Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DIRECT ACTIVATION OF ATIII IN WHOLE BLOOD AND PLASMA
RELATED APPLICATIONS
[0001] This application claims priority to provisional application U.S.S.N.
60/586,043,
filed July 7, 2004, the content of which application is specifically
incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Although vaccines are available to prevent many types of viral
infection, not all
viruses are able to be prevented by a vaccine and not all potential victims
are able to receive
vaccinations even if they are available. For example, the retrovirus human
immunodeficiency virus (HIV) causes Acquired Immunodeficiency Syndrome (AIDS),
an
incurable disease for which there is no vaccine in which the body's immune
system breaks
down leaving the victim vulnerable to opportunistic infections, e.g.,
pneumonia, and certain
cancers, e.g., Karposi's Sarcoma. Many patients with HIV are co-infected with
Hepatitis C
Virus (HCV), Hepatitis B (HBV), or other viruses. Although a vaccine is
available for
certain viruses such as HBV, many at-risk people do not receive or have access
to the
vaccine. Further, some data indicate that the HBV vaccine is not as effective
in people
already infected with HIV.
[0003] Viral infections, once established, are generally incurable. There are,
however, a
variety of anti-viral drugs that can prevent viruses from reproducing and
ravaging the
body's immune system, i.e., that slow the infection and lengthen the subject's
life.
However, such therapies often only partially effective, and it is unknown how
much viral
suppression is required to achieve durable virologic, immunologic, and
clinical benefits.
Anti-viral drugs are often highly toxic and can cause serious side effects,
including heart
damage, kidney failure, and osteoporosis.
[0004] For example, highly active antiretroviral drug therapy (HAART) is a
widely used
anti-HIV therapy that entails multiple-drug protease inhibitor-containing
regimens that can
completely suppress viral replication. Hepatic injury is a major concern as a
result of
antiretroviral therapy (HAART) and has been shown to occur with all classes of
antiretroviral therapy. The efficacy of current anti-HIV therapy is further
limited by the
complexity of regimens, pill burden, and drug-drug interactions. Compliance
with the toxic
effects of antiretroviral drugs make a lifetime of combination therapy a
difficult prospect
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and many patients cannot tolerate long-term treatment with HAART. Further,
poor
adherence to combination therapy regimes has led to the emergence of drug-
resistant strains
of HIV.
[0005] There is clearly a need for new anti-viral agents and other novel
approaches to
treating viral infection.
SUMMARY OF THE INVENTION
[0006] The present invention is based on the surprising finding that ATIII may
be activated
using saccharides such as heparin directly in situ in a blood product, such as
blood, plasma,
serum albumin, recombinant plasma, and the like, ad re-administered to a
subject. For
example, a blood product of a subject may be incubated with heparin or other
saccharide,
dialyzed, and re-administered.into the subject. Alternatively, a blood product
from another
source, such as a commercial source, may be incubated with heparin or other
saccharide,
dialyzed, and administered to a subject. The blood products so treated contain
activated
ATIII in an effective dose to treat viral infection. This novel treatment
eliminates the need
for activated ATIII preparation in advance. Such treated blood products as a
treatment may
be useful, for example, in treating patients having viral infections or
diseases or conditions
that are caused by or contributed to by thrombin activation. The methods of
treatment
provided by the present invention may result in shorter pre-clinical and
clinical testing
times. Further, these methods of treatment provide an alternative, or
supplement, to
existing treatment methods of viral infection using purified activated ATIII
pharmaceutical
preparations.
[0007] Other features and advantages of the invention will become apparent
from the
following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGURE 1 depicts the HIV-1 inhibitory activity in blood treated with
LMW heparin
(as described below in Example 4).
[0009] FIGURE 2 depicts the mechanism of action of activated heparin. Pure
ATIII from
Aventis (human source) and from GTC (recombinant) are inactive.
Oligosaccharide and
ATIII incubation and/or incubation with heat accelerate the interaction of
these molecules
to form a complex that undergoes a conformational change to produce an active
form.
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DETAILED DESCRIPTION OF THE INVENTION
[0010] 1. Definitions
[0011] For convenience, certain terms employed in the specification, examples,
and
appended claims are collected here. Unless defined otherwise, all technical
and scientific
terms used herein have the same meaning as commonly understood by one of
ordinary skill
in the art to which this invention belongs.
[0012] The articles "a" and "an" are used herein to refer to one or to more
than one (i.e., to
at least one) of the grammatical object of the article. By way of example, "an
element"
means one element or more than one element.
[0013] The term "activating ATIII" or "to activate ATIII" refers to treating
ATIII under
suitable conditions such that the ATIII becomes high molecular weight ATIII
and is able to
reduce viral load, i.e."
[0014] The term "administering" includes any method of delivery of a
pharmaceutical
composition or therapeutic agent into a subject's system or to a particular
region in or on a
subject. The phrases "systemic administration," "administered systemically,"
"peripheral
administration" and "administered peripherally" as used herein mean the
administration of a
compound, drug or other material other than directly into the central nervous
system, such
that it enters the patient's system and, thus, is subject to metabolism and
other like
processes, for example, subcutaneous administration. "Parenteral
administration" and
"administered parenterally" means modes of administration other than enteral
and topical
administration, usually by injection, and includes, without limitation,
intravenous,
intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular,
subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
[0015] The term "ATIII" refers to antithrombin III.
[0016] The term "blood product" refers to any product or substance that is
blood or is
derived from blood. For example, blood products include, but are not limited
to, whole
blood, plasma, serum, serum albumin preparations, and artificial preparations
of the same,
such as recombinant plasma.
[0017] The term "mammal" is known in the art, and exemplary mammals include
humans,
primates, bovines, porcines, canines, felines, and rodents (e.g., mice and
rats).
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[0018] The term "high molecular weight ATIII" means ATIII that has been
treated to
increase the molecular weight of the molecule relative to wildtype ATIII, e.g.
to about 60
Kda to about 240 kDa from 58 kDa.
[0019] A"patient," "subject" or "host" to be treated by the subject method may
mean either
a human or non-human animal.
[0020] The phrase "pharmaceutically acceptable" refers to those compositions
and dosages
thereof within the scope of sound medical judgment, suitable for use in
contact with the
tissues of human beings and animals without excessive toxicity, irritation,
allergic response,
or other problem or complication, commensurate with a reasonable benefit/risk
ratio.
[0021] The phrase "pharmaceutically-acceptable carrier" means a
pharmaceutically-
acceptable material, composition or vehicle, such as a liquid or solid filler,
diluent,
excipient, solvent or encapsulating material, involved in carrying or
transporting any
supplement or composition, or component thereof, from one organ, or portion of
the body,
to another organ, or portion of the body. The term "pharmaceutically
acceptable carrier"
refers to a carrier(s) that is "acceptable" in the sense of being compatible
with the other
ingredients of a composition and not deleterious to the recipient thereof.
Some examples of
materials which may serve as pharmaceutically-acceptable carriers include: (1)
sugars, such
as lactose, glucose and sucrose; (2) starches, such as corn starch and potato
starch; (3)
cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;
(8) excipients,
such as cocoa butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols,
such as propylene
glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12)
esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-
free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)
phosphate buffer
solutions; and (21) other non-toxic compatible substances employed in
pharmaceutical
formulations.
[0022] The term "saccharide" includes both monosaccharides and
polysaccharides.
[0023] The term "a saccharide able to activate ATIII" refers to any saccharide
able to
transform wildtype ATIII into activated and/or high molecular weight ATIII
that has the
ability to reduce viral load.
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[0024] The term "therapeutically effective amount" refers to that amount of
activated
ATIII, drug or other molecule which is sufficient to effect treatment when
administered to a
subject in need of such treatment. The therapeutically effective amount will
vary
depending upon the subject and disease condition being treated, the weight and
age of the
subject, the severity of the disease condition, the manner of administration
and the like,
which can readily be determined by one of ordinary skill in the art.
[0025] The term "treating" as used herein is intended to encompass curing as
well as
ameliorating at least one symptom or the progression of any condition or
disease.
decreased or prevented. Treatment may be performed either prophylactically, or
subsequent
to the initiation of a pathologic event.
100261 The term "viral load" refers to the concentration of a virus, such as
HIV, in the blood.
[0027] Unless otherwise indicated, all numbers expressing quantities of
ingredients,
reaction conditions, and so forth used in the specification and claims are to
be understood as
being modified in all instances by the term "about." Accordingly, unless
indicated to the
contrary, the numerical parameters set forth in this specification and
attached claims are
approximations that may vary depending upon the desired properties sought to
be obtained
by the present invention.
[002812. In Situ Blood Activation of ATIII
[0029] Antithrombin III (ATIII) is a glycoprotein present in blood plasma with
a well-
defined role in blood clotting. Specifically, ATIII is a potent inhibitor of
the reactions of
the coagulation cascade with an apparent molecular weight of between 54k Da
and 65 kDa
(Rosenberg and Damus, J. Biol. Chem. 248: 6490-505 (1973); Nordenman et al.,
Eur. J.
Biochem., 78: 195-204 (1977); Kurachi et al., Biochemistry 15: 373-7 (1976)),
of which
some ten percent is contributed by four glucosamine-base carbohydrate chains
(Kurachi et
al., Biochemistry 15: 373-7 (1976); Petersen et al., in The Physiological
Inhibitors of
Coagulation and Fibrinolysis (Collen et al., eds) Elsevier, Amsterdam, p. 48
(1979)).
[0030] Although its name implies that it works only on thrombin, ATIII
actually serves to
inhibit virtually all of the coagulation enzymes to at least some extent. The
primary
enzymes it inhibits are factor Xa, factor IXa, and thrombin (factor IIa). It
also has
inhibitory actions on factor XIIa, factor Xia and the complex of factor VIIa
and tissue factor
but not factor VIIa and activated protein C. ATIII also inhibits trypsin,
plasmin and
kallikrein (Charlotte and Church, Seminars in Hematology 28:3-9 (1995). Its
ability to
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limit coagulation through multiple interactions makes it one of the primary
natural
anticoagulant proteins.
[0031] ATIII acts as a relatively inefficient inhibitor on its own. However,
ATIII can be
activated by a simple template mechanism, or by an allosteric conformational
change
brought about by heparin binding (Skinner et al., J. Mol. Biol. 283: 9-14
(1998);
Huntington et al., J. Mol. Biol., 293: 449-55 (1999); Belar et al., J. Mol.
Biol. Chem., 275:
8733-41 (2000)). When ATIII binds heparin, the speed with which the reaction
that causes
inhibition occurs is greatly accelerated. This interaction is the basis of
heparin based
anticoagulation therapies.
[0032] Pure plasma derived or recombinant ATIII is inactive in lowering HIV
viral loads.
However, we have shown in U.S.S.N. 10/436,872 that ATIII that has been treated
to have a
higher molecular weight, or "activated," effectively reduces HIV viral loads
in infected
cells. This application, which is hereby incorporated by reference in its
entirety, features
pharmaceutical compositions comprising high molecular weight antithrombin III
(ATIII) in
an amount effective to treat a retroviral infection. Incubation of the ATIII
with heparin
and/or heat in a variety of combinations was shown to activate the ATIII and
inhibit
proliferation of the retroviral infection.
[0033] The present invention is based on the surprising finding that ATIII may
be activated
using saccharides such as heparin directly in situ in a blood product, such as
blood, plasma,
serum albumin, recombinant plasma, and the like. The blood product so treated
contains
activated or high molecular weight ATIII. This novel treatment eliminates the
need for
advance ATIII preparation, e.g. preparation, purification and formulation into
a
pharmaceutical composition. Such a treatment may be useful, for example, in
treating
patients having viral disease or diseases or conditions that are caused by or
contributed to
by thrombin activation.
[0034] For example, the patient's own blood or plasma or serum albumin in
combination
with heparin may be incubated at moderate conditions (about 37 to about 40 C),
(not
necessary if the saccharide concentration is much lower than the ATIII
equimolar
concentration in the treated blood), and the activated blood product injected
back into the
patient. The rationale behind this idea is that most patients will have
between about 100 to
about 150 mg ATIII per liter of blood, for which only a low heparin quantity
is needed for
activation.
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[0035] Accordingly, a method for preparing activated ATIII in situ in a blood
product may
comprise: (a) adding to a blood product comprising ATIII a saccharide able to
activate
ATIII in an amount sufficient to activate said ATIII; and (b) incubating the
resulting
mixture of the blood product and saccharide under conditions sufficient to
activate said
ATIII. In certain embodiments, the saccharide may be added in a quantity in
excess of that
quantity of ATIII in the blood product, for example, in an about 0.1 to about
1.0 equimolar
amount of the quantity of ATIII in the blood product.
[0036] The conditions sufficient to activate the ATIII are, in certain
embodiments about
37 C to about 40 C for about 24 to about 72 hours. In certain embodiments, the
temperature is about 37 C to about 38 C, about 38 C to about 39 C or about 39
C to about
40 C. In certain embodiments, the incubation time is about 24 to about 36
hours, about 36
to about 48 hours, about 48 to about 60 hours, or about 60 to about 72 hours.
[0037] The blood product may be any product or substance that is blood or is
derived from
blood. For example, blood products include, but are not limited to, whole
blood, plasma,
serum, serum albumin preparations, and artificial preparations of the same,
such as
recombinant plasma.
100381 The saccharide, may be for example, an oligosaccharide, such as any
form of
heparin, including low molecular weight heparin (about 2 to about 4 kDa), high
molecular
weight heparin (at least 12 kDa), and standard unfractionated heparin. In
certain
embodiments, the saccharide is a pentasaccharide. Further, the saccharide may
be an
oligosaccharide treated by a glycosidase or other restriction enzyme. In
certain
embodiments, the saccharide may be added in an about a 0.1 to about 0.2, about
0.2 to
about 0.3, about 0.3 to about 0.4, about 0.4 to about 0.5, about 0.5 to about
0.6, about 0.6 to
about 0.7, about 0.7 to about 0.8, about 0.8 to about 0.9, or about 0.9 to
about 1.0 equimolar
amount of the quantity of ATIII in the blood product. Saccharides that may be
used in the
methods of the present invention include, but are not limited to,
monosaccharides,
disaccharides, and polysaccharides (including penta-, hepta- and hexa-
saccharides), sugar
alcohols, and amino sugars. Examples of monosaccharides include glucose,
fructose,
galactose, mannose, arabinose, and inositol. Examples of disaccharides include
saccharose,
lactose, maltose, pectin. Examples of sugar alcohols include mannitol,
sorbitol, and xylitol.
Examples of amino sugars include glucosamine, galactosamine, N-acetyl-D-
glucosamine
and N-acetyl galactosamine, which are the building blocks that can form more
complex
oligosaccharides, such as aminoglycosides and heparin. In certain embodiments,
the
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oligosaccharides may be low molecular weight (2-4kDa) heparin, high molecular
weight (at
least l2kDa) heparin, standard unfractionated heparin, pectin,
pentasaccharides, and
aminoglycosides. In certain embodiments, the oligosaccharide has an affinity
for ATIII.
Saccharides as used herein can be derivatized with additional small molecules,
such as
biotin, avidin or streptavidin. Saccharides in certain embodiments may be
sulfated
oligosaccharides or oligosaccharides identified by glycosidase and other
restriction enzyme
reactions.
[0039] Blood products in which ATIII has been activated in situ are also
within the scope
of the present invention. Such blood products may be packaged, for example, in
sterile i.v.
or incubation bags.
[0040] 3. Methods of Treatiniz
[0041] The above-described methods of treating blood products to activate
ATIII in situ
and activated blood products produced thereby may be incorporated into methods
of
treating a disease in a subject. In certain embodiments, such methods of
treating a disease
in a subject in need of treatment may comprise (a) adding to a blood product
comprising
ATIII a saccharide able to activate ATIII in an amount sufficient to activate
said ATIII; (b)
incubating the resulting mixture of the blood product and saccharide under
conditions
sufficient to activate said ATIII; and (c) administering said mixture into
said subject to treat
a disease. Such methods may, in certain embodiments, further comprise
purifying said
mixture to remove unreacted saccharide prior to the infusing step, e.g. by
dialysis. The
methods of the present invention can be used to human and animal subjects such
as cows,
horses, dogs, cats, etc.
[0042] In certain embodiments, the disease is caused by a bacteria or virus.
Exemplary
viruses that may be treated using the methods of activating ATIII in situ or
with the blood
products of the invention include Hepatitis A Virus (HAV) infection, Hepatitis
B Virus
(HBV) infection, Hepatitis C Virus (HCV) infection, Human Immunodeficiency
Virus
(HIV) infection, corona virus infection, cytomegalovirus infection (CMV) and
severe acute
respiratory syndrome (SARS). In certain embodiments, the virus is a
retrovirus, such as
HIV. In other embodiments, the disease may be a disease or condition that is
caused by or
contributed to by thrombin activation.
[0043] Thrombin activation related diseases in a patient include sepsis,
trauma, acute
respiratory distress syndrome, thrombosis, stroke, and restenosis. The methods
may also be
used to treat patients at risk of a thrombin related pathological disease such
as reocclusion
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and restenosis in percutaneous trarisluminal coronary angioplasty; thrombosis
associated
with surgery, ischemia/reperfusion injury; and coagulation abnormalities in
cancer or
surgical patients. The treated blood product administered in the methods may
serve an anti-
coagulant in the treatment of, for example, congenital antithrombin III
deficiency which
leads to an increased risk of venous and arterial thrombosis, or acquired
antithrombin III
deficiency which results in disseminated intravascular coagulation,
microangiopathic
hemolytic anemias due to endothelial damage (i.e. hemolytic-uremic syndrome)
and veno-
occlusive disease (VOD). The methods and blood products may also be used to
treat semi-
chronic diseases like arterial thrombus and deep vein thrombosis.
[0044] Both methods of treating that are single-dose administrations and
longer term,
chronic, multi-dose treatments are within the scope of the present invention.
[0045] In certain embodiments, the methods of treating may further comprise
administering
other pharmaceutical compositions, such as an anti-viral drug, to said
subject. For example,
in certain embodiments, an anti-viral drug may be administered concurrently
with the blood
product. In other embodiments, however, an anti-viral drug may be administered
subsequent to administering the mixture. In still other embodiments, anti-
viral drug may be
administered both concurrently and subsequently with the blood product. An
anti-viral
drug may be added to the mixture, or administered as a separate composition.
[0046] Exemplary anti-viral drugs include reverse transcriptase inhibitors
such as
zidovudine, zalcitabine, didanosine, stavudine, lamivudine, abacavir,
tenofovir, nevirapine,
efavirenz, delavirdine; protease inhibitors such as saquinavir, ritonavir,
indinavir,
nelfinavir, amprenavir, lopinavir, and other agents such as adenine
arabinoside, adenine
arabinoside 5'-monophosphate, acyclovir, ganciclovir, famciclovir, lamivudine,
clevudine,
afedovir dipivoxil, entecavir, IFN-a-2b, IFN-a-2a, lymphoblastoid IFN,
consensus-IFN,
IFN-b, IFN-g, pegylated IFN-a-2a, corticosteroids, or thymosin al, IL-2, IL-
12, ribavirin,
cyclosporin or granulocyte macrophage colony stimulating factor, fusion
inhibitors such as
T-20 (enfuvirtide), zinc finger inhibitors, and ribavrin.
[0047] Highly Active Antiretroviral Therapy (HAART) is a recommended treatment
for
HIV and other viral infections. HAART combines two or more anti-viral
medications in a
daily regimen, also known as a "cocktail." Cocktails of anti-viral drugs are
well-known to
those of skill in the art. For example, effective known anti-HIV cocktails
include, but are
not limited to, combinations such as AZT, 3TC and efavirenz; nevirapine,
stavudine and
lamivudine; emtricitabine and tenofovir disoproxil fumarate; indinavir,
zidovudine and
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lamivudine (3TC); and zidovudine and 3TC. An effective anti-HCV cocktail is
ribavrin
and interferon.
[0048] The combined use of blood products of the present invention and other
anti-virals
may reduce the required dosage for any individual component because the onset
and
duration of effect of the different components may be complimentary. In such
combined
therapy, the different active agents may be delivered together or separately,
and
simultaneously or at different times within the day.
[0049] Other pharmaceutical compositions that may be administered either
concurrently,
subsequently, or both concurrently and subsequently include ATIII and
interferon or
interferon derived drugs. For example, the blood product may be supplemented
with
additional ATIII prior to adding the oligosaccharide to activate it. In
another embodiment,
anticoagulants may be used alone or in combination with saccharides such as
heparin or a
pentasaccharide to treat the blood products of the invention prior to their
use in a treatment
method in order to improve their efficacy.
[0050] Further, high molecular weight ATIII pharmaceutical compositions as
described in
Pending U.S. Patent Application 10/436,872 may be used to supplement the
method of
treatment, e.g. by administering it either concurrently, subsequently, or both
concurrently
and subsequently with administration of the treated blood products.
[0051] The blood products of the present invention may be administered
directly to a
subject or may be formulated in a conventional manner using one or more
physiologically
acceptable carriers or excipients. For such therapy, the compounds of the
invention can be
formulated for a variety of loads of administration, including systemic and
topical or
localized administration. Techniques and formulations generally may be found
in
Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For
systemic
administration, injection is preferred, including intramuscular, intravenous,
intraperitoneal,
and subcutaneous. The blood product may be formulated for parenteral
administration by
injection, e.g., by bolus injection or continuous infusion. For injection, the
blood products
of the invention can be formulated in liquid solutions, preferably in
physiologically
compatible buffers such as Hank's solution or Ringer's solution. In addition,
the blood
products may be formulated in solid form and redissolved or suspended
immediately prior
to use. Lyophilized forms of the blood products are also included.
[0052] Formulations for injection may be presented in unit dosage form, e.g.,
in ampoules
or in multi-dose containers, with an added preservative. The formulations may
take such
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forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively,
the active ingredient may be in powder form for constitution with a suitable
vehicle, e.g.,
sterile pyrogen-free water, before use.
[0053] Furthermore, as those skilled in the art will understand, the dosage of
any blood
product, agent, compound, drug, etc. used in the methods of the present
invention will vary
depending on the symptoms, age and body weight of the patient, the nature and
severity of
the disorder to be treated or prevented, the route of administration, and the
form of the
supplement. Any of the subject formulations may be administered in any
suitable dose,
such as, for example, in a single dose or in divided doses. Dosages for the
blood products
of the present invention, alone or together with any other compound of the
present
invention, or in combination with any compound deemed useful for the
particular disorder,
disease or condition sought to be treated, may be readily determined by
techniques known
to those of skill in the art, based on the present description, and as taught
herein. Also, the
present invention provides mixtures of more than one subject compound, as well
as other
therapeutic agents.
[0054] ATIII has been shown to be well-tolerated when administered at a dose
of
-100U/kg/day (Warren et al., JAMA 286: 1869-78 (2001)) and has an overall
elimination
half-life with 18.6 h was demonstrated (Ilias et al. Intensive Care Medicine
26: 7104-7115
(2000)). While the dose is appropriately determined depending on symptom, body
weight,
sex, animal species and the like, it is generally 1-1,000 units/kg body
weight/day,
preferably 10-500 units/kg body weight/day of ATIII (as contained in the
treated blood
products of the invention) for a human adult, which is administered in one to
several doses
a day. In the case of intravenous administration, for example, the dose is
preferably 10-100
units/kg body weight/day.
[0055] The precise time of administration and amount of any particular product
or
compound that will yield the most effective treatment in a given patient will
depend upon
the activity, pharmacokinetics, and bioavailability of a particular compound,
physiological
condition of the patient (including age, sex, disease type and stage, general
physical
condition, responsiveness to a given dosage and type of medication), route of
administration, and the like. The guidelines presented herein may be used to
optimize the
treatment, e.g., determining the optimum time and/or amount of administration,
which will
require no more than routine experimentation consisting of monitoring the
subject and
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adjusting the dosage and/or timing. In certain embodiments, the administration
regimen
may also be dependent on the relative amount of ATIII in the blood product,
for example,
when the blood product is the patient's blood..
[0056] While the subject is being treated, the health of the patient may be
monitored by
measuring one or more relevant indices at predetermined times during a 24-hour
period.
Treatment, including supplement, amounts, times of administration and
formulation, may
be optimized according to the results of such monitoring. The patient may be
periodically
reevaluated to determine the extent of improvement by measuring the same
parameters, the
first such reevaluation typically occurring at the end of four weeks from the
onset of
therapy, and subsequent reevaluations occurring every four to eight weeks
during therapy
and then every three months thereafter. Therapy may continue for several
months or even
years, with a minimum of one month being a typical length of therapy for
humans.
Adjustments to the amount(s) of agent administered and possibly to the time of
administration may be made based on these reevaluations. In embodiments
wherein the
blood products are administered along with a HAART regimen, the blood products
may be
administered in the HAART rest interval.
[0057] Treatment may be initiated with smaller dosages which are less than the
optimum
dose of the product or compound. Thereafter, the dosage may be increased by
small
increments until the optimum therapeutic effect is attained.
[005814. Kits
[0059] The present invention also provides kits comprising compositions of the
present
invention, and optionally instructions for their use. For example, kits for
the practice of
certain of the treatment methods of the invention may comprise a saccharide
able to activate
ATIII and instructions for use. Still other kits for the practice of certain
of the treatment
methods of the invention may comprise blood products in which ATIII has been
activated
in situ. Further, the present invention provides kits for the methods of
preparing activated
ATIII in situ in a blood product. For example, such kits may comprising an
oligosaccharide
able to activate ATIII and instructions for use.
[0060] Kit components may be packaged for either manual or partially or wholly
automated
practice of the foregoing methods. In other embodiments involving kits, this
invention
contemplates a kit including compositions of the present invention, and
optionally
instructions for their use. In other embodiments, a kit may further comprise
controls,
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reagents, buffers, and/or instructions for use. Such kits may have a variety
of uses,
including, for example, imaging, diagnosis, therapy, and other applications.
EXEMPLIFICATION
[0061] The invention now being generally described, it will be more readily
understood by
reference to the following examples, which are included merely for purposes of
illustration
of certain aspects and embodiments of the present invention, and are not
intended to limit
the invention.
[0062] Example 1: Preparation Procedures for In vitro Plasma ATIII Activation
Usin2
Standard, Unfractionated Heparin
[0063] Standard, unfractionated heparin for human use (1,000-10,000 units, or
about 2 to
about 40 mg) was added to 500 mL samples of commercially available human
plasma
(containing an average of about 120 to about 150 mg of ATIII) in a sterile
incubation bag.
The amount of heparin added to the bag depends on the viral load of the
patient and the
treatment regimen desired. The resulting mixture was incubated at room
temperature, 37 C
or 40 C, for 24-72 hours. Gentle mixing was applied as necessary during the
incubation
period. The incubated mixture may be infused into a patient with or without
further
purification to remove unreacted heparin. In these procedures, the heparin
dosage is very
low compared to the level of ATIII in the plasma so that free heparin in the
resultant
mixture is avoided, thus decreasing the risk for bleeding.
[0064] Example 2: Preparation Procedures for In vitro Blood ATIII Activation
Using
Standard, Unfractionated Heparin
[0065] Standard, unfractionated heparin for human use (1,000-20,000 units, or
about 2 to
about 40 mg) was added to 1,000 mL samples of blood drawn from HIV or HCV
patients
(containing an average of about 70 to about 150 mg of ATIII) in a sterile
incubation bag.
The resulting mixture was incubated at room temperature, 37 C or 40 C, for 24-
72 hours.
Gentle mixing was applied as necessary during the incubation period. The
incubated
mixture may be infused into a patient with or without further purification to
remove
unreacted heparin. In these procedures, the heparin dosage is very low
compared to the
level of ATIII in the plasma so that free heparin in the resultant mixture is
avoided, thus
decreasing the risk for bleeding.
[0066] Example 3: Preparation Procedures for In vitro Plasma ATIII Activation
Using
Low Molecular WeiEht Heparin
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[0067] Low molecular weight (LMW) heparin for human use (1,000-10,000 units,
or about
2 to about 20 mg) was added to 500 mL samples of commercially available human
plasma
(containing an average of about 70 to about 150 mg of ATIII) in a sterile
incubation bag.
The resulting mixture was incubated at room temperature, 37 C or 40 C, for 24-
72 hours.
Gentle mixing was applied as necessary during the incubation period. The
incubated
mixture may be infused into a patient with or without further purification to
remove
unreacted heparin. In these procedures, the heparin dosage is very low
compared to the
level of ATIII in the plasma so that free heparin in the resultant mixture is
avoided, thus
decreasing the risk for bleeding.
[0068] Example 4: Preparation Procedures for In vitro Blood ATIH Activation
Using
Low Molecular Weight Heparin
[0069] Low molecular weight (LMW) heparin for human use (1,000-20,000 units,
or about
2 to 20 mg) was added to 1,000 mL samples of blood drawn from HIV or HCV
patients
(containing an average of about 70 to 150 mg of ATIII) in a sterile incubation
bag. The
resulting mixture was incubated at room temperature, 37 C or 40 C, for 24-72
hours.
Gentle mixing was applied as necessary during the incubation period. The
incubated
mixture may be infused into a patient with or without further purification to
remove
unreacted heparin. In these procedures, the heparin dosage is very low
compared to the
level of ATIII in the plasma so that free heparin in the resultant mixture is
avoided, thus
decreasing the risk for bleeding.
[0070] Example 5: Activation of ATIII in the Blood or Blood Plasma Using
Heparin,
Heparin Derivatives, or other Oli2osaccharides Able to Activate ATIII
[0071] ATIII may also be activated in the blood or blood plasma using the
protocols
described in Examples 1-4 above using a 0.1 to 1.0 equimolar amount of any
heparin-
derived pentasaccharide or other oligosaccharide able to activate ATIII.
Examples of such
are provided in the Detailed Description above.
[0072] Example 6: Estimation of Protein Bound Heparin and Free Heparin in
Modified Forms
[0073] To estimate the free heparin and protein-bound heparin, both UV and
refractive
index (RI) integrations were used as follows.
[0074] To correct for the differences in the absorption detection by both UV
and RI
detectors, a correction coefficient was established from a pure protein
standard measure
with both detectors according to the following formula:
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[0075] K = UVprotein/Rlprotein or Rlprotein = UVprotein/K
[0076] The total RI integration may be expressed as follows:
[0077] Rltotal = Rlprotein + Rlheparin
[0078] Therefore, the bound heparin (Bheparin) is
[0079] %Bheparin = [(Rltotal - UVprotein/K)/Rltota1] x 100
[0080] Free heparin (Fheparin) is calculated according to:
[0081] %Fheparin = [Fheparin/(Fheparin + Bheparin)] X 100
100821 Example 7: Assay for Inhibition of HIV-1 Replication
[0083] X4 HTLV-IIIB (hereinafter X4 HIV; Chang et al., NATURE, 363: 466-9
(1993)), a
prototypical T-tropic strain of HIV (American Type Tissue Collection,
Monassass, VA,
USA; ATCC No. CRL-8543), was used to assess the effect of the ATIII activation
protocols on T-tropic HIV infection. The quantity of virus in a specified
suspension
volume (e.g. 0.1 ml) that will infect 50% of a number (n) of cell culture
microplate wells, or
tubes, is termed the Tissue Culture Infectious Dose 50 [TCIDSO]. TCID50 is
used as an
alternative to determining virus titer by plaqueing (which gives values as
PFUs or
plaque-forming units). Human T lymphoblastoid cells (H9 cells) expressing the
human
leukocyte antigen proteins (HLA) B6, Bw62, and Cw3 were acutely infected with
X4 HIV
at a MOI of 1 x 10"2 TCID50 per milliliter. The infected H9 cells were
resuspended to 5 x
105 cells/ml in R20 cell culture medium. Two milliliters of this suspension
was pipetted
into each well of a 24-well microtiter plate. These cells were then cultured
in the presence
or absence of samples having activated ATIII for up to 12 days. Every three
days (days 3,
6, 9 and 12), 1 ml cell supernatant was removed from test wells and replaced
with an equal
volume of R20 cell culture medium. Control wells were similarly sampled but
received
media containing samples that were untreated.
[0084] The concentration of the viral core protein p24 (gag) for HIV (Alliance
HIV-1 p24
ELISA kit, NEN Life Science, Boston MA, USA) was measured for each sample
obtained
at days 0, 3, 6, 9 and 12 respectively.
[0085] The results which are shown in FIGURE 1 demonstrate HIV-1 inhibitory
activity in
blood treated with LMW heparin.
EQUIVALENTS
[0086] While specific embodiments of the subject invention have been
discussed, the above
specification is illustrative and not restrictive. Many variations of the
invention will
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become apparent to those skilled in the art upon review of this specification
and the claims
below. For example, variants on the quantities of reactants given in the above
Examples
are within the scope of the invention, as are variants on the incubation time.
The full scope
of the invention should be determined by reference to the claims, along with
their full scope
of equivalents, and the specification, along with such variations.
[0087] All publications and patents mentioned herein, including those listed
below, are
hereby incorporated by reference in their entirety as if each individual
publication or patent
was specifically and individually indicated to be incorporated by reference.
In case of
conflict, the present application, including any definitions herein, will
control.
[0088] Wright, BIOASSAY, 18: 453-64 (1996); Skinner et al., J. Mol. Biol. 283:
9-14
(1998); Huntington et al., J. Mol. Biol. 293: 449-55 (1999); Deeks, JAMA, 286:
224-6
(2001); Stephenson, JAMA, 277: 614-6 (1997); Carr et al., Lancet, 351: 1881-3
(1998)
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