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rat brain activity with an IC50 of ~5 nM and human liver activity with an IC50 of 2.5 nM (Kadmus Pharmaceuticals, unpublished results).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]
The pharmacokinetic properties of KDS-4103 were assessed in rats following oral administration as a suspension. KDS-4103 was absorbed at a moderate rate with peak plasma concentrations (Cmax) achieved at 1.2 h after treatment. The oral elimination half-life of KDS-4103 was approximately 2 h. Linear exposure (Area Under the Curve, AUC, and Cmax) of KDS-4103 was observed at doses of 10 to 1000 mg
kg (Fig. 8). In the brain, peak concentrations and AUC values for KDS-4103 were similar to those observed in plasma. Maximum brain levels of KDS-4103 were reached approximately 1 hour after administration. Accordingly, FAAH inhibition in the brain was rapid (1 h), sustained at >90% through 12 h and >60% through 24 h after an oral dose of 10 mg
kg (Fig. 9).

PRECLINICAL SAFETY EVALUATION [1]
Formal preclinical safety studies to support an Investigational New Drug Application (IND) for KDS-4103 have been initiated. To date, single-dose and 7- 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 ongoing. Monkeys were chosen as the non-rodent species due to limited oral bioavailability in dogs. No signs of systemic toxicity have been noted at single oral doses up to 2000 mg
kg in rats or 1500 mg
kg in monkeys. In rats, this dose is at least 40-fold greater than that required to inhibit brain FAAH activity to less than 10% of baseline levels. Considering that plasma exposure at 1000 mg
kg p.o. is 20-fold greater than the effective dose, these results suggest a therapeutic index of at least 20. No treatment-related clinical observations were noted during repeated daily dosing at 1500 mg
kg in rats (28 days) and monkeys (through 21 days to date). Terminal evaluations (following 7 days in monkeys and 28 days in rats) including blood chemistry, hematology, and gross necropsy showed no signs of toxicity. CNS safety pharmacology assessment involving a complete functional observation battery in rats revealed no overt effects following oral doses up to 1500 mg
kg. No deficits were observed on the rotarod test in rats after treatment with KDS-4103 at doses up to 5 mg
kg, i.p. (33-fold over the ID50 for FAAH activity in the brain). Furthermore, in vitro bacterial cytotoxicity and Ames testing yielded negative results. Cardiovascular and respiratory safety pharmacology studies evaluating oral doses of 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 is a member of biphenyls. URB-597 is a small molecule drug with a maximum clinical trial phase of I and has 1 investigational indication.

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Analgesic Activity [1]
KDS-4103 was shown to exert moderate antinociceptive actions in the mouse hot-plate test, which measures the response of the animal to a noxious thermal stimulus. In this model, the compound modestly but significantly lengthened response latencies at a dose of 0.5 mg
kg, i.p.. These effects were prevented by the CB1 antagonist rimonabant (0.2 mg
kg, i.v.). More marked analgesic effects of acute doses of KDS-4103 (0.1–0.3 mg
kg, i.p.) were recently observed in the complete Freund’s adjuvant model of arthritis pain in the rat. These effects were prevented by the CB1 antagonist AM251, indicating that blockade of FAAH activity may result in CB1-mediated antiallodynic effects in an inflammatory pain model. Finally, administration of KDS-4103 enhanced a form of foot shock-induced analgesia in rats, which is mediated by release of anandamide and 2-AG in the brainstem periaqueductal gray Absence of Cannabinoid-Like Activity and Abuse Liability [1]
Though KDS-4103 increases brain anandamide levels, this compound does not reproduce the spectrum of pharmacological responses produced by exogenous anandamide or other cannabinoid agonists. Systemic doses of KDS-4103 (0.3 mg
kg, i.p.) that maximally block FAAH activity were found to produce no catalepsy (rigid immobility), hypothermia or hyperphagia, three typical signs of CB1 receptor activation. Even more importantly, KDS-4103 was shown to have no effect on two rat models of abuse liability, the conditioned place preference (CPP) test and the drug discrimination (DD) test. In the CPP test, acute administration of KDS-4103 (0.03–0.3 mg
kg, i.p.) produced no preference compared to vehicle or to the direct cannabinoid agonist Win-55212-2. Likewise, in the DD test KDS-4103 (0.1–3 mg
kg, i.p.) did not substitute for the plant-derived cannabinoid Ä9-THC or the synthetic cannabinoid agonist Win-55212-2 (Fig. 7). Additional Pharmacological Properties [1]
The availability of KDS-4103 has prompted a broad range of efforts to investigate the pharmacological properties of FAAH inhibitors. For example, experiments in spontaneously hypertensive rats have shown that by parenteral administration KDS-4103 (0.1–1 mg
kg, i.p.) reduces blood pressure, cardiac contractility, and vascular resistance to levels similar to those found in normotensive animals. These effects were prevented by administration of CB1 antagonists. Similar responses were observed in two mechanistically distinct models of hypertension — Dahl salt-sensitive rats and chronic angiotensin II infusion in rats — but not in normotensive control rats. These results suggest that KDS-4103 may normalize blood pressure in hypertensive rats by amplifying the cardiodepressor and vasodilator effects of endogenous anandamide and, as such, may offer a novel therapeutic strategy for the treatment of hypertension. In another study, KDS-4103 (0.1–3 mg
kg, i.p.) was found to reduce dose-dependently carrageenan-induced edema formation in mice, an effect that was blocked by the CB2 antagonist SR144528, but not by the CB1 antagonist AM251. A possible proinflammatory role of FAAH is further suggested by the reduced response to inflammatory stimuli observed in FAAH-null mice. [1]
KDS-4103 and its analogs represent a novel class of agents that prevent anandamide deactivation by targeting the intracellular enzymatic activity of FAAH. KDS-4103 inhibits FAAH activity with an IC50 value of approximately 5 nM in rat brain membranes in vitro, 0.5 nM in intact rat neurons in vitro, 3 nM in human liver microsomes in vitro, and an ID50 value of 0.15 mg
kg following i.p. administration in the rat. KDS-4103 has a remarkable selectivity for FAAH with no activity on other cannabinoid-related targets, including cannabinoid receptors, anandamide transport and monoglycerol lipase (the enzyme involved in the deactivation of the endocannabinoid ester 2-AG). Such target discrimination is matched by a lack of overt cannabimimetic effects in vivo. Thus, at doses that almost abolish FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke catalepsy, reduce body temperature or stimulate feeding, three key signs of cannabinoid receptor activation. Moreover, the compound does not display abuse liability in two animal models. KDS-4103 does elicit anxiolytic-like, antidepressant-like and analgesic responses, which parallel its ability to inactivate FAAH and are prevented by CB1 receptor blockade. These findings indicate that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB1 receptors, which may normally be engaged in controlling emotions and pain. In addition, KDS-4103 reduces carageenan-induced inflammation in mice and normalizes blood pressure in rat models of hypertension. To date, the ease of large-scale manufacturing, oral bioavailability, and safety profile indicate that KDS-4103 is a promising new therapeutic agent for a variety of significant medical conditions, 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.)