Impact of liver tumour burden, alkaline phosphatase elevation, and target lesion size on treatment outcomes with 177Lu-Dotatate: an analysis of the NETTER-1 study.

PURPOSE: To assess the impact of baseline liver tumour burden, alkaline phosphatase (ALP) elevation, and target lesion size on treatment outcomes with 177Lu-Dotatate. METHODS: In the phase 3 NETTER-1 trial, patients with advanced, progressive midgut neuroendocrine tumours (NET) were randomised to 177Lu-Dotatate (every 8 weeks, four cycles) plus octreotide long-acting release (LAR) or to octreotide LAR 60 mg. Primary endpoint was progression-free survival (PFS). Analyses of PFS by baseline factors, including liver tumour burden, ALP elevation, and target lesion size, were performed using Kaplan-Meier estimates; hazard ratios (HRs) with corresponding 95% CIs were estimated using Cox regression. RESULTS: Significantly prolonged median PFS occurred with 177Lu-Dotatate versus octreotide LAR 60 mg in patients with low (< 25%), moderate (25-50%), and high (> 50%) liver tumour burden (HR 0.187, 0.216, 0.145), and normal or elevated ALP (HR 0.153, 0.177), and in the presence or absence of a large target lesion (diameter > 30 mm; HR, 0.213, 0.063). Within the 177Lu-Dotatate arm, no significant difference in PFS was observed amongst patients with low/moderate/high liver tumour burden (P = 0.7225) or with normal/elevated baseline ALP (P = 0.3532), but absence of a large target lesion was associated with improved PFS (P = 0.0222). Grade 3 and 4 liver function abnormalities were rare and did not appear to be associated with high baseline liver tumour burden. CONCLUSIONS: 177Lu-Dotatate demonstrated significant prolongation in PFS versus high-dose octreotide LAR in patients with advanced, progressive midgut NET, regardless of baseline liver tumour burden, elevated ALP, or the presence of a large target lesion. Clinicaltrials.gov : NCT01578239, EudraCT: 2011-005049-11.

Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors.

BACKGROUND: Patients with advanced midgut neuroendocrine tumors who have had disease progression during first-line somatostatin analogue therapy have limited therapeutic options. This randomized, controlled trial evaluated the efficacy and safety of lutetium-177 (177Lu)-Dotatate in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors. METHODS: We randomly assigned 229 patients who had well-differentiated, metastatic midgut neuroendocrine tumors to receive either 177Lu-Dotatate (116 patients) at a dose of 7.4 GBq every 8 weeks (four intravenous infusions, plus best supportive care including octreotide long-acting repeatable [LAR] administered intramuscularly at a dose of 30 mg) (177Lu-Dotatate group) or octreotide LAR alone (113 patients) administered intramuscularly at a dose of 60 mg every 4 weeks (control group). The primary end point was progression-free survival. Secondary end points included the objective response rate, overall survival, safety, and the side-effect profile. The final analysis of overall survival will be conducted in the future as specified in the protocol; a prespecified interim analysis of overall survival was conducted and is reported here. RESULTS: At the data-cutoff date for the primary analysis, the estimated rate of progression-free survival at month 20 was 65.2% (95% confidence interval [CI], 50.0 to 76.8) in the 177Lu-Dotatate group and 10.8% (95% CI, 3.5 to 23.0) in the control group. The response rate was 18% in the 177Lu-Dotatate group versus 3% in the control group (P<0.001). In the planned interim analysis of overall survival, 14 deaths occurred in the 177Lu-Dotatate group and 26 in the control group (P=0.004). Grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia occurred in 1%, 2%, and 9%, respectively, of patients in the 177Lu-Dotatate group as compared with no patients in the control group, with no evidence of renal toxic effects during the observed time frame. CONCLUSIONS: Treatment with 177Lu-Dotatate resulted in markedly longer progression-free survival and a significantly higher response rate than high-dose octreotide LAR among patients with advanced midgut neuroendocrine tumors. Preliminary evidence of an overall survival benefit was seen in an interim analysis; confirmation will be required in the planned final analysis. Clinically significant myelosuppression occurred in less than 10% of patients in the 177Lu-Dotatate group. (Funded by Advanced Accelerator Applications; NETTER-1 ClinicalTrials.gov number, NCT01578239 ; EudraCT number 2011-005049-11 .).

Peptide receptor radionuclide therapy with 177Lu-DOTATATE for patients with somatostatin receptor-expressing neuroendocrine tumors: the first US phase 2 experience.

OBJECTIVE: Peptide receptor radionuclide therapy with radiolabeled somatostatin analogs is a novel method of treatment in patients with metastatic neuroendocrine tumors (NETs). For the first time in the United States, we present preliminary results of the treatment with Lutetium (177)(Lu) DOTATATE in patients with progressive NETs. METHODS: Thirty-seven patients with grade 1 and grade 2 disseminated and progressive gastroenteropancreatic NET were enrolled in a nonrandomized, phase 2 clinical trial. Repeated cycles of 200 mCi (7.4 GBq; ±10%) were administered up to the cumulative dose of 800 mCi (29.6 GBq; ±10%). RESULTS: Among 32 evaluable patients, partial response and minimal response to treatment were seen in 28% and 3%, respectively, and stable disease was seen in 41% of patients. A total of 28% had progressive disease. A response to treatment was significantly associated with lower burden of disease in the liver. No significant acute or delayed hematologic or kidney toxicity was observed. An impressive improvement of performance status and quality of life were seen after Lu-DOTATATE therapy. CONCLUSIONS: Treatment with multiple cycles of (177)Lu-DOTATATE peptide receptor radionuclide therapy is well tolerated. This treatment results in control of the disease in most patients, whereas systemic toxicities are limited and reversible. Quality of life is also improved.

Comparison of three radiolabelled peptide analogues for CCK-2 receptor scintigraphy in medullary thyroid carcinoma.

PURPOSE: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in a high percentage of medullary thyroid carcinomas (MTC). Analogous to somatostatin receptors, CCK-2 receptors might be viable targets for radionuclide scintigraphy and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed, and some have been carried through into clinical studies. However, these studies are mostly limited and difficult to compare. The aim of this study was to evaluate the diagnostic and therapeutic potential of three promising CCK-2 receptor-binding radiopeptides in patients with MTC. METHODS: (111)In-DOTA-(D: )Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) ((111)In-DOTA-CCK), a CCK analogue, and the gastrin-based ligands (99m)Tc-N(4)-Gly-(D: )Glu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-demogastrin 2) and (111)In-DOTA-(D: )Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((111)In-DOTA-MG11) were each administered to the same group of six patients. Planar images made at 3-5, 7 and 24 h p.i. were used for comparison of tumour visualisation and renal uptake. RESULTS: (99m)Tc-demogastrin 2 scintigraphy visualised all known lesions and new lesions in four of six patients. (111)In-DOTA-CCK and (111)In-DOTA-MG11 on the other hand missed several lesions; tumour uptake of these two radiopharmaceuticals was quite low. Comparison of retention of renal activity showed no major differences between the three radiopeptides. CONCLUSION: (99m)Tc-demogastrin 2 scintigraphy appeared most promising as a diagnostic tool in patients with MTC. Further studies are required to evaluate its value in patient management. Direct comparisons of the compounds studied strongly suggests that (111)In-DOTA-CCK and (111)In-DOTA-MG11 have less potential as imaging agents than (99m)Tc-demogastrin 2. These DOTA-linked compounds are considered unlikely to be useful for radionuclide therapy because of low tumour uptake.

Five Stabilized 111In-labeled neurotensin analogs in nude mice bearing HT29 tumors.

Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four (111)In-labeled DTPA-chelated NT analogs and one (111)In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

Molecular imaging of gastrin-releasing peptide receptor-positive tumors in mice using 64Cu- and 86Y-DOTA-(Pro1,Tyr4)-bombesin(1-14).

Bombesin is a tetradecapeptide neurohormone that binds to gastrin-releasing peptide receptors (GRPR). GRPRs have been found in a variety of cancers including invasive breast and prostate tumors. The peptide MP2346 (DOTA-(Pro(1),Tyr(4))-bombesin(1-14)) was designed to bind to these GRP receptors. This study was undertaken to evaluate radiolabeled MP2346 as a positron emission tomography (PET) imaging agent. MP2346 was radiolabeled, in high radiochemical purity, with the positron-emitting nuclides (64)Cu (t(1/2) = 12.7 h, beta+ = 19.3%, E(avg) = 278 keV) and (86)Y (t(1/2) = 14.7 h, beta+ = 33%, E(avg) = 664 keV). (64)Cu-MP2346 and (86)Y-MP2346 were studied in vitro for cellular internalization by GRPR-expressing PC-3 (human prostate adenocarcinoma) cells. Both (64)Cu- and (86)Y-MP2346 were studied in vivo for tissue distribution in nude mice with PC-3 tumors. Biodistribution in PC3 tumor-bearing mice demonstrated higher tumor uptake, but lower liver retention, in animals injected with (86)Y-MP2346 compared to (64)Cu-MP2346. Receptor-mediated uptake was confirmed by a significant reduction in uptake in the PC-3 tumor and other receptor-rich tissues by coinjection of a blockade. Small animal PET/CT imaging was carried out in mice bearing PC-3 tumors and rats bearing AR42J tumors. It was possible to delineate PC-3 tumors in vivo with (64)Cu-MP2346, but superior (86)Y-MP2346-PET images were obtained due to lower uptake in clearance organs and lower background activity. The (86)Y analogue demonstrated excellent PET image quality in models of prostate cancer for the delineation of the GRPR-rich tumors and warrants further investigation.

Anticancer activity of targeted proapoptotic peptides.

UNLABELLED: Tumor-induced angiogenesis can be targeted by RGD (Arg-Gly-Asp) peptides, which bind to alpha(v)beta(3)-receptors upregulated on angiogenic endothelial cells. RGD-containing peptides are capable of inducing apoptosis through direct activation of procaspase-3 to caspase-3 in cells. Additionally, tumor cells overexpressing somatostatin receptors can be targeted by somatostatin analogs. Radiolabeled somatostatin analogs are successfully used to image and treat such tumors via receptor-targeted scintigraphy and therapy. We combined these 2 peptides, RGD and somatostatin, to synthesize a new hybrid peptide, RGD-diethylenetriaminepentaacetic acid (DTPA)-octreotate (c(Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). An earlier study showed that tumor-bearing rats had high receptor-specific uptake of RGD-(111)In-DTPA-octreotate in somatostatin receptor subtype 2-positive tissues and tumors. Furthermore, RGD-(111)In-DTPA-octreotate showed a pronounced tumoricidal effect, which is probably the result of increased apoptosis, as is shown by an increased caspase-3 activity after incubation with (111)In-labeled RGD-DTPA-octreotate in comparison with the 2 monopeptides (111)In-DTPA-RGD and (111)In-DTPA-Tyr(3)-octreotate. In this study, we evaluated the biodistributions of RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate and investigated the caspase-3 activation of the unlabeled compound RGD-DTPA-octreotate in vitro. METHODS: Biodistribution studies on tumor-bearing rats were performed with RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate. The apoptotic activity, by activation of caspase-3 with RGD-DTPA-octreotate and RGD-octreotate, was examined using colorimetric and immunocytochemical assays. RESULTS: The radiolabeled compound, RGD-(111)In-DTPA-octreotate, showed high uptake and retention in the rats in which rat pancreatic CA20948 tumor had been implanted. A major drawback was high renal uptake. In vitro, the unlabeled peptide RGD-DTPA-octreotate induced a significant increase in caspase-3 levels in various cell lines in comparison with RGD and Tyr(3)-octreotate (P < 0.01). Caspase-3 activation was time dependent. To alter the elimination route, we examined the biodistribution of radioiodinated RGD-octreotate without DTPA [c(Arg-Gly-Asp-D-Tyr-Asp)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr], as a model of unlabeled RGD-octreotate, in tumor-bearing rats. (125)I-RGD-octreotate showed a much lower renal uptake than did RGD-(111)In-DTPA-octreotate. Furthermore, the affinity of RGD-octreotate increased in comparison with RGD-DTPA-octreotate (values of 1.4 x 10(-8) mol/L vs. 9.4 x 10(-8) mol/L, respectively, for inhibitory concentration of 50%). Finally, RGD-octreotate was still able to activate caspase-3, as was indicated with immunocytochemistry. CONCLUSION: Because of the high renal uptake, RGD-(111)In-DTPA-octreotate is unsuitable for radionuclide therapy. However, the unlabeled peptides, RGD-DTPA-octreotate and RGD-octreotate, also induced an increase in caspase-3 levels, indicating the therapeutic potential of this compound. Thus, the development of hybrid molecules can become a new approach in the treatment of cancer.

Novel bioactive and stable neurotensin peptide analogues capable of delivering radiopharmaceuticals and molecular beacons to tumors.

The prevalence of neurotensin receptor (NTR) in several human tumors makes it an attractive target for the delivery of cytotoxic drugs and imaging agents. Native neurotensin (NT) is a tridecapeptide that binds to NTR and induces tumor growth. Unfortunately, NT has a short plasma half-life, which hinders its use for in vivo biomedical applications. Numerous reports suggest that Arg(8)-Arg(9) and Tyr(11)-Ile(12) amide bonds are particularly susceptible to degradation by proteolytic enzymes. Predicated on this observation, we substituted Arg(8), Arg(9), and Ile(12) amino acids with the corresponding commercially available mimics. These surrogate amino acids are amenable to standard Fmoc peptide synthesis strategy, and the resulting compounds are stable in biological media for >4 h and bind to NTR with high affinity. Furthermore, conjugating DTPA to the new peptides and subsequent labeling with (111)In-DTPA for nuclear imaging or fluorescein for optical imaging did not diminish the NTR binding affinities of the peptides. In vivo biodistribution of a representative (111)In-DTPA-NT peptide analogue in SCID mice bearing NTR-positive human adenocarcinoma (HT29) xenograft shows that the compound was primarily retained in tumor tissue (2.2% ID/g) and the kidneys (4.8% ID/g) at 4 h postinjection. Coinjection of cold NT and the radiolabeled NT peptide analogue inhibited the tumor but not the kidney uptake, demonstrating that retention of the radiolabeled compound in tumor tissue was mediated by NTR specific uptake while it accumulates in the kidneys by a nonspecific mechanism. These findings show that the new NT peptide analogues are robust and can deliver imaging agents to NTR-positive tumors such as pancreatic cancer.

Optimising conditions for radiolabelling of DOTA-peptides with 90Y, 111In and 177Lu at high specific activities.

DOTA-conjugated peptides, such as [DOTA(0),Tyr(3)]octreotide (DOTATOC) and [DOTA(0),Tyr(3)]octreotate (DOTA-tate), can be labelled with radionuclides such as (90)Y, (111)In and (177)Lu. These radiolabelled somatostatin analogues are used for peptide receptor radionuclide therapy (PRRT). Radioligands for PRRT require high specific activities. However, although these radionuclides are produced without addition of carrier, contaminants are introduced during production and as decay products. In this study, parameters influencing the kinetics of labelling of DOTA-peptides were investigated and conditions were optimised to obtain the highest achievable specific activity. The effects of contaminants were systematically investigated, concentration dependently, in a test model mimicking conditions for labelling with minimal molar excess of DOTA-peptides over radionuclide. Kinetics of labelling of DOTA-peptides were optimal at pH 4-4.5; pH <4 strongly slowed down the kinetics. Above pH 5, reaction kinetics varied owing to the formation of radionuclide hydroxides. Labelling with (90)Y and (177)Lu was completed after 20 min at 80 degrees C, while labelling with (111)In was completed after 30 min at 100 degrees C. The effects of contaminants were systematically categorised, e.g. Cd(2+) is the target and decay product of (111)In, and it was found to be a strong competitor with (111)In for incorporation in DOTA. In contrast, Zr(4+) and Hf(4+), decay products of (90)Y and (177)Lu, respectively, did not interfere with the incorporation of these radionuclides. The following conclusions are drawn: (a) DOTA-peptides can be radiolabelled at high specific activity; (b) reaction kinetics differ for each radionuclide; and (c) reactions can be hampered by contaminants, such as target material and decay products.

The addition of DTPA to [177Lu-DOTA0,Tyr3]octreotate prior to administration reduces rat skeleton uptake of radioactivity.

Peptide receptor-targeted radionuclide therapy is nowadays also being performed with DOTA-conjugated peptides, such as [DOTA(0),Tyr(3)]octreotate, labelled with radionuclides like (177)Lu. The incorporation of (177)Lu is typically >/=99.5%; however, since a total patient dose can be as high as 800 mCi, the amount of free (177)Lu(3+) (= non-DOTA-incorporated) can be substantial. Free (177)Lu(3+) accumulates in bone with unwanted irradiation of bone marrow as a consequence. (177)Lu-DTPA is reported to be stable in serum in vitro, and in vivo it has rapid renal excretion. Transforming free Lu(3+) to Lu-DTPA might reroute this fraction from accumulation in bone to renal clearance. We therefore investigated: (a) the biodistribution in rats of (177)LuCl(3), [(177)Lu-DOTA(0),Tyr(3)]octreotate and (177)Lu-DTPA; (b) the possibilities of complexing the free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate to (177)Lu-DTPA prior to intravenous injection; and (c) the effects of free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate, in the presence and absence of DTPA, on the biodistribution in rats. (177)LuCl(3) had high skeletal uptake (i.e. 5% ID per gram femur, with localization mainly in the epiphyseal plates) and a 24-h total body retention of 80% injected dose (ID). [(177)Lu-DOTA(0),Tyr(3)]octreotate had high and specific uptake in somatostatin receptor-positive tissues, and 24-h total body retention of 19% ID. (177)Lu-DTPA had rapid renal clearance, and 24-h total body retention of 4% ID. Free (177)Lu(3+) in [(177)Lu-DOTA(0),Tyr(3)]octreotate could be complexed to (177)Lu-DTPA. Accumulation of (177)Lu in femur, blood, liver and spleen showed a dose relation to the amount of free (177)Lu(3+), while these accumulations could be normalized by the addition of DTPA. After labelling [DOTA(0),Tyr(3)]octreotate with (177)Lu the addition of DTPA prior to intravenous administration of [(177)Lu-DOTA(0),Tyr(3)]octreotate is strongly recommended.

Preclinical comparison of (111)In-labeled DTPA- or DOTA-bombesin analogs for receptor-targeted scintigraphy and radionuclide therapy.

UNLABELLED: The 14-amino-acid peptide bombesin (BN) has a high affinity for the gastrin-releasing peptide (GRP) receptor that is expressed by a variety of tumors. Recently, high densities of GRP receptors were identified by in vitro receptor autoradiography in human prostate and breast carcinomas using [(125)I-Tyr(4)]BN as radioligand. Radiometal-labeled diethylenetriaminepentaacetic acid (DTPA)-BN derivatives are potentially useful radioligands for receptor-targeted scintigraphy and radiotherapy of GRP receptor-expressing tumors. METHODS: [DTPA-Pro(1),Tyr(4)]BN (A), [DOTA-Pro(1),Tyr(4)]BN (B), [DTPA-epsilon-Lys(3),Tyr(4)]BN (C), and [DOTA-epsilon-Lys(3),Tyr(4)]BN (D) (where DOTA is dodecanetetraacetic acid) were synthesized and studied for competition with binding of [(125)I-Tyr(4)]BN to the GRP receptor. The (111)In-labeled BN analogs were studied in vitro for binding and internalization by GRP receptor-expressing CA20948 and AR42J pancreatic tumor cells as well as in vivo for tissue distribution in rats. Specific tissue binding was tested by coinjection of 0.1 mg [Tyr(4)]BN. RESULTS: All BN analogs competitively inhibited the binding of [(125)I-Tyr(4)]BN to the GRP receptor with 50% inhibitory concentration values in the range of 2-9 nmol/L. All (111)In-labeled analogs showed high and specific time- and temperature-dependent binding and internalization by CA20948 and AR42J cells. In in vivo studies, high and specific binding was found in GRP receptor-positive tissues such as pancreas (0.90, 1.2, 0.54, and 0.79 percentage injected dose per gram for A-D, respectively). In a rat model, the AR42J tumor could clearly be visualized by scintigraphy using [(111)In-DTPA-Pro(1),Tyr(4)]BN as the radioligand. Although [(111)In-DOTA-Pro(1),Tyr(4)]BN showed the highest uptake of radioactivity in GRP receptor-positive tissues as well as higher target-to-blood ratios, [(111)In-DTPA-Pro(1),Tyr(4)]BN was easier to handle and is more practical to use. Therefore, we decided to start phase I studies with this DTPA-conjugated radioligand. CONCLUSION: [(111)In-DTPA-Pro(1),Tyr(4)]BN is a promising radioligand for scintigraphy of GRP receptor-expressing tumors. We are currently performing a phase I study on patients with invasive prostate carcinoma.

DOTA-D-Tyr(1)-octreotate: a somatostatin analogue for labeling with metal and halogen radionuclides for cancer imaging and therapy.

The goal of this study was to evaluate a somatostatin receptor ligand, DOTA-D-Tyr(1)-octreotate (DOTA-DY1-TATE), that has the chelator 1,4,7,10-tetraazacyclotetradecane-N,N',N'',N'"-tetraacetic acid (DOTA) attached to the D-Tyr(1) residue, allowing radiolabeling with both radiohalogens and radiometals. A potential advantage of having a chelator attached to the Tyr(1) residue is that halogen radiolabels may residualize or remain trapped in tumor cells rather than clear from the tumor. DOTA-DY1-TATE was synthesized by solid-phase methods and radiolabeled with (61)Cu, (64)Cu, and (125)I in high radiochemical purity and specific activity. A competitive binding assay demonstrated that (nat)Cu-DOTA-DY1-TATE and DOTA-(nat)I-DY1-TATE had comparable affinity to (nat)In-DTPA-OC in AR42J rat pancreatic tumor cells membranes. (61)Cu-DOTA-DY1-TATE had a dissociation constant (K(d)) of 176.4 pM and a receptor concentration (B(max)) of 244.4 fmol/mg. A tumor uptake of 1.515 %ID/g was determined for (64)Cu-DOTA-DY1-TATE and 0.814 %ID/g for DOTA-(125)I-DY1-TATE in AR42J tumor bearing Lewis rats at 1 h postinjection. DOTA-(125)I-DY1-TATE remained in the tumor at a higher concentration out to 4 h postinjection, suggesting that the iodine may have residualized in the tumor cells. MicroPET imaging of (64)Cu-DOTA-DY1-TATE in AR42J tumor bearing rats and SCID mice at 2 h postinjection showed significant uptake and good contrast in the thigh tumors in the rat model and in the neck and thigh tumors of the mouse. This study demonstrates that DOTA-DY1-TATE is a somatostatin analogue that can be labeled with both metal and halogen radionuclides, and its (64)Cu- and (125)I-radiolabeled compounds showed somatostatin receptor-mediated uptake in normal and tumor tissues.

Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.