Helmut Maecke

Glucagon-like peptide-1 receptor imaging for localization of insulinomas.

CONTEXTnThe surgical removal of insulinomas is hampered by difficulties to localize it using conventional radiological procedures. Recently these tumors were shown to exhibit a very high density of glucagon-like peptide-1 receptors (GLP-1R) in vitro that may be used as specific targets for in vivo receptor radiolabeling.nnnOBJECTIVEnThe objective of the study was to test the 111In-labeled GLP-1R agonist 111In-DOTA-exendin-4 in localizing insulinomas using single photon emission computed tomography in combination with computed tomography images.nnnDESIGNnThis was a prospective open-label investigation.nnnSETTINGnThe study was conducted at three tertiary referral centers in Switzerland.nnnPATIENTSnPatients included six consecutive patients with proven clinical and biochemical endogenous hyperinsulinemic hypoglycemia.nnnINTERVENTIONn(111)In-DOTA-exendin-4 was administered iv at a dose of about 90 MBq (30 microg peptide) over 5 min. Whole-body planar images of the abdomen were performed at 20 min, 4 h, 23 h, 96 h, and up to 168 h after injection. After surgical removal of the insulinomas, GLP-1R expression was assessed in the tumor tissue in vitro by GLP-1R autoradiography.nnnMAIN OUTCOME MEASUREnThe detection rate of insulinomas was measured.nnnRESULTSnIn all six cases, the GLP-1R scans successfully detected the insulinomas identified using conventional methods in four cases. By using a gamma-probe intraoperatively, GLP-1R detection permitted a successful surgical removal of the tumors in all patients, diagnosed histopathologically as five pancreatic and one extrapancreatic insulinomas. In vitro GLP-1R autoradiography showed a high density of GLP-1R in all tested insulinomas.nnnCONCLUSIONnIn vivo GLP-1R imaging is an innovative, noninvasive diagnostic approach that successfully localizes small insulinomas pre- and intraoperatively and that may in the future affect the strategy of insulinoma localization.

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

PurposeRadiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as 111In and 68Ga.MethodsRM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC50 and Kd values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca2+ mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with 111In-RM2 and 68Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with 68Ga-RM2.ResultsRM2 and 111In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7±3.3xa0nmol/l for RM2; 9.3±3.3xa0nmol/l for natIn-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca2+ mobilization assays. 68Ga- and 111In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2±4.8%IA/g at 1xa0h; 11.7±2.4%IA/g at 4xa0h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6±4.7%IA/g at 1xa0h to 1.5±0.5%IA/g at 4xa0h.ConclusionRM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that 111In-RM2 and 68Ga-RM2 are ideal candidates for clinical SPECT and PET studies.

Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting.

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.

Targeted Radiotherapy of Prostate Cancer with a Gastrin-Releasing Peptide Receptor Antagonist Is Effective as Monotherapy and in Combination with Rapamycin

The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. Methods: To determine the effect of treatment with rapamycin and radiotherapy with a novel 177Lu-labeled GRPr antagonist (177Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and 177Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on 177Lu-RM2 tumor uptake, in vivo small-animal PET studies with 68Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of 177Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with 177Lu-RM2 alone or after pretreatment with rapamycin. Results: Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with 177Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of 177Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. Conclusion: Radiotherapy using a 177Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

Radiolabeled Bicyclic Somatostatin-Based Analogs: A Novel Class of Potential Radiotracers for SPECT/PET of Neuroendocrine Tumors

A variety of radiolabeled somatostatin analogs have been developed for targeting of somatostatin receptor (sst)–positive tumors. Bicyclic somatostatin-based radiopeptides have not been studied yet. Hypothesizing that the introduction of conformational constraints may lead to receptor subtype selectivity or may help to delineate structural features determining pansomatostatin potency, we developed and evaluated first examples of this new class of potential radiotracers for imaging or therapy of neuroendocrine tumors. Methods: The bicyclic peptides were synthesized by standard solid-phase peptide synthesis. DOTA was coupled to the resin-assembled peptide for labeling with 177Lu and 68Ga. Binding affinity and receptor subtype profile were determined using human ssts. Ca2+ flux, internalization, and efflux were studied in human embryonic kidney (HEK)-sst2 and HEK-sst3 cell lines. Biodistribution and PET/CT studies were performed in corresponding nude mice models. Results: Some of the new analogs showed high affinity for sst2 and sst3 and moderate affinity for sst1, sst4, and sst5, while exhibiting agonistic properties. The analog AM3, comprising an octreotide ring and a head-to-tail–coupled Arg-diaminobutyric acid(DOTA) cycle, showed the highest receptor affinity and agonist potency. 177Lu-AM3 showed high and receptor-mediated uptake in vivo in sst2 and sst3 tumors with low background. Kidneys were the only other tissue accumulating radioactivity that could be reduced by a preinjection of lysine. PET/CT studies of 68Ga-AM3 at 1 h after injection were characterized by clear localization of the tumor, visualization of the kidneys, and negligible background. Conclusion: The high rigidity of these new bicyclic somatostatin-based radiopeptides led to agonistic ligands with good affinity for all 5 ssts. The pharmacokinetic data of 177Lu/68Ga-AM3 make this peptide an excellent candidate as an imaging—and especially as a PET—radiotracer.

Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours.

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer.

The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

Does Imaging αvβ3 Integrin Expression with PET Detect Changes in Angiogenesis During Bevacizumab Therapy

In recent years, there has been a growing interest in molecular imaging markers of tumor-induced angiogenesis. Several radiolabeled RGD (arginine, glycine, aspartate) peptides have been developed for PET imaging of αvβ3 integrins in the tumor vasculature, but there are only limited data on how angiogenesis inhibitors affect the tumor uptake of these peptides. Methods: Changes in 68Ga-NODAGA-c(RGDfK) peptide uptake were measured using PET during bevacizumab therapy of 2 αvβ3-negative squamous cell carcinoma cell lines (A-431 and FaDu) that induce αvβ3-positive neovasculature when transplanted into nude mice. Tumor uptake of 68Ga-NODAGA-c(RGDfK) was correlated to microvascular density, vascular morphology, and permeability as well as αvβ3 integrin expression. Results: Bevacizumab significantly inhibited growth of A-431 tumors and caused a significant reduction in microvascular density and αvβ3 integrin expression within 7 d after start of therapy. Bevacizumab also caused a normalization of blood vessel morphology and decreased tumor necrosis. However, 68Ga-NODAGA-c(RGDfK) uptake was significantly increased at day 7 of therapy and did not decrease until after 3 wk of treatment. In Fadu xenografts, bevacizumab therapy caused only a minor inhibition of tumor growth and minor changes in 68Ga-NODAGA-c(RGDfK) uptake. Conclusion: Uptake of radiolabeled RGD peptides is not necessarily decreased by effective antiangiogenic therapy. Early in the course of therapy a decrease in the expression of αvβ3 integrins may not be reflected by a decrease in the uptake of RGD peptides.

Current status and future perspectives of PSMA-targeted therapy in Europe: opportunity knocks

Prostate cancer (PCA), the second most common cancer in men and the fourth most common malignancy overall, causes an estimated 90,000 deaths per year in Europe [1]. Castrationresistant PCA (CRPC) is defined according to the Prostate CancerWorking Group 2 criteria as PCAwith any progression occurring in the presence of castrate-level testosterone values. This progression may be biochemical, i.e. a rise in prostatespecific antigen (PSA) levels, or clinical, i.e. appearance of metastases on imaging [2]. The CRPC therapeutic landscape has changed dramatically over the last decade. In 2003, the only options for patients when medical or surgical castration and peripheral androgen blockade had failed were palliative chemotherapy with mitoxantrone or best supportive care, including symptomatic palliative radiation or corticosteroids. As of 2015, five compounds have been approved for treating CRPC. Each has been demonstrated in pivotal phase III trials to confer an overall survival benefit (Table 1) [3–10]. Docetaxel was the mainstay of therapy for several years, before abiraterone/prednisolone, enzalutamide, cabazitaxel and the alpha-emitter Ra entered the stage. Every one of these substances not only improves survival endpoints but also provides numerous palliative benefits, e.g. pain control, quality-of-life improvement, and prevention of skeletal events [3–10]. The optimal sequence of this variety of options, however, especially the optimal positioning of chemotherapy in relation to hormonal manipulation or Ra administration, remains unclear, as does the potential of combinations of compounds. Trials to investigate these questions are underway. Despite these advances, overall survival for patients with CRPC remains relatively short, e.g., a median 19 months for patients in 23 Phase 3 trials of novel therapies (n = 13909) [11]. Thus the search for CRPC treatments continues. The implementation of radiolabelled compounds targeting prostate-specific membrane antigen (PSMA) for both diagnostic and therapeutic applications is considered to be a milestone in the management of these patients. PSMA PET/CT offers an appealing combination of PCA specificity and high sensitivity at low tumour volumes. These characteristics have led to the evolution of PSMA PET/CT into an important diagnostic tool in the management of advanced PCA. In the course of this evolution, it has become apparent that PSMA expression persists in a high percentage of patients with CRPC – in contrast to the expression of * M. Luster luster@med.uni-marburg.de

Approaches to Improve the Pharmacokinetics of Radiolabeled Glucagon-Like Peptide-1 Receptor Ligands Using Antagonistic Tracers.

The glucagon-like peptide-1 (GLP-1) receptors are important biomarkers for imaging pancreatic β-cell mass and detection of benign insulinomas. Using GLP-1 receptor antagonists, we aimed to eliminate the insulin-related side effects reported for all GLP-1 receptor agonists. Additionally, using a nonresidualizing tracer, 125I-Bolton-Hunter-Exendin(9-39)NH2 (125I-BH-Ex(9-39)NH2), we aimed to reduce the high kidney uptake, enabling a better detection of insulinomas in the tail and head of the pancreas. Methods: The affinity and biodistribution of Ex(9-39)NH2-based antagonists, modified with DOTA or NODAGA chelators at positions Lys27 and Lys40 and labeled with 68Ga and 125I-BH-Ex(9-39)NH2, were compared with the reference GLP-1 receptor agonist [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. The inhibitory concentration of 50% (IC50) values were determined using autoradiography on human tissues with 125I-GLP-1(7-36)NH2 as a radioligand. Pharmacokinetics and PET imaging were studied in nude mice bearing rat Ins-1E tumors. Results: Conjugation of DOTA and NODAGA chelators at positions Lys27 and Lys40 of Ex(9-39)NH2 resulted in a distinct loss of affinity toward GLP-1 receptor in vitro. Among the studied antagonists, [Lys40(NODAGA-natGa)NH2]Ex(9-39) showed the lowest IC50 value (46.7 ± 16.3 nM). The reference agonist [Nle14,Lys40(Ahx-DOTA)NH2]Ex-4 demonstrated the highest affinity (IC50 = 0.9 ± 0.3 nM). Biodistribution of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 at 1 h after injection demonstrated 40.2 ± 8.2 percentage injected activity per gram (%IA/g) uptake in Ins-1E tumor, 12.5 ± 2.2 %IA/g in the pancreas, and 235.8 ± 17.0 %IA/g in the kidney, with tumor-to-blood and tumor-to-kidney ratios of 100.52 and 0.17, respectively. Biodistribution of [Lys40(NODAGA-68Ga)NH2]Ex(9-39) showed only 2.2 ± 0.2 %IA/g uptake in Ins-1E tumor, 1.0 ± 0.1 %IA/g in the pancreas, and 78.4 ± 8.5 %IA/g in the kidney at 1 h after injection, with tumor-to-blood and tumor-to-kidney ratios of 7.33 and 0.03, respectively. In contrast, 125I-BH-Ex(9-39)NH2 showed tumor uptake (42.5 ± 8.1 %IA/g) comparable to the agonist and 28.8 ± 5.1 %IA/g in the pancreas at 1 h after injection. As we hypothesized, the kidney uptake of 125I-BH-Ex(9-39)NH2 was low, only 12.1 ± 1.4 %IA/g at 1 h after injection. The tumor-to-kidney ratio of 125I-BH-Ex(9-39)NH2 was improved 20-fold. Conclusion: Our results suggest that iodinated Ex(9-39)NH2 may be a promising tracer for imaging GLP-1 receptor expression in vivo. Because of the 20-fold improved tumor-to-kidney ratio 125I-BH-Ex(9-39)NH2 may offer higher sensitivity in the detection of insulinomas and imaging of β-cell mass in diabetic patients. Further studies with 124I-BH-Ex(9-39)NH2 are warranted.