Pulsatile delivery of amoxicillin against Streptococcus pneumoniae

Raymond Cha1,2 and Michael J. Rybak1,2,*

1 Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy and Health Sciences and 2 School of Medicine, Wayne State University, Detroit, MI, USA

Received 27 May 2004; returned 11 July 2004; revised 24 August 2004; accepted 9 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective: ß-Lactam antimicrobials are dosed intermittently or continuously based on their short half-lives and concentration-independent activity. Based on the relationship between antimicrobial killing and bacterial growth cycle, the pharmacodynamics of a unique pulsatile strategy was investigated.

Methods: In vitro pharmacodynamic models with initial inocula of 6 log10 cfu/mL were utilized to simulate amoxicillin regimens against two Streptococcus pneumoniae isolates: 16891 (MIC = 4 mg/L) and ATCC 49150 (MIC = 0.016 mg/L). Time–kill profiles of pulsatile dosing of amoxicillin (total daily dose fractionated equally and given at 0, 2, 4 and 6 h for each 24 h cycle) were compared with regimens of every 8 h and every 12 h with the same 24 h drug exposure. Each regimen targeted cumulative peak concentrations of 30, 15 and 5 mg/L for each 24 h cycle. A t1/2 of 1 h was simulated for all experiments. Bacterial density was quantified over 96 h.

Results: Against 16891, every 8 h and every 12 h regimens exhibited minimal bacterial kill at all dosing levels. In contrast, pulsatile dosing at 30 mg/L/24 h resulted in an initial modest ~1 log10 cfu/mL kill with regrowth to growth control levels at 24 h but was immediately followed by a rapid ~2 log10 cfu/mL kill by 32 h. This pattern of kill and regrowth repeated at the same magnitude for each 24 h cycle for the 96 h study duration. Against the susceptible strain (ATCC 49150), both pulse and traditional dosing of amoxicillin resulted in rapid and significant kill to our detection limits for the entire study duration. A pattern of kill and regrowth was only observed at the lowest dose (0.05 mg/L) against ATCC 49150. At therapeutic levels, all regimens rapidly achieved undetectable limits against this strain for the study duration. No significant alterations in post-exposure MICs were noted. Overall bacterial density reduction was similar between the regimens for the susceptible isolate and greater with pulsatile regimens against the less susceptible strain.

Conclusion: Pulsatile dosing, which involves administration of the total daily dose over the first 6 h of the day, may represent a unique and alternative strategy for dosing ß-lactam antimicrobials.

Keywords: pharmacodynamics , intermediate resistance , controlled drug release , in vitro models


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Amoxicillin is a well-recognized and common aminopenicillin commonly used for streptococcal infections that demonstrates good bioavailability and low toxicity similar to other ß-lactam antimicrobials. Amoxicillin has been used to treat a variety of respiratory infections including otitis media, sinusitis, bronchitis and community-acquired pneumonia.13 The susceptibility breakpoint for amoxicillin against Streptococcus pneumoniae has been recently raised from 2 to 4 mg/L by the NCCLS.4 This change was based upon the differences in outcomes of patients who have CNS-related S. pneumoniae infections versus patients with upper respiratory infections. Using the higher breakpoints effectively lowers the resistance rates for this drug and increases its overall utility as a primary drug treatment of pneumococcal infections.

New technical advances in oral formulations for highly controlled release of drug in the gastrointestinal system allows for further evaluation of the effect of unique concentration exposures on the pharmacodynamics of amoxicillin (Donald Treacy, Advancis Pharmaceutical Corporation, personal communication). More specifically, this new oral formulation can be adjusted to release controlled drug quantities at specified timed intervals during its transit in the gastrointestinal tract to allow for once-daily dosing regimens of antibiotics that are traditionally administered multiple times per day. The rationale for this concentration exposure was indirectly based on one study where various modulation techniques of pulsatile UV exposure demonstrated potential improved bacterial eradication compared with non-modulated light.5 This study demonstrated significant bacterial density reductions as great as 106 log cfu/mL following numerous pulsatile UV exposures. Based on exposure modulation of antibacterial methods to potentially improve bacterial eradication, the potential of pulsatile antimicrobial exposures was investigated. The purpose of this study was to evaluate the pharmacodynamics of amoxicillin oral therapy, simulated as a pulsatile dose (equivalent 24 h dose, fractionated and given over the first 6 h of each 24 h dosing cycle) compared with traditional multiple dose regimens against S. pneumoniae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains and antimicrobials

Study strains included a clinical isolate of S. pneumoniae, 16891 (obtained from D.E. Low, Toronto, Canada), and the ATCC reference strain 49150. Amoxicillin analytical powder (Sigma, St Louis, MO, USA) was used throughout the study. Fresh stock solutions of amoxicillin were frequently prepared at concentrations of 2 mg/mL and were stored at 2–8°C between dosage administration times.

Susceptibility testing and media

MICs and MBCs were determined by broth microdilution using Mueller–Hinton Broth (BD, Sparks, MD, USA) supplemented with calcium, magnesium (25 and 12.5 mg/L, respectively) and 5% lysed horse blood (Rockland, Inc., Gilbertsville, PA, USA) (SMHB) according to the NCCLS guidelines.4 MICs were also carried out using Todd–Hewitt broth supplemented with 5% yeast extract (THB-Y; BD, Sparks, MD, USA). MIC values were confirmed by standard Etest methodology (AB Biodisk Solna, Sweden) using tryptic soy agar plates supplemented with 5% sheep blood (TSA-B; BD, Sparks, MD, USA). THB-Y was used for all in vitro model simulations. Samples were plated onto TSA-B and incubated in candle jars at 37°C for 24 h.

In vitro pharmacodynamic model

Previously described in vitro models consisting of a 250 mL one-compartment glass chamber with ports for the addition and/or removal of media, antibiotics, and samples were used to simulate amoxicillin regimens.6 Before each experiment, fresh colonies from an overnight growth on TSA-B agar were added to THB-Y as necessary to obtain a suspension of ~1 x 108 cfu/mL for each organism. A 2.5 mL volume of these suspensions was added to the chamber to produce a starting inoculum of ~1 x 106 cfu/mL. Amoxicillin dosing regimens were administered as pulsatile regimens (total drug for 24 h dose equally fractionated, and given over first 6 h of each 24 h dosing cycle) and as multiple dosing regimens to target a total 24 h daily exposure of 30, 15 and 5 mg/L, respectively: pulsatile = doses of 7.5, 3.75, 1.25 mg/L given at 0, 2, 4, 6, 24, 26, 28, 30, 48, 50, 52, 54, 72, 74, 76 and 78 h; twice daily = doses of 15, 7.5, 2.5 mg/L given every 12 h, and thrice daily = doses of 10, 5, 1.25 mg/L given every 8 h. Antibiotic doses were administered as a bolus into the models over 30 s using a hypodermic syringe. A peristaltic pump (Masterflex; Cole-Parmer Instrument Company, Chicago, IL, USA) was used to displace antibiotic-containing media to simulate a 1 h amoxicillin half-life for all simulations. All models were placed in a water bath and maintained at 37°C for the entire 96 h study period. Experiments were carried out in duplicate to ensure reproducibility.

Pharmacodynamic analysis

Samples were removed from each model at 0, 1, 2, 4, 6, 8, 24, 28, 32, 48, 56, 72, 80 and 96 h. Each sample was serially diluted in cold 0.9% sodium chloride and bacterial counts were determined by placing 20 µL aliquots of all dilutions onto TSA-B. Colonies were counted following incubation for 24 h at 37°C. We have determined previously that these methods have a limit of detection of 2 log10 cfu/mL.6 The total reduction in log10 cfu/mL over 96 h was determined by plotting time–kill curves. Bacterial densities over time and area under the time–kill curves were compared between regimens. Area under the time–kill curves was estimated by the trapezoidal rule with SigmaPlot. Antibiotic carryover was accounted for by incorporating ß-lactamase (Sigma–Aldrich, St Louis, MO, USA) into dilutions used for bacterial quantification. The development of raised amoxicillin MICs for each antibiotic regimen during experiments was investigated by plating 100 µL of the 48, 72 and 96 h samples onto TSA-B containing two- and four-fold the MIC for each strain. These plates were examined for growth after incubation for 48 h. Changes in MICs were also checked for each model from 96 h samples using Etest methodology.

Pharmacokinetic analysis

Samples obtained from 1, 2, 4, 6, 8, 24, 32, 48, 56, 72 and 96 h were reserved for determination of antibiotic concentrations. All samples were stored at –70°C until analysis. Concentrations for amoxicillin were determined by a microbioassay using Bacillus subtilis ATCC 6631 as the indicator organism.6 Paper diffusion discs (BD, Cockeysville, MD, USA) were spotted with 20 µL of samples or standards, and then placed in triplicate on Mueller–Hinton agar plates pre-swabbed with a 0.5 McFarland suspension (~7.0 log10 cfu/mL) of indicator organism. Inhibition zones were then measured following 24 h of incubation. Concentrations of amoxicillin standards were 5, 1.25 and 0.3125 mg/L. The inter-day coefficient of variation (% CV) for this assay is 3.1–6.8%. Peak concentrations, elimination rates and half-lives were estimated by the linear trapezoidal rule using PK Analyst software (Micromath, Salt Lake City, UT, USA).

Statistical analysis

Bacterial densities in log10 cfu/mL at all time points were compared by one-way ANOVA with Tukey's post-hoc test for multiple comparisons. A P value of ≤0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Susceptibility results

Strain 16891 and reference strain ATCC 49150 exhibited amoxicillin MICs of 4 mg/L and 0.016 mg/L, respectively. In addition, post-exposure susceptibility changes consisted of two-fold dilutional increases for all regimens against 16891. No significant dilutional changes were noted for post-exposure isolates obtained from experiments with 49150.

Pharmacokinetic results

Targeted pharmacokinetic profiles for each regimen are displayed in Figure 1. Actual achieved amoxicillin model concentrations are depicted in Figure 2.



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Figure 1. Antimicrobial target concentrations of study regimens (pulsatile, every 8 h, every 12 h, and continuous infusion) based on targeted peak concentrations, standard half-life of 1 h, and first-order exponential decay. The 4 mg/L dashed line depicts the MIC for the 16891 isolate.

 


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Figure 2. Concentrations (mg/L) of amoxicillin from pulsatile, every 12 h and every 8 h regimen simulations from in vitro pharmacodynamic models.

 
Pharmacodynamic results

Results from pharmacodynamic in vitro model studies are portrayed in Figure 3(a–. For the purpose of clarity, only select regimens (30 mg/L/24 h versus 16891, and 15 mg/L/24 h and 0.05 mg/L/24 h versus 49150) are presented. All initial inocula were within 1.0 log of the targeted value. Growth control experiments exhibited logarithmic growth over the first 8 h followed by a plateau of sustained bacterial densities for the remainder of model simulations. Against 49150, all regimens including standard and pulsatile at 30 mg/L per day exhibited rapid bactericidal activity reducing bacterial densities to undetectable limits within the first hour of simulations. Undetectable limits of bacterial quantification were maintained by all regimens against 49150 for the remainder of the study duration. For proof of theory, we retrospectively initiated further pharmacodynamic simulations against 49150 with similar regimens targeting significantly lower amoxicillin 24 h doses of 1, 0.15 and 0.05 mg/L to assess pharmacodynamic profile differences between regimens. Pharmacodynamic simulations at 1 and 0.15 mg/L/24 h cycle produced similar profiles as the 30 mg/L/24 h regimen, demonstrated by rapid bacterial kill to undetectable levels within 1 h. However, differences between regimens were noted when regimens targeting 24 h exposures of 0.05 mg/L were carried out. The pulsatile regimen exhibited bacterial reductions within the magnitude of 2 log10 cfu/mL which followed each completion of administration of pulsatile doses for every 24 h cycle. Against 16981, twice daily and thrice daily dosing of amoxicillin produced immediate bacterial kill within the magnitudes of less than 2 log10 cfu/mL over the first 8 h. This rapid kill was soon followed by significant regrowth to growth control levels for the remainder of the study simulations. Subsequent dosing exhibited minor bacterial kill with little overall impact on bacterial densities. Against both tested isolates, the pulsatile dosing regimen consistently exhibited 2–3 log kill after the completion of the administration of pulsatile doses followed by regrowth to initial inoculum levels. This was statistically lower (P < 0.05) than traditional regimens at these points. This pattern of modest kill with subsequent regrowth was observed for each 24 h cycle.



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Figure 3. Representative pharmacodynamic profiles of pulsatile, twice daily, and thrice daily amoxicillin against ATCC 49150 in (a) (30 mg/L/24 h) and (b) (0.05 mg/L/24 h) and against 16891 in (c) (30 mg/L/24 h), (d) (interchange of pulsatile and thrice daily regimen at 48 h) and (e) (continuous infusion at 8 mg/L for 6 h). Lower limit of detection was 2.0 log10 cfu/mL.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
S. pneumoniae is the primary pathogen responsible for causing significant morbidity and mortality in upper respiratory tract infections and in community-acquired pneumonia.1,79 The incidence of antibiotic-resistant S. pneumoniae has reached concerning levels worldwide. Recent surveillance data from various reported susceptibility studies and from the Centers for Disease Control demonstrate that penicillin resistance (intermediate and resistant) is as high as 40% in the United States.8 Despite this fact, ß-lactams remain one of the most prescribed oral agents for these syndromes and methods to improve their pharmacodynamic effects and reduce resistance are imperative. We investigated the comparative pharmacodynamics of a unique pulsatile regimen (the entire 24 h dose is administered in four equal pulse doses over the first 6 h of each day) to traditional regimens in an in vitro pharmacodynamic model.

Against the reference strain, no significant difference was noted between pharmacodynamic profiles of the various regimens within clinically applicable concentration exposures. This suggested that there was equivalent microbiological efficacy between pulsatile and traditional regimens against this susceptible strain. Additionally, experiments in which the pulsatile dose was lowered, indicated that as little as 0.15 mg/L/24 h was equivalent to a total dose 30 mg/L/24 h delivered in divided doses either twice or three times per day. Significantly different effects were noted by the pulsatile regimens against the intermediate-resistant strain evident by intermittent rapid bacterial kill that corresponded to dosing administration times. Each period of bacterial kill was immediately followed by exponential regrowth and this cycle repeated for the duration of the study. We also retrospectively carried out additional model simulations with amoxicillin at 30 mg/L/24 h as described above, but with slight alterations in the dosing scheme that involved interchange of standard thrice daily and pulsatile regimens at 48 h against the intermediate-resistant strain (Figure 3d). In other words, the model receiving the standard dosing regimen was switched to a pulsatile regimen at 48 h and the converse was carried out as well. Although at a slightly lesser magnitude of bacterial kill, the distinct cycle of bacterial kill with subsequent exponential bacterial regrowth was interestingly observed when the model was switched to a pulsatile regimen. Additionally, no further bacterial kill was noted when the second model was switched from a pulsatile regimen to a standard regimen. Overall, pulsatile regimens achieved at least equal bacterial burden reductions as traditional, established clinical regimens against the susceptible strain and greater bacterial burden reductions against the intermediate resistant strain. In order to try to understand the impact of pulse dosing further, we also retrospectively evaluated a continuous infusion (CI) regimen of 8 mg/L delivered over 0–6 h once daily for 96 h. The 8 mg/L CI regimen was chosen to try to match a similar overall AUC exposure to that over the pulse, twice daily and thrice daily amoxicillin regimens (Figure 2). The results (Figure 3e) demonstrate that the CI regimen impact on the 16891 isolate is similar to that of the pulse regimen emphasizing the importance of short interval exposures irrespective of t > MIC early in 24 h dosing cycles.

Early studies with penicillin also suggested favourable kill against streptococcus when exposure occurred during the early exponential growth phase to potentially optimize availability of antimicrobial target sites in new cells.2,10 Another possibility may be the difference between application of continued concentration exposures at a multifold of the MIC in the beginning of the 24 h dosing cycle instead of intermittent exposures (Figure 1) throughout the 24 h cycle. Given that the pharmacodynamic efficacy of amoxicillin and ß-lactams is independent of concentration ratios to the MIC (once a maximum of 4–5 times the MIC is obtained) and is related to the duration that the concentration remains above a multifold of the MIC as a function of the dosing interval (t > MIC), similar extent of kill may be expected.2,3,11 Despite the comparable t > MIC between regimens, we observed significant, though transient, kill with pulsatile regimens against the intermediate-resistant isolate. Further investigations in additional isolates with a variety of susceptibility profiles are warranted to explain whether this phenomenon is related to growth phase, cell wall changes, alteration in lytic or metabolic processes, or microenvironmental changes. Although there is insufficient information to conclude any differential resistance potential due to limited organisms and study duration, pulsatile regimens appear to produce overall comparable bacterial kill compared with traditional twice and thrice daily regimens and perhaps better bacterial kill against intermediate-resistant isolates. Pulsatile exposures of amoxicillin appears promising based on bacterial density reductions and further evaluation is warranted with studies against a variety of organisms with different susceptibility profiles followed by clinical and microbiological end point evaluations in in vivo studies.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by a grant from Advancis Pharmaceutical Corporation, Germantown, MD, USA.


    Footnotes
 
* Correspondence address. Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI 48201, USA. Tel: +1-313-577-4376; Fax: +1-313-577-8915; Email: m.rybak{at}wayne.edu


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Bartlett, J. G., Breiman, R. F., Mandell, L. A. et al. (1998). Community-acquired pneumonia in adults: guidelines for management. Clinical Infectious Diseases 26, 811–38.[ISI][Medline]

2 . Chambers, H. F. (2000). Penicillins. In Principles and Practice of Infectious Diseases, 5th edn (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp. 261–74. Churchill Livingstone, Philadelphia, PA, USA.

3 . Preston, S. L. & Drusano, G. L. (1999). Penicillins. In Antimicrobial Therapy and Vaccines (Yu, V. L, Merigan, T. C., Jr & Barriere, S. L., Eds), pp. 850–75. Williams & Wilkins, Baltimore, MD, USA.

4 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

5 . Rowan, N. J., MacGregor, S. J., Anderson, J. G. et al. (1999). Pulsed-light inactivation of food-related microorganisms. Applied and Environmental Microbiology 65, 1312–5.[Abstract/Free Full Text]

6 . Cappelletty, D. M. & Rybak, M. J. (1996). Bactericidal activities of cefprozil, penicillin, cefaclor, cefixime, and loracarbef against penicillin-susceptible and -resistant Streptococcus pneumoniae in an in vitro pharmacodynamic infection model. Antimicrobial Agents and Chemotherapy 40, 1148–52.[Abstract]

7 . Campbell, G. D., Jr & Silberman, R. (1998). Drug-resistant Streptococcus pneumoniae. Clinical Infectious Diseases 26, 1188–95.[ISI][Medline]

8 . Centers for Disease Control and Prevention. (1999). Defining the public health impact of drug-resistant Streptococcus pneumoniae: report of a working group. Morbidity and Mortality Weekly Report: Recommendations and Reports 45, RR-1, 1–20.

9 . Friedland, I. R. & McCracken, G. H., Jr (1994). Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. New England Journal of Medicine 331, 377–82.[Free Full Text]

10 . Tomasz, A. (1979). From penicillin-binding proteins to the lysis and death of bacteria: a 1979 view. Review of Infectious Diseases 1, 434–67.[ISI][Medline]

11 . Drusano, G. L. & Craig, W. A. (1997). Relevance of pharmacokinetics and pharmacodynamics in the selection of antibiotics for respiratory tract infections. Journal of Chemotherapy 9, Suppl. 3, 38–44.[ISI][Medline]