INTERACTIONS BETWEEN HMR-3647, A NEW KETOLIDE, AND HUMAN POLYMORPHONUCLEAR NEUTROPHILS

HMR 3647, a new ketolide, is active upon intracellular pathogens. We p reviously demonstrated that HMR 3004 (RU 64004), another ketolide, is highly concentrated by human polymorphonuclear neutrophils (PMNs), Thi s prompted us to evaluate whether the presence of a 3-keto group inste ad of an L-cladinose, a neutral sugar characteristic of erythromycin A derivatives, confers peculiar pharmacokinetic properties with regard to cellular accumulation and efflux. After incubation with the radiola belled drug, HMR 3647 uptake was determined by a velocity gradient cen trifugation technique. HMR 3647 was avidly concentrated by PMNs, witho ut saturation, over a 3-h incubation period, with cellular-to-extracel lular concentration ratios of 31 +/- 4.2 at 5 min and up to 338 +/- 27 .1 at 180 min. About 60% of HMR 3647 was located in the granular compa rtment; less than 6% was associated with the membranes. HMR 3647 gradu ally egressed from loaded cells placed in drug-free medium. Uptake was dependent on environmental temperature (activation energy, 128 +/- 9. 4 kJ/mol) but not on extracellular pH, HMR 3647 displayed Michaelis-Me nten saturation kinetics with a mean Vmax of 2315 ;ng/2.5 x 10(6) PMNs /5 min and a mean K-m of 117 mg/liter (144 mu M). As already observed with erythromycin A-derived macrolides, extracellular Ca2+ was necessa ry for optimal uptake of HMR 3647, Interestingly, verapamil increased the uptake of HMR 3647 at 5 min, but this was followed by gradual inhi bition at later incubation times, a phenomenon probably related to sti mulation of drug efflux, The impact of intracellular accumulation of H MR 3647 on PMN functions aas also investigated. In contrast to other e rythromycin A derivatives, HMR 3647 only weakly triggered granule exoc ytosis, but it inhibited superoxide anion production in a time- and co ncentration-dependent manner, with concentrations which inhibited 50% of control response of 55 (67 mu M) (5 min) and 30 (36 mu M) (30 min) mg/liter for formyl-methionyl-leucyl-phenylalanine stimulation and 117 (143 mu M) (5 min) and 44 (54 mu M) (30 min) mg/liter for phorbol myr istate acetate stimulation.