Journal of Natural Products
Article
analyses were performed on an UltiMate 3000RSLC (Dionex,
Benelux, Amsterdam, The Netherlands). Specifically, instrument
operation and chromatographic data acquisition and processing
were performed using the Chromeleon 7.2 chromatography data
system. All separations were performed by using (R,R)-Whelk-O1 and
(S,S)-Whelk-O1 CSPs, prepared according to a previously described
procedure starting from Kromasil 1.8 μm silica particles and slurry
packed into 100 × 4.6 mm (L × i.d.) stainless steel columns. Isocratic
conditions were set as follows: mobile phase: n-hexane/isopropanol
(99.5:0.5 v/v); flow rate: 1.0 mL/min; T = 30 °C; detection: UV 214
nm.
was measured with a 1450 Microbeta WallacTrilux Top counter after
the addition of 45 μL of scintillation cocktail. Specific binding was
calculated by subtracting the residual radioactivity signal obtained in
the presence of an excess of GTPγS, and the results were expressed as
percentage of vehicle control.
Enzymatic Assays. FAAH, MAGL, and ABHDs activity assays
were performed as previously described.22 Briefly, FAAH and MAGL
activity assays were performed using a U937 cell homogenate
(100 μg), which were diluted in 200 μL of 10 mM Tris-HCl and
1 mM EDTA, pH 8, containing 0.1% fatty-acid-free BSA. Compounds
were added at the screening concentration of 10 μM and incubated
for 30 min at 37 °C. Then, 100 nM AEA containing 1 nM
[ethanolamine-1-3H]AEA as a tracer for FAAH or 10 μM 2-oleoyl
glycerol (2-OG) containing 1 nM [glycerol-1,2,3-3H]2-OG was
added to the homogenates and incubated for 15 min at 37 °C. The
reaction was stopped by the addition of 400 μL of ice-cold CHCl3/
MeOH (1:1), and samples were vortexed and rapidly centrifuged at
16000g for 10 min at 4 °C. The aqueous phases were collected, and
the radioactivity was measured for tritium content by liquid
scintillation spectroscopy. hABHD6 and hABHD12 activities were
determined using cell homogenates from HEK-293 cells stably
transfected with hABHD6 and hABHD12. Compounds were
preincubated with 40 μg of cell homogenate for 30 min at 37 °C in
assay buffer (1 mM Tris and 10 mM EDTA plus 0.1% fatty-acid-free
BSA, pH 7.6). DMSO was used as vehicle control with 10 μM
WWL70 or 20 μM THL as positive controls for ABHD6 and
ABJHD12, respectively. Then, 10 μM 2-OG was added and incubated
for 5 min at 37 °C. The reaction was stopped by the addition of
400 μL of ice-cold CHCl3/MeOH (1:1). The samples were vortexed
and centrifuged (16000g, 10 min, 4 °C). Aliquots (200 μL) of the
aqueous phase were assayed for tritium content by liquid scintillation
spectroscopy. Blank values were recovered from tubes containing no
enzyme. Basal 2-OG hydrolysis occurring in nontransfected HEK293
cells was subtracted. The experiments were performed at least two
times in triplicate, and data are reported as mean values SD.
Animals. In vivo experiments were performed in accordance with
the Swiss Federal guidelines, which comply with the Institutional
Animal Care and Use Committee (IACUC) guidelines. In particular,
mice were handled according to Swiss Federal legislation, and
protocols were approved by the respective government authorities
(Veterinaramt Kanton Bern, experimental license BE-79/18). Male
BALB/c mice (8 to 10 weeks old) were provided by Janvier
Laboratories (St Berthevin, France). Mice were housed in groups of
five per cage in a specific pathogen-free unit under controlled 12 h
Enantioselective SFC. Synthetic (−)-Δ9-cis-THC and ( )-Δ9-
cis-THC were prepared according to previously published methods,
and standard solutions thereof were prepared in acetonitrile (0.4 mg/
mL).16,19 Extracts of two cannabis fiber hemp strains (Kompolti and
CRA_05 Fibranova) were purified by preparative thin-layer
chromatography (Merck silica gel 60 F254 TLC glass plates,
visualized with 254 nm light and cerium ammonium molybdate
solution followed by heating), isolating the ( )-Δ9-cis-THC-
containing fraction (Rf = 0.23, hexanes/ethyl acetate, 15:1). Solutions
of purified extracts were prepared in acetonitrile (1.4−1.6 mg/mL).
eSFC analyses were conducted on a Waters Acquity UPC2 analytical
SFC with a diode array detector. Data were analyzed and processed
using the Empower 3 software suite. Enantiomeric excess was
determined by eSFC; stationary phase: (R,R)-Whelk-O1 (5 μm, 250
× 4.6 mm L × i.d, Regis Technologies, Norton Grove, IL, USA);
mobile phase: CO2/isopropanol, 95.0:5.0, v/v; flow rate: 2.0 mL/min;
temperature = 40 °C, detection: UV 220 nm. The peaks were
assigned to (+)-Δ9-cis-THC (12.2 min) and (−)-Δ9-cis-THC (13.2
min) by co-injection of (−)-Δ9-cis-THC and ( )-Δ9-cis-THC.
CB1 and CB2 Binding Assay. The assay was performed as
previously described.22 Briefly, 15 μg of membrane preparation
obtained from CHO cells stably transfected with hCB1 or hCB2
receptors was resuspended in 300 μL of binding buffer [50 mM Tris-
HCl, 2.5 mM EDTA, 5 mM MgCl2, and fatty-acid-free bovine serum
albumin (BSA; 0.5 mg/mL) (pH 7.4)] in silanized glass tubes and co-
incubated with the test compounds at different concentrations (1 pM
to 100 μM) or vehicle and 0.5 nM [3H]CP55,940 (168 Ci/mmol) for
1.5 h at 30 °C. Nonspecific binding of the radioligand was determined
in the presence of 10 μM WIN55,512-2. After the incubation time,
membrane suspensions were rapidly filtered through a 0.5%
polyethylenimine-presoaked 96-well microplate bonded with GF/B
glass fiber filters (UniFilter-96 GF/B, PerkinElmer Life Sciences)
under a vacuum and washed 12 times with 150 μL of ice-cold washing
buffer. Filters were added to 45 μL of MicroScint-20 scintillation
liquid, and radioactivity was measured with the 1450 MicroBeta
Trilux top counter. Data were collected from at least three
independent experiments performed in triplicate, and the nonspecific
binding was subtracted. Results were expressed as [3H]CP55,940
bound as percentage of binding in vehicle-treated samples, and Ki
(inhibition constant) values were calculated applying the Cheng−
Prusoff equation.
light/12 h dark cycle (ambient temperature, 21
2 °C; humidity,
50−55%) with free access to standard rodent chow and water. The
mice were acclimatized to the animal house for 1 week before the
experiments.
Tetrad Test. Compounds were dissolved in pure DMSO and
administered intraperitoneally at different doses using five to eight
mice for each treatment group. (−)Δ9-trans-THC and (−)Δ9-cis-
THC were administered 1 h before assessing locomotion, catalepsy,
body temperature, and analgesia (collectively referred to as the tetrad
test). The rectal temperature was measured before (basal) and 1 h
after injection with a thermocouple probe (1 to 2 cm; Testo AG,
Switzerland), and the change in rectal temperature was expressed as
the difference between basal and postinjection temperatures.
Catalepsy was measured using the bar test, where mice were retained
in an imposed position with forelimbs resting on a bar 4 cm high; the
end-point of catalepsy was considered when both front limbs were
removed or remained over 120 s. Locomotion was determined using
the rotarod test; animals were placed on the rotarod (Ugo Basile,
Italy) at 6 rpm, and the latency to fall was measured with a cutoff time
of 120 s. Catalepsy and locomotion were measured in three trials. The
hot plate test was performed to evaluate analgesia using a 54−56 °C
hot plate (Thermo Scientific) with a Plexiglas cylinder. The latency to
the first nociceptive response (paw lick or foot shake) was measured.
Statistical Analysis. Data were collected from at least two
independent experiments each performed in triplicate. Results are
expressed as mean values and standard error deviation. The statistical
[35S]GTPγS Binding Assay. The assay was performed as
previously described.24 Briefly, 5 μg of clean membrane prepared
in-house from CHO-hCB2 and CHO-hCB1 cells was diluted in
silanized plastic tubes with 200 μL of GTPγS binding buffer [50 mM
Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, and 100 mM NaCl (pH 7.4)
supplemented with 0.5% fatty-acid-free BSA] in the presence of 10
μM GDP and 0.1 nM [35S]GTPγS (1250 Ci/mmol). The mixture was
kept on ice until the binding reaction was started by adding the test
compound, vehicle (negative control), or CP55,940 (positive
control). Nonspecific binding was measured in the presence of 10
μM GTPγS (Sigma). The tubes were incubated at 30 °C for 90 min
under shaking, and then they were put on ice to stop the reaction. An
aliquot (185 μL) of the reaction mixture was rapidly filtered through a
96-well microplate bonded with GF/B glass fiber filters (UniFilter-96
GF/B, PerkinElmer Life Sciences) previously presoaked with ice-cold
washing buffer [50 mM Tris-HCl (pH 7.4) plus 0.1% fatty-acid-free
BSA]. The filters were washed six times with 180 μL of washing buffer
under vacuum and dried under the air drier flow. The radioactivity
G
J. Nat. Prod. XXXX, XXX, XXX−XXX
Schafroth, Michael A.
