rat brain MGL(IC50=28±4 μM)

Without URB602, the maximum velocity (Vmax) is 1814±51 nmol min per mg of protein and the apparent Michaelis constant (Km) of MGL for 2-AG is 24±1.7 μM; with URB602, the Km is 20±0.4 μM and the Vmax is 541±20 nmol min per mg of protein (n=4). Rat forebrain organotypic slice cultures exhibit elevated concentrations of 2-arachidonoylglycerol (2-AG) stimulated by Ca2+ ionophore and baseline when incubated with URB602 (100 μM)[1]. The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is biologically deactivated by the serine hydrolase monoacylglycerol lipase (MGL), which is inhibited by URB602. Recombinant MGL (IC50=223±63 μM) is weakly inhibited by URB602 via a quick and noncompetitive mechanism[2].

Without URB602, the maximum velocity (Vmax) is 1814±51 nmol min per mg of protein and the apparent Michaelis constant (Km) of MGL for 2-AG is 24±1.7 μM; with URB602, the Km is 20±0.4 μM and the Vmax is 541±20 nmol min per mg of protein (n=4). Rat forebrain organotypic slice cultures exhibit elevated concentrations of 2-arachidonoylglycerol (2-AG) stimulated by Ca2+ ionophore and baseline when incubated with URB602 (100 μM)[1]. The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is biologically deactivated by the serine hydrolase monoacylglycerol lipase (MGL), which is inhibited by URB602. Recombinant MGL (IC50=223±63 μM) is weakly inhibited by URB602 via a quick and noncompetitive mechanism[2].

Samples containing 300 μM of URB602, 1.4 pM of MGL, or both URB602 and MGL are incubated in assay buffer for 30 minutes at 37°C. The reaction is halted at different times with an equivalent volume of ice-cold methanol, and the results are immediately examined using LC/MS in positive ionization mode. Utilizing a linear gradient of methanol in water containing 0.25% acetic acid and 5 mM ammonium acetate (from 60% to 100% of methanol in 8 min) at a flow rate of 0.5 mL/min and column temperature of 50°C, an SB-CN column (150×2.1 mm i.d., 5 μm) is eluted. The fragmentor voltage is 100V, and the capillary voltage is 4 kV.60 psi is the nebulizer pressure setting. At 350°C and a flow rate of 13 liters per minute, N2 is used as a drying gas. With the ESI in the positive mode, the entire scan spectrum from m/z 100 to 600 is obtained. URB602 ([M+H]+, m/z 296)[2] is quantified using extracted ion chromatograms.

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.[2]

Mice[3]
Mice on a C57BL/6 background are either male (5–6 wk; 20–26 g) or female (8 wk; 18–22 g) CB1-/- mice. As others have explained in detail, an oral marker is given to evaluate upper GI transit following an overnight fast (water ad libitum). An Evans blue marker (20 or 40 mg/kg) or vehicle (10% DMSO/Tween 80 in saline) is intraperitoneally (ip) administered, followed by an oral gavage (200 μL) of a mixture of 5% Evans blue and 5% gum arabic, 30 minutes later.After fifteen minutes, the animals are killed by cervical dislocation, and the intestine is immediately removed from the area of the ileocecal junction to the pyloric sphincter. The marker's travel distance is expressed as a percentage of the small intestine's overall length and is measured in centimeters.

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Rats[4]
Three hundred and seven adult male Sprague-Dawley rats weighing 275-350 g, at the time of testing, are used. In a first study, the dose-response curves for JZL184 and URB602 are determined using the AUC of Phase 1 or Phase 2 pain behaviour. In a second study, the antinociceptive effects of JZL184 (300 μg) and URB602 (600 μg) are evaluated following injection in the paw, ipsilateral or contralateral to formalin, to exclude the possibility that systemic leakage contributed to the pattern of results obtained. In a third study, antinociceptive effects of ED50 doses of JZL184 (0.03 μg i.paw) or URB602 (66 μg i.paw), in combination with 2-AG (ED50 dose of 1 μg i.paw), are quantified to evaluate the presence of additive or synergic effects of these drugs. In a fourth study, antinociceptive effects of JZL184 (at 10 μg i.paw, an analgesic dose) are studied in the presence or absence of either AM251 or AM630 to determine whether these effects are mediated through CB1 and/or CB2 receptors. The CB1 receptor antagonist AM251 exhibits 306-fold selectivity for CB1 over CB2 receptors, whereas the CB2 receptor antagonist AM630 exhibits 70-165-fold selectivity for CB2 over CB1 receptors. The doses employed (AM251 at 80 μg i.paw and AM630 at 25 μg i.paw) are those which block peripheral antinociceptive effects of URB602 in Wistar rats. For the first study (n=4-6 per group for URB602 and n=6-8 per group for JZL184) and for all the other behavioural studies (n=6 per group), drugs, administered either alone or in combination, are dissolved in the same total volume (50 μL) and injected into the right hind paw. Preliminary experiments (n=8 per group; data not shown) confirmed that formalin-induced pain behaviour did not change following intra-paw administration of either vehicle (PEG 300: Tween 80 in a 4:1 ratio or DMSO: ethanol: cremophor: 0.9% saline in a 1:1:1:17 ratio].

[1]. An endocannabinoid mechanism for stress-induced analgesia. Nature. 2005 Jun 23;435(7045):1108-12.

[2]. URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices. Chem Biol. 2007 Dec;14(12):1357-65.

[3]. Distribution and function of monoacylglycerol lipase in the gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol. 2008 Dec;295(6):G1255-65.

[4]. Peripheral antinociceptive effects of inhibitors of monoacylglycerol lipase in a rat model of inflammatory pain. Br J Pharmacol. 2011 Aug;163(7):1464-78.

BACKGROUND AND PURPOSE The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 µg), URB602 (0.001-600 µg), 2-AG (ED(50)), 2-AG + JZL184 (at their ED(50)), 2-AG + URB602 (at their ED(50)), AM251 (80 µg), AM251 + JZL184 (10 µg), AM630 (25 µg) or AM630 + JZL184 (10 µg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 ± 0.028; Phase 2: 0.03 ± 0.011 µg) produced greater antinociception than URB602 (ED(50) Phase 1: 120 ± 51.3; Phase 2: 66 ± 23.9 µg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor 2 (CB(2)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.[4]

C19H21NO2

295.38

295.16

C, 77.26; H, 7.17; N, 4.74; O, 10.83

565460-15-3

10979337

White solid powder

1.1±0.1 g/cm3

416.6±24.0 °C at 760 mmHg

122-123ºC

205.8±22.9 °C

0.0±1.0 mmHg at 25°C

1.595

5.59

38.33

O=C(OC1CCCCC1)NC2=CC(C3=CC=CC=C3)=CC=C2

HHVUFQYJOSFTEH-UHFFFAOYSA-N

InChI=1S/C19H21NO2/c21-19(22-18-12-5-2-6-13-18)20-17-11-7-10-16(14-17)15-8-3-1-4-9-15/h1,3-4,7-11,14,18H,2,5-6,12-13H2,(H,20,21)

cyclohexyl [1,1'-biphenyl]-3-ylcarbamate

URB602; URB-602; URB 602

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 : 59~100 mg/mL ( 199.74~338.55 mM )
Ethanol : ~59 mg/mL

Solubility in Formulation 1: 2.5 mg/mL (8.46 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), suspension solution; with sonication.
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 (8.46 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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Solubility in Formulation 3: 10% DMSO+90% Corn Oil: ≥ 2.5 mg/mL (8.46 mM)

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