Margret Schottelius

Molecular imaging targeting peptide receptors

Overexpressed neuropeptide receptors allow tumor visualization using e.g. radiolabeled peptidic receptor ligands and positron emission tomography (PET) or single photon emission computed tomography (SPECT). This review summarizes the development of radiolabeled probes for peptide receptor imaging since the emergence of the technology with a particular focus on peptide radiopharmaceuticals developed during the last 5 years, and highlights the different factors which are decisive during the process of tracer development and evaluation.

68Ga- and 177Lu-Labeled PSMA I&T: Optimization of a PSMA-Targeted Theranostic Concept and First Proof-of-Concept Human Studies

On the basis of the high and consistent expression of prostate-specific membrane antigen (PSMA) in metastatic prostate cancer (PC), the goal of this study was the development, preclinical evaluation, and first proof-of-concept investigation of a PSMA inhibitor for imaging and therapy (PSMA I&T) for 68Ga-based PET and 177Lu-based endoradiotherapeutic treatment in patients with metastatic and castration-resistant disease. Methods: PSMA I&T was synthesized in a combined solid phase and solution chemistry strategy. The PSMA affinity of natGa-/natLu-PSMA I&T was determined in a competitive binding assay using LNCaP cells. Internalization kinetics of 68Ga- and 177Lu-PSMA I&T were investigated using the same cell line, and biodistribution studies were performed in LNCaP tumor–bearing CD-1 nu/nu mice. Initial human PET imaging studies using 68Ga-PSMA I&T, as well as endoradiotherapeutic treatment of 2 patients with metastatic PC using 177Lu-PSMA I&T, were performed. Results: PSMA I&T and its cold gallium and lutetium analog revealed nanomolar affinity toward PSMA. The DOTAGA (1,4,7,10-tetraazacyclododecane-1-(glutamic acid)-4,7,10-triacetic acid) conjugate PSMA I&T allowed fast and high-yield labeling with 68GaIII and 177LuIII. Uptake of 68Ga-/177Lu-PSMA I&T in LNCaP tumor cells is highly efficient and PSMA-specific, as demonstrated by competition studies both in vitro and in vivo. Tumor targeting and tracer kinetics in vivo were fast, with the highest uptake in tumor xenografts and kidneys (both PSMA-specific). First-in-human 68Ga-PSMA I&T PET imaging allowed high-contrast detection of bone lesions, lymph node, and liver metastases. Endoradiotherapy with 177Lu-PSMA I&T in 2 patients was found to be effective and safe with no detectable side effects. Conclusion: 68Ga-PSMA I&T shows potential for high-contrast PET imaging of metastatic PC, whereas its 177Lu-labeled counterpart exhibits suitable targeting and retention characteristics for successful endoradiotherapeutic treatment. Prospective studies on larger cohorts of patients are warranted and planned.

First 18F-Labeled Tracer Suitable for Routine Clinical Imaging of sst Receptor-Expressing Tumors Using Positron Emission Tomography

Purpose: Despite excellent radionuclide characteristics, no 18F-labeled peptides are available for quantitative peptide receptor mapping using positron emission tomography (PET) so far, mainly due to time-consuming multistep radiosyntheses with limited overall yields. A newly developed two-step chemoselective conjugation method allows rapid and high-yield [18F]fluorination of peptides via oxime formation and was applied for the synthesis of new 18F-labeled carbohydrated Tyr3-octreotate (TOCA) analogs with optimized pharmacokinetics suitable for clinical routine somatostatin-receptor (sst) imaging. Experimental Design:18F-labeled glucose (Gluc-S-) and cellobiose (Cel-S-) derivatives of aminooxy-functionalized TOCA were synthesized via oxime formation with 4-[18F]fluorobenzaldehyde ([18F]FBOA-peptides). Both the in vitro internalization profile of Gluc-S-Dpr([18F]FBOA)TOCA and Cel-S-Dpr([18F]FBOA)TOCA in hsst2-expressing Chinese hamster ovary cells (dual tracer protocol) and their biodistribution in AR42J tumor-bearing mice were investigated and compared with two [18F]fluoropropionylated ([18F]FP) analogs, Gluc-Lys([18F]FP)TOCA and Gluc-S-Dpr([18F]FP)TOCA. Results: In contrast to [18F]FP-labeling (3 h), chemo-selective [18F]FBOA-formation (50 min) afforded the respective radiopeptides in high yields (65–85%). In vitro, Gluc-S-Dpr([18F]FBOA)TOCA and Cel-S-Dpr([18F]FBOA)-TOCA showed high internalization (139 ± 2 and 163 ± 8 of the reference [125I]Tyr3-octreotide, respectively), which was reflected by high tumor accumulation in vivo [21.8 ± 1.4 and 24.0 ± 2.5% of injected dose/g (1 h), respectively]. How-ever, only Cel-S-Dpr([18F]FBOA)TOCA and Gluc-S-Dpr([18F]FP)TOCA (tumor: 15.1 ± 1.5% of injected dose/g) with its very low accumulation in all of the nontarget organs showed improved tumor:organ ratios compared with Gluc-Lys([18F]FP)TOCA. For Cel-S-Dpr([18F]FBOA)TOCA,tumor:organ ratios (1 h) were 42:1, 27:1, 15:1, 3:1, and 208:1 for blood, liver, intestine, kidney, and muscle, respectively. Conclusion: Due to the fast and high-yield chemoselective radiofluorination strategy and to its excellent pharmacokinetics, Cel-S-Dpr([18F]FBOA)TOCA represents the first tracer suitable for routine clinical application in PET somatostatin receptor imaging.

PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent.

The overexpression of the chemokine receptor CXCR4 plays an important role in oncology, since together with its endogenous ligand, the stromal cell–derived factor (SDF1-α), CXCR4 is involved in tumor development, growth, and organ-specific metastasis. As part of our ongoing efforts to develop highly specific CXCR4-targeted imaging probes and with the aim to assess the suitability of this ligand for first proof-of-concept studies in humans, we further evaluated the new 68Ga-labeled high-affinity cyclic CXCR4 ligand, 68Ga-CPCR4-2 (cyclo(D-Tyr1-[NMe]-D-Orn2-[4-(aminomethyl) benzoic acid,68Ga-DOTA]-Arg3-2-Nal4-Gly5)). Methods: Additional biodistribution and competitions studies in vivo, dynamic PET studies, and investigations on the metabolic stability and plasma protein binding were performed in nude mice bearing metastasizing OH1 human small cell lung cancer xenografts. CXCR4 expression on OH1 tumor sections was determined by immunohistochemical staining. Results: natGa-CPCR4-2 exhibits high CXCR4 affinity with a half maximum inhibitory concentration of 4.99 ± 0.72 nM. 68Ga-CPCR4-2 showed high in vivo stability and high and specific tumor accumulation, which was reduced by approximately 80% in competition studies with AMD3100. High CXCR4 expression in tumors was confirmed by immunohistochemical staining. 68Ga-CPCR4-2 showed low uptake in nontumor tissue and particularly low kidney accumulation despite predominant renal excretion, leading to high-contrast delineation of tumors in small-animal PET studies. Conclusion: The small and optimized cyclic peptide CPCR4-2 labeled with 68Ga is a suitable tracer for targeting and imaging of human CXCR4 receptor expression in vivo. The high affinity for CXCR4, its in vivo stability, and the excellent pharmacokinetics recommend the further evaluation of 68Ga-CPCR4-2 in a proof-of-concept study in humans.

Chemoselective pre-conjugate radiohalogenation of unprotected mono- and multimeric peptides via oxime formation

Abstract As part of our ongoing efforts in the development of new 18F-labeled peptides for clinical PET imaging, a new two-step 18F-labeling methodology based on the chemoselective oxime formation between an unprotected aminooxy-functionalized peptide and a 18F-labeled aldehyde was investigated and optimized. 4-[18F]Fluorobenzaldehyde ([18F]FB-CHO) was prepared by direct n.c.a. fluorination of 4-formyl-N,N,N-trimethylanilinium triflate and purified by radio-HPLC or a strong-cation-exchange/reverse phase cartridge system. The aminooxyacetic acid (Aoa) modified model peptide LEF-NH2 (Leu-Glu-Phe-NH2) and monomeric, dimeric and tetrameric RGD-containing cyclopeptides were synthesized by solid phase peptide synthesis. Radiochemical yields of N-(4-[18F]fluorobenzylidene)-oxime-formation ([18F]FBOA) with the Aoa-modified unprotected peptides were investigated. Optimized reaction conditions (60 °C, 0.5 mM peptide, 15 min, aqueous solution, pH 2.5) resulted in 70%-90% conjugation yields for all unprotected peptides studied. Chemoselectivity was demonstrated in competition experiments with amino acid mixtures. Biodistribution in M21 melanoma bearing mice showed improved tumor uptake and excretion behaviour in the series c(RGDfE)HEG-Dpr-[18F]FBOA < (c(RGDfE)HEG)2K-Dpr-[18F]FBOA < ((c(RGDfE)HEG)2K)2K-Dpr-[18F]FBOA. Two hours p.i. the fraction of intact c(RGDfE)HEG-K-Dpr-[18F]FBOA in blood, liver, kidney and tumor was >90%, indicating high in vivo stability of the oxime linkage. Initial PET studies with ((c(RGDfE)HEG)2-K)2-K-Dpr-[18F]FBOA showed excellent imaging of M21-melanomas in mice. In conclusion, the new two-step chemoselective 18F-labeling fulfills all requirements for large scale syntheses of peptides in clinical routine. This methodology is also adaptable to other radioisotopes (e.g. radiohalogenation in general) and will thus offer a broad field of application.

Biodistribution and Radiation Dosimetry for a Probe Targeting Prostate-Specific Membrane Antigen for Imaging and Therapy

Prostate-specific membrane antigen (PSMA) is a promising target for diagnosis and treatment of prostate cancer. EuK-Subkff-68Ga-DOTAGA (68Ga-PSMA Imaging & Therapy [PSMA I&T]) is a recently introduced PET tracer for imaging PSMA expression in vivo. Whole-body distribution and radiation dosimetry of this new probe were evaluated. Methods: Five patients with a history of prostate cancer were injected intravenously with 91–148 MBq of 68Ga-PSMA I&T (mean ± SD, 128 ± 23 MBq). After an initial series of rapid whole-body scans, 3 static whole-body scans were acquired at 1, 2, and 4 h after tracer injection. Time-dependent changes of the injected activity per organ were determined. Mean organ-absorbed doses and effective doses were calculated using OLINDA/EXM. Results: Injection of 150 MBq of 68Ga-PSMA I&T resulted in an effective dose of 3.0 mSv. The kidneys were the critical organ (33 mGy), followed by the urinary bladder wall and spleen (10 mGy each), salivary glands (9 mGy each), and liver (7 mGy). Conclusion: 68Ga-PSMA I&T exhibits a favorable dosimetry, delivering organ doses that are comparable to (kidneys) or lower than those delivered by 18F-FDG.

Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer

BackgroundDue to its high expression in prostate cancer, PSMA (prostate-specific membrane antigen) represents an ideal target for both diagnostic imaging and endoradiotherapeutic approaches. Based on a previously published highly specific PSMA ligand ([68Ga]DOTA-FFK(Sub-KuE)), we developed a corresponding metabolically stable 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) construct for theranostic treatment of prostate cancer.MethodsAll ligands were synthesized by a combined solid phase and solution phase synthesis strategy. The affinity of the natgallium and lutetium complexes to PSMA and the internalization efficiency of the radiotracers were determined on PSMA-expressing LNCaP cells. The 68Ga- and 177Lu-labelled ligands were further investigated for lipophilicity, binding specificity, metabolic stability, as well as biodistribution and μPET in LNCaP-tumour-bearing mice.ResultsRadiochemical yields for 68Ga (3 nmol, 5.0 M NaCl/2.7 M HEPES (approximately 5/1), pH 3.5 to 4.5, 5 min, 95°C) and 177Lu labelling (0.7 nmol, 0.1 M NH4OAc, pH 5.5, 30 min, 95°C) were almost quantitative, resulting in specific activities of 250 to 300 GBq/μmol for the 68Ga analogues and 38 GBq/μmol for 177Lu complexes. Due to metabolic instability of l-amino acid spacers, d-amino acids were implemented resulting in a metabolically stable DOTAGA ligand. Compared to the DOTA ligand, the DOTAGA derivatives showed higher hydrophilicity (logP = -3.6 ± 0.1 and -3.9 ± 0.1 for 68Ga and 177Lu, respectively) and improved affinity to PSMA resulting in an about twofold increased specific internalization of the 68Ga- and 177Lu-labelled DOTAGA analogue. Especially, [68Ga]DOTAGA-ffk(Sub-KuE) exhibits favourable pharmacokinetics, low unspecific uptake and high tumour accumulation in LNCaP-tumour-bearing mice.ConclusionsThe pair of diagnostic/therapeutic PSMA-ligands [68Ga/177Lu]DOTAGA-ffk(Sub-KuE) possess remarkable potential for the management of prostate cancer.

In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma

CXCR4 is a G‐protein‐coupled receptor that mediates recruitment of blood cells toward its ligand SDF‐1. In cancer, high CXCR4 expression is frequently associated with tumor dissemination and poor prognosis. We evaluated the novel CXCR4 probe [68Ga]Pentixafor for in vivo mapping of CXCR4 expression density in mice xenografted with human CXCR4‐positive MM cell lines and patients with advanced MM by means of positron emission tomography (PET). [68Ga]Pentixafor PET provided images with excellent specificity and contrast. In 10 of 14 patients with advanced MM [68Ga]Pentixafor PET/CT scans revealed MM manifestations, whereas only nine of 14 standard [18F]fluorodeoxyglucose PET/CT scans were rated visually positive. Assessment of blood counts and standard CD34+ flow cytometry did not reveal significant blood count changes associated with tracer application. Based on these highly encouraging data on clinical PET imaging of CXCR4 expression in a cohort of MM patients, we conclude that [68Ga]Pentixafor PET opens a broad field for clinical investigations on CXCR4 expression and for CXCR4‐directed therapeutic approaches in MM and other diseases.

Introduction of functional groups into peptides via N-alkylation.

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.

Disclosing the CXCR4 Expression in Lymphoproliferative Diseases by Targeted Molecular Imaging

Chemokine ligand-receptor interactions play a pivotal role in cell attraction and cellular trafficking, both in normal tissue homeostasis and in disease. In cancer, chemokine receptor-4 (CXCR4) expression is an adverse prognostic factor. Early clinical studies suggest that targeting CXCR4 with suitable high-affinity antagonists might be a novel means for therapy. In addition to the preclinical evaluation of [68Ga]Pentixafor in mice bearing human lymphoma xenografts as an exemplary CXCR4-expressing tumor entity, we report on the first clinical applications of [68Ga]Pentixafor-Positron Emission Tomography as a powerful method for CXCR4 imaging in cancer patients. [68Ga]Pentixafor binds with high affinity and selectivity to human CXCR4 and exhibits a favorable dosimetry. [68Ga]Pentixafor-PET provides images with excellent specificity and contrast. This non-invasive imaging technology for quantitative assessment of CXCR4 expression allows to further elucidate the role of CXCR4/CXCL12 ligand interaction in the pathogenesis and treatment of cancer, cardiovascular diseases and autoimmune and inflammatory disorders.