Single Chelator-Minibody Theranostic Agents for 89Zr PET Imaging and 177Lu Radiopharmaceutical Therapy of PSMA-Expressing Prostate Cancer.

Here we describe an anti-prostate-specific membrane antigen (PSMA) minibody (IAB2MA) conjugated to an octadentate, macrocyclic chelator based on four 1-hydroxypyridin-2-one coordinating units (Lumi804 [L804]) labeled with 89Zr (PET imaging) and 177Lu (radiopharmaceutical therapy), with the goal of developing safer and more efficacious treatment options for prostate cancer. Methods: L804 was compared with the current gold standard chelators, DOTA and deferoxamine (DFO), conjugated to IAB2MA for radiolabeling with 177Lu and 89Zr in cell binding, preclinical biodistribution, imaging, dosimetry, and efficacy studies in the PSMA-positive PC3-PIP tumor-bearing mouse model of prostate cancer. Results: Quantitative radiolabeling (>99% radiochemical yield) of L804-IAB2MA with 177Lu or 89Zr was achieved at ambient temperature in under 30 min, comparable to 89Zr labeling of DFO-IAB2MA. In contrast, DOTA-IAB2MA was radiolabeled with 177Lu for 30 min at 37°C in approximately 90% radiochemical yield, requiring further purification. Using europium(III) as a luminescent surrogate, high binding affinity of Eu-L804-IAB2MA to PSMA was demonstrated in PC3-PIP cells (dissociation constant, 4.6 ± 0.6 nM). All 4 radiolabeled constructs showed significantly higher levels of internalization after 30 min in the PC3-PIP cells than in PSMA-negative PC3-FLU cells. The accumulation of 177Lu- and 89Zr-L804-IAB2MA in PC3-PIP tumors and all organs examined (i.e., heart, liver, spleen, kidney, muscle, salivary glands, lacrimal glands, carcass, and bone) was significantly lower than that of 177Lu-DOTA-IAB2MA and 89Zr-DFO-IAB2MA at 96 and 72 h after injection, respectively. Generally, SPECT/CT and PET/CT imaging data showed no significant difference in the SUVmean of the tumors or muscle between the radiotracers. Dosimetry analysis via both organ-level and voxel-level dose calculation methods indicated significantly higher absorbed doses of 177Lu-DOTA-IAB2MA in tumors, kidney, liver, muscle, and spleen than of 177Lu-L804-IAB2MA. PC3-PIP tumor-bearing mice treated with single doses of 177Lu-L804-IAB2MA (18.4 or 22.2 MBq) exhibited significantly prolonged survival and reduced tumor volume compared with unlabeled minibody control. No significant difference in survival was observed between groups of mice treated with 177Lu-L804-IAB2MA or 177Lu-DOTA-IAB2MA (18.4 or 22.2 MBq). Treatment with 177Lu-L804-IAB2MA resulted in lower absorbed doses in tumors and less toxicity than that of 177Lu-DOTA-IAB2MA. Conclusion: 89Zr- and 177Lu-L804-IAB2MA may be a promising theranostic pair for imaging and therapy of prostate cancer.

Evaluation of a bimodal, matched pair theranostic agent targeting prostate-specific membrane antigen.

BACKGROUND: Prostate cancer affects 1 in 6 men, and it is the second­leading cause of cancer-related death in American men. Surgery is one of the main treatment modalities for prostate cancer, but it often results in incomplete resection margins or complete resection that leads to nerve damage and undesirable side effects. In the present work, we have developed a new bimodal tracer, NODAGA-sCy7.5 PSMAi (prostate-specific membrane antigen inhibitor), labeled with the true matched theranostic pair 64Cu/67Cu and a near-infrared fluorescent dye. This agent could potentially be used for concomitant PET imaging, optical surgical navigation, and targeted radiopharmaceutical therapy. METHODS: A prostate-specific membrane antigen (PSMA)-targeting urea derivative was conjugated to NODAGA for copper radiolabeling and to the near-infrared fluorophore sulfo-Cy7.5 (sCy7.5). Binding studies were performed in PSMA-positive PC-3 PIP cells, as well as uptake and internalization assays in PC-3 PIP cells and PSMA-negative PC-3 wild type cells. Biodistribution studies of the 64Cu-labeled compound were performed in PC-3 PIP- and PC-3 tumor-bearing mice, and 67Cu biodistributions of the agent were obtained in PC-3 PIP tumor-carrying mice. PET imaging and fluorescence imaging were also performed, using the same molar doses, in the two mouse models. RESULTS: The PSMA conjugate bound with high affinity to PSMA-positive prostate cancer cells, as opposed to cells that were PSMA-negative. Uptake and internalization were rapid and PSMA-mediated in PC-3 PIP cells, while only minimal non-specific uptake was observed in PC-3 cells. Biodistribution studies showed specific uptake in PC-3 PIP tumors, while accumulation in PC-3 tumor-bearing mice was low. Furthermore, tumor uptake of the 67Cu-labeled agent in the PC-3 PIP model was statistically equivalent to that of 64Cu. PET and fluorescence imaging at 0.5 nmol per mouse also demonstrated that PC-3 PIP tumors could be clearly detected, while PC-3 tumors showed no tumor accumulation. CONCLUSIONS: NODAGA-sCy7.5-PSMAi was specific and selective in detecting PSMA-positive, as opposed to PSMA-negative, tumors in mouse models of prostate cancer. This bioconjugate could potentially be used for PET staging with 64Cu, targeted radiopharmaceutical therapy with 67Cu, and/or image-guided surgery with sCy7.5.

Albumin-Binding Lutetium-177-Labeled LLP2A Derivatives as Theranostics for Melanoma.

Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.

Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis.

BACKGROUND: CXCR3 is a chemokine receptor and is expressed in innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, detection of CXCR3 by positron emission tomography (PET) radiotracer can be a useful tool for detecting the development of atherosclerosis in a noninvasive manner. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, 18F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis. RESULTS: The reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured Ki values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [18F]1 was prepared by a two-step radiosynthesis with a decay-corrected radiochemical yield of 13 ± 2%, radiochemical purity > 99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (n = 6). The baseline studies showed that [18F]1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue in Apolipoprotein E (ApoE) knockout (KO) mice fed with a high-fat diet over 12 weeks. The uptake of [18F]1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [18F]1 in the abdominal aorta of C57BL/6 control mice fed with a normal diet were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. Immunohistochemistry studies demonstrated that [18F]1-positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [18F]1, and their CXCR3 expressions were minimal. CONCLUSION: [18F]1 was synthesized with good radiochemical yield and high radiochemical purity. In PET imaging studies, [18F]1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [18F]1 visualized CXCR3 expression in different regions in mice aligned with the tissue histology studies. Taken together, [18F]1 is a potential PET radiotracer for imaging CXCR3 in atherosclerosis.

Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis.

Background: CXCR3 is a chemokine receptor and is expressed on innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, the detection of CXCR3 by positron emission tomography (PET) radiotracer may be a useful tool to detect atherosclerosis development noninvasively. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, 18 F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis. Methods: The reference standard ( S )-2-(5-chloro-6-(4-(1-(4-chloro-2-fluorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)pyridin-3-yl)-1,3,4-oxadiazole ( 1 ) and its corresponding precursor 9 were synthesized using organic syntheses. The radiotracer [ 18 F] 1 was prepared in one-pot, two-step synthesis via aromatic 18 F-substitution followed by reductive amination. Cell binding assays were conducted using 1 , [ 125 I]CXCL10, and CXCR3A- and CXCR3B-transfected human embryonic kidney (HEK) 293 cells. Dynamic PET imaging studies over 90 min were performed on C57BL/6 and apolipoprotein E (ApoE) knockout (KO) mice that were subjected to a normal and high-fat diet for 12 weeks, respectively. Blocking studies were conducted with preadministration of the hydrochloride salt of 1 (5 mg/kg) to assess the binding specificity. Time-activity curves (TACs) for [ 18 F] 1 in both mice were used to extract standard uptake values (SUVs). Biodistribution studies were performed on C57BL/6 mice, and the distribution of CXCR3 in the abdominal aorta of ApoE KO mice was assessed by immunohistochemistry (IHC). Results: The reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured K i values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [ 18 F] 1 was prepared with decay-corrected radiochemical yield (RCY) of 13 ± 2%, radiochemical purity (RCP) >99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (EOS) ( n =6). The baseline studies showed that [ 18 F] 1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue (BAT) in ApoE KO mice. The uptake of [ 18 F] 1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [ 18 F] 1 in the abdominal aorta of C57BL/6 mice were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. IHC studies demonstrated that [ 18 F] 1 -positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [ 18 F] 1 , and their CXCR3 expressions were minimal. Conclusion: The novel radiotracer, [ 18 F] 1 was synthesized with good RCY and high RCP. In PET imaging studies, [ 18 F] 1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [ 18 F] 1 visualized CXCR3 expression in different regions in mice is in line with the tissue histology studies. Taken together, [ 18 F] 1 is a potential PET radiotracer for the imaging of CXCR3 in atherosclerosis.

Development and biodistribution studies of 77As-labeled trithiol RM2 bioconjugates for prostate cancer: Comparison of [77As]As-trithiol-Ser-Ser-RM2 vs. [77As]As-trithiol-Glu-Ser-RM2.

INTRODUCTION: Recent progress with the production of 72As (2.49 Mev ß+max (64%), 3.33 Mev ß+max (16%), 834 keV γ (81%), t1/2: 26 h) and 77As (0.683 Mev ß-max (97%), 239 keV γ (1.59%), t1/2: 38.8 h) has facilitated their evaluation as a potential "theranostic pair" for PET imaging and radiotherapy. Our 3rd generation trithiol chelate with two carboxylic acid groups was further developed as a bifunctional chelate for radioarsenic. METHODS: The As complex with the trithiol chelate was synthesized and characterized. No carrier added (nca) [77As][H2AsO4-] was used for radiolabeling studies. The trithiol chelate was conjugated to the RM2 peptide (DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) via solid phase peptide synthesis with two different linkers, Ser-Ser and Glu-Ser. The trithiol chelate and its RM2 bioconjugates were radiolabeled with nca 77As, and the RM2 bioconjugates were compared in initial biodistribution studies. RESULTS: The As diacid trithiol complex was characterized by 1H NMR, 13C NMR and HR-ESI-MS. The trithiol-RM2 precursor and As trithiol bioconjugates were characterized by HR-ESI-MS and/or LC-ESI-MS. Radiolabeling of the RM2 bioconjugates with 77As resulted in over 85% radiochemical yield for [77As]As-trithiol-Ser-Ser-RM2 ([77As]8) and 90% for [77As]As-trithiol-Glu-Ser-RM2 ([77As]9). Both radiotracers demonstrated excellent in vitro stability (≥ 90% remaining intact through 24 h in PBS buffer) and were more hydrophilic than previous analogues based on log D7.4 values. Biodistribution results of the two radiotracers in healthy CF-1 male mice demonstrated blockable pancreatic uptake at 1 h (82% for ([77As]8 and 78% for [77As]9) indicating specific gastrin-releasing peptide receptor (GRPR) uptake. The primary route of excretion was through the gastrointestinal system for both radiotracers. CONCLUSIONS: A new trithiol chelate with improved hydrophilicity was successfully conjugated to the RM2 peptide via two linkers, and high radiolabeling yield with nca 77As was achieved. In vivo biodistribution studies with both radiotracers demonstrated blockable pancreatic uptake suggestive of specific receptor uptake.

Development of Heterobivalent Theranostic Probes Having High Affinity/Selectivity for the GRPR/PSMA.

In this study, we describe the development of heterobivalent [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] radiotracers that display very high selectivity/specificity for gastrin-releasing peptide receptor (GRPR)-/prostate-specific membrane antigen (PSMA)-expressing cells. These studies include metallation, purification, characterization, and in vitro and in vivo evaluation of the new small-molecule-/peptide-based radiopharmaceuticals having utility for imaging and potentially therapy. Competitive displacement binding assays using PC-3 cells and LNCaP cell membranes showed high binding affinity for the GRPR or the PSMA. Biodistribution studies showed favorable excretion pharmacokinetics with high tumor uptake in PC-3 or PC-3 prostatic inhibin peptide (PIP) tumor-bearing mice. For example, tumor accumulation at the 1 h time point ranged from (4.74 ± 0.90) to (7.51 ± 2.61)%ID/g. Micro-single-photon emission computed tomography (microSPECT) molecular imaging investigations showed very high uptake in tumors with minimal accumulation of tracers in the surrounding collateral tissues in xenografted mice at 4 h postintravenous injection. In conclusion, [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] tracers displayed favorable pharmacokinetic and excretion profiles with high uptake and retention in tumors.

Design, synthesis and evaluation of 111In labeled DOTA-conjugated tetrapeptides having high affinity and selectivity for mu opioid receptors.

INTRODUCTION: Peripheral mu (µ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic 111In labeled DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated µ tetrapeptides. METHODS: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH2 (DAPO), where Dmt = 2',6'-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA). Constructs had DOTA conjugation at the Orn4 or Lys4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and 111In radiolabeling were accomplished by standard methods. Protein binding and Log D7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. RESULTS: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt1]DALDA showed good selectivity and subnanomolar affinity for µ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[111In]In-DOTA)-NH2, showed 25% specific binding in vivo to µ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. CONCLUSIONS: Visualizing peripheral µ opioid receptors using 111In labeled DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. ADVANCES IN KNOWLEDGE: This study shows in vivo labeling of peripheral µ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.

Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent.

The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst2-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)3]+ and [99mTc][Tc(CO)3]+. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst2-ANT peptide (3), to yield NSO-sst2-ANT (4). Two synthetic methods were used to prepare Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)3]+ complexation by 2. The resulting products demonstrated the expected molecular mass and nanomolar in vitro SSTR2 affinity (IC50 values under 30 nM, AR42J cells, [125I]iodo-Tyr11-somatostatin-14 radioligand standard). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [99mTc][Tc(OH2)3(CO)3]+ (99mTc; t½â€¯= 6 h, 141 keV γ) with 4 formed a third product, distinct from the Re analogues, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24 h), along with 75% stability in mouse serum through 4 h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [99mTc][Tc(CO)3]-labeled sst2-ANT derivative previously studied. Yet despite having adequate tumor uptake at 1 h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-saturating dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)3]+ core (M = Re, 99mTc) by disfavoring involvement of the N-triazole.

Synthesis and evaluation of a 99mTc tricarbonyl-labeled somatostatin receptor-targeting antagonist peptide for imaging of neuroendocrine tumors.

INTRODUCTION: A somatostatin receptor (SSTR)-targeting antagonist peptide (sst2-ANT) was radiolabeled with 99mTc tricarbonyl via a tridentate [N,S,N]-type ligand (L) to develop a radiodiagnostic agent, 99mTcL-sst2-ANT, for imaging of SSTR-expressing neuroendocrine tumors. METHODS: Receptor affinity was assessed in vitro with the nonradioactive analogue, ReL-sst2-ANT, via a challenge experiment in AR42J cells with 125I-SS-14 as the competing radioligand. Preparation of 99mTcL-sst2-ANT was achieved via reaction of [99mTc(CO)3(H2O)3]+ with L-sst2-ANT. To test the stability of the radiolabeled complex, challenge experiments were performed in phosphate-buffered saline solutions containing cysteine or histidine and also in mouse serum. Biodistribution and micro-SPECT/CT imaging studies were performed in AR42J tumor-bearing female ICR SCID mice. RESULTS: The half maximal inhibitory concentration (IC50 value) of ReL-sst2-ANT in AR42J cells was 15nM. Preparation of 99mTcL-sst2-ANT was achieved with ≥97% radiochemical yield (RCY) and was verified by HPLC co-elution with the ReL-sst2-ANT analogue. The radiolabeled complex remained intact for up to 24h in high concentration solutions of cysteine and histidine at 37°C. Furthermore, the radiotracer was 90% stable for 1h at 37°C in mouse serum. Micro-SPECT/CT images showed clear uptake in tumors and were supported by the biodistribution data, in which the 3.2% ID/g tumor uptake at 4h was significantly blocked by co-administration of nonradioactive SS-14. CONCLUSIONS: A [99mTc(CO)3(N,S,N)]+ chelate was employed for radiolabeling of an SSTR-targeting antagonist peptide. Synthesis of 99mTcL-sst2-ANT was achieved in high RCY, and the resulting complex displayed high in vitro stability. Somatostatin receptor affinity was retained in both cells and in tumor-bearing mice, where the complex successfully targeted SSTR-positive tumors via a receptor-mediated process. Advances in Knowledge and Implications for Patient Care. This first 99mTc-tricarbonyl-labeled SSTR antagonist peptide showed promising in vivo tumor targeting in mice. Future studies may lead to translation of a similar design into the clinic.

Cocaine occupancy of sigma1 receptors and dopamine transporters in mice.

Activation of sigma1 (σ1) receptors contributes to the behavioral and toxic effects of (-)-cocaine. We studied a key step, the ability of (-)-cocaine to occupy σ1 receptors in vivo, using CD-1(®) mice and the novel radioligand [(125) I]E-N-1-(3'-iodoallyl)-N'-4-(3",4"-dimethoxyphenethyl)-piperazine ([(125) I]E-IA-DM-PE-PIPZE). (-)-Cocaine displayed an ED50 of 68 µmol/kg for inhibition of specific radioligand binding in whole brain, with values between 73 and 80 µmol/kg for heart, lung, and spleen. For comparison, an ED50 of 26 µmol/kg for (-)-cocaine occupancy of striatal dopamine transporters (DAT) was determined by inhibition of [(125) I]3ß-(4-iodophenyl)tropan-2ß-carboxylic acid isopropyl ester ([(125) I]RTI-121) binding. A chief finding is the relatively small potency difference between (-)-cocaine occupancy of σ1 receptors and the DAT, although the DAT occupancy is likely underestimated. Interactions of (-)-cocaine with σ1 receptors were assessed further using [(125) I]E-IA-DM-PE-PIPZE for regional cerebral biodistribution studies and quantitative ex vivo autoradiography of brain sections. (-)-Cocaine binding to cerebral σ1 receptors proved directly proportional to the relative site densities known for the brain regions. Nonradioactive E-IA-DM-PE-PIPZE gave an ED50 of 0.23 µmol/kg for occupancy of cerebral σ1 receptors, and a 3.16 µmol/kg (i.p.) dose attenuated (-)-cocaine-induced locomotor hyperactivity by 30%. This effect did not reach statistical significance, but suggests that E-IA-DM-PE-PIPZE is a probable σ1 receptor antagonist. As groundwork for the in vivo studies, we used standard techniques in vitro to determine ligand affinities, site densities, and pharmacological profiles for the σ1 and σ2 receptors expressed in CD-1(®) mouse brain.

Relationship between cerebral sigma-1 receptor occupancy and attenuation of cocaine's motor stimulatory effects in mice by PD144418.

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (σ1) receptors. Selective σ1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity σ1 antagonist PD144418 (1,2,3,6-tetrahydro-5-[3-(4-methylphenyl)-5-isoxazolyl]-1-propylpyridine) has been reported not to inhibit cocaine-induced hyperactivity. To address this apparent paradox, we evaluated aspects of PD144418 binding in vitro, investigated σ1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for σ1 sites (Ki 0.46 nM) and 3596-fold selectivity over σ2 sites (Ki 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (Ki >100 µM), and the DAT interaction was weak (Ki 9.0 µM). In vivo, PD144418 bound to central and peripheral σ1 sites in mouse, with an ED50 of 0.22 µmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 µmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 µmol/kg) attenuated cocaine-induced hyperactivity in a dose-dependent manner in mice. There was good correlation (r(2) = 0.88) of hyperactivity reduction with increasing cerebral σ1 receptor occupancy. The behavioral ED50 of 0.79 µmol/kg corresponded to 80% occupancy. Significant σ1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 µmol/kg dose of PD144418.

A selective sigma-2 receptor ligand antagonizes cocaine-induced hyperlocomotion in mice.

Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.

Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer.

Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the (198)Au ß-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible (198)AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of (198)AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable (198)AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

Synthesis, radioiodination and in vitro and in vivo sigma receptor studies of N-1-allyl-N´-4-phenethylpiperazine analogs.

INTRODUCTION: Sigma-1 (σ(1)) receptor radioligands are useful for basic pharmacology studies and for imaging studies in neurology, psychiatry and oncology. We derived a hybrid structure, N-1-allyl-N´-4-phenethylpiperazine, from known ligands TPCNE and SA4503 for use as a scaffold for development of radioiodinated σ(1) receptor ligands. METHODS: E-and Z-N-1-(3'-iodoallyl)-N´-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-1 and Z-1), N-1-allyl-N´-4-(3',4'-dimethoxyphenethyl)-piperazine (2) and E-N-1-(3'-iodoallyl)-N´-4-(3″-methoxy-4'´-hydroxyphenethyl)-piperazine (3) were synthesized. Affinities for σ(1) and σ(2) receptors were determined. [(125)I]E-1 and [(125)I]Z-1 were prepared and evaluated in vivo in mice. [(125)I]E-1 was further evaluated in σ(1) receptor binding assays in vitro. RESULTS: E-1 displayed moderately high apparent affinity (15 nM) for σ(1) sites and 84-fold selectivity against σ(2) sites. Z-1 showed similar σ(1) affinity, but only 23-fold selectivity. In contrast, 2 exhibited poor binding to both subtypes, while 3 had good affinities but poor selectivity. E-1 profiled as a probable antagonist in the phenytoin shift assay. [(125)I]E-1 and [(125)I]Z-1 were prepared in good yields and with high specific radioactivities. Log D(7.4) values (2.25 and 2.27) fall within the optimal range for in vivo studies. Both radioligands selectively labeled σ(1) receptors in mouse brain and peripheral organs in vivo. [(125)I]E-1 showed a higher level of specific binding than [(125)I]Z-1 and displayed good metabolic stability. Further, [(125)I]E-1 selectively labeled σ(1) receptors in mouse brain homogenates (K(d) 3.79 nM; B(max)=599 fmol/mg protein). CONCLUSIONS: [(125)I]E-1 is a selective σ(1) receptor radioligand that exhibits properties amenable to in vitro and in vivo studies, with possible extension to single photon emission computed tomography using iodine-123.

Comparative evaluation of three 64Cu-labeled E. coli heat-stable enterotoxin analogues for PET imaging of colorectal cancer.

Analogues of the E. coli heat-stable enterotoxin (STh) are currently under study as both imaging and therapeutic agents for colorectal cancer. Studies have shown that the guanylate cyclase C (GC-C) receptor is commonly expressed in colorectal cancers. It has also been shown that STh peptides inhibit the growth of tumor cells expressing GC-C. The ability to determine GC-C status of tumor tissue using in vivo molecular imaging techniques would provide a useful tool for the optimization of GC-C-targeted therapeutics. In this work, we have compared receptor binding affinities, internalization/efflux rates, and in vivo biodistribution patterns of an STh analogue linked to N-terminal DOTA, TETA, and NOTA chelating moieties and radiolabeled with Cu-64. The peptide F(19)-STh(2-19) was N-terminally labeled with three different chelating groups via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with Cu-64 and used for in vitro internalization/efflux, in vivo biodistribution, and in vivo PET imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. Incorporation of DOTA-, TETA-, and NOTA-chelators at the N-terminus of the peptide F(19)-STh(2-19) resulted in IC(50)s between 1.2 and 3.2 nM. In vivo, tumor localization was similar for all three compounds, with 1.2-1.3%ID/g at 1 h pi and 0.58-0.83%ID/g at 4 h pi. The principal difference between the three compounds related to uptake in nontarget tissues, principally kidney and liver. At 1 h pi, (64)Cu-NOTA-F(19)-STh(2-19) demonstrated significantly (p < 0.05) lower uptake in liver than (64)Cu-DOTA-F(19)-STh(2-19) (0.36 +/- 0.13 vs 1.21 +/- 0.65%ID/g) and significantly (p < 0.05) lower uptake in kidney than (64)Cu-TETA-F(19)-STh(2-19) (3.67 +/- 1.60 vs 11.36 +/- 2.85%ID/g). Use of the NOTA chelator for coordination of Cu-64 in the context of E. coli heat-stable enterotoxin analogues results in higher tumor/nontarget tissue ratios at 1 h pi than either DOTA or TETA macrocycles. Heat-stable enterotoxin-based radiopharmaceuticals such as these provide a means of noninvasively determining GC-C receptor status in colorectal cancers by PET.

Radioactive gold nanoparticles in cancer therapy: therapeutic efficacy studies of GA-198AuNP nanoconstruct in prostate tumor-bearing mice.

Biocompatibility studies and cancer therapeutic applications of nanoparticulate beta-emitting gold-198 (198Au; beta(max) = 0.96 MeV; half-life of 2.7 days) are described. Gum arabic glycoprotein (GA)-functionalized gold nanoparticles (AuNPs) possess optimum sizes (12-18 nm core diameter and 85 nm hydrodynamic diameter) to target individual tumor cells and penetrate through tumor vasculature and pores. We report the results of detailed in vivo therapeutic investigations demonstrating the high tumor affinity of GA-198AuNPs in severely compromised immunodeficient (SCID) mice bearing human prostate tumor xenografts. Intratumoral administration of a single dose of beta-emitting GA-198AuNPs (70 Gy) resulted in clinically significant tumor regression and effective control in the growth of prostate tumors over 30 days. Three weeks after administration of GA-198AuNPs, tumor volumes for the treated animals were 82% smaller as compared with tumor volume of control group. The treatment group showed only transitory weight loss in sharp contrast to the tumor-bearing control group, which underwent substantial weight loss. Pharmacokinetic studies have provided unequivocal evidence for the optimum retention of therapeutic payload of GA-198AuNPs within the tumor site throughout the treatment regimen with minimal or no leakage of radioactivity to various nontarget organs. The measurements of white and red blood cells, platelets, and lymphocytes within the treatment group resembled those of the normal SCID mice, thus providing further evidence on the therapeutic efficacy and concomitant in vivo tolerance and nontoxic features of GA-198AuNPs. FROM THE CLINICAL EDITOR: In this study, the biocompatibility and cancer therapeutic applications of glycoprotein (GA) functionalized gold nanoparticles containing b-emitting Au-198 are described in SCID mice bearing human prostate tumor xenografts. The findings of significant therapeutic efficacy, good in vivo tolerance and non-toxic features make these particles ideal candidates for future human applications.

In vivo imaging of human colorectal cancer using radiolabeled analogs of the uroguanylin peptide hormone.

BACKGROUND: Uroguanylin is an endogenous peptide agonist that binds to the guanylate cyclase C receptor (GC-C). GC-C is overexpressed in human colorectal cancer (CRC), and exposure of GC-C-expressing cells to GC-C agonists results in cell cycle arrest and/or apoptosis, highlighting the therapeutic potential of such compounds. This study describes the first use of radiolabeled uroguanylin analogs for in vivo detection of CRC. MATERIALS AND METHODS: The peptides uroguanylin and E(3)-uroguanylin were N-terminally labeled with the DOTA chelating group via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with In-111 and used for in vivo biodistribution and SPECT imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. RESULTS: Alteration of the position 3 aspartate residue to glutamate resulted in increased affinity for GC-C, with IC(50) values of 5.0+/-0.3 and 9.6+/-2.9 nM for E(3)-uroguanylin and DOTA-E(3)-uroguanylin, respectively. In vivo, (111)In-DOTA-E(3)-uroguanylin demonstrated tumor uptake of 1.17+/-0.23 and 0.61+/-0.07% ID/g at 1 and 4 h post injection, respectively. The specificity of tumor localization was demonstrated by coinjection of 3 mg/kg unlabeled E(3)-uroguanylin, which reduced tumor uptake by 69%. Uptake in kidney, however, was dramatically higher for the uroguanylin peptides than for previously characterized radiolabeled E. coli heat-stable enterotoxin (STh) analogs targeting GC-C, and was also inhibited by coinjection of unlabeled peptide in a fashion not previously observed. CONCLUSION: Use of uroguanylin-targeting vectors for in vivo imaging of colorectal cancers expressing GC-C resulted in tumor uptake that paralleled that of higher affinity heat-stable enterotoxin peptides, but also resulted in increased kidney uptake in vivo.

Development of high-specific-activity (68)Ga-labeled DOTA-rhenium-cyclized alpha-MSH peptide analog to target MC1 receptors overexpressed by melanoma tumors.

INTRODUCTION: A previous report on (68)Ga-1,4,7,10-tetraazacyclodedecane-N,N',N'',N'''-tetraacetic acid (DOTA)-Re(Arg(11))CCMSH was shown to indicate the imaging agent's potency for early detection of metastatic melanoma. However, the main limiting factor to developing high-specific-activity (68)Ga-DOTA-Re(Arg(11))CCMSH is the short half-life of (68)Ga, which precludes further purification of the agent. To circumvent this problem, we incorporated the microwave technique to rapidly radiolabel the peptide with (68)Ga, thereby allowing enough time to include high-performance liquid chromatography (HPLC) purification in the overall procedure. METHODS: DOTA-Re(Arg(11))CCMSH was radiolabeled with (68)Ga in <1 min using a circular-cavity microwave apparatus. Reverse-phase HPLC purification was accomplished in less than 20 min. (68)Ga-DOTA-Re(Arg(11))CCMSH was then administered on B16/F1 murine melanoma-bearing C57 mice to study its biodistribution and positron emission tomography (PET) imaging capability. RESULTS: The production of high-specific-activity (68)Ga-DOTA-Re(Arg(11))CCMSH resulted in an improved tumor uptake [6.93+/-1.11%ID/g at 30 min postinjection (p.i.) and 6.27+/-1.60%ID/g at 1 h p.i.] and tumor retention (5.85+/-1.32%ID/g at 4 h p.i.). Receptor-mediated tumor uptake was verified by blocking studies. Furthermore, high-resolution PET images of the tumor were obtained, owing to high tumor-to-nontarget organ ratios at an early time point (i.e., at 1 h biodistribution: tumor/blood, 14.3; tumor/muscle, 89.6; tumor/skin, 12.3) and fast clearance of the labeled peptide from kidney and other healthy tissues. CONCLUSION: High-specific-activity (68)Ga-DOTA-Re(Arg(11))CCMSH may have a potential role in the early diagnosis of metastasized melanoma.

Synthesis and characterization of an (111)In-labeled peptide for the in vivo localization of human cancers expressing the urokinase-type plasminogen activator receptor (uPAR).

This study describes the synthesis and preliminary biologic evaluation of an (111)In-labeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-betaAla)(2)-K-K(DOTA)-NH(2) was synthesized and conjugated with the DOTA chelating moiety via conventional solid-phase peptide synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC(50) of 240 +/- 125 nM for the peptide, compared with IC(50) values of 0.44 +/- 0.02 and 0.75 +/- 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 h postinjection of (111)In-DOTA-peptide, and compared with data obtained using a scrambled control peptide as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the (111)-labeled peptide from the blood pool, with 0.12 +/- 0.06% ID/g remaining in blood at 4 h pi. Elimination was seen primarily via the renal/urinary route, with 83.9 +/- 2.2% ID in the urine at the same time point. Tumor uptake at this time was 0.53 +/- 0.11% ID/g, resulting in tumor/blood and tumor/muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo.