Selective Targeting of Procoagulant Platelets
20170326096 · 2017-11-16
Inventors
- Philip John Hogg (Malabar, New South Wales, AU)
- Vivien M.Y. Chen (Mosman, New South Wales, AU)
- Leonardo Pasalic (Glenfield, New South Wales, AU)
- Vu Minh Hua (Zetland, New South Wales, AU)
Cpc classification
G01N2500/04
PHYSICS
A61K31/00
HUMAN NECESSITIES
G01N33/86
PHYSICS
International classification
G01N33/86
PHYSICS
Abstract
The invention relates to a process for identifying procoagulant platelets both in vitro and in vivo, and the identification of compounds which selectively inhibit formation of procoagulant platelets.
Claims
1. A process for identifying procoagulant platelets, comprising contacting a platelet with a compound and determining whether the compound binds to the platelet, wherein the compound is an arsenoxide (or arsenoxide equivalent) compound.
2. A process for identifying a patient at risk of developing a prothrombotic or thrombotic condition, comprising contacting a platelet from said patient with a compound and determining whether the compound binds to the platelet, wherein the compound is an arsenoxide (or arsenoxide equivalent) compound.
3. The process according to claim 2, further comprising determining the proportion of procoagulant platelets.
4. The process according to any one of claim 1 or 3 wherein contacting the platelet with the compound occurs in vivo or in vitro.
5. The process according to any one of claims 1 to 4, wherein contacting the platelet with the compound occurs in whole blood.
6. The process according to any one of claims 1 to 5, wherein contacting the platelet with the compound occurs within a human subject.
7. The process according to any one of claims 1 to 6, wherein binding of the compound to the platelet indicates a prothrombotic or thrombotic condition.
8. The process according to any one of claims 2 to 7, wherein the prothrombotic or thrombotic condition is selected from the group consisting of: infection, sepsis, systemic inflammatory response syndrome, multi organ failure, thrombotic thrombocytopenia purpura, haemolyticuraemia syndrome, vascularisation, renal failure, ischaemic repercussion injury, solid organ transplant rejection, cardiovascular disease, stroke, venous thromboembolism, autoimmune disorders, sickle cell disease, inflammatory bowel disease, acute lung injury, malignancy, myocardial infarction (primary and secondary), embolic stroke, ischaemic stroke, thrombotic stroke, deep vein thrombosis (DVT), thromboembolism, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, arterial thrombosis and arterial embolis.
9. The process according to any one of claims 1 to 8, wherein the trivalent arsenoxide compound is a dithiol reactive compound.
10. The process according to any one of claims 1 to 9, wherein the compound is of the formula (I):
A-[(XBX′).sub.nB′—Y].sub.p (I) wherein A comprises at least one pendant group; (XBX′).sub.nB′ comprises a suitable linker group, wherein X is selected from the group consisting of —NR, —S(O)—, —S(O)O—, —S(O).sub.2—, —S(O).sub.2O—, —C(O)—, —C(S)—, —C(O)O—, C(S)O—, —C(S)S—, —P(O)(R.sub.1)—, and —P(O)(R.sub.1)O—, or is absent; B is selected from the group consisting of C.sub.1-C.sub.10 alkylene, C.sub.2-C.sub.10 alkenylene, C.sub.2-C.sub.10 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.5-C.sub.10 cycloalkenylene, C.sub.3-C.sub.10 heterocycloalkylene, C.sub.5-C.sub.10 heterocycloalkenylene, C.sub.6-C.sub.12 arylene, heteroarylene and C.sub.2-C.sub.10 acyl; X′ is selected from the group consisting of —NR—, —O—, —S—, —Se—, —S—S—, S(O)—, —OS(O)—, OS(O)O—, —OS(O).sub.2, —OS(O).sub.2O—, —S(O)O—, —S(O).sub.2—, —S(O).sub.2O—, —OP(O)(R.sub.1)—, —OP(O)(R.sub.1)O—, —OP(O)(R))OP(O)(R))O—, —C(O)—, —C(S)—, —C(O)O—, C(S)O—, —C(S)S—, —P(O)(R.sub.1)—, —P(O)(R))O—, and ##STR00024## or is absent; wherein E is O, S, Se, NR or N(R).sub.2.sup.+, B′ is selected from the group consisting of C.sub.1-C.sub.10 alkylene, C.sub.2-C.sub.10 alkenylene, C.sub.2-C.sub.10 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.5-C.sub.10 cycloalkenylene, C.sub.3-C.sub.10 heterocycloalkylene, C.sub.5-C.sub.10 heterocycloalkenylene, C.sub.6-C.sub.12 arylene, and heteroarylene or is absent; and wherein each R is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, OR, and C.sub.2-C.sub.10 acyl; R′ is the same as R or two R′ may be taken together with the nitrogen atoms to which they are attached to form a 5 or 6-membered saturated or unsaturated heterocyclic ring; each R.sub.1 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, halo, OR, and N(R).sub.2; each R.sub.2 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl and —C(O)R.sub.5; each R.sub.5 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, C.sub.1-C.sub.10 alkoxy, C.sub.3-C.sub.10 alkenyloxy, C.sub.3-C.sub.10 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.3-C.sub.10 heterocycloalkyloxy, C.sub.5-C.sub.10 heterocycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, heteroaryloxy, C.sub.1-C.sub.10 alkylthio, C.sub.3-C.sub.10 alkenylthio, C.sub.3-C.sub.10 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.3-C.sub.10 heterocycloalkylthio, C.sub.5-C.sub.10 heterocycloalkenylthio, C.sub.6-C.sub.12 arylthio, heteroarylthio, OH, SH and N(R).sub.2; wherein for each instance that B and/or B′ is arylene, the substituents directly attached to the respective arylene rings (including arsenoxide or arsenoxide equivalent) may be in a para-, meta- or ortho-relationship; and wherein each alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, arylene, heteroarylene and acyl may be independently substituted with hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, cyano, cyanate, isocyanate, OR.sub.2a, SR.sub.6, nitro, arsenoxide, —S(O)R.sub.3, —OS(O)R.sub.3, —S(O).sub.2R.sub.3, —OS(O).sub.2R.sub.3, —P(O)R.sub.4R.sub.4, —OP(O)R.sub.4R.sub.4, —N(R″).sub.2, —NRC(O)(CH.sub.2).sub.mQ, —C(O)R.sub.5; ##STR00025## wherein R, R.sub.1 and R.sub.5 are as defined above; and R.sub.2a is selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, —S(O)R.sub.3, —S(O).sub.2R.sub.3, —P(O)(R.sub.4).sub.2, N(R).sub.2 and —C(O)R.sub.5; each R.sub.3 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, C.sub.1-C.sub.10 alkoxy, C.sub.3-C.sub.10 alkenyloxy, C.sub.3-C.sub.10 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.3-C.sub.10 heterocycloalkyloxy, C.sub.5-C.sub.10 heterocycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, heteroaryloxy, C.sub.1-C.sub.10 alkylthio, C.sub.3-C.sub.10 alkenylthio, C.sub.3-C.sub.10 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.3-C.sub.10 heterocycloalkylthio, C.sub.5-C.sub.10 heterocycloalkenylthio, C.sub.6-C.sub.12 arylthio, heteroarylthio and N(R).sub.2, each R.sub.4 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, C.sub.1-C.sub.10 alkoxy, C.sub.3-C.sub.10 alkenyloxy, C.sub.3-C.sub.10 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.3-C.sub.10 heterocycloalkyloxy, C.sub.5-C.sub.10 heterocycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, heteroaryloxy, C.sub.1-C.sub.10 alkylthio, C.sub.3-C.sub.10 alkenylthio, C.sub.3-C.sub.10 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.3-C.sub.10 heterocycloalkylthio, C.sub.5-C.sub.10 heterocycloalkenylthio, C.sub.6-C.sub.12 arylthio, heteroarylthio, halo and N(R).sub.2; R.sub.6 is selected from the group consisting of C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.10 heterocycloalkenyl, C.sub.6-C.sub.12 aryl, heteroaryl, C.sub.1-C.sub.10 alkylthio, C.sub.3-C.sub.10 alkenylthio, C.sub.3-C.sub.10 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.3-C.sub.10 heterocycloalkylthio, C.sub.5-C.sub.10 heterocycloalkenylthio, C.sub.6-C.sub.12 arylthio, heteroarylthio, —S(O)R.sub.3, —S(O).sub.2R.sub.3 and —C(O)R.sub.5; R″ is the same as R or two R″ taken together with the N atom to which they are attached may form a saturated, unsaturated or aromatic heterocyclic ring system; Q is selected from halogen and —OS(O).sub.2Q.sub.1; wherein Q.sub.1 is selected from C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 perfluoroalkyl, phenyl, p-methylphenyl; and m is 1 to 5, n is an integer from 0 to 20 Y comprises at least one arsenoxide or arsenoxide equivalent; p is an integer from 1 to 10, and wherein the compound of formula (I) has more than 6 carbon atoms.
11. The process according to claim 10, wherein A is selected from the group consisting of natural, unnatural and synthetic amino acids, hydrophilic amines, peptides, polypeptides, sugar residues, oligosaccharides, and thiol containing proteins, small acid residues, hydroxyl containing residues, or a combination thereof.
12. The process according to claim 11, wherein said hydrophilic amine is selected from primary alkylamines, primary arylamines, primary aralkylamines, secondary alkylamines, secondary arylamines, secondary aralkylamines, tertiary alkylamines, tertiary arylamines and tertiary aralkylamines, and heterocyclic amines.
13. The process according to claim 11 or 12, wherein A is selected from the group consisting of dipeptides, tripeptides, tetrapeptides, pentapeptides, glutathione, glucosamine, saccharides, disaccharides, oligosaccharides, wherein the sulfur atom of each sulfur containing residue may be optionally oxidised to form a sulfoxide or sulfone.
14. The process according to claim 13, wherein A is selected from a peptide comprising one or more of cysteinylglycine, cysteic acid, aspartic acid, glutamic acid, lysine, and arginine; glucose, fructose, mannose, xylose, lyxose, galactose, hexose, sucrose, sorbose, galactosyl-sucrose, sorbitol, mannitol, and xylitol.
15. The process according to any one of claims 10 to 14, wherein X is selected from the group consisting of —C(O)—, —C(S)—, —C(O)O—, C(S)O—, and —C(S)S—, or is absent; B is selected from the group consisting of C.sub.1-C.sub.5 alkylene, C.sub.2-C.sub.5 alkenylene, C.sub.2-C.sub.5 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.5-C.sub.10 cycloalkenylene, C.sub.6-C.sub.12 arylene and C.sub.2-C.sub.5 acyl; X′ is selected from the group consisting of —O—, —S—, —NR—, —S—S—, —S(O)—, —P(O)(R.sub.1)—, —OP(O)(R.sub.1)—, OP(O)(R.sub.3)O—, —OP(O)(R.sub.1)OP(O)(R.sub.1)O—, —C(O)—, —C(S)—, —C(O)O—, C(S)O—, —C(S)S—, —Se—, ##STR00026## or is absent; wherein E is O, S or N(R).sub.2.sup.+; n is 0, 1 or 2; and B′ is C.sub.1-C.sub.5 selected from the group consisting of alkylene, C.sub.2-C.sub.5 alkenylene, C.sub.2-C.sub.5 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.5-C.sub.10 cycloalkenylene, and C.sub.6-C.sub.12 arylene, or is absent; and wherein each R is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, OR, and C.sub.2-C.sub.10 acyl; R′ is the same as R; each R.sub.1 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, halo, OR.sub.2 and N(R).sub.2; each R.sub.2 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, and —C(O)R.sub.5; each R.sub.5 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.5 alkenyloxy, C.sub.3-C.sub.5 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.5 alkenylthio, C.sub.3-C.sub.5 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.6-C.sub.12 arylthio, OH, SH, and N(R).sub.2; wherein for each instance that B and/or B′ is arylene, the substituents directly attached to the respective arylene rings (including arsenoxide or arsenoxide equivalent), may be in a para-, meta- or ortho-relationship, and wherein each alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and acyl may be independently substituted with hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, cyano, halo, cyanate, isocyanate, OR.sub.2a, SR.sub.6, nitro, arsenoxide, —S(O)R.sub.3, —OS(O)R.sub.3, —S(O).sub.2R.sub.3, —OS(O).sub.2R.sub.3, —P(O)R.sub.4R.sub.4, —OP(O)R.sub.4R.sub.4, —N(R″).sub.2, NRC(O)(CH.sub.2).sub.1nQ, —C(O)R.sub.5, ##STR00027## wherein R, R.sub.1 and R.sub.5 are as defined above; and R.sub.2a is selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, —S(O)R.sub.3, —S(O).sub.2R.sub.3, —P(O)(R.sub.4).sub.2, N(R).sub.2 and —C(O)R.sub.5; each R.sub.3 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.5 alkenyloxy, C.sub.3-C.sub.5 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.5 alkenylthio, C.sub.3-C.sub.5 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.6-C.sub.12 arylthio and N(R).sub.2; each R.sub.4 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.5 alkenyloxy, C.sub.3-C.sub.5 alkynyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.5 alkenylthio, C.sub.3-C.sub.5 alkynylthio, C.sub.3-C.sub.5 cycloalkylthio, C.sub.5-C.sub.5 cycloalkenylthio, C.sub.6-C.sub.12 arylthio, halo and N(R).sub.2; R.sub.6 is independently selected from the group consisting of C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.5 alkenylthio, C.sub.3-C.sub.5 alkynylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.6-C.sub.12 arylthio, —S(O)R.sub.3, —S(O).sub.2R.sub.3 and —C(O)R.sub.5, R″ is the same as R; Q is selected from the group consisting of halogen and —OS(O).sub.2Q.sub.1; wherein Q.sub.1 is selected from C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 perfluoroalkyl, phenyl, p-methylphenyl; m is 1 to 5.
16. The process according to any one of claims 10 to 15, wherein X is absent; B is selected from the group consisting of C.sub.1-C.sub.5 alkylene, C.sub.6-C.sub.12 arylene and C.sub.2-C.sub.5 acyl; X′ is selected from the group consisting of —O—, —S—, —NR—, —S—S—, —S(O)—, —S(O).sub.2—, —P(O)(R.sub.1)—, —C(O)—, —C(S)—, —C(O)O—, C(S)O—, —Se—, and ##STR00028## or absent; wherein E is O, S or N(R).sub.2.sup.+; n is 0, 1 or 2; and B′ is C.sub.1-C.sub.5 alkylene, C.sub.6-C.sub.12 arylene or is absent; and wherein each R is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, OR.sub.2 and C.sub.2-C.sub.5 acyl; R′ is the same as R; each R.sub.1 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, halo, OR.sub.2 and N(R).sub.2; each R.sub.2 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl and —C(O)R.sub.5; each R.sub.5 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.5 alkenyloxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.5-C.sub.10 cycloalkenyloxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.5 alkenylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.5-C.sub.10 cycloalkenylthio, C.sub.6-C.sub.12 arylthio, OH, SH and N(R).sub.2; wherein for each instance that B and/or B′ is arylene, the substituents directly attached to the respective arylene rings (including arsenoxide or arsenoxide equivalent) may be in a para-, meta- or ortho-relationship, and wherein each alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and acyl may be independently substituted with hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, halo, cyano, cyanate, isocyanate, OR.sub.2a, SR.sub.6, nitro, arsenoxide, —S(O)R.sub.3, —OS(O)R.sub.3, —S(O).sub.2R.sub.3, —OS(O).sub.2R.sub.3, —P(O)R.sub.4R.sub.4, —OP(O)R.sub.4R.sub.4, —N(R″).sub.2, —NRC(O)(CH.sub.2).sub.mQ, —C(O)R.sub.5, ##STR00029## wherein R, R.sub.1 and R.sub.5 are as defined above; and R.sub.2a is selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, —S(O)R.sub.3, —S(O).sub.2R.sub.3, —P(O)(R.sub.4).sub.2 and —C(O)R.sub.5; each R.sub.3 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.6-C.sub.12 arylthio and N(R).sub.2; each R.sub.4 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.3-C.sub.10 cycloalkyloxy, C.sub.6-C.sub.12 aryloxy, halo and N(R).sub.2; R.sub.6 is selected from the group consisting of C.sub.1-C.sub.5 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkylthio, C.sub.3-C.sub.10 cycloalkylthio, C.sub.6-C.sub.12 arylthio, —S(O)R.sub.3, —S(O).sub.2R.sub.3 and —C(O)R.sub.5, R″ is the same as R; Q is selected from halogen and —OS(O).sub.2Q.sub.1; wherein Q.sub.1 is selected from C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 perfluoroalkyl, phenyl, p-methylphenyl; and m is 1 to 5.
17. The process according to any one of claims 10 to 16, wherein X is absent; B is selected from the group consisting of C.sub.1-C.sub.5 alkylene, C.sub.6-C.sub.12 arylene and C.sub.2-C.sub.5 acyl; X′ is selected from the group consisting of —O—, —S—, —NR—, —C(O)—, and —C(O)O—, or is absent; n is 1; and B′ is C.sub.1-C.sub.5 alkylene, C.sub.6-C.sub.12 arylene or is absent; and R is selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl and C.sub.2-C.sub.5 acyl; wherein for each instance that B and/or B′ is arylene, the substituents directly attached to the respective arylene rings (including arsenoxide or arsenoxide equivalent), may be in a para-, meta- or ortho-relationship, and wherein each alkylene, arylene, and acyl may be independently substituted with hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.5 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.10 cycloalkenyl, C.sub.6-C.sub.12 aryl, halo, cyano, cyanate, isocyanate, OR.sub.2a, SR.sub.6, nitro, arsenoxide, —S(O)R.sub.3, —S(O).sub.2R.sub.3, —P(O)R.sub.4R.sub.4, —N(R″).sub.2, —NRC(O)(CH.sub.2).sub.mQ, —C(O)R.sub.5, ##STR00030## wherein each R is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl and C.sub.2-C.sub.5 acyl; R.sub.2a is selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, —S(O)R.sub.3, —S(O).sub.2R.sub.3, —P(O)(R.sub.4).sub.2 and —C(O)R.sub.5; each R.sub.3 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, and C.sub.6-C.sub.12 arylthio; each R.sub.4 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.6-C.sub.12 aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.6-C.sub.12 arylthio, halo and N(R).sub.2; each R.sub.5 is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, C.sub.6-C.sub.12, aryloxy, C.sub.1-C.sub.5 alkylthio, C.sub.6-C.sub.12 arylthio, OH, SH and N(R).sub.2; R.sub.6 is selected from the group consisting of C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkylthio, C.sub.6-C.sub.12 arylthio, —S(O)R.sub.3, —S(O).sub.2R.sub.3 and —C(O)R.sub.5, R″ is the same as R above; Q is selected from halogen and —OS(O).sub.2Q.sub.1; wherein Q.sub.1 is selected from C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 perfluoroalkyl, phenyl, p-methylphenyl; and m is, 2, 3, 4, or 5.
18. The process according to any one of claims 10 to 17, wherein X is absent; B is C.sub.2-C.sub.5 acyl; X′ is NR; n is 1; B′ is phenylene; and R is H; wherein the substituents directly attached to the phenylene ring may be in a para-, meta- or ortho-relationship.
19. The process of claim 18, wherein said compound is: ##STR00031## wherein R.sub.7 to R.sub.10 are independently selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, halogen, hydroxy, amino, nitro, carboxy, C.sub.1-C.sub.5 alkoxy, —OS(O).sub.2R.sub.3 and —NHC(O)CH.sub.2Q wherein Q is halogen, —OS(O).sub.2CH.sub.3, —OS(O).sub.2C.sub.6H.sub.5 and —OS(O).sub.2-p tolyl; and wherein, when any one of R.sub.7 to R.sub.10 is C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, —OS(O).sub.2R.sub.3 it is capable of forming a fused ring with the phenylene; and further wherein, at least one of R.sub.7 to R.sub.10 is C.sub.1-C.sub.5 alkyl, C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.5 alkoxy, or —OS(O).sub.2R.sub.3, in combination with at least any one other of R.sub.7 to R.sub.10, is capable of forming a fused ring with the phenylene.
20. The process of claim 19, wherein R.sub.7 to R.sub.10 are independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, nitro, cyano, carboxy, C.sub.1-C.sub.5 alkoxy, methyl, ethyl, isopropyl, tert-butyl, phenyl and —NHC(O)CH.sub.2Q wherein Q is halogen, —OS(O).sub.2CH.sub.3, —OS(O).sub.2C.sub.6H.sub.5 and —OS(O).sub.2-p tolyl.
21. The process of claim 18 or 19, wherein the arsenoxide (—As═O) group is at the 4-position of the phenylene ring.
22. The process of any one of claims 1 to 21 wherein the compound is selected from the group consisting of: ##STR00032## ##STR00033## ##STR00034##
23. The process of any one of claims 1 to 18 wherein the compound is represented by Formula (VI): ##STR00035## wherein G is selected from the group consisting of: hydrogen, halogen, hydroxy, amino, nitro, carboxy, C.sub.1-C.sub.5 alkoxy, C.sub.1-C.sub.5 alkyl and C.sub.6-C.sub.12 aryl and —NHC(O)CH.sub.2Q wherein Q is halogen, —OS(O).sub.2CH.sub.3, —OS(O).sub.2C.sub.6H.sub.5 or —OS(O).sub.2-p tolyl.
24. The process of claim 23, wherein G is selected from the group consisting of: hydrogen, halogen, hydroxy, amino, nitro, carboxy, C.sub.1-C.sub.5 alkoxy, methyl, ethyl, iso-propyl, tert-butyl, phenyl, and —NHC(O)CH.sub.2Q wherein Q is halogen, —OS(O).sub.2CH.sub.3, —OS(O).sub.2C.sub.6H.sub.5 or —OS(O).sub.2-p) tolyl.
25. The process of claim 23 or 24, wherein G is selected from the group consisting of hydroxy, fluorine, amino, and nitro.
26. The process according to any one of claims 1 to 9, wherein the compound is represented by formula (VI): ##STR00036## wherein the As(OH).sub.2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R.sup.1 is selected from hydrogen and C.sub.1-3 alkyl; R.sup.2 and R.sup.3 may be the same or different and are independently selected from hydrogen, optionally substituted C.sub.1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C.sub.2-3 alkenyl; and optionally substituted C.sub.1-3 alkoxy; R.sup.4 and R.sup.5 may be the same or different and are independently selected from hydrogen, optionally substituted C.sub.1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C.sub.2-3 alkenyl; and optionally substituted C.sub.1-3 alkoxy; m is an integer selected from 1, 2 and 3; n is an integer selected from 1, 2 and 3; * indicates a chiral carbon atom; and salts, hydrates, enantiomers and racemates thereof.
27. The process according to claim 26, wherein the As(OH).sub.2 group is ortho- or para- to the N-atom on the phenyl ring.
28. The process according to claim 26 or 27, wherein R.sup.1 is selected from hydrogen, methyl and ethyl.
29. The process according to any one of claims 26 to 28 wherein R.sup.1 is hydrogen.
30. The process according to any one of claims 26 to 29, wherein R.sup.2 and R.sup.3 are independently selected from hydrogen, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.1-3 alkoxy, halo-(C.sub.1-3)alkoxy, hydroxy(C.sub.1-3)alkyl and halo(C.sub.1-3)alkyl.
31. The process according to any one of claims 26 to 30, wherein R.sup.2 and R.sup.3 are independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, hydroxymethyl, CF.sub.3 and OCF.sub.3.
32. The process according to any one of claims 26 to 31, wherein R.sup.2 and R.sup.3 are independently selected from hydrogen, methyl and ethyl.
33. The process according to any one of claims 26 to 32 wherein R.sup.2 is methyl and R.sup.3 is hydrogen.
34. The process according to any one of claims 26 to 32, wherein R.sup.2 and R.sup.3 are both hydrogen.
35. The process according to any one of claims 26 to 34, wherein R.sup.4 and R.sup.5 are independently selected from hydrogen, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.1-3 alkoxy, halo-(C.sub.1-3)alkoxy, hydroxy-(C.sub.1-3)alkyl and halo(C.sub.1-3)alkyl.
36. The process according to any one of claims 26 to 35, wherein R.sup.4 and R.sup.5 are independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, hydroxy-(C.sub.1-3)alkyl, CF.sub.3 and OCF.sub.3.
37. The process according to any one of claims 26 to 36, wherein R.sup.4 and R.sup.5 are independently selected from hydrogen, methyl, ethyl and hydroxymethyl.
38. The process according to any one of claims 26 to 37, wherein R.sup.4 is hydrogen, methyl or ethyl and R.sup.5 is hydrogen.
39. The process according to any one of claims 26 to 38, wherein R.sup.4 is methyl and R.sup.5 is hydrogen.
40. The process according to any one of claims 26 to 38, wherein R.sup.4 and R.sup.5 are both hydrogen.
41. The process according to any one of claims 26 to 38, wherein R.sup.4 and R.sup.5 are both methyl.
42. The process according to claim 26, wherein the As(OH).sub.2 group is ortho- or para- to the N-atom on the phenyl ring; R.sup.1 is hydrogen or methyl; R.sup.2 and R.sup.3 are independently selected from hydrogen, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.1-3 alkoxy, halo-(C.sub.1-3)alkoxy, hydroxy(C.sub.1-3)alkyl and halo(C.sub.1-3)alkyl; R.sup.4 and R.sup.5 are independently selected from hydrogen, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.1-3 alkoxy, halo(C.sub.1-3)alkoxy, hydroxy(C.sub.1-3)alkyl and halo(C.sub.1-3)alkyl; m is 1 or 2; and n is 1 or 2.
43. The process according to claim 26, wherein the As(OH).sub.2 group is ortho- or para- to the N-atom on the phenyl ring; R.sup.1 is hydrogen or methyl; R.sup.2 and R.sup.3 are independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, CH.sub.2OH, CF.sub.3 and OCF.sub.3; R.sup.4 and R.sup.5 are independently selected from hydrogen, methyl, ethyl, CH.sub.2OH, methoxy, vinyl, CF.sub.3 and OCF.sub.3; m is 1; and n is 1.
44. The process according to claim 26, wherein the As(OH).sub.2 group is para- to the N-atom on the phenyl ring; R.sup.1 is hydrogen or methyl; R.sup.2 and R.sup.3 are independently selected from hydrogen, methyl and ethyl; R.sup.4 and R.sup.5 are independently selected from hydrogen, methyl and ethyl; m is 1; and n is 1.
45. The process according to claim 26, wherein the As(OH).sub.2 group is para- to the N-atom on the phenyl ring; R.sup.1 is hydrogen or methyl; R.sup.2 is hydrogen or methyl; R.sup.3 is hydrogen or methyl; R.sup.4 is hydrogen, methyl or ethyl; R.sup.5 is hydrogen or methyl; m is 1; and n is 1.
46. The process according to claim 26, wherein the As(OH).sub.2 group is para- to the N-atom on the phenyl ring; R.sup.1 is hydrogen; R.sup.2 is hydrogen or methyl; R.sup.3 is hydrogen; R.sup.4 hydrogen or methyl; R.sup.5 is hydrogen or methyl; in is 1; and n is 1.
47. The process according to claim 26, wherein the compound of formula (VI) is: ##STR00037## and salts, hydrates, enantiomers and racemates thereof.
48. The process according to claim 26, wherein the compound of formula (VI) is: ##STR00038## and salts, hydrates, enantiomers and racemates thereof.
49. The process according to claim 47 or 48, wherein the stereochemistry at the chiral carbon denoted * is (S), and salts and hydrates thereof.
50. The process of any one of claims 10 to 49, wherein the arsenoxide group (—As═O) is replaced by an arsenoxide equivalent as defined herein.
51. The process of claim 50, wherein the arsenoxide equivalent is any dithiol reactive species that shows essentially the same affinity towards dithiols as —As═O.
52. A process for identifying a compound which inhibits formation of procoagulant platelets, wherein said process comprises contacting a sample comprising platelets with a compound and determining whether the compound inhibits formation of procoagulant platelets.
53. A process for screening a plurality of compounds to identify a compound which selectively inhibits formation of procoagulant platelets, wherein said process comprises contacting a sample comprising platelets with the plurality of compounds, determining whether any of the compounds inhibit formation of procoagulant platelets, and if so, separately determining for each of the plurality of compounds whether the compound inhibits formation of procoagulant platelets.
54. The process according to claim 52 or 53, wherein inhibition of formation of procoagulant platelets occurs by inhibiting the mitochondrial necrosis pathway.
55. The process according to any one of claims 52 to 54, wherein inhibition of formation of procoagulant platelets is determined by comparing the proportion of procoagulant platelets before contacting the platelets with the compound with the proportion of procoagulant platelets after contacting the platelets with the compound.
56. The process according to claim 55, wherein the proportion of procoagulant platelets is determined by the process of any one of claims 1 to 51.
57. The process according to any one of claims 1 to 51, wherein the platelet is a P-selectin+ platelet.
58. The process according to any one of claims 52 to 56, wherein the platelets are P-selectin+ platelets.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DESCRIPTION OF EMBODIMENTS
[0217] The present invention relates to a process for identifying procoagulant platelets in vitro or in vivo, and the identification of compounds which selectively inhibit formation of procoagulant platelets.
[0218] The concept of the procoagulant platelet is central to the current model of hemostasis postulated by the inventors. It localizes coagulation activation and fibrin formation to the activated platelet clot. Until now, it has been difficult to directly measure the functional contribution of procoagulant platelets to clot formation due to the lack of an effective marker that can be used in vitro and in vivo. Other markers include annexin V, mitochondrial membrane dyes, Factor Va staining and direct morphological visualisation by differential interference contrast microscopy. These have been problematic for a number of reasons including the imperfect correlation between all of these markers and the procoagulant population. Furthermore, function blocking probes such as annexin V and lactadherin may perturb the system that is being measured.
[0219] The present inventors have demonstrated that arsenoxide compounds, such as GSAO, specifically, rapidly and stably marks procoagulant necrotic platelets in vitro and in vivo and does not affect their function.
[0220] Using GSAO the inventors were able to show that necrotic platelets are generated within a few seconds of agonist stimulation in vitro and are procoagulant. They are also generated in vivo during formation of occlusive murine thrombi and provide a procoagulant surface. In addition, analysis of platelets from human subjects receiving aspirin indicates that procoagulant necrotic platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway.
[0221] Arsenoxide compounds, such as GSAO, have several features that are particularly useful in a platelet necrosis marker. They complex with cytoplasmic proteins rather than DNA, the binding is independent of calcium and the interaction is covalent so the compound does not wash out. It also detects platelet membrane compromise within a few seconds and concentrates in the cytoplasm, which results in an exceptional signal to noise ratio. Importantly, arsenoxide compounds, such as GSAO, do not perturb measures of platelet function or coagulation in vitro or in vivo. In addition, an inert control compound, GSCA, provides for a high level of confidence with respect to specificity of GSAO labeling. Interestingly, the primary ligand identified in activated platelets, thromboxane A synthase-1, is a key regulator of haemostasis via the arachidonic acid pathway. This contrasts with the major ligand in necrotic mammalian cancer cells, heat shock protein-90.
[0222] In current models of hemostasis, the platelet surface provides a link between primary haemostasis and the localisation of coagulation and fibrin formation. Without the latter, bleeding complications occur, as evidenced by patients with Scott syndrome, a platelet function defect characterised by the failure to externalise phosphatidylserine and hence, failure to provide a surface for prothrombinase. The thrombogram profile in a study of cyclosporine treated platelets by the inventors mimicked that seen in Scott syndrome platelets with a significant decrease in peak thrombin and a lesser decrease in endogenous thrombin potential. However, while there is agreement with regards the functional importance of the ‘procoagulant’ platelet phenotype, there remains much debate about its identity.
[0223] The inventors have discovered that arsenoxide compounds, such as GSAO, in conjunction with the α-granule marker, P-selectin, can identify a population of platelets with features of necrosis that have a functionally procoagulant phenotype. The GSAO+ population (i.e. the population capable of biding to GSAO) has properties of the coated platelet, including high fibrinogen binding, and sustained high level intracellular calcium, which is characteristic of SCIP platelets. In addition, GSAO+ platelets undergo cyclophilin D-dependent opening of the mitochondrial permeability transition pore, which are the platelets described by Jobe and colleagues (Jobe et al., 2008). The time course studies indicate that the appearance of the necrotic platelet occurs within a few seconds after agonist stimulation. Indeed, a transition from the activated platelet to the necrotic platelet was not apparent. This suggests that platelets going down the necrotic pathway are committed early after stimulation and less likely to be merely the end stage of prolonged exposure to strong agonists.
[0224] The formation of necrotic platelets within the developing thrombus has been visualized in real time in murine arterioles. The necrotic platelets are spatially associated with sites of fibrin formation in the platelet aggregate, which implies that they are providing a procoagulant surface during thrombus formation. Furthermore, not all agonists initiating formation of the platelet aggregate result in platelet necrosis. It is interesting that the model resulting in an occlusive thrombus was associated with platelet necrosis, while very few necrotic platelets were generated in the non-occlusive thrombus model. It may be that necrotic platelets tip the balance from haemostatic thrombus formation to pathological occlusive thrombosis.
[0225] The use of a cell death imaging agent that complexes with cytoplasmic ligands, such as GSAO, to identify the apoptotic/necrotic cells in vitro and in vivo has previously been described (Park et al., 2011; Park et al., 2013; Xie et al., 2013). The in vitro studies were performed exclusively using washed human platelets. Methods based on washed platelets are very useful for defining mechanisms in in vitro studies, however a number of factors reduces the utility of this method in translational research (population based studies). Firstly, to isolate platelets, a relatively large volume of blood is needed, which can be particularly problematic when studying small rodent species (models of disease). Secondly, the preparation of washed platelets is a labor-intensive and time-consuming process that involves multiple washing and centrifugation steps. Technical expertise and constant practice are required to prevent introduction of processing artefacts. In addition, in washed platelet-based assays platelets are studied in a non-physiological matrix, and in the absence of other plasma and cellular factors, which are known to modulate platelet responses.
[0226] For example, platelets and leukocytes, primarily neutrophils and monocytes, feature a number of well-defined adhesion molecules (P-selectin/P-selectin glycoprotein ligand 1, GPlbα/αMβ2) and soluble factors (PF4-RANTES, CD40L TXA2), which enable a productive two-way communication. The cross-talk between platelets and leukocytes is precisely regulated and facilitates platelet and neutrophil activation/adhesion important in inflammation, hemostasis and thrombosis. Dysregulation of platelet-leukocyte interactions has been implicated in vascular injury in atherothrombosis. Potential pathogenetic role of dysregulated platelet-leukocyte collaboration has also been described in a number of disease models including deep vein thrombosis, immune mediated vasculitis, sickle cell disease, inflammatory bowel disease, acute lung injury and sepsis.
[0227] In one embodiment, the present invention provides a method to detect a procoagulant platelet subpopulation in whole blood using a novel marker of cell death and a rapid, flow cytometry-based assay. A notable advantage of this method is that it can be performed using as little as 100 μL, of whole blood without the need for specific technical skills, making it ideal for gaining new insights into the role of procoagulant platelets in health and disease. An innovation of the method is the use of an arsenoxide (or arsenoxide equivalent) compound, which in conjunction with the α-granule marker, P-selectin, can identify a population of platelets with features of necrosis that have a functionally procoagulant phenotype. Furthermore, this assay replicates native conditions more closely where platelets can productively interact with leukocytes and other constituents of whole blood.
[0228] The whole blood platelet necrosis assay was validated by measuring formation of procoagulant platelets in response to well-known agonist in humans and mice. The assay was able to reproducibly measure platelet propensity to necrosis in healthy human volunteers as well as in patients with ischaemic heart disease within a clinical setting. Consistent with previous reports, dual stimulation with thrombin and collagen resulted in the largest procoagulant platelet subpopulation, however the magnitude of this subpopulation was increased in patients compared to healthy volunteers. Furthermore, treatment with aspirin did not affect the necrotic platelet propensity, while combined treatment with aspirin and a P2Y12 inhibitor resulted in a blunted necrotic propensity in coronary artery disease patients after thrombin or thrombin/collagen stimulation.
[0229] As proof of principle the ability of bacteria to induce platelet necrosis in whole blood ex vivo was investigated. A methicillin resistant S. aureus strain (USA 300) enhanced the formation of procoagulant platelet subpopulation in response to thrombotic stimuli. This observation is in keeping with epidemiological evidence of increased risk of thrombosis in sepsis, and may be mediated by FcγRIIA, which is a key to platelet aggregation in response to bacteria, as well as a factor in hypercoagulability associated with systemic lupus erythematosus.
[0230] Accordingly, the whole blood platelet necrosis assay using arsenoxide (or arsenoxide equivalent) compounds is a favorable tool for detecting necrotic, procoagulant platelets rapidly and reproducibly by flow cytometry. It offers unprecedented potential for widespread application in studying the role of platelets in cardiovascular disease, venous thromboembolism, and other prothrombotic states such as malignancy, sepsis and autoimmune disorders.
[0231] Further advantages of the flow cytometry-based assay for detection of platelet necrosis in whole blood (WBPN) include a dramatic reduction in the volume of blood required, improvement of throughput, and enablement of the study of the behavior of platelets and interactions between platelets and leukocytes within a physiological matrix. These improvements make it more feasible to investigate potential pathological roles of necrotic platelets in large-scale clinical setting and will permit repeated measurement of the same animal resulting in less variability and reduction in the number of animals sacrificed. The application of the technique to quantifying platelet propensity to necrosis in humans and mice in several settings including cardiovascular and infectious disease is also demonstrated.
[0232] Disclosed herein is a process for identifying procoagulant platelets, comprising contacting a platelet with a compound and determining whether the compound binds to the platelet, wherein the compound is an arsenoxide (or arsenoxide equivalent) compound. The platelet may be a necrotic platelet, for example a P-selectin+ platelet. The platelet may not be an apoptotic platelet.
[0233] Also disclosed herein is a process for identifying a patient at risk of developing a prothrombotic or thrombotic condition, comprising contacting a platelet from said patient with a compound and determining whether the compound binds to the platelet, wherein the compound is an arsenoxide (or arsenoxide equivalent) moiety. The platelet may be a necrotic platelet, for example a P-selectin+ platelet. The platelet may not be an apoptotic platelet.
[0234] Platelets positive for P-selectin expression can be identified using standard techniques in the art (e.g. FACS, immunohistochemistry, and the like).
[0235] Arsenoxide (or arsenoxide equivalent) compounds used in accordance with the present invention and uses thereof have been disclosed in WO 01/21628, WO 02/74305, WO 03/39564, WO 04/42079, WO 08/052279 and WO 09/43114, the entire contents of which are incorporated herein by cross-reference.
[0236] Compounds used in accordance with the invention may themselves be labelled with a reporter compound, including hydrophilic fluorophores, a biotin label or radioisotopes. For example, fluorescent labelled compounds can be used or a fluorophore can be attached to the compound. Examples of suitable fluorophores include fluorescein and Cy™5.5. Sub-cellular localisation of compounds can be detected by standard techniques including confocal fluorescence microscopy. Alternatively, compounds can be attached to other detectable groups, eg biotin, and detected by staining with streptavidin, such as Streptavidin-Alexa Fluor 488.
[0237] A compound used in accordance with the invention may be administered in an amount effective to detect the presence of procoagulant platelets using standard imaging techniques known to those in the art. For example, such imaging techniques may include: single-photon emission computed tomography (SPECT), positron emission tomography (PET) and magnetic resonance imaging (MRI).
[0238] In a preferred embodiment, a compound used in accordance with the invention is administered by intravenous injection.
[0239] In one embodiment, the absolute number of procoagulant platelets in a given volume of blood is determined. The number of procoagulant platelets in the given volume of blood may be compared to the other platelets in the given volume. The relative number of procoagulant platelets may be indicative of a prothrombotic or thrombotic condition.
[0240] Also disclosed herein is a process for identifying a compound which inhibits formation of procoagulant platelets, wherein said process comprises contacting a sample comprising platelets with a compound and determining whether the compound inhibits formation of procoagulant platelets.
[0241] Further disclosed herein is a process for screening a plurality of compounds to identify a compound which selectively inhibits formation of procoagulant platelets, wherein said process comprises contacting a sample comprising platelets with the plurality of compounds, determining whether any of the compounds inhibit formation of procoagulant platelets, and if so, separately determining for each of the plurality of compounds whether the compound inhibits formation of procoagulant platelets.
[0242] Monitoring inhibition of formation of procoagulant platelets can be done using standard techniques known to those in the art. For example, such imaging techniques may include: single-photon emission computed tomography (SPECT), positron emission tomography (PET) and magnetic resonance imaging (MRI).
[0243] The necrotic platelet has implications for potential therapeutic intervention in thrombotic diseases. Ex vivo analysis of platelets from human subjects on aspirin therapy indicates that procoagulant necrotic platelets form despite aspirin therapy, but their numbers are reduced by inhibition of the mitochondrial necrosis pathway.
[0244] Blocking the procoagulant platelet is attractive in thrombosis, as it would not affect the platelet aggregate required for haemostasis, and unlike systemic anticoagulant therapy, the effect would be localised to those lesions that are more likely to be pathological. For example, patients that have persistent ischaemic events on anti-platelet therapy may benefit. In addition, testing peripheral blood for propensity to generate necrotic platelets may be a useful biomarker for prediction of recurrent ischaemic events, particularly for patients already on aspirin therapy.
[0245] Prothrombotic or thrombotic conditions may be venous or arterial in nature. These conditions may occur in mammals, including humans.
[0246] Examples of prothrombotic or thrombotic conditions include: infection, sepsis, systemic inflammatory response syndrome, multi organ failure, thrombotic thrombocytopenia purpura, haemolytic uraemia syndrome, vascularisation, renal failure, ischaemic repercussion injury, solid organ transplant rejection, cardiovascular disease, stroke, venous thromboembolism, autoimmune disorders, sickle cell disease, inflammatory bowel disease, acute lung injury, malignancy, myocardial infarction (primary and secondary), embolic stroke, ischaemic stroke, thrombotic stroke, deep vein thrombosis (DVT), thromboembolism, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, cerebral venous sinus thrombosis, arterial thrombosis and arterial embolis.
[0247] The present invention may also find use in identifying compounds which prevent thrombosis formation in the following situations: artificial/prosthetic vascular shunts and grafts; coronary stenting; prosthetic heart valves; cardiopulmonary bypass procedures; haemoperfusion and haemodialysis; venous thromboembolic disease following surgery, peripheral arterial disease; percutaneous transluminal coronary angioplasty, during pregnancy; during immobilization; after carotid surgery; cerebrovascular accidents such as transient ischaemic; progression of atherosclerosis; ischaemic conditions.
[0248] Whilst the arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention may be administered alone, it is generally preferable that the compound be administered as a pharmaceutical composition/formulation. In general pharmaceutical formulations may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.
[0249] The carriers, diluents and adjuvants must be “acceptable” in terms of being compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
[0250] Single or multiple administration of the arsenoxide (or arsenoxide equivalent) compounds used in accordance with the invention, or pharmaceutical compositions comprising said compounds, can be carried out with dose levels and pattern being selected by a physician. Regardless, the compounds or pharmaceutical compositions used in accordance with the present invention should be administered so as to provide a quantity of the compound sufficient to identify and/or quantify the presence of procoagulant platelets.
[0251] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of the arsenoxide (or arsenoxide equivalent) compounds or pharmaceutical compositions used in accordance with the invention which would be required to identify and/or quantify procoagulant platelets. Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750 mg per kg body weight per 24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250 mg per kg body weight per 24 hours; about 1.0 mg to about 250 mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.0 mg to about 200 mg per kg body weight per 24 hours; about 1.0 mg to about 100 mg per kg body weight per 24 hours; about 1.0 mg to about 50 mg per kg body weight per 24 hours; about 1.0 mg to about 25 mg per kg body weight per 24 hours; about 5.0 mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about 20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg per kg body weight per 24 hours.
[0252] Alternatively, an effective dosage may be up to about 500 mg/m.sup.2. Generally, an effective dosage is expected to be in the range of about 25 to about 500 mg/m.sup.2, preferably about 25 to about 350 mg/m.sup.2, more preferably about 25 to about 300 mg/m.sup.2, still more preferably about 25 to about 250 mg/m.sup.2, even more preferably about 50 to about 250 mg/m.sup.2, and still even more preferably about 75 to about 150 mg/m.sup.2.
[0253] In relation to arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention, an effective dosage is in the range of about 0.0001 mg to about 100 mg GSAO per kg body weight per 24 hours, preferably about 0.001 mg to about 100 mg GSAO per kg body weight per 24 hours, more preferably about 0.01 mg to about 50 mg GSAO per kg body weight per 24 hours, even more preferably about 0.1 mg to about 20 mg GSAO per kg body weight per 24 hours, even more preferably still about 0.1 mg to about 10 mg GSAO per kg body weight per 24 hours.
[0254] Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages of a compound used in a system of the present invention will be determined by the form, route and site of administration, and the nature of the patient. Also, such optimum conditions can be determined by conventional techniques.
[0255] Delayed release formulations are also included in the scope of the present invention.
[0256] Further, it will be apparent to one of ordinary skill in the art that the optimal quantity of a compound used in accordance with the present invention will be determined by the form, route and site of administration, and the nature of the particular patient. Also, such optimum conditions can be determined by conventional techniques.
[0257] Typically, salts of arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the compound or of the pharmaceutically acceptable salt thereof. By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.
[0258] For instance, suitable pharmaceutically acceptable salts of the arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically acceptable salts of the arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention therefore include acid addition salts.
[0259] For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
[0260] Also included within the scope of compounds useful in the processes of the present invention are prodrugs. Typically, prodrugs will be functional derivatives of the compounds used in the present invention which are readily converted in vivo to the required (active) compounds as used in the processes of the invention as active agents, such as therapeutic and/or diagnostic agents. Typical procedures for the selection and preparation of prodrugs are known to those of skill in the art and are described, for instance, in H. Bundgaard (Ed), Design of Prodrugs, Elsevier, 1985.
[0261] Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.
[0262] The pharmaceutical compositions representing a component of the processes of the invention may be administered by standard routes. In general, the compositions may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. Still generally, the compositions representing a component of the process of the invention may be in the form of a capsule suitable for oral ingestion, in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation.
[0263] For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2-propylene glycol.
[0264] Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin.
[0265] In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration of the capsule.
[0266] Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
[0267] Solid forms for oral administration may contain binders acceptable in pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
[0268] Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides, or mixtures thereof
[0269] Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate, and the like.
[0270] The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above, or natural gums such as guar gum, gum acacia or gum tragacanth.
[0271] The compositions for parenteral administration will commonly comprise an arsenoxide (or arsenoxide equivalent) compounds used in accordance with the processes of the present invention or a cocktail thereof dissolved in an acceptable carrier, such as water, buffered water, 0.4% saline, and 0.3% glycine etc, wherein such solutions are sterile and relatively free of particulate matter.
[0272] Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.
[0273] The invention will now be described in greater detail by reference to specific Examples which should not be construed as in any way limiting the scope of the invention.
EXAMPLES
Example 1—Rationale for the Platelet Necrosis Marker
[0274] GSAO is a tripeptide trivalent arsenical that has recently been characterized as an imaging agent for necrotic and late apoptotic nucleated cells (Park et al., 2011; Park et al., 2013; Xie et al., 2013) (
[0275] GSAO marking of dying/dead nucleated cells is compatible with a number of different reporter groups, including hydrophilic fluorophores, a biotin label or radioisotopes (Park et al., 2011; Park et al., 2013; Xie et al., 2013). Conjugates of GSAO with AF647, AF546 or Oregon Green were employed in this study. A GSAO control compound, GSCA (4-(N—((S-glutathionyl) acetyl)amino)benzoic acid), contains an inert carboxylic acid group in place of the chemically reactive As(III) in GSAO. GSCA has the same biodistribution as GSAO but does not react with proteins so washes out of cells.
Example 2—GSAO Labels a Subpopulation of Activated Platelets
[0276] Calcium ionophore treatment of platelets leads to a sustained rise in intracellular calcium and surface elaboration of phosphatidylserine, which are features of both the procoagulant and necrotic phenotype (Bevers et al., 1982). Washed human platelets were treated with calcium ionophore and labeled with GSAO-AF647 and an antibody against the α-granule marker, P-selectin. GSAO-AF647 was consistently retained in a subpopulation of platelets that coexpress P-selectin (
[0277] Dual stimulation with thrombin and collagen is known to be superior to either agonist alone in the generation of the procoagulant platelet (Schoenwaelder et al., 2009; Jobe et al., 2008; Fager et al., 2010). To determine whether GSAO labeling reflects this agonist profile, washed human platelets were stimulated with thrombin, collagen, or both thrombin and collagen. Thrombin generated minimal GSAO+ platelets, collagen stimulation resulted in a moderate proportion, while simultaneous stimulation with thrombin and collagen resulted in a marked increase in GSAO+ platelets (
Example 3—GSAO Rapidly Enters the Activated Platelet Subpopulation Via an Organic Anion-Transporting Polypeptide
[0278] The rate of GSAO+ platelet generation after agonist exposure was measured using time lapse flow cytometry. Results are reported for GSAO+ events based on lead in time prior to agonist exposure threshold. Resting platelets showed no increase in GSAO labeling over 10 min. Platelets exposed to thrombin demonstrate little GSAO labeling compared with unstimulated platelets. Collagen alone resulted in a gradual increase in GSAO labeling over 60 sec, while combination thrombin and collagen stimulation resulted in a rapid initial rise in GSAO labeling that peaked in 12 sec and was sustained over the 10 min of the experiment (
[0279] The organic anion-transporting polypeptide (OATP) family has been implicated in transport across the plasma membrane of organic anions in the same class as GSAO (Dilda et al., 2009; Dilda et al., 2008). Nine members of the OATP family have been reported in humans (Hagenbuch et al., 2003) and platelets express OATP2B1 (Niessen et al., 2009). DIDS is an inhibitor of OATP class B transporters (Kobayashi et al. 2003) and the present inventors tested its effect on GSAO labelling of agonist-stimulated platelets. DIDS inhibited GSAO labelling of P-selectin+ activated platelets (
Example 4—Labeling with GSAO and Exposure of P-Selectin Distinguishes Necrotic from Apoptotic Platelets
[0280] Bcl-X.sub.L dependent apoptosis is involved in regulation of platelet life span, although the role of platelet apoptosis in thrombosis is controversial. In order to determine if GSAO can differentiate agonist-induced necrotic versus apoptotic platelets, platelet apoptosis was triggered using the BH3 mimetic ABT-737. GSAO labelled P-selectin negative apoptotic platelets (
Example 5-GSAO+/P-Selectin+ Procoagulant Platelets have Features Consistent with Cyclophilin D-Dependent Regulated Necrosis, not Apoptosis
[0281]
TABLE-US-00001 GSAO+/P- Features Apoptosis Necrosis selectin+ platelet Membrane integrity Intact in early stages Early loss of Loss of membrane and loss in late stages membrane integrity integrity Initiating stimulus Ratio of Bcl-X.sub.L External ligands Physiological expression levels agonists Structural Shrinkage and Cellular swelling Cellular swelling morphology microparticle blebbing Phosphatidylserine Present Present Present exposure Effect of calcium No inhibition Inhibition Inhibition chelation Active caspases Dependent on Not dependent on Not dependent on execution of caspases caspases, but may be caspases, but may present be present Formation of mPTP Not characteristic An early event An early event Loss of Δψ.sub.m A late event An early event An early event Genetic ablation of Bax/Bak knockout CypD knockout CypD knockout factors RIP3 knockout Drug inhibition Caspase inhibitors Cyclosporine A Cyclosporine A
Example 6—GSAO+/Pselectin+ Marks Platelets Undergoing Cyclophilin D-Dependent Necrosis
[0282] The cyclophilin D-dependent regulated necrosis pathway is characterized by agonist stimulation, raised intracellular calcium, formation of the mitochondrial transition pore and loss of mitochondrial membrane potential. Dual agonist stimulation of platelets results in loss of mitochondrial transmembrane potential (
[0283] Cyclophilin D is involved in formation of the mitochondrial permeability transition pore, and inhibition of this protein blunts pore formation and necrosis (Nakagawa et al., 2005). Inhibiting cyclophilin D with cyclosporine A reduced GSAO labeling and procoagulant potential of agonist stimulated platelets (
Example 7—GSAO+ Platelets Form in Occluding Murine Thrombi and are Attenuated with Megakaryocyte Directed Deletion of the Cyclophilin D Gene
[0284] It is important that GSAO not interfere with thrombus formation for it to be used as an in vivo marker of procoagulant necrotic platelets. GSAO-AF647 or unconjugated GSAO does not trigger platelet apoptosis or necrosis (
[0285] Two methods of initiation of thrombosis in the murine cremaster arteriolar circulation were compared and contrasted to explore the functional relevance of GSAO+ platelets in vivo: (i) the endothelial stimulation laser injury model that is primarily dependent on thrombin for thrombus formation and not dependent on extravascular collagen exposure (Falati et al., 2002; Dubois et al., 2007), and (ii) the FeCl.sub.3 chemical injury model (Dubois et al., 2006). Dylight-conjugated anti-platelet CD42b antibody, GSAO-Oregon Green, GSAO-AF546, GSAO-AF647 or appropriately labelled control compound GSCA were introduced to the murine circulation and arterioles injured and thrombi imaged within 10-30 min. Thrombus formation was imaged in real time by three laser confocal microscopy and 2D orthogonal sections and 3D-high resolution images were reconstructed. GSAO-AF750 is detectable in the murine circulation up to 3 h after intravenous injection and persists within necrotic lesions for at least 6 h (Xie et al., 2013), so thrombi are imaged well before GSAO is cleared from the circulation. A 3000 Da dextran tracer has been shown to permeate the shell and core of laser-induced platelet thrombi (Vonorov et al., 2013; Stalker et al., 2013). GSAO-fluorophore has a molecular mass of ˜1500 Da and is expected to readily diffuse within thrombi.
[0286] GSAO-Oregon Green labelled a subset of platelets within thrombi induced by FeCl.sub.3 injury (
Example 8—GSAO Marks Functionally Procoagulant Platelets
[0287] The relationship between the activated and procoagulant phenotype of platelets remains controversial (Dale et al., 2002; Abaeva et al., 2013). Procoagulant platelets are characterised by presence of phosphatidylserine on the platelet surface, surface binding of plasma derived activated clotting factors, ability to generate thrombin and promotion of fibrin formation. Regardless of the agonist stimulus, 100% of GSAO+ platelets were positive for α-granule P-selectin, which implies that α-granule release is a prerequisite for GSAO labeling after agonist stimulation (
[0288] The synthetic covalent inhibitor of Factor Xa (FXa), FITC-labeled Glu-Gly-Arg chloromethyl ketone (GGACK-FITC), was used to detect this activated coagulation factor on the platelet surface. There was a high correlation (χ.sup.2=15793, p<0.000) between GSAO labeling and FXa on the agonist-activated platelet surface (
[0289] To determine if GSAO+ platelets support coagulation in vivo, the present inventors looked for localization of GSAO+ platelets with fibrin within thrombi induced by FeCl.sub.3 injury in murine cremaster arterioles. Fibrin is the end product of activation of the coagulation factors. Fibrin strands are shown adjacent to GSAO+ platelets in the 2D cross sectional sections (
Example 9—Aspirin Ingestion Sensitizes Platelets to Cyclophilin D Inhibition in Human Subjects
[0290] The standard pharmacological means for prevention of cardiovascular events is by inhibiting platelet cyclooxygenase-1 with aspirin, which suppresses platelet activation. To explore the possibility of targeting platelet activation (cyclooxygenase-1) and necrosis (cyclophilin D) pathways in combination, the effect of ex vivo cyclosporine A treatment on thrombin and collagen activation of platelets from healthy volunteers before and after 7 days of aspirin ingestion was compared. Aspirin ingestion resulted in no change in the GSAO+/P-selectin+ necrotic (
Example 10—Platelet Mitochondrial Membrane Potential
[0291] Mitochondrial membrane potential was measured using the JC-1 cationic dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide, Sigma) (Life Technologies). Washed platelets were incubated with JC-1 (2 μM) for 30 min in the dark at 37° C., washed once and analyzed immediately on a BDFACS Canto II. On some occasions, washed platelets were incubated with the mitochondrial membrane disrupter 2-[2-(3-chlorophenyl)hydrazinylyidene]propanedinitrile (CCCP, 4 μM) for 10 min.
Example 11—Platelet Surface FXa
[0292] Platelet surface FXa was measured using FITC-labeled Glu-Gly-Arg chloromethyl ketone (FITC-GGACK, Haematologic Technologies). Platelet rich plasma was diluted 1:5 with modified Hepes/Tyrodes buffer supplemented with fibrin polymerisation inhibitor Gly-Pro-Arg-Pro (2.5 mM, Sigma) and CaCl.sub.2 (4 mM), stimulated with thrombin and collagen as above, labeled with FITC-GGACK (6.5 μM) for 15 min then co-labeled with GSAO-AF647 or GSCA-AF647 (1 μIV). The labeled platelets were fixed (Pamfix, Platelet Solutions), washed and analyzed by flow cytometry on a BD Fortessa LSR.
Example 12—Platelet Procoagulant Potential
[0293] Thrombin generation capacity of agonist stimulated platelets was measured using the Calibrated Automated Thrombogram as described (Hemker et al., 2003) using the Fluorskan Ascent (Thermo Scientific). Agonist stimulated platelets were washed in phosphate-buffered saline and resuspended into the donor's own platelet poor plasma at 5×10.sup.4/μL and recombinant tissue factor (0.3 μM, Innovin, Dade Behring) was added. Assays were performed in triplicate.
Example 13—Imaging and Analysis of Murine Thrombi
[0294] This study was approved by the University of New South Wales Animal Care & Ethics Committee (ACEC 13/47A). C57BL/6J mice were obtained from The Jackson Laboratory. Cyclophilin D megakaryocyte-specific knockout mice were provided by Benjamin Kile (Walter & Eliza Hall Institute). The Cyclophilin D conditional knockout allele (Ppri.sup.flm1Mmos/J) has been previously described (Schinzel et al., 2005). Originally constructed in 129X1/SvJ-derived RW4 embryonic stem cells, mice carrying the Ppif.sup.flm1Mmos/J a mutation had been backcrossed several generations to C57BL/6 prior to being crossed with the Pf4-Cre transgenic strain (Tiedt et al., 2007). The latter was generated in, and maintained on, the C57BL/6 background.
[0295] Intravital video microscopy of the cremaster muscle microcirculation was performed using a VIVO Intravital Imaging System (Intelligent Imaging Innovations) consisting of an upright Zeiss Axio Examiner fitted with a motorised stage, piezo controlled z focus and 63x/1.0NA water immersion Plan Apo Objective (Zeiss). Imaging was though spinning disc confocal (Yokogawa, CSUX1-M1L-E) and digital images were captured with a Hamamatsu 9300 CCD camera connected to a VS4-1845 Image Intensifier GEN III (Video Scope International). Platelet marker Dylight 649-conjugated anti-CD42b antibody (0.1 μg/g mouse, Emfret Analytics), GSAO-Oregon Green or GSAO-AF546 (0.1 μg/g mouse) and rhodamine-2 (2 μg/g mouse, Life Technologies) or AF488-conjugated anti-fibrin antibody (0.5 μg/g mouse, clone 59D8 kindly supplied by Bruce and Barbara Furie, Beth Israel Deaconess Medical Centre, Harvard Medical School, MA), were injected into the murine circulation in different combinations. The animal was kept at 37° C. using a warming blanket; muscle was continuously bathed with a bicarbonate buffered physiological solution heated to 37° C. which also served as the imaging medium. Data was captured digitally from 3 fluorescence channels, using 488 nm, 561 nm and 640 nm laser stack excitation. The microscope system was controlled and images were analyzed using Slidebook 5.5 (Intelligent Imaging Innovations).
[0296] Thrombi were triggered in cremaster muscle arterioles using two modes of injury. A cremaster arteriolar (30- to 60-μm diameter) vessel wall was injured with a Vector galvanometer-based point 532 nm pulsed laser (Intelligent Imaging Innovations). Laser was focused through the microscope objective, parfocal with the focal plane. 4D data acquisition was initiated both prior to and after a single laser pulse for each injury. In separate experiments, filter paper saturated with 8% FeCl.sub.3 was applied to the mouse cremaster muscle for 3 min. The paper was removed and the tissue was washed using bicarbonate-buffered saline solution. 3D data acquisition was initiated prior to and after filter paper application. Z Stacks of XY planes (0.55 μm intervals) were imaged using a spinning-disk confocal scanner (Yokogawa CSU X1) using a pizeo focus drive. To quantitate signal in each thrombus, a threshhold value for each fluorescence channel was calculated independently within a non-thrombus involved portion of the vessel. A segment mask was created above threshold for each channel in each z plane and the cumulative integrated fluorescence calculated for each thrombus. Background value was multiplied by volume in voxels, background subtracted and the net integrated fluorescence calculated for each channel in each thrombus. 3D models of platelet aggregates and cross-sectional orthogonal views were rendered within Intelligent Imaging Innovation software.
Example 14—Aspirin Study Participants and Protocol
[0297] This study was approved by the University of New South Wales Human Research Ethics Advisory Committee (HC132219), conducted according to the Declaration of Helsinki and all volunteers gave written informed consent. Five healthy volunteers aged 25-45 years were recruited. Prior to starting the study, all volunteers abstained from aspirin or non-steroidal anti-inflammatory drugs (NSAIDS), or other anti-platelet therapies for 14 days. Volunteers received 100 mg enteric coated aspirin daily for 7 days. Blood was collected and washed platelets assessed at baseline, day 0, and again at day 7 of treatment.
Example 15—Statistical Analysis
[0298] Parametric paired t test was used to evaluate differences between different agonist stimulated groups and the effect of aspirin, cyclophilin D inhibition and pancaspase inhibition on stimulated platelets. Statistical significance was defined as p values of less than 0.05. Sensitivity and specificity were calculated using a 2×2 table and correlation measured by Pearson's Chi-squared analysis. Colocalization of signals in 3D confocal microscopy imaging of thrombi was determined using the Mander's overlap coefficient with a threshold set to the estimated background value (Image J JaCOP plugin software) (Bolte and Cordelieres, 2006).
Example 16—Whole Blood Assay for Necrotic Platelet Potential
[0299] Experiments using genetically modified mice indicate that formation of procoagulant platelets is dependent on the cyclophilin D necrosis pathway, but independent of the Bcl.sub.XL apoptosis pathway. In healthy volunteers, the strong platelet agonists, thrombin and collagen, induce formation of procoagulant platelets via a mechanism that is largely independent of protease activated receptors. Patients with coronary artery disease have a heightened procoagulant platelet potential and are sensitised to thrombin and ADP stimulation. The procoagulant platelet potential in these patients is insensitive to aspirin, which targets platelet aggregation, but modified by P2Y12 antagonists.
[0300] Blood from healthy human volunteers was drawn from an antecubital fossa vein using a 21-gauge needle into evacuated sterile collection tubes containing 0.109 M sodium citrate. Following no or minimal tourniquet application, the initial 2 mL of blood was discarded to avoid pre-analytical activation of platelets. The tube was gently mixed by inversion six times. Murine blood samples were collected by cardiac puncture under isoflurane anesthesia. Following midline abdominal and bilateral subcostal surgical incisions to expose the heart, blood was drawn from the left ventricle through a 25G needle into a 1 mL syringe containing sodium citrate (0.109 M) resulting in a blood to citrate ratio of 9:1. The needle was removed and the blood was gently expelled from the syringe into a sterile microcentrifuge tube and mixed gently by pipetting up and down 5 times.
[0301] Following collection, tubes were kept upright at room temperature and blood was assayed within 15 min of collection. Incubation and staining steps were performed in a sterile 96-well polypropylene plate. Citrated whole blood (15 μL) was diluted with Hepes buffered saline, pH 7.35 (30 μL) containing a final concentration of fibrin polymerization inhibitor GPRP (2.5 mM) and CaCl.sub.2 (2.5 mM). Agonist(s) were added in 5 μL volume to a final volume of 50 μL, incubated for 10 min at room temperature, and the reaction quenched by diluting with Hepes buffered saline to a final volume of 150 μL. Aliquots (20 μL) of diluted stimulated whole blood were labelled for 15 min at room temperature with GSAO-AF647 (2 μM), CD62P-PE, CD45-BUV395 and CD41a-BV510 in 100 μL final volume. GSCA-AF647 (2 μM) and mouse lgG.sub.1 κ isotype control PE served as controls for GSAO and anti-CD62P antibody, respectively. Cells were then fixed with two volumes of PamFix for 5 min. Fixed stained cells were transferred to 5 mL FACS tubes and washed once with ten volumes of Hepes buffered saline containing 0.35% human serum albumin. Cells were pelleted (1,500 g for 8 min) and resuspended in the same buffer.
[0302] Fixed cells were analyzed by flow cytometry 1 to 3 h after fixation at an average event rate of 2000 events per sec in an LSRFortessa flow cytometer, equipped with either four (405 nm, 488 nm, 561 nm and 640 nm) or five (395 nm) lasers (BD Biosciences). Using the lowest forward scatter threshold of 200, 5,000-10,000 events in the platelet gate (defined as CD41a positive but CD45 negative population) were acquired. Flow cytometry data were exported in the FSC 3.0 format and analysis was performed using FlowJo vX 10.0.7r2 (Tree Star). The gating strategy and analysis is described in detail in
Example 16—Assay for Procoagulant Platelet Potential in Human Blood
[0303] Thrombin is the most potent inducer of procoagulant platelets in human blood (
Example 17—Assay for Procoagulant Platelet Potential in Mouse Blood
[0304] Thrombin is also the most potent inducer of procoagulant platelets in murine blood (
Example 18—Coronary Artery Disease is Associated with a Heightened Procoagulant Platelet Potential
[0305] To examine the effect of CAD on procoagulant platelet potential, non-heparinized bloods from patients with exposure to either aspirin only or no antiplatelet medications within 7 days of the procedure were assayed. Compared to patients with no CAD, patients with CAD showed a heightened platelet procoagulant potential in response to thrombin or thrombin plus collagen (
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