Serum and WBC pharmacokinetics of 1500 mg of azithromycin when given either as a single dose or over a 3 day period in healthy volunteers

Guy W. Amsdena,b,c,d and Clare L. Grayd

a Clinical Pharmacology Research Center, b Research Institute, and Departments of c Pharmacy and d Medicine, Bassett Healthcare, Cooperstown, NY, USA

*Correspondence address. Clinical Pharmacology Research Center, Bassett Healthcare, One Atwell Road, Cooperstown, NY 13326, USA. Tel: +1-607-547-3680; Fax: +1-607-547-6914; E-mail: guy.amsden{at}bassett.org


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Owing to azithromycin's prolonged half-life, shorter and shorter dosage regimens are being studied for treatment of respiratory tract infections. Previous studies have concluded that the 3 and 5 day (1.5 g total) regimens not only provide at least equal serum and WBC exposures but also equal efficacy rates. An earlier clinical study using the entire 1.5 g dose at once or the current 3 day regimen in patients with atypical pneumonia noted equal efficacy. Similar trials are currently underway in both adult and paediatric populations. The goal of the present study was to investigate whether there were equal serum and WBC exposures when azithromycin was dosed as the current 3 day regimen or as a single large dose. Equal exposures would help validate future clinical trials of single dose regimens. Twelve healthy volunteers received both azithromycin regimens (1.5 g single dose and 500 mg/day for 3 days) in random order. Serum and WBC samples were collected at baseline and repeatedly for 10 days following the first dose of each regimen. Serum samples were assayed via HPLC (CV% < 10) and WBC samples via liquid chromatography/mass spectrometry (CV% < 10). Data were modelled using noncompartmental methods. Statistics were via ANOVA with significance defined as P < 0.05. All subjects completed both regimens with minimal incidence of adverse effects. Serum data [mean (range)] demonstrated no significant difference in exposure between the two regimens [single 13.1 (3.02–20.6) mg•h/L versus 3 day 11.2 (2.98–24.5) mg•h/L: P = 0.12], although it favoured the shorter regimen. WBC results demonstrated much higher exposures than seen with serum, but no significant difference between the two regimens was identified. These results suggest that a single oral 1.5 g regimen of azithromycin for respiratory tract infections should provide exposure at least equal to currently approved treatment regimens.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Owing to azithromycin's prolonged half-life of approximately 65 h, shorter and shorter dosage regimens are being studied for the treatment of community-acquired respiratory tract infections in both adults and children. Significant research has been conducted in the past to support the use of a 3 day regimen (500 mg once daily for 3 days) of azithromycin rather than the currently utilized American 5 day regimen (500 mg day 1, 250 mg/day days 2–5).1 Based on this research, this shorter regimen has been used successfully for a number of years in many countries throughout the world and is currently being evaluated for re-labelling in the United States.2,3 Beyond providing a safe, welltolerated antibiotic regimen that is active against the most common community-acquired respiratory pathogens, one of the most attractive properties of both of these regimens is the inherent potential for enhancement of patient compliance.

Azithromycin has shown excellent activity against certain pathogens, such as Chlamydia spp., when it is administered as a single dose for extra-pulmonary infections.4,5 Owing to this activity and a desire to enhance compliance even further, studies have been, and are being, conducted to investigate the clinical efficacy of utilizing single, large doses (1.5 g in adults, 30 mg/kg in children) of azithromycin for the treatment of community-acquired respiratory tract infections. Good initial results have been demonstrated with this regimen by Schönwald and coworkers,6 in adults who were treated for atypical pneumonia with either a single 1.5 g dose of azithromycin or the standard 3 day azithromycin regimen. Regardless of treatment group, 98% of patients were cured.

The current study was undertaken to characterize and compare the serum, granulocyte (PMN) and monocyte/ lymphocyte (M/L) pharmacokinetics and overall exposures of 1.5 g of azithromycin administered as a single large dose versus a standard 3 day regimen. Equal exposure between the two regimens would help support further study of this unique administration schedule.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was approved by the Institutional Review Board of Bassett Healthcare. Twelve subjects were enrolled and provided written informed consent. All subjects were healthy as determined by medical history, physical examination and laboratory screening (a complete blood count, serum chemistries and urine pregnancy tests in women of childbearing potential). Subjects were at least 18 years of age and within 30% of ideal body weight for their height and frame size. Women of childbearing potential used a barrier method of birth control for 3 months before the study and agreed to continue contraceptive use throughout the study and for the 3 months following completion of the study. Subjects were required to be free of any drug exposure for 10 days before the start of the study. Exclusion criteria included a sensitivity to macrolides, a recent history of drug or alcohol abuse, an intention to donate blood during or immediately before or after the study, and the use of nicotine or nicotine delivery devices within the past year.

This was an open-label, randomized, crossover study. An investigator-generated randomization scheme was used to assign subjects to the following dosage regimens in random order: (i) oral azithromycin, 500 mg (two 250 mg tablets) daily for 3 days; and (ii) oral azithromycin 1500 mg (six 250 mg tablets). All doses were administered with 240 mL of tap water after the subjects had consumed a standardized, low-fat breakfast. Subjects remained caffeine and alcohol free during the dosing and sampling periods, and there was an 8 week washout period between study arms.

Blood was sampled just before (baseline) the start of dosing for each regimen and then repeatedly over the next 240 h (10 days). Aggressive absorption and distribution profile sampling was conducted on the first day of both regimens as well as the third day of the 3 day regimen. After centrifugation, serum was harvested and stored at –80°C until assayed.

Additionally, before the first doses of both regimens and at 4, 72, 144 and 240 h after the first doses, 30 mL aliquots of blood were collected in tubes containing EDTA for white blood cell (WBC) harvesting. The blood was layered in 3.5 mL amounts on top of 3.5 mL of PMN Isolation Medium (Robbins Scientific Corporation, Sunnyvale, CA, USA) in borosilicate culture tubes and centrifuged at 1280 rpm for 30 min at 20°C. When centrifugation was completed, the samples were layered from top to bottom in the following order: plasma layer, M/L layer, medium layer, PMN layer, medium layer and packed erythrocytes. The M/L and PMN layers were then drawn off and pooled by cell type, subject and draw time. The collected cells were diluted with an equal volume of 0.45% sodium chloride solution to promote erythrocyte lysis and then re-centrifuged at 1280 rpm for 10 min at 20°C. The supernatant was then decanted, and the PMN and M/L pellets were resuspended in 3.0 mL of Hanks' buffered salts solution. A trypan blue exclusion test using a 100 cell count was conducted to ensure sample viability, with >=95% viability being acceptable. Cells were then diluted 1:10 with a gentian violet stain containing 3% acetic acid and were counted with a haemocytometer. Wright's stain smears were also created to assess WBC differentials and, to minimize error, all Wright's stain smears were interpreted by the institution's haematology department.

Serum samples were assayed for azithromycin, at the Infectious Diseases Pharmacokinetics Laboratory at the National Jewish Medical and Research Center in Denver, CO, USA, using a validated high-performance liquid chromatography (HPLC) assay procedure. The assay was performed using a Waters (Milford, MA, USA) model 510 pump and model 680 gradient controller and solvent select valve, a Spectra Physics (San Jose, CA, USA) model 8875 fixed volume autosampler, and an ESA (Bedford, MA, USA) Coulochem II electrochemical detector, a Macintosh 7100 computer (Apple Computers, Inc., Cupertino, CA, USA) and the Rainin (Woburn, MA, USA) Dynamax HPLC data management system. The standard curves for serum azithromycin ranged from 0.05 to 5.0 mg/L. The best fits of the standard curves were achieved with a weighting scheme of 1/Y2. The coefficients of determination (R2) for the standard curves all exceeded 0.99. The median recovery of azithromycin from serum was 85.8% (range, 70.7– 93.9%). Testing for azithromycin within-day precision produced a median coefficient of variation (CV) of 1.9% (low, 0% at 0.25 mg/L; high, 3.9% at 1.00 mg/L). Testing for azithromycin overall assay precision produced a median CV of 5.2% (low, 3.1% at 5.00 mg/L; high, 9.5% at 0.05 mg/L). Validation control sample CV values varied from 5.1% (0.21 mg/L) to 11.4% (1.50 mg/L).

All PMN and M/L cell samples were assayed at the Analytical Division of The Clinical Pharmacokinetics Laboratory in Buffalo, NY, USA, using a validated reverse phase, HPLC assay with tandem mass spectrometric (MS) detection. Erythromycin was used as the internal standard. The LC/MS system consisted of a Waters model 510 HPLC pump, a Waters model 717 autosampler with the sample storage compartment set at 4°C, a Waters model TCM column heater and a PE Sciex (Foster City, CA, USA) model API 365 mass spectrometric detector and MassChrom data system. The prepared samples were chromatographed over a Zorbax SB-CN HPLC column (Hewlett Packard, Wilmington, DE, USA) maintained at 30°C and using a mobile phase consisting of acetonitrile:methanol: 0.020 M ammonium bicarbonate at a flow rate of 0.5 mL/min. Azithromycin concentrations were calculated using peak area responses. Linearity was observed over the calibration curve range of 5.00–2000 ng/mL. The overall precision (percentage relative standard deviation) and accuracy (percentage analytical recovery) of the assay were determined from the quality control samples analysed during the analysis of study samples. The overall precision was calculated as 3.80% (3.14–4.19%) and the overall accuracy as 99.5% (95.3–105%).

All serum data were analysed by non-compartmental methods with the TopFit version 2.0 computer program and a weighting scheme of 1/Y2.7 Serum exposure curves were extrapolated from the last data point to the estimated time of reaching 0 mg/L (AUC0–{infty}) and calculated by the trapezoidal method. Other observed or calculated pharmacokinetic parameters included peak serum concentration (Cmax), total oral clearance [Clt/F (F denoting bioavailability)] and volume of distribution (Vd/F).

The concentration of azithromycin in PMNs and M/Ls was calculated by dividing the cell assay concentrations by the actual cell counts for the specific sample. This value was then divided by a composite cellular volume based on the actual percentages of PMNs, monocytes and lymphocytes and the cells' previously defined volumes.8 PMN and M/L exposure curves were then calculated by the trapezoidal method through the final sampling time point (AUC240).

All individual parameter data for all three biomatrices were log10 transformed and tested for normality using the Kolmogorov–Smirnov test with the SigmaStat Version 2.03 software package (SPSS, Inc., Chicago, IL, USA). After assuring data set normality, parameter data sets for the two dosage regimens were compared for significant differences using ANOVA and the same software package. If statistical significance (P < 0.05) was found using ANOVA, the sets were re-tested for identification of true significance using Tukey's HSD. Descriptive sample set and demographic data were created using the SYSTAT Version 7.0 software package (SPSS, Inc.).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Twelve healthy volunteers [six males, six females; mean (range) age: 41 (25–56) years; mean weight: 78 (56–104) kg] received both treatment regimens and completed the study. Five of the subjects (four female/one male) during six of the study arms (1 dose = 4; 3 dose = 2) complained of adverse events. The vast majority of symptoms reported involved the gastrointestinal tract and were of mild severity. One subject did report moderately severe hot flushes during the three dose arm and moderate nausea during the single dose arm. All subjects' symptoms resolved spontaneously without treatment. One subject did experience a severe adverse event that was not study related.

As anticipated, the peak serum concentrations (Table IGo) associated with the single dose regimen were significantly (P = 0.001) higher than those associated with the 3 day regimen (1.46 versus 0.54 mg/L, respectively). Although serum exposures for the two regimens (Table IGo and the FigureGo) did not differ significantly, subjects did have a higher mean azithromycin exposure (13.1 versus 11.2 mg•h/L) with the single dose regimen. Other than the significant difference in Cmax, no other serum pharmacokinetic parameter differed significantly between the two dosage regimens. When WBC peak concentrations and exposures were compared (Table IIGo) it was noted that although subjects generally had higher concentrations and exposure to azithromycin when it was dosed as a single oral bolus, the difference did not reach statistical significance. What was highly (P < 0.001) significant for both dosage regimens was that the M/L cells had higher concentrations and exposures to azithromycin than those associated with PMNs.


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Table I. Mean (range) serum azithromycin pharmacokinetic parameters when it is administered over a 1 or 3 day period
 


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Figure. Mean azithromycin serum concentrations versus time, when azithromycin is administered as either a single dose ({diamondsuit}) or a three dose ({square}) regimen.

 

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Table II. Mean (range) PMN and M/L peak azithromycin concentrations and sampling time exposure when it is given as one dose or as three doses
 
Both WBC types demonstrated significant retention of azithromycin during both dosage regimens. Mean concentrations in cells at 10 days after the start of the single dose and three dose regimens were, respectively, 18 and 17 mg/L for PMNs and 27 and 21 mg/L for M/Ls. These are in sharp contrast to the corresponding serum concentrations of <0.05 mg/L for both dosage regimens.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Azithromycin is an azalide antibiotic with unique pharmacokinetic properties that result in extremely high and prolonged tissue and phagocyte concentrations. These unique properties allow it to be administered in various shortened or infrequent dosage regimens for a wide variety of infections including community-acquired respiratory tract and AIDS-related opportunistic infections.1,9 The two dosage regimens most commonly used for community-acquired respiratory tract infections, including pneumonia, consist of 1.5 g of azithromycin administered over either 3 or 5 days. This is markedly different from the 10–14 day regimens utilized with all other antimicrobials to date.1 Beyond its high efficacy rates and good tolerance, one clear advantage of being able to administer azithromycin for such a short period of time is the inherently high compliance rate that would be expected with such an abbreviated regimen. Such compliance would most likely lead to less need for re-treatment of a recurrent infection, less impact on a person socially (i.e. less time away from work) and less resistance development.

The ability to achieve a 100% compliance rate is something that has been strived for for decades with all types of drugs, not just antimicrobials. A study in adult patients with atypical pneumonia by Schönwald et al.6 attempted this very concept by comparing a standard 3 day regimen of azithromycin with administering the entire 1.5 g dose at once. Both treatment groups had a 98% cure rate as well as a very small incidence of side effects (<5%). Studies involving this type of comparison are currently ongoing for other community-acquired respiratory tract infection indications and in other populations.

Although clinical data on a new dosage regimen are vital, pharmacokinetic data to support it are just as important. It was the purpose of this study to compare the two dosage regimens used by Schönwald in terms of both serum and phagocyte exposures to help lend further credence to additional studies of this unique dosing concept. Based on the analysis of the serum concentration data it was evident that although giving the azithromycin as one large oral bolus unsurprisingly resulted in significantly higher peak serum concentrations than those seen with 500 mg doses, the subjects' exposure to the drug did not differ significantly. As is demonstrated in Table IGo, though not significantly different, subjects actually tended to have higher serum exposures to azithromycin when it was dosed as a single dose. This is similar to what has been seen in other azithromycin dosage regimen comparisons. Both the work by Wildfeuer et al.,3 and our own previous data2 comparing the current standard 3 and 5 day regimens, have demonstrated that shorter courses (but the same total dose) of azithromycin have tended towards providing higher exposures. Why this consistent pattern occurs is unknown, but it is clear that doses of azithromycin of up to 1.5 g given at once do not saturate the intestinal transport system utilized by it and subjects are not at risk of receiving incomplete exposures to azithromycin when it is administered in this manner. What was interesting was that the exposure to the 3 day regimen in the present study was actually lower than that demonstrated in our previous study (11.2 versus 19.4 mg•h/L, respectively). Although one might suspect that the difference may be that the subjects were fed in the current study as opposed to having subjects fast in the previous one, this explanation would be unlikely as work has already been published on the lack of effect that food has on various dosage forms of azithromycin.10 It is more likely that this effect is due to the higher clearance rates of azithromycin that were demonstrated in the present subjects as compared with those in the previous study (125–153 L/h versus 91–103 L/h, respectively).

Much as has been published in the past, the current results demonstrate azithromycin's extremely high volume of distribution, which is due in part to the significant WBC uptake of the drug as is shown in Table IIGo.2 Both PMNs and M/Ls achieved high concentrations which were 1–2 log higher than corresponding serum concentrations. Even 10 days from the start of either therapy, subjects still demonstrated intracellular concentrations that were above the MIC90 of the vast majority of community-acquired respiratory pathogens.4 Because of the need to maintain azithromycin concentrations above the MIC of a pathogen for as long as possible (i.e. optimizing AUC:MIC ratio and/or time > MIC) these high, prolonged intracellular concentrations have been proposed as a reason for not only the drug's sustained activity after the last dose of drug, but also its lack of bacteraemic failures, and potentially, its lack of clinical failures due to moderately resistant pathogens.11,12 Consistent with our previous work with the 3 and 5 day regimens, the M/L fractions achieved significantly higher peak intracellular concentrations and exposures than the PMN fraction. Although the single dose regimen again achieved higher overall WBC exposures (PMN 26% higher, M/L 30% higher) to azithromycin than the 3 day regimen, these differences were not statistically significant and most likely not clinically significant.

As compared with the standard 3 or 5 day dosage regimens of azithromycin for community-acquired respiratory tract infections, higher dosages that are used for Mycobacterium avium complex prophylaxis, trachoma and gonorrhoea have been associated with a higher incidence of gastrointestinal (GI) adverse events.4 This incidence has been as high as 34% for nausea and 14% for diarrhoea in one study that utilized a single 2 g dose for the treatment of gonorrhoea.13 In our previous work with fasted subjects who had been administered the 3 day regimen, three of 12 (25%) subjects experienced GI side effects. In the present study the consumption of a low-fat breakfast before administration of each of the three doses in the 3 day regimen was the most likely cause of the lower incidence of documented adverse GI events [2/12 subjects (17%)]. Although this climbed to 33% when subjects received the 1.5 g dose after the same type of breakfast, we would have expected the incidence to be much greater if they had fasted before dose administration. Even though this incidence is similar to that experienced by patients who take a standard course of erythromycin for a respiratory tract infection, the severity is less as all of our patients only complained of mild GI symptoms.

In conclusion, this study demonstrates that subjects who receive 1.5 g of azithromycin as a single dose rather than over a 3 day period receive at least equal exposure to the drug. The administration of large doses of azithromycin in a fed rather than fasted state has the potential to greatly decrease the incidence and severity of GI side effects associated with azithromycin without having a negative effect on their exposure to it. These data provide pharmacokinetic support for the continued investigation of this new and unique azithromycin dosage regimen.


    Acknowledgments
 
This study was supported by the E. Donnall Thomas Resident Research Program in Internal Medicine of Bassett Healthcare and an unrestricted educational grant from the US Pharmaceuticals Group of Pfizer, Inc. (New York, NY, USA).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dunn, C. H. & Barradell, L. B. (1996). Azithromycin. A review of its pharmacological properties and use as a 3-day therapy in respiratory tract infections. Drugs 51, 483–505.[ISI][Medline]

2 . Amsden, G. W., Nafziger, A. N. & Foulds, G. (1999). Pharmacokinetics in serum and leukocyte exposures of oral azithromycin, 1500 mg, given over a 3- or 5-day period in healthy subjects. Antimicrobial Agents and Chemotherapy 43, 163–5.[Abstract/Free Full Text]

3 . Wildfeuer, A., Laufen, H., Leitold, M. & Zimmermann, T. (1993). Comparison of the pharmacokinetics of three-day and five-day regimens of azithromycin in plasma and urine. Journal of Antimicrobial Chemotherapy 31, Suppl. E, 51–6.[ISI][Medline]

4 . Ballow, C. H. & Amsden, G. W. (1992). Azithromycin: the first azalide antibiotic. Annals of Pharmacotherapy 26, 1253–61.[Abstract]

5 . Bailey, R. L., Arullendran, P., Whittle, H. C. & Mabey, D. C. W. (1993). Randomised controlled trial of single-dose azithromycin in treatment of trachoma. Lancet 342, 453–6.[ISI][Medline]

6 . Schönwald, S., Kuzman, I., Oreskovic, K., Burek, V., Skerk, V., Car, V. et al. (1999). Azithromycin: single 1.5 g dose in the treatment of patients with atypical pneumonia syndrome – a randomized study. Infection 27, 198–202.[ISI][Medline]

7 . Tanswell, P. & Koup, J. (1993). TopFit: a PC-based pharmacokinetic/pharmacodynamic data analysis program. International Journal of Clinical Pharmacology, Therapeutics and Toxicology 31, 514–20.

8 . Nibbering, P. H., Zomerdijk, T. P. L., Corsèl-Van Tilburg, A. J. & Van Furth, R. (1990). Mean cell volume of human blood leukocytes and resident and activated murine macrophages. Journal of Immunological Methods 129, 143–5.[ISI][Medline]

9 . Amsden, G. W., Peloquin, C. A. & Berning, S. E. (1997). The role of advanced generation macrolides in the prophylaxis and treatment of Mycobacterium avium complex (MAC) infections. Drugs 54, 69–80.[ISI][Medline]

10 . Foulds, G., Luke, D. R., Teng, R., Willavize, S. A., Friedman, H. & Curatolo, W. J. (1996). The absence of an effect of food on the bioavailability of azithromycin administered as tablets, sachet or suspension. Journal of Antimicrobial Chemotherapy 37, Suppl. C, 37–44.[ISI][Medline]

11 . Craig, W. A. (1997). Postantibiotic effects and the dosing of macrolides, azalides and streptogramins. In Expanding Indications for the New Macrolides, Azalides, and Streptogramins (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 27–38. Marcel Dekker, New York.

12 . Amsden, G. W. (1999). Pneumococcal macrolide resistance—myth or reality? Journal of Antimicrobial Chemotherapy 44, 1–6.[Free Full Text]

13 . Handsfield, H. H., Siegal, N. A. & Verdon, M. S. (1991). Single-dose azithromycin vs ceftriaxone for treatment of uncomplicated gonorrhea. In Program and Abstracts of the Thirty-First Interscience Conference of Antimicrobial Agents and Chemotherapy, Chicago, IL, p. 79. American Society for Microbiology, Washington, DC.

Received 6 March 2000; returned 11 July 2000; revised 17 August 2000; accepted 25 September 2000