Comparative pharmacodynamics of azithromycin and roxithromycin with S. pyogenes and S. pneumoniae in a model that simulates in vitro pharmacokinetics in human tonsils

Alexander A. Firsova,b,*, Stephen H. Zinnerc, Sergey N. Vostrova,b, Olga V. Kononenkoa, Yury A. Portnoya,b, Larissa V. Shustovaa and Igor B. Kadenatsia

a Department of Pharmacokinetics, Centre for Science & Technology LekBioTech, and b Department of Pharmacokinetics and Pharmacodynamics, Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, Moscow, Russia; c Mount Auburn Hospital, Harvard Medical School, Cambridge, MA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Most macrolides penetrate and persist in peripheral tissues, irrespective of plasma concentrations. For this reason, comparative pharmacodynamics of macrolides might be better based on tissue rather than plasma pharmacokinetics. The present study compares the antimicrobial effects of azithromycin and roxithromycin on Streptococcus pyogenes and Streptococcus pneumoniae using in vitro simulations of steady-state pharmacokinetics in human tonsils expected after a third 500 mg dose of azithromycin administered once a day and after a sixth 150 mg dose of roxithromycin administered twice a day. Clinical isolates of S. pyogenes and S. pneumoniae (MICs 0.12 and 0.47 mg/L of azithromycin, and 0.15 and 0.60 mg/L of roxithromycin, respectively) were used. More pronounced antistreptococcal effects were observed with azithromycin than with roxithromycin. Despite similar rates of initial killing of S. pyogenes and S. pneumoniae, the respective 12 h areas between the control growth curve and the time–kill curve of antibiotic-exposed bacteria (ABBCs) were 22% and 36% greater with azithromycin than roxithromycin. Moreover, with azithromycin, viable bacterial counts reached the theoretically achievable limit of detection (10 cfu/mL) 8–10 h after drug administration, with no regrowth within 48 h. In contrast to azithromycin, S. pyogenes and S. pneumoniae exposed to roxithromycin regrew 26 and 6 h, respectively, after initial reduction of the starting inoculum. Further in vitro simulations of tissue pharmacokinetics might be useful for pharmacodynamic comparisons among other macrolides.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Most macrolides introduced over the past decade exhibit an enhanced ability to penetrate and persist in peripheral tissues, irrespective of high (e.g. roxithromycin, clarithromycin) or relatively low (e.g. azithromycin, dirithromycin) plasma concentrations.1 Therefore, as a measure of tissue availability, the ratio of the area under the concentration–time curve (AUC) in peripheral tissue to the respective AUC in plasma differs among the macrolides. For example, the tissue availability of azithromycin is 55-fold higher in tonsils (17.7)2 and six-fold higher in sinus mucosa (6.2)2 than roxithromycin (0.323 and 0.95,4 respectively). These differences relate to comparable peak concentrations in tonsils and sinus with both drugs, despite lower azithromycin plasma concentrations,2–4 and help to explain the similar clinical efficacy of the two macrolides in upper respiratory tract infections.5

Similarities or differences in efficacy between these two macrolides might better correlate with their tissue concentrations than their plasma concentrations. Furthermore, a true understanding of comparative macrolide pharmacodynamics should be based on appropriate in vitro simulations of tissue rather than plasma pharmacokinetics, as reported in experiments with clarithromycin6 or erythromycin7 compared with azithromycin. The present study compares the antimicrobial effects of azithromycin and roxithromycin using an in vitro model simulating their pharmacokinetics in a specific peripheral tissue, human tonsils.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antimicrobial agents and bacterial strains

Azithromycin and roxithromycin powders were kindly provided by Roerig, a division of Pfizer Pharmaceuticals, (Groton, CT, USA) and Hoechst-Marion-Roussel (Bridgewater, NJ, USA), respectively. Clinical isolates of Streptococcus pyogenes and Streptococcus pneumoniae with similar susceptibility to both macrolides were used in the study. MICs determined by multiple serial dilutions were 0.12 and 0.47 mg/L of azithromycin and 0.15 and 0.60 mg/L of roxithromycin, respectively.

Simulated pharmacokinetic profiles

The simulated pharmacokinetic profiles reflected steady-state tonsillar concentrations of the macrolides expected after a third dose of azithromycin 500 mg od and after a sixth dose of roxithromycin 150 mg bd.

Azithromycin.
To calculate the steady-state concentrations of azithromycin in human tonsils, pharmacokinetic data from a 500 mg single dose study2 and a study that used two 250 mg doses administered 12 hourly8 were combined. The first study measured azithromycin concentrations in homogenized tonsils from 1 to 12 h post-dose, whereas the second study used data obtained between 13 and 178 h, without reference to the ascending limb of the concentration–time curve (Figure 1aGo). The combined data set was fitted by the Bateman function:

(1)
where C(t) is the drug concentration in the tonsils at the time t after administration, kin and kel are the rate constants of drug input and elimination, respectively, and A and B are coefficients.



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Figure 1. (a) Single dose pharmacokinetics of azithromycin in human tonsils compiled from reported studies ({square}2 and {blacksquare}8, respectively) fitted by equation 1Go. (b) Steady-state pharmacokinetics () simulated in an in vitro dynamic model over a 48 h time period and its breakdown into mono-exponential () and bi-exponential (– – –) profiles. (c) Steady-state pharmacokinetics of roxithromycin ({triangleup}3) fitted by equation 1Go (), and its breakdown into mono-exponential () and bi-exponential (– – –) profiles.

 
The lag-time (Tlag) of drug appearance in the tonsils was calculated by the equation:

(2)
Best fit estimates of these parameters obtained by a non-linear regression analysis using TopFit software [v. 1.1, Godecke (Freiburg), Schering (Berlin) and Thomae (Biberach-an-den-Riss, Germany)] are shown in the TableGo.


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Table. Parameters of equations 1 and 2GoGo, and those of the dynamic model
 
Assuming linear pharmacokinetics and using the superimposition principle, steady-state tonsillar concentrations of azithromycin were calculated based on the parameters of equation 1Go. The pharmacokinetic profile that can be expected over a 48 h time period after the third daily 500 mg dose of azithromycin is shown in Figure 1bGo. This profile is presented as the sum of the two superimposed profiles. One of these represents the mono-exponential time course of residual azithromycin concentrations following the preceding, i.e. second dose. The other represents azithromycin pharmacokinetics after a single 500 mg dose. These two profiles, simulated simultaneously, provide the net pharmacokinetic profile that mimics the third administration of azithromycin (see below).

Roxithromycin.
Steady-state concentrations of roxithromycin in homogenized tonsils have been reported in humans after twice daily administration of 150 mg for 3 days3 (Figure 1cGo). The concentration–time data were fitted by equation 1Go, taking into account the residual antibiotic concentration after its preceding dose, i.e. the fifth dose. This steady-state pharmacokinetic profile is presented as the sum of mono- and bi-exponential (Bateman) functions. The estimated parameters of equation 1Go are shown in the Table.

In vitro dynamic model

A slightly modified version of a previously described dynamic model9 was used. Pharmacokinetically, the model consists of two compartments: subcompartment 0, which mimics mono-exponential drug efflux from the systemic circulation to tonsillar tissue; and compartment 1, which mimics drug pharmacokinetics in tonsils, obeying the Bateman function. Mono-exponential efflux of antibiotic is provided by continuous dilution of antibiotic solution of volume in subcompartment 0 (V0) with fresh nutrient medium with a flow rate F. Bi-exponentially changing concentrations of antibiotic in compartment 1 of volume V1 are provided by continuous influx of antibiotic solution in nutrient medium from subcompartment 0 and by efflux of antibiotic solution from compartment 1 to the waste with the same flow rate F. Based on azithromycin and roxithromycin pharmacokinetic parameters, F, V0 and V1 were calculated as described elsewhere.10 The respective values of F, V0 and V1 are presented in the Table. To simulate the mono-exponential decay of residual antibiotic concentrations from the preceding dose, azithromycin or roxithromycin was administered into compartment 1 to achieve the desired level (8.4 and 1.0 mg/L, respectively). To simulate the lag-time in tonsillar drug appearance, antibiotics were administered into subcompartment 0 1 h after their administration into compartment 1.

Physically, the model was represented by three connected flasks: one containing fresh trypticase soy broth with 10% pooled horse serum; the second, subcompartment 0, containing the same broth; and the third, compartment 1, containing the broth plus a bacterial culture (Figure 2Go). The third flask had a magnetic stirrer and was incubated at 37°C. The system was filled with sterile broth. The medium in the third flask was inoculated with a 24 h culture of S. pyogenes or S. pneumoniae, and after a further 2 h incubation, when exponentially growing cultures approached c. 106 cfu/mL, azithromycin or roxithromycin was injected into the third flask and 1 h later into the second flask. Peristaltic pumps circulated fresh nutrient medium from the first flask to the second flask and then to the third flask, as well as from the third flask to the waste chamber. The volumes of fluids in the second and third flasks were maintained constant during the experiment.



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Figure 2. Schematic illustration of the dynamic model.

 
Validation of the dynamic model

The ability of the dynamic model to simulate the required pharmacokinetic profiles of azithromycin and roxithromycin was tested using ciprofloxacin, because levels of this drug can be measured easily with good reliability and sensitivity. The pharmacokinetic parameters of azithromycin and roxithromycin were produced in the model in triplicate using ciprofloxacin. To verify the suitability of the use of ciprofloxacin as a marker for the macrolides, parallel determinations of ciprofloxacin and roxithromycin were also performed when 10-fold greater concentrations of roxithromycin and ciprofloxacin with pharmacokinetic parameters of roxithromycin were simulated simultaneously.

Assays

Ciprofloxacin concentrations were assayed by highperformance liquid chromatography (HPLC) with a precolumn (50 x 4.6 mm) and on a column (250 x 4.6 mm) of Silasorb 5 C8 (Lachema, Czech Republic). The mobile phase was 0.02 M KH2PO4, ethanol and acetonitrile (70:20:10 v/v), pumped at a flow rate of 1.3 mL/min. Fluorometric detection (274 nm excitation, 418 nm emission) was used. The detection limit was 0.05 mg/L. The calibration curve was linear within the range 0.1–20 mg/L, and the coefficients of variation at ciprofloxacin 10 and 1 mg/L were 2.2% and 3.4%, respectively.

Roxithromycin concentrations were also determined by HPLC using a precolumn (50 x 4.6 mm) and on a column (250 x 4 mm) with Nucleosil 10 C18 (Macherey-Nagel, Germany). The mobile phase consisted of acetonitrile and 0.067 M phosphate buffer pH 4 (45:55 v/v), pumped at a flow rate of 1.1 mL/min. Detection was by UV absorption at 210 nm. The calibration curve was linear for roxithromycin concentrations ranging from 1 to 20 mg/L, the limit of detection was 0.5 mg/L and the coefficient of variation at roxithromycin 10 mg/L was 2.4%.

Quantification of bacterial growth and killing

In each experiment, 0.1 mL samples were withdrawn from bacteria-containing media in the central compartment (the third flask) throughout the observation period, initially every hour, then every 3 h, and again hourly during the last 6–7 h. These samples were subjected to serial 10-fold dilutions with chilled, sterile 0.9% NaCl and were plated in duplicate on trypticase soy agar supplemented with 10% pooled horse serum. After overnight incubation at 37°C in a 5% CO2 atmosphere, the resulting bacterial colonies were counted, and the numbers of cfu/mL were calculated. The limit of accurate quantification was 2 x 102 cfu/mL. A level of 10 cfu/mL was considered a theoretically achievable limit of detection.

The bacterial elimination rate constant (kelb) as a measure of the rate of initial killing was determined as described elsewhere.11 The area between the control growth curve and the time–kill curve of antibiotic-exposed bacteria (ABBC)12 as an integral measure of the antimicrobial effect was determined over the first 12 h (Figure 3Go).



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Figure 3. Determination of ABBC applied to the kinetics of the killing of S. pyogenes exposed to azithromycin. ABBC describes the shaded area between the control growth and the killing and regrowth curves, limited from above by a cut-off level of 109 cfu/mL and from below by the theoretical limit of detection (10 cfu/mL).

 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pharmacokinetic validation of the dynamic model

The suitability of ciprofloxacin as a marker for the macrolides was verified by simultaneous simulation of roxithromycin and ciprofloxacin with the pharmacokinetic parameters of roxithromycin. As seen in Figure 4aGo, the concentrations of ciprofloxacin in compartment 1 of the dynamic model matched those of roxithromycin. This validated the use of ciprofloxacin alone in the further model validation experiments. As seen in Figure 4 (b and cGo), the concentrations of ciprofloxacin determined with the pharmacokinetic parameters of azithromycin and roxithromycicn were close to the expected pharmacokinetic profiles.



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Figure 4. (a) Concentrations of roxithromycin ({triangleup}) and ciprofloxacin ({triangledown}) in compartment 1 of the model, determined simultaneously. (b) Roxithromycin profile () validated using ciprofloxacin ({triangledown}). (c) Azithromycin profile () validated using ciprofloxacin ({circ}).

 
Azithromycin and roxithromycin pharmacodynamics

The time–kill kinetics of S. pyogenes and S. pneumoniae exposed to the macrolides and the respective control growth curves are presented in Figure 5Go. Azithromycin produced rapid killing of S. pyogenes and S. pneumoniae, with no regrowth for at least 48 h; the viable counts reached the theoretical limit of detection 8–10 h after drug administration. In contrast to azithromycin, S. pyogenes and S. pneumoniae exposed to roxithromycin regrew 26 or 6 h, respectively, after initial rapid reduction of the starting inoculum. The differences between the antimicrobial effects of azithromycin and roxithromycin can be seen over the first 12 h, i.e. within the dosing interval of roxithromycin. Using ABBC as an endpoint of the antimicrobial effect, azithromycin is 22% more efficient against S. pyogenes and 36% more efficient against S. pneumoniae than roxithromycin (ABBC of 78 versus 64, and 72 versus 53 log cfu/mL•h, respectively). However, the rates of azithromycin- and roxithromycin-induced killing were similar—with S. pyogenes the respective kelbs were 1.4/h and 1.3/h, and with S. pneumoniae the kelbs were 1.3/h and 1.5/h, respectively.



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Figure 5. Kinetics of the killing of S. pyogenes (a) and S. pneumoniae (b) exposed to azithromycin ({square} and {blacksquare}) and roxithromycin ({triangleup} and {blacktriangleup}), respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In vitro simulations of tissue pharmacokinetics are relatively rare. Usually, such simulations are associated with dialysis models that expose bacteria to antibiotic in peripheral compartments where the observed pharmacokinetic profiles may approximate the time course of antibiotic concentrations in a peripheral compartment of a pharmacokinetic model, but they are not usually attributed to a specific tissue.10 The present study demonstrates an alternative approach to in vitro simulations in which specific tonsillar pharmacokinetic profiles are simulated directly in the central compartment (compartment 1) of the dynamic model. The reliability of these simulations was verified by using ciprofloxacin to mimic the pharmacokinetic parameters of azithromycin and roxithromycin, since it was a compound that could be easily assayed. The measured concentrations were close to the expected target concentrations.

Unfortunately, the realities of in vitro simulations of specific tissue pharmacokinetics are limited by the availability of in vivo data: all too often, reported tissue concentrations are too sparse to allow appropriate model fitting. Paradoxically, this applies to the new macrolides, although their excellent tissue penetration was a major driving force in their development. Moreover, most in vivostudies, including those used as a basis for our in vitrosimulations, report macrolide concentrations in homogenized tissues, i.e. the sum of relatively high interstitial and relatively low extracellular concentrations, which are in a dynamic equilibrium with plasma concentrations of free (i.e. non-protein-bound) drug.13 Although extracellular concentrations of macrolides are considered more predictive of their antimicrobial effect on most common respiratory tract pathogens,14 this may not explain reported similar efficacies of azithromycin, roxithromycin and clarithromycin in upper respiratory tract infections,5,15 and azithromycin and clarithromycin in streptococcal tonsillitis,15 despite the markedly lower extracellular concentrations of azithromycin.

This study demonstrated the more pronounced antimicrobial effects of simulated tonsillar concentrations of azithromycin compared with those of roxithromycin against S. pyogenes and S. pneumoniae. Despite similar rates of initial killing, the antibacterial effects as expressed by the ABBC determined over the first 12 h of antibiotic exposure were 22% and 36% greater with azithromycin than with roxithromycin, respectively. Moreover, no regrowth occurred with S. pyogenes and S. pneumoniae exposed to azithromycin, but bacterial regrowth was observed 26 or 6 h, respectively, after administration of roxithromycin.

Clearly, these differences would not have been seen with an in vitro simulation of plasma concentrations of these macrolides, which are much higher with roxithromycin than azithromycin. In this light, the more pronounced killing of bacteria exposed to plasma concentrations of clarithromycin compared with azithromycin reported by Bauernfeind et al.6 is quite predictable, because the simulated peak concentration-to-MIC ratios were 5–20 times higher for clarithromycin with four of the five organisms studied. Other conclusions might have been drawn if peripheral tissue pharmacokinetics of clarithromycin and azithromycin were simulated.

Overall, the results of pharmacodynamic comparisons among the tissue-selective macrolides might be highly dependent on whether systemic or peripheral pharmacokinetics are simulated. Care must be taken in deciding which model is most relevant to the clinical situation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by a grant from Roerig, a division of Pfizer Pharmaceuticals.


    Notes
 
* Correspondence address. Department of Pharmacokinetics and Pharmacodynamics, Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11 Bolshaya Pirogovskaya Street, Moscow, 119992 Russia. Tel: +7-095-332-3435; Fax: +7-095-332-3335; E-mail: firsov{at}dol.ru Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Lode, H., Boeckh, M., Schaberg, T., Borner, K. & Koeppe, P. (1993). Pharmacology. In The New Macrolides, Azalides, and Streptogramins, (Neu, H. C., Young, L. S. & Zinner, S. H., Eds), pp. 61–7. Marcel Dekker, Inc., New York.

2 . Firsov, A., Tichonov, V., Kuleshov, S. & Stratchunsky, L. (1993). Azithromycin penetration into sinus mucosa and tonsillar tissue. In Program and Abstracts of the Sixth European Congress of Clinical Microbiology and Infection Diseases, Seville, Spain, 1993. Abstract 306, p. 121. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland.

3 . Fraschini, G., Scaglione, F., Pintucci, G., Maccarinelli, G., Dugnani, S. & Demartini, G. (1991). The diffusion of clarithromycin and roxithromycin into nasal mucosa, tonsil and lung in humans. Journal of Antimicrobial Chemotherapy 27, Suppl. A, 61–5.

4 . Dewever, M. (1988). Determination of roxithromycin concentration in the mucosa of the maxillary sinus. British Journal of Clinical Practice 42, Suppl. 55, 81.

5 . Hoepelman, I. M. & Schneider, M. M. E. (1995). Azithromycin: the first of the tissue-selective azalides. International Journal of Antimicrobial Agents 5, 145–67. [ISI]

6 . Bauernfeind, A., Jungwirth, R. & Eberlein, E. (1995). Comparative pharmacodynamics of clarithromycin and azithromycin against respiratory pathogens. Infection 23, 316–20. [Medline]

7 . Hollander, J. G., den Knudsen, J. D., Mouton, J. W., Fuursted, K., Frimodt-Moller, N., Verbrugh, H. A. et al. (1998). Comparison of pharmacodynamics of azithromycin and erythromycin in vitro and in vivo. Antimicrobial Agents and Chemotherapy 42, 377–82. [Abstract/Free Full Text]

8 . Foulds, G. F., Chan, K. H., Johnson, J. T., Shepard, R. M. & Johnson, R. B. (1991). Concentrations of azithromycin in human tonsillar tissue. European Journal of Clinical Microbiology and Infectious Diseases 10, 853–6. [ISI][Medline]

9 . Firsov, A. A., Nazarov, A. D., Chernykh, V. M. & Navashin, S. M. (1988). Validation of optimal ampicillin/sulbactam ratio in dosage forms using in-vitro dynamic model. Drug Development and Industrial Pharmacy 14, 2425–42. [ISI]

10 . Firsov, A. A., Nazarov, A. D. & Chernykh, V. M. (1989). Pharmacokinetic approaches to rational antibiotic therapy. In Advances in Science and Engineering, (Nesterov, P. V., Ed.), Vol. 17. (In Russian), pp. 1–228. VINITI Publishers, Moscow.

11 . Firsov, A. A., Vostrov, S. N., Shevchenko, A. A. & Cornaglia, G. (1997). Parameters of bacterial killing and regrowth kinetics and antimicrobial effect examined in terms of area under the concentration–time curve relationships: action of ciprofloxacin against Escherichia coli in an in vitro dynamic model. Antimicrobial Agents and Chemotherapy 41, 1281–7. [Abstract]

12 . Firsov, A. A., Savarino, D., Ruble, M., Gilbert, D., Manzano, B., Medeiros, A. A. et al. (1996). Predictors of effect of ampicillin– sulbactam against TEM-1 ß-lactamase-producing Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC. Antimicrobial Agents and Chemotherapy 40, 734–8. [Abstract]

13 . Schentag, J. J. & Ballow C. H. (1991). Tissue-directed pharmacokinetics. American Journal of Medicine 91, Suppl. 3A, 5S–11S. [Medline]

14 . Carbon, C. & Poole, M. D. (1999). The role of newer macrolides in the treatment of community-acquired respiratory tract infections: a review of experimental and clinical data. Journal of Chemotherapy 11, 107–18. [ISI][Medline]

15 . Peters, D. H., Friedel, H. A. & McTavish, D. (1992). Azithromycin. A review of its antimicrobial activity, pharmacokinetic properties and clinical efficacy. Drugs 44, 750–99. [ISI][Medline]

Received 23 November 2000; returned 4 July 2001; revised 6 September 2001; accepted 17 September 2001