WO2022192503A1

WO2022192503A1 - Heptamethine carbocyanine dye-cross-bridged tetraamine cyclam (ctc) chelator conjugates, their stable complexes with copper-64, and uses thereof

Info

Publication number: WO2022192503A1
Application number: PCT/US2022/019695
Authority: WO
Inventors: Leland W.K. Chung; Ruoxiang Wang; Yi Zhang
Original assignee: Da Zen Theranostics, Inc.
Priority date: 2021-03-10
Filing date: 2022-03-10
Publication date: 2022-09-15

Abstract

Embodiments of the present invention relate to conjugates of the near infrared (NIR) heptamethine carbocyanine dye (HMCD) with chelator-radiometal complexes, radiopharmaceutical formulations comprising such complexes and their use, e.g. in internal radiotherapy and/or imaging of cancer. Provided are DZ-1-cross-bridged tetraamine cyclam (CTC) conjugates with optional linker L (DZ-1-(L)-CTC), wherein the CTC chelator is complexed with copper-64 (⁶⁴Cu). The CTC may be a chelator selected from the group comprising CB-TE2A, DiAmSar, derivatives or combinations thereof. Copper complexes comprising DZ-1-(L)-CTC may provide improved serum stability compared to previously known copper complexes while retaining tumor targeting and providing good imaging quality. The present invention also relates to methods of forming the complexes, pharmaceutical compositions comprising the complexes, methods of using the complexes or pharmaceutical compositions, methods of imaging and/or radiotherapy of cancer cells, tissues, soft and solid tumors, and/or their metastases, kits for imaging and/or radiotherapy, and the like.

Description

HEPTAMETHINE CARBOCYANINE DYE-CROSS-BRIDGED TETRAAMINE CYCLAM (CTC) CHELATOR CONJUGATES, THEIR STABLE COMPLEXES WITH COPPER-64, AND USES THEREOF

FIELD OF THE INVENTION

[0001] Embodiments of the present invention generally relate to conjugates of the near infrared (NIR) heptamethine carbocyanine dye (HMCD) with chelator-radiometal complexes, radiopharmaceutical formulations comprising such complexes and their use, e.g. in internal radiotherapy and/or imaging of cancer. In particular, in embodiments, provided are DZ-1 -cross- bridged tetraamine cyclam (CTC) conjugates with optional linker L (DZ-l-(L)-CTC), wherein the CTC chelator is complexed with copper-64 (⁶⁴Cu). More specifically the CTC may be a chelator selected from the group comprising CB-TE2A, DiAmSar, derivatives or combinations thereof. Copper complexes comprising DZ-1-(L)-CTC may provide improved serum stability compared to previously known copper complexes while retaining tumor targeting and providing good imaging quality. The present invention also relates to methods of forming the complexes, pharmaceutical compositions comprising the complexes, methods of using the complexes or pharmaceutical compositions, methods of imaging and/or radiotherapy of cancer cells, tissues, soft and solid tumors, and/or their metastases, kits for imaging and/or radiotherapy, and the like. Advantages of the embodiments may further include improved imaging and/or tumor detection, improved therapy of tumors, cancer, metastases, and pre-cancerous lesions, improved image guided therapy, and improved penetration of tumors, especially solid tumors. The complexes may allow to treat aggressive tumors that have few or no treatment options, particularly small round cell tumors, including tumors such as Ewing’s sarcoma (EWS), neuroblastoma, small cell lung cancer, Merkel cell tumors, and hormone-refractory prostate cancer (HRPC).

BACKGROUND

[0002] A limited number of diagnostic or therapeutic radiopharmaceuticals is available for imaging or treating cancer or tumors. Imaging takes advantage of the very low concentrations needed for detection, e.g. with Positron Emission Tomography (PET) detecting positron- emitters. Internal radiotherapy is a treatment in which a source of radiation is put inside the subject’s body for cancer therapy. Internal radiation therapy may be systemic, i.e. the treatment travels in the blood to tissues throughout the body to kill cancer cells through the radiation that the cells in proximity to the radiometal are exposed to. Radiometals can be delivered by chelating or complexing them with a large variety of chelator compounds and conjugating them to a targeting ligand.

[0003] Radionuclide complexes currently used for such targeted radiotherapy are mostly diagnostic, with a limited number of therapeutic applications. The latter include various antibodies, proteins, and some smaller compounds, in particular peptides that are able to target certain types of cancer.

[0004] Examples of the use of radionuclides and radiometals for tumor imaging or adding imaging functionality to targeted cancer drugs include certain dye conjugates, namely near infrared (NIR) heptamethine carbocyanine dye (HMCD) conjugates. These were described for targeted cancer drug therapy and imaging, e.g. in WO 2016106324 and US 10,307,489 and include double-conjugated dye-drug-chelator conjugates wherein the dye is conjugated to a) anti cancer drug gemcitabine as well as b) an imaging moiety; one such imaging moiety is ⁶⁴Cu- DOTA. The chelated/complexed radiometals are employed to provide imaging functionality to the cancer drug conjugates. A lysine-linked heptamethine carbocyanine dye conjugated with DOTA and complexed with Cu-64 (DZ-l-Lys-DOTA-Cu-64) was also described for imaging of cancer by Xiao et ah, Nuclear Medicine and Biology 40, p. 351-360 (2013) and e.g. in WO2016106324.

[0005] A problem with known radiometal complexes can be a lack of stability in the body, e.g. in blood and/or serum, which may result in insufficient performance, in particular in imaging performance and/or poor imaging quality (e.g. low signal strength, high noise, low signal/noise ratio, and/or resolution) when trying to detect small secondary tumors or metastases, as well as low efficacy and unnecessarily high radiation exposure with associated side effect and damage to non-cancerous tissues and organs.

[0006] Particular problems of copper radiometal chelates include poor bio-distribution and/or poor stability in vivo mediated by the chelator or radiometal or their combination, and which may be caused by dissociation of copper from the chelator and/or binding of non-target tissues. Some chelates are subject to retention in non-target tissues such as the liver, kidney or other organs. Dissociation and binding of ⁶⁴ Cu may prevent targeting to cancer cells or tumors as intended, and instead target, or rather, re-direct, ⁶⁴Cu to other non-cancerous parts of the body and expose these parts to harmful radiation. Some radiometal chelates (or their dissociated products) show activity in other organs, e.g. liver, kidney, blood, bone, and/or bone marrow. For example, upon dissociation, ⁶⁴Cu may bind to other proteins if the chelate-biomolecules were not sufficiently stable either kinetically and/or thermodynamically.

[0007] Still further problems with radiochemicals may include one or more of: lack of suitability for therapy for one or more cancer, insufficient sensitivity, insufficient selectivity for cancer cells, insufficient contrast between tumor and noncancerous tissues over time, insufficient biodistribution, insufficient tumor targeting, insufficient tissue and/or tumor penetration, insufficiently uniform distribution in tumor tissues, insufficient tumor accumulation ratios, insufficient retention in the tumor, slow clearance from the blood after administration, undesirably high radioactive exposure of tissues and organs, low clearance rate from important organs such as kidney, liver, heart etc., high radiation damage of non-targeted tissues or organs, in particular encapsulated tissues such as organs and dense tissues including tumor tissues, in particular calcified tumor tissues, slow establishment of steady state distribution in the body and organs including heart, liver, lungs and kidney, persistence of radioactivity in one or more organs including kidney and/or liver, no stable complex formation with one or more radiometal or difficulties to form stable complexes, low complex formation rates, low labelling efficiency with one or more radiometal, insufficient solubility in aqueous solutions, low stability in aqueous solutions, low stability at physiological pH, low stability in vivo , high rate of dissociation of the radiometal from the complex, in particular in vivo , slow tumor-specific targeting, high organ accumulation (e.g. kidney, spleen, liver, heart), slow clearance (e.g. from blood, liver, kidneys and other organs), and difficult or costly synthesis.

[0008] Various targeting ligand, chelator and radiometal combinations have been attempted for use in imaging, radiotherapy, or both, including matched pairs of the same targeting ligand for a theranostic approach. However, affinities of the targeting ligand for its receptor(s) have been found to be strongly dependent both on the chelator and radiometal used, thus their efficacy often is unpredictable. Similar unpredictability applies to stability of the radiometal complexes, as retaining the radiometal may be influenced by the targeting ligand, chelator, or the combination thereof, and in turn their combination with a desired radiometal.

[0009] Radiotherapy tends to be even more challenging than mere imaging applications and options for treatment by radiotherapy are thus much more limited. Careful integration is needed for a suitable radionuclide in combination with a suitable chelator and a suitable targeting ligand to provide the desired characteristics including affinity, stability and pharmacokinetics. Particular radiometals may emit one or more type of particles or radiation at particular different percentages, and for suitable targeting careful coordination of carrier with a given radiometal is needed so that the complex is stable, sufficiently effective to destroy the target cancer but arrives at the location without causing collateral radiation damage to the not targeted rest of the body and its organs and tissues.

[0010] Some therapeutics can provide both therapy and diagnostic imaging to a degree, though for improved performance of each, theranostic pairs are often employed. A “true” theranostic pair would be an identical matched pair, i.e. have the same carrier for the therapeutic radiometal to destroy the tumor, and the imaging radiometal to allow to diagnostic and/or guided therapy. However, both chelator and radiometal affect important performance characteristics and pharmacokinetics, thus a given carrier may not be able to provide an identically matched pair with suitable characteristics, stability and efficacy for a desired radiometal combination. As an alternative, a non-identical matched pair may be used to adjust characteristics, provided that the binding affinities do not present clinically significant differences.

[0011] Agents or pairs of agents that can provide theranostic functionality thus often lack true theranostic functionality or other desirable characteristics, i.e. the ability to provide both sufficient therapeutic effect and sufficiently high imaging sensitivity at the same time and at similar pharmacokinetics, as well as favorable general pharmacokinetics, a high cytotoxicity for cancer cells but a low toxicity for non-cancerous cells. [0012] A particular problem of radiochemicals is a high and persistent localization of the radioactivity being observed in certain organs, for example, without limitation, the kidneys, the liver, etc., which compromises tumor visualization in the region of the affected organ and limits therapeutic potential, e.g. for kidney or liver tumors and tumors localized in their area. Thus there is an interest in reducing organ radioactivity levels but not those in the target tissue(s). Radionuclide complexes also may encounter resorption by proximal tubules of the kidneys and/or adverse long residence times of radiometabolites in cells, particularly in renal cells, or cells of other organs, which may cause undesirably persistent radioactivity.

[0013] Therefore, there is a continued need in the art for radiopharmaceuticals that can provide improved radioimaging and/or improved radiotherapy, or both, and for radiopharmaceuticals with improved characteristics. In particular there is a need for radiopharmaceuticals with improved stability, in particular serum stability, reduced toxicity, increased effectiveness, and/or increased tumor penetration, favorable pharmacokinetics, among others. Further there is a need for theranostics that at allow both efficient therapy and imaging at the same time. Still further there is a need for therapeutics, imaging agents and theranostics that reduce or avoid damage to non-cancerous cells and tissues. These and other features and advantages of the present invention will be explained and will become apparent to one skilled in the art through the summary of the invention that follows.

SUMMARY OF THE INVENTION

[0014] In an embodiment, provided is a DZ-1-(L)-CTC conjugate, wherein the conjugate comprises a heptamethine carbocyanine dye (HMCD) moiety conjugated with an optional linker moiety L and a cross-bridged tetraamine cyclam (CTC) chelator residue R and as shown in FI below:

wherein L comprises one or more aminoacid residue, or alternatively, linker L is absent, and wherein the chelator forming the chelator residue R is selected from the group consisting of CB- TE2A, DiAmSar, or a derivative thereof, wherein the optional linker L is a residue selected from the group comprising a Lysine residue, and wherein the chelator residue is complexed with 64Cu.

[0015] In an embodiment, provided is a complex wherein R is CB-TE2A, and wherein L is a lysine residue which links DZ-1 to the chelator as shown in FII below:

[0016] In an embodiment, provided is a complex wherein R is DiAmSar as shown in Fill below:

Fill (DZl-DiAmSar).

[0017] In an embodiment, provided is a complex provided with one or more pharmaceutically acceptable excipient to form a pharmaceutical formulation.

[0018] In an embodiment, provided is a complex provided as a kit with one or more reagents for reconstitution of the complex in an administrable form.

[0019] In an embodiment, provided is a kit provided with instructions for mixing and complexing the conjugate and 64Cu in suitable amounts, optionally with one or more reagent, buffer or excipient, and optionally treating the resulting solution containing the formed complex to provide it in an administrable form.

[0020] In an embodiment, provided is a method for imaging or treating cancer wherein a DZ-1- Lys-chelator conjugate complex of formula FI is administered to a subject suffering from cancer or from a risk to develop cancer in a sufficient amount and for sufficient duration to allow imaging or treatment; wherein the conjugate comprises a heptamethine carbocyanine dye (HMCD) moiety conjugated with a chelator residue R via a lysine linker and as shown in formula FI herein-above, wherein L comprises one or more aminoacid residue, or alternatively, linker L is absent, and wherein the chelator forming the chelator residue R is selected from CB- TE2A, DiAmSar, or a derivative thereof, and wherein the chelator residue is complexed with 64Cu.

[0021] In an embodiment, provided is a method wherein R is CB-TE2A, and wherein L is a lysine residue which links DZ-1 to the chelator as shown in FII herein-above (DZl-Lys-CB- TE2A). [0022] In an embodiment, provided is a method wherein R is DiAmSar as shown in Fill herein above (DZl-DiAmSar).

[0023] In an embodiment, provided is a method wherein imaging is performed by Positron Emission Tomography (PET) or Single-Photon Emission Computerized Tomography (SPECT) to detect 64Cu radiation, and optionally additionally by Computer Tomography (CT).

[0024] In an embodiment, provided is a method wherein imaging is performed before and/or during one or more therapy time intervals to provide an image-guided therapy.

[0025] In an embodiment, provided is a method wherein the risk to develop cancer is one or more genetic alteration associated with EWS, and the alterations include one or more alteration to a member of the ETS family of transcription factors.

[0026] In an embodiment, provided is a method wherein the cancer is selected from the group comprising: Ewing’s Sarcoma (EWS), a small cell round tumor, adult neuroblastoma, neuroblastoma in children, small cell lung cancer, Merkel cell tumors, Merkel cell tumors of the skin, prostate cancer, hormone-refractory prostate cancer (HRPC), neuroendocrine differentiated HRPC (NE-HRPC), pre-B-cell acute lymphoblastic leukemia, and a cancer which is associated with alterations in one or more member of the ETS family of transcription factors.

[0027] In an embodiment, provided is a method wherein the cancer is Ewing’s Sarcoma (EWS).

[0028] In an embodiment, provided is a method wherein R is CB-TE2A, or a derivative thereof.

[0029] In an embodiment, provided is a method wherein R is DiAmSar, or a derivative thereof.

DESCRIPTION OF THE DRAWINGS

[0030] Fig. 1 illustrates a radio-HPLC analysis of ⁶⁴Cu-DZ-l-Lys-CB-TE2A. The top panel shows the UV channel at 780 nm, and the bottom panel shows the radio-channel. Fractions from 10 - 12 min contain the probe and were collected and pooled for use in Example 10. [0031] Fig. 2 illustrates the high serum stability of ⁶⁴Cu-DZ-l-Lys-CB-TE2A measured by radio- Thin Layer Chromatography (TLC) over time in hours in serum at 37°C (> 95%). The graph shows a high stability with no decline over the measured time period.

[0032] Fig. 3 illustrates the Standardized Uptake Value (SUV) observed in both mouse models bearing DU145vc (WT - left shoulder) and DU145sh (PTPN1 KD - right shoulder) xenografts, ranging from about 2.5 or more at 4h to between about 0.5 to about 2.5 at 24h, and about 0.125 at 48h (i.e. substantially clearance from the body and its organs).

[0033] Fig. 4 A illustrates a substantial tumor uptake and retention, good signal-to-noise ratio and high specificity observed at 4 h in both mouse models bearing DU145vc (WT - left shoulder) and DU145sh (PTPN1 KD - right shoulder) xenografts.

[0034] Fig. 4 B illustrates a substantial tumor uptake and retention, good signal-to-noise ratio and high specificity observed at 24 h in both mouse models bearing DU145vc (WT - left shoulder) and DU145sh (PTPN1 KD - right shoulder) xenografts.

[0035] Fig. 4 C illustrates an efficient clearance profile including from the kidneys at 48 h (compare to 24 h in Fig. 4 B).

[0036] Fig. 5 A illustrates a maximum intensity projection (MIP) which demonstrates the uptake and retention of ⁶⁴Cu-DZ-l-CB-TE2A in tumors at 4 h.

[0037] Fig. 5 B illustrates a maximum intensity projection (MIP) which demonstrates the uptake and retention of ⁶⁴Cu-DZ-l-CB-TE2A in tumors at 24 h.

[0038] Fig. 5 C illustrates a maximum intensity projection (MIP) which demonstrates the uptake and retention of ⁶⁴Cu-DZ-l-CB-TE2A in tumors at 48 h.

[0039] Fig. 6 illustrates low signal strength of Cu⁶⁴-DZ-l-NOTA in a PC3 subcutaneous model.

[0040] Fig. 7 A illustrates low signal-to-noise ratio and low specificity of Cu⁶⁴-DZ-l-NOTA in a PC3 subcutaneous mouse model at 4h. [0041] Fig. 7 B illustrates low signal-to-noise ratio and low specificity of Cu⁶⁴-DZ-l-NOTA in a PC3 subcutaneous mouse model at 24 h.

[0042] Fig. 7 C illustrates low signal-to-noise ratio and low specificity of Cu⁶⁴-DZ-l-NOTA in a PC3 subcutaneous mouse model at 48 h.

DETAILED SPECIFICATION

[0043] Embodiments of the present invention generally relate to conjugates of the near infrared (NIR) heptamethine carbocyanine dye (HMCD) with chelator-radiometal complexes, radiopharmaceutical formulations comprising such complexes and their use, e.g. in internal radiotherapy and/or imaging of cancer. In particular, in embodiments, provided are DZ-1- cross- bridged tetraamine cyclam (CTC) conjugates with an optional linker L (DZ-l-(L)-CTC) wherein the CTC chelator is complexed with copper-64 (⁶⁴Cu). More specifically the CTC may be a chelator selected from the group comprising CB-TE2A, DiAmSar, derivatives or combinations thereof. Copper complexes comprising DZ-1-(L)-CTC may provide improved serum stability compared to previously known copper complexes while retaining tumor targeting and providing good imaging quality. The present invention also relates to methods of forming the complexes, pharmaceutical compositions comprising the complexes, methods of using the complexes or pharmaceutical compositions, methods of imaging and/or radiotherapy of cancer cells, tissues, soft and solid tumors, and/or their metastases, kits for imaging and/or radiotherapy, and the like. Advantages of the embodiments may further include improved imaging and/or tumor detection, improved therapy of tumors, cancer, metastases, and pre-cancerous lesions, improved image guided therapy, and improved penetration of tumors, especially solid tumors. The complexes may allow to treat aggressive tumors that have few or no treatment options, particularly small round cell tumors, including tumors such as Ewing’s sarcoma (EWS), neuroblastoma, small cell lung cancer, Merkel cell tumors, and hormone-refractory prostate cancer (HRPC).

[0044] These DZ-l-(L)-CTC-⁶⁴Cu complexes include DZ-l-CTC-⁶⁴Cu and DZ-l-L-CTC-⁶⁴Cu (with or without a linker), and may be referred to herein as “conjugate complexes”, “dye complexes”, “dye-chelator complexes”, “HMCD-chelator complexes”, “DZ-1 -chelator complexes”, “radiometal complexes”, “64-Cu complexes”, “⁶⁴Cu complexes” or simply “complexes”.

[0045] Unlike other DZ-l-chelator conjugates with ⁶⁴Cu, the DZ-1 -cross-bridged tetraamine cyclam (CTC)-⁶⁴Cu-complexes as described herein may have a higher stability in serum, e.g. as compared to a DZ1-DOTA-⁶⁴CU complex (compare e.g. example 4).

[0046]Without wishing to be bound by theory, it is believed that the DZ1-CTC-⁶⁴CU complexes may be suitable for treatment of various tumors including in particular various aggressive and difficult to treat tumors that currently lack adequate treatment options, such as Ewing’s Sarcoma (EWS) and other small cell round tumors.

[0047] Copper-64 (⁶⁴Cu) is a positron emitter thus allowing use in PET imaging which can give real time images of physiological processes in the body in vivo thus allowing accurate monitoring, e.g. of drug distribution and biokinetics simultaneously.

[0048] Radiotherapy of cancer cells using ⁶⁴Cu which is a beta emitter may have the advantage of providing a beneficial ratio of substantial damage to the target cells while not harming non target tissues, due to its limited range of radiation. This however requires highly stable complexes, which limit the cytotoxic effects of the radioactive ⁶⁴Cu to its target. The stability of the ⁶⁴Cu complexes may depend on the chelator it is complexed to, and in turn the delivery ligand that the chelator is bound to, the linker used, as well as the combination of these, their size, and the type and location of tumors.

[0049] In embodiments, provided are methods for image-guided radiotherapy wherein the complexes provide cancer radiotherapy, radioimaging of cancer, or both. At the same time, these complexes are believed to be sufficiently effective in tumor targeting, tumor shrinking and/or cancer cell killing, thus allowing a true theranostic approach with a single (rather than a pair) of therapeutics providing both diagnostic and therapy, which due to radioimaging such as PET has a high sensitivity. In addition, the dye’s fluorescence may be used for parallel fluorescent monitoring. [0050] In embodiments, provided are radiometal complexes of a cross-bridged tetraamine cyclam (CTC) chelator selected from the group comprising CB-TE2A, DiAmSar, or derivatives thereof, conjugated, optionally via a linker such as a lysine linker (see formulae FI, FII and Fill shown herein below) to DZ-1, a heptamethine cyanine dye (HMCD), and compositions comprising these complexes, as well as compositions for making these complexes, e.g. in a test tube or kit, and the use of such compositions or complexes for the radiotherapy of cancer. The present invention also relates to methods of making the conjugates and complexes, pharmaceutical compositions including the complexes, methods of using the conjugates, complexes or pharmaceutical compositions, methods of imaging and/or radiotherapy of cancer cells, tissues, tumors, and/or their metastases, kits for imaging and/or radiotherapy, and the like. In addition, the present disclosure includes compositions used in and methods relating to non- invasive imaging, in particular PET or SPECT imaging, of the complexes in vivo. The complexes may be used for radio therapy of cancer cells and tissues, including of solid tumors. These compounds, including conjugates and resulting complexes, may have various advantages which may include an improved stability, reduced toxicity, and/or increased penetration of tumors.

[0051] In embodiments, the chelator conjugated to DZ-1 is a cross-bridged tetraamine cyclam (CTC), including CB-TE2A and DiAmSar, or derivatives thereof. Without wishing to be bound by theory, it is believed that cyclams, and in particular, cross-bridged cyclams, when in form of the conjugate, may contribute to the better stability of the resulting complexes with ⁶⁴Cu. The improvement in stability e.g. in serum does not appear to be shared by conjugates to non-cross- bridged cyclen chelators such as e.g. DOTA.

[0052] CB-TE2A is a macrocyclic chelator also known as 1,4,8,11- Tetraazabicyclo[6.6.2]hexadecane-4,ll-diacetic acid and has the chemical formula C₁₆H₃₀N₄O₄ 4HC1 (CAS #313229-90-2).

[0053] DiAmSar is a macrocyclic chelator also known as 1,8-Diamino-3,6,10,13,16,19- hexaazabicyclo[6,6,6]-eicosane and has the chemical formula C_I4H₃₄N₈ 5H₂0 (CAS # 91002- 72-1). [0054] The chelator may be conjugated with DZ-1 directly via its terminal COOH residue, or via a suitable linker, for example a lysine residue. Alternatively, other suitable linkers may include alkyl linkers, e.g., without limitation, C2 to C15, e.g. C2, C3, C4, C5 or longer linkers, and polyethylene glycol linkers of varied chain length, e.g. (PEG)n with n=l-24, e.g. n=2-24. Other suitable linkers may include aminoacids or peptides, e.g. peptides of one or more aminoacids, e.g. up to 2, 3, 4, 5 or more amino acids linked together. Aminoacids used either as the linker or as one or more of its component in a peptide. Such amino acids may be selected from one or more of alanine (ala), arginine (arg), asparagine (asn), aspartic acid (asp), cysteine (cys), glutamine (gin), glutamic acid (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), proline (pro), serine (ser), threonine (thr), tryptophan (trp), tyrosine (tyr), valine (val), or modifications or derivatives therof. Amino acids may be aliphatic (e.g. alanine, glycine, isoleucine, leucine, proline, valine), aromatic (phenylalanine, tryptophan, tyrosine), acidic (aspartic acid, glutamic acid), basic (arginine, histidine, lysine), or hydroxylic (serine, threonine), sulphur-containing (cysteine, methionine, or amidic (asparagine, glutamine). Preferred aminoacids may include lysine, glutamic acid, aspartic acid, cysteine, and preferred peptides may include peptides comprising one or more of lysine, glutamic acid, aspartic acid, cysteine.

[0055] Illustrative dye-linker-chelator compounds wherein R is a chelator as described herein and n is 1-20 (e.g. 1-10 or 1-5, e.g. 1, 2, 3, 4, or 5), are listed below:

[0056] In embodiments, provided is a DZ1-(L)-CTC radiometal complex which comprises a heptamethine carbocyanine dye (HMCD) moiety conjugated, optionally via a linker as described herein, with a CTC chelator residue R, wherein R is selected from the group consisting of CB- TE2A, DiAmSar, or derivatives thereof, for radiolabelling with a ⁶⁴Cu radiometal, and the resulting complex comprising ⁶⁴Cu.

[0057] Illustrative DZ-1-(L)-CTC conjugates suitable to form such complexes are shown in formula FI below:

wherein L represents an optional linker comprising lysine, and R is selected from CB-TE2A and DiAmSar.

[0058] In embodiments, provided is a DZl-Lys-CB-TE2A radiometal complex for radiolabelling with ⁶⁴Cu as shown below in formula FII:

[0059] In embodiments, provided is a DZl-Lys-DiAmSar conjugate for radiolabelling with ⁶⁴Cu as shown below in formula Fill:

[0060] It is believed that the complexes described herein may provide one or more advantages including formation of radiometal complexes that have a higher stability, including in serum. Further the complexes may provide an effective cancer therapy with low toxicity and good tumor penetration even of solid tumors. In particular, the complexes are believed to provide true theranostic agents that may be able to provide both a highly effective radiotherapy while providing highly sensitive imaging, thus allowing an image-guided therapy without the necessity to administer a matched theranostic pair. The complexes may further provide one or more of the following advantages: a more accurate quantitative analysis via PET or SPECT probe and/or optionally near infrared fluorescence (NIRF), a higher contrast between tumor and noncancerous tissues, a higher sensitivity, a better selectivity for cancer cells, in particular in vivo , a rapid steady state distribution in the body and organs including heart, liver, lungs and kidney, rapid tumor targeting, a uniform distribution in tumor tissues, a lack of persistence of radioactivity in one or more organs including kidney and/or liver. Further, these complexes may advantageously be more easily formed and/or at lower cost, may be easily labelled with a radiometal, may form stable complexes with one or more radiometal, may have high complex formation rates, a high radiochemical yield, a good labelling efficiency with one or more radiometal, a good solubility in aqueous solutions, a high stability in aqueous solutions, a high stability at physiological pH of about 7.4 (e.g. 7.2-7.6), a high stability in vivo , a low rate of dissociation of the radiometal from the complex, in particular in vivo , a good biodistribution, sufficient targeting of the complex to cancer tissues, fast cancer-specific targeting, sufficient targeting for tumor and/or metastases, sufficient targeting for larger tumors, a low organ accumulation (e.g. kidney, spleen, liver, heart), no or reduced radioactive exposure of tissues and organs, a rapid clearance rate from important or sensitive organs (e.g. from blood, liver, kidneys and other organs) after administration, sufficient tissue and tumor penetration, sufficient penetration of encapsulated tissues such as organs including dense tissues including tumor tissues, in particular calcified tumor tissues, improved and/or longer retention in the tumor.

[0061] Radiochemical yield for the isotopes of a specified element is the yield of a radiochemical separation expressed as a fraction or percentage of the activity originally present. Also called the recovery. In radiation chemistry, the number of species transformed by radiation per eV of absorbed energy: represented by the symbol G, the G-value. Radiochemical yield may be determined as will be apparent to a person of ordinary skill.

[0062] It is believed that the high stability of the complexes, including in serum, see examples herein-below, in addition to the combination with a CTC chelator and ⁶⁴Cu, may contribute to one or more advantages as described herein.

[0063] Complexes as described herein may be able to penetrate more easily and more deeply into a tumor. These radiopharmaceuticals may thus be better suited for solid tumors, in particular denser and/or larger and/or partially calcified tumors.

[0064] Complexes as described herein may be able to provide blood-brain-barrier (BBB) penetration and thus may be used for treatment of brain tumors and brain metastases.

[0065] Complexes as described herein may be able to provide higher tumor accumulation ratios and/or a fast blood clearance. Distribution in animals may show a favorable time-dependent clearance in their organs concomitant with specific accumulation in tumors (e.g. xenograft tumors in mice, see e.g. example 6).

[0066] Complexes as described herein may be able to provide an improved percentage and/or speed of tumor uptake, e.g. as measured by % of injected dose (ID) of e.g. FI, FII or Fill (20 pg/kg) and the tumor-to-blood ratio at points in time (e.g. at 4, 8, 16 and/or 24 hours), as will be apparent to a person of ordinary skill.

[0067] Complexes as described herein may be able to provide increased dosing options and less accumulation and/or improved organ clearance from organs including one or more of kidneys, liver and heart. Organs such as the kidneys often are dose-limiting organs in radiotherapy. Absorbed doses may be measured as will be apparent to a person of ordinary skill, and may be e.g. about 15 Grays (Gy) or less, e.g. 10, 5, 2, 1 Gy or less. [0068] Complexes as described herein may be able to provide a reduced absorbed dose to organs and/or soft tissues such as kidney, spleen, liver, and/or hematological toxicity (e.g. thrombocytopenia, neutropenia).

[0069] Complexes as described herein may be able to provide improved effectiveness against cancer at lower amounts of radioactivity. For example, less than 50 pCi, e.g. less than 20, 20, 5 or 2 pCi may be needed.

[0070] Complexes as described herein may be able to provide reduced toxicity at doses sufficient to treat cancer and/or significantly reduce tumor volume (e.g. by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%); thus a reduced LD50 and/or organ toxicity e.g. reduced kidney, spleen, liver, bone marrow, brain, heart or lung toxicity may be provided, and/or the complexes may allow to avoid or improve weight loss which often occurs during and after therapy.

[0071] Complexes as described herein may be able to provide reduced toxicity at doses sufficient to treat cancer and/or significantly reduce tumor volume while improving one or more of pain scores, bone pain scores, and median survival.

[0072] Complexes as described herein may be able to provide sufficient anti-cancer/anti-tumor effects at reduced total dose, for example less than 250 kBq/kg, e.g. less than 200, 150, 50, and 25 kBq/kg.

[0073] Complexes as described herein may be able to provide sufficient effectiveness to treat cancer and/or significantly reduce tumor volume while avoiding one or more side effects such as pain, dizziness, nausea, effects on the digestive tract, stomach pain, constipation, diarrhea, hair loss.

[0074] Complexes as described herein are believed to be particularly useful for Ewing’s sarcoma (EWS). EWS is an aggressive small round cell tumor of the bone and extra-osseous locations with metastases in lung, liver, brain. EWS is typically characterized by a signature chromosomal t(ll;22) translocation and EWS-FL1 fusion, typically affects young individuals (median age 15), and generally responds to radiation and poly chemotherapy. However, when EWS is disseminated or recurrent, attempts of therapy are largely unsuccessful.

[0075] In embodiments, the complexes described herein may administered to a subject diagnosed with EWS and/or presenting with one or more genetic alteration associated with EWS, for imaging, therapy, or both, including image-guided therapy.

[0076] Without limitation, typical genetic alterations of EWS include one of several possible reciprocal chromosomal translocations that generate the fusion between the gene encoding Ewing’s sarcoma breakpoint region 1 (EWSR1) and a gene encoding a member of the E- twentysix (ETS) family of transcription factors, other genetic alterations exist, some of which are described for illustration herein-below. About 85 to 90% of cases may bear the chromosomal translocation t(l I;22)(q24;ql2), which can lead to the fusion of EWSR1 to the gene encoding Friend leukemia virus integration 1 (FLI1)2 (Fig. 2). In about roughly a quarter of the cases, the only detectable genetic event is the chromosomal translocation, with the resulting fusion protein likely responsible for transformation. Other mutations of other genes include, without limitation, STAG2 and TP53. While the vast majority of Ewing’s sarcomas harbor a fusion protein containing EWS, about 1% of the tumors bear chromosomal translocations that implicate FUS or TAF15 which like EWS are RNA-binding proteins that share a structure composed of an intrinsically disordered, low-complexity, prionlike SYGQ-rich N-terminal transactivation domain, followed by three arginine-and-glycine-rich (RGG) repeats of different lengths. RGG1 and RGG2 are separated by an RNA recognition motif consisting of 87 amino acids, and RGG2 and RGG3 by a zinc-finger domain. EWSR1 can partner not only with genes encoding ETS family members but also with a broad range of non-ETS genes to generate fusion proteins implicated in the pathogenesis of diverse soft-tissue tumors. These include, without limitation, EWSR1-NFATC2, EWSR1-POUF1, EWSR1-PATZ1, EWSRl-SMARCA, and EWSR1-SP3 which can give rise to rare, undifferentiated round-cell tumors either resembling Ewing’s sarcoma or considered to be Ewing’s sarcomas. Other EWSRl-non-ETS fusions give rise to well-defined entities, including, without limitation, DSRCT (EWSR1-WT1), myxoid liposarcoma (EWSR1-DDIT3), clear-cell sarcoma (EWSR1-ATF1), and extraskeletal myxoid chondrosarcoma (EWSR1-NR4A3). Unrelated chromosomal translocations, which generate the non-FET-non-ETS gene fusions BCOR-CCNB329 and CIC-DETX4,30 may give rise to tumors with morphologic features resembling those of Ewing’s sarcoma. These tumors were initially classified as Ewing’s sarcoma, but their pathogenesis and biologic properties are now believed to be clearly distinct from those of Ewing’s sarcoma. For example, at least 5 of the 27 members of the ETS family can fuse to EWS to generate Ewing’s sarcoma, these include, without limitation: FLIl, 2 ERG, 32 FEV,33 ETV1,34 and E1AF35; FLU may be found in 85 to 90% of cases. ETS factors may also be implicated in the development of diverse cancers, including pre-B-cell acute lymphoblastic leukemia and prostate cancer. All members of the family share a DNA-binding domain that recognizes the consensus core 5'-GGAA/T-3' DNA motif, often referred to as the ETS binding motif. FLIl has two ETS-binding domains separated by an FLIl-specific (FLS) sequence. The 5' ETS domain and the FLS sequence form the N-terminal transactivating domain, which is substantially less potent than the EWS N-terminal transactivating domain by which it is replaced in the fusion protein. For example, after chromosomal translocation, the portion of FLIl containing the 3' ETS-binding domain that becomes fused to EWS may undergo a conformational change, which may allow it to activate a broader repertoire of genes than wild- type FLI.

[0077] Typical genetic alterations of EWS include several possible reciprocal chromosomal translocations that generate the fusion between the gene encoding Ewing’s sarcoma breakpoint region 1 (EWSR1) and a gene encoding a member of the E-twentysix (ETS) family of transcription factors. About 85 to 90% of cases bear the chromosomal translocation t(ll;22)(q24;ql2), which leads to the fusion of EWSR1 to the gene encoding Friend leukemia virus integration 1 (FLI1)2 (Fig. 2). In roughly a quarter of the cases, the only detectable genetic event is the chromosomal translocation, with the resulting fusion protein likely responsible for transformation. Other mutations of other genes include, without limitation, STAG2 and TP53.

[0078] Other cancer types that the complexes as described herein may be used to successfully treat include other small cell round tumors, including, without limitation, neuroblastoma, in particular neuroblastoma in children, small cell lung cancer, Merkel cell tumors, in particular Merkel cell tumors of the skin, and euroendocrine differentiated HRPC (NE-HRPC). [0079] Copper-64 (⁶⁴Cu) may be technically produced by several different reactions as will be apparent to a person of ordinary skill, with the most common methods using either a reactor or an accelerator. Thermal neutrons can produce ⁶⁴Cu in low specific activity (the number of decays per second per amount of substance) and low yield through the ⁶³Cu(n,y)⁶⁴Cu reaction. Alternatively, ⁶⁴Cu may be produced using high-energy neutrons via the ⁶⁴Zn(n,p)⁶⁴Cu reaction in high specific activity but low yield. Using a biomedical cyclotron the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction may produce large quantities of the nuclide with high specific activity.

[0080] In embodiments, radiotherapy as described herein may be improved by pairing such therapy with Positron Emission Tomography (PET) or SPECT and/or to provide image-guided therapy. Alternatively, the complexes as described herein may be used for imaging only, or therapy only. PET detects positron-emitters such as ⁶⁴Cu, and may optionally be combined with Computerized Tomography (CT) imaging, i.e. PET/CT. In CT, x-ray scans are taken from different angles with the various slices arranged in 3D by a computer which may be used as a map to overlay with signals detected by PET.

[0081] In embodiments, radiotherapy may be performed internally, i.e. a source of radiation may be provided inside the subject’s body for cancer therapy. Internal radiation therapy may be performed systemically, thus the treatment travels in the blood to tissues throughout the body to kill cancer cells in a targeted fashion trough the radiation the cells in proximity to the radiometal are exposed to. In embodiments, treatment may be through systemic or localized/site-specific administration, such as oral or intravenous injection for systemic administration, or localized injection or deposits, e.g. through use of seeds in brachytherapy of accessible tumors, as will be apparent to a person of ordinary skill. Using the stable complexes provided, the radiometals can be delivered to tumors and metastases in a targeted fashion.

[0082] In embodiments, one or more radiometal complex may be administered in a coordinated administration protocol.

[0083] In embodiments, imaging may be performed before and/or during one or more therapy time intervals to provide an image-guided therapy. The amount, concentration, duration and form of administration will be adjusted accordingly, as will be apparent to a person of ordinary skill. For example, imaging may allow to locate a suspected tumor, e.g. after genetic testing revealed a genetic alteration and pre-disposition, e.g. for a particular type of cancer, tumor or group of tumors, such as, without limitation, EWS. After location of the tumor or cancer, therapy may be performed, either systemically (e.g. systemic administration such as by IV or orally), or localized administration restricted to the tumor site and its surroundings (e.g. by localized injection). Alternatively, imaging and therapy may be performed in the same step. Still alternatively, imaging may be performed one or more times before and over the course of therapy to monitor tumor development and, upon therapy, shrinkage.

[0084] Without wishing to be bound by theory, it is believed that the DZ-1-(L)-CTC conjugates form complexes of high thermodynamic stability and kinetic inertness with ⁶⁴Cu, and will rapidly, quantitatively and stably coordinate with the radiometal at room temperature (at about 20 DEG C, or e.g. at less than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 DEG C), near neutral pH (at about 7, e.g. about 5 or more, about 6 or more, about 8 or less, about 7.5 or less), and at a low DZ-l-Lys-DOTA concentration, e.g. 0.1-100 micromolar or nanomolar, e.g. about 1 to about 20 micromolar, e.g. about 1 to about 10 micromolar, e.g. about 5-8 micromolar, allowing to easily provide radiopharmaceutical formulations.

[0085] Complexes as described herein may provide superior imaging during detection including e.g. PET, in particular one or more of better signal-noise ratio, stronger signal, and better resolution.

[0086] Complexes as described herein, may be suitable for tumors of all sizes with effects including tumor shrinkage, and, due to increased stability, in vivo distribution and/or imaging sensitivity, may be particularly effective to detect and destroy small tumors or metastases of a volume of less than e.g. about 5 to about 0.1 cm³ or less, e.g. about 5 cm³ or less, about 4 cm³ or less, about 3 cm³ or less, about 2 cm³ or less, about 1 cm³ or less, about 0.5 cm³ or less, about 0.1 cm³ or less. Where not otherwise indicated, tumor volumes apply to human patients typically having a weight of about 140 to 200 lb, e.g. about 170 lb. In non-human subjects tumor volumes will be less or more depending on body weight. Advantages may include a significant reduction or removal of metastases, higher absorbed doses, and/or a lower tissue penetration range. [0087] Complexes as described herein, due to increased stability and/or improved tumor penetration, may be particularly effective to also destroy larger tumors, in particular solid tumors, of a volume of more than e.g. about 5 cm³, e.g. about 5 cm³ to about 2000 cm³ or more, e.g. about 50, 100, 200, 500 or 1000 cm³ or more.

[0088] Complexes as described herein may be suitable for tumors of all sizes with effects including tumor shrinkage. Advantages may include a significant reduction or removal of metastases, higher absorbed doses, and/or a lower tissue penetration range.

[0089] In embodiments, Positron Emission Tomography (PET) may be used to provide data on radiometal distribution within target tissues by detection of gamma photons resulting from the decay of the radiometals. High spatial resolution of commonly available PET scanners allows to visually map radiometal decay events and thus provide an image which reflects the distribution of a radiometal in the body after administration of a radiometal complex. Such images provide anatomic and functional information to aid medical diagnosis and assist to track progress and allow adjustment of radiotherapy.

[0090] In embodiments, cancer as referred to herein includes precancerous and cancerous cells or tissues, tumors and their metastases, primary and secondary tumors. The conjugates and methods described herein may be particularly advantageous for use in brain cancers, brain tumors and their metastases. Without wishing to be bound by theory it is believed that CB-TE2A, DiAmSar, or derivatives thereof, in combination with DZ1 and ⁶⁴ Cu as described, and in particular when conjugated via a lysine linker, allows the radioactive conjugates to pass across the blood-brain barrier and allow imaging and therapy throughout the brain, including tumors located deep within the brain structure that often are not or not efficiently accessible by other methods and may be inoperable.

[0091] In embodiments, the cancer treated by the complexes may be selected from the group comprising: brain cancer, prostate cancer, lung cancer, Non-small-cell lung carcinoma (NSCLC), small-cell lung carcinoma (SCLC), pancreatic cancer, kidney cancer, lymphoma, colorectal cancer, skin cancer, HCC cancer, and breast cancer, squamous-cell carcinoma of the lung, anal cancers, epithelial tumors of the head and neck, bone cancer, carcinoma of the cervix, skin cancer, melanoma, hematopoietic cancers, lymphoma, and myeloma, or metastases of any thereof, including e.g., without limitation, metastases occurring in the brain, the bone, or other organs, brain tumors or their metastases, brain tumors and their brain, bone, lung or other organ metastases, bone tumors or their brain, bone, lung or other metastases, prostate tumors or their brain, bone, lung or other metastases, prostate tumors and their brain, bone, lung or other metastases, lung tumors and their brain, bone, lung or other metastases, and others.

[0092] In embodiments, the cancer may be a central nervous system (CNS) or brain tumor, or metastasis thereof, selected from the group comprising: acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma, glioblastoma, medulloblastoma, meningioma, oligodendroglioma, pituitary tumors, primitive neuroectodermal tumor, Schwannoma, brain stem glioma, ependymoma, juvenile pilocytic astrocytoma, optic nerve glioma, pineal tumor, rhabdoid tumor, adult Low-Grade (WHO Grade I or II) Glioma/Pilocytic, Infiltrative Supratentorial Oligodendroglioma, Anaplastic Gliomas/Glioblastoma, Adult Intracranial Ependymoma, Adult Medulloblastoma, Primary CNS Lymphoma, Primary Spinal Cord Tumors, Limited Brain Metastases, Extensive Brain Metastases, Leptomeningeal Metastases, and Metastatic Spine Tumors.

[0093] In embodiments, the cancer may be a Non-small-cell lung carcinoma selected from a Squamous-cell carcinoma, Adenocarcinoma (Mucinous cystadenocarcinoma), Large-cell lung carcinoma, Rhabdoid carcinoma, Sarcomatoid carcinoma, Carcinoid, Salivary gland-like carcinoma, Adenosquamous carcinoma, Papillary adenocarcinoma, and Giant-cell carcinoma. Alternatively, the cancer may be a small-cell lung carcinoma, including a Combined small-cell carcinoma. Alternatively, the cancer may be a non-carcinoma of the lung, including a Sarcoma, Lymphoma, Immature teratoma, and Melanoma.

[0094] In embodiments, a pharmaceutical composition comprising a radiometal complex, or for forming such complexes, is provided. The pharmaceutical composition may be for human or for veterinary use, and comprise one or more conjugate or complex of the invention (or a salt, solvate, metabolite, or derivative thereof) with one or more pharmaceutically acceptable carrier and/or one or more excipient and/or one or more active. The one or more carrier, excipient and/or active may be selected for compatibility with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. Such carriers are known in the art and may be selected as will be apparent to a person of ordinary skill in the art.

[0095] In embodiments, routes of administration for the compounds and pharmaceutical compositions include, but are not limited to: oral (e.g. in pill form), intravenous (i.e. injected into a subject’s vein), interstitially (i.e. inserted into a space in the body), intraperitoneal, subcutaneous, or intramuscular, and/or by brachytherapy (insertion of radioactive implants or seeds directly into the affected tissue, e.g. into or near a tumor location). Administration may be systemic (e.g. via blood circulation) or regional (e.g. localized to a particular organ of the body or part thereof). In some embodiments, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable excipient suitable for rendering the compound or mixture administrable via the above routes of administration.

[0096] In embodiments, the active ingredients can be admixed or compounded with a conventional, pharmaceutically acceptable excipient or carrier. A mode of administration, vehicle, excipient or carrier should generally be substantially inert with respect to the active agent, as will be understood by those of ordinary skill in the art. Illustrative of such methods, vehicles, excipients, and carriers are those described, for example, in Remington: The Science and Practice of Pharmacy (2020), ISBN-10: 0128200073, or in Handbook of Pharmaceutical Excipients, Ninth edition (2020), ISBN-10: 0857113755, the disclosures of which is incorporated herein by reference. The excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0097] In embodiments, the pharmaceutical formulations may be conveniently made available in a unit dosage form by any of the methods generally known in the pharmaceutical arts. Generally speaking, such methods of preparation comprise presenting the formulation in a suitable form for delivery, e.g., forming an aqueous suspension. The dosage form may optionally comprise one or more adjuvant or accessory pharmaceutical ingredient for use in the formulation, such as mixtures, buffers, and solubility enhancers. [0098] According to an embodiment of the present invention, parenteral dosage forms (i.e. that bypass the GI tract) of the pharmaceutical formulations include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, administration DUROS®-type dosage forms, and dose-dumping.

[0099] In embodiments, suitable vehicles that can be used to provide parenteral dosage forms of the compounds of the invention include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a compound of the invention as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0100] In embodiments, formulations for parenteral administration include aqueous and non- aqueous sterile injection solutions, which may further contain additional agents, such as anti oxidants, buffers, bacteriostats, and solutes, which render the formulations isotonic with the blood of the intended recipient. The formulations may include aqueous and non-aqueous sterile suspensions, which contain suspending agents and thickening agents.

[0101] In embodiments, injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0102] In embodiments, forms suitable for oral administration include tablets, troches, capsules, elixirs, suspensions, syrups, wafers, or the like prepared by art recognized procedures. The amount of active compound in such therapeutically useful compositions or preparations is such that a suitable dosage will be obtained. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier, for example, ethanol, glycerine or water, with a flavoring or coloring agent.

[0103] In embodiments, solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcelhdose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents.

[0104] In embodiments, solid compositions of a similar type can be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.

[0105] In embodiments, the active compounds conjugates or complexes can be in micro- encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound can be admixed with at least one inert diluent such as sucrose, lactose and starch. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms can also comprise buffering agents. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0106] In embodiments, the active compounds, conjugates or complexes can be present in form of salts, which may be particularly suitable for use in the treatment of cancer. The salts of the present invention may be administered to the patient in a variety of forms, depending on the route of administration, the salt involved, and the cancer being treated. For example, an aqueous composition or suspension of the salts may be administered by injection, or in the form of a pharmaceutical matrix by injection or surgical implantation, at a desired site. The particular technique employed for administering the matrix may depend, for example, on the shape and dimensions of the involved matrix. In some embodiments, the salt is introduced substantially homogeneously in a tumor to minimize the occurrence in the tumor of cold (untreated) areas. In certain embodiments, the salt is administered in combination with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to an excipient that can be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. A wide variety of pharmaceutically acceptable carriers or excipients are available and can be combined with the present salts, as will be apparent to one of ordinary skill in the art.

[0107] According to an embodiment of the present invention, effective amounts, toxicity, and therapeutic efficacy of the active compounds conjugates or complexes can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods exhibit large therapeutic indices. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the compound of the invention, which achieves a half- maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0108] In embodiments, the dosage of a pharmaceutical formulation as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule/regimen can vary, e.g. once a week, daily, or in particular predetermined intervals, depending on a number of clinical factors, such as the subject's sensitivity to each of the active compounds. [0109] In embodiments, an effective dose of a composition comprising radiometal complex can be administered to a patient once. Alternatively, an effective dose of a composition comprising a radiometal complex can be administered to a patient repeatedly. The radiometal complex can be administered over a period of time, such as over a 5 to 60 minute period, e.g. about 30 minutes. If warranted, the administration can be repeated, for example, on a regular basis, such as hourly, daily, bi-weekly or weekly, e.g. in a suitable time interval of e.g. about 6, 12, 24, 48 or 72 hours. In some instances, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after an initial administration, administration can be repeated once per week, month, six months or a year or longer.

[0110] In embodiments, the amount of the active compound, conjugate or complex in the pharmaceutical composition can be based on weight, moles, or volume. In some embodiments, the pharmaceutical composition comprises at least 0.0001%, 0.1%, 0.5% 1%, 2%, 3%, 4%, 5% or 10% of the active. In some embodiments, the pharmaceutical composition comprises 0.01%- 99% of the active, e.g. 0.05%-90%, 0.1%-85%, 0.5%-80%, l%-75%, 2%-70%, 3%-65%, 4%- 60% or 5%-50% of the active.

[0111] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the compound of the invention.

[0112] In embodiments, in addition to treating cancer (pre-cancerous or cancerous cells and tumors) in a subject in need thereof, such cancer is identified, imaged and/or localized. The method may comprise providing a radiometal complex; administering the complex to a subject; and optionally performing imaging, e.g. PET imaging, to detect the emitter released. This allows to visually follow tumor growth and/or shrinkage, e.g. to confirm or personalize an optimized dosage, and/or determine the location of tumor(s) and/or metastases. In various embodiments, imaging may be performed, for example, about 6 to 48 hours post administration. Imaging may be performed in comparison to normal tissue/cells to determine uptake rates differing in cancer/tumor tissues versus normal tissues.

[0113] In embodiments, in situ pharmacokinetic and pharmacodynamic analyses in a tumor or normal cell or tissue may be performed. The method can comprise providing the complex; contacting it with the cancer cells, tumor, or normal cell or tissue; and imaging the cancer cells, tumor, or normal cell or tissue, followed by pharmacokinetic and/or pharmacodynamics analyses, e.g. determining the signal (including radioactivity and/or NIR fluorescence, and changes thereof, at one or more point of time over time, e.g. before and after administration, and one or more times after administration.

[0114] In embodiments, a kit for providing or making a radiopharmaceutical preparation comprising a radiometal complex is provided. In general a vial containing the nonradiometal components of a radiopharmaceutical preparation, usually in the form of a sterilized, validated product to which the appropriate radiometal is added or in which the appropriate radiometal is diluted before use. One or more kit component may be provided in form of an optionally buffered solution, or may be provided for dissolution in an optionally buffered solution. For example, the kit may be a single or multidose vial, and the radiopharmaceutical preparation may require additional steps, e.g., without limitation, boiling, heating, filtration and/or buffering. Radiopharmaceutical preparations derived from kits generally are intended for immediate use after preparation, e.g. within 6-24 hours, e.g. within about 12 hours of preparation.

[0115] The kit can also further comprise conventional kit components, such as needles for use in injecting the compositions, one or more vials for mixing the composition components, and the like, as are apparent to those of ordinary skill. In addition, instructions, either as inserts or as labels, indicating quantities of the components, guidelines for mixing the components, and protocols for administration, can be included in the kits.

[0116] The concentration of the complexes employed in the pharmaceutical compositions and/or the amount administered to a patient or subject may vary and depends upon a variety of factors including, for example, the particular complex and/or pharmaceutically acceptable carrier employed, the particular disease being treated, the extent of the disease, the size and weight of the patient, and the like. Typically, the complex may be employed in the pharmaceutical compositions, and the compositions may be administered to a patient to provide initially lower levels of radiation dosages which may be increased until the desired therapeutic effect is achieved. Generally speaking, the complexes may be employed in pharmaceutical compositions which comprise an aqueous carrier to provide a concentration of absolute radioactivity which may range from about 4 MBq per milliliter (ml) (about 0.1 mCi/ml) or less to about 370 MBq/ml (about 10 mCi/ml), and all combinations and subcombinations of ranges therein. In embodiments, the concentration of the complex in the pharmaceutical compositions may be from about 37 MBq/ml (about 1 mCi/ml) to about 370 MBq/ml (about 10 mCi/ml). In addition, the compositions may be administered to a patient to provide a radiation dose which may range from about 1 KSv (about 1x105 Rem) to about 74 KSv (about 7.4 MRem), and all combinations and subcombinations of ranges therein. In embodiments, the compositions may be administered to a patient to provide a radiation dose of from about 7.4 KSv (about 7.4x105 Rem) to about 74 KSv (about 7.4 MRem). Such amounts are referred to herein as effective amounts or therapeutically effective amounts. In embodiments, the pharmaceutically acceptable carrier may further comprise a thickening agent.

[0117] Thickening agent refers to any of a variety of generally hydrophilic materials which, when incorporated in the present compositions, may act as viscosity modifying agents, emulsifying and/or solubilizing agents, suspending agents, and/or tonicity raising agents. Thickening agents which may be suitable for use in the present radiopharmaceutical compositions include, for example, gelatins, starches, gums, pectin, casein and phycocolloids, including carrageenan, algin and agar, semi -synthetic cellulose derivatives, polyvinyl alcohol and carboxyvinylates, and bentonite, silicates and colloidal silica. Other thickening agents would be apparent to one of ordinary skill.

[0118] The concentration of thickening agent may range from about 0.1 to about 500 milligrams (mg) per ml of pharmaceutical composition. In certain embodiments, the concentration of thickening agent may be from about 1 to about 400 mg/ml, e.g. from about 5 to about 300 mg/ml, e.g. from about 10 to about 200 mg/ml, e.g. from about 20 to about 100 mg/ml, or e.g. from about 25 to about 50 mg/ml. Compositions which may be prepared from the complexes, pharmaceutically acceptable carriers and optional thickening agents include, for example, suspensions, emulsions, and dispersions. In some embodiments, the complexes can be formulated and administered to a patient as a suspension.

[0119] Suspension may refer to a mixture, dispersion or emulsion of finely divided colloidal particles in a liquid. Suspensions may be obtained, for example, by combining the complexes with an inert solid support material. Particulate support materials which may be suitable for use as an inert solid support in the compositions of the present invention include, for example, materials derived from carbon, including those forms of carbon typically referred to as carbon black (lampblack) and/or activated carbon, as well as finely powdered oxides, Kieselguhr, and diatomaceous earth. In some embodiments, the support material comprises carbon black or activated carbon. The size of the particles of the particulate support material may vary and depends, for example, on the particular support material, complex, thickening agent, and the like, employed and may comprise particles ranging in size, for example, from about 0.1 micrometer (mm) to about 50 mm, e.g. the particle size may be from about 0.5 to about 25 mm, e.g. from about 1 to about 10 mm, e.g. from about 2 to about 5 mm.

[0120] Delivery of a therapeutically effective activity of a complex can be obtained via administration of a pharmaceutical composition comprising a therapeutically effective activity or dose of the complex, i.e. in a concentration that is sufficient to elicit the desired therapeutic or imaging effect according to the methods described herein. A therapeutically effective activity may be an activity effective to treat cancer, such as inhibiting or slowing growth of cancerous or precancerous tissue, or lowering the survival rate of cancer or pre-cancerous cells. The therapeutically effective dosage will vary somewhat patient to patient, and will depend upon the condition of the patient and the route of delivery. The effective activity of any particular compound would be expected to vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective dose can include, but are not limited to, the severity of the patient's condition, the disease or disorder being treated, the stability of the complex, and, if appropriate, any additional antineoplastic therapeutic agent being administered with the complex. Methods to determine efficacy and dosage are known to those of ordinary skill.

[0121] Exemplary illustrative embodiments

[0122] Example 1 - Synthesis of DZ-1-(L)-CTC chelator conjugate radiometal complexes

[0123] The synthesis of the DZ-1 dye and its derivatives has been previously described, e.g. in US 10,307,489, which is hereby incorporated herein in its entirety, and may be performed, for example, as described in detail in example la below. The conjugate may then be complexed with radiometals as described in example lb. The following chemicals and reagents may be used. [0124] The CTC chelators CB-TE2A and DiAmSar can be purchased from Macrocyclics (Plano, TX). All other chemicals mentioned herein, e.g. in the reaction schemes as shown, may also be purchased from various standard sources, for example VWR International (Radnor, PA) or Thermo Fisher Scientific (Waltham, MA), as will be apparent to a person of ordinary skill. Deionized ultrapure water (18.2 MW) may be used for making solutions which may be obtained from Milli-Q Direct Ultrapure Water System from Millipore (Billerica, MA, USA). Analytical reversed-phase (RP) high-performance liquid chromatography (HPLC) may be performed e.g. on an Agilent system, e.g. with a 1260 Infinity Diode-Array Detector, e.g. with an Apollo C18 RP column (5 pm, 150 x4.6 mm). For example, the mobile phase may change from 60% solvent A (e.g. 0.1% trifluoroacetic acid in 80% water) and 40% solvent B (e.g. 0.1% trifluoroacetic acid in 80% aqueous acetonitrile) to 100% solvent B over a period of e.g. about 30 min at a flow rate of about 1 ml/min monitoring at e.g. about 254 and/or about 780 nm). Electrospray Ionization Mass Spectrometry (ESI-MS) analysis may be performed on the synthesized compounds, e.g. on a Thermo TSQ Fortis Triple Quadrupole mass spectrometer system.

[0125] DZ-1 may be synthesized e.g. as described below, then it may be conjugated directly to a CTC, or alternatively, a linker may be used to link DZ-1 with a CTC, e.g. a lysine linker, e.g. as shown in scheme 2 herein-below, e.g. to form DZ-l-Lys-CB-TE2A, as shown e.g. in scheme 3 herein-below, or DZl-Lys may be linked to another CTC, e.g. without limitation, DZ-l-Lys- DiAmSar (not shown). Alternatively, as illustrated herein-below in scheme 5 for DiAmSar, DZ-1 may be directly linked to a CTC. The resulting DZ-1-(L)-CTC conjugate, with or without linker L, may then be subjected to ⁶⁴Cu radiolabeling (shown herein-below in schemes 4 and 6, respectively).

[0126] Example 1 - Synthesis of DZ-1-(L)-CTC conjugates

[0127] Scheme 1 shown below illustrates the first steps of conjugate synthesis forming a DZ-1- (L)-CTC conjugate, showing synthesis of dye compound 4, also referred to herein as “DZ-1”; alternative routes of synthesis will be apparent to a person of ordinary skill.

[0128] Scheme 1, synthesis of compound 4 (DZ-1):

la: R= -(CH₂)₄S0₃H lb: R= -(CH₂)₅COOH

[0129] Synthesis of compound la (compare scheme 1 above): The mixture 2, 3, 3- trimethylindolenine (5 g, 31.4 mmol) and 1,4-butane sultone (5.1 g, 37.7 mmol) may be heated with stirring at 120°C under argon for 5 h. The resulted reaction mixture may be cooled to RT (e.g. about 20°C) and the solid may be dissolved in a sufficient volume of solvent, e.g. and organic solvent, e.g. about 50 ml of methanol. Ethyl ether, e.g. about 200 ml, may be added to the methanol solution for precipitation, and the precipitate may be collected and washed with a sufficient volume a sufficient number of times of ethyl acetate (e.g. 15 ml, three times) and dried, e.g. under vacuum, to afford desired product 6.8 g (yield 73%) as a white solid. [0130] Synthesis of compound lb (compare scheme 1 above): To 6-Bromohexanoic acid (2.5 g, 13.0 mmol) may be added 2, 3, 3-trimethylindolenine (2.5 g, 15.7 mmol). The reaction mixture may be heated with stirring at a sufficiently elevated temperature, e.g. at about 110°C under a protective gas such as argon for 8 h. The resulting dark red solid may be dissolved in 50 ml of methanol. Ethyl ether 150 ml may be added. The precipitate may be filtered and washed a sufficient number of times and volume e.g. with ether (e.g. 15 ml, three times) followed by a sufficient number of volume and times washing with acetone (e.g. 15 ml, three times). The product obtained is a white solid (2.7 g, 58%).

[0131] Synthesis of compound 3 (compare scheme 1 above): To the mixture of la (2 g, 6.78 mmol) and compound 2 (3 g, 8.36 mmol) in EtOH (100ml) may be added CH₃COONa (0.28 g, 3.39 mmol), the resulted mixture may be heated at a sufficient temperature and duration, e.g. about 60 °C for about 18 hours. The precipitate may be filtered and sufficiently washed with cold ethanol (e.g. 20 ml, three times). The product may be dried under vacuum to afford desired product 3 as a dark blue solid (2.1 g, yield 58%). Mass spectrum (ESI) 525.19 [M+H]⁺.

[0132] Synthesis of compound 4, a heptamethine cyanine dye, also referred to herein as DZ- 1 (compare scheme 1 above): To the mixture of lb (0.67 g, 1.9 mmol) and compound 3 (1.0 g, 1.9 mmol) in EtOH (20 ml) may be added CH₃COONa (156 mg, 1.9 mmol), the resulting solution may be heated to reflux for a sufficient time, e.g. 3 h. The mixture may then be subjected to precipitation, e.g. kit may be poured into 100 ml of ice water. The solid may be filtered and crystallized from methanol-water to afford desired product 4 as a dark green solid (0.99 g, yield 74%). Mass spectrum (ESI) 705.31 [M+H]⁺.

[0133] Scheme 2 below shows a synthesis of a dye conjugated to a suitable linker moiety DZ-l-L, here of DZ-l-Lysine:

[0134] Synthesis of DZ-l-Lysine (compare scheme 2 above): DZ-1 4 (200 mg, 0.28 mmol) may be dissolved in 5 ml anhydrous CH2CI2. Ethyl chloroformate (CICOOC2H5) (46 mg, 0.42 mmol) and triethylamine (57 mg, 0.57 mmol) may be added. The mixture may be stirred for sufficient duration, e.g. about 2 hours, then N-a-Boc-Lysine 5 (70 mg, 0.28 mmol) in a sufficient volume, e.g. 2 ml of Dimethylformamide (DMF), may be added, and stirred for an additional sufficient duration, e.g. about 2 hours at room temperature (RT). The crude materials may be precipitated in cold diethyl ether (40 ml). Centrifugation for sufficient duration and rpm, e.g. about 5 min at about 3500 rpm, enabled recovery of the pellet which may be purified, e.g. by C18-RP silica chromatography elution with acetonitrile in aqueous NH4HCO3 solution (20 mM) to afford desired product DZ-l-(N-a-Boc)-Lysine as a dark green solid 109 mg (42%). DZ-1-(N- a-Boc)-Lysine may be dissolved in TFA (95%) 5 ml and the mixture may be sufficiently stirred, e.g. about 3 h at ambient temperature, e.g. at RT (about 20 degree centigrade). A sufficient amount of ethyl ether, e.g. 40 ml, may be added. The suspension may be centrifuged for sufficient time to achieve separation, allowing to decant the ether. The product may be dried, e.g. by placing it under high vacuum for sufficient duration, e.g. overnight. The resulting product 6 may be used without further purification for the steps described below.

[0135] Example 2 - Synthesis of DZ-l-L-CTC conjugates

[0136] Scheme 3 below illustrates one option for synthesizing a DZ-l-L-CTC, here DZ-1- Lysine-CB-TE2A 8:

[0137] Synthesis and radio-labeling of a DZ-1-(L)-CTC, here DZ-l-lysine-CB-TE2A 8 (compare scheme 3): A CTC, here CB-TE2A in a suitable form, e.g. one of its salt, e.g. a HC1 salt, may be mixed with suitable amounts of diisopropylcarbodiimide (DIC) and diisopropylethylamine (DIEA), or alternatively to DIEA, of Et3N. For example, CB-TE2A_*4HC1 salt 7 (50 mg, 0.11 mmol), diisopropylcarbodiimide (DIC) (14 mg, 0.11 mmol), and diisopropylethylamine (DIEA) (14 mg, 0.11 mmol) may be dissolved in a suitable volume of DMF, e.g. 2 ml Dimethylformamide (DMF). The mixture may be stirred for a suitable time to ensure complete solution and/or mixing, e.g. about 30 min. To the mixture, DZ-l-L (a conjugate with terminal aminocarboxyl functionality), e.g. DZ-l-Lysine 6 (92 mg, 0.11 mmol) may be added, and stirred for a suitable time to allow for complete reaction, e.g. about 5 hours at room temperature (RT), e.g. about 20 DEG C. The product is precipitated, e.g. in a suitable volume of cold diethyl ether (e.g. about 40 ml). Centrifugation for sufficient time and rpm separates the precipitated product, e.g. for about 5 min at about 3500 rpm. After precipitation, the crude product may be purified by a suitable method, e.g. by C18-RP semi-preparative, to afford the DZ1-L-CTC, here DZ-l-Lysine-CB-TE2A 8, as a dark green solid.

[0138] Scheme 4 below illustrates a suitable method to prepare ⁶⁴Cu-DZ-l-Lysine-CB-TE2A:

[0139] Radioactive labelling and preparation of a ⁶⁴Cu-DZ-l-L-CTC, here ⁶⁴Cu-DZ-l- lysine-CB-TE2A (compare scheme 4): To a reaction vial of suitable volume, e.g. 1.5 ml, the following may be added: about 5 pg of the DZ1-L-CTC, here the DZ-l-Lysine-CB-TE2A 8 in about 200 mΐ of 0.1 M MEOAc (pH about 5.5) solution, about 2 ~ 3 mCi of ⁶⁴CuCl2 in 0.1 M HC1. The reaction mixture may be carefully mixed, e.g. by shaking, and may be incubated at a suitably elevated temperature for sufficient time to allow for the radioactive labelling to occur, e.g. at about 75°C for about 0.5 h. The ⁶⁴Cu-DZ-l-lysine-CB-TE2A 9 complex may then be purified by a suitable method, e.g. by reversed-phase HPLC with a suitable column, e.g. an Apollo C18 RP column (5 m, 250 xlO mm). The column eluate may monitored by a suitable method, e.g. by ultraviolet absorbance at a suitable wavelength, e.g. about 254 nm, and/or with a Nal crystal detector. The mobile phase may change from 40% solvent A (e.g. about 0.1% trifluoroacetic acid in about 80% water) and 60% solvent B (e.g. about 0.1% trifluoroacetic acid in about 80% aqueous acetonitrile) to 100% solvent B at suitable time and flow rate, e.g. about 30 min at a flow rate of about 3 ml/min. The pure fraction of the ⁶⁴Cu-DZ-l-Lysine-CB-TE2A complex separated by HPLC may then be further concentrated and/or dried by gently blowing a positive flow of nitrogen. The residue left in test tube upon concentration may be reconstituted in a suitable volume of a suitable buffer, for example IX PBS buffer (e.g. about 1.0 ml).

[0140] Example 3 - Synthesis of DZ-l-CTC conjugates without linker [0141] Scheme 5 below illustrates the synthesis of a DZ1-CTC without linker, here DZ-1- DiAmSar:

[0142] Synthesis of DZ-l-DiAmSar (compare scheme 5): A mixture of suitable amounts of DZ-1 4 (e.g. about 50 mg, 0.071mmol), diisopropylcarbodiimide (DIC) (e.g. about 13.5 mg, 0.11 mmol) and hydroxybenzotriazole (HOBt) (e.g. about 11.5 mg, 0.085 mmol) may be dissolved in a suitable amount of DMF, e.g. about 2 ml DMF. The mixture is stirred for a suitable time to allow complete solution and mixing, e.g. about 30 min, then a CTC, here DiAmSar^»5H20 10 (29 mg, 0.071 mmol) may be added and stirred for a suitable amount of time to allow for reaction, e.g. about 5 hours at RT. The product may be precipitated in a suitable volume of medium and at a suitably cold temperature to allow for precipitation, e.g. cold diethyl ether (e.g. about 40 ml). The precipitate is separated e.g. by centrifugation at a suitably high rpm for a suitable time, e.g. for about 5 min at about 3500 rpm. The precipitated crude product may be purified by a suitable method, e.g. by C18-RP semi-preparative chromatography, to afford DZ-l-DiAmSar 11 as a dark green solid. [0143] Scheme 6 below illustrates the radioactive labelling of a DZ-l-CTC, here DZ-1- DiAmSar, and preparation of a ⁶⁴Cu-DZ-l-CTC, here ⁶⁴Cu-DZ-l-DiAmSar:

[0144] Preparation of a ⁶⁴Cu-DZ-l-CTC, here ⁶⁴Cu-DZ-l-DiAmSar: To a reaction vial of suitable volume, e.g. 1.5 ml, the following may be added: about 5 pg of a DZ1-CTC, here DZ-1- DiAmSar 11 in about 200 mΐ of about 0.1 M NTL_tOAc (pH about 5.5) solution, about 2 ~ 3 mCi of ⁶⁴CUC1₂ in about 0.1 M HC1. The reaction mixture may be shaken and incubated at a suitable temperature for a suitable time, e.g. about 75°C for about 0.5 h. The ⁶⁴Cu-DZ-l-DiAmSar 12 complex may be purified by a suitable method, e.g. by Reversed-phase HPLC with a suitable column, e.g. an Apollo C18 RP column (5 m, 250 xlO mm). The column eluate may monitored by a suitable method, e.g. by ultraviolet absorbance at a suitable wavelength, e.g. about 254 nm, and/or with a Nal crystal detector. The mobile phase may change from 40% solvent A (e.g. about 0.1% trifluoroacetic acid in about 80% water) and 60% solvent B (e.g. about 0.1% trifluoroacetic acid in about 80% aqueous acetonitrile) to 100% solvent B at suitable time and flow rate, e.g. about 30 min at a flow rate of about 3 ml/min. The pure fraction of the ⁶⁴Cu-DZ-l-DiAmSar complex separated by HPLC may then be further concentrated and/or dried by gently blowing a positive flow of nitrogen. The residue left in test tube upon concentration may be reconstituted in a suitable volume of a suitable buffer, for example IX PBS buffer (e.g. about 1.0 ml).

[0145] For radiolabeling, generally, a suitably radioactive amount and MBq/pg ratio of radiometal may be added to the DZ-1-(L)-CTC conjugates, as will be apparent to a person of ordinary skill. For example the ratio may be up to about 200 MBq/pg (radiometal xonjugate), about 4 to about 100 MBq/pg or about 8 to about 50 MBq/pg e.g. about 20 MBq/pg. For example, 100 MBq (2.7 mCi) of ⁶⁴Cu to 5 pg of DZ-l-CTC may be added to 0.1N ammonium acetate (pH 5.5) buffer, and the mixture may be incubated at a suitable temperature for a sufficient time to achieve complete labelling, for example at about 20°C to about 60°C, or about 30°C to about 50°C, e.g. at about 40°C for about 10 to about 60 minutes or about 20 to about 40 minutes, e.g. about 30 minutes.

[0146] For example, radiolabeling may be accomplished by addition of about 100 MBq (2.7 mCi) of ⁶⁴Cu to 5 pg of DZ-1-(L)-CTC in 0.1N ammonium acetate (pH 5.5) buffer, and incubating the mixture at about 40°C for about 30 min. The resulting ⁶⁴ Cu complex may be purified by Reversed-phase HPLC with an Apollo C18 RP column (5 p, 250 xlO mm). The column eluate may be monitored by ultraviolet absorbance at 254 nm and with a Nal crystal detector. The mobile phase may change from 40% solvent A (0.1% trifluoroacetic acid in 80% water) and 60% solvent B (0.1% trifluoroacetic acid in 80% aqueous acetonitrile) to 100% solvent B at 30 min at a flow rate of 3 ml/min. The pure fraction of the complex from the HPLC may be concentrated by gently blowing a positive flow of nitrogen for drying. The residue left in test tube upon concentration may be reconstituted in a suitable buffer depending on further testing or administration, e.g. IX PBS buffer (1.0 ml).

[0147] Example 4: Superior serum stability of DZ-l-(L)-CTC-⁶⁴Cu complexes

[0148] The serum stability of the complexes described herein may be determined as follows. A suitable amount, e.g. 50 microcuries of a DZ-l-(L)-CTC-⁶⁴Cu complex as described herein, e.g.

(e.g., without limitation, DZ-l-Lysine-CB-TE2A, DZ-l-Lysine-DiAmSar, DZ-1-CB-TE2A, DZ- 1-DiAmSar) may be added into a suitable volume, e.g. 100 mΐ, of fetal bovine serum (Invitrogen, Grand Island, NY). After incubation at 37°C for suitable time intervals, e.g. 1, 3, and 6 h, aliquots of the mixture may be removed and filtered, e.g. through a 0.2 mM microspin filter. The resulting filtrates may be analyzed by a suitable separation and detection method, e.g. by reverse- phase HPLC with a Bioscan Flow Count Radio-HPLC detector. The original peak represents an undegraded stable complex. Detection of any newly formed g-peaks in addition to the original peak identifies degraded products and thus a lack of stability of the complex in the serum. For comparison, analysis of DZ-l-Lysine-DOTA-⁶⁴Cu is performed. DZ-l-Lysine-DOTA-⁶⁴Cu lacks stability in serum. Due to lesser dissociated ⁶⁴Cu, the DZ1-(L)-CTC may provide clearer imaging and tumor detection in organs, and a better signal -to-noise ratio. Therapy may be more effective and have lesser side effects.

[0149] Example 5: In vivo stability and protein assays to detect undesirable ⁶⁴Cu binding

[0150] To determine the stability, or lack thereof, of a radiometal complex in vivo , a gel electrophoresis assay may be performed to detect separate and detect radionuclide-emitting SOD or any other protein, as will be apparent to a person of ordinary skill. If a complex is unstable, upon dissociation, the ⁶⁴Cu may bind to other proteins, including e.g., without limitation, superoxide dismutase (SOD) in liver, and any assay that allows separating protein-bound ⁶⁴Cu from complex/conjugate-bound ⁶⁴Cu and measuring the radionuclide-emission of the protein- bound ⁶⁴Cu may thus be used to determine a lack of stability and undesirable binding to proteins present in the organs of the body (liver, kidney, etc.). If excessive radiation is determined in particular non-tumorous organs, this may also indicate dissociation and a lack of stability. Similarly, organ-specific assays may be performed, e.g. on mouse or rat liver, kidney etc..

Example 6: PET/microPET imaging of mice with DZ-l-(L)-CTC-⁶⁴Cu complexes to determine blood clearance and organ distribution.

[0151] Prior to administration to subjects in need thereof, the specificity and tumors tissue penetration of the dye-CTC complexes described herein may be determined by any suitable conventional method, including e.g. PET/CT imaging, e.g. using a suitable animal model, as will be apparent to a person of ordinary skill. For example, a mammalian model such as a mouse model, in particular, a xenograft mammalian model having the cancer of interest, may be used for testing of the conjugates by imaging, in particular, PET imaging. Other mammalian models may be used, as will be apparent to a person of ordinary skill (e.g. rat, rabbit, dog, monkey, etc.).

[0152] MicroPET imaging (i.e. PET performed on small animals) may be performed as will be apparent to a person of ordinary skill. PET (or microPET) may be performed for imaging and/or therapy or both, to provide image guided therapy. Without wishing to be bound by theory, it is believed that the complexes due to their stability, clearance, efficacy, and advantageous pharmacokinetics are able to provide sufficient tumor uptake and organ clearance to allow for imaging and therapy. Doses may be adjusted for imaging/therapy, or one dose may be suitable for both imaging and therapy.

[0153] The DZ-l-(L)-CTC-⁶⁴Cu complex (e.g., without limitation, DZ-l-Lysine-CB-TE2A, DZ- 1-Lysine-DiAmSar, DZ-1-CB-TE2A, DZ-l-DiAmSar) may be administered intravenously to individual animals, e.g. mice, e.g. in a suitable number, e.g. a set of five, by injecting a suitable amount, e.g. about 300-500 pCi of the complex. The Transaxial microPET images may be collected in suitable intervals, e.g. hourly, e.g. at 1, 2 and 3 h (and/or 6, 9 and 12h, and/or 12, 24 and 48h, or any combination) post probe injection (pi) time points. The standardized uptake value (SUV) analyses may be performed as will be apparent to a person of ordinary skill, here on on cancer xenografts and muscles of individual mice as defined by CT scans and a tumor-to- muscle ratio may be calculated for each group at these time points, as will be apparent to a person of ordinary skill.

[0154] Blood clearance and organ distribution (e.g., without limitation: liver, kidney, blood, bone, bone marrow, brain) may be determined as follows. After anesthesia (e.g. by isoflurane 2- 3%), a group of mice, e.g. 5 mice, may be injected with one or more ⁶⁴Cu radiolabeled DZ1-(L)- CTC, e.g. DZ-l-Lysine-CB-TE2A or DZ-l-DiAmSar (e.g. about ~ 5 pCi), e.g. via the tail vein. Retro-orbital blood samples (e.g. 25 pi) may be collected at multiple time points or intervals, e.g. at 5, 15, 30, 60 and 180 min after injection, and radioactivity of all samples may be counted in a gamma counter (e.g. 1480 Wizard™, Perkin-Elmer™) and normalized to plot against injection time, followed by non-linear regression analysis, to determine the half-life time in blood (e.g. by Prism™ software, e.g. version Prism 9, publicly available from GraphPad™, San Diego, CA). Mice may be sacrificed immediately after the last blood sampling. Tumors and organs (including but not limited to e.g. heart, liver, lung, kidney, small intestine, stomach, bone, bone marrow, muscle, spleen, skin and brain) may be separately harvested and radioactivity may be determined in a gamma counter. Organ distribution data in duplicate may be obtained for each ⁶⁴Cu complex in mice at multiple time points in suitable time intervals, e.g. at three time points, e.g. at lh, 2h and 3h. Such determination may show a favorable blood and organ clearance for DZ1-(L)-CTC- ⁶⁴Cu. A comparison of these complexes to other less stable complexes, in particular e.g. to DZ-1- Lys-DOTA-⁶⁴ complexes, may show superior clearance.

[0155] Image registration and analysis may be performed as follows. PET/CT images may be processed following a standard protocol as will be apparent to a person of ordinary skill, e.g. using standard software as per the manufacturer’s guides, e.g. using the ASIPro™ software, Siemens Healthineers™, Erlangen, Germany. Pixel-wise standardized uptake values (SUVs) of PET may be calculated as a product of the pixel -wise activity divided by the injected dose and body weight. The tumor target may be delineated as 40% of maximum of SUV, anatomically overlaying with a CT image through image registration through the software used.

[0156] ⁶⁴Cu radiolabeled DZ1-(L)-CTC complexes as described herein are more stable compared to e.g. DZ-l-Lysine-DOTA, also compare example 4, and thus can provide a higher in vivo stability compared to e.g. DZl-Lys-DOTA-⁶⁴Cu, and/or increased specifity, which may be shown by PET imaging, e.g. the Standardized uptake values (SUV) in the tumor may be improved, tumor retention and/or tumor penetration may be increased e.g. as shown by PET/CT.. DZ1-(L)-CTC-⁶⁴CU may also advantageously provide a more rapid distribution and clearance that allows improved imaging and therapy and lower side effects. The distribution ratio between tumor and skeletal muscle and thus specificity may be determined by PET as described above and, for example, the tumor-to-muscle ratio after about 24 h of administration may be about 8 : 1 and may be significantly increased by about 20% or more (10:1), about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 120% or more (20:1), after about 36 to about 48 h of administration, or longer, as the tumor tissue retains the conjugate for an extended time, and such ratio may exceed that of DZ-l-Lysine-DOTA.

[0157] Example 7: DZ1-(L)-CTC-⁶⁴CU image-guided radiotherapy/PET, tumor weight reduction and retained body weight in mice, EWS

[0158] Ewing sarcoma (EWS) cell lines and in vivo models may be used to test efficacy against EWS, in particular against disseminated or recurrent EWS.

[0159] To assess the therapeutic effects, efficacy and toxicity of DZ1-(L)-CTC-⁶⁴CU, nude mice may be inoculated subcutaneously with human EWS cancer cells (e.g. ATCC® CRL-2971™, publicly available e.g. from ATCC). The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, ATCC 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum (ATCC 30-2020) to a final concentration of 10%.

[0160] ATCC® CRL-2971™ is a human clear cell sarcoma cell line with an EWS/ATF1 fusion gene produced by a consistent t(12;22)(ql3;ql2) chromosomal translocation.

[0161] Subsequently, nude mice with a subcutaneous tumor size of e.g. about -100 mm³ may be randomly assigned to several groups (e.g. n = 5 mice per group). For the therapeutic group, tumor-bearing mice will be administered intravenously (e.g. via the tail vein) with a suitable amount and volume for a suitable duration, e.g. about 0.2 ml of a DZ1-(L)-CTC-⁶⁴CU (e.g. about 5.55 GBq/kg). The treatment may be given in regular intervals, e.g. once a day or week for several days or weeks, e.g. once a week for 4 weeks. With the same treatment scheme, a control group may be injected via tail vein with vehicle solvent (e.g. saline of the same volume, e.g. about 0.2 ml). After the treatment, mice may be kept for another period, e.g. about 12 weeks. Endpoint of the study will be differential tumor growth between treatment and control group. Body weight may be monitored throughout.

[0162] Alternatively the therapeutic effects, efficacy and toxicity of DZ1-(L)-CTC-⁶⁴CU may be determined essentially using nude mice as described above except that inoculating with human cancer cells is performed intracranially, to determine crossing of the blood-brain-barrier, and the endpoint of the study will be differential animal death between treatment and control group. Body weight may be monitored throughout.

[0163] Tumor specific targeting may be determined with one or more methods, e.g. 1) near infrared fluorescence tumor imaging to detect accumulation of the DZ-1 moiety in the tumor, 2) SPECT/CT nuclear imaging in the same tumor, and/or 3) PET imaging (also see PET described herein-above). PET and SPECT imaging may determine higher clearance and a better signal-to- noise ratio, and allow for image-guided therapy. Tumor dimensions may be measured in regular intervals, e.g. daily or once or twice weekly, with a suitable method such as with calipers, e.g. digital calipers, and the tumor volume may be calculated using the formula: volume = 1/2 (length x width x width). To monitor potential toxicity, body weight may be measured. Mice may be euthanized when the tumor size exceeded the volume of 1,500 mm³ or the body weight lost >20% of original weight. Complexes as described herein may advantageously show a decreased tumor weight, indicative of efficacy, and retained body weight and indicative of a lack of toxicity, in contrast to e.g. DZ1-L-DOTA-⁶⁴CU.

[0164] Example 8: Image-guided therapy of prostate cancer

[0165] Male nude mice of 4 - 6 weeks of age may be inoculated subcutaneously with human prostate cancer cells, e.g. C4-2B (also known as ATCC® CRL-3315™ and publicly available from American Type Culture Collection (ATCC), Manassas, Virginia) or ARCaP_M, publicly available from Novicure™, Birmingham, AL (ARCaPM cells, Catalog Number: 3422, are human prostate cancer cells established from a parental mixed ARCaP cell population with high propensity for bone metastasis in mice. Histopathology of the tumors in bone is mainly of osteoblastic lesions that recapitulate human prostate cancer bone metastasis. ARCaPM cells (spindle-shape mesenchymal morphology) were derived by single cell cloning of the parental ARCaP cells. ARCaPM are highly aggressive prostate cancer metastatic cells. In one study, the incidence of bone metastasis for ARCaPM cells, after intracardiac injection, was determined to be 100% (9/9) with a respective latent period of 71- and 61 -days. ARCaPM cells can grow in culture using MCaP culture Medium available from Novicure™. ARCaPM cells may be used to study prostate cancer bone metastasis and the role of EMT in cancer metastasis. MCaP -medium was prepared using Dulbeccos modified eagle and F12K medium and contains essential and non- essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors and trace minerals and supplemented with several factors that are critical for the optimal growth of ARCaP cells in vitro. The medium is serum-free and should be supplemented with 5% heat- inactivated Fetal Bovine Serum. It is bicarbonate buffered and has a pH of 7.4 when equilibrated in an incubator with an atmosphere of 5% C0₂/95% air).

[0166] The mice may be kept for a suitably long duration, e.g. 2 weeks, for tumor formation to about 100 mm³ in volume. The probe solution for injection will be prepared in sterile phosphate buffered saline (PBS) (radioactive doses of probe DZ-l-Lysine-CTC-⁶⁴Cu complex). Mice may be administered intravenously (e.g. via tail vein) with the imaging probe in a suitable volume (e.g. a volume of about 100-150 pi), followed by procedures of blood collection, probe biodistribution, and fluorescence and PET imaging under inhalation anesthesia (e.g. 2% isoflurane in oxygen), as will be apparent to a person of ordinary skill.

[0167] As a first optional step, complexes as described herein may be administered e.g. as described herein to detect a primary tumor, and/or may be used to detect any residual metastases or secondary tumors thereof, e.g. after removal of a primary tumor, e.g. by surgery.

[0168] Therapy and/or imaging may be performed in parallel or subsequently on a first and/or one or more secondary tumor, or metastases of a first or one or more secondary tumor using the complexes as described herein, optionally with parallel detection/imaging of residual metastases/tumors, and optionally followed by a first or further tumor therapy, or in case a first tumor therapy has been performed, a second or further tumor therapy and/or image-guided radiotherapy/detection of metastases/secondary tumors by imaging. Complexes as described herein may be particularly advantageous for imaging, therapy, and/or image-guided therapy of prostate cancer, including e.g. an improved serum stability, reduced toxicity, and significantly reduced metastatic bone tumor formation.

[0169] Example 9: Image-guided therapy of brain tumors including glioblastoma [0170] To determine tumor suppression efficacy and/or animal survival and/or body weight etc., experiments may be essentially performed as described herein-above under example 2. For example, to assess tumor suppression efficacy, nude mice may be inoculated subcutaneously with human brain cancer cells (e.g. neuroblastoma, astrocytoma, or glioblastoma cells, publicly available from ATCC, e.g., without limitation, CRL-2271, CRL-2268, CRL-7674, and CRL- 7514 (neuroblastoma, human); HTB-12, HTB-13, HTB-14, HTB-17, CRL-7769, CRL-7903 (astrocytoma, human), HTB-15, HTB-16, CRL-2611, CRL-11543, CRL-11544 (glioblastoma, human)). Subsequently, nude mice with subcutaneous tumor size of -100 mm³ may be randomly assigned to several groups (e.g. n = 5 mice per group). To assess the efficacy on animal survival, the therapeutic effects, efficacy and toxicity may be determined by inoculating nude mice intracranially with human brain cancer cells (e.g. neuroblastoma, astrocytoma, or glioblastoma cells publicly available from ATCC). One week after inoculation, the subject mice will be randomly assigned to several groups (e.g. n = 5 mice per group).

[0171] The probe solution for injection may be prepared in sterile PBS buffer. Mice may be administered intravenously (via tail vein) with imaging probes (PBS volume e.g. 100-150 pi) for blood collection, probe biodistribution, and fluorescence and PET imaging by intravenous injection e.g. via the mouse tail vein, e.g. using a syringe (e.g. 1/2 cc U-100 Insulin Syringe) under inhalation anesthesia (2% isoflurane in oxygen). Radioactive doses, also known as probe, may be the DZ-1-(L)-CTC ⁶⁴ Cu complex, formed as will be apparent to a person of ordinary skill and as generally described herein-above.

[0172] One or more treatment administrations, optionally preceded or followed by one or more imaging step administration may be generally performed as described in example 5 herein above, with the following adjustments: The probe solution for injection may be prepared in sterile PBS buffer. Mice may be administered with imaging probes (PBS volume 100-150 mΐ) for blood collection, probe biodistribution, and fluorescence and PET imaging e.g. by intravenous injection e.g. via mouse tail vein e.g. using a syringe (e.g. 1/2 cc U-100 Insulin Syringe) under anesthesia (2% isoflurane in oxygen). [0173] In a first step, a brain tumor that is inoculated in the subcutaneous space may be surgically removed, optionally after detection using ⁶⁴Cu complexes as described herein. In a second step, residual disease may be determined post-surgery and/or at intervals after surgery to detect resurgence using ⁶⁴Cu complexes as described herein. Treatment may be in parallel at the same dose, or altematively/additionally, upon detection of residual or resurgent metastases or tumors, treatment and prolonged and optionally higher dosed ⁶⁴Cu complexes may be initiated.

[0174] Similarly, in animals bearing an intracranial brain tumor, the tumor may be detected with ⁶⁴Cu complexes as described herein, and may be administered in a therapeutic dose to prevent or slow tumor growth, prevent or postpone animal death, and/or cause tumor shrinkage.

[0175] Example 10: Stability and specificity of DZ-l-Lys-CB-TE2A with Copper-64

[0176] Radiolabeling with ⁶⁴ Cu may be performed as follows.

[0177] 37.5 pg DZ-l-Lys-CB-TE2A in 7.5 pL ethanol is added to a 0.4 M NaOAc solution (pH 5.5) containing 4.9 mCi of 64Cu(II). The solution mixture is heated to 65°C while being stirred and the temperature was maintained for 30 min. The reaction is run in the darkness and sampled to test the progress of the radiolabeling by radio-TLC (50 mM DTPA solution incubation 10 min; iTLC-SG plate, lx PBS as mobile phase). The radiochemical purity (RCP) of 64Cu-DZ-l- Lys-CB-TE2A is measured by radio-HPLC (62% A to 20% in 15min, lmL/min; A: lOmM NH40Ac, B: acetonitrile; Atlantis T3 column, 4.6*150nm, 3 pm). The HPLC fractions from 10 - 12 min are collected and pooled (RCP > 95%) for the following biological evaluation experiments.

[0178] Radio-HPLC analysis of 64Cu-DZ-l-Lys-CB-TE2A is shown in Fig. 1, with the top showing the UV channel at 780 nm, and the bottom showing the radio-channel.

[0179] A serum stability assay may be performed as follows.

[0180] Stability may be measured by radio-TLC in serum at 37°C. To 95 pL of serum 5 pL of 64Cu-DZ-l-Lys-CB-TE2A is added. The mixture was incubated at 37°C for 48 h. At time points of 2 h, 6 h, 24 h, and 48 h, the mixture was sampled for radio-TLC analysis, in which 400 pL of 50mM DTPA solution was used each time to assess the extent of 64Cu dissociation from 64Cu- DZ-l-Lys-CB-TE2A.

[0181] As shown in Fig. 2, the stability of 64Cu-DZ-l-Lys-CB-TE2A in serum maintained more than about 95% intact until at least about 48 h in serum at about 37°C.

[0182] In vivo PET imaging evaluation of ⁶⁴Cu-DZ-l-Lys-CB-TE2A may be performed as follows.

[0183] Studies in animal models, e.g. mouse, may be performed as follows.

[0184] Cancer cell lines, in particular, human cancer cell lines, more specifically prostate cancer cell lines such as human DU145 prostate cancer cell lines, e.g. DU145vc (PTPN1 WT) and DU145sh (PTPN1 knockdown (KD)), may be obtained from the American Type Culture Collection (ATCC). With regard to PTPN1 KD, its expression is upregulated in androgen receptor (AR)-negative prostate cancer (e.g., castration-resistant prostate cancer) as compared to AR-positive prostate cancer.

[0185] These or other cancer cell lines may be used to establish cancer xenograft models in suitable mice, in particular in SCID mice. For example, prostate cancer xenograft models in SCID mice may be used. About ~ 1 x 10⁶ cell suspensions of DU145vc and DU145sh may be injected subcutaneously, e.g. into the right and left shoulders of SCID mice, respectively. Post injection, mice are monitored for tumor growth in regular intervals.

[0186] DU145vc and DU145sh tumors started growing three weeks post-injection. DU145vc tumors grew much quicker than DU145sh. When DU145sh became palpable, the size of DU145vc was already in the range of 500 - 1000 mm³.

[0187] Small animal PET/CT Imaging may be performed as follows.

[0188] The imaging study may start at tumor sizes of about 500 mm³ or larger and may be performed on a suitable PET/CT imaging system, e.g. on a Siemens Inveon PET/CT Multimodality System (Knoxville, TN). Here, DU145sh tumor size at the start was about 500 to about 1,000 mm³, while DU145vc tumor size was between about 1,000 mm³ to about 2,000 mm³. PET is followed by CT data acquisition, which may be conducted at 80 kV and 500 mA with a focal spot of 58 pm. Static scans may be performed at suitable intervals, e.g. at 4 h, 24, and 48 h post-injection of e.g. about ~ 100 pCi of ⁶⁴Cu-DZ-l-Lys-CB-TE2A in 100 pL saline containing < 1% ethanol into each tumor-bearing mouse under anesthesia with 2% isoflurane in oxygen (at each time point, n = 2). Both CT and PET images are reconstructed with the manufacturer’s software. Reconstructed CT and PET images are fused for quantitative data analysis. Regions of interest (ROIs) were drawn as guided by CT and quantitatively expressed as standardized uptake values (SUV).

[0189] As shown in Fig. 3, Fig. 4 A-C and Fig. 5 A-C, for ⁶⁴Cu-DZ-l-Lys-CB-TE2A, substantial tumor uptake, good signal-to-noise ratio, high specificity and suitable clearance within 48 h to below 0.25 Standardized Uptake Value (SUV) is observed in both mouse models bearing DU145vc (WT - left shoulder) and DU145sh (PTPN1 KD - Right shoulder) xenografts. SUV range from about 2.5 or more at 4h to between about 0.5 to about 2.5 at 24h, and about 0.125 at 48h, see in particular Fig. 3. This shows sample signal strength within a practical time frame for imaging from about 4 h to about 24 hours or more, or 48 hours or more (note tumor retention with sufficient signal strength at maximum intensity even at about 48 hours, see in particular Fig. 5 C). Of note, ⁶⁴Cu-DZ-l-Lys-CB-TE2A also exhibited an efficient clearance profile from the kidneys, which is a high desirable feature, in particular for a copper radiotheranostic, compare in particular Fig. 4 B and Fig. 4C; these images at 24 h (B) and 48 h (C) post-injection are presented at the same signal intensity scale to show efficient renal clearance. In Fig. 5 A-C the three time point images are presented on different scales in the format of maximum intensity projection (MIP) which demonstrates the uptake and retention of ⁶⁴Cu-DZ-l-CB-TE2A in tumors. An excellent target-to-background ratio and a high specificity is notable.

[0190] Example 11: Comparative example with ⁶⁴Cu-DZ-l-NOTA shows its lack of specificity

[0191] The formula for DZ-l-NOTA-⁶⁴Cu is shown below.

DZ- 1 -NQTA-⁶⁴Cu

[0192] The experiment is performed essentially as described herein-above using a PC3 subcutaneous model (small tumor volume<100 mm3), and ⁶⁴Cu-DZ-l-NOTA (-100 uCi/animal). SCID mice were inoculated subcutaneously with lx lO⁶ PC3 cells. PC3 or PC-3 is a cell line initiated from a bone metastasis of a grade IV prostatic adenocarcinoma that is publicly available from ATCC (CRL-1435™). An IV injection is given and images are taken at various time points post-injection, as indicated (6, 24, 48h). A standardized uptake value (SUV) is calculated based on isotope activity (from PET)/ volume of region of interest (from CT) /body weight and is shown for the tumor in Fig. 6 and Fig. 7A, Fig. 7B, and Fig. 7C. The SUV was about 0.3 at 6 hours p.i., increased to about 0.4 SUV at 24h p.i. and decreased to about 0.3 at 48h. A low target-to-background ratio is notable, see Fig. 7A, Fig. 7B, and Fig. 7C, and a low specificity of the ⁶⁴Cu-DZ-l-NOTA is observed .

[0193] Complexes described herein may be particularly advantageous for imaging, therapy, and/or image-guided therapy of glioblastoma, including e.g. an improved serum stability, while retaining the ability to cross the blood-brain-barrier to improve animal survival.

[0194] Many suitable methods to make or use the complexes described herein are known in the art. According to an embodiment of the present invention, the complexes may be used for cancer therapy as described herein-above. Many different options of administration and treatments are available and can be selected and applied depending on the individual subject and determined treatment protocol, as will be apparent to a person of ordinary skill. Features of one embodiment may be employed with other embodiments as the skilled artisan would recognize, even if not explicitly stated herein. Descriptions of well-known components and processing techniques may be omitted so as to not unnecessarily obscure the embodiments.

[0195] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from this detailed description. The invention is capable of myriad modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the descriptions are to be regarded as illustrative in nature rather than restrictive.

Claims

1. A DZ-1-(L)-CTC conjugate, wherein the conjugate comprises a heptamethine carbocyanine dye (HMCD) moiety conjugated with an optional linker moiety L and a cross-bridged tetraamine cyclam (CTC) chelator residue R and as shown in FI below:

wherein L comprises one or more aminoacid residue, or alternatively, linker L is absent, and wherein the chelator forming the chelator residue R is selected from the group consisting of CB-TE2A, DiAmSar, or a derivative thereof, and wherein the chelator residue is complexed with ⁶⁴Cu.

2. The complex of claim 1 wherein R is CB-TE2A, and wherein L is a lysine residue which links DZ-1 to the chelator as shown in FII below:

3. The complex of claim 1 wherein R is DiAmSar as shown in Fill below:

Fill

(DZl-DiAmSar).

4. The complex of claim 1 provided with one or more pharmaceutically acceptable excipient to form a pharmaceutical formulation.

5. The complex of claim 4, provided as a kit with one or more reagents for reconstitution of the complex in an administrable form.

6. The complex of claim 5, wherein the kit is provided with instructions for mixing and complexing the conjugate and ⁶⁴Cu in suitable amounts, optionally with one or more reagent, buffer or excipient, and optionally treating the resulting solution containing the formed complex to provide it in an administrable form.

7. A method for imaging or treating cancer wherein a DZ-l-Lys-chelator conjugate complex of formula FI is administered to a subject suffering from cancer or from a risk to develop cancer in a sufficient amount and for sufficient duration to allow imaging or treatment; wherein the conjugate comprises a heptamethine carbocyanine dye (HMCD) moiety conjugated with a chelator residue R via a lysine linker and as shown in formula FI below:

wherein wherein L comprises one or more aminoacid residue, or alternatively, linker L is absent, and wherein the chelator forming the chelator residue R is selected from CB-TE2A, DiAmSar, or a derivative thereof, and wherein the chelator residue is complexed with ⁶⁴Cu.

8. The method of claim 7 wherein R is CB-TE2A, and wherein L is a lysine residue which links DZ-1 to the chelator as shown in FII below:

9. The method of claim 7 wherein R is DiAmSar as shown in Fill below:

Fill

(DZl-DiAmSar).

10. The method of claim 7, wherein imaging is performed by Positron Emission Tomography (PET) or Single-Photon Emission Computerized Tomography (SPECT) to detect ⁶⁴Cu radiation, and optionally additionally by Computer Tomography (CT).

11. The method of claim 7, wherein imaging is performed before and/or during one or more therapy time intervals to provide an image-guided therapy.

12. The method of claim 7, wherein the risk to develop cancer is one or more genetic alteration associated with EWS, and the alterations include one or more alteration to a member of the ETS family of transcription factors.

13. The method of claim 7, wherein the cancer is selected from the group comprising:

Ewing’s Sarcoma (EWS), a small cell round tumor, adult neuroblastoma, neuroblastoma in children, small cell lung cancer, Merkel cell tumors, Merkel cell tumors of the skin, prostate cancer, hormone-refractory prostate cancer (HRPC), neuroendocrine differentiated HRPC (NE-HRPC), pre-B-cell acute lymphoblastic leukemia, and a cancer which is associated with alterations in one or more member of the ETS family of transcription factors.

14. The method of claim 10, wherein the cancer is Ewing’s Sarcoma (EWS).

15. The method of claim 7, wherein R is CB-TE2A, or a derivative thereof.

16. The method of claim 7, wherein R is DiAmSar, or a derivative thereof.

17. The invention substantially as described herein.