Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide

Abstract

1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12 - 18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of approximately 5. Homologues with chain lengths < or = eight carbon atoms gave < 20% inhibition at 100 microM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 microM) and palmitoylisopropylamide (30 and 100 microM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB(1) or CB(2) receptors.

Figure 1

Structures of palmitoylethanolamide (C16:0), oleoylethanolamide (C18:1), palmitoylisopropylamide (C16:0) and R-palmitoyl-(2-methyl)ethanolamide (C16:0).

Figure 2

Inhibition of the hydrolysis by rat brain membranes of 2 μM [³H]-AEA by arachidonoyl-serotonin, AM404, arvanil and PTMK. Data are means±s.e. mean (when not enclosed by the symbol), n=3–6, with no preincubation with the compounds and membranes prior to addition of [³H]-AEA. Analysis of the data using a constant ‘top' value of 100 and variable slope (see Methods) gave ‘bottom' values (i.e. the plateau value of the remaining activity not inhibited by the compounds) of: arachidonoyl-serotonin, 3.7±2.2% (−0.8 to +8%); AM404, 1.3±2.1% (−3.1 to +5.7%); arvanil, 0.9±1.9% (−2.9 to +4.7%); PTMK, 0.3±1.4% (−2.5 to +3.0%) (means±s.e.mean, with 95% confidence intervals in parentheses). With the ‘bottom' values set to zero, the pI₅₀ values (means±s.e.mean) calculated from these data were 5.93±0.02, 5.44±0.02, 6.20±0.03 and 7.10±0.02 for arachidonoyl-serotonin, AM404, arvanil and PTMK, respectively.

Figure 3

Inhibition of the hydrolysis by rat brain membranes of 2 μM [³H]-AEA by a series of palmitoylethanolamide homologues (a) C16:0–C3:0 (b) C16:0–C18:0 and C18:1. Shown are means of 3–6 experiments, with no preincubation with the compounds and membranes prior to addition of [³H]-AEA. The data for palmitoylethanolamide are the same in both graphs.

Figure 4

Inhibition of the hydrolysis by rat brain membranes of 2 μM [³H]-AEA by a series of palmitoylethanolamide analogues. Shown are means of 3–4 experiments, with no preincubation with the compounds and membranes prior to addition of [³H]-AEA. The data for palmitoylethanolamide are the same as that shown in Figure 3.

Figure 5

Mode of inhibition of rat brain [³H]-AEA metabolism by (a) palmitoylisopropylamide and (b) oleoylethanolamide. Shown are means±s.e. mean of three experiments. Secondary replots of the mean data to illustrate the mixed-type nature of the inhibition are shown as inserts. The K_i(slope) and K_i(intercept) values were calculated from the mean data as described in Methods.

Figure 6

Effect of palmitoylethanolamide and related compounds upon the uptake of 10 μM [³H]-AEA into rat C6 glioma and RBL-2H3 basophilic leukaemia cells (a,b) shown are means±s.e.mean (n=3) of the total uptake in the absence and presence of cells. The numbers given above (or enclosed within) the columns are the specific uptake remaining in the presence of the test compounds, calculated as 100×(mean total uptake - mean uptake, no cells present)/(mean total uptake in the absence of compound - mean uptake, no cells present in the absence of compound). The dotted line shows the mean total uptake in the absence of test compound (i.e. ‘none'). (c) effects on the total uptake of [³H]-AEA into RBL-2H3 cells. Shown are means±s.e.mean, n=3. The dotted line shows the mean total uptake in the absence of test compound (i.e. 100%). The dashed line is a retroactive estimation of the mean percentage of the total [³H]-AEA uptake due to binding to the cell culture wells, determined from the data shown as ‘none' for the RBL-2H3 cells in panel a. The unfilled columns in the experiments shown in panel c were calculated from means of three separate experiments where the effects of the compounds upon the binding to [³H]-AEA to culture wells was determined. The observed figure was then normalized to 52% (i.e. the dashed line). Thus, for example, a compound producing a 34% inhibition of the binding to the well would have an unfilled column of 52×(0.66)=34% of total [³H]-AEA uptake.