WO2012094334A1 - One-step 18f labeling of biological substrates and uses of 18f labeled biological substrates - Google Patents

Abstract

Disclosed is a rapid one-step ¹⁸F radiolabeling method for biological substrates that contain a specific arene group activated for nucleophilic aromatic substitution with ¹⁸F. The method involves reacting a biological substrate having at least one 4-nitro-3-trifluorobenzamide moiety covalently bonded thereto with a ¹⁸F anion in the presence of a cryptand. The method of the present invention has one or more advantages: significantly shortens reaction and overall synthesis time; requires low amount of precursor; and provides acceptable yields of high specific activity product. Also disclosed are methods of identifying a cell or population of cells expressing a protein or peptide of interest and methods for diagnosing a disease in a subject. Further disclosed are ¹⁸F labeled compounds of formula (I): wherein X, n, R₁, R₂, and R₃ are as described herein.

Description

ONE-STEP ¹⁸F LABELING OF BIOLOGICAL SUBSTRATES AND USES OF ^{l 8}F

LABELED BIOLOGICAL SUBSTRATES

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent

Application No. 61/429,671 , filed January 4, 201 1, the disclosure of which is incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Labeling of biological substrates, such as peptides, proteins and other biomolecules with ¹⁸F traditionally has been accomplished in several radiosynthetic steps, using prosthetic groups which can be attached to the biological substrate via acylation, alkylation, amidation, imidation, oxime or hydrazone formations. The choice of the prosthetic (precursor) group for the labeling must take into account the complexity of its radiosynthesis, which generally requires several lengthy radiosynthetic steps, and therefore results in relatively low labeling yield. In addition, multi-step synthesis is also not amenable for automation.

[0003] One alternative route for labeling biological substrates is 1 ,3-dipolar cycloaddition of terminal alkynes and azides in the presence of Cu(I) as a catalyst, to give the corresponding triazole (click chemistry). 1 8 F-click chemistry has been used in several studies for labeling peptides. However, it requires the preparation of azide or alkyne functional group modified peptides, two radiochemical synthesis steps, and in some cases, it involves volatile 18 F-azide synthon.

[0004] There have been attempts at direct one-step labeling of peptides via 1 8 F-fluoride nucleophilic aromatic substitution using trimethyl ammonium as a leaving group. These prior art methods, such as displacement of aryl trimethyl ammonium moieties with ^I8F- fluoride, can result in the formation of dimethylamino aryl-conjugated peptide analogs as a by-product, which may not be separable by high-performance liquid chromatography (HPLC), leading to low chemical purity, and low effective specific activity of the labeled peptide. Other methods, such as one-step labeling with F-fluoride via ring opening of activated aziridines have been reported, but the F incorporation yield is rather low. [0005] The foregoing shows there still exists an unmet need more efficient methods for labeling biological substrates such as peptides and other biomolecules with F.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a novel, one-step method of labeling of biomolecules with ¹⁸F, by displacing a nitro group in an arene that is activated toward nucleophilic aromatic substitution by an ortho trifiuoromethyl group. The new labeling method of the present invention can be applied to any biological substrate, containing 4-

18

nitro-3-trifluoromethyl arene, as precursors for the direct F labeling.

[0007] In accordance with the present invention, the present invention provides a method of labeling a biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety, with a ¹⁸F anion in the presence of a cryptand molecule, to obtain the ¹⁸F biological substrate. This labeling method requires relatively low amounts of precursor and a short time of radiosynthesis, when compared to other direct peptide- labeling methods. In an embodiment, the method of the present invention is performed in the presence of a cryptand molecule, preferably, the cryptand molecule comprises

4,7, 13 , 16,21 ,24-hexaoxa- 1 , 10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222).

[0008] In another embodiment, the present invention provides a method of labeling a biological substrate, wherein the method includes an additional step of purifying the labeled biological substrate after reacting the biological substrate with the cryptand.

[0009] In a further embodiment, the present invention provides a method of preparation of a biological substrate labeled with ¹⁸F comprising reacting a biological substrate having at least one free amino group with a 4-nitro-3-trifluoromethylbenzoyl halide, obtaining a biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto, reacting the biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto with ¹⁸F anion in the presence of a cryptand and obtaining a ¹⁸F labeled biological substrate, and optionally purifying the ¹⁸F labeled biological substrate. In another embodiment, the reaction is heated to between about 100 °C and about 150 °C, preferably between about 120 °C and about 145 °C, and more preferably to about 130 °C. In accordance with the present invention, in an embodiment, the duration of the heating is between about 1 minute and 10 minutes.

[0010] In an embodiment, the present invention also provides a method of identifying a cell or a population of cells expressing a protein or peptide of interest comprising: contacting the cell or a population of cells expressing a protein or peptide of interest with a ^{l 8}F labeled targeting agent which selectively binds said protein or peptide of interest, wherein said targeting agent is made using the methods of the present invention, obtaining a

18

diagnostic image of the cell or population of cells, quantifying the amount of F labeled targeting agent molecule bound to the cell or population of cells, and correlating the amount of the bound ¹⁸F labeled targeting agent molecule with the amount of the protein or peptide of interest on the cell or population of cells. In another embodiment, the cell or population of cells expressing a protein or peptide of interest is a tumor cell or tumor cells.

[0011] In a further embodiment, the method of identifying a cell or a population of cells expressing a protein or peptide of interest, further provides that detecting the presence of the ¹⁸F labeled targeting agent binding said protein or peptide of interest on the cell or population of cells is performed by MRI, PET or SPECT imaging.

[0012] In an embodiment, the present invention also provides a method of diagnosing a disease in a patient comprising administering to a subject suspected of having said disease, a ¹⁸F labeled targeting agent made according to the above described methods of the present invention, which selectively binds a protein or peptide of interest in the subject's disease, obtaining a diagnostic image of the subject, determining the location of ¹⁸F labeled targeting agent bound to the protein or peptide of interest in the subject, correlating the location of the bound ¹⁸F labeled targeting agent with the location of the protein or peptide

18

of interest in the subject, and correlating the quantity of the bound F labeled targeting agent with the presence of disease in the subject.

[0013] In another embodiment, the present invention provides a compound of formula

(I):

(i); wherein Ri is a moiety derived from the biological substrate by covalently linking the biological substrate to the C=0 group through X, and X is O, NR', S, CH₂, NR'CO, or NR'CONH, where R'=H or lower alkyl, and where n=l to 100. R₂ is an electron

withdrawing group, including N0₂, CHO, COOR", COR", NR"₃+, wherein R"=H or lower alkyl, and CX'₃, wherein X' is F, CI or Br. R₃ is either H or an electron withdrawing group, including N0₂, CHO, COOR", COR", NR"₃+, wherein R"=H or lower alkyl, and CX'₃, wherein X' is F, CI or Br. In a preferred embodiment, R₂ is CF₃ and R₃ is H. In the compounds disclosed herein, including, e.g., formula I, examples of the lower alkyl are Ci_-6 alkyl group (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, etc.) and the like.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0014] Figure 1 is a schematic illustration of an embodiment of the method of the present invention. The figure depicts one-step F-labeling of RGD peptides 1 and 3.

[0015] Figure 2A shows HPLC (UV at 218 nm) and radioactivity chromatograms of peptide F-2 (injection of crude reaction products). Peptide F-2 eluted at retention time of 17.04 min. Figure 2B shows HPLC (UV at 218 nm) and radioactivity chromatograms of peptide F-4 (injection of crude reaction products). Peptide F-4 eluted at retention time of 12.75 min.

[0016] Figure 3 depicts % bound I-echistatin as a function of concentration in a competitive binding assay of RGD-modified peptides (peptides 2 and 4) in comparison to the non-modified peptides (c(RGDf ) (peptide 1) and E[c(RGDfK)]₂) (peptide 3) with ¹²⁵I- echistatin on MDA-MB-435 cells.

[0017] Figure 4 depicts representative PET images of an athymic nude mouse bearing orthotopic MDA-MB-435 tumor on the left mammary fat pad, at 0.5, 1 and 2 hours post- injection of 100 μθΐ (3.7 MBq) of peptide ¹⁸F-4, or co-injection with 300 μg of c(RGDfK). The upper row depicts ventral slices, and the arrows indicate MDA-MB-435 tumor. The lower row depicts dorsal slices.

[0018] Figure 5 A depicts the biodistribution of peptide ¹⁸F-4 in MDA-MB-435 tumor- bearing mice at 0.5, 1 , and 2 hours post-injection of the labeled peptide. Figure 5B depicts biodistribution of mice injected either with peptide F-4 or co-injection of peptide F-4 with nonradioactive c(RGDfK) (300 μg), 0.5 hour post-injection. Results are calculated from PET scans and are shown as averages of 5 mice ± SD, *P < 0.01 . Figure 5C depicts redistribution of peptide 1 8 F-4 (calculated from gamma counting of dissected organs) in MDA-MB-435 tumor-bearing mice at 2 hours post-injection of the labeled peptide. Results are shown as averages of 5 mice ± SD.

DETAILED DESCRIPTION OF THE INVENTION

[0019] In accordance with an embodiment, the present invention provides a method of labeling a biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto, with a ^{! 8}F anion (or a salt, e.g., alkali metal salt, of the ¹⁸F anion such as K¹⁸F) in the presence of a cryptand molecule, to obtain the ⁱ⁸F labeled biological substrate.

[0020] It will be understood that the term "biological substrate" as used herein can encompass various naturally-occumng, semi-synthetic, or synthetic molecules, such as, for example, is an amino acid, peptide, peptidomimetic, oligopeptide, protein, aptamer, oligonucleotide, DNA, RNA, antibody, receptor binding molecules, molecules having a receptor motif, haptens or small molecule, having a free amino group available to react with the 4-nitro-3-trifluoromethylbenzoyl halide in the methods of the present invention. In addition, the term "biological substrate" as used herein can encompass a peptide comprising from 4 to 100 amino acids.

[0021] In accordance with an embodiment of the invention, peptides or other biological substrates having reactive groups such as the phenolic OH of tyrosine can be modified to include ¹⁸F provided that these reactive groups are protected prior to carrying out the ^!8F fluorination reaction.

[0022] In accordance with an embodiment, the method of the present invention

1 8

provides a method of preparation of a biological substrate labeled with F comprising reacting a biological substrate wherein a biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto is:

(F-l) or

[0023] In accordance with an embodiment, the term "biological substrate" as used herein can also encompass placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate-specific membrane antigen, a-fetoprotein, B-lymphocyte surface antigen, truncated EGFR, idiotypes, gp95/gp97, N-CAM, cluster w4, cluster 5A, cluster 6, FLAP, CA-125 (lung and ovarian cancers), ESA, CD19, 22 or 37, 250 kD proteoglycan, P55, TCR-IgH fusion, blood group A B or O antigen, bombesin, somatostatin receptor specific peptides, somatostatin, the derivatives and related peptides thereof, neuropeptide Y, neuropeptide Yi, the derivatives and related peptides thereof, gastrin, gastrin releasing peptide, the derivatives and related peptides thereof, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1 , integrins (α₃βι, α_νβ₃, α_νβ₅, allb₃), LHRH agonists and antagonists, transforming growth factors, particularly TGF-a, angiotensin, cholecystokinin receptor peptides, cholecystokinin (CCK) and the analogs thereof; neurotensin and the analogs thereof, thyrotropin releasing hormone, pituitary adenylate cyclase activating peptide (PACAP) and the related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and the analogs thereof, tumor necrosis factor, interleukins (IL-1 , IL-2, 1L-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) RGB peptide, substance P, neuromedin-B, neuromedin-C, octreotide analogues, metenkephalin, neurotensin, melanocyte stimulating honrione and analogs thereof, follicle stimulating hormone and analogs thereof, luteinizing hormone and analogs thereof, human growth honrione and analogs thereof, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon,

erythropoietin, and colony-stimulating factor.

[0024] Analogs described above can include any suitable modification in the biological substrate, for example, where one or more amino acids of a peptide is replaced or substituted by a different amino acid or a different set of amino acids. For example, the replacement can be a replacement of a nonpolar side chain with another nonpolar side chain, a polar or neutral side chain with another polar or neutral side chain, an acidic side chain with another acidic side chain, and/or a basic side chain with another basic side chain.

[0025] As used herein, a "cryptand molecule" is a molecular entity comprising a cyclic or polycyclic assembly of binding sites that contains three or more binding sites held together by covalent bonds, and which defines a molecular cavity in such a way as to bind (and thus 'hide' in the cavity) another molecular entity, the guest (a cation, an anion or a neutral species), more strongly than do the separate parts of the assembly (at the same total concentration of binding sites). The adduct thus formed is called a 'cryptate'. Examples of cryptand molecules are bicyclic or oligocyclic molecular entities.

[0026] In accordance with an embodiment, the method further comprises heating the biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety in the presence of the ¹⁸F anion and the cryptand molecule. In another embodiment the reaction is heated to between about 100 °C and about 150 °C, preferably between about 120 °C and about 145 °C, and more preferably to about 130 °C.

[0027] In accordance with an embodiment, the duration of the heating is between about 1 minute and 10 minutes, for example, 2, 3, 4, 5, 6, 7, 8, or 9 minutes.

[0028] In an embodiment, the cryptand molecules complex the cation of the 1 8 F containing salt. Examples of cryptand molecules used in the methods of the present invention include 4,7,13, 18-tetraoxa-l ,10-diazabicyclo[8.5.5]-eicosane, 4,7,13,16,21 - pentaoxa-l ,10-diazabicyclo[8.8.5]-tricosane, (Kryptofix 221), 4,7, 13, 16,21 , 24-hexaoxa- l ,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222), and 4,7,10,16,19,24,27-heptaoxa- 1 ,13-diazabicyclo[ l 1 .8.8]-nonacosane. In a preferred embodiment, the cryptand molecule is 4,7, 13, 16,21 ,24-hexaoxa- l , 10-diazabicyclo[8.8.8]-hexacosane ( ryptofix 222).

[0029] In another embodiment, the present invention provides a method of labeling a biological substrate, wherein the method includes an additional step of purifying the labeled

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biological substrate after reacting the biological substrate with the cryptand, F anion, and heating same.

[0030] In a further embodiment, the method of the present invention provides a method of preparation of a biological substrate labeled with F comprising reacting a biological substrate having at least one free amino group with a 4-nitro-3-trifiuoromethylbenzoyl halide, obtaining a biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto, reacting the biological

18

substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety with F anion in

I

the presence of a cryptand and obtaining a F labeled biological substrate, and optionally

18

purifying the F labeled biological substrate.

[0031] In an embodiment, the invention provides a method of preparation of a

18

biological substrate labeled with F comprising reacting a biological substrate wherein a biological substrate having at least one free amino group is:

(F-l ') or

[0032] In an embodiment, the present invention provides a method of labeling a biological substrate, wherein the biological substrate is selected from the group consisting of nucleic acid, a nucleic acid targeting a complementary nucleic acid, aptamer, oligonucleotide, DNA, and RNA.

[0033] In another embodiment, the reaction is heated to between about 100 °C and about 150 °C, preferably between about 120 °C and about 145 °C, and more preferably to about 130 °C. In accordance with the present invention, in a further embodiment, the duration of the heating is between about 1 minute and 10 minutes, preferably from 2 to 8 minutes, and more preferably about 3.5 minutes.

[0034] In accordance with the present invention, in another embodiment, the present invention provides a method of labeling a biological substrate, wherein reaction step is performed in the presence of a cryptand. In a further embodiment, the cryptand molecule is 4,7,13,16,21 ,24-hexaoxa- 1 , 10-diazabicyclo [8.8.8] -hexacosane.

[0035] It is contemplated, in accordance with the present invention, that in an

embodiment, the biological substrate is a targeting agent. By "targeting agent" is meant any molecule that enables specific interaction with a target. The targeting agent can bind to a defined target population of cells, for example, through a receptor, a substrate, an antigenic determinant, or another binding site on the target cell population. Cell-surface molecules that are cancer specific antigens (or disease-specific antigens) can serve as targets.

[0036] Examples of a targeting agent include an "immunological agent," which is used herein to refer to an antibody, such as a polyclonal antibody or a monoclonal antibody, an immunologically reactive fragment of an antibody, an engineered immunoprotein and the like, a protein (target is receptor, as substrate, or regulatory site on DNA or RNA), a peptide (target is receptor), a nucleic acid (target is complementary nucleic acid), a steroid (target is steroid receptor), and the like. Derivatives and analogs of targeting agents that retain the ability to bind to a defined target can be used. Synthetic targeting agents can be designed, such as to fit a particular epitope.

[0037] In an embodiment, the present invention provides a method of labeling a biological substrate, wherein the biological substrate is selected from the group consisting of an antibody (e.g., scFv, F(ab^')₂, and Fab), a monoclonal antibody or monoclonal antibody fragment, an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A12 Ab, H17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B-lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab, cetuximab and an affibody.

[0038] Examples of cancer-specific, cell-surface molecules include placental alkaline phosphatase (testicular and ovarian cancer), pan carcinoma (small cell lung cancer), polymorphic epithelial mucin (ovarian cancer), prostate-specific membrane antigen, a- fetoprotein, B-lymphocyte surface antigen (B-cell lymphoma), truncated EGFR (gliomas), idiotypes (B-cell lymphoma), gp95/gp97 (melanoma), N-CAM (small cell lung carcinoma), cluster w4 (small cell lung carcinoma), cluster 5A (small cell carcinoma), cluster 6 (small cell lung carcinoma), FLAP (seminomas, ovarian cancer, and non-small cell lung cancer), CA-125 (lung and ovarian cancers), ESA (carcinoma), CD19, 22 or 37 (B-cell lymphoma), 250 kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion (childhood T-cell leukemia), blood group A antigen in B or O type individual (gastric and colon tumors), and the like.

[0039] Examples of cancer-specific, cell-surface receptors also include erbB-2, erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL-4 (lymphoma and leukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin (gliomas), tumor vasculature integrins, and the like. Examples of cancer specific, cell-surface receptors further include erbB-2 and tumor vasculature integrins, such as CD1 la, CD1 lb, CD1 l c, CD18, CD29, CD51 , CD61 , CD66d, CD66e, CD106, and CDwl45.

[0040] For example, erbB-2 receptor has been found in breast, ovarian, gastric, salivary gland and adeno-carcinomas and in non-small cell carcinomas of the lung. Over-expression of the erbB-2 receptor on such cancers has been found to correlate with poor prognosis. In vitro studies strongly suggest that over-expression of erbB-2 plays an important role in tumor progression. [0041] An example of a single-chain antibody scAb targeting agent is that which binds c- erbB-2 (see WO 93/16185). See, also, WO 93/21232 and H. Zola, Monoclonal Antibodies, BIOS Scientific Publishers, Oxfordshire, England (November 1994) for antibody sequences that can be used to construct scAbs.

[0042] A number of antibodies to cancer-specific, cell-surface molecules and receptors are known. C46 Ab (Amersham) and 85A12 Ab (Unipath) to carcino-embryonic antigen, H17E2 Ab (ICRF) to placental alkaline phosphatase, NR-LU-10 Ab (NeoRx Corp.) to pan carcinoma, HMFC1 Ab (ICRF) to polymorphic epithelial mucin, W14 Ab to B-human chorionic gonadotropin, RFB4 Ab (Royal Free Hospital) to B-lymphocyte surface antigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) to human melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP 7059588), and the like. ScAbs can be developed, based on such antibodies (see, for example, Bind et al., Science, 1988, 242, 423-426, and Whitlow et al., Methods, 1991 , 2(2), 97-105).

[0043] Examples of binding domains which can be used in targeting agents include the EOF domain of a-heregulin, a-integrin domain, tumor vasculature peptide motifs. Alpha- heregulin is a ligand with affinity for breast cancer cells expressing the human epidermal growth factor receptors erbB-2, erbB-3 and erbB-4. Heregulin can interact indirectly with erbB-2 via heterodimerization with erbB-3 or erbB-4.

[0044] The ¹⁸F labeled biological substrate targeting agents of the present invention, in an embodiment, can include any biological substrate with one, two, three, or more free amino groups, e.g., a free amino group, or a biological substrate where an amino group or groups can be added or introduced. In an embodiment, for example, the targeting agent of the present invention can be a protein targeting a receptor, a receptor antagonist, a protein targeting a substrate, a protein targeting a regulatory site on DNA, or its component nucleotides, adenine, guanine, cytosine and thymidine, or RNA or its component nucleotides, adenine, guanine, cytosine and uracil, a peptide, bombesin, gastrin-releasing peptide, RGB peptide, substance P, neuromedin-B, neuromedin-C, somatostatin, octreotide analogues, metenkephalin, a nucleic acid, a nucleic acid targeting a complementary nucleic acid, a steroid, a hormone, estradiol, neurotensin, melanocyte stimulating hormone, follicle stimulating hormone analogs, luteinizing hormone, human growth hormone, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon, erythropoietin, colony-stimulating factor, a glycoprotein, a glycolipid, a carbohydrate, a small molecular weight cell surface receptor agent, a growth factor hormone, epinephrine, an epinephrine derivative, a histamine, a prostaglandin, or a derivative thereof. In a preferred embodiment, the ¹⁸F labeled targeting agent is an antibody (e.g., scFv, F(ab^')₂, and Fab), a peptide, or a protein. For example, the F labeled targeting agent can be an antibody that binds with tumor cells. The monoclonal antibody or monoclonal antibody fragment can be related to an antibody such as an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A12 Ab, H17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B-lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab (e.g., Herceptin™) and cetuximab (e.g., Erbitux™, ImClone and Bristol-Myers-Squibb).

[0045] The ¹⁸F labeled targeting agents of the present invention can be affibodies. As used herein, the term Affibody^® (hereinafter "affibody") means molecules which are small highly robust proteins with specific affinities to target proteins. They can be designed and used, for example, like aptamers. Affibody molecules in accordance with the invention comprise a backbone derived from an IgG-binding domain of Staphylococcal Protein A (Protein A produced by S. aureus). The backbone can be derived from an IgG binding domain comprising the three alpha helices of the IgG-binding domain of Staphylococcal Protein A termed the B domain. The amino acid sequence of the B domain is described in Uhlen et al., J. Biol. Chem. 259: 1695-1702 (1984). Alternatively, the backbone can be derived from the three alpha helices of the synthetic IgG-binding domain known in the art as the Z domain, which is described in Nilsson et al., Protein Eng. 1 : 107-1 13 (1987). The backbone of an affibody comprises the amino acid sequences of the IgG binding domain with amino acid substitutions at one or more amino acid positions.

[0046] ¹⁸F labeled affibody molecules with specificity and selectivity for tumor markers can be used for non-invasive early detection of tumors, and to monitor disease by detecting tumor progression or regression in response to cancer therapy. The small size of the affibody molecules, compared to larger molecules, such as antibodies, gives access into solid tumors and rapid clearance from the blood stream. The affibody molecule constitutes a highly suitable carrier for directing radioisotopes and other toxins to tumor cells due to specific target binding and lack of irrelevant interactions, such as the Fc receptor binding displayed by some antibodies. High contrast tumor images can be visualized given the strong and/or specific binding of the ¹⁸F labeled affibody molecules in accordance with the embodiments of the present invention.

[0047] In an embodiment, the present invention also provides a method of identifying a cell or a population of cells expressing a protein or peptide of interest comprising contacting the cell or a population of cells expressing a protein or peptide of interest with a ¹⁸F labeled targeting agent which selectively binds said protein or peptide of interest, wherein said targeting agent is made in accordance with the above methods, obtaining a diagnostic image of the cell or population of cells, quantifying the amount of 18 F labeled targeting agent molecule bound to the cell or population of cells, and correlating the amount of the bound ¹⁸F labeled targeting agent molecule with the amount of the protein or peptide of interest on the cell or population of cells. In another embodiment, the cell or population of cells expressing a protein or peptide of interest is a tumor cell.

[0048] Accordingly, an embodiment of the present invention provides a method for obtaining a diagnostic image in a subject or patient. In particular, an embodiment of the

18

method comprises administering to the subject or patient, an F labeled targeting agent made using the methods of the present invention, in an amount effective to provide an image; and exposing the subject or patient to an energy source, whereupon a diagnostic image in the subject or patient is obtained. The diagnostic image can be, for example, magnetic resonance imaging (MRI), single photon emission computed spectroscopy (SPECT) image, positron emission tomography (PET) image, or the like.

[0049] Embodiments of the methods of the present invention can be used to prepare F labeled targeting agents or other labeled biological substrates to image cells, such as cancer cells, in the subject or patient. In an embodiment, the present invention provides a method of diagnosing a disease in a patient comprising administering to a subject suspected of having said disease, a ¹⁸F labeled targeting agent prepared according to the above methods, which selectively binds a protein or peptide of interest, obtaining a diagnostic image of the subject, determining the location of F labeled targeting agent bound to the protein or peptide of interest in the subject, and correlating the location of the bound 1 8 F labeled targeting agent with the location of the protein or peptide of interest in the subject. The spectroscopy can be, for example, SPECT, PET, gamma scintigraphy, or MRI. Preferably, the ¹⁸F labeled targeting agent is bound to a receptor on the surface of a cancer cell. In accordance with the present invention, it is contemplated that the above method of diagnosis of disease includes methods where the protein or peptide of interest is associated with tumor growth and the disease is cancer.

[0050] In accordance with an embodiment, the present invention provides a method of diagnosing a disease in a patient comprising obtaining a diagnostic image of a subject whose tissue or organ includes a ¹⁸F labeled targeting agent, wherein said targeting agent is a ¹⁸F labeled biological substrate prepared according to the above methods.

[0051] In a further embodiment, the detection of the presence of the probe binding said protein or peptide of interest is performed by MRI, PET or SPECT imaging.

[0052] In accordance with an embodiment, the present invention provides a method of diagnosing a disease wherein the ¹⁸F labeled targeting agent is

(F-3").

[0053] In another embodiment, the present invention provides a compound of formula (I):

wherein Ri is a moiety derived from the biological substrate by covalently linking the biological substrate to the C=0 group through X, and X is O, NR', S, C¾, NR'CO, or NR'CONH, where R'=H or lower alkyl, and where n=l to 100. R₂ is an electron

withdrawing group, including N0₂, CHO, COOR", COR", NR"₃ ⁺ (e.g., quaternary ammonium), wherein R"=H or lower alkyl, and CX'₃, wherein X' is F, CI or Br. R₃ is either H or an electron withdrawing group, including N0₂, CHO, COOR", COR", NR"₃+, wherein R"=H or lower alkyl, and CX'₃, wherein X' is F, CI or Br. In a preferred embodiment, R₂ is CF₃ and R₃ is H. In embodiments, n = 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, and 90.

[0054] In the compounds disclosed herein, including, e.g., formula I, examples of the lower alkyl are Ci_₆ alkyl group (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, etc.) and the like. In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), wherein R] is derived from a biological substrate moiety. In a further embodiment, the biological substrate is a peptide and X is NH.

[0055] In another embodiment, the present invention provides a compound of formula (I) where the biological substrate is

[0056] In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), wherein the biological substrate (Ri) is a moiety derived from an amino acid, peptide, peptidomimetic, oligopeptide, protein, aptamer, oligonucleotide, DNA, RNA, antibody, receptor binding molecules, molecules having a receptor motif, haptens or small molecule. In another embodiment, the present invention provides a compound of formula (I), wherein the biological substrate is a peptide comprising from 4 to 100 amino acids, and preferably 10 to 90 amino acids.

[0057] In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), where R] is a moiety derived from placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate-specific membrane antigen, a-fetoprotein, B-lymphocyte surface antigen, truncated EGFR, gp95/gp97, N-CAM, cluster w4, cluster 5 A, cluster 6, FLAP, CA-125, ESA, CD 19, CD22, CD37, 250 kD proteoglycan, P55, TCR-IgH fusion, blood group A antigen, DNA, or its component nucleotides, adenine, guanine, cytosme and thymidine, or RNA or its component nucleotides, adenine, guanine, cytosine and uracil, bombesin, gastrin-releasing peptide, RGB peptide, substance P, neuromedin-B, neuromedin-C, somatostatin, octreotide analogues, metenkephalin, estradiol, neurotensin, melanocyte stimulating hormone, follicle stimulating hormone analogs, luteinizing hormone, human growth hormone, interferon, erythropoietin, or colony-stimulating factor.

[0058] In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), wherein the biological substrate (Ri) is a moiety derived from those selected from the group consisting of placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate-specific membrane antigen, a-fetoprotein, B-lymphocyte surface antigen, truncated EGFR, idiotypes, gp95/gp97, N-CAM, cluster w4, cluster 5 A, cluster 6, FLAP, CA-125 (lung and ovarian cancers), ESA, CD 19, 22 or 37, 250 kD proteoglycan, P55, TCR-IgH fusion, blood group A B or O antigen, bombesin, somatostatin receptor specific peptides, somatostatin, the derivatives and related peptides thereof, neuropeptide Y, neuropeptide Yi, the derivatives and related peptides thereof, gastrin, gastrin releasing peptide, the derivatives and related peptides thereof, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1 , integrins (α₃βι, α_νβ₃, α_νβ₅, allb₃), LHRH agonists and antagonists, transforming growth factors, particularly TGF-a, angiotensin, cholecystokinin receptor peptides, cholecystokinin (CCK) and the analogs thereof; neurotensin and the analogs thereof, thyrotropin releasing hormone, pituitary adenylate cyclase activating peptide (PACAP) and the related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and the analogs thereof, tumor necrosis factor, interleukins (IL-1, IL-2, IL-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) RGB peptide, substance P, neuromedin-B, neuromedin-C, octreotide analogues, metenkephalin, neurotensin, melanocyte stimulating hormone, follicle stimulating hormone and analogs thereof, luteinizing hormone and analogs thereof, human growth hormone and analogs thereof, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon, erythropoietin, and colony-stimulating factor.

[0059] In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), wherein the biological substrate moiety (Ri) a moiety derived from those selected from the group consisting of nucleic acid, a nucleic acid targeting a complementary nucleic acid, aptamer, oligonucleotide, DNA, and RNA.

[0060] In accordance with the present invention, in a further embodiment, the present invention provides a compound of formula (I), wherein the biological substrate is an antibody (e.g., scFv, F(ab^')₂, and Fab), a monoclonal antibody or monoclonal antibody fragment, an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A 12 Ab, H 17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B-lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab, cetuximab and an affibody.

[0061] In another embodiment, the present invention provides pharmaceutical composition comprising any of the above identified compounds of formula I, and a pharmaceutically acceptable carrier.

[0062] The ¹⁸F labeled biological substrates made by the methods of the present invention are suitably used as diagnostic agents for a wide variety of diseases, including, for example, various cancers and neurological diseases and brain disorders. According to another embodiment of the present invention, a method is provided for diagnosing various cancers or neurological diseases and brain disorders in a subject, comprising administering

18

to the subject, at least one F labeled biological substrate of the present invention and/or its analog, derivative, isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, or N-oxide, or any combination thereof, in an amount effective to diagnose the cancer or neurological disease and brain disorder in the subject.

[0063] In a further embodiment, the present invention provides a pharmaceutical composition comprising a compound of ¹⁸F labeled biological substrates made by the above described methods of the present invention and a pharmaceutically acceptable carrier.

[0064] In an embodiment, the amino acid or amino acid derivative of the biological substrate of the present invention can include a diamino carboxylic acid (i.e., an organic molecule that contains two amino groups and one carboxylic acid group). For example, the amino acid can be lysine or the amino acid derivative can be a lysine derivative.

Alternatively, the amino acid derivative can be a maleimide active ester-functionalized amino acid. For example, the maleimide active ester can be V-[y-maleimidobutyryloxy] succinimide ester, ra-maleimidobenzoyl-N-hydrosuccinimide ester, succinimidyl-4-[7V- maleimidomethyl] cyclohexane-l-carboxy-[6-amido caproate], succinimidyl-6-[P- maleimidopropionamido] hexanoate, succinimidyl-4-[p-maleimidophenyl] butyrate, Ν-[ε- maleimidocaproyloxyj-succinimide ester, succinimidyl-4-[jV-maleimidomethyl] cyclohexane-l-carboxylate, N-[P-maleimidopropyloxy] succinimide ester, N-[a- maleimidoacetoxy] succinimide ester, and m-maleimidoaryl-N-hydrosuccinimide ester (Pierce Chemical). The amino acid derivative can be a maleimidomethyl cyclohexane carboxylate functionalized lysine. [0065] The term "subject" used herein includes animals such as humans, sheep, horses, cattle, pigs, monkeys, dogs, cats, rats, mice and other mammals.

[0066] In another embodiment, the term "contacting" means that the at least one F labeled biological substrate of the present invention is introduced into a subject in need of diagnosis for a disease, such as cancer, or a neurological disease or brain disorders, and the at least one labeled biological substrate is allowed to come in contact with the target in vivo.

[0067] The term "reacting" in the context of the embodiments of the present invention means placing compounds or reactants in proximity to each other, such as in solution, in order for a chemical reaction to occur between the reactants.

[0068] In accordance with the present invention, in an embodiment the present invention provides a method of locating a receptor that is overexpressed in a tissue or organ of a subject comprising administering to the subject a 18 F labeled compound of formula I, which selectively binds a protein or peptide of interest, obtaining a diagnostic image of the tissue or organ, determining the location of F labeled compound of formula I bound to the

18

tissue or organ, and correlating the location of the bound F labeled compound of formula I with the location of the receptor in the subject. In another embodiment, the present invention provides a method of locating a receptor that is overexpressed in a tissue or organ of a subject comprising obtaining a diagnostic image of a subject whose tissue or organ

1

includes F labeled targeting agent prepared according to the above methods, determining the location of ¹⁸F labeled compound of formula I bound to the tissue or organ, and correlating the location of the bound ^{I 8}F labeled compound of formula I with the location of the receptor in the subject.

[0069] In an embodiment, the present invention provides a method of measuring the quantity of a receptor that is overexpressed in a tissue or organ of a subject comprising administering to the subject a 18 F labeled compound of formula I which selectively binds a protein or peptide of interest, obtaining a positron emission tomography (PET) image of the tissue or organ, determining the amount of F labeled compound of formula I bound to the tissue or organ, and correlating the amount of the bound ¹⁸F labeled compound of formula I with the quantity of receptor in the subject. Preferably, the quantity of 18 F labeled compound of formula I is administered in an amount effective to provide an image. In a further embodiment, the diagnostic image can be a positron emission tomography (PET) image, a magnetic resonance image (MRI), a computerized tomography (CT) scan, an x-ray contrast image, single photon emission computed spectroscopy (SPECT) image, or a combination thereof.

[0070] In a further embodiment, the present invention provides method of measuring the quantity of a receptor that is overexpressed in a tissue or organ of a subject comprising obtaining a positron emission tomography (PET) image of the tissue or organ of a subject whose tissue or organ includes ¹⁸F labeled targeting agent prepared according to the above methods, determining the amount of ¹⁸F labeled compound of formula I bound to the tissue or organ, and correlating the amount of the bound ^{l 8}F labeled compound of formula I with the quantity of receptor in the subject.

[0071] ¹⁸F has a half-life (t_½) of 1 10 minutes, emits β+ particles at an energy of 635 keV, and is 97% abundant. ¹⁸F can be obtained from cyclotrons after bombardment of ¹⁸0-

18

enriched water with protons. The enriched water containing H- F can be neutralized with a base having a counter-ion that is any alkali metal (M), such as potassium or another

18

monovalent ion, and the water can be evaporated off to give a residue of M- F, which can be taken up in an organic solvent for further use. In general, the counter-ion is selected to enable the fluoride ion to react rapidly in an organic phase with a leaving group, such as a nitro group. Potassium is generally used as a counter-ion because it is cheaper than cesium.

However, with carrier- free F, trivial amounts of counter-ion are required, and the counter- ion cost is minimal.

[0072] Cesium is useful as a counter ion since it is a larger ion with a more diffuse charge than potassium. Accordingly, cesium has weaker ionic interactions with the small fluoride atom, and therefore does not interfere with the nucleophilic properties of the fluoride ion. For similar reasons, potassium is preferred to sodium, and, in general, the suitability of a Group la metal as a counter-ion in accordance with the present invention increases down the periodic table. Group lb reagents, such as silver, also are useful as counter-ions.

Further, organic phase transfer-type ions, such as tetraalkylammonium salts, also can be used as counter-ions.

[0073] Fluoride salts can have a tendency to become hydrated and lose their nucleophilic character. To minimize this aspect, the labeling reaction is preferably performed under anhydrous conditions. For example, fluoride (as potassium fluoride or as a complex with any of the other counter-ions discussed above) can be placed in an organic solvent, such as acetonitrile or tetrahydrofuran. With the help of cryptand agents which bind to the counter- ion, such as Kryptofix 2.2.2 (4,7,13, 16,21 , 24-hexaoxa-l ,10-diazabicyclo[8.8.8]- hexacosane), the fluoride ion is very nucleophilic in these solvents.

[0074] In accordance with an embodiment of the invention, the diagnostic image can be an MRI (magnetic resonance imaging). When administered to a subject, the ] 8 F labeled compound of formula (I) distributes to different tissues, and catalyzes the relaxation of protons in the tissues that have been excited by the absorption of radiofrequency energy from a magnetic resonance imager. The acceleration of the rate of relaxation of the excited protons provides for an image of different contrast when the subject is scanned with a magnetic resonance imager. The magnetic resonance imager is used to record images at various times, generally either before and after administration of the 18 F labeled compound of formula (I) or after administration only, and the differences in the images created by the presence of the radiolabeled compound of formula (I) in tissues are used in diagnosis. Guidelines for performing imaging techniques can be found in Stark et al., Magnetic Resonance Imaging, Mosbey Year Book: St. Louis, 1992, hereby incorporated by reference.

[0075] Single Positron Emission Computed Tomography (SPECT) is a non-invasive imaging method to localize the position of a target such as a cancer metastasis, based on radioactive substances that emit gamma radiation when decaying.

[0076] A CT (computer tomography) scan provides anatomical detail, such as size and location of the tumor or mass. Digital geometry processing is used to generate a three- dimensional image of the internals of an object from a large series of two-dimensional X- ray images taken around a single axis of rotation. CT produces a volume of data which can be manipulated, through a process known as windowing, in order to demonstrate various structures based on their ability to block the X-ray beam. Combined techniques such as PET/CT and PET/MRI are also suitable for use in the invention.

[0077] PET (positron emission tomography) is a non-invasive imaging method to localize the position of a target such as a cancer metastasis. In PET, 51 1 keV gamma photons produced during positron annihilation decay are detected. A positron-emitting

radionuclide, such as 1 ^, 8⁰F, is introduced, usually by injection, and accumulates in the target tissue or organ. As it decays it emits a positron, which promptly combines with a nearby electron resulting in the simultaneous emission of two identifiable gamma rays in opposite directions. These are detected by a PET camera and give very precise indication of their origin. A PET scan can provide in vivo physiology such as metabolic detail (e.g., cellular activity) of the tumor or mass. The diagnosis is at a molecular level thereby providing detection of a tumor or mass at an early stage.

[0078] With respect to pharmaceutical compositions described herein, the

pharmaceutically acceptable carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use.

[0079] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0080] Solid carriers or diluents include, but are not limited to, gums, starches (e.g., com starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0081] For liquid formulations, phamiaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0082] Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, com oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0083] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or

intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0084] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0085] In addition, in an embodiment, the labeled peptides, proteins, and biomolecules or derivatives thereof, of the present invention may further comprise, for example, binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium,

crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., cremophor, glycerol, polyethylene glycerol, benzlkonium chloride, benzyl benzoate, cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates), and/or adjuvants.

[0086] The choice of earner will be determined, in part, by the particular biological substrate, as well as by the particular method used to administer the same. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the peptide, protein or biomolecule, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0087] Suitable soaps for use in parenteral formulations include, for example, fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include, for example, (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- -aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0088] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the peptide, protein or biomolecule, in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants, for example, having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include, for example, polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

[0089] The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0090] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15^th ed., pages 622-630 (2009)).

[0091] For purposes of the invention, the amount of the labeled biological substrate administered should be sufficient to produce a diagnostic response in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular labeled biological substrate and the condition of a human, as well as the body weight of a human to be diagnosed. The "effective amount" can be defined, for example, as the amount sufficient to allow the target tissue, cell, or organ, or cell population to be visualized using one or more of the various imaging techniques described herein.

[0092] The dose of the labeled biological substrate, also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular biological substrate. Typically, an attending physician will decide the dosage of the labeled biological substrate to administer to the patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, and biological substrate, to be administered, route of administration, and the target tissue, or organ, cell, or cell population to be visualized. By way of example, and not intending to limit the invention, the dose of the labeled biological substrate, can be about 0.001 to about 1000 mg/kg body weight of the subject/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day.

[0093] The labeled biological substrate compositions of the present invention may also include incorporation of the active ingredients into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[0094] The following examples further illustrate the invention, but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0095] This example illustrates a methodology employed in carrying out embodiments of the present invention. Kryptofix 2.2.2 was purchased from EMD Chemicals (Darmstadt, Germany). All other solvents and chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO). c(RGDfK) and E[c(RGDfK)₂] were purchased from Peptide International

1 8

(Louisville, KY). ' -fluoride was obtained from the National Institutes of Health (NIH)

Clinical Center (CC) cyclotron facility from irradiation of an 1 8 O-water target by the

1 8 0(p,n) 18 F nuclear reaction. Ci₈ cartridges (Waters Corporation, Milford, MA) were each activated with 5 ml of ethanol and 10 ml of water. [0096] Mass spectrometric analysis. The LC/MS analysis employed a Waters LC-MS system (Waters, Milford, MA) that included an Acquity UPLC system coupled to the Waters Q-Tof Premier high resolution mass spectrometer. An Acquity BEH Shield RP18 column (150 x 2.1 mm) was employed for chromatography. Elution was achieved with a binary mixture of two components. Solution A was composed of 2 mM ammonium formate, 0.1% formic acid, and 5% acetonitrile (ACN); solution B was composed of 2 mM ammonium formate and 0.1% formic acid in ACN. The elution profile, at 0.35 ml/minute, had the following components: initial condition at 100% (v:v) A and 0% B; gradient 0-40% B over 5 minutes; isocratic elution at 40% B for an additional 5 minutes; 40-80% B over 2 minutes; and re-equilibrated with A for an additional 3 minutes. The retention time for peptides 1 and 3 were 9.8 minutes and 5.8 minutes, respectively. The retention time for

18 18

peptides F-2 and F-4 were 9.2 minutes and 5.4 minutes, respectively. The injection volume was 10 μΐ. The entire column elute was introduced into the Q-Tof mass spectrometer. Ion detection was achieved in ESI mode using a source capillary voltage of 3.5 kV, source temperature of 1 10°C, desolvation temperature of 200 °C, cone gas flow of 50 L/Hr (N₂), and desolvation gas flow of 700 L/Hr (N₂) (data not shown).

[0097] MDA-MB-435 Cell Culture. MDA-MB-435 cell line was purchased from American Type Culture Collection (ATCC) and grown in Leibovitz's L-15 medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) under a 100% air atmosphere at 37 °C.

[0098] Integrin α_νβ₃ receptor binding assay. MDA-MB-435 cells were scraped off and resuspended with binding buffer [25 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol, hydrochloride (Tris-HCl), pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 1 mM MnCl₂, and 0.1% bovine serum albumin (BSA)]. Incubation was conducted in a 96- well plate with a total volume of 200 μΙ_, in each well containing 2xl0⁵ cells, 0.02 μθι (0.74 kBq) ¹²⁵I-echistatin (Perkin-Elmer, Waltham, MA) and 0-5000 nM of c(RGDf ) or unlabeled peptide 2, or 0-500 nM of E[c(RGDfK)₂] or unlabeled peptide 4, for 2 hours on a shaker at room temperature. After incubation, cells were washed three times with cold phosphate buffer saline (PBS) with 0.1 % BSA. Thereafter, the plate was heated to 40 °C and dried. The dried filter membranes were punched off from the wells and collected in polystyrene culture test tube (12x75 mm).

[0099] Cell bound radioactivity was measured using a gamma counter (1480 Wizard 3, Perkin-Elmer). The IC50 values were calculated by nonlinear regression analysis using the GraphPad Prism computer-fitting program (GraphPad Software, Inc., San Diego, CA).

Each data point is a result of the average of duplicate wells.

[0100] Tumor xenograft model. Athymic nude mice were purchased from Harlan (Frederick, MD) and housed under pathogen free conditions. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals, and under protocols approved by the NIH Clinical Center Animal Care and Use Committee (CC/ACUC). The MDA-MB-435 tumor model was generated by orthotopical injection of 5x 10⁶ cells in the left mammary fat pad of female athymic nude mice. The mice were ready for use in 2-3 weeks after tumor inoculation when the tumor size reached 100-300 mm .

[0101] Small animal PET studies. Tumor-bearing mice were anesthetized using isoflurane/0₂ (1.5-2% v/v) and injected with 100 μθΐ (3.7 MBq) of ¹⁸F-4 (See Fig. 1). PET scans were performed using an Inveon DPET scanner (Siemens Medical Solutions, Malvern, PA) at 0.5, 1 and 2 hours post-injection (n = 5/group). For blocking experiments, 100 μθϊ (3.7 MBq) of ¹⁸F-4 were co-injected with 300 μg of c(RGDfK) (n = 5). The images were reconstructed by a two-dimensional ordered subsets expectation maximum (2D-OSEM) algorithm, and no correction was applied for attenuation or scatter. Image analysis was done using ASI Pro VM™ software (Siemens Medical Solutions).

[0102] Biodistribution. Each tumor-bearing mouse was injected 100 μθί (3.7 MBq) of peptide ¹⁸F-4 in a volume of 100 μΐ phosphate-saline buffer through the tail vein. At 2 hours post-injection after the PET scans were completed, blood was drawn from the heart and the mice were then sacrificed. Liver, muscle, kidneys, intestine and tumor were removed (n = 5). The organs were wet weighed and assayed for radioactivity using a gamma counter.

[0103] Statistical analysis. Results were expressed as mean ± SD. Two-tailed paired and unpaired Student's t tests were used to test differences within groups and between groups, respectively. P values <0.05 were considered statistically significant.

EXAMPLE 2

[0104] This example illustrates a method of synthesis of precursor peptides 1 and 3 (Fig. 1) in accordance with an embodiment of the invention. 4-Nitro-3-trifluoromethylbenzoyl chloride was synthesized via chlorination of the corresponding acid, using , a - dichloromethyl methyl ether as a chlorination agent (Kiesewetter, D. O., et al., Nucl. Med. Bio.1 30: 1 1 -24(2003)). The conversion to the desired benzoyl chloride was efficient, with chemical yield of 80%.

[0105] Synthesis of 4-nitro-3-trifluoromefhylbenzoyl chloride. 4-Nitro-3- trifluoromethybenzoic acid (1.06 g, 4.5 mmol) was treated with a, a-dichloromethyl methyl ether (0.6 ml, 6.75 mmol). One milliliter of dichloroethane was added, and the reaction was heated at 50 °C for 4 hours. Full conversion to the acid chloride was verified by GC-MS. The solvent and excess reagents were evaporated and the residual oil was distilled in a Kugelrohr apparatus (140° C, 1 mm) to give 0.9 g (3.55 mmol) of the desired 4-nitro-3- trifluoromethylbenzoyl chloride. The oil solidified upon standing and was used without any additional manipulations. 1H-NMR (300 MHz, CDC1₃): δ 8.04-7.99 (d, 2H), 8.52-8.43 (dd, 2H), 8.57 (d, 2H). GC-MS (CI-CH₄) 253.95 (M⁺).

[0106] Synthesis of 4-nitiO-3-trifluoromethylbenzoyl-c(RGDfK) (peptide 1) and 4-nitro- 3-trifluoromethylbenzoyl-E[c(RGDfK)₂] (peptide 3, Fig. 1 ). The conjugation procedure of 4- nitro-3-trifluoromethylbenzoyl chloride with monomeric RGD peptide c(RGDf ) and dimeric RGD peptide E[c(RGDfK)]₂ was conducted by dissolving the peptide (8-10 mg) in 0.3-0.5 ml Ν,Ν-dimethylformamide. Then, 1.2 eq. of 4-nitro-3-trifluoromethylbenzoyl chloride were added, followed by adding 10 eq. of triethylamine. The reaction was mixed at room temperature for several hours. Purifications of peptides 1 and 3 were conducted on a reversed-phase HPLC system using a Higgins Cj₈ column (5 μηι, 20 x 250 mm). The flow was set at 12 ml/minute using two gradient systems; for peptide 1 , starting from 95% of solvent A (0.1 % TFA in water) and 5% of solvent B (0.1 % TFA in ACN) and increasing to 35% solvent A and 65% solvent B at 32 minutes. The retention time of peptide 1 on this system was 27.1 minutes. For peptide 3, the gradient started from 95% of solvent A and 5% of solvent B and increasing to 35% solvent A and 65% solvent B at 35 minutes. The retention time of peptide 3 on this system was 26.3 minutes. The desired functionalized conjugated peptides (compounds 1 and 3) were collected and the solvent was lyophilized. The purities of peptides 1 and 3 were determined by injection into analytical HPLC, using Phenomenex Ci₈ column (Luna, 5 μηι, 250 x 4.6 mm) at flow rate of 1 ml/minute and a gradient system starting from 70% of solvent A and 30% of solvent B and increasing to 60% solvent A and 40% solvent B at 35 minutes. The retention times of peptides 1 and 3 on this system were 17.3 minutes and 13.1 minutes, respectively. LC-MS also confirmed the mass of the conjugated peptides: 1 , 821 [M+H]⁺; 3, 1535 [M+H]⁺. [0107] Synthesis of 4-fluoro-3-trifluoromethylbenzoyl-c(RGDfK) (peptide 2) and 4- fluoro-3-trifluoromethylbenzoyl-E[c(RGDfK)₂] (peptide 4). Non-radioactive standards for l ⁸F-2 and ¹⁸F-4 were prepared by conjugation of c(RGDfK) and E[c(RGDfK)₂] with the commercially available 4-fluoro-3-trifluorobenzoyl chloride, under the same chemical conditions as described above. The conjugation of 4-fluoro-3-trifluorobenzoyl chloride with the peptides was confirmed by LC-MS analysis: peptide 2, 794 [M+H⁺]; peptide 4, 1509 [M+H⁺]. The purities of peptides 2 and 4 were determined by the same analytical HPLC conditions described above for peptides 1 and 3. The retention time of peptides 2 and 4 on this system were 16.9 minutes and 12.5 minutes, respectively.

[0108] Synthesis of 4-¹⁸F-fluoro-3-trifluoromethylbenzoyl-c(RGDfK) (¹⁸F-2) and 4-¹⁸F- fluoro-3-trifluoromethylbenzoyl-E[c(RGDfK.)₂] ( F-4). Reactive F-fluoride ion was prepared by adding to ¹⁸F/H₂ ¹⁸0 solution, K₂C0₃ (1.38 mg, 10 μπιοΐ), and Kryptofix in ACN (7.52 mg, 20 pmol). Azeotropic removal of water and ACN was achieved by heating under a stream of argon. The dried K¹⁸F^»Kryptofix 2.2.2 complex was then dissolved in 300 μΐ anhydrous dimethylsulfoxide (DMSO) and added to 440-800 μg (~ 0.5 μιηοΐ) of the modified peptides (1 and 3) in a screw-cap test tube. The tube was capped, vortexed and heated in the microwave for 3.5 minutes at 130 °C.

[0109] After cooling the reaction vial to ambient temperature in a water bath, the vial content was diluted with 10 ml of water and loaded onto an activated C]₈ Sep-Pak cartridge.

The cartridge was washed with water (10 ml) and the desired labeled peptide ( 18 F-2 or 18 F-4) was eluted with 10 mM HC1 in ethanol (1 ml) into a glass test tube. The ethanol was evaporated for 2-3 minutes under a stream of argon at 60 °C and then the crude labeled peptide was diluted with 0.1 % TFA/H₂0 and injected into reversed-phase HPLC using a Phenomenex Ci₈ column (Luna, 5 μηι, 250 x 10 mm). The flow was set at 4 ml/minute using a gradient system starting from 70% of solvent A (0.1% TFA in water) and 30% of solvent B (0.1 % TFA in ACN) and increasing to 60% solvent A and 40% solvent B at 35 minutes. ¹⁸F- 2 and ¹⁸F-4 were eluted with a retention time of 17 minutes and 12.7 minutes, respectively. ^{i 8}F-2 and ¹⁸F-4 were analyzed using HPLC and compared to nonradioactive standards by co- injection (Figure 2).

[0110] 4-Nitro-3-trifluoromethylbenzoyl chloride was conjugated to both RGD monomer c(RGDfK) and dimer E[c(RGDf )]₂ in the presence of tri ethyl amine at room temperature to give peptides 1 and 3, which were purified on a reversed-phase HPLC system. Eight to ten mg of c(RGDfK) and E[c(RGDfK)]₂ were used for the conjugation to give functionalized conjugated peptides 1 and 3, respectively, which were achieved in reasonable chemical yield of 50-60 % and were sufficient for several radioactive runs. The chemical purity of peptides 1 and 3 from the above reaction was greater than 99%, as determined by analytical HPLC and LC-MS analysis.

EXAMPLE 3

[0111] This example illustrates synthesis of peptide standards 2 and 4 in accordance with an embodiment of the present invention. The synthesis of unlabeled standards for the fluorination was done in a similar way to peptides 1 and 3 using the commercially available 4-fluoro-3-trifluoromethylbenzoyl chloride. Peptides 2 and 4 were achieved in slightly lower yield (42-46 %) than peptides 1 and 3. The chemical purity of peptides 2 and 4 was found to be greater than 99% as determined by analytical HPLC and LC-MS analyses.

EXAMPLE 4

[0112] This example illustrates a method of synthesis of peptides 1 8 F-2 and 18 F-4 in accordance with an embodiment of the present invention. 18 F-fluoride displacement of the nitro group in peptides 1 and 3, was done rapidly using a microwave device set to a temperature of 130 °C and low amount of peptide precursors (~ 0.5 μιηοΐ, Fig. 1). After ¹⁸F- fluoride displacement, the unreacted ¹⁸F-fluoride was washed out using activated C-18 sep- pak cartridge and the crude labeled peptides were injected into reversed-phase HPLC system. The incorporation of ¹⁸F-fluoride into peptide 1 resulted in one major radioactive peak of the desired labeled peptide 1 8 F-2 with a retention time of 17 minutes. Peptide 1 8 F-4 was eluted from the HPLC with retention time of 12.7 minutes (Figure 2). The monomeric peptide 1 8 F-2

1 8 was achieved in higher radiochemical yield (19 ± 4%, n = 6) than the dimeric peptide F-4 (9 ± 2%, n = 4). Both have radiochemical purity greater than 99%, with high specific activity of 79 ± 13 ΟΒ /μιτιοΙ (SOS). The overall radio-synthesis time to get peptides ^{! 8}F-2 or ¹⁸F-4 formulated and ready for injection was approximately 40 minutes.

[0113] It is of note that attempts to perform this direct fluorination using an oil bath at 130 °C for 20 minutes with the same small amount of conjugated peptides, were also successful and gave similar radiochemical yields.

1 8 1 8

[0114] Peptide ^{, 0}F-2 was achieved in higher radiochemical yield than peptide F-4.

Direct fluorination on monomeric RGD peptide 1 , to yield 18 F-2, gave lesser radioactive by-

1 8

products than fluorination on the dimeric RGD, peptide 3, to yield F-4 (Figures 2A and 2B). [0115] The amino acid sequence is important for this type of fluorination. Attempts to label modified c(RGDyk) led to little to no desired product (data not shown), hence tyrosine containing peptides may not be suitable substrates for this type of fluorination as such;

however, peptides or other biological substrates having reactive groups such as the phenolic OH of tyrosine can be modified provided that these groups are protected prior to carrying out the ¹⁸F fluorination reaction. The ¹⁸F-fluoride displacement reaction was conducted at relatively high temperature (130 °C), which resulted in slight decomposition of peptides 1 and 3, as detected by UV at 218 nm (Figures 2 A and 2B) but had minimal effect on the labeling efficiency. The specific activity of the final product is thus related to the amount of precursor and radioactivity used for the reaction.

EXAMPLE 5

125

[0116] This example illustrates a competitive binding assay with radiolabeled I- echistatin. The affinity of peptides 2 and 4 for integrin α_νβ₃ was tested using the human breast carcinoma cell line MDA-MB-435, which is known to express medium level of integrin α_νβ₃. Binding affinities of the modified-RGD peptides, 2 and 4, were compared with the non-modified RGD peptides, c(RGDfK) and E[c(RGDfK)₂]. The IC₅₀ values of peptides 2 and 4 binding to MDA-MB-435 cells were 1 19 nM and 63 nM, which were comparable with those of c(RGDf ) (67 nM) and E[c(RGDfK)₂] (33 nM), respectively (Figure 3).

[0117] In order to verify that the introduction of substituted arene into the peptide does not affect its biological behavior in vitro, the nonradioactive peptide standards 2 and 4 were tested for binding to integrin α_νβ₃ expressing MDA-MB-435 cells in a competitive binding assay against ¹²⁵I-echistatin. Modification of RGD peptides did not change significantly the biological binding affinities in comparison to RGD monomer c(RGDfK) and dimer

E[c(RGDfK)]₂ (Figure 3).

EXAMPLE 6

[0118] This example illustrates a method of PET imaging and biodistribution using the ¹⁸F labeled biological substrates in accordance with an embodiment of the invention. Since peptide 4 showed higher affinity for integrin α_νβ₃ in binding assay than peptide 2, it was

18 further evaluated in vivo in orthotopic MDA-MB-435 tumor mice. PET images of F-4 were acquired at 0.5, 1 and 2 hours post-injection (Figure 4). Peptide ^{l 8}F-4 had initial high tumor uptake (3.8 ± 0.16 %ID/g) and good tumor-to-background contrast at 0.5 hour post-injection, which was slightly increased at the 2 hour time point (4.43 ± 0.6 %ID/g, Figures 4 and 5A). At 0.5 hour post-injection, peptide ^{l 8}F-4 uptake in metabolic organs such as liver and intestine was low (2.5 ± 0.3 and 0.87 ± 0.09 %ID/g, respectively, Figures 4 and 5a). At 1 hour post-injection, peptide 18 F-4 uptake in the liver decreased to 1.67 ± 0.34 %ID/g, however the uptake in the intestine increased (2.43 ± 0.04 %ID/g, Figures 4 and 5A). At 2 hours post-injection, the uptake in the intestine increased to 4.19 ± 1.1 %ID/g (Figure 5 A) and some uptake in the gallbladder was detected (Figure 4).

[0119] Blocking studies were carried out by co-injection of peptide 18 F-4 with an excess

18

(0.5 μηιοΐ) of monomelic c(RGDfK). Peptide F-4 tumor uptake was reduced by 82 % (Figures 4 and 5B) at 0.5 hour, which suggested that the peptide F-4 uptake in the tumor is due to a specific binding to integrin α_νβ₃. The inhibition of peptide 18 F-4 uptake in other organs and tissue expressing low level of integrin α_νβ₃ was also found, which was consistent with previously reported observations. See, Wu, Z.,et al., Eur. J. Nucl. Med. Mol. Imaging 34: 1823-31 (2007); Chen, X. et al., Multimodality imaging of tumor integrin ανβ3 expression. Mini Rev Med Chem 6, 227-34 (2006).

[0120] Peptide ¹⁸F-4 has higher integrin binding affinity than peptide ¹⁸F-3 and was further evaluated for its usability as a PET imaging agent by injection into MDA-MB-435 tumor-bearing mice. Biodistribution of peptide F-4 was analyzed at 0.5, 1 and 2 hours post- injection using PET scans of live animals. At the 0.5 hour time point, 18 F-4 showed very clear image with high tumor to background contrast (Figure 4), high tumor uptake (3.81 ± 0.16 %ID/g) and low accumulation in metabolic organs such as liver and intestine. The tumor uptake elevated at 1 and 2 hours post-injection (Figure 5A). In addition to the PET scans, biodistribution of peptide ^{1 8}F-4 by organ dissection was performed at 2 hours post- injection. The results achieved by dissection of the organs and counting using a gamma counter (Figure 5C). Similar to the PET scan data, the uptake in the tumor was 4.57 ± 0.67 %ID/g and the uptake in the intestine was relatively high (5.04 ± 0.39 %ID/g) at 2 hours post- injection. Low uptake (0.68 ± 0.28 %ID/g) was detected in the bone (Figure 5C). However, accumulation of peptide ¹⁸F-4 was higher in the intestine at 2 hours post-injection (Figures 4 and 5a), suggesting a hepatobiliary clearance of peptide ^{l 8}F-4. The ¹⁸F-labeled RGD peptides are metabolically stable with little to no defluorination as evidenced by very low bone uptake at 2 hour time point (Figure 5C) Co-injection of ¹⁸F-4 with c(RGDf ) effectively blocked the uptake in the tumor (Figures 4 and 5A) which indicates specific binding of peptide 18 F-4 to integrin α_νβ₃. The PET quantification (Figure 5A) was consistent with the results obtained from direct tissue sampling (Figure 5C).

[0121] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0122] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0123] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those prefen'ed embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of labeling a biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto, comprising:

reacting the biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto with a 18 F anion in the presence of a cryptand molecule; and obtaining the ¹⁸F labeled biological substrate.

2. The method of claim 1 , wherein the biological substrate having at least one 4- nitro-3 -trifluoromethylbenzamide moiety covalently bonded thereto is an amino acid, peptide, peptidomimetic, oligopeptide, protein, aptamer, oligonucleotide, DNA, RNA, antibody, receptor binding molecules, molecules having a receptor motif, haptens or small molecule, each having at least one 4-nitro-3 -trifluoromethylbenzamide moiety covalently bonded thereto.

3. The method of claim 2, wherein the biological substrate having at least one 4- nitro-3 -trifluoromethylbenzamide moiety covalently bonded thereto is a peptide comprising from 4 to 100 amino acids and having at least one 4-nitro-3 -trifluoromethylbenzamide moiety covalently bonded thereto.

4. The method of claim 1 or 2, wherein the biological substrate having at least one 4-nitro-3 -trifluoromethylbenzamide moiety covalently bonded thereto is selected from the group consisting of placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate- specific membrane antigen, a-fetoprotein, B-lymphocyte surface antigen, truncated EGFR, idiotypes, gp95/gp97, N-CAM, cluster w4, cluster 5A, cluster 6, FLAP, CA-125 (lung and ovarian cancers), ESA, CD19, 22 or 37, 250 kD proteoglycan, P55, TCR- IgH fusion, blood group A B or O antigen, bombesin, somatostatin receptor specific peptides, somatostatin and derivatives thereof, neuropeptide Y, neuropeptide Y] and derivatives thereof, gastrin, gastrin releasing peptide and derivatives thereof, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1 , integrins (α₃βι, α_νβ₃, α_νβ₅, allb₃), LHRH agonists and antagonists, transforming growth factors, TGF-a, angiotensin, cholecystokinin receptor peptides, cholecystokinin (CCK) and analogs thereof; neurotensin and analogs thereof, thyrotropin releasing hormone and analogs thereof, pituitary adenylate cyclase activating peptide (PACAP) and related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and the analogs thereof, tumor necrosis factor, interleukins (IL- 1 , IL-2, IL-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) RGB peptide, substance P, neuromedin-B, neuromedin-C, octreotide analogues, metenkephalin, neurotensin, melanocyte stimulating hormone and analogs thereof, follicle stimulating hormone and analogs thereof, luteinizing hormone and analogs thereof, human growth hormone and analogs thereof, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon, erythropoietin, and colony-stimulating factor, each having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto.

5. The method of claim 1 or 2, wherein the biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto is selected from the group consisting of nucleic acid, a nucleic acid targeting a complementary nucleic acid, aptamer, oligonucleotide, DNA, and RNA, each having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto.

6. The method of any of claims 1 -3, wherein the biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto is an antibody (e.g., scFv, F(ab')₂, and Fab), a monoclonal antibody or monoclonal antibody fragment, an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A12 Ab, H17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B-lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab, cetuximab and an Affibody, each having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto.

7. The method of any of claims 1 -6, wherein the reacting step further comprises heating the biological substrate having at least one 4-nitro-3-trifiuoromethylbenzamide moiety in the presence of the 1 8 F anion and the cryptand molecule.

8. The method of any of claims 1 -7, further comprising purifying the ¹⁸F labeled biological substrate.

9. The method of any of claims 1 -8, wherein the cryptand molecule is selected from the group consisting of: 4,7,13, 18-tetraoxa-l ,10-diazabicyclo[8.5.5]-eicosane;

4,7, 13, 16,21 -pentaoxa-l ,10-diazabicyclo[8.8.5]-tricosane, (Kryptofix 221); 4,7,13, 16,21 ,24- hexaoxa-l ,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222); and 4,7,10,16,19,24,27- heptaoxa- 1 ,13 -diazabicyclo[ 1 1.8.8]-nonacosane.

10. The method of any of claims 1-9, wherein the cryptand molecule is

4,7,13,16,21 ,24-hexaoxa-l ,10-diazabicyclo[8.8.8]-hexacosane.

1 1. The method of any of claims 7-10, wherein the temperature of the reaction during heating is between 100 °C and 150 °C.

12. The method of any of claims 7-1 1 , wherein the duration of the heating is between about 1 minute and 10 minutes.

13. The method of any of claims 1-12, wherein the biological substrate having at least one 4-nitro-3-trifluoromethylbenzamide moiety covalently bonded thereto is:

(F-l) or

14. A method of preparation of a biological substrate labeled with 18 F comprising: a) reacting a biological substrate having at least one free amino group with a 4-nitro- 3-trifluoromethylbenzoyl halide;

b) obtaining a biological substrate having at least one 4-nitro-3- trifluoromethylbenzamide moiety covalently bonded thereto;

c) reacting the biological substrate having at least one 4-nitro-3-

18

trifluoromethylbenzamide moiety covalently bonded thereto with F anion in the presence of a cryptand and obtaining a F labeled biological substrate; and optionally

d) purifying the F labeled biological substrate.

15. The method of claim 14, wherein the biological substrate having at least one free amino group is an amino acid, peptide, peptidomimetic, oligopeptide, protein, aptamer, oligonucleotide, DNA, RNA, antibody, receptor binding molecules, molecules having a receptor motif, haptens or a small molecule.

16. The method of claim 14 or 15, wherein the biological substrate having at least one free amino group is a peptide comprising from 4 to 100 amino acids.

17. The method of any of claims 14-16, wherein the biological substrate having at least one free amino group is selected from the group consisting of placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate-specific membrane antigen, -fetoprotein, B-lymphocyte surface antigen, truncated EGFR, idiotypes, gp95/gp97, N-CAM, cluster w4, cluster 5 A, cluster 6, FLAP, CA-125 (lung and ovarian cancers), ESA, CD19, 22 or 37, 250 kD proteoglycan, P55, TCR-lgH fusion, blood group A B or O antigen, bombesin, somatostatin receptor specific peptides, somatostatin and derivatives thereof, neuropeptide Y, neuropeptide Y| and derivatives thereof, gastrin, gastrin releasing peptide, the derivatives thereof, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1 , integrins (α₃βι, α_νβ₃, α_νβ₅, allb₃), LHRH agonists and antagonists, transforming growth factors, particularly TGF-a, angiotensin, cholecystokinin receptor peptides, cholecystokinin (CCK) and analogs thereof; neurotensin and analogs thereof, thyrotropin releasing hormone and analogs thereof, pituitary adenylate cyclase activating peptide (PACAP) and related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and the analogs thereof, tumor necrosis factor, interleukins (IL-1, IL-2, IL-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) RGB peptide, substance P, neuromedin-B, neuromedin-C, octreotide analogues, metenkephalin, neurotensin, melanocyte stimulating hormone and analogs thereof, follicle stimulating hormone and analogs thereof, luteinizing hormone and analogs thereof, human growth hormone and analogs thereof, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon, erythropoietin, and colony-stimulating factor.

18. The method of claim 15, wherein the biological substrate having at least one free amino group is selected from the group consisting of nucleic acid, a nucleic acid targeting a complementary nucleic acid, aptamer, oligonucleotide, DNA, and RNA.

19. The method of claim 15 or 16, wherein the biological substrate having at least one free amino group is an antibody (e.g., scFv, F(ab^')₂, and Fab), a monoclonal antibody or monoclonal antibody fragment, an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A12 Ab, FI17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B- lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab, cetuximab and an affibody.

20. The method of any of claims 14-19, wherein the reacting step further comprises heating the biological substrate having at least one free amino group in the presence of the ¹⁸F anion and the cryptand molecule.

21. The method of any of claims 14-20, further comprising purifying the F labeled biological substrate.

22. The method of any of claims 14-21 , wherein the cryptand molecule is selected from the group consisting of: 4,7,13, 18-tetraoxa-l ,10-diazabicyclo[8.5.5]-eicosane;

4,7, 13, 16,2 l -pentaoxa-l , 10-diazabicyclo[8.8.5]-tricosane, (Kryptofix 221); 4,7,13,16,21 ,24- hexaoxa-l ,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222); and 4,7,10,16,19,24,27- heptaoxa-l ,13-diazabicyclo[l 1.8.8]-nonacpsane.

23. The method of any of claims 14-22, wherein the cryptand molecule is 4,7, 13,16,21 ,24-hexaoxa-l ,10-diazabicyclo[8.8.8]-hexacosane.

24. The method of any of claims 20-23, wherein the temperature of the reaction during heating is between 100 °C and 150 °C.

25. The method of any of claims 20-24, wherein the duration of the heating is between about 1 minute and 10 minutes.

26. The method of any of claims 14-25, wherein a biological substrate having at least one free amino group is

(F-l ') or

27. A method of identifying a cell or a population of cells expressing a protein or peptide of interest comprising:

a) contacting the cell or a population of cells expressing a protein or peptide of interest with a ¹⁸F labeled targeting agent which selectively binds said protein or peptide of interest, wherein said ¹⁸F targeting agent is a ¹⁸F labeled biological substrate made according to the method of any of claims 1-26;

b) obtaining a diagnostic image of the cell or population of cells,

c) quantifying the amount of ¹⁸F labeled targeting agent bound to the cell or population of cells, and

d) correlating the amount of the bound ¹⁸F labeled targeting agent with the amount of the protein or peptide of interest on the cell or population of cells.

28. The method of claim 27, wherein the cell or population of cells is a tumor cell or tumor cells.

1 8

29. The method of claim 27, wherein detecting the presence of the ^{, 0}F labeled targeting agent binding said protein or peptide of interest on the cell or population of cells is performed by MRI, PET or SPECT imaging.

30. A method of diagnosing a disease in a patient comprising:

a) administering to a subject suspected of having said disease, a F labeled targeting agent, wherein said targeting agent is a 1 8 F labeled biological substrate prepared according to any of claims 1 -26, which selectively binds a protein or peptide of interest, b) obtaining a diagnostic image of the subject;

c) determining the location of F labeled targeting agent bound to the protein or peptide of interest in the subject, and

d) correlating the location of the bound 18 F labeled targeting agent with the location of the protein or peptide of interest in the subject.

31. The method of claim 30, wherein the protein or peptide of interest is associated with tumor growth and the disease is cancer.

32. The method of claim 30, wherein detecting the presence of the F labeled targeting agent binding said protein or peptide of interest is performed by MRI, PET or SPECT imaging.

33. A method of diagnosing a disease in a patient comprising:

obtaining a diagnostic image of a subject whose tissue or organ includes a F labeled targeting agent, wherein said targeting agent is a F labeled biological substrate prepared according to any of claims 1-26.

34. The method of claim 33, wherein detecting the presence of the targeting agent binding said protein or peptide of interest is performed using MRI, PET or SPECT imaging.

35. The method of any of claims 27-34, wherein the ¹⁸F labeled targeting agent is

(F-l ") or

(F-3").

36. A compound of formula (I):

wherein Ri is a moiety derived from the biological substrate by covalently linking the biological substrate to the C=0 group through X, and wherein X is O, NR', S, CH₂, NR'CO, or NR'CONH, where R'=H or lower alkyl, where n=l to 100, where R₂ is an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR'V (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br, and R"=H or lower alkyl, R₃ is H, or an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR"₃ ⁺ (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br.

37. The compound of claim 36, wherein the biological substrate is a peptide and X is NH.

38. The compound of claim 36 or 37, wherein R₂ is CF₃ and R₃ is H.

39. The compound of any of claims 36 to 38, wherein the compound is

(F-3").

40. The compound of claim 36, wherein the biological substrate is an amino acid, peptide, peptidomimetic, oligopeptide, protein, aptamer, oligonucleotide, DNA, RNA, antibody, receptor binding molecules, molecules having a receptor motif, haptens or small molecule.

41. The compound of claim 36, wherein the biological substrate is a peptide comprising from 4 to 100 amino acids.

42. The compound of claim 36, wherein the biological substrate is selected from the group consisting of placental alkaline phosphatase, pan carcinoma, polymorphic epithelial mucin, prostate-specific membrane antigen, a-fetoprotein, B-lymphocyte surface antigen, truncated EGFR, idiotypes, gp95/gp97, N-CAM, cluster w4, cluster 5A, cluster 6, FLAP, CA-125 (lung and ovarian cancers), ESA, CD19, 22 or 37, 250 kD proteoglycan, P55, TCR- IgH fusion, blood group A B or O antigen, bombesin, somatostatin receptor specific peptides, somatostatin and derivatives thereof, neuropeptide Y, neuropeptide Yi, and derivatives thereof, gastrin, gastrin releasing peptide, derivatives thereof, epidermal growth factor (EGF of various origin), insulin growth factor (IGF) and IGF-1, integrins (α₃βι, α_νβ₃, α_νβ₅, llb₃), LHRH agonists and antagonists, transforming growth factors, TGF-a, angiotensin, cholecystokinin receptor peptides, cholecystokinin (CCK) and the analogs thereof;

neurotensin and analogs thereof, thyrotropin releasing hormone, pituitary adenylate cyclase activating peptide (PACAP) and related peptides thereof, chemokines, substrates and inhibitors for cell surface matrix metalloproteinase, prolactin and analogs thereof, tumor necrosis factor, interleukins (IL-1 , 1L-2, IL-4 or IL-6), interferons, vasoactive intestinal peptide (VIP) RGB peptide, substance P, neuromedin-B, neuromedin-C, octreotide analogues, metenkephalin, neurotensin, melanocyte stimulating hormone and analogs thereof, follicle stimulating hormone and analogs thereof, luteinizing hormone and analogs thereof, human growth hormone and analogs thereof, a serum protein, a fibrinolytic enzyme, a biological response modifier, interleukin, interferon, erythropoietin, and colony-stimulating factor.

43. The compound of claim 36, wherein the biological substrate is selected from the group consisting of nucleic acid, a nucleic acid targeting a complementary nucleic acid, aptamer, oligonucleotide, DNA, and RNA.

44. The compound of any of claims 36 to 38, wherein the biological substrate is an antibody (e.g., scFv, F(ab')₂, and Fab), a monoclonal antibody or monoclonal antibody fragment, an antibody or single chain antibody (scAb) to c-erbB-2, C46 Ab, 85A12 Ab, H17E2 Ab, NR-LU-10 Ab, HMFC1 Ab, W14 Ab, RFB4 Ab to B-lymphocyte surface antigen, A33 Ab, TA-99 Ab, trastuzumab, cetuximab and an affibody.

45. A pharmaceutical composition comprising a compound of any of claims 36-44 and a pharmaceutically acceptable carrier.

46. A method of locating a receptor that is overexpressed in a tissue or organ of a subject comprising: a) administering to the subject a ^{l 8}F labeled compound of formula I which selectively binds a protein or peptide of int

(I),

wherein Ri is a moiety derived from the biological substrate by covalently linking the biological substrate to the C=0 group through X, and wherein X is O, NR', S, CH₂, NR'CO, or NR'CONH, where R'=H or lower alkyl, where n=l to 100, where R₂ is an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR'V (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br, and R"=H or lower alkyl, R₃ is H, or an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR"₃ ⁺ (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br; b) obtaining a diagnostic image of the tissue or organ;

18

c) determining the location of F labeled compound of formula I bound to the tissue or organ; and

d) correlating the location of the bound 18 F labeled compound of formula I with the location of the receptor in the subject.

47. A method of locating a receptor that is overexpressed in a tissue or organ of a subject comprising:

a) obtaining a diagnostic image of a subject whose tissue or organ includes Ϊ 8 F labeled targeting agent prepared according to any of claims 1-35;

b) determining the location of 18 F labeled targeting agent bound to the tissue or organ; and

c) correlating the location of the bound ¹⁸F labeled targeting agent with the location of the receptor in the subject.

48. A method of measuring the quantity of a receptor that is overexpressed in a tissue or organ of a subject comprising:

a) administering to the subject a ^{l 8}F labeled compound of formula I which selectively binds a protein or peptide of interest

(I),

wherein Rj is a moiety derived from the biological substrate by covalently linking the biological substrate to the C=0 group through X, and wherein X is O, NR', S, CH₂, NR'CO, or NR'CONH, where R'=H or lower alkyl, where n=l to 100, where R₂ is an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR"₃ ⁺ (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br, and R"=H or lower alkyl, R₃ is H, or an electron withdrawing group selected from the group consisting of N0₂, CHO, COOR", COR", NR"₃ ⁺ (quaternary ammonium), and CX'₃, wherein X' is F, CI or Br; b) obtaining a positron emission tomography (PET) image of the tissue or organ, c) determining the amount of F labeled compound of formula I bound to the tissue or organ, and

d) correlating the amount of the bound 18 F labeled compound of formula I with the quantity of receptor in the subject.

49. The method of claim 48, wherein the F labeled compound of formula I is administered in an amount effective to provide an image.

50. The method of claim 48, wherein the diagnostic image is positron emission tomography (PET) image, a magnetic resonance image (MRI), a computerized tomography (CT) scan, an x-ray contrast image, single photon emission computed spectroscopy (SPECT) image, or a combination thereof.

51. A method of measuring the quantity of a receptor that is overexpressed in a tissue or organ of a subject comprising:

a) obtaining a positron emission tomography (PET) image of the tissue or organ of a subject whose tissue or organ includes F labeled targeting agent prepared according to any of claims 1 -35;

b) determining the amount of ¹⁸F labeled targeting agent bound to the tissue or organ, and

c) correlating the amount of the bound ¹⁸F labeled targeting agent with the quantity of receptor in the subject.