Penetration, efflux and intracellular activity of tigecycline in human polymorphonuclear neutrophils (PMNs)

Christine T. Ong1, Chinedum P. Babalola1, Charles H. Nightingale1 and David P. Nicolau1,2,*

1 Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour Street, Hartford, CT 06102, USA; 2 Division of Infectious Diseases, Hartford Hospital, Hartford, CT 06102, USA


* Correspondence address. Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour Street, Hartford, CT 06102, USA. Tel: +1-860-545-3940; Fax: +1-860-545-3992; E-mail: dnicola{at}harthosp.org

Received 22 November 2004; returned 10 May 2005; revised 21 June 2005; accepted 8 June 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To evaluate the penetration, efflux and intracellular activity of tigecycline in human polymorphonuclear neutrophils (PMNs).

Methods: PMNs were isolated from fresh whole blood and tested for viability and purity prior to use. Tigecycline drug uptake was evaluated by incubating 5 x 106 cells/mL at 37°C up to 3 h at tigecycline concentrations of 1, 2, 5 and 10 mg/L. Drug efflux from PMNs was determined following a 2 h incubation with tigecycline at 10 mg/L. Its intracellular activity against Staphylococcus aureus was evaluated following tigecycline extracellular exposures of 1 mg/L.

Results: Tigecycline uptake was rapid and achieved high concentrations within PMNs with maximal penetration noted at 1 h of incubation. At 1 h, dose-dependent intracellular concentrations ranged from 15.83 ± 11.09 mg/L to 264 ± 54.60 mg/L at tigecycline 1 and 10 mg/L, respectively. At these exposures, intracellular drug concentrations were ~20 and 30 times higher at 1 h than extracellular concentrations. By 3 h, tigecycline displayed sustained high intracellular exposures. Tigecycline cell efflux followed first order kinetics with a half-life of 1.39 h. Tigecycline was bacteriostatic against intracellular S. aureus.

Conclusions: Tigecycline rapidly achieved high intracellular concentrations in PMNs and exhibited static activity against S. aureus supporting its potential clinical utilization.

Keywords: glycylcyclines , intracellular concentration , phagocytes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability of Staphylococcus aureus to survive within phagocytic cells has been reported to contribute to their pathogenicity and their common predisposition to cause persistent and recurrent infections.1,2 Therapeutic failures despite appropriate antibiotic treatments could in part be due to the inability of certain antibiotics to reach intracellularly localized S. aureus. Hence, antibiotics with intracellular activity may play a critical role in treating such infection.

Tigecycline, a tetracycline derivative, belongs to a novel class of antibiotics known as the glycylcyclines. Tigecycline possesses broad-spectrum antibacterial activities against Gram-positive, Gram-negative and anaerobic bacteria including methicillin-resistant Staphylococci aureus, penicillin-resistant Streptococcus pneumoniae and vancomycin-resistant Enterococcus.35 In this study, we evaluated the intracellular pharmacokinetics and activity of tigecycline in human polymorphonuclear cells (PMNs).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tigecycline (Wyeth Pharmaceuticals, Wayne, NJ, USA) analytical grade standards were utilized. Stock solutions were prepared in Hank's buffered salt solution, pH 7.2 (HBSS; Sigma Chemical Co., St Louis, MO, USA) at concentrations ranging from 0.1–10 mg/L and stored at –80°C prior to use. Stock solutions were used within 3 months of preparation.

PMNs were recovered from heparinized venous blood of healthy donors using the One-StepTM Polymorph (Accurate Chemical and Scientific Corp., Westbury, NY, USA) velocity gradient technique.3 The harvested PMNs were purified using an ammonium chloride solution to lyse residual erythrocytes. The purity and viability of the PMN preparations were >90% for both as determined by the Trypan Blue exclusion test. Final cell count was determined using a Coulter counter and adjusted to 5 x 106 PMNs/mL prior to each experiment.

The ability of PMNs to accumulate tigecycline was tested per subject (n = 6) in duplicate at concentrations of 0.1, 1, 2, 5 and 10 g/L. The PMNs were incubated with tigecycline at 37°C at various time intervals from 0–3 h. At each respective time point using methods described previously,6,7 the cells were separated from the drug-containing supernatant by high speed centrifugation at 10 000g for 5 min at 4°C. The cells were then quickly washed with ice-cold HBSS to quench the kinetic reaction prior to storage at –80°C.

For the efflux study, the PMNs (as isolated above from six donors) were divided into 1 mL aliquots with HBSS containing 106 cells/mL. All aliquots were placed in a vibrating water bath at 37°C for 2 h with 10 mg/L of tigecycline. The tigecycline-loaded PMNs were centrifuged, washed with cold medium and resuspended with 1 mL of HBSS. Prior to reincubation of the samples, 0 h samples were collected and processed while the rest were assessed for drug efflux at 0.08, 0.16, 0.5, 1 and 2 h. The supernatant and the cell pellet were separated and stored at –80°C until analysis. All experiments were conducted in triplicate.

The intracellular activity of tigecycline against S. aureus ATCC 29213 (MIC = 0.25 mg/L) was evaluated using previously described methods.8 The PMNs and freshly transferred bacteria were combined using a 1 : 10 ratio in a series of polypropylene tubes and incubated in a water bath shaker for 1 h at 37°C.4 After incubation, the mixtures were washed three times with 1 mL of cold phosphate-buffered saline (PBS, pH = 7.2) to remove the extracellular bacteria. The infected PMNs were resuspended in 0.5 mL of cold RPMI 1640 (Sigma Chemical Co.), supplemented with 10% {gamma}-irradiated fetal bovine serum (Sigma Chemical Co.) and 2 mM L-glutamine (Sigma Chemical Co.). Since tigecycline's clinical dosing regimen provides for a 100 mg loading dose, which achieves peak concentrations of ~1 mg/L, a final tigecycline concentration of 1 mg/L was used in this experiment and added to each tube. Time 0 h samples were processed while the remaining samples were incubated at 37°C. The infected PMNs were harvested, washed with cold PBS, and lysed with sterile water for bacterial release and quantitative assessment at 1, 2, 3, 4, 6 and 24 h.9 Plates were incubated at 37°C for 24 h. Experiments were conducted in duplicate from PMNs obtained from three different donors. Controls (without antibiotic) were also included for each evaluated period.

Drug concentrations in the cell pellet and supernatant were determined using a validated high-performance liquid chromatography (HPLC) technique.10 Intracellular drug release was ensured by the addition of 5% trichloroacetic acid during the HPLC extraction procedure.10 Tigecycline assays in both matrices were linear (r = 1.0) over the concentration range of 0.05–5 mg/L and 0.05–2 mg/L, respectively. The interday quality control samples (%CV) ranged from 3.4–5.4% for the HBSS and 3.2–5% for the human PMNs. Intraday values were 2.0–6.4% and 0.7–5.0%, respectively. The measured intracellular concentrations were adjusted for both the number of PMNs and the average cell volume (334 fL) as determined previously using the following equation:11,12 corrected PMN concentration = (PMN concentration)/ (average cell volume) x (WBC count).

Data were expressed as means ± standard deviation. The ratio of intracellular to extracellular cell concentrations (C/E) ratios was determined. Non-compartmental analysis (WinNonlin, version 4.2, Pharsight Corp., Mountain View, CA, USA) was performed to determine the rate constant (Kefflux), and intracellular half-life (t1/2) of tigecycline. Bacterial density studies were reported as a change in log cfu from the 0 h controls. Bacteriostatic is defined as no net change from 0 h controls. Bactericidal effects are defined as ≥1 log reduction from the 0 h controls.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tigecycline uptake was rapid and achieved high concentrations within PMNs with maximal penetration noted at 1 h of incubation for all concentrations studied (Figure 1). At 1 h, dose-dependent intracellular concentrations ranged from 15.83 ± 11.09 mg/L and 264 ± 54.60 mg/L using 1 and 10 mg/L, respectively. At these exposures, drug penetration resulted in C/E ratios of 21.11 ± 13.70 and 29.47 ± 5.20 at 1 h. Although these ratios were slightly reduced at 3 h, they remained high at 15.46 ± 13.54 for 1 mg/L and 28.07 ± 5.40 for 10 mg/L exposures. Since intracellular tigecycline concentrations could not be detected in the 0.1 mg/L treatment group, a C/E ratio could not be determined in the current analysis.



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Figure 1. Tigecycline intracellular concentrations in PMNs isolated from six donors (presented as mean ± SD).

 
Tigecycline exhibited gradual egression from loaded PMNs with a percent release of 18.10 ± 3.79 at 0.08 h, 45.98 ± 4.9 at 0.5 h and 66.53 ± 2.98 at 2 h (Figure 2). Drug release followed a linear regression and first order kinetics with Kefflux of 0.4998 h–1 and t1/2 of 1.39 h. In PMNs infected with S. aureus, tigecycline displayed bacteriostatic activity throughout the evaluated period of 24 h (Figure 3).



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Figure 2. Tigecycline cell-associated drug efflux in PMNs from six donors (presented as mean ± SD).

 


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Figure 3. Intracellular activity of tigecycline (TGC) against S. aureus (ATCC 29213) (presented as mean ± SD).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The persistence and recurrence of S. aureus infection despite antibiotic therapy may be attributed to its internalization and survival within phagocytic cells. Targeting these infection sites may prove critical in improving clinical response.

In this study, tigecycline displayed rapid intracellular penetration into PMNs with concentrations reaching ~20–30 times higher compared with extracellular drug concentrations at 1 h. Intracellular drug concentrations remained elevated at 3 h under continuous exposure of the PMNs to tigecycline. The evaluation of tigecycline's efflux from the cells resulted in a shorter intracellular t1/2 than what is observed clinically in plasma (1.4 versus ~50 h).13 The incubation of drug-loaded PMN in an antibiotic-free medium may have accelerated this drug release. Although the current study did not evaluate the mechanisms behind drug uptake and efflux, the sustained intracellular concentrations observed under continued extracellular exposures during the uptake study suggested a passive mechanism. The conditions of prolonged exposures accompanied with the gradual drug elimination (as seen in vivo) would likely sustain drug intracellular concentrations for an extended period. Hence, the protracted half-life of the compound based on the efflux study should only represent a conservative estimate of its intracellular kinetics after systemic exposure in vivo.

Tigecycline is currently being investigated for the treatment of complicated skin and soft tissue infections and intra-abdominal infections.14,15 Clinical data have shown efficacy with dosing regimens of 25 or 50 mg administered intravenously every 12 h with an initial loading dose of 50 or 100 mg, respectively. Multiple doses exhibited linear kinetics with mean peak, AUCss and t1/2 in the range 0.32–1.17 mg/L, 1.48–4.98 mg·h/L and 49.3–66.5 h (25–100 mg given twice daily).16 Tigecycline's pharmacodynamic parameter for optimal bacterial activity can be achieved with sustained concentrations at 50% of the time above the MIC (t > MIC).17 Twice-daily dosing regimens, long plasma t1/2, high intracellular concentrations and prolonged intracellular bacteriostatic effects favourably support tigecycline's intracellular pharmacodynamic profile.

Several considerations should be kept in mind when interpreting these results. The intracellular penetration and concentration of tigecycline can be affected by various elements such as physiochemical factors (e.g. pH) and varying extracellular drug exposures. Although this study only evaluated one set of experimental conditions, it was conducted using a pH and incubation temperature to approximate in vivo conditions. Unlike an in vivo system, however, this study was limited by an observance at constant drug concentrations over a short period rather than changing drug concentrations over time as seen in vivo. Moreover, the effect of protein binding (tigecycline is ~70%)5 on cellular uptake was not evaluated in this study.

Tigecycline achieved high intracellular PMN concentrations while displaying intracellular bacteriostatic activity against S. aureus. While the clinical impact of such activity remains to be determined, these data support the clinical utilization of tigecycline for intracellular pathogens with the potential for tigecycline-loaded PMNs to deliver additional drug to the site of infection.


    Acknowledgements
 
We would like to thank Christina Sutherland, Chonghua Li, and Toral Patel for their bioanalytical and technical assistance in this study. Financial support for this study was provided by Wyeth Pharmaceuticals.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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4. Onyeji CO, Nicolau DP, Nightingale CH et al. Interferon – {gamma} effects on activities of gentamicin and vancomycin against Enterococcus faecalis resistant to the drugs: an in vitro study with human neutrophils. Int J Antimicrob Agents 1999; 11: 31–7.[CrossRef][ISI][Medline]

5. Zhanel GG, Homenuik K, Nichol K et al. The glycylcyclines: A comparative review with the tetracyclines. Drugs 2004; 64: 63–88.[ISI][Medline]

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8. Petersen PJ, Jacobus NV, Weiss WJ et al. In vitro and in vivo antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother 1999; 43: 738–44.[Abstract/Free Full Text]

9. Carryn S, Van Bambeke F, Mingeot-Leclercq M et al. Comparative intracellular (THP-1 macrophage) and extracellular activities of ß-lactams, azithromycin, gentamicin, and fluoroquinolones against Listeria monocytogenes at clinically relevant concentrations. Antimicrob Agents Chemother 2002; 46: 2095–103.[Abstract/Free Full Text]

10. Li C, Sutherland C, Nightingale CH et al. Quantitation of tigecycline, a novel glycylcycline by liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2005; 819: 201.[ISI]

11. Nibbering PH, Zomerdijk PL, Corsel-Van Tilburg AJ et al. Mean cell volume of human blood leukocytes and resident and activated murine macrophages. J Immunol Methods 1990; 129: 143–5.[CrossRef][ISI][Medline]

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14. Postier RG, Green SL, Klein SR et al. Results of a multicenter, randomized, open-label efficacy and safety study of two doses of tigecycline for complicated skin and skin-structure infections in hospitalized patients. Clin Ther 2004; 26: 704–14.[CrossRef][ISI][Medline]

15. Murray J, Wilson S, Klein S et al. The clinical response to tigecycline in the treatment of complicated intra-abdominal infections in hospitalized patients, a phase 2 clinical trial. In Programs and Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract 739. American Society for Microbiology, Washington, DC, USA.

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