Søren Østergaard

68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

INTRODUCTION The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. METHODS Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. RESULTS In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga-NODAGA-AE105-NH(2) 60 min postinjection. CONCLUSIONS The use of (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the (64)Cu-labeled version ((64)Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH(2)) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and (68)Ga-NODAGA-AE105-NH(2) is the favored tracer as it provides the highest tumor-to-background ratio.

A Parallel Semisynthetic Approach for Structure–Activity Relationship Studies of Peptide YY

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N‐terminal truncated version PYY(3–36) decreases food intake and has potential as an anti‐obesity agent. The anorectic effect of PYY(3–36) is mediated through Y2 receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C‐terminal tetrapeptide sequence of PYY(3–36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C‐terminally modified PYY(3–36) analogues. By using an intein‐based expression system, PYY(3–29) was generated as a C‐terminal peptide α‐thioester. Heptapeptides bearing an N‐terminal cysteine and modifications at one of the four C‐terminal positions were synthesized in a 96‐well plate by parallel solid‐phase synthesis. In the plate format, an array of [Ala30]PYY(3–36) analogues were generated by ligation, desulfurization, and subsequent solid‐phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non‐proteinogenic amino acids, were tested in a functional Y2 receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y2 receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.

Metabolism of peptide YY 3–36 in Göttingen mini-pig and rhesus monkey

Peptide YY 3-36-amide (PYY3-36) is a peptide hormone, which is known to decrease appetite and food-intake by activation of the Y2 receptor. The current studies were designed to identify the metabolites of PYY3-36 in mini-pig and rhesus monkey. Plasma samples were analyzed by high resolution LC-MS (and MS/MS) in order to unambiguously identify the metabolites of PYY3-36. In summary, the metabolism of PYY3-36 was similar in mini-pig and rhesus monkey. Several metabolites were identified and PYY3-34 was identified at the highest levels in plasma. In addition, mini-pigs were also dosed with PYY1-36-amide, PYY3-35, PYY3-34 and [N-methyl 34Q]-PYY3-36-amide in order to investigate the mechanisms by which PYY was metabolized. PYY3-35 was rapidly converted to PYY3-34 whereas dosing of PYY3-34 to mini-pigs only showed circulating degradation products at low levels, i.e., PYY3-34 was metabolically more stable than PYY3-36 and PYY3-35. [N-methyl 34Q]-PYY3-36-amide was hypothesized to be stable toward cleavage between 34Q and 35R and after i.v. administration to mini-pigs, one major cleavage product was identified as [N-methyl 34Q]-PYY3-35. Overall, this showed that cleavage between 35R and 36Y was possible as well as between 34Q and 35R (as shown for PYY3-35), which indicated that metabolism of PYY3-36 to PYY3-34 may be a two-step process. PYY1-36 was also dosed to mini-pigs, which showed that PYY1-36 was metabolized in the C-terminal as PYY3-36. The overall degradation pattern of PYY1-36 was more complex due to the simultaneous enzymatic degradation in the N-terminal to form PYY2-34/36 and PYY3-34/36. In vitro incubations with heparin stabilized plasma showed that PYY3-36 was degraded with a half-life of 175 min, whereas incubations with PYY3-35 (half-life of 6 min) showed a rapid formation of PYY3-34. In conclusion, the present studies showed that PYY3-36 underwent enzymatic degradation in the C-terminal part and that the major circulating metabolite was PYY3-34. Furthermore, it may be a sequential two-step process leading to the formation of PYY3-35 and subsequently the metabolically more stable PYY3-34.

Binding mode of the peptide YY carboxyterminus to the human Y2 receptor

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment (32TRQRY36-amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln2886.55 and Tyr2195.39, while Gln1303.32 contributes to interactions with Q34 in the peptide and T32 is close to the tip of TM7 in the receptor. This leaves the core, α-helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y2 system and can be used as a basis for optimization of Y2 receptor agonists.

Abstract 5280: PET imaging of proteolysis: Evaluation of 68Ga-DOTA and 68Ga-NODAGA chelates of an uPAR-specific peptide in a human glioblastoma xenograft model

Background & Aim: The urokinase-type plasminogen activator receptor (uPAR) has been shown to facilitate cancer cell invasion and metastasis. uPAR is over-expressed in various cancers, including human breast, prostate and colorectal cancer, and over-expression has been shown to correlate with poor prognosis. The established correlation between uPAR expression and disease progression provides an opportunity to develop an in vivo imaging agent, which could identify cancer patients at risk. In the present study, we produced and compared two new PET tracers based on 68Ga, the chelators DOTA and NODAGA and the uPAR antagonist peptide AE105 in a human glioblastoma xenograft model using microPET/CT. Method: Both compounds were synthesized using standard Fmoc solid-phase peptide chemistry and subsequently N-terminal conjugated with either DOTA or NODAGA. The conjugated peptides were radiolabeled with 68Ga using a 68Ge/68Ga generator system, which render the production of the tracers independent of a onsite cyclotron. The compounds were evaluated in vitro by surface plasmon resonance against human uPAR and in vivo using micro-positron emission tomography in a mouse model bearing human glioblastoma U87MG xenografts. All findings were validated using uPAR ELISA on tumor tissue and uPAR immnohistochemistry on tumor slides. Results: Both compounds were labeled under mild conditions with high yield and radiochemical purity (>95%). Both compounds showed excellent uPAR binding (IC50 values of 10-15 nM) in vitro, which was identical to the un-conjugated peptide AE105. In vivo, they displayed different tumor uptake, with 68Ga-DOTA-AE105 and 68Ga-NODAGA-AE105 having an uptake of 0.48±0.3 and 2.58±0.02 %ID/g 0.5 hr post injection, respectively. Besides tumor uptake, high uptake in the kidneys was also observed for both compounds. Dynamic PET scans illustrated higher stability in vivo for 68Ga-NODAGA-AE105 compared to 68Ga-DOTA-AE105, thus generating the best tumor-to-background contrast. Conclusion: A new 68Ga-based PET tracer for tumor imaging of human uPAR has been developed. Based on head-to-head comparison, NODAGA seems to be the optimal chelator, giving the highest uptake in tumor tissue. The use of generator-produced 68Ga could further support translation into the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2011-5280

Abstract 5237: Non-invasive detection of urokinase-type plasminogen activator receptor (uPAR) expression in four human cancer xenograft mouse models using microPET/CT

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background & Aim The urokinase-type plasminogen activator receptor (uPAR) has been shown to facilitate cancer cell invasion and metastasis. uPAR is over-expressed in various cancers, including human breast, prostate and colorectal cancer, and these increased levels correlate to poor patient prognosis. With a view to future tailoring of uPAR-targeted therapies and the proven prognostic significance, we have developed an in vivo imaging positron-emission-tomography (PET) agent, which could identify cancer patients at increased risk and whom may benefit from such an adjuvant treatment. Methods The peptide antagonist AE1051 was characterized in vivo after DOTA conjugation and radio-labeling with 64Cu, in four different human cancer xenograft mouse models: H727 (lung carcinoid), HT-29 (colorectal carcinoma), A2780 (ovarian carcinoma) and U87MG (glioblastome) with a different expression of uPAR (n=10 mice, 2 tumors/mice). Nude mice carrying the above xenotransplants were injected intravenously with the tracer and a 10 min static PET scan were performed 1, 4.5 and 22 hrs post injection, followed by compute-tomography (CT) scan. Tumors were subsequently removed and their lysates were used to assess uPAR expression levels using standard uPAR ELISA. Another group of mice were PET scanned with 18F-FDG (n=10) and the results were compared with the results obtained with the uPAR tracer. In addition, a separate group of mice were subjected to biodistribution analysis of 64Cu-DOTA-AE105 (n=3 mice) and a mutated non-binding version of the tracer (n=3 mice) using a well-counter, to investigate the uptake in various organs and tissue 4.5 hrs p.i. Results A high and specific tumor uptake of 18F-FDG were observed in all four xenograft models but no correlation between uptake and uPAR expression was found (p=0.30, r=0.24). In contrast, a significant correlation was found for 64Cu-DOTA-AE105 tumor uptake and uPAR expression after both 1.0 hr (p=0.01, r=0.53), 4.5 hrs (p=0.0075, r=0.57) and 22 hrs (P<0.0001, r=0.76) across all four cancer xenograft models (n=20 tumors). The specificity of the tracer was validated using a mutated non-binding version of the peptide. A significant decrease in tumor uptake based on well-counter analysis (n=6 tumors) in the U87MG xenograft was found (p=0.028) compared with 64Cu-DOTA-AE105 tumor uptake after 4.5 hrs post injection, thus validating the specificity of the tracer towards uPAR. Conclusion Our data demonstrate a significant quantitative correlation between uPAR expression and tumor uptake and thus, that the PET tracer 64Cu-DOTA-AE105 is a promising radiotracer for non-invasive detection of uPAR in different human cancers. The results indicate a different tumor uptake profile compared with standard 18F-FDG and thus additional new tumor-specific information. 1Ploug et al 2001, Biochemistry 40, 12157-12168. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5237.