High-yielding 18F radiosynthesis of a novel oxytocin receptor tracer, a probe for nose-to-brain oxytocin uptake in vivo

Probing the mechanism of intranasal oxytocin brain uptake through generation and validation of a novel peptide PET tracer.

to 20 µmol of dry peptide bound resin), unless otherwise stated. After cleavage, crude mixtures were precipitated using ice cold TBME, centrifuged at 4000 rpm for 10 minutes at 4 o C, supernatants were discarded and the remaining peptides were washed with a fresh aliquot of TBME. The process was repeated. Crudes were subsequently dried in a desiccator over silica gel after which the solids were dissolved in a water methanol mixture and purified by RP-LCMS. Pure fractions were combined and lyophilized.

NODA-dLVT
After peptide synthesis, the resin was retrieved, washed and swelled in DMF for 1 h. Solvent was removed and a solution of 2% v/v hydrazine monohydrate in DMF was added. The mixture was agitated for 1 h, the deprotection mixture removed, and the resin washed. This process was repeated and resin bound peptide was used directly for conjugation. For this, HATU (44 mg, 115 µmol) in DMF (200 µL) was added dropwise to a mixture of NODA-MPAA (61 mg, 120 µmol), 2 and DIPEA (21 µL, 120 µmol) in DMF (400 µL). The mixture was agitated for 5 min then added to a DMF slurry of DIPEA (21 µL, 120 µmol) and resin bound dLVT (40 µmol) with the free lysine residue. The mixture was agitated for a further 2.5 h, after which the solution was removed by suction and washed. Resin was subsequently dried in a desiccator overnight then deprotected and cleaved using a mixture of TFA/DTT/H 2 O/TIS (91 : 3 : 3 : 3) for 3 h. Crude mixture was precipitated, centrifuged and dried in a dessicator, after which time the solid was dissolved and purified by RP-LCMS. Following lypolization a white solid was afforded (16 mg, 29%). Cyclisation was achieved using phosphate buffer (0.01 M, pH 7.5), at room temperature with 15% v/v DMSO (at a peptide concentration of 1 mg/ml) for 48 h. The peptide was purified by preparative RP-LCMS, affording NODA-dLVT as a white solid after lyophilisation (77%).

Synthesis of OT and peptide analogues
Synthesis of reduced open chain peptides followed the general procedure. Once completed, peptides were cleaved using the usual deprotection mixture and work up. Peptides were dissolved in a water methanol mixture and purified by RP-LCMS. Oxidation, when required, was performed in air with 0.1 M ammonium hydrogen carbonate buffer (peptide concentration of 0.1 mg/ml) for up to 3 days, after which the solution was concentrated, then lyophilised to yield pure peptides.

NODA-dLVT labelling with Al 18 F
Care was taken to ensure reactants were prepared in metal free conditions. A stock solution of NODA-dLVT in water was made. Aliquots (72 nmol) were removed and placed in eppendorfs for lyophilization. For the preparation of buffer solutions, any pH adjustment made used metal free glacial acetic acid.

Preparation of Na 18 F
In a typical procedure a QMA cartridge was washed with 0.5 M sodium acetate buffer (10 ml, pH 8.4), followed by deionized water (10 ml), as described previously. 4 [ 18 F]fluoride solution (6-10 mL; 1 GBq) was loaded onto the cartridge, washed with water (2 ml) and Na 18 F was eluted with 20% saline solution into 100 µL fractions. The fraction containing the highest activity (790 MBq) was subsequently used for radiolabelling.
Radiolabelling time: The length of reaction was studied using a molar ratio of 0.6:1 aluminium chloride to peptide with 20% saline (n=3). 18 F yields were not improved through extending the reaction time past 15 min: at 5 min, 18 F yield was 40 ± 8% and at 15 min the labelling efficiency was 84 ± 2%. After 30 min 18 F yields remained at 84 ± 3%.

Tissue Preparation for rat brain autoradiography
Male Wistar rats (250 g; n=3) were stunned then decapitated. Brains were rapidly removed, incubated in 20% sucrose solution for 2 h, and immediately frozen (-80 o C). Tissues were stored at -80 o C until required. Rat brains were sectioned in the sagittal plane (20 µm thickness; according to the atlas of Paxinos and Watson, 1998). Tissues were cut using a cryostat microtome (CM3050S, Leica, UK), and thaw-mounted onto glass microscope slides. Slides were stored at -80 o C until use.

Autoradiography Procedure
Tissue was thawed to room temperature, dried then washed with ice cold buffer (50 mM Tris, pH 7.4). Sections were air dried and incubated for 1 h at room temperature with [ 18 F]AlF-NODA-dLVT (3 nM) in buffer (50 mM Tris pH 7.4, 10 mM MgCl 2 ). For nonspecific binding, OT (10 µM) was co-incubated with [ 18 F]AlF-NODA-dLVT (3 nM) over the duration of the experiment. After incubation, slides were washed with ice-cold buffer (2 min × 2), followed by a final wash in ice-cold distilled water (2 min). Slides were dried in a cool airstream then exposed to 18 F sensitive phosphor screens (Amersham, UK) with [ 18 F]AlF-NODA-dLVT standards in X-ray cassettes overnight. Phosphor screens were imaged using a Typhoon 8600 scanner (Molecular Dynamics) and images were analysed with OptiQuant 5.0 (Perkin Elmer) and FIJI. Values were converted to relative binding percentages using calibrated [ 18 F]AlF-NODA-dLVT standards. Binding potential (BP) is defined as the binding of tracer in brain region/total activity incubated as determined by the standard. Specific binding in each region is calculated by comparing the total and non-specific BP by the equation: ((total BP -nonspecific BP)/total BP) × 100.

i.n. administration
A 0.3 mL insulin syringe, containing a 29-gauge needle, was fitted with a PE10 cannula (15 mm). The cannula was fitted so that it covered approximately 3 mm of the needle tip.
Radioligand was added to the syringe and the activity measured before and after administration to calculate the injected dose (ID). Anesthesia was induced using a mixture of 5% isoflurane and oxygen (Animalcare, UK) at a flow rate of 1 L/min and maintained at 2.5% isoflurane.
Once unresponsive, the rat was held in an almost vertical position and radioactivity was administered through one nare at the back of the nasal cavity.

Biodistribution
Rats were sacrificed by removal of the heart under isoflurane anaesthesia at 10 min following tail vein injection or 30 min following i.n. administration. Major organs were excised, washed with saline, weighed and counted in a gamma counter (LKB Wallac 1282), together with standards of the injection mixture. Organ uptake was calculated as percentage injected dose per gram [%ID/g] of tissue mass. Data are reported as mean ± SD.

Statistical analysis
Statistical analysis was carried out using the unpaired Student's t test algorithm on GraphPad Prism software. Findings were deemed significant if p values were less than 0.05.

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