Fatty-acid amide hydrolase (FAAH)

At an IC50 value of approximately 0.50 nM, URB-597 (KDS-4103) inhibits the hydrolysis of [3H]anandamide by primary cultures of rat cortical neurons, which is catalyzed by FAAH[1].

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When FAAH activity was assayed in membrane fractions using [3H]anandamide (anandamide[ethanolamine-3H]) as a substrate, URB-597 (KDS-4103) was found to inhibit rat brain activity with an IC50 of ~5 nM and human liver activity with an IC50 of 2.5 nM (Kadmus Pharmaceuticals, unpublished results). URB-597 (KDS-4103) was also found to prevent the FAAH-catalyzed hydrolysis of [3H]anandamide by primary cultures of rat cortical neurons with an IC50 value of ~0.50 nM. Notably, KDS-4103 selectively blocks the breakdown of [3H]anandamide without reducing its carrier-mediated uptake, causing non-metabolized [3H]anandamide to accumulate in, and eventually exit from, the neurons. Thus, after a 4-min incubation with [3H]anandamide, the intracellular content of non-metabolized [3H]anandamide is markedly higher in inhibitor-treated than in control neurons (Fig. 2A). Similar results were obtained with KDS-4101, a carbamate-based FAAH inhibitor that is weaker than KDS-4103 at inhibiting FAAH activity (Fig. 2A). Importantly, the anandamide transport blocker N- (4-hydroxyphenyl) eicosa-5,8,11,15-tetraenamide (AM404) has an opposite effect, significantly reducing [3H]anandamide internalization (Fig. 2A). When neurons treated with URB-597 (KDS-4103) are exposed for 4 min to [3H]anandamide and then incubated for 15 min in an [3H]anandamide-free solution, ~43% of the accumulated [3H]anandamide is released back into the medium (Fig. 2B). This process is linear with time (Fig. 2B) and is not inhibited by AM404 (Fig. 2A), suggesting that it is due to passive diffusion rather than reverse transport. No such time-dependent release is observed in control untreated neurons, the medium of which only contains residual levels of [3H]anandamide carried over from the preincubation period. Together, these studies demonstrate that KDS-4103 is potent at blocking anandamide hydrolysis in rat brain neurons without having any inhibitory effect on anandamide transport. As a result of its ability to block FAAH activity, URB-597 (KDS-4103) causes extracellular levels of anandamide to increase [1].

In rodents that are either FAAH-null (-/-) or wild-type (+/+), URB-597 (KDS-4103) suppresses rat brain FAAH activity following intraperitoneal (ip) treatment at a median inhibitory dosage (ID50) of 0.15 mg/kg[1]. Treatment with CB1 receptor antagonists prevents the strong anxiolytic, antidepressant, and analgesic effects that KDS-4103 (0.1-0.3 mg/kg, ip) elicits in rats and mice[1]. Rats and cynomolgus monkeys may consume KDS-4103 orally[1]. After taking 10 mg/kg orally, URB-597 inhibits FAAH in the brain quickly (about an hour), maintaining >90% through 12 hours, and >60% through 24 hours.[1]

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By intraperitoneal (i.p.) injections URB-597 (KDS-4103) produced a profound dose-dependent inhibition of brain FAAH activity in rats, which is half-maximal at ~0.15 mg
kg. After injection of URB-597 (KDS-4103) (0.3 mg
kg, i.p.), FAAH inhibition is rapid in onset (<15 min), persistent (>12 h) (Fig. 3A), and accompanied by significant elevations in the brain content of anandamide (Fig. 3B) and other fatty-acid ethanolamides that are substrates for FAAH (14). Similar changes in FAAH activity and fatty-acid ethanolamide levels are also observed in peripheral tissues. Significantly elevated anandamide levels are also observed in the brains of FAAH–
– mice, and URB-597 (KDS-4103) (0.3 mg
kg, i.p.) does not further alter these levels. In agreement with its inability to affect monoglyceride lipase (MGL) activity (Table 1), URB-597 (KDS-4103) does not change the brain content of the endocannabinoid 2-arachidonoylglycerol (2-AG), a primary substrate for this enzyme. As previously observed in mutant FAAH–
– mice, FAAH inhibition is associated with increased sensitivity to the administration of exogenous anandamide. Thus, KDS-4103 (0.3 mg
kg, i.p.) markedly enhances the hypothermic response produced by a sub-threshold dose of anandamide (5 mg
kg, i.p.), although it has no effect on body temperature when injected alone (Fig. 4A). The effect of anandamide plus KDS-4103 is mediated by CB1 receptors, since it is prevented by the CB1 antagonist rimonabant. Notably, KDS-4103 does not further increase the sensitivity to anandamide in FAAH–
– mice (Fig. 4B), supporting the notion that FAAH inhibition plays an exclusive role in mediating the actions of KDS-4103 [1].

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In vivo, URB-597 (KDS-4103) inhibits rat brain FAAH activity after intraperitoneal (i.p.) administration with a median inhibitory dose (ID(50)) of 0.15 mg/kg. The compound does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice URB-597 (KDS-4103) elicits significant, anxiolytic-like, antidepressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB(1) receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1,500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were observed in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1,500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.

In vitro pharmacology screening showed that URB-597 (KDS-4103) does not affect the activities of various serine hydrolases including human and electric-eel acetylcholinesterase, horse plasma butyryl cholinesterase, and rat brain MGL (Table 1). As noted above, the lack of MGL inhibition is particularly important because of the involvement of this enzyme in the biological inactivation of the endocannabinoid 2-AG. In vitro screening for interactions with other members of the endocannabinoid system demonstrated that KDS-4103 has no effect on anandamide transport or CB1 and CB2 receptor binding (see above). Furthermore, at a test concentration of 10 ìM, KDS-4103 was found not to significantly interact with a broad panel of receptors, ion channels, neurotransmitter transporters and enzymes (Table 1), cytochrome P450 isoforms (Table 2), or HERG channel activity (not shown). In a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain protein extracts, URB-597 (KDS-4103) was found to prevent FPR binding to triacylglycerol hydrolase (TGH) with an IC50 of 192 nM. However, direct in vitro measurements of enzyme activity showed that KDS-4103 has no effect on either TGH (rat liver) or triacylglycerol lipase (rat white adipose tissue) activity at concentrations as high as 10 ìM. Moreover, in vivo administration of KDS-4103 (3 mg
kg, i.p.) failed to alter triacylglycerol levels in rat liver and white adipose tissue, although it significantly inhibited FAAH activity in the same tissues (Clapper and Piomelli, unpublished results). Collectively, these results indicate that KDS-4103 is remarkably selective for FAAH [1].

Animal/Disease Models: Wistar rats[1]
Doses: 250, 500, 750, 1000, 1250 mg/kg (pharmacokinetic/PK Analysis)
Route of Administration: Oral administration
Experimental Results: Absorbed at a moderate rate with peak plasma concentrations (Cmax) achieved at 1.2 h after treatment. The oral elimination half-life was approximately 2 h.

Pharmacokinetics [1] This study evaluated the pharmacokinetic properties of KDS-4103 suspension in rats after oral administration. KDS-4103 was absorbed at a moderate rate, reaching peak plasma concentration (Cmax) 1.2 hours after administration. The oral elimination half-life of KDS-4103 was approximately 2 hours. The exposure of KDS-4103 (area under the curve, AUC, and Cmax) was linear in the dose range of 10 to 1000 mg·kg (Figure 8). In brain tissue, the peak concentration and AUC of KDS-4103 were similar to those in plasma. The maximum concentration of KDS-4103 in brain tissue was reached approximately 1 hour after administration. Therefore, after oral administration of 10 mg/kg, FAAH inhibition in the brain was rapid (1 hour), with an inhibition rate >90% within 12 hours and >60% within 24 hours (Figure 9).

Preclinical Safety Assessment [1] Formal preclinical safety studies have been initiated to support the Investigational New Drug (IND) application for KDS-4103. To date, single-dose, 7-day, and 28-day repeated-dose studies have been completed in rats; single-dose and 7-day repeated-dose studies have been completed in cynomolgus monkeys, and a 28-day repeated-dose study is currently underway. Monkeys were chosen as a non-rodent model due to the limited oral bioavailability in dogs. No signs of systemic toxicity were observed in rats at single oral doses up to 2000 mg/kg and in monkeys up to 1500 mg/kg. In rats, this dose is at least 40 times the dose required to inhibit FAAH activity in the brain to below 10% of baseline levels. These results suggest a therapeutic index of at least 20, given that plasma exposure at an oral dose of 1000 mg/kg is 20 times the effective dose. No treatment-related clinical symptoms were observed during repeated daily administration of 1500 mg/kg in rats (28 days) and monkeys (currently 21 days). Final assessments (7 days after monkeys, 28 days after rats), including blood chemistry, hematology, and necropsy, revealed no signs of toxicity. Central nervous system safety pharmacology assessments in rats (including complete functional observations) showed no significant adverse reactions after oral administration at doses up to 1500 mg/kg. No defects were observed in the rotarod test after intraperitoneal injection of KDS-4103 at doses up to 5 mg/kg (equivalent to 33 times the brain's FAAH activity ID50). Furthermore, in vitro bacterial cytotoxicity tests and Ames tests were negative. Cardiovascular and respiratory safety pharmacology studies evaluating oral doses of KDS-4103 at 50, 275, and 1500 mg/kg will be completed in the first half of 2006.

[1]. Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597). CNS Drug Rev. Spring 2006;12(1):21-38.

URB597 belongs to the biphenyl class of compounds. URB-597 is a small molecule drug currently in Phase I clinical trials and has one investigational indication.

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Analgesic activity[1]
In the mouse hot plate test, KDS-4103 showed moderate analgesic activity. This test is used to measure the response of animals to harmful thermal stimuli. In this model, intraperitoneal injection of 0.5 μg/kg of KDS-4103 slightly but significantly prolonged the latency of response. These effects were blocked by the CB1 receptor antagonist rimonaban (0.2 μg/kg, intravenous injection). More significant analgesic effects of acute doses of KDS-4103 (0.1–0.3 μg/kg, intraperitoneal injection) were recently observed in a fully Freund's adjuvant-induced rat model of arthritis pain. These effects were blocked by the CB1 antagonist AM251, suggesting that the blockade of FAAH activity may lead to CB1-mediated anti-ectopic pain effects in inflammatory pain models. Finally, administration of KDS-4103 enhanced the analgesic effect induced by foot shock in rats, which was mediated by the release of arachidonic acid ethanolamine (anandamide) and 2-arachidonic acid glyceride (2-AG) from the periaqueductal gray matter of the brainstem. Lack of cannabinoid-like activity and abuse risk [1] Although KDS-4103 increases the level of arachidonic acid ethanolamine in the brain, the compound does not reproduce the pharmacological response profile produced by exogenous arachidonic acid ethanolamine or other cannabinoid agonists. Systemic injection of KDS-4103 (0.3 mg/kg, intraperitoneal injection) maximally blocked FAAH activity but did not cause rigidity (immobility), hypothermia, or overeating, all three of which are typical manifestations of CB1 receptor activation. More importantly, KDS-4103 had no effect on either of the two abuse-prone rat models—the conditioned position preference (CPP) test and the drug discrimination (DD) test. In the CPP test, acute intraperitoneal injection of KDS-4103 (0.03–0.3 mgu/kg) did not show any preference compared to solvent or direct cannabinoid agonist Win-55212-2. Similarly, in the DD test, intraperitoneal injection of KDS-4103 (0.1–3 mgu/kg) also failed to replace plant-derived cannabinoid α9-THC or synthetic cannabinoid agonist Win-55212-2 (Figure 7). Other pharmacological properties [1] The advent of KDS-4103 has spurred extensive research to explore the pharmacological properties of FAAH inhibitors. For example, in experiments with spontaneously hypertensive rats, administration of KDS-4103 (0.1–1 mg u0001 kg) via parenteral route (intraperitoneal injection) reduced blood pressure, myocardial contractility, and vascular resistance to levels similar to those in normotensive animals. CB1 receptor antagonists blocked these effects. Similar responses were observed in two hypertension models with different mechanisms—Dahl salt-sensitive rats and chronic angiotensin II infusion rats—but not in normotensive control rats. These results suggest that KDS-4103 may normalize blood pressure in hypertensive rats by enhancing the cardioinhibitory and vasodilatory effects of endogenous anandamide, thus potentially providing a novel therapeutic strategy for hypertension. In another study, KDS-4103 (0.1–3 mgu0001kg, intraperitoneal injection) was found to dose-dependently reduce carrageenan-induced edema in mice. This effect was blocked by the CB2 receptor antagonist SR144528 but not by the CB1 receptor antagonist AM251. FAAH may have pro-inflammatory effects, further suggested by the reduced response to inflammatory stimuli observed in FAAH knockout mice. [1] KDS-4103 and its analogues represent a new class of drugs that inhibit the inactivation of arachidonic acid ethanolamine by targeting the intracellular enzyme activity of FAAH. The IC50 value of KDS-4103 inhibiting FAAH activity in rat meninges in vitro was approximately 5 nM, approximately 0.5 nM in intact rat neurons in vitro, and approximately 3 nM in human liver microsomes in vitro. The ID50 value in rats after intraperitoneal administration was 0.15 mg/kg. KDS-4103 exhibits significant selectivity for FAAH and inactivity against other cannabinoid-related targets, including cannabinoid receptors, arachidonic acid ethanolamine (AEA) transporters, and monoglyceride lipases (enzymes involved in the inactivation of endocannabinoid esters 2-AG). This target selectivity is consistent with the lack of pronounced cannabinoid effects in vivo. Therefore, at doses that almost completely inhibit FAAH activity and significantly increase brain AEA levels, KDS-4103 does not induce rigidity, hypothermia, or food stimulation—three key hallmarks of cannabinoid receptor activation. Furthermore, the compound did not show abuse potential in either animal model. KDS-4103 elicits anxiolytic, antidepressant, and analgesic responses consistent with its ability to inhibit FAAH and can be inhibited by CB1 receptor blockers. These findings suggest that the mechanism of action of KDS-4103 is through enhancing the sustained action of arachidonic acid ethanolamine on certain CB1 receptors, which may normally be involved in controlling mood and pain. In addition, KDS-4103 can reduce carrageenan-induced inflammation in mice and restore blood pressure to normal in a hypertensive rat model. To date, KDS-4103 is easy to mass-produce, has good oral bioavailability and safety, indicating that it is a promising new therapeutic agent for the treatment of a variety of important diseases, including anxiety, depression and pain. [1]

C20H22N2O3

338.4003

338.163

C, 70.99; H, 6.55; N, 8.28; O, 14.18

546141-08-6

1383884

White to off-white solid powder

1.2±0.1 g/cm3

533.2±50.0 °C at 760 mmHg

276.3±30.1 °C

0.0±1.4 mmHg at 25°C

1.618

3.51

2

3

5

25

462

0

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

ROFVXGGUISEHAM-UHFFFAOYSA-N

InChI=1S/C20H22N2O3/c21-19(23)16-8-4-6-14(12-16)15-7-5-11-18(13-15)25-20(24)22-17-9-2-1-3-10-17/h4-8,11-13,17H,1-3,9-10H2,(H2,21,23)(H,22,24)

[3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate

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)

Solubility in Formulation 1: 10 mg/mL (29.55 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 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 (7.39 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 (7.39 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 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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