Potential anti-inflammatory actions of the elmiric (lipoamino) acids

doi:10.1016/j.bmc.2007.03.026

Bioorganic & Medicinal Chemistry

Volume 15, Issue 10, 15 May 2007, Pages 3345-3355

https://doi.org/10.1016/j.bmc.2007.03.026 Get rights and content

Abstract

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a marked increase in media levels of i15-deoxy-PGJ₂ with minimal effects on PGE production. It is reported in the literature that prostaglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating a connection between them.

Graphical abstract

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and tested for their possible use as anti-inflammatory agents. The compounds were assayed in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation.

Introduction

Acid congeners of anandamide or lipoamino acids, here named elmiric acids, exist as endogenous substances and may have a role in regulating tissue levels of anandamide.¹ This hypothesis received support from a study that showed that such a substance, N-arachidonylglycine (NAGly, Fig. 1), is indeed an endogenous constituent of many tissues and occurs at higher concentrations than anandamide.² An interesting property of NAGly is its potent inhibitory effect on FAAH, the enzyme primarily responsible for the termination of anandamide action via hydrolysis. As could be expected, NAGly treatment in both in vitro and in vivo models leads to a robust increase in anandamide concentrations.³ It was also observed that several of the elmiric acids profoundly reversed the prostaglandin (PG) profile from a pro inflammatory to an anti-inflammatory state in a cell culture model. This has formed the basis for a hypothesis on the mechanism for the potential anti-inflammatory action of these compounds. At this point, it is assumed that these two effects occur independently of one another, however, the possibility that the amidase inhibition and PG ratio effects are somehow interrelated cannot be ruled out.

The older literature on this topic is mainly concerned with lipoamino acids of bacterial origin.4, 5, 6, 7, 8 These involve amino acid conjugation with complex and unusual fatty acids, and little is known about their function in bacteria. More recently, attention has been given to the lipoamino acids present in mammalian species in part because of their possible relationships to the endocannabinoids. The origin of NAGly in vivo is not well understood, however, it is an endogenous substance found in rat brain and other sites that occurs in amounts greater than the closely related endocannabinoid, anandamide.² In this same report, it was suggested that NAGly might have analgesic properties similar to those reported for anandamide9, 10 but would be inactive in assays for psychotropic action such as the ring test.¹¹ The latter was in agreement with a report showing a lack of affinity by NAGly for the cannabinoid receptor, CB1.¹² This activity profile is reminiscent of that observed for THC-11-oic acid and its analog ajulemic acid.¹³ Based on preliminary data, NAGly appears to be the endogenous ligand for the orphan G-protein-coupled receptor GPR18.14, 15 Like anandamide, NAGly is also a substrate for COX-2 giving rise to amino acid conjugates of the prostaglandins.¹⁶ Other amino acid conjugates have been found in diverse tissues¹⁷ and N-arachidonoyl-l-serine has recently been isolated from rat¹⁷ and from bovine brain and reported to have vasodilatory effects in rat mesenteric arteries.¹⁸ Finally, NAGly inhibits the glycine transporter, GLYT2a, which is expressed in pre synaptic glycinergic neurons.¹⁹

We have defined the term elmiric acids as compounds that conform to the general structure shown in Figure 2 for which a short hand nomenclature system is proposed. Using this system N-arachidonylglycine would be written as: EMA-1 (20:4). EMA stands for elmiric acid; each amino acid constituent is assigned a number, for example, 1 = glycine; 2 = alanine, etc. The identity of the acyl substituent is indicated in parentheses; for example, (20:4) = arachidonoyl; (16:0) = palmitoyl, etc. This system would also accommodate any number of unnatural amino acids. We are proposing this nomenclature to simplify the naming of these compounds; it has not been approved or adopted by any official body.

In this report, we have screened in vitro a library of elmiric acids consisting of glycine and l-alanine conjugates with a series of ten fatty acids. The objective was to determine whether any of these compounds are candidates for further study as potential anti-inflammatory agents. Positive findings would support further studies in which the amino acid moieties would be expanded not only to include naturally occurring molecules but also synthetic examples. The ultimate goal is to discover a potent and efficacious molecule for the treatment of conditions characterized by acute and chronic inflammation.

Section snippets

Chemistry

Several of the elmiric acids were synthesized in the university laboratories using the procedures described below and shown in Scheme 1. Briefly this involved condensation of the acid chloride with the amino acid methyl ester in the presence of triethylamine followed by saponification with lithium hydroxide to yield the elmiric acid. However, the majority of the compounds came from a commercially available library of endocannabinoids obtained from Biomol International LP (Plymouth Meeting, PA

Effects on cell proliferation

Initially, the library shown in Table 1 was screened for anti-proliferative activity in an in vitro assay using the mouse macrophage cell line RAW264.7 as the drug target. Table 2 shows the results of this study in which each compound was tested at three concentrations, 0.1, 1.0, and 10 μM, and compared with vehicle treated cells as a control. Based on these limited data, it is not possible to assign a rank order for this group of substances, however, it is clear that many produced around 90%

Summary and conclusions

The goal of this project was to discover a structure–activity relationship for the elmiric (lipoamino) acids as potential anti-inflammatory agents. To this end, a screening procedure was developed to allow the selection of molecules for testing in intact animal models for anti-inflammatory activity. The procedure is based on the concept that prostaglandins can mediate either pro or anti-inflammatory actions depending on their molecular composition. While PGE₂ is dominant during the development

General chemical methods

All materials were obtained from commercial sources and used without further purification. For TLC, Silica Gel 60 F₂₅₄ plates from Merck were used with detection by UV light or iodine vapor chamber. Acid chlorides were obtained from Nu-chek Prep, Inc. (Elysian, MN). Amino acids and esters were from Sigma–Aldrich.

General procedure for synthesis

Compounds not available commercially in the Biomol library were prepared as follows.

Esterification: The amino acid (200 mg) is dissolved in 30 ml of methanol saturated with HCl and

Acknowledgments

This publication was made possible by Grants DA17969 (SHB) and DA13691 (RBZ) from National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD. Its contents are solely the responsibility of the author and do not necessarily represent the official views of the National Institute on Drug Abuse. We thank Dr. Akbar Ali for obtaining the NMR spectra of several of the elmiric acids used in this report and James and Barbara Evans for the mass spectral data.

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Potential anti-inflammatory actions of the elmiric (lipoamino) acids

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Effects on cell proliferation

Summary and conclusions

General chemical methods

General procedure for synthesis

Acknowledgments

Prostaglandins Other Lipid Mediat.

J. Biol. Chem.

Biochem. Pharmacol.

Biochim. Biophys. Acta

Chem. Phys. Lipids

FEMS Immunol. Med. Microbiol.

Prog. Neurobiol.

Pharmacol. Ther.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Life Sci.

Bioorg. Med. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Life Sci.

Biochem. Pharmacol.

Int. J. Immunopharmacol.

J. Immunol. Methods

J. Immunol. Methods

Adv. Exp. Med. Biol.

J. Bacteriol.

Proc. Natl. Acad. Sci. U.S.A.

Br. J. Pharmacol.

J. Med. Chem.