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
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Abstract |
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Introduction |
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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.
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Materials and methods |
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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 5.002000 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% (3.144.19%) and the overall accuracy as 99.5% (95.3105%).
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) 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 KolmogorovSmirnov 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.).
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Results |
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As anticipated, the peak serum concentrations (Table I) 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 I
and the Figure
) did not differ significantly, subjects did have a higher mean azithromycin exposure (13.1 versus 11.2 mgh/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 II
) 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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Discussion |
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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 I, 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 mgh/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 (125153 L/h versus 91103 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 II.2 Both PMNs and M/Ls achieved high concentrations which were 12 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.
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Acknowledgments |
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References |
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2
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Amsden, G. W. (1999). Pneumococcal macrolide resistancemyth or reality? Journal of Antimicrobial Chemotherapy 44, 16.
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