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

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-PGJ(2) 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.

Fig. 1. The structure of the endocannabinoid anandamide and its endogenous analog, N-arachidonylglycine (NAGly)

Several amino acid – fatty acid combinations have been found in tissue extracts .

Fig. 2. The general structure for the elmiric acids

This name has been chosen to designate the entire family of molecules that includes both the naturally occurring lipoamino acids such as NAGly and synthetic analogs where R₁ is a long chain alkyl group and R₂ and R₃ can be a wide range of substituents.

Figure 3. Effects of the elmiric acids in the mouse paw edema model

The compounds were dissolved in safflower oil and administered orally at 30 mg/kg. After 60 min, 50 ul of the 0.5% arachidonate in pH 8.3 carbonate buffer was injected into the plantar surface of the left (ipsilateral) hind paw of each mouse. The right (contra lateral) paws were injected with 50 ul of the buffer vehicle. This step was done with mice under deep halothane anesthesia. After 2 hours, mice were euthanized with halothane, both hind paws severed at the ankle joint and the weights recorded. Values shown are the means ± s.e.m. p<0.05 Plots and statistical analyses were done using Prism by GraphPad.

Figure 4. Effects of elmiric acids in the mouse subcutaneous pouch assay

Subcutaneous pouches were generated in male CD-1 mice (30–35g) by injection of air on two-three successive occasions. Panel A. Drugs were given orally in safflower oil (50ul) at a dose of 20mg/kg. After 60 min, cytokines TNF-a and IL1-b were injected into the pouches with mice under halothane anesthesia. After 90 min, mice were euthanized with halothane, cells harvested and counted. Panel B. One hour after oral dosing at the indicated levels, 2% carrageenan was injected into the pouches. The pouch contents were allowed to incubate for 4 hrs following which the mice were euthanized with halothane and the cells harvested and counted. Plots and statistical analyses were done using Prism by GraphPad. N=4 mice/group. P<0.05 by ANOVA

Figure 5. Reduction of arachidonic acid (A) phorbol ester (B) induced ear edema in the mouse

CD-1 male mice between 34 and 40g were treated as described in Experimental. Treatment was initiated by the application of vehicle (10ul), dexamethasone (10ug/ear) or EMA-1(20:4) and EMA-23(20:4) (200ug/ear) in 10ul acetone. Values shown are expressed as the differences between the ipsi lateral (induced) and contra lateral (vehicle control) ears in microns. Paired t-test results were: p<0.02 for vehicle vs. dexamethasone and p<0.007 for vehicle vs. EMA-1(20:4). Plots and statistical analyses were done using Prism by GraphPad. N = 8. Note: EMA-23 = 1-aminocyclopropane-carboxylic acid

Figure 6. Dose related reduction of phorbol ester induced ear edema in the mouse

Treatment was initiated by the application of vehicle (10ul acetone), or drug in 10ul acetone to the ipsi lateral ears and only acetone to all contra lateral ears. 30 min after drug treatment 10ul of the PMA (phorbol ester) solution in acetone was applied to all ipsi lateral ears and 10ul of acetone to all of the contra lateral ears. After 4 hrs, all ear thicknesses were measured using a digital micrometer. Data are expressed as the differences between the ipsi and contra lateral ears in microns. Plots and statistical analyses were done using Prism by GraphPad. N = 4; p<0.05. Note: EMA-22 = 1,1-dimethylglycine; EMA-25 = 1-aminocyclohexane-carboxylic acid.

Figure 7. Dose related reduction of phorbol ester induced ear edema in the mouse

All of the conditions were the same as in Figure 6 except that the drugs were dissolved in dimethylacetamide (DMA) instead of acetone. p<0.05. Note: EMA-8 = methionine.

Scheme 1

General synthetic procedure for the elmiric acids.

Scheme 2. Putative mechanism of action for the elmiric acids

During an inflammatory response, cells are activated to release arachidonic acid. Activity of the bi functional enzyme COX-2 oxidizes arachidonic acid to PGG₂ and a subsequent peroxidase reaction yields PGH₂. Then, under the influence of terminal synthases, PGD₂ and PGE₂ are formed. PGD₂ is then converted to the PGJ series in an unregulated manner. Resolution of inflammation occurs when the PGJ series predominates over the PGE series. The elmiric acids, which are not COX-2 inhibitors, apparently up regulate PGD synthase without affecting PGE synthase.