miRNA-187-3p-Mediated Regulation of the KCNK10/TREK-2 Potassium Channel in a Rat Epilepsy Model.

Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.

(R)-[11C]PK11195 brain uptake as a biomarker of inflammation and antiepileptic drug resistance: evaluation in a rat epilepsy model.

Neuroinflammation has been suggested as a key determinant of the intrinsic severity of epilepsy. Glial cell activation and associated inflammatory signaling can influence seizure thresholds as well as the pharmacodynamics and pharmacokinetics of antiepileptic drugs. Based on these data, we hypothesized that molecular imaging of microglia activation might serve as a tool to predict drug refractoriness of epilepsy. Brain uptake of (R)-[11C]PK11195, a ligand of the translocator protein 18 kDa and molecular marker of microglia activation, was studied in a chronic model of temporal lobe epilepsy in rats with selection of phenobarbital responders and non-responders. In rats with drug-sensitive epilepsy, (R)-[11C]PK11195 brain uptake values were comparable to those in non-epileptic controls. Analysis in non-responders revealed enhanced brain uptake of up to 39% in different brain regions. The difference might be related to the fact that non-responders exhibited higher baseline seizure frequencies than responders indicating a more pronounced intrinsic disease severity. In hippocampal sections, ED1 immunostaining argued against a general difference in microglia activation between both groups. Our data suggest that TSPO PET imaging might serve as a biomarker for drug resistance in temporal lobe epilepsy. However, it needs to be considered that our findings indicate that the TSPO PET data might merely reflect seizure frequency. Future experimental and clinical studies should further evaluate the validity of TSPO PET data to predict the response to phenobarbital and other antiepileptic drugs in longitudinal studies with scanning before drug exposure and with a focus on the early phase following an epileptogenic brain insult.

Synthesis and in vitro and in vivo evaluation of SiFA-tagged bombesin and RGD peptides as tumor imaging probes for positron emission tomography.

Gastrin-releasing-peptide (GRP)-receptors and αvß3-integrins are widely discussed as potential target structures for oncological imaging with positron emission tomography (PET). Favored by the overexpression of receptors on the surface of tumor cells good imaging characteristics can be achieved with highly specific radiolabeled receptor ligands. PEGylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (one-letter codes for arginine-glycine-aspartic acid) peptides as specific αvß3 binders were synthesized and tagged with a silicon-fluorine-acceptor (SiFA) moiety. The SiFA synthon allows for a fast and highly efficient isotopic exchange reaction at room temperature giving the [(18)F]fluoride labeled peptides in up to 62% radiochemical yields (d.c.) and ≥99% radiochemical purity in a total synthesis time of less than 20 min. Using nanomolar quantities of precursor high specific activities of up to 60 GBq µmol(-1) were obtained. To compensate the high lipophilicity of the SiFA moiety various hydrophilic structure modifications were introduced leading to significantly reduced logD values. Competitive displacement experiments with the PESIN derivatives showed a 32 to 6 nM affinity to the GRP receptor on PC3 cells, and with the RGD peptides a 7 to 3 µM affinity to the αvß3 integrins on U87MG cells. All derivatives proved to be stable in human plasma over at least 120 min. Small animal PET measurements and biodistribution studies revealed an enhanced and specific accumulation of the RGD peptide (18)F-SiFA-LysMe3-γ-carboxy-d-Glu-RGD (17) in the tumor tissue of U87MG tumor-bearing mice of 5.3% ID/g whereas the PESIN derivatives showed a high liver uptake and only a low accumulation in the tumor tissue of PC3 xenografts. Stability studies with compound 17 provided further information on its metabolism in vivo. These results altogether demonstrate that the reduction of the overall lipophilicity of SiFA tagged RGD peptides is a promising approach for the generation of novel potent (18)F-labeled imaging agents.

PESIN multimerization improves receptor avidities and in vivo tumor targeting properties to GRPR-overexpressing tumors.

The gastrin releasing peptide receptor (GRPR), being overexpressed on several tumor types, represents a promising target for specific noninvasive in vivo tumor imaging using positron emission tomography. Many of the radiolabeled bombesin analogs being applied in tumor imaging, however, suffer from shortcomings such as limited in vivo stability and poor tumor to background ratios. These obstacles can be overcome by peptide multimerization, as this approach results in constructs comprising several copies of the same peptide, thus retaining the ability to specifically bind to the target structure even if one peptide is cleaved. Furthermore, peptide multimers can result in increased binding avidities to the target, which can entail higher absolute tumor uptakes and also tumor to background levels. We therefore synthesized monomers and multimers of the peptide PESIN on dendrimer scaffolds comprising linkers of different lengths. The monomers/multimers were functionalized with the chelator NODAGA, efficiently radiolabeled with (68)Ga and evaluated in vitro regarding their GRPR binding affinity. The results show that shorter distances between the peptide moieties result in substantially higher binding affinities/avidities of the monovalent/multivalent PESIN ligands to the GRPR. Furthermore, the bivalent ligands gave the best results in terms of binding avidity, achieving a 2.5-fold increase in avidity compared to the respective monomer. Moreover, the most potent bivalent ligand showed an about 2-fold higher absolute tumor uptake and twice as high tumor-to-background ratios than the monomeric reference DOTA-PESIN in an initial animal PET study in tumor-bearing mice. Thus, besides high avidities, multivalency also positively influences the in vivo pharmacokinetics of peptide multimers.