Synthesis and Evaluation of Candidate PET Radioligands for Corticotropin-Releasing Factor Type-1 Receptors

Abstract

Introduction

A radioligand for measuring the density of corticotrophin-releasing factor subtype-1 receptors (CRF₁ receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF₁ receptor ligands based on a core 3-(phenylamino)pyrazin-2(1H)-one scaffold.

Methods

CRF₁ receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF receptors (displacement of [¹²⁵I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF₁ receptor density (B_max) was assessed in rhesus brain cortical and cerebellum membranes with the CRF receptor ligand, [³H]BMS-728300.

Results

The three ligands selected for development showed high binding affinity (IC₅₀ values, 0.3–8 nM) at CRF₁ receptors and moderate lipophilicity (LogD, 2.8–4.4). [³H]BMS-728300 and the two ¹⁸F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF₁ receptor B_max in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [¹⁸F]BMS-721313, [¹⁸F]BMS-732098 and [¹¹C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro.

Conclusions

Candidate CRF₁ receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF₁ receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF₁ receptors.

Introduction

Corticotropin-releasing factor (CRF), a 41-amino acid peptide, plays a preeminent role in the regulation of the physiological and behavioral responses to stress [1–4]. Moreover, chronically elevated levels of CRF have been proposed to underlie the development of psychiatric disorders such as anxiety and depression, especially in individuals with a genetic vulnerability to such disorders [5]. Knock-out studies have implicated the CRF₁ receptor as the primary mediator of the behavioral and hypothalamic-pituitary axis (HPA) responses to CRF. CRF₁ receptor deletion in mice results in decreased anxiety-like behavior and a reduced HPA axis response to stress [6]. Moreover, conditional knock-out of CRF₁ receptors only in anxiety-related limbic pathways results in less anxiety-like behavior but leaves the HPA system responsive to stress [7]. Consistent with these findings, CRF₁ receptor-selective antagonists produce anxiolytic-like behavioral efficacy in a broad group of behavioral models [8,9]. Antidepressant-like behavioral efficacy has also been reported with some antidepressant models [10,11].

The recognition of the central role of CRF and CRF₁ receptors in mediating stress responses has led to the development of many selective CRF₁ receptor antagonists. However, CRF₁ antagonists have failed to show efficacy in human trials for anxiety (pexacerfont; [12]) or depression (ONO-2333Ms, verucerfont, pexacerfont and CP-316,311 [9,13]). Interestingly, however, CRF₁ receptor antagonists may have utility for the treatment of the negative emotional symptoms of drug or alcohol withdrawal and stress-induced relapse [13]. Additionally, they may prove useful in the prevention or amelioration of the symptoms of PTSD [14].

A key issue in the clinical development of CRF₁ receptor antagonists has been the absence of a suitable radioligand for use with positron emission tomography (PET) to establish target engagement and facilitate dose selection. A PET CRF₁ receptor radioligand would also be valuable for neuropsychiatric investigations. Several groups have attempted to develop CRF₁ receptor radioligands for PET imaging, but without success. These radioligands include radiofluorinated analogues of the high-affinity non-peptide CRF₁ receptor antagonist antalarmin [15], radiofluorinated and radioiodinated derivatives of CP-154,526, another prototype non-peptide CRF₁ receptor antagonist [16], and [⁷⁶Br]MJL-1-109-2, a brominated derivative of CP-154,526 [17]. More recently, Jagoda et al. [18] evaluated another ⁷⁶Br-labeled radioligand, 4-[⁷⁶Br]BMK-152. This radioligand had improved affinity (K_D = 0.23–0.3 nM) and decreased lipophilicity (cLogP, 2.6) relative to earlier compounds [17,18] and exhibited higher specific binding signals in rat and monkey brain slice autoradiography. Three ¹¹C-labeled CRF₁ receptor ligands (SN003, DPC696 and R121920) have been evaluated in PET studies using baboons [19, 20]; all three showed a uniform distribution throughout the brain with no enrichment in areas of high CRF₁ receptor density. Using in vitro membrane binding and autoradiographic analyses, specific CRF₁ receptor binding with [¹²⁵I]Tyr⁰-sauvagine was undetectable in baboon occipital cortex and cerebellar cortex, suggesting that CRF₁ receptor density in baboon brain is insufficient for PET imaging [20].

The present study selected novel candidate CRF₁ receptor ligands from a group having a common core 3-(phenylamino)pyrazin-2(1H)-one scaffold for development as possible PET radioligands. Initial selection was based of their CRF₁ receptor potency, selectivity and physicochemical properties and then followed up with an evaluation of their specificity using in vitro autoradiography and biodistribution studies in rat. Three promising candidates were identified for evaluation with PET in rhesus monkey. However, none proved to be effective for imaging brain CRF₁ receptors in vivo.

Materials and Methods

Materials

Kryptofix 2.2.2 (K 2.2.2; 98%), ammonium formate (99.995%), potassium carbonate (99%), sodium hydroxide (97%), sodium bicarbonate (99.7%), acetonitrile (anhydrous, 99.8%), iodomethane (99%), bromoiodomethane, dibromomethane (99%), DMF (anhydrous, 99.8%), DMSO (anhydrous, 99.9%), methanol (99.93%), and 18-crown-6 (99.5%) were purchased from Aldrich (Milwaukee, WI). High purity acetonitrile (Burdick & Jackson; Muskegon, MI) was used for HPLC. TBAH (1 M solution in methanol) was diluted to 0.167 M with methanol (high purity solvent; Burdick & Jackson). Ethanol (Aaper Alcohol and Chemical Company; Shelbyville, KY) and 0.9% saline (USP; APP Inc.; Schaumburg, IL) were used to formulate the radioligands. DMP-696, DMP904 and the other CRF₁ receptor ligands and precursors were provided by Neuroscience Chemistry, Bristol-Myers Squibb. [³H]BMS-728300 (specific activity = 87.5Ci/mmol) was synthesized by the Bristol-Myers Squibb Radiochemistry Group. ¹⁸F radiochemistry was conducted at both Bristol-Myers Squibb (Wallingford, CT) and NIMH (Bethesda, MD) whereas ¹¹C radiochemistry was only conducted at NIMH.

Animal care

All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and the National Institutes of Health Animal Care and Use Committee [21].

Cyclotron Production of isotopes

Fluorine-18 was purchased from PETNET Pharmaceutical Services at Milstein Hospital (New York, NY) for the Site 1 (Bristol-Myers Squibb) studies; in vitro, ex vivo and rat biodistribution studies. Lower fluorine-18 specific activities were achieved in these studies relative to those attained at Site 2. This was due in part to the longer transportation times for the radioactive starting materials (PETNET production vs. on-site production). Carbon-11 and fluorine-18 for the PET studies (Site 2; Molecular Imaging Branch, National Institute of Mental Health) were produced with a proton beam from a PETtrace cyclotron (GE Medical Systems; Milwaukee, WI).

Radiochemistry

Site 1: Radiosynthesis of [¹⁸F]BMS-721313 and [¹⁸F]BMS-732098

All radiochemistry was performed in a lead-shielded hot-cell for personnel protection from radiation using an in-house designed and constructed remote-controlled radiosynthesis module. Radioactivity was measured with a calibrated CRC 15R dose calibrator (Capintec, Inc; Ramsey, NJ) and corrected for physical decay. Radio-HPLC was performed on a Rainin Dynamax System equipped with a Dynamax UV D-2 absorbance detector, and a INUS γ-ram flow-through radioactivity detector. HPLC columns were purchased from VWR (Radnor, PA).

No-carrier-added (NCA) aqueous [¹⁸F]fluoride ion (200 mCi), prepared from the ¹⁸O(p,n)¹⁸F reaction (PETNET Pharmaceutical Services), was applied to a previously activated QMA cartridge contained within the remotely controlled synthesis system. After passing nitrogen through the cartridge, the activity was eluted into a 5-mL silanized microvial with potassium carbonate solution (6 mg/mL in deionized water; 0.1 mL), then with an aliquot (1 mL) of a stock solution composed of potassium carbonate (30 mg; 0.22 mmol) and K 2.2.2 (165 mg; 0.44 mmol) in deionized water (1 mL) plus acetonitrile (14 mL), and finally with acetonitrile (1 mL). The eluate was evaporated by placing the vial in an oil-bath (90 °C) and applying a gentle stream of nitrogen under partial vacuum. Once the liquid volume had been reduced to < 0.3 mL, acetonitrile (0.5 mL) was added and the solution reduced by azeotropic distillation. This process was repeated three times to remove all traces of water. During the final distillation, the vial was removed from the oil-bath before the solution had reached dryness and was placed under full vacuum at room temperature for 6 min. Bromoiodomethane (0.5 mL) was added to the residue and the vial was placed in an oil-bath (90 °C) while also allowing a low flow of nitrogen to sweep the volatile [¹⁸F]bromofluoromethane gently into another conical vial containing cesium carbonate (6.4 mg; 19.6 μmol) in a solution of phenolic precursor (2.1 mg; BMS-720328 for [¹⁸F]BMS-721313, BMS-729393 for [¹⁸F]BMS-732098) in DMF (0.5 mL), immersed in an ethylene glycol-dry ice-bath (–30 to –10 °C). Radioactivity detectors were used to monitor radioactivity transfer, and when the distillation was completed the vial was sealed and heated in an oil-bath (100 °C) for 10 min. The vial was then cooled in an ethylene glycol-dry ice cold bath to ≤ 40 °C, as indicated by external infrared sensors. The reaction mixture was diluted with water (~ 2 mL) plus MeCN-H₂O (50: 50 v/v; 1 mL) and the desired product separated by HPLC on a Zorbax Rx-C18 column (5 μm; 9.4 × 250 mm) eluted with MeCN-0.1% TFA (55: 45 v/v) at 4.6 mL/min. Eluate was monitored for absorbance at 260 nm and for radioactivity. The fraction containing either [¹⁸F]BMS-721313 (R_t = 12.1 min) or [¹⁸F]BMS-732098 (R_t = 11.5 min) was collected and then diluted at least three-fold with water before being applied to a HLB glass cartridge (200 mg; Waters, Milford, MA). The cartridge was rinsed with water (5 mL) and then carefully rinsed with ethanol (1 mL). The radioligand was then eluted from the cartridge with dichloromethane (2 mL). The dichloromethane was reduced to < 0.1 mL with nitrogen under partial vacuum, and before dryness, ethanol (0.5 mL) was added and the solution again concentrated to < 0.1 mL, at which time, more ethanol was added to bring the final solution volume to 1.0 mL. This method gave 1.0-2.9 mCi of [¹⁸F]BMS-721313 and 1.1-3.25 mCi of [¹⁸F]BMS-732098.

After 20 min, the radioligand solution was analyzed with HPLC on a Zorbax Rx-C18 column (5 μm; 4.6 × 250 mm) eluted with MeCN-0.1% TFA (55: 45 v/v) at 1.0 mL/min. Eluate was monitored for absorbance at 260 nm and for radioactivity. No radioactive impurities were detected. The specific activity was found to be 0.750-0.464 Ci/μmol for [¹⁸F]BMS-721313 and 0.24-0.10 Ci/μmol for [¹⁸F]BMS-732098.

Site 2: Radiosynthesis of [¹⁸F]BMS-721313 , [¹⁸F]BMS-732098 and [¹¹ C]BMS-709460

All radiochemistry was performed in a lead-shielded hot-cell for personnel protection from radiation. Radioactivity was measured with a calibrated AtomlabTM 300 dose calibrator (Biodex Medical Systems; Shirley, NY) and corrected for physical decay. Radio-HPLC was performed on a Gold HPLC module (System Gold 126 solvent module equipped with a 166 UV absorbance detector; Beckman Coulter, Fullerton, CA) and with a Flow-count radioactivity detector (pin-diode or sodium iodide; Bioscan; Washington, DC). HPLC columns were purchased from Phenomenex (Torrance, CA).

NCA [¹⁸F]fluoride ion was produced through the ¹⁸O(p,n)¹⁸F nuclear reaction by irradiating [¹⁸O]water (1.8 mL, 95% isotopic enrichment) with protons (17 MeV; 20 αA) for 120 min. Potassium carbonate (0.5 mg) and K 2.2.2 (5.0 mg) in H₂O-MeCN (1: 9 v/v; 100 αL) were added to the radioactive solution and delivered to a TRACERlab Fx_FN module (GE Medical Systems) equipped with a secondary Peltier-cooling and heating device [22]. This solution was dried by two cycles of addition and evaporation of acetonitrile (2 mL). Dibromomethane (100 μL) in acetonitrile (1 mL) was then added to the radioactive solution and heated at 110 °C for 15 min. The reaction mixture was then cooled to 35 °C. A stream of nitrogen was used to transfer the generated [¹⁸F]bromofluoromethane through a series of four silica gel cartridges (Sep-Pak Plus; Waters) and into a pre-cooled (–5 °C) vessel which had been charged with phenolic precursor (BMS-720328; 0.3 mg), potassium carbonate (2 mg; 15 μmol) and 18-crown-6 (5 mg; 19 μmol) in DMF (0.8 mL). When trapping of radioactivity had maximized, the sealed reaction mixture was heated at 110 °C for 20 min. The reaction mixture was then concentrated to about 0.5 mL and diluted with mobile phase A (H₂O-MeCN-TFA, 95: 5: 0.1 by vol.; 0.7 mL). This solution was injected onto a Prodigy C18 column (10 μm, 100 Å, 10 × 250 mm) eluted at 9 mL/min with mobile phase A increased linearly over 15 min to A-MeCN (45: 55 v/v) with eluate monitored for absorbance at 260 nm and for radioactivity. The [¹⁸F]BMS-721313 (R_t = 39 min) fraction was collected.

[¹⁸F]BMS-732098 was prepared as described for [¹⁸F]BMS-721313, but using phenolic precursor (BMS-729393; 0.3 mg), potassium carbonate (2 mg) and 18-crown-6 (5 mg) in DMF (0.7 mL) in the labeling reaction. [¹⁸F]BMS-732098 (R_t = 41 min) was separated under the same HPLC conditions as described for [¹⁸F]BMS-721313.

NCA [¹¹C]carbon dioxide (~ 1 Ci) was produced according to the ¹⁴N(p,α)¹¹C reaction [23] by irradiating nitrogen (initial pressure 160 psi; 75-mL volume), containing 1% O_2, with a proton beam (16.5 MeV; 45 αA) for 20 min. The [¹¹C]carbon dioxide was converted into [¹¹C]iodomethane via [¹¹C]methane [24]. Thus, [¹¹C]carbon dioxide was delivered through stainless steel tubing (1/16 in i.d.) over 2 min to a PETtrace MeI module (GE Medical Systems), trapped on molecular sieve (13X) and reduced to [¹¹C]methane over nickel at 360 °C. The [¹¹C]methane was recirculated over iodine at 720 °C to generate [¹¹C]iodomethane, which became trapped on Porapak Q held in the recirculation path. Phenolic precursor (BMS 736646; 1.0 mg) was dissolved in anhydrous DMSO (0.3 mL) in a 1.1-mL V-vial. Aqueous sodium hydroxide solution (5 M; 5 μL) was added and the vial sealed and placed in a Synthia apparatus [25]. After 3 minutes, [¹¹C]iodomethane was released from the PETrace module in a stream (17 mL/min) of helium and trapped in the V-vial. When the trapped radioactivity had maximized (after 4–6 min), the reaction vessel was sealed and heated at 90 °C for 5 min. The reaction mixture was then diluted with water (0.5 mL) and injected onto a Prodigy C18 column (10 μm, 100 Å, 10 × 250 mm i.d.) eluted at 6 mL/min with MeCN-0.1 M HCOONH₄ (70: 30 v/v) with eluate monitored for absorbance at 254 nm and radioactivity. The fraction containing [¹¹C]BMS-709460 (R_t = 7 min) was collected.

The collected radioligand HPLC fraction was directed into a modified semiautomated rotary evaporator and taken to dryness under reduced pressure. The residue was dissolved in sterile saline for injection, USP (3–10 mL) containing ethanol (5% v/v) and filtered through a sterile Millex-GV filter (0.22 μm, 33 mm diameter; Millipore, MA) into a sterile dose vial.

Formulated [¹⁸F]BMS-721313 (100 μL) was analyzed for radiochemical purity on a Luna C18 column (10 μm, 100 Å, 4.6 × 250 mm i.d.) eluted with A-MeCN (45: 55 v/v) at 2 mL/min with eluate monitored for absorbance at 260 nm and for radioactivity (R_t = 7.9 min). Formulated [¹⁸F]BMS-732098 was analyzed similarly but with a mobile phase of A-MeCN (37: 63 v/v) with eluate monitored for absorbance at 254 nm and radioactivity (R_t = 3.6 min). Formulated [¹¹C]BMS-709460 (R_t = 3.5 min) was analyzed on a Prodigy C18 column (10 μm, 100 Å, 4.6 × 250 mm i.d.) eluted with MeCN-0.1 M HCOONH₄ (70: 30 v/v) at 3 mL/min with eluate monitored for absorbance at 260 nm and radioactivity. All radioligands were identified by their co-elution with the respective non-radioactive reference compound.

In vitro CRF₁ receptor binding

Rat and rhesus monkey brain tissues were obtained from Analytical Biological Services, Inc. (Wilmington, DE). Frontal cortex (rat or rhesus monkey) was homogenized in assay buffer containing 50 mM Hepes (pH 7.0 at 23°C), 10 mM MgCl₂, 2 mM EGTA, 1 μl/ml aprotinin, 1 μl/ml leupeptin, 1 μl/ml pepstatin A, 0.005% Triton X-100, 10U/ml bacitracin and 0.1% ovalbumin. The suspension was centrifuged at 32000 × g for 30 min. The resulting supernatant was discarded and the pellet resuspended by homogenization in assay buffer and centrifuged again. The supernatant was discarded and the pellet resuspended by homogenization in assay buffer and aliquots frozen at −70°C. On the day of the experiment aliquots were thawed quickly and added (25 μl/well) to a mixture containing [¹²⁵I]ovine-CRF (PerkinElmer Life Sciences; Boston, MA) plus the compound to be evaluated in a total volume of 100 μl of assay buffer. Competition binding experiments were performed using a single concentration of [¹²⁵I]ovine-CRF (150 pM; ovine-CRF is >100 fold selective for CRF₁ vs. CRF₂) in the presence of 10 increasing concentrations (in duplicate) of test compound (3 pM – 100 nM). The assay mixture was incubated for 2 h at 21°C. Bound and free radioligand were then separated by rapid filtration using glass fiber filters (Whatman GF/B, pretreated with 0.3% PEI) on a Brandel Cell Harvester. Filters were then washed multiple times with ice-cold wash buffer (PBS without Ca²⁺ and Mg²⁺, 0.01% Triton X-100; pH 7.0 at 23°C). Non-specific binding was defined using 1 μM DMP696 (Li et al., 2005). Filters were then counted in a Wallac Wizard gamma counter. IC₅₀ values were determined using non-linear regression four-parameter logistic equation, y = A + (B-A)/(1+(C/x)^D) where A=0% inhibition, B=100% inhibition, C=log IC₅₀ and D=slope factor (ActivityBase; IDBS, Surrey, UK).

Saturation binding of [³H]BMS-728300 to rat and rhesus monkey frontal cortex membranes was determined using the methodology described above (competition studies) but with 6 increasing concentrations of [³H]BMS-728300 (no test compound present), and with non-specific binding defined at each point using 5 μM DMP696. K_D and B_max values were estimated using the nonlinear regression “one site binding” (hyperbola) from Graphpad Prism (version 5).

Lipophilicity: shake-flask LogD method

LogD at pH 7.4 was measured by a shake-flask method described earlier [27].

In vitro binding autoradiography

Brains from adult male Sprague–Dawley rats (n=2; 200–300 g; Charles River Laboratories; Wilmington, MA) and rhesus monkeys (n=2; Analytical Biological Services, Inc.; Wilmington, DE) were used. Separate sections from the same animals were employed to characterize multiple radioligands to avoid inter-animal differences. Thus, sections used for each radiolabeled ligand were not always from the immediately adjacent level. Frozen brain tissues were sectioned (20 μm) on a cryostat and mounted on slides. Sets of the slides were preincubated in assay solution containing 50 mM HEPES, 10 mM MgCl₂ and 200 mM EGTA (pH 7.4) for 10 min, followed by incubation in the assay solution containing 1.3 nM [¹⁸F]BMS-721313, [¹⁸F]BMS-732098 or [³H]BMS-728300 for 40 min at 22–24 °C. Nonspecific binding was defined by incubation of adjacent sections with the corresponding radioligand in the presence of DMP696 (1 μM), a selective CRF₁ receptor antagonist. After incubation, the slides were washed in ice-cold phosphate-buffered saline (PBS; pH 7) andsubsequently dried under a stream of cold air. The slides were then placed in cassettes against either multi-purpose or ³H-sensitive storage phosphor-imaging screens (Packard Instrument Co.; now PerkinElmer, Waltham, MA); overnight for ¹⁸F-labeled ligands and 7 to 10 days for ³H-labeled ligands. The screens were then scanned with a Cyclone storage phosphor-imaging system (Packard Instruments Co). Captured images were analyzed with OptiQuant Acquisition and Analysis software (Packard Instruments Co). Radioligand binding in a given brain region was measured as digital light units per millimeter squared (DLU/mm²), and the specific binding density was calculated by subtraction of the non-specific binding from the total binding values.

Pre-block of [¹⁸F]BMS-732098 binding in vitro by DMP696 administrated in vivo

Rats were dosed orally with either DMP696 (10 mg/kg) or the vehicle (0.25% methocel). After 60 min, the rats were sacrificed, and the brains were collected, frozen and sectioned as described above. The slides of brain sections were incubated with [¹⁸F]BMS-732098 (2 nM) for 20 min at 22–24 °C, after which they were briefly rinsed in cold PBS andsubsequently dried under a stream of cold air. The nonspecific binding was determined by incubation of adjacent sections with [¹⁸F]BMS-732098 in the presence of DMP696 (1 μM). The slides were then processed for storage phosphor-imaging as described above. The percent inhibition of specific [¹⁸F]BMS-732098 binding by orally-dosed DMP696 was calculated as (specific binding in vehicle-treated–specific binding in DMP696-treated)/(specific binding in vehicle-treated) × 100%, and defined as DMP696 occupancy at CRF₁ receptors.

Biodistribution of [¹⁸F]BMS-721313 and [¹⁸F]BMS-732098 in rats

Rats (male Sprague–Dawley, 220–260 g) were obtained either from Hilltop Lab Animals (Scottsdale, PA) with a surgically placed polyurethane jugular vein cannula or from Harlan Sprague Dawley (Indianapolis, IN) and then cannulated in-house with a silicone jugular vein cannula. The cannula was flushed with saline just before use. Awake rats were injected intravenously via the jugular cannula with [¹⁸F]BMS-721313 or [¹⁸F]BMS-732098 (0.2–0.4 mCi) using Hamilton glass syringes. The rats were sacrificed 5, 20, 40 or 90 min after injection of [¹⁸F]BMS-732098 and 15 or 60 min after injection of [¹⁸F]BMS-721313. Blood, brain, heart, kidney, liver, lung, muscle, spleen and tibia were collected, weighed and counted to determine the radioactivity. Data were corrected for decay and reported as mean percentage of the injected dose per gram of tissue (% ID/g tissue).

PET experiments in monkeys

Six male rhesus monkeys (Mucacca mulatto) were used. For each scanning session, the subject monkey was immobilized with ketamine and maintained under anesthesia with 1.5–2.5% isoflurane in oxygen. An intravenous perfusion line, filled with saline (0.9% w/v), was used for bolus injection of radioligand (> 99.9% radiochemical purity). PET serial dynamic images of brain were obtained for up to 150 min on an Advance (GE Medical Systems, WI) or High Resolution Research Tomograph (HRRT; Siemens/CPS, Knoxville, TN, USA). Comparisons between baseline and binding blockade scans were always performed using the same scanner.

Decay-corrected time-activity curves were obtained for several irregular volumes of interest (VOIs) in brain (cerebellum, frontal cortex, temporal cortex, parietal cortex, caudate, putamen, thalamus, hippocampus, pituitary). Radioactivity levels in VOIs were normalized for injected dose and monkey weight by expression as a percentage of the standardized uptake value (%SUV) where %SUV = [(% injected dose per mL of tissue) × body weight in g]. Results from PET experiments in which radioligand was given as a bolus injection plus constant infusion were fitted to a two tissue compartmental model with a metabolite corrected arterial input function to derive regional values of total distribution volume (V_T).

A baseline scan was performed with [¹⁸F]BMS-721313 (6.76 mCi; carrier 2.77 nmol) in one monkey (15 kg). In a displacement experiment, another monkey (11 kg) was dosed intravenously with the CRF₁ receptor antagonist DMP904 (1.11 mg/kg) at 15 min after [¹⁸F]BMS-721313 injection (4.77 mCi; carrier 19.8 nmol).

A baseline scan was performed with [¹⁸F]BMS-732098 (5.42 mCi; carrier 12.7 nmol) in one monkey (13 kg). Another monkey (12 kg) was dosed intravenously with DMP904 (3 mg/kg) at 20 min after [¹⁸F]BMS-732098 injection (5.76 mCi; carrier 6.9 nmol).

The following scans were performed with [¹¹C]BMS-709460. One monkey (13 kg) was scanned at baseline after bolus intravenous injection of [¹¹C]BMS-709460 (3.66 mCi; carrier, 2.35 nmol). In a pre-block experiment, the same monkey was scanned with [¹¹C]BMS-709460 (3.91 mCi; carrier 3.19 nmol) at 2 h after intravenous injection with non-radioactive BMS-709460 (1 mg/kg).

Another monkey (14 kg) was studied at baseline with [¹¹C]BMS-709460 administered as an intravenous bolus injection (3.29 mCi; 6.45 mL/min over 2 min) followed by a constant infusion (1.82 mCi; 0.047 mL/min for 150 min) (B/I = 4.5 h); the total mass of injected carrier was 3.39 nmol. Arterial blood samples were withdrawn during the course of this experiment to determine the time-course of the concentration of non-metabolized radioligand in plasma, (i.e. the metabolite-corrected arterial input function) by radio-HPLC, as described below. The same monkey was studied again at about 3 h from the first radioligand injection after giving [¹¹C]BMS-709460 (specific radioactivity at start of injection, 989 mCi/μmol) and a dose of BMS-709460 by bolus injection (3.55 mL/min for 2 min) and constant infusion (0.1075 mL/min) over 120 min (B/I = 2.4 h). The bolus injection contained [¹¹C]BMS-709460 (3.54 mCi) plus BMS-709460 (15.0 mg), and the infusion contained [¹¹C]BMS-709460 (2.89 mCi) and BMS-709460 (27.3 mg). The metabolite-corrected arterial input function was determined as in the baseline experiment.

Stability of [¹¹C]BMS-709460 in monkey plasma in vitro

[¹¹C]BMS-709460 was incubated for 30 min in whole monkey blood (0.5 mL) at room temperature. A sample (0.20 mL) was removed and added to water (0.3 mL) to lyse the cells. Another aliquot (0.45 mL) was then removed and added to acetonitrile (0.7 mL), and centrifuged. The supernatant liquid was analyzed by radio-HPLC on a Nova-Pak C18 column (4 μm; 10 × 800 mm; Waters) housed in a radial compression module and eluted with MeOH: H₂O: Et₃N (80: 20: 0.1 by vol.) at 2.5 mL/min.

Emergence of radiometabolites of [¹¹C]BMS-709460 in monkey plasma in vivo

During PET scans, in which [¹¹C]BMS-709460 was given as a bolus injection followed by a constant infusion, blood samples were drawn periodically on 17 (baseline experiment) and 16 occasions (receptor block experiment) from the monkey femoral artery and collected in heparin-treated Vacutainer tubes. The samples were centrifuged and the plasma separated. A sample of plasma (0.45 mL) was mixed with acetonitrile (0.7 mL) and centrifuged. The supernatant liquid was analyzed with radio-HPLC on a Novapak C18 column (4 μm; 8 × 100 mm) eluted at 2.5 mL/min with MeOH/H₂O/Et₃N (80: 20: 0.1 by vol.). The time-courses for percentages of radioactivity in plasma represented by parent radioligand and its radiometabolites were calculated [28].

Results

In vitro brain section binding autoradiography with [¹⁸F]BMS-721313, [¹⁸F]BMS-732098 and [³H]BMS-728300

Three compounds, BMS-721313, BMS-732098 and BMS-728300, that each exhibited a low CRF₁ IC₅₀ value (IC₅₀ for the displacement of [¹²⁵I]ovine-CRF from rat cortical membranes ≤ 10 nM) and a relatively moderate lipophilicity (LogD, 2.78–3.13; Table 1) were ¹⁸F- or ³H-labeled for autoradiographic evaluation of specific binding to CRF₁ receptors in brain sections from rat and rhesus monkey. Figures 1 (rat) and 2 (rhesus monkey) show representative autoradiograms of in vitro binding of [¹⁸F]BMS-721313 and [¹⁸F]BMS-732098. Figure 3 shows the autoradiograms for [³H]BMS-728300 in both the rat and rhesus brain sections. The total/non-specific binding ratios of the three ligands in rat and monkey brain regions are plotted in Figure 4. The regional specific binding pattern among all three ligands was similar, with higher density in the cerebellum and frontal and limbic cortices than in thalamus and brainstem. However, [¹⁸F]BMS-732098 (LogD, 2.87) and [³H]BMS-728300 (LogD, 2.78) exhibited higher ratios of total/non-specific binding than [¹⁸F]BMS-721313 (LogD, 3.13).

Table 1.

Measured lipophilicities and binding IC₅₀ values (displacement of [¹²⁵I]ovine-CRF) of ligands. R in the structure indicates the location of the radiolabelled group or its precursor, or the location of the group in the corresponding "cold" ligand.

Ligand	Structure	“Cold” ligand	LogD (n)	IC50, nM (n)	Radiolabel	Precursor
BMS-606581		R=CH3	3.36 (2)	4.9±0.7 (2)	R=CT3	R=H BMS-720328
BMS-721313		R=CH2F	3.13 (1)	5.2±0.6 (3)	R=CH218F	R=H BMS-720328
BMS-665330		R=CH3	4.35 (1)	0.3±0.04 (5)	R=CT3	R=H BMS-716500
BMS-728300		R=CH3	2.78 (1)	7.9±1.4 (3)	R=CT3	R=H BMS-729393
BMS-732098		R=CH2F	2.87 (1)	4.3±0.7 (6)	R=CH218F	R=H BMS-729393
BMS-626493		R=CH3	3.35 (1)	1.2±0.2 (7)	R=CT3	R=H BMS-736585
BMS-709460		R=CH3	3.7 (2)	0.8±0.1 (5)	R=11CH3	R=H BMS-736646

Fig. 1.

Representative autoradiograms of [¹⁸F]BMS-721313 and [¹⁸F]BMS-732098 binding in brain sections of rat (top: sagittal; bottom: coronal). Sagittal sections on the left panel are ~2 mm lateral to the midline; sagittal sections on the right panel are ~3 mm lateral to the midline. Pseudo-color in red indicates moderate/high levels of binding. Total: total binding; NS: non-specific binding. Bs: brainstem; Cb: cerebellum; FC: frontal cortex; PC: parietal cortex; OC: occipital cortex; St: striatum; Th: thalamus;

Fig. 2.

Representative autoradiograms of [¹⁸F]BMS-721313 and [¹⁸F]BMS-732098 binding in the coronal brain sections of rhesus monkey (Note: the [¹⁸F]BMS-721313 sections were ~1 mm rostral to the [¹⁸F]BMS-732098 sections). Pseudo-color in yellow/red indicates moderate/high levels of binding. Total: total binding; NS: non-specific binding. Bs: brainstem; Cb: cerebellum; FC: frontal cortex; St: striatum; TC: temporal cortex.

Fig 3.

Representative autoradiograms of [³H]BMS-728300 binding in the brain of rat (top; sagittal sections ~2 mm lateral to the midline) and rhesus monkey (bottom, coronal sections ~1 mm caudal to the [¹⁸F]BMS-732098 sections shown in Figure 2). Pseudo color in blue/yellow indicates moderate/high levels of binding. Total: total binding; NS: non-specific binding. Am: amygdala; Bs: brainstem; Cb: cerebellum; FC: frontal cortex; St: striatum; TC: temporal cortex.

Fig. 4.

Total/non-specific (NS) binding ratios of [¹⁸F]BMS-721313, [¹⁸F]BMS-732098 and [¹⁸F]BMS-728300 in five brain regions in rat (n=2) and rhesus monkey (n=2).

Pre-block of [¹⁸F]BMS-732098 binding in vitro by DMP696 administrated in vivo

Previously, we demonstrated that DMP696, a selective CRF₁ receptor antagonist, when administered in vivo blocks in vitro binding of [¹²⁵I-Tyr°]sauvagine, a peptide radioligand for CRF₁ receptors [29]. Here we evaluated whether the in vivo blockade of CRF₁ receptors by DMP696 could also be measured by in vitro binding of [¹⁸F]BMS-732098. Specifically, rats were dosed orally with DMP696 (10 mg/kg) or vehicle, and the brains were collected 60 min after dosing and processed for in vitro binding of [¹⁸F]BMS-732098. The result showed that 10 mg/kg DMP696 blocked 90 ± 3% (n = 3) of the specific binding of [¹⁸F]BMS-732098 in the cortex, a finding consistent with previous observations [29].

Binding density of CRF₁ receptors in rat and rhesus monkey brain

The tritiated ligand [³H]BMS-728300 was used to measure the B_max of CRF₁ receptors in the rat and rhesus brain using an equilibrium binding assay. BMS-728300 has a CRF₁ receptor IC₅₀ of 7.9 ± 1.4 nM (n = 3; Table 1) and is > 10,000-fold selective for binding to CRF₁ vs. CRF₂ receptors (data not shown). The specificity of this ligand for CRF₁ receptors was further substantiated by brain section autoradiographic imaging of [³H]BMS-728300 in rat and cynomolgus monkey (Figure 3) where the highest levels of [³H]BMS-728300 binding were observed in CRF₁ receptor-rich regions, and were absent from CRF₂ receptor-rich regions such as the choroid plexus [29, 31, 32]. In membranes isolated from the rat and rhesus brain, [³H]BMS-728300 bound to a single population of saturable high affinity sites in the brain regions examined. Figure 5 shows the binding of [³H]BMS-728300 in frontal cortex membranes from rat and rhesus monkey; in the rat frontal cortex B_max and K_D were 117 fmol/mg protein and 8 nM, respectively (n=1; triplicate determinations). Table 2 summarizes the B_max and K_D values measured in selected regions of the rhesus monkey brain.

Fig. 5.

Saturation isotherm of [³H]BMS-728300 binding in frontal cortex membranes. A: rhesus monkey (representative experiment; triplicate determinations) and B: rat (representative experiment; triplicate determinations).

Table 2.

Binding density (B_max) and dissociation constant (K_D) of [³H]BMS-728300 in rhesus monkey brain (n = 3).

Brain region	Bmax (fmol/mg protein)	KD(nM)
Parietal cortex	52 ± 5.5	13.5 ± 2.1
Cerebellum	121 ± 15	15.1 ± 3.1
Frontal cortex	43 ± 0.7	11.4 ± 2.6
Temporal cortex	136 ±2 8	10.8 ± 1.0

Biodistribution of [¹⁸F]BMS-732098,F] and [¹⁸F]BMS-721313 rat

The distributions of radioactivity in selected organs of the rat after injection of either [¹⁸F]BMS-721313 or [¹⁸F]BMS-732098 are summarized in Table 3. The uptake of [¹⁸F]BMS-721313 was measured at 5, 20, 40 and 90 min whereas that of [¹⁸F]BMS-732098 was at 15 and 60 min after injection. Both radioligands produced moderate radioactivity uptake in the brain that subsequently declined at a rate similar to the rate of radioactivity loss from the blood. Radioactivity uptake in the periphery was initially highest in the liver, kidney and lung. Radioactivity in tibia increased with time, indicating some degree of radioligand defluorination.

Table 3.

Biodistribution of [¹⁸F]BMS-732098 (A) and [¹⁸F]BMS-721313 (B) in rat.

A.
Organ	% ID/g
	5 min	20 min	40 min	90 min
Blood	0.49±0.10	0.41±0.08	0.23±0.04	0.10±0.02
Brain	0.51±0.7	0.26±0.06	0.21±0.07	0.10±0.01
Heart	1.05±0.28	0.45±0.04	0.25±0.04	0.06±0.02
Kidney	1.25±0.29	1.14±0.13	0.57±0.07	0.29±0.01
Liver	1.64±0.50	1.27±0.14	0.54±0.06	0.20±0.04
Lung	1.02±0.13	0.61±0.01	0.30±0.05	0.13±0.04
Muscle	0.47±0.05	0.28±0.04	0.18±0.03	0.07±0.01
Spleen	0.52±0.17	0.30±0.01	0.28±0.15	0.08±0.02
Tibia	0.41±0.05	1.73±0.18	2.68±1.17	3.92±1.3
n =2 rats; mean±SD
B.
Organ	% ID/g
	15 min	60 min
Blood	0.53±0.17	0.21±0.08
Brain	0.27±0.07	0.07±0.02
Kidney	1.06±0.30	0.50±0.21
Liver	1.85±0.55	0.57±0.24
Lung	0.95±0.21	0.31±0.09
Muscle	0.34±0.10	0.23±0.22
Tibia	0.72±0.28	2.37±1.68
n =4 rats; mean±SD

Radiochemistry for monkey PET experiments

Three ligands, namely BMS-732098, BMS-721313 and BMS-709460, were selected for evaluation of their efficacies as brain CRF₁ receptor radioligands in vivo. In addition to binding IC₅₀, lipophilicity and suitability for ¹⁸F- or ¹¹C-labeling, selection of BMS-732098 and BMS-721313 was based on their CRF₁ receptor specificity using in vitro autoradiography and biodistribution studies in rat. BMS-732098 and BMS-721313 were labeled by treating the corresponding desfluoromethyl precursors with [¹⁸F]bromofluoromethane, itself generated from cyclotron-produced [¹⁸F]fluoride ion [22]. Ligand BMS-709460 was labeled by treating the phenolic analog with [¹¹C]iodomethane. Each radioligand was readily separated in high radiochemical purity (> 99%) by reverse phase HPLC and readily formulated for intravenous injection. Each procedure gave formulated radioligand in adequate yields and specific radioactivities for PET experiments in monkey (Table 4). From the end of radionuclide production, the radiosyntheses of [¹⁸F]BMS-732098, [¹⁸F]BMS-721313, and [¹¹C]BMS-709460 required 145, 145 and 35 min, respectively.

Table 4.

Radiochemical yields and specific activities for radioligands prepared for PET imaging.

Radioligand	n	Radiochemical yield		Specific activityb
		(mCi)	(%)	(Ci/μmol)
[18F]BMS-721313	2	13.7	3.8	2.64
[18F]BMS-732098	2	13.4	3.6	1.06
[11C]BMS-709460	7	20.2 ± 13.4a	n.d.	2.05 ± 0.79

^aFrom a 45 μA × 20 min cyclotron irradiation for carbon-11 production. n.d. not determined.

^bAt end of synthesis.

Monkey PET experiments

In a PET experiment in monkey in which [¹⁸F]BMS-732098 was injected as a bolus, radioactivity rapidly entered the brain to a high level (Figure 6A). Moderately high radioactivity uptakes were observed in all examined brain regions soon after injection. Thereafter, radioactivity level declined quite rapidly to a similarly low level in all regions. In a repeat experiment in another monkey in which the selective CRF₁ receptor antagonist DMP904 [8] was administered intravenously at 15 min the pattern of high initial radioactivity uptake followed by fast washout from all regions was very similar to that in the first experiment (Figure 6B). Qualitatively similar results were seen with [¹⁸F]BMS-721313 for baseline and blocking experiments (Figure 7)

Fig. 6.

Time-activity curves for brain regions in monkey after injection of [¹⁸F]BMS-732098 under baseline conditions (A) and in an experiment in another monkey in which the CRF₁ receptor antagonist DMP904 (3 mg/kg, i.v.) was administered 20 min after the radioligand (B).

Fig. 7.

Time-activity curves for brain regions in monkey after injection of [¹⁸F]BMS-721313 under baseline conditions (A) and in an experiment in another monkey in which the CRF₁ receptor antagonist DMP904 (1.11 mg/kg, i.v.) was administered 15 min after the radioligand (B).

[¹¹C]BMS-709460 was initially studied with PET in a single monkey first at baseline after intravenous injection of radioligand alone and then 3 h later after predosing the monkey with a high dose of BMS-709460 at about 5 min before radioligand injection to block CRF₁ receptors. Uptake of radioactivity into brain in the baseline experiment was fast and maximally very high (Figure 8A). This uptake declined rapidly from all regions down to a similarly low value. Ratios of radioactivity in putatively CRF₁ receptor-rich regions (e.g. cerebellum) to that in receptor-poor regions (e.g. caudate) were small (generally < 1.3) following maximal brain radioactivity uptake. The same pattern of brain regional radioactivity kinetics was observed in the second experiment (Figure 8B).

Fig. 8.

Time-activity curves for brain regions in monkey after injection of [¹¹C]BMS-709460 under baseline conditions (A) and in an experiment in the same monkey in which the CRF₁ receptor antagonist BMS-709460 was administered before radioligand (B).

In the two experiments in a single monkey in which [¹¹C]BMS-709460 was given as a bolus injection followed by a constant infusion, the arterial input function for unchanged [¹¹C]BMS-709460 was measured, so allowing the regional distribution volume (V_t) to be assessed under baseline and CRF₁ receptor blocking conditions. The acquired data fitted well to a two-tissue compartmental model. No appreciable differences were seen in regional distribution volumes between baseline and CRF₁ receptor blocking conditions (Table 5). In these experiments, plasma radiometabolite analysis revealed several findings. [¹¹C]BMS-709460 was stable in plasma in vitro (> 99.5% intact after 30 min at r.t.). The recovery of radioactivity from plasma into acetonitrile for HPLC analysis was generally high (> 91%) throughout the time-course of blood sampling. Two significant radiometabolites (¹¹C]A, R_t = 2.3 ± 0.3 min; n = 28; [¹¹C]B, R_t = 3.7 ± 0.4 min; n = 26) were detected each eluting earlier than [¹¹C]BMS-709460 (R_t = 5.5 ± 0.4 min; n = 28). Radiometabolites constituted a greater proportion of total radioactivity in plasma in the baseline experiment than in the receptor block experiment (Figure 9).

Table 5.

Distribution volumes for [¹¹C]BMS-709460 in brain regions of monkey under baseline and BMS-709460 pre-block conditions.

Region	VT
	Baseline	Pre-Block
Cerebellum	8.7	8.2
Putamen	7.2	9.2
Thalamus	10.0	9.1
Hippocampus	8.5	9.6
Caudate	8.0	8.7

Fig. 9.

Distribution of radioactivity between [¹¹C]BMS-709460 and its radiometabolites during baseline (A) and receptor pre-block (B) PET experiments in the same monkey with the radioligand administered intravenously by bolus injection followed by constant infusion.

Discussion

Several CRF₁ receptor antagonists were selected for evaluation as possible PET ligands based primarily on binding IC₅₀, moderate lipophilicity, results of in vitro autoradiography and suitability for labeling with carbon-11 or fluorine-18. With regard to selecting ligands for development as PET radioligands, high affinity for the imaging target is essential for achieving a target specific signal whereas moderate lipophilicity is important for promoting adequate brain entry without encountering adverse effects of high lipophilicity, such as low brain entry, low free fraction, high non-specific binding, and lipophilic radiometabolites. We therefore selected ligands that presented high affinity together with moderate lipophilicity. Thus, among all the possible candidates (Table 1), BMS-709460 was selected for labeling with ¹¹C, because of its combination of moderate lipophilicity and second highest affinity; although BMS-665330 had marginally higher affinity, its lipophilicity was also much higher. Both ¹⁸F-labeled ligands, besides having quite high affinity and moderate lipophilicity were successful for use in autoradiography in vitro, and therefore both were advanced to evaluation with PET in monkey.

The ligand ([¹²⁵I]CRF) concentration (150 pM; determined experimentally, data not shown) used in the rat frontal cortex binding assays was below the K_D of the ligand for the tissue (450 pM). Thus, if the CRF₁ receptor antagonists were competitive inhibitors the IC₅₀ would be very close to the K_i value. However, the CRF₁ receptor inhibitors appear to be strong negative modulators; i.e., allosteric inhibitors [26, 30], that can “masquerade” as competitive antagonists (for a review see [33]) in both functional and binding assays. Caution must therefore be applied in extrapolating affinity from the IC₅₀ values. Additionally, very slow CRF₁ receptor dissociation rates have been reported for some CRF₁ ligands [34,35] indicating that binding equilibrium may not have been reached in our competition binding studies. Moreover, kinetic studies conducted at 37°C revealed that affinity was increased for some CRF₁ compounds but was decreased for others [35]. Nevertheless, the IC₅₀ values determined by competition binding for CRF₁ compounds representing multiple chemotypes have consistently shown a good concordance with the in vivo IC₅₀ (i.e. free concentration of compound required for 50% CRF₁ receptor occupancy; see [26, 36] for examples).

A structurally-related CRF₁ receptor ligand was labeled with ³H to allow the determination of B_max and K_D in membranes prepared from isolated brain regions of the rhesus and rat, and to facilitate further autoradiographical studies without the need to resynthesize ¹⁸F-labeled compounds. The observation that the IC₅₀ for the displacement of [¹²⁵I]CRF from the orthosteric site by BMS-728300 (8 nM; Table 1) was comparable to the K_D for [³H]BMS-728300 binding (11–15 nM; Table 2; allosteric site) presumably reflects the strong interaction between the orthosteric and allosteric sites.

The CRF₁ radioligands [¹⁸F]BMS-732098, [¹⁸F]BMS-721313 and [³H]BMS-728300 were prepared for autoradiographic evaluation of their binding in rat and rhesus brain sections with the view that a lack of specific binding would disqualify the compounds from further evaluation. The three radioligands were found to exhibit similar, and regionally specific, binding patterns, i.e. a higher density of binding in frontal and limbic cortex, and cerebellum than in thalamus and brainstem. This binding pattern corresponds well with previously described CRF₁ receptor distribution in the rat [29,31] and rhesus brain [32], and substantiates the CRF₁ receptor selectivity of these compounds. [¹⁸F]BMS-732098 (LogD, 2.87) and [³H]BMS-728300 (LogD, 2.78) appeared to exhibit a higher degree of specific binding than [¹⁸F]BMS-721313 (LogD, 3.13) in rat and rhesus brain sections. Given their similar CRF₁ receptor binding affinities (IC₅₀, 4–6 nM), the improved specific binding obtained with [¹⁸F]BMS-732098 and [³H]BMS-728300 can likely be attributed to their lower lipophilicities. Moreover, the ratios of CRF₁ receptor-specific to non-specific binding exhibited by [¹⁸F]BMS-732098 (~ 2.1) and [³H]BMS-728300 (~ 2.3) were improved over the earlier CRF₁ ligands [³H]SN003 with a ratio of about 1 [37], and [⁷⁶Br]MJL-1-109-2 which showed little regional specificity (similar high binding level in cerebellum and brainstem [17]. In contrast, the CRF₁ receptor radioligand 4-[⁷⁶Br]BMK gave a higher specific/non-specific binding ratio (~ 2–4) than the current ligands. Nevertheless, the quality of the autoradiographic images produced by [¹⁸F]BMS-732098 and [³H]BMS-728300 are comparable to those generated by 4-[⁷⁶Br]BMK. Notably, the substantially longer half-life and high imaging resolution of the non-peptide small-molecule CRF₁ receptor antagonist [³H]BMS-728300 makes this radioligand valuable for the further investigation of CRF₁ receptor expression and regulation in normal and disease states.

Biodistribution experiments in the rat showed that both ¹⁸F-labeled compounds ([¹⁸F]BMS-721313 and [¹⁸F]BMS-732098) readily entered the brain, consistent with other structurally-related and structurally-distinct CRF₁ receptor ligands [9]. Both [¹⁸F]BMS-732098 and [¹⁸F]BMS-721313 were taken forward for evaluation in PET studies. [¹¹C]BMS-709460 was also evaluated based on its higher CRF₁ receptor binding potency (IC_50, 0.8 nM), acceptable lipophilicity (LogD, 3.7) and ease of labeling. Autoradiography was not performed with [¹¹C]BMS-709460 because ¹¹C was unavailable at the site where autoradiography was conducted.

[¹⁸F]BMS-732098 and [¹⁸F]BMS-721313 gave acceptably high radioactivity uptake in rhesus monkey brain. However, the baseline studies showed relatively little difference in uptake between putatively receptor-rich regions and receptor-poor regions, especially after the initial distribution phase; i.e. there was little or no receptor-specific signal. The findings were similar in displacement experiments where the selective CRF₁ receptor antagonist DMP904 was given 20 min after [¹⁸F]BMS-732098 or 15 min after [¹⁸F]BMS-721313. Specifically, the displacing agent had little or no effect on washout; i.e., no specific signal was evident.

In rhesus monkey PET experiments, [¹¹C]BMS-709460 gave higher brain radioactivity uptake than [¹⁸F]BMS-721313 or [¹⁸F]BMS-732098, but exhibited similar washout kinetics. The appreciably higher CRF₁ receptor binding potency of [¹¹C]BMS-709460 may have been expected to give a more promising radioligand, but again there was no evidence of a receptor-specific signal. A follow-up experiment in the same monkey, in which CRF₁ receptors were blocked with DMP904 preceding bolus radioligand injection, showed no appreciable difference in kinetics, although the initial radioactivity uptake was higher. This pair of experiments provided no clear evidence of receptor specific binding, only of good brain entry. Therefore, bolus plus constant infusion experiments with [¹¹C]BMS-709460 were performed with measurement of a metabolite-corrected arterial input function under both baseline and receptor block conditions, followed by regional compartmental analysis to derive volumes of distribution that reflect specific plus non-specific binding. Bolus plus constant infusion instead of bolus injection was applied because we suspected that the rapid decline of brain activity may have been caused by a rapid decline of the parent concentration in arterial plasma. In such a case, bolus plus constant infusion may produce higher levels of parent in plasma and brain, and make the blood analysis easier. Blockade of CRF₁ receptors was found to cause a negligible decrease in V_T (Table 5), confirming the absence of a receptor-specific signal in the baseline experiment. Additionally, these experiments revealed that [¹¹C]BMS-732098 was metabolized into two less lipophilic unidentified radiometabolites. Radiometabolites in brain have the potential to obscure receptor-radioligand specific PET signals and to confound successful receptor measurement [38]. It is unknown whether these radiometabolites entered the brain. Interestingly, the emergence of these radiometabolites in plasma was reduced in the receptor block study potentially reflecting a reduced availability of metabolizing enzyme activity for [¹¹C]BMS-709460 due to presence of a relatively high concentration of DMP904. Similarly, the increase in uptake in the [¹⁸F]BMS-721313 blocking study might be explained by reduced radioligand metabolism or reduced radioligand sequestration by CRF₁ receptors in the periphery and higher input from plasma into brain.

Thus, PET experiments showed good brain radioactivity uptake for all three radioligands, yet no indication of specific (or non-specific) binding. Similar findings have been reported elsewhere for other candidate CRF₁ receptor radioligands [16,19,20]. In the studies conducted by Kumar and colleagues [19] and Sullivan and colleagues [20], the lack of a specific signal appeared to be due to the low level of CRF₁ receptors in the baboon brain. In contrast, our in vitro binding studies demonstrate the presence of CRF₁ receptors in the rhesus cortex and cerebellum although the question still remains whether there is sufficient CRF₁ receptor density to support PET studies. Thus, it is generally considered that the ratio of receptor density (B_max) to radioligand affinity (K_D), where each is expressed as nM, should well exceed 5 for a PET radioligand to show efficacy [39]. Very high ratios may be required for radioligands having very low plasma free fraction (f_p) [40]. Here we found that B_max in rhesus monkey brain cerebellum was 121 fmol/mg protein which equates to about 12 nM, assuming the presence of about 100 mg protein per mL of brain tissue. One caution with this estimate is that the CRF₁ receptor density/protein concentration in the membrane preparation may have been altered to some degree during the membrane isolation process. Our B_max finding for rhesus brain corresponds well with a previous report where the CRF₁ receptor density was found to be 87, 123 and164 fmol/mg protein in the frontal cortex, temporal cortex and cerebellum of the adult rhesus monkey, respectively (41). In the same study, a higher density of 302 fmol/mg protein was found in pituitary. If we assume the IC₅₀ values approximate the K_D, as was the case for BMS-728300, we can estimate the B_max/”K_D” ratios for [¹⁸F]BMS-732098, [¹⁸F]BMS-721313 and [¹¹C]BMS-709460 in cerebellum to be 2.8, 2.3 and 15, respectively. Other factors may have contributed to the lack of specific binding in the current PET studies: 1. The association of the radioligand with the CRF₁ receptor may have been slow relative to loss from the brain (i.e., the ligand affinity in vivo could be lower than predicted by the in vitro studies); 2. Occupation of the CRF₁ receptor by CRF or other related peptides in vivo may have resulted in direct blockade of radioligand binding to the receptor or a reduction in apparent affinity of the receptor for the radioligand caused by allosteric modulation by CRF or related peptides. This situation may have contributed to the discrepant results between the specificity observed in vitro, where CRF and other ligands would have been lost or diluted, and the lack of a specific signal in the PET studies. Autoradiography is also a fairly sensitive methodology. We know from previous studies, with efficacious doses of CRF₁ receptor antagonists, that unoccupied CRF₁ receptors are readily measured under stressful conditions, where CRF would be elevated, such as are encountered in the situational anxiety test [26, 29] or the elevated-plus maze test [42]. However, tracer levels of CRF₁ ligand may not behave in the same manner as pharmacological concentrations of ligand. Additionally, the concentration of circulating CRF and related peptides and the tone of the stress system in anesthetized rhesus monkey may be dissimilar to that of the awake rat; 3. Lack of access to the binding site. This appears unlikely given the large number of structurally diverse CRF₁ antagonists that produce behavioral efficacy, presumably by binding to central CRF₁ receptors (e.g., [8]). Additionally, the PET studies showed that all the ligands readily entered the brain. We estimate that the peak concentrations of each radioligand in monkey brain in baseline or displacement experiments were, with one exception, near or below the radioligand IC₅₀ values. As this peak concentration likely contains a major component of non-specifically bound radiotracer, peak free radioligand concentrations were likely much lower than the IC₅₀ values. Hence, CRF₁ receptors are not expected to have been fully saturated by carrier. The exact extents of receptor occupancy by carrier remain unknown, but do not explain the absence of appreciable specific signal in the monkey PET experiments.

Finally, given its high binding potency at CRF₁ receptors, [¹¹C]BMS-709460 may merit testing in humans where the CRF₁ receptor density may be higher than in rhesus brain. Thus, CRF receptor density in human brain tissue has been reported as 680 fmol/mg protein [43] and ≥ 1200 fmol/mg protein [44]. However, it is unclear how many of these CRF binding sites were CRF₁ receptors. In a later study, the CRF₁ receptor-specific binding density was determined to be 60–150 fmol/mg protein [20], similar to the B_max in rhesus monkey cortex/cerebellum measured in the current study.