N-Amino acid linoleoyl conjugates: anti-inflammatory activities

Abstract

Several N-linked amino acid-linoleic acid conjugates were studied for their potential as anti inflammatory agents. The parent molecule, N-linoleoylglycine was tested in an in vivo model, the mouse peritonitis assay where it showed activity in reducing leukocyte migration at doses as low as 0.3mg/kg when administered by mouth in safflower oil. Harvested peritoneal cells produced elevated levels of the inflammation-resolving eicosanoid 15-deoxy-Δ(13,14)-PGJ(2). These results are similar to those obtained in earlier studies with N-arachidonoylglycine. An in vitro model using mouse macrophage RAW cells was used to evaluate a small group of structural analogs for their ability to stimulate 15-deoxy-Δ(13,14)-PGJ(2) production. The d-alanine derivative was the most active while the d-phenylalanine showed almost no response. A high degree of stereo specificity was observed comparing the d and l alanine isomers; the latter being the less active. It was concluded that linoleic acid conjugates could provide suitable templates in a drug discovery program leading to novel agents for promoting the resolution of chronic inflammation.

Figure 1. The structure of NAgly

There are three regions of the molecule that are of pharmacological interest. Region 1 confers a high degree of specificity of action. Polyunsaturated residues produce molecules with analgesic and anti- inflammatory action whereas saturated structures are inactive. Region 2 is related to metabolic stability since the EMAs are degraded by FAAH (fatty acid amide hydrolase) activity. Region 3, the amino acid residue, can modulate the analgesic/anti-inflammatory activities depending on steric factors and the chiral nature of the amino acid.

Figure 2

Structures of the novel elmiric acids

Figure 3. N-linoleoylglycine inhibits leukocyte migration (A) and increases PGJ production (B) in the mouse peritonitis model

A. The indicated treatments were administered by mouth and after 30 min, the mice were injected i.p. with 1 ml (sterile filtered) 8% BBL Fluid Thioglycollate Medium. Cells were harvested from the peritoneal cavity after 3 hours, exposed to lysing buffer for 2 minutes to remove erythrocytes, suspended in PBS/BSA and differential cell counts obtained. Control mice were given safflower oil. N=8. B. Peritoneal cells were collected and maintained in culture fo 18 hrs. The media were then harvested and their PGJ levels measured by ELISA assay. N=4 Study A was carried out by BRM, Inc., (Worcester, MA). All animal studies were performed according to institutional, local, state, federal and NIH guidelines for the use of animals in research under Institutional Animal Use and Care Committee (IACUC)-approved protocols at BRM and The University of Massachusetts. Medical School.

Figure 4. Electronic and steric effects on PGJ responses in RAW cells

Cells used were RAW264.7 and were obtained from ATCC. The base medium is Gibco DMEM with 10% fetal bovine serum added. Cells are grown in a T-75 flask in 15 ml of medium; medium is replaced on day four and sub cultured on day seven. Cells are removed by scraping without the aid of trypsin. A sub cultivation ratio of 1:3 to 1:6 was used. Elisa assay kits for PGJ were obtained from Assay Designs, Inc., (Ann Arbor, MI). The identity of the PGJ analyte in the culture medium was confirmed by mass spectrometry (Wood and Makriyannis, unpublished data). Treatments were carried out in 48 well plates with 50,000 cells/500ul DMEM+FCS media/well and TNFα (10 nM) added. Cells were incubated for 20 hrs at 37°C and 5% CO₂. Media were changed to 500ul of serum free DMEM, treated for 2 hrs and100 ul removed for assays. NAgly [10 μM] control; 16,300 pg/ml. DMSO control: <16.0 pg/ml. N=4

Figure 5. Estimated rank order responses in PGJ RAW cell model

Cells were treated as in Figure 4. All values were obtained at 10uM concentrations of each EMA analog. Control: 1% DMSO vehicle. N=4 *ANOVA gave a p value of less than 0.05.