Biological activities of synthetic lipid A analogs: pyrogenicity, lethal toxicity, anticomplement activity, and induction of gelation of Limulus amoebocyte lysate

Abstract

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble. Strong biphasic pyrogenic responses with a maximal increase in body temperature of 1 to 2 degrees C were obtained with 50 micrograms/kg doses of 3 disaccharide preparations of 15 tested. With two preparations (50 micrograms/kg) moderate pyrogenicity with monophasic fever curves and a maximal temperature increase of about 0.6 degrees C was obtained. Lethal toxicity tests were carried out in galactosamine-sensitized mice. Of 15 synthetic preparations, 4 exhibited lethal toxicity under these conditions. The effective doses of the lipid A analogs in both in vivo tests were, however, several hundred times higher than those of bacterial lipid A. For the activities in vivo, hydroxyacyl residues seemed to be important. Anticomplement activity was demonstrable in seven preparations, one of which expressed an activity comparable to that of lipid A. Preparations containing non-hydroxylated fatty acids seemed to be most active in this test. None of the synthetic preparations was found to exhibit gelation activity for Limulus amoebocyte lysate when tested in doses up to 0.4 micrograms, whereas bacterial free lipid A was active in doses of about 2 pg. None of the monosaccharide derivatives exhibited any of these activities.