Materials
Precursor, HAPTHI ((S)-1-(4-amino-3-hydroxybutyl)-3-phenyl-1,3-dihydrobenzo[c][1, 2, 5]thiadiazole 2,2-dioxide, and cold reference compound Me@HAPTHI ((S)-1-(3-hydroxy-4-(methylamino)butyl)-3-phenyl-1,3-dihydrobenzo[c][1, 2, 5]thiadiazole 2,2-dioxide) were custom-synthesized by ABX Advanced Biochemical Compounds (Radeberg, Germany). Briefly, synthesis of (2S)-4-(2,2-dioxido-3-phenyl-2,1,3-benzothiadiazol-1(3H)-yl)-1-(methylamino)butan-2-ol followed the route described by Neill et al. [
20,
21]. For more details, see Additional file
1.
2-Butanone (MEK, <99.0 % ACS reagent), acetonitrile (ACN, HPLC grade), dimethylsulfoxide (DMSO), tetrabutylammonium hydroxide 30-hydrate (TBAH), ammonium formate, ammonium acetate, sodium hydroxide, triethylamine and ethanol (absolute) were purchased from Sigma-Aldrich (Vienna, Austria) in the highest available grades. In addition, iodine (sublimated grade for analysis; ACS, Pharm. Eur.) was obtained from Merck (Darmstadt, Germany). Silver triflate impregnated carbon was prepared by reaction of 1 g of silver trifluoromethanesulfonate (Sigma Aldrich, Vienna, Austria) in 20 mL ACN with 3 g of Graphpac-GC (80/100 mesh, Alltech, Deerfield, USA). The suspension was stirred under protection from light and in an argon atmosphere for 30 min. After removal of the solvent, the resulting powder was dried under protection from light for further 2 h under reduced pressure.
For formulation of the product, 0.9 % saline solution from B. Braun (Melsungen, Germany), 3 % saline solution (Landesapotheke Salzburg, Austria) and sodium dihydrogenphosphate-monohydrate and disodiumhydrogenphosphate-dihydrate (both from Merck, Darmstadt, Germany) were used. Sterile water was purchased from Meditrade Medicare Medizinprodukte (Kufstein, Austria). Phosphate buffer (125 mM) was prepared by dissolving 0.224 g sodium dihydrogenphosphate-monohydrate and 1.935 g disodiumhydrogenphosphate-dihydrate in 100 mL sterile water. For solid phase extraction, C18 plus SepPak® cartridges were purchased from Waters (Waters® Associates, Milford, USA). Low-protein binding Millex® GS 0.22 μm sterile filters were obtained from Millipore (Bedford, USA).
All other chemicals and solvents for the radiosyntheses were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Vienna, Austria) with at least analytical grade and used without further purification.
NET, DAT and SERT expressing membrane preparations were obtained from Perkin Elmer (MA, USA). An ODP-50 column (20 × 4.0 mm, 5 μm) was purchased from Shodex® (Showa Denko Europe GmbH, Munich, Germany). For prediction of BBB penetration, a Redistech IAM.PC.DD2 column (Regis Technologies Inc., Morton Grove, USA) was used.
Microsomal preparations (human/rat liver microsomes) for stability testing were obtained from BD Bioscience (NJ, USA). Pooled human and rat plasma was obtained from Innovative Research (MI, USA).
The human postmortem tissue (7–9 h postmortem time, no history of neurological diseases) was obtained from the Neurobiobank of the Medical University of Vienna and approved by the local ethics committee (“Molecular neuropathologic investigation of neurodegenerative diseases” Nr.396/2011) following the principles of the Helsinki Declaration. Wild-type male rats were deeply anesthesized by isoflurane and sacrificed by decapitation. The organs of interest (i.e. brain, heart and testis) were removed and quick-frozen in i-pentan. Research using animal tissue was carried out under institutional approval in accordance with the Austrian Animal Care Law. Tissues were cut at −20 °C in a micro-cryotome (Microm HM 560, Thermo Scientific, Austria). Frozen slices were thaw-mounted onto superfrost slides (Menzel-Gläser SUPERFROST plus microscopy slides, Thermo Scientific, Germany). A barrier pen (Mini PAP Pen, Invitrogen, USA) was used for immunohistochemistry only. For detection of autoradiography, a Cyclone Phospho-Imager (Cyclone Plus Storage Phosphor System, Perkin Elmer, Germany) and Phosphor Imager plates (Multisensitive Phosphor Screens Long Type MS, PPN 7001724, Perkin Elmer, Germany) were used. The lead shielded and light-protected cassettes (Fisher Biotech Autoradiography Cassette FBCS 1417) were purchased from Fisher Scientific (PA, USA).
The NET-antibody (SLC6A2 Antibody H-67, sc-67216) was purchased from Santa Cruz Biotechnology (TX, USA). An endogenous Avidin-Biotin blocking kit (ab64212) as well as the DAB (=3,3′-diaminobenzidine) substrate kit (94665) was obtained from abcam (Cambridge, UK). A rabbit primary antibody isotype control was purchased from Invitrogen (CA, USA). A peroxidase-based Vectastain ABC kit (Rabbit IgG, PK-4001) was obtained from Vector Laboratories (CA, USA). Phosphate buffered saline (PBS pH 7.4, tenfold concentrate, 11237) was obtained from Morphisto Evolutionsforschung und Anwendung GmbH (Germany). Mayer’s Hemalaun solution was purchased from Merck Millipore (Germany). Histofluid (Marienfeld Superior, Germany) was used as a mounting medium. Coverslips from Menzel Gläser (24 × 60 mm, Thermo Fisher Scientific, Germany) were used for conservation of mounted slides. All other chemicals were obtained from Sigma-Aldrich.
Instrumentation
[11C]CO2 was produced within a GE PETtrace cyclotron (General Electric Medical System, Uppsala, Sweden) by a 14 N(p,α)11C nuclear reaction under irradiation of a gas target filled with N2 (+1 % O2) (Air Liquide Austria GmbH, Schwechat, Austria).
The evaluation of the reaction conditions was performed manually with starting activities <2 GBq. After optimization of the reaction parameters, [11C]Me@HAPTHI-synthesis was transferred to the TRACERlab™ FX C Pro synthesizer and a fully automated synthesis was established.
Crude [11C]Me@HAPTHI was purified by semi-preparative reversed phase HPLC using the built-in semi-preparative HPLC system equipped with a radioactivity and a UV detector (Linear Instruments Model 200 Detector UV/VIS) and a LaPrep HPLC pump (VWR International, Radnor, USA). A SupelcosilTM LC-ABZb, 5 μm, 250 × 10 mm (Supelco®, Bellefonte, PA, USA) column was used with a mobile phase of ACN/0.1 M ammonium acetate 40/60 v/v% at a flow rate of 6 mL/min.
The analytical HPLC was performed on a Merck-Hitachi LaChrom HPLC system (L-7100 pump; LaChrom L-7400 UV detector) using a NaI radio-detector (Bertholdt Technologies, Bad Wildbach, Germany) and a GinaStar® processing software (Raytest, Straubenhardt, Germany). A Phenomenex® Prodigy, Phenyl-3(PH-3), 5 μm, 250 × 4.6 mm (Phenomenex®, Aschaffenburg, Germany) column with a mobile phase consisting of ACN/0.1 M ammonium formate 50/50 v/v% at a flow rate of 2 mL/min was used while detection of the cold compounds was performed at 280 nm.
The osmolality of the final sterile product was measured with a Wescor osmometer Vapro® 5600 (Sanova Medical Systems, Vienna, Austria).