Fig 1.
Fatty acid ethanolamides and FAAH inhibitor URB597.
Fig 2.
General formulas of PEA prodrugs reported in Table 1.
Table 1.
Stability data for palmitoylethanolamide (PEA, 1) and compounds 3–5, 7–9, 19–24, 26–31, 33.
Fig 3.
Synthesis of acyloxymethylcarbonates 3–5.
Reagents and conditions: (a) chloromethyl chloroformate, pyridine, DCM, 0°C, 1 h; (b) RCOOCs, tetra-butyl ammonium iodide, acetonitrile, 80°C, 12 h.
Fig 4.
Reagents and conditions: (a) p-nitrophenyl chloroformate, pyridine, DCM, 0°C to rt, 3 h; (b) RCH(NH2)COOH, Na2CO3, tert-butanol, water, 50°C, 12 h.
Fig 5.
Synthesis of amino acid esters of PEA.
Reagents and conditions: (a) RCH(NHBoc)COOH, DCC, DCM, 23°C, 12 h; (b) HCl, AcOEt, rt, or TFA/DCM 1:1 v/v, 0°C, 1–3 h, then HCl in AcOEt; (c) H2, Pd/C 10% w, HCl, EtOH, rt; (d) N-Boc-1-O-Bn-L- aspartic acid, DCC, DCM, 23°C, 12 h.
Fig 6.
Synthesis of L-Val-(R)-α-methyl-PEA hydrochloride (33).
Reagents and conditions: (a) N-Boc-L-Valine, DCC, DCM, 23°C, 12 h; (b) TFA/DCM 1:1 v/v, 0°C, 3 h then HCl, rt.
Fig 7.
L-Val-PEA (20) biotransformation and PEA release in rat plasma.
Time course of L-Val-PEA (20, ●) hydrolysis in 80% v/v rat plasma and corresponding release of PEA (1,○).
Fig 8.
Role of amidase activity on compound degradation in rat liver homogenate.
PEA levels in rat liver homogenate after addition of 50 nm PEA (left) or 50 nm L-Val-PEA prodrug (right) and pre-treatment with FAAH inhibitor URB597 at the final concentration of 100 nm (○) and 10 μm (▲), if compared to controls (●); data are expressed as means ± SEM, n = 3. * P<0.05; ** P<0.005.
Fig 9.
Docking of L-Val-PEA (20) into rat FAAH active site.
Representation of L-Val-PEA (20) docked into rat FAAH active site. The amide group undertakes hydrogen bonds with the two NH groups of I238 and G239 belonging to the oxyanion hole and with the backbone carbonyl of M191 (not shown). The catalytic triad S217, S241 and K142 is also displayed. The palmitoyl portion fits the acyl chain binding (ACB) channel and the valine occupies the cytosolic port.
Fig 10.
Prodrug and PEA plasma levels after oral administration to rats.
a-c) Plasma levels of PEA (●), 20 (□) and 28 (■) after a single oral administration to male Wistar rats of equimolar doses (PEA: 100 mg kg-1; 20 and 28: 145 mg kg-1). d-e.) Plasma levels of released PEA after a single oral administration of 20 (□) and 28 (■). Data are expressed as means ± SEM, n = 9.
Table 2.
Plasma concentrations (nM) after p.o. administration of PEA (1), 20 and 28.a