Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF PROPHYLAXIS AGAINST LARGE MYOCARDIAL INFARCTIONS
BACKGROUND
Technical Field
This disclosure relates to methods of determining the effectiveness of anti-
inflammatory compounds in reducing incidence of myocardial infarction. Methods
of
prophylaxis against myocardial infarctions which exhibit CK-MB levels greater
than
about 50 nano-gramslml in a subject are also described.
Backgiround of Related Art
Coronary artery disease is often characterized by lesions or occlusions in the
coronary arteries which may result in inadequate blood flow to the myocardium,
or
1o myocardial ischemia, which is typically responsible for such complications
as angina
pectoris, necrosis of cardiac tissue (myocardial infarction), and sudden
death. In some
cases, coronary artery disease may be treated by the use of drugs and by
modifications
in behavior and diet. In other cases, dilatation of coronary arteries may be
achieved by
such procedures as angioplasty, laser ablation, atherectomy, catheterization,
and
intravascular stents.
For certain patients, coronary artery bypass grafting (CABG) is the preferred
form of treatment to relieve symptoms and often increase life expectancy. CABG
consists of direct anastomosis of a vessel segment to one or more of the
coronary
arteries. For example, a reversed segment of the saphenous vein may be grafted
at
one end of the ascending aorta as an arterial blood source and at the other
end to a
coronary artery at a point beyond the arterial occlusion. Alternatively, the
internal
mammary artery is located in the thoracic cavity adjacent the sternum and is
likewise
suitable for grafting to a coronary artery, such as the left anterior
descending artery.
During the CABG surgery, the heart is usually stopped from beating, to
facilitate
the anastomosis procedures. While the heart is not beating, extracorporeal
circulation
of the blood supports most of the patient's body (excluding the heart and, to
some
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extent, the lungs). A cardiopulmonary bypass (CPB) machine receives
deoxygenated
blood from the patient's body, adds oxygen and various nutrients to the blood,
and
pumps the oxygenated blood back into the patient's body.
Although CABG surgery has substantially improved the therapeutic outcome of
patients with advanced myocardial ischemia, the recovery period may be often
traumatic to the patient with significant attendant risks. For example, it is
known that
CPB elicits a systemic infilammatory response that causes tissue injury and
contributes
to significant perioperative and long-term clinical morbidity. During CPB,
exposure of
blood to bioincompatible surfaces of the extracorporeal circuit, as well as
tissue
ischemia and reperfusion associated with the procedure, induces the activation
of
several major humoral pathways of inflammation. Clinical manifestations
attributed to
this systemic inflammatory response may include myocardial injury which may
manifest
as myocardial infarction (heart cell death) or as severe ventricular
dysfunction requiring
circulatory assist.
~5 Techniques to measure damage to the heart, using blood chemistry are known
in
the art. When heart cells die, certain enzymes that are normally kept inside
viable cells
are released into the circulating blood. One such enzyme is creative kinase
(CK),
which catalyzes the reversible transfer of a phosphate group from ATP to
creative. It
exists as a dimer composed of two subunits commonly identified as the M-
subunit and
2o the B-subunit. CK-MB is associated with myocardial infarction, and is
present in serum
in only trace concentrations in the absence of such an episode (other
isoenzymes CK-
MM and CK-BB are found in skeletal muscle and brain cells). Appearance of CK-
MB
isoenzyme in serum is, therefore, indicative of myocardial infarction.
Therefore, it is known in the art that, if a drug can reduce CK-MB levels in
blood
25 during and/or after CABG surgery involving CPB, the reduction in blood CK-
MB levels
indicates that the drug helped protect the heart against cell death and tissue
damage.
Fitch et al., Pharmacology and Biological Efficacy of a Recombinant,
Humanized,
Single-Chain Antibody C5 Complement Inhibitor in Patients Undergoing Coronary
Artery Bypass Graft Surgery With Cardiopulmonary Bypass (Circulation, 1999;
ao 100:2499.), disclose that h5G1.1-scFv, a recombinant single-chain antibody
C5
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inhibitor, proved to be a potent inhibitor of systemic complement activation,
inhibiting
both complement-dependent hemolytic activity and, more imporfiantly, the
generation of
the proinflammatory activation product C5-9 and C5b-9 in patients undergoing
CPB.
Fitch et al. further disclose that the potent complement inhibitory and anti-
infilammatory
s ~ activities of h5G1.1-scFv were associated with significant reductions in
postoperative
CK-MB release, new cognitive deficits, and blood loss. The potent inhibitory
and anti-
inflammatory effects of h5G1.1-scFv were associated with significant
reductions in
postoperative myocardial injury. In addition, Fitch measured CD11 b on
activated
neutrophils and monocytes, and reported that in doses sufficient to completely
block
~o hemolytic activity and soluble C5b-9 generation (e.g. 1.0 and 2.0 mg/kg),
h5G1.1-scFv
significantly attenuated peak leukocyte CDllb expression compared with the
placebo.
Nonetheless, Fitch et al. (citing Gray et al., Circulation, 1982:66:1185-1189;
Calliff et al.
J.AmCoII Cardiol, 1998:31:241-251; Abdelmeguid et al. Circulation,
1995:91:2733-
2741; and Kong et al. JAMA, 1997:277:461-466) state that "there does not
appear to be
~s a threshold effect, but rather, it is apparent that the greater the release
of CK-MB, the
greater the subsequent morbidity, cost, and mortality,...(and that] it is
likely that
significant reductions in postoperative myocardial injury might be associated
with
improved outcomes".
However, no method of detecting and/or differentiating inflammatory damage
zo from traumatic damage in patients having undergone CABG involving CPB based
on
postoperative CK-MB peak levels in the blood exists in the art. Hence, no
method of
testing the efficacy of an anti-inflammatory drug by monitoring CK-MB peak
levels in
such patients exists. Accordingly, no method of prophylaxis against large
myocardial
infarction (which more often result in mortality, e.g., such as those which
exhibit CK-MB
zs levels greater than about 50 nano-grams/ml) is known in the art. Further,
the relative
utility of anti-inflammatory drugs to limit larger, as opposed to smaller,
post-CABG
myocardial infarctions is not known.
It would be advantageous to provide a method of testing the efficacy of an
anti-
inflammatory drug by monitoring CK-MB peak levels in patients having undergone
so CABG involving CPB. It would be of further advantage to provide a method of
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prophylaxis against large myocardial infarctions as indicated by peak CK-MB
levels of
about 50 nano-grams/ml or more.
SUMMARY
A method of determining effectiveness of an anti-inflammatory compound in
reducing incidence of post-CABG myocardial infarction has now surprisingly
been
found. This method includes administering an anti-inflammatory compound to a
subject
group including at least one patient undergoing a procedure involving
cardiopulmonary
bypass; comparing incidence of infarctions in the subject group to incidence
of
infarctions in a control sample of patients for a given peak CK-MB level in
the blood
~o (such as, for example, greater than 50 nano-gramslml) in both groups;
wherein a
decrease in the incidence of infarctions in the subject group indicates
effectiveness of
the compound.
In another embodiment a method of prophylaxis against myocardial infarctions
which exhibit peak CK-MB levels greater than about 50 nano-grams/ml in a
subject is
~5 provided. This method includes administering to the subject undergoing a
procedure
involving cardiopulmonary bypass an effective myocardial infarction reducing
amount of
an anti-inflammatory compound.
BRIEF DECRIPTIPON OF THE DRAWINGS
Figure 1 is a graph summarizing the reduction of incidence of myocardial
2o infarction which exhibit various CK-MB levels that was provided by an anti-
C5 antibody,
namely h5G1.1-scFv in a controlled, randomized clinical fiest of patients
undergoing
CABG with CPB.
Figure 2 is a graphical presentation of the data of Table One showing the
reduction in peak CK-MB values resulting from the use of h5G1.1-scFv.
25 DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The methods of determining effectiveness of an anti-inflammatory compound in
reducing incidence of myocardial infarction in accordance with this disclosure
includes
the step of administering an anti-infilammatory compound to a subject group
including at
least one patient undergoing a procedure which involves cardiopulmonary
bypass.
so Such procedures include, but are not limited to CABG and heart transplant.
The level
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of CK-MB in the patients' blood is measured and the incidence of myocardial
infarctions
which exhibit peak CK-MB levels greater than about 50 nano-grams/ml is
determined.
The incidence of such infarctions in the subject group is then compared to the
incidence
of infarctions exhibiting a comparable level of CK-MB in a control sample of
patients. A
s decrease in the incidence of infarctions in the subject group indicates
effectiveness of
the compound. .
Anti-inflammatory compounds which can be evaluated by the methods described
herein include non-steroidal anti-inflammatory actives or drugs (NSAIDS). The
NSAIDS
can be selected from the following categories: propionic acid derivatives;
acetic acid
to derivatives; fenamic acid derivatives; biphenylcarboxylic acid derivatives;
and oxicams.
Ali of these NSAIDS are fully described in the U.S. Pat. No. 4,985,459 to
Sunshine et
al., issued Jan. 15, 1991, incorporated by reference herein. Most preferred
are the
propionic NSAIDS including, but not limited to aspirin, acetaminophen,
ibuprofen,
naproxen, benoxaprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen,
indoprofen,
15 pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen,
suprofen,
alminoprofen, tiaprofenic acid, fluprofen and bucloxic acid. Another useful
class of anti-
inflammatory compounds include inhibitors of cyclooxygenase-1 (COX-1 ) and
inhibitors
of cyclooxygenase-2 (COX-2). Also useful are the steroidal anti-inflammatory
drugs
including hydrocortisone and the like. Particularly useful are anti-
inflammatory
2o compounds which reduce neutrophil activation or monocyte activation by
greater than
about 30%.
Preferred anti-inflammatory compounds are compounds which bind to or
otherwise block the generation and/or activity of complement components. A
specific
class of such compounds which are particularly useful are antibodies specific
to a
2s human complement component.
The complement system acts in conjunction with other immunological systems of
the body to defend against intrusion of cellular and viral pathogens. There
are at least
25 complement proteins, which are found as a complex collection of plasma
proteins
and membrane cofactors. The plasma proteins make up about 10% of the globulins
in
so vertebrate serum. Complement components achieve their immune defensive
functions
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by interacting in a series of intricate but precise enzymatic cleavage and
membrane
binding events. The resulting complement cascade leads to the production of
products
with opsonic, immunoregulatory, and lytic functions. A concise summary of the
biologic
activities associated with complement activation is provided, for example, In
The Merck
Manual, 16t" Edition.
The complement cascade progresses via the classical pathway or the alternative
pathway. These pathways share many components, and while they differ in their
initial
steps, they converge and share the same "terminal complement" components (C5
through C9) responsible for the activation and destruction of target cells.
o The classical complement pathway is typically initiated by antibody
recognition of and
binding to an antigenic site on a target cell. The alternative pathway is
usually antibody
independent, and can be initiated by certain molecules on pathogen surfaces.
Additionally, the lectin pathway is typically initiated with binding of
mannose-binding
lectin (MBL) to high mannose substrates. These pathways converge at the point
where
~5 complement component C3 is cleaved by an active protease (which is
different in each
pathway) to yield C3a and C3b. Other pathways activating complement attack can
act
later in the sequence of events leading to various aspects of complement
function.
C3a is an anaphylatoxin (see discussion below). C3b binds to bacterial and
other
cells, as well as to certain viruses and immune complexes, and tags them for
removal
2o from the circulation. (C3b in this role is known as opsonin.) The opsonic
function of C3b
is generally considered to be the most important anti-infective action of the
complement
system. Patients with genetic lesions that block C3b function are prone to
infection by a
broad variety of pathogenic organisms, while patients with lesions later in
the
complement cascade sequence, i.e., patients with lesions that block C5
functions, are
25 found to be more prone only to Neisseria infection, and then only somewhat
more
prone (Fearon, in Intensive Review of Internal Medicine, 2"d Ed. Fanta and
Minaker,
eds. Brigham and Women's and Beth Israel Hospitals, 1983).
C3b also forms a complex with other components unique to each pathway to
form classical or alternative C5 convertase, which cleaves C5 into C5a and
CSb. C3 is
3o thus regarded as the central protein in the complement reaction sequence
since it is
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essential to both the alternative and classical pathways (Wurzner, et al.,
Complement
Inflamm. 8:328-340, 1991 ). This property of C3b is regulated by the serum
protease
Factor I, which acts on C3b to produce iC3b. While still functional as
opsonin, iC3b
cannot form an active C5 convertase.
s ~ C5a is another anaphylatoxin (see discussion below). C5b combines with C6,
C7, and C8 to form the C5b-8 complex at the surface of the target cell. Upon
binding of
several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal
complement complex--TCC) is formed. When sufficient numbers of MACs insert
into
target cell membranes the openings they create (MAC pores) mediate rapid
osmotic
lysis of the target cells. Lower, non-lytic concentrations of MACs can produce
other
effects. In particular, membrane insertion of small numbers of the C5b-9
complexes into
endothelial cells and platelets can cause deleterious cell activation. In some
cases
activation may precede cell lysis.
As mentioned above, C3a and C5a are anaphylatoxins. These activated
complement components can trigger mast cell degranulation, which releases
histamine
and other mediators of inflammation, resulting in smooth muscle contraction,
increased
vascular permeability, leukocyte activation, and other inflammatory phenomena
including cellular proliferation resulting in hypercellularity. C5a also
functions as a
chemotactic peptide that serves to attract pro-inflammatory granulocytes to
the site of
2o complement activation.
Any compounds which bind to or otherwise block the generation and/or activity
of
any of the human complement components, such as, for example, antibodies
specific to
a human complement component are useful herein. Some compounds include 1 )
antibodies directed against complement components C-1, C-2, C-3, C-4, C-5, C-
6, C-7,
2s C-8, C-9, Factor D, Factor B, Factor P, MBL, MASP-1, AND MASP-2 and 2)
naturally
occurring or soluble forms of complement inhibitory compounds such as CR1, LEX-
CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, y bind protein,
complestatin, and K76 COOH. Suitable compounds for use herein are antibodies
that
reduce, directly or indirectly, the conversion of complement component C5 into
so complement components C5a and CSb. One,class of useful antibodies are those
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having at least one antibody-antigen binding site and exhibiting specific
binding to
human complement component C5, wherein the specific binding is targeted to the
alpha chain, of human complement component C5. Such an antibody 1 ) inhibits
complement activation in a human body fluid; 2) inhibits the binding of
purified human
complement component C5 to either human complement component C3 or human
complement component C4; and 3) does not specifically bind to the human
complement activation product for CSa. Particularly useful complement
inhibitors are
compounds which reduce the generation of C5a and/or C5b-9 by greater than
about
30%. A particularly useful anti-C5 antibody is h5G1.1-scFv. Methods for the
preparation of h5G1.1-scFv are described in U.S. Patent Application No.
081487,283
filed June 7, 1995 now U.S. patent no. and "Inhibition of Complement
Activity by Humanized Anti-C5 Antibody and Single Chain Fv", Thomas et al.,
Molecular
Immunology, Vol. 33, No. 17/18, pages 1389-1401', 1996, the disclosures of
which are
incorporated herein in their entirety by this reference.
The following non-limiting example is included to illustrate the present
invention
but is not intended to limit the scope thereof.
EXAMPLE
RANDOMIZED, DOUBLE-BLIND, PLACEBO CONTROLLED STUDY OF THE EFFECT
OF h5G1.1-scFv ON TOTAL MORTALITY AND ADVERSE CARDIOVASCULAR
2o ISCHEMIC OUTCOMES IN PATIENTS UNDERGOING CARDIOPULMONARY
BYPASS
A randomized, multi-center, double-blind, placebo-controlled study was
conducted of h5G1.1-scFv administered to patients at moderately increased risk
of
adverse post-operative ischemic events undergoing CPB as part CABG.
2s ~ The study population consisted of individuals who elected to undergo non-
emergent coronary-artery bypass graft (CABG) surgery, without valve surgery,
which
required the use of a cardiopulmonary bypass (CPB) machine. There were
approximately 270 patients°for each of the three treatment arms.
Patients were randomized to receive one of the following three treatment
ao combinations: i) Bolus 2.0 mglkg h5G1.1-scFv followed by 0.05 mg/kglhr
h5G1.1-scFv
for 24 hours; ii) Bolus 2.0 mglkg h5G1.1-scFv; and iii) Placebo. The h5G1.1-
scFv or
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matching placebo was provided as a solution for injection in 30 m( vials with
a
concentration of 2 mglml. Patients received the bolus of study medication fen
(10)
minutes before the initiation of cardio-pulmonary bypass via a unique line.
The drug
was not to be combined with other medication given via this route. The
infusion began
immediately following bolus administration, and continued for 24 hours at a
constant
drip rate.
Patients were evaluated at an initial screening visit, which occurred within
14
days prior to the first administration of study medication. Blood pressure and
heart rate
were recorded every 15 minutes throughout the intraoperative period, beginning
at the
o induction of anesthesia.
For purposes of CK-MB measurements, intra- and post-operative blood draws
were pertormed at 4, 8,16, 20, 24, 30 and 36 hours post-CPB. Additionally, the
post
operative day (POD) 2 CK-MB draw was at 48 hours post-CPB. There was a 30
minute
window for each of these blood draws except for those drawn in the OR, which
were
~5 exact. The POD 4 CK-MB draw was collected with routine blood draws.
Measurements
of CK-MB were made using a microparticle enzyme immunoassay that is
commercially
available under the tradename AxSYM system from Abboft Laboratories, (Abbott
Park,
Illinois).
The study was conducted in accordance with standard operating procedures
2o designed to ensure adherence to Good Clinical Practice (GGP) as required by
the
following: Directive 91/507/EEC: The Rules Governing Medicinal Products in the
European Community; Code of Federal Regulations dealing with clinical studies
(21
CFR including parts 50 and 56 concerning informed consent and IRB
regulations);and
Declaration of Helsinki, concerning medical research in humans
("Recommendations
2s Guiding Physicians in Biomedical Research Involving Human Subjects,"
Helsinki 1964,
amended Tokyo 1975, Venice 1983, Hong Kong 1989 and Somerset West 1996). Note
for Guidance on Good Clinical Practice in valid version approved by CPMP in
July
1996. International Conference on Harmonisation; Good Clinical Practice:
Consolidated
Guideline; Notice of Availability, in Federal Register, May 9,1997.
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The distribution of peak CK-MB levels for each patent group was subjected to
conventional statistical analysis to calculate percentiles. The results are
summarized in
Figures 1 and 2. As seen in Figure 1, a significant decrease in the incidence
of
myocardial infarctions which exhibit CK-MB levels of each of >60 ng/ml, >70
ng/ml, >80
ng/ml, >90 ng/ml, >110 ng/ml and, >120 ng/ml was provided by h5G1.1-scFv. As
seen
in Figure 2, the effectiveness of administering the anti-inflammatory compound
surprisingly is observed to be significant only in patients experiencing
myocardial
infarction which exhibits a peak CK-MB value of greater than about 50 nglml.
In another aspect, a novel method for testing the anti-inflammatory drug
efficacy
~o and formulation of endpoints in CABG clinical trials has been discovered.
Specifically,
by using the methods disclosed herein endpoints in CABG trials with myocardial
infarction defined, in part, by CK-MB peak levels of >50, >60, >70, >80, >90,
>100 or
>120 can be effectively utilized to evaluate anti-inflammatory drugs.
Because the anti-inflammatory compound evaluated using the methods herein
~5 may be determined to reduce the incidence of myocardial infarctions of such
severity to
exhibit a CK-MB level of greater than 50 ng/ml, in another aspect, this
disclosure
contemplates a method of prophylaxis against myocardial infarctions which
exhibit CK-
MB levels greater than about 50 nano-gramslml in a subject. This method
includes
administering to the subject undergoing a procedure which involves CPB an
effective
2o myocardial infarction reducing amount of an anti-inflammatory compound.
Ascertaining
what amount constitutes an effective myocardial infarction reducing amount of
the anti-
inflammatory compound can be ascertained using the novel screening procedure
described hereinabove, or by any technique known to those skilled in the art.
The
dosage of the anti-inflammatory compound that constitutes an effective
myocardial
25 infarction reducing amount will depend on a number of factors, including,
for example,
the specific anti-inflammatory compound selected and its method of operation.
However, typically the anti-inflammatory compound can be administered in an
amount
ranging from about 0.01 mg/kg to about 20.Omglkg, preferably from about
0.10mg/kg to
about 10.Omg/kg.
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Any anti-inflammatory compound evaluated using the methods herein and
determined to reduce the incidence of myocardial infarctions may be used in
the
present method of prophylaxis. Any compounds which bind to or otherwise block
the
generation and/or activity of any of the human complement components, such as,
for
example, antibodies specific to a human complement component are useful for
prophylaxis. Some compounds include 1 ) antibodies directed against complement
components C-1, C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-9, Factor D, Factor B,
Factor P,
MBL, MASP-1, AND MASP-2 and 2) naturally occurring or soluble forms of
complement
inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra
o venom factor, FUT-175, y bind protein, complestatin, and K76 COOH: Suitable
compounds for use herein are antibodies that reduce, directly or indirectly,
the
conversion of complement component C5 into complement components C5a and CSb.
One class of useful antibodies are those having at least one antibody-antigen
binding
site and exhibiting specific binding to human complement component C5, wherein
the
specific binding is targeted to the alpha chain of human complement component
C5.
Such an antibody 1 ) inhibits complement activation in a human body fluid; 2)
inhibits the
binding of purified human complement component C5 to either human complement
component C3 or human complement component C4; and 3) does not specifically
bind
to the human complement activation product for CSa. A particularly useful anti-
C5
2o antibody is h5G1.1-scFv.
Although preferred and other embodiments of the invention have been described
herein, further embodiments may be perceived by those skilled in the art
without
departing from the scope of the invention as defined by the following claims.
11
