N-Amino acid linoleoyl conjugates: anti-inflammatory activities.

A class of lipoamino acids, long chain fatty acids covalently coupled to amino acids, which we have termed elmiric acids (EMA)1, 2, are emerging as an important family of endogenous signaling molecules 3 that act as physiological regulators of pain and inflammation4. The existence of these endogenous substances was first predicted in 1997 when synthetic examples were produced and shown to exhibit anti inflammatory and analgesic activity in mice5. Subsequent studies identified several naturally occurring EMAs in rat brain extracts 6. To date, about 50 naturally occurring members as well as several synthetic analogs of the EMA family have been identified15, 16, 2. Their actions include analgesia 4, 7, 8, anti-inflammatory effects 2, selective inhibition of cancer cell proliferation 9, vasodilation 10, cell migration11, 12 and calcium ion mobilization 13, 14. The prototypic EMA, N-arachidonylglycine (NAgly) shown in Figure 1, is found in rat brain, spinal cord, and other tissues where it occurs in amounts greater than the closely related endocannabinoid, anandamide4. Early reports4, 5, suggested that NAGly possessed analgesic properties but lacked the psychotropic activity of the cannabinoids. It has been shown that NAgly has low affinity for the cannabinoid CB1 receptor 17, however, it appears to activate the orphan G-protein coupled receptor (GPCR), GPR1814, 18. In this report, we have focused on lipoamino acids containing a linoleoyl residue for the purpose of discovering a template structure better suited toward the design of promising drug candidates (Figure 2). The simplest member of this series is N-linoleoylglycine (LINgly) or, using the elmiric acid system, EMA-1 (18:2). The notations used in this report are as follows: Amino acid EMA names are: Glycine, EMA-1; Alanine, EMA-2; Phenylalanine, EMA-9 and Tyrosine, EMA-10. Fatty acid abbreviations are: Palmitic, 16:0; Linoleic, 18:2; Arachidonic, 20:4, For a more complete list see reference 1. Figure 1 The structure of NAgly Figure 2 Structures of the novel elmiric acids A widely used test for anti inflammatory action is the mouse peritonitis assay. This test is based on the migration of leukocytes into the peritoneal cavity following the injection of a pro inflammatory agent into the cavity. The inhibition of this migration is considered to be a measure of the anti inflammatory potential of a compound. Figure 3 Panel A shows the results obtained when LINgly24 at doses ranging from 0.3 to 20 mg/kg was subjected to this test. The values obtained suggest that the ED-50 is less than 0.3 mg/kg, which would make it a relatively potent anti inflammatory agent. Since it was administered orally, it is also indicative of its stability and good bioavailability. Thus the prototypic structure for this study, LINgly, appears to have been well chosen. The data shown in Panel B relates to a putative mechanism of action for the anti-inflammatory activity of LINgly and will be discussed in below. Figure 3 N-linoleoylglycine inhibits leukocyte migration (A) and increases PGJ production (B) in the mouse peritonitis model Literature reports indicate that the elevations of tissue concentrations of PGJ are associated with the resolution of an inflammatory response19. This was suggested to come about through the binding and activation of the transcription factor PPAR-γ followed by increased expression of anti inflammatory factors. Our previous studies showed a positive correlation between PGJ levels and an anti inflammatory action of the EMAs in vivo 1. A robust stimulation of PGJ by LINgly was observed in RAW cells over a concentration range of 0.5 to 10 μM (Table 1). The response was comparable to that shown by NAgly indicating no sacrifice of potency when going from four to two double bonds and decreasing the chain length by two carbon atoms. However, earlier reports showed that the palmitoyl analog PALgly had no anti inflammatory effect either in vitro 1 or in vivo 18 representing a dramatic change with the saturated, shorter chain analog. This is in contrast to an earlier report that describes PALgly as a modulator of calcium influx and nitric oxide production in sensory neurons 20. An explanation for such differences could be related to the binding affinities of these EMAs to GPR18, however, these data are currently not available. Table 1 LINgly shows similar potency compared to NAgly in the RAW cell model. The available data allow some comments to be made on a possible mechanism for the anti inflammatory effects of the linoleoyl sub family of EMAs reported on here. In previous publications, we have proposed a putative mechanism of action involving the activation of the arachidonic acid cascade leading to an elevation of eicosanoid products 1, 2, 18, 21. The initial step is the stimulation of the release of free arachidonic acid that can then promote the synthesis of specific anti inflammatory eicosanoids such as PGJ and LXA4. How these eicosanoids bring about a resolution of chronic inflammation is uncertain and is a topic currently being studied19, 22, 23. A possible role for GPR18 in the EMA promoted release reaction should be considered. Stereoisomeric preferences in a response are often an indication of receptor binding involvement in a particular response. Thus, we have compared the D and L enantiomers of N-linoleoylalanine in the PGJ model for anti inflammatory activity. The data shown in Table 2 indicate that there is, in fact, a considerable difference in activity with the D isomer being the more active. The decreases in cell count with increased drug are probably due to a decrease in proliferation rather than cell viability since we did not see many dead cells in the culture media. This high level of chiral preference suggests that a specific receptor may mediate the observed response. Again, based on our recent report 18, we would speculate that GPR18 is a likely mediator for this response. Table 2 Stereospecificity of EMA action in the RAW cell model. Figure 4 depicts a dramatic loss of activity when N-linoleoyl-D-phenylalanine (D-EMA-9(18:2)), and to a lesser extent N-linoleoyl-D-tyrosine, (D-EMA-10(18:2)) is substituted for N-linoleoyl-D-alanine (EMA-2 (18:2)). This could be attributed to steric volume increases or, possibly, to electronic effects. Further studies would be needed to distinguish between these possibilities. Figure 4 Electronic and steric effects on PGJ responses in RAW cells To summarize, a rank order (N-linoleoyl-D-alanine > N-linoleoylglycine > N-arachidonylglycine > N-linoleoyl-D-tyrosine > N-linoleoyl-L-alanine > N-linoleoyl-D-phenylalanine) of anti inflammatory action showing a complete range of responses was found even with the limited series of analogs studied in this report (Figure 5). Moreover, it may be concluded that linoleic acid analogs of NAgly can serve as lead molecules in a drug discovery program aimed at producing a stable and easily accessible drug candidate. By comparison, arachidonoyl derivatives are expensive and inherently less stable than linoleoyl derivatives and show a greater tendency to be non crystalline in nature Figure 5 Estimated rank order responses in PGJ RAW cell model