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Updated: Aug 22, 2026
Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Published on: August 23, 2013
Cellular uptake of two fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils in vitro
H Abdelghaffar1, D Vazifeh, M T Labro
1INSERM U479, CHU Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.
Abstract:
We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (V(max), about 2,300 ng/2.5 x 10(6) PMN/5 min; K(m), about 50 microg/ml); (iii) the inhibitory effects of Ni(2+) (a blocker of the Na+-Ca(2+) exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system.
Insights
New fluoroketolides, HMR 3562 and HMR 3787, show rapid cellular accumulation in human polymorphonuclear neutrophils (PMN). These compounds are primarily stored in PMN granules and exhibit characteristics of an active transport system.
Area of Science:
- Pharmacology
- Cell Biology
- Drug Discovery
Background:
- Fluoroketolides are a class of antibiotics with potential clinical applications.
- Understanding the cellular pharmacokinetics of novel drug candidates is crucial for their development.
Purpose of the Study:
- To analyze the in vitro cellular accumulation and pharmacokinetics of two novel fluoroketolides, HMR 3562 and HMR 3787.
- To investigate the mechanism of cellular uptake and distribution of these compounds in human polymorphonuclear neutrophils (PMN).
Main Methods:
- In vitro analysis of cellular accumulation in human PMN.
- Determination of cellular-to-extracellular concentration ratios (C/E) over time.
- Investigation of compound localization within PMN.
- Assessment of uptake sensitivity to pH and activation energy.
- Evaluation of transport system characteristics including saturation kinetics and inhibition studies.
Main Results:
- Both HMR 3562 and HMR 3787 demonstrated rapid uptake by PMN, reaching high C/E ratios (>300).
- Compounds were predominantly localized in PMN granules (~75%) with slow egress due to reuptake.
- Uptake kinetics suggested an active, carrier-mediated transport system, influenced by pH and exhibiting moderate activation energy.
- Inhibitory effects of Ni(2+), phorbol myristate acetate, and H89 provided further evidence for an active transport mechanism.
Conclusions:
- HMR 3562 and HMR 3787 exhibit distinct cellular pharmacokinetics in PMN, characterized by rapid uptake and significant intracellular accumulation.
- The data strongly suggest the involvement of an active, possibly carrier-mediated, transmembrane transport system for these fluoroketolides.
- Despite structural differences, these findings reinforce the concept that erythromycin A derivatives share a common transmembrane transport system.
