Lower Kidney Uptake Research Articles

Preparation, Biological Evaluation, and First-in-Human Single-Photon Emission Computed Tomography (SPECT) Study of 99mTc-Labeled Prostate-Specific Membrane Antigen (PSMA)-Targeted Radiotracers Containing Triazole with Reduced Kidneys Accumulation.

Prostate-specific membrane antigen (PSMA), a well-established biological marker for prostate cancer (PCa) imaging and therapy, is overexpressed on the surface of prostate cancer lesions. In this study, a triazole ring was introduced into the linker by click chemistry to generate a HYNIC-derived ligand (T), which exhibited good PSMA affinity (Ki = 2.23 nM). Eight stable 99mTc-labeled complexes, [99mTc]Tc-T-Mn (n = 1-8), with hydrophilic properties were synthesized by incorporating different coligands at high radiochemical yields and purities without purification. The radioligands were concentrated in the kidneys of healthy Kunming male mice and were significantly blocked by the PSMA inhibitor ZJ-43. The uptake of the optimized complex [99mTc]Tc-T-M2 was correlated with PSMA, and it had good PSMA affinity (Kd = 5.42 nM). [99mTc]Tc-T-M2 accumulated on LNCaP (PSMA++) tumors and was significantly blocked by ZJ-43 at 2 h p.i., indicating high PSMA specificity. Relatively suitable kidney uptake was beneficial for reducing kidneys exposure in patients. SPECT/CT imaging of [99mTc]Tc-T-M2 in LNCaP (PSMA++) or 22Rv1 (PSMA+) tumor-bearing mice revealed high tumor uptake, low background uptake (especially low kidney uptake (49.06 ± 9.20 %ID/g) at 2 h p.i.), and obvious inhibition by ZJ-43, whereas PC-3 (PSMA-) tumors were undetectable. A freeze-dried [99mTc]Tc-T-M2 kit was successfully developed (T-M2 kit). Preliminary clinical trials showed that [99mTc]Tc-T-M2 clearly identified small prostate cancer lesions and has potential for clinical application.

Effects of Polyethylene Glycol and 8-Aminooctanoic Acid Linkers on Melanoma Uptake of [99mTc]Tc-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized α-MSH Peptides.

The purpose of this study was to evaluate the effect of linkers on tumor targeting and biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex {[99mTc]Tc(CO)3-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-CONH2} and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex {[99mTc]Tc(CO)3-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on B16/F10 melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and radiolabeled with [99mTc]Tc via the {[99mTc]Tc(CO)3(OH2)3}+ intermediate. The biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiochemical yields and exhibited MC1R-specific binding on B16/F10 melanoma cells. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex exhibited a higher tumor uptake than [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2, 4, and 24 h postinjection. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 13.63 ± 1.13, 31.93 ± 2.57, 20.31 ± 3.23, and 1.33 ± 0.15% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 1.6 and 3.4 times the tumor uptake of [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2 and 4 h postinjection, respectively. Meanwhile, the normal organ uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was lower than 1.8% ID/g at 2 h postinjection. The renal uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was only 1.73 ± 0.37, 0.73 ± 0.14, and 0.03 ± 0.01% ID/g at 2, 4, and 24 h postinjection, respectively. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios at 2 h postinjection. Single-photon emission computed tomography imaging revealed that the B16/F10 melanoma lesions could be clearly visualized by [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex at 2 h postinjection. Overall, the high tumor uptake and low kidney uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex highlighted its potential for melanoma imaging and warranted the future evaluation of [188Re]Re(CO)3-NOTA-PEG2Nle-CycMSHhex for melanoma therapy.

A Dimeric FAP-Targeting Small-Molecule Radioconjugate with High and Prolonged Tumor Uptake.

Imaging procedures based on small-molecule radioconjugates targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumors. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and invivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with an extended tumor residence time and favorable tumor-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analog were assayed against recombinant human FAP. Preclinical experiments with 177Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and 177Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed on mice bearing FAP-positive HT-1080 tumors. Results: OncoFAP and BiOncoFAP displayed comparable subnanomolar dissociation constants toward recombinant human FAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumor uptake than 177Lu-OncoFAP (∼20 vs. ∼4 percentage injected dose/g, respectively, at 24 h after injection). Notably, 177Lu-BiOncoFAP showed favorable tumor-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent antitumor efficacy when administered at therapeutic doses to tumor-bearing mice. Conclusion: 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable invivo tumor-to-organ ratios, a long tumor residence time, and potent anticancer efficacy.

Novel 64Cu-Labeled NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Enhanced Tumor to Kidney Uptake Ratios.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 64Cu-NOTA-PEG2Nle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-NOTA-AocNle-CycMSHhex {64Cu-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and purified by HPLC. The melanocortin-1 (MC1) receptor binding affinities of the peptides were examined on B16/F10 melanoma cells. The biodistributions of 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 64Cu-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake than 64Cu-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 melanoma cells. 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiolabeling yields and showed MC1R-specific binding on B16/F10 cells. 64Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 64Cu-NOTA-AocNle-CycMSHhex at 0.5, 2, 4, and 24 h post-injection. The tumor uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69, and 8.78 ± 2.29% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2% ID/g at 2 h post-injection except for kidney uptake. The renal uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 3.66 ± 0.52, 3.27 ± 0.52, and 1.47 ± 0.56 ID/g at 2, 4, and 24 h post-injection, respectively. 64Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using 64Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 2 h post-injection. High tumor uptake and low kidney uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.

Novel Al18F-NOTA-Conjugated Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptides with Enhanced Melanoma Uptake.

The purpose of this study was to evaluate the effect of linker on tumor targeting and biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex {Al18F-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-poly(ethylene glycol)-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and Al18F-NOTA-AocNle-CycMSHhex {Al18F-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of Al18F-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake and lower renal uptake than that of Al18F-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2/AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex were readily prepared with more than 55% of radiolabeling yields and displayed melanocortin-1 receptor (MC1R)-specific binding on B16/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake and lower kidney and liver uptake than Al18F-NOTA-AocNle-CycMSHhex at 1 and 2 h post injection. The tumor and renal uptakes of Al18F-NOTA-PEG2Nle-CycMSHhex were 17.44 ± 0.76 and 2.07 ± 0.43% ID/g at 1 h post injection, respectively. Al18F-NOTA-PEG2Nle-CycMSHhex showed the high tumor to normal organ uptake ratios after 1 h post injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using Al18F-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 1 and 2 h post injection. Overall, high tumor uptake, low kidney and liver uptake, and fast urinary clearance of Al18F-NOTA-PEG2Nle-CycMSHhex highlighted its potential as an MC1R-targeted imaging probe for melanoma detection.

The Effect of Albumin-Binding Moiety on Tumor Targeting and Biodistribution Properties of 67Ga-Labeled Albumin Binder-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

Background: The purpose of this study was to examine the effect of 4-p-(tolyl)butyric acid as an albumin-binding (ALB) moiety on tumor targeting and biodistribution properties of 67Ga-labeled albumin binder-conjugated alpha-melanocyte-stimulating hormone peptides. Materials and Methods: DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(ALB)-Gly/GlyGly/GlyGlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} were synthesized with 4-p-(tolyl)butyric acid serving as an ALB moiety. The melanocortin-1 receptor (MC1R)-binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 67Ga-DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 67Ga-DOTA-Lys(ALB)-GGNle-CycMSHhex {67Ga-ALB-G2} were determined on B16/F10 melanoma-bearing C57 mice. Results: The IC50 value of DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {ALB-G1, ALB-G2, ALB-G3} was 0.67 ± 0.07, 0.5 ± 0.09 and 0.51 ± 0.03 nM on B16/F10 cells, respectively. 67Ga-ALB-G2 was further evaluated as a lead peptide because of its higher tumor uptake (30.25 ± 3.24%ID/g) and lower kidney uptake (7.09 ± 2.22%ID/g) than 67Ga-ALB-G1 and 67Ga-ALB-G3 at 2 h postinjection. The B16/F10 melanoma uptake of 67Ga-ALB-G2 was 15.64 ± 4.55, 30.25 ± 3.24, 26.76 ± 3.23, and 10.71 ± 1.21%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 67Ga-ALB-G2 as an imaging probe at 2 h postinjection. Conclusions: The introduction of 4-p-(tolyl)butyric acid as an ALB moiety increased the blood retention, and resulted in higher tumor/kidney ratio of 67Ga-ALB-G2 as compared with its counterpart without an albumin binder. However, the resulting high uptake of 67Ga-ALB-G2 in blood and liver need to be further reduced to facilitate its therapeutic application when replacing 67Ga with therapeutic radionuclides.

The Dependence of Renal 68Ga[Ga]-DOTATOC Uptake on Kidney Function and Its Relevance for Peptide Receptor Radionuclide Therapy with 177Lu[Lu]-DOTATOC.

Background: In addition to its SSTR-specific binding in tumors and healthy tissues, DOTATOC analogues accumulate in kidney parenchyma. Renal tracer uptake might be a surrogate of kidney function or dysfunction. This study aimed to evaluate if kidney function can be estimated from 68Ga[Ga]-DOTATOC uptake in PET/CT and its impact on the nephrotoxicity of 177Lu[Lu]-DOTATOC PRRT. Methods: Two cohorts of patients (A: 128 diagnostic patients; B: 32 PRRT patients) were evaluated retrospectively. SUV values of the kidneys, physiologically SSTR-expressing organs and in background compartments were assessed. Kidney function was calculated as eGFR by CKD-EPI creatinine equation. Pearson’s correlation coefficients and treatment-induced changes of uptake and kidney function were assessed and compared. Results: Kidney function and renal DOTATOC uptake showed a significant inverse correlation (R2 = 0.037; p = 0.029). Evaluated models of PET/CT measurements were not able to predict kidney function sufficiently. The uptake of other organs did not depend on eGFR. While the renal uptake increased after PRRT (p < 0.001), the kidney function did not change significantly (p = 0.382). Neither low pre-therapeutic eGFR nor high pre-therapeutic kidney uptake were risk factors of PRRT-induced deterioration in kidney function. Conclusion: The relevance of kidney function for renal 68Ga[Ga]-DOTATOC uptake is limited. The nephrotoxicity of 177Lu[Lu]-DOTATOC PRRT might be low and cannot be reliably predicted by pre-therapeutic measurements.

Potential of three-step pretargeting radioimmunotherapy using biotinylated bevacizumab and succinylated streptavidin in triple-negative breast cancer xenograft.

Pretargeting radioimmunotherapy (PRIT) is a promising approach that can reduce long-time retention of blood radioactivity and consequently reduce hematotoxicity. Among the PRIT strategies, the combination of biotin-conjugated mAb and radiolabeled streptavidin (StAv) is a simple and convenient method because of its ease of preparation. This study performed three-step (3-step) PRIT using the sequential injection of (1) biotinylated bevacizumab (Bt-BV), (2) avidin, and (3) radiolabeled StAv for the treatment of triple-negative breast cancer (TNBC). Four biodistribution studies were performed using 111In in tumor-bearing mice to optimize each step of our PRIT methods. Further, a therapeutic study was performed with optimized 3-step PRIT using 90Y-labeled StAv. Based on the biodistribution studies, the protein dose of Bt-BV and avidin was optimized to 100μg and 10 molar equivalent of BV, respectively. Succinylation of StAv significantly decreased the kidney accumulation level (with succinylation (6.96 ± 0.91) vs without succinylation (20.60 ± 1.47) at 1h after injection, p < 0.0001) with little effect on the tumor accumulation level. In the therapeutic study, tumor growth was significantly suppressed in treatment groups with optimized 3-step PRIT using 90Y-labeled succinylated StAv compared to that of the no-treatment group (p < 0.05). The 3-step PRIT strategy of this study achieved fast blood clearance and low kidney uptake with little effect on the tumor accumulation level, and a certain degree of therapeutic effect was consequently observed. These results indicated that the pretargeting treatment of the current study may be effective for human TNBC treatment.

In situ Generated 212Pb-PSMA Ligand in a 224Ra-Solution for Dual Targeting of Prostate Cancer Sclerotic Stroma and PSMA-positive Cells.

Background: New treatments combating bone and extraskeletal metastases are needed for patients with metastatic castration-resistant prostate cancer. The majority of metastases overexpress prostate-specific membrane antigen (PSMA), making it an ideal candidate for targeted radionuclide therapy.Objective: The aim of this study was to test a novel liquid 224Ra/212Pb-generator for the rapid preparation of a dual-alpha targeting solution. Here, PSMA-targeting ligands are labelled with 212Pb in the 224Ra-solution in transient equilibrium with daughter nuclides. Thus, natural bone-seeking 224Ra targeting sclerotic bone metastases and 212Pb-chelated PSMA ligands targeting PSMA-expressing tumour cells are obtained.Methods: Two PSMA-targeting ligands, the p-SCN-Bn-TCMC-PSMA ligand (NG001), specifically developed for chelating 212Pb, and the most clinically used DOTA-based PSMA-617 were labelled with 212Pb. Radiolabelling and targeting potential were investigated in situ, in vitro (PSMA-positive C4-2 human prostate cancer cells) and in vivo (athymic mice bearing C4-2 xenografts).Results: NG001 was rapidly labelled with 212Pb (radiochemical purity >94% at concentrations of ≥15 µg/ml) using the liquid 224Ra/212Pb-generator. The high radiochemical purity and stability of [212Pb]Pb-NG001 were demonstrated over 48 hours in the presence of ascorbic acid and albumin. Similar binding abilities of the 212Pb-labelled ligands were observed in C4-2 cells. The PSMA ligands displayed comparable tumour uptake after 2 hours, but NG001 showed a 3.5-fold lower kidney uptake than PSMA-617. Radium-224 was not chelated and, hence, showed high uptake in bones.Conclusion: A fast method for the labelling of PSMA ligands with 212Pb in the 224Ra/212Pb-solution was developed. Thus, further in vivo studies with dual tumour targeting by alpha-particles are warranted.

Novel [99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake.

The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for 99mTc(CO)3+ radiolabeling. NOTA/NODAGA-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The IC50 values of NOTA/NODAGA-GGNle-CycMSHhex were 0.8 ± 0.1 and 0.9 ± 0.1 nM on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were readily prepared via the [99mTc(CO)3(OH2)3]+ intermediate and displayed MC1R-specific binding on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62% ID/g) and lower kidney uptake (1.59 ± 0.52% ID/g) at 2 h postinjection than 99mTc(CO)3-NODAGA-GGNle-CycMSHhex. The B16/F10 melanoma uptake of 99mTc(CO)3-NOTA-GGNle-CycMSHhex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81, and 3.16 ± 2.28% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. 99mTc(CO)3-NOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h postinjection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 99mTc(CO)3-NOTA-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. High tumor uptake, low kidney uptake, and fast urinary clearance of 99mTc(CO)3-NOTA-GGNle-CycMSHhex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)3-NOTA-GGNle-CycMSHhex for melanoma therapy.

Development of 18F-Fluoroglycosylated PSMA-Ligands with Improved Renal Clearance Behavior

The prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is highly expressed in the malignant human prostate epithelium. Therefore, PSMA has emerged as a very attractive target for developing radiopharmaceuticals for the diagnosis, e.g., by positron emission tomography (PET) imaging, and radiotherapy of prostate cancer. The aim of this study was to develop 18F-labeled PSMA ligands bearing different 18F-glycosyl moieties to study the effect on the in vivo clearance behavior of radiotracers in addition to their tumor binding ability. Therefore, we applied click chemistry-based 18F-fluoroglcosylation using 2-deoxy-2-[18F]fluoroglucosyl azide or 6-deoxy-6-[18F]fluoroglucosyl azide as prosthetic groups for the radiosynthesis of the 18F-fluoroglycosylated glutamate-urea-lysine-based PSMA inhibitors 2-[18F]FGlc-PSMA ([18F]7) and 6-[18F]FGlc-PSMA ([18F]8). The PSMA inhibitory potencies were determined by competitive radioligand binding assays using 99mTc-MIP-1404 and PSMA-expressing PC-3 PIP cells, revealing moderate PSMA inhibitory potencies for [18F]7 (IC50 = 234 nM) and [18F]8 (IC50 = 59 nM). Biodistribution and small-animal PET studies were performed using PSMA-positive PC-3 PIP and PSMA-negative PC-3 tumor-bearing nude mice. PSMA inhibitors [18F]7 and [18F]8 were obtained in high radioactivity yields of 19-22% (nondecay-corrected, referred to [18F]fluoride) and with molar activities of 71-136 GBq/μmol. In the biodistribution studies, the uptake levels of [18F]7 and [18F]8 in PC-3 PIP tumors were 13 ± 3%ID/g and 6 ± 5%ID/g at 60 min p.i., respectively. PSMA-negative PC-3 tumors and all other tissues had negligible low uptake values. Interestingly, [18F]7 had high uptake in the kidneys, with remarkable retention from 30 to 60 min p.i. (74 to 72%ID/g). In contrast, [18F]8 revealed a low uptake of 7.5%ID/g in the kidneys at 30 min p.i. and was rapidly cleared through the kidney (0.9%ID/g at 120 min p.i.). In direct comparison to a 68Ga-PSMA-11 PET scan of the same mouse, [18F]7 and [18F]8 showed 2- to 3-fold higher uptake values in PC-3 PIP tumors. Both radiotracers were solely cleared via the kidneys and not via the hepatobiliary pathway. The regional kidney distribution pattern of the tracers in the kidneys revealed that 68Ga-PSMA-11 and 2-[18F]FGlc-PSMA([18F]7) mainly accumulated in the cortex of the kidneys, whereas 6-[18F]FGlc-PSMA([18F]8) showed a 10-fold lower kidney uptake with accumulation in the inner medulla or pelvis of the kidneys. Overall, the developed 6-fluoroglucosyl derivative [18F]8, with its considerably low kidney uptake and fast clearance, demonstrated high uptake in PSMA-positive tumors in vivo. This candidate could, therefore, be valuable for translation into the clinic.

Preparation of the alpha-emitting prostate-specific membrane antigen targeted radioligand [212 Pb]Pb-NG001 for prostate cancer.

Prostate-specific membrane antigen (PSMA) is the most promising target for radioligand therapy of prostate cancer. The aim of this study was to prepare a small molecular ligand p-SCN-Bn-TCMC-PSMA (NG001) and compare it with the commonly used DOTA-based PSMA-617. The PSMA-targeting ability of the 212 Pb-labelled ligands was evaluated using PSMA-positive C4-2 human prostate cancer cells. Lead-212 is an in vivo generator of alpha particles by its daughter nuclides 212 Bi and 212 Po. NG001 was synthesized by conjugating the isothiocyanato group of p-SCN-Bn-TCMC to the amino group of a glutamate-urea-based PSMA-binding entity. Molecular size, chelator unit and chelator linking method are different in NG001 and PSMA-617. Both ligands were efficiently labelled with 212 Pb using a 224 Ra/212 Pb-solution generator in transient equilibrium with progeny. Lead-212-labelled NG001 was purified with a yield of 85.9±4.7% and with 0.7±0.2% of 224 Ra. Compared with [212 Pb]Pb-PSMA-617, [212 Pb]Pb-NG001 displayed a similar binding and internalization in C4-2 cells, with comparable tumour uptake in mice bearing C4-2 tumours, but almost a 2.5-fold lower kidney uptake. Due to the rapid normal tissue clearance and tumour cell internalization, any significant translocalization of 212 Bi was not detected in mice. In conclusion, the obtained results warrant further preclinical studies to evaluate the therapeutic efficacy of [212 Pb]Pb-NG001.

In Vitro and In Vivo Characterization of an 18F-AlF-Labeled PSMA Ligand for Imaging of PSMA-Expressing Xenografts.

The aim was to compare the prostate-specific membrane antigen (PSMA)-targeting characteristics of PSMA-11, radiolabeled on the basis of chelation of 18F-AlF, with those of 68Ga-PSMA-11 to image PSMA-expressing xenografts. Methods: Labeling of 18F-AlF-PSMA-11 via 18F-AlF-complexation was performed as described by Boschi et al. and Malik et al. with minor modifications. Several conditions for the quality control of the labeling of 18F-AlF-PSMA-11 via 18F-AlF-complexation were evaluated to characterize the influence of ethanol, acetonitrile, and trifluoroacetic acid on the stability of the labeled product. Internalization kinetics of 18F-AlF-PSMA-11 were compared with those of 68Ga-PSMA-11 using PSMA-expressing LNCaP tumor cells. Biodistribution of 18F-AlF-PSMA-11 (0.26 nmol/mouse, 8-9 MBq/mouse) in male BALB/c nude mice with PSMA-expressing subcutaneous LS174T-PSMA tumors was compared with that of 68Ga-PSMA-11 at 1 and 2 h after injection. In addition, 18F-AlF-PSMA-11 PET/CT and 68Ga-PSMA-11 PET/CT imaging were performed at 1 and 2 h after injection. Results: In contrast to 68Ga-PSMA-11, 18F-AlF-PSMA-11 was not stable in water (radiochemical purity was 64.5% immediately after purification and 52.7% at 120 min after purification). 18F-AlF-PSMA-11 remained relatively stable in 25 mM NH4OAc, pH 6.9, and radiochemical purity decreased from 98.5% at purification to 96.3%, 94.7%, and 92.5% at 60, 120, and 180 min after purification. In vitro, the 18F- and 68Ga-labeled compounds showed rapid internalization in LS174T-PSMA cells. The highest tumor uptake (percentage injected dose [%ID]) was observed at 2 h after injection (10.8 ± 2.3 %ID/g and 7.9 ± 1.3 %ID/g for 18F-AlF-PSMA-11 and 68Ga-PSMA-11, respectively [P > 0.05]). Renal tracer uptake peaked at 2 h after injection (43.5 ± 5.7 %ID/g and 105.8 ± 13.8 %ID/g for 18F-AlF-PSMA-11 and 68Ga-PSMA-11, respectively, P < 0.05). Bone uptake of 18F-AlF-PSMA-11 was 3.3 ± 0.6 at 1 h after injection and 5.0 ± 0.6 %ID/g at 2 h after injection and was dependent on the radiochemical purity at the time of injection. Bone uptake of 68Ga-PSMA-11 reached 0.1 ± 0.0 %ID/g at 1 and 2 h after injection. PSMA-expressing xenografts could be visualized using both 68Ga-PSMA-11- and 18F-AlF-PSMA-11 PET/CT. Conclusion: 18F-AlF-PSMA-11 using direct labeling with aluminum fluoride can be produced in NH4OAc, pH 6.9; shows a high internalization rate; and visualizes PSMA-expressing tumors with similar tumor uptake. Lower kidney uptake than with 68Ga-PSMA-11 may be advantageous for tumor detection. However, the limited instability and consequent Al18F uptake in bone might hamper the visualization of small PCa bone metastases.

Development of a New Folate-Derived Ga-68-Based PET Imaging Agent.

The folate receptor (FR) has emerged as an interesting diagnostic and therapeutic drug target with many potential applications in oncologic and inflammatory disorders. It was therefore the aim of this study to develop a folate-derived Ga-68-based positron emission tomography (PET) imaging tracer that is straightforward to radiolabel and could be broadly used in clinical studies. We validated its target binding affinity and specificity and compared it to [99mTc]EC20, the folate single-photon emission computed tomography (SPECT) imaging tracer that has been most extensively studied clinically so far. The new folic acid-derived PET imaging agent is linked via a polyethyleneglycol linker to the chelator 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA). This new compound, NOTA-folate, was labeled with gallium-68. We tested the probe's stability in human plasma and its selectivity in vitro, using the FR-positive KB cell line as well as the FR-negative A549 cell line. The pharmacokinetic profile of [68Ga]NOTA-folate was evaluated in FR-positive KB mouse xenografts. Following intravenous injection of [68Ga]NOTA-folate (383±53μCi), PET/computed tomography (CT) imaging studies as well as biodistribution studies were performed using KB tumor-bearing mice (n=3). In vitro as well as in vivo studies were performed in parallel with the SPECT imaging tracer [99mTc]EC20. In comparison to [99mTc]EC20 (radiochemical yield (RCY)=82.0±2.9%, 91.8±2.0% purity), similar radiochemical yield (87.2±6.9%) and radiochemical purity (95.6±1.8%) could be achieved for [68Ga]NOTA-folate. For both tracers, we observed high affinity for FR-positive cells in vitro and high plasma stability. In PET/CT and biodistribution studies, [68Ga]NOTA-folate appeared to display slightly superior in vivo performance in comparison to [99mTc]EC20. In detail, 68Ga-NOTA-folate showed very good tumor uptake and retention (6.6±1.1%ID/g), relatively low kidney uptake (21.7±1.1%ID/g), and very low liver uptake (0.38±0.08%ID/g). In vivo blocking studies using a fivefold excess of EC20 reduced the tumor uptake to 2.5±0.7%ID/g, confirming receptor specific binding of [68Ga]NOTA-folate in vivo. We validated a new Ga-68 folate-based PET imaging agent with excellent pharmacokinetics and tumor uptake. Based on a head-to-head comparison between both tracers, [68Ga]NOTA-folate is a suitable imaging probe for the delineation of FR-positive tumors and a promising candidate for clinical translation.

Low kidney uptake of GLP-1R-targeting, beta cell-specific PET tracer, 18F-labeled [Nle14,Lys40]exendin-4 analog, shows promise for clinical imaging.

BackgroundSeveral radiometal-labeled, exendin-based tracers that target glucagon-like peptide-1 receptors (GLP-1R) have been intensively explored for β cell imaging. The main obstacle has been the high uptake of tracer in the kidneys. This study aimed to develop a novel GLP1-R-specific tracer, with fluorine-18 attached to exendin-4, to label β cells for clinical imaging with PET (positron emission tomography). We hypothesized that this tracer would undergo reduced kidney uptake. 18F-labeled [Nle14,Lys40]exendin-4 analog ([18F]exendin-4) was produced via Cu-catalyzed click chemistry. The biodistribution of [18F]exendin-4 was assessed with ex vivo organ γ-counting and in vivo PET imaging. We also tested the in vivo stability of the radiotracer. The localization of 18F radioactivity in rat and human pancreatic tissue sections was investigated with autoradiography. Receptor specificity was assessed with unlabeled exendin-3. Islet labeling was confirmed with immunohistochemistry. The doses of radiation in humans were estimated based on biodistribution results in rats.Results[18F]exendin-4 was synthesized with high yield and high specific activity. Results showed specific, sustained [18F]exendin-4 uptake in pancreatic islets. In contrast to previous studies that tested radiometal-labeled exendin-based tracers, we observed rapid renal clearance of [18F]exendin-4.Conclusions[18F]exendin-4 showed promise as a tracer for clinical imaging of pancreatic β cells, due to its high specific uptake in native β cells and its concomitant low kidney radioactivity uptake.

Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.

A new transplantable ovarian tumor model is presented using a novel folate receptor (FR) positive, murine ovarian cancer cell line that emulates the human disease and induces widespread intraperitoneal (i.p.) tumors in immunocompetent mice within 4–8 weeks of implantation. Tumor development was monitored using a new positron emission tomography (PET) FR-targeting reporter with PET/computerized tomography (PET/CT) and fluorescence molecular tomography (FMT) using a commercial FR-targeting reporter. Conventional structural magnetic resonance imaging (MRI) was also performed. Adult female C57BL/6 mice were injected i.p. with 6 × 106 MKP-L FR+ cells. Imaging was performed weekly beginning 2 weeks after tumor induction. The albumin-binding, FR-targeting ligand cm09 was radiolabeled with the positron emitter 68Ga and used to image the tumors with a small animal PET/CT. The FR-reporter FolateRSense 680 (PerkinElmer) was used for FMT and flow cytometry. Preclinical MRI (7 T) without FR-targeting was compared with the PET and FMT molecular imaging. Tumors were visible by all three imaging modalities. PET/CT had the highest imaging sensitivity at 3–3.5 h postadministration (mean %IA/g mean > 6) and visualized tumors earlier than the other two modalities with lower kidney uptake (mean %IA/g mean < 17) than previously reported FR-targeting agents in late stage disease. FMT showed relatively low FR-targeted agent in the bladder and kidneys, but yielded the lowest anatomical image resolution. MRI produced the highest resolution images, but it was difficult to distinguish tumors from abdominal organs during early progression since a FR-targeting MRI reporter was not used. Nevertheless, there was good correlation of imaging biomarkers between the three modalities. Tumors in the mouse ovarian cancer model could be detected using FR-targeted imaging as early as 2 weeks post i.p. injection of tumor cells. An imaging protocol should combine one or more of the modalities, e.g., PET/CT or PET/MRI for optimal tumor detection and delineation from surrounding tissues.

Abstract 103: 64-Cu-NOTA-panitumumab F(ab')2 fragments for PET/CT imaging of pancreatic cancer

Abstract Background: Pancreatic cancer is a leading cause of cancer related death. Radiolabeled panitumumab (Pmab) has potential as an imaging agent for pancreatic cancer because it targets over-expression of human epidermal growth factor receptor (EGFR) commonly found in the disease. Our aim was to develop a Pmab-based radioimmunoconjugate (RIC) for use for PET imaging of pancreatic cancer. Methods: Pmab Fab fragments (PmabFab), F(ab')2 fragments (PmabF(ab')2), intact IgG (PmabIgG) and irrelevant anti-CD20 rituximab F(ab')2 (RmabF(ab')2) were derivatized with NOTA then 64Cu-labeled to ≥95 % radiochemical purity. RIC immunoreactivity was assessed by binding assays with MDA-MB-468 breast cancer cells (1 × 10E6 EGFR/cell), PANC-1 pancreatic cancer cells (4 × 10E5 EGFR/cell), and a primary pancreatic tumour cell line, OCIP-23 (4 × 10E5 EGFR/cell). RICs (4 MBq, 10 µg) were injected i.v. (tail vein) into non-tumour bearing Balb/c mice and biodistribution was assessed at 18 h post injection (p.i). Tumours were established in NOD-Scid or CD1 nude mice by s.c. inoculation with 1 × 10E7 PANC-1 cells or 1 × 10E7 MDA-MB-468 cells. RICs were injected i.v. (tail vein) into tumour-bearing mice (30 MBq, 10 µg) then PET/CT imaging and biodistribution studies were performed at 24 and 48 h p.i. Results: MDA-MB-468 cell binding assays showed 89.0 ± 0.7 % of applied 50 nM 64Cu-PmabFab and 88.8 ± 4.5 % of 64Cu-PmabF(ab')2 was displaced by 100-fold molar excess PmabIgG, demonstrating specificity for EGFR. Assessment of 64Cu-PmabF(ab')2 binding to pancreatic cell lines showed that 90.1 ± 2.0 % in PANC-1 cells and 80.2 ± 1.0 % in OCIP-23 cells of applied RIC was displaced by PmabIgG. In Balb/c mice, kidney uptake of 64Cu-PmabFab was significantly higher at 90.8 ± 9.0 % ID/g than that of 64Cu-PmabF(ab')2 and PmabIgG with 6.9 ± 0.7 % ID/g and 11.2 ± 1.0 % ID/g, respectively; (p≤0.05). Liver uptake of 64Cu-PmabF(ab')2 was lower at 5.4 ± 0.6 % ID/g than 64Cu-PmabIgG at 8.7 ± 0.9 % ID/g (p≤0.05). 64Cu-PmabF(ab')2 was selected for PET/CT imaging and biodistribution studies. The tumour:blood ratio in MDA-MB-468 xenografted mice was 1:1 at 24 h, but increased at 48 h to 5:1, with tumour uptake of 8.0 ± 0.8 % ID/g. 64Cu-RmabF(ab')2 tumour uptake in MDA-MB-468 xenografted mice at 48 h was 0.5 ± 0.1 % ID/g, significantly lower than that of 64Cu-PmabF(ab')2 (p≤0.005). The tumour:blood ratio in PANC-1 xenografted mice was 1:1 at 24 h, and improved to 2.4:1 at 48 h, with 6.2 ± 1.1 % ID/g tumour uptake. MDA-MB-468 and PANC-1 tumours were imaged by PET/CT. Studies with primary pancreatic tumour OCIP-23 xenografts are in progress. Conclusions: F(ab')2 is the optimal form of Pmab for PET/CT imaging due to lower kidney uptake than Fab fragments, and lower liver uptake than intact IgG. EGFR-positive PANC-1 and MDA-MB-468 xenografts were well visualized with 64Cu-PmabF(ab')2 at 48 h p.i. Supported by a grant from the Canadian Cancer Society Research Institute with funds from the Canadian Cancer Society. Citation Format: Amanda J. Boyle, David W. Hedley, Sachdev S. Sidhu, Mitchell A. Winnik, Raymond M. Reilly. 64-Cu-NOTA-panitumumab F(ab')2 fragments for PET/CT imaging of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 103. doi:10.1158/1538-7445.AM2014-103

Pretargeted immunoPET of prostate cancer with an anti-TROP-2 x anti-HSG bispecific antibody in mice with PC3 xenografts.

Pretargeting with bispecific antibodies and radiolabeled hapten-peptides could be used to specifically target tumors with high target-to-background ratios. TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-HSG (histamine-succinyl-glycine) Fab fragment. The TROP-2 antigen is expressed in many epithelial cancers, including prostate cancer (PC), and therefore, this bispecific antibody can be used for pretargeting of PC. In this study, the potential for pretargeted radioimmunoPET with TF12 and the (68)Ga-labeled di-HSG peptide IMP288 in mice with human PC xenografts was investigated using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) as a reference. The potential of pretargeted immunoPET with TF12 and the (68)Ga-labeled di-HSG hapten-peptide, IMP288, was studied in mice with subcutaneous PC3 tumors using [(18)F]FDG as a reference. Furthermore, the use of this pretargeting system for imaging PC lesions was evaluated in mice with intraperitoneally growing tumors with [(18)F]FDG as a reference. [(68)Ga]lMP288 showed rapid accumulation in the TF12 pretargeted subcutaneous tumor (7.2 ± 1.1% ID/g) with low uptake in the kidneys (1.8 ± 0.5% ID/g) and high tumor-to-blood ratios (17.4 ± 11.2) at 1h p.i. Accumulation of [(18)F]FDG in the s.c. tumors was significantly lower (3.4 ± 0.9% ID/g, P = 0.008), with lower tumor-to-blood ratios (3.0 ± 1.9, P = 0.011). ImmunoPET/CT images clearly visualized both subcutaneous and intraperitoneal tumors as small as 5mm(3) with low blood levels and kidney uptake as early as 1h p.i. Pretargeted immunoPET with TF12 in combination with a (68)Ga-labeled hapten-peptide is an efficient system for rapid, sensitive, and specific imaging of prostate cancer.

In vivo bacteriophage peptide display to tailor pharmacokinetics of biological nanoparticles.

Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance. In vivo phage display was performed to identify peptides displayed on phage that were excreted rapidly into the urine of mice. A novel in vitro assay using kidney cells, developed to predict in vivo kidney retention, and in vivo pharmacokinetic analyses were performed to characterize selected peptides/phage clones. Forty-three renal clearance clones (RCC) were identified. In vivo mixing experiments and in vitro kidney cell assays identified RCC1-02 as the lead compound. In vivo analysis of fluorescently labeled phage clones demonstrated the ability of RCC1-02 peptide to redirect the biodistribution of the large phage particle towards excretion via the kidney. Pharmacokinetic analysis of [(111)In]-radiolabeled peptides revealed that kidney retention of the control ErBB-2-avid peptide, [(111)In]DOTA-KCCYSL, at 2-h postinjection was 5.7 ± 0.7 %ID/g. In comparison, [(111)In]DOTA-RCC1-02 had kidney retention values of 1.66 ± 0.43 %ID/g, respectively. In vivo phage display can identify phage and corresponding peptides that rapidly clear the renal system. In the future, these peptides may be used to impart favorable pharmacokinetics onto a wide range of radioimaging or therapeutic macromolecules.

In Vivo and In Vitro Studies on Renal Uptake of Radiolabeled Affibody Molecules for Imaging of HER2 Expression in Tumors

Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody ZHER2:342 was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the (111)In- and (68)Ga-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: KD1=0.8 nM, Bmax1=71,500 and KD2=9.2 nM, Bmax2=367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.