Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to examine the biodistribution of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH. ^99mTc-RAD-Arg-(Arg¹¹)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to ^99mTc-RAD-Lys-(Arg¹¹)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using ^99mTc-RAD-Arg-(Arg¹¹)CCMSH as an imaging probe.     

Very Late Antigen-4 (α4β1 Integrin) Targeted PET Imaging of Multiple Myeloma

Biomedical imaging techniques such as skeletal survey and ¹⁸F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30–50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α₄β₁ integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, ⁶⁴Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of ⁶⁴Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with ⁶⁴Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, ⁶⁴Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using ⁶⁴Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.     

Melanoma targeting property of a Lu-177-labeled lactam bridge-cyclized alpha-MSH peptide

The purpose of this study was to determine the melanoma targeting property of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice. ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex exhibited high receptor-mediated melanoma uptake and fast urinary clearance. The tumor uptake of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex was 20.25 ± 4.59 and 21.63 ± 6.27% ID/g at 0.5 and 2 h post-injection, respectively. Approximately 83% of injected dose cleared out the body via urinary system at 2 h post-injection. ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex were 2.76 and 1.74 at 2 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex underscored its potential for melanoma treatment in the future.     

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.     

Evaluation of 111In-DOTA-F56 peptide targeting VEGFR1 for potential non-invasive gastric cancer xenografted tumor mice Micro-SPECT imaging

Non-invasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in the early diagnosis of tumors, especially in gastric cancer. Here, we designed and evaluated a novel ¹¹¹In-DOTA-F56 peptide as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind VEGFR1. It was obtained by radiolabeling DOTA-F56 with ¹¹¹InCl₃ with 98% radiochemical purity and 1.4 ± 0.4 GBq/µmol specific activity. ¹¹¹In-DOTA-F56 was obtained by the reaction of DOTA-F56 (10 µg) with ¹¹¹InCl₃ in pH 4.0 sodium acetate buffer at 85 °C for 20 min. ¹¹¹In-DOTA-F56 shows good stability in 0.01 M Phosphate Buffered Saline (PBS) and 5% Human Serum Albumin (HSA). ¹¹¹In-DOTA-F56 has a high binding affinity for human gastric cancer BGC-823 cells. Bio-distribution studies of ¹¹¹In-DOTA-F56 were performed in nude mice xenografted with human gastric cancer BGC-823 cells and the results revealed tumor uptake accumulation. A blocking dose of DOTA-F56 significantly reduced the tumor uptake of ¹¹¹In-DOTA-F56. Tumors were observed with Micro-SPECT images, and the uptake in the tumor increased with time from 4 h to 24 h. The MIP of the Micro-SPECT also showed that the excess DOTA-F56 can specifically block ¹¹¹In-DOTA-F56 in a mouse tumor model. We successfully synthesized the ¹¹¹In-DOTA-F56 VEGFR1-targeted peptide as a non-invasive molecule with fine radiochemical properties. Micro-SPECT indicates tumor uptake, which can be further blocked by excess of the F56 peptide, indicating that ¹¹¹In-DOTA-F56 peptide has potential for early detection of VEGFR1 positive gastric cancer and is worthy of further clinical investigations.     

Synthesis and characterization of 64Cu- and Cy5.5-labeled tetraiodothyroacetic acid derivatives for tumor angiogenesis imaging

It was previously reported that tetraiodothyroacetic acid (tetrac) inhibits angiogenesis by binding to the cell surface receptor for thyroid hormone on integrin α_Vβ₃. Therefore, we synthesized and evaluated two ⁶⁴Cu-labeled tetrac derivatives and a Cy5.5-labeled tetrac derivative for tumor angiogenesis imaging. Tetrac was structurally modified to conjugate with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) via its hydroxy or carboxylic acid end, and the resulting DOTA-conjugated tetrac derivatives were then labeled with ⁶⁴Cu. Tetrac was also conjugated with Cy5.5 via its carboxylic acid end. All three tetrac derivatives (1–3) exhibited greater inhibitory activity than tetrac against endothelial cell tube formation. The U87MG cell binding of [⁶⁴Cu]2 showed a time-dependent increase over 24 h and it was inhibited by 38% at 4 h in the presence of tetrac, indicating specificity of [⁶⁴Cu]2 to the thyroid hormone receptor site on integrin α_Vβ₃. Positron emission tomography (PET) images of U87MG tumor-bearing mice injected with [⁶⁴Cu]1 and [⁶⁴Cu]2 revealed that high radioactivity accumulated in the tumors, and that the tumor uptake and tumor-to-nontarget uptake ratio were higher in small tumors than in large tumors. In addition, the Cy5.5-labeled tetrac derivative (3) displayed a strong near-infrared (NIR) signal in the tumors. Taken together, these results suggest that these ligands hold promise as imaging agents for visualization of tumor angiogenesis.     

Evaluation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model

Recently, we selected a novel anti-hPD-L1-specific HCAb named Nb6 with high affinity (EC₅₀ = 0.65 ng/mL) for potential hPD-L1 targeted non-invasive PET imaging. In this research, Nb6 was conjugated with the bifunctional chelator NCS-Bz-NOTA ((2-[(4-Isothiocyanophenyl) methyl]-1,4,7-triazacy-clononane-1,4,7-triacetic acid)) and further labeled with radio-nuclide ⁶⁴Cu. ⁶⁴Cu-NOTA-Nb6 was prepared with over 95% labeling yield, over 99% radiochemical purity and 14–16 GBq/μmol specific activity after PD-10 column purification. It shows good stability in 0.01 M PBS and 5% HSA solutions. ⁶⁴Cu-NOTA-Nb6 has a high binding affinity to 3.60 nM which was tested by human lung adenocarcinoma A549 cell lines. Tumor lesion can be clearly observed from 20 h to 38 h by Micro-PET equipment after ⁶⁴Cu-NOTA-Nb6 administration. The study revealed that ⁶⁴Cu-NOTA-Nb6 has good lesion detection ability, high ratios between tumor and non-tumor signal and can specifically target A549 xenografted tumor model. Taken together of good stability, high binding affinity, and tumor detection ability, ⁶⁴Cu labeled Nb6 is a promising radio-tracer in diagnosing of hPD-L1 overexpression tumor, supposed to monitor PD-L1overexpression tumor progression and guide targeted therapy with PET molecular imaging.     

A cell permeable peptide analog as a potential-specific PET imaging probe for prostate cancer detection

Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH₂GR₁₁) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR₉, NHGR₁₁, and NHGR₁₃) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH₂GR₁₁ showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR₉, FITC-NHGR₁₁, and FITC-NHGR₁₃ performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR₁₁ in the prostate tissue. Further in vivo evaluation of FITC-NHGR₁₁ in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with ⁶⁴Cu-labeled NH₂GR₁₁ showed that the peptide was rapidly cleared from the blood (t _1/2 = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of ⁶⁴Cu-labled NH₂GR₁₁ was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.     

Complete ablation of small squamous cell carcinoma xenografts with186Re-labeled monoclonal antibody E48

In previous studies we have shown that in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts, radioimmunotherapy (RIT) with¹⁸⁶Re-labeled MAb E48 resulted in complete regressions in one-third of the tumors (followup >150 d). MAb E48 was labeled with¹⁸⁶Re following a novel labeling procedure developed at our institute. The injected dose was 600 μCi, which was the maximum tolerated dose (MTD; <15% wt loss) in these studies. The mean size of the tumors was 140±60 mm³. To investigate whether the therapeutic efficacy of RIT in our xenograft model would be improved when treating smaller xenografts, mice bearing 2 HNSCC xenografts with a vol of 75±17 mm³ (number of mice,n=6; number of tumors,t=12) were treated with 600 μCi of¹⁸⁶Re-labeled MAb E48 IgG. All tumors completely regressed and did not regrow during followup (>150 d). In all mice, weight loss did not exceed 10%. to obtain biodistribution data, mice bearing two xenografts with a vol of 58±31 mm³ were injected with 100 μCi of¹⁸⁶Re-labeled MAb E48 IgG. The maximum uptake in blood was 26.4% injected dose/g (%ID·g⁻¹) at 2 h pi and was 53.1%ID·g⁻¹ in the tumor at d 7 pi. In normal tissues, no nonspecific accumulation was observed. Based on these biodistribution data, the absorbed cumulative radiation dose was calculated. The accumulated dose in blood and tumor was 2004 cGy and 8580 cGy, respectively. In other tissues, the dose was less than 8.1% of the dose delivered to tumor. These data implicate that RIT with¹⁸⁶Re-labeled MAb E48 may be especially suited to be used as adjuvant therapy for the treatment of head and neck cancer patients with minimal residual disease.     

Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT

We previously reported that microSPECT/CT imaging with ¹¹¹In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab′)₂ of pertuzumab modified with NOTA chelators for complexing ⁶⁴Cu. The effect of the administered mass (5–200 µg) of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to ¹¹¹In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to ¹¹¹In-labeled pertuzumab.     

Development of a Novel PET Tracer [18F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

Background and Objective

The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.

Methods

C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested in vitro and in vivo.

Results

The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.

Conclusions

[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.     

Evaluation of 99mTc-probestin for imaging APN expressing tumors by SPECT

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP = 2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG₂) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that ^99mTc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.     

A three-time-point method for assessing kinetic parameters of 64Cu-labeled Ramucirumab trapping in VEGFR-2 positive lung tumors

ObjectiveTo describe a three-time-point method for estimating kinetic parameters involved in ⁶⁴Cu-labeled Ramucirumab (⁶⁴Cu-NOTA-RamAb) trapping of VEGFR-2 positive lung tumors.Materials and methodsPositron emission tomography (microPET) data of tumor-bearing mice for ⁶⁴Cu-NOTA-RamAb trapping in VEGFR-2 positive HCC4006 tumor were used, involving tissue activity measurements acquired at 3, 24 and 48 h post-injection, without and with administration of RamAb blocking dose. A kinetic model provided an analytical formula describing the tissue time-activity-curve, involving ⁶⁴Cu-NOTA-RamAb uptake (Ki), release rate constant (k_R) and fraction of free tracer in blood and interstitial volume (F).ResultsFitting analytical formula outcomes on mean microPET data yielded values of the kinetic parameters: Ki = 0.0314/0.0123 gram of blood per hour per gram of tissue, k_R = 0.0387/0.0313 h⁻¹ and F = 0.2075/0.2007 gram of blood per gram of tissue, without/with RamAb blocking dose, respectively (R = 0.99999 for the graph displaying microPET versus theoretical data; P < .01).ConclusionsThree independent kinetic parameters (Ki, k_R and F) can be assessed from three data points acquired at early, mid and late imaging, i.e., at 3, 24 and 48 h post-injection, for further characterization of ⁶⁴Cu-NOTA-RamAb trapping in VEGFR-2 positive lung tumors.     

Effect of Recombinant Human Thyrotropin on the Uptake of Radioactive Iodine (123I) in Dogs with Thyroid Tumors

In humans, recombinant human thyrotropin (rhTSH) enhances radioactive iodine uptake (RAIU) in patients with differentiated thyroid cancer. No studies have been performed in veterinary medicine to optimize radioiodine treatment of thyroid cancer. The aim of this study was to evaluate the effect of rhTSH on the uptake of radioiodine-123 (¹²³I) in dogs with thyroid tumors. Nine dogs with thyroid neoplasia were included in this prospective cross-over study. The dogs were divided in 2 groups. In one group, ¹²³I was administered for a baseline RAIU determination in week 1. In week 2 (after a washout period of 2 weeks), these dogs received rhTSH (100 μg IV) 24 h before ¹²³I injection. In the other group the order of the protocol was reversed. For each scan, the dogs received 37 MBq (1 mCi) of ¹²³I intravenously (IV) and planar scintigraphy was performed after 8 and 24 h for tumor RAIU calculation. Overall, rhTSH administration caused no statistically significant change on thyroid tumor RAIU at 8 h (p = 0.89) or at 24 h (p = 0.98). A significant positive correlation was found between the effect of rhTSH on tumor 8h-RAIU and rhTSH serum concentrations at 6 h (τ = 0.68; p = 0.03), at 12 h (τ = 0.68; p = 0.03) and at 24 h (τ = 0.78; p = 0.02) after rhTSH injection. This study suggests that IV administration of 100 μg rhTSH 24 h before ¹²³I has an inconsistent effect on thyroid tumor RAIU. Further studies are necessary to determine the best protocol of rhTSH administration to optimize thyroid tumor RAIU.     

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the 99mTc(CO)3+ core

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH₂) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)₃(H₂O)₃]⁺ (M = ^99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new ^99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.     

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors

Purpose

The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with ¹¹¹In for SPECT and intraoperative applications as well as ⁶⁸Ga and ⁶⁴Cu for PET imaging.

Methods

The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)₃), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with ¹¹¹In, ⁶⁸Ga and ⁶⁴Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with ⁶⁸Ga-HBED-CC-PSMA (HBED-CC: N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) and ¹¹¹In-PSMA-617 for comparison.

Results

⁶⁸Ga-CC34, ⁶⁴Cu-CC34, and ¹¹¹In-CC34 were prepared in radiochemical purity >95%. ^68/natGa-CC34, ^64/natCu-CC34, ^111/natIn-CC34, ^68/natGa-HBED-CC-PSMA, and ^111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with K_d values of 19.3±2.5 nM, 27.5±2.7 nM, 5.5±0.9 nM, 2.9±0.6 nM and 5.4±0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. ⁶⁸Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of ⁶⁸Ga-CC34 (14.5±2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to ⁶⁸Ga-HBED-CC-PSMA (15.8±1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of ⁶⁸Ga-CC34 were also comparable to ⁶⁸Ga-HBED-CC-PSMA (P>0.05). Tumor uptake of ¹¹¹In-CC34 (28.5±2.6% IA/g) at 1 h p.i. was lower than ¹¹¹In-PSMA-617 (52.1±6.5% IA/g) (P = 0.02). The acquisition of PET-images with ⁶⁴Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.

Conclusions

⁶⁸Ga-CC34 is comparable to ⁶⁸Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. ⁶⁴Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. ⁶⁴Cu-CC34 is currently being prepared for clinical translation.     

Replacement of the Lys linker with an Arg linker resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide

The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide through structural modification or l-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, d-Phe⁷, Arg¹¹] α-MSH_3-13 {(Arg¹¹)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg¹¹)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of l-lysine co-injection on the renal uptake was determined through the co-injection of l-lysine with ^99mTc-RGD-Arg-(Arg¹¹)CCMSH or ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, as well as increasing the tumor uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH compared to ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. ^99mTc-RGD-Arg-(Arg¹¹)CCMSH exhibited high tumor uptake (21.41 ± 3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81 ± 3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were 40.14–64.08% of those of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH (p <0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of l-lysine was effective in decreasing the renal uptakes of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH by 27.7% and ^99mTc-RGD-Lys-(Arg¹¹)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, warranting the further evaluation of ¹⁸⁸Re-labeled RGD-Arg-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

Synthesis,in vitro and in vivo characterization of <Superscript>64</Superscript>Cu(I) complexes derived from hydrophilic tris(hydroxymethyl)phosphane and 1,3,5-triaza-7-phosphaadamantane ligands

Four novel ⁶⁴Cu complexes ([⁶⁴Cu(thp)₄]⁺ (1), [⁶⁴Cu(TPA)₄]⁺ (2), [HC(CO₂)(pz^Me2)₂ ⁶⁴Cu(thp)₂] (3) and [HC(CO₂)(tz)₂ ⁶⁴Cu(thp)₂] (4), [where thp is tris(hydroxymethyl)phosphine, TPA is 1,3,5-triaza-7-phosphaadamantane, pz^Me2 is 3,5-dimethylpyrazole and tz is 1,2,4-triazole] were successfully synthesized and characterized. The complexes were produced in high radiochemical purity and yield (more than 98%) without the need for further purification. Their logP values and serum stabilities were measured and in vitro behavior was observed in cultured EMT-6 cells. The logP values (± standard deviation) obtained were −2.26 ± 0.04 (1), 0.01 ± 0.01 (2), −1.24 ± 0.03 (3) and −2.06 ± 0.03 (4). Complex 3 demonstrated the highest serum stability, with approximately 33% of the complex still intact after 1-h incubation. Complex 2 showed a rapid cell-association with EMT-6 cells, with more than 8.5% association at 2 h. This association was significantly higher (P < 0.001) than for the other three compounds after a 2-h incubation (1, 1.21%; 3, 0.63%; 4, 2.75%). Biodistribution and small-animal positron emission tomography/computed tomography was undertaken with 1 in mice bearing EMT-6 tumors. EMT-6 tumor uptake was high at 1 h (7.71 ± 2.17 %ID/g) and decreased slowly over 24 h (4 h, 4.90 ± 0.78 %ID/g; 24 h, 3.74 ± 0.73 %ID/g). The PET/CT images show that the EMT-6 tumors can be visualized at all time points. In this proof-of-concept study, we have successfully synthesized and characterized a novel series of versatile water-soluble Cu(I) complexes containing monophosphine ligands. We also report the use of 1 as a building block for new radiopharmaceuticals, perhaps the first time such a method has been used in the production of copper radiopharmaceuticals.     

Quantifying Metabolic Heterogeneity in Head and Neck Tumors in Real Time: 2-DG Uptake Is Highest in Hypoxic Tumor Regions

Purpose

Intratumoral metabolic heterogeneity may increase the likelihood of treatment failure due to the presence of a subset of resistant tumor cells. Using a head and neck squamous cell carcinoma (HNSCC) xenograft model and a real-time fluorescence imaging approach, we tested the hypothesis that tumors are metabolically heterogeneous, and that tumor hypoxia alters patterns of glucose uptake within the tumor.

Experimental Design

Cal33 cells were grown as xenograft tumors (n = 16) in nude mice after identification of this cell line''s metabolic response to hypoxia. Tumor uptake of fluorescent markers identifying hypoxia, glucose import, or vascularity was imaged simultaneously using fluorescent molecular tomography. The variability of intratumoral 2-deoxyglucose (IR800-2-DG) concentration was used to assess tumor metabolic heterogeneity, which was further investigated using immunohistochemistry for expression of key metabolic enzymes. HNSCC tumors in patients were assessed for intratumoral variability of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) uptake in clinical PET scans.

Results

IR800-2-DG uptake in hypoxic regions of Cal33 tumors was 2.04 times higher compared to the whole tumor (p = 0.0001). IR800-2-DG uptake in tumors containing hypoxic regions was more heterogeneous as compared to tumors lacking a hypoxic signal. Immunohistochemistry staining for HIF-1α, carbonic anhydrase 9, and ATP synthase subunit 5β confirmed xenograft metabolic heterogeneity. We detected heterogeneous ¹⁸F-FDG uptake within patient HNSCC tumors, and the degree of heterogeneity varied amongst tumors.

Conclusion

Hypoxia is associated with increased intratumoral metabolic heterogeneity. ¹⁸F-FDG PET scans may be used to stratify patients according to the metabolic heterogeneity within their tumors, which could be an indicator of prognosis.     

In vivo monitoring of the therapeutic efficacy of a CXCR1/2 inhibitor with 18F-FDG PET/CT imaging in experimental head and neck carcinoma: A feasibility study

The chemokine receptors CXCR1/2 play a key role in the aggressiveness of several types of cancers including head and neck squamous cell carcinomas (HNSCCs). In HNSCCs, CXCR1/2 signaling promotes cell proliferation and angiogenesis leading to tumor growth and metastasis. The competitive inhibitor of CXCR1/2, C29, inhibits the growth of experimental HNSCCs in mice. However, a non-invasive tool to monitor treatment response is essential to implement the use of C29 in clinical practices. ¹⁸F-FDG PET/CT is a gold-standard tool for the staging and the post-therapy follow-up of HNSCCs patients. Our study aimed to perform the first in vivo monitoring of C29 efficacy by non-invasive ¹⁸F-FDG PET/CT imaging. Mice bearing experimental HNSCCs (CAL33) were injected with ¹⁸F-FDG (T0) and thereafter treated (n = 7 mice, 9 tumors, 50 mg/kg by gavage) or not (n = 7 mice, 10 tumors) with C29 for 4 consecutive days. Final ¹⁸F-FDG-tumor uptake was determined at day 4 (TF). The average relative change (TF-T0) in ¹⁸F-FDG tumor uptake was +25.85 ± 10.93 % in the control group vs ?5.72 ± 10.07 % in the C29-treated group (p < 0.01). These results were consistent with the decrease of the tumor burden and with the decrease of tumor proliferating Ki67+ cells. These results paved the way for the use of ¹⁸F-FDG to monitor tumor response following C29 treatment.