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Research ArticleNeuropharmacology

Quantification of ONO-2952 Occupancy of 18-kDaTranslocator Protein in Conscious Monkey Brains using Positron Emission Tomography

Katsukuni Mitsui, Noriko Morimoto, Tomohiro Niwa, Yoshiyuki Yamaura, Hiroyuki Ohba, Hideo Tsukada and Seishi Katsumata
Journal of Pharmacology and Experimental Therapeutics March 2017, 360 (3) 457-465; DOI: https://doi.org/10.1124/jpet.116.238568

Abstract

We have previously shown that ONO-2952, a novel 18-kDa translocator protein (TSPO) antagonist, inhibits stress-induced accumulation of neurosteroids and noradrenaline release in the rat brain and alleviates the subsequent symptomatic responses with a brain TSPO occupancy of 50% or more. In this study, we measured ONO-2952 brain TSPO occupancy in conscious rhesus monkeys using positron emission tomography (PET) with 11C-PBR28 as ligand for translational research to clinical application. PET scans were performed after single and repeated oral administration of ONO-2952 at several dose levels for each animal, with sequential arterial blood sampling. In vitro binding studies showed that ONO-2952 potently binds to brain TSPO in monkeys with an affinity equivalent to that in rats. ONO-2952, given orally before PET scans, dose dependently decreased 11C-PBR28 uptake without marked brain region specificity. Results of the quantitative analysis using arterial input function revealed that TSPO occupancy after ONO-2952 single and repeated oral administration tended to increase in parallel with its plasma concentration, reaching the highest level of 100%. These findings indicate that ONO-2952 has sufficient brain distribution in primates and that ONO-2952 TSPO occupancy in humans can also be determined using PET.

Introduction

The 18-kDa translocator protein (TSPO), which has five transmembrane domains, is widely distributed throughout the body and is particularly prevalent in steroid-producing tissues, including the brain, where this protein is predominantly located in glial cells (Marangos et al., 1982; Gavish et al., 1999; Cosenza-Nashat et al., 2009; Rupprecht et al., 2010). Within cells, TSPO is located in the outer mitochondrial membrane and forms a channel-like structure that allows the transport of cholesterol from intracellular sources into mitochondria, a pathway known as the rate-limiting step in steroidogenesis (Anholt et al., 1986; Rupprecht et al., 2010; Rone et al., 2012). Neurosteroids, which are endogenous steroids produced in the central nervous system (CNS), act as allosteric modulators of excitatory and/or inhibitory neurotransmission, including adrenergic, GABAergic, glutamatergic, and cholinergic neurotransmission (Belelli and Lambert, 2005; Strous et al., 2006). In this regard, an unbalance in the levels of neurosteroids in the CNS would have significant pathophysiological consequences. Consistent with this idea, TSPO expression in the brain or peripheral blood cells, as well as neurosteroid concentration in the cerebrospinal fluid or plasma, have been found to be altered in patients with stress-related or neurologic disorders, such as depression, generalized anxiety disorder, post-traumatic stress disorder, Alzheimer disease, and schizophrenia (Rasmusson et al., 2006; Rupprecht et al., 2010; Da Pozzo et al., 2012). Taken together, these findings indicate that drugs that can act on TSPO in the brain would have beneficial effects on stress-related disorders; however, the clinical use of TSPO ligands for treatment of stress-related disorders has not been fully explored.

ONO-2952 is a novel TSPO antagonist that exhibits high affinity for the rat TSPO (K i = 0.33 nmol/liter) with high selectivity over other receptors, transporters, ion channels, and enzymes. Pharmacologic studies in rats exposed to acute stress have shown that ONO-2952 inhibits stress-induced neurosteroid accumulation and noradrenaline release in the brain, resulting in antistress effect (Mitsui et al., 2015). In addition, our ex vivo binding study in rats showed that ONO-2952 produces its antistress effect with brain TSPO occupancy of more than 50%. As we postulate that a TSPO occupancy over 50% would be needed for ONO-2952 to exert its antistress effect in humans, a relationship between the plasma concentration of ONO-2952 and its brain TSPO occupancy in human is necessary to predict this compound’s clinical effective doses.

Positron emission tomography (PET) is used as a tool to assess the relationship between the compound plasma concentration and its target protein occupancy, thereby allowing selection of the optimal clinical dosage (Matthews et al., 2012). TSPO has historically been used as a marker for reactive gliosis, which is closely related to neuronal dysfunction associated with a number of CNS diseases. Accordingly, several structurally different TSPO ligands have been synthesized as imaging tools for the detection of brain injury (Chauveau et al., 2008; Chen and Guilarte, 2008). Among these ligands, aryloxyanalide-based 11C-PBR28 has been shown to be a useful TSPO-specific PET radioligand not only in nonhuman primates but also in humans (Brown et al., 2007; Fujita et al., 2008; Imaizumi et al., 2008; Owen et al., 2014). As a step to establish a relationship between ONO-2952 plasma concentration and its brain TSPO occupancy in human, we measured ONO-2952 brain TSPO occupancy in conscious rhesus monkeys using PET with 11C-PBR28 as a radioligand. In vitro binding studies using 3H-PBR28 in membrane fractions from rat and monkey brain were also conducted to confirm the validity of 11C-PBR28 as a PET probe for determination of ONO-2952 TSPO occupancy.

Materials and Methods

Drugs and Chemicals

ONO-2952 (1-[(1S)-1-(4-chloro-2-methoxyphenyl)-5-fluoro-1,9-dihydrospiro[β-carboline-4,1'-cyclopropan]-2(3H)-yl]ethanone, purity: ≥95%), PBR28 (N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine), and O-desmethyl PBR28 were synthesized in our laboratories. PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide), a selective TSPO antagonist (Le Fur et al., 1983), was purchased from Sigma-Aldrich (St. Louis, MO), and 3H-PBR28 (specific radioactivity: 3.02 TBq/mmol) was purchased from Sekisui Medical Co., Ltd. (Tokyo, Japan). All other chemicals were purchased from Sigma-Aldrich unless otherwise noted. Compounds used for the binding study were first dissolved in 0.5% dimethylsulfoxide and then added to the assay buffer. ONO-2952 was suspended in 0.5% (w/v) methylcellulose (MC) solution for oral administration at a dosing volume of 5 ml/kg.

Animals

Male Wistar rats (Slc:Wistar, Japan SLC, Inc.; 11 weeks old) were used in this study. Rats were housed in groups of less than five animals per cage in a temperature- and humidity-controlled animal room (temperature 24 ± 2°C, relative humidity 55% ± 15%) under 12-hour light/dark cycles (lights on from 8:00 to 20:00). Three male rhesus monkeys (Macaca mulatta, weight 4.1–5.6 kg; Shin Nippon Biomedical Laboratories, Ltd., Tokyo, Japan) were used for in vitro TSPO-binding study. Six male rhesus monkeys (M. mulatta, weight 4.5–8.0 kg; Hamri Co., Ltd., Ibaraki, Japan) were used for the PET study. Monkeys were housed one animal per cage in a temperature- and humidity-controlled animal room (temperature 24 ± 4°C, relative humidity 50% ± 20%) under a 14-hour light/10-hour dark cycles (lights on from 7:00 to 21:00). All experimental procedures were approved by the institutional animal care and use committee of ONO Pharmaceutical Co., Ltd., Osaka, Japan, and Central Research Laboratory, Hamamatsu Photonics K.K.

In Vitro Binding Assay

ONO-2952 and PK11195 inhibition constants (K i) for displacement of 3H-PBR28 TSPO binding in mitochondrial membrane fractions prepared from rat whole brain and monkey brain (hippocampus and occipital cortex) were determined as described in our previous report (Mitsui et al., 2015) with minor modifications. Protein concentration in each brain homogenate was adjusted to 1 mg/ml with HEPES buffer, and the homogenate (50 μl) was incubated for 90 minutes at 4°C with 0.5 nmol/liter 3H-PBR28 (final concentration) in a final reaction volume of 200 µl. Nonspecific binding was defined as binding in the presence of unlabeled PK11195 (20 μmol/liters). 3H-PBR28 saturation binding assay was also performed for each membrane fraction.

To confirm whether ONO-2952 competitively inhibits PBR28 binding to TSPO in monkey brain tissue, dissociation constants (K d) and maximum binding (B max) of 3H-PBR28 were determined with increasing concentrations of nonradioactive test compound in homogenates derived from the occipital cortex. Competitive inhibition was defined as significant increase in K d values with no influence on B max values (Obata et al., 1992). When the 95% confidence interval of K d or B max did not overlap between assays (i.e., without and with ONO-2952), the change was considered significant.

All assays were performed in duplicate. GraphPad Prism software (version 5.01; GraphPad Software Inc., La Jolla, CA) was used to calculate K i values of test compounds and K d and Bmax values of 3H-PBR28 in each membrane fraction.

Determination of ONO-2952 TSPO Occupancy in the Rat Brain: Ex Vivo Experiment

The relationship between ONO-2952 plasma concentration and TSPO occupancy in the brain was evaluated in rats. ONO-2952 or vehicle (0.5% MC) was orally administered once to each rat. Three hours after ONO-2952 administration, approximately 0.5 ml of blood samples were collected via the jugular vein using a heparinized syringe, and the plasma was separated by centrifugation and frozen at −80°C until use. The rats were then decapitated, and the cerebral cortex and hippocampus were dissected. The brain was weighed and homogenized in 5× (w/v) ice-cold 50 mmol/liter HEPES buffer. Protein concentration in the homogenate was adjusted with HEPES to 10 mg/ml for use in the determination of ONO-2952 TSPO occupancy. TSPO occupancy of ONO-2952 was determined from 3H-PBR28 specific binding to TSPO in the brain homogenates. Each homogenate (50 μl) was incubated for 90 minutes at 4°C with 3H-PBR28 (final concentration, 0.5 nmol/liter) in a final reaction volume of 200 µl. Assays were performed in duplicate, and ONO-2952 TSPO occupancy was calculated using eq. 1:Embedded Image (1)where X is the specific binding level in each brain homogenate, and Y is the mean specific binding level in the control group treated with the vehicle.

The plasma concentration of ONO-2952 was determined by positive turbo ion spray liquid chromatography-tandem mass spectrometry with solid-phase extraction. The lowest limit of quantitation was 0.1 ng/ml.

Evaluation of ONO-2952 TSPO Occupancy in Conscious Monkey Brain: PET Study

The relationship between the plasma concentration of ONO-2952 and its TSPO occupancy in the brain of conscious rhesus monkeys was determined using 11C-PBR28 as a PET ligand. The PET study was conducted by Central Research Laboratory, Hamamatsu Photonics K.K.

Synthesis of 11C-PBR28.

11C-PBR28 was synthesized as reported previously (Brown et al., 2007). Positron-emitting carbon-11 was produced by 14N(p,α)11C nuclear reaction using a cyclotron (HM-18; Sumitomo Heavy Industries, Ltd., Tokyo, Japan). 11C-PBR28 was radiolabeled by methylation of its desmethyl precursor using 11C-methyl triflate and purified by high-performance liquid chromatography (Megapak SIL C18-10 7.6 × 250 mm; JASCO Corporation, Tokyo, Japan) with a mobile phase of 500/500 acetonitrile/30 mmol/liter ammonium acetate at a flow rate of 6 ml/min. After a fraction corresponding to 11C-PBR28 was collected and the eluate evaporated, the residue was dissolved in physiologic saline to obtain the final 11C-PBR28 product. Radioactivity of the final product was measured using a curiemeter (IGC-3; Aloka, Tokyo, Japan), and a portion was analyzed by high-performance liquid chromatography (Finepack C18-S 4.6 × 150 mm; JASCO Corporation, Tokyo, Japan) using 500/500/2 acetonitrile/30 mmol/liter ammonium acetate/acetic acid at a flow rate of 2 ml/min (wavelength, 254 nm). The product radiochemical purity was more than 99%, and its specific radioactivity was more than 150 GBq/μmol.

Imaging Method.

PET scans were performed as reported previously (Tsukada et al., 2000; Noda et al., 2003). At least 1 month before the start of chair training, an acrylic plate, by which the test animal was fixed to a monkey chair, was attached to the top of the monkey’s head with the monkeys under pentobarbital anesthesia. The monkeys were then trained to sit on the monkey chair twice a week for more than 3 months to acclimate and relieve the stress of head fixation during PET measurement. After being fasted overnight, the test animals were anesthetized with 2.5% sevoflurane, and a cannula was placed in the cephalic vein or saphenous vein for i.v. administration of the test compounds. Another cannula was placed in the femoral artery or posterior tibial artery for arterial blood sampling. The animal was then fixed on the monkey chair. After confirmation of full recovery from anesthesia, the animal was transferred to a high-resolution animal PET scanner (SHR-7700; Hamamatsu Photonics K.K., Hamamatsu, Japan), and its head was fixed into a stereotactic apparatus (SFCT-RB-PR-2, Hamamatsu Photonics K.K.). A transmission scan was then performed for 30 minutes using a 68Ge/68Ga calibration source. Next, an emission scan (121 minutes total) was started simultaneously with the start of the 11C-PBR28 injection.

For quantitative analysis of 11C-PBR28, PET scans were performed during arterial blood sampling. Approximately 0.5 ml of arterial blood was collected at 20 time points (i.e., 8, 16, 24, 32, 40, 48, 56, 64, 90, and 150 seconds and 4, 6, 10, 20, 30, 45, 60, 75, 90, and 120 minutes after 11C-PBR28 injection). The blood samples were centrifuged to separate the plasma, and the radioactivity was measured. To the plasma (100 μl) separated from blood samples collected at 16, 40, and 64 seconds, and 6, 10, 30, 45, 60, 75, 90, and 120 minutes after 11C-PBR28 injection, ethanol (200 μl) was added, and the mixture was centrifuged for metabolites analysis. The resulting supernatant was developed on a thin-layer chromatography plate (AL SIL G/UV; Whatman, Kent, Buckinghamshire, UK) with a mobile phase of 45/5/1 chloroform/methanol/triethylamine. The ratio of metabolites to the parent 11C-PBR28 was determined using an imaging plate (BAS-IIIs; Fujifilm Corporation, Tokyo, Japan). Arterial input function was defined as time-course change in radioactivity concentration of the parent 11C-PBR28 in arterial plasma.

For the ONO-2952 experiment, PET scans were performed at 2 and/or 24 hours after single or repeated (twice daily for 3 days and once on day 4) oral administration of ONO-2952 at several doses ranging from 0.3 to 30 mg/kg. The vehicle (0.5% MC) was administered orally once 2 hours before PET scans. Venous blood was collected for measurement of ONO-2952 plasma levels immediately before injection of 11C-PBR28. ONO-2952 plasma concentrations were determined by the method described already herein. For the PK11195 experiment, PET scans were performed 1 minute after i.v. administration of PK11195 (0.1 mg/kg) or vehicle (5% ethanol-0.5% Tween80 saline solution).

Images Analysis and Quantification.

PET images were reconstructed with a Filtered Back Projection method using a Hanning filter of 4.5 mm in SHR Control II program (Hamamatsu Photonics K.K.). PMOD program (PMOD Technologies Ltd., Zurich, Switzerland) was used to calculate time-activity curves (TACs) of 11C-PBR28 in the brain. The regions of interest (ROIs) (cerebellum, hippocampus, corpus striatum, thalamus, occipital cortex, temporal cortex, frontal cortex, and parietal cortex) were drawn on PET reconstructed images of individual animals with reference to brain magnetic resonance imaging images previously obtained for each animal using a 3T magnetic resonance imaging scanner (Allegra; Siemens, Erlangen, Germany) to determine the TAC of 11C-PBR28 in each ROI.

The standardized uptake value (SUV), which represents mean brain radioactivity accumulation between 91 and 121 minutes postdose normalized for the injected dose of radioactivity and the animal’s body weight, was calculated according to eq. 2:Embedded Image (2)Using metabolite-corrected arterial input function, the total distribution volume (V t) of 11C-PBR28 was determined from the TACs of each ROI by the Two-Tissue Compartment (2TC) model (Innis et al., 2007; Fujita et al., 2008), using a calculation tool for PMOD. In addition, V t images were generated from the reconstructed PET images blurred by three-dimensional Gauss filter of 4 mm full width at half maximum and input function by 2TC analysis using PMOD.

TSPO occupancy in the whole brain was defined as the slope of linear regression of the V tbase versus (V tbase − V tdrug) in each brain ROI, where V tbase and V tdrug were the regional distribution volumes after vehicle and drug administration, respectively, termed the Lassen plot method (Cunningham et al., 2010; Owen et al., 2014).

Statistical Analysis

Except where noted, data are expressed as mean ± S.E.M. Statistical analyses were performed using GraphPad Prism software (version 5.01; GraphPad Software Inc.). Nonlinear regression analysis was performed with a least-squares method.

Results

In Vitro Binding of ONO-2952 to Rat and Monkey TSPO

As shown in Table 1, the K d value of 3H-PBR28 in mitochondrial membrane fractions prepared from rat brain was equivoundalent to that in the fractions prepared from monkey hippocampus or occipital cortex; however, the B max value in the rat fractions was lower than that in the monkey fractions. ONO-2952 bound to rat TSPO with a K i value comparable to or slightly lower than that for binding to monkey TSPO. No significant differences were found in K i values between ONO-2952 and PK11195 binding to rat TSPO. As for the affinity of these two test compounds for monkey TSPO, PK11195 showed higher K i values than did ONO-2952 in each brain region.

View this table:
TABLE 1

K i values of ONO-2952 for binding to rat and monkey TSPO

Competitive binding assay was performed using 3H-PBR28.

Effect of ONO-2952 on Kd and Bmax Values for 3H-PBR28 Binding to Monkey Brain TSPO

Saturation binding of 3H-PBR28 in the absence or presence of ONO-2952 in membrane fraction from monkey occipital cortex revealed that ONO-2952 significantly and concentration dependently increased 3H-PBR28 K d value without affecting B max values (Table 2).

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TABLE 2

Effect of ONO-2952 on K d and B max values for 3H-PBR28 binding to monkey brain TSPO

Saturation binding assay for three separate experiments was performed with membrane fractions prepared from monkey occipital cortex using 3H-PBR28.

Relationship between ONO-2952 Plasma Concentration and its Ex Vivo TSPO Occupancy in the Rat Brain

ONO-2952 plasma concentration and TSPO occupancy in the rat brain 3 hours after oral administration are shown in Table 3. ONO-2952 occupancy of TSPO in the rat cerebral cortex and hippocampus increased with increasing plasma concentration, reaching a maximum of approximately 90% in the cerebral cortex at a dose of 3 mg/kg. The estimated IC50 value of ONO-2952 in the cerebral cortex and hippocampus were 9.78 and 18.6 ng/ml, respectively.

View this table:
TABLE 3

ONO-2952 plasma concentration and TSPO occupancy in the rat brain

Plasma concentration is expressed as the mean ± S.D. TSPO occupancy was determined by ex vivo binding using 3H-PBR28. TSPO occupancy in the cerebral cortex and hippocampus is expressed as the mean ± S.E. (n = 8 in each group). Plasma concentrations required for 50% of maximal (fixed at 100%) TSPO occupancy in the cerebral cortex and hippocampus were 9.78 and 18.6 ng/ml, respectively.

PET Study in Conscious Monkeys

Effect of PK11195 on 11C-PBR28 TAC in the Monkey Brain

11C-PBR28 uptake peaked 25 to 35 minutes after vehicle administration in all brain regions with an SUV of 1.5 or more, except in the cerebellum (Fig. 1). The highest SUV was observed in the occipital cortex, the lowest in the cerebellum (Fig. 1A). As shown in Fig. 1B, pretreatment with PK11195 shifted peak 11C-PBR28 uptake to within 5 minutes after injection, followed by a rapid decrease in lower levels in all brain regions at times later than 20 minutes after injection compared with the corresponding levels after vehicle administration.

Fig. 1.
Fig. 1.

Typical 11C-PBR28 TACs in each ROI of conscious monkey brain after (A) vehicle and (B) PK11195 dosing. PET scans were performed 1 minute after i.v. administration of PK11195 (0.1 mg/kg) or vehicle (5% ethanol-0.5% Tween80 saline solution). The uptake values in each ROI were normalized for the injected dose of radioactivity and for animal body weight. Cere, cerebellum; Hippo, hippocampus; Str, corpus striatum; Thal, thalamus; OccCtx, occipital cortex; TmpCtx, temporal cortex; FrtCtx, frontal cortex; ParCtx, parietal cortex.

TSPO Occupancy by PK11195 in the Brain

Quantitative PET images demonstrated that PK11195 (0.1 mg/kg, i.v.) decreased 11C-PBR28 Vt across broad brain regions (Fig. 2 and Table 4). The averaged TSPO occupancy of PK11195 as determined by using the slope of the Lassen plots was 49.2% (Fig. 2C and Table 4).

Fig. 2.
Fig. 2.

Typical MR and PET images of 11C-PBR28 in conscious monkey after (A) vehicle and (B) PK11195 dosing. PET scans were performed 1 minute after i.v. administration of PK11195 (0.1 mg/kg) or vehicle (5% ethanol-0.5% Tween80 saline solution) with sequential arterial blood sampling. Color scale indicates V t. (C) Linear regression of the V tbase versus (V tbase − V tdrug) in each brain region of interest (Lassen plot).

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TABLE 4

Effect of PK11195 on 11C-PBR28 Vt in conscious monkeys

PET scans were performed 1 minute after i.v. administration of PK11195 (0.1 mg/kg) or vehicle (5% ethanol-0.5% Tween80 saline solution) with sequential arterial blood sampling. V t in each ROI is expressed as the mean ± S.E. (n = 3). Averaged TSPO occupancy of three monkeys as determined by using the Lassen plots was 49.2 ± 29.66%.

Effect of ONO-2952 on 11C-PBR28 TAC in the Monkey Brain

As shown in Fig. 3, pretreatment with ONO-2952 (0.1 mg/kg) resulted in a slight increase in the uptake of 11C-PBR28 in the brain compared with pretreatment with the vehicle (Fig. 3A). Oral administration of ONO-2952, at doses of 1 and 10 mg/kg, dose dependently shifted the peak in 11C-PBR28 uptake to approximately 15 and 8 minutes after injection, respectively (Fig. 3, B and C). TACs in ONO-2952–treated monkeys decreased thereafter in a dose-dependent manner compared with those in the vehicle-treated monkeys. Similar effects on 11C-PBR28 TACs were observed 2 hours or 24 hours after repeated administration of ONO-2952 (Fig. 4A).

Fig. 3.
Fig. 3.

Typical 11C-PBR28 TACs in each ROI of conscious monkey brains after vehicle and ONO-2952 dosing. PET scans were performed 2 hours after single oral administration of ONO-2952 at (A) 0.1 mg/kg, (B) 1 mg/kg, and (C) 10 mg/kg or vehicle (0.5% MC). Uptake values in each ROI were normalized for the injected dose of radioactivity and for animal body weight. Cere, cerebellum; Hippo, hippocampus; Str, corpus striatum; Thal, thalamus; OccCtx, occipital cortex; TmpCtx, temporal cortex; FrtCtx, frontal cortex; ParCtx, parietal cortex.

Fig. 4.
Fig. 4.

Typical 11C-PBR28 (A) TACs in each ROI and (B) PET images in conscious monkey after (a) vehicle and (b–e) ONO-2952 dosing. PET scans were performed (a–c) 2 hours and (d, e) 24 hours after the final administration of ONO-2952 or vehicle (0.5% MC) with sequential arterial blood sampling. ONO-2952 at doses of (b, d) 1 mg/kg and (c, e) 10 mg/kg was orally administered twice daily for 3 days and once on day 4. Uptake values in each ROI were normalized for the injected dose of radioactivity and for animal body weight. Cere, cerebellum; Hippo, hippocampus; Str, corpus striatum; Thal, thalamus; OccCtx, occipital cortex; TmpCtx, temporal cortex; FrtCtx, frontal cortex; ParCtx, parietal cortex. Color scale indicates total distribution volume (V t). (C) Linear regression of the V tbase versus (V tbase − Vtdrug) in each brain ROI (Lassen plot).

TSPO Occupancy by ONO-2952 in the Brain

Pretreatment with ONO-2952 (1 and 10 mg/kg) 2 hours before PET scans dose dependently decreased 11C-PBR28 V t in the whole brain (Fig. 4B and Table 5). This trend continued even 24 hours after dosing with no inter-regional differences (Fig. 4B and Table 5). The regression lines in each brain ROI of a representative monkey are shown in Fig. 4C. Overall, no marked brain region specificity for ONO-2952 TSPO occupancy was noted at any dose. In addition, ONO-2952 TSPO occupancy was dependent on the plasma concentration of this compound irrespective of the time from the final dosing (Table 5).

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TABLE 5

Effects of ONO-2952 on 11C-PBR28 V t and ONO-2952 plasma concentration in conscious monkeys

Relationship between ONO-2952 Plasma Concentration and its Brain TSPO Occupancy in Monkeys

TSPO occupancy of ONO-2952 in the whole brain determined by PET generally increased with increasing plasma concentration, reaching a maximal occupancy as high as 100.2% (Fig. 5). Analysis of brain TSPO occupancy plotted as function of plasma concentration revealed that the two parameters correlated well. In the fit with maximal occupancy fixed at 100%, the estimated IC50 value of ONO-2952 in the PET study was 19.6 ng/ml.

Fig. 5.
Fig. 5.

Relationship between plasma concentration of ONO-2952 and its TSPO occupancy in the brain of conscious monkeys. PET scans were performed after single and repeated oral administration of ONO-2952 at three or four dose levels for each animal with sequential arterial blood sampling. Plasma concentration was determined from blood withdrawn 2 hours or 24 hours after oral administration of ONO-2952 at several doses ranging from 0.3 mg/kg to 30 mg/kg, immediately before i.v. administration of 11C-PBR28. TSPO occupancy in the whole brain was defined as the slope of the Lassen plot. Dotted line represents the regression curve of TSPO occupancy-plasma concentration in PET study. IC50, plasma concentration required for 50% of maximal (fixed at 100%) TSPO occupancy in PET study. Single: ONO-2952 was orally administered once; repeated: ONO-2952 was orally administered twice daily for 3 days and once on day 4. 2 h: Assessed 2 hours after the final administration; 24 h: Assessed 24 hours after the final administration.

Discussion

Using conscious monkeys, we confirmed in this PET study that ONO-2952, a novel TSPO antagonist, has sufficient brain distribution, with a clear relationship between its plasma concentration and TSPO occupancy in the brain.

In vitro binding experiment using 3H-PBR28 as a radioactive ligand showed that ONO-2952 exhibits high affinity for monkey TSPO compared with PK11195, a well characterized TSPO ligand (Table 1). These findings are in agreement with those showing that PK11195 exhibits interspecies variability in its affinity for TSPO, with K i values of 4.35 nmol/liter and 0.73 nmol/liter for monkey and rat TSPOs, respectively (Briard et al., 2008). Although the K i values of ONO-2952 and PBR28 showed a small interspecies variability, the binding affinity of these compounds to TSPO is considered to be similar across species. As shown in Table 2, K d value of 3H-PBR28 increased with increasing concentration of ONO-2952, although the B max value did not change. These findings indicate that these two compounds mutually compete for TSPO binding sites in the monkey brain. In addition, PBR28 reportedly has good selectivity for TSPO against 36 off-targets (Imaizumi et al., 2008), and 11C-PBR28 exhibits sufficient signal and appropriate kinetics for imaging TSPO in nonhuman primate brain (Briard et al., 2008; Imaizumi et al., 2008). Based on these findings, we decided to evaluate TSPO occupancy of ONO-2952 in the brain using 11C-PBR28 as a radiolabeled ligand for the PET study.

Our ex vivo experiment, aimed at measuring TSPO occupancy of ONO-2952 in the rat brain, revealed that TSPO occupancy of this compound increases with increasing plasma concentration. This finding is in agreement with our previous work using 3H-PK11195 as a radioactive ligand and showing that TSPO occupancy in the rat cerebral cortex and hippocampus treated with 0.3 mg/kg of ONO-2952 reaches about 50% at plasma concentration of 6.30 ng/ml (Mitsui et al., 2015); however, a dose-independent relationship was found between 0.03 and 0.1 mg/kg in the hippocampus in the current study. This result might have been due to variability associated with administration of very low dose of ONO-2952. Our PET study in monkeys, on the other hand, demonstrates the need for a relatively higher plasma concentration than in the rat to achieve target occupancy levels (Fig. 5 and Table 3). In our hands, in vitro protein binding levels of ONO-2952 (100 ng/ml) in rat and monkey sera were 98.7% and 99.7%, respectively (unpublished data). Considering the small interspecies difference in the affinity of ONO-2952 for TSPO in rat and monkey, the difference in protein binding of two species may result in a higher requirement of blood exposure in the study in monkeys.

Combining the results obtained from the in vitro and ex vivo binding studies, 11C-PBR28 was considered a useful ligand for determining TSPO occupancy of ONO-2952 in the monkey PET study. Consistent with the reported brain kinetics of 11C-PBR28 in anesthetized monkeys (Imaizumi et al., 2008), this ligand showed wide and sufficient distribution in our PET study using conscious monkeys. In addition, pretreatment with PK11195 shifted peak 11C-PBR28 uptake in all brain regions and then rapidly decreased this uptake to levels lower than those detected in the vehicle-treated monkeys (Fig. 1). It is interesting to note that similar effects on 11C-PBR28 TACs were observed after oral administration of ONO-2952 in a dose-dependent manner (Fig. 3). These findings indicate that 11C-PBR28 recognizes and specifically binds to biomolecules, including TSPO, that are inhibited by both PK11195 and ONO-2952. TSPO has been found to be expressed not only in the CNS but also in peripheral organs and peripheral blood cells (Gavish et al., 1999; Kreisl et al., 2010). A blocking study has shown that plasma levels of unmetabolized 11C-PBR28 increase after pretreatment with nonradioactive TSPO ligand (Imaizumi et al., 2008). Therefore, the high 11C-PBR28 uptake observed in the PK11195-treated monkey (Fig. 1), as well as in the ONO-2952-treated monkey (Fig. 3 and Fig. 4) may be explained by the increase in unbound 11C-PBR28 resulting from increased binding of cold ligand to peripheral TSPO, which in turn penetrates the brain. As a result of these peripheral effects on 11C-PBR28 kinetic, and since no suitable reference brain region free of specific binding sites for 11C-PBR28 was found, we used metabolite-corrected arterial input function to determine V t values and TSPO occupancy of ONO-2952. As shown in Fig. 2 and Table 4, a significant reduction in V t after PK11195 dosing was observed in all ROIs in the monkey brain, with TSPO occupancy of about 50%. ONO-2952 also decreased the V t of 11C-PBR28 in a dose-dependent manner (Fig. 4). The current PET occupancy study shows that ONO-2952 bound to TSPO in all brain ROIs, including the amygdala and hippocampus, both of which are key structures associated with physiologic responses to stress (Guan et al., 2003; Myers and Greenwood-Van Meerveld, 2009; Belujon and Grace, 2011). These findings support our hypothesis that ONO-2952 may modify stress response in humans through its binding to TSPO in the brain. Similar inhibitory effects on V t images characterize brain distribution of ONO-2952 after oral administration 2 hours or 24 hours before PET scans. Although TSPO occupancy of ONO-2952, as determined by quantitative PET assessment 24 hours after administration, was slightly less than that 2 hours after dosing, plasma concentration of ONO-2952 and its TSPO occupancy in the whole brain roughly correlated, suggesting that ONO-2952 reversibly binds to TSPO with no excessive accumulation in the brain. As shown in Fig. 5, a good correlation between ONO-2952 pharmacokinetic and its TSPO occupancy was observed irrespective of the dosing time, which further supports the reversible binding property of this compound.

We have previously shown that ONO-2952 exerts antistress effects with brain TSPO occupancy of 50% or more in rats exposed to acute stress (Mitsui et al., 2015). In addition, the relationship between plasma concentration of ONO-2952 and its brain occupancy in rats is not changed by 1-hour stress exposure (unpublished data). Based on these findings, we assume the target occupancy of this protein for a similar effect in human would be 50% or greater. Although the contribution of peripheral TSPO to the beneficial effect of ONO-2952 in rats has not been examined, pharmacologic profile points at central TSPO as the principal target involved in the mechanism of action of this compound (Mitsui et al., 2015). Nevertheless, it is important to note that there is a marked difference in the PK occupancy relationship between the two species, as discussed earlier. Taken together, the results show that a minimal therapeutic dose of ONO-2952, as well as its optimal dose level for clinical use, could be estimated using a human PET study by targeting a brain occupancy of 50% rather than using this compound’s pharmokinetic profile. More recently, we have published a study showing that ONO-2952 exhibits clinical efficacy in patients with irritable bowel syndrome at whole-brain occupancy level of 77.4% (Whitehead et al., 2017). As we did not investigate here the relationship between the antistress effect of ONO-2952 and its TSPO occupancy in the monkey brain, the reason why higher whole-brain occupancy is needed in this POC study compared with rats studies remains unclear. To clarify this remaining question, determination of expression levels of TSPO in the brain of patients with stress-related disorders would be necessary.

In conclusion, this study shows that ONO-2952 sufficiently penetrates conscious monkey brain with a clear relationship between its plasma concentration and TSPO occupancy.

Acknowledgments

The authors thank the staff of the Central Research Laboratory of Hamamatsu Photonics K.K. for their assistance in performing the PET study.

Authorship Contributions

Participated in research design: Mitsui, Tsukada, Katsumata.

Conducted experiments: Mitsui, Morimoto, Niwa, Yamaura, Ohba, Tsukada, Katsumata.

Performed data analysis: Mitsui, Morimoto, Niwa, Yamaura, Ohba, Tsukada, Katsumata.

Wrote or contributed to the writing of the manuscript: Mitsui, Morimoto, Tsukada, Katsumata.

Footnotes

Abbreviations

CNS
central nervous system
MC
methylcellulose
PET
positron emission tomography
ROI
region of interest
SUV
standardized uptake value
TAC
time-activity curve
TSPO
18 kDa translocator protein
Vt
total distribution volume

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Research ArticleNeuropharmacology

ONO-2952 Brain TSPO Occupancy in Conscious Monkeys

Katsukuni Mitsui, Noriko Morimoto, Tomohiro Niwa, Yoshiyuki Yamaura, Hiroyuki Ohba, Hideo Tsukada and Seishi Katsumata
Journal of Pharmacology and Experimental Therapeutics March 1, 2017, 360 (3) 457-465; DOI: https://doi.org/10.1124/jpet.116.238568
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