DETECTION OF ANTAGONIST-DEPENDENT GPIIb/IIIa RECEPTOR ANTIBODIES

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: TITLE OF THE INVENTION DETECTION OF ANTAGONIST-DEPENDENT GPIIb/IIIa RECEPTOR ANTIBODIES BACKGROUND OF THE INVENTION 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 Hb/IHa 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 Ilb 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/HIa 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: GPIIb/IIIa receptor antagonist complex indicates the presence of interaction between the GPIIb/πia receptoπGPIIbΛHa 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/IlIa 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 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 antagonistφlasma antibody complex with a secondary anti-human detectable antibody to form a GPIIb/IIIa receptor:GPIIb/πia receptor antagonistφlasma antibody:secondary anti- human detectable antibody complex, and detecting the presence of the secondary anti-human detectable antibody in the GPIIb/IIIa receptor:GPIIb/πia receptor antagonisCplasma antibody:secondary anti- human detectable antibody complex. In the method of the invention, the GPIIb/IIIa receptor:GPπb/πia 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 antagonistφlasma antibody complex. Plasma which does not contain antagonist-dependent antibodies will not form a GPIIb/IIIa receptor: GPIIb/IIIa receptor antagonisCplasma 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 antagonistφlasma 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/πia receptor antagonistφlasma antibody complex forms following addition of the patient's plasma, then the secondary anti-human detectable antibody will complex with the GPIIb/πia receptoπGPϋb/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/πia 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 antibody secondary 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 antagonis plasma antibody:secondary anti-human detectable antibody complex indicates that the GPIIb/IIIa receptoπGPIIb/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 labled 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 labled 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/πia receptor antagonist complex to form a first reaction product, reacting the first reaction product with a secondary antihuman 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 Ilb/IIIa receptor antagonist suitable for the methods of the invention is any antagonist which is useful for inhibiting fibrinogen binding to the GP Ub/IIIa platelet receptor. Such antagonists are well known in the art Antagonists for the GP Ub/IIIa receptor have been described in, for example, United States Patents 5,470,849, 5,463,01 1, 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-Tφ-Phe-Cys-NH2, Mpr-(Acetimidyl-Lys)-Gly-Asp-Tφ-Phe-Pen-NH2, Mpr-(Phenylimidyl- Lys)-Gly-Asp-Tφ-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-dioxobuty 1] amino] -4-pentynoate), 5,227,490, 5,206,373, 4,703,036 (e.g. N-Methyl-D-phenylalanyl-N-[(lS)- l-formyl-4-guanidinobutyl]-L-prolinamide), EP 505 868 (e.g. ((l-(2-((4 (aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-l-oxopropyl)-4- piperidinyl)oxy)-(S)-acetic acid), WO 931 1152 (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-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin- 4-y l)ethy 1] -4H-pyrazolo- [ 1 ,5 -a] [ 1 ,4] diazepin-2-y 1] carbonyl] - amino]propionic acid), WO 9514683 (e.g. methyl-N 3 -[2-{3-(4- formamidinophenyl)-isoxazolin-5(R)-yl } -acetyl]-N2-(n-butyloxycarbonyl)- 2,3-(_ )-diaminopropionate acetate salt), EP 333 356 and WO 9422820. They are described as useful for inhibiting fibrinogen binding and inhibiting clot formation. EXAMPLE 1 GP Ilb/IIIa antagonist treatment and method for identifying a patient at risk to developing GP Ilb/πia antagonist-induced thrombocytopenia 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 Ilb/IHa antagonist 2(S)-[(p- Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4- yl)ethyl]-4H-pyrazolo-[l ,5-a][l,4]diazepin-2-yl]carbonyl]-amino]propionic acid (compound 1-1). described in WO 94/18981. Prior to initiation of treatment with compound ±, 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 receptorxompound 1-1 complex to bind antagonist-dependent plasma antibodies to the complex and form a GPIIb/πia receptorxompound l-l :plasma antibody complex. The GPIIb/IIIa receptorxompound l-l :plasma antibody complex is incubated with horseradish peroxidase anti-human IgG to form a GPIIb/IIIa receptorxompound l-l :plasma antibody :horseradish peroxidase anti- human IgG complex. The presence of the horseradish peroxidase antihuman IgG in the GPIIb/IIIa receptorxompound l-l :plasma antibody:horseradish peroxidase anti-human IgG complex is detected by observing a horseradish peroxidase-induced enzyme reaction, which confirms the presence of plasma antibodies associated with a thrombocytopenic reaction to the GPIIb/IIIa receptorxompound 1 -1 complex. Thus, the patient is determined to be at risk to developing antagonist-induced thrombocytopenia. EXAMPLE 2 GP Ilb/IIIa antagonist treatment and method for identifying a patient not at risk to developing GP Ilb/IIIa antagonist-induced thrombocytopenia 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 Ilb/IIIa antagonist 2(S)-[(p- Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4- yl)ethyl]-4H-pyrazolo-[l ,5-a][l ,4]diazepin-2-yl]carbonyl]-amino]propionic acid (compound 1-1). described in WO 94/18981. Prior to initiation of treatment with compound LJL, 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 receptorxompound 1-1 complex to attempt to bind antagonist- dependent plasma antibodies to the complex and form a GPIIb/IIIa receptorxompound l-l :plasma antibody complex. However, no patient plasma antibodies are present, and the GPIIb/IIIa receptorxompound 1 - 1 :plasma antibody complex does not form. The GPIIb/πia receptorxompound 1-1 complex is incubated with horseradish peroxidase anti-human IgG. Since no plasma antibody complex formed, the horseradish peroxidase anti-human IgG does not associate with the GPIIb/πia receptorxompound 1-1 complex. The horseradish peroxidase anti-human IgG is washed away because it does not complex with the GPIIb/IIIa receptorxompound 1-1 complex. Exposure of the GPIIb/πia receptorxompound 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.

Claims: WHAT IS CLAIMED IS: 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 receptoπGPIΪb/IIIa receptor antagonist complex to form a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:plasma antibody complex, incubating the GPIIb/IIIa receptor:GPπb/IIIa receptor antagonist:plasma antibody complex with a secondary anti-human detectable antibody to form a GPIIb/IIIa receptor:GPIIb/nia receptor antagonisCplasma antibody secondary antihuman detectable antibody complex, and detecting the presence of the secondary anti-human detectable antibody in the GPIIb/IIIa receptor:GPIIb/πia receptor antagonist:plasma antibody:secondary antihuman detectable antibody complex. 2. A method of claim 1 wherein the antagonist is selected from the group consisting of Mpr-(Acetimidyl-Lys)-Gly-Asp-Tφ-Phe-Cys-NH2, Mpr-(Acetimidyl-Lys)-Gly-Asp-Tφ-Phe-Pen-NH2, Mpr-(Phenylimidyl-Lys)-Gly-Asp-Tφ-Phe-Pen-NH2, Mpr-(Phenylimidyl-Lys)-Gly-Asp-Tφ-Phe-Cys-NH2, N-Methyl-D-phenylalanyl-N-[(lS)-l-formyl-4-guanidinobutyl]-L- prolinamide, ((l-(2-((4-(aminoiminomethyl)benzoyl)amino)-3-(4- hydroxyphenyl)- 1 -oxopropyl)-4-piperidinyl)oxy)-(S)-acetic acid, Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-l ,4- dioxobutyl]amino]-4-pentynoate, N-(2-(2-(((3-((aminoiminomethyl)amino)propyl)amino)carbonyl)-l - 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]-l,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/πia receptor:GPIIb/πia 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.

Inventors: BEDNAR BOHUMIL, GOULD ROBERT J, BEDNAR, BOHUMIL, GOULD, ROBERT, J.
Applicants: BEDNAR BOHUMIL, GOULD ROBERT J, MERCK & CO INC, BEDNAR, BOHUMIL, GOULD, ROBERT, J., MERCK & CO., INC.
Application year: 1997
Main classification: G01N33
IPC codes: G01N33