Synthesis and biological evaluation of a novel non-peptidic brain-penetrant F-18-labeled oxytocin receptor ligand


Synthesis and biological evaluation of a novel non-peptidic brain-penetrant F-18-labeled oxytocin receptor ligand

Wenzel, B.; Mollitor, J.; Kranz, M.; Deuther-Conrad, W.; Günther, R.; Teodoro, R.; Fischer, S.; Ludwig, F.-A.; Smits, R.; Steinbach, J.; Hoepping, A.; Brust, P.

1. Introduction
Oxytocin is a neurohypophysial peptide hormone, synthesized in the hypothalamus and stored in the posterior pituitary gland for release into the bloodstream. It acts as neurotransmitter and neuromodulator to regulate a diverse range of CNS functions including emotional, parental, and sexual behaviors. Its receptor (OTR) is expressed in peripheral organs as well as in specific brain areas related to cognitive function and psychiatric diseases such as schizophrenia and depression. So far, investigation of the distribution of OTR in brain in vivo is hampered by the lack of suitable radiotracers. Thus, the development of a brain-penetrant PET ligand with high affinity and selectivity to the OTR would promote the development of non-invasive and quantitative imaging of OTR expression in healthy and diseased brain. Therefore, we synthesized a series of fluorinated non-peptidic OTR ligands and performed radiofluorination of one selected candidate to investigate its in vivo properties by organ distribution and dynamic PET imaging studies in mice and pigs.

2. Materials & Methods
Binding affinities of the novel compounds to the human OTR were determined by radioligand displacement studies using stably transfected hOTR-HEK293 cells and [3H]oxytocin. The radiosynthesis of the selected candidate [18F]ABX163 was performed in a two-step procedure using the methoxymethyl (MOM)-protected tosylate precursor ABX185. 18F was incorporated using K[18F]F-Kryptofix 222-carbonate complex at 90°C in ACN within 15 min followed by removal of the two MOM protecting groups with 1M HCl at 90°C within 15 min. The radiotracer was isolated by semi-preparative HPLC (Reprosil-Pur AQ column, 250x10mm), ACN/aqu. 20 mM NH4OAc) followed by final purification with a Sep-Pak C18 Plus light cartridge and formulation in isotonic saline containing 10% ethanol. Specific binding of [18F]ABX163 was assessed by in vitro autoradiography on mouse brain slices. Metabolism and organ distribution of the radiotracer were studied in female CD-1 mice at 30 and 60 min p.i. Dynamic PET scans were performed in mice (animal PET/MR; 60 min) and in one female piglet (PET; 120 min), the latter one accompanied by chromatographic analysis of plasma radio-metabolites.

3. Results
Based on the biphenyl-benzopyrrolodiazepine derivative WAY-162720, described as OTR selective and brain-penetrant ligand [1], three fluoro-containing reference compounds were synthesized which retained high affinity towards the OTR (Ki=14-22 nM). Radiolabeling of the selected candidate [18F]ABX163 was obtained in a two-step radiosynthesis with a RCY(DC) of 20-25%, radiochemical purity >98%, and specific activity of 35-133 GBq/µmol.[18F]ABX163 was stable for at least 60 min in saline, PBS, and pig plasma at 37°C. A logDoctanol/PBS value of 2.9 ± 0.2 (n=3) was determined by shake flask method.
Both organ distribution and dynamic PET imaging studies revealed limited uptake of the radiotracer in mouse brain (mean SUV value of 0.04). Besides, significant uptake in the pituitary gland was observed (SUV=0.85 at 55 min p.i.), which indicates target-specific binding of [18F]ABX163. By a dynamic PET study in one piglet, a mean SUV of 0.43 was estimated for whole brain at 120 min p.i. Most remarkable was the elevated uptake in the olfactory bulb with SUV120=0.73, a region with high expression of OTR. Metabolite analysis of pig plasma by radio-HPLC demonstrated moderate metabolism of [18F]ABX163 with non-metabolized tracer accounting for 44% of total radioactivity at 30 min p.i.

4. Discussion & Conclusion
Radiofluorination of a novel non-peptidic oxytocin receptor ligand [18F]ABX163 was achieved with appropriate radiochemical yield and specific activity. Evidence was obtained, that uptake of [18F]ABX163 in the pituitary gland of mouse brain and olfactory bulb of pig brain reflects target-specific binding. Differences in brain uptake between mice and pigs may be caused by species-specific expression of efflux transporters in the blood-brain barrier. With the development and evaluation of [18F]ABX163 we could demonstrate for the first time the potential of non-peptidic oxytocin receptor ligands for imaging of OTR in brain by PET. To further improve brain uptake, we are currently working on structural modifications of [18F]ABX163.

[1] Ring RH, Malberg JE, Potestio L, Ping J, Boikess S, Luo B, Schechter LE, Rosenzweig-Lipson S, [2006] Psychopharmacology 218-225.

  • Lecture (Conference)
    ESRR'2014 - 17th European Symposium on Radiopharmacy and Radiopharmaceuticals, 24.-27.04.2014, Pamplona, Spain

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Publ.-Id: 19719