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Studies were undertaken to demonstrate the efficacy of erythromycin in killing Legionella pneumophila organisms that have been ingested by human polymorphonuclear leukocytes. With use of an electron microscope-autoradiography method in conjunction with an antibiotic-uptake assay, it was shown that [3H]-labeled erythromycin A base penetrated into the cytoplasm of human polymorphonuclear leukocytes in concentrations up to 24 times higher than the extracellular drug level. Similar studies using [14C]benzyl penicillin revealed that this antibiotic penetrated human polymorphonuclear leukocytes very poorly. Removal of erythromycin from the extracellular environment resulted in rapid leakage of radioactivity from the cells; an assay for bioactivity showed that this material was unaltered drug. Human polymorphonuclear leukocytes were permitted to ingest L. pneumophila and were then treated with erythromycin A base. The erythromycin killed the ingested legionellae, as evidenced by bacterial plate counts and morphological destruction of ingested organisms. These data clearly demonstrate the efficacy of erythromycin in killing susceptible intracellular pathogens.

The ability of erythromycin A base to penetrate and accumulate in tissue culture cells of human origin was investigated. The antibiotic was highly concentrated by early passage ceUls of normal bronchus, kidney, liver, lung, and skin and by cancer ceUs derived from breast, liver, and lung. Intracellular levels 4 to 12 times that of the extracellular milieu were obtained in both early-passage and transformed cells. The total quantity of erythromycin accumulated depended on the extracellular concentration of antibiotic, but the cellular/ extracellular ratios were, for the most part, independent of the initial extracellular drug concentration. In all cell types tested, the accumulated antibiotic rapidly egressed when cells were incubated in antibiotic-free medium. Bioactivity assays demonstrated that the expelled drug was unmetabolized, fully active antibiotic. The concentration of erythromycin by a variety of human cell types probably accounts, in part, for the effectiveness of the antibiotic against intracellular parasites such as Legionella and Chlamydia spp.Erythromycin has been widely, successfully, and safely used for over two decades as treatment for infections caused by susceptible pathogens. Although erythromycin has been recognized as an effective drug, investigators have been puzzled by the low and unpredictable levels in plasma that are usually observed after administration of the antibiotic. The paucity of drug in the vasa may be due to tissue accumulation, since distribution studies on tissue samples excised from dogs (14) and rats (15) have shown that erythromycin is concentrated well above blood levels in organs such as the liver, spleen, kidney, lung, and pancreas and in submaxillary and adrenal glands.Infections attributed to Legionella spp., which are facultative intracellular bacteria, and Chlamydia spp., which are obligate intracellular parasites, are effectively treated with erythromycin (1). Both organisms are known to parasitize a large number of human cell types. The ability of erythromycin to control these organisms suggests that the antibiotic penetrates and exerts its action within the invaded host cell. Indeed, recent studies have shown that both human polymorphonuclear leukocytes (PMNs) (20, 23) and alveolar macrophages (8,12) The resulting [3H]erythromycin was extracted with methylene chloride and crystallized from acetonitrile. Further purification by high-pressure liquid chromatography on a C18 column with a mobile phase consisting of acetonitrile (1,350 ml), methanol (300 ml), 0.2 M ammonium formate (300 ml), water (1,050 ml), and ammonium hydroxide (0.6 ml) (pH 7.5 to 8.0) gave [3H]erythromycin A base (specific activity, 19.5 mCi/mmol; >98% radio pure

The nature of the carbonyl and nitrogen substituents of hydroxamic acids has a major influence on the biological profile of these compounds. Hydroxamates with small groups such as methyl appended to the carbonyl and relatively large nitrogen substituents generally have longer duration in vivo, produce greater plasma concentrations, and often are more potent inhibitors of in vivo leukotriene biosynthesis than hydroxamic acids with the opposite arrangement. The structure-activity relationships that describe in vitro 5-lipoxygenase inhibitory activity and in vivo leukotriene biosynthesis inhibitory potency for a group of these hydroxamic acids were investigated. While most of the compounds examined were potent in vitro inhibitors of 5-lipoxygenase, their in vivo potencies varied widely. This discrepancy was usually attributable to differences in bioavailability. Substitution patterns are described that produce potent, orally active inhibitors of leukotriene biosynthesis.

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