Peptide PET / SPECT probes specific to tumor proteins in the extracellular matrix of tumor cells

Abstract

To provide peptide positron emission tomography (PET) / single photon emission computed tomography (SPECT) probes to oncoproteins in tumor and / or cancer extracellular matrix.SOLUTION: A PET / SPECT probe includes the following formula: P-L-C, where P is an EDB-FN targeting peptide, C is a PET / SPECT contrast agent; and L is an optional linker that covalently links the peptide to the contrast agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 793,789, filed on 17 January 2019, the subject matter of which is incorporated herein by reference in its entirety.

[0002] government funds This invention was made with government support under grant numbers CA211762 and CA194518, awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention. [Background technology]

[0003] Cancer detection and treatment are hampered by the inability to distinguish between cancer cells and normal cells. Superior detection tools for imaging cancer or tumors are needed for the early diagnosis of cancer. Molecular recognition of tumor cells would facilitate induced surgical resection. To improve surgical resection, targeted imaging tools must specifically label tumor cells not only in the major tumor but also along the edges of the tumor and in small tumor cell clusters dispersed throughout the body. Targeted imaging tools designed to label molecules accumulating in the tumor microenvironment may also be effective as therapeutic targeting agents, as they can identify both the major tumor cell population and regions with invasive cells involved in tumor recurrence. The ability to directly target tumor cells and / or their microenvironment would increase both the specificity and sensitivity of current therapies and thus reduce the nonspecific side effects of chemotherapeutic agents that affect cells throughout the body.

[0004] Positron emission tomography (PET) imaging is primarily based on the elevated glucose metabolism in prostate cancer compared to normal tissue. 18 It is used in clinical testing for prostate cancer using F]-FDG. However, 18[F]-FDG PET has not demonstrated the ability to distinguish benign prostate cancer from invasive cancer. PSMA-specific PET probes have recently been developed for prostate cancer. Clinical trials have demonstrated the ability of PSMA probes in the effective detection of PSMA-positive prostate tumors. However, recent trials have cautioned that PSMA probes may not be able to distinguish benign tissue from prostate cancer. PET probes are needed to detect invasive cancers and stratify risk, meeting the clinical demand for non-invasive diagnostic modalities for accurate clinical management of cancer. [Overview of the Initiative]

[0005] The embodiments described herein relate to peptide positron emission tomography (PET) / single-photon emission computed tomography (SPECT) probes for oncoplastic proteins in tumors and / or cancer extracellular matrices, which can be used to detect the location and / or distribution of cancer in a target tissue, the invasiveness of cancer in a target, and / or the effectiveness of cancer treatments and / or cancer therapies administered to a target requiring cancer treatment and / or cancer therapy.

[0006] In some embodiments, the PET / SPECT probe may include the following formula: [ka] In the formula, P is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 20, 21, 22, 23, 24, and their retroinverso-type amino acid sequences; C is a PET or SPECT contrast agent; and L is an optional linker that covalently binds the peptide to the PET / SPECT contrast agent.

[0007] In some embodiments, the linker is a non-peptide linker. The non-peptide linker can be a non-peptide aliphatic, heteroaliphatic, cyclic, and / or heterocyclic linker. The non-peptide linker may include, for example, alkylene, alkylene oxide, arylene, or alkylene-arylene linkers that covalently bond the peptide to the contrast agent.

[0008] PET / SPECT contrast agents may contain at least one of a metal chelating agent or a metallic fullerene and a positron- or gamma-ray emitting radionuclide. Examples of metal chelating agents include diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazadodecanetetraacetic acid (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetic acid (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetic acid (DO3MA), and N,N',N'',N'''-tetraphosphonatomethyl-1,4,7,10-tetraa This may include at least one of the following: zacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenemethylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephenylphosphonic acid) (DOTPP), N,N'-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N'-bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N'-diacetic acid (HBED-CC), and their derivatives. Positron- or gamma-ray emitting radionuclides may include, for example, 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89Zr, 90 Y, 153 Sm, or 89 Sr can be included.

[0009] In some embodiments, the PET / SPECT probe can have the following formula:

Chemical Formula

[0010] In still other embodiments, the PET / SPECT probe can be systemically administered to a subject to detect the distribution and / or location of cancer in the subject, as well as the invasiveness of the cancer. The cancer may include, for example, at least one of breast cancer, liver cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, brain cancer, head and neck cancer, or skin cancer.

Brief Description of Drawings

[0011] [Figure 1] A schematic diagram illustrating the synthesis of ZD2-DA-(64Cu-DOTA) is shown. [Figure 2] Macroscopic bright-field and 3D volume-rendered PET / CT images of two representative mice carrying LNCaP and PC3 tumors are shown at 4 and 22 hours after injection of ZD2-DA-(64Cu-DOTA). [Figure 3] Quantitative tracer uptake was observed in muscle, liver, heart, and LNCaP and PC3 tumors at 4 and 22 hours after ZD2-DA-64Cu(DOTA) injection (N=4). [Figure 4] The graph shows the biodistribution of ZD2-DA-(64Cu-DOTA) in different tissues 24 hours after injection. Data are expressed as mean ± sem (N=3). [Figure 5] Images showing immunofluorescence staining of EDB-FN in LNCaP and PC3 prostate tumor sections are shown. Scale bar: 50 μm. [Figure 6] An outline of the synthesis procedure for ZD2-(Ga-NOTA)(4) is shown below. [Figure 7] (A-B) shows Western blots (A) and fluorescence confocal images (B) illustrating the expression of EDB-FN in BXPC3, Capan-1, Panc10.05, and Panc-1 human pancreatic cancer cells (A) and tumor xenografts in mice (B). Tissue slides are stained with BC-1 anti-EDB-FN monoclonal antibody and secondary antibodies labeled with AF-488 and DAPI. [Figure 8] Images showing the specific binding of ZD2-Cy5.5 to EDB-FN in BXPC3, Capan-1, Panc10.05, and Panc-1 human pancreatic cancer xenograft specimens. The BC-1 / ZD2 column indicates that the binding of ZD2-Cy5.5 was blocked by pre-incubation of the specimen with BC-1 antibody. [Figure 9] Fluorescence images showing the binding patterns of ZD2-Cy5.5 to EDB-FN in specimens of human pancreatic cancer, pancreatic intraepithelial neoplasm, and normal pancreas are shown. [Figure 10] Two-dimensional coronal PET / CT images of mice carrying Capan-1 and BXPC3 human pancreatic cancer xenografts, taken 1 and 2 hours after intravenous injection of ZD2-(68Ga-NOTA) at a dose of 300 μCi / mouse. T: tumor, B: bladder. [Figure 11] Three-dimensional PET images of mice carrying Capan-1 and BXPC3 human PaCa xenografts, taken one hour after intravenous injection of ZD2-(68Ga-NOTA) at a dose of 300 μCi / mouse. T: tumor, K: kidney, B: bladder. [Figure 12] A schematic diagram illustrating the synthesis of ZD2-HBED-CC is shown. [Figure 13] A schematic diagram illustrating the synthesis of ZD2-AH-HBED-CC is shown. [Figure 14] A schematic diagram illustrating the synthesis of ZD2-(Ga-HBED-CC) is shown. [Figure 15] A schematic diagram illustrating the synthesis of ZD2-AH-(Ga-HBED-CC) is shown. [Figure 16] This image shows PET / CT images of mice carrying BXPC3 and Capan-1 human pancreatic tumor xenografts, taken with ZD2-(68Ga-HBED-CC). [Figure 17] This image shows PET / CT images of mice carrying BXPC3 and Capan-1 human pancreatic tumor xenografts, taken with ZD2-AH-(68Ga-HBED-CC). [Modes for carrying out the invention]

[0012] Methods including conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodological treaties such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as generally understood by those skilled in the art to which this application belongs. Generally understood definitions of molecular biology terms can be found, for example, in Rieger et al., Glossary of Genetics, Classical and Molecular, 5th Edition, Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

[0013] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "one element" means one or more elements.

[0014] The terms “comprise,” “comprising,” “include,” “including,” “have,” and “have” are used in an inclusive and open sense, meaning that additional elements may be included. The terms “e.g.,” and “for example” as used herein are non-restrictive and for illustrative purposes only. “Include” and “include, but not limited to” are used interchangeably.

[0015] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly indicates otherwise.

[0016] The term "pharmaceutical" is used herein to refer to chemical compounds, mixtures of chemical compounds, biological polymers, or extracts comprising biological materials.

[0017] The terms “cancer” or “tumor” refer to any neoplastic growth in a subject, including initial tumors and any metastases. Cancer can be either a liquid or solid tumor type. Liquid tumors include tumors of hematological origin, such as myeloma (e.g., multiple myeloma), leukemia (e.g., Waldenström syndrome, chronic lymphocytic leukemia, and other leukemias), and lymphoma (e.g., B-cell lymphoma, non-Hodgkin lymphoma). Solid tumors can occur in organs and include cancers of the lungs, brain, breast, prostate, ovaries, colon, kidneys, and liver.

[0018] The terms "cancer cell" or "tumor cell" can refer to cells that divide at an abnormal (i.e., increased) rate. Cancer cells include carcinomas such as squamous cell carcinoma, non-small cell carcinoma (e.g., non-small cell lung cancer), small cell carcinoma (e.g., small cell lung cancer), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonic carcinoma, breast cancer, gastrointestinal cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, and squamous cell carcinoma of the head and neck region; fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordosarcoma, angiosarcoma, endosarcoma, rhino This includes, but is not limited to, sarcomas such as mphangiosarcoma, synovial sarcoma, and mesosarcoma; hematological malignancies such as myeloma, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphoma (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmacytoma, reticular sarcoma, or Hodgkin's disease), and tumors of the nervous system, including glioma, glioblastoma pleomorphontoma, meningioma, medulloblastoma, schwannoma, and epidermioma.

[0019] As used herein, the term “isolated” refers to a molecule isolated from other DNA, RNA, polypeptides, or proteins, respectively, that is present in a naturally occurring source of macromolecules. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free from cellular material, or culture media when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Furthermore, “isolated nucleic acid” or “isolated peptide” means a nucleic acid fragment or peptide fragment that does not exist naturally as a fragment and is not found in its natural state.

[0020] The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as an equivalent, analogues of RNA or DNA made from nucleotide analogues, and, as applicable to the embodiments described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0021] The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably in this specification.

[0022] The phrases "parenteral administration" and "administered parenterally" are, This is a term recognized in the art and includes methods of administration other than enteral and local administration, such as injection, and includes, but is not limited to, injections and infusions of intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal cells.

[0023] Terms such as “patient,” “subject,” and “mammalian host” are used interchangeably in this specification and refer to mammals, including humans and veterinary subjects.

[0024] The term "polypeptide" refers to polymers consisting of amino acid residues associated with naturally occurring structural variants and their non-naturally occurring synthetic analogs linked via peptide bonds or modified peptide bonds (i.e., peptide isosteres), associated naturally occurring structural variants and their non-naturally occurring synthetic analogs, glycosylated polypeptides, and all "mimicking" and "peptide-mimicking" polypeptide forms. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. This term can refer to oligopeptides, peptides, polypeptides, or protein sequences, or any fragment, part, or subunit thereof. The term "protein" usually refers to a large polypeptide. The term "peptide" usually refers to a short polypeptide.

[0025] A polypeptide or "part" of a protein refers to at least approximately three consecutive amino acid residues of the polypeptide. It is understood that a part of a polypeptide may contain all of the amino acid residues of the polypeptide.

[0026] A “mutant,” “derivative,” or “variant” of a polypeptide (or the DNA that encodes it) is a polypeptide that is modified or altered by one or more amino acids (or one or more nucleotides) so that the peptide (or nucleic acid) is not identical to the wild-type sequence but has homology to the wild-type polypeptide (or nucleic acid).

[0027] A "mutation" of a polypeptide (or the DNA that encodes it) is a modification or alteration of one or more amino acids (or one or more nucleotides) such that the peptide (or nucleic acid) is not identical to any sequence listed herein, but has homology to a wild-type polypeptide (or nucleic acid).

[0028] As used herein, "recombinant" means that the protein originates from a prokaryotic or eukaryotic expression system.

[0029] As used herein, the terms “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” mean the administration of a compound, drug, or other substance indirectly to a specific tissue, organ, or region of the target being treated (e.g., the brain), which enters the animal’s system and is subjected to metabolism and other similar processes, such as subcutaneous administration.

[0030] The term "wild-type" refers to a naturally occurring polynucleotide sequence that codes for a protein or part thereof, or a protein sequence or part thereof, which is usually present in vivo.

[0031] Throughout this specification, where a composition is described as having, including, or comprising, certain components, the composition is also essentially composed of, or intended to be composed of, the listed components. Similarly, where a method or process is described as having, including, or comprising, certain process steps, the process is also essentially composed of, or composed of, the listed processing steps. Furthermore, it should be understood that, as long as the compositions and methods described herein are implementable, the order of steps or the order in which to perform a particular operation is not important. In addition, two or more steps or operations can be performed simultaneously.

[0032] Embodiments described herein relate to peptide positron emission tomography (PET) / single-photon emission computed tomography (SPECT) probes for oncoplastic proteins in tumors and / or cancer extracellular matrix that can be used to detect, monitor, and / or image the distribution and / or location of cancer in a subject, and / or metastasis, migration, and / or invasion of cancer cells; to detect and / or monitor the invasiveness and / or malignancy of cancer cells in a subject; and / or to determine and / or monitor the effectiveness of cancer treatments and / or cancer therapies administered to a subject requiring cancer treatment and / or cancer therapy.

[0033] The PET / SPECT probes described herein contain targeted peptides having peptide sequences that specifically bind to and / or form complexes with carcinoembryonic fibronectin (onfFN) isoforms, extradomain-B fibronectin (EDB-FN), or extradomain-A fibronectin (EDA-FN). Cancer, particularly malignant cancers, have a unique tumor microenvironment that promotes cancer cell survival, proliferation, and metastasis. The presence of onfFN has been observed in various human cancer types, including prostate cancer, breast cancer, and pancreatic cancer. High expression of onfFN, EDB-FN, and / or EDA-FN has correlated with cancer aggressiveness and inversely correlated with patient survival. Using PET / SPECT probes containing targeted peptides that specifically bind to EDB-FN and / or EDB-FN, it has been shown that cancer cells in the tissue of interest can be detected, monitored, and / or imaged, and similarly, the aggressiveness, malignancy, metastasis, migration, dispersion, and / or invasion of cancer cells can be determined.

[0034] PET / SPECT probes containing targeted peptides can be systemically administered to subjects by intravenous or parenteral administration, and can readily target extracellular matrix proteins EDB-FN and / or EDA-FN to clarify the location, distribution, and / or invasiveness of cancer cells in the subject, as well as the tumor margins.

[0035] In some embodiments, the PET / SPECT probe may include the following formula: [ka] In the formula, P is a targeted peptide, C is a PET / SPECT contrast agent, and L is an optional linker that covalently binds the peptide to the contrast agent.

[0036] In some embodiments, the targeted peptide can specifically bind to EDB-FN. Targeted peptides that specifically bind to EDB-FN may include linear peptides having the amino acid sequences of TVRTSAD (SEQ ID NO: 1), NWGDRIL (SEQ ID NO: 2), NWGKPIK (SEQ ID NO: 3), SGVKSAF (SEQ ID NO: 4), GVKSYNE (SEQ ID NO: 5), IGKTNTL (SEQ ID NO: 6), IGNSNTL (SEQ ID NO: 7), IGNTIPV (SEQ ID NO: 8), and LYANSPF (SEQ ID NO: 9); cyclic peptides having the amino acid sequences of CTVRTSADC (SEQ ID NO: 10), CNWGDRILC (SEQ ID NO: 11), CNWGKPIKC (SEQ ID NO: 12), CSGVKSAFC (SEQ ID NO: 13), CGVKSYNEC (SEQ ID NO: 14), CIGKTNTLC (SEQ ID NO: 15), CIGNSNTLC (SEQ ID NO: 16), CIGNTIPVC (SEQ ID NO: 17), or CLYANSPFC (SEQ ID NO: 18); linear peptides with cysteine ​​linkers; or retroinverso peptides having the retroinverso amino acid sequences of those linear peptides.

[0037] In other embodiments, the targeted peptide can specifically bind to EDA-FN. Targeted peptides that specifically bind to EDA-FN may include linear peptides having the amino acid sequence of WNYPFRL (SEQ ID NO: 19), SNTSYVN (SEQ ID NO: 20), SFSYTSG (SEQ ID NO: 21), WSPAPMS (SEQ ID NO: 22), TREHPAQ (SEQ ID NO: 23), or ARIIDNA (SEQ ID NO: 24); cyclic peptides having the amino acid sequence of CWNYPFRLC (SEQ ID NO: 25), CSNTSYVNC (SEQ ID NO: 26), CSFSYTSGC (SEQ ID NO: 27), CWSPAPMSC (SEQ ID NO: 28), CTREHPAQC (SEQ ID NO: 29), or CARIIDNAC (SEQ ID NO: 30); linear peptides with a cysteine ​​linker; or retroinverso peptides having the retroinverso amino acid sequence of any of these linear peptides.

[0038] Targeted peptides can be subjected to various modifications, substitutions, insertions, and deletions, and such modifications offer specific advantages in their use. In this regard, a targeted peptide that binds to and / or forms a complex with EDB-FN and / or EDA-FN can be substantially homologous rather than identical to the sequence of the listed peptides, with one or more modifications made, while maintaining its ability to specifically bind to and / or form a complex with EDB-FN and / or EDA-FN.

[0039] Targeted peptides can be any of the various forms of polypeptide derivatives, including amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, retroinverso peptides, analogs, fragments, chemically modified polypeptides, and similar derivatives.

[0040] Retroinverso peptides are linear peptides in which the amino acid sequence is reversed and the chirality of the α-center of the amino acid subunits is also reversed. These types of peptides are designed by including D-amino acids in a reverse sequence to help maintain a similar side-chain topology to the original L-amino acid peptide and to make them more resistant to proteolysis. D-amino acids exhibit a conformational mirror image of the native L-amino acids found in native proteins present in biological systems. Peptides containing D-amino acids have advantages over peptides containing only L-amino acids. In general, these types of peptides are less susceptible to proteolysis and have a longer shelf life when used. Furthermore, the insertion of D-amino acids in selected sequence regions, either alone or with intermediate L-amino acids, allows for the design of targeted peptides that are not only resistant to proteolysis but also bioactive and have increased bioavailability. Moreover, when properly designed, retroinverso peptides can have similar binding properties to L-peptides.

[0041] The term “analog” includes any peptide having a substantially identical amino acid sequence to the sequences specifically described herein, in which one or more residues are conservedly substituted with functionally similar residues, and which specifically bind to and / or form complexes with the EDB-FN and / or EDA-FN described herein. Examples of conservative substitutions include substitution of one nonpolar (hydrophobic) residue with another, such as isoleucine, valine, leucine, or methionine; substitution of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine; substitution of one basic residue with another, such as lysine, arginine, or histidine; or substitution of one acidic residue with another, such as aspartic acid or glutamic acid.

[0042] The term "conservative substitution" also includes the use of chemically derivatized residues instead of non-derivatized residues, provided that such peptides exhibit the required binding activity.

[0043] A "chemical derivative" refers to a target peptide having one or more residues that are chemically derivatized by the reaction of a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, or formyl group. A free carboxyl group may be derivatized to form a salt, methyl and ethyl ester, or other type of ester or hydrazide. A free hydroxyl group may be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine may be derivatized to form N-benzylhistidine. Polypeptides containing one or more naturally occurring amino acid derivatives from the 20 standard amino acids are also included in chemical derivatives. For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine in place of lysine, 3-methylhistidine in place of histidine, homoserine in place of serine, and ornithine in place of lysine. The peptides described herein also include any peptides whose sequences have one or more additions and / or deletions or residues from the sequences of the peptides shown herein, provided that they maintain the required binding specificity or activity.

[0044] The term "fragment" refers to any target peptide having an amino acid residue sequence shorter than that of the polypeptides shown herein.

[0045] Any polypeptide or compound may also be used in the form of a pharmaceutically acceptable salt. Acids that can form salts with polypeptides include inorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphate acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, and sulfanilic acid.

[0046] Bases that can form salts with polypeptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, as well as organic bases such as mono-, di-, and tri-alkyl and aryl-amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.).

[0047] Targeted peptides can be synthesized by any technique known to those skilled in the polypeptide field, including recombinant DNA technology. Synthetic chemical techniques such as solid-phase Merrifield synthesis may be used for reasons such as purity, antigen specificity, freedom from undesirable by-products, and ease of manufacture. A summary of many available techniques is provided for solid-phase peptide synthesis, including Steward et al., "Solid Phase Peptide Synthesis", WH Freeman Co., San Francisco, 1969; Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Patent No. 4,244,946; and for conventional solution synthesis, Schroder et al., "The Peptides", Vol. 1, Academic Press (New These may be recognized in JFWMcOmie, 1965, each of which is incorporated herein by reference. Suitable protecting groups available for use in such synthesis are described in the above text and in JFWMcOmie, "Protective Groups in Organic Chemistry," Plenum Press, New York, 1973, which is incorporated herein by reference.

[0048] Generally, the intended solid-phase synthesis method involves the sequential addition of one or more amino acid residues, or appropriately protected amino acid residues, to a growing peptide chain. Typically, either the amino group or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. Different selectively removable protecting groups are used for amino acids containing reactive side groups, such as lysine.

[0049] Using solid-phase synthesis as an example, protected or derivatized amino acids can be bound to an inert solid support via their unprotected carboxyl or amino groups. Next, the protecting group of the amino or carboxyl group is selectively removed, and the following amino acid in the sequence, having a well-protected complementary (amino or carboxyl) group, is mixed and reacted under conditions suitable for forming amide bonds with residues already bound to the solid support. Then, the protecting group of the amino or carboxyl group can be removed from this newly added amino acid residue, and the next amino acid (well-protected) is added, and so on. After all the desired amino acids have been linked in the appropriate sequence, the remaining terminal and side-group protecting groups (and the solid support) can be removed sequentially or simultaneously to obtain the final linear polypeptide.

[0050] Furthermore, the targeted peptides described herein can be used as a starting point for developing higher affinity small molecules, peptides, antibodies, and / or antibody fragments with similar ligand-binding capabilities. For example, the development and screening of small molecules from peptide pharmacophores using computer screening can be easily carried out, and the binding affinity of such identified molecules can be easily screened against the targeted peptides using the assay described herein for selecting small molecule drugs.

[0051] Additional residues may also be added to any end of the peptide for the purpose of providing a “linker” that can conveniently link and / or immobilize the peptide to other polypeptides, proteins, detectable moieties, labels, solid matrices, or carriers.

[0052] The amino acid residue linker is usually at least one residue, and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking include glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid. Furthermore, targeted peptide agents can differ in the modified sequence by terminal modifications such as terminal NH2 acylation, e.g., acetylation or thioglycolic acid amidation, or terminal carboxyl amidation, e.g., ammonia, methylamine. As is well known, terminal modifications are useful in reducing sensitivity to proteinase digestion and thus help extend the half-life of polypeptides in solution, especially in biological fluids where proteases may be present. In this regard, polypeptide cyclization is also a useful terminal modification and is particularly preferred due to the stable structure formed by cyclization and from the viewpoint of the biological activity observed for cyclic peptides such as those described herein.

[0053] If the linker is a peptide linker, the polypeptide linker can be generated as a single recombinant polypeptide using conventional molecular biological / recombinant DNA methods.

[0054] For example, the targeted peptide may contain lysine, which can react with a carbonyl-containing group such as an anhydride or acid halide, or with an alkyl group containing a good leaving group (e.g., a halide). The targeted peptide may also contain cysteine, which facilitates chemical coupling via thiol-selective chemistry (e.g., a maleimide-activated compound). Furthermore, the targeted peptide may contain tyrosine, which can be modified using a diazonium coupling reaction. In exemplary embodiments, the amino acid residue linker is a cysteine-glycine (CG) linker.

[0055] In other embodiments, chemical binding groups can be used. These binding groups can work to increase the chemical reactivity of the substituent on either the targeted peptide or the compound or molecule to which the targeted peptide is bound, thereby increasing the coupling efficiency. Binding chemistry can include maleimidyl binders that can be used to bind to thiol groups, isothiocyanate and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) binders that can bind to free amine groups, diazoniums that can be used to bind to phenols, and amines that can be used to bind to free acids such as carboxylic acid groups using carbodiimide activation.

[0056] Useful functional groups are present in the targeted peptide based on the specific amino acids present, and additional groups can be designed. It will be apparent to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homofunctional and heterofunctional (such as those listed in the catalog of Pierce Chemical Co., Rockford, Ill.), can be used as binding groups. Coupling can be carried out, for example, via amino groups, carboxyl groups, sulfhydryl groups, or oxidized carbohydrate residues.

[0057] Other types of bonding chemistry are also available. For example, methods for conjugating polysaccharides into peptides include, but are not limited to, methods using diester squalate (1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al. Bioconjug Chem. 2:148-153 (1991)), coupling to NaIO4-activated oligosaccharides via α or ε-amino groups (Bocher et al., J.Immunol. Methods 27,191-202 (1997)), coupling via peptide binders in which the polysaccharide has a reducing end and does not contain carboxyl groups (U.S. Patent No. 5,342,770), and coupling with synthetic peptide carriers derived from human heat shock protein hsp65 (U.S. Patent No. 5,736,146). Further methods for conjugating polysaccharides, proteins, and lipids into peptides are described in U.S. Patent No. 7,666,624.

[0058] In some embodiments, the linker is a non-peptide linker. The non-peptide linker can be a non-peptide aliphatic, heteroaliphatic, cyclic, and / or heterocyclic linker. The non-peptide linker may include, for example, alkylene, alkylene oxide, arylene, or alkylene-arylene linkers that covalently bond the peptide to the contrast agent.

[0059] In other embodiments, the linker may be a PEG molecular linker. The PEG molecules may have varying lengths and molecular weights, including, for example, PEG200, PEG1000, PEG1500, PEG4600, PEG10,000, or combinations thereof.

[0060] PET / SPECT contrast agents can be directly conjugated to the targeted peptide or linked to it via a linker. The role of the contrast agent is to facilitate the detection step of a detection or diagnostic method by enabling visualization of the complex formed when a PET / SPECT probe containing the targeted peptide binds to EDB-FN and / or EDA-FN. The contrast agent can be selected to produce a signal that can be measured and whose intensity is related (preferably proportional) to the amount of PET / SPECT probe bound to the tissue being analyzed.

[0061] In certain embodiments, the contrast agent comprises a chelating agent and a metal ion. The chelating agent generally has one or more groups that can form a covalent bond with a linker. Many different chelating agents known in the art can be used herein. In one embodiment, the chelating agent comprises an acyclic or cyclic compound comprising at least one heteroatom (e.g., oxygen, nitrogen, sulfur, phosphorus) having a lone pair of electrons that can coordinate with the imaging agent. Examples of metal chelating agents include diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazadodecanetetraacetic acid (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetic acid (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetic acid (DO3MA), and N,N',N'',N'''-tetraphosphonatomethyl-1,4,7,10-tetraa The following are examples of compounds that may be included: Zacyclododecane (DOTP), 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetrakis(methylenemethylphosphonic acid) (DOTMP), 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetrakis(methylenephenylphosphonic acid) (DOTPP), N,N'-Ethylenedi-L-Cysteine, 1,4,7-Triazacyclononane-1,4,7-Triazacyclononane (NOTA), 1,4,7-Triazacyclononane (TACN), N,N'-Bis(2-Hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N'-Diacetic acid (HBED-CC), and at least one of their derivatives. The term "derivative" is defined herein as the corresponding salt of a chelating agent and its ester.

[0062] The selection of metal ions can vary depending on the detection technique (e.g., PET or SPECT). Metal ions useful for PET and SPECT imaging include: 67 Ga, 68Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 It contains Sr.

[0063] In some embodiments, the PET / SPECT probe may have the following formula: [ka] During the ceremony, P1 is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, WNYPFRL (SEQ ID NO: 19), SNTSYVN (SEQ ID NO: 20), SFSYTSG (SEQ ID NO: 21), WSPAPMS (SEQ ID NO: 22), TREHPAQ (SEQ ID NO: 23), ARIIDNA (SEQ ID NO: 24), and their retroinverso amino acid sequences. R 1 This is optional and, if present, includes alkylene, alkylene oxide, arylene, or alkylene arylene linker, for example, -(CH2) n -,-(OCH2CH2) n Examples include , or Arirene, where n is an integer from 1 to 18. M is 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 A metal selected from the group consisting of Sr, or a salt thereof.

[0064] In another embodiment, the PET / SPECT probe is given by the following formula: [ka]

[0065] In yet another embodiment, the PET / SPECT probe is given by the following formula: [ka] and 67 Ga, 68 Ga, 64 Cu, 99m Tc, 111 In, 89 Zr, 90 Y, 153 Sm, or 89 It may have PET / SPECT radionuclides selected from the group consisting of Sr, or salts thereof.

[0066] The PET / SPECT probes described herein can be administered to a subject by, for example, systemic, topical, and / or parenteral administration methods. These methods include, for example, injection, infusion, deposition, implantation, or topical administration, or any other administration method where access to tissue by the molecular probe is desired. In one example, administration of the molecular probe may be by intravenous injection of the molecular probe into the subject. The probe may be administered as a single or multiple dose. As used herein, “administered” means providing or delivering the molecular probe in an amount and duration effective for labeling cancer cells in the subject.

[0067] The PET / SPECT probes containing the targeted peptides described herein can be administered to a patient in a detectable amount of a pharmaceutical composition containing the molecular probe or a pharmaceutically acceptable water-soluble salt thereof.

[0068] "Detectable amount" means that the amount of molecular probe administered is sufficient to enable detection of the binding or complex formation of the probe to EDB-FN and / or EDA-FN expressed by cancer cells or other cells in the cancer cell microenvironment. "Imaging effective amount" means that the amount of PET / SPECT probe administered is sufficient to enable imaging of the binding or complex formation of the molecular probe to EDB-FN and / or EDA-FN of cancer cells or other cells in the cancer cell microenvironment.

[0069] The formulation of the PET / SPECT probe to be administered will vary depending on the chosen route of administration (e.g., solution, emulsion, capsule, etc.). Suitable pharmaceutically acceptable carriers may contain inactive components that do not excessively inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, and free from other undesirable reactions upon administration to the subject. Standard formulation techniques, such as those described in Remington's Pharmaceutical Sciences, ibid., can be used. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic physiological saline (physiological saline containing approximately 0.9% mg / ml benzyl alcohol), phosphate-buffered physiological saline, Hanks' solution, and Ringer's lactate solution.

[0070] The preparation of pharmacological compositions containing dissolved or dispersed active ingredients is well understood in the art. Typically, such compositions are prepared as injections, either as liquid solutions or suspensions, although solid forms suitable for solutions or suspensions can also be prepared in liquid form before use. The formulations differ depending on the chosen route of administration (e.g., solutions, emulsions, capsules).

[0071] Any polypeptide or compound may also be used in the form of a pharmaceutically acceptable salt. Acids that can form salts with polypeptides include inorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphate acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, and sulfanilic acid.

[0072] Bases that can form salts with polypeptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, as well as organic bases such as mono-, di-, and tri-alkyl and aryl-amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.).

[0073] The PET / SPECT probes described herein can be used in a manner to detect and / or determine the presence, location, and / or distribution of cancer cells expressing EDB-FN and / or EDA-FN in an organ, tissue, or body region of interest. The presence, location, and / or distribution of the probes in animal tissue, for example, prostate tissue, can be visualized (e.g., using the in vivo imaging modalities described above). As used herein, “distribution” refers to a spatial characteristic that is scattered over an area or volume. In this case, “distribution of cancer cells” refers to the spatial characteristic of cancer cells scattered over an area or volume contained in an animal tissue, for example, prostate tissue. The distribution of the molecular probes may correlate with the presence or absence of cancer cells in the tissue. The distribution may be a clue to the presence or absence of cancer cells, or may be combined with other factors and symptoms by those skilled in the art to clearly detect the presence or absence of migrating or dispersing cancer cells, cancer metastases, or to clarify tumor margins in the subject.

[0074] In one embodiment, a PET / SPECT probe may be administered to a subject to assess the distribution of malignant or metastatic cancer cells in the subject and correlate the distribution to specific locations. Surgeons routinely use stereotactic techniques and intraoperative MRI (iMRI) in surgical resection. This allows them to specifically identify and sample tissue from different regions of the tumor, such as the tumor edge or tumor center. Often, they also sample areas of tissue at the tumor margin outside the tumor edge, which may appear normal to the naked eye but are infiltrated by dispersed tumor cells upon histological examination.

[0075] Using PET / SPECT probes that specifically bind to and / or form complexes with EDB-FN and / or EDA-FN associated with malignant or metastatic cells in intraoperative imaging techniques can induce surgical resection and eliminate the surgeon's "knowledge and experience guesswork" regarding the location of tumor margins. Previous studies have determined that wider surgical resection improves patient survival. Therefore, probes that function as diagnostic molecular imaging agents have the potential to increase patient survival rates.

[0076] In some embodiments, intraoperative imaging (IOI) techniques can be combined with systemically or locally administered PET / SPECT probes as described herein to identify and facilitate the removal of cancer cells. The PET / SPECT probe, upon administration to a subject, can target, detect, and / or determine the presence, location, and / or distribution of cancer cells, i.e., cancer cells associated with EDB-FN and / or EDA-FN expression, within a patient's organ or body region. In one example, the probe can be combined with IOI to identify malignant cells that have infiltrated and / or are beginning to infiltrate the tumor margin. This method can be performed in real time during surgery. This method may include the local or systemic application of a PET / SPECT probe containing a detectable portion, such as a PET or SPECT contrast agent. Imaging modalities can then be used to detect and subsequently collect image data. The obtained image data can be used, at least partially, to determine surgical and / or radiological treatment. Alternatively, this image data can be used, at least partially, to control automated surgical devices (e.g., lasers, scalpels, micromachines) or to assist in manual guidance of surgery. Furthermore, image data can be used to plan and / or control the delivery of therapeutic drugs (e.g., by microelectronic devices or micromachines).

[0077] Another embodiment described herein relates to a method for determining the invasiveness or malignancy of cancer cells in a subject. The binding strength of a PET / SPECT probe to cancer was found to correlate with the invasiveness of the cancer. Enhanced binding correlated with more aggressive cancers, while decreased or reduced binding correlated with less aggressive or benign tumors. In one example, the binding of a probe to a prostate tumor section correlated with the Gleason score based on the invasiveness of the tumor, with enhanced binding strength of the molecular probe correlated with aggressive or malignant prostate cancer and distinguished from benign prostatic hyperplasia, which showed lower probe binding. The methods and molecular probes described herein can be used to monitor and / or compare the invasiveness of cancer in a subject before, during, or after the administration of cancer treatment or cancer therapy regimens.

[0078] Another embodiment described herein relates to a method for monitoring the effectiveness of a cancer treatment or cancer therapy administered to a subject. Using the method described herein and a PET / SPECT probe, the invasiveness, invasion, migration, dispersion, and metastasis of cancer in a subject before, during, or after administration of a cancer treatment or cancer therapy regimen can be monitored and / or compared.

[0079] As used herein, “cancer treatment” or “cancer therapy” may include any agent or treatment regimen that can negatively affect cancer in animals by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or extending the lifespan of animals with cancer. Cancer treatment may include one or more therapies, including but not limited to chemotherapy, radiotherapy, hormone therapy, and / or biological / immunotherapy. For example, a reduction in the volume, proliferation, migration, and / or dispersion of cancer in a subject may indicate the effectiveness of a given therapy. This can provide a direct clinical efficacy endpoint measurement of cancer treatment. Thus, in another embodiment, a method for monitoring the effectiveness of cancer treatment is provided. More specifically, embodiments of this application provide a method for monitoring the effectiveness of cancer therapy.

[0080] A method for monitoring the effectiveness of cancer treatment may include the steps of administering a PET / SPECT probe in vivo to an animal as described herein, then visualizing the distribution of the probe in the animal (e.g., using an in vivo imaging modality as described herein), and then correlating the probe distribution with the effectiveness of the cancer treatment. The administration steps are intended to occur before, during, and after the course of the treatment regimen to determine the effectiveness of the selected treatment regimen. One method for evaluating the effectiveness of cancer treatment is to compare the distribution of the probe before and after cancer therapy.

[0081] In some embodiments, a PET / SPECT probe bound to and / or complexed with EDB-FN and / or EDA-FN is detected in a subject to detect and / or provide information about the invasiveness, location, and / or distribution of cancer cells in the subject. The invasiveness, location, and / or distribution of cancer cells in the subject can then be compared to a control to determine the effectiveness of cancer treatment and / or cancer therapy. The control may be the location and / or distribution of cancer cells in the subject before administration of cancer treatment and / or cancer therapy. The location and / or distribution of cancer cells in the subject before administration of cancer treatment and / or cancer therapy can be determined by administering the probe to the subject and detecting the probe that has bound to and / or complexed with cancer cells in the subject before administration of cancer treatment and / or cancer therapy.

[0082] In certain embodiments, the method and PET / SPECT probe described herein can be used to measure the effectiveness of a therapeutic agent administered to a subject for treating metastatic or invasive cancer. In this embodiment, the probe can be administered to the subject before, during, or after administration of a treatment regimen to image the distribution of cancer cells and determine the effectiveness of the treatment regimen. In one example, the treatment regimen may include surgical resection of metastatic cancer, and the probe can be used to clarify the distribution of metastatic cancer before and after surgery to determine the effectiveness of the surgical resection. Optionally, the method and probe can be used in intraoperative surgical procedures, such as surgical tumor resection, to more easily clarify and / or image the mass or volume of cancer cells during surgery.

[0083] In another embodiment, the targeted peptide can be conjugated with a therapeutic agent and administered to a subject to treat cancer such as metastatic cancer. In this embodiment, the targeted peptide conjugated with a therapeutic agent can be administered to a subject, and metastatic cells can be targeted with the therapeutic agent.

[0084] Therapeutic agents may include antiproliferative agents that exert antitumor, chemotherapeutic, antiviral, antimitotic, antitumorogenic, and / or immunotherapeutic effects, for example, by directly preventing neoplastic cell development, maturation, or spread in tumor cells through cell proliferation inhibition or cytodestructive effects, without indirectly involving mechanisms such as alteration of biological responses. Numerous antiproliferative agents are available for commercial use, clinical evaluation, and preclinical development. For the convenience of discussion, antiproliferative agents are classified into the following classes, subtypes, and species. ACE inhibitors, alkylating agents, angiogenesis inhibitors, angiostatins, anthracyclines / DNA intercalators, anticancer antibiotics or antibiotic-type drugs, antimetabolites, antitransferase compounds, asparaginase, bisphosphonates, cGMP phosphodiesterase inhibitors, calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNA topoisomerase, endostatins, epipodophyllotoxin, genistein, hormonal anticancer agents, hydrophilic bile acids (URSOs), immunomodulators or immunological agents, integrin antagonists, interferon antagonists or drugs, MMP inhibitors, various antineoplastic agents, monoclonal antibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors, pBATT, radioactive / chemosensitizers / protective agents, retinoids, selective inhibitors of endothelial cell proliferation and migration, selenium, stromelycin inhibitors, taxanes, vaccines, and vinca alkaloids.

[0085] The main categories into which several antiproliferative agents are classified include antimetabolites, alkylating agents, antibiotic-type drugs, hormonal anticancer agents, immunological agents, interferon-type drugs, and various antineoplastic agents. Some antiproliferative agents function through multiple or unknown mechanisms and can therefore be classified into two or more categories.

[0086] In some embodiments, targeted peptides can be bound to therapeutic agents using linking molecules. These linking molecules may be linkers, or they may be non-peptide linkers. [Examples]

[0087] Example 1 The inventors of the present invention have identified ZD2 64 We developed a Cu-DOTA conjugate as a PET probe for EDB-FN and evaluated its effectiveness in PET imaging in mice carrying xenografts of aggressive PC3 and slow-growing LNCaP human prostate tumors. We showed that EDB-FN was highly expressed in aggressive PC3 tumors and negligibly expressed in slow-growing, non-metastatic LNCaP tumors. MRI using the EDB-FN-targeted contrast agent ZD2-Gd (HP-DO3A) showed stronger contrast enhancement in PC3 tumors than in LNCaP tumors. 64 The use of Cu is particularly attractive due to its half-life of 12.74 hours, providing an extended imaging timeframe for cancer detection in the prostate, especially with minimal background estimation from the bladder. The PET probe conjugates the ZD2 peptide to DOTA, a macrocyclic ligand, and then... 64 The probe was synthesized by complex formation with CuCl2. The ability of the PET probe to detect cancer and characterize tumor invasiveness was evaluated in mice carrying PC3 and LNCaP tumors.

[0088] material and method Synthesis of ZD2-PEG-DOTA and its chelate Unless otherwise specified, reagents used in chemical synthesis were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Fmoc-protected amino acids and 2-chlorotrityl chloride resin were obtained from Chem-Impex International, Inc. (Wood Dale, IL). The spacer, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-NH-(CH2CH2O)2-CH2COOH), was obtained from Chempep (Wellington, FL). 1,4,7,10-tetraaza-cyclododecane-1,4,7-tris-tert-butylacetate-10-acetic acid (DOTA-tris(t-Bu)) was purchased from TCI America (Port-land, OR).

[0089] The precursor ZD2-DA-DOTA, containing the ZD2 peptide (sequence: TVRTSAD), two repeats of NH2-(CH2CH2O)2-CH2COOH, and DOTA, was synthesized by sequentially adding the corresponding protecting amino acid, Fmoc-NH-(CH2CH2O)2-CH2COOH, and t-Bu-DOTA on a solid-phase resin using standard Fmoc-peptide chemistry. The product was then cleaved from the resin using trifluoroacetic acid / triisopropylsilane / H2O (96.5:1:2.5), stirred at room temperature for 3 hours, and precipitated in ether to obtain the crude product. The final product was purified using preparative HPLC on an Agilent 1100 HPLC system equipped with a semi-preparative C18 column (Agilent Technologies, Santa Clara, CA). ZD2-PEG-DOTA was characterized by MALDI-TOF mass spectrometry using a Voyager DE-STR spectrometer (PerkinElmer, Waltham, MA) in linear mode with R2,5-dihydroxybenzoic acid as the matrix (M+1: 1425.8, experimentally; 1425.7, calculated).

[0090] Cell cultures and animal models The animal studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University (CWRU), and all subjects signed informed consent forms. PC3 and LNCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Roswell Park Memorial Institute medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin in a humidified incubator maintained at 37°C and 5% CO2. Male athymoid nude mice (4-6 weeks old) were obtained from the Case Comprehensive Cancer Center (Cleveland, OH, USA) and housed at the CWRU Animal Core Facility. 3 million cells in high-concentration Matrigel (Corning, Tewksbury, MA) were used for tumor inoculation. LNCaP cells were subcutaneously inoculated into the left flank of mice. Four weeks later, PC3 cells were inoculated into the right flank of the same mice for PET imaging.

[0091] radioactive label radioactive isotope 64 Cu(II) was obtained from the University of Wisconsin-Madison (Madison, WI). Chelation of ZD2-PEG-DOTA with Cu(II) was first tested using non-radioactive CuCl2 in a 0.1N HCl aqueous solution under the same conditions as for radiolabeling. Equimolar amounts of ZD2-DA-DOTA and CuCl2 solution in PBS buffer (pH 7.4) were mixed and stirred at 45°C for 30 minutes. The formation of ZD2-DA-(Cu-DOTA) was verified by MALDI-TOF mass spectrometry (M+1: 1487.8, experimental; 1486.04, calculated). For radiolabeling, 10 mCi 64 Cu(II) was dissolved in 200 μL of 0.1N HCl. 20 microliters64 A Cu(II) solution (approximately 1 mCi) was mixed with 480 μL of ZD2-DA-DOTA (0.05 mg / mL, large excess, PBS) in a 1.5 mL microcentrifuge tube. The container was then heated and maintained at 45°C for 30 minutes with intermittent shaking. The final pH of the solution was adjusted to neutral using NaOH solution before injection.

[0092] PET imaging All in vivo imaging studies were performed according to protocols and guidelines approved by the CWRU Animal Research Committee. Mice were anesthetized with 2% isoflurane in oxygen and given approximately 200 μCi [approximately 7.4 MBq] of ZD2-DA- 64 Cu(DOTA) was injected via the tail vein. Mice underwent PET scans with 4-hour and 22-hour uptake periods (Inveon microPET, Siemens Medical Solutions USA Inc.), followed by a 10-minute static PET scan. Images were reconstructed using 3D-OSEM with a 3D histogram and a zoom factor of 1.0 (MAP of 2 replicates followed by 18 replicates). CT scans (Siemens Medical Solutions USA Inc.) were performed after PET procedures for anatomical concurrent registration. PET / CT images were analyzed using AMIDE version 1.0.557 and AMIRA software. ROIs were plotted for PC3 and LNCaP tumors, and the ratio of specific binding to nonspecific (muscle) binding was calculated.

[0093] Biological distribution Three mice were euthanized 22 hours after the final micro-PET / CT imaging, and their organs and blood were collected and weighed. Radioactivity was determined using a gamma counter. The percentage of the injected volume per gram of tissue was calculated using a standard containing 2% of the injected volume.

[0094] Histological analysis After image acquisition, the mice were euthanized. Tumors were harvested, embedded in an optimal cutting temperature medium, frozen at -80°C, and frozen-sectioned to 5 μm thickness. The tissues were then permeabilized with cold acetone. The tissues were blocked with bovine serum albumin (1%) in PBS at room temperature for 1 hour. Anti-EDB-FN BC1 antibody (Abcam, Cambridge, MA) was incubated with tissue sections of PC3 and LNCaP tumors. After extensive washing, secondary anti-mouse Alexa Fluor488 antibody was incubated for 1 hour. The tissue sections were counterstained with Prolong Gold anti-fading mounting medium supplemented with 4'6-diamidino-2-phenyl-indole (Thermo Fisher, Waltham, MA). The stained tissues were imaged using an Olympus FV1000 confocal laser scanning microscope.

[0095] result ZD2 64 Cu-DOTA conjugates are produced by using solid-phase peptide chemistry to conjugate the ZD2 peptide to the macrocyclic chelate DOTA, followed by... 64 The target ligand ZD2-DA-DOTA was synthesized by complex formation with CuCl2 (Figure 1). A short spacer with two repeats of 8-amino-3,6-dioxaoctanoic acid was introduced between the peptide and the chelating agent. The targeted ligand ZD2-DA-DOTA was purified by preparative high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry [m / z = 1425.8 (M+1), experimental; 1425.5, calculated]. Preparation of the targeted PET probe was demonstrated by complex formation of equimolar amounts of ZD2-DA-DOTA in phosphate-buffered saline (PBS) buffer (pH 7.4) and non-radioactive CuCl2 in dilute HCl (0.1N) at 45°C for 30 minutes, and the same conditions were used for radiolabeling. The formation of ZD2-DA-(Cu-DOTA) was verified by MALDI-TOF mass spectrometry [m / z=1487.8(M+1), experimental; 1486.04, calculated].

[0096] Next, ZD2-DA-( in prostate cancer PET imaging) 64The efficacy of Cu-DOTA was investigated in male nude mice carrying both PC3 and LNCaP human prostate cancer xenografts. We have previously shown that EDB-FN is highly expressed in aggressive PC3 tumors and negligibly expressed in slow-growing, non-metastatic LNCaP tumors. Using tumor models, we tested the probe's ability to detect and stratify aggressive prostate cancer by representing high-risk and low-risk prostate tumors. Radiolabeling was performed in 20 μL of a 1.5 mL microcentrifuge tube. 64 The reaction was performed by mixing Cu(II) solution (0.1N HCl, approximately 1 mCi or 37 MBq) with 480 μL of ZD2-DA-DOTA (0.05 mg / mL, large excess, PBS, pH=7.4) and maintaining at 45°C for 30 minutes with intermittent shaking. The reaction mixture was then diluted in PBS in a 1:2 ratio and tested with pH test strips to ensure a neutral pH for intravenous injection. The radioactive tracer was intravenously injected at a dose of 7.4 MBq (200 μCi) per mouse. PET images of the mice were acquired at 4 and 22 hours post-injection in groups of four mice carrying both PC3 and LNCaP tumor xenografts.

[0097] Figure 2 shows ZD2-DA-( 64 Representative three-dimensional (3D) volume renderings and axial PET / computed tomography (CT) images of two tumor-bearing mice at 4 and 22 hours after injection of Cu-DOTA are shown. Invasive PC3 tumors showed a visually stronger signal than slow-growing LNCaP tumors. The location and size of the PC3 tumors were clearly depicted in the PET images. Tracer uptake or signal intensity was quantitatively analyzed in the region of interest (ROI) at 4 and 22 hours. As shown in Figure 3, ZD2-DA-( 64Cu-DOTA resulted in higher probe uptake in PC3 tumors than in LNCaP tumors. At 22 hours, PET revealed more than twice the accumulation of PET tracers in aggressive PC3 tumors (7711±1994 Bq / mL) compared to less aggressive LNCaP tumors (3213±1511 Bq / mL) (N=4, P<0.05, two-sided Student's t-test). Other organs that showed substantial uptake of radiotracers were the liver, stomach, and kidneys, indicating clearance of radiotracers via hepatic and renal pathways.

[0098] The biodistribution of the radiotracer was measured 24 hours after injection, after frightening the mice (Figure 4). The biodistribution pattern was consistent with PET imaging results, showing strong uptake in tumors, liver, and kidneys. Other organs, such as the brain and muscles, showed low uptake of the radiotracer, which is a desirable characteristic of the radiotracer. A comparison of radiotracer uptake in PC3 and LNCaP tumors showed that radiotracer accumulation in PC3 (1. 64 The ID% / g value was shown to be higher in invasive PC3 tumors (0.86 ID% / g) than in LNCaP tumors (N=3, P=0.32, two-sided Student's t-test), confirming that the probe preferentially accumulates in invasive PC3 tumors rather than non-metastatic LNCaP tumors.

[0099] EDB-FN expression in prostate tumors was determined by immunofluorescence staining of tissue sections of PC3 and LNCaP tumors with the anti-EDB-FN monoclonal antibody BC1 after PET imaging. BC1 antibody and EDB-FN were stained using Alexa Fluor488 conjugate anti-mouse antibody. Figure 5 shows fluorescence images of tumor sections acquired with an Olympus FV1000 confocal laser scanning microscope. Strong fluorescence staining was visually evident in PC3 tumor sections, while almost no staining was observed in LNCaP tumors. Consistently, we have previously shown that EDB mRNA levels in LNCaP cells were lower than those in PC3 cells. EDB-FN expression levels in the two different prostate tumors correlated well with observations by PET molecular imaging. The results were ZD2-DA-( 64 This suggests that Cu-DOTA is effective for highly sensitive and quantitative visualization of EDB-FN expression in prostate cancer.

[0100] In this example, the peptide probe ZD2-DA-( 64 The possibility of PET imaging of the ECM oncoplasmic protein EDB-FN using Cu-DOTA was demonstrated. The inventors have already shown that EDB-FN is highly expressed in rapidly proliferating PC3 tumors and low in slow-proliferating LNCaP tumors. ZD2 peptide-targeted MRI contrast agent was able to produce a stronger signal in PC3 tumors than in LNCaP tumors. ZD2-DA-( 64 The results of PET molecular imaging with Cu-DOTA (EDB-FN) were consistent with MR molecular imaging using ZD2 peptide-targeted MRI contrast, particularly 22 hours post-injection. Comparing probe uptake in tumors, a strong PET signal was detected in rapidly proliferating PC3 tumors with higher EDB-FN expression than in slow-proliferating LNCaP tumors. However, significant signal intensity was still observed in LNCaP tumors on PET images. This suggests that... 64 This may be due to the relatively low chelation stability of Cu-DOTA monoamide. 64Cu(II) has been shown to accumulate in prostate tumors in animal models. The relatively high signal intensity in LNCaP tumors is due to free Cu(II) released from the probe. 64 This may be due to Cu(II) accumulation. Nevertheless, the targeting effect of the probe's ZD2 peptide still resulted in significantly higher signal intensity in PC3 tumors than in LNCaP tumors. Compared to MR molecular imaging, PET imaging provides highly sensitive and quantitative visualization and measurement of EDB-FN expression levels in prostate cancer, offering more accurate risk stratification of invasive prostate cancer.

[0101] Generally, PET imaging with probes with relatively short half-lives suffers from significant signal inference from the bladder when imaging primary tumors in the prostate due to the limited imaging window. 64 Cu is important for early detection of primary tumors in the prostate, as it allows sufficient time to empty the bladder and minimize potential signal interference from the bladder. Substantial signaling was still visually apparent in the tumor 22 hours after injection, with little to no signaling from the bladder. Significant signal intensity was observed with ZD2-DA-( 64 This was observed in the liver by Cu-DOTA, and may be due to the relatively low stability of Cu-DOTA monoamide. Release from chelate. 64 The release of Cu-(II) can lead to the nonspecific accumulation of radioactive isotopes in the liver.

[0102] Antibodies and antibody fragments are being developed targeting EDB-FN for the detection of cancers, including prostate cancer. This study demonstrated that small peptide-targeted PET probes specific to EDB-FN also have potential for prostate cancer imaging. Compared to antibody-based probes, small peptide PET probes have several advantages, including cost-effective generation, superior tumor penetration through diffusion and perfusion, and rapid excretion of unbound probes from circulation.

[0103] Example 2 The inventors have shown that EDB-FN is highly expressed in human pancreatic cancer (PaCa) specimens and PaCa tissue from mouse PaCa models, and is not expressed in normal pancreatic tissue in either case. The presence of EDB-FN in the PaCa tumor ECM enables rapid and specific binding of targeted tracers for highly sensitive molecular imaging and PaCa diagnosis. The peptide sequence of the EDB fragment is conserved across all mammalian species.

[0104] We identified the peptide ZD2 (Thr-Val-Arg-Thr-Ser-Ala-Asp), which specifically binds to EDB-FN. The ZD2 peptide showed strong binding affinity to high-grade prostate tumors, weak binding affinity to low-grade tumors, and non-binding in normal tissue. This example demonstrates that the ZD2 peptide can be used to develop a PET probe for highly sensitive and quantitative molecular imaging of EDB-FN for accurate detection and risk stratification of pancreatic cancer. A ZD2 peptide-targeted Ga(III) PET probe was designed and synthesized by conjugating NOTA to the ZD2 peptide using the linker 6-aminohexanoic acid. The efficacy of the targeted Ga(III) tracer was evaluated for PET imaging in male nude mice carrying invasive, rapidly proliferating PC3 and slow-proliferating LNCaP prostate cancer xenografts.

[0105] experiment material Protecting amino acids for peptide synthesis were purchased from Novabiochem (Burlington, MA, USA). N,N-diisopropylethylamine (DIPEA) was purchased from MP Biomedical LLC (Santa Ana, CA, USA). O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU) was purchased from Anaspec Inc (Fremont, CA, USA). Fmoc-6-aminohexanoic acid was purchased from Chem-IMPEX International (WD, IL, USA). t-butylbromoacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemical reagents were purchased from Thermo Fisher. 1 ¹H-NMR spectra were acquired using a 500 MHz Varian Inova NMR spectrometer (supplier and address) with TMS as an internal standard. Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectra were acquired using a Voyager DE-STR spectrometer (PerSeptive BioSystems) in linear mode with 2,5-dihydroxybenzoic acid as the matrix. An Agilent 1100 with a ZORBAX 300 SB-C18 column semi-preparative HPLC was used for ligand purification under the following conditions: Eluent A, H₂O / TFA (0.1%); B, MeCN / TFA (0.1%); 15 min at 0%B, 30 min at 0-50%B, 5 min at 50%B, 2 min at 50-100%B, 5 min at 100%B, flow rate 2 mL / min, UV detection at 210 nm. Ga was eluted with 0.1 M HCl. 68 Ge / 68 Ga was obtained from a Ga generator (ITG isotope technologies Garching GmbH, Germany).

[0106] synthesis Synthesis of 1,4-bis(tert-butoxycarbonylmethyl)-1,4,7-triazanonanes 1,4,7-Triazacyclononane (1.5 g, 11.62 mmol) was dissolved in anhydrous CHCl3 (15 mL) in an ice bath, and tert-butyl bromoacetate (4.98 g, 25.56 mmol) in CHCl3 (30 mL) was slowly added over 1.5 hours. The mixture was stirred at room temperature for 24 hours to remove the solvent. The residue was treated with DI water (15 mL), adjusted to pH 3 with 1 M HCl, and extracted with ether (50 mL x 2). The organic phase was removed, and the aqueous phase was adjusted to pH 8-9 with 1 M NaOH and extracted again with CH2Cl2 (25 mL x 3). Finally, the organic phase was evaporated to obtain the product. Yield: 36%. 1 H NMR (500MHz, CDCl3): δ=1.48(s,18H),2.79(s,4H),3.03~3.07(m,4H),3.24(s,4H),3.37(s,4H),9.46(s,H).

[0107] Synthesis of NOTA-bis(t-Bu ester) 1,4-Bis(tert-butoxycarbonylmethyl)-1,4,7-triazanonanane (0.3 g, 0.84 mmol) and bromoacetic acid (0.415 g, 3 mmol) were dissolved in methanol (3 mL), and K2CO3 (0.53 g, 3.84 mmol) in water (3 mL) was added. The mixture was stirred overnight at room temperature and concentrated. Next, the residue was dissolved in water and adjusted to pH 4 with 1 M HCl. Water was removed by rotary evaporation, and the product was purified by flash chromatography (methanol:ethyl acetate 6.5:3.5). Yield: 64 %, 1 H NMR (500MHz, D2O): δ=1.48(s,18H),2.84(s,4H),3.08(m,4H),3.35(s,4H),3.47(s,4H).

[0108] Synthesis of ZD2-HA-NOTA ZD2-HA was synthesized using solid-phase chemistry. A mixture of 6-aminocaproic acid (1.5 equivalents), HBTU (1.5 equivalents), and DIPEA (1.5 equivalents) in 10 mL of anhydrous DMF was added to the resin at the end of peptide synthesis (0.5 mmol peptide), and the mixture was shaken until ninhydrin no longer changed color (Kaiser test). Next, the resin was washed using DMF (10 mL × 3) and DCM (10 mL × 3). Subsequently, ZD2-HA was cleaved from the resin for 3 hours using a cocktail of TFA:H2O:TIBS (96.5:2.5:1). ZD2-HA was precipitated in cold ethyl ether, centrifuged, and lyophilized. The product was characterized by MALDI-TOF mass spectrometry: calculated m / z for [M], C 47 H 83 N 15 O 18 , 1146.25; found (M+H + ), 1147.56.

[0109] Synthesis of non-radioactive Ga-ZD2-HA-NOTA To a solution of ZD2-HA-NOTA (0.11 g, 0.1 mmol) dissolved in 10 mL of NaAc-Ac buffer (0.1 M, pH 5.5), Ga(NO3)3 (0.076 g, 0.3 mmol) was added. The solution was stirred overnight at room temperature, and finally, the product was purified using preparative HPLC and lyophilized to obtain a flocculent white powder. Yield: 43%. The product was characterized by MALDI-TOF mass spectrometry: calculated m / z for [M], C 47 H 81 GaN 15 O 18 , 1212.51; found (M+H + ), 1213.54.

[0110] Results and Discussion Chemistry and Radiochemistry The synthesis of ZD2-HA-NOTA is shown in Figure 6. NOTA-bis(t-Bu ester) was prepared from TACN as a starting material by two substitutions. Next, the precursor ZD2-HA-NOTA was successfully synthesized by conjugation of NOTA-bis(t-Bu ester) and ZD2-HA using a solid-phase peptide and purified by RP-HPLC. The purified ZD2-HA-NOTA was characterized by MALDI-TOF (m / z = 1147.56) and HPLC (purity: approximately 98%). Nat Ga-ZD2-HA-NOTA was also prepared and characterized by MALDI-TOF (m / z=1213.54) and RP-HPLC (purity: approximately 96%).

[0111] Non-radioactive ZD2-(Ga-NOTA) was initially synthesized according to the procedure shown in Figure 6. The macrocyclic ligand NOTA was used under relatively mild conditions. 68 This is because it can easily form a stable chelate with Ga(III), which is important for maintaining the peptide's binding properties. The ZD2 peptide was synthesized using standard solid-phase peptide synthesis, and then 6-aminohexanoic acid (HA) was conjugated to the N-terminus of the peptide as a spacer. NOTA-bis(t-Bu ester) was finally conjugated to the amino group on the resin, and the targeted ligand ZD2-NOTA was obtained by treating the resin with a cocktail of TFA:H2O:TIBS (96.5:2.5:1). The final product was purified by preparative HPLC. The purified ZD2-NOTA was characterized by MALDI-TOF (m / z = 1147.56 [M+1], experimentally; 1146.25, calculated) and had a purity of approximately 98% (HPLC), Figure 7A, B. Next, ZD2-( Nat Ga-NOTA was prepared by reacting the ligand with excess GaCl3 in acetate buffer (0.1 M, pH 5.5) at room temperature. ZD2-( Nat Ga-NOTA) was purified using preparative HPLC and characterized by MALDI-TOF (m / z = 1213.54 [M+1], measured; 1212.51, calculated), with a purity of approximately 96% (HPLC), Figure 43C, D. Peptides and ZD2-(Nat Ga-NOTA) has high water solubility, which is an advantageous property for minimizing non-specific tissue binding.

[0112] Radiotracer ZD2-( 68 Ga-NOTA) was radiosynthesized in the radiopharmaceutical laboratory of University Hospitals, Cleveland (UH) in collaboration with Dr. Avril by reacting ZD2-NOTA with GaCl3 in sodium acetate buffer solution (0.1M, pH 5.5) at 90°C for 15 minutes. The pH of the reaction solution was finally adjusted with NaOH. The radiochemical yield was approximately 77% as determined by HPLC (water + 0.1% TFA / acetonitrile + 0.1% TFA gradient, UV at 220 nm) equipped with a radio detector and a Zorbax Eclipse C18 column. The radiolabeled tracer was purified using reverse-phase HPLC with a C-18 column prior to imaging. ZD2-( 68 The HPLC chromatogram of Ga-NOTA) is shown in Figure 8, and the product parameters are summarized in Table 3. The small peaks around the main peak are probably 68 attributable to complex formation between Ga(III) and the peptide, which is commonly observed for radiolabeled peptide products. The radiolabeling yield is comparable to that of clinical tracers. The purity of the product is also equivalent to that of clinical-grade products. [Table 1]

[0113] Expression of EDB-FN in human pancreatic cancer cells and tumor xenografts EDB-FN expression was first demonstrated by Western blotting in four different human pancreatic cancer cell lines, including BXPC3, Capan-1, Panc10.05, and Panc-1 cells. These human PaCa cell lines are commonly used to develop mouse PaCa cancer models in preclinical studies. All cancer cell lines tested exhibited high expression of EDB-FN, Figure 9A. The tumor model was developed by subcutaneous transplantation of cancer cells in the flank of female nude mice, as described by ATCC. EDB-FN expression was demonstrated in tumor xenografts of human PaCa cells using immunofluorescence staining with BC-1 anti-EDB-FN monoclonal antibody. As shown in Figure 9B, substantial expression of EDB-FN was observed in all four PaCa subtypes, and no expression was observed in normal pancreas and muscle, consistent with reported results. High expression of EDB-FN was observed in the ECM of PaCa tumors. The results indicate that EDB-FN is highly expressed by PaCa cells and tumors, making it a promising oncoprotein target for molecular imaging and detection of PaCa.

[0114] ZD2 peptide that binds to EDB-FN in PaCa tumors The ZD2 peptide (Thr-Val-Arg-The-Ser-Ala-Asp) targeted fluorescent tracer ZD2-Cy5.5 was synthesized according to a reported method for evaluating the peptide's binding to EDB-FN in PaCa tumors. The binding specificity of the ZD2 peptide to EDB-FN in pancreatic cancer was tested by incubation of ZD2-Cy5.5 with tumor slides of the tumor xenografts described above. Strong binding of ZD2-Cy5.5 (red) was observed in all four tumor tissues tested, as shown in Figure 10, as well as immunofluorescence staining in Figure 9B. No significant binding of ZD2-Cy5.5 to normal pancreas and muscle was observed. The strong binding of ZD2-Cy5.5 to EDB-FN in PaCa was blocked by the BC-1 anti-EDB-FN monoclonal antibody (BC-1 / ZD2). In PaCa specimens pre-incubated with BC-1 antibody, followed by ZD2-Cy5.5 (BC-1 / ZD2), little red fluorescence staining was observed. The results suggest that both ZD2-Cy5.5 and BC-1 specifically bind to the same EDB-FN protein target in tumor tissue. The ZD2 peptide is a promising targeting agent for the specific binding of EDB-FN in PaCa tumors.

[0115] EDB-FN expression in human PaCa tumors EDB-FN expression in human pancreatic cancer is indicated by staining human pancreatic cancer specimens with ZD2-Cy5.5. As shown in Figure 11, strong red fluorescence was observed in human PaCa specimens, and almost no fluorescence was observed in normal pancreas, although some fluorescence intensity was shown in precancerous pancreatic intraepithelial neoplasms. The fluorescence intensity suggests high EDB-FN expression in PaCa, low expression in precancerous tissue, and no expression in normal pancreas.

[0116] ZD2-( 68 PET imaging of PaCa using Ga-NOTA ZD2-( in high-sensitivity molecular imaging of EDB-FN and detection of PaCa) 68 Effectiveness of Ga-NOTA The tracer was evaluated by micro-PET / CT in mouse models carrying Capan1 and BXPC3 human PaCa xenografts. Tumor models were developed in female nude mice, similar to C.1. Tracers synthesized using the method described above were intravenously injected at a dose of 300 μCi per mouse. Figure 12 shows representative 2D coronal PET / CT images showing the tumor at 1 and 2 hours post-injection of the tracer. Strong uptake of the tracer was observed in both the tumor and bladder at both time points. In normal tissues and organs, particularly the brain, liver, and lungs, little uptake was observed at 1 hour post-injection. Background noise increased slightly at 2 hours post-injection, likely due to decreased radioactivity and prolonged scan time. The signal intensity in both tumors was approximately 5 times that in muscle at 1 and 2 hours post-injection. Three-dimensional PET images also showed strong uptake into the tumor with little nonspecific uptake into surrounding normal tissues and organs other than the kidney and bladder, Figure 13. High signal intensity in the kidneys and bladder indicates that the tracer is primarily excreted via renal filtration. These results suggest that ZD2-( is useful for molecular imaging of EDB-FN and early detection of PaCa.) 68 We will demonstrate the efficacy and high specificity of Ga-NOTA and further verify the specific expression of EDB-FN in PaCa. ZD2 peptide targeting. 68 Ga chelates show promise for highly sensitive early detection of pancreatic cancer in clinical practice.

[0117] Example 3 Synthesis of ZD2-HBED-CC The ZD2 peptide was synthesized using standard solid-phase chemistry. Next, HBED-CC-tris(tBu) ester was conjugated to the N-terminus of the ZD2 peptide on a resin. The peptide was then cleaved from the resin using a TFA / water / TIBS(96.5 / 2.5 / 1) cocktail. The product was precipitated in ethyl ether, purified by preparative HPLC, lyophilized, and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, experimentally 1264.02; calculated for C55H82N12O22, 1264.32.

[0118] Synthesis of ZD2-AH-HBED-CC The ZD2 peptide was synthesized using standard solid-phase chemistry. Next, Fmoc-6-aminohexanoic acid was conjugated to the N-terminus of the ZD2 peptide on a resin. Subsequently, HBED-CC-tris(tBu) ester was reacted with the peptide, and cleavage from the resin was continued using a TFA / water / TIBS(96.5 / 2.5 / 1) cocktail. The product ZD2-AH-HBED-CC was precipitated in ethyl ether, purified by preparative HPLC, lyophilized, and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, experimentally 1377.1; calculated for C61H93N13O23, 1377.48.

[0119] Synthesis of ZD2-(Ga-HBED-CC) Ligand- and linker-free ZD2-HBED-CC and gallium nitrate were mixed in PBS at 90°C for 2 minutes. The product ZD2-(Ga-HBED-CC) was then purified by preparative HPLC and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, experimentally 1329.8; calculated for C55H79GaN12O22, 1329.47.

[0120] Synthesis of ZD2-AH-(Ga-HBED-CC) ZD2-HBED-CC with ligands and linkers, and gallium nitrate were mixed in PBS at 90°C for 2 minutes. The product ZD2-AH-(Ga-HBED-CC) was then purified by preparative HPLC and characterized by MALDI-TOF mass spectrometry. (M+1) m / z, experimentally 1442.9; calculated for C61H90GaN13O23, 1442.55.

[0121] PET imaging of mice with tumors All in vivo imaging studies were performed according to protocols and guidelines approved by the CWRU Animal Research Committee. Mice carrying BxPC3 or Capan-1 human pancreatic xenografts were anesthetized with 2% isoflurane in oxygen. Tracer ZD2-(68 Ga-HBED-CC) or ZD2-AH-( 68 Ga-HBED-CC was injected via tail vein at doses of 100–300 μCi [5.3–13.0 MBq]. Mice then underwent a 10 or 20-minute static PET scan (Inveon microPET, Siemens Medical Solutions USA Inc.) after a 30 or 60-minute uptake period. CT scans were followed after all PET procedures for simultaneous anatomical registration. PET / CT images were analyzed using Inveon Research Workplace version 3.0 and Horos software. Regions of interest (ROIs) were plotted for tumors, major organs, and muscles to calculate specific and nonspecific tissue uptake ratios. Images were processed with 3D reconstruction at a zoom factor of 1.0 using 3D-OSEM with two repeats, followed by MAP with 18 repeats.

[0122] ZD2-( in high-sensitivity molecular imaging of EDB-FN and detection of pancreatic cancer) 68 The efficacy of Ga-HBED-CC was evaluated by micro-PET / CT in mouse models carrying Capan-1 and BxPC3 human pancreatic cancer xenografts. The figure shows representative 2D and 3D whole-body PET / CT images of tumor-bearing mice 30 or 60 minutes post-injection. Strong tracer uptake was observed in the tumors, kidneys, and bladder 30 or 60 minutes post-injection, as shown in the whole-body PET images. Tracer uptake in both tumors was significantly higher than in normal organs and tissues, including the brain, heart, liver, and muscle. High signal intensities in the kidneys and bladder indicate that the tracer is primarily excreted via renal filtration.

[0123] Quantitative analysis revealed that uptake of both BxPC3 and Capan-1 in tumors was significantly higher than in normal tissues, including the brain, heart, liver, and muscle, at 30 or 60 minutes post-injection. ZD2-AH-( 68In Ga-HBED-CC, tumor uptake was approximately 18.3 times and 13 times higher in BxPC3 and Capan-1 tumors than in muscle at 60 minutes post-injection (p<0.01). Linker-less ZD2-( 68 In Ga-HBED-CC, tumor uptake was approximately 10.2 times and 7.3 times higher in BxPC3 and Capan-1 tumors compared to muscle 60 minutes after injection (p<0.01). Tumor uptake was significantly higher than in normal tissue in both tumor models (p<0.05). These results suggest that ZD2-( 68 Ga-HBED-CC) and ZD2-AH-( 68 Both Ga-HBED-CC demonstrate high specificity for pancreatic cancer tumors with minimal uptake in normal tissues, including the liver.

[0124] From the above description of the present invention, those skilled in the art will recognize improvements, changes, and modifications. Such improvements, changes, and modifications within the art are intended to be covered by the appended claims. All references, publications, and patents cited in this application are incorporated herein by reference in their entirety. As described in the present invention, the following is claimed:

Claims

1. A PET / SPECT probe for use in detecting, monitoring, and / or imaging cancer cells and / or cancer cell invasion, wherein the following formula: 【Chemistry 1】 or having salts thereof, During the ceremony, P １ However, it is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, R １ However, it is an optional linker, and if present, it is alkylene, alkylene oxide, arylene, or alkylene arylene linker. M is a probe that emits positrons or gamma rays.

2. M is, ６７ Ga, ６８ Ga, ６４ Cu, ９９ｍ Tc, １１１ In, ８９ Zr, ９０ Y, １５３ Sm, or ８９ Sr, the probe according to claim 1, which is a metal selected from the group consisting of.

3. The probe according to claim 1, wherein the cancer includes at least one of breast cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, testicular cancer, glioblastoma, sarcoma, bone cancer, brain cancer, head and neck cancer, or skin cancer.

4. The probe according to claim 1, formulated for in vivo administration.

5. The probe according to claim 1, formulated for systemic administration to a subject having or suspected of having cancer.

6. A probe according to claim 1 for detecting, monitoring, and / or imaging pancreatic cancer cells and / or pancreatic cancer cell invasiveness.

7. P １ The probe according to claim 1, wherein the amino acid sequence is the sequence of sequence number 1.

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