The current translational investigation was aimed at demonstrating target receptor engagement for ORM-12741 as well as establishing the utility of [11C]ORM-13070 as a suitable PET tracer for assessment of α2C-AR occupancy in rat and human brain.
α2-AR subtype binding and antagonist characteristics in vitro
Receptor binding assays were performed at Cerep (Celle l’Evescault, France) according to their standard procedures, using stably transfected cell lines. Inhibition constants (Ki) were calculated using the Cheng-Prusoff equation (10). CHO cells transfected to express human α2-AR subtypes were used to determine antagonist properties of ORM-12741 in a calcium ion based fluorescent assay as described previously (11). Adrenaline and noradrenaline were used as agonists, and changes in intracellular calcium were monitored with a FLEXstation bench top scanning fluorometer equipped with an integrated fluid transfer workstation (Molecular Devices, San Jose, CA, USA) and SOFTmax PRO version 3.2 software. ORM-12741 (Orion Pharma, Espoo, Finland; 10-2 M) was dissolved in DMSO and subsequently diluted in Probenecid Ringer buffer.
Radiosynthesis of [11C]ORM-13070
[11C]ORM-13070 was synthesized at Turku PET Centre Radiopharmaceutical Laboratory, Turku, Finland as described previously (4) and was dissolved in a mixture of propylene glycol/ethanol/0.1 M phosphate buffer (7/3/45, v/v/v), pH 7.4 (4).
Rat brain ex vivo autoradiography
An ex-vivo autoradiography method, as described in (4), based on specific displacement of [11C]ORM-13070 binding in the caudate-putamen nucleus, was used to determine α2C-AR occupancy in rat brain. Male Sprague-Dawley rats (n = 4-6/group) were treated with vehicle (PEG 300/5 % glucose) or ORM-12741 (dissolved in PEG 300 and diluted with 5% glucose solution; 2, 10, 50 or 1000 µg/kg, s.c.) 10 min before injection of [11C]ORM-13070 (38-81 MBq) into the tail vein. Body temperature was kept stabile using a heating mattress. At 10 or 30 min after [11C]ORM-13070 administration, the rats were stunned by CO2 asphyxiation and terminal blood samples were taken for determination of plasma levels of ORM-12741. Brains were frozen by immersion in isopentane chilled on CO2 ice. Cryosections (40 µm) of the brain were prepared and regions of interest (caudate-putamen/cerebellum) were analysed by autoradiography as described previously by the Aida 2D densitometry program (4). Occupancy calculations were done similarly to the clinical study described below but instead of the baseline, average values in the vehicle group were used.
Animal care complied with the guidelines of the International Council of Laboratory Animal Science. The Animal Experiment Board of the Province of Southern Finland approved the methodologies used in this study.
α2C-AR occupancy in human brain in vivo
The clinical trial was an open label, single dose, uncontrolled study performed at a single centre. The primary objective of the study was to determine the extent of brain α2C-AR occupancy after different single oral doses of ORM-12741 and to describe the relationship of α2C-AR occupancy as a function of ORM-12741 dose and drug concentration in plasma. The study design involved dose ranging with adaptive selection of doses and assessment time points. The study protocol was approved by the Ethics Committee of the Hospital District of Southwest Finland and the Finnish Medicines Agency (EudraCT 2008-004929-42), and the trial was registered in the ClinicalTrials.gov database (NCT00829907).
Healthy male volunteers were enrolled after informed consent. Concomitant medications that could have affected the outcome of the study were prohibited within 2 weeks prior to the first PET scan or less than 5 times the elimination half-life of the medication. The use of nicotine containing products were forbidden during the stay at the study centre. Drug abuse and alcohol breath test were performed prior to PET scans and had to be negative. Each subject had three visits: a screening visit, a treatment visit and an end-of-study safety visit. A brain MRI scan was obtained for an individual anatomical reference map. All included subjects had a baseline PET scan ([11C]ORM-13070 alone) and 1 - 3 scans at set time points after different doses of ORM-12741 (Table 1).
Soft gelatin capsules containing ORM-12741 (0.1 mg, 1 mg and 10 mg) were produced by Orion Pharma (Espoo, Finland), and each study subject received a single oral dose (0.3, 1, 10, 30 or 60 mg) (Table 1).
Each [11C]ORM-13070 dose (target radioactivity 550 MBq; <10 μg of ORM-13070) was given as a rapid intravenous bolus injection (1 - 10 mL) at the start of the PET scan. PET imaging was performed as described previously (7). In brief a high-resolution research tomograph (HRRT; Siemens Medical Solutions,Knoxville, TN) with the subject’s head fixed in a head holder with an individually prepared thermoplasticmask was used. In addition, head movements were recorded with an infrared camera (Vicra®; Northern Digital Inc., Waterloo, ON, Canada). Slices of approximately 1.22 mm thickness covered the whole brain (axial field of view 25.3 cm). The camera was used in 3-D mode with scatter correction. The HRRT achieved transaxial and axial spatial resolution (full-width at half-maximum) of 2.5 mm. Before each PET scan, a transmission scan was done for attenuation correction with a 137Cs rotating point source. Regions-of-interest (ROIs) were manually drawn on the co-registered MRI scans using Imadeus software (version 1.1, Forima, Turku, Finland), checked to match the summated PET images and then transferred onto the dynamic PET image, from which regional time-activity curves were obtained for the following selected regions of the left and right brain hemispheres: caudate nucleus, cerebellar cortex, hippocampal region, lateral frontal cortex, occipital cortex, parietal cortex, putamen and thalamus.
Tracer uptake in the ROIs was described with areas under the curves (AUC) in the scan time window of 5 - 30 min after tracer injection. As the cerebellum has been reported to be devoid of α2C-ARs, it was used as a reference region for correction of non-specific uptake. A binding parameter (BiP) was calculated for each ROI as the ratio of specific binding (AUCregion – AUCcerebellar cortex) and the AUC in the cerebellar cortex. Receptor occupancy by [11C]ORM-13070 was negligible at the employed tracer doses (< 10 μg) (7). Receptor occupancy in the target regions was calculated according to the equation:
where BiPbaseline = pre-drug baseline BiP value, BiPdrug = BiP value following ORM-12741.
Left- and right-side receptor occupancy estimates were averaged to a single value for each ROI.
Liquid chromatography-tandem mass spectrometry was used for the determination of concentrations of ORM-12741 in plasma extracted from venous blood samples that were collected before and 10min, 40min, 60min 90min, 2h, 3.5 h, 4h, 6h, 6,5h,12h, 12.5h and 24 h after ORM-12741 dosing. Plasma PK variables (Cmax: peak concentration, tmax: time to peak concentration, AUCt: area under the drug plasma concentration-time curve from time zero to the last observed concentration, AUC∞: area under the drug plasma concentration-time curve from time zero to infinity, t1/2: terminal half-life) for ORM-12741 were calculated by non-compartmental analysis using the WinNonlin® Professional software package version 5.0.1 (Pharsight Corporation, Mountain View, CA, USA). The actual time points for blood sampling were used in the PK calculations.
Non-linear regression analysis was used to evaluate the relationships between ORM-12741 plasma levels and receptor occupancy (Sigmoid Emax model):
where Emax is a maximum receptor occupancy estimate, EC50 is a half maximal effective concentration estimate and h is a slope factor. Temporal occupancy patterns were estimated with a regression model. Statistical analyses were performed with SAS® for Windows (SAS Institute Inc., Cary, NC, USA) on observed cases only.
The safety of the subjects was evaluated by recording of adverse events (AEs), supine heart rate and blood pressure, 12-lead electrocardiogram, laboratory safety assessments and physical examination findings.