Effects of alatrofloxacin, the parental prodrug of trovafloxacin, on phagocytic, anti-inflammatory and immunomodulation events of human THP-1 monocytes

Biomedicine & Pharmacotherapy 57 (2003) 359–365 www.elsevier.com/locate/biopha

Original article

Effects of alatrofloxacin, the parental prodrug of trovafloxacin, on phagocytic, anti-inflammatory and immunomodulation events of human THP-1 monocytes Iris H. Hall a,*, Ute E. Schwab b, E. Stacy Ward a, Timothy J. Ives c,d a

Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27559-7360, USA b Department of Medicine, Cystic Fibrosis/Pulmonary Research and Clinical Treatment Center, University of North Carolina, Chapel Hill, NC 27599-7360, USA c Division of Pharmacotherapy, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27559-7360, USA d Division of General Internal Medicine, Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7360, USA Received 12 February 2003; accepted 18 March 2003

Abstract Alatrofloxacin functions similar to other fluoroquinolone antibiotics in that it not only has antibiotic activity to kill invading organisms by interfering with DNA synthesis, it possesses immunosuppressive activity. In the first hour after bacteria have been phagocytosed by THP-1 monocytes, the drug activates a lytic mechanism involving the release of c-AMP, tumor necrosis factor (TNFa), interleukin-1 (IL-1), IL-6 and nitric oxide, with elevations in lysosomal hydrolytic enzyme activities. This effect reverses between 2 and 4 h. At this time, all of these inflammatory processes are returned to normal values or below suggesting that alatrofloxacin reduces the spread of infection and destruction of tissue related to inflammation. © 2003 Published by Éditions scientifiques et médicales Elsevier SAS. Keywords: Fluoroquinolone; Antibiotics; Immunomodulation; Anti-inflammation

1. Introduction Recently a number of fluoroquinolone antibiotics have been shown to modulate cytokine synthesis and release from immune and inflammatory cells. These properties have led to speculation that these agents may not only afford antibiotic activity, but may be useful in suppressing the spread of infection and the destruction of tissue [18]. Since a number of immune phagocytic cells concentrate fluoroquinolones intracellularly, it has been suggested that the cells act as a vehicle to conduct the antibiotic to infected sites in the body [10,11]. Both macrolides and fluoroquinolones have been shown in human THP-1 monocytes to activate initially the oxygen burst and phagocytic process followed at a later time by suppression of cytokine and free radical release, hydrolytic lysosomal enzyme activity and tissue lipid peroxidation [1,13–15,18].

* Corresponding author. E-mail address: [email protected] (I.H. Hall). © 2003 Published by Éditions scientifiques et médicales Elsevier SAS. doi:10.1016/S0753-3322(03)00054-4

Alatrofloxacin, the parenteral prodrug of trovafloxacin, is a fluoronaphthyridone which contains an L-alanyl-L-alanyl salt. Trovafloxacin has demonstrated activity against a wide range of gram-positive and gram-negative bacteria [6,9,19,33] as well as anaerobic organisms [9,32]. It has been found to be useful in organisms that have demonstrated resistance to levofloxacin [9,19,20,25]. It is more active than temafloxacin, ciprofloxacin and ofloxacin against grampositive organism [9]. Administration of alatrofloxacin to rats with intra-abdominal abscesses caused by Bacteroides fragilis or Escherichia coli demonstrated that it was more effective as a single drug, compared to a combination of clindamycin and gentamicin [32]. The mode of action of trovafloxacin in bacteria is the inhibition of DNA gyrase and DNA topoisomerase IV activities. The antibiotic is taken up by macrophages 28-fold over its extracellular concentration level and kills intracellular organisms [6]. Changes in cytokine levels have also been noted after in vivo treatment with the antibiotic [32]. The purpose of the current study was to examine the effects of alatrofloxacin on the early activation

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events of phagocytosis of human monocytes to determine the antibiotic effects on the generation of free radicals, activation of hydrolytic enzymes and cytokine levels as they relate to killing of foreign organism and suppression of inflammation and the immune response. 2. Material and methods 2.1. Source of material Alatrofloxacin mesylate was obtained from Pfizer Pharmaceuticals Production Corp. (Ringaskiddy, Co. Cork, Republic of Ireland). All other supplies unless otherwise noted were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Cell culture techniques THP-1 acute monocytes (ATCC TIB-202; American Type Culture Collection, Rockville, MD, USA) were maintained in RPMI-1640 growth medium (GIBCO, Grand Island, NY, USA), 10% heat-inactivated fetal calf serum (Flow Laboratories, McLean, VA, USA), and 3 × 10–5 M b-mercaptoethanol and penicillin (100 U/ml)/streptomycin (100 µg/ml) at 37 °C in a 5% CO2 incubator [18]. Cells were fed fresh growth medium without antibiotics P/S 18 h before each study. 2.3. Effects of alatrofloxacin in zymogen A or bacteria stimulated THP-1 monocytes on metabolic events THP-1 monocytes (106 cells) were pre-treated with zymogen A (0.5 mg/ml) for 1 h, or Staphylococcus aureus for 2 h (bacteria: monocyte ratio of ~10:1) and then incubated with alatrofloxacin at concentrations of 0.004, 0.04, 0.4 or 4 µg/ml in 96-well plates from 0 to 4 h, after which a number of biochemical assays were performed [18]. For changes in pH and phagocytosis, these preparations were incubated with acridine orange (14.4 mg/100 ml) at pH 7.2 for 20 min and quenched with crystal violet (50 mg/100 ml) for 1 min. Using a Cytofluor 2350 Fluoresence Measurement System (Millipore Corp., Bedford, MA, USA) with excitation at 450 nm, the cellular pH change from pH 7.4 (control value) was determined at 520 nm and phagocytosis was determined at 620 nm [5,12]. NADPH oxidase activity was determined as the rate of cytochrome C reduction at 550 nm [30] using a visible 96-well plate reader (Molecular Devices Corp., Sunnyvale, CA, USA). Protein kinase C activity was determined via ELISA immunoassay techniques (kit #539484; Calbiochem, San Diego, CA). Nitric oxide (NO) release was determined spectrophotometrically (kit #482650; Calbiochem) and read at 560 nm. Hydrogen peroxide release was measured with a Bioxytech H2O2 –560 kit (Oxis International, Inc., Portland, OR, USA). Glutathione reductase activity was determined spectrophotometrically by measuring the oxidation of NADPH at 340 nm (kit #359962; Calbio-

chem). Superoxide dimutase (SOD) activity was assayed using a kit (#574600; Calbiochem) and read at 490 nm. Cathepsin D activity was determined with an ELISA kit (#QIA-29; Calbiochem). N-acetyl glucosaminidase (NAG) activity was determined with p-nitrophenyl-N-acetyl-b-Dglucosaminide [8] as the substrate. The enzyme reaction was terminated with glycine buffer, pH 10.6 and read at 409 nm using a visible 96-well plate reader with p-nitrophenol as the standard. Lipid peroxidation after antibiotic exposure was determined by colorimetric methods (Bioxytech LPO-586; Oxis). 2.4. Effects of alatrofloxacin on TNF␣, IL-1, IL-6, and IL-8 release Zymogen A-stimulated THP-1 monocytes (106 cells/ml) were exposed to different concentrations of alatrofloxacin (0.004, 0.04, 0.4 and 4 µg/ml) over 4 h. Cell-free extracts were obtained and used to evaluate c-AMP, tumor necrosis factor (TNFa), interleukin-1 (IL-1), IL-6, and IL-8 release via ELISA immunoassays (Quantikine kits #DE0450, DTA50, DLA50, D6050 and D1500, respectively, from R&D Systems, Minneapolis, MN, USA).

2.5. Co-incubation studies with inhibitors of the phagocytic process THP-1 monocytes (106 cells) were incubated with alatrofloxacin at 4 µg/ml as well as one of the following agents: NaF at 10 µM, which blocks glycolysis and the pentose phosphate shunt reducing energy for the phagocytic process; NH4Cl at 10 mM which blocks the fusion of the phagosome and lysosome vacuoles; and carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 50 µM), a partial inhibitor of the pH gradient-activated chloride ion uptake of phagosomes [18]. THP-1 monocytes pH, phagocytosis, NAG activity and hydrogen peroxide release were evaluated to determine if the effects of alatrofloxacin on these parameters were reversed by these inhibitors over 4 h. 2.6. Inhibition of DNA synthesis after exposure to S. aureus for 2 or 24 h Human THP-1 monocytes (106 cells) were pre-incubated with S. aureus (bacteria: monocyte ratio of ~10:1) for 2 h and non-phagocytosed bacteria were removed by lysostaphin treatment as described below. Alatrofloxacin and 100 µl of [methyl-3H]-thymidine (65.3 mCi/mmol; Moravek Biochemicals, Brea, CA, USA) were added and incubated for 2 or 24 h [16]. The reaction was stopped with 10% percholoric acid and the acid soluble precipitate was collected on GF/A filters (Fischer Scientific, Atlanta, GA, USA) by vacuum suction, counted and corrected for quenching.

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Table 1 Effects of alatrofloxacin on phagocytic processes of human THP-1 monocytes pretreated with zymogen A for 1 h Assay (N = 6) c-AMP

Protein kinase C

NADPH oxidase

NO release

H2O2 release

SOD

Glutathione reductase

Cell pH change

Phagocytosis

Time (h) 0.25 0.5 0.75 1 0.25 0.30 1 0.25 0.5 1 2 4 0.25 0.5 1 2 4 0.25 0.50 1 2 4 0.25 0.5 1 2 4 0.25 0.5 1 2 0.25 0.5 1 2 4 0.25 0.5 1 2 4

Control 100 ± 5 100 ± 5 100 ± 4 100 ± 3 100 ± 3 100 ± 4 100 ± 4 100 ± 5 100 ± 4 100 ± 3 100 ± 4 100 ± 5 100 ± 4 100 ± 4 100 ± 4 100 ± 3 100 ± 5 100 ± 3 100 ± 4 100 ± 5 100 ± 3 100 ± 4 100 ± 3 100 ± 4 100 ± 4 100 ± 2 100 ± 4 100 ± 4 100 ± 4 100 ± 5 100 ± 3 100 ± 2 100 ± 3 100+3 100 ± 4 100 ± 3 100 ± 3 100 ± 5 100 ± 2 100 ± 4 100 ± 4

4 µg/ml 117 ± 4 * 148 ± 4 * 110 ± 3 96 ± 3 189 ± 5 * 201 ± 6 * 126 ± 4 * 194 ± 5 * 163 ± 6 * 134 ± 5 * 131 ± 4 * 124 ± 3 * 144 ± 5 * 145 ± 3 * 110 ± 3 58 ± 3 * 58 ± 4 * 116 ± 3 159 ± 4 * 117 ± 3 * 116 ± 3 * 89 ± 3 135 ± 4 * 123 ± 4 * 145 ± 5 * 125 ± 4 * 120 ± 4 * 417 ± 8 * 169 ± 6 * 106 ± 4 55 ± 4 * 103 ± 4 105 ± 3 101 ± 3 51 ± 3 * 47 ± 4 * 105 ± 3 103 ± 4 99 ± 3 69 ± 4 * 47 ± 3 *

0.4 µg/ml 115 ± 4 * 119 ± 4 * 109 ± 4 92 ± 3 159 ± 4 * 161 ± 6 * 101 ± 3 121 ± 4 * 139 ± 5 * 133 ± 4 * 107 ± 4 121 ± 4 * 124 ± 3 * 121 ± 4 * 106 ± 4 56 ± 4 * 53 ± 3 * 110 ± 4 138 ± 5 * 107 ± 4 99 ± 4 85 ± 3 120+4 * 99 ± 4 129 ± 3 * 109 ± 4 95 ± 3 229 ± 4 * 118 ± 4 * 104 ± 5 46 ± 3 * 102 ± 4 104 ± 3 100 ± 1 49 ± 4 * 45 ± 1 * 104 ± 3 102 ± 3 98 ± 3 66 ± 4 * 45 ± 4 *

0.04 µg/ml 102 ± 3 107 ± 3 107 ± 2 87 ± 3 130 ± 4 * 130 ± 4 * 90 ± 3 104 ± 4 134 ± 4 * 125 ± 3 * 103 ± 4 110 ± 4 99 ± 4 98 ± 4 78 ± 3 * 51 ± 4 * 52 ± 4 * 108 ± 3 126 ± 4 * 103 ± 3 96 ± 3 82 ± 3 92 ± 4 97 ± 3 118 ± 3 * 95 ± 3 88 ± 2 173 ± 5 * 118 ± 3 * 100 ± 5 37 ± 3 * 101 ± 3 103 ± 2 100 ± 3 48 ± 2 * 45 ± 3 * 104 ± 4 103 ± 3 98 ± 2 66 ± 5 * 45 ± 4 *

0.004 µg/ml 79 ± 2 104 ± 3 101 ± 3 86 ± 3 117 ± 3 * 93 ± 3 67 ± 3 * 100 ± 3 123 ± 4 * 118 ± 4 * 97 ± 5 102 ± 4 77 ± 4 * 89 ± 4 69 ± 3 * 48 ± 4 * 27 ± 3 * 104 ± 3 123 ± 4 * 85 ± 4 91 ± 3 70 ± 3 90 ± 3 97 ± 4 114 ± 4 99 ± 4 86 ± 4 151 ± 4 * 104 ± 3 93 ± 4 37 ± 5 * 101 ± 6 102+4 99 ± 2 47 ± 3 * 44 ± 2 * 103 ± 5 101 ± 4 97 ± 4 65 ± 3 * 44 ± 3 *

* P = 0.001.

2.7. Intracellular and extracellular activity of alatrofloxacin against S. aureus Human THP-1 monocytes were stimulated with S. aureus, as described above. Then, non-phagocytosed bacteria were removed by incubating the suspension with lysostaphin (10 µg/ml) for 15 min at 37 °C [7]. The suspension was then centrifuged and the monocytes with intracellular bacteria were incubated in the absence (control) or presence of different concentrations of alatrofloxacin. At this time, the number of monocyte-associated microorganisms was approximately 5 × 104 cfu/ml. After 2 or 24 h of incubation, samples were removed and the monocytes disrupted by brief sonication. Serial dilutions were performed and plated onto agar to

determine the number of viable intracellular bacteria. To assess the activity of alatrofloxacin against staphylococci in THP-1 cell-free medium, pre-opsinized S. aureus bacteria (~106 cfu/ml) were exposed to drug at 37 °C in the absence of monocytes. After 2 or 24 h, samples were removed and serial dilutions plated onto agar. The assay was repeated with differences between assays being <1 log 10 cfu/ml. Fig. 7 is a representative example of the results. 2.8. Statistical analysis Data is presented in the tables and figures as the percent of control with standard deviations. The probable significance

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Table 2 Effects of alatrofloxacin on immunologic cytokines after treatment of human THP-1 monocytes with zymogen A for 1 h Assay (N = 6) TNFa

IL-1 IL-6 IL-8

Time (h) 0.5 1 2 4 1 2 2 4 1 2 4

Control 100 ± 4 100 ± 3 100 ± 5 100 ± 2 100 ± 5 100 ± 3 100 ± 4 100 ± 5 100 ± 3 100 ± 2 100 ± 4

4 µg/ml 222 ± 8 * 168 ± 5 * 121 ± 4 * 116 ± 3 * 106 ± 5 121 ± 4 * 132 ± 5 * 104 ± 4 149 ± 5 * 312 ± 7 * 209 ± 5 *

0.4 µg/ml 173 ± 5 * 166 ± 6 * 108 ± 3 88 ± 4 92 ± 4 119 ± 4 * 110 ± 5 102 ± 3 145 ± 4 * 299 ± 6 * 205 ± 4 *

0.04 µg/ml 113 ± 5 106 ± 4 88 ± 4 59 ± 5 * 87 ± 6 79 ± 5 * 105 ± 4 100 ± 5 127 ± 6 * 187 ± 6 * 167 ± 5 *

0.004 µg/ml 110 ± 4 99 ± 3 84 ± 4 59 ± 4 * 79 ± 4 * 65 ± 4 * 90 ± 4 100 ± 2 85 ± 4 102 ± 5 156 ± 4 *

* P = 0.001.

difference between the control and treated raw data was determined by the Student’s t-test. 3. Results The zymogen-stimulated human THP-1 monocytes after incubation with alatrofloxacin showed that c-AMP levels were elevated at 15 and 30 min in a concentration-dependent manner but were normal after 1 h (Table 1). Protein kinase C activity was elevated at 15 and 30 min but returned to normal levels after 1 h at the lower concentration of alatrofloxacin. NADPH oxidase activity over the first 2 h was elevated but was returning to normal levels at 4 h. NO release was significantly elevated at 15 and 30 min, but was normal by 1 h. At 2 and 4 h, NO was significantly reduced below normal levels. Hydrogen peroxide release was significantly elevated at all tested concentrations of alatrofloxacin at 30 min, but returned to normal by 1 h, and was below normal at 4 h. At 4 µg/ml, SOD activity was elevated from 15 min to 4 h. At lower concentrations of the antibiotic, the SOD activity was normal. Glutathione reductase activity was elevated at 15 and 30 min, but returned to normal levels at 1 h and was below

normal levels at 2 h. THP-1 monocyte pH was significantly acidic at 2 and 4 h. Phagocytosis was also inhibited after 2 and 4 h of incubation with alatrofloxacin. TNFa levels were elevated at 30 min through 4 h at the highest antibiotic concentration (i.e. 4 µg/ml), and at 30 min for the remaining concentrations (Table 2). TNFa levels were lower at 4 h at 0.004–0.4 µg/ml of drug. IL-1 and IL-6 release was elevated at 4 µg/ml at 2 h, whereas IL-8 levels were elevated at 1, 2 and 4 h from 0.4 to 4 µg/ml concentrations of drug. NAG activity was elevated significantly at all concentrations at 30 min, but returned to normal levels at 2 h and was below normal levels at 4 h (Table 3). Cathepsin D activity was significantly higher for the first 4 h and then returned to normal at 6 h. Lipid peroxidation was evident for the first 2 h of drug incubation, but returned to normal levels at 4 h. Studies on the incubation of alatrofloxacin with an inhibitor of energy, a blocker of the phagosome–lysosome merger and an inhibitor of the membrane proton pump, showed that these agents had little influence on the effect of alatrofloxacin on cell pH or phagocytosis (Figs. 1 and 2). The agents did suppress the antibiotic’s effects on THP-1 monocyte NAG activity (Fig. 3) and hydrogen peroxide release (Fig. 4). When the THP-1 monocytes

Table 3 Effects of alatrofloxacin on lysosomal enzymes and lipid peroxidation of human THP-1 monocytes after pre-treatment with zymogen A for 1 h Assay (N = 6) NAG

Cathepsin D

Lipid peroxidation

* P = 0.001.

Time (h) 0.25 0.5 1 2 4 0.25 0.5 1 2 4 6 0.25 0.5 1 2 4

Control 100 ± 4 100 ± 3 100+4 100 ± 2 100 ± 3 100 ± 4 100 ± 3 100 ± 6 100 ± 5 100 ± 4 100 ± 4 100 ± 3 100 ± 5 100 ± 3 100 ± 4 100 ± 4

4 µg/ml 179 ± 5 * 308 ± 7 * 172 ± 6 * 103 ± 4 90 ± 4 137 ± 4 * 171 ± 5 * 159 ± 4 * 151 ± 4 * 130 ± 5 * 107 ± 5 133 ± 5 * 204 ± 5 * 152 ± 6 * 149 ± 5 * 101 ± 4

0.4 µg/ml 144 ± 3 * 210 ± 5 * 124 ± 4 * 97 ± 3 81 ± 4 133 ± 4 * 150 ± 5 * 139 ± 5 * 139 ± 4 * 127 ± 4 * 100 ± 3 126 ± 2 * 204 ± 6 * 141 ± 4 * 138 ± 4 * 100 ± 3

0.04 µg/ml 98 ± 3 183 ± 6 * 110 ± 5 92 ± 5 69 ± 3 * 122 ± 3 * 145 ± 5 * 137 ± 4 * 132 ± 6 * 112 ± 5 * 100 ± 5 122 ± 4 * 139 ± 5 * 128 ± 4 * 114 ± 4 74 ± 4 *

0.004 µg/ml 92 ± 4 127 ± 4 * 92 ± 3 87 ± 3 74 ± 4 * 114 ± 4 133 ± 4 * 136 ± 4 * 133 ± 3 * 91 ± 4 73 ± 4 113 ± 5 138 ± 5 * 110 ± 3 107 ± 5 73 ± 3 *

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Fig. 1. Effects of alatrofloxacin on zymogen A-stimulated human THP-1 monocyte cell pH incubated with inhibitors over 4 h (N = 6; all standard deviations were within 2.7%).

were stimulated with S. aureus and incubated for 2 h, there was a transient increase in cell pH followed by a general decrease to the acidic pH over 4 h (Fig. 5). Phagocytosis was slightly decreased by alatrofloxacin over 4 h. NAG activity and release of hydrogen peroxide were elevated at 15 and 30 min, and subsequently returned to normal, while NAG activity was reduced below normal control values at 4 h.

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Fig. 4. Effects of alatrofloxacin on zymogen A-stimulated human THP-1 monocyte hydrogen peroxide release incubated with inhibitors over 4 h (N = 6; all standard deviations were within 4.5%).

DNA synthesis in THP-1 monocytes was not decreased by alatrofloxacin over 4 h; in fact, it was slightly elevated (Fig. 6). DNA synthesis in S. aureus alone was suppressed by the antibiotic in a concentration-dependent manner at 2 h, but was more significant suppressed at 24 h. DNA synthesis of THP-1-phagocytosed S. aureus was marginally reduced at 2 h in a concentration-dependent manner, and was more significantly inhibited at 24 h of incubation. The degree of inhibition of DNA synthesis lagged behind slightly in the bacteria phagocytosed by THP-1 cells compared to the bacteria alone. Examination of the number of S. aureus bacteria after exposure to the antibiotic showed significant killing at 4 and 40 µg/ml at 24 h (Fig. 7a). When alatrofloxacin was tested against S. aureus phagocytosed by THP-1 monocytes, killing of the organism was marginal at 0.4 µg/ml, but reached 100% at 40 µg/ml (Fig. 7b).

4. Discussion

Fig. 2. Effects of alatrofloxacin on zymogen A-stimulated human THP-1 monocyte phagocytosis incubated with inhibitors over 4 h (N = 6; all standard deviations were within 3.2%).

Fig. 3. Effects of alatrofloxacin on zymogen A-stimulated human THP-1 monocyte NAG activity incubated with inhibitors over 4 h (N = 6; all standard deviations were within 4.1%).

Pathogenic bacteria and other infectious agents can activate macrophages or monocytes directly, initiating a cytokine cascade in the inflammation process [2,4,17,28,31], and

Fig. 5. Effects of alatrofloxacin at 4 µg/ml on S. aureus-stimulated human THP-1 monocyte, cell pH, phagocytosis, NAG activity, and hydrogen peroxide release over 4 h (N = 6; all standard deviations were within 3.8%).

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Fig. 6. Effects of alatrofloxacin on DNA synthesis of THP-1 monocytes, S. aureus, and THP-1 monocytes with phagocytosed S. aureus, at 2 and 24 h.

the immunological response. Stimulated monocytes release a broad spectrum of cytokines, which can induce the subsequent cytokine cascade. IL-1, TNFa, and IL-6 are biologically active peptides produced by monocytes, induced by

endotoxin [4,26] and other stimuli. Soluble factors such as these can initiate and maintain the acute phase of the inflammatory response [26] with the concurrent release of free radicals and lysosomal hydrolytic enzymes. Inflammation, tissue destruction and systemic spread of the infection are conveyed by these cellular processes. Fluoroquinolones and macrolides have been reported to inhibit the production of IL-1 or TNFa in lipopolysaccharide-stimulated human monocyte cultures [2,3,17,23,24,27,29]. Independent of its antimicrobial activity, clarithromycin has demonstrated in vitro immunomodulatory activity during the activation of human lymphocytes and monocytes [18,21,23–25]. After treatment with clarithromycin, azithromycin, moxifloxacin, gemifloxacin or grepafloxacin human THP-1 monocytes stimulated with either zymogen A or S. aureus demonstrated modulation of free radicals and cytokine release with reduction in lysosomal hydrolytic enzyme activity and tissue lipid peroxidation [13–15,18]. The effects of these fluoroquinolones including the naphthal derivatives appear to be in two stages. Initially, there is a rapid release of TNFa, c-AMP, NO and hydrogen peroxide and activation of protein kinase C and membrane NADPH oxidase activity in the first 30–60 min. These effects could cause a cidal attack on any bacteria ingested by these phagocytic cells for destruction as lysosomal hydrolytic activity and lipid peroxidation are elevated at this time. At about 1–2 h, there is a reversal of these effects and these biochemical parameters return to normal or below normal levels, suggesting that this type of cidal effect has ended. Certain antiinflammatory cytokines, e.g. IL-6 and IL-8 have been suggested to reverse this process [22] and these interleukins are elevated in THP-1 monocytes after an hour in the presence of alatrofloxacin inhibition of DNA synthesis of the extracellular bacteria or the bacteria phagocytosed by THP-1 monocytes was modest at 2 h, but was more evident at 24 h. Bacterial killing at these times is more consistent with the bacteriostatic function of the fluoroquinolones (i.e. via interference with bacterial DNA synthesis).

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