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Purpose: Positron emission tomography with the radioligand [¹¹C]D-deprenyl has shown an increased signal at the location of pain in patients with ankle sprains, rheumatoid arthritis and chronic whiplash injury, but the mechanism of this tracer uptake and its exact binding site in inflammation or tissue injury is still unclear. The aim of this study was to further evaluate [¹¹C]D-deprenyl´s usefulness as a marker of acute inflammation.

Methods: An animal PET/CT study was performed three days after the induction of a rat model of inflammatory or surgical pain. Fourteen adult male Sprague-Dawley rats and three tracers [¹¹C]D-deprenyl, [¹¹C]L-dideuterumdeprenyl and [¹⁸F]fluorodeoxyglucose were used.

Results: No [¹¹C]D-deprenyl accumulation was seen in a rat model of musculoskeletal pain. In the rat model of inflammatory pain all three ligands were shown to visualize the inflamed ankle joint with much lower uptake in the control ankle joint. The uptake was largest with [¹¹C]D-deprenyl and [¹¹C]L- dideuteriumdeprenyl, where approximately 1 % of the injected dose could be found in the affected ankle joint during the first minutes, whereas the uptake of [¹⁸F]FDG was approximately 0.5 % of the injected dose. However, the ratio of uptake of the injected ankle joint versus the control ankle joint was much higher for [¹⁸F]FDG (around 10 fold increase) than for the two deprenyl enantiomers (2 – 3 fold increase). The uptake pattern of [¹¹C]D-deprenyl and [¹¹C]L-dideuteriumdeprenyl did not show signs of specific binding or irreversible trapping.

Conclusions: Contrary to our expectations, of the three tracers only [¹⁸F]FDG may be used as markers of peripheral inflammation in a rat model of inflammatory pain. However, as a high site-specificity is required, [¹¹C]D-deprenyl and [¹¹C]L-dideyteriumdeprenyl deserve further exploration regarding sensitivity, specificity and uptake mechanisms in human pain syndromes.

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of ¹⁸⁸Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule Z_HER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all ¹⁸⁸Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The ¹⁸⁸Re-Z_HER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of ¹⁸⁸Re-Z_HER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental ¹⁸⁸Re-Z_HER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of ¹⁸⁸Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

Receptor tyrosine kinases (RTK) are transmembrane receptors regulating cellular proliferation, differentiation, apoptosis, motility and recruitment of the vasculature. Aberrant expression and/or function of RTK have been detected in many malignant tumors and are considered to be a part of the transformed phenotype. The action of several classes of anti-cancer drugs is based on specific recognition of RTK. Monoclonal antibodies target extracellular binding domains, while tyrosine kinase inhibitors (TKI) bind to intracellular kinase domains to suppress RTK signaling. The issues regarding the efficient use of RTK targeting are the inter- and intra-patient heterogeneity of RTK expression and the changes of expression levels during the course of disease and in response to therapy. Radionuclide molecular imaging of RTK expression may aid in selecting patients who would benefit from RTK-targeting therapy and in identifying non-responders. Therefore, the therapy would be more personalized. Currently, radiolabeled proteins (monoclonal antibodies and their fragments, natural peptides ligands to RTK and de novo selected affinity proteins) and TKI and their analogues are under development for the visualization of RTK. In this review, we discuss the advantages and disadvantages of these approaches.

Introduction

Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.

Methods

The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z_HER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 were studied. Biodistribution of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 and [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 was compared in mice.

Results

The affinity of [MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 binding to HER2 was 67 pM. The ¹¹¹In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 due to higher clearance rate from normal tissues.

Conclusions

MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

Affibody molecules constitute a new class of probes for radionuclide tumor targeting. The small size of affibody molecules is favorable for rapid localization in tumors and clearance from circulation. However, high renal re-absorption of affibody molecules prevents the use of residualizing radiometals, including a number of promising low energy beta- and alpha-emitters, for radionuclide therapy. We tested a hypothesis that affibody-based pretargeting mediated by a bioorthogonal interaction between trans-cyclooctene (TCO) and tetrazine would provide higher accumulation of radiometals in tumor xenografts than in the kidneys.

Methods:

TCO was conjugated to the anti-HER2 affibody molecule Z2395. DOTA-tetrazine was labeled with indium-111 and lutetium-177. In vitro pretargeting was studied in HER2-expressing SKOV-3 and BT474 cell lines. In vivo studies were performed on BALB/C nu/nu mice bearing SKOV-3 xenografts.

Results:

¹²⁵I-Z2395-TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45±16 pM. ¹¹¹In-tetrazine bound specifically and selectively to Z2395-TCO pre-treated cells. In vivo studies demonstrated HER2-specific ¹²⁵I-Z2395-TCO accumulation in xenografts. TCO-mediated ¹¹¹In-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z2395-TCO. At 1 h post injection, the tumor uptake of ¹¹¹In-tetrazine and ¹⁷⁷Lu-tetrazine was ca. 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of ¹¹¹In in comparison with direct targeting.

Conclusion:

The feasibility of affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pretargeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy.

Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide ⁶⁸Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant Z_HER2:2395 HER2-binding Affibody molecule with ⁶⁸Ga. DOTA and NODAGA were site-specifically conjugated to the Z_HER2:2395 Affibody molecule having a C-terminal cysteine and labeled with ⁶⁸Ga and ¹¹¹In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for ⁶⁸Ga than for ¹¹¹In. The tumor uptake of ⁶⁸Ga-NODAGA-Z_HER2:2395 and ⁶⁸Ga-DOTA-Z_HER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for ⁶⁸Ga-NODAGA- Z_HER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody Z(HER2:342) was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the In-111- and Ga-68-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: K-D1 = 0.8 nM, B-max1 = 71,500 and K-D2 = 9.2 nM, B-max2 = 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.

Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of ^99mTc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide–based chelator (designated Z_HER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate ¹⁸⁸Re-Z_HER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)–expressing tumors.

Methods:

Z_HER2:V2 was labeled with ¹⁸⁸Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.

Results:

Binding of ¹⁸⁸Re-Z_HER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that ¹⁸⁸Re-Z_HER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.

Conclusion:

¹⁸⁸Re-Z_HER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

We describe the successful implementation of palladium-aryl oxidative addition complexes as stoichiometric reagents in carbonylation reactions with (CO)-C-11 to produce structurally challenging, pharmaceutically relevant compounds. This method enables the first C-11-carbonyl labeling of an approved PET tracer, [C-11]raclopride, for the dopamine D2/D3 receptor by carbonylation with excellent radiochemical purity and yield. Two other molecules, [C-11]olaparib and [C-11]JNJ 31020028, were efficiently labeled in this manner. The technique distinguishes itself from existing methods by the markedly improved purity profiles of the tracer molecules produced and provides access to complex structures in synthetically useful yields, hereby offering a viable alternative to other C-11-labeling strategies.

Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa are limited and in many cases only palliative. In a later, androgent independent, stage of PCa androgen receptors can be activated without interaction with ligand, i.e., by receptors of tyrosine kinase (RTK) family in the outlaw pathway. Human epidermal growth factor receptors HER2 and EGFR belong to RTK-family. HER2 is one of the main actors in the outlaw pathway with EGFR as the preferable heterodimerizing partner. We hypothesized that information on HER2 expression in advanced PCa could be useful for selection of patients for anti-RTK therapy and monitoring of therapy response. A panel of PCa cell lines (LNCap, PC3, DU-145) was subjected to a 8-week treatment using drugs influencing the RTK: trastuzumab (anti‑HER2), 17-DMAG (Hsp90 inhibitor), alone or in combination, and their HER2 and EGFR expressions were compared with non-treated cells. Treatment with trastuzumab decreased proliferation of LNCap and DU-145 cell lines, while 17-DMAG and trastuzumab/17‑DMAG combination affected all three cell lines. HER2 expression was significantly increased in PC3 cells, the most resistant cell line. On the contrary, in responding cells (LNCap and DU-145) HER2 expression decreased, accompanied by increased EGFR expression. However, additional treatment of cells with cetuximab (anti‑EGFR) did not give any additive effect to trastuzumab. In this study the response to anti-RTK therapy proved to vary between different PCa cell lines. We have demonstrated that RTK targeting treatments may affect the phenotypic profile of PCa tumor cells that correlates with therapy outcome. Observation of such changes during treatment could be used for monitoring and an improved therapy outcome.

Prostate cancer (PCa) is the most common type of cancer among males. Human epidermal growth factor receptor type 2 (HER2) expression in PCa has been reported by several studies and its involvement in the progression towards androgen-independent PCa has been discussed. External irradiation is one of the existing therapies, which has been demonstrated to be efficient in combination with androgen deprivation therapy for the treatment of advanced PCa. However, 20-40% of patients develop recurrent and more aggressive PCa within 10 years. The current study investigates the involvement of HER2 in survival and radioresistance in PCa cells and we hypothesized that, by monitoring HER2 expression, treatment may be personalized. The PCa cell lines, LNCap, PC3 and DU-145, received a 6 Gy single dose of external irradiation. The number of PC3 cells was not affected by a single dose of radiation, whereas a 5-fold decrease in cell number was detected in LNCap (P<0.00001) and DU-145 (P<0.0001) cells. The HER2 expression in PC3 exhibited a significant increase post irradiation, however, the expression was stable in the remaining cell lines. The administration of trastuzumab post-irradiation resulted in a 2-fold decrease in the PC3 cell number, while the drug did not demonstrate additional effects in LNCap and DU-145 cells, when compared with that of irradiation treatment alone. The results of the present study demonstrated that an increase in membranous HER2 expression in response to external irradiation may indicate cell radioresistance. Furthermore, imaging of HER2 expression prior to and following external irradiation may present a step towards personalized therapy in PCa.

Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium-111 (In-111) and assessed in vitro and in vivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with In-111 provided stable conjugates. In vitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT-474 breast carcinoma cells. In mice bearing BT-474 xenografts, the tumor uptake of the two conjugates was receptor-specific. Direct in vivo comparison of In-111-HEHEHE-Z08698-NOTA and In-111-HEHEHE-Z08699-NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for In-111-HEHEHE-Z08698-NOTA [12 +/- 3 vs. 8 +/- 1,4 h post injection (p.i)] due to more efficient blood clearance. In-111-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

The d-amino acid oxidase activator (DAOA) protein regulates the function of d-amino oxidase (DAO), an enzyme that catalyzes the oxidative deamination of d-3,4-dihydroxyphenylalanine (D-DOPA) and d-serine. D-DOPA is converted to l-3,4-DOPA, a precursor of dopamine, whereas d-serine participates in glutamatergic transmission. We hypothesized that DAOA polymorphisms are associated with dopamine, serotonin and noradrenaline turnover in the human brain. Four single-nucleotide polymorphisms, previously reported to be associated with schizophrenia, were genotyped. Cerebrospinal fluid (CSF) samples were drawn by lumbar puncture, and the concentrations of the major dopamine metabolite homovanillic acid (HVA), the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the major noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. Two of the investigated polymorphisms, rs3918342 and rs1421292, were significantly associated with CSF HVA concentrations. Rs3918342 was found to be nominally associated with CSF 5-HIAA concentrations. None of the polymorphisms were significantly associated with MHPG concentrations. Our results indicate that DAOA gene variation affects dopamine turnover in healthy individuals, suggesting that disturbed dopamine turnover is a possible mechanism behind the observed associations between genetic variation in DAOA and behavioral phenotypes in humans.

The development of positron emission tomography (PET) from being an exclusive and expensive research tool at major research institutes to a clinically useful modality found at most major hospitals around the world is largely dependent on radiochemistry and synthesis technology achievements by a few pioneer researchers starting their PET careers 40 to 50years ago. Especially, the introduction of [C-11]methyl iodide resulted in a quantum jump in the history of PET tracer development enabling the smooth labelling of a multitude of useful tracers. A more recent and still challenging methodological improvement is transition metal mediated C-11-carbonylations, having a large synthetic potential that has, however, not yet been realized in the clinical setting. This mini-review focuses on the history of carbon-11 radiochemistry and related technology developments and the role this played in PET tracer developments, especially emphasizing radiolabelling of endogenous compounds. A few examples will be presented of how the use of radiolabelled endogenous substances have provided fundamental information of in vivo biochemistry using the concept of position-specific labelling in different positions in the same molecule.

Cardiac amyloidosis is a differential diagnosis in heart failure and is associated with high mortality. There is currently no noninvasive imaging test available for specific diagnosis. N-[methyl-¹¹C]2-(4′-methylamino-phenyl)-6-hydroxybenzothiazole (¹¹C-PIB) PET is used in the evaluation of brain amyloidosis. We evaluated the potential use of ¹¹C-PIB PET in systemic amyloidosis affecting the heart.

Methods:

Patients (n = 10) diagnosed with systemic amyloidosis—including heart involvement of either monoclonal immunoglobulin light-chain (AL) or transthyretin (ATTR) type—and healthy volunteers (n = 5) were investigated with PET/CT using ¹¹C-PIB to study cardiac amyloid deposits and with ¹¹C-acetate to measure myocardial blood flow to study the impact of global and regional perfusion on PIB retention.

Results:

Myocardial ¹¹C-PIB uptake was visually evident in all patients 15–25 min after injection and was not seen in any volunteer. A significant difference in ¹¹C-PIB retention in the heart between patients and healthy controls was found. The data indicate that myocardial amyloid deposits in patients diagnosed with systemic amyloidosis could be visualized with ¹¹C-PIB. No correlation between ¹¹C-PIB retention index and myocardial blood flow as measured with ¹¹C-acetate was found on the global level, whereas a positive correlation on the segmental level was seen in a single patient.