Stability Evaluation and Stabilization of a Gastrin-Releasing Peptide Receptor (GRPR) Targeting Imaging Pharmaceutical.

The prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are identified as important targets on prostate cancer. Receptor-targeting radiolabeled imaging pharmaceuticals with high affinity and specificity are useful in studying and monitoring biological processes and responses. Two potential imaging pharmaceuticals, AMBA agonist (where AMBA = DO3A-CH2CO-G-[4-aminobenzyl]- Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) and RM1 antagonist (where RM1 = DO3A-CH2CO-G-[4-aminobenzyl]-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), have demonstrated high binding affinity (IC50) to GRP receptors and high tumor uptake. Antagonists, despite the poor tumor cell internalization properties, can show clearer images and pharmacokinetic profiles by virtue of their higher tumor uptake in animal models compared to agonists. For characterization, development, and translation of a potential imaging pharmaceutical into the clinic, it must be evaluated in a series of tests, including in vitro cell binding assays, in vitro buffer and serum stability studies, the biodistribution of the radiolabeled material, and finally imaging studies in preclinical animal models. Data related to acetate buffer, mouse, canine, and human sera stability of 177Lu-labeled RM1 are presented here and compared with the acetate buffer and sera stability data of AMBA agonist. The samples of 177Lu-labeled RM1 with a high radioconcentration degrade faster than low-radioconcentration samples upon storage at 2-8 °C. Addition of stabilizers, ascorbic acid and gentisic acid, improve the stability of 177Lu-labeled RM1 significantly with gentisic acid being more efficient than ascorbic acid as a stabilizer. The degradation kinetics of 177Lu-labeled AMBA and RM1 in sera follow the order (fastest to slowest): mouse > canine > human sera. Finally, 177Lu-labeled RM1 antagonist is slower to degrade in mouse, canine, and human sera than 177Lu-labeled AMBA agonist, further suggesting that an antagonist is a more promising candidate than agonist for the positron emission tomography (PET) imaging and therapy of prostate cancer patients.

Novel Peptide NIRF Optical Surgical Navigation Agents for HNSCC.

Head and neck squamous cell carcinoma (HNSCC) survival rates have not improved in a decade, with a 63% 5-year recurrence rate after surgery, making HNSCC a compelling indication for optical surgical navigation (OSN). A promising peptide, HN1, targeted and internalized in human HNSCC cells in multiple laboratories, but was slow (24 h) to accumulate. We modified HN1 and explored structural variables to improve the uptake kinetics and create IRdye800 adducts useful for OSN. Eleven new molecules were synthesized and characterized chemically, in human HNSCC cells (Cal 27), and in HNSCC xenograft mice. Cal 27 flank xenografts in Balb/c nude mice were imaged for 3-48 h after 40 nmol intravenous doses of IR800-labeled molecules. Cell uptake kinetics in the 1-2 h window incubated at 1-10 µM were independent of the dye label (FITC, Cy5, or IR800), but increased markedly with additional N-terminal lipophilic substitution, and after resequencing the peptide to separate polar amino acids and move the lysine-dye more centrally. Microscopy confirmed the strong Cal 27 cell binding and demonstrated primarily cytosolic and membrane localization of the fastest peptide, 4Iphf-HN17. 4Iph-HN17-IR800 showed 26-fold greater rate of uptake in cells than HN1-IR800, and far stronger OSN imaging intensity and tumor to background contrast in mice, suggesting that the new peptide is a promising candidate for OSN of HNSCC.

Development of an orthotopic canine prostate cancer model expressing human GRPr.

BACKGROUND: Ace-1 canine prostate cancer cells grow orthotopically in cyclosporine immunosuppressed laboratory beagles. We previously transfected (human Gastrin-Releasing Peptide Receptor, huGRPr) into Ace-1 cells and demonstrated receptor-targeted NIRF imaging with IR800-G-Abz4-t-BBN, an agonist to huGRPr. Herein, we used the new cell line to develop the first canine prostate cancer model expressing a human growth factor receptor. METHODS: Dogs were immunosuppressed with cyclosporine, azathioprine, prednisolone, and methylprednisolone. Their prostate glands were implanted with Ace-1huGRPr cells. The implantation wounds were sealed with a cyanoacrylic adhesive to prevent extraprostatic tumor growth. Intraprostatic tumors grew in 4-5 week. A lobar prostatic artery was then catheterized via the carotid artery and 25-100 nmol IR800-Abz4-t-BBN was infused in 2 mL followed by euthanasia in dogs 1-2, and recovery for 24 h before euthanasia in dogs 3-6. Excised tissues were imaged optically imaged, and histopathology performed. RESULTS: Dog1 grew no tumors with cyclosporine alone. Using the four drug protocol, Dogs 2-6 grew abundant 1-2 mm intracapsular and 1-2 cm intraglandular tumors. Tumors grew >5 cm when the prostate cancer cells became extracapsular. Dogs 4-6 with sealed prostatic capsule implantation sites had growth of intracapsular and intraglandular tumors and LN metastases at 5 weeks. High tumor to background BPH signal in the NIRF images of sectioned prostate glands resulted from the 100 nmol dose (∼8 nmol/kg) in dogs 2-4 and 50 nmol dose in dog 5, but not from the 25 nmol dose in Dog 6. Imaging of mouse Ace-1huGRPr tumors required an intravenous dose of 500 nmol/kg body wt. A lymph node that drained the prostate gland was detectable in Dog 4. Histologic findings confirmed the imaging data. CONCLUSION: Ace-1huGRPr cells created viable, huGRPr-expressing tumors when implanted orthotopically into immune-suppressed dogs. Local delivery of an imaging agent through the prostatic artery allowed a very low imaging dose, suggesting that therapeutic agents could be used safely for treatment of early localized intraglandular prostate cancer as adjuvant therapy for active surveillance or focal ablation therapies, or for treating multifocal intraglandular disease where focal ablation therapies are not indicated or ineffective.

A human GRPr-transfected Ace-1 canine prostate cancer model in mice.

BACKGROUND: A versatile drug screening system was developed to simplify early targeted drug discovery in mice and then translate readily from mice to a dog prostate cancer model that more fully replicates the features of human prostate cancer. METHODS: We stably transfected human cDNA of the GRPr bombesin (BBN) receptor subtype to canine Ace-1 prostate cancer cells (Ace-1(huGRPr) ). Expression was examined by (125) I-Tyr(4) -BBN competition, calcium stimulation assay, and fluorescent microscopy. A dual tumor nude mouse xenograft model was developed from Ace-1(CMV) (vector transfected Ace-1) and Ace-1(huGRPr) cells. The model was used to explore the in vivo behavior of two new IRDye800-labeled GRPr binding optical imaging agents: 800-G-Abz4-t-BBN, from a GRPr agonist peptide, and 800-G-Abz4-STAT, from a GRPr antagonist peptide, by imaging the tumor mice and dissected organs. RESULTS: Both agents bound Ace-1(huGRPr) and PC-3, a known GRPr-expressing human prostate cancer cell line, with 4-13 nM IC50 against (125) I-Tyr(4) -BBN, but did not bind Ace-1(CMV) cells (vector transfected). Binding was blocked by bombesin. Ca(2+) activation assays demonstrated that Ace-1(huGPRr) expressed biologically active GRPr. Both Ace-1 cell lines grew in the flanks of 100% of the nude mice and formed tumors of ∼0.5 cm diameter in 1 week. In vivo imaging of the mice at 800 nm emission showed GRPr+: GRPr- tumor signal brighter by a factor of two at 24 h post IV administration of 10 nmol of the imaging agents. Blood retention (4-8% ID at 1 h) was greater by a factor >10 and cumulative urine accumulation (28-30% at 4 h) was less by a factor 2 compared to a radioactive analog of the t-BBN containing agent, (177) LuAMBA, probably due to binding to blood albumin, which we confirmed in a mouse serum assay. CONCLUSIONS: The dual tumor Ace-1(CMV) /Ace-1(huGRPr) model system provides a rapid test of specific to nonspecific binding of new GRPr avid agents in a model that will extend logically to the known Ace-1 orthotopic canine prostate cancer model. Prostate 76:783-795, 2016. © 2016 Wiley Periodicals, Inc.

Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

Design, synthesis, and structure-activity relationship of novel CCR2 antagonists.

A series of novel 1-aminocyclopentyl-3-carboxyamides incorporating substituted tetrahydropyran moieties have been synthesized and subsequently evaluated for their antagonistic activity against the human CCR2 receptor. Among them analog 59 was found to posses potent antagonistic activity.

Use of a leukocyte-targeted peptide probe as a potential tracer for imaging the tuberculosis granuloma.

Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG12 to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG12-Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis.

Biological Evaluation of a Fluorescent-Imaging Agent for Medullary Thyroid Cancer in an Orthotopic Model.

Context: The primary and definitive treatment of medullary thyroid cancer (MTC) is surgical resection. Recurrent or residual disease is typically a result of incomplete surgical removal. Objective: Our objective is to develop a compound that assists in intraoperative visualization of cancer, which would have the potential to improve surgical cure rates and outcomes. Results: We report the biological characterization of Compound-17, which is labeled with IRdye800, allowing fluorescent visualization of MTC mouse models. We found that the agent has high affinity for two human MTC cell lines (TT and MZ-CRC1) in vitro and in vivo. We further tested the affinity of the compound in a newly developed MTC orthotopic xenograft model and found that Compound-17 produces fluorescent signals within MTC-derived orthotopic xenografts in comparison with a sequence-jumbled control compound and surrounding normal tissues. Conclusions: Compound-17 is a unique and effective molecule for MTC identification that may have therapeutic potential.

A high-affinity near-infrared fluorescent probe to target bombesin receptors.

PURPOSE: This study aimed to create new optical surgical navigation NIRF probes for prostate and breast cancers. PROCEDURES: IR800-linker-QWAVGHLM-NH2 with linker = GSG, GGG, and G-Abz4 were synthesized and characterized. IC50 for bombesin receptors (BBN-R) in PC-3 prostate and T47D breast cancer cells, fluorescence microscopy in PC-3 cells, and NIRF imaging in mice PC-3 tumor xenografts were studied. RESULTS: GGG, GSG, and G-Abz4 derivatives had IC50 (nM) for BBN-R+ PC-3 cells = 187 ± 31, 56 ± 5, and 2.6 ± 0.2 and T47D cells = 383 ± 1, 57.4 ± 1.2, and 3.1 ± 1.1, respectively. By microscopy the Abz4 derivative showed the highest uptake, was competed with by BBN, and had little to no binding to BBN-R- cells. In NIRF imaging the G-Abz4 probe was brighter than GGG probe in BBN-R+ tissues in vivo and tissues, tumors, and tumor slices ex vivo. Uptake could be partially blocked in BBN-R+ pancreas but not visibly in tumor. CONCLUSIONS: Linker choice can dominate peptidic BBN-R binding. The G-Abz4 linker yields a higher affinity and specific BBN-R binder in this series of molecules.

Development of a calcium-sensing receptor molecular imaging agent.

BACKGROUND: Calcium-sensing receptor (CaSR) is expressed by parathyroid cells and thyroid C-cells (from which medullary thyroid carcinoma [MTC] is derived). A molecular imaging agent localizing to the CaSR could improve the detection of parathyroids and MTC preoperatively or intraoperatively. We synthesized a novel compound containing a fluorine residue for potential future labeling and demonstrated that the compound inhibited CaSR function in vitro. METHODS: We synthesized compound M, a derivative of a known calcilytic compound, Calhex-231. Human embryonic kidney cells transfected with green-fluorescent protein-tagged CaSR or control vector were preincubated with compound M before the addition of calcium. Immunoblotting for total mitogen-activated protein kinase (MAPK: ERK1/2), activated MAPK (phosphorylated ERK1/2), and glyceraldehyde 3-phosphate dehydrogenase was performed. RESULTS: Synthesis of compound M was confirmed by mass spectrometry. Inhibition of the MAPK signaling pathway by compound M was demonstrated in a dose-dependent manner by a decrease in phosphorylated ERK1/2 with no change in total ERK1/2 levels. Compound M inhibited MAPK signaling slightly better than the parent compound. CONCLUSION: We have developed a novel molecule which demonstrates functional inhibition of CaSR and has a favorable structure for labeling. This compound appears to be appropriate for further development as a molecular imaging tool to enhance the surgical treatment of parathyroid disease and MTC.

Localization of CaSR antagonists in CaSR-expressing medullary thyroid cancer.

OBJECTIVE: Image-based localization of medullary thyroid cancer (MTC) and parathyroid glands would improve the surgical outcomes of these diseases. MTC and parathyroid glands express high levels of calcium-sensing receptor (CaSR). The aim of this study was to prove the concept that CaSR antagonists specifically localize to CaSR-expressing tumors in vivo. DESIGN: We synthesized two isomers of a known CaSR calcilytic, Calhex 231, and four new analogs, which have a favorable structure for labeling. Their antagonistic activity was determined using immunoblots demonstrating decreased ERK1/2 phosphorylation after calcium stimulation in human embryonic kidney cells overexpressing CaSR. Compound 9 was further radiolabeled with (125)I and evaluated in nude mice with and without heterotransplanted xenografts of MTC cell lines, TT and MZ-CRC-1, that do and do not express CaSR, respectively. RESULTS: Two newly synthesized compounds, 9 and 11, exhibited better antagonistic activity than Calhex 231. The half-life of (125)I-compound 9 in nude mice without xenografts was 9.9 hours. A biodistribution study in nude mice bearing both tumors demonstrated that the uptake of radioactivity in TT tumors was higher than in MZ-CRC-1 tumors at 24 hours: 0.39 ± 0.24 vs 0.18 ± 0.12 percentage of injected dose per gram of tissue (%ID/g) (P = .002), with a ratio of 2.25 ± 0.62. Tumor-to-background ratios for TT tumors, but not MZ-CRC-1 tumors, increased with time. Tumor-to-blood values increased from 2.02 ± 0.52 at 1 hour to 3.29 ± 0.98 at 24 hour (P = .015) for TT tumors, and 1.7 ± 0.56 at 1 hour to 1.48 ± 0.33 at 24 hour (P = .36) for MZ-CRC-1 tumors. CONCLUSIONS: Our new CaSR antagonists specifically inhibit CaSR function in vitro, preferentially localize to CaSR-expressing tumors in vivo, and therefore have the potential to serve as scaffolds for further development as imaging pharmaceuticals.

Pharmacokinetic properties of intravitreal I-124-aflibercept in a rabbit model using PET/CT.

PURPOSE: To determine the anatomic characteristics and pharmacokinetic properties of intravitreally-placed aflibercept in a rabbit model. MATERIALS AND METHODS: Four Dutch-belted rabbits underwent intravitreal injection with I-124-aflibercept. Serial imaging with PET/CT imaging was performed on days 0, 2, 5, 7, 14, 21, 28 and 35. Measured radioactivity emission in becquerels/mL was used to calculate the half-lives for aflibercept. RESULTS: I-124 aflibercept was confined within the vitreous cavity for the duration of the study. I-124 aflibercept could be detected in the vitreous cavity until day 28 and the average retention time with standard error after correction for radioactive decay was 4.58 ± 0.07 days. CONCLUSIONS: I-124 aflibercept was only visible in the vitreous cavity by PET/CT imaging following intravitreal injection. The retention properties were found to be comparable to those measured by other reported methods.

4-Amino-2-alkyl-butyramides as small molecule CCR2 antagonists with favorable pharmacokinetic properties.

A systematic examination of the central aromatic portion of the lead (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-(4-fluorophenyl)-4-(1'H-spiro[indene-1,4'-piperidin]-1'-yl)butanamide (9) led to the discovery of a novel class of CCR2 receptor antagonists, which carry small alicyclic groups such as cyclopropyl, cylobutyl, or cyclopropylmethyl attached at C2 of the carbon backbone. The most potent compound discovered, namely (2S)-N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-[(1R,3'R)-3'-methyl-1'H-spiro[indene-1,4'-piperidin]-1'-yl]butanamide (29), showed very high binding affinity (IC50 = 4 nM, human monocyte) and excellent selectivity toward other related chemokine receptors. The excellent pharmacokinetic profile of this new lead compound allows for extensive in vivo evaluation.