TECHNICAL FIELD

The invention relates to methods of detecting, diagnosing, imaging and treating cancer in a subject.

BACKGROUND OF INVENTION

The cholecystokinin B receptor (also known as CCK2R, CCKBR, and the gastrin receptor) is a trans-membrane. G protein-coupled receptor. CCK2R is primarily expressed in (i) the brain and central nervous system, and (ii) the mucosa of the gastrointestinal tract, including parietal and ECL cells. It is reported to be most abundantly expressed in the cerebral cortex of the mammalian brain where it has been implicated in the regulation of memory/learning and response to stress1. In the alimentary canal, its presence has been detected in the GI tissues, as well as exocrine and endocrine pancreas. Conflicting reports suggest the receptor is also expressed in peripheral tissues such as adipocytes and pancreas2-9, along with CCK1R, a G protein-coupled receptor with 50% homology to CCK2R. The natural ligands of CCK2R are gastrin and cholecystokinin peptides1.

A number of different types of human tumors have been found that overexpress or ectopically express CCK2R in high densities or at high frequencies including medullary thyroid cancers, insulinomas, small cell lung cancers, non small cell lung cancers, bronchial and ileal carcinoids, GIST tumors, and colon cancers, hepatocellular carcinomas, and pancreatic cancers10-18. One manner in which disregulation of CCK2R signaling may contribute to tumor formation or growth relates to gastrin signaling through the receptor. Gastrin is a peptide hormone that stimulates secretion of gastric acid (HCl) by the parietal cells of the stomach upon binding of CCK2R. Gastrin has been reported to be an inhibitor of cancer cell apoptosis, likely through its ability to induce activation of the serine/threonine protein kinase PKB/Akt19-21. Several reports have shown that inhibiting the CCK2R receptor and the gastrin autocrine loop induces apoptosis and inhibits the proliferation of the cancer cell lines in vivo.

CCK2 receptors found in tumor tissue have been reported to include both normal protein as well as a constitutively active a CCK2R-receptor splice variant (CCK2i4svR) that has the fourth intron inappropriately retained, resulting in the addition of 69 amino acids in the third intracellular loop domain of the receptor, the domain known to be important for signal transduction24-29.

Cytotoxic agents that target CCK2R might serve to block uncontrolled activation of the receptor in tumors. Helpfully, because CCK2R is expressed in normal brain tissue, the blood brain barrier will block polar compounds that could affect normal activity of the receptor in the brain. Therefore, where CCK2R and its splice variant are expressed outside of the brain by a tumor, the tumor will be the only tissue targeted by CCK2R and CCK2i4svR-specific cytotoxic agents.

CCK2R-specific antagonists have been developed and extensive structure-activity relationships have reported. Both in vitro and in vivo studies have demonstrated that the growth potentiating effects of gastrin can be abolished in the presence of selective CCK2R antagonists. One such antagonist is Z-360, an orally-active CCK2R antagonist (Zeria Pharmaceuticals Co., Ltd., Tokyo, Japan) that has a Ki=0.47 nmol/L with a selectivity ratio over CCK1R=672. Preclinical studies have shown that oral administration of Z-360 along with the chemotherapeutic gemcitabine significantly inhibited subcutaneous PANC-1 tumor growth compared with either agent alone (27.1% inhibition) and significantly increased survival compared with the vehicle. This antagonist is currently in Phase II human clinical trials for treatment of pancreatic cancer in combination therapy with gemcitabine2-31,34-35. The modest anti-tumor effect of the antagonist may be due to several factors. First, many cancers involve multiple genetic mutations that are not easily treated by a single agent. There exist multiple redundancies, cross talk and possible compensatory mechanisms between signaling pathways. Consequently blocking one part of a pathway may not always provide enough improvement/antitumor activity that could be translated to improved survival outcome. Secondly, ongoing mutations in the primary molecular target of the drug may also result in drug resistance towards some of these therapies. Third, the presence of a constitutively active splice variant CCK2i4svR means that blocking CCK2R activity using an antagonist may be less effective.

The development of additional agents that selectively target CCK2R and that have the ability to deliver payloads (cytotoxic or diagnostic) would broaden the arsenal of agents that could be used in the treatment and diagnosis of tumors in which CCK2R is expressed. Such agents may also be used in the diagnosis and imaging of cancer in a subject.

BRIEF SUMMARY OF INVENTION

The present invention relates to a small group of highly related benzazepine compounds that selectively target and bind CCK2R and the splice variant CCK2i4svR. The benzazepine compounds are linked to one or more therapeutic or imaging agents, and these conjugates are used in the treatment, diagnosis and imaging of tumors expressing CCK2R and the splice variant.

The invention is thus directed to conjugates comprising a targeting ligand linked to an active moiety. The targeting ligand is a benzazepine compound that selectively binds to CCK2R and/or CCK2i4svR. The active moiety is a therapeutic agent or imaging agent. The targeting ligand and the active moiety are joined by a linker as described herein.

In a first embodiment, the invention is directed to conjugates comprising
B-[L-D]n
wherein B is a targeting ligand, L is a linker, D is an active moiety, and n is an integer of between 1 and 5. In a particular aspect, n=1.

The targeting ligand Z-360 is an excellent example of a targeting ligand that may be used in the conjugates of the invention.



Additional, non-limiting examples of targeting ligands of the present invention are compounds of Formula I


wherein:
the bond between X and Y is a single bond or a double bond;
R1=H, CH3or heterocycle with R2;
R2=heterocycle with R1, CH3, CH2CON(Et)2, CH2COC(CH3)3, CH2CONHC(CH3)3,


X=CR3or NR4;
R3=phenyl, cyclohexyl, CH3, CH2—CH3,


R4=phenyl, cyclohexyl,


Y=CH2, N (dashed=double bond), C═O;
Z=


R5=absent, phenyl, Cl, CO2H, CH3, OCH3, NHCH3, F, SCH2CO2H, SCH2CO2Et,


and analogs, derivatives, salts and esters thereof.

The active moiety D is a therapeutic agent or an imaging agent. Therapeutic agents include, but are not limited to, radio-therapeutic agents, immunotherapeutic agents, photodynamic therapy agents and chemotherapeutic agents. Imaging agents include, but are not limited to, radio-imaging agents, optical imaging agents. PET imaging agents, MRI contrast agents. CT contrast agents, and FRET imaging agents. In some aspects, the therapeutic agent is tubulysin B hydrazide or desacetyl vinblastine monohydrazide. In some aspects, the imaging agent is fluorescein (FITC), rhodamine, or a cyanine-based near infrared dye such as S0456. IR800CW, or LS288.

The linker L is a bivalent or polyvalent hydrophilic spacer comprised of charged or polar amino acids, sugars or sugar-containing oligomers, or a hydrophilic polymer such as polyethylene glycol. Specific examples of the linker are L1, L2 or L3

L1: HN-Glu-Arg-Asp-CO

L2: HN-Glu-PS-Glu-PS-CO

L3: HN-Octanoyl-Glu-PS-Glu-PS-CO

wherein PS is the following formula



In one aspect, the embodiment includes conjugates of the following formula
B-[L-D]n
wherein B is a targeting ligand of Formula I



wherein:

the bond between X and Y is a single bond or a double bond;

R1=H, CH3or heterocycle with R2;

R2=heterocycle with R1, CH3, CH2CON(Et)2, CH2COC(CH3)3, CH2CONHC(CH3)3,



X=CR3or NR4;

R3=phenyl, cyclohexyl, CH3, CH2—CH3,



R4=phenyl, cyclohexyl,



Y=CH2, N (dashed=double bond), C═O;

Z=,



R5=absent, phenyl, Cl, CO2H, CH3, OCH3, NHCH3, F, SCH2CO2H, SCH2CO2Et,



and analogs, derivatives, salts and esters thereof.

L is a bivalent or polyvalent linker,

D is an active moiety, wherein each active moiety is individually selected from therapeutic agents and imaging agents, and n is an integer of between 1 and 5.

In a second embodiment, the invention is directed to methods for detecting a tumor in a subject, comprising administering a conjugate as defined herein to a subject suspected of having a tumor and detecting the conjugate in the subject, wherein the active moiety D of the conjugate is an imaging agent.

In this embodiment, imaging agents include, but are not limited to, radio-imaging agents, optical imaging agents, PET imaging agents, MRI contrast agents, CT contrast agents, and FRET imaging agents. In some aspects, the imaging agent is fluorescein (FITC), rhodamine, S0456, IR800CW, or LS288. In other aspects, the imaging agent is radio-imaging agent comprising111In,99mTc,64Cu,67Cu,67Ga or68Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry, confocal microscopy, fluorescence activated cell sorters, a fluorescence imaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a third embodiment, the invention is directed to methods for diagnosing cancer in a subject, comprising administering a conjugate as defined herein to a subject suspected of having cancer and detecting the conjugate in the subject, wherein the active moiety D of the conjugate is an imaging agent.

In this embodiment, imaging agents include, but are not limited to, radio-imaging agents, optical imaging agents, PET imaging agents, MRI contrast agents, CT contrast agents, and FRET imaging agents. In some aspects, the imaging agent is fluorescein (FITC), rhodamine, S0456, IR800CW, or LS288. In other aspects, the imaging agent is radio-imaging agent comprising111In,99mTc,64Cu,67Cu,67Ga or68Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry, confocal microscopy, fluorescence activated cell sorters, a fluorescence imaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a fourth embodiment, the invention is directed to methods for imaging cancer in a subject, comprising administering a conjugate as defined herein to a subject suspected of or having cancer and detecting the conjugate in the subject, wherein the active moiety D of the conjugate is an imaging agent.

In this embodiment, imaging agents include, but are not limited to, radio-imaging agents, optical imaging agents. PET imaging agents. MRI contrast agents, CT contrast agents, and FRET imaging agents. In some aspects, the imaging agent is fluorescein (FITC), rhodamine, S0456. IR800CW, or LS288. In other aspects, the imaging agent is radio-imaging agent comprising111In,99mTc,64Cu,67Cu,67Ga or68Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry, confocal microscopy, fluorescence activated cell sorters, a fluorescence imaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a fifth embodiment, the invention is directed to methods for treating a subject having cancer, comprising administering a therapeutically-effective amount of a conjugate as defined herein to a subject having cancer, wherein the active moiety D of the conjugate is a therapeutic agent.

In this embodiment, therapeutic agents include, but are not limited to, radio-therapeutic agents, immunotherapeutic agents, photodynamic therapy agents and chemotherapeutic agents. In some aspects, the therapeutic agent is tubulysin B hydrazide or desacetyl vinblastine monohydrazide.

In certain aspects of this embodiment, the conjugate will be in a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable diluent or carrier.

In relevant embodiments of the invention, cells of the tumor express CCK2R or CCK2i4svR, or both. The tumor may be, but is not limited to, medullary thyroid cancers, insulinomas, small cell lung cancers, non small cell lung cancers, astrocytoma, gastric cancer, bronchial and ileal carcinoids, GIST tumors, and colon cancers, prostate cancer, hepatocellular carcinomas, and pancreatic cancers.

In relevant embodiments of the invention, the cancers that may be diagnosed, imaged or treated include those that express CCK2R or CCK2i4svR, or both. The cancers include, but are not limited to, medullary thyroid cancers, insulinomas, small cell lung cancers, non small cell lung cancers, astrocytoma, gastric cancer, bronchial and ileal carcinoids, GIST tumors, and colon cancers, prostate cancer, hepatocellular carcinomas, and pancreatic cancers.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

