Kidney Uptake Research Articles

New Radiolabeled Exendin Analogues Show Reduced Renal Retention.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex {67Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 67Cu-NOTA-GGNle-CycMSHhex {67Cu-NOTA-GlyGlyNle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized and purified by HPLC. The biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex was determined in B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 67Cu-NOTA-PEG2Nle-CycMSHhex was further examined in B16/F10 melanoma-bearing C57 mice. 67Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection. The tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 27.97 ± 1.98, 24.10 ± 1.83, and 9.13 ± 1.66% ID/g at 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2.6% ID/g at 4 h post-injection, except for kidney uptake. The renal uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 6.43 ± 1.31, 2.60 ± 0.79, and 0.90 ± 0.18% ID/g at 2, 4, and 24 h post-injection, respectively. 67Cu-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 single photon emission computed tomography (SPECT) using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 4 h post-injection. The favorable tumor targeting and biodistribution properties of 67Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment.

Preclinical Evaluation of a PSMA-Targeting Homodimer with an Optimized Linker for Imaging of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) targeting radiopharmaceuticals have been successfully used for diagnosis and therapy of prostate cancer. Optimization of the available agents is desirable to improve tumor uptake and reduce side effects to non-target organs. This can be achieved, for instance, via linker modifications or multimerization approaches. In this study, we evaluated a small library of PSMA-targeting derivatives with modified linker residues, and selected the best candidate based on its binding affinity to PSMA. The lead compound was coupled to a chelator for radiolabeling, and subject to dimerization. The resulting molecules, 22 and 30, were highly PSMA specific (IC50 = 1.0-1.6 nM) and stable when radiolabeled with indium-111 (>90% stable in PBS and mouse serum up to 24 h). Moreover, [111In]In-30 presented a high uptake in PSMA expressing LS174T cells, with 92.6% internalization compared to 34.1% for PSMA-617. Biodistribution studies in LS174T mice xenograft models showed that [111In]In-30 had a higher tumor and kidney uptake compared to [111In]In-PSMA-617, but increasing T/K and T/M ratios at 24 h p.i. Tumors could be clearly visualized at 1 h p.i. by SPECT/CT after administration of [111In]In-22 and [111In]In-PSMA-617, while [111In]In-30 showed a clear signal at later time-points (e.g., 24 h p.i.).

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 Comparative Study of 18F-FAPI-42 and 18F-FDG PET/CT for Evaluating Acute Kidney Injury in Cancer Patients.

Compare the value of imaging using positron 18F-labeled fibroblast activation protein inhibitor-42 (18F-FAPI-42) and 18F-labeled deoxyglucose (18F-FDG) for assessment of AKI. This study analyzed cancer patients who received 18F-FAPI-42 and 18F-FDG PET/CT imaging. Eight patients had AKI with bilateral ureteral obstruction (BUO), eight had BUO (CKD1-2) with no acute kidney disease (AKD), and eight had no ureteral obstruction (UO) with normal renal function. The average standardized uptake value (SUVave) of the renal parenchyma (RP-SUVave), the blood pool SUVave (B- SUVave), SUVave in the highest region of the renal collective system (RCS-SUVave), and the highest serum creatinine level (top SCr) were recorded. The 18F-FAPI-42 and 18F-FDG results showed that radiotracer of renal parenchyma was more concentrated in the AKI group than in the other two groups, whereas the RP-SUVave from 18F-FAPI-42 was higher than that from 18F-FDG in the AKI group (all P < 0.05). 18F-FAPI-42 imaging in the AKI group showed uptake by the renal parenchyma with a diffuse increase, but very little radiotracer in the renal collecting system, similar to a "super kidney scan." The renal parenchyma also had an increase of SUVave, with accumulation of radiotracer in the renal collecting system. AKI was more severe when a patient had a "super kidney scan" in both kidneys (P < 0.05). The B-SUVave level was higher in the AKI group than in the other two groups in 18F-FAPI-42 (both P < 0.05). 18F-FAPI-42 imaging had higher RP-SUVave than 18F-FDG imaging in cancer patients who had BUO with AKI. An increased renal parenchyma uptake in both kidneys and low radiotracer distribution in the collecting system suggest more severe AKI.

Intravascular Large B-Cell Lymphoma Identified by PET/CT.

Intravascular large B-cell lymphoma is a rare subtype of extranodal invasive non-Hodgkin lymphoma, and the diagnosis is challenging. Here we report findings of 18F-FDG PET/CT study in a case of intravascular large B-cell lymphoma invading bilateral lungs and kidneys in a 63-year-old woman. The PET/CT images showed diffuse increased FDG uptake in bilateral lungs and kidneys. Unexpectedly, there was no abnormal density on the CT images. The 18F-FDG PET/CT seems to be valuable and sensitive in the diagnosis of intravascular large B-cell lymphoma.

Abstract 5037: DARPins as powerful targeting agents for radioligand therapeutics

Abstract The development of effective radioligand therapeutics (RLTs) is frequently hampered by the lack of high-quality targeting agents that selectively deliver radioactive payloads to the site of disease while sparing healthy tissues. Antibodies can have high affinity and specificity to tumor targets, but their large size results in limited tumor penetration and long systemic half-life is frequently causing haematological toxicities. Alternatively, targeting agents with low molecular weight such as small molecules and peptides often suffer from limited affinity and specificity to the tumor target, resulting in off-target effects and limited tumor retention. DARPins (Designed Ankyrin Repeat Proteins) developed by Molecular Partners combine small size (15 kDa) and ideal binding properties. Due to their rigid-body target binding mode DARPins combine very high affinity and specificity and unless engineered accordingly, DARPins have very short systemic half-lives. Thanks to a simple and robust architecture, DARPins can be efficiently coupled with radioactive payloads, even at elevated temperatures; and they can tolerate sequence-engineering approaches, which are not compatible with other protein scaffolds. To establish the DARPin platform for RLT, we have used DARPin candidates against different tumor targets. We have previously shown that increasing affinity to the tumor target correlates with elevated tumor uptake and long tumor residence in preclinical mouse models. We now also show that DARPins exhibit a homogeneous and deep tumor penetration in vivo that is highly superior to antibody benchmarks. Globular proteins below 60 kDa in size are typically cleared from the bloodstream via the renal pathway. This generally results in a strong kidney accumulation of small sized, protein-based targeting agents and their coupled residualizing radionuclides, leading to dose-limiting kidney toxicities. To overcome this limitation, we have undertaken an extensive engineering approach of the DARPin scaffold. Our results show that sequence engineering strongly reduces kidney uptake of DARPins without affecting their tumor uptake. This effect was confirmed with independent DARPin candidates suggesting a general applicability of the approach. Combined with other orthogonal strategies, we are able obtain favourable tumor to kidney ratios in preclinical mouse models. These results show that our proprietary optimized DARPin platform offers an attractive solution to the limitations of protein-based targeting agents for RLT applications. Together with the fact that high-affinity DARPins can be generated against a large variety of tumor targets, we conclude that our platform provides a powerful basis for the development of next-generation RLTs. Several DARPin-RLT programs in indications with high unmet medical need are currently in development. Citation Format: Andreas Bosshart, Stephan Wullschleger, Martin Behe, Alain Blanc, Stefan Imobersteg, Alexandra Neculcea, Jacqueline Blunschi, Liridon Abduli, Sarah Schütz, Julia Wolter, Christian Reichen, Amelie Croset, Alessandra Villa, Christian Lizak, Anne Goubier, Roger Schibli, Daniel Steiner. DARPins as powerful targeting agents for radioligand therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5037.

Synthetic PET via Domain Translation of 3-D MRI.

Historically, patient datasets have been used to develop and validate various reconstruction algorithms for PET/MRI and PET/CT. To enable such algorithm development, without the need for acquiring hundreds of patient exams, in this article we demonstrate a deep learning technique to generate synthetic but realistic whole-body PET sinograms from abundantly available whole-body MRI. Specifically, we use a dataset of 56 18F-FDG-PET/MRI exams to train a 3-D residual UNet to predict physiologic PET uptake from whole-body T1-weighted MRI. In training, we implemented a balanced loss function to generate realistic uptake across a large dynamic range and computed losses along tomographic lines of response to mimic the PET acquisition. The predicted PET images are forward projected to produce synthetic PET (sPET) time-of-flight (ToF) sinograms that can be used with vendor-provided PET reconstruction algorithms, including using CT-based attenuation correction (CTAC) and MR-based attenuation correction (MRAC). The resulting synthetic data recapitulates physiologic 18F-FDG uptake, e.g., high uptake localized to the brain and bladder, as well as uptake in liver, kidneys, heart, and muscle. To simulate abnormalities with high uptake, we also insert synthetic lesions. We demonstrate that this sPET data can be used interchangeably with real PET data for the PET quantification task of comparing CTAC and MRAC methods, achieving ≤ 7.6% error in mean-SUV compared to using real data. These results together show that the proposed sPET data pipeline can be reasonably used for development, evaluation, and validation of PET/MRI reconstruction methods.

New Long-Acting [89Zr]Zr-DFO GLP-1 PET Tracers with Increased Molar Activity and Reduced Kidney Accumulation.

Positron emission tomography (PET) imaging is used in drug development to noninvasively measure biodistribution and receptor occupancy. Ideally, PET tracers retain target binding and biodistribution properties of the investigated drug. Previously, we developed a zirconium-89 PET tracer based on a long-circulating glucagon-like peptide 1 receptor agonist (GLP-1RA) using desferrioxamine (DFO) as a chelator. Here, we aimed to develop an improved zirconium-89-labeled GLP-1RA with increased molar activity to increase the uptake in low receptor density tissues, such as brain. Furthermore, we aimed at reducing tracer accumulation in the kidneys. Introducing up to four additional Zr-DFOs resulted in higher molar activity and stability, while retaining potency. Branched placement of DFOs was especially beneficial. Tracers with either two or four DFOs had similar biodistribution as the tracer with one DFO in vivo, albeit increased kidney and liver uptake. Reduced kidney accumulation was achieved by introducing an enzymatically cleavable Met-Val-Lys (MVK) linker motif between the chelator and the peptide.

Increased renal uptake and urine excretion of oxidized LDL is possibly associated with formation of large calcium oxalate nephrolithiasis: a preliminary study

Growing evidence have suggested an association between nephrolithiasis and cardiovascular disease (CVD)with unclear mechanism. Oxidized low-density lipoproteins (oxLDL) induces atherosclerosis and was found to be the possible link between these two diseases. Our study aimed to examine the serum, urine and kidney expression of oxLDL in relation to large calcium oxalate (CaOx) renal stone disease. A total of 67 large CaOx dominant renal stone patients and 31 stone-free controls were enrolled in the prospective case-control study. All participants were without known CVD history. Serum, urine, and kidney biopsy were collected before and during percutaneous nephrolithotomy, respectively. Enzyme-linked immunosorbent assays were used to assess serum and urine oxLDL, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and high-sensitivity C-reactive protein (hsCRP). There was no significantly difference in circulating oxLDL, but serum hsCRP was significantly near two-fold higher in nephrolithiasis patients. Serum hsCRP was also correlated with stone maximal length. Urine oxLDL was significantly higher in the nephrolithiasis group and correlated with serum hsCRP and stone maximal length. Increased oxLDL uptake in kidney was found in nephrolithiasis patients, whereas no significantly renal expression of oxLDL was observed in controls. The renal uptake of oxLDL with increased oxLDL excretion from large CaOxrenal stone formers, independent of increased circulating oxLDL, is a novel pathological finding in kidney stone disease and brings attention to the possible involvement of renal steatosis in the process of urolithiasis formation.

A Systematic Investigation into the Influence of Net Charge on the Biological Distribution of Radiometalated Peptides Using [68Ga]Ga-DOTA-TATE Derivatives.

Recently, several radiometalated peptides have been approved for clinical imaging and/or therapy (theranostics) of several types of cancer; nonetheless, the primary challenge that most of these peptides confront is significant renal uptake and retention, which is often dose limiting and can cause nephrotoxicity. In response to this, numerous methods have been employed to reduce the uptake of radiometalated peptides in the kidneys, and among these is adding a linker to modulate polarity and/or charge. To better understand the influence of net charge on the biodistribution of radiometalated peptides, we selected the clinically popular construct DOTA-TATE (NETSPOT/LUTATHERA) as a model system. We synthesized derivatives using manual solid-phase peptide synthesis methods including mechanical and ultrasonic agitation to effectively yield the gold standard DOTA-TATE and a series of derivatives with different net charges (+2, +1, 0, -1, -2). Dynamic PET imaging from 0 to 90 min in healthy female mice (CD1) revealed high accumulation and retention of activity in the kidneys for the net-neutral (0) charged [68Ga]Ga-DOTA-TATE and even higher for positively charged derivatives, whereas negatively charged derivatives exhibited low accumulation and fast renal excretion. Ex vivo biodistribution at 2 h post injection demonstrated a significant retention of [68Ga]Ga-DOTA-TATE (∼74 %ID/g) in the kidneys, which increased as the net positive charge per molecule increased to +1 and +2 (∼272 %ID/g and ∼333 %ID/g, respectively), but the -1 and -2 net charged molecules exhibited lower renal uptake (∼15 %ID/g and 16 %ID/g, respectively). Interestingly, the net -2 charged [68Ga]Ga-DOTA-(Glu)2-PEG4-TATE was stable in blood serum but had much higher healthy organ uptake (lungs, liver, spleen) than the net -1 compound, suggesting instability in vivo. Although the [68Ga]Ga-DOTA-PEG4-TATE derivative with a net charge of 0 also showed a decrease in kidney uptake, it also showed instability in blood serum and in vivo. Despite the superior pharmacokinetics of the net -1 charged [68Ga]Ga-DOTA-Glu-PEG4-TATE in healthy mice with respect to kidney uptake and overall profile, dynamic PET images and ex vivo biodistribution in male mice (NSG) bearing AR42J (SSTR2 overexpressing) subcutaneous tumor xenografts showed significantly diminished tumor uptake when compared to the gold standard [68Ga]Ga-DOTA-TATE. Taken together, these findings indicate unambiguously that kidney uptake and retention are significantly influenced by the net charge of peptide-based radiotracers. In addition, it was illustrated that the negatively charged peptides had substantially decreased kidney uptake, but in this instantiation the tumor uptake was also impaired.

Visualizing increased uptake of [18F]FDG and [18F]FTHA in kidneys from obese high-fat diet fed C57BL/6J mice using PET/CT ex vivo.

It is known that high-fat diet (HFD) and/or diabetes may influence substrate preferences and energy demands in the heart preceding diabetic cardiomyopathy. They may also induce structural glomerular changes causing diabetic nephropathy. PET/CT has been utilized to examine uptake of energy substrates, and to study metabolic changes or shifts before onset of metabolic disorders. However, conventional PET/CT scanning of organs with relatively low uptake, such as the kidney, in small animals in vivo may render technical difficulties. To address this issue, we developed a PET/CT ex vivo protocol with radiolabeled glucose and fatty acid analouges, [18F]FDG and [18F]FTHA,to study substrate uptake in mouse kidneys. We also aimed to detect a possible energy substrate shift before onset of diabetic nephropathy. The ex vivo protocol reduced interfering background as well as interindividual variances. We found increased uptake of [18F]FDG and [18F]FTHA in kidneys after HFD, compared to kidneys from young mice on standard chow. Levels of kidney triglycerides also increased on HFD. Lipoprotein lipase (LPL) activity, the enzyme responsible for release of fatty acids from circulating lipoproteins, is normally increased in postprandial mice kidneys. After long-term HFD, we found that LPL activity was suppressed, and could therefore not explain the increased levels of stored triglycerides. Suppressed LPL activity was associated with increased expression of angiopoietin-like protein4, an inhibitor of LPL. HFD did not alter the transcriptional control of some common glucose and fatty acid transporters that may mediate uptake of [18F]FDG and [18F]FTHA. Performing PET/CT ex vivo reduced interfering background and interindividual variances. Obesity and insulin resistance induced by HFD increased the uptake of [18F]FDG and [18F]FTHA and triglyceride accumulation in mouse kidneys. Increased levels of [18F]FDG and [18F]FTHA in obese insulin resistant mice could be used clinically as an indicator of poor metabolic control, and a complementary test for incipient diabetic nephropathy.

The effect of long-acting somatostatin analogues on the uptake of [177Lu]Lu-HA-DOTATATE.

According to IAEA/EANM/SNMMI guidelines, long-acting somatostatin analogues (LA-SSAs) should be discontinued 4-6weeks prior to peptide receptor radionuclide therapy (PRRT) to prevent somatostatin receptor saturation. The aim of this study was to determine the effect of continued use of long-acting SSAs during PRRT on the uptake of [177Lu]Lu-HA-DOTATATE on SPECT/CT. Consecutive patients with neuroendocrine tumours who were treated with PRRT receiving 7.4GBq of [177Lu]Lu-HA-DOTATATE were included. Patients were divided into 3 groups: (1) control (LA-SSA stopped > 6weeks prior to PRRT), or continued treatment with (2) long-acting octreotide < 6weeks prior to PRRT, or (3) long-acting lanreotide < 6weeks prior to PRRT. The uptake of [177Lu]Lu-HA-DOTATATE was quantified in healthy tissues (spleen, liver, kidneys, bone marrow) and tumour lesions on SPECT/CT performed 24h after PRRT. A Mann-Whitney U test was used to determine differences in uptake between the long-acting octreotide and long-acting lanreotide groups compared to the control group. Forty-two patients with 135 cycles of PRRT were included: 28 with lanreotide, 50 with octreotide, and 57 cycles without LA-SSAs. Uptake of [177Lu]Lu-HA-DOTATATE was significantly decreased in liver parenchyma in patients with lanreotide (p < 0.001) and in the spleen in patients with either octreotide or lanreotide (both p < 0.001). No differences were observed for uptake in kidneys, bone marrow, and blood pool. Uptake of [177Lu]Lu-HA-DOTATATE in tumours was the same in patients with lanreotide compared to the control (p = 0.862) and in patients with octreotide compared to the control (p = 0.201), independent of tumour location. Long-acting octreotide and lanreotide do not interfere with the uptake of [177Lu]Lu-HA-DOTATATE in tumour lesions 24h post-injection. Uptake in healthy liver parenchyma significantly decreases after lanreotide administration prior to PRRT, while uptake in healthy spleen tissue significantly decreases with both octreotide and lanreotide administration.

Lys-urea-Aad, Lys-urea-Cmc and Lys-urea-Cms as potential pharmacophores for the design of PSMA-targeted radioligands to reduce off-target uptake in kidneys and salivary glands.

High kidney and salivary gland uptake is a common feature of prostate-specific membrane antigen (PSMA)-targeted radioligands derived from the lysine-urea-glutamic acid (Lys-urea-Glu) pharmacophore. In this study we investigated if radioligands derived from lysine-urea-2-aminoadipic acid (Lys-urea-Aad), lysine-urea-S-carboxylmethylcysteine (Lys-urea-Cmc) and lysine-urea-O-carboxylmethylserine (Lys-urea-Cms) pharmacophores with/without an albumin binder could retain good PSMA-targeting capability but with minimized kidney and salivary gland uptake. Methods: HTK03177 and HTK03187 were obtained by replacing Aad in the previously reported Lys-urea-Aad-derived HTK03149 with Cmc and Cms, respectively. HTK03170, HTK04048 and HTK04028 were derived from HTK03149, HTK03177 and HTK03187, respectively, with the conjugation of an albumin-binding moiety, 4-(p-methoxyphenyl)butyric acid. In vitro competition binding assays were conducted using PSMA-expressing LNCaP prostate cancer cells and [18F]DCFPyL as the radioligand. Imaging and biodistribution studies of 68Ga-labeled HTK03177 and HTK03187, and 177Lu-labeled HTK03170, HTK04048 and HTK04028 were performed in LNCaP tumor-bearing mice. Radioligand therapy study of [177Lu]Lu-HTK03170 was carried out in LNCaP tumor-bearing mice and [177Lu]Lu-PSMA-617 was used for comparison. Results: The calculated Ki(PSMA) values of Ga-HTK03177, Ga-HTK03187, Lu-HTK03170, Lu-HTK04048 and Lu-HTK04028 were 5.0±2.4, 10.6±2.0, 1.6±0.4, 1.4±1.0 and 13.9±3.2 nM, respectively. PET Imaging and biodistribution studies at 1 h post-injection showed that both [68Ga]Ga-HTK03177 and [68Ga]Ga-HTK03187 had high uptake in LNCaP tumor xenografts (24.7±6.85 and 21.1±3.62 %ID/g, respectively) but minimal uptake in normal organs/tissues including kidneys (7.76±1.00 and 2.83±0.45 %ID/g, respectively) and salivary glands (0.22±0.02 and 0.16±0.02 %ID/g, respectively). SPECT imaging and biodistribution studies showed that the LNCaP tumor uptake of 177Lu-labeled HTK03170, HTK04048 and HTK04028 peaked at 4-24 h post-injection at ~43-65 %ID/g and was relatively sustained over time. Their peaked average uptake in kidneys (≤ 17.4 %ID/g) and salivary glands (≤ 2.92 %ID/g) was lower and continuously reduced over time. Radioligand therapy study showed that compared with [177Lu]Lu-PSMA-617 (37 MBq), a quarter dose of [177Lu]Lu-HTK03170 (9.3 MBq) led to a better median survival (63 vs 90 days). Conclusions: Our data demonstrate that that Lys-urea-Aad, Lys-urea-Cmc and Lys-urea-Cms are promising pharmacophores for the design of PSMA-targeted radioligands especially for radiotherapeutic applications to minimize toxicity to kidneys and salivary glands.

68Ga]Ga-DATA5m-LM4, a PET Radiotracer in the Diagnosis of SST2R-Positive Tumors: Preclinical and First Clinical Results

Radiolabeled somatostatin subtype 2 receptor (SST2R)-antagonists have shown advantageous profiles for cancer theranostics compared with agonists. On the other hand, the newly introduced hybrid chelator (6-pentanoic acid)-6-(amino)methyl-1,4-diazepinetriacetate (DATA5m) rapidly binds Ga-68 (t1/2: 67.7 min) at much lower temperature, thus allowing for quick access to "ready-for-injection" [68Ga]Ga-tracers in hospitals. We herein introduce [68Ga]Ga-DATA5m-LM4 for PET/CT imaging of SST2R-positive human tumors. LM4 was obtained by 4Pal3/Tyr3-substitution in the known SST2R antagonist LM3 (H-DPhe-c[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2) and DATA5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). [67Ga]Ga-DATA5m-LM4 was evaluated in HEK293-SST2R cells and mice models in a head-to-head comparison with [67Ga]Ga-DOTA-LM3. Clinical grade [68Ga]Ga-DATA5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA5m-LM4 displayed high SST2R binding affinity. [67Ga]Ga-DATA5m-LM4 showed markedly higher uptake in HEK293-SST2R cells versus [67Ga]Ga-DOTA-LM3 and was stable in vivo. In HEK293-SST2R xenograft-bearing mice, it achieved longer tumor retention and less kidney uptake than [67Ga]Ga-DOTA-LM3. [68Ga]Ga-DATA5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, [68Ga]Ga-DATA5m-LM4 has shown excellent prospects for the PET/CT diagnosis of SST2R-positive tumors, further highlighting the benefits of Ga-68 labeling in a hospital environment via the DATA5m-chelator route.

Biologic Evaluation of a Heterodimeric HER2-Albumin Targeted Affibody Molecule Produced by Chemo-Enzymatic Peptide Synthesis.

Targeted molecular radiation therapy is a promising emerging treatment modality in oncology, and peptide synthesis may shorten the time to reach the clinical stage. In this study, we have explored Chemo-Enzymatic Peptide Synthesis, or CEPS, as a new means of producing a therapeutic HER2 targeted Affibody® molecule, comprising a C-terminal albumin binding domain (ABD) for half-life extension and a total length of 108 amino acids. In addition, a DOTA moiety could be incorporated at N-terminus directly during the synthesis step and subsequently utilized for site-specific radiolabeling with the therapeutic radionuclide 177Lu. Retained thermodynamic stability as well as retained binding to both HER2 and albumin was verified. Furthermore, HER2 binding specificity of the radiolabeled Affibody molecule was confirmed by an in vitro saturation assay showing a significantly higher cell-bound activity of SKOV-3 (high HER2 expression) compared with BxPC3 (low HER2 expression), both in the presence and absence of HSA. In vivo evaluation in mice bearing HER2 expressing xenografts also showed specific tumor targeting as well as extended time in circulation and reduced kidney uptake compared with a HER2 targeted Affibody molecule without the ABD moiety. To conclude, we have demonstrated that CEPS can be used for production of Affibody-fusion molecules with retained in vitro and in vivo functionality.

Avalanche Photodiode-Based γ-Photon Scintillation Detectors for Personalized Dosimetry in Targeted Radionuclide Therapy

<h3>Purpose/Objective(s)</h3> Targeted radionuclide therapy holds great promise as shown in neuroendocrine and prostate cancer, but highly variable biodistribution and tumor uptake requires personalized dosimetry for each patient. Optimal dosimetry requires the measurement of total integrated dose, demanding dose measurements over multiple half-lives of the radionuclide. The current state-of-art, SPECT, is often only available to a small subset of patients and is rarely done more than once. Therefore, a method for continuous radionuclide dosimetry is needed. Highly-sensitive γ-photon detectors placed on the body could solve this problem by monitoring γ decays indicative of the activity within tumors and organs at risk (OAR). We have developed a system capable of detecting γ-photons externally, in real time, with high sensitivity and dynamic range. Here we present our results for photon detection as well as a methodology to use these signals to monitor the activity in tumors and OAR in real time. <h3>Materials/Methods</h3> Y₂O₃ Eu-doped phosphor was compacted at the interface of a bare optical fiber to scintillate incoming γ-photons into 610 nm (red) light. The optical fiber was coupled to avalanche photodiodes (APDs) with a 60% peak detection efficiency at 610 nm and dead time of 50ns. The transistor-transistor logic signals from each APD are conveyed to an FPGA to compute counts per minute (CPM). The system was tested by measuring the emitted γ-photons from a titration of ¹⁷⁷Lu. Six vials were prepared by serially diluting ¹⁷⁷Lu to activities per mL ranging from 0.6∼566.2µCi/mL. The detector was placed on the outside of the vials, and the vials were incrementally changed. The CPM were recorded in real time. To determine the ability to reconstruct a SPECT image in tumors and OAR, behavioral simulations were conducted. %ID/g in these organs, paralleling the measured activity in the ¹⁷⁷Lu dilutions, was varied and the ability of the sensors to track these variations was evaluated. <h3>Results</h3> The mean CPM for the six vials ranged from 30.8 CPM (0.6µCi/mL) to 47,240 CPM (566.2µCi/mL) and was highly linear with activity. The least-squares regression fit between µCi/mL and CPM was CPM=82.28 ⨉ Concentration(µCi/mL) with an R² value of 0.9970. This range of activities is consistent with tumor and kidney uptake of an 800µCi ¹⁷⁷Lu injection in a mouse model. With this mapping from µCi/mL to CPM, the behavioral simulation demonstrated that the temporally varying trend of %ID/g of tumors and OAR could successfully be tracked in real time. It was shown that sensor placement can be optimized to predictably quantify and prevent excessive cross-talk between signals from different organs. <h3>Conclusion</h3> An APD-based γ-photon scintillation detector was developed and verified with a ¹⁷⁷Lu titration. We envision that the proposed work could be used to create a sensor network on the body capable of real time dosimetry of tumors and OAR.

Cross-reactivity to glutamate carboxypeptidase III causes undesired salivary gland and kidney uptake of PSMA-targeted small-molecule radionuclide therapeutics.

Recently, Pluvicto™ ([177Lu]Lu-PSMA-617), a small-molecule prostate-specific membrane antigen (PSMA) radioligand therapeutic, has been approved by the FDA in metastatic castration-resistant prostate cancer. Pluvicto™ and other PSMA-targeting radioligand therapeutics (RLTs) have shown side effects due to accumulation in certain healthy tissues, such as salivary glands and kidney. Until now, the molecular mechanism underlying the undesired accumulation of PSMA-targeting RLTs had not been elucidated. We compared the sequence of PSMA with the entire human proteome to identify proteins closely related to the target. We have identified glutamate carboxypeptidase III (GCPIII), N-acetylated alpha-linked acidic dipeptidase like 1 (NAALADL-1), and transferrin receptor 1 (TfR1) as extracellular targets with the highest similarity to PSMA. The affinity of compound 1 for PSMA, GCPIII, NAALADL-1, and TfR1 was measured by fluorescence polarization. The expression of the putative anti-target GCPIII was assessed by immunofluorescence on human salivary glands and kidney, using commercially available antibodies. A fluorescent derivative of Pluvicto™ (compound 1) bound tightly to PSMA and to GCPIII in fluorescence polarization experiments, while no interaction was observed with NAALADL-1 and TfR1. Immunofluorescence analysis revealed abundant expression of GCPIII both in healthy human kidney and salivary glands. We conclude that the membranous expression of GCPIII in kidney and salivary gland may be the underlying cause for unwanted accumulation of Pluvicto™ and other Glu-ureido PSMA radio pharmaceuticals in patients.

Comparison of PSMA-TO-1 and PSMA-617 labeled with gallium-68, lutetium-177 and actinium-225

BackgroundPSMA-TO-1 (“Tumor-Optimized-1”) is a novel PSMA ligand with longer circulation time than PSMA-617. We compared the biodistribution in subcutaneous tumor-bearing mice of PSMA-TO-1, PSMA-617 and PSMA-11 when labeled with 68Ga and 177Lu, and the survival after treatment with 225Ac-PSMA-TO-1/-617 in a murine model of disseminated prostate cancer. We also report dosimetry data of 177Lu-PSMA-TO1/-617 in prostate cancer patients.MethodsFirst, PET images of 68Ga-PSMA-TO-1/-617/-11 were acquired on consecutive days in three mice bearing subcutaneous C4-2 xenografts. Second, 50 subcutaneous tumor-bearing mice received either 30 MBq of 177Lu-PSMA-617 or 177Lu-PSMA-TO-1 and were sacrificed at 1, 4, 24, 48 and 168 h for ex vivo gamma counting and biodistribution. Third, mice bearing disseminated lesions via intracardiac inoculation were treated with either 40 kBq of 225Ac-PSMA-617, 225Ac-PSMA-TO-1, or remained untreated and followed for survival. Additionally, 3 metastatic castration-resistant prostate cancer patients received 500 MBq of 177Lu-PSMA-TO-1 under compassionate use for dosimetry purposes. Planar images with an additional SPECT/CT acquisition were acquired for dosimetry calculations.ResultsTumor uptake measured by PET imaging of 68Ga-labeled agents in mice was highest using PSMA-617, followed by PSMA-TO-1 and PSMA-11. 177Lu-PSMA tumor uptake measured by ex vivo gamma counting at subsequent time points tended to be greater for PSMA-TO-1 up to 1 week following treatment (p > 0.13 at all time points). This was, however, accompanied by increased kidney uptake and a 26-fold higher kidney dose of PSMA-TO-1 compared with PSMA-617 in mice. Mice treated with a single-cycle 225Ac-PSMA-TO-1 survived longer than those treated with 225Ac-PSMA-617 and untreated mice, respectively (17.8, 14.5 and 7.7 weeks, respectively; p < 0.0001). Kidney, salivary gland, bone marrow and mean ± SD tumor dose coefficients (Gy/GBq) for 177Lu-PSMA-TO-1 in patients #01/#02/#03 were 2.5/2.4/3.0, 1.0/2.5/2.3, 0.14/0.11/0.10 and 0.42 ± 0.03/4.45 ± 0.07/1.8 ± 0.57, respectively.ConclusionsPSMA-TO-1 tumor uptake tended to be greater than that of PSMA-617 in both preclinical and clinical settings. Mice treated with 225Ac-PSMA-TO-1 conferred a significant survival benefit compared to 225Ac-PSMA-617 despite the accompanying increased kidney uptake. In humans, PSMA-TO-1 dosimetry estimates suggest increased tumor absorbed doses; however, the kidneys, salivary glands and bone marrow are also exposed to higher radiation doses. Thus, additional preclinical studies are needed before further clinical use.

Boosting Bismuth(III) Complexation for Targeted α‐Therapy (TAT) Applications with the Mesocyclic Chelating Agent AAZTA**

AbstractTargeted α therapy (TAT) is a promising tool in the therapy of cancer. The radionuclide 213BiIII shows favourable physical properties for this application, but the fast and stable chelation of this metal ion remains challenging. Herein, we demonstrate that the mesocyclic chelator AAZTA quickly coordinates BiIII at room temperature, leading to a robust complex. A comprehensive study of the structural, thermodynamic and kinetic properties of [Bi(AAZTA)]− is reported, along with bifunctional [Bi(AAZTA‐C4‐COO−)]2− and the targeted agent [Bi(AAZTA‐C4‐TATE)]−, which incorporates the SSR agonist Tyr3‐octreotate. An unexpected increase in the stability and kinetic inertness of the metal chelate was observed for the bifunctional derivative and was maintained for the peptide conjugate. A cyclotron‐produced 205/206Bi mixture was used as a model of 213Bi in labelling, stability, and biodistribution experiments, allowing the efficiency of [213Bi(AAZTA‐C4‐TATE)]− to be estimated. High accumulation in AR42J tumours and reduced kidney uptake were observed with respect to the macrocyclic chelate [213Bi(DOTA‐TATE)]−.