The entry of antibiotics into human monocytes

Effective therapy of infections due to facultative intracellular micro-organisms, which persist after ingestion by mononuclear phagocytic cells, requires the use of antibiotics with the ability to inactivate these intra-phagocytic bacteria. Since entry of antibiotics into mononuclear phagocytes is a pre-requisite for activity against such intracellular organisms, we have determined the uptake of 11 radiolabelled antibiotics by human peripheral blood monocytes. beta-Lactams (penicillin G, cefamandole and cefotaxime), gentamicin, and metronidazole had a limited ability to enter monocytes, achieving cellular concentrations which were equal to or less than extracellular levels (C/E less than or equal to 1). Imipenem, a novel beta-lactam antibiotic, rapidly bound to monocytes, but cell-associated drug progressively declined during further incubation. Chloramphenicol, a lipid-soluble drug, and trimethoprim were concentrated three-fold by human monocytes. In comparison with the other antibiotics, roxithromycin (C/E = 14), clindamycin (C/E = 6 to 7) and erythromycin propionate (C/E = 4 to 5) were markedly concentrated by human monocytes. In contrast to our findings in other phagocytes, there was no evidence that active membrane transport was involved in monocyte uptake of clindamycin, erythromycin and roxithromycin. We have demonstrated that several antibiotics are highly concentrated within human monocytes, and it will be important to evaluate the effects of this antibiotic uptake on various monocyte functions, including intra-phagocytic antibacterial activity.     

Contrasts between phagocyte antibiotic uptake and subsequent intracellular bactericidal activity.

An ideal antibiotic for therapy of infections due to facultative intracellular organisms would enter phagocytes readily and kill intracellular bacteria. We have examined the consequences of antibiotic uptake by human polymorphonuclear lymphocytes (PMN) on intraphagocytic bactericidal activity, using antibiotics which differ markedly in their ability to enter PMN. After ingestion of Staphylococcus aureus, PMN were evaluated in regard to uptake of antibiotics and survival of intraphagocytic bacteria in the presence or absence of these drugs. Except for erythromycin, the uptake of which was slightly decreased, the entry of tested antibiotics into PMN was increased or unchanged after ingestion of S. aureus. Clindamycin and erythromycin, which achieved high cellular levels in PMN, failed to produce a significant reduction in viable intraphagocytic S. aureus during 3 h of antibiotic exposure. In contrast, rifampin, which was concentrated severalfold by phagocytes, was able to kill intracellular staphylococci. Gentamicin and penicillin G penetrated PMN rather poorly. However, while gentamicin demonstrated efficient intraphagocytic killing of bacteria, penicillin had no intracellular effect during the 3-h incubation period. These observations document that the ability to enter phagocytes is only one of the factors which determine the intracellular antibacterial activity of an antibiotic.     

Entry of roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim, and metronidazole into human polymorphonuclear leukocytes.

Entry of antibiotics into phagocytes is necessary for activity against intracellular organisms. Therefore, we examined the uptake of five of the newer antibiotics--roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim, and metronidazole--by human polymorphonuclear leukocytes (PMN). Antibiotic uptake by PMN was determined by a velocity gradient centrifugation technique and expressed as the ratio of the cellular concentration of antibiotic to the extracellular concentration (C/E). Cefotaxime, like other beta-lactam antibiotics, was taken up poorly by phagocytes (C/E less than or equal to 0.3). The metronidazole concentration within PMN was similar to the extracellular level. Imipenem bound rapidly to phagocytes (C/E = 3), but cell-associated drug progressively declined during the incubation period. Trimethoprim was well concentrated by PMN (C/E = 9 to 13), and uptake was unexpectedly greater at 25 degrees C than at 37 degrees C. The most striking finding was that roxithromycin was more avidly concentrated by PMN (C/E = 34) than any other antibiotic we studied. Entry of roxithromycin into phagocytes was an active process and displayed saturation kinetics characteristic of a carrier-mediated membrane transport system. Ingestion of microbial particles by PMN slightly decreased the ability of these cells to accumulate roxithromycin (C/E = 24 to 31). These studies identified two antibiotics, trimethoprim and especially roxithromycin, which are markedly concentrated within human PMN and may prove useful in treatment of infections caused by susceptible intracellular organisms.     

Uptake of antibiotics by human polymorphonuclear leukocyte cytoplasts.

Enucleated human polymorphonuclear leukocytes (PMN cytoplasts), which have no nuclei and only a few granules, retain many of the functions of intact neutrophils. To better define the mechanisms and intracellular sites of antimicrobial agent accumulation in human neutrophils, we studied the antibiotic uptake process in PMN cytoplasts. Entry of eight radiolabeled antibiotics into PMN cytoplasts was determined by means of a velocity gradient centrifugation technique. Uptakes of these antibiotics by cytoplasts were compared with our findings in intact PMN. Penicillin entered both intact PMN and cytoplasts poorly. Metronidazole achieved a concentration in cytoplasts (and PMN) equal to or somewhat less than the extracellular concentration. Chloramphenicol, a lipid-soluble drug, and trimethoprim were concentrated three- to fourfold by cytoplasts. An unusual finding was that trimethroprim, unlike other tested antibiotics, was accumulated by cytoplasts more readily at 25 degrees C than at 37 degrees C. After an initial rapid association with cytoplasts, cell-associated imipenem declined progressively with time. Clindamycin and two macrolide antibiotics (roxithromycin, erythromycin) were concentrated 7- to 14-fold by cytoplasts. This indicates that cytoplasmic granules are not essential for accumulation of these drugs. Adenosine inhibited cytoplast uptake of clindamycin, which enters intact phagocytic cells by the membrane nucleoside transport system. Roxithromycin uptake by cytoplasts was inhibited by phagocytosis, which may reduce the number of cell membrane sites available for the transport of macrolides. These studies have added to our understanding of uptake mechanisms for antibiotics which are highly concentrated in phagocytes.     

Antibiotic uptake by alveolar macrophages of smokers.

Cigarette smoking, particularly when associated with chronic pulmonary disease, increases the risk of respiratory tract infection. Thus, we elevated the uptake of antibiotics by alveolar macrophages (AM) obtained by bronchoalveolar lavage from persons who smoke and have associated pulmonary abnormalities, circumstances which adversely affect certain macrophage functions. The entry of radiolabeled drugs into AM was determined by a velocity-gradient centrifugation technique, and uptake was expressed as the ratio of cellular to extracellular antibiotic concentration (C/E). Cefamandole and penicillin G were taken up poorly by the AM obtained from smokers (C/E less than or equal to 1). Cellular levels of isoniazid, gentamicin, and tetracycline were similar to their extracellular concentrations. The lipid-soluble drugs lincomycin, chloramphenicol, and rifampin were concentrated severalfold by the AM from smokers (C/E = 3 to 11). Ethambutol also entered macrophages readily (C/E = 11). Erythromycin and clindamycin were massively concentrated by the AM from smokers (C/E = 23 to 56). The AM of smokers accumulated a lipid-soluble antibiotic (rifampin) and actively transported agents (erythromycin propionate, clindamycin) more avidly than did the AM of nonsmokers. Augmented uptake of these antibiotics by the AM of smokers may be related to structural and functional alterations induced by smoking.     

Membrane transport of clindamycin in alveolar macrophages.

The use of antibiotics which can penetrate phagocytic cells and kill intracellular organisms is desirable in the treatment of chronic facultative bacterial infections. Recently, we reported that several antibiotics were selectively concentrated by rabbit alveolar macrophages. Clindamycin accumulation was especially marked. In the present study we evaluated the plasma membrane transport (initial uptake) of clindamycin in alveolar macrophages. The transport of clindamycin is an active process, as documented by requirements for cellular viability, elevated environmental temperature, metabolic energy, and establishment of the 40- to 50-fold cellular/extracellular gradient. Energy for membrane transport of the drug depended at least in part upon mitochondrial oxidative respiration and cell membrane Na-K pump activity. Kinetic analysis of active clindamycin transport revealed it to be saturable, with a high binding affinity (Km = 1 mM) and a high velocity of uptake (Vmax = 15.8 nmol/45 s per 10(6) cells). Clindamycin uptake was not influenced by the presence of hexose or amino acids, but was inhibited by nucleosides (adenosine, puromycin). Decreased clindamycin transport in the presence of puromycin was typical of competitive inhibition (increased Km, unchanged Vmax). Conversely, competitive inhibition of adenosine transport by clindamycin was documented. Thus, clindamycin is transported into alveolar macrophages via the nucleoside system. The potential biological consequences of this unique antibiotic transport mechanism are of interest.     

Antibiotic inhibition of the respiratory burst response in human polymorphonuclear leukocytes.

Recently we found that certain antibiotics which are markedly concentrated by human polymorphonuclear leukocytes (PMN) failed to kill susceptible, intraphagocytic Staphylococcus aureus, even though cellular drug levels were quite high. The possibility that specific antibiotics might adversely affect phagocyte antibacterial function was considered. Thus, we studied the effects of multiple antibiotics and adenosine, a known modulator of the PMN respiratory burst response, on neutrophil antibacterial function. At nontoxic concentrations, these drugs had no effect on degranulation in stimulated PMN. Adenosine was a potent inhibitor of formyl-methionyl-leucyl-phenylalanine (FMPL)-stimulated superoxide and hydrogen peroxide generation in PMN but produced less inhibition of microbial particle-induced respiratory burst activity. Three of the tested antibiotics, all of which reach high concentrations in phagocytic cells, had a marked modulatory effect on the PMN respiratory burst. Clindamycin, which enters phagocytes by the cell membrane adenosine (nucleoside) transport system, had only a modest effect on FMLP-mediated superoxide production but inhibited the microbial particle-induced response by approximately 50%. Roxithromycin and trimethoprim were efficient inhibitors of PMN superoxide generation stimulated by FMLP and concanavalin A (also inhibited by erythromycin) but had less effect on zymosan-mediated respiratory burst activity. Antibiotics which entered phagocytes less readily had no effect on the respiratory burst response in PMN. These results, as well as those of experiments with inhibitors of cell membrane nucleoside receptors, indicated that the antibiotic effect is mediated through intraphagocytic pathways. The possibility that antibiotic-associated inhibition of the PMN respiratory burst response might alter leukocyte antimicrobial and inflammatory function deserves further evaluation.     

Penetration of antibiotics into bovine neutrophils and their activity against intracellular Staphylococcus aureus

The penetration of three antibiotics, penicillin, chloramphenicol and erythromycin into bovine neutrophils, either alone or containing previously ingested Staphylococcus aureus, was determined, and their intracellular activity against these bacteria was measured. Uptake of radiolabelled antibiotics was assessed by rapidly separating neutrophils from extracellular antibiotic by centrifugation through silicone oil. Intracellular activity was estimated by comparing the numbers of bacteria surviving intracellularly in neutrophils exposed to antibiotic for 3 h at ten times the MBC, with those surviving intracellularly in untreated neutrophils. Penicillin was slightly concentrated within the neutrophils, reaching a maximum intracellular concentration 1.75 times that of the extracellular concentration; this is the C/E ratio. Chloramphenicol entered to a greater extent with a maximum C/E ratio of 7.08. Erythromycin became highly concentrated within the neutrophils with a C/E ratio of 11.46 after 90 min incubation. The presence of ingested staphylococci significantly reduced the uptake of chloramphenicol, but had no significant effect on the penetration of the other antibiotics. Intracellular activity studies indicated that, at ten times MBC, only penicillin had any significant activity against intracellular staphylococci, reducing survival by 28%. This work demonstrates that penetration of certain antibiotics can be altered by the presence of ingested staphylococci and that high intracellular levels of antibiotics do not necessarily ensure good intracellular activity against pathogenic micro-organisms.     

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.     

Uptake of flurithromycin by human polymorphonuclear phagocytes: partial characterization of the entry mechanism.

The ability of flurithromycin and erythromycin to enter human polymorphonuclear phagocytes were studied and compared by a velocity centrifugation gradient technique. Both macrolides were markedly concentrated by human cells and attained cellular to extracellular concentration ratios (C/E) > or = 10. The incorporation was rapid and essentially complete after 60 min incubation. When PMNs were pretreated with formaldehyde, or incubated at low temperatures (4-25 degrees C) or at low pH, the transport ratios of both molecules were reduced. Sodium fluoride and 2,4-dinitrophenol, which decreased erythromycin uptake, did not affect flurithromycin penetration. Perturbation of cell membrane by phorbol myristate acetate, but not by formyl methionyl leucyl peptide, affected C/E ratios of both antibiotics. The addition of amino acids or nucleosides did not influence their transfer into PMNs.     

Entry of lomefloxacin and temafloxacin into human neutrophils, peritoneal macrophages, and tissue culture cells.

The uptake of lomefloxacin (difluoroquinolone) and temafloxacin (trifluoroquinolone) by human polymorphonuclear leukocytes (PMNs), peritoneal macrophages (PM phis), and two tissue culture cells (McCoy and Vero) was measured by a fluorometric assay. Both antimicrobials reached high intracellular concentrations in PMNs [cellular to extracellular ratio (C/E) greater than 4], in PM phis (C/E greater than 3) and lower in tissue culture cells (C/E greater than 1) at an extracellular concentration of 5 mg/L. Lomefloxacin uptake by PMNs was more rapid than that of temafloxacin. Entry of both quinolones into PMNs was environmental temperature-dependent, but not affected by cell viability. Ingestion of opsonized Staphylococcus aureus did not affect the ability of PMNs to concentrate these antimicrobials. Ingestion of opsonized zymosan or stimulation with phorbol myristate acetate significantly increased the PMN association of both quinolones, this effect being particularly marked with temafloxacin. It is concluded that both lomefloxacin and temafloxacin are markedly concentrated within human phagocytes and tissue culture cells, although this phenomenon is not dependent on the degree of fluorination of the molecule.     

Effect of clindamycin on the intracellular bactericidal capacity of human peritoneal macrophages

Survival and growth of bacteria within peritoneal macrophages has been implicated as a cause of recurrence and relapse of Staphylococcus epidermidis peritonitis during peritoneal dialysis. We studied the effect of orally administered clindamycin (known to enter and concentrate in phagocytes) on the intracellular killing of S. epidermidis by human peritoneal macrophages. Clindamycin (300 mg qid) was taken for one day by eight CAPD patients. Peritoneal macrophages were isolated from the effluents and their capacity to phagocytose and kill S. epidermidis was measured. In effluents containing clindamycin, the macrophages showed better uptake (32 vs 17%, P less than 0.01) and intracellular killing (70 vs 42%, P less than 0.01) of S. epidermidis compared with control after 1 h incubation. After 18 h S. epidermidis within peritoneal macrophages incubated with clindamycin, showed a further decrease in viability (-0.33 decrease in log cfu/ml). In contrast, control phagocytes allowed numbers of S. epidermidis to increase over 18 h (+1.46 increase in log cfu/ml; P less than 0.01 compared to clindamycin). Antibiotics with the ability to suppress intracellular bacterial growth should be studied for treatment of CAPD-related S. epidermidis peritonitis.     

Intraphagocytic penetration of antibiotics

The penetration of several antibiotics into human polymorphonuclear leucocytes was measured with a bioassay. The aminoglycosides (gentamicin, netilmicin), oxacillin and LY146032, a new lipopeptidic antibiotic, had a penetration which was generally less than 60%, whereas new fluoro-quinolones (enoxacin, ciprofloxacin, CI934, Ro236240) and rifamycins (rifampicin, LM427) were concentrated 2.4 to 14.2-fold. The concentration of vancomycin and teicoplanin associated with the neutrophils appeared to be saturable over the range of extracellular concentrations tested (5-20 mg/l). Coumermycin, an inhibitor of DNA-gyrase, was highly concentrated (11.3 to 16.6-fold) within the neutrophils. The penetration of clindamycin and erythromycin was low (0.60- to 1.48-fold).     

Role of extracellular calcium in in vitro uptake and intraphagocytic location of macrolides.

We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism.     

In vitro demonstration of transport and delivery of antibiotics by polymorphonuclear leukocytes.

Several antibiotics are concentrated inside polymorphonuclear leukocytes (PMN). To investigate whether PMN could act as vehicles for delivery of antibiotics, we combined an assay measuring PMN chemotaxis under agarose with a bioassay measuring levels of antibiotic in agar. Double-layer plates were made by pouring a layer of chemotaxis agarose into tissue culture plates and then adding a thin layer of Trypticase soy agar. Neutrophils were incubated with antibiotic for 1 h and then were washed and placed in wells made in the plates. After allowing PMN to migrate under the agar toward a chemoattractant well containing formyl-methionine-leucine-phenylalanine for 3 h, Streptococcus pyogenes was streaked on top of the agar and grown overnight. PMN migration and zones of inhibition of bacterial growth were measured. Neutrophils migrated 2.51 +/- 0.16 mm toward the chemoattractant well and 1.48 +/- 0.12 mm toward the medium well; migration was not significantly affected by any of the antibiotics used. Plates with PMN incubated without antibiotic showed insignificant inhibition of bacterial growth toward chemoattractant and medium wells (0.38 +/- 0.18 and 0.14 +/- 0.12 mm, respectively; for both, P > 0.05 for difference from 0). PMN incubated with oxacillin (3 micrograms/ml), a drug not concentrated in PMN, caused a similar lack of inhibition (0.28 +/- 0.09 mm toward chemoattractant; 0.14 +/- 0.03 mm toward medium). Incubation with 30 microns of ciprofloxacin per ml resulted in inhibition that was similar in both directions (1.40 +/- 0.16 versus 1.18 +/- 0.13 mm). However, for PMN incubated with azithromycin (3 micrograms/ml), an agent highly concentrated inside phagocytes, there was a large degree of inhibition which was significantly greater in the direction of chemoattractant than in the direction of medium (3.47 +/- 0.30 versus 1.89 +/- 0.25 mm; P < 0.001), indicating that release of bioactive azithromycin by neutrophils occurred after migration. Likewise, after incubation with rifampin (10 micrograms/ml), which is also concentrated by PMN, inhibition was significantly greater in the direction of chemoattractant than in the direction of medium (1.54 +/- 0.24 versus 0.81 +/- 0.28 mm; P = 0.001). We conclude that for certain antibiotics, PMN may act as vehicles for transport and delivery of active drug to sites of infection.     

Antibiotic action on phagocytosed bacteria measured by a new method for determining viable bacteria

Antibiotics such as rifampin that act on phagocytosed bacteria have clinical advantages. The investigation of such intraphagocytic activity of antibiotics is, however, hampered by the time-consuming and cumbersome procedures necessary for enumerating viable bacteria. We have developed a semiautomatic biophotometric method for this purpose that permits the processing of relatively large numbers of samples. Using this method with Staphyloccus aureus Wood 46 as a test organism, we studied the dose dependence of the activities of a number of antibiotics against intraphagocytic bacteria. We could confirm the very good intracellular activity of rifampin. Its activity at low concentrations was slightly better against intracellular than against extracellular bacteria. In contrast, clindamycin and erythromycin, both of which have been reported to accumulate within phagocytes, did not exhibit a correspondingly enhanced intracellular activity, erythromycin being active intracellularly only at high concentrations. Clindamycin and vancomycin were effective against intracellular organisms, but only at relatively high concentrations owing to their low bactericidal activity against S. aureus Wood 46. Penicillin G, ampicillin, gentamicin, and streptomycin exhibited no useful intracellular activity. These results demonstrate that radioactive accumulation studies of antibacterial agents are not sufficient to show intracellular activity. Intracellular activity must be demonstrated in a functional test, preferably with graded concentrations of the test substance.     

Uptake and Intracellular Activity of Moxifloxacin in Human Neutrophils and Tissue-Cultured Epithelial Cells

The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 ± 1.0 and 8.7 ± 1.0, respectively (20 min; 37°C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 μg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanide m-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonized Staphylococcus aureus significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular S. aureus in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.     

Interactions of dirithromycin with human polymorphonuclear leukocytes.

Dirithromycin, a new macrolide antibiotic, achieves prolonged, high levels in tissue. We previously demonstrated that certain macrolides are highly concentrated within phagocytic cells. This background information prompted us to evaluate the interactions of dirithromycin and human polymorphonuclear leukocytes (PMNs). After incubation with radiolabeled dirithromycin, antibiotic uptake by PMNs was determined by a velocity-gradient centrifugation technique and was expressed as the ratio of the cellular to the extracellular drug concentration (C/E). Dirithromycin was avidly accumulated by PMNs (C/E, 5 at 15 min, 10 at 30 min, 19 at 1 h, and 35 at 2 h). Uptake was dependent on cell viability, physiologic environmental temperature, and pH (optimum 8.6), but was not influenced by potential competitive inhibitors of membrane transport. Incubation with sodium cyanide caused an increase in dirithromycin accumulation by PMNs. Ingestion of microbial particles (mimicking in vivo infection) modestly inhibited the entry of dirithromycin into PMNs. After removal of extracellular drug, the efflux (release) of dirithromycin from PMNs was slow; only 10% was released within the first 30 min. This prolonged retention of dirithromycin within phagocytic cells might allow delivery and release of accumulated drug at sites of infection. The impact of intraphagocytic dirithromycin on cellular function was also evaluated. In a manner similar to that of other highly concentrated, weakly basic antibiotics, dirithromycin inhibited the respiratory burst response (superoxide production) in stimulated PMNs. The presence of dirithromycin slightly increased the intraphagocytic killing of Staphylococcus aureus in human PMNs. These interactions of dirithromycin with phagocytic cells may promote the extraphagocytic, and possibly the intraphagocytic, killing of infecting organisms.     

Influence of pentoxifylline and its derivatives on antibiotic uptake and superoxide generation by human phagocytic cells.

Pentoxifylline modulates multiple activities of stimulated polymorphonuclear neutrophils (PMNs), including the respiratory burst response and membrane transport of certain substances (e.g., nucleosides). We found that several weakly basic antibiotics are highly concentrated by human PMNs and that these drugs also inhibit the respiratory burst response (by a mechanism different from that of pentoxifylline). Since both pentoxifylline and antibiotics will be administered to some patients with serious infections, we have evaluated several types of interactions between these drugs and human PMNs and have attempted to identify the mechanisms that produce alterations in cellular function. Roxithromycin, dirithromycin, and clindamycin were avidly concentrated by PMNs. Pentoxifylline and two derivatives (HWA-448 [torbafylline] and HWA-138 [albifylline]) increased the uptake of these antibiotics by PMNs, both in the resting state and during phagocytosis. Pentoxifylline, HWA-448, HWA-138, and the highly concentrated antibiotics each exerted an inhibitory effect on the stimulated respiratory burst response in PMNs. The combination of both pentoxifylline and a modulatory antibiotic (roxithromycin or clindamycin) inhibited superoxide production to a greater extent than either agent alone. This additive effect might be expected, since pentoxifylline and the modulatory antibiotics influence the respiratory burst activation pathway at different sites. The ability of pentoxifylline to augment the entry of antibiotics into neutrophils has important therapeutic implications. The consequences of this phenomenon might include improved intracellular bactericidal activity as well as efficient antibiotic delivery and release at sites of infection.     

Uptake and intracellular activity of sparfloxacin in human polymorphonuclear leukocytes and tissue culture cells.

The penetration of sparfloxacin into human neutrophils (PMN) and different tissue culture cells (HEp-2 and McCoy) was evaluated. The cellular to extracellular concentration ratios (C/E) of sparfloxacin were always higher than 4 at extracellular concentrations ranging from 0.5 to 25 mg/liter. The uptake of sparfloxacin by PMN was rapid, nonsaturable, reversible, not energy dependent, and significantly reduced at pH 8. The penetration of this agent into PMN was similar when viable and Formalin-killed cells were used and was not affected by environmental temperature. Ingestion of opsonized zymosan significantly increased the amount of PMN-associated sparfloxacin. Sparfloxacin at a concentration of 0.5 mg induced a significant reduction in the survival of intracellular Staphylococcus aureus. It is concluded that sparfloxacin reaches intracellular concentrations within leukocytic cells much higher than extracellular concentrations, while remaining active intracellularly.