The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell DP4/8/9/2, dipeptidyl peptidases 4/8/9/2; EC, endothelial cell; FAP, fibroblast activating protein alpha; GEMSA, 2-Guanidinoethylmercaptosuccinic acid; hK1, tissue kallikrein; Kal, plasma kallikrein; MEE, external zone of median eminence; MEI, internal zone of median eminence; ME, median eminence; Mep-A/B, meprin A/B; MGTA, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid; NEP, neprilysin; NPY, neuropeptide Y; P32/98, Isoleucine thiazolidide hemifumarate; PEP, prolyl oligopeptidase; Pl, plasmin; sAmpP/mAmpP, aminopeptidases P1/P2; TOP, thimet oligopeptidase; uPA, urokinase; Y 1/2/5 -R, Y 1/2/5 -receptor. Ganong 2000). Apart from being involved in cardiovascular, body-fluid, food-intake, immunology, and temperature regulation, they are also associated with energy metabolism and stress-response as the median eminence is part of the hypothalamic-pituitary-adrenal axis, whereas the area postrema is linked to the neuro-autonomal-sympathetic stress axis via the nucleus tractus solitary. Although EM and AP lack the BBB, they tightly control transport/signals from blood/CSF to CNS and vice versa via tanycytes, membrane transporter systems and receptors such as Y2-R (Rodriguez et al.

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Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with (18)F and organ/tumor uptake was followed by μPET imaging and ex vivo biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy.

Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

Metabolic acidosis is a common feature of tumor microenvironment and may affect the phenotype of tumor cells, including invasive capacity and formation of metastases. We tested whether previous exposure to an acidic environment alters metastatic potential of two rat carcinoma cell lines in the animal model. In addition, we determined the effect of an acidic environment on motility and invasive capacity of AT-1 prostate carcinoma cells in culture. Exposure of tumor cells to an acidic environment (pH 6.6, 5 % CO2, 6 h) prior to tail vein injection in rats enhanced formation of lung metastases significantly. In culture, acidosis increased cellular motility of AT-1 cells. When the tumor cells were transferred back to pH 7.4, enhanced motility persisted for at least 3 h but vanished after longer periods (24 h), therefore presenting a "short-term memory effect." Although acidosis augmented phosphorylation of ERK1/2 and p38, and inhibition of ERK1/2 phosphorylation or of p38 kinase activity reduced basal motility at pH 7.4, acidosis-induced increase in motility was not dependent on ERK1/2 or p38 kinase. Src family kinases were not involved either. By contrast, scavenging reactive oxygen species (ROS), known to be increased in AT-1 cells under acidic conditions, blunted acidosis-induced motility increase. Our data indicate that tumor cells may acquire enhanced motility in an acidic micromilieu, at least in part due to enhanced ROS formation. Because enhanced motility persists for at least 3 h after leaving the acidic environment, this may promote metastasis formation, as observed in our in vivo model.