Effect of protein binding on the in vitro activity and pharmacodynamics of faropenem

F. J. Boswell, J. P. Ashby, J. M. Andrews and R. Wise*

Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK

Received 29 November 2001; returned 22 April 2002; revised 5 June 2002; accepted 21 June 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The influence of protein binding upon different aspects of the in vitro activity of faropenem on recently isolated Staphylococcus aureus and respiratory pathogens was determined. The protein binding of faropenem was investigated in inactivated human serum and albumin by ultrafiltration. The effect of the presence of inactivated human serum and albumin on the in vitro activity of faropenem and amoxicillin was established and the influence of protein binding on the pharmacodynamic properties of faropenem and amoxicillin was compared. The protein binding of faropenem was 96% and 95% in pooled inactivated human serum and 99% and 98% in 45 mg/L human albumin, at 8 and 25 mg/L, respectively. The presence of inactivated human serum (20% and 70%) increased the mean faropenem MICs by two dilution steps and albumin increased the mean faropenem MICs by three dilution steps. The mean amoxicillin MICs were less affected than faropenem by the presence of either inactivated human serum or albumin. Faropenem and amoxicillin exhibited similar time-dependent kinetics. Faropenem was bacteriostatic on Moraxella catarrhalis, Haemophilus influenzae and group A streptococci, and bactericidal for Streptococcus pneumoniae (after 4 h with concentrations equivalent to 5 x and 10 x MIC) in Iso-Sensitest broth. In 70% inactivated human serum faropenem was slowly bactericidal against M. catarrhalis, H. influenzae (one strain) and S. pneumoniae (one strain) but not group A streptococci and the other S. pneumoniae strain. A significant inoculum effect was observed with all strains except S. pneumoniae. Both faropenem and amoxicillin appeared more active in 70% inactivated human serum than in Iso-Sensitest broth.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Significant worldwide antibiotic resistance has emerged, particularly among those agents used to treat respiratory tract infections (RTIs). Antimicrobials selected for the treatment of RTIs should ideally possess activity against common and atypical pathogens (when treating community-acquired pneumonia) and have optimal pharmacokinetic and pharmacodynamic parameters that facilitate convenient dosage schedules and good tissue penetration. The previous penems shared structural similarities with both penicillins and cephalosporins and were characterized by a broad antibacterial spectrum with good ß-lactamase stability; however, they exhibited short plasma elimination half-lives.1 Despite previous reports on orally available penem antibiotics1 none have been marketed to date. Faropenem-daloxate is a novel oral penem with a potent broad spectrum of activity against Gram-negative, Gram-positive and certain anaerobic bacteria and has been shown to be highly stable against a number of ß-lactamases.2

The aim of this study was to investigate the influence of protein binding upon different aspects of the in vitro activity of faropenem—the active moiety of faropenem-daloxate—and amoxicillin (protein binding 95% and 17%, respectively), a broad spectrum aminopenicillin that is unstable to ß-lactamases.3 Routinely, in vitro antibacterial activities are most often described by determining their MICs. MICs are possible predictors of potency, but do not represent bactericidal activity or provide any data on the time course of antimicrobial activity. Time–kill curves are pharmacodynamic examples of bactericidal activity expressed as the rate of killing by a fixed concentration of an antimicrobial. Time–kill kinetic techniques have demonstrated better correlation with in vivo efficacy than other bactericidal activity determination methods and are also the most reliable method for determining and differentiating tolerance.4 Although faropenem has been investigated previously2,5,6 the effects of protein binding on its pharmacodynamics have not been studied.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains: susceptibility testing

A total of 34 recent non-replicate clinical isolates were studied, consisting of two methicillin-susceptible Staphylococcus aureus (MSSA), 10 Moraxella catarrhalis (five ß-lactamase-producing strains), 10 Haemophilus influenzae (five ß-lactamase-producing strains), two group A streptococci and 10 Streptococcus pneumoniae (including macrolide-, penicillin- and quinolone-resistant strains).

Bacterial strains: time–kill kinetics

A total of 12 of the above isolates was studied, consisting of four M. catarrhalis (two ß-lactamase-producing strains), four H. influenzae (two ß-lactamase-producing strains), two group A streptococci and two S. pneumoniae.

Antimicrobial agents

The antimicrobial agents investigated and their sources were: faropenem (Bayer, West Haven, CT, USA) and amoxicillin (GlaxoSmithKline, Worthing, UK). Both antimicrobial agents were prepared and stored throughout these investigations following the manufacturers’ guidelines.

Protein binding

The protein binding of faropenem was determined at 8 and 25 mg/L in pooled inactivated human serum (Tissue Culture Services, Botolph Claydon, UK) and 45 g/L human albumin (fraction V) (Sigma Chemicals, Poole, UK) by ultrafiltration7 employing Centrifree ultrafiltration units (Amicon, Stonehouse, UK). Ultrafiltrates were assayed against faropenem phosphate buffer (pH 6) calibrators by microbiological plate assay using antibiotic medium No 1 (Oxoid, Basingstoke, UK) and S. aureus NCTC 6571 as the indicator organism.

Effect of protein binding on in vitro activity

The effect of inactivated human serum and albumin upon the in vitro activity (MICs and MBCs) of faropenem and amoxicillin was studied. A microdilution method was employed using either Iso-Sensitest broth (Oxoid) alone or Iso-Sensitest broth supplemented with 20 or 70% (v/v) inactivated human serum or 32 g/L human albumin (albumin level of normal serum, 45 g/L).8 These broths were further supplemented with 20 mg/L nicotinamide adenine dinucleotide (NAD) (Sigma Chemicals), 20 mg/L haemin (Sigma Chemicals) and 5% laked horse blood (Oxoid) (for streptococci), and a final inoculum of 5 x 105 cfu/mL was employed.9 Following incubation for 18–24 h at 35–37°C in an atmosphere enriched with 4–6% carbon dioxide for H. influenzae and S. pneumoniae, 20 µL of broth culture was subcultured on to appropriate antibiotic-free media for MBC determination. The MIC was defined as the lowest concentration at which there was no visible growth and the MBC was defined as the lowest concentration at which no colonies were observed (equivalent to 99.9% lethality).9

Effect of protein binding on time–kill kinetics

Faropenem and amoxicillin concentrations equivalent to 2 x, 5 x and 10 x MIC were added to logarithmic phase cultures of ~105 and 107 cfu/mL in Iso-Sensitest broth and 70% (v/v) inactivated human serum (both supplemented with 5% laked horse blood, 20 mg/L NAD and 20 mg/L haemin). Non-antibiotic-exposed growth controls were included for both inocula. Viable counts were determined (three replicates) at 0, 2, 4, 6 and 24 h after the addition of faropenem and amoxicillin on Columbia agar plates (Oxoid) [supplemented with 5% defibrinated horse blood (Tissue Culture Services) and 20 mg/L NAD as necessary] following appropriate serial dilution in phosphate-buffered saline pH 7.3 (Oxoid).10 The bacteria were enumerated after 48 h incubation at 35–37°C in an atmosphere enriched with 4–6% carbon dioxide and the time–kill kinetics were plotted as log10 cfu/mL against time. Since bactericidal activity was defined as a 3.0 log10 decrease in cfu/mL (99.9% kill),4 we defined bacteriostatic activity as <99.9% kill. The lower limit of bacterial enumeration was 2.7 log10 cfu/mL.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein binding

The protein binding of faropenem at 8 and 25 mg/L in pooled inactivated human serum was 96% and 95%, and 99% and 98% in 45 mg/L human albumin, respectively.

Effect of protein binding on in vitro activity

Table 1 demonstrates the effect of 20% and 70% inactivated human serum and 32 g/L human albumin upon the MICs and MBCs of faropenem and amoxicillin. Table 2 demonstrates the mean significant (more than one dilution step) increase in faropenem and amoxicillin MICs in the presence of 20% or 70% inactivated human serum or human albumin compared with supplemented Iso-Sensitest broth. Increases in MBC were similar to the increases in MIC.


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Table 1.  In vitro activity [MIC/MBC (mg/L)] of faropenem and amoxicillin
 

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Table 2.  Mean significant increase in faropenem and amoxicillin MICs
 
Effect of protein binding on time–kill kinetics

The two ß-lactams exhibited time-dependent kill kinetics, amoxicillin being modestly more bactericidal than faropenem at equipotent concentrations. Bactericidal activity was defined as a 3 log10 decrease in cfu/mL; however, in some instances when studying the 105 cfu/mL inoculum a 3 log10 decrease could not be determined (due to the lower limit of detection), but a decrease of >2.3 log10 was observed. In these cases if no regrowth was observed for the remaining duration of the experiment it was termed bactericidal. Bactericidal rates (change in log10 cfu/mL) of faropenem and amoxicillin at concentrations equivalent to 2 x, 5 x, 10 x MIC in supplemented Iso-Sensitest broth and 70% inactivated human serum at inocula of 107 and 105 cfu/mL are shown in Tables 3 and 4. Figures 1 and 2 depict graphically the time–kill kinetics of faropenem and amoxicillin in Iso-Sensitest broth and 70% inactivated human serum for one strain of each species studied at an inoculum of ~105 cfu/mL. Against M. catarrhalis and H. influenzae strains (inocula of 105 and 107 cfu/mL) no bactericidal effect was observed with faropenem in Iso-Sensitest broth. However, a bactericidal effect was observed in 70% inactivated human serum (inocula of 105 cfu/mL) after 6 h with concentrations equivalent to 10 x MIC for M. catarrhalis except for strain R109 and H. influenzae (two strains, one ß-lactamase producing and one non-producing). No bactericidal effect was observed with either faropenem and amoxicillin against group A streptococci (inocula 105 and 107 cfu/mL) in either Iso-Sensitest broth or 70% inactivated human serum. In Iso-Sensitest broth at inocula of 105 cfu/mL a bactericidal effect was observed with faropenem against S. pneumoniae after 4 h with concentrations equivalent to 5 x and 10 x MIC; however, this effect was observed only with one strain in 70% inactivated human serum.


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Table 3.  Bactericidal rates (change in log10 cfu/mL) of faropenem and amoxicillin at concentrations equivalent to 2 x, 5 x, 10 x MIC in supplemented Iso-sensitest broth, inocula 107/105 cfu/mL
 

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Table 4.  Bactericidal rates (change in log10 cfu/mL) of faropenem and amoxicillin at concentrations equivalent to 2 x, 5 x, 10 x MIC in supplemented 70% inactivated human serum, inocula 107/105 cfu/mL
 


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Figure 1. Faropenem time–kill kinetics for M. catarrhalis R111 (a and e), H. influenzae A52 (b and f), group A streptococci P334 (c and g) and S. pneumoniae P16 (d and h) in Iso-Sensitest broth and 70% inactivated human serum, respectively, at inocula of ~105 cfu/mL.

 


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Figure 2. Amoxicillin time–kill kinetics for M. catarrhalis R111 (a and e), H. influenzae A52 (b and f), group A streptococci P334 (c and g) and S. pneumoniae P16 (d and h) in Iso-Sensitest broth and 70% inactivated human serum, respectively, at an inoculum of ~105 cfu/mL.

 
Against M. catarrhalis (inocula 105 and 107 cfu/mL) no bactericidal effect was observed with amoxicillin in Iso-Sensitest broth. However, a bactericidal effect was observed in 70% inactivated human serum after 6 h with concentrations equivalent to 2 x to 10 x MIC (two strains, one ß-lactamase producing and one non-producing). Against H. influenzae (one strain, inoculum 105 cfu/mL) a bactericidal effect was observed with amoxicillin in Iso-Sensitest broth after 6 h, with concentrations equivalent to 5 x and 10 x MIC. However, in 70% inactivated human serum a bactericidal effect was observed after 6 h, with concentrations equivalent to 10 x MIC (three strains). In Iso-Sensitest broth at an inoculum of 105 cfu/mL a bactericidal effect was observed with amoxicillin against S. pneumoniae after 6 h, with concentrations equivalent to 10 x MIC. In 70% inactivated human serum a bactericidal effect was observed with amoxicillin and S. pneumoniae (one strain) after 4 h with concentrations equivalent to 10 x MIC.

Generally, a significant inoculum effect on bactericidal activity was observed with M. catarrhalis, H. influenzae and group A streptococci but less inoculum effect was observed with S. pneumoniae strains.

Both faropenem and amoxicillin appeared more active in 70% inactivated human serum than in Iso-Sensitest broth against M. catarrhalis (three strains), H. influenzae and S. pneumoniae (one strain) but not the group A streptococci. This increase in bactericidal activity was most marked during the initial phase of exposure from 0 to 4 h but it was less impressive at 6 h (Tables 3 and 4).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The majority of ß-lactams, including the penems, have to date exhibited moderate to low protein binding, for example, amoxicillin protein binding is <20%9 and the investigational penem FCE 22101’s protein binding was 35–40%, and consequently a negligible serum effect was observed on MIC/MBC.1 However, the higher protein binding of faropenem up to 95% (A. Dalhoff, personal communication) and 95–96% as reported here is noteworthy. No significant differences were observed between 70% inactivated human serum-supplemented Iso-Sensitest broth and 32 g/L human albumin-supplemented Iso-Sensitest broth due to the fact that antimicrobials bind mainly to the serum protein, albumin, alone. In addition, only the unbound fraction of the antimicrobial is active against bacteria and usually significant reductions in MIC (eight-fold) are only observed when antimicrobial protein binding is >90%.11

All the strains examined had faropenem MICs of <=1 mg/L, demonstrating significant activity against S. aureus and respiratory pathogens reported previously.2 Also as reported previously2 faropenem, unlike amoxicillin, had significant activity against both the ß-lactamase-producing and non-producing strains of M. catarrhalis and H. influenzae. For both faropenem and amoxicillin the majority of MICs and MBCs were equal to or within one dilution of each other, therefore demonstrating no tolerance.

The effect of protein binding upon the antibacterial activity depended on species tested, being more marked in ß-lactamase-producing strains of M. catarrhalis (a three dilution-step increase in MIC in 20% and 70% inactivated human serum and human albumin) than S. aureus (a two dilution-step increase in 70% inactivated human serum). Amoxicillin MICs were affected less by the presence of human serum and albumin due presumably to lower protein binding. However, the effect of protein binding on the in vitro activity of faropenem was not substantial (a two or three dilution-step increase in MIC) these results correlate with those observed previously.2 The increase in faropenem MICs was lower than the increase in MICs noted for other highly protein bound antimicrobials.11

Pharmacodynamics describe the relationship between an antimicrobial and its effect and thus provide a rational basis for optimizing dosing regimens. The pharmacodynamics of faropenem and amoxicillin were expressed as the rate of killing by fixed concentrations. In general, faropenem and amoxicillin exhibited the pharmacodynamic properties expected of ß-lactams, i.e. concentration-independent activity.5 The results we obtained for faropenem are similar to those obtained by others, e.g. Morosini et al.12 noted a bactericidal effect of faropenem on S. pneumoniae after 6 h. When regrowth was observed at 24 h, these cultures were morphologically identical to the controls. Regrowth may be due to either the selection of resistant mutants, inactivation of the antimicrobial or susceptible bacterial cells escaping the antimicrobial activity by adhering to the culture vessel walls.4 The clinical importance of regrowth is unclear, particularly if it occurs after the usual dosing interval.4 Antibiotic carryover was not deemed to be a problem in our determinations as this occurs at concentrations > 16 x MIC4 and serial dilution was employed, which minimizes residual antimicrobial effects.

The effect of inoculum on pharmacodynamics was also evaluated; a significant inoculum effect was observed with all strains except S. pneumoniae. In addition, a modest concentration effect was observed such that, in general, higher concentrations (5 x and 10 x MIC) exhibited reductions in viable counts similar to each other but greater than the reduction in viable count achieved at 2 x MIC as observed previously.5 Also as reported previously5 the production of ß-lactamase by strains of M. catarrhalis and H. influenzae had no apparent effect on the bactericidal activity of faropenem. The presence of inactivated human serum enhances the antimicrobial bactericidal effect observed with both faropenem and amoxicillin. These data indicate that an unidentified phenomenon has an impact on faropenem’s antibacterial activity in the presence of serum. Schmidt et al.13 also found faropenem to have highly effective bactericidal activity against S. pneumoniae and the presence of 50% fetal calf serum did not decrease the efficacy of faropenem despite its high protein binding.

However, the clinical relevance of the higher protein binding of faropenem upon the pharmacokinetics of the compound needs further assessment.


    Acknowledgements
 
We would like to thank A. Dalhoff of Bayer for financial support and advice.


    Footnotes
 
* Corresponding author. Tel: +44-121-507-4255; Fax: +44-121-551-7763; E-mail: r.wise{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Wise, R. & Ballard, R. C. (1989). Review of the in vitro evaluation of FCE 22101. Journal of Antimicrobial Chemotherapy 23, Suppl. C, 7–16.[Abstract]

2 . Woodcock, J. M., Andrews, J. M., Brenwald, N. P., Ashby, J. P. & Wise, R. (1997). The in vitro activity of faropenem, a novel oral penem. Journal of Antimicrobial Chemotherapy 39, 35–43.[Abstract]

3 . Sutherland, R. (1997). ß-Lactams: penicillins. In Antibiotics and Chemotherapy, 7th edn (O’Grady, F., Lambert, H. P., Finch, R. G. & Greenwood, D., Eds), pp. 256–305. Churchill Livingstone, New York, NY, USA.

4 . National Committee for Clinical Laboratory Standards. (1992). Methods for Determining Bactericidal Activity of Antimicrobial Agents: Tentative Guideline M26-T. NCCLS, Villanova, PA, USA.

5 . Boswell, F. J., Andrews, J. M. & Wise, R. (1997). Pharmacodynamic properties of faropenem demonstrated by studies of time–kill kinetics and postantibiotic effect. Journal of Antimicrobial Chemotherapy 39, 415–8.[Abstract]

6 . Cormican, M. G. & Jones, R. N. (1995). Evaluation of the in vitro activity of faropenem (SY 5555 or SUN 5555) against respiratory tract pathogens and ß-lactamase producing bacteria. Journal of Antimicrobial Chemotherapy 35, 535–9.[Abstract]

7 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In vitro activity of Bay 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 101–6.[Abstract]

8 . Lentner, C., Ed. (1984). Geigy Scientific Tables. Volume 3, 8th edn, p. 141. Ciba-Geigy Ltd, Basle, Switzerland.

9 . Working Party of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

10 . Miles, A. A., Misra, S. S. & Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene 38, 732–49.

11 . Wise, R. (1986). The clinical relevance of protein binding and tissue concentrations in antimicrobial therapy. Clinical Pharmacokinetics 11, 470–82.[ISI][Medline]

12 . Morosini, M. I., Negri, M. C., Loza, E. & Baquero, F. (1995). In vitro activity of RU 67655, a new oral penem antibiotic against Streptococcus pneumoniae. In Program and Abstracts of the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Abstract F130, p. 135. American Society for Microbiology, Washington, DC, USA.

13 . Schmidt, A., Ammelung, P., Bach, R., Morciszek, D. & Dalhoff, A. (2000). Activity of faropenem against Streptococcus pneumoniae under in vitro test conditions that mimic pharmacokinetic properties in man. In Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 498, p. 16. American Society for Microbiology, Washington, DC, USA.