NeuroImage xxx (2012) xxx–xxx

YNIMG-08930; No. of pages: 9; 4C: 5, 7

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NeuroImage

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Characterization of [11C]RO5013853, a novel PET tracer for the glycine transportertype 1 (GlyT1) in humans

Dean F. Wong a,b,c,d,e,⁎,1, Susanne Ostrowitzki f,1, Yun Zhou d, Vanessa Raymont d, Carsten Hofmann f,Edilio Borroni f, Anil Kumar d, Nikhat Parkar f, James R. Brašić d, John Hilton d,Robert F. Dannals d, Meret Martin-Facklam f

a The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 601 North Caroline Street, Room 3245,Johns Hopkins Outpatient Center, Baltimore, MD 21287 -0807, USAb Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21287–0807, USAc Department of Environmental Health Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287–0807, USAd Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21287 -0807, USAe Honorary Professor of Neuroscience and Pharmacology, University of Copenhagen, Denmarkf F. Hoffmann-La Roche Ltd., Pharmaceutical Division, Grenzacherstrasse 124, CH-4070 Basel, Switzerland

⁎ Corresponding author at: 601 N. Caroline St., JHOMedical Institutions, Baltimore, MD 21287–0807, USA. F

E-mail address: dfwong@jhmi.edu (D.F. Wong).1 These two authors contributed equally.

1053-8119/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.neuroimage.2011.11.052

Please cite this article as: Wong, D.F., et al.,in humans, NeuroImage (2012), doi:10.101

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 July 2011Revised 8 November 2011Accepted 14 November 2011Available online xxxx

Keywords:Positron emission tomography (PET)ReceptorsN-methyl-D-aspartate (NMDA)Glycine transporter type 1 (GlyT1)Glycine reuptake inhibitor (GRI)Radioligand assaysSchizophrenia

We characterize a novel radioligand for the glycine transporter type 1 (GlyT1), [11 C]RO5013853, in humans.Ten healthy male volunteers, 23–60 years of age, were enrolled in this PET study; seven subjects participatedin the evaluation of test-retest reliability and three subjects in whole body dosimetry. Subjects were admin-istered intravenous bolus injections of approximately 1100 MBq (30 mCi) [11 C]RO5013853 with a high spe-cific activity of about 481 GBq (13 Ci)/μmol. Standard compartmental model analysis with arterial plasmainput function, and an alternative noninvasive analysis method which was evaluated and validated by occu-pancy studies in both baboons and humans, were performed. Mean parameter estimates of the volumes ofdistribution (VT) obtained by a 2-tissue 5-parameter model were higher in the cerebellum, pons, and thala-mus (1.99 to 2.59 mL/mL), and lower in the putamen, caudate, and cortical areas (0.86 to 1.13 mL/mL), withestimates showing less than 10% difference between test and retest scans. Tracer retention was effectivelyblocked by the specific glycine reuptake inhibitor (GRI), bitopertin (RG1678). [11 C]RO5013853 was safeand well tolerated. Human dosimetry studies showed that the effective dose was approximately0.0033 mSv/MBq, with the liver receiving the highest absorbed dose.In conclusion, quantitative dynamic PET of the human brain after intravenous injection of [11C]RO5013853attains reliable measurements of GlyT1 binding in accordance with the expected transporter distribution inthe human brain. [11C]RO5013853 is a radioligand suitable for further clinical PET studies. Full characteriza-tion of a novel radiotracer for GlyT1 in humans is provided. The tracer has subsequently been used to assessreceptor occupancy in healthy volunteers and to estimate occupancy at doses associated with best efficacy ina clinical trial with schizophrenic patients with predominantly negative symptoms.

© 2011 Elsevier Inc. All rights reserved.

Introduction

Glycine transporter type 1 (GlyT1) is a target of interest in the de-velopment of novel drugs for schizophrenia. GlyT1 inhibition is one ofthe key strategies to address N-methyl-D-aspartate receptor (NMDA-R) hypofunction which is postulated to play an important role in thepathophysiology of schizophrenia (Harrison and Weinberger, 2005;Hashimoto, 2011; Krystal et al., 1994; Millan, 2005; Sanger, 2004;

C Room 3245, Johns Hopkinsax: +1 410 955 0696.

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Characterization of [11C]RO506/j.neuroimage.2011.11.052

Stone, 2009). Promising clinical findings were recently reported inschizophrenic patients with predominantly negative symptoms whowere treated with the glycine reuptake inhibitor (GRI) bitopertin(RG1678) (Umbricht et al., 2010). Positron emission tomography(PET) data indicate that low to medium target occupancy was associ-ated with the best efficacy (Umbricht et al., 2011).

A radioligand specific for GlyT1 with appropriate molecular prop-erties for a PET imaging probe is a key tool for the clinical developmentof a GRI. Such a radioligand allows assessment of brain penetration ofcandidate drugs and enables the quantification and characterization oftransporter occupancy. RO5013853 ([5-methanesulfonyl-2-((S)-2,2,2-trifluoro-1-methyl-ethoxy)-phenyl]-[5-(tetrahydro-pyran-4-yl)-1,3-dihydro-isoindol-2-yl]-methanone) was identified as a high

13853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

Table 1Injected radioactivity dose, mass, mass per kg, and specific activity per subject per scan[n=7 subjects].

Datasubject

ID Scannumber

Injecteddose(MBq)[mCi]

Mass(μg)

Massper kg(ng/kg)

Specificactivity(GBq/μmol)[Ci/μmol]

1 101 1 1095.94[29.62]

2.83 30.43 191[5]

2 1084.47[29.31]

1.65 17.74 324[9]

2 102 1 1079.29[29.17]

2.35 30.92 227[6]

2 1175.86[31.78]

1.73 22.76 335[9]

3 106 1 1135.53[30.69]

2.47 33.38 227[6]

2 1091.5[29.50]

0.72 9.73 746[20]

4 107 1 1013.80[27.40]

1.16 13.18 430[12]

2 1072.26[28.98]

1.47 16.70 359[10]

5 109 1 1143.67[30.91]

0.43 4.67 1301[35]

2 1025.64[27.72]

1.08 11.74 468[13]

6 108 1 1161.80[31.40]

0.93 9.69 614[17]

2 1107.04[29.92]

1.74 18.12 313[8]

7 110 1 1147.37[31.01]

1.18 14.75 480[13]

2 1025.27[27.71]

0.76 9.50 667[18]

2 D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

affinity ligand for GlyT1 suitable for PET radiochemistry and was suc-cessfully radiolabeled yielding [11C]RO5013853 (Pinard et al., 2011).Furthermore, it was selected as the better of two PET ligands evaluatedin a study in non-human primates presented in this issue (Borroniet al., 2012, this issue).

Here we report the full characterization of [11C]RO5013853 as anovel PET radioligand for GlyT1 in humans. Objectives were as fol-lows: (1) to examine and analyze the specific uptake and kinetics of[11 C]RO5013853 in the human brain; (2) to assess the test–retest re-liability of quantitative PET measurements between two sessions; (3)to determine human radiation dosimetry following injection of a sin-gle microdose of [11 C]RO5013853; (4) to report rodent radiation do-simetry for suitability of the microdose of the radiotracer for humanuse; and (5) to report the acute safety and tolerability of [11C]RO5013853 administered intravenously at microdose levels inhealthy humans.

Materials and methods

PET study

Subjects and designThis single-center, open label, non-randomized study was con-

ducted in accordance with the Declaration of Helsinki Principles.The study received ethical approval from the Johns Hopkins Schoolof Medicine Institutional Review Board (Baltimore, MD) and Chesa-peake Research Review, Inc. (Columbia, MD) and was conductedunder an exploratory U.S. IND following appropriate animal toxicolo-gy studies of the unlabeled ligand and radiation dosimetry. All partic-ipants provided written, informed consent after receiving oral andwritten descriptions of study procedures and aims.

The study was conducted in 10 healthy male volunteers, 23 to60 years of age, with a body mass index ranging from 21.6 to29.7 kg/m2. Eligibility criteria included: no significant medical or sur-gical history; no history of head trauma with prolonged loss of con-sciousness; no neurologic conditions; no history of alcohol and/ordrug abuse or addiction within the last 2 years; heart rate between40 and 100 beats per minute; no family history of congenital longQT syndrome or sudden death; and a clinically unremarkable brainmagnetic resonance imaging (MRI) scan at screening. Subjects wereto be medication-free for 14 days or 5 times the elimination half-life(whichever was longer) prior to the first PET scan and throughoutthe study. Subjects entered the clinic the day before the PET scanand remained in-house until the day after the PET scan. Seven sub-jects were to be scanned twice, in order to study test-retest reliabilityof outcome parameters, and 3 subjects underwent whole body PETscans for evaluation of human radiation dosimetry.

Safety assessmentsSafety assessments included: continuous medical monitoring dur-

ing and following the radioligand injection for approximately 24hours, adverse event reporting, blood chemistry profile, includingliver and renal function tests, complete blood count, electrocardio-gram, urinalysis, and a physical examination.

Radioligand[11 C]RO5013853 is a highly selective ligand for GlyT1 and was

synthesized as previously described (Pinard et al., 2011). Briefly, anappropriate desmethyl precursor was radiolabeled with [11C]methyliodide and purified by reverse-phase high-performance liquid chro-matography (HPLC). A typical synthesis produced 4132.9±1465.2 MBq (111.7±39.6 mCi) (mean±SD, hereafter) of [11C]RO5013853. The end of synthesis specific radioactivity was 494±296 GBq (13±8 Ci)/μmol. Analytical HPLC showed a radiochemicalpurity of 100%. [11C]RO5013853 was provided as a sterile, pyrogen-free injectable solution (14:1 normal saline:ethanol).

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO5in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

Table 1 lists the radioactivity dose (MBq [mCi]), mass (μg), mass perkg (ng/kg), and specific activity (GBq [Ci]/μmol) for individual subjectsand scans. For the first PET session, subjects (n=7) received an averagedose of 1111.11±52.17 MBq (30.03±1.41 mCi) (mean±SD), withmass of 1.62±0.91 μg, mass per kg of 19.57±11.70 ng/kg, and specificactivity of 496±388 GBq (13±10 Ci)/μmol. The administered dose forthe second PET session averaged 1082.99±51.8 MBq (29.27±1.40 mCi), with mass of 1.31±0.45 μg, mass per kg of 15.19±4.98 ng/kg, and specific activity of 459±178 GBq (12±5 Ci)/μmol.The administered doses, masses, and specific activities of [11C]RO5013853 were not significantly different across the two sessions.The injected radioactivity was consistent with ALARA (as low absorbedradiation as possible) principles for the scientific question asked andbased on rodent and verified by human radiation dosimetry.

The maximal allowed mass dose per injection was 20 μgRO5013853 which fulfilled the definition of a microdose (Food andDrug Administration, 2006).

Blood sampling and metabolite analysisFor derivation of the plasma input function, arterial blood was ini-

tially sampled frequently with increasingly prolonged intervalsthroughout the scan for 90 minute post injection. Total radioactivitywas measured in more than 30 samples using a gamma counterthat was cross-calibrated against the PET activity measurements. Se-lected blood samples collected at 0, 5, 15, 30, 45, 60, and 90 minuteswere analyzed by HPLC for the presence of [11 C]RO5013853 and itsradiolabeled metabolites using a general method developed previ-ously for PET radiotracers (Hilton et al., 2000).

PET scanningFollowing placement of a radial arterial catheter (for the input

function) and a venous catheter (radioligand injection), seven

013853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

3D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

subjects received two single injections of [11C]RO5013853 each,1–3 weeks apart. In five of these subjects, both test and retest scanswere acquired on the GE Advance PET scanner. To examine whethera high-resolution research tomograph (HRRT®, CPS Innovations,Inc., Knoxville, TN, USA) scanner could improve the quantitative PETmeasurements of [11C]RO5013853 uptake in brain tissue, an addition-al two subjects' first scan was acquired on the HRRT and the secondon the GE Advance PET scanner.

The head of the subject was fitted with a thermoplastic mask tominimize head motion. A transmission scan with a Ge-68 (GE Ad-vance) or Cs-137 (HRRT) point source was initially performed for at-tenuation correction. The 90-minute dynamic emission scan of the GEAdvance scanner was acquired (30 frames: 4×0.25, 4×0.5, 3×1,2×2, 5×4, and 12×5 min) in three-dimensional (3D) mode andstarted with radioligand injection over 1 minute. Dynamic imageswere reconstructed using filtered back-projection with a ramp filter(35 slices with image matrix 128×128; pixel size: 2×2 mm2; inter-slice spacing 4.25 mm), resulting in a spatial resolution of5.5×5.5×4.25 mm full-width at half-maximum at the center of thefield of view. For the dynamic HRRT scans, the images were recon-structed in 3D mode with high-resolution span-3 modality with thesame frame schedule as the one used for the GE Advance scanner.The statistical 3D reconstruction of each frame sinogram used six iter-ations of the OP­OSEM algorithm (16 subsets), followed by 2 mmGaussian post-smoothing. Crystal dead time, decay, and calibrationfactor corrections were applied, producing a quantitative dynamicimage (207 slices with image matrix 256×256; pixel size:1.22×1.22 mm2, inter-slice spacing 1.22 mm), resulting in a spatialresolution of 2.5×2.5×2.5 mm full-width at half-maximum at thecenter of the field of view (Rahmim et al., 2004). HRRT has beenused and validated in the Johns Hopkins PET center over the past5 years (Horti et al., 2006; Wilcox et al., 2008; Wong et al., 2010).

MRI dataEach participant underwent a brain MRI scan (GE 1.5 T Sigma;

spoiled gradient-recalled [SPGR]) sequence and a double echo (pro-ton density and T2-weighted) sequence as part of the eligibility as-sessment for this study. Additionally, the MRI (matrix size:256×256, pixel size 0.938×0.938 mm2, 124 slices with 1.5 mm slicethickness) was used for co-registration with the PET data to enhanceanatomical definitions of regions of interest (ROIs).

Image analysisThe means of 90-minute dynamic PET images were used for MRI

to PET co-registration using Statistical Parametric Mapping 2 software(SPM2; Friston, 2002). The ROIs including cerebellum, pons, thala-mus, putamen, caudate, and cingulate, occipital, parietal, temporal,orbital frontal, prefrontal, and superior frontal cortices were manuallydrawn on the co-registered MRI images (Zhou et al., 2007). The ROItime-activity curves (TACs) were obtained by applying the ROIs tothe dynamic PET images. A standardized uptake value (SUV) was cal-culated for the normalization of ROI TACs:

SUVðTACÞ ¼ TACðMBq=mLÞ= injected tracer dose MBqð Þ=body weightðgÞð Þ¼ TAC μCi=mLð Þ= injected tracerdose μCið Þ=body weight gð Þð Þ:

Tracer kinetic modelingThe ROI tracer kinetics were analyzed by compartmental models

with the measured metabolite-corrected plasma input function. A 1-tissue 3-parameter (1T3P; K1, k2′, VP) as well as 2-tissue 5­parameter(2T5P; K1, k2, k3, k4, VP) model in series configuration were used to fitthe measured ROI TACs (Huang et al., 1986; Innis et al., 2007; Koeppeet al., 1991; Schmidt and Turkheimer, 2002). To reduce the variationof tracer total distribution volume (VT) resulting from the estimatesof k4, a nonlinear model fitting algorithm with k4 was coupled over

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO50in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

all ROIs for each dynamic PET study (Cunningham et al., 2004; Zhouet al., 2007, 2010). The final selection of the appropriate tracer kineticmodel was based on the Akaike information criterion (AIC; Akaike,1976; Carson et al., 1993, 1997; Gunn et al., 2001; Turkheimer et al.,2003). A lower AIC predicts a better fit; therefore, the model associat-ed with the lower AIC is considered the best model.

VT in tissue was calculated after model fitting as VT=K1/k2′ andVT=(K1/k2)(1+k3/k4) for 1T3P and 2T5P, respectively. The non-displaceable binding potential (BPND), an index of tracer specificbinding in ligand-receptor PET studies was estimated as:

BPND=VT/VT(reference tissue)−1 (Innis et al., 2007; Koeppe et al.,1991),

where the superior frontal cortex was used as a reference tissue (asthis region exhibits minor specific binding only). This method wasevaluated in full human occupancy studies (unpublished data). BPNDwas also estimated by a simplified reference tissue model (SRTM;Lammertsma and Hume, 1996; Zhou et al., 2003).

Parametric image of VT

To evaluate voxel-wise tracer distribution in brain, a multi-graphical analysis method (Zhou et al., 2010) using a relativeequilibrium-based graphic analysis (RE plot; Zhou et al., 2009) withthe Gjedde-Patlak plot (GP plot; Gjedde, 1981; Patlak et al., 1983;Patlak and Blasberg, 1985) (RE–GP plots) was used to generate para-metric VT images. The novel graphic analysis method is a model-independent quantitative approach.

Reproducibility metricsThe test-retest reliability (percent difference) of an outcome vari-

able P was calculated as:

100� Ptest–Pretestj jPtest

where P can be K1, VT, and BP ROI estimates, and Ptest and Pretest areestimated from the first and second dynamic PET scans, respectively.

Specificity of [11C]RO5013853

In a second study (unpublished data), healthy volunteer subjectswere dosed with a high-affinity, selective GRI, bitopertin (RG1678)(Alberati et al., 2010; Pinard et al., 2010), until steady state plasmaconcentrations were reached. Subjects underwent dynamic PET scan-ning with a standard protocol pre-dose and on the last day of treat-ment at steady state. Included for illustrative purposes here are PETdata from one subject (baseline and post-baseline after once daily ad-ministration with 175 mg bitopertin over 12 days) to show proof-of-principle for bitopertin to block [11 C]RO5013853 binding.

Radiation dosimetry (rodents and humans)

Rodent radiation dosimetry was carried out by intravenous injec-tion of the radiotracer into the tail vein in CD-1 mice (triplicates).After sacrifice and organ dissection, radioactivity was measured in agamma spectrometer at varying sacrifice times at 5, 15, 30, 60, 90,120, and 180 minutes post injection. Results were used to estimatethe human effective dose in this study. For human dosimetry, threesubjects received an average injected activity of 1076.7 MBq (range:1065.6–1084.1) [29.1 mCi (range: 28.8–29.3)] followed by whole-body PET-computed tomography scans on a GE Discovery VCT. Radio-activity concentrations were measured by PET at three to four timepoints up to 60 min after radioligand injection. Time integrals of ac-tivity (Stabin and Siegel, 2003) were entered into the OLINDA/

13853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

Fig. 1. Mean time activity curves (TACs) in standardized uptake values (SUVs) for rep-resentative regions of test-retest scans performed on GE Advance scanner (n=10) infive healthy volunteers demonstrating initial rapid decline in SUV followed by slowerelimination phase. Other cortical regions examined (cingulate, occipital, parietal, tem-poral, orbital frontal, and prefrontal cortices) had TACs (data not shown) similar to thatof the superior frontal cortex.

4 D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

EXM® software (Vanderbilt University, Nashville, TN, USA; Stabin etal., 2005) using the adult male model for both species.

Autoradiographic studies in healthy human brain

Tritiated RO5013853 was used for this study. Sections (10 μm)from a healthy human brain (48 year old female, University of Auck-land) were cut in a cryostat and thaw-mounted on adhesion glassslides (Paul Marienfeld GmbH, Lauda-Königshofen, Germany). Brainsections (3 sections per region of interest) were incubated at roomtemperature for 10 min in Ringer buffer (NaCl 120 mM, KCl 5 mM,CaCl2 2 mM, MgCl2 1 mM, Tris–HCl 50 mM, pH 7.4), followed by60 min in Ringer buffer containing 1 nM radioligand. Non-specificbinding (NSB) was measured using an additional two sections incu-bated with Ringer buffer containing 1 nM radiotracer and 1 μM bito-pertin (60 min at 37 °C). Sections were rinsed 2×5 min and1×15 min in Ringer buffer and dipped three times in distilled waterat 4 °C. Slide-mounted brain sections were dried and exposed to aFuji Imaging Plate (BAS-TR 2025, Fujifilm, Dielsdorf, Switzerland)with a [3H]microscale (RPA-510, GE Healthcare, Glattbrugg, Switzer-land) for five days. The imaging plate was scanned in a Fujifilmhigh-resolution plate scanner (BAS-5000, Bucher Biotec AG, Basel,Switzerland). The total amount of radioligand bound to the brainareas of interest (TB) was measured using the MCID™ image analysisprogram (version 7; InterFocus Imaging GmbH, Mering, Germany)and expressed as fmol of bound radioligand per mg of protein. Theamount of radioligand specifically bound to the GlyT1 (SB) was calcu-lated according to the formula SB=TB−NSB. NSB was very low andranged from 0.9% to 4% of the TB measured.

Results

Safety

[11C]RO5013853 was well tolerated and no clinically relevantchanges in vital signs, electrocardiogram, or laboratory parameterswere observed. Four out of ten subjects reported a total of six adverseevents of mild intensity. Two of the adverse events were judged re-motely related (diarrhea, abdominal pain), and four unrelated to[11C]RO5013853. The highest single mass dose administered of2.83 μg RO5013853 was well below the maximal allowed dose of20 μg and consistent with the definition of microdose.

Whole-body radiation dosimetry (rodent and human)

The mouse radiation dosimetry demonstrated that the liver hadthe highest uptake of any single organ, around 65%, and received adose of approximately 0.05 mSv/MBq, as did the small intestine. Theradiation effective dose was calculated to be 0.0047 mSv/MBq(0.0174 rem/mCi).

The human dosimetry data fit well with one or two exponentialfunctions. Most organs appeared to receive around 0.002–0.005 mSv/MBq (0.074 to 0.019 rem/mCi). The liver appeared to receive the high-est dose of 0.016 mSv/MBq (0.059 rem/mCi), and the gallbladder0.01 mSv/MBq (0.037 rem/mCi). The effective dose was 0.0033 mSv/MBq (0.012 rem/mCi), therefore approximately 1.4 fold lower thanthe estimated effective dose in mice.

Time activity curves (TACs)

Average SUVs over time from test-retest scans (two scans oneach of five participants, all performed on the GE scanner) showedthat the tracer rapidly enters the brain. The peak was reached ataround 1 min, with an initial fast decline in SUV followed by aslower elimination phase in the areas of high GlyT1 binding

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO5in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

(Fig. 1). Cerebellum, pons, and thalamus showed the highest andcortical areas the lowest SUVs.

Distribution of GlyT1 in human brain (autoradiography)

The regional distribution of RO5013853 binding sites was further in-vestigated at higher resolution by autoradiography using tritiatedRO5013853. High binding density was observed in the pons, the superiorand inferior colliculi, and various thalamic nuclei (data not shown). In thecerebellum, a higher density of [3H]RO5013853 binding sites wasdetected in the granular layer and a lower density in the molecularlayer and the white matter (Fig. 2A). Moderate binding site density wasseen in the caudate nucleus, the dentate gyrus, the putamen, and the CA1–3 regions of the hippocampus (Fig. 2B). Low binding was observed inall cortical regions investigated. Co-incubation of the radioligand withthe specific GRI, bitopertin (RG1678), completely abolished the binding,confirming the specificity of RO5013853 for GlyT1 (data not shown).

Radiotracer metabolism

Fig. 3 shows the average metabolite ratios for all dynamic scans(two scans on each of 7 participants). At 30 and 90 min after [11C]RO5013853 injection, ~30% and ~50% of the radiotracer were metab-olized, respectively. Both scans showed very similar plasma metabo-lite profiles, with b4% difference in the mean metabolite ratiothroughout the entire duration of the scan. All metabolites weremore polar than the parent as they eluted rapidly in the HPLC.

Kinetic model for quantification of binding

Fig. 4 shows a representative observed TAC from a single study forthe superior frontal cortex and thalamus with fitted curves as pre-dicted by the 1T3P and the 2T5P models. Note that the predictedcurves fit the data points better for the 2T5P model than of the 1T3Pmodel, particularly in the early phase of tracer kinetics (paired t-test for AICs, pb0.001 for all ROIs; Table 2). Therefore, we used the2T5P model for further data analysis.

Parameter estimates

Overall, the estimates of K1 and VT were consistent across both GEAdvance and HRRT scanners. Estimates of K1 were fairly homogenousacross all regions of interest (ROIs), with values between 0.02 and

013853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

Fig. 2. Pseudocolor images of [3H]RO5013853 autoradiographs of human brain sections. Red indicates high binding site density whereas blue indicates low binding sites density (seecalibration scale). Two brain regions are shown in this figure, the cerebellum (A) and the hippocampus (B). In the cerebellum, the highest density of binding sites is observed in thegranular layer (gl), with lower densities observed in the white matter and the molecular layer. In the hippocampus, moderate densities of binding sites are observed in the dentategyrus (DG) and in the CA1, CA2 and CA3 regions.

5D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

0.08 mL/min/mL (0.04±0.01 [mean±SD hereafter], n=12×14) fordata from both scanner types. VT ranged from 1.86 to 2.89 mL/mL incerebellum, pons, and thalamus (pons being highest: 2.59±0.23 mL/mL, n=10; GE data), from 0.94 to 1.26 mL/mL in striatum(putamen and caudate; 1.12±0.09 mL/mL, n=2×10), and from0.78 to 1.09 mL/mL in the cortical areas (0.90±0.06 mL/mL,n=7×10; Fig. 5). In the two subjects also scanned on the HRRT, VT

ranged from 1.61 to 2.15 mL/mL in cerebellum, pons, and thalamusand was approximately 0.86 mL/mL in the cortical areas.

BPND derived by the 2T5P model in cerebellum, pons, and thala-mus was 1.26±0.19, 1.93±0.30, and 1.43±0.22, respectively(n=10; GE data). For comparison, BPND values in the two subjectsalso scanned on the HRRT were 0.85–0.99 in cerebellum, 1.44–1.47in pons, and 1.29–1.45 in thalamus. The 2T5P GE values were overallcomparable to the BPND derived by SRTM (1.02±0.14 [cerebellum],1.50±0.18 [pons], and 1.47±0.32 [thalamus], [n=10]). In all otherareas, BPND was below 0.30 for both methods used with high coeffi-cient of variation and were thus not reliable.

Test-retest reliability

The parameters K1 and VT were used for test–retest analysis. Thepercentage difference between the two scans for the K1 estimates

Fig. 3. Metabolite ratio of [11C]RO5013853 (%) in plasma of healthy volunteers for alldynamic scans (n=7 each, mean±SD) as well as the average curve for all scans show-ing that about 30% and 50% of the radiotracer were metabolized after 30 and 90 min,respectively.

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO50in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

(GE data) ranged from 14.6±12.8% in the thalamus to 26.0±20.7%in the caudate (n=5).

VT estimates from GE data showed a percentage difference over allROIs of 4.3±2.9% (n=5×12), with ranges from 2.4±1.8% in the cer-ebellum to 6.1±2.7% in the prefrontal cortex (n=5).

The percentage difference for the BPND estimates of cerebellum,pons, and thalamus were 12.6±12.3%, 7.9±6.9%, 14.4±11.3%, and7.3±7.3%, 6.0±2.6%, 26.1±13.3% for the 2T5P and SRTMmodels, re-spectively (n=5).

Parametric images

Consistent with the estimates of VT using kinetic modeling, voxel-wise parametric mapping showed that the cerebellum, pons, andthalamus were the regions with the highest VT, while the cortical re-gions had the lowest VT (Fig. 6).

Transporter specificity of the tracer

Following 12 days of once daily dosing with 175 mg bitopertin, adrug candidate that binds selectively and with high affinity toGlyT1, tracer binding is almost completely blocked (GlyT1 occupancyby bitopertin: 92% in the thalamus, Fig. 7; and unpublished data).

Fig. 4. Representative time activity curve (TAC) from a single dynamic PET scan for thesuperior frontal cortex and the thalamus and the fitted curves as predicted by the 1-tissue 3-parameter (1T3P) and 2-tissue 5-parameter (2T5P) compartment model.

13853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

Table 2Mean (SD) AIC values by each ROI for the GE advance scanner (n=10).

Regions Models

1T3P 2T5P

Cerebellum −223.88 (8.92) −268.11 (25.51)Pons −214.64 (8.03) −239.29 (17.62)Thalamus −179.37 (13.79) −196.62 (21.08)Putamen −187.24 (9.99) −198.89 (15.03)Caudate −181.50 (14.49) −187.49 (13.24)CingulateCx −196.50 (10.69) −215.77 (18.82)OcciptalCx −203.96 (6.74) −235.24 (16.25)ParietalCx −213.38 (8.31) −253.18 (23.99)TemporalCx −222.21 (10.74) −252.92 (23.06)OrbitalFrontalCx −217.07 (9.05) −244.18 (18.09)PreFrontalCx −214.99 (12.18) −239.80 (24.41)SuperiorFrontalCx −209.77 (7.97) −235.23 (23.63)

6 D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

Discussion

Since the successful imaging of dopamine and D2/D3, serotonin5HT2A, and mu opiate receptors (Frost et al., 1985; Wagner et al.,1983; Wong et al., 1984) and subsequent imaging of dopamine(Wong et al., 1993) and serotonin transporters (Szabo et al., 1995),the imaging of glutamate/glycine receptors has been an area of in-tense interest in basic and translational neuroscience. The imagingof this system including the GlyT1 is of great value for understandingnormal and pathological receptor distribution and expression levelsin neuropsychiatric illnesses. In addition, imaging is paramount inthe development of putative novel drugs involving these targets, astarget occupancy measurements are now considered an integralpart of clinical drug development in neuroscience. In this paper, wedocument for the first time feasibility and reliability of the novel ra-diotracer [11C]RO5013853, a highly selective inhibitor of the GlyT1in humans. A recent clinical trial showed an improvement in negativesymptoms in patients with schizophrenia who were treated with theGRI, bitopertin (RG1678) for 8 weeks (Umbricht et al., 2010). Usingthis novel tracer, it was shown that low to medium target occupancyseems sufficient to achieve the strongest clinical effects, while highoccupancy levels were associated with a lack of efficacy (Umbrichtet al., 2011). This finding underscores the significant contributionPET studies can make to the understanding of a drug's mechanismof action.

Microdose levels of [11C]RO5013853 were safe and well toleratedin this first in human study. [11 C]RO5013853 is primarily retained inthe cerebellum, the pons, and the thalamus, with little uptake in

Fig. 5. Tracer distribution volume (VT, mean±SD) for scans acquired on the GE ad-vance scanner for each ROI, showing highest values for cerebellum, pons, andthalamus.

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO5in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

cortical regions. This is consistent with human autoradiography datapresented here, and with the known distribution of the GlyT1 innon-human primate and pig brain PET studies (Borroni et al., 2012,this issue; Hamill et al., 2011; Passchier et al., 2010; Toyohara et al.,2011) and also with results from previous immunohistochemical, au-toradiography, and tissue distribution studies in rodents and non-human primates (Borroni et al., 2012, this issue; Cubelos et al.,2005; Toyohara et al., 2011; Zafra et al., 1995; Zeng et al., 2008).

Examination with the gold standard arterial input function dem-onstrated characteristics associated with many successful PET radio-tracers, including reasonable brain uptake (VT was 2.24±0.29 mL/mL in target areas [means over cerebellum, pons, and thalamus,n=3×10] versus 0.95±0.11 mL/mL in others, n=9×10) and a rela-tively high test/retest reliability (b10% difference between scans forVT, a parameter used to measure tissue tracer uptake and to derivetracer specific binding in tissue BPND). We determined that quantifi-cation with the 2T5P model was suitable for all kinetic analyses,which is consistent with prior baboon studies presented in thisissue (Borroni et al., 2012, this issue). We subsequently found in a re-lated receptor occupancy study that full range of dose occupancy upto near 100% could be achieved (unpublished data) and that a refer-ence region approach is quite acceptable, with the superior frontalcortex having been identified as the reference region.

Test-retest reliability of BPND derived from the SRTM was compa-rable to the standard compartmental model. This suggests that in fu-ture studies using this tracer, data can appropriately be quantifiedusing the SRTM method, and thus no arterial sampling would berequired.

Characterization of the kinetics of another GlyT1 PET tracer, [11C]GSK931145, in dynamic PET with standard compartmental analysiswas published recently (Gunn et al., 2011, Synapse). In this study,the regional distribution of tracer binding in humans (increased up-take in cerebellum, brain stem, and thalamus) was similar to thatshown here. Furthermore, the authors reported that the [11C]GSK931145 cerebral K1 value was 0.025±0.0097 mL/min/mL inhuman studies which was lower than the [11C]RO5013853 K1 value(0.037±0.0076 mL/min/mL, n=12 ROIs×10 scans) obtained in ourstudy. The direct comparison of VT and BPND estimates between[11 C]GSK931145 (Gunn et al., 2011, Synapse) and [11 C]RO5013853from this study are listed in Supplemental Table 1. As compared to[11C]GSK931145, the [11C]RO5013853 tracer has higher VT with re-markably higher test-retest reliability. The BPND estimates of [11C]RO5013853 in cerebellum, pons, and thalamus are comparable tothose in [11C]GSK931145 studies (see Supplemental Table 1). Thusinitial brain uptake, binding, and test–retest reliability are betterwith [11 C]RO5013853.

In a brief, preliminary report from a second GlyT1 PET tracer, [18F]CFpyPB, the highest binding was reported in the brain stem, followedby cerebellum, thalamus and midbrain, and white matter, and thelowest binding in the striatum and cortical gray matter, with VT rang-ing from 7 down to 2 mL/mL. Test–retest variability was noted asbelow 12% (n=3, Sanabria-Bohorquez et al., 2009; Williams et al.,2008). In comparison, for [11C]RO5013853, while we observed a mag-nitude of binding that was overall lower (VT ranged from approxi-mately 3 down to 2 mL/mL in cerebellum, pons, and thalamus, andless than 1 mL/mL in cortical areas) and slightly different ranking inregional biodistribution sites (i.e., thalamus higher than cerebellum),we obtained a substantially stronger VT test–retest reliability (meanpercentage difference over all regions below 5%).

Specificity of [11C]RO5013853 for GlyT1 was shown, as tracerbinding is blocked by prior administration of a selective GRI (bitoper-tin, Fig. 7; and unpublished data).

Additional favorable characteristics of this novel radiotracer in-clude (i) the results from human radiation dosimetry which allowat least three injections of 1110 MBq (30 mCi) each to total3330 MBq (90 mCi) under local institutional and FDA rules and

013853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

Fig. 6. Transverse parametric images of tracer distribution volume (VT; [mL/mL], means, n=10) generated by applying the RE-GP plots to the [11C]RO5013853 dynamic PETimages acquired from GE Advance scanner, with the pons and the thalamus showing the highest VT, and the cortical regions, the lowest VT. The parametric images of VT

were spatially normalized to the standard space (voxel size: 2×2×2 mm3) using SPM2, and the mean of 90-min dynamic PET images were used to determine the parametersof spatial normalization.

7D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

which indicate a lower human effective dose 0.00032 rem/MBq(0.012 rem/mCi) compared to the radiotracers [11C]GSK9311450.00041 rem/MBq (0.015 rem/mCi; Murthy et al., 2008; Bullich etal., Online First™, 21.August 2010) and [18F]CFpyPB 0.0025 rem/MBq (0.093 rem/mCi; Sanabria-Bohorquez et al., 2009), (ii) modestradiotracer metabolism, and (iii) a reproducible synthesis which is akey element to any PET study where timely availability and reliabilityof the tracer are critical.

There are some limitations to the radiotracer, however, as theoverall brain uptake is sufficient but modest. For [11C]RO5013853,this limitation can be overcome by injection of as much as1110 MBq (30 mCi) per PET scan, which was considered safe givenresults from the radiation dosimetry studies.

We also tested whether using the HRRT rather than the GE Ad-vance scanner would benefit the imaging results from these studies.The HRRT scanner has improved resolution compared to the GE Ad-vance (2.5 mm compared to ~5 mm full width at half maximum res-olution) with comparable sensitivity; however, the HRRT iscomposed of LSO scintillator-crystals which emit radioactivity. Inphantom studies, careful consideration of the LSO background activi-ty in the field of view indicates that for any ROI in which concentra-tion becomes around 370–555 Bq/mL (10–15 nCi/mL), backgroundactivity may potentially interfere with the measurements (it is possi-ble to compensate for the resulting bias, but noise levels introduced

Fig. 7. Tracer distribution volume VT (mL/mL) axial images of a representative partici-pant at baseline (middle) and following once daily dosing over 12 days with 175 mg ofbitopertin at steady state (right) at the level of the thalamus demonstrating almostcomplete blockage of tracer binding. T1-weighted MRI (left) used for co-registeringPET images to better delineate ROIs.

Please cite this article as: Wong, D.F., et al., Characterization of [11C]RO50in humans, NeuroImage (2012), doi:10.1016/j.neuroimage.2011.11.052

by background contamination will remain substantial). Thus, if theROI has a low radioactive concentration (less than around 7400 Bq/mL [200 nCi/mL]) early on in a 90 min [11C] PET study, the activityof the ROI could fall below the background threshold at the end ofthe study, and the observed TAC would not be accurate because ofcontamination with the background activity. Because of the modestbrain uptake of [11C]RO5013853 (although good regional brain distri-bution, and occupancy [Fig. 7; unpublished data), we elected to con-tinue studies with the GE Advance to avoid possible artifacts in ourfuture occupancy studies.

This does not exclude the HRRT for future use, however. Furtherstudies may be advisable to determine if the HRRT could be usefulto discriminate GlyT1 binding in small brain regions when an evenhigher radioactivity is injected. For the current application, i.e.,GlyT1 occupancy, larger brain regions are of primary interest, andhence scanning at high resolution (HRRT) was not beneficial.

Conclusions

We found that [11C]RO5013853 is a suitable PET imaging agent forhuman studies of the GlyT1 transporter. [11C]RO5013853 readily pen-etrates the blood–brain barrier and binds to brain regions known toexpress GlyT1. Brain signal is modest; however, test-retest reliabilityfor estimated key parameters is sufficiently high to afford reasonableprecision in tracer binding determinations and is superior to otherexisting human GlyT1 PET tracers. Finally, evidence is presentedthat shows that radioligand binding can be effectively blocked by ahighly specific GlyT1 inhibitor. [11C]RO5013853 can thus be used infurther occupancy studies.

Supplementary materials related to this article can be found onlineat doi:10.1016/j.neuroimage.2011.11.052.

Acknowledgements

Special thanks to Arman Rahmin, Ph.D, the HRRT physicist incharge, and Maria Thomas, Ph.D, for scientific editorial assistance, aswell as to Ron Goldwater, MD, Principal Investigator at PAREXEL,and Michael Stabin, Ph.D. (Vanderbilt University) for analysis of do-simetry data. This study was funded by F. Hoffmann-La Roche,

13853, a novel PET tracer for the glycine transporter type 1 (GlyT1)

8 D.F. Wong et al. / NeuroImage xxx (2012) xxx–xxx

contract to JHU. JHU faculty receive salary support through a numberof sponsored research sources including Wong NIH career award K24DA000412, and none receive direct funding from Roche except viasponsored JHU contracts. Support for third-party editorial assistancefor this manuscript, furnished by Veronica Porkess, Ph.D. (CompleteHealthVizion) and by archimed medical communication ag, was pro-vided by F. Hoffmann-La Roche Ltd.

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Zhou, Y., Resnick, S.M., Ye, W., Fan, H., Holt, D.P., Klunk, W.E., Mathis, C.A., Dannals, R.,Wong, D.F., 2007. Using a reference tissue model with spatial constraint to quantify[11C]Pittsburgh compound B PET for early diagnosis of Alzheimer's disease. Neuro-image 36, 298–312.

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DATA QUOTED AS UNPUBLISHED:

Martin-Facklam, M., Pizzagalli, F., Zhou, Y., Ostrowitzki, S., Raymont, V., Brašić, J.R.,Parkar, N., Umbricht, D., Dannals, R.F., Goldwater, R., Wong, D.F., submitted forpublication. Glycine transporter Type 1 (GlyT1) occupancy by RG1678: A positronemission tomography study in healthy volunteers.

13853, a novel PET tracer for the glycine transporter type 1 (GlyT1)