Roland Haubner

Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [18F]Galacto-RGD

Background The integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [18F]Galacto-RGD, a 18F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET). Methods and Findings Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [18F]Galacto-RGD in the tumor correlates with αvβ3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize αvβ3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [18F]Galacto-RGD with PET enables noninvasive quantitative assessment of the αvβ3 expression pattern on tumor and endothelial cells in patients with malignant tumors. Conclusions Molecular imaging with [18F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases.

Positron Emission Tomography Using [18F]Galacto-RGD Identifies the Level of Integrin αvβ3 Expression in Man

Purpose: The integrin αvβ3 plays a key role in angiogenesis and tumor cell metastasis and is therefore an important target for new therapeutic and diagnostic strategies. We have developed [18F]Galacto-RGD, a highly αvβ3-selective tracer for positron emission tomography (PET). Here, we show, in man, that the intensity of [18F]Galacto-RGD uptake correlates with αvβ3 expression. Experimental Design: Nineteen patients with solid tumors (musculoskeletal system, n = 10; melanoma, n = 4; head and neck cancer, n = 2; gliobastoma, n = 2; and breast cancer, n = 1) were examined with PET using [18F]Galacto-RGD before surgical removal of the tumor lesions. Snap-frozen specimens (n = 26) were collected from representative areas with low and intense standardized uptake values (SUV) of [18F]Galacto-RGD. Immunohistochemistry was done using the αvβ3-specific antibody LM609. Intensity of staining (graded on a four-point scale) and the microvessel density of αvβ3-positive vessels were determined and correlated with SUV and tumor/blood ratios (T/B). Results: Two tumors showed no tracer uptake (mean SUV, 0.5 ± 0.1). All other tumors showed tracer accumulation with SUVs ranging from 1.2 to 10.0 (mean, 3.8 ± 2.3; T/B, 3.4 ± 2.2; tumor/muscle ratio, 7.7 ± 5.4). The correlation of SUV and T/B with the intensity of immunohistochemical staining (Spearmans r = 0.92; P < 0.0001) as well as with the microvessel density (Spearmans r = 0.84; P < 0.0001) were significant. Immunohistochemistry confirmed lack of αvβ3 expression in normal tissue (benign lymph nodes, muscle) and in the two tumors without tracer uptake. Conclusions: Molecular imaging of αvβ3 expression with [18F]Galacto-RGD in humans correlates with αvβ3 expression as determined by immunohistochemistry. PET with [18F]Galacto-RGD might therefore be used as a new marker of angiogenesis and for individualized planning of therapeutic strategies with αvβ3-targeted drugs.

[18F]Galacto-RGD Positron Emission Tomography for Imaging of αvβ3 Expression on the Neovasculature in Patients with Squamous Cell Carcinoma of the Head and Neck

Purpose: [18F]Galacto-RGD has been developed for positron emission tomography (PET)–imaging of αvβ3 expression, a receptor involved in angiogenesis and metastasis. Our aim was to study the feasibility of PET imaging with [18F]Galacto-RGD in patients with squamous cell carcinoma of the head and neck (SCCHN). Experimental Design: Eleven patients with primary diagnosis of SCCHN were examined. After injection of 140 to 200 MBq [18F]Galacto-RGD, static emission scans 60 min post injection from the head to the abdomen (n = 11) and dynamic scans >60 min covering the tumor region (n = 6) for kinetic modeling were acquired. Standardized uptake values (SUV) were measured in tumors, muscle and oral mucosa. Immunohistochemistry was done using an αvβ3-specific antibody (n = 7). Image fusion with magnetic resonance imaging and/or computed tomography (CT) scans (n = 8) and calculation of tumor subvolumes based on SUVs was done using the iPlan software (BrainLAB). Results: [18F]Galacto-RGD PET identified 10 of 12 tumors, with SUVs ranging from 2.2 to 5.8 (mean, 3.4 ± 1.2). Two tumors <5 mm were missed. Tumor/blood and tumor/muscle ratios were 2.8 ± 1.1 and 5.5 ± 1.6, respectively. Tumor kinetics was consistent with a two-tissue compartmental model with reversible specific binding. Immunohistochemistry confirmed αvβ3 expression in all tumors with αvβ3 being located on the microvessels in all specimens and additionally on tumor cells in one specimen. Image fusion of [18F]Galacto-RGD PET with magnetic resonance imaging/multislice CT and definition of tumor subvolumes was feasible in all cases. Conclusions: [18F]Galacto-RGD PET allows for specific imaging of αvβ3 expression in SCCHN with good contrast. Image fusion and definition of tumor subvolumes is feasible. This technique might be used for the assessment of angiogenesis and for planning and response evaluation of αvβ3-targeted therapies.

Radiolabeled Tracers for Imaging of Tumor Angiogenesis and Evaluation of Anti-Angiogenic Therapies

A variety of therapeutic strategies in oncology are focused on the inhibition of tumor-induced angiogenesis. Thus, there is a keen interest in methods which allow non-invasive monitoring of molecular targets involved in angiogenesis which would support information for planning and controlling corresponding therapies. Moreover, such techniques would provide an insight into the formation of new sprouting blood vessels, the involved processes and regulatory mechanisms in patients. At the moment, development of radiotracer based techniques is mainly concentrated on three different targets which include peptidic and non-peptidic alpha v beta 3-integrin binding antagonists, matrix metalloproteinase inhibitors and single chain anti-fibronectin antibody fragments. Development of radiolabeled MMP inhibitors is based on either the decapeptide Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys resulting from a phage display library or small molecular weight compounds. The in vitro data for these tracers are very promising. However, more detailed in vivo data are necessary to evaluate the potency of MMP-inhibitors for in-vivo imaging. The radiolabelled anti-ED-B single chain antibody fragment scFv L-19 shows selective accumulation in the tumor vasculature in a murine tumour model. In a first patient study a selective localisation of the (123)I-labeled tracer in lesions of different tumours was found. On the basis of the lead structure cyclo(-Arg-Gly-Asp-dPhe-Val) a variety of different radiolabeled RGD-peptides has been developed for the non-invasive determination of the alpha v beta 3 expression. These developments include peptides labeled with minimum structural alteration, peptide carbohydrate conjugates, peptidomimetics based on the RGD-structure as well as heterodimeric, homodimeric and homotetrameric ligand systems. Many of the tracers show high alpha v beta 3-affinity and selectivity in vitro and receptor selective tumour accumulation with high image contrast in different murine tumour models. Further studies have to demonstrate that this approach can be translated to clinical settings allowing visualisation of alpha v beta 3-positive tumours and alpha v beta 3 expression during tumour-induced angiogenesis in patients.

Imaging of integrin αvβ3 expression in patients with malignant glioma by [18F] Galacto-RGD positron emission tomography

Inhibitors targeting the integrin alpha(v)beta(3) are promising new agents currently tested in clinical trials for supplemental therapy of glioblastoma multiforme (GBM). The aim of our study was to evaluate (18)F-labeled glycosylated Arg-Gly-Asp peptide ([(18)F]Galacto-RGD) PET for noninvasive imaging of alpha(v)beta(3) expression in patients with GBM, suggesting eligibility for this kind of additional treatment. Patients with suspected or recurrent GBM were examined with [(18)F]Galacto-RGD PET. Standardized uptake values (SUVs) of tumor hotspots, galea, and blood pool were derived by region-of-interest analysis. [(18)F]Galacto-RGD PET images were fused with cranial MR images for image-guided surgery. Tumor samples taken from areas with intense tracer accumulation in the [(18)F]Galacto-RGD PET images and were analyzed histologically and immunohistochemically for alpha(v)beta(3) integrin expression. While normal brain tissue did not show significant tracer accumulation (mean SUV, 0.09 +/- 0.04), GBMs demonstrated significant but heterogeneous tracer uptake, with a maximum in the highly proliferating and infiltrating areas of tumors (mean SUV, 1.6 +/- 0.5). Immunohistochemical staining was prominent in tumor microvessels as well as glial tumor cells. In areas of highly proliferating glial tumor cells, tracer uptake (SUVs) in the [(18)F]Galacto-RGD PET images correlated with immunohistochemical alpha(v)beta(3) integrin expression of corresponding tumor samples. These data suggest that [(18)F] Galacto-RGD PET successfully identifies alpha(v)beta(3) expression in patients with GBM and might be a promising tool for planning and monitoring individualized cancer therapies targeting this integrin.

Radionuclide Imaging: A Molecular Key to the Atherosclerotic Plaque

Despite primary and secondary prevention, serious cardiovascular events such as unstable angina or myocardial infarction still account for one-third of all deaths worldwide. Therefore, identifying individual patients with vulnerable plaques at high risk for plaque rupture is a central challenge in cardiovascular medicine. Several noninvasive techniques, such as magnetic resonance imaging, multislice computed tomography, and electron beam tomography are currently being tested for their ability to identify such patients by morphological criteria. In contrast, molecular imaging techniques use radiolabeled molecules to detect functional aspects in atherosclerotic plaques by visualizing their biological activity. Based upon the knowledge about the pathophysiology of atherosclerosis, various studies in vitro and in vivo and the first clinical trials have used different tracers for plaque imaging studies, including radioactive-labeled lipoproteins, components of the coagulation system, cytokines, mediators of the metalloproteinase system, cell adhesion receptors, and even whole cells. This review gives an update on the relevant noninvasive plaque imaging approaches using nuclear imaging techniques to detect atherosclerotic vascular lesions.

68Ga- and 111In-labelled DOTA-RGD peptides for imaging of αvβ3 integrin expression

Purposeαvβ3 integrins are important cell adhesion receptors involved in angiogenic processes. Recently, we demonstrated using [18F]Galacto-RGD that monitoring of αvβ3 expression is feasible. Here, we introduce 68Ga- and 111In-labelled derivatives and compare them with [18F]Galacto-RGD.MethodsFor radiolabelling, cyclo(RGDfK(DOTA)) was synthesised using SPPS. For in vitro characterisation determination of partition coefficients, protein binding, metabolic stability, αvβ3 affinity and cell uptake and for in vivo characterization, biodistribution studies and micro positron emission tomography (PET) imaging were carried out. For in vivo and in vitro studies, human melanoma M21 (αvβ3 positive) and M21-L (αvβ3 negative) cells were used.ResultsBoth tracers can be synthesised straightforward. The compounds showed hydrophilic properties and high metabolic stability. Up to 23% protein-bound activity for [68Ga]DOTA-RGD and only up to 1.4% for [111In]DOTA-RGD was found. Cell uptake studies indicate receptor-specific accumulation. This is confirmed by the biodistribution data. One hour p.i. accumulation in αvβ3-positive tumours was 2.9 ± 0.3%ID/g and in αvβ3-negative tumours 0.8 ± 0.1%ID/g for [68Ga]DOTA-RGD ([111In]DOTA-RGD: 1.9 ± 0.3%ID/g and 0.5 ± 0.2%ID/g; [18F]Galacto-RGD: 1.6 ± 0.2%ID/g and 0.4 ± 0.1%ID/g). Thus, tumour uptake ratios were comparable. Due to approx. 3-fold higher blood pool activities for [68Ga]DOTA-RGD, tumour/blood ratios were higher for [111In]DOTA-RGD and [18F]Galacto-RGD. However, microPET studies demonstrated that visualisation of αvβ3-positive tumours using [68Ga]DOTA-RGD is possible.ConclusionsOur data indicate that [68Ga]DOTA-RGD allows monitoring of αvβ3 expression. Especially, the much easier radiosynthesis compared to [18F]Galacto-RGD would make it an attractive alternative. However, due to higher blood pool activity, [18F]Galacto-RGD remains superior for imaging αvβ3 expression. Introduction of alternative chelator systems may overcome the disadvantages.

αvβ3-integrin imaging: a new approach to characterise angiogenesis?

OverviewThe field of angiogenesis research is one of the most rapidly growing biomedical disciplines. Great efforts are being made to develop anti-angiogenesis drugs for treatment of cancer as well as non-oncological diseases. Thus, imaging techniques allowing non-invasive monitoring of corresponding molecular processes will be of great interest. One target structure involved in the angiogenic process is the integrin αvβ3, which mediates the migration of activated endothelial cells during vessel formation.Materials and methodsA variety of radiolabelled RGD peptides have been introduced for monitoring of αvβ3 expression using nuclear medicine tracer techniques.ObjectivesThis review discusses tracer development and highlights some strategies for tracer optimisation. It summarises the preclinical and clinical data and discusses the potential of this class of tracer to characterise angiogenesis.

Novel 64Cu- and 68Ga-Labeled RGD Conjugates Show Improved PET Imaging of ανβ3 Integrin Expression and Facile Radiosynthesis

PET with 18F-labeled arginine-glycine-aspartic acid (RGD) peptides can visualize and quantify ανβ3 integrin expression in patients, but radiolabeling is complex and image contrast is limited in some tumor types. The development of 68Ga-RGD peptides would be of great utility given the convenience of 68Ga production and radiolabeling, and 64Cu-RGD peptides allow for delayed imaging with potentially improved tumor-to-background ratios. Methods: We used the chelators DOTA,1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) to radiolabel the cyclic pentapeptide c(RGDfK) with 68Ga or 64Cu. NODAGA-c(RGDfK) was labeled at room temperature with both radionuclides within 10 min. Incubation at 95°C for up to 30 min was used for the other conjugates. The affinity profile of the metallopeptides was evaluated by a cell-based receptor-binding assay. Small-animal PET studies and biodistribution studies were performed in nude mice bearing subcutaneous U87MG glioblastoma xenografts. Results: The conjugates were labeled with a radiochemical purity greater than 97% and specific activities of 15–20 GBq/μmol. The affinity profile was similar for all metallopeptides and comparable to the reference standard c(RGDfV). In the biodistribution studies, all compounds demonstrated a relatively similar tumor and normal organ uptake at 1 h after injection that was comparable to published data on 18F-labeled RGD peptides. At 18 h after injection, however, 64Cu-NODAGA-c(RGDfK) and 64Cu-CB-TE2A-c(RGDfK) showed up to a 20-fold increase in tumor-to-organ ratios. PET studies demonstrated high-contrast images of the U87MG tumors at 18 h, confirming the biodistribution data. Conclusion: The ease of radiolabeling makes 68Ga-NODAGA-c(RGDfK) an attractive alternative to 18F-labeled RGD peptides. The high tumor-to-background ratios of 64Cu-NODAGA-c(RGDfK) and 64Cu-CB-TE2A-c(RGDfK) at 18 h warrant testing of 64Cu-labeled RGD peptides in patients.

Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography

In the field of molecular imaging, positron emission tomography (PET) has emerged as an imaging modality with excellent sensitivity for in vivo studies. PET labeling is challenging since short-lived positron-emitting isotopes such as F and C are used as labeling agents. The optimization and efficient application of rapid and reliable labeling strategies are prerequisites for obtaining access to new radiopharmaceuticals for both research and clinical trials. Bioactive peptides that specifically address molecular targets in vivo represent an important class of PET tracers to facilitate predictive imaging and PET-guided therapy. Diverse strategies for the synthesis of peptide-based radiopharmaceuticals using F-labeled prosthetic groups have been elaborated, including chemoselective oxime conjugation and the use of F-labeled maleimide derivatives as cysteinereactive reagents. Following the concept of click chemistry introduced by Sharpless et al., the Huisgen [3+2] azide– alkyne cycloaddition has been adapted to F-radiosynthetic methods in order to take advantage of its selectivity, reliability, and speed under aqueous mild Cupromoted reaction conditions. The versatility of peptide imaging agents is frequently hampered by their instability in vivo because of rapid degradation by endogenous peptidases. As an example, the synthesis of radiolabeled peptide-based imaging agents for the neurotensin receptor-1 (NTR-1), which is overexpressed in a number of human cancers, requires modifications to improve the metabolic stability. Synthetic approaches to RGD tracers targeting avb3 integrin, which plays a key role in angiogenesis, capitalize on the pioneering studies by Kessler et al. , who successfully developed cyclic pentameric RGD peptides that selectively recognize integrin avb3. [9] Various radiolabeled cyclic RGD peptides have been described. Among these, [F]galactoRGD has been extensively evaluated in clinical studies. Since glycosylation of peptides is known to frequently improve the biokinetic and in vivo clearance properties, [F]galacto-RGD and further radiopeptides have been approached. 13] However, the multistep radiosynthesis of [F]galacto-RGD is time-consuming and laborious. In proposals to overcome this drawback, F-labeling by 2-deoxy-2[F]fluoroglucose (FDG) has been discussed. 15] The major disadvantages of the F-peptide-labeling strategies currently used are 1) harsh reaction conditions, 2) laborious multiple-step syntheses with a limited decayuncorrected radiochemical yield (RCY), which would complicate the automation for large-scale production, and 3) lipophilic derivatization, which impair the biokinetic properties of the tracer. Based on our previous work on click chemistry in drug discovery and the synthesis of b-mannosyl azides, we herein present an efficient strategy toward F-labeling with concomitant glycosylation for the synthesis of F-glycopeptides as imaging agents for PET. We combined this strategy with the development of a metabolically stable glycopeptoid analogue of NT(8–13), which is the highly potent C-terminal hexapeptide of the natural agonist neurotensin (NT). As a proof of concept, two F-glycopeptides derived from NT(8– 13) and c(RGDfPra), respectively, were applied to biodistribution studies and mPET for imaging NTR and avb3-integrin expression in vivo using xenograft nude mice models. In detail, 2-deoxy-2-fluoroglucosyl azide (3) could be obtained starting from tetraacetylated 2-deoxy-2-fluoroglucose. The glucosyl azide 3 was applied for the Cu-catalyzed azide–alkyne coupling with a series of alkyne-functionalized peptides to evaluate the influence of the appended glycosyl residue on receptor recognition. Commercially available propargylglycine (Pra) was introduced by solid-phase-supported synthesis at position X into the sequence of the bioactive peptide c(RGDfX) and at the N terminus of NT(8– 13) and metabolically stabilized derivatives thereof. Considering our studies on the influence of peptide backbone modifications and ligand conformation on affinity changes for a series of NT(8–13) analogues, metabolic stabilization was envisioned by alteration of three amino acids in the sequence [*] Dr. S. Maschauer, Dr. C. Hocke, Prof. T. Kuwert, Dr. O. Prante Nuklearmedizinische Klinik, Labor f r Molekulare Bildgebung Friedrich-Alexander-Universit t Erlangen-N rnberg Schwabachanlage 6, 91054 Erlangen (Germany) Fax: (+ 49)9131-853-4325 E-mail: olaf.prante@uk-erlangen.de