CA2271684A1 - Detection of antagonist-dependent gpiib/iiia receptor antibodies - Google Patents
Detection of antagonist-dependent gpiib/iiia receptor antibodies Download PDFInfo
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- CA2271684A1 CA2271684A1 CA002271684A CA2271684A CA2271684A1 CA 2271684 A1 CA2271684 A1 CA 2271684A1 CA 002271684 A CA002271684 A CA 002271684A CA 2271684 A CA2271684 A CA 2271684A CA 2271684 A1 CA2271684 A1 CA 2271684A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
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Abstract
The present invention is a method for identifying a patient at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which 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 secondary anti-human detectable antibody to form a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:plasma antibody:secondary 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 antibody:secondary anti-human detectable antibody complex.
Description
WO 98!22821 PCTIUS97/20954 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 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 .._~..~~__,r _.w_. _._ . ... a_ W~ 98/22821 PCT/US97/20954 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 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 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.
WO 98/22821 PCT/US97l20954 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 _~~..r...r . _ .~
WO 98!22821 PCT/US97120954 (7_ 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.
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 -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.
1 _5
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 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 .._~..~~__,r _.w_. _._ . ... a_ W~ 98/22821 PCT/US97/20954 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 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 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.
WO 98/22821 PCT/US97l20954 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 _~~..r...r . _ .~
WO 98!22821 PCT/US97120954 (7_ 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.
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 -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.
1 _5
Claims (6)
1. A method for identifying a patient at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which 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 secondary anti-human detectable antibody to form a GPIIb/IIIa receptor:GPIIb/IIIa 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.
2. A method of claim 1 wherein the antagonist is selected from the group consisting of 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, Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2, N-Methyl-D-phenylalanyl-N-[(1S)-1-formyl-4-guanidinobutyl]-L-prolinamide, ((1-(2-((4-(aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-1-oxopropyl)-4-piperidinyl)oxy)-(S)-acetic acid, Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate, N-(2-(2-(((3-((aminoiminomethyl)amino)propyl)amino)carbonyl)-1-piperidnyl)-1-(cyclohexylmethyl)-2-oxoethyl)-(R,S)-glycine, (2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]propionic acid hydrochloride, Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate, 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, (R)-methyl-3-[[[3-[4-(aminoiminomethyl)phenyl]-4,5-dihydro-5-isoxazolyl]acetyl]amino]-N-{butoxycarbonyl)-L-alanine monoacetate, and pharmaceutically acceptable salts thereof
3. A method of claim 2 wherein the secondary anti-human detectable antibody is selected from 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, and enzyme labeled IgA.
4. A method of claim 3 wherein the fluorescence-labeled F(ab')2 anti-IgG, fluorescence-labeled F(ab')2 anti-IgM, fluorescence-labeled F(ab')2 anti-IgA antibodies are labeled with flourescein.
5. A method of claim 3 wherein the enzyme labeled IgG, enzyme labeled IgM, and enzyme labeled IgA antibodies are labeled with horseradish peroxidase.
6. A method for identifying a patient not at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which comprises incubating patient plasma with a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex to form a first reaction product, incubating 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.
Applications Claiming Priority (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US3166196P | 1996-11-21 | 1996-11-21 | |
| US60/031,661 | 1996-11-21 | ||
| US3546197P | 1997-01-14 | 1997-01-14 | |
| US60/035,461 | 1997-01-14 | ||
| GBGB9702822.9A GB9702822D0 (en) | 1997-02-12 | 1997-02-12 | Anticoagulant test |
| GB9702822.9 | 1997-02-12 | ||
| GBGB9705856.4A GB9705856D0 (en) | 1997-03-21 | 1997-03-21 | Anticoagulant test |
| GB9705856.4 | 1997-03-21 | ||
| PCT/US1997/020954 WO1998022821A1 (en) | 1996-11-21 | 1997-11-17 | DETECTION OF ANTAGONIST-DEPENDENT GPIIb/IIIa RECEPTOR ANTIBODIES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2271684A1 true CA2271684A1 (en) | 1998-05-28 |
Family
ID=27451602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002271684A Abandoned CA2271684A1 (en) | 1996-11-21 | 1997-11-17 | Detection of antagonist-dependent gpiib/iiia receptor antibodies |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0939900A4 (en) |
| JP (1) | JP2001504584A (en) |
| CA (1) | CA2271684A1 (en) |
| WO (1) | WO1998022821A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6623981B2 (en) * | 1998-01-27 | 2003-09-23 | Bristol-Myers Squibb Company | Detection of patients at risk for developing integrin antagonist/agonist mediated disease states |
| AU2971600A (en) * | 1999-01-26 | 2000-08-07 | Du Pont Pharmaceuticals Company | A facs method for detection of gpiib/iiia inhibitor dependent activators in plasma samples |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4717654A (en) * | 1984-06-19 | 1988-01-05 | Akzo N.V. | Process for solid phase platelet antibody assay |
| US5318899A (en) * | 1989-06-16 | 1994-06-07 | Cor Therapeutics, Inc. | Platelet aggregation inhibitors |
| FR2665769B1 (en) * | 1990-08-09 | 1994-04-29 | Serbio | DETERMINATION OF THROMBOPENIA ESPECIALLY INDUCED BY HEPARIN BY MEANS OF THE PLATELET FACTOR 4. |
| US5585243A (en) * | 1993-09-15 | 1996-12-17 | The Blood Center Of Southeastern Wisconsin, Inc. | Method of detecting cytopenia that is mediated by drug-dependent antibody binding to blood cells |
-
1997
- 1997-11-17 CA CA002271684A patent/CA2271684A1/en not_active Abandoned
- 1997-11-17 WO PCT/US1997/020954 patent/WO1998022821A1/en not_active Application Discontinuation
- 1997-11-17 EP EP97947553A patent/EP0939900A4/en not_active Withdrawn
- 1997-11-17 JP JP52378398A patent/JP2001504584A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO1998022821A1 (en) | 1998-05-28 |
| EP0939900A1 (en) | 1999-09-08 |
| JP2001504584A (en) | 2001-04-03 |
| EP0939900A4 (en) | 2003-01-08 |
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