Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain.

INTRODUCTION: Two 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABAA/benzodiazepine receptor by comparing them to [(11)C]flumazenil in rhesus monkey. METHODS: Radiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [(18)F]AH114726 and [(18)F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil. RESULTS: [(18)F]GEH120348 and [(18)F]AH114726 were obtained in 13-24% yields (end of synthesis) with high chemical (>95%) and radiochemical (>99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [(18)F]AH114726 and [(18)F]GEH120348 were determined in a non-human primate and directly compared with [(11)C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [(11)C]flumazenil and the known concentrations of GABAA/benzodiazepine receptors in the monkey brain. [(18)F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [(11)C]flumazenil. In contrast, [(18)F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil. CONCLUSION: The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [(11)C]flumazenil, support that [(18)F]AH114726 is a suitable option for imaging the GABAA receptor.

Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3).

Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH(2) (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.

Automated synthesis, characterization and biological evaluation of [(68)Ga]Ga-AMBA, and the synthesis and characterization of (nat)Ga-AMBA and [(67)Ga]Ga-AMBA.

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.

177Lu-AMBA biodistribution, radiotherapeutic efficacy, imaging, and autoradiography in prostate cancer models with low GRP-R expression.

UNLABELLED: (177)Lu-DO3A-CH(2)CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH(2) ((177)Lu-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, (177)Lu-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 (177)Lu-AMBA in low GRP-R models of prostate cancer and determine how reduced expression affects (177)Lu-AMBA radiotherapy efficacy. METHODS: The LNCaP and DU145 cell lines were used to determine the binding (K(d)), retention, and efflux of (177)Lu-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: (177)Lu-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 (177)Lu-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 (177)Lu-AMBA identify tumors in vivo. Despite having lower uptake, (177)Lu-AMBA demonstrated radiotherapeutic efficacy and decreased proliferation in the LNCaP and DU145 xenografts; in the LNCaP model, (177)Lu-AMBA normalized the phenotype of microvasculature, reducing tumoral blood pooling. CONCLUSION: (177)Lu-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.

In vitro and in vivo metabolism of Lu-AMBA, a GRP-receptor binding compound, and the synthesis and characterization of its metabolites.

The metabolism of (177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-(4-aminobenzoyl)-QWAVGHLM-NH(2)), a radiotherapeutic 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 (177)Lu-AMBA). The primary metabolites were Lu-DO3A-CH(2)CO-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 (177)Lu-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 (nat)Lu and (177)Lu 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 (177)Lu-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact (177)Lu-AMBA. IC(50) values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against (177)Lu-AMBA. No direct binding to PC-3 cells was observed with (177)Lu-labeled A-C, confirming IC(50) results. (177)Lu-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 (approximately 78-87%) within 1 h. These results demonstrate that the tumor uptake observed with (177)Lu-AMBA is due to parent drug and not due to any of its identified metabolites.

Isolation of a 177Hf complex formed by beta-decay of a 177Lu-labeled radiotherapeutic compound and NMR structural elucidation of the ligand and its Lu and Hf complexes.

(177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is being developed for the radiotherapeutic treatment of tumors that express the gastrin-releasing peptide receptor (GRP-R). In this study we investigated the fate of the (177)hafnium ((177)Hf) that forms upon the decay of (177)Lu while the latter is complexed with AMBA. When decayed solutions of (177)Lu-AMBA were analyzed, it was found that (177)Hf is retained in the DO3A monoamide chelator, forming a pair of interconverting isomers. We report the synthesis and full characterization of (nat)Lu-AMBA and the studies performed to demonstrate its correspondence to radioactive (177)Lu-AMBA. We also report the synthesis and characterization of Hf-AMBA and, by NMR studies, show structural analogies between Hf-AMBA, its parent compound Lu-AMBA, and the unmetallated AMBA ligand. In the NMR spectra of both the metallated and unmetallated AMBA ligand, a stacking interaction between the amino benzoyl residue in the linker and a tryptophan in the truncated bombesin [BBN(7-14)-NH(2)] peptide targeting group was found.

Synthesis, stabilization and formulation of [177Lu]Lu-AMBA, a systemic radiotherapeutic agent for Gastrin Releasing Peptide receptor positive tumors.

A robust formulation was developed for [(177)Lu]Lu-AMBA ((177)Lu-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)), a Bombesin-like agonist with high affinity for Gastrin Releasing Peptide (GRP) receptors. During optimization of labeling, the effect of several radiostabilizers was evaluated; a combination of selenomethionine and ascorbic acid showed superiority over other tested radiostabilizers. The resulting two-vial formulation maintains a radiochemical purity (RCP) of >90% for at least 2 days at room temperature. The method of stabilization should be useful for other methionine-containing peptide radiopharmaceuticals in diagnostic and therapeutic applications.

177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer.

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.

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor.

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.