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NEUROPHARMACOLOGY
-Aminobutyric AcidA Receptors Measured Using in Vivo [3H]Flumazenil (8-Fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic Acid Ethyl Ester) Binding and [11C]Flumazenil Micro-Positron Emission TomographyMerck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, United Kingdom (J.R.A., P.S.-S.); Departments of Anesthesia (J.S.B.), Clinical Neurosciences (J.L.H.) and Wolfson Brain Imaging Centre (T.D.F., M.C.C., J.-C.B., J.C.C., F.I.A.), Addenbrookes Hospital, Cambridge, United Kingdom; and Imaging Research and Pharmacology, Merck Research Laboratories, West Point, Pennsylvania (R.J.H.)
Received for publication
October 2, 2006
Accepted
December 11, 2006.
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Abstract |
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).
The methods generally used to measure occupancy in the brain of preclinical species and in man are very different, raising the possibility that methodological differences may be a factor in the translation of occupancy measurements from preclinical species into man. Hence, in preclinical species, occupancy is generally assessed by measuring the extent to which predosing an animal with test compound inhibits either the degree of binding of a radioligand administered to the live animal (in vivo binding; e.g., Atack et al., 1999) or the binding of radioligand incubated with brain tissue after the animal is killed (ex vivo binding; e.g., Duffy et al., 2002), with, for methodological reasons, in vivo binding being preferred to ex vivo binding (Li et al., 2006). With in vivo binding, the quantitation of radioactivity in the brain necessitates removal of the brain, homogenization, and filtration and washing, the latter of which removes free ligand and low-affinity nonspecific binding followed by liquid scintillation counting. On the other hand, in PET studies, brain radioactivity is detected in live subjects and comprises radioactivity in the specific and nonspecific binding, free radioligand, and blood compartments.
With the availability of small animal scanners, such as the micro-PET (Tai et al., 2001; Yang et al., 2004; Cheery and Chatziioannou, 2005), it is now possible to perform PET in rodents. The purpose of the present study was to measure occupancy using micro-PET techniques and compare these data with those obtained by traditional in vivo binding methods. More specifically, lorazepam occupancy of the benzodiazepine binding site of rat brain GABAA receptors was measured using the inhibition of the in vivo binding of radiolabeled flumazenil in terminal homogenization and filtration or in vivo micro-PET assays using [3H]flumazenil or [11C]flumazenil, respectively. In addition, a bridging study was performed in which lorazepam occupancy was measured under simulated micro-PET conditions but using [3H]flumazenil in vivo binding methods. Because these methods employed different dosing routes (oral for in vivo [3H]flumazenil binding and i.v. for [11C]flumazenil micro-PET), plasma-occupancy relationships were determined because these are independent of drug dose route, and the plasma lorazepam concentration required to give 50% occupancy (EC50) was calculated.
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Drugs. Lorazepam was obtained from Sigma-Aldrich (Gillingham, UK), whereas bretazenil was synthesized at Merck Research Laboratories (Harlow, UK). [3H]Flumazenil ([3H]Ro 15-1788; 87 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA).
[3H]Flumazenil in Vivo Binding. Male CD (Sprague-Dawley-derived) rats (250–300 g; Charles River UK Ltd, Ramsgate, Kent, UK; n = 5–10/group) received vehicle (0.5% methyl cellulose p.o. with a dosing volume of 5 ml/kg), lorazepam (0.1, 0.3, 1, 3, 5, or 10 mg/kg), or, to define the level of nonspecific binding, bretazenil (5 mg/kg i.p. in 100% polyethylene glycol 300). This dose of bretazenil was chosen because it gives 100% occupancy of the benzodiazepine binding site of rat brain GABAA receptors (Atack et al., 2006). Twenty-seven minutes later, animals received an i.v. tail vein injection of [3H]flumazenil (diluted 1:150 with saline and dosed at 1 µl/g; equivalent to a dose of 2 µCi of [3H]flumazenil/300-g rat) and, after an additional 3 min, were culled by decapitation. Trunk blood was collected into lithium-heparin tubes and centrifuged, and plasma was retained for subsequent analysis for drug concentrations (see below). Brains were removed and rapidly homogenized in 10 volumes of ice-cold buffer (50 mM potassium phosphate, pH 7.4, containing 100 mM KCl), and 300-µl aliquots were filtered and washed with 10 ml of buffer over Whatman GF/B filters (Whatman, Brentford, UK). The filters were then placed in scintillation vials, scintillation fluid was added, and radioactivity was counted on a Beckman LS6500 scintillation counter (Beckman Coulter, High Wycombe, UK). The radioactivity in bretazenil-treated animals (approximately 100 dpm) was subtracted from each of the other groups to give specific binding that was in the region of 2000 dpm in vehicle-treated rats. In lorazepam-treated animals, the extent to which radioactivity was reduced relative to the vehicle group represented the extent to which lorazepam occupied the benzodiazepine binding site of rat brain GABAA receptors.
Measurement of Plasma Lorazepam Concentrations. Aliquots of plasma (50 µl) were prepared for analysis by protein precipitation using 3 volumes of acetonitrile containing diazepam as an internal standard. After centrifugation, the supernatants were transferred to a high-performance liquid chromatography vial and analyzed by liquid chromatography-tandem mass spectrometry (Micromass Micro triple quadrupole mass spectrometer; Water Corp. Ltd., Manchester, UK). The system consisted of a 5-cm x 3.2-mm Kromasil KR-100 C18 column (Hichrom, Theale, UK) and employed a mobile phase of acetonitrile and 25 mM ammonium formate, pH 3. A ballistic gradient was used (time and acetonitrile concentrations = 0 min, 20%; 0.5 min, 90%; 3 min, 90%; 3.1 min, 20%; 6 min, 20%) with a flow rate of 0.5 ml/min. Lorazepam and diazepam were detected by multiple reaction monitoring of the transitions of m/z 321 to 193 and 285.3 to 154.2, respectively.
Intravenous Pharmacokinetics of Lorazepam. The design of the micro-PET study was such that two doses of lorazepam were to be administered after brain radioactivity reached a peak (
10 min) and before levels of radioactivity dropped to levels at which the signal/noise ratio became an issue (50 min). Although a low-dose single bolus injection of lorazepam would be sufficient to achieve sustained low plasma concentrations, a combination of a higher dose bolus injection with constant infusion would be required to rapidly achieve sustained higher plasma concentrations of lorazepam. Based on the [3H]flumazenil in vivo binding data, a plasma lorazepam concentration of greater than 500 ng/ml was targeted to give high (>80%) occupancy. The concentration of the infusion solution required to achieve this target plasma lorazepam level was calculated using eq. 1:
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The i.v. pharmacokinetics of lorazepam were investigated in male CD rats (300 g), in which a jugular vein cannula was exteriorized at the nape of the neck under isoflurane anesthesia. Animals were allowed to recover for a minimum of 36 h during which they were freely moving and singly housed. Lorazepam, formulated as an aqueous solution in 2% DMSO/10% hydroxypropyl-
-cyclodextrin, was administered via a bolus tail vein injection (1 ml/kg) at 0.05, 0.1, or 0.5 mg/kg, one rat per dose level, and blood was serially sampled via the indwelling cannula up to 2 h postdose. Pharmacokinetic parameters were calculated from plasma versus time data by model-independent methods using in-house software.
After determination of the plasma clearance of lorazepam, a dosing regime was constructed to achieve the plasma lorazepam concentrations required for 80% receptor occupancy. To examine the utility of this dosage regime, dual jugular and femoral vein cannulated male CD rats (300 g) were surgically prepared under isoflurane anesthesia. Rats freely moving and tethered to protect the exteriorized infusion and blood sampling cannulae were singly housed and allowed to recover for a minimum of 36 h. Rats (n = 3 per dose group) were dosed with a bolus loading dose of lorazepam (0.5 mg/kg and 1 ml/kg) and/or an infusion of lorazepam (0.2 mg/ml) administered via the femoral vein cannula and delivered at a rate of 6 ml/h/kg by a Harvard Model S infusion pump (Harvard Apparatus Inc., Edenbridge, UK). Lorazepam was formulated as aqueous solutions in 2% DMSO/10% hydroxypropyl--cyclodextrin. Blood was serially sampled via the jugular vein cannula at time points up to 1 h after the start of the infusion.
Micro-PET Simulation Studies. To examine the potential effect of anesthetic on both the pharmacokinetics of lorazepam and receptor occupancy measurements, a study was conducted in terminally anesthetized male CD rats (300 g) to mimic the micro-PET protocol. Rats were maintained under isoflurane anesthesia (1.5%) throughout the surgical procedure and subsequent micro-PET simulation. Following a small skin incision above the right femoral triangle, the femoral artery and vein (for blood sampling and compound administration, respectively) were cannulated with polypropylene cannulae, the patency was checked, and the cannulae were flushed with heparinized saline (50 IU/ml) before suturing of the skin incision.
After a 10-min postsurgical nonrecovery period, two doses of lorazepam were administered as follows: low-dose lorazepam, 0.075 mg/kg i.v. bolus, t = 10 min; and high-dose lorazepam, 0.5 mg/kg i.v. bolus followed by continuous infusion of 0.2 mg/ml lorazepam (6 ml/h/kg) at t = 21 min. Blood was sampled via the femoral artery cannula at 11, 15, 20, 22, 23, 27, 32, 42, and 52 min postsurgery. To define levels of total and nonspecific binding, two separate groups of anesthetized rats were dosed with vehicle (1 ml/kg, i.v., n = 5) or bretazenil (5 mg/kg, 1 ml/kg i.p., 100% polyethylene glycol 300, n = 3), respectively, and culled 30 min postdose. All lorazepam formulations were made up as aqueous solutions in 2% DMSO/10% hydroxypropyl--cyclodextrin. At each time point, one rat was culled, and at the 15-, 20-, 27-, 32-, 42-, and 52-min times, occupancy was determined following before i.v. administration of [3H]flumazenil, as described above. Aliquots of plasma were frozen and stored at -20°C before subsequent analysis of drug concentrations.
Micro-PET Studies. [11C]Flumazenil was produced from [11C]iodomethane using a modified captive solvent method (Cleij et al., 2007). Radiochemical yields of 8 to 10 GBq and specific radioactivities of 520 to 600 GBq/µmol were achieved at the end of the syntheses. The [11C]flumazenil was formulated in 1 ml of saline containing 5% v/v ethanol with the molar amount of coinjected unlabeled flumazenil kept constant at 0.3 nmol by adjusting the amount of injected radioactivity, which varied between 59 and 90 MBq.
An overview of the design of the micro-PET study is presented in Fig. 1. The micro-PET experiments were designed to allow each animal to receive two doses of either vehicle (n = 2 rats) or lorazepam (n = 5) and, therefore, generate two data points per animal. During this period, plasma samples were taken for subsequent analysis of lorazepam concentrations (Fig. 1). In order to estimate the extent of nonspecific binding plus free radioligand, at t = 46 min, rats were given a dose of unlabeled flumazenil (0.25 mg/kg i.v.).
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In brief, male CD rats (300 g) were prepared as described above for the micro-PET simulation studies with an additional cannula implanted into the left femoral vein for administration of the [11C]flumazenil. The cannulated rats were placed in the micro-PET P4 scanner (Concorde Microsystems Inc., Knoxville, TN) and maintained under isoflurane anesthesia throughout the duration of the experiment. The experiment was initiated by the injection of [11C]flumazenil (injected over 30 s using a syringe pump). The scanner had a 22- x 7.8-cm field of view with a resolution of 2 mm at the center, where the brain of the rat was located.
Data for lorazepam dosed PET scans were acquired over a 72-min period (16 x 0.5 min, then 64 x 1-min frames). The list mode PET data were plotted into 3D sinograms with dead time, and random corrections were applied. Images were reconstructed from the 3D sinogram data into 0.5- x 0.5- x 0.5-mm voxels in a 180 x 180 x 151 array using a version of 3D filtered back-projection (Kinahan and Rogers, 1990) with corrections for background, normalization, sensitivity, and decay.
A cortical region of interest (ROI) was defined for each subject using the threshold tool in Analyze on an image produced by summing the dynamic images over the period t = 6 to 12 min. This ROI was then applied to the dynamic images to produce a cortical time-activity curve (TAC). To produce the mean control TAC, the TAC for control 2 was scaled and shifted to produce a least-squares fit with the TAC for control 1, and the TACs were then averaged.
To calculate occupancy, the TAC from a displacement study was scaled and shifted to achieve the least-squares fit to the mean control TAC over the range t = 4 to 8 min using second order polynomial functions. Secondly, over the period t = 60 to 72 min, an exponential was fitted to the displacement study TAC to estimate the nonspecifically bound and free signal in the cortical ROI (the high-dose flumazenil injection at t = 46 min was assumed to have removed the specifically bound cortical signal by t = 60 min). Occupancy was then averaged over the t = 15 to 25 min (low-dose lorazepam) and t = 36 to 46 min (high-dose lorazepam) time periods using eq. 2:
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To more specifically analyze the relationship between lorazepam occupancy and plasma drug concentrations suggested by the dose dependence of these two parameters, occupancy was plotted as a function of plasma drug concentrations for each individual animal. As can be seen from Fig. 3, the relationship between lorazepam occupancy and plasma drug concentrations was a sigmoidal relationship, best fitted by a curve with a Hill slope of 0.86 and an EC50 of 134 ng/ml (95% confidence intervals = 119–151 ng/ml).
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Based upon these parameters, two infusion paradigms were evaluated; the first, a 0.5 mg/kg bolus followed by a 0.2 mg/ml infusion at a rate of 6 ml/h/kg; and the second, for comparative purposes, a 0.2 mg/ml infusion alone (i.e., without the bolus loading dose). As can be clearly seen from Fig. 4B, the bolus followed by infusion achieved relatively constant plasma lorazepam concentrations in the region of 400 to 600 ng/ml. On the other hand, although the infusion alone ultimately gave plasma concentrations that approached those of the bolus/infusion method (500 ng/ml), the time taken to achieve these concentrations, around 1 h, was inappropriate for the micro-PET experiment.
Micro-PET Simulation. Because the pharmacokinetic studies described above were performed in awake animals, yet the micro-PET experiments would be performed in anesthetized rats, we performed a micro-PET simulation experiment with the purpose of measuring plasma lorazepam concentrations in anesthetized animals. In addition, certain animals were given [3H]flumazenil to measure the lorazepam occupancy under these conditions. Figure 5 shows that 5 to 10 min after administration of the low dose of lorazepam (0.075 mg/kg i.v.), plasma concentrations were in the region of 40 ng/ml, which were comparable with the plasma concentrations achieved in conscious animals at 0.05 and 0.1 mg/kg doses (concentrations at 10 min = 20 and 45 ng/ml, respectively; Fig. 4A). The high dose (0.5 mg/kg bolus followed by 0.2 mg/ml infusion) achieved plasma concentrations (580–830 ng/ml) slightly higher than those observed in conscious animals (500 ng/ml; Fig. 4B), presumably due to the lower liver blood flow (and hence reduced clearance) and changes in the distribution of lorazepam in anesthetized animals (Bell et al., 1985; Hansen et al., 1991).
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; Atack et al., 1999). Administration of a low dose of lorazepam followed by a high dose produced sequential reductions in brain [11C]flumazenil, consistent with lorazepam displacing [11C]flumazenil radioligand from the benzodiazepine site of GABAA receptors. Finally, i.v. administration of unlabeled flumazenil produced essentially a complete inhibition of [11C]flumazenil binding, thereby demonstrating that [11C]flumazenil has very low levels of nonspecific binding. Representative images of brain [11C]flumazenil in vehicleand high-dose lorazepam-treated rats (Fig. 7B, inset) show that in control animals, the uptake of [11C]flumazenil into the brain was widespread and consistent with the distribution of benzodiazepine binding sites in the brain and that lorazepam produced a qualitatively equivalent reduction in [11C]flumazenil in different regions of the brain, consistent with its equivalent affinity for the different GABAA receptor subtypes.
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Quantitation of the inhibition of [11C]flumazenil binding under the low- and high-dose conditions showed (Fig. 8A) that lorazepam produced occupancy of 23 ± 3% and 86 ± 1%, respectively, which compares well with occupancy measured in the micro-PET simulation study (33 ± 2% and 83 ± 3%, respectively; Fig. 6A). The plasma-occupancy relationship for the micro-PET data was fitted by a curve with a Hill slope of 1.13 and an EC50 of 96 ng/ml (95% confidence intervals = 74–124 ng/ml). These data are consistent with the EC50 values of 134 and 94 mg/ml in the [3H]flumazenil in vivo binding and the [3H]flumazenil micro-PET simulation studies, respectively (Table 2).
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; Yang et al., 2004; Cheery and Chatziioannou, 2005). Applications for such devices include, for example, following tumor progression or the fate of grafts (Phelps, 2000; Lu et al., 2006). More specifically, within the CNS, small animal PET imaging in rodents allows pathological changes to be monitored, for instance, after lesioning (Hume et al., 1996; Strome et al., 2006), transplantation (Torres et al., 1995), stroke (Takasawa et al., 2007), or in transgenic animals (Wang et al., 2005). In addition, small animal PET studies permit the measurement of the occupancy of drugs. This is of particular interest because the demonstration that a potential drug crosses the blood-brain barrier and binds to its target should be a key step in the process of drug discovery. Furthermore, occupancy is a parameter that can be translated across species because PET can be used in rodents, primates, and man (Phelps, 2000). The purpose of the present study, therefore, was to validate occupancy measured using micro-PET in relation to the more established terminal method of measuring occupancy in small animals, namely the ability of a compound to occupy receptors and therefore inhibit the binding of radioligand, with radioactivity measured ex vivo.
For these studies, we chose to examine the occupancy of lorazepam at the benzodiazepine binding site of GABAA receptors because not only is there high expression of GABAA receptors within the brain but the prototypic PET ligand for these receptors, [11C]flumazenil, is well described (Maziere et al., 1984; Abadie et al., 1992). Hence, [11C]flumazenil readily penetrates through the blood-brain barrier in vivo, whereas its major 11C-metabolite, the carboxylic acid [11C]desethyl-flumazenil, is hydrophilic and does not (Debruyne et al., 1991). Importantly, [11C]flumazenil also has high and displaceable specific binding (Samson et al., 1985; Pappata et al., 1988; Abadie and Baron, 1990; Fryer et al., 2002), which becomes flow-independent within a few minutes after i.v. administration (Koeppe et al., 1991). It should be emphasized that flumazenil binds with comparable affinity to GABAA receptors containing an 
1, 2, 3, or 5 subunit (the subunit being a key determinant of the affinity of the benzodiazepine binding site; Atack, 2005). Because flumazenil is not subtype-selective, the inhibition of [3H]flumazenil or [11C]flumazenil binding by a compound such as lorazepam that also is not subtype-selective represents equivalent occupancy of the 1, 2, 3, and 5 subtypes. For example, a 75% inhibition of [3H]flumazenil or [11C]flumazenil by lorazepam represents equivalent (75%) occupancy at each of the four subtypes.
The measurement of lorazepam occupancy using in vivo [3H]flumazenil binding and [11C]flumazenil micro-PET differs in several key respects. Thus, the former involves oral predosing of lorazepam to awake animals and then injecting radioligand and measuring the extent by which in vivo [3H]flumazenil binding, measured ex vivo, is reduced relative to a group of vehicle-treated animals; in the latter, [11C]flumazenil is administered first, and then this binding is displaced by i.v. administration of lorazepam, with the extent of this displacement being calculated using an estimate of the [11C]flumazenil time-activity curve had lorazepam not been administered. Despite these many methodological differences, the plasma EC50 values for the [3H]flumazenil in vivo binding and [11C]flumazenil micro-PET studies were very similar, 134 and 96 ng/ml, respectively. Moreover, a hybrid of these two methods in which the micro-PET dosing and anesthetic conditions were mimicked but [3H]flumazenil rather than [11C]flumazenil was used as the radioligand and radioactivity was detected ex vivo rather than in vivo using PET, also gave a similar EC50 value (94 ng/ml).
The similarity of the plasma lorazepam EC50 values for conscious animals in the [3H]flumazenil in vivo binding assay and isoflurane-anesthetized rats in the micro-PET studies suggests that under the conditions used, isoflurane does not appreciably alter the binding of radiolabeled flumazenil to the benzodiazepine binding site of the GABAA receptor. This is in contrast to sevoflurane and propofol, which have both been reported to enhance [11C]flumazenil binding in man (Salmi et al., 2004), and isoflurane, which under the conditions used (Hansen et al., 1991) increased in vivo [3H]flumazenil binding, although the latter was considered to be an effect on reduced hepatic clearance (Hansen et al., 1991) rather than a direct effect on the GABAA receptor (Gyulai et al., 2001). Nevertheless, it is somewhat surprising that in the present study, isoflurane does not seem to significantly alter the binding of [3H]flumazenil given that GABAA receptors have a distinct recognition site for isoflurane (Mihic et al., 1997; Jenkins et al., 2001; Schofield and Harrison, 2005), and isoflurane can alter the binding of radioligands to the GABA and benzodiazepine binding sites of native GABAA receptors (Harris et al., 1993, 1994). However, at the very least, these data highlight the need to consider the possible effects of anesthetics when performing in vivo radioligand binding performed under the anesthetized, immobile conditions required for micro-PET studies.
A dose of 0.075 mg/kg i.v. produced 33 or 23% occupancy in the simulation and actual micro-PET studies (Figs. 6 and 8), corresponding to ED50 values of 0.15 and 0.25 mg/kg, respectively. These data are comparable with the ED50 for lorazepam of 0.34 mg/kg i.v. measured in baboon using [123I]iomazenil single-photon emission computed tomography (Sybirska et al., 1993). The similarity of the ED50 values of lorazepam across species is mirrored by the comparable potency of flumazenil at inhibiting the binding of [11C]flumazenil in baboons and man in which the ED50 values were 9 and 20 µg/kg i.v., respectively, the latter being calculated on the basis of a 1.5-mg dose producing 55% occupancy (Brouillet et al., 1991; Savic et al., 1991).
In baboons, a therapeutically relevant dose of lorazepam (0.03 mg/kg i.v.) produced negligible occupancy (<5%) of benzodiazepine binding sites as measured using [123I]iomazenil single-photon emission computed tomography (Sybirska et al., 1993). Likewise, in man, 1 mg of lorazepam produced occupancy, depending on brain region, of 6 to 9% (Lingford-Hughes et al., 2005). This level of occupancy was achieved with a mean exposure over a 90-min period of 9100 ng/min/ml, which is equivalent to an average plasma lorazepam concentration of 101 ng/ml. The fact that in man, a plasma concentration of 100 ng/ml produced occupancy of <10%, whereas in rat, it is equivalent to 50% occupancy emphasizes that although plasma-occupancy relationships can for some compounds be relatively comparable across species, including man, this is not a general rule, and plasma occupancy relationships need to be established in man using PET as part of the clinical development program.
In summary, we have shown that two very different methods of determining receptor occupancy, namely the inhibition of in vivo binding of radioligand by predosing of test compound followed by ex vivo detection of radioactivity or the displacement of already bound radioligand with in vivo detection using PET, gave very similar results. These two methods, therefore, validate each other and demonstrate the utility of micro-PET as an in vivo pharmacological tool. Moreover, not only do the more traditional occupancy in vivo binding methods validate micro-PET but vice versa micro-PET validates "old-fashioned" occupancy measurements. These latter methodologies are much less expensive, higher throughput, and do not require the infrastructure (e.g., proximity to a cyclotron, dedicated radiochemistry support, etc.) associated with micro-PET, yet the current data suggest that they are an equally valid approach to measuring occupancy in preclinical species and that such data can be compared with occupancy data generated using human PET studies irrespective of the marked methodological differences.
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ABBREVIATIONS: CNS, central nervous system; PET, positron emission tomography; lorazepam, 7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one; flumazenil, 8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester; DMSO, dimethyl sulfoxide; 3D, three-dimensional; ROI, region of interest; TAC, time-activity curve.
Address correspondence to: Dr. John R. Atack, Neuroscience, Johnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, La Jolla, CA 92121. E-mail: JAtack{at}prdus.jnj.com
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