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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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PF-3845 is a potent, selective and irreversible FAAH inhibitor with Ki of 230 nM. |
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Targets
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FAAH |
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IC50 |
230 nM (Ki) [1] |
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In Vitro
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PF-3845 selectively inhibits FAAH by carbamylating FAAH’s serine nucleophile. [1] |
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In Vivo
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PF-3845 treated mice (10 mg/kg, i.p.) shows rapid and complete inactivation of FAAH in the brain, as judged by competitive activity-based protein profiling (ABPP) with the serine hydrolase-directed probe fluorophosphonate (FP)-rhodamine. PF-3845 shows a long duration of action up to 24 hour. PF-3845-treated mice also shows dramatic (>10-fold) elevation in brain levels of AEA and other NAEs (N-pamitoyl ethanolamine [PEA] and N-oleoyl ethanolamine [OEA]). FAAH is AEA-degrading enzyme fatty acid amide hydrolase. PF-3845 (1–30 mg/kg, oral administration [p.o.]) causes a dose dependent inhibition of mechanical allodynia with a minimum effective dose (MED) of 3 mg/kg (rats are analyzed at 4 hour post dosing with PF-3845). At higher doses (10 and 30 mg/kg), PF-3845 inhibits pain responses to an equivalent, if not greater,degree than the nonsteroidal anti-inflammatory drug naproxen (10mg/kg, p.o.). [1] PF-3845 (10 mg/kg, i.p.) significantly reverses LPS-induced tactile allodynia, but doesn’t modify paw withdrawal thresholds in the saline-injected paw. [2] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| FAAH Assay |
The GDH-coupled FAAH assay is performed for determination of potencies (Kinact/ Ki values) of irreversible inhibitors. For the development of structure-activity relationship,the FAAH assay is performed in 384-well microplates with a final volume of 50μL. In the 384-well format assay, the overall potency, Kinact/ Ki values, are generally calculated from the slope, Kinact/[ Ki (1 + [S]/ Km)], which is obtained from the kobs versus [I] linear lines under the conditions of [I] <>i as described (from the kobs values fitted to equation 3 in PNAS 2008). In order to obtainKinact and Ki values separately for PF-3845 and URB597, the FAAH assay is performed in the 96-well format with a final volume of 200μL. Due to its more frequent reading capabilities (every 10 and 30 seconds for the 96- and 384-well formats, respectively), the 96- well format allows a measurement of Kobs values at higher concentrations of inhibitors. The Kinact and Ki values are obtained by fitting the kobs versus [I] curves to the equation, Kobs = Kinact [I]/{[I] + Ki (1 + [S]/ Km}. FAAH inactivation rates in the absence of inhibitors are subtracted from all kobs values obtained in the presence of inhibitors. The ratio Kinact/ Ki calculated from the individual Kinact and Ki values obtained separately agrees well with the Kinact/ Ki value obtained from the slope described above. |
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Animal Study
[1]
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| Animal Models |
Male C57BL/6 mice |
| Formulation |
PF-3845 is dissolved at 1 mg/ml by sonication and vortexing directly into a solution of 18:1:1 v/v/v saline:emulphor:ethanol. |
| Doses |
10 mg/kg or 1-30 mg/kg |
| Administration |
Administered via i.p. 1 hour before sacrificed by CO2 or oral administration. |
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