CA2382150A1 - An in vitro phagocytosis method to predict the in vivo potential of integrin antagonists/agonists to induce thrombocytopenia - Google Patents

Abstract

This invention relates to the use of an in vitro cell-based phagocytosis method to determine the potential of integrin antagonists or agonists to induce thrombocytopenia. The method measures the extent of phagocytosis in samples containing (i) platelets, (ii) an integrin antagonist or agonist and (iii) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent anti-platelet antibodies. The phagocytosis process in the method is preferably measured using chemiluminescence or dyes. The metho d is useful for screening integrin antagonists/agonists, such as glycoprotein IIb/IIIa receptor (GPIIb/IIIa) antagonists, for their phagocytotic and therefore thrombocytopenic potential in the presence of drug-dependant antibodies. The present methods are also useful for identifying patients who may be at risk to develop thrombocytopenia mediated by treatment with an integrin antagonist or agonist. This invention is also useful for the identification of new integrin antagonists or agonists which do not mediate the binding of drug-dependent antibodies to platelets and which do not media te thrombocytopenia.

Description

TITLE
An In Vitro Phagocytosis Method to Predict the In Vivo Potential of Integrin Antagonists/Agonists to Induce Thrombocytopenia FIELD OF THE INVENTION
This invention relates the use of an in vitro cell-based phagocytosis method to determine the potential of integrin antagonists or agonists to induce thrombocytopenia. The method measures the extent of phagocytosis in samples containing (i) platelets, (ii) an integrin antagonist or agonist and (iii) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent anti-platelet antibodies. The phagocytosis process in the method is preferably measured using chemiluminescence or dyes.
The method is useful for screening integrin antagonists or agonists, such as glycoprotein IIb/IIIa receptor (GPIIb/IIIa) antagonists, for their phagocytotic and therefore thrombocytopenic potential in the presence of drug-dependant antibodies. The present methods are also useful for identifying patients who may be at risk to develop thrombocytopenia mediated by treatment with an integrin antagonist or agonist. This invention is also useful for the identification of new integrin antagonists or agonists which do not mediate the binding of drug-dependent antibodies to platelets and which do not mediate thrombocytopenia.
BACKGROUND OF THE INVENTION
Thromboembolic diseases, including stable and unstable angina pectoris, myocardial infarction, stroke, and lung embolism are the major cause of disability and mortality in most developed countries. Recently, therapeutic strategies aimed at interfering with the binding of ligands to the integrin GPIIb/IIIa have been explored to treat these patient groups. Platelet GPIIb/IIIa is the main platelet receptor for fibrinogen and other adhesive glycoproteins, including fibronectin, vitronectin and von Willebrand factor.

Interference of ligand binding with this receptor has been proven beneficial in animal models of thromboembolic disease (Coller, B.S. GPIIb/IIIa Antagonists: Pathophysiologic and Therapeutic Insights from Studies of C7E3 FAB. Thromb.
Haemost. 78: 1, 730-735, 1997), and in limited studies involving human subjects (White, H.D. Unmet Therapeutic Needs in the Management of Acute Ixchemia. Am. J. Cardiol. 80: 4A, 2B-10B, 1997; Tcheng, J.E. Glycoprotein IIb/IIIa Receptor Inhibitors: Putting EPIC, IMPACT II, RESTORE, and EPILOG
Trials into Perspective. Am. J. Cardiol. 78: 3A, 35-40, 1996).
A number of cell surface receptor proteins, referred to as integrins or adhesion protein receptors, have been identified which bind to extracellular matrix ligands or other cell adhesion protein ligands thereby mediating cell-cell and cell-matrix adhesion processes. The integrins are encoded by genes belonging to a gene superfamily and are typically composed of heterodimeric transmembrane proteins containing 0c-and (3-subunits. Integrin subfamilies contain a common (3-subunit combined with different oc-subunits to form adhesion protein receptors with different specificities. In addition to GPIIb/IIIa, a number of other integrin cell surface receptors have been identified. For example, members of the (31 subfamily, 0C4(31 and 0c5(31, have been implicated in various inflammatory processes, including rheumatoid arthritis, allergy, asthma and autoimmune disorders.
The integrin GPIIb/IIIa, also referred to as the platelet fibrinogen receptor, is the membrane protein mediating platelet aggregation. GPIIb/IIIa in activated platelets is known to bind four soluble RGD containing adhesive proteins, namely fibrinogen, von Willebrand factor, fibronectin, and vitronectin. The term "RGD" refers to the amino acid sequence Arg-Gly-Asp. The binding of fibrinogen and von Willebrand factor to GPIIb/IIIa causes platelets to aggregate. The binding of fibrinogen is mediated in part by the RGD
recognition sequence that is common to the adhesive proteins that bind GPIIb/IIIa. RGD-peptidomimetic GPIIb/IIIa antagonist compounds are known to block fibrinogen binding and prevent platelet aggregation and the formation of platelet thrombi. GPIIb/IIIa antagonists represent an important new approach for anti-platelet therapy for the treatment of thromboembolic disorders.
It has been reported that certain individuals receiving certain GPIIb/IIIa antagonists may develop life-threatening thrombocytopenia. The principal cause of these thrombocytopenias is thought to be immune-mediated, due to the presence of drug-deper~dent anti-platelet antibodies (Berkowitz, S.D., Harrington, R.A., Rund, M.M., and Tcheng, J.E. Acute Profound Thrombocytopenia after C7E3 FAB
(abciximab) Therapy. Circulation 95:809-813, 1997).
The general phenomenon of drug-dependent thrombocytopenia/thromboembolic complications is well known.
Clinically important examples are heparin-induced thrombocytopenia (HIT) (Amiral, J., Bridley, F., Wolf, M., et al., Antibodies to macromolecular platelet factor IV-heparin complexes in heparin-induced thrombocytopenia: A study of 44 cases. Thromb. Haemost. 1995, 73:21-28; Ansell, J., Deykin, D., Heparin-induced thrombocytopenia and recurrent thromboembolism. Am. J. Hematol. 1980, 8:325-332), and heparin-induced thrombotic thrombocytopenia (HITT), though other drugs have been implicated (Kelton, J.G., Sheridan, D.P., Santos, A.V., et al. Heparin-induced thrombocytopenia:
Laboratory studies. Blood, 1988, 72:925-930; Chong, B., Berndt, M. Heparin induced thrombocytopenia. Blut 1989, 58:53-57; Curtis, B.R., McFarland, J.G., Wu, G-G., Visentin, G.P., and Aster, R.H., Antibodies in sulfonamide-induced immune thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa complex. Blood, 1994 84:176-183). HIT and HITT are thought to be of immune origin involving binding to the platelet of drug-dependent anti-platelet antibodies induced by the formation of heparin/platelet Factor IV/antibody complexes (Karpatikin, S., Drug-induced thrombocytopenia. 1971, Amer. J. Medical Sciences, 262:68-78). Platelet clearance is believed mediated W~ X1/27617 CA 02382150 2002-02-15 PCT/US00/28149 by the reticuloendothelial system (RES). In some cases such drug/antibody complexes are reported to activate platelets, leading directly to platelet secretion and aggregation (Amiral, J., Wolf, M., Fisher, A.M., Boyer-Neumann, C., Vissac, A.M., and Meyer, D. Pathogenicity of IgA and/or IgM
antibodies to heparin-platelet Factor IV complexes in patients with heparin-induced thrombocytopenia. British J. of Haem.
1996, 92:954-959).
Cases of thrombocytopenia of unknown origin are referred to as idiopathic thrombocytopenic purpura (ITP). In most patients this disorder is thought to be caused by autoantibodies against platelet membrane glycoproteins (Gonzalez-Conejero, R., Rivera, J., Rosillo, M.C., Lozano, M.L., and Garcia, V.V.; Comparative study of three methods to detect free plasma antiplatelet antibodies. Acta Haematol., 96:135-139, 1996; Stockelber, D., Hou, M., Jacobson, S., Kutti, J., Wadenvik, H., Detection of platelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP). A
comparative study using flow cytometry, a whole platelet ELISA, and an antigen capture ELISA. Eur. J. Haematol., 56:72-77, 1996) and possibly glycolipids (Arnout, J. The pathogensis of the antiphospholipid syndrome: A hypothesis based on parallelisms with heparin-induced thrombocytopenia.
Thrombosis and Haemostasis, 75:536-541, 1996; Cuadrado, M.J., Mujic, F., Munoz, E., Khamashta, M.A., Hughes, G.R.V., Thrombocytopenia in the antiphospholipid syndrome. Annals of the Rheumatic Diseases, 56:194-196, 1997), with removal of IgG-sensitized platelets by the RES.
The complications associated with the use of GPIIb/IIIa antagonists may limit their utility and integrin antagonist or agonists in general, because patients may develop a thrombocytopenic episode mediated by GPIIb/IIIa antagonist-dependent drug-dependent antibodies (GPIIb/IIIa DDABs) (as defined below) and/or other drug-dependent mechanisms.
It follows from the foregoing considerations that a sensitive and specific assay that can detect such GPIIb/IIIa DDABs or other integrin DDABs may be beneficial in identifying WO 01/27617 CA 02382150 2002-02-15 PCT/USO~/2g149 patients with DDABs that are present prior to treatment with the GPIIb/IIIa antagonist, and/or antibodies that develop and increase in titer following administration of the GPIIb/IIIa antagonist, which patients may be at risk to develop thrombocytopenia in response to the GPIIb/IIIa antagonist.
The present invention provides such assay methods for the detection of anti-platelet integrin DDABs which methods are useful in the design of integrin antagonists or agonists and are useful for use in conjunction with the administration of an integrin antagonist or agonist drug.
SUMMARY OF THE INVENTION
This present invention provides an in vitro cell-based phagocytosis method to determine the potential of integrin antagonists or agonists to induce thrombocytopenia. The method measures the extent of phagocytosis in samples containing (i) platelets, (ii) an integrin antagonist or agonist and (iii) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent anti-platelet antibodies. The phagocytosis process in the method is preferably measured using chemiluminescence or dyes.
The method is useful for screening integrin antagonists or agonists, such as glycoprotein IIb/IIIa receptor (GPIIb/IIIa) antagonists, for their phagocytotic and therefore thrombocytopenic potential in the presence of drug-dependant antibodies. The present methods are also useful for identifying patients who may be at risk to develop thrombocytopenia mediated by treatment with an integrin antagonist or agonist. This invention is also useful for the identification of new integrin antagonists or agonists which do not mediate the binding of drug-dependent antibodies to platelets and which do not mediate thrombocytopenia.
This invention provides treatment methods and procedures for identifying patients at risk for integrin antagonist or agonist mediated disease states. The present invention provides assays and methods useful for the detection, in a patient bodily fluid sample, of drug-dependent antibodies that _5_ bind to cells in the presence of an integrin antagonist or agonist. The present invention provides sensitive, specific and easy-to-use assays that may be used in conjunction with integrin antagonist or agonist treatment. These assays are capable of detecting low levels of integrin antagonist or agonist-dependent antibodies that bind to cells which may be present in an individual prior to the administration of an integrin antagonist or agonist, and/or for the detection of developing integrin-antagonist or agonist-dependent substances following treatment with the integrin antagonist or agonist.
An object of the present invention provides assays and methods for the detection in a patient bodily fluid sample, of DDABs that bind to platelets in the presence of a GPIIb/IIIa antagonist. The present invention provides a method of using an in vitro cell-based system to simulate the in vivo phagocytotic process of the reticuloendothelial system (RES) that occurs during platelet clearance. This invention also relates to the use of different GPIIb/IIIa preparations to identify patients at risk for early-onset thrombocytopenia/thromboembolic complications upon treatment with GPIIb/IIIa antagonists, thereby increasing the specificity of antibody detection.
This invention also relates to the use of DDABs and humanized chimeric antibodies as a standard for DDAB assays.
The synthesis and characterization of humanized chimeric antibodies specific for GPIIb/IIIa:antagonist complexes is described and exemplified in commonly-owned PCT Application No. PCT/US99/01640, published July 29, 1999 the contents and disclosure of which is hereby incorporated by reference in its entirety.
The present assays may be used to identify patients at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or thromboembolic complications and/or to identify patients who are not at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or thromboembolic complications.

DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below.
The term "integrin" as used herein refers to any of the many cell surface receptor proteins, also referred to as adhesion protein receptors, which have been identified which bind to extracellular matrix ligands or other cell adhesion protein ligands thereby mediating cell-cell and cell-matrix adhesion processes.
The term "integrin antagonists" as referred to herein (also referred to herein as integrin inhibitors) includes compounds (including proteins, peptides and peptidomimetic compounds and other small molecule compounds) which act as inhibitors of the binding of the integrin protein to endogenous protein ligands of such integrin. Preferred integrin inhibitors used in the present invention are RGD-peptidomimetic compounds.
The term "integrin agonists" as referred to herein includes compounds which act as stimulators of the binding of the integrin protein to endogenous proteins ligands of such integrin.
As used herein, the term "RGD-peptidomimetic compounds"
refers to chemical compounds which bind to the RGD-binding region of the integrin and which block RGD-mediated binding of one or more adhesive proteins to such integrin. Preferred in the present invention are antagonists of the GPIIb/IIIa integrin.
As used herein, the term "integrin drug-dependent antibodies" or "integrin DDABs" or refers to antibodies that (a) bind to platelets in the presence of an integrin antagonist or agonist but do not bind to platelets in the absence of the antagonist or agonist, or (b) which bind to platelets in the absence of the integrin antagonist or agonist, but whose binding or ability to induce platelet activation is potentiated by the integrin antagonist or agonist.

W~ ~l/27617 CA 02382150 2002-02-15 PCT/USO~/28149 As used herein, the term "GPIIb/IIIa antagonist-dependent drug-dependent antibodies" or "GPIIb/IIIa DDABs" or "GPIIb/IIIa drug-dependent antibodies" refers to antibodies that (a) bind to platelets in the presence of a GPIIb/IIIa antagonist but do not bind to platelets in the absence of a GPIIb/IIIa antagonist, or (b) which bind to platelets in the absence of a GPIIb/IIIa antagonist, but whose binding or ability to induce platelet activation is potentiated by GPIIb/IIIa antagonists. GPIIb/IIIa DDABs may bind, for example, to stable neoepitopes in GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complexes, which are mediated or induced by the binding of the GPIIb/IIIa antagonist to GPIIb/IIIa. The GPIIb/IIIa DDABs may also bind to unstable neoepitopes requiring the constant presence of GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complex, and the antagonist, or to structural entities consisting of GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complexes, and the antagonist itself.
"Compound A" referred to herein is 2(S)-[(n-butoxycarbonyl)amino]-3-[[[3-[4-(aminoiminomethyl)phenyl]
isoxazolin-5(R)-yl]methylcarbonyl]amino]propionic acid or its methyl ester. The preparation of Compound A is disclosed in PCT Patent Application Publication Number WO 95/14683, and is incorporated herein by reference in its entirety.
"Compound B" referred to herein is 2(S)-[[(3,5-dimethylisoxazol-4-yl)sulfonyl]amino]-3-[[[3-[4-(aminoimino methly)phenyl]isoxazolin-5(R)-yl]methylcarbonyl]amino]
propionic acid. The preparation of Compound B is disclosed in PCT Patent Application Publication Number WO 96/37482, published November 28, 1996, and is incorporated herein by reference in its entirety.
"Compound C" referred to herein is to 2(S)-[(4-methylphenylsulfonyl)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. The preparation of Compound C is disclosed in PCT Patent Application Publication Number WO
_g-94/18981, and is incorporated herein by reference in its entirety.
"Compound D" referred to herein is 5-[2-(piperdin-4-yl)ethyl]thieno[2,3-b]thiophene-2-N-(3-2(S)-(3-pyridinylsulfonylamino)propionic acid]carboxamide. The preparation of Compound D is disclosed in PCT Patent Application Publication Number WO 95/14351, and is incorporated herein by reference in its entirety.
The term "antibody" as used herein includes antibody from a monoclonal or polyclonal or recombinant source that is produced in response to an antigen or which is selected by binding to a selected binding site, as well as fragments, chimeric forms, altered forms and derivatives of such antibody, as well as chemically and recombinantly produced forms thereof. The source of the antibody sample to be tested in the assays of the present invention may be any bodily fluid or tissue or cells containing such antibody, with the preferred source of such antibody sample being blood or plasma.
As used herein, the term "recombinant" refers to any DNA, RNA, protein or peptide that is derived directly or indirectly through molecular cloning or any organism or virus containing such DNA, RNA, protein or peptide.
The term "opsonin" as used herein refers to a substance, generally an antibody that makes a cell or microorganism more susceptible to the engulfing action of phagocytes. The term "opsonization", as used herein refers to a process in which an antigen is combined with the opsonin antibody on a cell to make the cell more susceptible to the engulfing action of phagocytes. The term "opsonizing antibody", as used herein refers to an antibody which causes the cell to be more susceptible to the engulfing action of phagocytes.
The term "THP-1" as used herein refers to a commercially available monocyte-like cell line (American Type Culture Collection (ATCC) Number TIB-202; Depositor: S. Tsuchiya).
The term "oxidative burst" as used herein refers to the activation of the oxidative metabolism, the respiratory burst, and the resultant generation of reactive oxygen metabolites, such as superoxide anion (Oz), hydrogen peroxide (HZOZ), in cells as a consequence of phagocytosis.
The term "CL detection agent" as used herein refers to a compound that converts the energy of the respiratory burst associated with phagocytosis into light emission or chemiluminescence. Examples of compounds of this type include but are not limited to luminol and lucigenin.
DETAILED DESCRIPTION OF THE INVENTION
This present invention provides an in vitro cell-based phagocytosis method to determine the potential of integrin antagonists or agonists to induce thrombocytopenia. The method measures the extent of phagocytosis in samples containing (i) platelets, (ii) an integrin antagonist or agonist and (iii) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent anti-platelet antibodies. The phagocytosis process in the method is preferably measured using chemiluminescence or dyes.
The method is useful for screening integrin antagonists/agonists, such as glycoprotein IIb/IIIa receptor (GPIIb/IIIa) antagonists, for their phagocytotic and therefore thrombocytopenic potential in the presence of drug-dependant antibodies. The present methods are also useful for identifying patients who may be at risk to develop thrombocytopenia mediated by treatment with an integrin antagonist or agonist. This invention is also useful for the identification of new integrin antagonists or agonists which do not mediate the binding of drug-dependent antibodies to platelets and which do not mediate thrombocytopenia.
The present invention describes procedures for identifying patients at risk for thrombocytopenia or other adverse reactions mediated by treatment with integrin antagonists or agonists. This invention provides procedures for identifying patients at risk for integrin antagonist or agonist mediated thrombocytopenia or other adverse reactions prior to treatment and during treatment. The present VVO ~l/27617 CA 02382150 2002-02-15 PCT/US00/28149 invention provides assays and methods useful for the detection in a patient bodily fluid sample of drug-dependent antibodies (DDABs) that recognize an integrin in the presence of an integrin antagonist or agonist. The present invention provides sensitive, specific and easy-to-use assays which may be used in conjunction with integrin antagonist or agonist treatment, such assays being capable of detection of low levels of integrin DDABs which may be present in an individual prior to the administration of an integrin antagonist or antagonist and/or for the detection of developing integrin DDABs following treatment with the integrin antagonist or agonist.
Patients with pre-existing or developing DDAB titer may have a greater risk of undergoing thrombocytopenic episodes following administration of an GPIIb/IIIa antagonist.
Patients that are determined to have pre-existing DDABs may either be excluded from therapy with GPIIb/IIIa antagonists, or may be treated with a compound that is less prone to potentiate the binding of DDABs. Alternatively, if a DDAB
titer should develop, the therapy can be stopped prior to the onset of a clinically significant thrombocytopenic episode.
Patients with pre-existing DDABs may be at risk of developing a thrombocytopenic episode upon treatment with GPIIb/IIIa antagonist.
Low titers of pre-existing DDABs may be present in a significant percentage of the general population. It follows that procedures aimed at identifying patients in the DDAB-positive population that are at increased risk for thrombocytopenia/thromboembolic complications will facilitate the exclusion of this "high risk" population from therapy with a specific GPIIb/IIIa antagonist, treatment with chemically distinct GPIIb/IIIa antagonists, or identify patients in need of extensive monitoring during treatment.
In patients with developing or increasing DDAB titer, the identification of such an increase at the earliest time point is necessary to terminate therapy with a specific GPIIb/IIIa WO 01/27617 CA 02382150 2002-02-15 PCT/US00/2g149 antagonist prior to the development of a clinically significant thrombocytopenic episode. Different methods exist for measuring the levels of drug-dependant antibodies associated with thrombocytopenia, including various immunoassays .
The present invention provides a different approach for detecting drug-dependant antibodies associated with thrombocytopenia which involves utilizing a functional assay rather than immunoassays. Platelet clearance is believed mediated by the reticuloendothelial system (RES). The present invention provides an in vitro cell-based system which is based upon the in vivo phagocytotic process of the reticuloendothelial system, which occurs during platelet clearance. The reticuloendothelial system includes certain more or less strongly phagocytic cells (including leukocytes, macrophages, histiocytes, and microglia) distributed throughout the body, but primarily found in lymph nodes and in blood and lymph sinuses in the liver, spleen, and bone marrow.
The RES functions as a defense system by identifying and destroying invading, or foreign substances. For example, platelets presenting on their surface certain drug-dependant antibodies are recognized by phagocytic leukocytes, which engulf the platelets leading to their destruction.
When phagocytic cells interact with particulate or soluble stimuli, cells respond with an activation of the oxidative metabolism, the respiratory burst. As a consequence, cells generate a number of reactive oxygen metabolites, such as superoxide anion (Oz-) and hydrogen peroxide (H202). Generation of oxygen metabolites can be detected as light emission or chemiluminescence using a CL
detection agent (Johansson et al. J. Leukocyte Biol. 45: 444-451, 1989). An oxidative burst occurs upon phagocytosis and this is captured by a CL detection agent such as luminol, that converts this energy to chemiluminescence. Luminol added to a system can be used to detect the phagocytotic process by acting as a bystander substrate for oxygen metabolites generated during activation of the phagocytes (Allen et al.

Biochem Biophys. Res. Commun. 69: 245, 1976). The amount, extent and onset of chemiluminescence is indicative of phagocytic activity. Chemiluminescence light units are measured and the amplitude of response allows the present methods to be carried out so as to compare different integrin antagonist or agonist compounds or compare different patient plasma samples which may contain integrin DDABs The present assay method has shown certain advantages relative to immunoassay-based (ELISA) assays for the identification of GPIIb/IIIa DDABs in human plasma samples and the identification of GPIIb/IIIa antagonist compounds which have reduced potential to be associated with thrombocytopenia.
For example, the assay method of the present invention identified a GPIIb/IIIa antagonist compound as being positive in inducing platelet phagocytosis, where such compound was negative in an immunoassay test for DDABs, and where the compound was subsequently shown to result in many positives for DDAB when tested against normal human plasma samples.
This result indicates that the present assay may provide improved methods for the prediction of the propensity of drug candidate compounds to elicit DDAB reactions.
Monitoring the phagocytic activity of phagocytic cells in the presence of platelets coated with integrin drug-dependant antibodies therefore represents a useful assay for identifying those antibodies capable of promoting platelet destruction and inducing a thrombocytopenic response. Thus, the present invention provides an assay for detection of drug-dependant antibodies that are functionally active in promoting platelet phagocytosis.
The present invention describes assays and methods for the detection in a patient bodily fluid sample of DDABs that recognize the platelet integrin GPIIb/IIIa in the presence of a GPIIb/IIIa antagonist. The present assays may be used to identify patients at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or other adverse reactions and/or to identify patients who are not at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or other adverse reactions.
The present invention describes an in vitro cell-based system using phagocytic cells, selected GPIIb/IIIa antagonists and samples known to contain or which may contain GPIIb/IIIa DDABs. The in vitro cell-based system of the present invention detects pre-existing GPIIb/IIIa DDABs (i.e., DDABs which are pre-existing in the patient prior to the patient being administered the GPIIb/IIIa antagonist). The method of the present invention also detects GPIIb/IIIa DDABs for which an antibody titer develops following the GPIIb/IIIa antagonist being administered to the patient, such GPIIb/IIIa DDABs being potentiated by the presence of the GPIIb/IIIa antagonists.
The present assays and methods may be used to identify individuals having GPIIb/IIIa antagonist-induced DDABs and may be used to exclude, terminate, and/or change therapeutic modalities with GPIIb/IIIa antagonists prior to the onset of thrombocytopenia or other adverse thromboembolic complications.
It has been found in the present invention that use of different GPIIb/IIIa antagonists in the in vitro cell-based system detect different DDABs recognizing different epitopes.
Thus, different GPIIb/IIIa antagonists in the present system differ in their ability to induce the formation of epitopes that are recognized by DDABs in a patient. Thus the present assays may be employed to identify integrin antagonists or agonists which may be less likely to induce DDABs or induce epitopes which are recognized by pre-existing DDABs.
Samples which may contain GPIIb/IIIa DDABs may be obtained from, for example, plasma samples from individuals that exhibit thrombocytopenia or thromboembolic complications, from untreated individuals having pre-existing DDABs or from treated individuals that develop DDABs after administration of a GPIIb/IIIa antagonist. In addition, GPIIb/IIIa DDABs may be obtained from an individual or organism immunized with GPIIb/IIIa in the presence or absence of a GPIIb/IIIa antagonists. The assays of the present invention can be used WO 01/27617 CA 02382150 2002-02-15 pCT/US00/28149 to rapidly identify such DDABs. The assays of the present invention are also useful for identifying integrin antagonists/agonists that inhibit the integrin receptor but do not potentiate the binding of DDABs to the integrin and are therefore less likely to potentiate a DDAB response.
The present invention provides methods and assays useful for the detection, in patient body fluid samples, of antibodies that recognize an integrin. The present invention provides sensitive, specific and easy-to-use assays which may be used in patients to elucidate the involvement of antibodies to integrins in the disease state, such assays being capable of detecting low levels of integrin-directed DDABs. These antibodies may be present in patients' blood, body fluids and tissues without drug therapy. Typical examples include auto-antibodies directed to platelet surface antigens, such as GPIIb/IIIa, which can be encountered in patients with idiopathic thrombocytopenic purpura. In addition, such assays are useful for the detection of low levels of DDABs directed to integrins on the platelet surface, on megakaryocytes or their progenitor cells. These DDABs may be present in an individual prior to administration of drug therapy, including treatment with integrin antagonist or agonist drug, and may increase or develop following treatment with the drug.
Integrin DDABs may be obtained from, for example, whole blood from individuals that exhibit thrombocytopenia or other thromboembolic complications, from untreated individuals having preexisting antibodies or from treated individuals that develop DDABs after administration of an integrin antagonist or agonist drug or other medications.
Persons skilled in the art are aware that the following methods can be practiced by contacting the components of the methods in varying order. The order of the steps given in the following methods is illustrative of the invention and not intended to be limiting.
The present invention provides a method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising:

(a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:
(i) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, (ii) an integrin antagonist or agonist;
(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c).
A preferred integrin antagonist or agonist in any of the methods set forth herein is a GPIIb/IIIa antagonist.
It is preferred in any of the methods set forth herein that the sample containing platelets is platelet-rich plasma.
It is preferred in any of the methods set forth herein that the level of phagocytosis is measured using a chemiluminescence agent or dye that detects oxidative burst in the sample.
The phagocytic cells in any of the methods set forth herein may be THP-1 cells.
It is preferred in any of the above methods that the sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, is a plasma sample obtained from a subject who has been administered an integrin antagonist or agonist drug or from a subject who is being pre-screened prior to being administered an integrin antagonist or agonist drug.
It is preferred in any of the above methods that the sample containing platelets and the sample known to contain or which may contain integrin antagonist or agonist drug-W~ ~l/27617 CA 02382150 2002-02-15 PCT/LTS~O/28149 dependent antibodies, are obtained from the same subject who has been administered an integrin antagonist or agonist drug or who is being pre-screened prior to being administered an integrin antagonist or agonist drug.
The present invention provides a method for identifying or characterizing integrin antagonist or agonist drug candidates having reduced potential to cause thrombocytopenia mediated by integrin drug-dependent antibodies, said method comprising:
(a) preparing a sample by contacting the following:
(i) a sample containing platelets;
(ii) a selected integrin antagonist or agonist compound;
(iii) a sample containing integrin antagonist or agonist drug-dependent antibodies;
(iv) phagocytic cells;
(b) measuring the level of phagocytosis in the sample of step (a) ;
(c) performing steps (a) and (b) using different selected integrin antagonist or agonist compounds;
(d) comparing the level of phagocytosis obtained for each of the different selected integrin antagonist or agonist compounds;
(e) selecting integrin antagonist or agonist compounds which result in the lowest level of phagocytosis.
The present invention also provides a method for the treatment of subjects with a GPIIb/IIIa antagonist comprising pre-screening the subjects prior to administering the GPIIb/IIIa antagonist, said pre-screening comprising the steps of (a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:
(i) a plasma sample from the subject, (ii) a GPIIb/IIIa antagonist;

(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c) .
The present invention also provides a method for the treatment of a subject with a GPIIb/IIIa antagonist comprising testing the subject concurrently with administering the GPIIb/IIIa antagonist to the subject, said testing comprising the steps of:
(a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:
(i) a plasma sample from the subject, (ii) a GPIIb/IIIa antagonist;
(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c) .
It is preferred in any of the above methods wherein the GPIIb/IIIa antagonist used in the treatment is a compound, or pharmaceutically acceptable salt forms thereof, selected from:
2(S)-[(n-butoxycarbonyl)amino]-3-[[[3-[4-(aminoiminomethyl)phenyl] isoxazolin-5(R)-yl]methylcarbonyl]amino]propionic acid or its methyl ester;
2(S)-[[(3,5-dimethylisoxazol-4-yl)sulfonyl]amino]-3-[[[3-[4-(aminoimino methly)phenyl]isoxazolin-5(R)-yl]methylcarbonyl]amino] propionic acid;
2(S)-[(4-methylphenylsulfonyl)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;

5-[2-(piperdin-4-yl)ethyl]thieno[2,3-b]thiophene-2-N-(3-2(S)-(3-pyridinylsulfonylamino)propionic acid]carboxamide.

The present invention also provides a method for detecting in a subject integrin drug-dependent antibodies, comprising:
(a) obtaining a sample of platelet-rich plasma;
(b) contacting the sample of step (a) with one or both of the following:
(i) an integrin antagonist or agonist compound, (ii) a plasma sample which contains or may contain integrin drug-dependent antibody;
(c) contacting a sample of phagocytic cells with a CL
detection agent;
(d) contacting the sample of step (b) with the sample of step (c); and (e) measuring the level of chemiluminescence in the sample of step (d).
The present invention provides a method for detecting the formation or increase of integrin drug-dependent antibodies in a subject, comprising:
(a) prior to administering to the subject an integrin antagonist or agonist, testing the subject using the above method;
(b) administering to the subject an integrin antagonist or agonist;
(c) concurrently with administering to the subject the integrin antagonist or agonist, testing the subject using the above method; and (d) comparing the results of step (a) with the results of step (c).
It is preferred in any of the above methods wherein the integrin antagonist or agonist which is administered is an GPIIb/IIIa antagonist.
It is preferred in any of the above methods wherein the integrin antagonist or agonist compound of step (b) is the W~ ~l/27617 CA 02382150 2002-02-15 PCT/US00/28149 active form or active metabolite of the integrin antagonist or agonist that is used to treat the subject.
It is preferred in any of the above methods wherein the integrin antagonist of step (b) is selected from the following group of compounds or an active metabolite form thereof:
2(S)-[(n-butoxycarbonyl)amino]-3-[[[3-[4-(aminoiminomethyl)phenyl] isoxazolin-5(R)-yl]methylcarbonyl]amino]propionic acid or its methyl ester;
2(S)-[[(3,5-dimethylisoxazol-4-yl)sulfonyl]amino]-3-[[[3-[4-(aminoimino methly)phenyl]isoxazolin-5(R)-yl]methylcarbonyl]amino] propionic acid;
2(S)-[(4-methylphenylsulfonyl)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;
5-[2-(piperdin-4-yl)ethyl]thieno[2,3-b]thiophene-2-N-(3-2(S)-(3-pyridinylsulfonylamino)propionic acid]carboxamide.
It is preferred in any of the above methods wherein the method is used to determine the existence of integrin drug-dependent antibodies prior to or during integrin antagonist or agonist therapy and is used to select a treatment which is less likely to result in thrombocytopenia or other adverse effects of platelet depletion.
It is preferred in any of the above methods wherein the sample of step (b)(ii) is substituted with a sample containing a recombinant human chimeric or humanized antibody specific for an integrin bound with an integrin antagonist or agonist, said sample serving as a positive control.
The present invention also provides a pharmaceutical test kit for detecting the presence of integrin drug-dependent antibodies in a biological sample, comprising:
(a) a phagocytic cell preparation, (b) a CL detection agent, (c) at least one integrin antagonist or agonist, and W~ ~l/27617 CA 02382150 2002-02-15 PCT/US~O/28149 (d) a positive control antibody specific for an integrin bound with an integrin antagonist or agonist.
It is preferred in the above pharmaceutical test kit wherein the positive control antibody is a recombinant human chimeric or humanized antibody which recognizes an integrin bound with an integrin antagonist or agonist.
The present invention provides a method for detecting the formation or increase in integrin drug-dependent antibodies prior to or during integrin antagonist or agonist therapy, to identify a treatment which is less prone to side-effects, said method comprising any of the methods described above.
The present invention provides a method of identifying a subject having increased risk of developing integrin drug-dependent antibody associated adverse side effects, including thrombocytopenia or thromboembolic complications, following treatment with an integrin antagonist or agonist, comprising any of the methods described above.
The present invention provides a method of identifying a subject having increased risk of thrombocytopenia or thromboembolic complications within the first week of treatment with GPIIb/IIIa antagonist comprising any of the methods described above.
It is preferred in any of the above methods wherein the sample containing antibody is obtained from the subject and the method is performed prior to treatment of the subject with the integrin antagonist or agonist.
The present invention provides a method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising: obtaining a sample containing platelets;
optionally contacting the sample with a sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, or an integrin antagonist or agonist; contacting with phagocytotic cells; measuring the level of phagocytosis.
The present invention provides a method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising:
(a) obtaining a sample containing platelets;
(b) contacting the sample with phagocytotic cells;
(c) measuring the level of phagocytosis in the sample.
Representative integrin antagonist compounds, including GPIIb/IIIa antagonists, are disclosed in the following patents and patent applications, which are incorporated herein by reference: PCT Patent Application 95/14683; PCT Patent Application 95/32710; U.S. Patent 5,334,596; U.S. Patent 5,276,049; U.S. Patent 5,281,585; European Patent Application 478,328; European Patent Application 478,363; European Patent Application 512,831; PCT Patent Application 94/08577; PCT
Patent Application 94/08962; PCT Patent Application 94/18981;
PCT Patent Application 93/16697; Canada Patent Application 2,075,590; PCT Patent Application 93/18057; European Patent Application 445,796; Canada Patent Application 2,093,770;
Canada Patent Application 2,094,773; Canada Patent Application 2,101,179; Canada Patent Application 2,074,685; Canada Patent Application 2,094,964; Canada Patent Application 2,105,934;
Canada Patent Application 2,114,178; Canada Patent Application 2,116,068; European Patent Application 513,810; PCT Patent Application 95/06038; European Patent Application 381,033; PCT
Patent Application 93/07867; and PCT Patent Application 94/02472.
Other GPIIb/IIIa inhibitor compounds are described in the following patents and patent applications: GB 2 271 567 A; GB
2 292 558 A; EP 0 645 376 A1; EP 0 668 278 A1; EP 0 608 759 A2; EP 0 635 492 A1; WO 94/22820; US 5,340,798 and WO
94/09029; US 5,256,812; US 5,084,466; WO 94/01396 and US
5,272,162; WO 94/21602; WO 94/22444; W094/29273; WO 95/18111;
WO 95/18619; WO 95/25091; WO 94/18162; US 5,220,050 and WO

W~ 01/27617 CA 02382150 2002-02-15 PCT/US00/28149 93/16038; US 4,879,313 and EP 0 352 249 B1; US 5,227,490, US
5,229,616 and WO 94/12181; US 5,258,398 and W0 93/11759; WO
93/08181 and EP 0 537 980 A1; WO 93/09133; EP 0 530 505 B1; EP
0 566 919 A1; EP 0 540 334 B1; EP 0 560 730 A2; WO 93/10091;
EP 0 542 363 A2 and WO 93/14077; EP 0 505 868 B1; EP 0 614 664 A1; US 5,358,956; WO 94/26745; WO 94/12478; WO 94/14776; WO
93/00095; WO 93/18058; US 5,239,113, US 5,344,957 and EP 0 542 708 A1; WO 94/22825; US 5,250,679 and WO 93/08174; US
5,084,466; EP 0 668 278; US 5,264,420; EP 0 529 858; US
5,389,631; EP 0 632 016; EP 0 503 548; WO 92/19595; EP 0 525 629; EP 0 604 8000; EP 0 587 134; EP 0 623 615; EP 0 655 439;
US 5,446,056 and WO 95/14682; US 5,399,585; WO 93/12074; EP 0 512 829; EP 0 372 486 and US 5,039,805; EP 0 632 020 and US
5,494,922; US 5,403,836; WO 94/22834; WO 94/21599; WO
94/17034; WO 96/20192; WO 96/19223; WO 96/19221; WO 96/19222;
EP 727425; EP 478362; EP 478363; US 5,272,158; US 5,227,490;
US 5,294,616; US 5,334,596; EP 645376; EP 711770; US
5,314,902; WO 94/00424; US 5,523,302; EP 718287; DE 4446301;
WO 96/22288; WO 96/29309; EP 719775; EP 635492; WO 96/16947;
US 5,602,155; WO 96/38426; EP 712844; US 5,292,756; WO
96/37482; WO 96/38416; WO 96/41803;and WO 97/11940. Each of these references is incorporated herein by reference in its entirety.
Preferred GPIIb/IIIa antagonists include the following:
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(phenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-methyl-phenyl-sulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(butanesulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(propanesulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(ethanesulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(methyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;

N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(ethyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(1-propyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-propyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(R)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(R)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(1-(2-methyl)-propyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-(2-methyl)-propyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(benzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(benzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(benzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-methylbenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-methoxybenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;

WO ~l/27617 CA 02382150 2002-02-15 PCT/USO~/28149 N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-chlorobenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-bromobenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-fluorobenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-phenoxybenzyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-(methyloxyethyl)-oxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-pyridinylcarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-pyridinylcarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-pyridinyl-carbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-(2-pyridinyl)-acetyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(2-(3-pyridinyl)-acetyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-(4-pyridinyl)-acetyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-pyridyl-methyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-pyridyl-methyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(4-pyridyl-methyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(4-butyloxyphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;

N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-thienylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(3-methylphenylsulfonyl)-2,3-(R, S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(R)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(R)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(R)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-iodophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-trifluoromethylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-chlorophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-2-methoxycarbonylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2,4,6-trimethylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]
N2-(2-chlorophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-trifluoromethylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;

N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-trifluoromethylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-fluorophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-fluorophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-methoxyphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2,3,5,6-tetramethylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-cyanophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-chlorophenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-propylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-phenylethylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-isopropylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-phenylpropylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-pyridylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(phenylaminosulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(benzylaminosulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(dimethylaminosulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(2-fluoro-4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;

W~ 01/27617 CA 02382150 2002-02-15 PCT/US00/28149 N3-[2-{3-(2-formamidino-5-pyridinyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(2-formamidino-5-pyridinyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(3-formamidino-6-pyridinyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(3-formamidino-6-pyridinyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(phenylaminocarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(4-fluorophenylaminocarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(1-naphthylaminocarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(benzylaminocarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-bromo-2-thienylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(3-methyl-2-benzothienylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(2-cyclopropylethoxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(2-cyclopropylethoxycarbonyl)-2,3-(S)-diaminopropanoic acid;

VV~ 01/27617 CA 02382150 2002-02-15 N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(S)-yl}-acetyl]-N2-(2-cyclopropylethoxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-guanidinophenyl)-isoxazolin-5(R,S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-guanidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{3-(4-guanidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-(S)-diaminopropanoic acid;
N3-[2-{5-(4-formamidinophenyl)-isoxazolin-3(R,S)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropanoic acid;
or a propionate ester prodrug form of said compound, wherein the hydrogen of the hydroxy group of the diaminopropanoic acid moiety is substituted with a group selected from:
methyl;
ethyl;
isopropyl;
methylcarbonyloxymethyl-;
ethylcarbonyloxymethyl-;
t-butylcarbonyloxymethyl-;
cyclohexylcarbonyloxymethyl-;
1-(methylcarbonyloxy)ethyl-;
1-(ethylcarbonyloxy)ethyl-;
1-(t-butylcarbonyloxy)ethyl-;
1-(cyclohexylcarbonyloxy)ethyl-;
i-propyloxycarbonyloxymethyl-;
cyclohexylcarbonyloxymethyl-;
t-butyloxycarbonyloxymethyl-;
1-(i-propyloxycarbonyloxy)ethyl-;
1-(cyclohexyloxycarbonyloxy)ethyl-;
1-(t-butyloxycarbonyloxy)ethyl-;
dimethylaminoethyl-;
diethylaminoethyl-;
(5-methyl-1,3-dioxacyclopenten-2-on-4-yl)methyl-;
(5-(t-butyl)-1,3-dioxacyclopenten-2-on-4-yl)methyl-;
(1,3-dioxa-5-phenyl-cyclopenten-2-on-4-yl)methyl-;
1-(2-(2-methoxypropyl)carbonyloxy)ethyl-.

Further preferred integrin antagonists useful in the present invention are compounds, or enantiomeric or diasteriomeric forms thereof, or mixtures of enantiomeric or diasteriomeric forms thereof, or active metabolites thereof, and salt forms thereof, selected from:
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(phenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-methyl-phenyl-sulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(butanesulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(propanesulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(ethanesulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(methyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(ethyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(1-propyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-propyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(1-(2-methyl)-propyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-(2-methyl)-propyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(benzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-methylbenzyloxycarbonyl)-2,3-diaminopropanoic acid;

N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-methoxybenzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-chlorobenzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-bromobenzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-fluorobenzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-phenoxybenzyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-(methyloxyethyl)-oxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-pyridinylcarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-pyridinylcarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-pyridinyl-carbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-(2-pyridinyl)-acetyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-(3-pyridinyl)-acetyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-(4-pyridinyl)-acetyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-pyridyl-methyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-pyridyl-methyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-pyridyl-methyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-butyloxyphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-thienylsulfonyl)-2,3-diaminopropanoic acid;

N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-iodophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-trifluoromethylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-chlorophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-methoxycarbonylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2,4,6-trimethylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-chlorophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-trifluoromethylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-trifluoromethylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-fluorophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-fluorophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-methoxyphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2,3,5,6-tetramethylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-cyanophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-chlorophenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-propylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-phenylethylsulfonyl)-2,3-diaminopropanoic acid;

W~ ~l/27617 CA 02382150 2002-02-15 PCT/USO~/28149 N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-isopropylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-phenylpropylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-pyridylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(phenylaminosulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(benzylaminosulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(dimethylaminosulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(2-fluoro-4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(2-formamidino-5-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(2-formamidino-5-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(3-formamidino-6-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(3-formamidino-6-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(phenylaminocarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-fluorophenylaminocarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(1-naphthylaminocarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(benzylaminocarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-bromo-2-thienylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methyl-2-benzothienylsulfonyl)-2,3-diaminopropanoic acid, WO 01/27617 CA 02382150 2002-02-15 PCT/USO~/28149 N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(isobutyloxycarbonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-cyclopropylethoxycarbonyl)-2,3-diaminopropanoic acid, N3-[2-{3-(4-guanidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-guanidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropanoic acid;
N3-[2-{5-(4-formamidinophenyl)-isoxazolin-3-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-diaminopropanoic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-bromo-phenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(2-methyl-phenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(3-formamidino-6-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(2-formamidino-5-pyridinyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(2-fluoro-4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-methylphenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(3-bromo-phenylsulfonyl)-2,3-diaminopropionic acid;
N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5-yl}-acetyl]-N2-(4-bromo-phenylsulfonyl)-2,3-diaminopropionic acid;
or a propionate ester prodrug form of said compound, wherein the hydrogen of the hydroxy group of the propanoic acid moiety is substituted with a group selected from:
methyl;
ethyl;
isopropyl;
methylcarbonyloxymethyl-;

ethylcarbonyloxymethyl-;
t-butylcarbonyloxymethyl-;
cyclohexylcarbonyloxymethyl-;
1-(methylcarbonyloxy)ethyl-;
1-(ethylcarbonyloxy)ethyl-;
1-(t-butylcarbonyloxy)ethyl-;
1-(cyclohexylcarbonyloxy)ethyl-;
i-propyloxycarbonyloxymethyl-;
cyclohexylcarbonyloxymethyl-;
t-butyloxycarbonyloxymethyl-;
1-(i-propyloxycarbonyloxy)ethyl-;
1-(cyclohexyloxycarbonyloxy)ethyl-;
1-(t-butyloxycarbonyloxy)ethyl-;
dimethylaminoethyl-;
diethylaminoethyl-;
(5-methyl-1,3-dioxacyclopenten-2-on-4-yl)methyl-;
(5-(t-butyl)-1,3-dioxacyclopenten-2-on-4-yl)methyl-;
(1,3-dioxa-5-phenyl-cyclopenten-2-on-4-yl)methyl-;
1-(2-(2-methoxypropyl)carbonyloxy)ethyl-.
Further preferred GPIIb/IIIa antagonists useful in assays of the present invention are Compounds A, B, C and D listed above, and salt forms, prodrug forms and metabolites thereof.
The invention now being generally described can be more readily understood by reference to the following examples that are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Example 1 General method for detection of phagocytosis of platelets Platelets are obtained by drawing blood into citrate Vacutainer~ tubes. The preferred species is human, but any mammalian species could be utilized. Platelets may be from the same or different individual from which the test plasma or bodily fluid is obtained. Platelet-rich plasma (PRP) is obtained by centrifugation at 600 g for 10 minutes.
Alternatively, platelet preparations can be obtained by gel-filtration purification. Platelets then may or may not be washed in a buffer consisting of 9 mM EDTA, 150 mM NaCl, 25 mM
Na2HP0. Other washing solutions could be used, for example any isotonic physiological buffer containing an agent to maintain the platelets in an inactive state, such as EDTA or PGE1 (Prostaglandin E1, Sigma-Aldrich. Platelets are counted in a Coulter counter and the platelet number adjusted by the addition of physiologically isotonic buffer, the preferred concentration is 1x10e/mL.
Platelets are incubated with compound, antibody, bodily fluid or combination for one hour at 37°C; 5% CO2, 95% air.
The preferred antibody is a monoclonal antibody that recognizes a neoepitope formed via interaction of drug and receptor. Preferred bodily fluid is human plasma that is to be tested for DDAB.
Phagocytic cells are then prepared. Preferred phagocytic cells include undifferentiated THP-1 cells (ATCC) (maintained in the presence of (3-mercaptoethanol), although any phagocytic cell or cell line could be used, such as leukocytes, differentiated THP-1 cells, or purified monocytes or macrophages. THP-1 cells are pelleted by centrifugation at 1000x g for 10 minutes and resuspended at a preferred concentration of 5x106/mL in RPMI medium (Gibco/BRL) or any other isotonic physiological buffer solution, such as PBS, Hank's BSS, M199, and DMEM, Waymouth's media.
Cyclic hydrazide 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) is utilized to detect the respiratory burst such that chemiluminescence is generated during phagocytosis.
Other substrates for detection are possible, such as reduction of nitroblue tetrazolium (NBT).
THP-1 cells, 5x105/well and luminol, final concentration in well 0.2 mM, are added together in a tube or plate, a 96 well plate is preferred. The constituents are incubated for 10 minutes, 37°-C; 5o CO2, 95o air. The platelet preincubation mix is then added to the THP-1/luminol mixture. Readings are made in a luminometer at various timepoints. Phagocytosis is evidenced by an increase in measured light over time. Data are analyzed by plotting light units versus time. Readings are taken out to 60 minutes. A negative sample would exhibit no increase in emitted light over this time course, while a positive sample would typically increase emitted light beginning at approximately 5 minutes and peaking around 30 minutes.
Example 2 Utilizing the platelet phagocytosis assay to identify improved GPIIb/IIIa antagonists In this case the drugs are GPIIb/IIIa antagonists and the objective is to identify a class or individual compounds that appear not to elicit an immune response. Monoclonal antibodies are derived as previously described and possess an epitope which corresponds to GPIIb/IIIa receptor + compound complex. Blood is drawn from human donors in citrate VacutainerTM tubes and PRP is obtained by centrifugation at 200x g, 10 min. Platelets are counted in a Coulter counter and used at 3 x 10' platelets per well of a 96-well microliter plate. Platelets and test compound are incubated for 15 minutes at 37-°C before the addition of antibody (2-10 ug/mL).
Incubation is continued at 37-°C for an additional 45 minutes.
THP-1 cells (ATCC) grown in RPMI medium (Gibco-BRL) in the presence of 10o fetal bovine serum and (3-mercaptoethanol are harvested, counted and adjusted to a concentration of 5 x 106/mL. Luminol (Sigma) is made in RPMI medium and used at a final concentration of 0.2 mM. THP-1 cells (5 x 105/well) and luminol are added together in a 96-well microliter assay plate and incubated for 10 minutes at 37-°C, 5.0% COZ/95% air.
Platelet, drug, antibody mixture is added to micotiter plate wells and readings are taken every 15 minutes in a luminometer. Plate is kept in the incubator between readings.
Data are analyzed by plotting light units versus time. A
negative sample would exhibit no increase in emitted light WO Ol/2761~ CA 02382150 2002-02-15 PCT/USO~/28149 over time. Such a negative result would be indicative of a desirable compound structure or chemical class.
Example 3 Utilizing the platelet phaaocytosis assay to determine the presence of drug-dependent antibodies in human plasma Blood is drawn from human donors in citrate Vacutainer tubes and PRP is obtained by centrifugation at 200x g, 10 min.
Platelets are counted in a Coulter counter and used at 3 x 10' platelets per well of a 96-well microliter plate. Test plasma is tested neat and at various dilutions, e.g. 1:2; 1:5; and 1:10. Platelets are preincubated with drug for 15 minutes at 37°-C. Subsequently the test plasma is added and the mixture incubated an additional 45 minutes at 37°-C. THP-1 cells (ATCC) grown in RPMI medium (Gibco-BRL) in the presence of 10%
fetal bovine serum and (3-mercaptoethanol are harvested, counted and adjusted to a concentration of 5 x 106/mL. Luminol (Sigma) is made in RPMI medium and used at a final concentration of 0.2 mM. THP-1 cells (5 x 105/well) and luminol are added together in a 96-well microtiter assay plate and incubated for 10 minutes at 37°-C, 5.0% COz/95% air. Test plasma, drug, platelet mixture is added to microtiter plate wells containing the THP-1 cells and readings are taken every 15 minutes in a luminometer. Plate is kept in the incubator between readings. Data are analyzed by plotting light units versus time. A phagocytic response, as evidenced by increased emission of light over time, would be indicative of the presence of drug-dependent antibodies in the test plasma sample.

Claims (26)

WHAT IS CLAIMED IS:

1. A method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising:
(a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:
(i) a sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, (ii) an integrin antagonist or agonist;
(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c).

2. A method of Claims 1, 6-10, 12, 13, or 26 wherein the integrin antagonist or agonist is a GPIIb/IIIa antagonist.

3. A method of Claims 1, 6-10, 12, 13, or 26 wherein the sample containing platelets is platelet-rich plasma.

4. A method of Claims 1, 6-10, 12, 13, or 26 wherein the level of phagocytosis is measured using a chemiluminescence agent or dye that detects oxidative burst in the sample.

5. A method of Claims 1, 6-10, 12, 13, or 26 wherein the phagocytic cells are THP-1 cells.

6. A method of Claim 1 wherein the sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, is a plasma sample obtained from a subject who has been administered an integrin antagonist or agonist drug or from a subject who is being pre-screened prior to being administered an integrin antagonist or agonist drug.

7. A method of Claim 1 wherein the sample containing platelets and the sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, are obtained from the same subject who has been administered an integrin antagonist or agonist drug or who is being pre-screened prior to being administered an integrin antagonist or agonist drug.

8. A method for identifying or characterizing integrin antagonist or agonist drug candidates having reduced potential to cause thrombocytopenia mediated by integrin drug-dependent antibodies, said method comprising:
(a) preparing a sample by contacting the following:
(i) a sample containing platelets;
(ii) a selected integrin antagonist or agonist compound;
(iii) a sample containing integrin antagonist or agonist drug-dependent antibodies;
(iv) phagocytic cells;
(b) measuring the level of phagocytosis in the sample of step (a);
(c) performing steps (a) and (b) using different selected integrin antagonist or agonist compounds;
(d) comparing the level of phagocytosis obtained for each of the different selected integrin antagonist or agonist compounds;
(e) selecting integrin antagonist or agonist compounds which result in the lowest level of phagocytosis.

9. A method for the treatment of subjects with a GPIIb/IIIa antagonist comprising pre-screening the subjects prior to administering the GPIIb/IIIa antagonist, said pre-screening comprising the steps of:
(a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:

(i) a plasma sample from the subject, (ii) a GPIIb/IIIa antagonist;
(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c).

10. A method for the treatment of a subject with a GPIIb/IIIa antagonist comprising testing the subject concurrently with administering the GPIIb/IIIa antagonist to the subject, said testing comprising the steps of:
(a) obtaining a sample containing platelets;
(b) contacting the sample of step (a) with one or both of the following:
(i) a plasma sample from the subject, (ii) a GPIIb/IIIa antagonist;
(c) contacting the sample resulting from step (b) with phagocytotic cells;
(d) measuring the level of phagocytosis in the sample of step (c).

11. A method of Claim 9 or 10 wherein the GPIIb/IIIa antagonist used in the treatment is a compound, or pharmaceutically acceptable salt forms thereof, selected from:
2(S)-[(n-butoxycarbonyl)amino]-3-[[[3-[4-(aminoiminomethyl)phenyl] isoxazolin-5(R)-yl]methylcarbonyl]amino]propionic acid or its methyl ester;
2(S)-[[(3,5-dimethylisoxazol-4-yl)sulfonyl]amino]-3-[[[3-[4-(aminoimino methly)phenyl]isoxazolin-5(R)-yl]methylcarbonyl]amino] propionic acid;
2(S)-[(4-methylphenylsulfonyl)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; and 5-[2-(piperdin-4-yl)ethyl]thieno[2,3-b]thiophene-2-N-(3-2(S)-(3-pyridinylsulfonylamino)propionic acid]carboxamide.

12. A method for detecting in a subject integrin drug-dependent antibodies, comprising:
(a) obtaining a sample of platelet-rich plasma;
(b) contacting the sample of step (a) with one or both of the following:
(i) an integrin antagonist or agonist compound, (ii) a plasma sample from a subject which contains or may contain integrin drug-dependent antibody;
(c) contacting a sample of phagocytic cells with a CL
detection agent;
(d) contacting the sample of step (b) with the sample of step (c); and (e) measuring the level of chemiluminescence in the sample of step (d).

13. A method for detecting the formation or increase of integrin drug-dependent antibodies in a subject, comprising:
(a) prior to administering to the subject an integrin antagonist or agonist, testing the subject using the method of Claim 12;
(b) administering to the subject an integrin antagonist or agonist;
(c) concurrently with administering to the subject the integrin antagonist or agonist, testing the subject using the method of Claim 12; and (d) comparing the results of step (a) with the results of step (c).

14. A method of Claim 13, wherein the integrin antagonist or agonist which is administered is an GPIIb/IIIa antagonist.

15. A method of Claim 13, wherein the integrin antagonist or agonist compound of step (b) is the active form or active metabolite of the integrin antagonist or agonist that is used to treat the subject.

16. A method of Claim 13, wherein the integrin antagonist of step (b) is selected from the following group of compounds or an active metabolite form thereof selected from:
2(S)-[(n-butoxycarbonyl)amino]-3-[[[3-[4-(aminoiminomethyl)phenyl] isoxazolin-5(R)-yl]methylcarbonyl]amino]propionic acid or its methyl ester;
2(S)-[[(3,5-dimethylisoxazol-4-yl)sulfonyl]amino]-3-[[[3-[4-(aminoimino methly)phenyl]isoxazolin-5(R)-yl]methylcarbonyl]amino] propionic acid;
2(S)-[(4-methylphenylsulfonyl)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; and 5-[2-(piperdin-4-yl)ethyl]thieno[2,3-b]thiophene-2-N-(3-2(S)-(3-pyridinylsulfonylamino)propionic acid]carboxamide.

17. A method of Claim 13, wherein the method is used to determine the existence of integrin drug-dependent antibodies prior to or during integrin antagonist or agonist therapy and is used to select a treatment which is less likely to result in thrombocytopenia or other adverse effects of platelet depletion.

18. A method of Claim 12 or 13, wherein the sample of step (b)(ii) is substituted with a sample containing a recombinant human chimeric or humanized antibody specific for an integrin bound with an integrin antagonist or agonist, said sample serving as a positive control.

19. A pharmaceutical test kit for detecting the presence of integrin drug-dependent antibodies in a biological sample, comprising:
(a) a phagocytic cell preparation, (b) a CL detection agent, (c) at least one integrin antagonist or agonist, and (d) a positive control antibody specific for an integrin bound with an integrin antagonist or agonist.

20. A pharmaceutical test kit of Claim 19, wherein the positive control antibody is a recombinant human chimeric or humanized antibody which recognizes an integrin bound with an integrin antagonist or agonist.

21. A method for detecting the formation or increase in integrin drug-dependent antibodies prior to or during integrin antagonist or agonist therapy, to identify a treatment which is less prone to side-effects, said method comprising the method of Claim 1.

22. A method of identifying a subject having increased risk of developing integrin drug-dependent antibody associated adverse side effects, including thrombocytopenia or thromboembolic complications, following treatment with an integrin antagonist or agonist, comprising the method of Claim 1.

23. A method of identifying a subject having increased risk of thrombocytopenia or thromboembolic complications within the first week of treatment with GPIIb/IIIa antagonist comprising the method of Claim 1.

24. A method of Claim 1 wherein the sample containing antibody is obtained from a subject and the method is performed prior to treatment of the subject with an integrin antagonist or agonist.

25. A method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising:
obtaining a sample containing platelets; optionally contacting the sample with a sample known to contain or which may contain integrin antagonist or agonist drug-dependent antibodies, or an integrin antagonist or agonist; contacting with phagocytotic cells; measuring the level of phagocytosis.

26. A method for detecting integrin antagonist or agonist drug-dependent antibodies in a sample comprising:

(a) obtaining a sample containing platelets;
(b) contacting the sample with phagocytotic cells;
(c) measuring the level of phagocytosis in the sample.