Cyclic GRPR binding peptides

Abstract

Described herein are gastrin releasing peptide receptor (GRPR) binding peptide ligands having the formula: R1-R2-R3-(Q)-Z; wherein R1 is absent or is a chelator moiety, optionally chelated to a radioactive atom moiety; R2 is absent or is a linker; R3 is selected from the group consisting of an amino acid sequence having between 1-5 amino acids, or is absent; "Q" is a cyclic peptide sequence having between 4-7 amino acids; "Z" is a linear peptide sequence having between 4- 8 amino acids. Described herein are exemplary GRPR-binding peptide ligands and methods for diagnosis and treatment using them, in particular in relation to various types of cancer associated with GRPR expression.

Description

3296 / 4.2CYCLIC GRPR BINDING PEPTIDESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] Benefit is claimed to US Provisional Patent Application 63 / 709,458, filed October 20, 2024; the contents of which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing is submitted as an XML file named 3296_4_2_sequence_listing created October 18, 2025, about 201 Kilobytes, which is incorporated by reference herein in its entirety.FIELD

[0003] Embodiments of the invention relate to gastrin-releasing peptide receptor (GRPR) binding peptides, compositions comprising same, and methods of using such compositions.BACKGROUND

[0004] The GRPR is a G-protein-coupled receptor (GPCR) which is activated by the nanomolar high-affinity endogenous ligand gastrin-releasing peptide (GRP). GRPR is expressed in various normal healthy tissues that include the pancreas, gastrointestinal tract, adrenal gland, and brain. Notably, the GRPR is found to be overexpressed in a variety of human tumors, including prostate and breast cancer, small cell lung cancer, gastrinomas, gastrointestinal stromal tumors, and others. The GRP peptide-ligand is a linear 27 -residue-long neuropeptide. The GRP regulates gastrointestinal hormone release, gastrointestinal motility, and epithelial cell proliferation by binding and activating GRPR. The GRP also plays a role in the central nervous system, influencing fear learning, social interactions, and memory. It acts as a stress-activated neuromodulator and has been linked to various diseases, including cancer, where its expression and receptor are often aberrant. Additionally, GRP is involved in immune responses and inflammation, and its receptor antagonists are being investigated as potential therapeutic candidates for these conditions in addition to cancers. The GRPR is also activated by the high-affinity ligands that include the endogenous Neuromedin C, and the amphibian Bombesin (BBN). These GRPR ligands act as GRPR agonists, and similarly to GRP, they are linear peptides that share the same carboxy-terminal amino acid sequence and are amidated at3296 / 4.2 the C-terminus by the carboxamide end group. Neuromedin C (alias GRP- 10, GRP 18-27) is a 10-residue-long neuropeptide produced from the C-terminal tail of the GRP. Bombesin (BBN), a 14-residue-long peptide, isolated from the skin of the frog Bombina bombina. GRP, Neuromedin C, BBN, and many of their analogs elicit mitogenic and stimulatory effects in the body, such as bowel movement and secretion of intestinal hormones. They are also involved in regulating various processes in the central nervous system and gastrointestinal tract. For this reason, various peptide-based ligands have been developed specifically as GRPR antagonists as potential biomarkers and therapeutic candidates in cancer.

[0005] Being overexpressed in cancerous tissues, GRPR binding modalities have attracted attention as possible radioligands to treat and diagnose cancer. Much of the research has been focused on the highly conserved C-terminus of GRP and BBN, including N-amide alkylations, and experimentation around the two terminal amino acids. However, the prominent challenge with the known GRPR modalities is the significant toxicity and unfavorable biodistribution, affected by the physiological abundance of the receptor. Many analogs are excreted via hepatobiliary route, and there have been reports of abdominal toxicity when some of the analogs were administered with therapeutic amounts of lutetium- 177. Additionally, GRPR-binding peptidic analogs have demonstrated pronounced lack of metabolic stability in plasma and organs.

[0006] Therefore, there exists a need in the art to provide further GRPR analogs exhibiting low physiological binding, and / or improved metabolic stability, without reducing, and preferably, with increased efficacy in treating various cancers, in comparison to known analogs.SUMMARY

[0007] Described herein are gastrin releasing peptide receptor (GRPR)-binding peptide ligands having the formula:R1-R2-R3-(Q)-Z wherein R1is absent or is a chelator moiety, optionally chelated to a radioactive atom moiety (radionuclide); R2is absent or is a linker; R3is selected from the group consisting of an amino acid sequence having between 1-5 amino acids, or is absent; “Q” is a cyclic peptide sequence having between 4-7 amino acids; and having a structure X1-A1-X2, wherein XI and X2 are each independently amino acids that bond to each other forming a cyclic ring, and Al is a3296 / 4.2 sequence comprising between 2 and 5 amino acids; wherein Al comprises at least one of A 1(a): selected from the group consisting of: glutamine, citrulline, methionine, D-methionine, asparagine, histidine, or glutamic or aspartic acid; and at least one of Al(b): selected from the group consisting of tryptophan, isotryptophan, alpha-methyl tryptophan, 5-hydroxy- tryptophan, kynurenine, histidine, tyrosine, phenylalanine, D-tryptophan, D-histidine, D- tyrosine, and D-phenylalanine; “Z” is a linear peptide sequence having between 4-8 amino acids, and comprising the sequence Val-X2-His-X3-X4; or Phg-X2-His-X3-X4; wherein X2 is Gly, DSer, [NMe]Thr or bAla; X3 is selected from the group consisting of: Sta, Chg, Leu, Phe, [NMe]Leu, He, [NMe]Ile, Ac6c, HyPro, Om, Ala, and Iva; or is absent, and X4 is selected from the group consisting of: Leu, Met, Gly, GlyS2[SMe], hSer[Me], [NMe]hSer[Me], Nle, Ac6c, Chg, Cys[Me], Ala and Tza; or is absent, wherein GlyS2[SMe] is N-(2- (methylthio)ethyl)glycine; hSer[Me] is O-methyl-L-homoserine; Nle is norleucine; Ac6c is 1- aminocyclohexanecarboxylic acid; Tza is 3-thiazolylalanine; HyPro is hydroxyproline; Cys[Me] is S-methyl-L-cysteine, [NMe] is N-methyl threonine, Om is ornithine, [NMe]Ile is N-methyl isoleucine, and Iva is isovaline; and wherein the C-terminal amino acid of Z is optionally either amidated, optionally with a C1-C10 linear or branched alkyl, or is reduced; and wherein, when Q has 6-7 amino acids, Z has the sequence His-Leu-X5 wherein X5 is Met, GlyS2[Me] or is absent.

[0008] Further described herein are GRPR-binding peptide ligands according to the formula:R1-R2-R3-(Q)-Z-NH2; wherein R1is absent or is a chelator moiety, optionally chelated to a radioactive atom moiety; R2is absent or is a linker; R3is selected from the group consisting of an amino acid sequence having between 1-3 amino acids, or is absent; “Q” is a cyclic peptide sequence having between 4-6 amino acids; “Z” is a linear peptide sequence having between 4- 8 amino acids, having at least a 70%, at least 80%, at least 90%, at least 100% sequence homology to a C terminus of a peptide, selected from the group consisting of: bombesin, GRP, and neuromedin C; and NH2 stands for C-terminal amidation where the free carboxyl group at the C-terminus has been replaced with an amide group.

[0009] In addition, methods for diagnosis and treatment of disease using compositions comprising GRPR-binding peptide ligands are disclosed. In particular compositions comprising GRPR peptides may be used for detection, imaging, diagnosis of physiological and3296 / 4.2 pathophysiological conditions where the GRPR is overexpressed, and treatment of cancers expressing GRPR.

[0010] Optionally, the GRPR-binding peptide ligands are conjugated to moieties which can be detected using imaging techniques such as positron emission tomography (PET), positron emission tomography-computed tomography (PET-CT), single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI). Additionally, these peptides may bear a tyrosine moiety suitable for binding halogen radioactive isotopes, as described in greater detail below.

[0011] The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE FIGURES

[0012] Fig. 1 shows the structural formula of GRPR-binding peptide ligand Bl;

[0013] Fig. 2 shows the structural formula of GRPR-binding peptide ligand B2;

[0014] Fig. 3 shows the structural formula of GRPR-binding peptide ligand B3;

[0015] Fig. 4 shows the structural formula of GRPR-binding peptide ligand B4;

[0016] Fig. 5 shows the structural formula of GRPR-binding peptide ligand D28;

[0017] Fig. 6 shows the structural formula of GRPR-binding peptide ligand D5;

[0018] Fig. 7 shows the structural formula of GRPR-binding peptide ligand D4;

[0019] Fig. 8 shows the structural formula of GRPR-binding peptide ligand D27;

[0020] Fig. 9 shows the structural formula of GRPR-binding peptide ligand D7;

[0021] Fig. 10 shows the structural formula of GRPR-binding peptide ligand D21;

[0022] Fig. 11 shows the structural formula of GRPR-binding peptide ligand D41;

[0023] Fig. 12 shows the structural formula of GRPR-binding peptide ligand D8;

[0024] Fig. 13 presents representative maximum intensity projection images obtained from PET-CT of [68Ga]NeoB and [68Ga]D4 at 2.5 hr after i.v. injection to Foxnlnu mice bearing PC-3 (human prostate) tumor, highly expressing GRPR;

[0025] Fig. 14 depicts a survival plot of the xenograft-bearing animals after three weekly administrations (starting from Day 0) of vehicle alone and GRPR-binding peptides;

[0026] Fig. 15 demonstrates tumor xenograft growth curves for animals receiving vehicle in a mouse xenograft model;3296 / 4.2

[0027] Fig. 16 demonstrates tumor xenograft growth curves for animals receiving NeoB in a mouse xenograft model;

[0028] Fig. 17 demonstrates tumor xenograft growth curves for animals receiving RM2 in a mouse xenograft model;

[0029] Fig. 18 demonstrates tumor xenograft growth curves for animals receiving D7 peptide in a mouse xenograft model;

[0030] Fig. 19 demonstrates tumor xenograft growth curves for animals receiving D5 peptide in a mouse xenograft model;

[0031] Fig. 20 demonstrates tumor xenograft growth curves for animals receiving D8 peptide in a mouse xenograft model;

[0032] Fig. 21 demonstrates tumor xenograft growth curves for animals receiving D28 peptide in a mouse xenograft model; and

[0033] Fig. 22 demonstrates tumor xenograft growth curves for animals receiving D4 peptide in a mouse xenograft model.DETAILED DESCRIPTION

[0034] Terms

[0035] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” “Consisting essentially of’ indicates a composition, method, or process that includes only those listed features as the active or essential elements but can include non-active elements in addition. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0036] It is appreciated that certain features in the present specification, which are, for brevity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, combinations of various features which are,3296 / 4.2 for clarity and demonstration, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination, or as suitable in any other described embodiment unless inoperative. Various other features as described herein for the aspect of the peptides are applicable mutatis mutandis to methods of diagnosis and / or treating of conditions, the compositions and / or dosage forms for use in these methods according to the teachings herein and vice versa.

[0037] In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.

[0038] Administration: The introduction of a composition into a subject by a chosen route. Administration of an active compound or composition can be by any route known to one of skill in the art.

[0039] In the context of present disclosure, administering a composition to a patient in need thereof, the administration includes any route for providing a composition comprising a GRPR-binding peptide of the present disclosure. For diagnostic and therapeutical applications, systemic administration is preferred, that is, administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intravenous and intra-arterial administration. Preferably, the administration includes intravenous administration. Depending on the desired dose and patient- specific considerations, the systemic administration may include a bolus administration, i.e., the mode of administering the composition wherein the whole volume comprising desired dose is administered within a short time frame, e.g., between several seconds and several minutes, e.g., within 5 to 10 minutes. Alternatively, particularly when systemic administration of a preparation of high specific activity is contemplated, or high total activity, the systemic administration may include infusion, e.g., by gravity method, peristaltic pump, or syringe pump, i.e., metered infusion calculated to deliver the dose of the compound within longer time intervals, such as between 20 to 120 minutes, preferably between 30 to 60, or 30 to 40 minutes.

[0040] Amino Acid: As used herein, the term “amino acid” is used to refer to any organic molecule that contains at least one amino group and at least one carboxyl group. Typically, at least one amino group is at the alpha position relative to a carboxyl group. The amino acids may be naturally occurring. Naturally occurring amino acids include, for3296 / 4.2 example, the twenty most common levorotatory (L) amino acids normally found in mammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). Other naturally occurring amino acids include, for example, amino acids that are synthesized in metabolic processes not associated with protein synthesis. For example, the amino acids ornithine and citrulline are synthesized in mammalian metabolism during the production of urea. Another example of a naturally occurring amino acid includes hydroxyproline (Hyp or HyPro). Additionally, the amino acids may be “self-capped”, i.e., may contain cyclic amino acids such as pyroglutamic acid (5-oxo-Pro), as found naturally in several peptides. The peptides described herein may contain one or more non-naturally occurring amino acids. Non- naturally occurring amino acids are those amino acids that typically are not synthesized in normal metabolic processes in living organisms, and do not naturally occur in proteins. In addition, the non-naturally occurring amino acids suitably are also not recognized by common proteases. The non-naturally occurring amino acids may be levorotary (L-), dextrorotatory (D- ), or both of the stereoisomers may be used in the same sequence. The non- naturally occurring amino acid can be present at any position in the peptide. For example, the non-naturally occurring amino acid can be at the N-terminus, the C-terminus, or at any position between the N-terminus and the C-terminus. The non-natural amino acids may, for example, comprise alkyl, aryl, or alkylaryl groups that are not found in natural amino acids. Some examples of non-natural alkyl amino acids include a-aminobutyric acid, P- aminobutyric acid, y-aminobutyric acid, 5-aminovaleric acid, and s-aminocaproic acid. Some examples of non-natural aryl amino acids include ortho, meta, and para-aminobenzoic acid. Some examples of non-natural alkylaryl amino acids include ortho-, meta-, and paraaminophenylacetic acid, and y-phenyl-P-aminobutyric acid. Non- naturally occurring amino acids include derivatives and / or metabolites of naturally occurring amino acids. The derivatives of naturally occurring amino acids may, for example, include the addition of one or more chemical groups to the naturally occurring amino acid. An amino acid may be N-substituted, for example with an optionally substituted alkyl group. The optionally substituted alkyl group for N-substituted non-naturally occurring amino acids may be similar or identical to the residues of the naturally3296 / 4.2 occurring amino acids. The optionally substituted alkyl group for N-substituted non-naturally occurring amino acids may also be an alkyl group bearing at any suitable position, e.g., at a position distal to the N-atom, a functional group involved in the formation of a bond selected from the group consisting of an S-S bond, and amide bond, a click chemistry bond, a urea bridge, and a “stapling” bond (catalyst-assisted ring-closure metathesis product, known under this name); for example, such functional groups include inter alia a thiol, a carboxylic group or an amine, an alkyne or an azide, a urea precursor, or an alkene. The following amino acid residues, and their abbreviations are used inter alia herein: ornithine (Om) statine (Sta, (3S,4S)- 4-amino-3-hydroxy-6-methylheptanoic acid), norleucine (Nle), N-(2- (methylthio)ethyl)glycine (GlyS2[SMe]), N-thioethyl glycine (GlyS2), N-acetyl tyrosine (ac- Tyr), 2-piperazin-l-ylacetic acid (PZACM), cyclohexyl glycine (Chg), reduced Leucine (Leu- ol), 1 -aminocyclohexanecarboxylic acid (Ac6c), beta-Alanine (bAla), citrulline (Cit), kynurenine (Kyn), alpha-methyl tryptophan ([aMe]Trp), S-methyl-L-cysteine (Cys[Me]), isovaline (Iva), hydroxyproline (hyPro), O-methyl-L-homoserine (hSer[Me]), N-methyl leucine ([NMe]Leu), N-methyl methoxyhomoserine [NMe]hSer[Me], 3- thiazolylalanine (Tza), 5-hydroxy-tryptophan (Trp[5OH]), phenylglycine (Phg), N-methyl isoleucine ([NMe]Ile), (S)-2-amino-3-(naphthalen-l-yl)propanoic acid (INal), and N-methyl threonine ([NMe]Thr).

[0041] AMTG: A GRPR antagonist having the sequence DOT A — (4-amino-l- carboxymethyl-piperidine)-DPhe-Gln-[aMe]Trp-Ala-Val-Gly-His-Sta-Leu-NH2, disclosed in the publication of PCT application with publication number WO2021121734, as “AMTG” in Table IB.

[0042] Bombesin: a 14-amino acid peptide isolated from the skin of the European fire- bellied toad, and having the structure: pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His- Leu-Met-NH2.

[0043] Cancer: The product of neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not invade the surrounding tissue and / or metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and / or can metastasize is referred to as “malignant.” According to some embodiments, the types of cancer treated and / or diagnosed may be any cancer type which cells or vasculature or microenvironments overexpresses GRPR, preferably being selected from the group consisting of: prostate cancer, breast cancer, small cell lung cancer,3296 / 4.2 non-small-cell lung cancer, colon carcinoma, including colorecta carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cell carcinoma, gastroenteropancreatic neuroendocrine tumor, esophageal squamous cell tumor, neuroblastoma, glioma, glioblastoma, head and neck squamous cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, and pancreatic cancer. Optionally, the cancer expressing GRPR in a patient may be selected according to a biopsy of the patient examined as known in the art for positive expression and / or overexpression of GRPR. Additionally, the cancer expressing GRPR in a patient may be selected according to positive PET imaging of the tumor by a GRPR specific radioligand, e.g., by a cyclic peptide according to the present disclosure.

[0044] Chelator: An agent moiety useful in delivering specific isotopes, e.g., metals, into cells, the moiety binding metal ions by chelation. Any suitable chelator may be used in implementing the teachings herein, for example but not limited to derivatives of DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), NOTA (2-(4,7-bis(2-(tert-butoxy)- 2-oxoethyl)-l,4,7-triazonan-l-yl) acetic acid), NODA (4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)- l,4,7-triazacyclononan-l-yl)-5-(tert-butoxy)-5-oxopentanoic acid), DOTAM (1,4,7,10- T etraazacyclododecane- 1 ,4,7 , 10-tetraacetamide) , DOT AGA (1,4,7, 10-tetraazacyclododecane- 1-glutaric acid-4,7, 10-triacetic acid), sarcophagine (3,6,10,13,16,19-hexaazabicycloicosane), NOD AGA (1,4, 7 -triazacyclononane- 1,4, 7 -triacetic acid- 1 -glutaric acid), CB-TE2A (1,4,8,11- Tetraazabicyclo[6.6.2]hexadecane-4,l l-diacetic acid), HBED (N,N'-bis(2- hydroxybenzyl)ethylenediamine-N,N'-diacetic acid), macropa chelators, like p-NH2-Macropa (6-(( 16-((6-carboxypyridin-2-yl)methyl)- 1,4,10, 13-tetraoxa-7, 16-diazacyclooctadecan-7 - yl)methyl)-4-[2-(4-aminophenyl)ethoxy)]picolinic acid), Bispa2 (6,6'-((9-hydroxy-l,5- bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3 ,7 -diazabicyclo [3.3.1 ]nonane-3 ,7 -diy l)bis- (methylene))dipicolinic acid), DTPA (Diethylenetriamine pentaacetate), or EDTA (ethylenediaminetetraacetic acid). Additional chelators are described in PCT Application publication number WO 03 / 006070. The chelators may also be selected in accordance with the desired radioactive atom to be used in the GRPR-binding peptide ligands in accordance with the present disclosure. For example, it is known that technetium-99m is frequently bound via an N3S chelator, such as S-acetylmercaptoacetyl-triglycine (MAG3), or an N4 chelator, such as 6-carboxy-l,4,8,l l-tetraazaundecane. Similar chelators, including N2S2, like (bis)aminoethanethiols, may also be used for rhenium- 188. DOTA and DOTAGA may be particularly used when the chelated atom is gallium-68, yttrium-90, or lutetium- 177. DOTAM3296 / 4.2 chelator may be also particularly useful for atoms like lead-212. Additionally, smaller acyclic chelators may be used for indium- 111, copper-64, and sometimes also for gallium-68. Macropa or bispa2 chelators may be used for actinium-225. Other chelators may be used as suitable and known in the art. The term “chelator” as used herein also includes tyrosine and optionally its analogs that are capable of binding halogen atoms.

[0045] Cyclic peptide: amino acid sequence comprising a cyclic ring structure, which may be formed by linking one end of the peptide backbone or side chain to another end of the peptide backbone or another side chain, using an amide bond or other chemically stable bond such as lactone, ether, thioether, reduced amide, amine, triazine, a carbon-carbon double bond, a carbamate (urea bridge), or disulfide bond. A cyclic peptide may comprise some amino acids in linear form and some in cyclic form. In the frame of present disclosure, the cyclic peptide is as described herein.

[0046] Diagnostic imaging: the process of creating visual representations of the structures and / or processes inside a living body, e.g., of a patient in need thereof, while using minimally invasive techniques. Particularly preferred diagnostic imaging in conjunction to the present disclosure are positron-emission tomography (PET), and single-photon emission computed tomography (SPECT). Diagnostic images may usually be obtained by administering to the patient in need thereof of an effective amount of imaging moiety, i.e., the GRPR-binding peptide bearing a suitable imaging-enhancing or image-producing atom, and scanning the patient at a predetermined time point to localize the signal. Ideally, most of the excess of tracer is cleared from the body by the predetermined time point whereas the diagnostic target retains the tracer, resulting in clear diagnostic image. In some cases, the intensity of the tracer uptake may be expressed as standardized uptake value, known as SUV. The principles of diagnostic imaging are readily known in the art, and some further aspects in conjunction to the present disclosure are disclosed herein.

[0047] Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound can be administered in a single dose, or in several doses, for example weekly, or biweekly, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, the radiodosimetry characteristics of the subject, and the manner of administration of the compound.3296 / 4.2

[0048] GRP: Gastrin-releasing peptide, a 27 amino acid-containing C-amidated peptide that stimulates the release of gastrin from the G cells of the stomach, and having the structure: Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly- Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2.

[0049] Linker: Generally, one or more linear aliphatic chains or aromatic moieties, or amino acids, that serve as a spacer between two molecule regions, such as between two amino acid molecules or two peptide chains or between an agent, such as a chelator, or an active agent and the peptide. The linker, as a spacer, can improve the binding of the chelator-peptide conjugate by reduction of the steric effect of the chelator on the binding of the peptide ligand to the target receptor, and support the assumption of a favorable secondary structure / conformation. The linker can act as pharmacokinetic agent by means of enhancer or reducer of renal or hepatobiliary clearance, plasma protein binding, distribution of the conjugate in human body or as enhancer of tumor uptake. A non-limited example of linkers are natural and non-natural amino acids, optionally having the N-terminus capped, aromatic or aliphatic omega-amino-C4-C10-alkyl carboxylic acids, dicarboxylic acids, and combinations thereof. Further suitable linkers are described in greater detail herein below.

[0050] NeoB: A GRPR antagonist previously known as NeoBOMBl having a CAS number 1589488-43-6, described in PCT Application Publication WO 2014 / 052471 (SEQ ID No: 1 thereof), having DOTA as a chelator and p-aminobenzylamine di-glycolic acid as a linker.

[0051] Neuromedin B: a mammalian c- amidated decapeptide present in human central nervous system and gastrointestinal tract and having the structure: Gly-Asn-Leu-Trp- Ala-Thr-Gly-His-Phe-Met-NH2.

[0052] Neuromedin C: a mammalian c- amidated decapeptide present in human central nervous system and gastrointestinal tract and having the structure: Gly-Asn-His-Trp- Ala-Val-Gly-His-Leu-Met-NH2 which is derived from the C-terminal sequence GRP (GRP 18- 27).

[0053] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in context of this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes the principles of compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed. In general, parenteral formulations for systemic administration usually comprise injectable sterile and preferably isotonic fluids that include pharmaceutically3296 / 4.2 and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, optionally containing alcohols such as glycerol or ethanol, or the like as a vehicle. For solid compositions (for example, powder, for reconstitution), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, trehalose, and others. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate, or sorbitan monolaurate, as well as gentisic acid, citric acid, or ascorbic acid.

[0054] Preventing or treating a disease: Preventing a disease refers to inhibiting the full development of a disease, for example inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, and may also include preventing of further decline or progression of the condition.

[0055] Radioactive atom moiety (radionuclide): an isotope of radioactive element having utility in nuclear oncology. Many radionuclides are used in medicine, including but not limited to fluorine-18, chromium-51, cobalt-57, cobalt-58, iron-59, copper-64, copper-67, gallium-68, selenium-75, rubidium-82, strontium-89, zirconium-89, yttrium-90, technetium- 99m, indium-i l l, iodine-123, iodine-124, iodine-131, samarium-153, terbium-161, holmium- 166, erbium-169, lutetium-177, rhenium-186, rhenium-188, thallium-201, lead-203, astatine- 211, lead-212, bismuth-212, bismuth-213, radium-223, radium-224, actinium-225, and thorium-227. For example, diagnostic radioactive isotopes (radioisotopes) include positionemitting radioisotopes, such as copper-64, gallium-68, zirconium-89, iodine-124, and fluorine- 18. Radioisotopes detectable by SPECT and therapeutic radioisotopes useful in conjunction with the present disclosure also include beta-emitting radioisotopes or mixed beta and gamma emitting radioisotopes include yttrium-90, technetium-99m, indium- 111, iodine-123, iodine- 131, samarium-153, holmium-166, lutetium-177, rhenium-186, rhenium-188, and copper-67. SPECT radioisotopes that are not routinely used for therapeutics include thallium-201, lead- 203. Therapeutic radioisotopes that are not routinely used for imaging but only for therapy further include beta emitting strontium-89, erbium-169, beta and Auger-electron-emitting terbium-161, alpha emitting astatine-211, actinium-225, lead-212, bismuth-212, radium-223, radium-224, and thorium-227. Metallic radioisotopes may be used with chelators, whereas3296 / 4.2 halogenic radioisotopes (fluorine, iodine, astatine) may be used, inter alia, via tyrosine chemistry. Of these, the commonly used radionuclides include fluorine- 18, gallium-68, yttrium-90, technetium-99m, indium-i l l, iodine-123, iodine-131, and lutetium-177; some further radionuclides are also being investigated, such as copper-64, copper-67, zirconium-89, terbium-161, holmium-166, astatine-211, lead-212, actinium-225, and thorium-227. In context of the present disclosure, the currently preferably radionuclides include copper-64, copper-67, gallium-68, yttrium-90, indium-i l l, terbium-161, lutetium-177, astatine-211, lead-212, and actinium-225; most preferably gallium-68, lutetium-177, astatine-211, lead-212, and actinium- 225.

[0056] RM2: A GRPR antagonist having the sequence DOTA- 4-amino-l- carboxymethyl-piperidine -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, published as Compound 2 of PCT patent application with publication number W02009109332.

[0057] Therapeutically effective amount: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound may be administered in a single dose, or in several doses, for example daily, weekly, bi-weekly, etc., during a course of treatment. However, the effective amount will be dependent on the compound applied, the subject being treated, the severity and type of the disease, and the dosimetry profile of the compound, biodistribution, and the limiting exposure values to off- target organs as known in the art.

[0058] GRPR-Binding Peptide Ligands

[0059] Previously known peptides having GRPR (BB2) binding capabilities have been tested in diagnosis and treatment of cancers associated with elevated GRPR levels. One of those previously known GRPR described herein, known as NeoB, is a GRPR antagonist ligand, i.e., it antagonizes the physiological effects of GRP hormone. NeoB, in addition to other previously known compounds, were disadvantageous in that they were associated with hepatobiliary excretion into the intestines and led to intestinal wall off-targeting and pancreatic targeting. These characteristics made such known compounds less than optimal for use as diagnostic agents or agents for treatment, as administration to patients in need thereof led to accumulation in non-tumor organs, thereby decreasing the efficacy of imaging in patients suffering from orthotopic tumors, and potentially impairing the treatment of such tumors if attempted.3296 / 4.2

[0060] The GRPR-binding peptide ligands when conjugated to a chelator and a diagnostic radioisotope, as described herein, when administered to immunosuppressed mice in xenograft (CDX) models were found to have clear imaging of the tumors as a result of reduced off-targeting of the peptides to healthy organs such as the intestines. Additionally, as seen in the examples below, the GRPR-binding peptide ligands demonstrated antitumor efficacy in the model, that is equipotent or superior to that of NeoB.

[0061] The GRPR-binding peptide ligands described herein preferably comprise a chelator, configured to bind metal ions by chelation. Any suitable chelator may be used in implementing the teachings herein, for example derivatives of DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid), NOTA (2-(4,7-bis(2-(tert-butoxy)-2- oxoethyl)-l,4,7-triazonan-l-yl) acetic acid), NODA (4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)- l,4,7-triazacyclononan-l-yl)-5-(tert-butoxy)-5-oxopentanoic acid), EDTA(ethylenediaminetetraacetic acid), DOT AM l,4,7,10-Tetraazacyclododecane-l,4,7,10- tetraacetamide, sarcophagine 3,6,10,13,16,19-hexaazabicycloicosane, or DTPA (Diethylenetriamine pentaacetate). Additional suitable chelators are described herein. In some embodiments, the chelator is chelating a radionuclide, which is generally an atom having a radioactive isotope, as described below. The chelator may or may not have the radioactive atom moiety bound to it. When the radioactive atom is not present, the GRPR-binding peptide ligands may be conveniently handled as precursors from the manufacture point to the clinical site, where the radionuclide may be introduced into them prior to administration to the subject in need thereof (by a process of radiolabeling). In those embodiments in which a chelator and a radionuclide is present, subsequent to administration to a patient in need thereof, the GRPR- binding peptide ligand will concentrate in cells overexpressing GRPR, thereby allowing for enhanced imaging and / or treatment of those cells.

[0062] In some embodiments, the radionuclide includes one or more radioactive atoms, for example, atoms selected from the group consisting of copper-64, copper-67, gallium-68, yttrium-90, indium-i l l, terbium-161, lutetium-177, astatine-211, lead-212, and actinium-225; preferably gallium-68, lutetium-177, astatine-211, lead-212, and actinium-225. Additional radioactive atoms according to their intended use are described herein.

[0063] According to some embodiments, the GRPR-binding peptide ligands described herein comprise a linker, connecting a GRPR-binding peptide ligand moiety with a chelator moiety. Various linkers may be used according to embodiments. A linker may comprise 1, 2,3296 / 4.23, 4, or 5 amino acids, optionally in combination with another linker moiety. Some linkers may include more than one moiety, same or different. Some embodiments include the following linkers, as in Table 1:

[0064] Table 1:3296 / 4.23296 / 4.23296 / 4.23296 / 4.20065] Although the moiety “DOT A” is mentioned in Table 1, it is noted that this is to show the linker structure as it connects to the chelator DOTA. As such, the linker-chelator combinations with DOTA may include but are not limited to: Lys[DOTA]- (S)-2,2',2"-(10-(2- ((5-amino-5-carboxypentyl)amino)-2-oxoethyl)- 1,4,7, 10-tetraazacyclododecane- 1,4,7- triyl)triacetic acid; Bz-Lys[DOTA]- (S)-2,2',2"-(10-(2-((5-benzamido-5- carboxypentyl)amino)-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7 -triyl)triacetic acid; AdamA-Lys[DOTA]- 2,2',2"-(10-(2-(((S)-5-((3S,5S,7S)-adamantane-l-carboxamido)-5- carboxypentyl)amino)-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7 -triyl)triacetic acid; and Hex-Lys[DOTA]- (S)-2,2',2"-(10-(2-((5-(6-aminohexanamido)-5-carboxypentyl)amino)- 2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7 -triyl)triacetic acid.

[0066] Additional linkers which may be used include: 2-(2-((4- (aminomethyl)phenyl)amino)-2-oxoethoxy)acetic acid (PAMA_DGA), 4- (aminomethyl)benzoic acid (PAMBA), 7-aminoheptanoic acid (AHeptA), pentane-di- carboxylic acid (PndA), and gamma-amino-beta-hydroxybutyric acid (GABOB).

[0067] Thus, the linker may be a natural or unnatural amino acid, e.g., Glu, Asp, Gly, Lys, Phe, beta- Ala (bAla), and hydroxyproline (HyPro). The amino acid may be N-capped with a suitable carboxylic acid. The amino acid bearing functional groups may be connected to a chelator also via the side chain thereof, e.g., Lys[DOTA], Bz-Lys[DOTA], Hex-Lys[DOTA], and AdamA-Lys[DOTA], where [DOTA] is an exemplary chelator connected via the side chain of Lys, and Bz-, Hex-, AdamA- stand for residues of benzoic, hexanoic, and adamantic acids, respectively. The linker may be an amino-carbamic acid thereby forming a urea bridge, e.g., an amino-(C2-C6)alkyl-carbamic acid, such as 2-aminoethylcarbamic acid. The linker may also be functionalized short polymeric chain, e.g., PEG2 or PEG4, as shown in Table 1 above.3296 / 4.2The linker may also be an amino-carboxylic acid moiety having the amine and carboxylic acid separated by between 4 and 10 atoms, preferably carbon atoms, optionally forming a part of an aromatic system, e.g., 4-(aminomethyl)benzoic acid (PAMBA), gamma-aminobutyric acid (GABA), 4-aminobenzoic acid (PABA), gamma-amino-beta-hydroxybutyric acid (GABOB), 6-aminohexanoic acid (AHexA), 7-aminoheptanoic acid (AHeptA), 8-amino-octanoic acid, 2- piperazin-l-ylacetic acid (PZACM), 2-(l,4-diazepan-l-yl)acetic acid (DzpA), 4-(piperazin-l- ylmethyl)-benzoic acid (PZBzA), or 2-(4-aminopiperidin-l-yl) acetic acid (PPACM). The linker may also be a diamine, e.g., p-aminobenzylamine , or a di-carboxylic acid, e.g., glutaric acid (alias pentane-di-carboxylic acid, or PndA), or diglycolic acid (DGA), or a derivative of a diamine or dicarboxylic acid, e.g., 2-(2-oxo-2-piperazin-l-ylethoxy)-acetic acid (PZ_DGA). The linker may also be a 6-hydrazinylpyridine-3-carboxylic acid (HYNIC) moiety. The linker may also be a group of several linkers, e.g., p-aminobenzylamine di-glycolic acid, alias 2-(2- ((4-(aminomethyl)phenyl)amino)-2-oxoethoxy)acetic acid (PAMA_DGA), 2-[4-(3- aminopropanoyl)-piperazin-l-yl]acetic acid (bAla-PZACM), (2-(4-aminobutanamido)- ethyl)carbamic acid (GABA-Urea bridge), (R)-4-amino-5-(4-(carboxymethyl)piperazin-l-yl)- 5 -oxopentanoic acid (DGlu-PZACM), 2-(2-(4-(3-amino-propanoyl) piperazin- l-yl)-2- oxoethoxy)-acetic acid (bAla-PZ_DGA), 3-(2-(2-(6-hydrazineyl-nicotinamido) ethoxy)- ethoxy)propanoic acid (HYNIC_PEG-2), or 6-(3-aminopropanoyl-amino) hexanoic acid (bAla-AHexA).

[0068] Preferably, the linker may be selected from the group consisting of Lys[DOTA],Bz-Lys[DOTA], Hex-Lys[DOTA], AdamA-Lys[DOTA], 6-aminohexanoic acid (AHexA), 7- aminoheptanoic acid (AHeptA), 2-piperazin-l-ylacetic acid (PZACM), 2-(l,4-diazepan-l- yl)acetic acid (DzpA), 4-(piperazin-l-ylmethyl)-benzoic acid (PZBzA), 2-(4-aminopiperidin- 1-yl) acetic acid (PPACM), 2-aminoethylcarbamic acid (Urea_bridge), 2-(2-((4- (aminomethyl)phenyl)amino)-2-oxoethoxy)acetic acid (PAMA_DGA), 2-(2-oxo-2-piperazin- l-ylethoxy)-acetic acid (PZ_DGA), 2-[4-(3-aminopropanoyl)-piperazin-l-yl]acetic acid (bAla-PZACM), 4-(aminomethyl)benzoic acid (PAMBA), gamma-aminobutyric acid (GABA), pentane-di-carboxylic acid (PndA), and 3-(2-(2-(6-hydrazineyl-nicotinamido) ethoxy)-ethoxy)propanoic acid (HYNIC_PEG-2).

[0069] According to a specific preferred embodiment, the linker is p- aminobenzylamine di-glycolic acid (PAMA_DGA). Preferably, the chelator moiety is bound via the methylamine of the p-aminobenzylamine linker end and the D-phenylalanine forms an3296 / 4.2 amide bond with the di-glycolic acid linker end. According to further specific embodiments, the linker is 2-piperazin-l-ylacetic acid (PZACM), 2-aminoethylcarbamic acid (Urea_bridge), 2-(2-oxo-2-piperazin-l-ylethoxy)-acetic acid (PZ_DGA), 7-aminoheptanoic acid (AHeptA), bAla, bAla-PZACM, or bAla-PZ_DGA. Preferably the linker is bound to the chelator moiety via the amine, e.g., 4-N of piperazine or amine nitrogen, and the carboxylic acid to the R3moiety of the peptide chain.

[0070] It has now been unexpectedly found that it may be possible to produce GRPR- binding peptides with similar or superior binding properties, and superior biodistribution and / or metabolic stability, by providing the peptides having a cyclic portion, as described in greater detail below, and a linear portion protruding from the cyclic portion. This key-like form, without being bound by a particular theory, enhances both the metabolic stability of the peptide, and stabilizes amino acid residues that are important for binding in a favorable conformation. As described above, the cyclic GRPR-binding peptides have a general formula of Rx-R2-R3- (Q)-Z. The terminal amino acids that form a bond in a manner other than via the carboxylic acid, may have the free carboxylic acid terminated as known in the art, e.g., with an amide, an alkyl amide, an ester, a reduction to hydroxyl, or with a further protection group.

[0071] When the peptide has no chelator moiety, R1is preferably absent, and when the peptide is synthesized to be able to chelate a radioactive atom moiety, R1is a chelator moiety. The R1chelator moiety may optionally be chelated to a radioactive atom moiety, particularly at or close to the point of care of a patient in need thereof. R1may also be a tyrosine or an analogue thereof suitable for reacting with a halogen, such as fluorine, iodine, and astatine.

[0072] The R2may be absent from the peptide; in these peptides the optionally present chelator moiety is bound directly to the peptide. Conversely, R2may be present in the peptide even if R1is absent. However, preferably, R2is present and is a linker as described generally herein above.

[0073] The R3-(Q)-Z- part of the molecule forms the peptidic part of the GRPR-binding peptide, with a cyclic portion Q being optionally preceded (in the direction of the N-terminus) by an R3short sequence and succeeded (in the direction of the C-terminus) with the Z moiety. Generally, R3is selected from the group consisting of an amino acid sequence having between 1-5 amino acids, e.g., 1-3 amino acids, or R3may be absent. Preferably, when present and Q does not contain a phenylalanine, R3is an aromatic amino acid, for example, an aromatic amino acid in D-formation, further preferably, a phenylalanine, e.g., D-Phe.3296 / 4.2

[0074] The “Q” is a cyclic peptide sequence having between 4-7, preferably between 4-6 amino acids, with the terminal (i.e., the first and the last of the Q-sequence) amino acids forming a ring-closing bond between themselves, while also being a part of the cyclic peptide sequence Q. The sequence Q may have a structure of X1-A1-X2, with XI and X2 being each amino acids having R-groups or N-alkyl groups which are bound to each other; and Al is a peptide having between 2 and 5 amino acids, between 2 and 4 amino acids, or preferably 2 amino acids. These terminal amino acids XI and X2 may bear inter-reactive groups in relation to each other, either as naturally occurring residues, non-naturally occurring residues, or as part of N-alkylation of these amino acids. The optionally substituted alkyl group for N-substituted amino acids may be similar or identical to the residues of the naturally occurring amino acids. The optionally substituted alkyl group for N-alkylated amino acids may also be an alkyl group bearing at any suitable position, e.g., at a position distal to the N-atom, a functional group involved in formation of a bond, preferably selected from the group consisting of an S-S bond, an amide bond, a thio-ether bond, a click chemistry bond (bridging 1,2,3-triazole group), a thiol-maleimide link, a urea bridge, and a “stapling” bond (catalyst-assisted ring-closure metathesis product containing an alkene bond, known under this name); for example, such functional groups include inter alia a thiol, a carboxylic group or an amine, an alkyne or an azide, a urea precursor, or an alkene. Thus, XI and X2 may bear pairs of functional groups, one on each, as in Table 2.

[0075] Table 2:3296 / 4.2

[0076] The central (non-terminal) part of the Q sequence Al preferably comprises a group of glutamine, citrulline, methionine, asparagine, histidine, or glutamic or aspartic acid; and tryptophan or an isostere thereof. Generally, preferably at least one of the amino acids of Al is an amino acid selected from the group consisting of tryptophan, isotryptophan, alpha methyl tryptophan, 5-hydroxy-tryptophan, kynurenine, histidine, tyrosine, phenylalanine, D- tryptophan, D-histidine, D-tyrosine and D-phenylalanine. Preferably, Al comprises at least one of Al(a): selected from the group consisting of: glutamine, citrulline, methionine, D- methionine, asparagine, histidine, or glutamic or aspartic acid; and at least one of A 1(b): selected from the group consisting of tryptophan, isotryptophan, alpha- methyl tryptophan, 5- hydroxy-tryptophan, kynurenine, histidine, tyrosine, phenylalanine, D-tryptophan, D-histidine, D-tyrosine, and D-phenylalanine. The position of this central (non-terminal) part may preferably be located in the direction of the C-terminus relative to the “Z” linear part. When the Q sequence comprises 4-7 amino acids, it may also contain alanine, valine, and / or glycine in the part of Q sequence that is adjacent to the “Z” linear part, e.g., -Gln-Trp-Ala-Val-X2, preferably wherein X2 may be N-mercaptoalkyl glycine, e.g., GlyS2 (N-thioethyl glycine), or cysteine.

[0077] The part “Z” is a linear peptide sequence having between 4-8 amino acids. Optionally, the “Z” linear peptide may be Val-Gly-His-Sta-Leu-, wherein Sta is statine. Additionally, “Z” linear peptide may be -Val-Gly-His-Chg-Met-, wherein Chg is cyclohexyl glycine; -Val-Gly-His-Chg-Leu-; Val-Gly-His-Leu-GlyS2[SMe]-, wherein -GlyS2[SMe] is N- (2-(methylthio)ethyl)glycine; -Val-Gly-His-Leu-hSer[Me], wherein hSer[Me] is O-methyl-L- homoserine; -Val-Gly-His-Leu-[NMe]hSer[Me]-; -Val-Gly-His-Phe-Met; -Val-Gly-His-Sta- Nle-, wherein Nle is norleucine; -Val-Gly-His-Leu-Nle-; -Val-Gly-His-[NMe]Leu-Met-; -Val- Gly-His-Ile-Met; -Val-Gly-His-Chg-Ac6c-, wherein Ac6c is 1 -aminocyclohexanecarboxylic acid; -Val-Gly-His-Ac6c-Met-; -Val-Gly-His-Ac6c-Chg-; -Val-Gly-His-Ac6c-Met-; -Val- Gly-His-Ac6c-Tza-, wherein Tza is 3-thiazolylalanine; -Val-Gly-His-Hy Pro-Met-, wherein HyPro is hydroxyproline; -Val-Gly-His-Iva-Met-, wherein Iva is isovaline. Alternatively, “Z” linear peptide may also be -Val-bAla-His-Phe-Nle-, wherein bAla is beta alanine. Further alternatively, “Z” linear portion may be -Val-Gly-His-2,6-dimethylheptan-4-amine. Further, specifically, wherein Q sequence comprises -Gln-Trp-Ala-Val-X2, “Z” linear sequence may be -His-Leu-[SMe]GlyS2.3296 / 4.2

[0078] The C-terminus may be amidated, such as a primary amide -NH2. While NH2 represents an amide group bonded directly to the C terminus amino acid carboxylic group, other forms of amidation are contemplated including Cl -CIO amidation. In addition, other forms carboxylic termination are contemplated, such as reduction to hydroxyl, an ester, or even a free carboxylic residue.

[0079] GRPR-binding peptide ligands according to an embodiment, may optionally have a formula of R1-R2-R3-(Q)-Z-NH2, wherein Rxand R2are as defined above, R3comprises D-Phe or Gly, preferably D-Phe, Q is Xl-R4-R5-X2, wherein R4is Gin or Glu, and R5is tryptophan, alfa methyl tryptophan, kynurenine, histidine, or tyrosine, and Z is -Val-Gly-His- R6-R7-, wherein R6and R7are present and are independently selected from Leu, He, Nle, [NMe]Leu, [NMe]Ile, Met, Sta, Phe, Chg, Gly, GlyS2[SMe], hSer[Me], [NMe]hSer[Me], Ac6c, HyPro, Om, Ala, Iva, Cys[Me], or Tza.

[0080] Some particularly preferred GRPR-binding peptide ligands may be selected according to their pharmacological properties, including, but not limited to, binding to GRPR, displacement of GRPR-binding ligand, interaction with cells expressing GRPR, secondary pharmacological (effector) assays, metabolic assays, specificity, and others as known in the art. Particularly, the GRPR-binding peptides may be selected based on displacement binding screening. For this purpose, a substrate expressing GRPR is used, e.g., a cell membrane preparation, or cells expressing GRPR. Similarly, an artificial support supporting recombinant GRPR may also be used. The substrate is exposed to radioactive binder of GRPR (tracer), as known in the art and described herein, for example, to a radioactive derivative of bombesin, neuromedin C, GRP, NeoB, RM2, AMTG, and the like. The preferred radioactive tracer may include [125I][Tyr4]-bombesin, or [177Lu]NeoB. The radioactive tracer is used at a concentration equal to the predetermined dissociation constant of the ligand, predetermined in a separate assay as known in the art. The GRPR-binding peptides may be screened using this system at a selected appropriate concentration, e.g., between 0.1 nM and 100 nM, such as 10 nM, or 1 nM if significant binding is expected from previous experiments. The percent of inhibition of radioactive tracer specific binding is then determined. The preferred peptides may usually demonstrate at least 25% inhibition efficiency, preferably, above 50% inhibition, or even above 70% inhibition. Preferred GRPR-binding peptide ligands demonstrate above 50% inhibition at 10 nM concentration. Specifically, these peptides demonstrate this efficacy when measured using either membranes preparation of HEK-293 transfected with BB2 receptor and3296 / 4.2 using [125I][Tyr4] -bombesin in 0.04 nM concentration, or using PC-3 cells naturally overexpressing GRPR and using [177Lu]NeoB in 1 nM concentration.

[0081] For selected peptides, the percent of inhibition of radioactive tracer specific binding at increasing concentrations of the displacing GRPR-binding peptide may then be fitted to a suitable pharmacological model to furnish binding parameters, such as IC50, Ki, and Hill’s coefficient of the tested peptide, if desired.

[0082] Preferred GRPR-binding peptide ligands

[0083] GRPR-binding peptide ligands were prepared having the structures shown inTable 3. Parentheses indicate amino acids which form cyclic moieties.Table 3:3296 / 4.23296 / 4.23296 / 4.2

[0085] Table 4:3296 / 4.23296 / 4.23296 / 4.23296 / 4.23296 / 4.23296 / 4.23296 / 4.2

[0086] Particularly preferred GRPR-binding peptide ligands include D28, D4, D5, D7, D8, D12, and D41.

[0087] Methods for Diagnosis and Treatment

[0088] Further described herein are methods for diagnosis of a cancer associated with GRPR comprising: administering an effective amount of a GRPR-binding peptide ligand to a subject in need thereof, and imaging the subject, particularly wherein the peptides are chelated to a diagnostic radioactive atom. Optionally, diagnostic methods are performed using a GRPR binding peptide ligand having 100-300 megabecquerel (MBq) activity per patient, preferably 150-250 MBq. Optionally, diagnostic methods are performed using a GRPR binding peptide ligand in an amount of 10-100 micrograms (pg) per dose, preferably 25-75 pg. Optionally, the radioactive atom is gallium-68.

[0089] Further described herein are methods for treatment of a cancer associated with GRPR comprising: administering an effective amount of a GRPR binding peptide ligand to a subject in need thereof. Optionally, treatment is performed using a GRPR binding peptide ligand having 5-12 giga-Becquerel (GBq) activity per treatment, preferably 7-10 GBq. Optionally, treatment methods are performed using a GRPR binding peptide ligand in an amount in 100-300 pg of peptide, preferably 150-250 pg per treatment. Optionally, the radionuclide is lutetium- 177 or actinium-225.

[0090] According to an embodiment provided is a gastrin releasing peptide receptor (GRPR)-binding peptide ligand having the formula: RX-R2-R3-(Q)-Z; wherein R1is absent or is a chelator moiety, optionally chelated to a radioactive atom moiety; R2is absent or is a linker; R3is selected from the group consisting of an amino acid sequence having between 1-5 amino3296 / 4.2 acids, or is absent; Q” is a cyclic peptide sequence having between 4-7 amino acids; and having a structure X1-A1-X2, wherein XI and X2 are each independently amino acids that bond to each other forming a cyclic ring, and Al is a sequence comprising between 2 and 5 amino acids; wherein Al comprises at least one of A 1(a): selected from the group consisting of: glutamine, citrulline, methionine, D-methionine, asparagine, histidine, or glutamic or aspartic acid; and at least one of Al(b): selected from the group consisting of tryptophan, isotryptophan, alpha-methyl tryptophan, 5 -hydroxy-tryptophan, kynurenine, histidine, tyrosine, phenylalanine, D-tryptophan, D-histidine, D-tyrosine, and D-phenylalanine; “Z” is a linear peptide sequence having between 4-8 amino acids, and comprising the sequence Val-X2-His- X3-X4; or Phg-X2-His-X3-X4; wherein X2 is Gly, DSer, [NMe]Thr, or bAla; X3 is selected from the group consisting of: Sta, Chg, Leu, Phe, [NMe]Leu, He, [NMe]Ile, Ac6c, HyPro, Om, Ala, and Iva; or is absent, and X4 is selected from the group consisting of: Leu, Met, Gly, GlyS2[SMe], hSerfMe], [NMe]hSer[Me], Nle, Ac6c, Chg, Cys[Me], Ala, and Tza; or is absent, wherein GlyS2[SMe] is N-(2-(methylthio)ethyl)glycine; hSer[Me] is O-methyl-L-homoserine; Nle is norleucine; Ac6c is 1 -aminocyclohexanecarboxylic acid; Tza is 3 -thiazolylalanine; HyPro is hydroxyproline; Cys[Me] is S-methyl-L-cysteine, [NMe] is N-methyl threonine, Orn is ornithine, [NMe]Ile is N-methyl isoleucine, and Iva is isovaline; and wherein the C-terminal amino acid of Z is optionally either amidated, optionally with a Cl -CIO linear or branched alkyl, or is reduced; and wherein, when Q has 6-7 amino acids, Z has the sequence His-Leu- X5 wherein X5 is Met, GlyS2[Me] or is absent. Optionally, Z comprises a sequence selected from the group consisting of: Val-Gly-His-Sta-Leu; Val-Gly-His-Chg-Met; Val-Gly-His-Chg- Leu; Val-Gly-His-Leu-GlyS2[SMe]; Val-Gly-His-Leu-hSer[Me]; Val-Gly-His-Leu- [NMe]hSer[Me]; Val-Gly-His-Phe-Met; Val-Gly-His-Sta-Nle; Val-Gly-His-Leu-Nle; Val- Gly-His-[NMe]Leu-Met; Val-Gly-His-Ile-Met; Val-Gly-His-Chg-Ac6c; Val-Gly-His-Ac6c- Met; Val-Gly-His-Ac6c-Chg; Val-Gly-His-Ac6c-Met; Val-Gly-His-Ac6c-Tza; Val-Gly-His- Hy Pro-Met; Val-Gly-His-Iva-Met; and Val-bAla-His-Phe-Nle. Optionally, Z is Val-Gly-His- Sta-Leu-NH2. Optionally, at least one of the Al amino acids is tryptophan. Optionally, each of XI and X2 are each cysteine. Optionally, Q is composed of the sequence (Cys-Gln-Trp- Cys). Optionally, R3 is absent or is one amino acid. Optionally, R3 is D-phenylalanine. Optionally, R3 is absent. Optionally, R1is a chelator moiety and is selected from the group consisting of: DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), NOTA (2- (4,7-bis(2-(tert-butoxy)-2-oxoethyl)-l,4,7-triazonan-l-yl) acetic acid), NODA (4-(4,7-bis(2-3296 / 4.2(tert-butoxy)-2-oxoethyl)-l,4,7-triazacyclononan-l-yl)-5-(tert-butoxy)-5-oxopentanoic acid), DOTAM (l,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetamide), DOTAGA (1,4,7,10- tetraazacyclododecane-1 -glutaric acid-4,7, 10-triacetic acid), sarcophagine (3,6,10,13,16,19- hexaazabicycloicosane), NODAGA (l,4,7-triazacyclononane-l,4,7-triacetic acid- 1 -glutaric acid), EDTA (ethylenediaminetetraacetic acid), and DTPA (Diethylenetriamine pentaacetate). Optionally, the chelator is DOTA, DOTAGA, or DOTAM. Optionally, R2is a linker. Optionally, the linker is selected from the group consisting of: an amino acid sequence having between 1 and 3 amino acids, Lys[DOTA], Bz-Lys[DOTA], Hex-Lys[DOTA], AdamA- Lys[DOTA], 6-aminohexanoic acid (AHexA), 7-aminoheptanoic acid (AHeptA), 2-piperazin- 1-ylacetic acid (PZACM), 2-(l,4-diazepan-l-yl)acetic acid (DzpA), 4-(piperazin-l-ylmethyl)- benzoic acid (PZBzA), 2-(4-aminopiperidin-l-yl) acetic acid (PPACM), 2- aminoethylcarbamic acid (Urea_bridge), 2-(2-((4-(aminomethyl)phenyl)amino)-2- oxoethoxy)acetic acid (PAMA_DGA), 2-(2-oxo-2-piperazin-l-ylethoxy)-acetic acid (PZ_DGA), 2-[4-(3-aminopropanoyl)-piperazin-l-yl]acetic acid (bAla-PZACM), 2-(2-(4-(3- amino-propanoyl) piperazin- l-yl)-2-oxoethoxy)-acetic acid (bAla-PZ_DGA), 4- (aminomethyl)benzoic acid (PAMBA), gamma-aminobutyric acid (GABA), pentane-di- carboxylic acid (PndA), and 3-(2-(2-(6-hydrazineyl-nicotinamido) ethoxy)-ethoxy)propanoic acid (HYNIC_PEG-2). Optionally, R2comprises a linker selected from the group consisting of: 2-piperazin-l-ylacetic acid, 2-aminoethylcarbamic acid, 2-(2-oxo-2-piperazin-l-ylethoxy)- acetic acid, 7-aminoheptanoic acid, bAla, 2-[4-(3-aminopropanoyl)-piperazin-l-yl]acetic acid, and 2-(2-(4-(3-amino-propanoyl) piperazin- l-yl)-2-oxoethoxy)-acetic acid. Optionally, the chelator is chelated to a radioactive atom. Optionally, the radioactive atom is selected from the group consisting of: copper-64, copper-67, gallium-68, yttrium-90, indium-i l l, terbium-161, lutetium- 177, astatine-211, lead-212, and actinium-225. Optionally, the radioactive atom is gallium-68. Optionally, the radioactive atom is lutetium- 177. Optionally, the radioactive atom is actinium-225. Optionally, the radioactive atom is astatine-211 or lead-212. Optionally, the GRPR-binding peptide ligand has the sequence:DOTA-PAMA_DGA-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; DOTA-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; DOTA-Urea_bridge-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; DOTA-bAla-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; DOTA-bAla-PZ_DGA-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;3296 / 4.2D0TA-DGlu-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; or DOTA-bAla-AHeptA-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2. Optionally, the GRPR- binding peptide ligand ace, at a concentration of 10 nM, shows greater than 25% displacement of a radiolabeled GRPR ligand at a concentration equal to its dissociation constant, from GRPR expressed on a substrate, wherein: the radiolabeled GRPR ligand is

[1251] [ Tyr4]-bombesin, and the substrate is cell membrane homogenate of HEK-293 cells transfected with human BB2; or the radiolabeled GRPR ligand is [177Lu]-NeoB, and the substrate is PC-3 (human prostatic adenocarcinoma cancer) cells. Optionally, the GRPR peptide at a concentration of 10 nM, shows greater than 50% displacement of the radiolabeled GRPR ligand. Optionally, the GRPR peptide at a concentration of 10 nM, shows greater than 70% displacement of the radiolabeled GRPR ligand.

[0091] Further described herein is a pharmaceutical composition comprising at least one GRPR-binding peptide ligand as described above, and at least one pharmaceutically acceptable excipient.

[0092] Further described herein is a GRPR-binding peptide ligand as described above for use in treatment or diagnosis of a disease associated with GRPR by administering an effective amount of the GRPR-binding peptide ligand to the patient in need thereof. Optionally, the disease associated with GRPR is GRPR+cancer. Optionally, the cancer is selected from the group consisting of: prostate cancer, breast cancer, small cell lung cancer, colon carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cell carcinoma, gastroenteropancreatic neuroendocrine tumor, esophageal squamous cell tumor, neuroblastoma, head and neck squamous cell carcinoma, ovarian cancer, endometrial cancer, and pancreatic cancer.

[0093] Further described herein is a method for diagnosis of a GRPR+tumor in a subject comprising administering to a subject an amount of the GRPR-binding peptide ligand, as described above, and imaging the subject. Optionally, the imaging is selected from the group consisting of: positron emission tomography (PET), positron emission tomography-computed tomography (PET-CT), and single photon emission computed tomography (SPECT).

[0094] The following examples are provided to illustrate certain particular features and / or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.3296 / 4.2EXAMPLES

[0095] Example 1A: Preparation of GRPR-binding peptide ligands B l, B2, and B3

[0096] GRPR-binding peptide ligands Bl, B2, and B3 were prepared as follows:

[0097] Step 1: Swelling: 2.65 g of RINK-amide MBHA resin (substitution- 0.68 mmol / g; 100-200 mesh, 1% DVB; “chem-Impex”; Lot # 000954-1212. Total scale; 1.8 mmol, was transferred to a reactor. Next, 30 ml DMF was added, and the reactor was left to shake overnight, at room temperature (rt).

[0098] Step 2: Fmoc Deprotection: First, 28 ml of 20% piperidine in DMF was transferred to the reactor and shaken for 20 minutes twice, at rt. Next, the resin was washed with DMF, 7 times, for 1 minute each, at rt. Subsequently, a Ninhydrin test was conducted resulting in positive results.

[0099] Step 3: Coupling of AA: A preactivation step was performed by mixing the AA (1.8 mmol, 3 eq), 0.827 g HOBt*H2O (1.8 mmol, 3 eq), and 0.845 ml DIC (1.8 mmol, 3 eq) with 25.5 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt. The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A Ninhydrin test was performed, resulting in negative results.

[0100] Steps 2 and 3 were repeated according to the peptide sequence: Val-Gly-His- Leu-Met (AA calculation in Table 5).

[0101] Table 5, (1.8 mmol scale).

[0102] The peptide -resin was split 1: 1 into reactors A and B, 0.9 mmol each. ReactorB was stored at 2-8°C. Reactor A, 0.9 mmol scale.

[0103] Step 2- Fmoc deprotection was performed with 14 ml 20%piperidine / DMF.3296 / 4.2

[0104] Step 3: Coupling of AA: A preactivation step was performed by mixing the AA (0.9 mmol, 3 eq), 413.5 mg HOBt*H2O (0.9 mmol, 3 eq), and 0.423 ml DIC (0.9 mmol, 3 eq) with 12.5 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt.

[0105] The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A Ninhydrin test was performed, resulting in negative results. Steps 2 and 3 were repeated according to the peptide sequence: Trp-Cys (AA calculation in Table 6).

[0106] Table 6, Reactor A, 0.9 mmol scale.

[0107] The peptide-resin was split 1:2 into reactors B3, B2, and Bl, 0.3 mmol and 0.6 mmol, accordingly.

[0108] Reactor B3, 0.3 mmol scale.

[0109] Step 2- Fmoc deprotection was performed with 5 ml 20%piperidine / DMF.

[0110] Step 3: Coupling of AA: A preactivation step was performed by mixing the AA(0.3 mmol, 3 eq), 138 mg HOBt*H2O (0.3 mmol, 3 eq), and 0.141 ml DIC (0.3 mmol, 3 eq) with 4.25 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt. The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A Ninhydrin test was performed, resulting in negative results.

[0111] Steps 2 and 3 were repeated according to the peptide sequence His-Trp (AA calculation in Table 7).3296 / 4.2

[0112] Table 7, reactor B3 (0.3 mmol).

[0113] Scale reactor B2 and Bl - 0.6 mmol.

[0114] Step 2- Fmoc deprotection was performed with 5 ml 20% piperidine / DMF.

[0115] Step 3: Coupling of AA: A preactivation step was performed by mixing the AA (0.3 mmol, 3 eq), 138 mg HOBt*H2O (0.3 mmol, 3 eq), and 0.141 ml DIC (0.3 mmol, 3 eq) with 4.25 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt. The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A Ninhydrin test was performed, resulting in negative results. Steps 2 and 3 were repeated according to the peptide sequence Gln-Cys (AA calculation in Table 8).

[0116] Table 8, reactor B2, B 1 (0.6 mmol).

[0117] The peptide-resin was split 1: 1 into reactors B3, B l, 0.3 mmol each.

[0118] Scale reactor B2 0.3 mmol.

[0119] Step 2- Fmoc deprotection was performed with 5 ml 20% piperidine / DMF.

[0120] Step 3: Coupling of AA: A preactivation step was performed by mixing the AA (0.3 mmol, 3 eq), 138 mg HOBt*H2O (0.3 mmol, 3 eq), and 0.141 ml DIC (0.3 mmol, 3 eq) with 4.25 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt. The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A Ninhydrin3296 / 4.2 test was performed, resulting in negative results. Steps 2 and 3 were repeated according to the peptide sequence D-Phe (AA calculation in Table 9).

[0121] Table 9, reactor B2 (0.3 mmol).reactors B3, B2, and Bl. The next cyclization and cleavage steps were performed on reactors B3,B 2 and Bl

[0123] Cyclization step: Before the cyclization step, the resin was pre-washed three times with a solution of DMF / H2O (4: 1), for 1.5 minutes each time. Then, iodine was weighed, dissolved in the pre-washing solution, and added to the reactor all at once. The reactor was left to shake for 1 h at rt. Following that, a series of resin post-washings was performed in the following order:- DMF / H2O (4: 1) solution: 8 times for 1.5 minutes each.- DMF: 5 times for 1.5 minutes each with 3 ml DCM.- 2% Ascorbic acid in DMF solution: 5 times for 1.5 minutes each.- DMF: 5 times for 1.5 minutes each.The resin was then transferred to a 15 ml Falcon tube, dried in a vacuum for 40 min, and stored at 2-8 °C.

[0124] Cleavage and total deprotection: In a 50 ml Falcon tube, the cocktail solution was prepared. The cocktail and the peptide resin were separately cooled to 0 °C, combined, and left undisturbed for an additional 15 min in the ice bath. Then, the reaction mixture was shaken for 3.15 h at rt.

[0125] Work up: The resin was filtrated into a new 15 ml pre-weighed Falcon. The remaining resin was washed with 0.5- 1.0 ml cocktail solution. Next, the filtrate was evaporated by an N2 stream resulting in an oily residue. The residue was treated with cooled methyl t- butyl ether (MTBE). Upon pouring the ether, a white solid precipitation was obtained. The mixture underwent centrifugation (5 m, 40,000 rpm), and was decantated 3 times.

[0126] Finally, the resulting crude was dried over a vacuum for 3.5 h, to obtain the crude peptides in the following yields: B3: 314 mg; B2: 361 mg; and B l 301 mg.

[0127] Example IB: Preparation of GRPR-binding peptide ligand B43296 / 4.2

[0128] GRPR-binding peptide ligand was prepared generally according to the following process. First, the scale of the synthesis was determined by the calculation of RINK- amide MB HA substitution. Then, the resin was transferred to the reactor, DMF was added, and the reactor was left to shake for 5-12 h at rt. Fmoc deprotection step was carried out twice for 15 minutes each time using 20% piperidine in DMF. Subsequently, the resin was washed with DMF, 7 times, for 1 minute each at rt. Following the deprotection step, the Ninhydrin test was conducted. If the result was positive, the next step was performed. If it was negative, the deprotection step was repeated.The amino acid coupling process was performed according to the peptide sequence Gly-(Asp- His-Trp-Orn)-Val-Gly-His-Leu-Met-NH2.

[0129] The amino acids (3 eq), and the additive, OxymaPure, (3 eq) were weighed and dissolved in DMF (205 mmol / 1000 mL). DIC (3 eq) was added and the resulting mixture and transferred to the reactor. The reactor was left to shake over 1.5 - 2 h at rt followed by washing with DMF, 5 times, 2 min each. After the coupling step, the Ninhydrin test described in Step 2 was performed. Steps 2 and 3 were repeated according to the peptide sequence.

[0130] The allyl and alloc protecting groups of Asp and Orn were removed with Pd°(PPh3)4 catalyst and DMBA (dimethyl benzyl amine) in dichloromethane.

[0131] The cyclization was achieved by reacting the resulting peptide with PyBOP (benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate) and DIEA (diisopropyl ethylamine) in DMF.

[0132] Cleavage of the peptides from the resin was performed as follows. In a 50 ml Falcon tube, a cleavage cocktail solution was prepared. The cocktail and the peptide resin were separately cooled to 0 °C, combined, and left undisturbed for an additional 15 min in the ice bath. Then, the reaction mixture was shaken for 3.15 h at rt. The resin was filtrated into a new 15 ml pre- weighed Falcon. The remaining resin was washed with 0.5- 1.0 ml cocktail solution. Next, the filtrate was evaporated by an N2 stream resulting in an oily residue. The residue was treated with cooled methyl t-butyl ether (MTBE). Upon pouring the ether, a white solid precipitation was obtained. The mixture underwent centrifugation (5 min, 40,000 rpm), and was decantated 3 times. The resulting crude was dried over a vacuum for 3.5 h.

[0133] Purification was performed by HPLC (Preparative System) using the preparative column (Gemini C18 5um 110A 150*4.6mm) mobile phase: A:0.1% TFA in H2O B:0.075% TFA in ACN.3296 / 4.2

[0134] Example 2: In vitro testing of GRPR-binding peptide ligands B l, B2, B3

[0135] Cell-free membrane displacement binding of Bl, B2, and B3 to membranous GRPR (derived from transfected HEK-293 cells with human recombinant GRPR (BB2) was tested using the following method. Cell membrane (4 pg protein) were incubated for 60 min at 22°C with 0.04 nM [125I][ Tyr4]-bombesin in the absence or presence of the test compound in a buffer containing 20 mM HEPES / NaOH (pH 7.4), 3 mM MgCl2, 1 mM EDTA and 0.3% BSA. Nonspecific binding was determined in the presence of 1 pM bombesin.

[0136] Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF / B, Packard) presoaked with 0.3% PEI and rinsed several times with ice- cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried and then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).

[0137] The results were expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is GRP, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

[0138] The results are shown in Table 10.

[0139] Table 10:0140] The data show that all three peptides showed specific binding to GRPR.

[0141] The data described herein indicate that B l, B2, and B3 are likely to be effectiveGRPR-binding peptide ligands having therapeutic and diagnostic potential.

[0142] Example 3: Preparation of additional GRPR-binding peptide ligands

[0143] Further peptides have been synthesized and tested according to the general procedure above, and as elaborated below.

[0144] The following general procedure was used:

[0145] Step 1: Swelling: 2.65 g of RINK-amide MB HA resin (substitution- 0.68 mmol / g; 100-200 mesh, 1% DVB; “chem-Impex”). Total scale; 1.8 mmol, was transferred to a reactor. Next, 30 ml DMF was added, and the reactor was left to shake overnight, at room temperature (rt).3296 / 4.2

[0146] Step 2: Fmoc Deprotection: First, 28 ml of 20% piperidine in DMF was transferred to the reactor and shaken for 20 minutes twice, at rt. Next, the resin was washed with DMF, 7 times, for 1 minute each, at rt. Subsequently, a Ninhydrin test was conducted resulting in positive results.

[0147] Step 3: Coupling of Amino Acid (AA): A pre-activation step was performed by mixing the AA (1.8 mmol, 3 eq), 0.827 g HOBt*H2O (1.8 mmol, 3 eq), and 0.845 ml DIC (1.8 mmol, 3 eq) with 25.5 ml DMF. The resulting reaction mixture was left to shake for 20 min, at rt. The reaction mixture was transferred to the reactor and shaken for an additional l-2h, at rt. The washing step was conducted by washing the resin with DMF, 5 times for 1 min. A ninhydrin test was performed, resulting in negative results.

[0148] Repetition of steps 2-3 was performed according to the amino acid sequences as in Tables 3 and 4.

[0149] Step 4: On-resin cyclization step: Before the cyclization step, the resin was prewashed three times with a solution of DMF / H2O (4: 1), for 1.5 minutes each time. Then, iodine was weighed, dissolved in the pre-washing solution, and added to the reactor all at once. The reactor was left to shake for 1 h at rt. Following that, a series of resin post-washings was performed in the following order:

[0150] - DMF / H2O (4: 1) solution: 8 times for 1.5 minutes each.

[0151] - DMF: 5 times for 1.5 minutes each with 3 ml DCM.

[0152] - 2% Ascorbic acid in DMF solution: 5 times for 1.5 minutes each.

[0153] - DMF: 5 times for 1.5 minutes each.

[0154] The resin was then transferred to a 15 ml Falcon tube, dried in a vacuum for 40 min, and stored at 2-8 °C.

[0155] Step 5: Cleavage and total deprotection: In a 50 ml Falcon tube, the cocktail solution was prepared (94.0% TFA / 3% TIS / 3% H2O). The cocktail and the peptide resin were separately cooled to 0 °C, combined, and left undisturbed for an additional 15 min in the ice bath. Then, the reaction mixture was shaken for 3.15 h at rt.

[0156] Step 6: Work up: The resin was filtrated into a new 15 ml pre-weighed Falcon. The remaining resin was washed with 0.5- 1.0 ml cocktail solution. Next, the filtrate was evaporated by an N2 stream resulting in an oily residue. The residue was treated with cooled methyl t-butyl ether (MTBE). Upon pouring the ether, a white solid precipitation was obtained.3296 / 4.2The mixture underwent centrifugation (5 m, 40,000 rpm) and was decantated 3 times. The resulting crude product was dried over a vacuum for 3.5 h, to obtain the crude peptides.

[0157] Step 7 : Purification: Purification was performed by HPLC (Preparative System) using the preparative column (Gemini C 185um 110A 150*4.6mm) mobile phase: A:0.1% TFA in H2O B:0.075% TFA in ACN.

[0158] For each individual case where additional deprotections were necessary, for example, in peptides comprising Lys[DOTA], these were performed according to the general procedure described above.

[0159] Example 4: Binding Assay

[0160] The displacement binding of the selected GRPR-binding peptide ligands was tested as described in Example 2. The results are shown in Table 11 below. The displacement binding was expressed in qualitative manner, with binding of 90% and above being indicated as A, binding of between 70% and 90% indicated as B, and binding of between 50% and 70% indicated as C.

[0161] Table 11:3296 / 4.2

[0162] In summary, several GRPR-binding peptide ligands were found to have favorable displacement binding of above 50%, including peptides with and without linker and chelator moieties.

[0163] Example 5: Radiolabeling of selected peptides

[0164] [68Ga]GaCh solution was obtained by eluting the 68Ge / 68Ga generator with 4 ml of 0.05 M HC1 (flow rate of ~1 ml per min). An aliquot of about 10 pg of accurately determined amount of the peptide was dissolved in ultrapure water to the concentration of 1 mg / mL, and a 10 pL aliquot was mixed with 0.9 mL of 1 M sodium acetate solution and 1.5 mL of [68Ga]GaCh, bringing to the total volume of 2.41 mL. The pH of the reaction solution was verified to be within the limits of 3.7-3.9. The obtained reaction solution was incubated at 85-90 °C for 10 min in a preheated heating block. Meanwhile, the C-18 cartridge was preconditioned with 5 mL ethanol + 5 mL N2 + 5 mL MQ water + 5 mL N2. At the end of the reaction period, the reaction mixture was cooled down to 30-40 °C and transferred to the preconditioned C-18 cartridge. The cartridge was rinsed with 2 mL MQ water + 2 mL N2, and 68Ga-labeled product was eluted from the cartridge with 0.1 % [v / v] AcOH in ethanol (1 mL) into a product vial. The obtained solution of 68Ga-labeled product was dried under a N2 flow at 70-75 °C for 5-10 min, to complete dryness. Thereafter, the final product was redissolved in 1 mL of saline / ethanol (90 / 10, [v / v]), radiochemical purity and specific activity were determined by Radio High Performance Liquid Chromatography (Radio-HPLC) to ensure the minimal accepted purity of >95%. The final product was diluted to the predefined final amount / concentration for dose administration using saline.3296 / 4.2

[0165] Radiolabeling with 177-lutetium was performed by dissolving about 50 pg of the peptide in water to a final concentration of about 1 mM in a low-binding Eppendorf tube, and radiolabeling buffer containing 0.4 M ammonium acetate and 0.325 M of gentisic acid at pH 4.1 mixed with [177Lu] LuCh 0.05 M solution in HC1, in a volume ratio of 2.8: 1 was added, to the desired specific activity. The reaction mixture was incubated for 30 minutes at 90 °C using a thermomixer system. At the end, the reaction vial was centrifuged and the contents assayed for radiopurity with radioactive HPLC. (C- 18, 2.6 pm, 50x2.1 mm; gradient of 95% to 5%, of 0.1% TFA in water and 0.1% TFA in acetonitrile).

[0166] Example 6 - Cellular binding assays

[0167] Using synthetic procedures described above, peptides were synthesized, radiolabeled, and tested in a cell-binding assay performed using PC-3 (human prostatic adenocarcinoma cancer) cells, with or without the use of 10 pM of GRP as a blocking agent. The cells were expanded at 37°C and 5% CO2 in complete medium. Following expansions, the cells were detached from the culturing flask with Trypsin lx, by incubating for 3-5 min at 37°C and 5% CO2 and recollecting with double volume of fresh complete medium. The cells were then counted with an automated cell counter and centrifuged (5 min, 300 g, 21 °C) to remove the supernatant and resuspended with fresh complete medium to reach the desire concentration. The cells were re-seeded in 6-well plate, in triplicate, 2 mE / well.

[0168] On the day after seeding, the old media was removed by aspiration, and 177- Eu- radiolabeled peptide was added (prepared according to the procedures as described in Example 5), at the desired concentration in complete fresh media (1 mE / well, with 1 nM concentrations of radiolabeled compound). For the total binding experiments, only the radioactive peptide was supplied, for non-specific binding determination, an aliquot of blocking peptide was added to block the specific interactions. The cells were then incubated at 37°C, 5% CO2, for over 1 h and up to 4h. Following the incubation, the supernatant was collected into the gamma counter tube and labeled as “S” and diluted to 2 mL with PBS. The wells were then washed twice with 1 mL of cold PBS lx and collected in gamma counter tubes labeled “W”. The cells were then lysed by adding 1 mL of NaOH IM / well and incubating 10 min at RT. The cell fraction washing was collected into gamma counter tubes labeled “C”, followed by another short wash of the cells with 1 mL of NaOH IM, collecting the supernatant into the same tube.3296 / 4.2

[0169] The fraction bound was calculated by dividing the amount of activity found in C by the total activity found in all three fractions, as in formula Fb = C / (S + W + C), and was expressed in percentage of total activity. The results were reported as Fb in total binding and Fb in non-specific binding. The specific binding was then calculated by subtracting the nonspecific binding from total binding.

[0170] To compare between various runs, the percentage of specific binding for each peptide was calculated by dividing the specific binding of the peptide by the specific binding of NeoB on the same run. The results of selected peptides are presented in the table below. The specific binding was expressed in qualitative manner, with 90% and above being indicated as A, between 70% and 90% indicated as B, and between 50% and 70% indicated as C.

[0171] The binding of the peptides is shown in table 12 below.

[0172] Table 12:

[0173] As evident from the results above, several 177-Lu radioligands exhibit specific binding to GRPR, with affinity in the nanomolar range, in a cell-based assay.

[0174] Example 7 -Cellular displacement test

[0175] Peptides were synthesized using the procedures described above. Peptides were tested for displacement capability of 177-lutetium labeled NeoB. The cellular system used for this example was described in Example 6, but in this displacement mode the GRP blocker was replaced with the non-radioactive tested peptides in a concentration of 10 nM, as elaborated below. The results are summarized in the tables below.3296 / 4.2

[0176] NeoB was synthesized using solid-phase synthesis employing an Fmoc-based approach. BAL resin (1% DVB, 0.3 mmol) was swelled in DMF, drained, and activated by shaking for 10 min in 4 mL of 47.5:47.5:5 methanol / DMF / acetic acid solution. 2,6- Dimethylheptane-4-amine (10 equiv) in 2 mL of 1: 1 methanol / DMF solution was added and the mixture was shaken for 1 h. Sodium cyanoborohydride (10 equiv) was added, and the mixture was shaken for 16 h. The reaction vial was drained and washed with dichloromethane and DMF. Fmoc-His(Trt)-OH (3 equiv) preactivated with HATU (3 equiv), HOAt (3 equiv) and DIEA (8 equiv) in DMF (6 mL) was then added to the reaction vial and shaken for at least 1 h. Fmoc-deprotection was performed using 20% piperidine in DMF. Using a similar procedure, Fmoc-Gly-OH (HATU and HOAt substituted by HBTU and HOBt), Fmoc-Val- OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-DPhe-OH, Fmoc- protected p-aminobenzylamine di-glycolic acid, and DOTA(OtBu)3 was subsequently coupled to the peptide sequence. The peptide was cleaved with a mixture of 82.5 / 5 / 2.5 / 5 / 5 TFA / water / EDT / thioanisole / phenol and purified by HPLC (Agilent 1260 Infinity II Preparative System) using the preparative column (Gemini C18 5um 110A 150*4.6mm) mobile phase: A:0.1% TFA in H2O B:0.075% TFA in ACN.

[0177] The displacement efficiency of NeoB was tested by percentile of retained radioactivity in presence of displacing (tested) non-radioactive peptide in concentration of 10 nM, versus 1 nM of [177Lu]NeoB radioligand. In Table 13 below, the binding efficiency is expressed in qualitative manner, with 90% and above being indicated as A, between 70% and 90% of [177Lu]NeoB displacement being indicated as B, and between 50% and 70% indicated as C.

[0178] Table 13:3296 / 4.23296 / 4.2

[0179] Example 8 - Metabolic stability of selected peptides

[0180] Metabolic stability of selected GRPR binding cyclic peptides was tested in various biological media. Renal homogenate has been chosen as the most aggressive proteolytic medium, for this comparative study. Briefly, tested peptides were subjected to the renal homogenate, and the concentration of the peptide in the medium was followed using HPLC-MS / MS, for time intervals up to 6 hours. Male rat kidneys were homogenized with 100 mM PBS in 1:3 ratio, 1 g of untreated kidney and 3 mL of 100 mM PBS, at 1450 rpm with tissue homogenizer, for at least 2 rounds of 3 minutes each, at 4 °C. Protein concentration was determined for each run (about 38 mg / mL). Pooled homogenate was aliquoted and stored frozen, thawed before experiment, and diluted to about 1 mg / mL protein content with PBS.

[0181] For the test, a working solution of tested peptide was prepared in DMSO (100 pM). A 96-well plate was used. Aliquots of 98 pL of the homogenate were supplied per well, followed by 2 pL of tested peptide solution. For positive control, somatostatin working solution in DDW was used. Each well was used per time point, a separate plate was used, and several peptides were tested together on each plate. For each plate, the specimens included the blank, and the time points of 0, 60 minutes, 120 minutes, 240 minutes, and 360 minutes.

[0182] The plates were kept on water bath at 37°C. At each time point the relevant plate was removed from the bath, and 500 pL of stop solution (methanol with tolbutamide and labetalol as internal standards) were introduced into each well, centrifuged at 3220xg for 20 minutes, and 150 pL of supernatant was collected and transferred for bioanalysis. The samples were stored sealed at 4°C until the analysis with HPLC-MS / MS.

[0183] Percentage remaining of test compound was calculated as a ratio between the readings at time points and time zero, of the analyte peak area normalized by the peak area of3296 / 4.2 the internal standard. The decline was fitted to exponential function to determine the half-life, to serve the basis for the comparative stability. NeoB reference peptide was synthesized as described above. Additionally, two other known reference peptides were prepared: RM2, and AMTG, for comparison with the cyclic GRPR-binding peptide ligands prepared and described herein.

[0184] The results are summarized in table 14 below. The half-life that could not be determined during the test period is shown as above 867 minutes.

[0185] Table 14:3296 / 4.2

[0186] It can be readily seen that many of the GRPR-binding peptide ligands disclosed herein possess significantly better metabolic stability characteristics than three other GRPR- binding peptides previously known in the art.

[0187] Example 9: PET imaging and biodistribution studies

[0188] PET-CT imaging was performed using gallium-68 labelled peptides, according to the table below. Animals were used at different times after tumor inoculation, according to the tumor volume. Athymic nude mice bearing PC3 xenograft tumors were scanned. To acquire the images, anesthesia was induced by inhalation of 3% isoflurane in pure O2 and maintained by 1.5-2% isoflurane in 100% O2. With the animal under anesthesia, the labeled compound was injected via one of the lateral tail veins. Dynamic, whole body 60-min PET imaging sessions were immediately started after administration of the labeled compound using a MOLECUBES P-CUBE scanner. After the PET scan, whole-body high-resolution CT acquisitions were performed on the MOLECUBES X-CUBE scanner to provide anatomical information of each animal as well as the attenuation map for the later reconstruction of the PET images. Static PET images were also acquired at t = 2.5h and t=4h after administration of the labelled compound. In all cases, PET images were reconstructed using a 3D OSEM reconstruction algorithm and applying random, scatter and attenuation corrections. PET-CT images of the same mouse were co-registered and analyzed using a PMOD image processing tool. Volumes of interest (VOIs) were drawn on selected organs as well as on the heart in order3296 / 4.2 to get an estimation of the concentration of radioactivity in blood. Time-activity curves (decay corrected) were obtained as counts per second (cps) / cm3in each organ. Curves were transformed into real radioactivity (Bq / cm3) curves by using a calibration factor, obtained from previous scans performed on a phantom (micro-deluxe, Data spectrum Corp.) under the same experimental conditions (isotope, reconstruction algorithm and energetic window).

[0189] To compare images of different runs (different peptides) on a unified scale, the following procedure was performed. A radioactive tracer was injected, followed by performing a PET scan to detect the tracer. Using a PMOD image processing tool SUVs (Standardized Uptake Values) were calculated to measure the amount of tracer that has accumulated in each tissue, and therefrom MIP (Maximum Intensity Projection) images were generated to provide a 3D overview of the entire volume of the body, highlighting the areas of highest tracer uptake. These MIP images afford unified scale of SUVs for comparison of different runs.

[0190] Selected radiation absorbed in xenografts obtained in the PET scans are presented in Table 15 below.

[0191] Table 15:3296 / 4.2

[0192] It can be readily seen that many GRPR-binding peptide ligands described herein exhibit favorable tumor uptake values and some show high tumor retention over time.

[0193] A representative comparative image of NeoB and D4 is presented in Fig. 13. Representative MIP images obtained from PET-CT of [68Ga]NeoB (0.48 nmol, upper) and [68Ga]D4 (0.67 nmol) (lower)at 2.5 hr after i.v. injection to Foxnlnu mice bearing PC-3 (human prostate) tumor, highly expressing GRPR. First, SUV (Standardized Uptake Value), a metric used to quantify the concentration of the radioactive tracer in the body's tissues, were calculated to measure the amount of tracer that has accumulated in different tissues. It normalized the uptake by the injected dose and the patient's size, allowing for more consistent comparisons between patients and scans. Thereafter, the MIP (Maximum Intensity Projection) was constructed by 3D rendering method that creates a composite image from a series of 2D images by showing the brightest pixels from all the slices along a particular projection path. MIP represent the entire radioactive signal that comes from all organs in a single image. It can be readily seen that despite the larger injected dose, [68Ga]D4 showed favorable biodistribution with significantly less off-target (non-specific) uptake, particularly from hepatobiliary excretion and other GIT uptake, while the specific accumulation in the tumor was similar to NeoB (tumors indicated by “T” and white arrows).

[0194] Example 10: SPECT-CT Imaging

[0195] SPECT-CT imaging was performed using lutetium-177 labelled peptides. Animals were used at different times after tumor inoculation, based on the tumor volume. Athymic nude mice bearing PC3 xenograft tumors were scanned. To acquire the images, anesthesia was induced by inhalation of 3% isoflurane in pure O2 and maintained by 1.5-2%3296 / 4.2 isoflurane in 100% O2. With the animal under anesthesia, the labeled compound was injected via one of the lateral tail veins.

[0196] Static, whole body 30-min SPECT imaging sessions were carried out at t=4h, 24h, and 48h after administration. Some animals also were subjected to an additional session at 72h and 168h after administration. After each SPECT scan, whole-body high-resolution CT acquisitions were performed on the MOLECUBES X-CUBE scanner to provide anatomical information of each animal. In all cases, a vial with a known concentration of radioactivity was introduced in the field of view, in order to obtain absolute quantification values. SPECT images were reconstructed, co-registered with CT images and analyzed using PMOD image processing tool. Volumes of interest (VOIs) were drawn on selected organs (namely: lungs, liver, kidneys, bladder, tumor, stomach, intestine and muscle), as well as the heart in order to get an estimation of the concentration of radioactivity in blood. A VOI was also drawn in the vial to normalize radioactivity values. Time-activity curves (decay corrected) were obtained as cps / cm3 in each organ and transformed into real radioactivity (Bq / cm3) using the values obtained for the VOI drawn at the vial.

[0197] Peptides were selected based on favorable biodistribution profile obtained withSPECT-CT scans. The results are summarized in Table 16 below.

[0198] Table 16:3296 / 4.2

[0199] It can be readily seen that several GRPR-binding peptide ligands exhibit favorable overall biodistribution with generally minimal off-target background, and therefore could be successfully used as therapeutic agents.

[0200] Example 11 - Efficacy study in xenograft model

[0201] Athymic Foxnlnu female nude mice (average weight 24.2 g, ±1.78 SD, 21.1- 28.1) were used for induction of the cell-line-derived-xenograft (CDX) with the human PC3 cells (metastatic castration resistant prostate cancer - mCRPC)tumors. On day 0 the animals were inoculated with 5 million cells subcutaneously in the shoulder. Cells viability was over 93.2%. The xenografts were allowed to grow to an average of 178 mm3(±71 SD, 68-383) in 16-24 days. Thereafter, animals received 40 MBq of 177-Lu- peptides, dosed at 0.4 nanomoles, or vehicle (n=6). Tested peptides included D28 (n=3), D4 (n=3), D5 (n=3), D7 (n=4), D8 (n=3), as well as NeoB (n=4), and RM2 (n=3). The animals received second injection one week after the first one, and a third injection one week after the second one. Tumor volumes were followed until reaching the humane euthanasia endpoint (1500 mm3), or xenograft-related adverse event, like ulcer of the xenograft.

[0202] A survival plot is presented in Figure 14. In the Figure, percentage of the initial subjects remaining in the study in ordinate axis is presented versus the time elapsed from the first injection, presented in abscissa axis. Animals receiving vehicle (denoted “Vehicle” and corresponding to the marker (♦) - filled diamond), were compared to the animals receiving3296 / 4.2NeoB (denoted “NeoB” and corresponding to the marker (■) - filled square), RM2 (denoted “RM2” and corresponding to the marker (A) - filled triangle), peptide D28 (denoted “D28” and corresponding to the marker (x) - slant cross), peptide D4 (denoted “D4” and corresponding to the marker (*) - stylized asterisk), peptide D5 (denoted “D5” and corresponding to the marker (•) - filled circle), peptide D7 (denoted “D7” and corresponding to the marker (|) - vertical dash), and peptide D8 (denoted “D8” and corresponding to the marker (-) - horizontal dash). It is readily seen that peptides D28 and D4 outperformed both reference peptides NeoB and RM2. As seen in the individual graphs (Figures 15-22), wherein tumor volume is plotted versus time elapsed from first injection), one of animals treated with D28 was in stable disease and did not progress until the end of the experiment, and all three animals treated with D4 demonstrated surprisingly low variability and outperformed at least RM2 in terms of delaying disease progression.

[0203] A further experiment has been conducted using single-injection of 225-actinium as radioactive isotope. Mice bearing 256 mm3xenografts (±62.4, 176.4-361.4) were divided into groups to receive either vehicle (n=7), 25 or 100 kBq in 1 nanomole of NeoB (n=4 each), or 25 or 100 kBq in 1 nanomole of D4 (n=3 and n=4, respectively).

[0204] Vehicle group doubled the initial volume in between 10-13 days and reached end point by the days 23-37. One animal had spontaneous resolution of the xenograft.

[0205] NeoB high-dose group doubled initial volume in between 23 and 27 days, and reached end point by days 41-61 (40, 43, 45, 61), whereas high-dose D4 doubled initial volume in between 20 to 27 days, and reached end point by days 44-49 (2+2). Low doses did not differ significantly from vehicle.

[0206] It can be therefore cautiously concluded that at least D4 is as efficacious as NeoB in the conducted studies, and has potential to outperform it in diagnosis and treatment of cancer due to low off-target uptake.3296 / 4.2

[0207] Example 12: Internalization of a selected peptide

[0208] Distribution between membrane-bound and internalized fractions in a cellular binding test was evaluated. Cells culturing and preparation was performed using procedures as in Example 6.

[0209] The 177-Lu-radiolabeled NeoB and D4 peptide ligands were added to the cells at 1 nM concentrations. The cells were then incubated at 37°C, 5% CO2, for 1.5 h, then the plate was placed on ice to stop internalization. The supernatant was collected into the gamma counter tube and labeled as “S” and diluted to 2 mL with PBS. The wells were then washed twice with 1 mL of cold PBS lx and collected in gamma counter tubes labeled “W”. In the next step, the membrane-bound fraction was detached by 3 acidic washes (5 minutes each) using Glycine 0.05 M, pH 2.8. After each cycle the supernatant was collected in gamma counter tubes labeled Ml, M2, M3. To collect the internalized fraction, the cells were then lysed by adding 1 mL of NaOH IM and incubating 10 min at RT. The cell fraction was collected into gamma counter tubes labeled “C”, followed by another short wash of the cells with 1 mL of NaOH IM, collecting the supernatant into the same tube.

[0210] For each peptide the percentage of membrane bound fraction and the internalized fraction out of the total binding was calculated. The membrane-bound fraction was determined by dividing the amount of activity found in Ml -3 fractions by the total activity found in all fractions, as in formula Fm= (Ml + M2 + M3) / (S + W + Ml + M2 + M3 + C), and was expressed in percentage of total activity. The internalized fraction was determined by dividing the amount of activity found in C fraction by the total activity found in all fractions, as in formula Fi = C / (S + W + Ml + M2 + M3 + C), and was expressed in percentage of total activity. The total binding of each peptide is the sum of the membrane-bound and internalized fractions. Tb=Fm + Fi. In the last step the percentage (proportion) of internalized and membrane-bound fractions out of the total binding was calculated, as an indication of agonist / antagonist behavior. % membrane-bound fraction=Fm / Tb X 100. % internalized fraction=Fi / Tb X 100.

[0211] The results are for NeoB: 77% bound to membranes and 23% internalized, and for D4 78% bound to membrane and 22% internalized.

[0212] Example 13: Pharmacological characterization of D43296 / 4.2

[0213] Binding of D4 was tested as in Example 2, in concentrations between 0.137 nM and 100 nM. Each concentration was run in duplicate. The results of inhibition percent of control specific binding are shown in Table 17 below:Table 17:

[0214] The results were fitted to competition curves with Hill coefficient using nonlinear regression, using the equationwhere B is specific binding, Bmax is maximal binding without the inhibitor (fitted as the difference of the upper and lower asymptotes and normalized by adding the lower asymptote value to the B), C is the displacer concentration, nH is the Hill coefficient which indicates on the cooperativity of the binding, and IC50 is the concentration of the displacer causing 50% of inhibition. The upper and lower asymptotes were fitted as 95 and -4, respectively. Ki was then calculated using Cheng-Prusoff equationtaking Kd of bombesin (the radioligand) as 0.04 nM as known in the art.

[0215] The IC50 was found to be 3.0 nM, and Ki 1.5 nM, with Hill coefficient 1.0.

[0216] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of3296 / 4.2 the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

3296 / 4.2CLAIMS1. A gastrin releasing peptide receptor (GRPR) -binding peptide ligand having the formula:R1-R2-R3-(Q)-Z wherein R1is absent or is a chelator moiety, optionally chelated to a radioactive atom moiety;R2is absent or is a linker;R3is selected from the group consisting of an amino acid sequence having between 1- 5 amino acids, or is absent;“Q” is a cyclic peptide sequence having between 4-7 amino acids; and having a structure X1-A1-X2, wherein XI and X2 are each independently amino acids that bond to each other forming a cyclic ring, and Al is a sequence comprising between 2 and 5 amino acids; wherein Al comprises at least one of A 1(a): selected from the group consisting of: glutamine, citrulline, methionine, D-methionine, asparagine, histidine, or glutamic or aspartic acid; and at least one of Al(b): selected from the group consisting of tryptophan, isotryptophan, alpha- methyl tryptophan, 5-hydroxy-tryptophan, kynurenine, histidine, tyrosine, phenylalanine, D-tryptophan, D-histidine, D-tyrosine, and D-phenylalanine;“Z” is a linear peptide sequence having between 4-8 amino acids, and comprising the sequence Val-X2-His-X3-X4; or Phg-X2-His-X3-X4; wherein X2 is Gly, DSer, [NMe]Thr, or bAla;X3 is selected from the group consisting of: Sta, Chg, Leu, Phe, [NMe]Leu, He, [NMe]Ile, Ac6c, HyPro, Om, Ala, and Iva; or is absent, andX4 is selected from the group consisting of: Leu, Met, Gly, GlyS2[SMe], hSer[Me], [NMe]hSer[Me], Nle, Ac6c, Chg, Cys[Me], Ala, and Tza; or is absent, wherein GlyS2[SMe] is N-(2-(methylthio)ethyl)glycine; hSer[Me] is O-methyl-L- homoserine; Nle is norleucine; Ac6c is 1 -aminocyclohexanecarboxylic acid; Tza is 3- thiazolylalanine; HyPro is hydroxyproline; Cys[Me] is S-methyl-L-cysteine, [NMe] is3296 / 4.2N-methyl threonine, Orn is ornithine, [NMe]Ile is N-methyl isoleucine, and Iva is isovaline; and wherein the C-terminal amino acid of Z is optionally either amidated, optionally with a C1-C10 linear or branched alkyl, or is reduced; and wherein, when Q has 6-7 amino acids, Z has the sequence His-Leu-X5 wherein X5 is Met, GlyS2[Me] or is absent.

2. The GRPR-binding peptide ligand according to claim 1, wherein Z comprises a sequence selected from the group consisting of:Val-Gly-His-Sta-Leu;Val-Gly-His-Chg-Met;Val-Gly-His-Chg-Leu;Val-Gly-His-Leu-GlyS2[SMe];Val-Gly-His-Leu-hSer[Me];Val-Gly-His-Leu-[NMe]hSer[Me] ;Val-Gly-His-Phe-Met;Val-Gly-His-Sta-Nle;Val-Gly-His-Leu-Nle;Val-Gly-His-[NMe] Leu-Met;Val-Gly-His-Ile-Met;Val-Gly-His-Chg-Ac6c;Val-Gly-His-Ac6c-Met;Val-Gly-His-Ac6c-Chg;Val-Gly-His-Ac6c-Met;Val-Gly-His-Ac6c-Tza;Val-Gly-His-Hy Pro-Met;3296 / 4.2Val-Gly-His-Iva-Met; andVal-bAla-His-Phe-Nle.

3. The GRPR-binding peptide ligand according to claim 1 or 2 wherein Z is Val-Gly-His- Sta-Leu-NH2.

4. The GRPR-binding peptide ligand according to any one of the previous claims wherein at least one of the Al amino acids is tryptophan.

5. The GRPR-binding peptide ligand according to any one of the previous claims wherein each of XI and X2 are each cysteine.

6. The GRPR-binding peptide ligand according to any one of the previous claims wherein Q is composed of the sequence (Cys-Gln-Trp-Cys).

7. The GRPR-binding peptide ligand according to any one of the previous claims wherein R3is absent or is one amino acid.

8. The GRPR-binding peptide ligand according to claim 7 wherein R3is D-phenylalanine.

9. The GRPR-binding peptide ligand according to claim 7 wherein R3is absent.

10. The GRPR-binding peptide ligand according to any one of the previous claims wherein said R1is a chelator moiety and is selected from the group consisting of: DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), NOTA (2-(4,7-bis(2-(tert- butoxy)-2-oxoethyl)-l,4,7-triazonan-l-yl) acetic acid), NODA (4-(4,7-bis(2-(tert- butoxy)-2-oxoethyl)-l,4,7-triazacyclononan-l-yl)-5-(tert-butoxy)-5-oxopentanoic acid), DOT AM (l,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetamide), DOT AGA (1,4,7, 10-tetraazacyclododecane-l -glutaric acid-4,7, 10-triacetic acid), sarcophagine (3,6,10,13, 16, 19-hexaazabicycloicosane), NODAGA (1,4,7 -triazacyclononane- 1,4,7- triacetic acid- 1 -glutaric acid), EDTA (ethylenediaminetetraacetic acid), and DTPA (Diethylenetriamine pentaacetate) .

11. The GRPR-binding peptide ligand according to claim 10 wherein the chelator is DOTA, DOTAGA, or DOTAM.

12. The GRPR-binding peptide ligand according to any one of the previous claims, wherein R2is a linker.3296 / 4.

213. The GRPR-binding peptide ligand according to claim 12 wherein the linker is selected from the group consisting of: an amino acid sequence having between 1 and 3 amino acids, Lys[DOTA], Bz-Lys[DOTA], Hex-Lys[DOTA], AdamA-Lys[DOTA], 6- aminohexanoic acid (AHexA), 7-aminoheptanoic acid (AHeptA), 2-piperazin-l- ylacetic acid (PZACM), 2-(l,4-diazepan-l-yl)acetic acid (DzpA), 4-(piperazin-l- ylmethyl)-benzoic acid (PZBzA), 2-(4-aminopiperidin-l-yl) acetic acid (PPACM), 2- aminoethylcarbamic acid (Urea_bridge), 2-(2-((4-(aminomethyl)phenyl)amino)-2- oxoethoxy)acetic acid (PAMA_DGA), 2-(2-oxo-2-piperazin-l-ylethoxy)-acetic acid (PZ_DGA), 2-[4-(3-aminopropanoyl)-piperazin-l-yl]acetic acid (bAla-PZACM), 2-(2- (4-(3-amino-propanoyl) piperazin- l-yl)-2-oxoethoxy)-acetic acid (bAla-PZ_DGA), 4- (aminomethyl)benzoic acid (PAMBA), gamma-aminobutyric acid (GABA), pentane- di-carboxylic acid (PndA), and 3-(2-(2-(6-hydrazineyl-nicotinamido) ethoxy)- ethoxy)propanoic acid (HYNIC_PEG-2).

14. The GRPR-binding peptide ligand according to claim 13 wherein R2comprises a linker selected from the group consisting of: 2-piperazin-l-ylacetic acid, 2- aminoethylcarbamic acid, 2-(2-oxo-2-piperazin-l-ylethoxy)-acetic acid, 7- aminoheptanoic acid, bAla, 2-[4-(3-aminopropanoyl)-piperazin-l-yl]acetic acid, and 2- (2-(4-(3-amino-propanoyl) piperazin- l-yl)-2-oxoethoxy)-acetic acid.

15. The GRPR-binding peptide ligand according to any one of the previous claims wherein the chelator is chelated to a radioactive atom.

16. The GRPR-binding peptide ligand according to claim 15 wherein the radioactive atom is selected from the group consisting of: copper-64, copper-67, gallium-68, yttrium-90, indium-i l l, terbium-161, lutetium-177, astatine-211, lead-212, and actinium-225.

17. The GRPR-binding peptide ligand according to claim 16 wherein the radioactive atom is gallium-68.

18. The GRPR-binding peptide ligand according to claim 16 wherein the radioactive atom is lutetium-177.

19. The GRPR-binding peptide ligand according to claim 16 wherein the radioactive atom is actinium-225.3296 / 4.

220. The GRPR-binding peptide ligand according to claim 16 wherein the radioactive atom is astatine-211 or lead-212.

21. The GRPR-binding peptide ligand according to any one of claims 1-7 or 10-20 and having the sequence:DOTA-PAMA_DGA-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;DOTA-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;DOTA-Urea_bridge-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;DOTA-bAla-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;DOTA-bAla-PZ_DGA-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2;DOTA-DGlu-PZACM-DPhe-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2; orDOTA-bAla-AHeptA-(Cys-Gln-Trp-Cys)-Val-Gly-His-Sta-Leu-NH2.

22. The GRPR-binding peptide ligand according to any one of claims 1-20, wherein the GRPR-binding peptide ligand, at a concentration of 10 nM, shows greater than 25% displacement of a radiolabeled GRPR ligand at a concentration equal to its dissociation constant, from GRPR expressed on a substrate, wherein: a. the radiolabeled GRPR ligand is [125I] [ Tyr4] -bombesin, and the substrate is cell membrane homogenate of HEK-293 cells transfected with human BB2; or b. the radiolabeled GRPR ligand is [177Lu] NeoB, and the substrate is PC-3 (human prostatic adenocarcinoma cancer) cells.

23. The GRPR-binding peptide ligand according to claim 22, wherein the GRPR peptide at a concentration of 10 nM, shows greater than 50% displacement of the radiolabeled GRPR ligand.

24. The GRPR-binding peptide ligand according to claim 23, wherein the GRPR peptide at a concentration of 10 nM, shows greater than 70% displacement of the radiolabeled GRPR ligand.3296 / 4.

225. A pharmaceutical composition comprising at least one GRPR-binding peptide ligand according to any one of claims 1-24, and at least one pharmaceutically acceptable excipient.

26. The GRPR-binding peptide ligand according to any one of claims 1-24 for use in treatment or diagnosis of a disease associated with GRPR by administering an effective amount of the GRPR-binding peptide ligand to the patient in need thereof.

27. The GRPR-binding peptide ligand according to claim 26 wherein the disease associated with GRPR is GRPR+cancer.

28. The GRPR-binding peptide ligand according to claim 27 wherein the cancer is selected from the group consisting of: prostate cancer, breast cancer, small cell lung cancer, colon carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cell carcinoma, gastroenteropancreatic neuroendocrine tumor, esophageal squamous cell tumor, neuroblastoma, head and neck squamous cell carcinoma, ovarian cancer, endometrial cancer, and pancreatic cancer.

29. A method for diagnosis of a GRPR+ tumor in a subject comprising administering to a subject an amount of the GRPR-binding peptide ligand, according to any one of claims 1-24 and imaging the subject.

30. The method according to claim 26, wherein the imaging is selected from the group consisting of: positron emission tomography (PET), positron emission tomography- computed tomography (PET-CT), and single photon emission computed tomography (SPECT).

Images