Jacobson et al., page 1
Supplementary methods
Membrane preparation. A CHO-K1 recombinant cell line stably expressing human
CB1 receptor was purchased from Euroscreen s.a., (cat# ES-110-C; Brussels, Belgium). The
CHO recombinant cell line stably expressing human CB2 receptor was established in-house.
Cells were grown and collected from 15 cm tissue culture plates and then centrifuged down. Cell
pellets were washed once with cold PBS, and resuspended in buffer A (~0.23 mL/plate of cells;
20 mM HEPES, pH 7.4, 10 mM EDTA, EDTA-free complete protease inhibitor cocktail [cat#
1873580, 1 tablet/25 mL; Roche, Nutley, NJ, USA). The cell suspension was homogenized on
ice using a Polytron homogenizer (Polytron PT-2100; Kinematica AG, Littau-Luzern,
Switzerland) at 25,000 rpm in three 15-s intervals with a 1-min pause in between. The
homogenate was then centrifuged at 2,000 rpm on a tabletop low speed centrifuge for 10 min.
The supernatant, after passing through a cell strainer, was then centrifuged at 50,000 × g for 25
min at 4 °C and was resuspended in buffer B (~0.026 mL/plate of cells; 15 % glycerol, 20 mM
HEPES, pH 7.4, 0.1 mM EDTA, EDTA-free complete protease inhibitor cocktail [1 tablet/10
mL]). Protein concentration of the resulting preparation was determined using the BCA Protein
Assay kit and bovine serum albumin (BSA) as the standard. The membranes were aliquoted and
kept frozen at -80 °C at 10-20 µg/µL concentration.
Conditions for radioligand binding assay. Solutions of test compounds ranging from
0.01 nM to 10 mM were prepared in DMSO. The desired amount of membrane preparation was
diluted with ice-cold assay buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.05 % BSA,
pH 7.4) and was vortexed. Two µL or less of compound was distributed into each well of a
round-bottom 96-well polystyrene assay plate, followed by addition of 100 µL of diluted
membranes (3-10 µg/well) and kept on ice until the addition of [3H]-CP55940. [3H]-CP55940
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was diluted with cold (unlabeled) assay buffer and 100 µL was added into each well. The
reaction was carried out at room temperature for 120 min before the membranes were harvested
onto a PerkinElmer Unifilter GF/B-96 filter plate using a Packard Filtermate Harvester
(PerkinElmer). After nine washes with buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl2,
0.05 % BSA, pH 7.4), the filter was dried in a 37 ˚C oven for 30 min. MicroScint-20 was added
and the plate sealed for scintillation counting on a TopCount (Packard Instrument Company,
Meriden, CT, USA).
Protein determination in brain tissue. In studies comparing plasma and brain
concentrations of Compound 64 and rimonabant, whole brains were collected and snap-frozen in
liquid nitrogen until analysis. Brains were weighed, and for every gram of brain, 3 mL of icecold homogenization buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 4.7 mM KCl) was added.
The brains were homogenized in a 50 mL conical tube in ice water bath using a Polytron
homogenizer at 23,000 rpm at three intervals of 10 s each with 1 min pause in between. The
pellet was then resuspended in 50 mM Tris-HCl, pH 7.4, 300 mM KCl, 10 mM EDTA and
incubated for 1 hr on ice. The homogenate was centrifuged at 50,000 × g for 25 min at 4 °C and
resuspended in ice-cold 50 mM Tris-HCl, pH 7.4, and this procedure was repeated again. Final
resuspension of the pellet was with buffer containing 20 mM HEPES, 2 mM MnCl2, 0.1 % BSA,
and protease inhibitor cocktail (1 tablet/50 mL). Protein concentration of the preparation was
determined using the BCA Protein Assay kit using BSA as the standard. The brain preparation
was aliquoted and kept frozen at -80 °C at 10-20 µg/µL concentration.
Analysis of test compounds using liquid or high performance chromatography/mass
spectrometry/mass Spectrometry (LC/MS/MS and HPLC/MS/MS). Two methods of
detection for plasma concentrations were used for Compound 64. The first utilized an
Jacobson et al., page 3
LC/MS/MS method for quantification with terfenadine in acetonitrile as an internal standard
(IS). Samples (20 L) were injected onto a Zorbax SB-C8 analytical column (2.1x50 mm, 5 m,
Agilent Technologies Inc., Palo Alto, CA, USA) and the target analyte and internal standard
were eluted using a gradient method with a mobile phase consisting of 0.05% formic acid in
water (solvent A) and 0.05% formic acid in acetonitrile (solvent B) at retention time 3.26 and
2.87 min, respectively. The flow rate was 600 L/min. Mass spectral analyses were carried out
using turbo ion spray (TIS) ionization source in the positive ion mode. Compound 64 (477.04 >
292.25) and the IS (473.1 > 437.3) were monitored using multiple reaction monitoring (MRM).
Data collection and peak integration were performed using Analyst TM 1.2 software. The standard
curve range used for plasma for this method of analysis was 1 to 2,500 ng/mL. The quality
control samples were 8, 80 and 400 ng/mL Whole mouse brains were homogenized with a
solution of 5% acetonitrile in water (diluting brain weight to volume 1:9) using an Ultra-Turrax
T8 Homogenizer (IKA Works, Inc., Staufen, Germany). Brain concentrations of Compound 64
were quantified using a similar LC/MS/MS assay as described as above. The standard curve
range used was 11 to 27,500 ng/g of brain tissue. The quality control samples were 88, 880 and
4400 ng/g brain tissue.
The second method used to quantify plasma Compound 64 concentrations employed
HPLC/MS/MS method with 3-fluoro-4-[7-(3-fluoro-benzyl)-5-isobutyl-6-oxo-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-5-yl]-benzonitrile as the IS, manufactured in-house. The internal standard
(25 µL) and 0.5 mL of acetonitrile/ethanol/acetic acid (90/10/0.1, v/v/v) were added to plasma
samples (100 µL). The mixture was vortexed, sonicated (5 min), and centrifuged (4,000 rpm, 5
min). Supernatant (200 µL) was evaporated to dryness under nitrogen at 45 °C. The residue was
reconstituted in 200 µL of 25% acetonitrile/water containing 0.1% acetic acid and 10 µL was
Jacobson et al., page 4
injected onto a ACE3 C18 30 x 2.1 mm, 3 m (MAC MOD) column. Samples were eluted using a
gradient method with Solvent A consisting of 0.2% formic acid in water and Solvent B
consisting of 0.2% formic acid in acetonitrile (flow rate = 0.5 µL/min). Mass spectral analysis
was carried out using electrospray ionization in the positive ion mode. The lower limit of
quantification was 1.05 nM and control plasma was spiked at concentrations ranging from 0.5 to
10,000 ng/mL. For this approach, the IS parent and product m/z were 421.2 and 341.0,
respectively. The quantification for rimonabant concentrations in plasma (parent m/z, 436.1,
product m/z, 381.1) were done similarly with the exception that the plasma sample volume was
25 µL and at the end of the sample preparation, samples were not taken to dryness.