11C-LY2428703 was synthesized as previously described  and according to our Investigational New Drug Application #112,494, submitted to the US Food and Drug Administration. The radioligand was obtained in high radiochemical purity (>99%) and specific activity (specific activity at injection is reported below). Labeling was done with 11C, because it does not modify the basic structure of the molecule.
Saturation binding assay
P2 membrane fractions were prepared from the cerebella of male Sprague–Dawley rats (n = 3), male rhesus monkeys (n = 3), and female humans (n = 4). For saturation analyses, 100 μg of cerebellar membrane protein and increasing concentrations of [3H]LY456066  were added to quadruplicate wells in the presence of binding buffer (10 mM K2HPO4, 1.2 mM KH2PO4, 100 mM KCl, 0.1% DMSO, 0.05% ethanol and 0.1% BSA, pH 7.6). Nonspecific binding was defined by heterologous displacement of 1 μM LY480084 (mGlu1 antagonist) in select wells. Plates were incubated on ice for 75 min and membranes harvested on 0.3% polyethylenimine pretreated Wallac filter mats using a Tomtec Harvester MachII (Tomtec, Hamden, CT, USA). Dissociation constant (K
D) and receptor density (B
max) values were obtained in GraphPad Prism using standard nonlinear one-site binding equations. Data are presented as mean ± SEM (n = 3 to 4); one-way analysis of variance followed by Tukey's post hoc test.
Plasma analyses were performed on a reverse-phase chromatography C18 column (Novapak, 4 μm, 100 × 8 mm, Waters Corp. Milford, MA, USA) using Radial-Pak® compression module RCM-100 with a sentry pre-column. Human plasma analyses were performed with a mobile phase of MeOH/H2O/Et3N, 65:15:0.1 by volume, at a flow rate of 2.5 mL/min; nonhuman primate plasma samples were analyzed with a mobile phase of MeOH/H2O/Et3N, 72.5:27.5:0.1 by volume, at a flow rate of 1.5 mL/min. To compare the outcome from both systems, elution volume is reported rather than retention time.
In vitro stability of 11C-LY2428703 in human and nonhuman primate blood
Nonradioactive anticoagulated blood was obtained on the day of study from either human or nonhuman primates. Aliquots of 11C-LY2428703 (about 150 kBq) were added to 0.5 mL of whole blood or 0.5 mL of plasma. Samples were incubated at room temperature for 30 min, after which either 450 μL of the radioactive plasma was added to 720 μL of acetonitrile or 200 μL of the radioactive whole blood was added to 300 μL of water. The tube containing the blood suspension was shaken well to mix and lyse the blood cells. Seven hundred and twenty microliters of acetonitrile was subsequently added and mixed well to precipitate the proteins, followed by 100 μL of water, which was again re-mixed. Radioactivity in the tubes was measured in a gamma counter and then centrifuged at 10,000×g for 1 min. The clear supernatant liquid was injected into the high-performance liquid chromatography (HPLC) column and the precipitate counted to calculate the recovery of radioactivity into the acetonitrile.
The stability of the radioligand in whole blood or plasma was calculated as the ratio of the HPLC % composition of samples divided by the radiochemical purity of the radioligand.
Radiometabolites of 11C-LY2428703 in human and nonhuman primate plasma in vivo
Arterial plasma was analyzed as previously described . Briefly, after 11C-LY2428703 injection into either human or nonhuman primate, the input function was determined by periodically drawing anticoagulated blood samples. Blood aliquots were removed so that the γ-counter could provide information necessary for vascular correction. The remaining blood was then centrifuged, and plasma (450 μL) aliquots were removed and placed in acetonitriles (720 μL/ea) spiked with carrier LY2428703. The samples were mixed well, 100 μL water aliquots were added, and then, the sample was further mixed. The samples were counted in the γ-counter and centrifuged at 10,000×g for 1 min. The clear supernatant liquids were injected onto the radio-HPLC as described above. The precipitates were then counted for radioactivity to allow the calculation of the efficiency of recovery of radioactivity in the acetonitrile. Human plasma samples were separated with a mobile phase of higher polarity (MeOH/H2O/Et3N, 65:15:0.1 by volume) to ensure that the parent radiochromatographic peak comprised a single radiochemical species.
Plasma free fraction
The plasma free fraction (f
p) of 11C-LY2428703 was measured by ultrafiltration through membrane filters (Centrifree; Millipore, Billerica, MA, USA) as previously described . Briefly, about 740 kBq of 11C-LY2428703 (approximately 5 μL) was added to 650 μL of plasma. The mixture was incubated at room temperature for 10 min and then processed as described previously . When radioactivities for the filter components were high, they were allowed to decay until they were within the optimal range of the γ-counter. Determining the free fraction during preblocking was done by drawing blood samples after the preblocking treatments were initiated but immediately before administering the radioligand.
Four male rhesus monkeys (Macaca mulatta, 11.7 ± 2.9 kg) were imaged in 13 PET scans. Seven scans were performed at baseline, three scans were performed after receptor blocking to calculate nondisplaceable uptake (V
ND), and three scans were performed after blockade of the efflux transporters at the blood–brain barrier. Receptors were blocked by mixing 11C-LY2428703 with an mGluR1 antagonist - either nonradioactive LY2428703 (2.0 mg/kg) or LY2332084 (0.5 mg/kg) - in the same syringe. Efflux transporters were blocked in two scans by injecting DCPQ, which selectively blocks the efflux transporter ABCB1 (P-gp), and in one scan by injecting elacridar, which blocks both ABCB1 (P-gp) and ABCG2 (BCRP).
All studies were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
PET images were acquired using either the high-resolution research tomograph (HRRT) or the FOCUS 220 scanner (Siemens/CPS, Knoxville, TN, USA) for 120 min in 33 frames, with frame durations ranging from 30 s to 5 min, and correcting for attenuation and scatter. About 2 h before the PET scan, anesthesia was initiated with ketamine (10 mg/kg IM) and then maintained with 1% to 3% isoflurane. Electrocardiograph, body temperature, heart rate, and respiration rate were measured throughout the experiment. At baseline, the injected activity was 210 ± 51 MBq (specific activity at time of injection 43.2 ± 25.6 GBq/μmol, mass dose 6.9 ± 4.7 nmol or 0.62 ± 0.35 nmol/kg). Tissue time-activity curves were obtained from semi-automatic regions of interest encompassing the whole cerebellum and the rest of the brain. Brain uptake was expressed as standardized uptake value (SUV). Arterial input function was obtained for eight scans: four at baseline, two after blocking with nonradioactive LY2428703, one after blocking with DCPQ, and one after blocking with elacridar. Blood samples (0.5 mL each) were drawn through a port connected to the femoral artery at 15-s intervals until 2 min, followed by 1-mL samples at 3, 5, 10, 30, 60, 90, and 120 min. The plasma time-activity curve was corrected for the fraction of unchanged radioligand following the procedure detailed above.
To assess the effects of blocking agents on the f
P of 11C-LY2428703, f
P was measured in three monkeys both at baseline and after blockade. The first monkey was blocked with cold LY2428703 and f
P was measured 2.5 min after blocker injection (which corresponds to the maximal concentration in plasma of LY2428703), and again at 40 min. The other two monkeys were blocked with DCPQ and elacridar, respectively.
Whole-body biodistribution and radiation dosimetry
Two male rhesus monkeys (14.1 and 19.9 kg) were scanned after intravenous injection of 318 and 274 MBq of 11C-LY2428703, respectively. Dynamic two-dimensional scans were acquired on the GE Advance tomograph (GE Medical Systems, Milwaukee, WI, USA) in five bed positions of the body (head to upper thigh), with frames of increasing duration (15 s to 4 min) for a total scan time of up to 115 min.
Images were analyzed by placing regions of interest on the dynamic tomographic images. Regions were drawn in identifiable source organs: brain, heart, lungs, liver, spleen, gallbladder, kidneys, lumbar vertebrae, small intestine, testes, and urinary bladder. Because the images did not include the body below mid-thigh, organ uptake was corrected for this recovery. The mean recovery of activity in the body above the thigh measured by PET was 85.6% of the injected activity measured by a dose calibrator. This recovered total activity in the body was used as the new injected activity for each scan.
At each time point, the activity measured within the organs was converted into the fraction of the injected dose by dividing the organ activities by the recovered injected activity. The area under the curve (AUC) of each organ was calculated by the trapezoidal method until acquisition ended. The area under the curve after the acquisition of the last image (i.e., to infinity) was calculated by assuming that the decline in radioactivity after this time point occurred only via physical decay, without any further biological clearance.
Residence times from the monkeys were converted into corresponding human values by multiplying with a factor to scale organ and body weights: (b
m) × (o
h), where b
m and b
h are the body weights of monkey and human, respectively, and o
m and o
h are the organ weights of monkey and human, respectively.
Radiation absorbed doses were calculated from the residence times for each source organ. We used the model for a 70-kg adult male in the OLINDA/EXM computer program .
Three healthy females participated in the study (29 ± 2-year old, 69 ± 13 kg). All were free from current medical and psychiatric illnesses, as assessed by medical history, physical examination, electrocardiogram, urinalysis including drug screening, and blood tests (complete blood count, serum chemistries, thyroid function test, and antibody screening for syphilis, HIV, and hepatitis B). The National Institutes of Health Central Nervous System Institutional Review Board approved the protocols and the consent forms. Written informed consent was obtained from all subjects.
PET images were acquired using the GE Advance scanner (GE Healthcare, Milwaukee, WI, USA) for 120 min. An 8-min 68Ge transmission scan was obtained before the injection of the radiotracer for attenuation correction. The mean injected activity was 328 ± 12 MBq. The specific activity at the time of injection was 87.2 ± 34.7 GBq/μmol, which corresponded to 5.5 ± 2.3 nmol (0.082 ± 0.037 nmol/kg) of carrier. Blood samples (1 mL each) were drawn from the radial artery at 15-s intervals until 150 s, followed by 3-mL samples at 3, 4, 6, 8, 10, 15, 20, 30, 40, and 50 min, and 4.5 mL at 60, 75, 90, and 120 min. The unchanged parent fraction in plasma and f
P of 11C-LY2428703 were determined as described above.
Magnetic resonance imaging
To identify brain regions, magnetic resonance (MR) images were obtained using a 3-T GE Signa device (GE Healthcare, Milwaukee, WI, USA). T1-weighted structural images were acquired with a voxel size of 0.86 mm × 0.86 mm × 1.2 mm. The image acquisition sequences were the time of repetition (7.3 ms), echo time length (2.8 ms), and flip angle (6°).
The average PET image created from all frames was first coregistered to the individual MR image. Then, both MR and all PET images were spatially normalized to a standard anatomic orientation (Montreal Neurological Institute (MNI) space) based on transformation parameters from the MR images. A volume-of-interest template  as implemented in PMOD (PMOD Technologies Ltd, Zurich, Switzerland) was used to obtain brain time-activity curves.
Volumes of distribution (V
T) were obtained by nonlinear compartmental analysis. Goodness-of-fit by nonlinear least squares analysis was evaluated using the Akaike Information Criterion (AIC) and Model Selection Criterion (MSC). The most appropriate model is that with the smallest AIC and the largest MSC score. Goodness-of-fit by the compartment models was compared with F statistics . A value of P < 0.05 was considered significant for F statistics. The identifiability of kinetic variables was calculated as standard error obtained from the diagonal of the covariance matrix  and expressed as a percentage of the rate constant.