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.

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.

Molecular imaging and personalized medicine: an uncertain future.

The Food and Drug Administration has described their view of the role that imaging will play in the approval, and perhaps postapproval, use of new therapeutic drugs. The therapeutic drug industry and regulatory authorities have turned to imaging to help them achieve better efficiency and efficacy. We must extend this initiative by demonstrating that molecular imaging can also improve the efficiency and efficacy of routine treatment with these same drugs. The role of molecular imaging in personalized medicine, using targeted drugs in oncology, is very attractive because of the regional information that it provides (in many cases, with a functional or dynamic component), which cannot be provided by in vitro methods ("regional proteomics"). There is great potential for molecular imaging to play a major role in selecting appropriate patients and providing early proof of response, which is critical to addressing the conflict between the high price of treatment and limited reimbursement budgets. This is a new venture in both molecular imaging and targeted drugs. However, there are various regulatory, financial, and practical barriers that must be overcome to achieve this aim, in addition to the normal scientific challenges of drug discovery. There is an urgent need to reduce the cost (i.e., time and money) of developing imaging agents for routine clinical use. The mismatch between the current regulations and personalized medicine includes molecular imaging and requires the engagement of the regulatory authorities to correct. Therapeutic companies must be engaged early in the development of new targeted drugs and molecular imaging agents to improve the fit between the two drug types. Clinical trials must be performed to generate data that not only shows the efficacy of imaging plus therapy in a medical sense, but also in a financial sense. Molecular imaging must be accepted as not just good science but also as central to routine patient management in the personalized medicine of the future.

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.

The cost of developing imaging agents for routine clinical use.

The objective of this study was to estimate the financial cost of developing new imaging agents for clinical use and to discuss the effects of these costs on the future clinical imaging agent environment. Publicly available financial data from the annual reports of major companies developing and selling imaging agents were examined and the data used to develop cost estimates. These estimates were compared with the in-depth data and analyses available for the development costs of therapeutic drugs. The cost of developing a drug for diagnostic imaging to commercialization is in the 100 dollars to 200 million dollars range, whereas a blockbuster imaging drug has current sales of 200 dollars to 400 million dollars. Most of these blockbuster imaging agents have been on the market for some time. The majority provide morphologic images with general indications in a slowly changing section of the market. Future agents will most likely address smaller markets and be in the rapidly developing molecular imaging field. The costs are high and are a significant brake on the development of imaging agents for commercialization. If new imaging agents are to realize their commercial potential, ways must be found to make the financials more attractive. The prices per dose are currently low so they must either be greatly increased for new imaging agents, with a corresponding increase in the value of the information they provide, or the use of imaging agents must be widened and/or their development made less costly in time and money. Without addressing these issues, the commercialization of new imaging agents will continue to be slow and may get slower. This will impact the progress of imaging agents toward use as validated biomarkers.

Identification of ideal peptides for heterovalent ligands.

Recent use of hetero-dimerization to improve the affinity of peptide ligands has made peptides an attractive alternative to small molecules and proteins. Using this strategy, we have developed peptides with affinities comparable to antibodies and specificities often better than small molecules or antibodies. These peptides can be used as a delivery vehicle for drugs or diagnostics, especially in the case of tumor targeting cytotoxic drugs or targeted diagnostics. We describe here an assay to identify an ideal pair of peptides suitable for heterovalent ligands.

Use of ultrasound contrast agent microbubbles in preclinical research: recommendations for small animal imaging.

Ultrasound contrast imaging techniques represent a real opportunity to improve efficiency in the preclinical drug discovery and development process. Ultrasound contrast agents (UCAs) combined with specific ultrasound contrast detection modes provide real-time, high spatial resolution of both organ and lesion blood perfusion, the so-called dynamic contrast-enhanced ultrasound imaging. With the advent of targeted UCA, ultrasound molecular imaging is gaining momentum in molecular imaging, particularly because of the simultaneous real-time anatomical and functional/molecular imaging capabilities. In preclinical research, contrast-enhanced ultrasound imaging, with either nontargeted or targeted UCA, is a fast-growing imaging modality that has not yet been standardized compared with other imaging modalities. Contrast-enhanced ultrasound imaging is an operator-dependent imaging modality, requiring adherence to rigorous step-by-step protocols. In this article, which is intended for advanced, hands-on researchers, we report key factors that can lead to variability in preclinical results and recommend some preventive methods to limit or cancel their effect on the final results. Standardized procedures are a prerequisite for acceptance of new contrast-enhanced ultrasound imaging methods to eliminate factors that could distort results, improve the reproducibility between different centers and studies, and, therefore, allow translation to clinical application.

Selective induction of cell adhesion molecules by proinflammatory mediators in human cardiac microvascular endothelial cells in culture.

Pro-inflammatory mediators can dramatically alter many responses of cultured endothelial cells in vitro, which are relevant to understanding the role played by the endothelium in inflammation in vivo. The aim of this study was to determine the ability of a comprehensive array of pro-inflammatory stimuli to modulate Cell Adhesion Molecule (CAM) expression in cultures of human microvascular cardiac endothelial cells (HMVEC.c). Cell ELISA, immunocy-tochemistry and flow cytometry were used to measure the CAM expressions in HMVEC.c in response to interleukins, TNF-α and LPS. Passage matched HMVEC.c from different donors showed different CAM expression profiles, confirming inherent variability in endothelial cells. Endothelial cells from different parts of the vasculature are exposed to different cytokines and thus different protein expression profiles. A thorough understanding of these innate differences in expression pattern of the microvasculatures of cardiac tissues might allow us the opportunity to target these tissues selectively.

Upregulation of coronary endothelial P-selectin in a monkey heart ischemia reperfusion model.

The design of targeted ultrasound contrast agents for molecular imaging of myocardial ischemia-reperfusion (IR) requires the availability of an adequate in vivo model in a species in which cross reactivity with the target occurs. P-selectin (Psel) is an activation-dependent endothelial receptor that supports rapid and reversible cell adhesion in a flowing system. Together with E- and L-selectins it constitutes the selectin family of adhesion molecules. We investigated the myocardial expression of selectins in a closed chest minimally invasive monkey myocardial IR model. Catheter-based occlusion (30-50 min) followed by reperfusion (3 or 5 h) of left anterior descending artery (LAD) was performed in anesthetised monkeys. At the end of each procedure animals were killed, and their hearts were excised. The tissues were analyzed immunohistochemically using an anti-human Psel antibody (AK-6 clone) that cross reacts with rhesus monkey. Histopathological features confirm the presence of IR injuries in myocardial tissues. There was significant increase in the Psel expression in vessels from the IR areas. However, significantly higher Psel immunoreactivity was also seen in areas which are distant from IR injuries.

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.

In vitro binding evaluation of 177Lu-AMBA, a novel 177Lu-labeled GRP-R agonist for systemic radiotherapy in human tissues.

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.

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.

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.

Phase 1 study to identify tumour hypoxia in patients with head and neck cancer using technetium-99m BRU 59-21.

The aim of this study was to assess the safety and biodistribution of technetium-99m BRU 59-21, a novel radioactively labelled 2-nitro-imidazole hypoxic marker, in head and neck cancer patients and to correlate uptake with pimonidazole staining. (99m)Tc-BRU 59-21 was administered intravenously (mean dose 824 MBq, range 780-857 MBq) to ten head and neck cancer patients scheduled for primary surgery, and whole-body images and SPET scans were then obtained. Uptake of radioactivity in the regions of interest was determined and tumour to normal tissue ratios were calculated after correlative evaluation with MRI/CT. Twelve to 16 h before surgery (up to 2 weeks after the scan), patients received pimonidazole intravenously. Tumour sections were stained immunohistochemically for pimonidazole binding. No serious adverse events were reported. In five patients there were ten adverse events, which were mild in intensity and resolved completely without intervention. Uptake of (99m)Tc-BRU 59-21 was observed in eight of the ten primary tumours. Tumour to normal tissue ratios on the SPET scans for primary tumour and lymph nodes increased from 1.8 (range 0.9-2.7) to 2.1 (range 0.8-3.7) between 30 min and 3 h post injection. Tumour to normal tissue ratios in the primary tumour were significantly correlated with pimonidazole staining for SPET scans performed 30 min and 3 h post injection ( P=0.016 and P=0.037, respectively). When primary tumour and involved lymph nodes were considered in conjunction, correlation between the tumour to normal tissue ratio and pimonidazole staining was observed for early ( P<0.001) but not for late SPET scans ( P=0.076). However, late scans showed better tumour delineation than early scans. Administration of (99m)Tc-BRU 59-21 in head and neck cancer patients appears to be safe and feasible. Uptake and retention in tumour tissue was observed, suggestive of tumour hypoxia, and this was supported by correlations with staining for the hypoxic marker pimonidazole.