WO2015135082A1 - Bradykinin receptor b1 targeting probes - Google Patents

Abstract

Provided in the present disclosure are peptidic imaging probes (radiotracers) useful for the non- invasive detection of cancers and other conditions or disorders characterized by expression or over- expression of the bradykinin B1 (B1R) receptor. The present peptidic probes may, for example, be ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958. The imaging probes of the disclosure are peptidic compositions, bind with high affinity and selectivity to the B1R and are ¹⁸F- labelled and suitable for imaging procedures such as positron emission tomography (PET). These agents can be used for the early diagnosis, staging and prognosis of B1R-positive cancers or other disease conditions. The B1R imaging probes can also be used to identify B1R-positive cancer patients who may benefit from emerging B1R-targeted therapies.

Description

BRADYKININ RECEPTOR Bl TARGETING PROBES

FIELD

This disclosure relates to novel compositions and methods for non-invasively imaging tissues expressing or overexpressing bradykinin B l receptor. In an embodiment, the disclosure provides radio-isotope labelled peptidic compositions for imaging and diagnosis of cancers expressing the bradykinin B l receptor (B1R).

BACKGROUND

Cancer is the leading cause of death in the developed countries including Canada. To reduce the mortality rate, new tools for early diagnosis and characterization of cancer, and new specific and effective treatment options are urgently needed. A large number of cancers over express a high density of receptors to endogenous regulatory peptides on their cell surface that can be used as diagnostic/prognostic markers and therapeutic targets [Reubi et al, Endocrine Reviews 2003, 24, 389-427; Khan et al, Anti-Cancer Agents in Medicinal Chemistry 2008, 8, 186-199; Heppeler et al, Current Medicinal Chemistry 2000, 7, 971 -994]. The ability to noninvasively quantify the expression of these receptors in tumours will improve cancer diagnosis, staging and prognosis, and facilitate the development of receptor-targeted therapies. The successful application of radiolabeled somatostatin [Krenning et al, pp. 1-50, Freeman LM, ed, Nuclear Medicine Annual, 1995, Raven Press, New York] and vasoactive intestinal peptide [Virgolini et al, New England Journal of Medicine 1994, 331, 1 116-1121 ] analogs for imaging receptor-expressing tumours has promoted the development of other regulatory peptides as potential imaging agents. Bradykinin (BK) Bl receptor (B 1R) has been reported to be over expressed in a variety of cancers but not in normal tissues. Activation of B 1R promotes cancer cell growth and invasion, and antagonism of B 1R results in inhibition of cancer growth in both in vitro and in vivo models. Antagonism of B 1R have been proposed as potential anti- cancer therapy [Fernandes et al, Peptides 201 1, 32: 1849-1854; Gera et al, International Immunopharmacology 2008, 8, 289-292; Taub et al, Cancer Research 2003, 63, 2037-2041 ; Molina et al, Breast Cancer Research and Treatment 2009, 118, 499-510].

Bradykinin Bl and B2 receptors (B1R and B2R) are G protein-coupled receptors (GPCRs), and are known to have a role in pain and inflammation pathways [Campos et al, TRENDS in Pharmacological Sciences 2006, 27, 646-651 ; Calixto et al, British Journal of Pharmacology 2004, 143, 803-818]. The peptides, BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and kallidin (Lys-BK, Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), are produced by enzymatic cleavage of kininogens, and act as the endogenous agonists for the constitutively expressed and widely distributed B2R [Leeb-Lundberg et al, Pharmacological Reviews 2005, 57, 27-77]. The removal of the C-terminal Arg from BK and kallidin by carboxypeptidase N generates [des- Arg⁹]BK and [des-Arg¹⁰]kallidin, respectively, which are the natural agonists for the inducible B1R [Leeb-Lundberg et al, Pharmacological Reviews 2005, 57, 27-77]. The B1R is normally not expressed in most tissues but has been observed undergoing up-regulation in a variety of cancers. By examining human breast tumours, Molina et al. [Breast Cancer Research and Treatment 2009, 118, 499-510] reported that B 1 R was highly expressed in ductal carcinoma in situ (4/4) and invasive ductal carcinoma (11/13). Using immunohistochemical staining on 16 prostate cancer biopsies, Taub et al. [Cancer Research 2003, 63, 2037-2041] reported that B1R was detected in all of prostatic intraepithelial neoplasia and malignant lesions, but not in benign prostate tissues, whereas B2R was ubiquitously expressed. By looking at different subtypes, Chee et al. [Biological Chemistry 2008, 389, 1225-1233] reported that B1R and B2R were expressed in majority of the lung cancers. Expression of both B1R and B2R has also been found in condrosarcomas [Yang et al, Journal of Cellular Biochemistry 2010, 109, 82-92] and astrocytic tumours [Raidoo et al, Immunopharmacology 1999, 43, 255-263]. In additional, expression of B2R was reported in hepatoma, lymphoma, and duodenal carcinoid as well [Wu et al, International Journal of Cancer 2002, 98, 29-35].

The involvement of BK in tumour growth and metastasis is thought to be a multifaceted including possible roles in proliferation, migration and metastasis, survival, and angiogenesis. It has been postulated that B1R/B2R Antagonists could be promising anti-cancer agents and several groups are investigating this hypothesis.

In spite of efforts engaged in the development of B1R antagonists, there is currently no commercially available non-invasive technology to select BIR-positive cancer patients for treatment. New compositions, methods and procedures for the non-invasive in-vivo imaging of BIR-positive cancers such as prostate cancer, breast cancer and lung cancer are desired. This would allow for the earlier diagnosis and prognosis of patients with BIR-positive cancers as well as enable rationale selection of patients who will benefit from treatment with BIR-targeted therapies. There is a need for novel radiotherapies that target cells and tissues inappropriately expressing B1R. SUMMARY

The present disclosure provides at least in part ¹⁸F-labelled peptide based imaging probes which may be useful for PET-based diagnostic imaging of cancer or other disease/disorders whereby the imaging probes of the disclosure bind with high affinity and selectivity to the Bradykinin (BK) Bl receptor (BIR) that is expressed by the targeted cells or tissues.

The present disclosure further provides peptide based compositions suitable for treatment/therapy of cancer or other diseases/disorders whereby said peptide based compositions bind with high affinity and selectivity to Bradykinin (BK) Bl receptors (BIR) expressed by the targeted cells or tissues.

In an aspect, the present disclosure relates to radio-labeled bradykinin Bl receptor (BIR) targeting compounds and their use in medical imaging applications for imaging tissues or tumours expressing BIR, or in therapy for treatment of a disease or condition in which BIR is expressed or over-expressed. Accordingly, in certain embodiments, the disclosure relates to radio-labeled BIR targeting compounds comprising a peptidic compound that selectively binds to BIR and a radiolabel suitable for imaging or radiotherapy, and to precursors of such BIR targeting compounds that may subsequently be radio-labeled.

In an aspect, radio-labeled peptide-based imaging probes are provided which selectively bind to BIR and are suitable for imaging, for example positron emission tomography (PET) or single photon emission computed tomography (SPECT) based imaging, of patients having a disease or disorder in which BIR is expressed such as, for example, cancer, an inflammatory condition, an infection or cardiovascular disease.

In an aspect, the present disclosure relates to radio-labeled peptide and non-peptide based compositions, which selectively bind to BIR and are suitable for treatment of patients having a disease or disorder in which BIR is expressed such as, for example, cancer, an inflammatory condition, an infection or cardiovascular disease.

Certain embodiments of the present disclosure relate to precursors of the above probes and compositions, which can subsequently be radio-labeled and used as probes or therapeutic compositions. The radiolabel may be introduced, for example, via a group comprised by the precursor which can be modified to incorporate a radiolabel by readily available synthetic procedures, such as "click" chemistry, or via a chelating moiety comprised by the precursor which is capable of chelating a suitable radiolabel.

In certain embodiments, the disclosure relates to the use of the peptidic imaging probes for the detection and early diagnosis of breast cancer, prostate cancer, lung cancer or other malignancies. In some embodiments, the disclosure relates to the use of the peptidic imaging probes as adjunct imaging agents for the diagnosis of breast cancer.

In certain embodiments, the disclosure relates to the use of the peptidic imaging probes for monitoring response to therapy for a disease or condition in which BIR is expressed such as, for example, cancer, inflammatory disease, infection or cardiovascular disease.

In certain embodiments, the disclosure relates to the use of the peptidic imaging probes in diagnostic procedures (non-invasive detection of BIR expression by diagnostic imaging) for predicting response of patients to treatment with BIR antagonists and selecting patients for treatment accordingly.

This summary does not necessarily describe all the aspects or embodiments of the present invention. Further aspects or embodiments may become apparent from consideration of the ensuing description of preferred embodiments of the disclosure. A person skilled in the art will realise that other embodiments of the disclosure are possible and that the details of the disclosure can be modified in a number of respects, all without departing from the inventive concept. Thus, the following drawings, descriptions and examples are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

Figure 1 presents a schematic design of radiolabeled peptides targeting BIR.

Figure 2 presents a schematic diagram of lentiviral vector carrying human BIR (BDKRBl) and fusion antibiotic Blasticidin (Bsd) and red fluorescence protein (RFP) dual markers. Fluorescence microscopy of HEK293T: :GFP: :hB 1R cells using red or yellow filter. Figure 3 presents the chemical structure of F-AmBF₃-B9858. The amino acid sequence (Lys- Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic) represents the receptor binding domain. The linker 4- amino-(l -carboxymethyl)piperidine (Pip) separates the receptor binding domain from the radiolabel.

Figure 4 presents an image 1-hr. post- injection PET (left) and biodistribution data (right, n = 4) of ¹⁸F-AmBF₃-B9858 in NSG mice bearing mice bearing B 1R positive HEK293:hBlR tumour (right arrow) and B1R negative HEK293 tumour (left arrow).

Figure 5 presents a pathway for synthesis of (A) AmBF₃-B9858 and (B) the intermediate alkyne-conjugated trifluoroborate (1).

Figure 6 presents a pathway for synthesis of AmBF₃-B9858, AmBF₃-B9958, and their ¹⁸F- labeled derivatives.

Figure 7 presents HPLC chromatograms of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958. Left column: Radio- (upper) and UV (set at 229 nm) chromatograms of 18F-AmBF3-B9858. Right column: radio- (upper) and UV (set at 229 nm) chromatograms of 18F-AmBF3-B9958.

Figure 8 presents representative displacement curves of [³H]-[Leu⁹,des-Arg^,0]kallidin by AmBF₃-B9858 and AmBF₃-B9958.

Figure 9 presents HPLC chromatograms of plasma stability assays of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958. Left column: radio-chromatograms of ¹⁸F-AmBF₃-B9858 after being incubated in mouse plasma at 37 °C for 1 and 2 h. Right column: radio-chromatograms of ¹⁸F- AmBF₃-B9958 after being incubated in mouse plasma at 37 °C for 1 and 2 h.

Figure 10 presents MIP (maximum intensity projection) PET images of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 in mice bearing both B 1R (pointed by right arrows) and B 1 R^" tumors (pointed by left arrows) tumors without (upper row) and with (lower row) co-injection of cold standard (100 μg).

Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense. DETAILED DESCRIPTION

Unless defined otherwise, 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 invention belongs.

5a It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods and uses of the invention. As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

The terms "subject" and "patient" as used herein refer to an animal in need of treatment. The term "animal," as used herein, refers to both human and non-human animals, including, but not limited to, mammals, birds and fish, and encompasses domestic, farm, zoo, laboratory and wild animals, such as, for example, cows, pigs, horses, goats, sheep and other hoofed animals; dogs; cats; chickens; ducks; non-human primates; guinea pigs; rabbits; ferrets; rats; hamsters and mice.

The use of the word "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one." As used herein, the terms "comprising," "having," "including" and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term "consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term "consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.

The present disclosure provides at least in part ¹⁸F-labelled peptide based imaging probes which may be useful for PET-based diagnostic imaging of cancer or other disease/disorders whereby the imaging probes of the disclosure bind with high affinity and selectivity to the Bradykinin (BK) Bl receptor (B1R) that is expressed by the targeted cells or tissues.

6 The present disclosure further provides peptide based compositions suitable for treatment/therapy of cancer or other diseases/disorders whereby said peptide based compositions bind with high affinity and selectivity to Bradykinin (BK) Bl receptors (B1R) expressed by the targeted cells or tissues.

One embodiment disclosure provides a peptidic BIR-specific PET imaging probe ¹⁸F-AmBF₃- B9858 comprised of the following amino-acid sequence: BF₃-DMAm-Pip-Lys-Lys-Arg-Pro- Hyp-Gly-Igl-Ser-DIgl-Oic. Unlabeled probe (designated AmBF₃-B9858) may be labelled at the BF₃ moiety though, for example, an aqueous isotopic exchange reaction as disclosed herein, to provide ¹⁸F-AmBF₃-B9858. This probe may be useful for specifically imaging cancers (including but not limited to cancers of the breast, prostate or lung) or other disease conditions or tissues (including but not limited to inflammation, infection and cardiovascular disease) where the tumour or affected/diseased tissue expresses B1R.

Another embodiment provides a novel peptidic BIR-specific compound AmBF₃-B9858 comprised of the following amino-acid sequence: BF₃-DMAm-Pip-Lys-Lys-Arg-Pro-Hyp-Gly- Igl-Ser-DIgl-Oic. This compound may be useful for treatment/therapy of cancers (including but not limited to cancers of the breast, prostate or lung) or other disease conditions (including but not limited to inflammation, infection and cardiovascular disease) whereby the cancer or disease condition involves the expression or over-expression of B1R.

Another aspect of the present disclosure provides for certain amino acids of the novel peptidic BIR-specific probe BF₃-B9858 (whether used for PET imaging or therapeutic applications) disclosed herein to be replaced with alternative amino acids to alter the biological properties of the probe for desired diagnostic or therapeutic end uses. In a non-limiting example, the amino acid proline (Pro) may be replaced with the unnatural amino acid hydroxyproline (Hyp) and/or the amino acid arginine (Arg) may be replaced with the unnatural amino acid homoarginine (Har). In another non-limiting example L-form/conformation amino-acids in AmBF₃-B9858 may be replaced with D-form/conformation amino-acids.

Another embodiment provides novel peptidic BIR-specific PET imaging probes or compounds for diagnosis or treatment of diseases (including but not limited to cancer, inflammation, infection and cardiovascular disease) whereby the amino-acid sequence of the probes is about

7 70 % or more, about 80% or more, about 90% or more, about 95% or more, about 99% or more, homologous to the amino-acid sequences of AmBF₃-B9858 as disclosed herein.

One embodiment disclosure provides a peptidic BIR-specific PET imaging probe ¹⁸F- AmBF₃- B9958 comprised of the following amino-acid sequence: BF₃-DMAm-Pip-Lys-Lys-Arg-Pro- Hyp-Gly-Cpg-Ser-(D-Tic)-Cpg. Unlabeled probe (designated BF3-B9958) may be labelled at the BF₃ moiety though, for example, an aqueous isotopic exchange reaction as disclosed herein, to provide ¹⁸F-AmBF₃-B9958. This probe may be useful for specifically imaging cancers (including but not limited to cancers of the breast, prostate or lung) or other disease conditions or tissues (including but not limited to inflammation, infection and cardiovascular disease) where the tumour or affected/diseased tissue expresses B1R.

Another embodiment provides a novel peptidic BIR-specific compound AmBF₃-B9958 comprised of the following amino-acid sequence: BF₃-DMAm-Pip-Lys-Lys-Arg-Pro-Hyp-Gly- Cpg-Ser-(D-Tic)-Cpg. This compound may be useful for treatment/therapy of cancers (including but not limited to cancers of the breast, prostate or lung) or other disease conditions (including but not limited to inflammation, infection and cardiovascular disease) whereby the cancer or disease condition involves the expression or over-expression of B1R. Another aspect of the present disclosure provides for certain amino acids of the novel peptidic BIR-specific probe AmBF₃-B9958 (whether used for PET imaging or therapeutic applications) disclosed herein to be replaced with alternative amino acids to alter the biological properties of the probe for desired diagnostic or therapeutic end uses. In a non-limiting example, the amino acid proline (Pro) may be replaced with the unnatural amino acid hydroxyproline (Hyp) and/or the amino acid arginine (Arg) may be replaced with the unnatural amino acid homoarginine (Har). In another non-limiting example L-form/conformation amino-acids in AmBF₃-B9958 may be replaced with D-form/conformation amino-acids.

Another embodiment provides novel peptidic BIR-specific PET imaging probes or compounds for diagnosis or treatment of diseases (including but not limited to cancer, inflammation, infection and cardiovascular disease) whereby the amino-acid sequence of the probes is about 70 % or more, about 80% or more, about 90% or more, about 95% or more, about 99% or more, homologous to the amino-acid sequences of AmBF₃-B9958 as disclosed herein.

8 Another aspect of the disclosure provide for the amino-acid sequences comprising AmBF₃- B9858 or AmBF₃-B9958 to be reversed relative to the amino-acid sequence orientation of AmBF₃-B9858 or AmBF₃-9958 as explicitly disclosed herein. The present ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 may be useful for the detection and early diagnosis of breast, prostate, lung, or other malignancies. The present ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 may be useful as an adjunct imaging agent for the diagnosis of breast cancer. The present ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 may be useful for monitoring response to therapy for diseases/conditions where BIR is expressed such as, for example, cancer, inflammatory disease, infection or cardiovascular disease. The present ¹⁸F-AmBF₃- B9858 or ¹⁸F-AmBF₃-B9958 may be useful in diagnostic procedures (e.g. non-invasive detection of BIR expression by diagnostic imaging) for predicting or analyzing the response of patients to treatment with BIR antagonists. The present ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃- B9958 may be useful as a tool to assist with precise localization of primary or recurrent prostate cancer in order to guide and assist with targeting therapy such as, for example, focal ablative therapies.

The present ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 may be useful for the detection of damage to the endovascular intima that can occur in autoimmune vasculitis or atherosclerosis. In a non-limiting example, ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 can be used detect and image unstable plaques in blood vessels to predict the risk of and localize cardiovascular disease. In another non-limiting example of cardiovascular imaging applications of ¹⁸F-AmBF₃- -B9858 or ¹⁸F-AmBF₃-B9958 may be useful to guide intervention in patients with abdominal aortic aneurysm since intimal damage (as detected by this probe) may be a precursor to aneurysm rupture. In another non-limiting example of cardiovascular imaging applications of ¹⁸F-AmBF₃-B9858 or ¹⁸F-AmBF₃-B9958 may be useful in procedures for detection of and prediction of unstable plaques in coronary artery disease in order to assess the risk or likelihood of myocardial infarction in patients with borderline coronary stenoses. While not wishing to be bound by theory, the present radio-labelled BIR compounds are expected to show high contrast, rapid renal clearance, minimal non-target organ uptake, and high tumour to normal tissue ratios, which properties make these compounds useful as diagnostic imaging agents or for radiotherapy applications.

9 Naturally occurring amino acids are identified throughout by the conventional three- or one- letter abbreviations indicated in Table 1 below, which are as generally accepted in the peptide art and recommended by the IUPAC-IUB commission in biochemical nomenclature. Table 1. Amino acid codes

Name 3-Letter 1-Letter Name 3-Letter 1-Letter

Code Code Code Code

Alanine Ala A Leucine Leu L

Arginine Arg R Lysine Lys K

Asparagine Asp N Methionine Met M

Aspartic Acid Asp D Phenylalanine Phe F

Cysteine Cys C Proline Pro P

Glutamic Acid Glu E Serine Ser S

Glutamine Gin Q Threonine Thr T

Glycine Gly G Tryptophan Trp w

Histidine His H Tyrosine Tyr Y

Isoleucine lie I Valine Val V

The peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. By convention, L-amino acids are represented by upper case letters and D-amino acids by lower case letters or preceded by the designation "D."

Modified amino acid sequences include, for example, sequences that differ from a parental amino acid sequence in that they comprise one or more amino acid substitutions, additions and/or deletions. Substitutions include substitution of a naturally occurring amino acid with a different naturally occurring amino acid, as well as substitution of a naturally occurring amino acid with a non-naturally occurring amino acid. The non-naturally occurring amino acid may provide the same functionality as the amino acid it replaces or it may provide a different or additional functionality. When the sequence contains substitution of a naturally occurring amino acid with a different naturally occurring amino acid, this can be a "conservative" substitution or "non-conservative" substitution. A conservative substitution involves the replacement of one amino acid residue by another residue having similar side chain properties. As is known in the art, the twenty naturally occurring amino acids can be grouped according to the physicochemical properties of their side chains. Suitable groupings include alanine, valine,

10 leucine, isoleucine, proline, methionine, phenylalanine and tryptophan (hydrophobic side chains); glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine (polar, uncharged side chains); aspartic acid and glutamic acid (acidic side chains) and lysine, arginine and histidine (basic side chains). Another grouping of amino acids is phenylalanine, tryptophan, and tyrosine (aromatic side chains). A conservative substitution involves the substitution of an amino acid with another amino acid from the same group. A non-conservative substitution involves the replacement of one amino acid residue by another residue having different side chain properties, for example, replacement of an acidic residue with a neutral or basic residue, replacement of a neutral residue with an acidic or basic residue, replacement of a hydrophobic residue with a hydrophilic residue, and the like. In certain embodiments, when a BIR targeting moiety comprises substitution of a naturally occurring amino acid with a different naturally occurring amino acid, the substitution is a conservative substitution. Additions and deletion that may be comprised by a modified amino acid sequence include addition or deletion of one or more amino acid at the N-terminus, the C-terminus or both termini of the parental peptide, as well as addition or deletion of one or more internal amino acids.

The bradykinin Bl receptor (BIR) targeting compounds described herein bind with high affinity to BIR. The BIR targeting compounds are peptidic compounds having general Formula (II):

B-L-Xaa²-Xaa²-Arg-Pro-Xaa³-Gly-Xaa⁴-Ser-Xaa⁵-Xaa⁶ (II) wherein:

B is a radio-labeled moiety B is ¹⁸F-trifluoroborate;

L is an amino-heterocyclic based linker linker such as amino-(l- carboxymethyl)piperidine;

Xaa² is absent, Lys or D-Arg;

Xaa³ is Pro or Hyp;

Xaa⁴ is Phe, Cha, Thi, (a-Me)Phe, Igl or Cpg;

Xaa⁵ is Pro, D-Tic, D-Hyp, D-PNal or D-Igl, and

Xaa⁶ is Leu, He, D-Phe, Cpg or Oic.

In certain embodiments, the BIR targeting moiety for radio-labelling is derived from a potent peptidic compound with high binding affinity and selectivity for BIR. In certain embodiments, the BIR targeting moiety used to prepare the radio-labeled peptidic compounds is a modified

11 version of one of the natural BIR agonists: [des-Arg9]-BK and [des-ArglO]-kallidin. Non- limiting examples of such compounds are provided in Table 2.

Table 2:

¹ Abbreviations for non-naturally occurring amino acids are as follows:

Cha: β-cyclohexylalanine;

Cpg: a-cyclopentylglycine

Hyp: hydro xyproline

Igl: 2-indanylglycine

(aMe)Phe: a-methylphenylalanine

PNal: β-napthylalanine

Oic: octahydroindole-2-carboxylic acid

Orn: ornithine

12 Sar: sarcosine

Thi: 2-thienylalanine

Tic: l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

Certain embodiments of the disclosure relate to peptidic B IR-specific PET/SPECT imaging or radio-therapeutic probes for diagnosis or treatment of diseases (including but not limited to cancer, inflammation, infection and cardiovascular disease) having an amino-acid sequence that is at least about 70 %, about 80%, about 90%, about 95% or about 99% identical to the amino- acid sequences of the compounds explicitly disclosed herein (Table 2). Certain embodiments of the disclosure relate to peptidic B 1R targeting compounds in which the amino-acid sequences are reversed relative to the amino-acid sequence orientation of the compounds explicitly disclosed herein (Table 2). The present peptidic compounds are believed to be stable in vivo and have high binding affinity for B 1R giving them the potential to be potent and long-acting antagonists of B1R. The present probes may be radiolabeled. Examples of radiolabels include, but are not limited to ¹⁸F, ⁶⁸Ga, and ⁿC. Preferred for use here is ¹⁸F which is readily available from most medical cyclotron facilities and has appropriate nuclear properties for PET/SPECT imaging, including high positron emission per integration (97%), low positron energy (0.635 MeV), and a suitable physical half-life of 109.7 min. The radiolabel may be added to the probe via any suitable method. For example, via a one-step ¹⁸F-labeling approach via an ¹⁸F-¹⁹F isotope exchange reaction on an ammoniomethyl-trifluoroborate (AmBF₃) moiety. While not wishing to be bound by theory, it is believed that ¹⁸F trifluoroborate as the radiolabel provides benefits in terms of imaging performance such as, for example, aiding in the binding affinity or reducing the signal to noise ratio.

The present disclosure provides two ¹⁸F-labeled B IR-targeting tracers, ¹⁸FAmBF₃-B9858 and ¹⁸F-AmBF₃-B9958.

The peptidic B 1R targeting compounds may be radio-labelled at a position which is not required for receptor binding and which is separated from the B 1R targeting moiety via a linker of appropriate length to reduce the chance of interference of the radiolabeled moiety with receptor binding. The present linker may be a amino-heterocycle wherein the hetercycle is, for example, a five or six-membered ring. The present linker may be a piperidine-based linker such as amino-(l-carboxymethyl)piperidine (Pip). While not wishing to be bound by theory it is believed that the tertiary nitrogen affords a positive charge which could improve affinity. Other

13 linkers may be utilized herein such as, for example, 6-aminohexanoic acid (Axh), 8- aminooctanoic acid (Aoc), Glycine-glycine (gly-gly), and 8-amino-3,6-dioxa-octanoic acid (PEG2). While not wishing to be bound by theory, the combination of a ¹⁸F trifluoroborate radiolabel, a piperidine-based linker and a BIR targeting peptide is believed to offer superior performance for imaging applications.

Non-limiting examples of radio-labeled peptidic BIR targeting compounds and precursors include compounds of general Formula (I):

B-L-X (I)

wherein:

B is ¹⁸F-trifluoroborate trifluoroborate moiety such as dimethylammoniomethy- trifluoroborate (AmBF₃);

L is a linker such as the piperidine-based 4-amino-(l-carboxymethyl)piperidine (Pip); X is a peptide selected from those of general Formula (II) such as Lys-Lys-Arg-Pro- Hyp-Gly-Igl-Ser-(D-Igl)-Ile or Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-(D-Tic)-Cpg.

In certain embodiments, the disclosure relates to conjugates of the above-described peptidic compounds, in which the peptidic compound is conjugated to one or more additional chemical or biochemical moieties that provide additional functionality to the peptide, for example, increased stability, improved bioavailability or improved pharmacokinetics and/or that assist in delivery of the peptide to the appropriate tissue(s) or organ(s). Conjugates include peptidic compounds fused to one or more biological moieties as well as peptidic compounds in which the amino -terminus and/or carboxy-terminus and/or one or more amino acid side chain has been derivatized with a suitable chemical substituent group for conjugation to one or more chemical or biological moieties. Examples of such chemical or biological moieties include, but are not limited to, various carriers, lipophilic moieties, antibodies and other biological ligands, liposomes, polymeric matrices, non-polymeric matrices, particles such as gold particles, microdevices and nanodevices, and nano-scale semiconductor materials.

The radio-labelled peptidic BIR targeting compounds according to the disclosure may be prepared by standard peptide and synthetic chemistry procedures from commercially available starting materials. Exemplary, non-limiting procedures are provided in the Examples.

14 The radio-labelled BIR targeting compounds are typically formulated for administration to a patient. Certain embodiments of the disclosure thus relate to pharmaceutical compositions comprising one or more of the radio-labelled BIR targeting compounds and a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients.

The pharmaceutical compositions comprising the radio-labelled BIR targeting compounds are typically formulated for parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intra-articular, intravenous, intraperitoneal, intramuscular, intravascular, intrasternal, intrathecal injection or infusion techniques.

In certain embodiments, the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension. Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000). In certain embodiments, the disclosure relates to the use of the radio-labelled BIR targeting compounds in medical imaging applications or in radiotherapy in patients having a disease or disorder associated with expression or over-expression of BIR.

15 Certain embodiments relate to diagnostic applications of the radio-labelled B IR targeting compounds for imaging a cancer or tissue in which B IR is expressed or over-expressed, for example, in oncology, inflammation or cardiovascular disease. Overexpression of BIR has been demonstrated in many malignancies, including early breast and prostate cancers (prostatic intraepithelial neoplasia and malignancy), lung cancers and brain cancers. Certain embodiments of the disclosure thus contemplate that the radio-labelled B IR targeting compounds could be used as imaging probes for cancers of the breast, prostate, lung and brain.

In the breast, ductal carcinomas in situ also overexpress the B IR receptor. Overexpression of B IR has been observed in 76% of primary breast cancers. In the prostate, benign prostate lesions do not overexpress B IR. Accordingly, certain embodiments of the disclosure contemplate that the radio-labelled BIR compounds will find use as probes for the diagnosis of early stage breast cancer, prostate cancer and other malignancies. Based on the data provided in the Examples, radio-labelled B IR compounds are expected to show high contrast, rapid renal clearance, minimal non-target organ uptake, and high tumour to normal tissue ratios, which properties make these compounds well-suited for use as imaging agents for cancer diagnosis, including diagnosis of early stage cancer.

Certain embodiments of the disclosure contemplate the use of the radio-labelled B IR compounds as adjunct imaging agents for the diagnosis of breast cancer. In some embodiments, the radio-labelled B IR compounds can be labelled with positron emitters and could be used with positron emission mammography (or breast gamma imaging) to detect abnormal breast lesions at an early stage, and/or be used to characterize equivocal lesions on mammography or breast MRI, which would be followed up with repeat examinations rather than biopsy.

Certain embodiments of the disclosure contemplate that the radio-labelled B IR compounds may be used as probes to localize primary or recurrent prostate cancers in patients with elevated tumour markers (such as elevated PSA). Such imaging agents could find use, for example, to confirm the diagnostic of malignancy, guide focal ablative treatment if the disease is localized, or guide salvage treatment in the case of prostate cancer recurrence. The disclosure also contemplates the use of unlabeled compounds AmBF₃-B9858 or AmBF₃-B9958 as a therapeutic treatment for cancers or other diseases/disorders characterized by expression of B IR.

16 In some embodiments of the disclosure, it is contemplated that the radio-labelled BIR compounds may be used as PET/SPECT imaging probes to assist with precise localization of primary or recurrent prostate cancer in order to guide and assist with focal ablative therapies.

In some embodiments, the disclosure contemplates that the radio-labelled BIR compounds could be used to monitor response to therapy, by providing an independent assessment of the residual cellular content of a tumour known to overexpress BIR. Overexpression of BIR may be an indicator of angiogenesis in tumours, as BIR is known to have antiangiogenic activity. In certain embodiments, therefore, the radio-labelled BIR compounds could find use to predict or monitor response to anti-angiogenic medications, such as Avastin.

There is some evidence that BIR antagonists might cause growth inhibition in some cancers. In certain embodiments, BIR expression and receptor blockage could be detected by imaging with the radio-labelled BIR compounds, which could then act as a predictive biomarker for treatment success.

In some embodiments, the use of the radio-labelled BIR compounds in multimodality imaging of cancers is contemplated, for example, combined functional imaging and anatomical imaging, such as PET/CT or SPECT/CT. Multimodality imaging may be useful in situations in which a cancer is present, but the uptake of imaging agent is low.

Inflammation and infection can cause local tissue damage, which leads to the overexpression of BIR, which involved in the inflammatory and nociceptive response. Certain embodiments of the disclosure contemplate that the radio-labelled BIR compounds could be used to provide images outlining sites of active inflammation or infection, and a quantitative assessment disease involvement, in inflammatory disorders of the joints. In some embodiments, the radio-labelled BIR compounds could be used to monitor inflammatory disease activity and response to therapy.

BIR has been reported to be overexpressed when the endovascular intima is damaged. Certain embodiments contemplate the use of the radio-labelled BIR compounds to detect endovascular damage, such as can occur with autoimmune vasculitis or atherosclerosis. Some embodiments of the disclosure contemplate that the radio-labelled BIR compounds could be used, for example, to guide intervention in patients with abdominal aortic aneurysm - the evidence of

17 intimal damage could be a precursor for aneurysm rupture, as a predictor of unstable plaques in coronary artery disease, in order to predict the likelihood of myocardial infarction in patients with borderline coronary stenoses and/or as a guide to whether carotid endarterectomy is needed in patients with stenotic carotid arteries.

Certain embodiments relate to therapeutic applications of the radio-labelled BIR targeting compounds in cancer. Cancers that are BIR positive could be amenable to treatment by radionuclide therapy. For example, the present BIR targeting compounds may be used to document target expression to predict response to other BIR compounds labelled with therapeutic radioisotopes and calculate radiation dosimetry

Certain embodiments of the disclosure relate to pharmaceutical packs or kits containing one or more BIR targeting compounds, for example, therapeutic or diagnostic packs or kits. The compounds may be provided radio-labelled or as precursors suitable for radio-labelling, in which case the kit may optionally include additional reagents for radio-labelling the compounds.

In certain embodiments, one or more of the components of the kit can be lyophilized and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized components. Individual components of the kit would typically be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.

In certain embodiments, the compound(s) are provided in the kit in the form of pharmaceutical compositions suitable for administration to a subject. In this case, if desired, the container may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to the subject.

To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.

18 EXAMPLES

All chemicals and solvents were obtained from commercial sources and used without further purification.

EXAMPLE 1

N-Propargyl-NN-dimethylammoniomethyl-trifluoroborate (1) was synthesized as previously reported (Liu, Z. et al. Dual mode fluorescent ¹⁸F-PET tracers: efficient modular synthesis of rhodamine-[cRGD]₂-[¹⁸F]-organotrifluoroborate, rapid and high yielding ¹⁸F-labeling at high specific activity with correlated in vivo PET Imaging and ex vivo fluorescence. Bioconjugate Chem. 2014, 25, 1951-1962).

CHO-K1 cell membranes overexpressing recombinant hBlR were obtained from PerkinElmer (Waltham, MA). ¹⁸F-fluoride Trap & Release Columns were purchased from ORTG Inc. (Oakdale, TN). CI 8 light Sep-Pak cartridges (1 cm³, 50 mg) were obtained from Waters (Milford, MA). Balb/c mouse plasma for stability studies was obtained from Innovative Research (Novi, MI). BIR-targeting peptides were synthesized using a solid-phase approach on an Aapptec (Louisville, KY) Endeavor 90 peptide synthesizer. Mass analyses were performed using a Bruker (Billerica, MA) Esquire-LC/MS system with ESI ion source. Purification and quality control of cold and ¹⁸F-labeled peptides were performed on an Agilent HPLC system equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector, and a Bioscan (Washington, DC) Nal scintillation detector. The radiodetector was connected to a Bioscan B-FC-1000 flow-count system, and the output from the Bioscan flowcount system was fed into an Agilent 35900E interface, which converted the analog signal to a digital signal. The operation of the Agilent HPLC system was controlled using Agilent ChemStation software. Radioactivity of ¹⁸F-labeled peptides was measured using a Capintec (Ramsey, NJ) CRC^®- 25R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies was counted using a Packard (Meriden, CT) Cobra II 5000 series autogamma counter. HPLC Analysis. The HPLC conditions used for purification and quality control are summarized in Table 3.

Synthesis of Azidoacetyl-Pip-B9858 (2). Azidoacetyl-Pip-B9858 was synthesized via a N^a- Fmoc solid-phase peptide synthesis strategy starting from 2-chlorotrityl chloride resin. The resin was treated with 20% piperidine in DMF to remove the N"-Fmoc protecting group. The

19 following Fmoc-protected amino acids (3 equiv) including Fmoc-Oic-OH, Fmoc-D-Igl-OH, Fmoc-Ser(iBu)-OH, Fmoc-Igl-OH, Fmoc-Gly-OH, Fmoc-Hyp(iBu)-OH, Fmoc-Pro-OH, Fmoc- Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and the linker Fmoc-4-amino-(l -carboxymethyl)piperidine (Fmoc-Pip-OH) were subsequently coupled to the sequence according to their order. The coupling was carried out in NMP with standard in situ activating reagent HBTU (3 equiv) in the presence of DIEA (6 equiv). Bromoacetic acid (40 equiv) was preactivated with DIC (20 equiv) in DCM for 10 min, filtered, and then coupled to the peptide sequence. Finally, the resin was treated with sodium azide (27.5 equiv) in DMSO to provide the azide functional group at the N-terminus for the click reaction. The peptide was deprotected and simultaneously cleaved from the resin by treatment with a cocktail of trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5). After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The crude product was filtered, dried, and purified by HPLC using condition

1 (Table 3). Azidoacetyl-Pip-B9858 2 was obtained in 13% yield. ESI-MS: calcd [M + H]⁺ for azidoacetyl-Pip-B9858 (2)

1505.8; found, 1505.8.

Synthesis of Azidoacetyl-Pip-B9958 (3). Pip-B9958 was synthesized following similar procedures as those for the preparation of Pip-B9858 but replacing both Igl6 and Oic9 with Cpg and replacing D-Igl8 with D-Tic. Azidoacetic acid (40 equiv) was preactivated with DIC (20 equiv) in DCM for 10 min, filtered, and then coupled to the peptide sequence to provide the azide functional group at the N-terminus for the click reaction. The peptide was deprotected and cleaved from the resin using a cocktail of trifluoroacetic acid/water/triisopropylsilane/ethanedithiol/thioanisol/phenol (81.5:5: 1 :2.5:5:5). After filtration to remove resin, the crude product was precipitated by the addition of cold diethyl ether. The crude product was filtered, dried, and purified by HPLC using condition 2 (Table 3). Azidoacetyl-Pip-B9958 3 was obtained in 20% yield. ESI-MS: calcd [M + H]⁺ for azidoacetyl-Pip-B9958 (3) C₆₆H₁₀5N₂oOi5, 1417.8; found, 1418.2.

Synthesis of AmBF₃-B9858. An Eppendorf tube (1.5 mL) was loaded with a mixture of 1 (3.5 mg, 22.6 μπιοΐ), CuS0₄ (1.0 M, 5.0 μΓ), sodium ascorbate (1.0 M, 12.5 μΓ), 5% NH₄OH (MeCN/H20 = 1 : 1, 50 μΐ,), and 2 (5.3 mg, 3.5 μπιοΐ). The reaction was allowed to proceed for

2 h at 45 °C. Purification was performed by HPLC using condition 3 (Table 3) to isolate 3.6 mg of AmBF₃-B9858 (62%). ESI-MS: calcd [M + H]⁺ for AmBF3-B9858 C₇9H₁₂oBF₃N₂₁Oi₅, 1670.9; found, 1671.1.

20 Synthesis of AmBF₃-B9958. An Eppendorf tube (1.5 mL) was loaded with a mixture of 1 (3.5 mg, 22.6 umol), CuS0₄ (1.0 M, 5.0 uL), sodium ascorbate (1.0 M, 12.5 μΓ), 5% NH₄OH (MeCN/H₂0 = 1 : 1, 50 uL), and 3 (2.8 mg, 2.0 umol). The reaction was allowed to proceed for 2 h at 45 °C and purified by HPLC using condition 4 (Table 3) to obtain 1.5 mg of AmBF₃-B9958 (47%). ESI-MS: calcd [M + H]⁺ for AmBF₃-B9958 C₇2H116BF3N21O1₅, 1582.9; found, 1583.1.

In Vitro Competition Binding Assays. The binding affinity of AmBF₃-B9858 and AmBF₃-B9958 to B 1R was measured using competition binding assays on B IR-expressing CHO-K1 cell membranes as reported previously (Lin, K. et. al. In vivo imaging of bradykinin receptor Bl , a widely overexpressed molecule in human cancer. Cancer Res. 2014, 75, 387-393).

Radiolabeling. ¹⁹F-AmBF₃-B9858 (or ¹⁹F-AmBF₃-B9958, 100 nmol) was dissolved in a mixture of aqueous pyridazine-HCl buffer (20 pL, 1M, pH 2) and DMF (20 μί) in a 4.5 mL Falcon tube. No carrier-added ¹⁸F-fluoride was obtained by bombarding H₂ ¹⁸0 with 18 MeV protons, followed by trapping on an anion exchange (9 mg, QMA, chloride form) column. ¹⁸F- fluoride was eluted off the column with 100 μΐ_^ of saline into the Falcon tube containing ¹⁹F- AmBF₃-B9858 (or ¹⁹F-AmBF₃-B9958). The tube was placed in a heating block and heated at 80 °C for 15 min. The reaction mixture was subsequently quenched with 5% aqueous NH₄OH (2 mL) and loaded onto a CI 8 light Sep-Pak cartridge. Free ¹⁸F-fluoride was removed by washing the Sep-Pak cartridge with DI water (2 mL ^χ 2). ¹⁸F-AmBF₃-B9858 (or ¹⁸F- AmBF₃-B9958) was then eluted off the cartridge with 4: 1 ethanol/saline (0.5 mL) and diluted with saline for in vitro plasma stability and in vivo biodistribution and PET/CT imaging studies. A small sample was removed for quality control analysis by HPLC using condition 5 (Table 3).

The chemical identity of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 was confirmed by coinjecting the radiolabeled product with their respective nonradiolabeled standard.

In Vitro Plasma Stability Assay. Twenty microliters of ¹⁸F-labeled peptide was added to 500 μΐ_^ of mouse plasma and incubated at 37 °C for 1 and 2 h. At the end of incubation period, the reaction mixture was quenched by MeCN (1 mL). The quenched reaction mixture was centrifuged, and the supernatant was aspirated, filtered, and analyzed by HPLC using condition 5 (Table 3).

21 Cell Culture: Human embryonic kidney cell line HEK293T was purchased as part of Lenti-X Expression System from Clontech Laboratories Inc. (Mountain View, CA). Cells were cultured in high glucose DMEM or RPMI 1640 (StemCell Technologies, Vancouver, BC) supplemented by 10% FBS, 2mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator containing 5% CO₂. Cells at 80-90% confluences were washed with sterile phosphate-buffered saline (lx PBS pH7.4) and harvested with 0.25%) trypsin. Number of cells collected was counted using a Scepter handheld automated cell counter (Millipore, Billerica, MA). Gene Transfer and Expression Using Recombinant Lentiviruses: We used the Lenti-X Lentiviral Expression Systems provided by Clontech Laboratories Inc to induce expression of Green Fluorescent Protein (GFP) and human bradykinin Bl receptor (BKRB1) on human embryonic kidney cells (HEK293). The lentiviral expression vector carrying GFP, pGIPz(GFP) (Open Biosystems, Rockford, IL) was used to induce GFP expression. Pre-made inducible lentiviral particles at lxlO⁷ IFU/mL for expressing human BDKRB1 (bradykinin Bl receptor/BlR) were obtained from GenTarget Inc., San Diego, CA (cat. no. LVP291) to induce BDKB1R expression. An antibiotic blasticidin (Bsd) - RFP (red fluorescence protein) fusion marker under RSV promoter is also present in the expression vector to allow the selection of successfully transduced cells. Figure 2 shows schematic representation for lentiviral vector carrying human B1R (BDKRB1) provided by GenTarget Inc. Fluorescence microscopy of HEK293T::hBlR cells showed a membranous and cytoplasmic fluorescence of RFP, suggesting successful B1R over-expression.

Biodistribution and PET/CT Imaging Studies. These studies were performed as previously reported (Lin, K. et. al.). Male immunodeficient NODSCID IL2RKO mice were obtained from a breeding colony at the Animal Resource Centre of the BC Cancer Research Centre. All experiments were conducted in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia. Wild-type HEK293T and HEK293T::hBlR tumours were inoculated by subcutaneous injection of 1 ^x 10⁶ cells on each dorsal flank of the mice. Each mouse had both a B1R⁺ and B1R tumour. After 2 weeks of growth, palpable tumours measuring approximately 7 mm in diameter were obtained. Mice were injected with either 0.37 or 3.7 MBq of ¹⁸F-labeled peptides, depending on whether the mice were used only for biodistribution or used for imaging followed by biodistribution. For blocking experiments, the radioactive compound was coinjected with 100 μg of the same nonradioactive peptide. After 60 min of uptake, the mice

22 were anesthetized by isoflurane inhalation, followed by C(¾ asphyxiation. Blood was promptly withdrawn, and the organs of interest were harvested, rinsed with normal saline, blotted dry, and weighed. The radioactivity of the collected mouse tissues was counted and expressed as the percentage of the injected dose per gram of tissue (% ID/g).

PET imaging experiments were conducted using a Siemens (Erlangen, Germany) Inveon microPET/CT scanner. Mice bearing two tumors derived from HEK293T and HEK293T::hBlR cells, as described above, were also used for those experiments. The mice were sedated with 2% isoflurane inhalation for i.v. injection of the radiotracer with or without the presence of a 100 μg excess unlabeled peptide and then were allowed to recover and roam freely in their cages for 55 min. At that point, the mice were sedated with 2% isoflurane inhalation and placed in the scanner. A baseline CT scan was obtained for localization and attenuation correction using 60 kV X-rays at 500 μΑ, using three sequential bed positions with 33% overlap and 220° continuous rotation. Body temperature was maintained by a heating pad during acquisition. A single static emission scan was acquired for 10 min. PET data were acquired in list mode acquisition, reconstructed using the 3d-OSEM-MAP algorithm with CT-based attenuation correction, and coregistered for alignment. The mice were euthanized afterward, and their organs were harvested for biodistribution analysis. Table 3 : HPLC Conditions for the Purification and Quality Control of Peptides HPLC

Azidoacetyl-Pip-B9858 2 and azidoacetyl-Pip-B9958 3 were prepared in 13 and 20% yields, respectively. After conjugation with alkyne 1 (Fig. 6) and HPLC purification, AmBF₃-B9858 and AmBF₃-B9958 were obtained in 62 and 47% yields, respectively. The identities of these four peptides were confirmed by mass spectrometry analysis. ¹⁸F labelling was performed via a

23 ¹⁸F-¹⁹F isotope exchange reaction, and the final purified product was obtained within a synthesis time of 30 min. Starting with 21.9-28.4 GBq of ¹⁸F-fluoride, ¹⁸F-AmBF₃-B9858 was obtained in 27.9 ± 5.4% (n = 3) nondecay-corrected radiochemical yield. Starting with 21.5-33.3 GBq of ¹⁸F-fluoride, ¹⁸F-AmBF₃-B9958 was obtained in 24.3 ± 1.8% (n = 3) non- decay-corrected radiochemical yield. Both ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 were obtained with >99% radiochemical purity (Fig. 7). The specific activity of ¹⁸F- AmBF₃-B9858/¹⁸F-AmBF₃-B9858 was in the range of 43-87 GBq/μπιοΙ (n =6) at the end of synthesis. BIR Binding Affinity. AmBF₃-B9858 and AmBF₃-B9958 effectively inhibited the binding of [³H]-[Leu⁹,des-Arg¹⁰]-kallidin to hBlR expressed on CHO-K1 cells in a dose-dependent manner. The representative displacement curves are shown in Fig. 8. The Ki values of AmBF₃-B9858 and AmBF₃-B9958 to BIR were 0.09 ± 0.08 and 0.46 ± 0.03 nM, respectively. Stability in Mouse Plasma. The stability of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 was assessed in mouse plasma and analyzed by HPLC (Fig. 9). No significant metabolites were observed in the HPLC chromatograms, and >95% of ¹⁸F-AmBF₃-B9858 and ¹⁸F- AmBF₃-B9958 remained intact after a 2 h incubation at 37 °C. Biodistribution in Tumor-Bearing Mice. To assess the pharmacokinetics and in vivo B1R- targeting capability of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958, biodistribution studies were performed in mice bearing both B1R⁺ and BIR- tumors (Table 5).

Both ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 exhibited fast renal excretion and low background activity. Only kidneys and B1R⁺ tumours showed higher uptake (>1% ID/g) in the control mice at 1 h p.i., and the uptake values were 36.2 ± 5.78 and 3.94 ± 1.24% ID/g for ¹⁸FAmBF₃-B9858 and 30.9 ± 6.74 and 4.20 ± 0.98% ID/g for ¹⁸F-AmBF₃-B9958, respectively. The B1R⁺ tumor-to-BlR^~ tumour, B1R⁺ tumour-to-blood, and B1R⁺ tumour-to-muscle ratios were 12.2 ± 3.02, 6.69 ± 3.60, and 21.3 ± 4.33 for ¹⁸F-AmBF₃-B9858 and 23.4 ± 7.77, 14.7 ± 3.56, and 48.6 ± 10.7 for ¹⁸F-AmBF₃-B9958, respectively. Coinjection with 100 μg of cold standard significantly reduced the uptake of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 in B1R⁺ tumours to 0.79 ± 0.15 and 0.47 ± 0.13% ID/g, respectively. Compared with the control mice, the uptake of ¹⁸F-AmBF₃-B9958 in the blocked mice remained relatively unchanged for all tissues/organs other than the B1R⁺ tumor. On the contrary, significantly higher uptake of

24 ¹⁸F-AmBF₃-B9858 in the blocked cohort was observed in blood, fat, large intestine, liver, lung, B1R tumor, muscle, bone, and brain.

Table 5: Biodistribution (%ID/g) and uptake ratios of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃- B9958 at 1 h p.i. in tumor-bearing mice with/without co-injection of cold standard (100 μg). Data are presented as mean ± SD (n = 3 - 5).

F-AmBF₃-B9858 '°F-AmBF₃-B9958

Control Blocked Control Blocked

Blood 0.69 ± 0.34 1.59 ±0.21" 0.29 ± 0.06 0.37 ± 0.07

Fat 0.13 ±0.04 0.22 ± 0.07* 0.05 ± 0.02 0.06 ±0.01

Testes 0.24 ± 0.08 0.35 ± 0.04 0.11 ±0.01 0.11 ± 0.02

Large intestine 0.31 ±0.07 0.65 ± 0.27* 0.26 ±0.18 0.15 ±0.07

Small intestine 0.93 ± 0.62 0.43 ± 0.20 0.35 ±0.18 0.19 ±0.09

Spleen 0.43 ± 0.25 0.63 ±0.21 0.32 ±0.14 0.20 ± 0.09

Liver 0.58 ± 0.09 1.01 ±0.36* 0.25 ± 0.04 0.22 ± 0.05

Pancreas 0.18 ±0.07 0.28 ±0.16 0.07 ±0.01 0.09 ±0.01

Adrenal glands 0.34 ±0.19 0.55 ± 0.32 0.15±0.15 0.17±0.13

Kidney 36.2 ± 5.78 29.8 ± 6.88 30.9 ± 6.74 26.5 ±4.61

Lungs 0.70 ± 0.23 1.10 ±0.04* 0.31 ± 0.05 0.33 ± 0.06

Heart 0.39 ±0.14 0.58 ± 0.04 0.13 ±0.02 0.16 ±0.04

B1 R- tumor 0.32 ± 0.06 0.61 ±0.19* 0.19 ±0.08 0.19 ±0.07

B1R+ tumor 3.94 ± 1.24 0.79 ±0.15** 4.20 ± 0.98 0.47 ±0.13***

Muscle 0.19 ±0.05 0.47 ±0.13** 0.09 ±0.01 0.09 ± 0.02

Bone 0.48 ±0.13 0.96 ±0.41* 0.33 ±0.11 0.47 ±0.12

Brain 0.02 ±0.01 0.03 ±0.01* 0.01 ±0.00 0.01 ±0.00

B1R+T:B1R-T 12.2 ±3.02 1.37 ± 0.42*** 23.4 ± 7.77 2.67 ±0.71***

B1R+T:Blood 6.69 ± 3.60 0.50 ± 0.08* 14.7 ±3.56 1.36 ± 0.64***

B1 R+T:Muscle 21.3 ±4.33 1.70 ± 0.14*** 48.6 ± 10.7 5.41 ±2.41***

*, **, and *** indicate the P values for the uptake difference between control and blocked groups are < 0.05, < 0.01 and < 0.001, respectively.

PET/CT Imaging. Images of ¹⁸F-AmBF₃-B9858 and ¹⁸F-AmBF₃-B9958 taken at 1 h p.i. were taken (Fig. 10). These images are consistent with the data obtained from the biodistribution studies. Higher uptake was observed in B1R⁺ tumor, kidney, and bladder, and the uptake in BlPv⁺ tumor was blocked by coinjecting the tracer with 100 μg of nonradioactive standard. Higher background activity was observed when using ¹⁸F-AmBF₃-B9858. Coinjection of nonradioactive standard visibly increased background activity levels, and this was particularly noticeable with ¹⁸F-AmBF₃-B9858.

EXAMPLE 2

Synthesis Strategy: BF₃-DMAm moiety was appended to the B9858 peptide via a cationic linker (Pip) to provide the compound BF₃-B9858 (Figure 3). BF3-B9858 is readily radiolabeled to high specific activity in one-step with ¹⁸F via an aqueous ¹⁸F-¹⁹F isotope-exchange reaction

25 on the trifluoroborate (BF₃) group to give the final tracer and lead candidate ¹⁸F-BF₃-B9858. The synthetic scheme is shown in Figure 5.

Synthesis of BF₃-B9858: The amino acid sequence of B9858, the linker 4-amino-(l- carboxymethyl)piperidine, and azidoacetic acid are assembled sequentially on solid phase using an inhouse Endeavor 90 peptide synthesizer (See Table 1 for amino acid sequence). At the end of elongation, the peptide is cleaved and concomitantly de-protected via trifluoroacetic acid treatment. The intermediate peptide is coupled with alkyne-conjugated trifluoroborate (Fig. 5) via a Cu -catalyzed click reaction. The final product BF₃-B9858 is purified by semi-preparative high performance liquid chromatography (HPLC). The identity of BF₃-B9858 is confirmed by high-resolution mass spectrometry and amino acid analysis, and the purity of BF₃-B9858 is determined by analytical HPLC. The novel structure of BF₃-B9858 is shown in Figure 3.

¹⁸F-labeling of B9858: The ¹⁸F-containing H₂[¹⁸0]0 is transferred from the cyclotron target station to an ORTG ¹⁸F trap/release cartridge (#500-101) set up in a hot cell. The ¹⁸F-fluoride (~ 30 GBq) is trapped in the cartridge, and eluted off with saline (100 μί) into a 4-mL polypropylene vial containing BF₃-B9858 (50 μπιοΐε) in aqueous pyridazine buffer (1M, 60μΙ,, pH = 2.5). The vial is heated at 80 °C in a heated sand bath for 20 min, and then quenched with 2 mL 5% ammonium hydroxide. The quenched reaction mixture is passed through a CI 8 light Sep-Pak cartridge which is preconditioned with ethanol (10 mL) and water (10 mL). The Sep- Pak cartridge is washed with water (2 mL), and then ¹⁸F-BF3-B9858 (~ 7.5 GBq) is eluted off the Sep-Pak cartridge with 1 : 1 ethanol/saline into a 10-mL product vial. ¹⁸F-BF3-B9858 is diluted with saline to a concentration of 1 mCi/mL for the dosimetry study. Cell Culture: Human embryonic kidney cell line HEK293T was purchased as part of Lenti-X™ Expression System from Clontech Laboratories Inc. (Mountain View, CA). Cells were cultured in high glucose DMEM or RPMI 1640 (StemCell Technologies, Vancouver, BC) supplemented by 10% FBS, 2mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator containing 5% CO₂. Cells at 80-90% confluences were washed with sterile phosphate-buffered saline (lx PBS pH7.4) and harvested with 0.25%) trypsin. Number of cells collected was counted using a Scepter handheld automated cell counter (Millipore, Billerica, MA).

Gene Transfer and Expression Using Recombinant Lentiviruses: We used the Lenti-X Lentiviral Expression Systems provided by Clontech Laboratories Inc to induce expression of

26 Green Fluorescent Protein (GFP) and human bradykinin B l receptor (BKRB 1) on humen embryonic kidney cells (HEK293). The lentiviral expression vector carrying GFP, pGIPz(GFP) (Open Biosystems, Rockford, IL) was obtained from Dr. Samuel Aparicio's laboratory at BCCRC. pGIPz(GFP) carries antibiotic resistance selection marker against puromycin (PuroR). To generate recombinant lentivirus carrying GFP, Lenti-X HT Packaging Systems (Clontech Laboratories Inc.) were transfected together with pGIPz(GFP) into HEK293T cell line. Cells were allowed to grow for 72 hours before growth medium containing lentiviruses were harvested and used to infect mammalian cells. In summary, 100,000 cells were cultured in 6-well plate up to 70% confluences. Hexadimethrine bromide (polybrene) (Sigma-Aldrich Canada Ltd, Oakville, ON) at 8 μg/mL final concentrations in 0.9% NaCl was used to reduce charge repulsion between cell membrane and the lentivirus. Growth medium of the overnight cell culture post-lentivirus infection was refreshed and cells were allowed to grow for 48 hours before antibiotic (puromycin) screening started. Cells survived in the presence 10 μg/mL of puromycin were cross-checked under fluorescence microscopy for green fluorescence detection. Some cells are not expressing GFP despite resistance against puromycin. Lentivirus works were done in Biosafety Level 3 facility at Terry Fox Laboratory, BCCRC.

Pre-made inducible lentiviral particles at lxl 0⁷ IFU/mL for expressing human BDKRBl (bradykinin Bl receptor/BlR) was obtained from GenTarget Inc., San Diego, CA (cat. no. LVP291). The B IR open reading frame is constitutively expressed under a tetracycline inducible suCMV promoter. An antibiotic blasticidin (Bsd) - RFP (red fluorescence protein) fusion marker under RSV promoter is also present in the expression vector to allow the selection of successfully transduced cells. HEK293T: :GFP cells were cultured in a 96-well microplate in quadruplicate with the following cells number: lx (-20,000 cells/well), l/2x, l/4x, and l/20x in 100 iL of RPMI growth medium with 1 % FBS. Duplicate wells received 5x10⁴ IFU lentivirus carrying human B IR and Bsd-RFP with or without 8 μg/mL of polybrene. The plate was briefly centrifuged at 500 rpm for 5 minutes and incubated at 37°C for 6 hours before replacement of the culture medium. Blasticidin at 10 μg/mL final concentration was added to growth medium 72 hours later to select transduced cells. Growth medium was refreshed every two days in the presence of blasticidin for about a week before trypsinization and transfer of the cells in larger flasks, consecutively from 6-well plate, T25, T75, and T225. Cells resistant to blasticidin also expressed red fluorescence protein. Figure 2 shows schematic representation for lentiviral vector carrying human B IR (BDKRB l) provided by GenTarget Inc. Fluorescence microscopy of HEK293T: :GFP: :hB lR cells shows a membranous and cytoplasmic fluorescence of RFP, suggesting the localization of B IR over-expression.

27 Preparation of Cell Membrane: Ready to use cell membrane (12.5 μg/μL) from CHO-K1 cells over-expressing human B1R was obtained from Perkin Elmer Inc., Waltham, MA (cat. no. ES- 091-M400UA). We also tested binding using cell membranes from HEK293T::GFP::hBlR. In the latter case, the membranes were prepared as follows: 90% confluent cells grown in large flask, T225, were detached and pelleted in a 50 mL conical tube using centrifuge at 1 ,200 rpm for 5 minutes. Afterward, cells were re-suspended in 20-30 mL of cold 50 mM Tris-HCl pH 7.4. Ice cold cells were disrupted for 15 seconds at 15,000 rpm with a Polytron PT3100 (Kinematica AG, Lucerne, Switzerland). The solution was aliquoted and centrifuged at 4°C at 17,000xg for 30 minutes. The supernatant was discarded, and then the pellet was dissolved in 1 - 2 mL of cold salt solution containing 50 mM Tris-HCl pH 7.4 and 100 mM NaCl and incubated for 1 hour on ice. The cell membranes were pelleted by centrifugation at 4°C at 17,000xg for 30 minutes and resuspended in 200-600 of 50 mM Tris-HCl pH 7.4. The cell membrane extract was stored in -80°C. A Bradford assay (cat. no. R1271, Fermentas, Thermo Fisher Scientific, Burlington, ON) was done to determine the protein concentration. A 50 ug of cell membrane per well was used for competitive binding assay. The control bradykinin used was H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH (BK-ANT) obtained from Bachem Americas Inc., Torrance, CA (cat. no. H-2582). Radiochemical bradykinin used was H-3 labeled [Leu⁹,desArg¹⁰]Kallidin obtained either from American Radiolabeled Chemicals Inc, St. Louis, MO (cat. no. ART1609, lot no. 111103, specific activity: 83.8 Ci/mmol, original dilution 1.0 μΰί/μί, half-life: 4537 days, stock concentration: 11.906 μΜ) or Perkin Elmer Inc., Boston, MA (cat. no. NET1096250UC, lot no. 1586889, specific activity: 76.0 Ci/mmol, original dilution: 1.0 μΰί/μί, half-life: 4537 days, stock concentration: 13.086 μΜ). Saturation Binding Assays: Assays were done using 96-wells MultiScreen_HTs with glass fiber filter and PVDF support obtained from Millipore Corp, Billerica, MA (cat. no. MSFB N6B, lot no. R8AN42424). Wells of the MultiScreen plate was pre-soaked with 0.5% of cold Poly(ethyleneimine) (PEI) (cat. no. P3143, Sigma-Aldrich, St. Louis, MO) for 30 minutes. Afterward, wells were washed once with 50 mM of Tris-HCl pH 7.4. Total volume per wells was 200 μΐ,. Increasing concentrations of the radioligand (H-3 labeled [Leu⁹,desArg¹⁰]Kallidin) from 0.005 to 10 nM concentration were incubated in the presence of cell membranes (50 μg/well), with and without the addition of BK-ANT as a competitor (50 μΜ). The assay buffer contained 50 mM of Tris-HCl pH 7.4 and 5 mM of MgCl₂. The Multiscreen plate was incubated at 27°C for 15 minutes. The assay was stopped by suctioning the reaction solution through the PVDF membrane filter, and washing of the filter with ice cold

28 50 mM Tris-HCl pH 7.4. Scintillation fluid was added to each well, and the radioactivity was measured using a MicroBeta Trilux Microplate Scintillation and Luminescence Counter (Perkin Elmer Inc., Shelton, CT). Graphpad Prism 5 was used to calculate the affinity constant (Kd) and the receptor concentration. (Bmax)

Kd for ³H-Des-Arg¹⁰,Leu⁹-Kallidin binding to B 1R was 6-fold lower in CHO-K1 : :hB lR (Kd = 0.5742 nM) than in HEK293T: :GFP: :hB lR (Kd = 3.459 nM), although the latter results will need to be confirmed in further experiments Competitive Binding Assays: Assays were done using 96-wells MultiScreen_HTs with glass fiber filter and PVDF support obtained from Millipore Corp, Billerica, MA (cat. no. MSFB N6B, lot no. R8AN42424). Wells of the MultiScreen plate was pre-soaked with 0.5% of cold Poly(ethyleneimine) (PEI) (cat. no. P3143, Sigma-Aldrich, St. Louis, MO) for 30 minutes. Afterward, wells were washed once with 50 mM of Tris-HCl pH 7.4. Total volume per wells was 200 yL. A fixed concentration of the hot radioligand (1 , 3, 5 or 12.5 nM depending on the radioligand) was incubated with progressively higher concentrations (10^"n to 10^"4 M) of the non radioactive BF3-B9858 in the presence of cell membranes (50 μg/well) and the assay buffer (50 mM of Tris-HCl pH 7.4 and 5 mM of MgCl₂). The MultiScreen plate was then incubated in 27°C for 15 minutes with agitation at 300 rpm. The assay was stopped by suctioning the reaction solution through the bottom PVDF membrane filter and discarded. The membranes were washed 7x with 150 μίΛνεΙΙ of cold 50 mM Tris-HCl pH 7.4. Radioactivity was counted using 1450 MicroBeta TriLux - Microplate Scintillation and Luminescence Counter (Perkin Elmer Inc., Shelton, CT). Tumor implantation: Imaging and biodistribution experiments were performed using NODSCID IL2RKO mice. Each cage, equipped with enrichments, contained three or four mice. The mice were maintained and the experiments were conducted in accordance with the guidelines established by Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. Mice were housed under pathogen-free conditions and kept on twelve hour light and twelve hour dark cycle in the Animal Research Centre, British Columbia Cancer Research Centre, Vancouver, Canada.

Mice were anesthetised briefly with 2.5% isoflurane in 2.0 L/min of oxygen during cells implantation. After wiping the skin surrounding the injection site with an alcohol prep pad, a 31-Gauge needle was used to inject 10⁷ HEK293T: :GFP and HEK293T: :hBlR cells in matrigel

29 (1 : 1) on the back of the mouse. Imaging was performed once tumor lump was visible. Tumor size was measured by CT and calculated using the ellipsoid formula as follows: V³^ = width x length x thickness x 0.524. PET/CT Imaging: Each tumor bearing mouse was injected -3.7 MBq of ¹⁸F-BF3-B9858 through the tail vein. The mice were imaged using the BC Cancer Agency Inveon microPET/CT scanner that has a 1.3 mm spatial resolution and a high sensitivity. Briefly, a localization CT scan was first obtained using 3 overlapping position to cover the entire mouse. This CT scan was used for attenuation and scatter correction after segmentation for reconstructing the PET images. A dynamic acquisition was then performed for 60 minutes, with the mouse under isoflurane sedation. The animals were kept warm by a monitoring system with an integrated heating pad and physiological acquisition system. At the end of the acquisition, the mice were euthanized, and major organs were collected, weighed, and counted to determine the % injected dose per gram of tissue (%ID/g). The biodistribution data are shown in Figure 4 (right panel). The images were reconstructed using OSEM/3DMAP iterative reconstruction, and typical images of ¹⁸F-BF3-B9858 at 1 h post-injection are shown in Figure 4 (left panel).

BF3-B9858 bound B1R with high affinity (Ki = 0.083 ± 0.074 nM). 18F-BF3-B9858 was prepared in 30 min synthesis time, and isolated in -25% (n=3) radiochemical yield with >99% radiochemical purity and >100 GBq/μπιοΙ specific activity. ¹⁸F-BF3-B9858 was stable in plasma with <1% decomposition after 2 hr incubation at room temperature. PET imaging and biodistribution showed that radioactivity was cleared rapidly from most organs/tissues, and excreted mainly through the renal pathway (see Figure 4).. High uptake in B1R+ tumor (7.46±3.97 %ID/g) was observed at lh post-injection, and the bone uptake (0.51±0.20 %ID/g) was minimal. Uptake ratios of B1R+ tumor to liver, blood, muscle and B1R- tumor at lh post- injection were 10.8±4.6,11.7±3.4, 41.4±22.7 and 14.8±3.5, respectively. Surprisingly, 18F- BF3-B9858 had a much higher binding affinity to the B1R compared to B9858 itself (14 nM). BF3-B9858 (Ki= 0.08 nM) also outperformed other Ga-labeled B1R targeting peptide analogs that we have designed to date.

The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference. Although the invention has been described with reference to certain specific embodiments, various

30 modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

31

Claims

CLAIMS:

1. A radio-labeled BIR targeting compound, or precursor thereof, of general Formula (I):

B-L-X (I)

wherein:

B is a ¹⁸F-trifluoroborate moiety;

L is a piperidine-based linker; and

X is a peptide having the sequence Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-(D-Igl)-Ile.

2. A radio-labeled BIR targeting compound, or precursor thereof, of general Formula (I):

B-L-X (I)

wherein:

B is a ¹⁸F-trifluoroborate moiety;

L is a piperidine-based linker; and

X is Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-(D-Tic)-Cpg.

3. A radio-labeled BIR targeting compound, or precursor thereof, of general Formula (II):

B-L-Xaa²-Xaa²-Arg-Pro-Xaa³-Gly-Xaa⁴-Ser-Xaa⁵-Xaa⁶ (II) wherein:

B is a ¹⁸F-trifluoroborate moiety;

L is an amino-heterocyclic based linker;

Xaa² is absent, Lys or D-Arg;

Xaa³ is Pro or Hyp;

Xaa⁴ is Phe, Cha, Thi, (a-Me)Phe, Igl or Cpg;

Xaa⁵ is Pro, D-Tic, D-Hyp, D-PNal or D-Igl, and

Xaa⁶ is Leu, He, D-Phe, Cpg or Oic.

4. Use of the compound of any of claims 1 to 3, for imaging of a tissue or cancer expressing or overexpressing BIR.

5. The use according to claim 4, wherein the cancer or tissue is a cancer associated with BIR overexpression.

6. The use according to claim 4, wherein the cancer is a breast cancer, prostate cancer, lung cancer or brain cancer.

32

7. The use according to claim 4, wherein the cancer is an early stage breast or prostate cancer.

8. The use according to claim 4, wherein the cancer is primary or recurrent prostate cancer.

9. The use according to claim 4, wherein the tissue is damaged endovascular intima.

10. Use of a compound according to any of claims 1 to 3 for treatment of a disease or condition in which BIR is expressed or overexpressed.

11. The use of claim 10 wherein the disease is cancer.

12. The use of claim 11 wherein the cancer is associated with BIR overexpression.

13. The use of claim 11 wherein the cancer is an early stage breast or prostate cancer.

14. Use of a compound according to any of claims 1 to 3 for the manufacture of a medicament for the treatment of a of a disease or condition in which BIR is expressed or overexpressed.

15. A method for imaging a tissue or cancer expressing or overexpressing bradykinin B 1 receptor (BIR) in a patient, comprising administering to the patient a radio-labelled BIR targeting compound according to any of claims 1 to 3; and imaging the tissue or cancer.

16. The method according to claim 15, wherein the cancer or tissue is a cancer associated with BIR overexpression.

17. The method according to claim 15, wherein the cancer is a breast cancer, prostate cancer, lung cancer or brain cancer.

18. The compound according to any of claims 1 to 3 wherein the ¹⁸F-trifluoroborate moiety such as dimethylammoniomethy-trifluoroborate (AmBF₃).

19. The compound according to any of claims 1 to 3 wherein the linker is 4-amino-(l- carboxymethyl)piperidine (Pip).

33