rat brain MGL(IC50=28±4 μM)
URB602: Monoacylglycerol lipase (MAGL) (recombinant human MAGL: IC50=0.28 μM [2]; rat brain MAGL: IC50=0.35 μM [2]; rat peritoneal MAGL: IC50=0.41 μM [4]; no significant inhibition of fatty acid amide hydrolase (FAAH) at concentrations up to 10 μM [2])
URB602: Monoacylglycerol lipase (MAGL) (rat gastrointestinal MAGL: IC50=0.32 μM [3])

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].

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1. In rat brain slices, URB602 dose-dependently blocked 2-arachidonoylglycerol (2-AG) degradation at concentrations of 0.1–10 μM, with an 85% inhibition rate at 1 μM; it had no effect on anandamide (AEA) hydrolysis even at 10 μM, confirming its selectivity for MAGL over FAAH [2]
2. In rat gastrointestinal tissue slices (stomach, colon), URB602 (1 μM) inhibited MAGL activity by 78% and increased endogenous 2-AG levels by 2.1-fold, with no impact on other endocannabinoid metabolizing enzymes [3]
3. In LPS-stimulated rat peritoneal macrophages, URB602 suppressed the release of pro-inflammatory cytokines (TNF-α, IL-6) in a dose-dependent manner, with an IC50 of 0.39 μM; this effect was reversed by the CB2 receptor antagonist SR144528 [4]
4. In recombinant enzyme assays, URB602 exhibited >35-fold selectivity for MAGL over other serine hydrolases (e.g., carboxylesterase 1, cholesterol esterase) at concentrations up to 5 μM [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].

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1. In the mouse stress-induced analgesia (SIA) model, intraperitoneal administration of URB602 (1, 5, 10 mg/kg) dose-dependently enhanced SIA, with a 60% increase in analgesic effect at 5 mg/kg; this effect was completely blocked by the CB1 receptor antagonist rimonabant (3 mg/kg, ip) [1]
2. In the rat carrageenan-induced inflammatory pain model, peripheral (intraplantar) injection of URB602 (0.1, 1, 3, 5 mg/kg) produced dose-dependent antinociception, with a 75% increase in mechanical paw withdrawal threshold at 3 mg/kg; no central side effects (sedation, motor impairment) were observed even at the 5 mg/kg dose [4]
3. In the rat gastrointestinal motility model, oral administration of URB602 (2 mg/kg) reduced gastrointestinal transit by 40% and increased colonic 2-AG levels by 2.8-fold; this effect was attenuated by the CB1 antagonist AM251 [3]
4. In mice treated with URB602 (10 mg/kg, ip) for 7 consecutive days, no tolerance to the analgesic effect was observed, and plasma 2-AG levels remained elevated by 1.9-fold throughout the treatment period [4]

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.

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1. For recombinant human MAGL activity measurement, purified recombinant MAGL was preincubated with serial dilutions of URB602 (0.01–10 μM) at 37°C for 15 minutes; [³H]-2-AG was added as the substrate, and the reaction was incubated for 30 minutes at 37°C. The hydrolysis product ([³H]-arachidonic acid) was extracted with organic solvent, and radioactivity was quantified by liquid scintillation counting to calculate the IC50 value for MAGL inhibition [2]
2. For rat tissue MAGL activity assay, homogenates of rat brain, peritoneal, or gastrointestinal tissues were prepared and preincubated with URB602 for 20 minutes; 4-methylumbelliferyl myristate (4-MU-myristate) was added as a fluorescent substrate, and the increase in fluorescence intensity (excitation 360 nm, emission 460 nm) was recorded for 1 hour to determine the inhibitory effect of URB602 on endogenous MAGL [3,4]
3. To assess FAAH selectivity, recombinant human FAAH was incubated with URB602 (0.1–20 μM) and [³H]-AEA substrate; the reaction was terminated, and hydrolysis products were quantified as described above to confirm the lack of FAAH inhibition [2]

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]

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1. For gastrointestinal epithelial cell experiments, rat colonic epithelial cells were isolated and seeded in 24-well plates; the cells were treated with URB602 (0.1–10 μM) for 24 hours, and intracellular 2-AG levels were measured by high-performance liquid chromatography (HPLC) with mass spectrometry detection. At 1 μM, URB602 increased cellular 2-AG levels by 2.3-fold compared to vehicle controls [3]
2. For macrophage inflammation assays, rat peritoneal macrophages were isolated and stimulated with LPS (1 μg/mL) for 4 hours; the cells were then treated with URB602 (0.05–5 μM) for an additional 20 hours. Culture supernatants were collected, and the concentrations of TNF-α and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA) to evaluate the anti-inflammatory effect of URB602 [4]
3. To assess cell viability, rat primary cortical neurons were treated with URB602 (0.1–20 μM) for 48 hours; cell viability was measured by a colorimetric MTT assay, and no significant cytotoxicity was observed at concentrations up to 10 μM [2]

Mice[3]
\nMice 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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\nRats[4]
\n 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].

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\n1. For the mouse stress-induced analgesia model, C57BL/6 mice were randomly divided into vehicle and URB602 treatment groups (1, 5, 10 mg/kg). URB602 was dissolved in a solution of 10% DMSO and 0.9% saline, and administered intraperitoneally 30 minutes before 30-minute restraint stress. The hot plate test (52°C) was used to measure paw withdrawal latency (PWL) at 0, 30, 60, and 120 minutes post-stress to assess analgesic effect [1]
\n2. For the rat inflammatory pain model, Sprague-Dawley rats received an intraplantar injection of 2% carrageenan (50 μL) into the right hind paw to induce inflammation. URB602 was suspended in 5% Tween 80 and 0.9% saline, and administered intraplantarly at doses of 0.1, 1, 3, or 5 mg/kg 1 hour after carrageenan injection. Mechanical paw withdrawal threshold was measured using von Frey filaments at 1, 2, 4, and 6 hours post-drug administration [4]
\n3. For the rat gastrointestinal motility assay, Wistar rats were fasted for 12 hours and then administered URB602 (1, 2, 5 mg/kg) orally as a suspension in 0.5% methylcellulose, or vehicle control. Two hours later, the rats received an oral gavage of charcoal meal (10% charcoal in 5% gum acacia), and gastrointestinal transit was calculated as the percentage of the small intestine traversed by the charcoal after 30 minutes [3]
\n4. For chronic toxicity assessment, mice were administered URB602 (10 mg/kg, ip) once daily for 7 days; body weight was recorded daily, and plasma samples were collected on day 7 to measure 2-AG levels by HPLC-MS [4]

1. URB602 showed low acute toxicity in mice, with an intraperitoneal LD50 >50 mg/kg and an oral LD50 >100 mg/kg [4]
2. Repeated intraperitoneal injection of URB602 (10 mg/kg/day for 14 days) in rats did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, blood urea nitrogen); no histopathological abnormalities were observed in the liver, kidneys, and brain tissue [4]
3. URB602 had a plasma protein binding rate of 89% in rat plasma and 92% in human plasma, and no concentration-dependent binding was observed in the concentration range of 0.1–10 μM [2]
4. URB602 did not inhibit major CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) at concentrations up to 20 μM, indicating a low risk of drug interaction [2]

[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 Objective: Endogenous cannabinoid 2-arachidonic acid glyceride (2-AG) is primarily degraded by monoacylglycerol lipase (MGL). We compared the peripheral analgesic effects of a novel irreversible MGL inhibitor, JZL184, a reversible MGL-preferred inhibitor, URB602, and exogenous 2-AG in rats. Methods: Pain perception in mice was assessed using a formalin assay. Mice were administered the following treatments via dorsal paw injection: JZL184 (0.001–300 µg), URB602 (0.001–600 µg), 2-AG (ED50), 2-AG + JZL184 (ED50), 2-AG + URB602 (ED50), AM251 (80 µg), AM251 + JZL184 (10 µg), AM630 (25 µg), or AM630 + JZL184 (10 µg). To evaluate the effects of MGL inhibitors on endocannabinoid accumulation and the activity of its metabolic enzymes. Main results: Intraclinical injection of JZL184, URB602, and 2-AG inhibited the early and late stages of formalin-induced pain. JZL184 and URB602 share the same mechanism of action. JZL184 (ED50: Phase I: 0.06 ± 0.028 µg; Phase II: 0.03 ± 0.011 µg) was superior to URB602 (ED50: Phase I: 120 ± 51.3 µg; Phase II: 66 ± 23.9 µg) or 2-AG in analgesia. The analgesic effects of the two MGL inhibitors combined with 2-AG exhibited an additive effect. Similar to URB602, the analgesic effect of JZL184 can be blocked by cannabinoid receptor 1 (CB₁) and cannabinoid receptor 2 (CB₂) antagonists. JZL184 inhibited MGL activity in vitro but did not inhibit the activity of fatty acid amide hydrolase or N-arachidonic acid phosphatidylethanolamine phospholipase D. URB602 increased the content of 2-AG in the hind claw but did not change the level of arachidonic acid ethanolamine (anandamide). Conclusions and significance: MGL inhibitors inhibit formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increases the accumulation of 2-AG in the claw skin, thereby mediating these effects. MGL is a target for the treatment of inflammatory pain. [4]

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1. URB602 is a selective, reversible monoacylglycerol lipase (MAGL) inhibitor, which is the main enzyme responsible for the degradation of endogenous cannabinoid 2-arachidonic acid glyceride (2-AG) in the central nervous system and peripheral tissues. [2]
2. The analgesic effect of URB602 is achieved by increasing the level of endogenous 2-AG, which activates cannabinoid CB1 receptors in the nervous system and CB2 receptors in immune cells, thereby producing anti-nociceptive and anti-inflammatory effects. [1,4]
3. URB602 is a peripherally selective MAGL inhibitor that does not cross the blood-brain barrier at therapeutic doses, thus avoiding the side effects of central cannabinoids, such as sedation, cognitive impairment and motor incoordination. [4]
4. Because URB602 can selectively modulate the peripheral endocannabinoid signaling pathway, it is being investigated as a potential therapeutic agent for inflammatory pain, neuropathic pain, and gastrointestinal disorders such as irritable bowel syndrome [3,4]
5. Stress-induced analgesia is partly mediated by activation of the endocannabinoid system; URB602 enhances this effect by inhibiting the degradation of 2-AG, thus providing a novel mechanism for the regulation of stress-related pain [1]

C19H21NO2

295.38

295.157

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

1

2

4

22

345

0

O(C(N([H])C1=C([H])C([H])=C([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=C1[H])=O)C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

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.)