Human THP-1 monocyte uptake and cellular disposition of 14C-grepafloxacin

J Infect Chemother (2004) 10:11–18 DOI 10.1007/s10156-003-0289-8

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2004

ORIGINAL ARTICLE Iris H. Hall · Ute E. Schwab · E. Stacy Ward John C. Rublein · John D. Butts · Timothy J. Ives

Human THP-1 monocyte uptake and cellular disposition of 14C-grepafloxacin

Received: June 17, 2003 / Accepted: October 30, 2003

Abstract Uptake of 14C-grepafloxacin into human mononuclear (THP-1) cells was determined at pH 7.4, 6.8, or 5.0 over a 4-log antibiotic concentration. Grepafloxacin was taken up by THP-1 monocytes rapidly by both a passive and an active transport mechanism at pH 7.4. Its uptake was initially linear, with equilibrium being reached after ~1 h. Efflux followed first-order clearance and was complete within 1 h, suggesting no longterm sequestering of the antibiotic occurred. Neither cell number nor serum protein binding appeared to have any effect on antibiotic uptake. High intracellular concentrations were achieved and the ratios of cellular to extracellular antibiotic concentration (IC/EC) were between 529 and 644 at 0.04 µg/ml at pH 7.4 and 6.8, suggesting that monocytes may contain sufficient levels of grepafloxacin for affecting bacteriostatic killing. Grepafloxacin disposition within the THP-1 monocytes showed large amounts present in the nucleus and cell sap in stimulated and unstimulated cells, and its presence was evenly distributed throughout the cytosol, nuclei, lysosomes, mitochondria, and ribosomes. After stimulation by zymogen A, Staphylococcus aureus, or Streptococcus pneumoniae, increased amounts of grepafloxacin were found within THP-1 monocytes and isolated phagosome vacuoles. No antibiotic sequestration occurred inside stimu-

I.H. Hall · E.S. Ward Division of Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA U.E. Schwab Cystic Fibrosis/Pulmonary Research and Clinical Treatment Center, Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC, USA J.C. Rublein · J.D. Butts Department of Pharmacy, University of North Carolina Hospitals, University of North Carolina, Chapel Hill, NC, USA T.J. Ives (*) Division of Pharmacotherapy, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA Tel. ⫹1-919-843-0391; Fax ⫹1-919-962-0644 e-mail: [email protected]

lated monocytes, although a sufficient intracellular grepafloxacin concentration was available to kill phagocytized bacteria. Metabolic inhibitors, suppressors of K⫹/Cl⫺ and Cl⫺ transporters, inhibitors of the phagocytic process, low temperature, and low pH inhibited grepafloxacin uptake by THP-1 monocytes. Key words Grepafloxacin · Fluoroquinolone · THP-1 cell · Transport

Introduction Grepafloxacin, a once-daily fluoroquinolone with broad activity against gram-positive and -negative bacteria,1 has a wide distribution, with greater concentration in tissue than plasma, except in the central nervous system (CNS), with a higher uptake in human polymorphonuclear leukocytes (PMNs), as compared to levofloxacin, ofloxacin, and the R isomer of ofloxacin.2 Some bacteria are destroyed extracellularly by macrophages, while others are phagocytized intracellularly and fuse with lysosomal vacuoles for enzymatic hydrolytic destruction.3 In select cases, bacteria actually inhibit the lysosomal-phagosome fusion to survive and may be responsible for some subsequent serious human infections.4 As macrophages and monocytes undergo phagocytosis of pathogenic bacteria through internalization, it is of interest to know the pharmacokinetic properties of the macrophage handling of grepafloxacin under different conditions, in which this antibiotic can exert its bactericidal activity. Certain suspended phagocytic cells have been suggested to concentrate antibiotics and act as a vehicle to transport the antibiotic to sites of infection in the body.5,6 This study reports the ability of unstimulated human monocyte cells to accumulate and efflux grepafloxacin across a range of external concentrations and pHs. Human THP-1 monocytes, which are capable of bacterial phagocytosis and fluoroquinolone cellular uptake, were used. Grepafloxacin concentration and its disposition in THP-1 monocytes were determined to compare to that in human

12

PMNs. While the ratio of the intracellular to the extracellular drug concentration (IC/EC) for fluoroquinolone antibiotics in human monocytes has been examined,7–10 as well as that for β-lactams, macrolides, quinolones, rifampicin, and microcycline,11 there have been no reports on the effects of pH over a 4-log concentration of grepafloxacin over 48 h and its concentration within subcellular organelles of THP1 phagocytic cells.

Materials and methods Reagents C-Grepafloxacin (81.2 µCi/mg POC-17116) was provided by GlaxoWellcome (Research Triangle Park, NC, USA) and Otsuka Pharmaceutical (Osaka, Japan) and maintained at ⫺40°C until used. All other substrates and reagents were obtained from Sigma Chemical (St Louis, MO, USA). Radioactive counting was performed in Ecosint-H with a β-liquid scintillation counter (Packard Tricarb 2100-TR; Packard Instrument, Meriden, CT, USA) and corrected for quenching.

14

performed with dead THP-1 cells. THP-1 monocytes were heated at 60°C, treated for 30 min with 10% formalin, washed with RPMI-1640 twice and incubated with antibiotic, as indicated above.6,9,13,14 Grepafloxacin was not found to be associated with these dead THP-1 monocytes. To determine the influence of temperature on antibiotic uptake by the monocytes, initial uptake studies were conducted from 0 to 2 h at 37°C and 0°C, where it was determined that FCS had no effect on grepafloxacin uptake. Effect of metabolic energy blockers on grepafloxacin uptake by THP-1 monocytes Conditions identical to those described above for the uptake studies were employed, except that the THP-1 monocytes were co-incubated for 0.5, 1, 2, 4, or 6 h with 14 C-grepafloxacin (0.4 µg/ml) and one of the following metabolic blockers: sodium azide (10 µM) or sodium cyanide (1.5 mM), which block the mitochondrial electron transport system;6 sodium fluoride (NaF; 10 µM), which blocks glycolysis;6,9,13,14 adenosine (100 µM), a competitor for the nucleotide transporter;6,14 ouabain (5 µM), which blocks Na-ATPase;15 or ammonium chloride (NH4Cl; 10 mM), which blocks the fusion of lysosomal-phagosome vacuoles.15

Cell culture techniques A human acute monocytic leukemia cell line (THP-1; ATCC TIB-202) was obtained from the American Tissue Culture Collection (Rockville, MD, USA). These monocytes can be stimulated and do undergo phagocytosis when challenged.7,8 Suspended THP-1 acute monocytes were grown and maintained in a pH-adjusted growth medium (RPMI-1640; Sigma) with 10% heat-inactivated fetal calf serum (FCS; Sigma), plus 3 ⫻ 10⫺5 M β-mercaptoethanol and penicillin (100 units/ml)/streptomycin (100 µg/ml) at 37°C in a 5% CO2 incubator. Cells were fed fresh growth medium 18 h before each study. Grepafloxacin uptake by human THP-1 monocytes THP-1 monocytes (106 cells) were suspended in RPMI-1640 growth medium supplemented with 10% heat-inactivated FCS and 3 ⫻ 10⫺5 M β-mercaptoethanol adjusted to pH 7.4, 6.8, or 5.0. Serial dilutions of 14C-grepafloxacin were prepared in growth medium RPMI-1640 at 0.04, 0.4, 4.0, and 40 µg/ml. These concentrations were selected based on the minimum inhibitory concentration (MIC)50 values (µg/ml) for a variety of strains of Staphylococcus aureus.12 Grepafloxacin was incubated for 0.5, 1, 2, 4, and 24 h at 37°C in 5% CO2, after which the cells were collected by centrifugation (Beckman Coulter centrifuge; rotar GH-3B; Beckman Coulter, Fullerton, CA, USA) at 3500 rpm for 3 min. To lyse the cells, the pellet was passed through a layer of 0.5 ml of silicone oil and 20 µl of 88% formic acid at the appropriate pH and centrifuged (Eppendorf Micro centrifuge, model 5415D; Brinkmann Instruments, Westbury, NY, USA). Aliquots were counted. Corrections for any incidental binding of the drug to the outer membrane were

Effect of ion channel blockers on grepafloxacin uptake by THP-1 monocytes Conditions identical to those described above for the uptake studies were employed, except that THP-1 monocytes were co-incubated for 0.5, 1, 2, 4, or 6 h with 14Cgrepafloxacin (0.4 µg/ml) and one of the following agents suspended in isotonic phosphate-buffered saline (PBS) at pH 7.4: two calcium channel antagonists,16 (⫹)-verapamil HCl (10 µM; Sigma; V-4629),17 or (R)-(⫹)-Bay K 8644 (5 µM; Sigma; B-132); a potassium/chloride ion co-transport inhibitor, R-(⫹)-butyl indazone ((R)-(⫹)-DIOA; 10 µM; Sigma; D-129); a chloride ion channel inhibitor, R-(⫹)methyl indazone (IAA-94; 2 µM; Sigma; I-117); a selective inhibitor of the Na⫹/H⫹ antiporter, 5-(N-ethyl-N-isopropyl) amiloride HCl (EIPA; 30 µM; Sigma; A-171), or a partial inhibitor of the pH gradient-activated chloride ion uptake, carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 50 µM; Sigma; C-2759).10 The effects of concentration of grepafloxacin and time on these inhibitory effects on the uptake processes were examined. 14

C-Grepafloxacin efflux from THP-1 monocytes

THP-1 monocytes (0.2 ⫻ 106 cells) were incubated with 14Cgrepafloxacin at all four concentrations. After 1 h, cells were harvested by centrifugation at 3500 rpm for 6 min, washed once in the appropriate pH medium, and the pooled extracellular media was discarded.8 The pelleted cells were resuspended in fresh RPMI-1640 with 10% heat-inactivated FCS and β-mercaptoethanol adjusted to pH 7.4, 6.8, or 5.0. Samples of the extracellular medium were collected at 0.5,

13

1, 2, 4, and 8 h, and at each time point, the cells were resuspended in fresh medium. The extracellular medium was layered onto 150 µl of silicone oil (top layer) and 20 µl of 88% formic acid (bottom layer) and then subjected to velocity-gradient centrifugation.8,13 Whole cells were trapped in the formic acid layer. The pellet was treated with polyethylene glycol to verify that no extracellular drug remained in the pellet. The extracellular medium was counted and corrected for quenching. In similar experiments, THP-1 monocytes (106 cells) were co-incubated with R-(⫾)-verapamil (10 µM), a pglycoprotein transport inhibitor, at pH 7.4.15 The extracellular 14C-grepafloxacin concentration was determined, using the procedure described above. Subcellular disposition of grepafloxacin in THP-1 monocytes with/without zymogen A stimulation THP-1 monocytes (106 cells) were incubated with 14Cgrepafloxacin (0.4 µg/ml) for 1 or 4 h at 37°C with/without zymogen A (0.5 mg/ml) treatment.8 Cells were collected by centrifugation, homogenized, and fractionated by ultracentrifugation into subcellular organelles; nuclei (600 g ⫻ 10 min), mitochondria (9000 g ⫻ 15 min), lysosomes (40 000 g ⫻ 40 min), ribosomes with endoplasmic reticulum (ER) (105 000 g ⫻ 1 h), and the remaining cellular sap.8 Each fraction was washed in 0.25 M sucrose buffer and 0.001 M ethylene diamine tetraacetic acid (EDTA) at pH 7.4, and analyzed for radioactive content, the total radioactivity in that fraction was calculated. Stimulation of THP-1 monocytes by S. aureus, S. pneumoniae, zymogen A, lipopolysaccharide (LPS), or latex beads S. aureus (ATCC 29213) was grown overnight on sheep blood agar. Single colonies were inoculated into PBS and this suspension was adjusted to an optical density (OD)470nm of 0.5 (3 ⫻ 108 CFU/ml).8 For opsonization, the bacterial suspension was pelleted, suspended in 100 µl of 20% human serum complement, and left for 30 min at 37°C. Next, opsonized bacteria were washed, suspended in 1 ml of serumfree RPMI, and added to THP-1 cells (5 ⫻ 105/ml) at a bacteria/monocyte ratio of ~10 : 1. In some experiments, bacteria/monocyte ratios of ~100 : 1 and 200 : 1 were used. After 2 h of incubation, the suspension was centrifuged, re-suspended in fresh RPMI medium, and incubated with different concentrations of radiolabeled grepafloxacin. Additionally, S. pneumoniae (ATCC 6301) was opsonized as described above and incubated with THP-1 cells at a bacteria/monocyte ratio of ~10 : 1. Human THP-1 monocytes (106 cells) were stimulated by incubating with zymogen A (0.5 mg/ml),8,14 LPS Salmonella (1 mg/ml; Sigma; L-6143), or latex beads (1.07 microns, 10 µl/ml) for 18 h, as described above. Stimulated THP-1 monocytes were incubated with grepafloxacin (0.4 µg/ml), and the uptake was measured (as noted above) over 4 h. Untreated human THP-1 monocytes served as a control.

Isolation of phagosome-lysosome vacuoles containing grepafloxacin in pretreated THP-1 monocytes THP-1 monocytes (106 cells) were incubated with 14Cgrepafloxacin at 0.4 µg/ml at 37°C with or without zymogen A (0.5 mg/ml) treatment for 4 or 24 h, or with S. aureus for 1 and 2 h (bacteria/monocyte ratio ~10 : 1). Cells were homogenized in 0.25 M sucrose, 20 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES)/KOH (pH 7.2), and 0.5 mM ethylene glycol tetraacetic acid (EGTA), and centrifuged, and the post-nuclear supernate was brought to a final concentration of 39% sucrose (prepared w/v in 20 mM HEPES/KOH, pH 7.2; and 0.5 mM EGTA, pH 8.0). To isolate the phagosome-lysosome vacuoles, a sucrose gradient was used, in that 2 ml of 55% sucrose was layered onto 1 ml of a 65% sucrose cushion in an ultracentrifuge tube to which was added 2 ml of 32.5% sucrose, 2 ml of 25% sucrose, and 2 ml of 10% sucrose solution.18 The sucrose gradient was centrifuged for 60 min at 100 000 g at 4°C. Phagosome-lysosome vacuoles were collected at the 55% to 65% interface, including the 65% sucrose solution.17 Aliquots were analyzed for radioactivity to determine the antibiotic concentration in that fraction. Calculations and modeling The uptake and efflux of grepafloxacin over time and at a range of different pHs are presented as the average percentage of the extracellular concentration. Calculation of the intracellular grepafloxacin concentrations (Table 1) and IC/ EC ratios (Table 2) for the THP-1 human monocytic cells was based upon a volume of 10.9 µl/107 cells.19 A compartmental modeling approach was used to describe the initial uptake of grepafloxacin into THP-1 cells. Grepafloxacin cellular concentration vs time data were plotted to determine the rate of cellular uptake; the resulting rate vs concentration data were analyzed with a two-compartment model. A differential equation, based upon the mass balance of grepafloxacin in the compartment, was resolved by nonlinear least-squares regression with WinNonlin 3.1 (Pharsight, Mountain View, CA, USA). Several models, using linear, saturable, or mixed linear/saturable processes were fitted to the data. The goodness of fit of each model was assessed by visual examination of the distribution of residuals, the condition number, and Akaike’s information criterion.19,20 Treated and control groups were averaged and the SD determined. Differences between the treated and control groups were compared by Student’s t-test.

Results Uptake of grepafloxacin at 0.4 µg/ml by THP-1 monocytes was linear at all concentrations over the first 30 min, but reached a plateau within 1 h at pH 7.4, 6.8, or 5.0 (Fig. 1). From 2 to 48 h, at the lowest concentration (i.e., 0.04 µg/ml), an increase in grepafloxacin uptake was observed at all three pHs used, but this was not observed for the other

14 Table 1. Intracellular concentration of grepafloxacin in THP-1 human monocytes (µg/ml) Time (h) µg/ml

pH 7.4

pH 6.8

pH 5.0

0.04

0.4

4.0

40

0.04

0.4

4.0

40

0.04

0.4

4.0

40

0.5 1 2 4 24 48

2.90 4.22 4.99 7.15 13.8 20.9

28.9 33.0 71 26 23.0 21.6

195 217 209 186 145 128

697 1912 2019 1519 1211 1160

2.86 10.9 14.1 18.7 22.7 25.7

15.0 16.5 26.8 27.8 28.9 50.2

139 152 170 224 185 169

1108 1140 1215 2360 1430 1400

3.15 3.52 3.34 4.08 13.9 23.5

33.0 33.3 34.4 37.1 27.7 21.9

111 113 133 158 109 28

889 1074 1148 1222 29.0 26.7

Table 2. Intracellular/Extracellular (IC/EC) ratios of grepafloxacin at different pHs over 48 h Time (h) µg/ml

pH 7.4

pH 6.8

pH 5.0

0.04

0.4

4.0

40

0.04

0.4

4.0

40

0.04

0.4

4

40

0.5 1 2 4 24 48

72.5 105 150 179 344 529

83 178 191 64 58 54

48.8 54.3 52.3 46.4 31.8 54.0

47.8 50.5 17.4 28.9 30.3 37.9

69.6 182.6 352.5 467.5 566.5 643.5

38.2 41.32 67.0 69.5 72.3 125.5

34.8 38.0 42.5 56.0 46.3 42.2

27.7 28.5 30.4 59.0 35.7 35.0

78.6 88.0 83.5 102 346 588

82.5 83.3 83.5 92.5 69.5 54.5

27.8 27.9 33.3 39.4 27.2 7.0

22.2 26.9 28.7 30.6 0.72 0.67

Table 3. Percent intracellular uptake of grepafloxacin by THP-1 human monocytes (n ⫽ 4) Time (h) µg/ml

pH 7.4

pH 6.8

0.04

0.4

4.0

40

0.04

0.4

4.0

40

0.04

0.4

4.0

40

0.5 1 2 4 24 48

0.79 1.15 1.36 1.90 3.72 5.70

0.79 0.90 1.15 0.70 0.63 0.59

0.53 0.61 0.87 0.86 0.39 0.35

0.19 0.53 0.55 0.41 0.33 0.32

0.78 2.99 3.85 5.10 6.17 6.98

0.41 0.45 0.72 0.76 0.78 1.37

0.38 0.41 0.46 0.61 0.50 0.46

0.27 0.31 0.33 0.64 0.39 0.38

0.85 0.90 0.95 1.10 3.57 6.35

0.90 0.90 0.93 1.00 0.75 0.59

0.30 0.31 0.36 0.43 0.22 0.08

0.24 0.29 0.31 0.33 0.08 0.07

three higher concentrations employed (i.e., 0.4, 4, and 40 µg/ml), where the uptake remained at approximately 1% of the external concentrations (Table 3). Using an estimated monocyte volume (as noted above), the intracellular grepafloxacin concentration increased with the external concentration, for all pHs and times (Table 1), except at pH 5.0 at 40 µg/ml at 24 and 48 h. The IC/EC ratio decreased with increasing extracellular antibiotic concentrations at all three pHs (Table 2). At the lowest concentration, of 0.04 µg/ ml, the IC/EC ratio increased over time for all pHs, and the ratio decreased as the extracellular pH decreased. Based on model selection criteria described in “Materials and methods”, the uptake of grepafloxacin at pH 7.4 by the THP-1 monocytes is best described by a “mixed” process (i.e., saturable plus linear; Fig. 2). The associated parameter estimates indicate that the process is carrierdependent at pH 7.4. At pH 5.0 and 6.8, the cellular uptake of grepafloxacin becomes better described by a linear equation, indicating that only passive diffusion occurs. Over 48 h, THP-1 cells stimulated with zymogen A or latex beads demonstrated an increase in grepafloxacin uptake at 0.4 µg/ml, but not those stimulated with LPS. Zymogen A treatment reached a peak at 24 h, with a 3.5-fold

pH 5.0

increase in the uptake of grepafloxacin, which was timedependent, while the latex beads achieved a twofold increase at 1 h. Antibiotic uptake by THP-1 monocytes in the presence of S. aureus was increased 4.3-fold at 4 h and 6.4fold at 6 h, and with S. pneumoniae, there was a 2.4-fold increase at 2 h (P ⱕ 0.001). Ratios of bacteria to monocytes of 100 : 1 and 200 : 1 afforded the best stimulation in uptake of the antibiotic (data not shown) and the uptake was timedependent. The distribution of 14C-grepafloxacin in the subcellular organelles was mainly in the cell sap (~70%), with only a small amount in the nucleus (13%), mitochondria (6%), lysosomes (7%), or microsomes or endoplasmic reticulum (ER; 3.6%) of unstimulated THP-1 cells. Stimulation from 1 to 4 h with zymogen A led to increased drug concentration within the cell, as well in as the subcellular organelles, particularly the ER, to 27.92% (P ⱕ 0.001), thus decreasing the amount of drug in the cell sap at 1 h to 51% and at 4 h to 39%. Uptake of the antibiotic into isolated phagosome vacuoles was increased in 4 h after zymogen A treatment, by 110% of control values, but it was reduced to 23% of the control values by 24 h. Two hours of the co-incubation of THP-1 monocytes and S. aureus demonstrated a lower con-

15 Fig. 1. Effects of pH on grepafloxacin uptake by THP-1 monocytes. Closed circles, pH 5; closed triangles, pH 6.8; closed squares, pH 7.4

Kinetic parameter estimates for grepafloxacin uptake in THP-1 monocytes at pH7.4, 6.8 and 5.0 Km

k

pH

µM/min

µM

min⫺1

5.0

–

–

6.8

_

_

0.006 ⫹ 0.0002

7.4

1.56 ⫾ 0.09

12.18 ⫾ 21.3

0.031 ⫾ 0.0039

0.22 ⫾ 0.01

0 °C 37 °C

Percent Uptake

centration of grepafloxacin, i.e., 38% of the control value in the isolated phagosome vacuoles, but the concentration at 1 h was significantly increased, by 169% of the control value (P ⱕ 0.001). THP-1 uptake studies at 0° and 37°C (pH 7.4) demonstrated a slower uptake of grepafloxacin at 0.4 µg/ml (0°C) over 120 min (Fig. 2), than the uptake at 37°C. Azide and cyanide treatment caused a significant reduction, of 29%– 52% and 34%–81%, respectively, in drug uptake over 6 h (Table 4), suggesting that part of the cellular uptake was energy-dependent. Treatment with NaF significantly reduced the drug uptake at 0.4 µg/ml over 6 h, by 22%–42%, signifying that the oxygen-dependent phagocytosis of the monocytes was partially blocked with agents which block glycolysis, pentose phosphate shunt, and oxidative phosphorylation. NaF blocked the uptake of the antibiotic in stimulated THP-1 monocytes by zymogen A significantly, by ~50% over 24 h. Several parameters can be inhibited in the phagocytic processes by known chemicals. Ouabain, which blocks the Na⫹/ ATPase pump; CCCP, which blocks the proton pump; and NH4Cl, which blocks the fusion of phagosomes and lysosomes, caused a decrease in grepafloxacin uptake by human THP-1 monocytes over 6 h (Tables 4 and 5). Examination of the effects of ion channel blockers showed that the sodium/hydrogen antiport blocker

Vmax

1 0.5 0 0

50

100

minutes Fig. 2. Effects of temperature on THP-1 percent uptake of grepafloxacin (0.4 µg/ml) at pH 7.4 over 2 h (n ⫽ 4; SDs were all within 0.2% of the original data points)

amiloride (EIPA) had minimum effect on grepafloxacin uptake, whereas the calcium blockers Bay K-8664, and (⫾) verapamil HCl (L-channel blocker) caused a marginal reduction of grepfloxacin uptake over 6 h (Table 4). The potassium/chloride co-transporter R(⫹)-DIOA and the chloride channel blocker R(⫹)-IAA-94 markedly reduced grepafloxacin uptake by THP-1 monocytes which was both time-dependent (Table 4) and concentration-dependent (Table 5).

16 Table 4. Percentage uptake of grepafloxacin (0.4 µg/ml) by THP-1 monocytes in the presence of energy blockers and transport channel blockers over 6 h (n ⫽ 6)

Grepafloxacin Energy blockers ⫹ Azide ⫹ NaF ⫹ Ouabain ⫹ Cyanide ⫹ Adenosine Transport channel blockers ⫹ EIPA ⫹ R(⫹)Bay ⫹ DIOA ⫹ IAA94 ⫹ CCCP ⫹ Verapamil (⫾)

0.5 h

1h

2h

4h

6h

100 ⫾ 4

100 ⫾ 3

100 ⫾ 3

100 ⫾ 2

100 ⫾ 3

65 ⫾ 2* 58 ⫾ 3* 148 ⫾ 6* 66 ⫾ 4* 61 ⫾ 3*

71 ⫾ 72 ⫾ 77 ⫾ 56 ⫾ 56 ⫾

4* 3* 3* 4* 2*

44 ⫾ 78 ⫾ 57 ⫾ 35 ⫾ 47 ⫾

3* 4* 3* 3* 3*

52 73 28 22 43

⫾ 3* ⫾ 5* ⫾ 2* ⫾ 3* ⫾ 4*

48 ⫾ 71 ⫾ 8⫾ 19 ⫾ 39 ⫾

4* 4* 1* 2* 3*

124 ⫾ 5 146 ⫾ 6* 27 ⫾ 3* 117 ⫾ 4 55 ⫾ 3* 101 ⫾ 4

113 ⫾ 117 ⫾ 29 ⫾ 75 ⫾ 22 ⫾ 83 ⫾

3 3 3* 4 3* 4

67 ⫾ 21 ⫾ 15 ⫾ 57 ⫾ 13 ⫾ 69 ⫾

3* 4* 3* 3* 1* 3*

71 ⫾ 4* 17 ⫾ 2* 4 ⫾ 2* 13 ⫾ 2* 4 ⫾ 1* 75 ⫾ 5

100 ⫾ 8⫾ 6⫾ 4⫾ 3⫾ 88 ⫾

3 4* 3* 2* 1* 4

* P ⬍ 0.001, Student’s test EPIA, amiloride; R(⫹) Bay, Bay K 8644; DIOA, butyl indazone; IAA94, methyl indazone; CCCP, carbonyl cyanide m-chlorophenyl hydrazone

Table 5. Percentage uptake of grepafloxacin (from 0.004 to 40 µg/ml) by THP-1 monocytes over 2 h in the presence of energy blockers and transport channel blockers (n ⫽ 6)

Grepafloxacin Energy blockers ⫹ NaF ⫹ Ouabain ⫹ Cyanide Transport channel blockers ⫹ EIPA ⫹ R(⫹)Bay ⫹ DIOA ⫹ IAA94 ⫹ CCCP ⫹ Verapamil S(⫺) Lysosomal infusion inhibitor ⫹ NH4Cl

0.004 µg/ml

0.04 µg/ml

0.4 µg/ml

4.0 µg/ml

40 µg/ml

100 ⫾ 3

100 ⫾ 3

100 ⫾ 4

100 ⫾ 3

100 ⫾ 2

78 ⫾ 4 72 ⫾ 3* 66 ⫾ 4* 124 ⫾ 117 ⫾ 57 ⫾ 93 ⫾ 71 ⫾ 192 ⫾

6* 4 4* 5 5* 6*

104 ⫾ 5

77 ⫾ 5 64 ⫾ 4* 56 ⫾ 4* 113 ⫾ 52 ⫾ 50 ⫾ 55 ⫾ 64 ⫾ 269 ⫾

3* 4* 4* 4* 5* 6*

114 ⫾ 4

73 ⫾ 4* 57 ⫾ 3* 35 ⫾ 3* 64 21 30 48 25 279

⫾ 6* ⫾ 2* ⫾ 3* ⫾ 4* ⫾ 4* ⫾ 5*

130 ⫾ 6*

72 ⫾ 4* 28 ⫾ 2* 22 ⫾ 3* 66 ⫾ 14 ⫾ 20 ⫾ 26 ⫾ 19 ⫾ 321 ⫾

5* 2* 3* 3* 2* 5*

107 ⫾ 4

52 ⫾ 3* 9 ⫾ 1* 19 ⫾ 2* 45 ⫾ 3⫾ 11 ⫾ 17 ⫾ 9⫾ 381 ⫾

3* 1* 2* 2* 2* 7*

13 ⫾ 2*

* P ⬍ 0.001, Student’s test

Efflux of grepafloxacin at all three pHs was rapid and essentially complete within 1 h, whether the cells were preloaded for 30 min or 1 h with drug. The P-glycoprotein transport inhibitor, R-(⫹)-verapamil, retarded grepafloxacin efflux by ~10% at 0.5 and 1 h when drug concentrations were 4 and 40 µg/ml, but had no effect beyond this time or at a lower drug concentration.

Discussion Uptake of 14C-grepafloxacin and its efflux by human THP-1 monocytic cells appeared to be rapid, and the antibiotic did not sequester within these cells. Sufficient amounts were taken up to kill phagocytzed bacteria, and with IC/EC ratios from 17 to 191 at pH 7.4. THP-1 monocytes may be able to transport the antibiotic to the site of infection, similar to that seen with azithromycin in PMNs and fibroblasts.6 Azithromycin only produced an IC/EC ratio of 61 in monocytes,7 compared to grepafloxacin with an IC/EC ratio of

100–500 at 0.04 µg/ml in THP-1 monocytes. Previously, it has been reported that pH and presence of serum had no effect on the uptake of the antibiotic,14 consistent with observations with grepafloxacin in THP-1 monocytes. The lowest external concentration of the antibiotic resulted in the highest percentage uptake by THP-1 monocytes, suggesting that saturation of the uptake process occurred at a very low concentration of grepafloxacin and that any further addition of the drug to the extracellular medium did not improve the drug uptake rate. Based upon model selection criteria, as described in “Materials and methods,” uptake of grepafloxacin at pH 7.4 by the THP-1 monocytes is best described as a mixed process (i.e., saturable and linear) indicating both a carrier-mediated process and a passive diffusion mechanism at pH 7.4. At pH 5.0 and 6.8, the cellular uptake of grepafloxacin becomes better described by a linear equation, indicating that only passive diffusion occurs. The associated parameter estimates indicate that the uptake process is carrier-dependent. This form of uptake has been observed for both azithromycin and clarithromycin in THP-1 monocytes,7,8 but not for

17

moxifloxacin.30 A mixed transport has also been suggested for macrolides in PMNs21,22 and in THP-1.8,29 Previous studies by Hara et al.29 in THP-1 monocytes with grepafloxacin demonstrated similar findings over 60 min at 20 µg/ml. They showed the drug was taken up rapidly by passive diffusion and, in part, by an active transport mechanism, achieving an IC/EC ratio of 11.9. The uptake was temperature-dependent and decreased at lower extracellular pHs. Stimulants such as phorbol myristate acetate (PMA) increased the uptake of grepafloxacin in THP-1 monocytes. Grepafloxacin uptake in these cells exceeded that of ciprofloxacin, levofloxacin, tosufloxacin, and sparfloxacin, as determined by an HPLC assay. The isotope method used in this study would reflect all labeled species of the drug and would be independent of extraction errors. Studies with moxifloxacin in THP-1 monocytes show similar differences in IC/EC ratios, of 4.39 by HPLC techniques28 and more than 50 by the isotope method.30 Further, the present study demonstrates in THP1 monocytes that the IC/EC ratio varies with time, pH, stimulation, and drug concentration. As grepafloxacin contains a carboxyl group and a piperazine ring, with a pKa level of 7.1 and 8.8, respectively, ionization of these groups may affect the binding of grepafloxacin to a transport carrier protein at pH 6.8 and 5.0. Ionization of the antibiotic may also lead to drug trapping in subcellular organelles, which typically are more acidic (e.g., lysosomes). Previous studies with grepafloxacin in human PMN cells found an I/E ratio of 66.2, which was higher than that of other drugs in this class (i.e., levofloxacin, ofloxacin, and (R)-ofloxacin), and it was found that this ratio decreased at lower pH levels.3 The PMN uptake and efflux of grepafloxacin was rapid and temperature-dependent. KF and 2,4-dinitrophenol caused a partial reduction in uptake of the antibiotic in PMNs, suggesting that nucleoside transport may play a role in the uptake of grepafloxacin, as well as passive mechanisms.3 Ouabain, which blocks sodium pump ATPase activity, had no effect on the transport of grepafloxacin into PMNs.3 Drug uptake was both temperature- and energydependent, which has also been observed for macrolides and fluoroqinolones in THP-1 monocytes.8,30 Phagocytic functions of macrophages and monocytes are temperatureand energy-dependent. This process is reduced by NaF and cyanide or azide, which block glucose metabolism and oxidative phosphorylation, respectively. Grepafloxacin uptake into THP-1 monocytes was reduced in the presence of these blocking agents in much the same way that the uptake of macrolides is reduced in both PMNs and THP-1 monocytes. As the membrane undergoes morphological changes for endocytosis, temperature and energy may affect the amount of drug uptake into THP-1 monocytes at pH 7.4, as well as its transport to the phagosome-lysosomal vacuoles. The current studies at pH 7.4, however, suggest that grepafloxacin uptake into human THP-1 monocytes is effected by a membrane transporter involving ion channel protein, most likely, potassium, potassium/chloride, or proton channel transporter(s). As noted, R-(⫹)-butyl indazone blocks po-

tassium/chloride channels, R(⫹) IAA-94 blocks chloride transporters, and CCCP blocks proton ion channels. The calcium channel blockers, (R)-(⫹)-Bay K8644 (slow channel) and (⫾)-verapamil HCl (L-type) only had a marginal effect on grepafloxacin uptake by THP-1 cells. Calcium depletion from the extracellular medium and calcium ion channel blockers (e.g., Ca2⫹ and Ni2⫹) have been reported to reduce uptake of the macrolides in PMNs, as did verapamil, but the organic calcium blocker, nifedipine, did not.16 It has been suggested that macrolides and fluoroquinolones require a nucleoside transport for their uptake, and in this study, adenosine competed with grepafloxacin for its uptake at pH 7.4. Monocyte subcellular disposition of grepafloxacin in unstimulated THP-1 cells occurred, although only 1.2% of the extracellular concentration showed no preferential accumulation in any of the organelles and was concentrated in the cell sap. Antibiotic efflux appeared to be passive and was independent of the p-glycoprotein transport system, as it was not inhibited by the presence of R-(⫾)-verapamil, which is consistent with the efflux of macrolides from THP1 monocytes.7,8 Stimulated THP-1 cells demonstrated a higher uptake of grepafloxacin with zymogen A and latex beads, but not with LPS. These agents should stimulate phagocytic processes, and more drug was engulfed into the cells by this process. NaF, which blocks the energy production of monocyte phagocytosis, also blocked this stimulated antimicrobial uptake. Subcellular disposition of grepafloxacin in THP-1 cells stimulated by zymogen A demonstrated higher intracellular drug concentrations, with subcellular organelles also having accumulated more of the drug, particularly in the nuclei and ER. These vacuoles of THP-1 monocytes, when co-incubated with S. aureus, demonstrated an elevated drug concentration at 1 h, with a decrease of antibiotic at 2 h. Certain pathogenic bacteria can survive even after phagocytosis by macrophages and cause chronic infections (e.g., Listeria monocytogenes, Mycobacterium tuberculosis, Legionella pneumophilia, S. aureus, Brucella, and Salmonella).22–27 Listeria monocytogenes escapes from the vacuoles,22,23 whereas Mycobacterium modifies the maturation of the phagosomes.24–26 Vacuoles containing Mycobacterium avium fail to become acidified at pH 6.5 because of the lack of a proton pump or a failure to fuse with lysosomes.24 Ideal antimicrobial therapy would be for the agent to accumulate in the phagosome, causing intracellular killing of the organisms when fusion with lysosomes containing hydrolytic enzymes occurred. Some antibiotics fail to be effective because intracellular influx does not occur to a significant extent, while others are dependent upon energy transport or nucleoside transport mechanisms for intracellular uptake. These studies with unstimulated human THP-1 monocytes demonstrated an IC/EC ratio of 192; however, in the presence of bacterial stimulation or phagocytosis, this ratio increased to 619, 864, and 418. As the concentration of S. aureus increased, the concentrations of grepafloxacin in the cell increased, indicating sufficient quantity of antibiotic to render bactericidal effects.

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The present studies with THP-1 cells demonstrated that the disposition of grepafloxacin was similar to the results found with PMNs. The IC/EC ratios were slightly less in THP-1 monocytes at the higher grepafloxacin concentrations, but at 0.4 µg/ml, they were higher than those observed in PMNs. Grepafloxacin is taken up both actively and passively, and it appears to require an energy process for phagocytosis in monocytes to kill bacteria. Acknowledgments Active radiolabeled drug and a supporting grant to cover laboratory expenses were provided by GlaxoWellcome and Otsuka.

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