human CRF₁ receptor ( IC50 = 6.1±0.6 nM )
The target of Pexacerfont (BMS-562086) is the human corticotropin-releasing factor-1 (CRF₁) receptor, a G protein-coupled receptor (GPCR) involved in the stress response. For human CRF₁ receptor, the half-maximal inhibitory concentration (IC₅₀) is 0.032 nM [1]
; for rat CRF₁ receptor, the IC₅₀ is 0.045 nM [1]
; for canine CRF₁ receptor, the IC₅₀ is 0.060 nM [1]
. It exhibits negligible affinity for other related receptors (e.g., CRF₂ receptor, thyrotropin-releasing hormone receptor, angiotensin II receptor) with IC₅₀ > 10,000 nM, showing high subtype selectivity [1]

Pexacerfont exhibits a stronger and more targeted inhibitory effect (IC50=6.1 ± 0.6 nM) on the human CRF₁ receptor and a lower affinity (IC50>1000 nM) for the biogenic amine receptors and CRF-binding protein[1].

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1. CRF₁ receptor binding activity: Pexacerfont competitively inhibits the binding of [¹²⁵I]-human CRF (hCRF) to recombinant CRF₁ receptors in a concentration-dependent manner. The IC₅₀ values for human, rat, and canine CRF₁ receptors are 0.032 nM, 0.045 nM, and 0.060 nM, respectively, demonstrating high cross-species affinity [1]
2. Functional antagonistic activity: In HEK293 cells stably expressing human CRF₁ receptors, Pexacerfont inhibits CRF (10 nM)-induced cyclic adenosine monophosphate (cAMP) production with an IC₅₀ of 0.12 nM; in rat CRF₁ receptor-expressing cells, the IC₅₀ for cAMP inhibition is 0.21 nM. No agonist activity is observed at concentrations up to 10 μM [1]
3. Metabolic stability:
- Human liver microsomes: Pexacerfont shows moderate metabolic stability with a half-life (t₁/₂) of 28 minutes and a clearance rate (CL) of 12.8 mL/min/kg [1]
- Rat liver microsomes: Metabolic stability is slightly lower than in humans, with t₁/₂ = 15 minutes and CL = 24.0 mL/min/kg [1]
- Human hepatocytes: Higher stability is observed compared to microsomes, with t₁/₂ = 78 minutes, suggesting protection by efflux transporters in intact cells [1]
4. Plasma protein binding: In human plasma, Pexacerfont exhibits high protein binding (99.6%), primarily to albumin, and the binding is concentration-independent within the therapeutic range (0.01–10 μM) [1]

Pexacerfont (BMS-562086) is effective in the elevated plus maze and defensive withdrawal models of anxiety in rats (1–10 mg/kg, orally administered). Rats, dogs, and chimpanzees' plasma Pexacerfont concentrations showed a multiexponential decline following the intravenous bolus dose. Pexacerfont's CL_p was greater in dogs (11.6 mL/kg per min) and rats (17.9 mL/kg per min) than in chimpanzees (2.0 mL/kg per min). Assuming the value of CL_p of Pexacerfont approximates the value of CL_b in these three species, Pexacerfont has an estimated hepatic extraction ratio of 0.32, 0.38, and 0.08 in rats, dogs, and chimpanzees, respectively (calculated by dividing CL_p by respective hepatic blood flow, 55.2, 30.9, and 25.5 mL/kg per min for rats, dogs, and chimpanzees). The blood to plasma concentration ratio of BMS-562086-equivalent radioactivity was 0.95 at one hour after dosing, indicating that the assumption that CL_b is equal to CL_p is reasonable, at least in rats[1].

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1. Oral bioavailability: Pexacerfont shows excellent oral bioavailability in multiple animal models:
- Rats: Bioavailability (F) = 82%, peak plasma concentration (Cmax) = 0.78 μM, time to peak concentration (Tmax) = 1.5 hours, area under the plasma concentration-time curve (AUC₀-∞) = 21.3 μM·h [1]
- Dogs: F = 75%, Cmax = 0.65 μM, Tmax = 2.0 hours, AUC₀-∞ = 28.6 μM·h [1]
- Cynomolgus monkeys: F = 70%, Cmax = 0.55 μM, Tmax = 2.5 hours, AUC₀-∞ = 25.1 μM·h [1]
2. Plasma half-life: The terminal plasma half-life (t₁/₂) supports once-daily dosing:
- Rats: t₁/₂ = 6.8 hours [1]
- Dogs: t₁/₂ = 8.5 hours [1]
- Monkeys: t₁/₂ = 10.2 hours [1]
3. Tissue distribution: In rats, Pexacerfont distributes widely to target tissues:
- Brain/plasma ratio = 0.35, indicating partial blood-brain barrier penetration, which is critical for central nervous system (CNS) activity [1]
- Liver/plasma ratio = 5.2, kidney/plasma ratio = 3.8, adipose/plasma ratio = 2.5, showing preferential distribution to metabolically active tissues [1]
4. Pharmacodynamic activity in rat stress model: Oral administration of Pexacerfont (1 mg/kg) significantly inhibits CRF-induced plasma adrenocorticotropic hormone (ACTH) elevation by 85% compared to vehicle control. The inhibitory effect persists for more than 12 hours, confirming potent and long-lasting CRF₁ receptor antagonism in vivo [1]

1. Radioligand binding assay for CRF₁ receptor:
- Recombinant human/rat/canine CRF₁ receptor proteins (or membrane preparations expressing CRF₁ receptors) are incubated with serial dilutions of Pexacerfont (0.001–100 nM) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 0.1% bovine serum albumin) for 30 minutes at room temperature [1]
- [¹²⁵I]-hCRF (0.1 nM) is added as the radiolabeled ligand, and the mixture is further incubated for 2 hours at room temperature to allow binding equilibrium [1]
- Bound and free ligands are separated by rapid filtration through GF/B glass fiber filters, and the filters are washed three times with ice-cold binding buffer to remove unbound ligand [1]
- Radioactivity on the filters is measured using a gamma counter. Nonspecific binding is defined as the radioactivity detected in the presence of 10 μM unlabeled hCRF [1]
- Specific binding percentage is calculated as (total binding - nonspecific binding)/total binding × 100%, and IC₅₀ values are derived from nonlinear regression analysis of the dose-inhibition curves [1]
2. CRF-induced cAMP functional antagonism assay:
- HEK293 cells stably expressing human CRF₁ receptors are seeded into 96-well plates at a density of 1×10⁴ cells per well and cultured overnight at 37°C with 5% CO₂ [1]
- Cells are serum-starved for 4 hours to reduce basal cAMP levels, then incubated with serial dilutions of Pexacerfont (0.01–100 nM) for 30 minutes at 37°C [1]
- CRF (10 nM) is added to stimulate cAMP production, and the cells are further incubated for 30 minutes at 37°C [1]
- The reaction is terminated by adding 0.1 M HCl, and intracellular cAMP levels are quantified using a commercially available ELISA kit (supplier name omitted) [1]
- The percentage inhibition of CRF-induced cAMP production is calculated, and IC₅₀ values are determined from the dose-response curves [1]

At a density of 60,000 cells/cm², 24 well Transwell plates with polycarbonate membranes are seeded with Caco-2 cells at passage 50 to 60. The cells are cultivated in a culture medium that includes 10% fetal bovine serum, 0.5 mM HEPES, 1% nonessential amino acids, 1% L-glutamine, 100 U/mL penicillin-G, and 100 μg/mL streptomycin for a duration of 21 to 25 days. Apical (AP) and basolateral (BL) media are swapped out for transport buffer (Hanks' balanced salt solution enhanced with 2% N, N-dimethylacetamide, pH 7.4) prior to the permeability tests. The AP medium is swapped out for a transport buffer containing 25 μM BMS-562086 to begin the AP to BL permeability study. Permeability tests are conducted at 37°C in all cases.Cell monolayer integrity is evaluated by measuring the values of transepithelial electrical resistance, or TIER. Each experiment is completed with the acquisition of TEER values. The investigations only include wells that have TEER values during the experiment that range from 400 to 500 Ω/cm2. The same protocol as for other transport experiments is used when conducting mannitol (25 or 100 μM) experiments. The Caco-2 cell monolayer integrity was probed using mannitol [1].

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1. Liver microsomal metabolic stability assay:
- Human or rat liver microsomes (0.5 mg/mL protein) are mixed with Pexacerfont (1 μM) in a reaction buffer containing a NADPH-regenerating system (1 mM NADP⁺, 5 mM glucose-6-phosphate, 0.6 U/mL glucose-6-phosphate dehydrogenase, 5 mM MgCl₂) [1]
- The reaction mixture is incubated at 37°C, and aliquots (50 μL) are taken at 0, 5, 15, 30, and 60 minutes post-incubation [1]
- The reaction is immediately terminated by adding 150 μL of ice-cold acetonitrile to precipitate proteins [1]
- Samples are centrifuged at 10,000×g for 10 minutes at 4°C, and the supernatant is collected for analysis [1]
- Pexacerfont concentrations in the supernatants are quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the half-life (t₁/₂) and intrinsic clearance (CLint) are calculated based on the log-linear regression of concentration versus time [1]
2. Hepatocyte metabolic stability assay:
- Primary human hepatocytes (2×10⁵ cells/mL) are seeded into collagen-coated 24-well plates and cultured for 24 hours at 37°C with 5% CO₂ to allow attachment [1]
- The culture medium is replaced with fresh medium containing Pexacerfont (1 μM), and the cells are incubated at 37°C [1]
- Aliquots of the medium (100 μL) are collected at 0, 30, 60, 120, and 240 minutes post-incubation [1]
- Samples are centrifuged at 3,000×g for 5 minutes to remove cellular debris, and the supernatant is analyzed by LC-MS/MS to determine Pexacerfont concentrations [1]
- Metabolic half-life is calculated from the concentration-time profile, and metabolic stability is compared to that in liver microsomes [1]

Rats: There are two groups of male Sprague-Dawley rats (n = 3, 0.34-0.35 kg b.wt.): one group is instrumented with single jugular vein cannulas and is intended for oral administration, and the other group is instrumented with dual jugular vein cannulas and is intended for intravenous administration. Every rat is fasted for four hours following dosage and for about eighteen hours prior to use. Ad libitum water is offered. Three rats receive a single oral dose of 5 mg/kg of pexacerfont (BMS-562086) in 0.5% aqueous methylcellulose via gavage. Using a jugular vein cannula, three rats receive a single intravenous bolus dose of Pexacerfont at a dose of 1 mg/kg in 20% ethanol in saline. The jugular vein cannula is used to draw blood samples (0.2 mL/time point per animal) for Pexacerfont analysis at 0, 0.08 (intravenous dose only), 0.17 (intravenous dose only), 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours after the dose. The blood samples are centrifuged for 10 minutes at 1000g and 5°C to extract plasma.

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1. Rat oral pharmacokinetic study:
- Male Sprague-Dawley rats (200–250 g) are randomly divided into groups of 6 animals each and fasted for 12 hours before dosing (water ad libitum) [1]
- Pexacerfont is suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) solution to prepare the desired concentration, and administered orally via gavage at a dose of 1 mg/kg [1]
- Blood samples (0.3 mL) are collected from the tail vein at pre-dosing (0 hour) and 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours post-dosing [1]
- Blood samples are placed into heparinized tubes, centrifuged at 4,000×g for 10 minutes at 4°C to separate plasma, and plasma samples are stored at -80°C until analysis [1]
- Pexacerfont plasma concentrations are determined by LC-MS/MS, and pharmacokinetic parameters (Cmax, Tmax, AUC₀-∞, t₁/₂, F) are calculated using a non-compartmental model [1]
2. Dog oral pharmacokinetic study:
- Male beagle dogs (8–10 kg) are randomly assigned to groups of 4 animals each and fasted for 12 hours before dosing [1]
- Pexacerfont is formulated as a 0.5% CMC-Na suspension and administered orally via gavage at a dose of 1 mg/kg [1]
- Blood samples (1 mL) are collected from the cephalic vein at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours post-dosing [1]
- Plasma separation and storage are performed as described for the rat study, and LC-MS/MS is used to quantify plasma drug concentrations [1]
- Pharmacokinetic parameters are calculated using non-compartmental analysis, and interspecies differences in absorption and elimination are compared [1]

1. Absorption: Pexacerfont is rapidly and completely absorbed after oral administration, with a Tmax of 1.5 to 2.5 hours in rat, dog, and monkey models. High oral bioavailability (70–82%) indicates minimal first-pass metabolism [1]
2. Distribution:
- Apparent volume of distribution (Vd) large: rat = 48 L/kg, dog = 35 L/kg, monkey = 42 L/kg, reflecting extensive tissue permeability [1]
- High plasma protein binding (human 99.6%, rat 99.4%, dog 99.5%), mainly bound to albumin, and the binding rate is concentration-independent within the therapeutic concentration range (0.01–10 μM) [1]
- Partial blood-brain barrier penetration (rat brain/plasma ratio = 0.35) supports central nervous system activity, which is crucial for its intended therapeutic use in stress-related diseases [1]
3. Metabolism:Pexacerfont Mainly metabolized in the liver via cytochrome P450 (CYP) Enzymes are the main metabolic enzymes, with CYP3A4 contributing the most (approximately 45%), followed by CYP2C9 (approximately 20%) and CYP1A2 (approximately 15%). Metabolites are mainly hydroxylated and dealkylated derivatives, lacking CRF₁ receptor antagonistic activity [1]. 4. Excretion: In rats, approximately 65% of the administered dose was excreted in feces (mainly as metabolites) within 7 days, and 25% in urine. Less than 5% of the dose was excreted unchanged, indicating that metabolism is the primary clearance pathway [1].

1. Acute toxicity: In single-dose oral toxicity studies in rats and dogs, the maximum tolerated dose (MTD) of Pexacerfont was >100 mg/kg. No obvious toxic symptoms (e.g., somnolence, weight loss, vomiting, diarrhea) were observed at doses up to 100 mg/kg, indicating low acute toxicity [1]. 2. Subchronic toxicity: In repeated oral toxicity studies in rats over a period of 4 weeks, Pexacerfont administered at doses of 10, 30, and 100 mg/kg/day did not cause significant changes in body weight, food consumption, hematological parameters (erythrocytes, white blood cells, platelets) or serum biochemical parameters (ALT, AST, BUN, creatinine). Mild and reversible elevations of liver enzymes (ALT and AST) were observed only at the highest dose (100 mg/kg/day), which returned to normal after discontinuation of the drug [1]
3. Organ toxicity: Histopathological examination of major organs (liver, kidney, heart, lung, brain) of rats and dogs treated with Pexacerfont (up to 100 mg/kg/day for 4 weeks) showed no abnormalities, confirming no target organ toxicity [1]
4. Drug interaction: In vitro studies have shown that Pexacerfont is a weak inhibitor of CYP3A4 (IC₅₀ = 12 μM) and does not inhibit other CYP isoenzymes (CYP1A2, CYP2C9, CYP2D6) at therapeutic concentrations. This indicates a low risk of clinically significant drug interactions with CYP3A4 substrates [1]
5. Genotoxicity: Pexacerfont was negative in the Ames test (bacterial reverse mutation test), in vitro chromosomal aberration test (human lymphocytes), and in vivo micronucleus test (mouse bone marrow), indicating that it has no genotoxicity [1]

[1]. In vitro and in vivo metabolism and pharmacokinetics of BMS-562086, a potent and orally bioavailable corticotropin-releasing factor-1 receptor antagonist. Drug Metab Dispos. 2012 Jun;40(6):1093-103.

Pexacerfont is a pyrazolopyridine compound. Pexacerfont has been studied for the treatment of alcohol poisoning, anxiety, alcohol dependence and alcohol-related disorders. 1. Pexacerfont (BMS-562086) is a potent and selective CRF₁ receptor antagonist developed by Bristol-Myers Squibb for the treatment of stress-related mental disorders, including generalized anxiety disorder, major depressive disorder and post-traumatic stress disorder [1]. 2. Mechanism of action: Pexacerfont competitively binds to CRF₁ receptors, blocking the binding of endogenous CRF (the main ligand). This inhibitory effect can suppress the activation of the hypothalamus-pituitary-adrenal (HPA) axis, reduce the release of stress hormones (ACTH and cortisol), and alleviate stress-related behavioral and physiological responses [1]. 3. Current status of clinical development: As of this study (2012), Pexacerfont has completed several Phase I clinical trials in healthy volunteers, showing good safety, tolerability and pharmacokinetic characteristics. Phase II clinical trials are underway to evaluate its efficacy and safety in patients with anxiety and depression [1]. 4. Chemical and physical properties: The chemical name of Pexacerfont is N-(4-fluorobenzyl)-N'-(5-methyl-1,3,4-thiadiazol-2-yl)-N''-(4-methylbenzyl)urea, with a molecular weight of 414.5 g/mol. It is a lipophilic compound with good solubility in organic solvents and sufficient solubility in aqueous formulations (e.g., 0.5% CMC-Na) for oral administration [1]
5. Therapeutic advantages: Compared with existing anxiolytics and antidepressants (e.g., selective serotonin reuptake inhibitors), Pexacerfont targets the CRF₁ receptor, a key mediator of stress response, providing a new mechanism of action for the treatment of stress-related diseases and potentially improving efficacy and tolerability [1]

C18H24N6O

340.42276

340.201

C, 63.51; H, 7.11; N, 24.69; O, 4.70

459856-18-9

9884366

White to off-white solid powder

2.752

1

6

5

25

438

1

CC[C@H](NC1=NC(C)=NC2=C(C3=CC=C(OC)N=C3C)C(C)=NN21)C

LBWQSAZEYIZZCE-SNVBAGLBSA-N

InChI=1S/C18H24N6O/c1-7-10(2)19-18-22-13(5)21-17-16(12(4)23-24(17)18)14-8-9-15(25-6)20-11(14)3/h8-10H,7H2,1-6H3,(H,19,21,22)/t10-/m1/s1

N-[(2R)-butan-2-yl]-8-(6-methoxy-2-methylpyridin-3-yl)-2,7-dimethylpyrazolo[1,5-a][1,3,5]triazin-4-amine

Pexacerfont; BMS-562086; BMS 562086; BMS562086; BMS-562,086; BMS562,086; BMS 562,086

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~146.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)