Interaction of the New Ketolide ABT-773 (Cethromycin) with Human Polymorphonuclear Neutrophils and the Phagocytic Cell Line PLB-985 In Vitro

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Abstract
A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (Vmax, about 10 000 ng/2.5 × 106PMNs/5 min;Km, about 60 μg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni2+) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (VmaxandKmvalues were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.