain the proper active agent level in the animal's
body.
The compositions ofthe present invention can be stered to a patient at
dosage levels in the range of from about 0.01 to about 100 mg/kg per day. For a normal
adult human, a dosage in the range offiom about 0.01 to about 100 mg/kg is typically
sufficient, with 0.1 to 10 mg/kg per day a preferred dosage. However, some variability in
the general dosage range may be required depending upon the age and weight ofthe
subject being treated, the intended route of administration, the particular compound being
administered and the like. The determination of dosage ranges and optimal dosages for a
particular patient is well within the ability of one of ordinary skill in the art having the
benefit of the instant sure. It is also noted that the compounds of the present
ion can be used in sustained release, controlled release, and delayed release
formulations, which forms are also well known to one of ry skill in the art.
It is not critical whether the compositions of the present ion are
administered directly to the cell, to a tissue comprising the cell, a body fluid that contacts
the cell, or a body location from which the compound can e or be transported to the
cell. It is sufficient that the composition is administered to the patient in an amount and
by a route whereby an amount of the composition sufficient to mobilize lipids in the cell
arrives, directly or indirectly at the cell. The minimum amount varies with the identity of
the compositions.
The specific dosage and dosage range that can be used s on a number
of factors, including the requirements of the patient, the ty of the condition being
treated, and the pharmacological activity of the active ingredient being administered. The
determination of dosage ranges and optimal dosages for a particular patient is well within
the ordinary skill of one in the art in View of this disclosure. It is understood that the
rily d physician, dentist, or veterinarian will readily ine and prescribe
an effective amount of the composition to bring about the desired treatment in the
subject. In so proceeding, the physician or veterinarian can, for example, prescribe a
vely low dose at first, subsequently increasing the dose until an appropriate response
is obtained. It is further understood, r, that the specific dose level for any
particular human will depend upon a variety of factors including the activity of the
specific composition employed, the age, body , general health, gender, and diet of
the human, the time of administration, the route of administration, the rate of excretion,
any drug combination, and the severity of any disorder being treated.
In certain embodiments, apoaequorin is formulated in the form of a
pharmaceutical injectable dosage, including apoaequorin combination with an injectable
carrier system. As used herein, inj ectable and infusion dosage forms (i.e., parenteral
dosage forms) include, but are not limited to, liposomal inj ectables or a lipid bilayer
vesicle having olipids that encapsulate an active drug substance. Injection
includes a sterile preparation intended for parenteral use.
Five distinct classes of injections exist as defined by the USP: emulsions,
lipids, powders, solutions and sions. Emulsion injection includes an emulsion
comprising a sterile, pyrogen—free preparation intended to be administered parenterally.
Lipid complex and powder for solution injection are sterile preparations intended for
reconstitution to form a solution for parenteral use. Powder for suspension injection is a
e preparation intended for reconstitution to form a suspension for parenteral use.
Powder lized for mal sion injection is a sterile freeze dried preparation
intended for reconstitution for parenteral use that is ated in a manner allowing
incorporation of liposomes, such as a lipid bilayer vesicle having phospholipids used to
encapsulate an active drug substance within a lipid bilayer or in an aqueous space,
whereby the formulation may be formed upon reconstitution. Powder lyophilized for
solution injection is a dosage form intended for the solution prepared by lyophilization
("freeze drying"), whereby the process involves removing water from products in a
frozen state at extremely low pressures, and whereby subsequent addition of liquid
creates a solution that conforms in all respects to the requirements for ions. Powder
lized for suspension injection is a liquid preparation intended for parenteral use that
contains solids suspended in a le fluid medium, and it ms in all respects to
the requirements for Sterile Suspensions, whereby the medicinal agents intended for the
suspension are prepared by lyophilization. Solution injection involves a liquid
preparation containing one or more drug substances dissolved in a suitable solvent or
mixture of mutually miscible solvents that is suitable for injection. Solution concentrate
injection involves a sterile preparation for eral use that, upon addition of suitable
solvents, yields a solution conforming in all respects to the requirements for injections.
Suspension injection involves a liquid preparation ble for injection) containing solid
particles sed throughout a liquid phase, whereby the particles are insoluble, and
whereby an oil phase is dispersed throughout an aqueous phase or Vice-versa.
Suspension liposomal injection is a liquid preparation (suitable for injection) having an
oil phase dispersed throughout an s phase in such a manner that liposomes (a lipid
bilayer vesicle usually ning phospholipids used to ulate an active drug
substance either within a lipid bilayer or in an aqueous space) are formed. Suspension
sonicated injection is a liquid ation (suitable for injection) containing solid
particles dispersed hout a liquid phase, whereby the particles are insoluble. In
addition, the product may be sonicated as a gas is bubbled through the suspension
resulting in the formation of microspheres by the solid particles.
The parenteral carrier system es one or more pharmaceutically le
excipients, such as solvents and co-solvents, solubilizing agents, wetting agents,
suspending agents, thickening agents, emulsifying , chelating agents, buffers, pH
adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents,
protectants, tonicity adjusters, and special additives.
s exemplary embodiments of compositions and methods according to
this invention are now described in the following examples. The following examples are
offered for illustrative purposes only and are not intended to limit the scope of the present
invention in any way. , various modifications ofthe ion in addition to those
shown and described herein will become apparent to those skilled in the art from the
ing description and the following es and fall within the scope of the
appended claims.
III. EXAMPLES
Example 1: Time course and effectiveness of apoaequorin in altering
expression levels of IL-10.
In this example, the inventors demonstrate the effectiveness of apoaequorin
increasing mRNA expression ofthe anti-inflammatory cytokine IL—10. Preconditioning
is a phenomenon in which a brief non-lethal ischemic episode attenuates the damage
caused by a subsequent more severe ischemic insult. Preconditioning reduces expression
of inflammatory ors (e.g., IL-lB and IL—6;) compared to that seen in response to
brain injury. IL-10 is an anti-inflammatory cytokine and has been shown to be increased
following administration of a Cay-channel blocker. Preconditioning increases the
production of IL-1 0, and IL-10 is ated with neuroprotection. One way it may be
neuroprotective is by indirectly reducing IL-6 through reduction of TNF-a production.
MATERIALS AND METHODS
Animals. 50 male F344 adult rats (mean age = 3.4 i 0.2 mo.) were used. Rats
were kept on a 14/1 O-hr day/night cycle with free access to food and water.
Surgery. Rats were anesthetized and mounted on a taxic apparatus.
Under aseptic ions, the scalp was incised and retracted to the side, and the head
was leveled between bregma and lambda. Each rat was prepared with bilateral stainless
steel guide cannulae aimed at the dorsal hippocampus (dhpc) using stereotaxic
coordinates (3.5 mm posterior, d: 2.6 mm l, 3.0 mm ventral) relative to bregma.
Cannulae were secured to the skull with stainless steel screws and epoxy. A stainless
steel cap remained in place when the rats were not being injected to prevent the guide
cannulae from becoming occluded.
Drugs. Apoaequorin (AQ; 0, 0.4, 1, and 4%, Quincy Bioscience) was
prepared in zero Ca2+-aCSF (artificial cerebral spinal fluid) with 6% DMSO added to
facilitate neuronal uptake. Rats were given an infusion of AQ in one hemisphere and
vehicle in the other hemisphere 1 hr, 1 day, 2 days, 3 days, or 5 days prior to
decapitation. ral infusions (0.5 ul/side) were given over 60 sec and the injection
cannulae remained in place for an additional 2 min to ensure diffusion. The infusion
cannulae were cut to extend 0.5 mm beyond the guide cannulae.
Oxygen-Glucose Deprivation. l slices (400 um) were ed using
standard procedures. Following l-hour slice recovery in aCSF, in vitro ischemia was
induced by transferring slices to fructose-CSF (glucose replaced with fructose and
bubbled with 95% N2 — 5% C02 instead of a 95% 02 — 5% C02) for 5 min. The slices
were then returned to oxygenated aCSF containing 0.2% trypan blue for 30 min
reperfusion. Trypan blue readily penetrates dead cells and stains them blue while leaving
living cells unstained. The slices were rinsed in oxygenated room temperature aCSF
twice then fixed in 10% neutral ed formalin overnight in the refrigerator. Slices
were then cryoprotected, cut on a at (40 um), and mounted onto subbed slides.
Cell Counts. Slices were examined under an s microscope (equipped
with a digital camera) at 10X, and pictures were taken. Trypan blue stained neurons
within CA1 (about an 800 um section) were counted by an menter blind to
experimental conditions. Statistical analyses were med using Statview (v 5.0; SAS
Institute, Inc., Cary, NC). An ANOVA was used to evaluate a drug effect. Asterisk
indicates p < .05.
Western Blots. Rats were given a bilateral injection of 4% AQ and brains
were removed at one ofthe following time points: 1 hour, 1 day, 2 days, or 3 days. Brains
were rapidly frozen and stored at -80°C. The dhpc and ventral hippocampus (vhpc) were
dissected out and homogenized tely. s were centrifuged and the supernatant
removed and measured using a Bradford protein assay kit (Bio-Rad). Protein samples
were normalized and loaded for SDS-PAGE (10%). Proteins were transferred onto PVDF
membranes using a semidry transfer apparatus (Bio-Rad). Membranes were then
incubated in blocking buffer (2 hours), y antibody (overnight at 4 °C; 1:5000
mouse anti-aequorin con] or 1:1000 rabbit anti-B-actin [Cell Signaling
Technology]), and secondary antibody (90 min; 1:5000 goat anti—mouse [Santa Cruz
hnology] or 1:5000 goat anti-rabbit [Millipore]). Membranes were then washed,
placed in a uminescence solution (Santa Cruz Biotechnology), and exposed to
autoradiography film (Hyperfilm MP). Images were taken and densitometry was
performed using NIH Image J re. A band was considered positive if the optical
density value of the band (minus the background for each lane) was greater than 2
standard deviations above the mean of the vhpc bands.
PCR. For RT-PCR, rats were given an infusion of 4% AQ in one hemisphere
and vehicle in the other hemisphere. One hour, 1 day or 2 days post—infusion, the
hippocampi were removed and immediately placed into Trizol. The tissue was
homogenized using a 25-gauge needle and syringe, and samples were frozen and stored at
-80°C until RNA isolation. RNA was isolated using Qiagen RNeasy mini kit protocol.
Isolated RNA was dissolved in 50 ul RNase free H20. RNA purity was calculated based
on the absorbance ratio of 260 and 280 nm, and an absorbance reading between 1.8 and
2.1 was considered pure RNA. Reverse ription was conducted to produce cDNA
using the Qiagen RT2 HT First Stand Kit. IL-10 and n primers were purchased
from Qiagen and used in ance with the Qiagen RT2 qPCR Primer Assay.
Amplification of cDNA was ed by fluorescence of SYBR green (nonspecific
DNA stain). Samples were run in triplicate in 96 well plates using StepOne Real Time
PCR system and software. Gene expression of the housekeeping protein B-actin, served
as a quantification baseline for cytokine expression. Gene expression changes were
analyzed using the Pfaffl method. Primer efficiency was calculated based on two
randomly selected samples for each of B-actin and IL-10.
As shown in Figure l, anti—inflammatory cytokines sed following
apoaequorin infusion. In particular, IL-10 mRNA was elevated as early as 1 hour post-
infusion. No change in B-actin expression was detected between groups. In view ofthe
well known onship n increased level of anti-inflammatory nes and
inflammation reduction, it can be concluded that apoaequorin is a usefiil agent for
reducing inflammation in a medically relevant animal model system, one that is relevant
and immediately applicable to the human setting.
Example 2: Apoaequorin protects neurons from ischemia and alters
cytokine mRNA levels in rat hippocampus.
Referring to Fig. 2A, the inventors demonstrated that an intrahippocampal
injection of uorin (AQ, the CaBP component of aequorin) significantly protects
hippocampal s from ischemic cell death. The neuroprotective s ofAQ were
first observed at 24 hours and endured for up to 48 hours (Fig. 2B and 2C). Interestingly,
western blot analysis suggested that AQ protein levels dramatically decrease over this
time period (Fig. 2D). Although the neuroprotective effects ofAQ were strongest at 48
hours, the relatively low level ofAQ n present at that time suggests that other
factors may play a role in the ability ofAQ to protect neurons from ischemic cell death.
One possibility is that AQ administration leads to a change of levels of anti-
inflammatory cytokines. Interleukin-10 (IL-10), an anti—inflammatory ne, has
previously demonstrated the ability to protect neurons from hypoxia in vitro. To test the
hypothesis that cytokine expression may play a role in the neuroprotective effects of AQ,
cytokine mRNA levels were measured at different time points following a , intrahippocampal
administration ofAQ. Twelve male F344 rats were bilaterally ated in
the dorsal hippocampus. Following recovery, 4% AQ was injected unilaterally (vehicle in
the other side, counterbalanced) and after 1, 24, or 48 hrs, the brains were d and
the hippocampi dissected, placed on dry ice, and homogenized. RNA was isolated using
Qiagen’s RNeasy mini kit. Reverse transcription was performed using Qiagen’s RT2 HT
First Strand Kit-96 and qPCR was used to quantify IL—10 and B-actin. As shown in Fig.
3A, IL-10 was significantly increased in the AQ treated hemispheres at 1 hour (t(14) =
.30, p < .05), but not 24 or 48 hours (p > .05) post injection. AQ treatment did not affect
B-actin mRNA levels (Fig. 3B).
Further PCR array analyses with ’s RT2 Profiler Arrays for cytokines
and chemokines were performed in an effort to evaluate whether other cytokines and
ines are activated at these time points (Fig. 3C). rative s are depicted in
Fig. 4 and suggest that multiple nflammatory cytokine and chemokine mRNA levels
change in a time—dependent manner following AQ administration.
Fig. 5 depicts a schematic showing the interplay of immune response genes
examined by the inventors. Accordingly, the inventors parsed the assayed genes by
function, and Figure 6 depicts graphs showing (A) change in acute inflammatory
response genes ing AQ injection; (B) change in T/B cell activators following AQ
injection; (C) change in angiogenesis/cell proliferator genes following AQ injection; (D)
pattern of change in macrophage attracting genes following AQ injection; (E) change in
m or genes ing AQ injection; and (F) genes grouped by function
change with AQ ion. Table 1 ts this data in tabular form. Collectively, these
results suggest that AQ is neuroprotective and that this neuroprotection involves a
neuroimmunomodulatory mechanism.
Summary
The data presented in this example demonstrate that the neuroprotective
effects of apoaequorin are time-dependent. An injection ofAQ into area CA1 of dorsal
hippocampus significantly decreases cell death when administered 24 and 48, but not 1
hour prior to ischemic insult. Interestingly, AQ shows an inverse relationship between
availability and efficacy.
The present data further demonstrate that the neuroprotective effects of
apoaequorin may involve a neuroimmunomodulatory mechanism. IL-lO tion post-
injection suggests the immune system plays a role in its protective effect. Time-
dependent activation of other cytokines and chemokines support this hypothesis.
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Other embodiments and uses ofthe invention will be apparent to those d
in the art from consideration from the specification and practice of the invention
disclosed herein. All references cited herein for any , including all journal
citations and U.S./foreign patents and patent applications, are specifically and entirely
incorporated herein by reference. It is tood that the invention is not d to the
specific reagents, formulations, reaction conditions, etc., herein illustrated and described,
but embraces such modified forms thereof as come within the scope ofthe following
claims.
REFERENCES
1. Ara, J., Fekete, 8., Frank, M., Golden, J. A., Pleasure, D., Valencia, I. (2011).
Hypoxic-preconditioning induces neuroprotection against hypoxia—ischemia in
newborn piglet brain. Neurobiology ofDisease, 43, 473 -485.
2. Baimbridge, K. G., Celio, M. R., & Rogers, J. H. (1992). Calcium-binding
proteins in the nervous system. Trends in Neuroscience, 15(8), 8
3. Bano, D., Young, K. W., Guerin, C. J., Lefeuvre, R., Rothwell, N. J., Naldini, L.,
et a1. (2005). Cleavage of the plasma membrane Na+/Ca2+ exchanger in
excitotoxicity. Cell, 120(2), 275-285.
4. Chard, P. S., Bleakman, DJ, Christakos, S., Fullmer, C. S., & Miller, R. J. (1993).
Calcium buffering properties of calbindin D28k and parvalbumin in rat y
neurones. Journal ofPhysiology, 472, 341-357.
. Choi, D. W. (1992). Excitotoxic cell death. Journal of Neurobiology, 23(9),
1261-1276.
6. Detert, J. A., Hochstetter, E. L., Lescher, J. L., Van Langendon, T. M., & Moyer,
J. R., Jr. (2011) Time course and effectiveness of aqoaequorin as a
neuroprotectant in the brain, Society for Neuroscience Abstracts, m No.
781.04.
7. Detert, J. A., r, J. D., Hochstetter, E. L., Van don, T. M., & Moyer,
J. R., Jr. . Neuroprotection of hippocampal CA1 neurons from ischemic
cell death using the m binding protein aequorin, Society for Neuroscience
Abstracts, 35, Program No. 52.24.
8. Fan, Y., Shi, L., Gu, Y., Zhao, Y., Xie, J., Qiao, J., et a1. (2007). Pretreatment
with lbindin D 28k alleviates rat brain injury induced by ischemia and
reperfusion. Journal ofCerebral BloodFlow and Metabolism, 27(4), 8.
Gary, D. S., Sooy, K., Chan, S. L., Christakos, S., & Mattson, M. P. (2000).
Concentration— and cell type-specific s of calbindin D28k on vulnerability
of hippocampal neurons to seizure-induced injury. Brain Research. Molecular
Brain Research, 75(1), 89-95.
. Hochstetter, E. L., Detert, J. A., r, J. D., and Moyer, J. R., Jr. (2012).
Apoaequorin protects neurons from ia and alters cytokine mRNA levels in
rat hippocampus. yfor Neuroscience Abstracts, 38, Program No. .
11. Jones, N. M., & Bergeron, M. . Hypoxic preconditioning induces changes
in HIF-l target genes in neonatal rat brain. Journal ofCerebral Blood Flow and
Metabolism, 21(9), 1 105-1114.
12. Kristian, T., & Siesjo, B. K. (1998). Calcium in ischemic cell death. Stroke,
29(3), 705-718.
13. Lee, J. M., , G. J., & Choi, D. W. (1999). The changing landscape of
ischaemic brain injury mechanisms. Nature, 399(6738 Suppl), A7-l4.
14. Rehni, A. K., & Singh, T. G. (2012). Involvement of COR-2 chemokine receptor
activation in ischemic preconditioning and postconditioning of brain in mice.
Cytokine, 60, 83-89.
. Roger, V. L., Go, A. S., Lloyd-Jones, D. M., Benjamin, B. J., Berry, J. D.,
Borden, W. B., Bravata, D. M.,...Turner, M. B. (2012). Heart disease and stroke
statistics-2012 update: a report from the American Heart Association.
Circulation, 125:e2—e220. doi: 10.1l61/CIR.0b013e31823ac046.
16. Wei, D., Ren, C., Chen, X., Zhao, H. (2012). The chronic protective effects of
limb remote preconditioning and the underlying mechanisms involved in
atory factors in rat stroke. PLoS ONE, 7(2): e30892. doi:
.1371/journal.pone.003892.
17. Yenari, M. A., , M., Sun, G. H., Meier, T. J., Kunis, D. M., McLaughlin,
J. R., et a1. (2001). Calbindin d28k overexpression protects striatal neurons from
transient focal cerebral ia. Stroke, 32(4), 1028-1035.
Claims (4)
1. Use of Apoaequorin in the cture of a medicament for preconditioning neurons to reduce neuronal injury due to brain ia in a subject by administering the apoaequorin to the subject 48 hours before the brain ischemia occurs.
2. Use of Apoaequorin according to claim 1, wherein the apoaequorin is formulated for administration by injection.
3. Use of Apoaequorin according to claim 2, wherein the injection is an intra-hippocampal injection.
4. Use of uorin according to any one of claims 1 to 3, wherein the subject is human.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161559816P | 2011-11-15 | 2011-11-15 | |
US61/559,816 | 2011-11-15 | ||
PCT/US2012/065291 WO2013074798A1 (en) | 2011-11-15 | 2012-11-15 | Apoaequorin for reducing neuronal injury due to ischemia |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ624736A NZ624736A (en) | 2016-05-27 |
NZ624736B2 true NZ624736B2 (en) | 2016-08-30 |
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