Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
DETECTION OF ANTAGONIST-DEPENDENT GPIIb/IIIa RECEPTOR ANTIBODIES
BACKGROUND OF THE TNVENTION
Drug-induced thrombocytopenia contributes to morbitity and,
occasionally, mortality in patients treated with a wide range of medications
(Karpatkin, Am. J. Med. Sci. 262:68 ( 1971 )). More than 100 different
medications have been implicated in drug-induced thrombocytopenia,
including heparin, quinine, quinidine and sulfonamide antibiotics (Shulman
et al. Hemostasis and Thrombosis (ed 2) Philadelphia, PA, Lippincott
( 1987) p.452, and Kracke et al. JAMA 122:168 (1943).
Drug-dependent antibodies reactive with platelets have been
identified in only a few instances. Curtis et al., Blood, vol. 84, no.l (July
1) 1984: pp. 176-183, applied flow cytometry to the detection of such
antibodies induced by sulfonamide antibiotics. Visentin et al. Transfusion
Oct. 1990 30 (8) pp. 694-700 describes detection of drug-dependent,
platelet-reactive antibodies by antigen-capture ELISA and flow cytometry.
The present invention is a means for identifying, in a patient,
the presence of one or more antibodies in GP IIb/IIIa receptor (fibrinogen
receptor) antagonist-induced thrombocytopenia which recognize GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist complexes formed with purified
platelets or purified GPIIb/IIIa receptor and a selected GPIIb/IIIa receptor
antagonist. Identification of such antibodies identifies the patient as being
at risk to development of thrombocytopenia resulting from administration
to the patient of the selected GP IIb/IIIa receptor antagonist.
SUMMARY OF THE INVENTION
The invention is a method for identifying a patient at risk to
developing fibrinogen receptor antagonist-induced thrombocytopenia
resulting from treatment of the patient with a selected fibrinogen receptor
antagonist, which comprises reacting patient plasma with a GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist complex to form a first reaction
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product, reacting the first reaction product with a secondary anti-human
detectable antibody to form a second reaction product, and detecting in the
second reaction product the level of binding between the secondary anti-
human detectable antibody and the GPIIb/IIIa receptor:GPIIb/IIIa receptor
antagonist complex. An indication that the secondary anti-human
detectable antibody binds to the GPIIb/IIIa receptor:GPITb/IIIa receptor
antagonist complex indicates the presence of interaction between the
GPIIb/BIa receptor:GPIIb/IIIa receptor antagonist complex and a patient
plasma antibody. The presence of such interaction identifies the patient as
at risk to developing thrombocytopenia following treatment with the
selected fibrinogen receptor antagonist.
DETAILED DESCRIPTION OF THE INVENTION
In the method, the patient at risk is one who is receiving
treatment to inhibit thrombosis by administration of a selected fibrinogen
receptor antagonist which inhibits the binding of fibrinogen to the
GPIIb/IIIa receptor.
The objective of the method is to determine whether the
patient's plasma contains an antibody to the GPIIb/IIIa receptor:GPIIb/IIIa
receptor antagonist complex formed when the selected fibrinogen receptor
antagonist binds to the GPIIb/IIIa receptor. The GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist complex may be prepared for
purposes of the method by coating commercially available GPIIb/IIIa
platelet receptors, including but not limited to those on platelets or
purified
2_5 platelets, and GPIIb/IIIa platelet receptors and purified GPIIb/IIIa
platelet
receptors, with a selected fibrinogen receptor antagonist to form a
GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex.
The method for identifying a patient at risk comprises
incubating patient plasma with a GPIIb/IIIa receptor:GPIIb/IIIa receptor
antagonist complex to form a GPIIb/IIIa receptor:GPIIb/IIIa receptor
antagonist:plasma antibody complex, incubating the GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist:plasma antibody complex with a
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secondary anti-human detectable antibody to form a GPIIb/IIIa
receptor:GPIIb/ITIa receptor antagonist:plasma antibodyaecondary anti-
human detectable antibody complex, and detecting the presence of the
secondary anti-human detectable antibody in the GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist:plasma antibodyaecondary anti-
human detectable antibody complex.
In the method of the invention, the GPIIb/IIIa
receptor:GPIIb/ITIa receptor antagonist complex is incubated with the
patient's plasma. Patient plasma which contains antagonist-dependent
antibodies will induce formation of a GPIIb/IIIa receptor:GPIIb/IIIa
receptor antagonist:plasma antibody complex. Plasma which does not
contain antagonist-dependent antibodies will not form a GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist:plasma antibody complex after this
incubation step. The material formed following the incubation step is
washed to remove substances which do not associate with the formed
GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:plasma antibody
complex.
The material formed following the above-described incubation
step is then incubated with a secondary anti-human detectable antibody (e.g.
antihuman IgG, antihuman IgM, antihuman IgA, associated with a
detectable marker such as a fluorescent label or an enzyme (e.g.
horseradish peroxidase, which induces a detectable reaction when exposed
to a substrate that is acted upon by the enzyme)). If a GPIIb/IIIa
receptor:GPIIb/IIIa receptor antagonist:plasma antibody complex forms
following addition of the patient's plasma, then the secondary anti-human
detectable antibody will complex with the GPIIb/IIIa receptor:GPIIb/IIIa
receptor antagonist:plasma antibody complex, and will not be washed away
during a washing step. If a GPIIb/IIIa receptor:GPIIb/IIIa receptor
antagonist:plasma antibody complex does not form following addition of
the patient's plasma, then the secondary antibody will not complex with the
GPIIb/ITIa receptor:GPIIb/IIIa receptor antagonist complex, and will be
washed away during a subsequent washing step. Detection of the presence
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of the GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:plasma
antibodyaecondary anti-human detectable antibody complex is an
indication that the patient does have antagonist-dependent antibodies
reactive with platelets and that the patient is at risk to developing
thrombocytopenia following consumption of the selected fibrinogen
receptor antagonist. The absence of the GPIIb/IIIa receptor:GPIIb/IIIa
receptor antagonist:plasma antibodyaecondary anti-human detectable
antibody complex indicates that the GPIIb/IIIa receptor:GPIIb/IIIa receptor
antagonist:plasma antibody complex did not form, and that the patient is
not at risk to developing fibrinogen receptor antagonist induced
thrombocytopenia.
More particularly, the secondary anti-human detectable
antibody is, for example, a fluorescence-labeled F(ab')2 anti-IgG,
fluorescence-labeled F(ab')2 anti-IgM, fluorescence-labeled F(ab')2 anti-
IgA, enzyme labeled IgG, enzyme labeled IgM, or enzyme Tabled IgA. The
fluorescence-labeled F(ab')2 anti-IgG, fluorescence-labeled F(ab')2 anti-
IgM, fluorescence-labeled F(ab')2 anti-IgA antibodies may be suitably
labeled with flourescein. The enzyme labeled IgG, enzyme labeled IgM,
and enzyme Tabled IgA antibodies may be suitably labeled with an enzyme
such as horseradish peroxidase.
The invention also is a method for identifying a patient not at
risk to developing fibrinogen receptor antagonist-induced
thrombocytopenia which comprises reacting patient plasma with a
GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex to form a first
reaction product, reacting the first reaction product with a secondary anti-
human detectable antibody to form a second reaction product, washing
substances from the second reaction product which do not complex with the
GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex to form a
washed second reaction product, and detecting the absence of the secondary
anti-human detectable antibody in the washed second reaction product.
The selected GP IIb/IIIa receptor antagonist suitable for the
methods of the invention is any antagonist which is useful for inhibiting
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fibrinogen binding to the GP IIb/IIIa platelet receptor. Such antagonists
are well known in the art
Antagonists for the GP IIb/IIIa receptor have been described
in, for example, United States Patents 5,470,849, 5,463,011, 5,455,243,
5,451,578, 5,446,056, 5,441,952, 5,422,249, 5,416,099, 5,405,854,
5,397,791, 5;393,760, 5,389,631, 5,380,713, 5,374,622, 5,358,956,
5,344,783, 5,340,798, 5,338,7235,334,596, 5,321,034, 5,318,899 (e.g.
cyclic heptapeptides Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2,
Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH2, Mpr-(Phenylimidyl-
Lys)-Gly-Asp-Trp-Phe-Pen-NH2,and Mpr-(Phenylimidyl-Lys)-Gly-Asp-
Trp-Phe-Cys-NH2, wherein Mpr is mercapto propionyl), 5,312,923,
5,294,616, 5,292,756 (e.g. 2-S-(n-Butylsulfonylamino)-3[4-piperidin-4-
yl)butyloxyphenyl]propionic acid hydrochloride), 5,281,585 5,272,158,
5,264,420, 5,260,307, 5,239,113 (e.g. Ethyl 3-[[4-[[4-
(aminoiminomethyl)phenyl)amino]-1,4-dioxobutyl]amino]-4-pentynoate),
5,227,490, 5,206,373, 4,703,036 (e.g. N-Methyl-D-phenylalanyl-N-[( 1 S)-
1-formyl-4-guanidinobutyl]-L-prolinamide), EP 505 868 (e.g. (( 1-(2-((4
(aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-1-oxopropyl)-4-
piperidinyl)oxy)-(S)-acetic acid), WO 9311152 (e.g. N-(2-(2-(((3-
{(aminoiminomethyl )amino)propyl)amino)-carbonyl)-1-piperidnyl)-1-
(cyclohexylmethyl)-2-oxoethyl)-(R,S)-glycine), WO 9418981 (e.g. 2(S)-
[(p-Toluencsulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-
4-yl)ethyl ~-4H-pyrazolo-[ 1,5-a][ 1,4]diazepin-2-yl]carbonyl]-
amino]propionic acid), WO 9514683 (e.g. methyl-N3-[2-{3-(4-
formamidinophenyl)-isoxazolin-5(R)-yl }-acetyl]-N2-(n-butyloxycarbonyl)-
2,3-(S)-diaminopropionate acetate salt), EP 333 356 and WO 9422820.
They are described as useful for inhibiting fibrinogen binding and
inhibiting clot formation.
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EXAMPLE I
GP IIb/IIIa antagonist treatment and method for identifying a patient at
risk to developing GP IIb/IIIa antagonist-induced thrombocv~enia
A patient with acute coronary ischemic syndromes receives
coronary revascularization with angioplasty. Aspirin is administered in a
dose of 32S mg at least two hours before angioplasty, and daily thereafter.
Heparin is given intravenously in an initial bolus dose of 10,000 to 12,000
units followed by incremental bolus doses of up to 3000 units at 1 S-minute
intervals, but no more than 20,000 units is given during the procedure.
The goal is to keep the activated clotting time between 300 and 3S0 seconds
during the operation. Heparin is continued by constant infusion for at least
12 hours to maintain the activated partial-thromboplastin time at l.S to 2.S
times the control value. Aspirin is required at discharge in a dose of 32S
1 S mg per day.
The patient is scheduled to receive an oral tablet containing 1 S
mg of the fibrinogen receptor gp IIb/IIIa antagonist 2(S)-[(p-
Toluenesulfonyl)amino]-3-[ [[S,f ,7,8-tetrahydro-4-oxo-S-[2-{piperidin-4-
yl)ethyl]-4H-pyrazolo-[ l ,S-a] ( 1,4]diazepin-2-yl] carbonyl]-amino]propionic
acid (compound 1-1 ), described in WO 94/18981. Prior to initiation of
treatment with compound 1-1, the patient is screened to determine whether
the patient is at risk to developing thrombocytopenia induced by compound
1-1. A plasma sample is drawn from the patient and incubated with a
GPIIb/IIIa receptor:compound 1-1 complex to bind antagonist-dependent
2S plasma antibodies to the complex and form a GPIIb/ITIa
receptor:compound 1-l:plasma antibody complex. The GPIIb/IIIa
receptor:compound 1-l:plasma antibody complex is incubated with
horseradish peroxidase anti-human IgG to form a GPIIb/IIIa
receptor:compound 1-l:plasma antibody:horseradish peroxidase anti-
human IgG complex. The presence of the horseradish peroxidase anti-
human IgG in the GPIIb/IIIa receptor:compound 1-l:plasma
antibody:horseradish peroxidase anti-human IgG complex is detected by
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observing a horseradish peroxidase-induced enzyme reaction, which
confirms the presence of plasma antibodies associated with a
thrombocytopenic reaction to the GPIIb/IIIa receptor:compound 1-1
complex. Thus, the patient is determined to be at risk to developing
antagonist-induced thrombocytopenia.
EXAMPLE 2
GP IIb/IIIa antagonist treatment and method for identifying a patient not at
risk to developing GP IIb/IIIa antagonist-induced ~thrombocYt_openia
A patient with acute coronary ischemic syndromes receives
coronary revascularization with angioplasty. Aspirin is administered in a
dose of 325 mg at least two hours before angioplasty, and daily thereafter.
Heparin is given intravenously in an initial bolus dose of 10,000 to 12,000
units followed by incremental bolus doses of up to 3000 units at 15-minute
intervals, but no more than 20,000 units is given during the procedure.
The goal is to keep the activated clotting time between 300 and 350 seconds
during the operation. Heparin is continued by constant infusion for at least
12 hours to maintain the activated partial-thromboplastin time at 1.5 to 2.5
times the control value. Aspirin is required at discharge in a dose of 325
mg per day.
The patient is scheduled to receive an oral tablet containing 15
mg of the fibrinogen receptor gp IIb/IIIa antagonist 2{S)-[(p-
Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-
yl)ethyl]-4H-pyrazolo-[ 1,5-a] [ 1,4]diazepin-2-yl]carbonyl]-amino]propionic
acid {compound 1-1 ), described in WO 94/18981. Prior to initiation of
treatment with compound I - I , the patient is screened to determine whether
the patient is at risk to developing thrombocytopenia induced by compound
1-1. A plasma sample is drawn from the patient and incubated with a
GPIIb/IIIa receptor: compound 1-1 complex to attempt to bind antagonist-
dependent plasma antibodies to the complex and form a GPIIb/IIIa
receptor:compound 1-! :plasma antibody complex. However, no patient
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plasma antibodies are present, and the GPIIb/IIIa receptor:compound 1-
l:plasma antibody complex does not form. The GPIIb/IiIa
receptor:compound 1-1 complex is incubated with horseradish peroxidase
anti-human IgG. Since no plasma antibody complex formed, the
_S horseradish peroxidase anti-human IgG does not associate with the
GPIIb/IIIa receptor:compound 1-1 complex. The horseradish peroxidase
anti-human IgG is washed away because it does not complex with the
GPIIb/IIIa receptor:compound 1-1 complex. Exposure of the GPIIb/IIIa
receptor:compound 1-1 complex to conditions that would identify the
presence of the horseradish peroxidase anti-human IgG indicate that no
enzyme is present and that the patient plasma does not have plasma
antibodies associated with compound 1-1-induced thrombocytopenia, and
that the patient is not at risk to developing thrombocytopenia.
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