Macrophages are a class of white blood cells tasked with the elimination of diseased or dying cells and foreign substances, and as a result, play an important role in maintaining the health of the biological systems in which they are employed. Unrestrained, they have also been shown to cause tissue damage in a number of auto-immune diseases, including: rheumatoid arthritis, psoriasis, Crohn’s disease, lupus, and atherosclerosis. In addition, activated macrophages have also been shown to release pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1, promoting and intensifying the disease.<br/>Activated macrophages are known to over-express folate receptor (FR) isoform β, a cell-surface glycoprotein which binds with high affinity to the vitamin folic acid (FA). Our group has previously shown that activated macrophages and hence, sites of inflammation, can be visualized by FA conjugates of the radioimaging agent <sup>99m</sup>Tc. Treatment of Lewis rats with a conjugate of the anti-folate aminopterin (AMT) hydrazide, a more active but also more toxic structural relative of the ubiquitous anti-inflamatory methotrexate (MTX), to FA through a reducible disulfide linker system resulted in the reduction of symptoms seen in adjuvant induced arthritis (AIA), autoimmune uveitis (EAU), and autoimmune encephalomyelitis (EAE).<br/>Herein, the synthesis of a series of FA-AMT conjugates which link AMT to various FA-peptidic/sacchro-peptidic spacers via various cysteine/penacillamine/cysteinamine derived linker systems is described. As designed, the linker allows for two distinct sites of release: 1) a reductively labile disulfide bond, the stability of which can be increased by increasing the adjacent stearic bulk, and 2) an enzymatically-cleavable (Γ-glutamyl hydrolase) amide bond, the stability of which is effected by the substituents on the α-carbon as well as the steric bulk at the β-position relative to the amide. Testing in AIA models showed L-cysteine methyl ester with dimethyl cysteine (penicillame) providing additional steric bulk on the spacer side of the disulfide bond was optimal.

Targeted folate-aminopterin anti-inflammatory conjugates: Optimization of a reductively/enzymatically labile cysteine-derived linker system

254th National Meeting (2017)

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