60/504, 118 filed September 19,2003 and Provisional Patent Application Serial No.

60/506, 638 filed September 26,2003, the contents of which are hereby incorporated by reference.

Background of the Invention Development of PET (positron emission tomography) and SPECT (single photon emission computed tomography) ligands as diagnostic imaging agents is an active research field. Diagnostic and radionuclides can be diveded into two major categories: positron-emitting (e. g, 18F) and single photon-emitting radionuclides (e. g., 1311 and 1230.

A positron is a positively charged particle (fol+) emitted by a radionuclide. This particle quickly interacts with a neighboring electron and two 511 keV gamma photons are produced. A single photon is a gamma radiation emitted by a radionuclide, usually in random directions.

Positron emission tomography is a technique for measuring the concentrations of positron-emitting radioisotopes. PET is a high resolution, non-invasive, imaging technique for the visualization of human disease. In the clinical setting, fluorine-18 (18F) is one of the most widely used positron-emitting nuclides. 18F has a half-life (tl/2) of 110 minutes, and emits zip particles at an energy of 635 keV.

The short half-life of 18F has limited or precluded its use with longer-lived specific targeting vectors such as antibodies, antibody fragments, recombinant antibody constructs and longer-lived receptor-targeted peptides. In addition, complicated chemistry has been required to link the inorganic fluoride species to such organic targeting vectors. In typical synthesis methods, an intermediate is radiofluorinated, and the 18F-labeled intermediate is purified for coupling to protein amino groups (Lang et al., Appl. Radiat. Isol., 45 (12) : 1155-63 (1994) ; Vaidyanathan et al., Bioconj. Chem., 5: 352-56 (1994)).

These methods are tedious to perform and require the efforts of specialized professional chemists. They are not amenable to kit formulations for use in a clinical setting. Multiple purifications of intermediates are commonly required, and the final step, involving linkage to protein lysine residues, usually results in 30-60% yields, necessitating a further purification step prior to patient administration.

Summary of the Invention The invention is based, at least in part, on the discovery that targeting molecules can be labeled with radioactive halogens (e. g., l8F, 124I, l25I, l23I, l3lI, etc. ) through the conversion of halogenated deoxy glucose to the corresponding aldehyde, and reacting this aldehyde with a hydrazine derivatized targeting molecule. This method has wide applicability to a wide range of proteins, nucleic acids, and other targeting molecules which can be advantageously labeled. The labeled targeting molecules may then be administered to a subject and analyzed in vivo through the use of an imaging method, for example, positron emission tomography.

In one embodiment, the invention pertains to a radiolabeled targeting molecule of the formula: (G-L).-M (I) wherein: each G is an independently selected halogenated deoxy-saccharide moiety ; each L is an independently selected hydrazino linking moiety; M is a targeting molecule ; and z is an integer from 1 to 50, and pharmaceutically acceptable salts, esters, and prodrugs of.

The invention also pertains, at least in part, to radiolabeled targeting molecules of the formula (II): wherein X is an attachment moiety; W is a halogen; M is a protein; and z is 1,2, or 3, and pharmaceutically acceptable esters, prodrugs, and salts thereof.

In another embodiment, the invention pertains, at least in part, to a method for radiolabeling a targeting molecule. The method includes contacting a hydrazine derivatized targeting molecule with an halogenated deoxy saccharide aldehyde compound, and allowing the halogenated deoxy saccharide aldehyde compound to react with the hydrazine derivatized targeting molecule such that the targeting molecule is

radiolabeled.

In yet another embodiment, the invention features a method for radioimaging a subject in vivo. The method includes administering to the subject a composition comprising a radiolabeled targeting molecule of the invention, and obtaining a radioimage of the subject.

In another embodiment, the invention includes a method for imaging cell death in a mammalian subject in vivo. The method includes administering to the subject a composition comprising a radiolabeled targeting molecule of the invention; and obtaining a radioimage. Preferably, the radioimage is a representation of cell death in the mammalian subject.

In a further embodiment, the invention pertains to a method for synthesizing a halogenated deoxy saccharide aldehyde compound (e. g. , a fluoro deoxy glucose aldehyde compound). The method includes contacting a halogenated deoxy saccharide (e. g. , a fluoro deoxy glucose) with an effective amount of periodate under appropriate conditions, such that a halogenated deoxy saccharide aldehyde compound (e. g. , a fluoro deoxy glucose aldehyde compound) is formed.

The invention also pertains, at least in part, to a composition comprising a radiolabeled targeting molecule of the invention and a pharmaceutically acceptable carrier.

In another further embodiment, the invention pertains, at least in part to a method of treating a subject with radiotherapy. The method includes administering an effective amount of a radiolabeled targeting molecule of the invention to a subject, such that said subject is treated. In certain embodiments, the radiolabeled targeting molecule is radiolabeled withal.

Other features and advantages of the invention will be apparent from the following detailed description and the claims.

Brief Description of the Invention 1. Methods for Radio Labeling Targeting Molecules and Radiolabeled Targeting Molecules In one embodiment, the invention pertains to a method for radiolabeling a targeting molecule. The method includes contacting a hydrazine derivatized targeting molecule with a halogenated deoxy glucose aldehyde compound under appropriate conditions, and allowing the halogenated deoxy glucose aldehyde compound to react with the hydrazine derivatized targeting molecule.

The term"targeting molecule"includes any targeting molecule which can be

advantageously labeled and used in the methods of the invention. Examples of targeting molecules include proteins, nucleic acids, fats, fatty acids, carbohydrates, and fragments thereof. In certain embodiments, the targeting molecule may be a targeting molecule which is associated with a disease. The targeting molecule may be biologically produced, native, or chemically synthesized. It also may be chemically modified any manner which allows the radiolabeled target molecule to perform its function.

Modifications include esters, prodrugs, salts, other labels, etc. The modifications may be made synthetically or through biological processes.

The targeting molecule of the invention may be a cytokine, e. g., tumor necrosis factor a (TNF a), tumor necrosis factor (3 (TNF ß), Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-12 (IL-12), Interleukin-18 (IL-18) or Interferon- (INF-y); or growth factor, e. g., Epidermal Growth Factor (EGF), Platelet-Derived Growth Factor (PDGF), Fibroblast Growth Factors (FGFs), Transforming Growth Factors-/3 (TGFs-ß), Transforming Growth Factor-a (TGF-a), Erythropoietin (EPO), Insulin-Like Growth Factor-I (IGF-1) or Insulin-Like Growth Factor-11 (IGF-11). Additional examples of targeting molecules include cell surface bound proteins, such as receptors, adhesion molecules, signaling molecules or extracellular matrix molecules. Non limiting examples of membrane bound targeting molecules include integrins, GPCRs and various cellular determinant molecules of known or unknown function, such as CD-4, CD-8, and the like.

Examples of targeting molecules, and conditions associated with these targeting molecules, are set forth in Table I below.

TABLE I Target Disease/Condition IL-1 receptor IL-la and (3 Inflammation, Arthritis, inflammatory bowel disease IL-4 asthma IL-4 Allergic airway disease IL-5 Allergic airway disease IL-5 asthma IL-6 inflammation IL-6 Kaposi's sarcoma IL-7 Immune response IL-8 Inflammatory disease IL-8 Crohn's disease IL-18 arthritis IL-9 asthma IL-10 colitis IL-11 Crohn's disease TNF receptor Inflammation, arthritis, autoimmune thyroid disease, ischemic heart disease TNF-a and and Inflammation, arthritis, autoimmune thyroid disease, ischemic heart disease 11 2 or Ischemic heart disease IL-II Ischemic heart disease EGF receptor Cancer Vascular endothelial growth factor Arthritis receptor VEGF Arthritis VEGF Cancer VEGF receptor Cancer aldosterone receptor Cardiovascular heart disease Aldosterone Cardiovascular heart disease Somatostatin receptor Grave's disease Somatostatin Grave's disease Fibronectin receptor Ullrich's disease Fibronectin Ullrich's disease angiotensin receptor Heart disease Angiotensin Heart disease Amyloid beta-peptide [Abeta (1-42)] Alzheimer's disease Transthyretin Alzheimer's disease Erythropoietin benign erythrocytosis Prions Neurodegeneration Prostaglandin Neurodegeneration cholesterol Heart disease Huntington protein Huntington disease Interferon a and (3Immune response PPARY hepatogastroenterological diseases Retinoid X hepatogastroenterological diseases Adrenocorticotropic Hormone Cushing's disease Hepatocyte growth factor Cardiovascular disease estrogen receptor Coronary heart disease apolipoprotein B-100 Coronary heart disease Estrogen receptor Liver disease glucose-6-phosphatase Glycogen storage disease type 1 erythrocyte antioxidant enzyme Behcet's disease Homocysteine Cardiovascular disease transforming growth factor-beta graft-versus-host disease transforming growth factor-betal type graft-versus-host disease It receptor Transforming Growth Factor-beta Coeliac Disease transforming growth factor betal renal disease insulin-like growth factor binding macrovascular disease and protein-1 (IGFBP-1) hypertension in type 2 diabetes Hepatocyte growth factor periodontal disease vascular endothelium growth factor Eales'disease vascular endothelial growth factor-A Paget's disease (VEGF-A) androgen receptor Paget's disease platelet-derived endothelial cell Paget's disease growth factor/thymidine phosphorylase (PD-ECGF/TP) Insulin-like growth factor I (IGF-I) inflammatory bowel disease IGF binding protein-3 inflammatory bowel disease Rb Cancer P16 Cancer P21 Cancer P53 Cancer HIF-1 Cancer Insulin Diabetes NF kappa B Inflammatory disease NF kappa B Cell Death I kappa kappa Beta Immune response Apolipoprotein A1 Heart disease Apolipoprotein CII Hyperlipidemia Apolipoprotein CII heart disease Apolipoprotein E Cardiovascular disease Apolipoprotein E Alzheimer's disease CD4 Immune response CD4 receptor Immune response/HIV infection CCR5 Immune response/HIV infection SBR1 HDL receptor/coronary heart disease Annexin V Clot formation Annexin V Apoptosis EGF Oncogenesis EGF Wound healing Fibrin Wound healing Fibrin Clot formation Vasoactive intestinal peptide inflammation

In another embodiment, the targeting molecule is annexin or a fragment thereof, e. g., aimexin V or a fragment thereof. Annexin V is normally found in high levels in the cytoplasm of a number of cells including placenta, Lymphocytes, monocytes, biliary and renal (cortical) tubular epithelium. Although the physiological function of annexins has not been fully elucidated, several properties of annexins make them useful as diagnostic and/or therapeutic agents. In particular, it has been discovered that annexins possess a very high affinity for anionic phospholipid surfaces, such as a membrane leaflet having an exposed surface of phosphatidylserine (PS). Also included are fragments of annexin which may be advantageously labeled using the methods of the invention. Also included are mutants and derivatives of annexin. Examples of derivatives include annexins which have been chemically modified, for example, pegylated.

For example, the PS-binding site in domain 1 of annexin V (described in Montvaille P. et al. (2002) JB. C 277 (27); 24684-24693) may be labeled using the methods of the invention. Molecules (e. g. , small molecules peptidomimetics, etc. ) that are capable of binding to phospholipids may also be labeled using the methods of the invention. Exemplary molecules include small molecules or peptidomimetics that are designed based on the phospholipid binding domains of annexin V.

In one embodiment, the targeting molecule is annexin. Purified native, recombinant, or synthetically-prepared annexin may be used. Annexin V, for example, may be conveniently purified from human placenta (as described in Funakoshi, et al.

(1987) Biochemistry 26: 5572, the contents of which are incorporated herein by reference). Recombinant annexin offers several advantages, however, including ease of preparation and economic efficiency. A number of different annexins have been cloned from humans and other organisms. Their sequences are available in sequence databases, including GenBank.

The invention may be practiced using annexin V. Annexin V is one of the most abundant annexins, (ii) it is simple to produce from natural or recombinant sources, and (iii) it has a high affinity for phospholipid membranes. Human annexin V has a molecular weight of 36 kd and a high affinity (kd = 7 nmol/L) for phosphatidylserine (PS). The sequence of human annexin V can be obtained from GenBank under accession numbers U05760-U05770.

An exemplary expression system suitable for making annexin for use with the present invention employs the pET12a expression vector (Novagen, Madison,

Wisconsin) in E. coli. (described in Wood, et al. (1996) Blood 88: 1873-1880, incorporated herein by reference).

Other bacterial expression vectors may be utilized as well. They include, e. g., the plasmid pGEX (Smith, et al. (1988) Gene 67: 31) and its derivatives (e. g., the pGEX series from Pharmacia Biotech, Piscataway, NJ). These vectors express the polypeptide sequences of a cloned insert fused in-frame with glutathione-S-transferase.

Recombinant pGEX plasmids can be transformed into appropriate strains of E. coli and fusion protein production can be induced by the addition of IPTG (isopropyl-thio galactopyranoside). Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using glutathione agarose affinity chromatography according to standard methods (described in, for example, Ausubel, et al. Current Protocols in Molecular Biology (John Wiley and Sons, Inc., Media, PA). Other commercially-available expression systems include yeast expression systems, such as the Pichia expression kit from Invitrogen (San Diego, CA); baculovirus expression systems (Reilly, et al. in Baculovirus Expression Vectors : A Laboratory Manual (1992) ; Clontech, Palo Alto CA); and mammalian cell expression systems (Clontech, Palo Alto CA; Gibco-BRL, Gaithersburg MD).

A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. The recombinantly produced polypeptides are typically isolated from lysed cells or culture media.

Isolated recombinant polypeptides produced as described above may be purified by standard protein purification procedures, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. Protein preparations can also be concentrated by, for example, filtration (Amicon, Danvers, Mass.).

The targeting molecule may also be a molecule which has an affinity for a second molecule. For example the targeted molecule may be a tag that has high affinity for a protein/construct or other molecule, which may, for example, localize in a particular organ, e. g.,"pretargeting."Pretargetting is discussed in more detail in Paganelli, Eur. J. Med. Mol. Imaging (2003) 30: 773-776.

Pretargeted therapy has been found to be advantagous in the treatment of solid tumors. One method of treating solid tumors would be to transfect target tumor tissues with a new fusion gene containing intracellular and membrane spanning domains of scavenger receptors linked to avidin, which would form the extracellular domain of the fusion protein. Then, only the tumor target cells will express avidin on their surface,

thus allowing for the biotinylated compounds (e. g. , halogenated deoxy saccharide compounds comprising biotin) to home on the tumor cells.

In a further embodiment, the targeting molecule is a molecule which has an affinity for a second molecule. The second molecule may comprise a label (e. g., fluorescent or radioactive label) or may have a particular affinity for a particular organ.

The second molecule may comprise, for example, a binding part and a organ localization part.

The binding part may be any moiety or molecule which binds with high affinity to the targeting molecule. Examples of binding parts include proteins (e. g., biotin, avidin, antibodies, RNase/HuS (see e. g., Backer MV et al., Protein Expr Purif. 2002 Dec; 26 (3): 455-61), nucleotides and analogues thereof (see, e. g. , Liu, G. et al. Nucl Med Commun., 2003 Jun; 24 (6): 697-705 ; Liu G et al., JNucl Med. 2002 Mar ; 43 (3): 384-91), or other molecules with a high affinity for a particular targeting molecule. Other examples of binding parts include those described in Goldberg et al. (Eur. J. Nucl. Med.

Mol. Imaging, (2003) 30: 776-780).

In one embodiment, the targeting molecule comprises avidin, and the second molecule comprises biotin. In another embodiment, the targeting molecule comprises biotin and the second molecule comprises avidin. In another embodiment, the targeting molecule comprises a nucleic acid and the binding part comprises an anti-sense nucleic acid.

In a further embodiment wherein M comprises a protein, the appropriate conditions are such that M is not denatured. In a further embodiment, the temperature of the reaction is between 20 C and 40C, or between room temperature and 37 C.

In a further embodiment, wherein said targeting moiety has an affinity for a second molecule, the appropriate temperature may be higher.

The term"hydrazine derivatized targeting molecule"includes targeting molecules which comprise at least one hydrazine (NH2-NH-) moiety. This moiety may be attached to the targeting molecule through an attachment moiety. The targeting molecule may naturally comprise the hydrazine moiety or may be synthetically obtained.

For example, in the Exemplification, a method for derivatizing the targeting molecule annexin with Hynic (Hydrazino Nicotinamide) is described. However, the methods of the invention also include other methods for attaching hydrazino functionality to targeting molecules and other attachment moieties.

In one embodiment, the hydrazine derivatized targeting molecule is of the formula:

wherein: each X is an independently selected attachment moiety; M is a targeting molecule; z is an integer from 1 to 50.

In a further embodiment, z is 1,2, 3,4, 5,6, 7,8, 9 or 10. In certain embodiments, z is 1,2, 3 or 4.

The attachment moiety can be any moiety which is capable of attaching the hydrazino functionality to the targeting molecule. Examples of attachment moieties include chains of, for example, 0 to 30 carbon and/or heteroatoms which may include straight chain, branched and cyclic moieties, each of which may be optionally substituted. The attachment moiety may include aryl, alkenyl, alkynyl, alkyl, peptidyl, thioamide, and acyl moieties, or may be a covalent bond. In one embodiment, the attachment moiety is aryl, e. g. , heteroaryl, e. g., nicotinamide.

The term"halogenated deoxy saccharide aldehyde compound"includes two, three, four, five, and six carbon sugars in the aldehyde form, wherein at least one hydrogen or hydroxyl group is replaced with a halogen, advantageously a radioactive fluorine, bromine, chlorine, or iodine isotope such as l8F, l24I, l25I, l23I, l3lI, 76Br, 77Br, etc. In a further embodiment, the sugar is a glucose derivative, such as 2-fluoro-2-deoxy glucose, although the fluorine may also replace other hydroxyl groups which allow the radiolabeled targeting molecule to perform its intended function. For example, 2,3- difluoro-2,3-deoxyglucose, 3-fluoro-3-deoxy glucose, 4-fluoro-4-deoxyglucose, and 5- fluoro-5-deoxyglucose are also included. In other embodiments, the halogenated deoxy saccharide aldehyde compound comprises two aldehyde functional groups. In one embodiment, the halogenated deoxy saccharide aldehyde compound is a halogenated deoxy glucose aldehyde compound, such as for example, a fluoro deoxy glucose aldehyde compound which comprises one or more 18F isotopes. Also included are esters, prodrugs and salts of the halogenated deoxy saccharide aldehyde compounds.

One example of a halogenated deoxy glucose aldehyde compound is:

wherein W is a halogen.

In a further embodiment, the invention pertains to a radiolabeled targeting molecule of the formula: (G-L) Z-M (I) wherein: each G is an independently selected halogenated deoxy-saccharide moiety; each L is an independently selected hydrazino linking moiety; M is a targeting molecule; and z is an integer from 1 to 50, and pharmaceutically acceptable salts, esters, and prodrugs of.

In a further embodiment, z is 1,2, 3,4, 5,6, 7, 8,9 or 10. In another embodiment, z is 1,2, 3, or 4.

The term"halogenated deoxy-saccharide moiety"includes halogenated deoxy saccharide aldehyde compounds which have been reacted with the hydrazine derivatized targeting molecules, such that a hydrazone bond is formed. Preferably, the halogenated deoxy saccharide moiety is comprised of one or more radioactive halogen isotopes, such as radioactive bromine (e. g., 76Br or 77Br), iodine (e. g., 1241, l25I, l23I, or 131p, chlorine, or fluorine isotopes (e. g., 18F). In a further embodiment, the halogenated deoxy saccharide moiety is a halogenated deoxy glucose moiety. In a further embodiment, the halogenated deoxy-saccharide moiety may be treated with an oxidizing agent such as permaganate or periodate such that an additional aldehyde moiety is formed. This aldehyde moiety may be further derivatized in any manner known in the art which allows the resulting radiolabeled targeting molecule to perform its intended function. In a further embodiment, the halogenated deoxy-saccharide moiety is of the formula: wherein W is a halogen.

In a further embodiment, the halogenated deoxy saccharide moiety is a fluoro deoxy glucose moiety, of the formula:

The term"hydrazino linking moiety"includes moieties which link the targeting molecule to the fluoro deoxy glucose moiety. Preferably, the hydrazino linking moiety is a moiety of the formula : wherein X is an attachment moiety. Generally, the hydrazino linking moiety is formed by reacting the hydrazine derivatized targeting molecule with the fluoro deoxy glucose aldehyde compound.

In a further embodiment, the hydrazino linking moiety comprises hydrazino nicotinamide (e. g., HYNIC).

In a further embodiment, the invention pertains, at least in part, to a radiolabeled targeting molecule of the formula (IT) : wherein X is an attachment moiety; W is a halogen; M is a protein; and z is 1,2, 3,4, 5,6, 7,8, 9, or 10 and pharmaceutically acceptable esters, prodrugs, and salts thereof.

In a further embodiment, the invention also pertains to a composition comprising

a radiolabeled targeting molecule of the invention (e. g. , a molecule of formula I, II, or otherwise described herein) and a pharmaceutically acceptable carrier.

The term"alkyl"includes saturated aliphatic groups, including straight-chain alkyl groups (e. g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (e. g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (e. g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e. g., Cl-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term Cl-C6 includes alkyl groups containing 1 to 6 carbon atoms.

Moreover, the term alkyl includes both"unsubstituted alkyls"and"substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e. g. , with the substituents described above. An"alkylaryl"or an "arylalkyl"moiety is an alkyl substituted with an aryl (e. g., phenylmethyl (benzyl)).

The term"alkyl"also includes the side chains of natural and unnatural amino acids.

The term"aryl"includes groups, including 5-and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term"aryl"includes multicyclic aryl groups, e. g., tricyclic, bicyclic, e. g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzthiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, diazapurine, or indolizine. Those aryl groups having

heteroatoms in the ring structure may also be referred to as"aryl heterocycles", "heterocycles,""heteroaryls"or"heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e. g., tetralin).

The term"alkenyl"includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.

For example, the term"alkenyl"includes straight-chain alkenyl groups (e. g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. The term alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e. g., C2-C6 for straight chain, C3-C6 for branched chain).

Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkenyl includes both"unsubstituted alkenyls"and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino,

arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term"alkynyl"includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.

For example, the term"alkynyl"includes straight-chain alkynyl groups (e. g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e. g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both"unsubstituted alkynyls"and "substituted alkyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including, e. g., alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified,"lower alkyl"includes alkyl groups as defined above, but having from one to five carbon atoms in its backbone structure."Lower alkenyl"and"lower alkynyl"have chain lengths of, for example, 2-5 carbon atoms.

The term"acyl"includes compounds and moieties which contain the acyl radical (CH3CO-) or a carbonyl group. The term"substituted acyl"includes acyl groups where one or more of the hydrogen atoms are replaced by, for example, alkyl groups, alkynyl

groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term"alkoxy"includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.

Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term"amine"or"amino"includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term"alkylamino"includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term"dialkylamino"includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term"arylamino"and"diarylamino"include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively.

The term"alkylarylamino,""alkylaminoaryl"or"arylaminoalkyl"refer s to an amino group which is bound to at least one alkyl group and at least one aryl group. The term "alkylaminoalkyl"refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.

The term"amide"or"aminocarboxy"includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl

group. The term includes"alkylaminocarboxy"groups which include alkyl, alkenyl, or alkynyl groups bound to an amino group bound to a carboxy group. It includes arylaminocarboxy groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms "alkylaminocarboxy,""alkenylaminocarboxy,""alkynylaminocarbo xy,"and "arylaminocarboxy"include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group.

The term"carbonyl"or"carboxy"includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. Examples of moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term"ether"includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes "alkoxyalkyl"which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.

The term"ester"includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group.

The term"ester"includes alkoxycarbony groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are as defined above.

The term"hydroxy"or"hydroxyl"includes groups with an-OH or-0'.

The term"halogen"includes fluorine, bromine, chlorine, iodine.

The term"heteroatom"includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

It will be noted that the structure of some of the compounds of this invention includes stereogenic carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e. g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof.

2. Methods of Radioimaging In one embodiment, the invention pertains to a method for radioimaging a subject in vivo. The method includes administering to the subject a composition comprising a radiolabeled targeting molecule of the invention as described above (e. g. , a

compound of formula (I), (II), or otherwise described herein), and obtaining a radioimage of said subject. In a further embodiment, the radioimage is obtained through the use of positron emission tomography, e. g. , when the halogen is 18F. In another embodiment, the radioimage is obtained through single photon emitted computed tomography (SPECT), e. g. when the halogen is 123 1. The particular targeting molecule labeled may be selected to correspond to a particular condition the subject is suffering from or is at risk of suffering from. The particular choice of labeled targeting molecule may be chosen to elucidate the path of a particular targeting molecule within the subject's body or the location of a particular area of interest, e. g. , a tumor. One of skill in the art will appreciate that the methods and areas of the body for imaging will be selected according to the needs of a particular subject. For example, an organ of a subject or a portion or specimen thereof (e. g., brain, heart, liver lung, pancreas, colon) or a gland of a subject or a portion thereof (e. g. , prostate, pituitary or mammary gland) may be chosen for imaging. In addition, the imaging may be conducted using surgical or needle biopsy of a subject after administration of the compound of the invention to the subject; or by the use of a catheter that may detect radiation in a vessel of a subject.

The term"subject"includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human.

The term"administering"to a subject includes dispensing, delivering or applying a composition of the invention to a subject by any suitable route for delivery of the composition to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

If it is desired to follow the localization and/or the signal over time, for example, to record the effects of a treatment on the distribution and/or localization of cell death, the imaging can be repeated at selected time intervals to construct a series of images.

The intervals can be as short as minutes, or as long as days, weeks, months or years.

Images, such as positron emission tomographs, generated by methods of the present invention may be analyzed by a variety of methods. They range from a simple visual examination, mental evaluation and/or printing of a hardcopy, to sophisticated digital image analysis.

The radioimage may be obtained at any time after administration of the radiolabeled targeting molecule of the invention. The length of time after administration of the molecule prior to the taking of the radioimage may depend on the particular radioactive isotope used. For example, an 18F positron emission tomograph may be

obtained from 1 to 500 minutes after administration. In another embodiment, the positron emission tomograph is obtained 1, 2,3, 4,5, 10,15, 20,25, 30,35, 40,45, 50, 55,60, 65,70, 75,80, 85,90, 95,100, 105,110, 115, or 120 minutes after the administration of the composition of the invention to the subject. In another embodiment, the image is obtained about 10-30,15-25, 20-25, or 20-30 hours after the administration of the composition of the invention to the subject. In another embodiment, an 123I SPECT maybe obtained from 0. 01 to 130 hours after administeration. In an embodiment, the image is obtained at a plurality of time points, thereby monitoring changes in the location of the targeting molecule or accumulation of the targeting molecule. In certain embodiments, the images obtained at a plurality of time points may be used to determine the number of cells undergoing cell death or to monitor changes in the location of cells undergoing cell death.

The compositions of the present invention may be administered to a subject using standard protocols, such as protocols for the administration of radiolabeled compounds.

The compositions of the invention may be administered to a subject in an amount effective, at dosages and for periods of time necessary, to achieve the desired result. An effective amount of the compositions of the invention may vary according to factors such as the age and weight of the subject, and the ability of the composition to elicit a desired response in the subject. An effective amount is also one in which any toxic or detrimental effects (e. g., side effects) of the compositions are outweighed by the diagnostically or therapeutically beneficial effects. The compositions of the invention may be administered at a concentration of O. Ol. g-1000 g targeting molecule/kg, 0.1- 900 ug targeting molecule/kg, 0. 2-800 ug targeting molecule/kg, 10-700 ug targeting molecule/kg, 10-600 llgtargetingmolecule/kg, 10-500, ugtargetingmolecule/kg, 10-400 pg targeting molecule/kg, 10-300 llg targeting molecule/kg, 10-200 Zg targeting molecule/kg, or 10-100 ug targeting moleculelkg.

The levels of a particular targeting molecule may be selected such that the levels are lower than the levels which cause a pharmacological effects in a subject. For example, annexin V begins to have pharmacological effects (anti-coagulant effects) at doses greater than about 300 ug/kg. Accordingly, certain diagnostic methods of the present invention (which involve annexin V and seek to avoid pharmacological effects of the labeled annexin) are practiced at doses lower than 300 ug/kg, typically less than about 50, ug/kg. Such tracer doses (e. g. , 0. 01 uglkg to 50 uglkg) have no reported pharmacologic or toxic side effects in animal or human subjects.

In addition, if a second molecule is used, the second molecule may be administered concurrently with the radiolabeled targeting molecule, before the

radiolabeled targeting molecule, or after the radiolabeled targeting molecule. Methods of pretargeting are described in greater detail in, for example, Eur. J. Nucl. Med. Mol.

Imaging, (2003) 30: 773-776, Eur. J Med. Mol. Imaging (2003) 30: 776-780; and J. Nucl.

Med. 28: 1294-1302 (1987).

The compositions of the invention are typically suspended in a suitable delivery vehicle, such as sterile saline. The vehicle may also contain stabilizing agents, carriers, excipients, stabilizers, emulsifiers, and the like, as is recognized in the art.

The compositions of the invention may be administered to a subject by any suitable route for administration. A preferred method of administration is intravenous (i. v. ) injection. It is particularly suitable for imaging of well-vascularized internal organs, such as the heart, liver, spleen, and the like. Methods for i. v. injection of, e. g., radiopharmaceuticals are known. For example, it is recognized that a radiolabeled pharmaceutical is typically administered as a bolus injection using either the Oldendorf/Tourniquet method or the intravenous push method (see, e. g. , Mettler and Guierbteau, (1985) Essentials Of Nuclear Medicine Imaging, Second Edition, W. B.

Saunders Company, Philadelphia, PA).

For imaging the brain, the compositions of the invention can be administered intrathecally. Intrathecal administration delivers a compound directly to the sub-arachnoid space containing cerebral spinal fluid (CSF). Delivery to spinal cord regions can also be accomplished by epidural injection to a region of the spinal cord exterior to the arachnoid membrane.

For bronchoscopy applications, the annexin compounds of the present invention may be administered by inhalation. For example, the compositions of the invention may be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e. g., a gas such as carbon dioxide, or a nebulizer.

Other modes of administration include intraperitoneal (e. g. , for patients on kidney dialysis), and intrapleural administration. For specific applications, the invention contemplates additional modes of delivery, including intraarterial injection, intramuscular injection, subcutaneous, intralymphatic, insufflation, oral, intravaginal and/or rectal administration.

After the compositions of the invention are administered, they are allowed to localize to the target tissue or organ. Localization in this context refers to a condition when either an equilibrium or a pseudo-steady state relationship between bound, "localized", and unbound, "free"compound within a subject has been achieved. The amount of time required for such localization is typically on the order of minutes to tens of minutes and may be estimated by the serum half-life of the compound. The localization time also depends on the accessibility of the target tissue to the compound.

This, in turn, depends on the mode of administration, as is recognized in the art.

Imaging is preferably initiated after most of the compound has localized to its target (s). One of skill in the art will appreciate, however, that it may be desirable to perform the imaging at times less than or greater than the half-life timepoints. For example, in imaging cell death due to blood vessel injury, the accessibility of the target tissue is very high, such that a strong signal can be obtained from the target site in only a few minutes, especially if a low dose of labeled composition is administered gradually to minimize signal from circulating label.

In all of the above cases, a reasonable estimate of the time to achieve localization may be made by one skilled in the art. Furthermore, the state of localization as a function of time may be followed by imaging the positron emission signal from the labeled composition according to the methods of the invention.

The present invention also pertains to methods and compositions for imaging cell death in vivo. In one embodiment, the invention pertains, at least in part to annexin derivatized with fluoro deoxy glucose, which allows for the efficient and effective detection of cells undergoing cell death using positron emission tomography.

In another aspect, the present invention provides a method for the in vivo imaging of cell death, e. g., cell death caused by apoptosis, in a mammalian subject. The method includes administering to the subject a composition of the invention comprising annexin coupled fluoro deoxy glucose; and obtaining a radio image, e. g. , a positron emission tomograph, wherein said image is a representation of cell death in the mammalian subject.

The term"cell death"includes the processes by which mammalian cells die.

Such processes include apoptosis (both reversible and irreversible) and processes thought to involve apoptosis (e. g., cell senescence), as well as necrosis. "Cell death"is used herein to refer to the death or imminent death of nucleated cells (e. g., neurons, myocytes, hepatocytes and the like) as well as to the death or imminent death of anucleate cells (e. g., red blood cells, platelets, and the like). Cell death is typically manifested by the exposure of PS on the outer leaflet of the plasma membrane.

In a further embodiment, the invention pertains to a method for synthesizing a halogenated deoxy saccharide aldehyde compound (e. g. , a fluoro deoxy glucose aldehyde compound). The method includes contacting a halogenated deoxy saccharide (e. g. , a fluoro deoxy glucose) with an effective amount of periodate under appropriate conditions, such that a halogenated deoxy saccharide aldehyde compound (e. g. , a fluoro deoxy glucose aldehyde compound) is formed.

3. Methods of Radiotherapy

In a further embodiment, the invention pertains to a method of treating a subject with radiotherapy. The method includes administering an effective amount of a radiolabeled targeting molecule of the invention (e. g. , a compound of formula I, II, or otherwise described herein) to a subject, such that the subject is treated, wherein the radiolabeled targeting molecule comprises one or more therapeutic radioisotopes.

The radiolabeled targeting molecules of the invention may be useful for treating all types of cancers including all solid tumors, i. e. , carcinomas e. g., adenocarcinomas, and sarcomas. Adenocarcinomas are carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures. Sarcomas broadly include tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. Examples of carcinomas which may be treated using the methods of the invention include, but are not limited to, carcinomas of the prostate, breast, ovary, testis, lung, colon, and breast. The methods of the invention are not limited to the treatment of these tumor types, but extend to any solid tumor derived from any organ system. Examples of cancers include, but are not limited to, colon cancer, bladder cancer, breast cancer, melanoma, ovarian carcinoma, prostatic carcinoma, lung cancer, and a variety of other cancers. The methods of the invention may also be used in the inhibition of cancer growth in adenocarcinomas, such as, for example, those of the prostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the invention pertains to a method for treating a subject suffering or at risk of suffering from cancer, by administering to the subject an effective amount of a radiolabeled targeting molecule of the invention, such that inhibition of cancer cell growth occurs, e. g., cellular proliferation, invasiveness, metastasis, or tumor incidence is decreased, slowed, or stopped. In a further embodiment, the molecules of the invention may be administered in combination with one or more standard cancer therapies, such as, but not limited to, chemotherapeutic agents, radiation therapy (e. g., total body irradiation and targeted external irradiation) and apoptosis inducing agents.

The term"therapeutic radioisotopes"include radioisotopes such as 1311 which may have therapeutic effect when administered to a particular subject. For example, the subject could be suffering from cancer or a tumor and the radioisotope would be selected such that the radiation emitted is therapeutic to the particular subject.

The term"treated, ""treating"or"treatment"includes therapeutic and/or prophylactic treatment. The treatment includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated.

For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.

The language"in combination with"a second agent or treatment includes co- administration of the molecule of the invention with the second agent or treatment; administration of the molecule of the invention first, followed by the second agent or treatment; and administration of the second agent or treatment first, followed by the molecule of the invention. The second agent may be any agent which is known in the art to treat, prevent, or reduce the symptoms of a disease being treated in the subject with the radioisotope, such as, for example, cancer. Furthermore, the second agent may be any agent of benefit to the patient when administered in combination with the administration of a molecule of the invention.

The language"chemotherapeutic agent"is art known and includes chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable or which otherwise treat at least one resulting symptom of such a growth. Chemotherapeutic agents are well known in the art (see e. g., Gilman A. G., et al., She Pharmacological Basis of Therapeutics, 8th Ed. , Sec 12: 1202- 1263 (1990}), and are typically used to treat neoplastic diseases. Examples of chemotherapeutic agents include: bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex), granisetron (Kytril), irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), tretinoin (Retin- A), Triapine, vincristine, and vinorelbine tartrate (Navelbine).

Other examples of chemotherapeutic agents include alkylating drugs such as Nitrogen Mustards (e. g., Mechlorethamine (HN2), Cyclophosphamide, Ifosfamide, Melphalan (L-sarcolysin), Chlorambucil, etc.) ; ethylenimines, methylmelamines (e. g., Hexamethylmelamine, Thiotepa, etc.) ; Alkyl Sulfonates (e. g. , Busulfan, etc.), Nitrosoureas (e. g., Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl- CCNU), Streptozocin (streptozotocin), etc.), triazenes (e. g, Decarbazine (DTIC; dimethyltriazenoimi-dazolecarboxamide)), Alkylators (e. g., cis- diamminedichloroplatinum II (CDDP)) and the like.

Other examples of chemotherapeutic agents include antimetabolites such as folic acid analogs (e. g., Methotrexate (amethopterin) ); pyrimidine analogs (e. g., fluorouracil ('5-fluorouracil ; 5-FU) ; floxuridine (fluorode-oxyuridine) ; FUdr; Cytarabine (cyosine arabinoside) ); purine analogs (e. g., Mercaptopurine (6-mercaptopurine; 6-MP); Thioguanine (6-thioguanine; TG); Pentostatin (2'-deoxycoformycin)) and the like.

Other examples of chemotherapeutic agents also include dimethyl busulfan, cyclophosphamide, bischloroethyl nitrosourea, cytosine arabinoside, vinca alkaloids

(e. g., Vinblastin (VLB) and Vincristine) ; topoisomerase inhibitors (e. g., Etoposide, Teniposide, Camptothecin, Topotecan, or 9-amino-campotothecin CPT-11) ; antibiotics (e. g. , Dactinomycin (actinomycin D), adriamycin, daunorubicin, doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin (mitomycin C), Taxol, or Taxotere); enzymes (e. g., L-Asparaginase); and biological response modifiers (e. g., interferon or interleukin 2). Other chemotherapeutic agents include cis-diaminedichloroplatinum II (CDDP); Carboplatin; Anthracendione (e. g., Mitoxantrone); Hydroxyurea; Procarbazine (N-methylhydrazine); and adrenocortical suppressants (e. g., Mitotane or aminoglutethimide).

Other chemotherapeutic agents include adrenocorticosteroids (e. g., Prednisone); progestins (e. g., Hydroxyprogesterone caproate,; Medroxyprogesterone acetate, Megestrol acetate, etc.) ; estrogens (e. g., diethylstilbestrol ; ethenyl estradiol, etc.) ; antiestrogens (e. g. Tamoxifen, etc.) ; androgens (e. g., testosterone propionate, Fluoxymesterone, etc.) ; antiandrogens (e. g., Flutamide) ; and gonadotropin-releasing hormone analogs (e. g., Leuprolide, Cetrolelix or Abarelix).

In addition, the method may be used in conjunction with biologically active anti- cancer agents and apoptosis inducing agents such as TNF, TRAIL or Fas or with antibodies, small molecules or pharmacophores which bind these receptors and also induce apoptosis.

4. Applications In summary, the compositions and methods of the present invention provide a number of clinical and diagnostic benefits. For example, using the methods of the invention, the response of individual patients to established therapeutic anti-cancer regimens may be efficiently and timely evaluated; the anti-neoplastic activity of new anti-cancer drugs may be evaluated; the optimal dose and dosing schedules for new anti- cancer drugs may be identified; and the optimal dose and dosing schedules for existing anti-cancer drugs and drug combinations may be identified. In addition, using the methods of the invention, cancer patients in clinical trials may be categorized efficiently into responders and non-responders to therapeutic regimens.

The methods of the invention provide, among other things, a non-invasive technique for evaluating the early response of individual patient tumors to chemotherapy. This facilitates the selection of effective treatment by allowing rapid identification of ineffective treatments whose side effects might not be balanced by expected benefits.

Major uses for the annexin containing compositions of the invention include the detection of inappropriate apoptosis in disease states where it should not occur, e. g.,

immune disorders such as Lupus, transplant rejection, or in cells subject to severe ischemia; and the detection of insufficient apoptosis when it should occur, e. g., tumors or cells infected with a virus.

The annexin containing compositions of the invention may be employed in a variety of clinical settings in which apoptotic and/or necrotic cell death need to be monitored, such as, without limitation, organ and bone marrow transplant rejection or injury, infectious and non-infectious inflammatory diseases, autoimmune disease, cerebral and myocardial infarction and ischemia, cardiomyopathies, atherosclerative disease, neural and neuromuscular degenerative diseases, sickle cell disease, 0-thalassemia, cancer therapy, AIDS, myelodysplastic syndromes, and toxin-induced liver disease, and the like. The annexin containing compositions of the invention may also be useful as a clinical research tool to study the normal immune system, embryological development, and immune tolerance and allergy.

The annexin containing compositions of the invention can be used, for example, to image and quantify apoptotic cell death in normal and malignant tissues undergoing treatment. Monitoring apoptosis with serial imaging studies using these compounds can be used for the rapid testing and development of new drugs and therapies in a variety of diseases. In addition, the methods may be used to monitor the progress of treatment, monitor the progress of disease, or both. Further, they may be used to aid in early detection of certain diseases.

An advantage of the above method is that, by imaging at selected intervals, the method can be used to track changes in the intensity of the emission from the subject over time, reflecting changes in the number of cells undergoing cell death. Such an approach may also be used to track changes in the localization of the compositions of the invention in the subject over time, reflecting changes in the distribution of cells undergoing cell death.

The invention is further illustrated by the following Example which should not be construed as limiting. All patents, patent applications, and literature references cited herein are hereby expressly incorporated by reference.

Exemplification of the Invention Example 1: Preparation of Hynic-Annexin V rh-Annexin V was concentrated to between 1-5 mg/mL and modified with a 10-fold molar excess of succinimidyl 6-hydrazinopyridine-3-carboxylate

hydrochloride (Hynic) in 100 mM phosphate buffer at pH 7.4. After a 2 hour incubation with gentle stirring, the sample was purified on a Sephadex G-25 gel permeation column using 113 mM Tricine buffer. Paranitrobenzaldehyde reaction (see King et al, Biochemistry, 25: 5774 (1986)) indicated a derivatization of 0.3 to 0.8 mol of Hynic per mol of Annexin V.

Preparatio) z of 18F-Annexin V Method 1: To 0.5 mg of Hynic-Annexin V protein in a 0.1 M phosphate pH 5.2 buffer is added 0.5 mL of 18F Fluorodeoxyglucose, FDG (0.5 to 15 mCi). The reaction mixture is incubated at room temperature for up to 60 min. The product is then purified by passing on a gel permeation column (PD-10) using phosphate buffered saline, and is collected in 1 mL fractions, in greater than 80% radiochemical purity as determined by ITLC SG using a citric acid dextrose mobile phase. The product is then injected in an animal with B cell lymphoma treated with cyclophosphamide (100 mg/Kg) and used to image the area of the treated tumor (see Blankenberg et al., Proc. Natl. Acad. Sci. USA, 95: 6349 (1998)).

Method 2: To 0.5 mL of 18F Fluorodeoxyglucose, FDG (0.5 to 15 mCi) is added 0.1 mL of a 1 mg/mL solution of sodium periodate freshly prepared in water. 0.5 mg of Hynic-Annexin V is added and the reaction is incubated for up to 60 min. The product is then purified by passing on a gel permeation column (PD-10) using phosphate buffered saline, and is collected in 1 mL fractions, in greater than 80% radiochemical purity as determined by ITLC SG using citric acid dextrose mobile phase. The product is then injected in an animal with B cell lymphoma treated with cyclophosphamide (100 mg/Kg) and used to image the area of the treated tumor.

Example 2: rh-Annexin is coupled to biotin using the Hnatowich et al approach (D. J.

Hnatowich et al., J. Nucl. Med., 28: 1294-1302 (1987) ). Avidin is then added to the biotin-Annexin to form the targeting Avidin-Annexin. Hynic-Biotin is prepared by reacting a solution of 10 mg/mL biocytin, a lysine conjugate of biotin with an available primary amine for conjugation, with a 1: 1 molar ratio of succinimidyl 6- hydrazinopyridine-3-carboxylate hydrochloride (Hynic) in 100 mM bicarbonate pH 8.2, aliquoted and is kept frozen until use.

To 0.5 mL of F-18 Fluorodeoxyglucose, FDG (0.5 to 15 mCi) is added 0.1 mL of a 1 mg/mL solution of sodium periodate freshly prepared in water. 0.1 mg of Hynic- Biotin is added and the reaction is incubated for up to 60 min, using a waterbath at 80oC if necessary. The reaction mixture is then mixed with Avidin-Annexin, and the product is purified by passing on a gel permeation column (PD-10) using phosphate buffered saline, and is collected in 1 mL fractions. The amount of radioactivity on the Annexin product is determined by ITLC SG using citric acid dextrose mobile phase or by gel permeation chromatography. The product is then injected in an animal with B cell lymphoma treated with cyclophosphamide (100 mg/Kg) and used to image the area of the treated tumor (as above).

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.