Introduction: AMBA is a bombesin analogue that binds to GRPr. In a mouse model of estrogen-dependent human breast cancer, we tested whether Ga-68-AMBA can be used for PET detection of GRPr-expressing tumors and could be more accurate than F-18-FDG to monitor tumor response to hormone therapy.Methods: The radiolabeling of Ga-68-AMBA was automated using a R&D Synchrom module. ZR75-1, a breast cancer cell line, was xenografted in nude mice. Ga-68-AMBA tumor uptake was compared with that of F-18-FDG before and after treatment with tamoxifen.Results: AMBA was Ga-68-radiolabelled in 30 min with 95.3% yield and purity >= 98%. Prior to treatment, (68)GaAMBA was highly concentrated into tumors (tumor to non-tumor ratio = 2.4 vs. 1.3 with F-18-FDG). With tamoxifen treatment (n = 6) Ga-68-AMBA uptake plateaued after 1 week and decreased after 2 weeks, with a significant reduction compared to controls (n = 4). In contrast the effect of tamoxifen treatment could not be appreciated using F-18-FDG.Conclusions: Ga-68-AMBA appeared better than F-18-FDG to visualize and monitor the response to hormone treatment in this breast cancer model. (C) 2014 Elsevier Inc. All rights reserved.

Ga-AMBA (Ga-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is a bombesin-like agonist with high affinity for gastrin releasing peptide receptors (GRP-R). Syntheses for (nat)Ga-AMBA, [(67)Ga]Ga-AMBA and [(68)Ga]Ga-AMBA were developed. The preparation of HPLC-purified and Sep-Pak purified [(68)Ga]Ga-AMBA were fully automated, using the built-in radiodetector of the Tracerlab FX F-N synthesizer to monitor fractionated (68)Ge/(68)Ga generator elution and purification. The total synthesis time, including the fractional elution of the generator, was 20 min for Sep-Pak purified material and 40 min for HPLC-purified [(68)Ga]Ga-AMBA. Both [(67)Ga]Ga-AMBA and [(177)Lu]Lu-AMBA showed comparable high affinity for GRP-R in the human prostate cancer cell line PC-3 in vitro (k(D)=0.46+/-0.07; 0.44+/-0.08 nM), high internalization (78; 77%) and low efflux from cells at 2 h (2.4+/-0.7; 2.9+/-1.8%). Biodistribution results in PC-3 tumor-bearing male nude mice showed comparable uptake for [(177)Lu]Lu-, [(111)In]In-, [(67)Ga]Ga- and [(68)Ga]Ga-AMBA.

The metabolism of Lu-177-AMBA (AMBA = DO3A-CH3CO-G-(4-aminobenzoyl)-QWAVGHLM-NH2), a radio-therapeutic compound in clinical development that binds to GRP and NMB receptors, was studied in vitro (mouse, rat and human plasma, mouse kidney homogenate) and in vivo (by analysis of mouse and rat plasma and urine following IV injection of Lu-177-AMBA). The primary metabolites were Lu-DO3A-CH2CO-G-Abz4-R, where R = -Q-OH (A), -QW-OH (B), and -QWAVGH-OH (C). Minor amounts of (D) where R = -QWAVGHLM-OH and (E) -QWAVGHL-OH were also observed. Clearance of Lu-177-AMBA and of radioactivity from mouse and rat blood was rapid in vivo. In mouse and rat urine, only metabolites Lu-A and Lu-B were found-no parent drug was excreted. Unmetalated ligands and Lu-nat. and Lu-177 complexes for Lu-AMBA metabolites A-E were synthesized, characterized by HPLC and MS, and used to perform in vitro competition and direct binding studies on GRP receptor-positive PC-3 (human prostate) cancer cells. Biodistribution studies with Lu-177-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact Lu-177-AMBA. IC50 values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against Lu-177- AMBA. No direct binding to PC-3 cells was observed with Lu-177-labeled A-C, confirming IC50 results. Lu-177-labeled metabolites A-E showed no uptake in GRP-receptor positive tumor or pancreas in PC-3 tumor bearing mice. All metabolites were rapidly excreted via the renal route (similar to 78-87%) within I It. These results demonstrate that the tumor uptake observed with Lu-177-AMBA is due to parent drug and not due to any of its identified metabolites.

Lu-177-DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (Lu-177-AMBA) is a radiolabeled bombesin derivative that is bound and internalized by cells expressing the G-protein-coupled gastrin-releasing peptide receptor (GRP-R) and is currently in phase I clinical trials. In previous radiotherapy studies with PC-3 xenografted mice, Lu-177-AMBA treatment significantly increased survival and reduced tumor growth rates. The PC-3 tumor cell line has an elevated expression of GRP-Rs (2.5 x 10(5)/cell), whereas LNCaP-a prostate cancer metastatic cell line representing the early androgen-sensitive stage of prostate cancer-and DU145-an androgen-insensitive metastatic line-express lower receptor numbers (5.9 x 10(3) and 1.2 x 10(4)/cell, respectively). Because of tumor heterogeneity, the high number of receptors in the PC-3 line may not represent the clinical situation, and little definitive work on the GRP-R status of primary prostate tumors and metastases exists. We sought to evaluate the tumor binding and imaging potential of Lu-177-AMBA in low GRP-R models of prostate cancer and determine how reduced expression affects Lu-177-AMBA radiotherapy efficacy. Methods: The LNCaP and DU145 cell lines were used to determine the binding (K-d), retention, and efflux of Lu-177-AMBA. Biodistribution radiotherapy, imaging, and autoradiography studies were performed in LNCaP, DU145, or PC-3 tumor-bearing male nude mice. Immunohistochemistry was used to determine the proliferative state in LNCaP and DU145 models and the vascular phenotype of LNCaP radiotherapy tumors. Results: Lu-177-AMBA binds to GRP-R in these cell lines with high affinity (K-d of LNCaP, 0.65 +/- 0.2 nM; K-d of DU145, 0.53 +/- 0.1 nM). The uptake of Lu-177-AMBA is at least 10-fold less in LNCaP and DU145 cell lines than it is in the PC-3 cell line. Autoradiography identifies activity concentrated in areas of viable tumor tissue, and gamma-images of Lu-177-AMBA identify tumors in vivo. Despite having lower uptake, Lu-177-AMBA demonstrated radiotherapeutic efficacy and decreased proliferation in the LNCaP and DU145 xenografts; in the LNCaP model, Lu-177-AMBA normalized the phenotype of microvasculature, reducing tumoral blood pooling. Conclusion: Lu-177-AMBA is a single radiolabeled agent that combines targeted radiotherapy after imaging dosimetry with the potential for single-agent or multimodality therapy for prostate cancer.

4216 177Lu-AMBA is a radiolabeled bombesin derivative that is bound and internalized by cells expressing the gastrin-releasing peptide receptor (GRP-R), a G-protein-coupled receptor. In our previous report of radiotherapy studies with immunocompromised mice bearing PC-3 xenografts, 177Lu-AMBA treatment significantly increased survival and reduced tumor growth rates. The PC-3 tumor cell line has a high number of GRP receptors (2.5 x 105 /cell), whereas LNCaP, a prostate cancer metastatic cell line thought to be representative of the early androgen-sensitive stage of prostate cancer, expresses a lower receptor number (5.9 x 103 / cell). While the high number of receptors per cell in the PC-3 line may not be representative of the clinical situation, there is little definitive work on the GRP receptor status of primary prostate tumors and metastases. Therefore we sought to evaluate the binding and efficacy of 177Lu-AMBA in a low GRP receptor number model of prostate cancer, and determine how reduced GRP receptor expression affects 177Lu-AMBA radiotherapy treatment efficacy and survival. Biodistribution studies confirmed the binding of 177Lu-AMBA to LNCaP xenografts in a receptor density-dependent manner when compared to PC-3. At 1h, LNCaP demonstrated 1.52 ± 0.94 % ID/g tumor as compared to 6.35 ± 2.23 %ID/g tumor in PC-3; and 0.83 ± 0.18 %ID/g tumor at 24 h, compared to 3.39 ± 0.85 %ID/g tumor in PC-3 at 24 h. There was no difference in survival between treated and controls for LNCaP at the end of the study (60-day; n=12), in contrast to PC-3 bearing mice (n=32) which demonstrated a two-fold increase in survival over controls with treatment. However, with the same 0.1 mL administered dose of 177Lu-AMBA (1.11 GBq/kg; 27.75 MBq/0.1 mL; peptide mass ~ 41.3 mg/m2), the Mean Time to Progression of tumor growth was increased by 100% in both LNCaP and PC-3 xenografts. Immunohistochemistry for ki67 showed a reduction in proliferation in treated tumors compared to control. In addition, the characteristic extravasation of blood (ecchymosis) of LNCaP xenografts was significantly reduced and confirmed histologically. The LNCaP cell line internalized a 177Lu-AMBA dose sufficient to reduce abnormal vascular phenotypes, mimicking androgen ablation and creating a more normal vessel route for further treatment delivery. These results confirm that 177Lu-AMBA, which has both gamma-emissions suitable for imaging and medium energy beta-emissions for radiotherapy and is currently in Phase I trials, has the potential to be clinically effective as a therapeutic and imaging agent, or as an adjunct to chemotherapy in prostate cancer, even in cases where the relative expression of GRP levels in the primary tumor or metastases is low.

Members of the gastrin-releasing peptide (GRP) family and its analogs bombesin (BBN) have been implicated in the biology of several human cancers including prostate, breast, colon and lung. To date, three mammalian GRP/BBN receptor subtypes have been cloned and characterized: the neuromedin B receptor (NMBR), the GRP receptor (GRPR) and the BBN-receptor subtype 3 (BB(3)). The fourth BBN receptor subtype, BB(4), has only been identified in amphibian and at present no mammalian equivalent of this receptor has been described. GRPR analogs have been used as carriers to deliver drugs, radionuclides and cytotoxins to target various cancer types that are GRPR positive. We investigated the in vitro binding properties of (177)Lu-AMBA, a novel radiolabelled BBN analog currently undergoing clinical trial as systemic radiotherapy for hormone refractory prostate cancer (HRPC) patients. Pharmacological analyses of the (177)Lu-AMBA was determined using in vitro binding studies using membrane target system containing specific receptor subtypes. We investigated the distribution of binding sites for (177)Lu-AMBA by receptor autoradiography on human neoplastic and non-neoplastic tissues. Pharmacological characterizations of (177)Lu-AMBA shows, high affinity towards NMB and GRP receptors, while little or no affinity towards BB(3) receptor. Among the 40 different types of non-neoplastic tissues tested seven of them showed limited but specific binding of (177)Lu-AMBA. Fourteen of 17 primary prostate cancers, six of 13 primary breast cancers expressed binding sites for (177)Lu-AMBA. Furthermore, no apparent differences in (177)Lu-AMBA-binding sites expression were observed between matched pairs (primary vs. secondary) of prostate and breast cancer tissues. These data represent the molecular basis for clinical applications of (177)Lu-AMBA for diagnosis and treatment of GRP-R and NMB-R positive tumors.

266 Objectives: We compared the biodistribution of 111In-, 67Ga- (a surrogate for 68Ga-), and 177Lu-AMBA in male nude mice xenographed with human (PC-3) prostate cancer cells [AMBA=DO3A-CH2CO-G-[4-aminobenzoyl]-QWAVGHLM-NH2]. This cell line expresses Gastrin-Releasing Peptide receptors (GRP-R), which are over-expressed in many cancers. 111In or 68Ga analogs of Lu-AMBA may have value for GRP-R positive tumor diagnosis, patient selection and evaluation of 177Lu-AMBA radiotherapeutic treatment effects. Methods: Compounds were prepared by reaction of radiometals with excess AMBA at 100oC and HPLC purified. PC-3 tumor-bearing mice (n=4, 4 and 9 per group for In-, Ga- and Lu-AMBA) were administered 5 μCi (0.1 mL) of test compound via i.v. tail vein, sacrificed after 1 and 24 h and tissues counted for residual radioactivity. Results: All compounds exhibited similar biodistributions and uptake in target and non-target tissues. Tumor uptake was favorable (4-6% ID/g) for all agents at 1 h, with 2.5-3% ID/g remaining at 24 h. The route of excretion was renal (~50-60% within 1 h). Kidney retention was low (≦1% ID at 24h) for all three compounds. Conclusions: Based on the comparable biodistribution results obtained for 111In, 67Ga and 177Lu-AMBA in PC-3 tumor bearing mice, and their high uptake and retention in GRP-R positive tissues, we anticipate that 68Ga-AMBA labeled for PET imaging or 111In-AMBA for gamma scintigraphy will demonstrate similar distribution clinically to the radiotherapeutic compound 177Lu-AMBA. The monosubstituted DOTA chelate in the AMBA ligand allows ready substitution of +3 radioisotopes useful for planar, SPECT, PET and radiotherapeutic applications.

Genentech Inc and Novartis Ophthalmics AG have developed and launched the humanized anti-VEGF antibody fragment ranibizumab, a 48-kDa humanized antibody fragment that inhibits all forms of biologically active VEGF-A, for the treatment of age-related macular degeneration by intravitreal administration. Phase I to III clinical trials to confirm the role of ranibizumab in the treatment of choroidal neovascularization (phase II and III), diabetic macular edema (phase II and III), retinal venous occlusion (phase II and III), telangiectasia (phase I and II), central serous chorioretinopathy (phase I), polypoidal choroidal vasculopathy (phase I/II), conjunctival neoplasms (phase I) and von Hippel-Lindau syndrome (phase I) are ongoing.

UNLABELLED:Gastrin-releasing peptide receptors (GRP-R) are upregulated in many cancers, including prostate, breast, and lung. We describe a new radiolabeled bombesin (BBN) analog for imaging and systemic radiotherapy that has improved pharmacokinetics (PK) and better retention of radioactivity in the tumor.METHODS:DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA) was synthesized and radiolabeled. The human prostate cancer cell line PC-3 was used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Receptor specificity was determined by in vitro autoradiography in human tissues. PK and radiotherapy studies were performed in PC-3 tumor-bearing male nude mice.RESULTS:177Lu-AMBA has a high affinity for the GRP-R (Kd, 1.02 nmol/L), with a maximum binding capacity (Bmax) of 414 fmol/10(6) cells (2.5 x 10(5) GRP-R/cell). Internalization was similar for 177Lu-AMBA (76.8%), 177Lu-BBN8 (72.9%), and 125I-[Tyr4]-BBN (74.9%). Efflux was markedly lower for 177Lu-AMBA (2.9%) compared with 177Lu-BBN8 (15.9%) and 125I-[Tyr4]-BBN (46.1%). By receptor autoradiography, Lu-AMBA binds specifically to GRP-R (0.8 nmol/L) and to the neuromedin B receptor (NMB-R) (0.9 nmol/L), with no affinity for the bb3 receptor (>1,000 nmol/L). 177Lu-AMBA was renally excreted (55 %ID 1 h [percentage injected dose at 1 h]); tumor uptake at 1 and 24 h was 6.35 %ID/g and 3.39 %ID/g, respectively. One or 2 doses of 177Lu-AMBA (27.75 MBq/dose) significantly prolonged the life span of PC-3 tumor-bearing mice (P < 0.001 and P < 0.0001, respectively) and decreased PC-3 tumor growth rate over controls. When compared using World Health Organization criteria, mice receiving 2 doses versus 1 dose of 177Lu-AMBA demonstrated a shift away from stable/progressive disease toward complete/partial response; by RECIST (Response Evaluation Criteria in Solid Tumors), median survival increased by 36% and time to progression/progression-free survival increased by 65%.CONCLUSION:177Lu-AMBA binds with nanomolar affinity to GRP-R and NMB-R, has low retention of radioactivity in kidney, demonstrates a very favorable risk-benefit profile, and is in phase I clinical trials.

A novel bifunctional maleimido CHX-A″ DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with 111In. The resulting 111In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A″ DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A″ DTPA for imaging and/or therapies of cancer.