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JAC Advance Access originally published online on April 9, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):156-160; doi:10.1093/jac/dkn133
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

In vitro pharmacodynamics of novel rifamycin ABI-0043 against Staphylococcus aureus

Brian T. Tsuji1,2,*, Jenny C. Yang1, Alan Forrest1, Pamela A. Kelchlin1 and Patrick F. Smith1,{dagger}

1 Laboratory for Antimicrobial Pharmacodynamics, School of Pharmacy and Pharmaceutical Sciences and The New York State Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, State University of New York, NY 14260, USA 2 Roswell Park Cancer Institute, Departments of Pharmacy and Medicine, Buffalo, NY 14263, USA

* Correspondence address. School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, NY, USA. Tel: +1-716-881-7543; Fax: +1-716-849-6890; E-mail: btsuji{at}buffalo.edu

Received 27 November 2007; returned 20 December 2007; revised 15 February 2008; accepted 16 February 2008


   Abstract
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Objectives: ABI-0043 is a novel benzoxazinorifamycin derivative, which derives its potent bactericidal activity by the specific inhibition of bacterial RNA polymerase. We evaluated the in vitro pharmacodynamics and bactericidal activity of ABI-0043 against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA).

Methods: Using time–kill studies at a wide range of concentrations of ABI-0043, we evaluated the killing activity against four clinical isolates of S. aureus over 24 h. An integrated pharmacokinetic/pharmacodynamic area measure was applied to all cfu data and was fitted to a Hill-type mathematical model to evaluate pharmacodynamics.

Results: Bacterial killing for ABI-0043 occurred rapidly and in a concentration-dependent manner. Bactericidal activity was achieved within 4 h at ≥16x MIC against all isolates. Bacterial reductions were greatest at ≥64x MIC against MRSA and MSSA isolates, as a >4 log10 cfu/mL reduction was observed as early as 2 h, and sustained throughout 24 h. The pharmacodynamics of ABI-0043 was well described by a Hill-type model, with a steep sigmoidicity constant and a low EC50 against all isolates.

Conclusions: ABI-0043 displayed rapid and sustained bactericidal activity against S. aureus clinical isolates. ABI-0043 represents a promising antistaphylococcal agent to combat serious S. aureus infections. Further, pharmacokinetic, pharmacodynamic and in vivo studies are warranted to determine its ultimate place in antibacterial therapy.

Keywords: S. aureus , rifalazil , benzoxazinorifamycin


   Introduction
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ABI-0043 is a novel benzoxazinorifamycin that derives its potent bactericidal activity by the specific inhibition of bacterial RNA polymerase.1,2 ABI-0043 is closely related to rifalazil, also referred to as KRM-1648 or ABI-1648, but displays more potent in vitro activity against Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Propionibacterium acnes and Chlamydia spp. and has improved activity against rifampicin-resistant isolates of S. aureus and S. pyogenes.2–4 ABI-0043 has displayed efficacy against S. aureus in several animal models including the neutropenic murine thigh model,3 the mouse septicaemia model3 and a foreign body infection model.5 Limited data exist on the pharmacodynamics and bactericidal activity of ABI-0043 against clinical S. aureus isolates.


   Materials and methods
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Clinical isolates utilized in the study included methicillin-resistant S. aureus (MRSA; S285 and S293) and methicillin-susceptible S. aureus (MSSA; S288 and S290). All isolates were obtained from the blood of infected patients from the VA Medical Center (Buffalo, NY, USA) and were susceptible to rifampicin. S. aureus ATCC 29213 was utilized as a control.

ABI-0043 was provided by Activbiotics Inc., Lexington, MA, USA. ABI-0043 powder was dissolved in 100% dimethyl sulphoxide (DMSO) (Sigma Chemical Company, St Louis, MO, USA) by bath sonication for 30 min to prepare a stock solution of 50 mg/mL. One volume of stock solution was added to four volumes of diluted liquid fill,3 resulting in a dosing solution containing ABI-0043 in 20% DMSO. The 20% DMSO diluent alone showed no antibacterial effect on S. aureus.3,5 Fresh working solutions of ABI-0043 were made prior to each experimental run.

MICs and minimum bactericidal concentrations (MBCs) were determined in quadruplicate using a microdilution technique in accordance with CLSI criteria.6 Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) (SMHB) was used for all broth microdilution susceptibility testing and selective time–kill experiments. The bactericidal activity of ABI-0043 was assessed by time–kill experiments. Briefly, fresh bacterial colonies from an overnight growth were added to normal saline and adjusted spectrophotometrically to provide a standard suspension. This suspension was diluted with SMHB and a standard antibiotic stock solution to achieve a starting inoculum of ~106 cfu/mL. Each 10 mL culture was placed in test tubes that were incubated in a water bath at 35°C with constant shaking, and 0.1 mL samples were withdrawn for determination of bacterial counts at 0, 1, 2, 4, 8 and 24 h. The following time–kill experiments were evaluated: growth control and 0.5x, 2x, 8x, 16x, 64x and 128x MIC of ABI-0043. Colony counts were determined by plating 50 µL of each diluted sample onto Trypticase soy agar [TSA; with 5% sheep blood (Becton–Dickinson, Sparks, MD, USA)] with an automated spiral dispenser (WASP; Don Whitley Scientific Limited, West Yorkshire, UK) and incubated at 35°C to confirm colony counts. This method results in a lower limit of detection of 2.0 log10 cfu/mL. Antimicrobial carryover was minimized by serial dilution (10- to 100 000-fold) and centrifuging the bacterial samples at 10 000 g for 5 min and reconstituting with sterile normal saline to their original volumes. All time–kill experiments were completed in duplicate to quadruplicate. Development of resistance was evaluated at 24 h for all experiments in duplicate. Samples from each time point were plated on TSA plates containing 4- and 8-fold the MIC of the respective antibiotic to assess the development of resistance and examined for growth after 48 h of incubation at 35°C. Bactericidal activity was defined as a reduction of ≥3 log10 cfu/mL (99.9% killing) in bacterial density from the starting inoculum.

To accommodate all available data generated for each regimen tested and avoid conclusions based on cfu counts at a single time point, an integrated pharmacokinetic/pharmacodynamic area measure (log ratio area) was applied to all cfu data. For each regimen tested, the area under the cfu versus time curve from 0 to 24 h (AUCcfu0–24) was calculated via the trapezoidal rule for both the growth control (AUCcfugrowth control) and drug-containing regimens (AUCcfudrug). The AUCcfu0–24 was normalized by the AUCcfu0–24 of the growth control and the logarithm of this ratio was used to quantify the drug effect as shown in Equation (1). Additionally, the traditional approach (log ratio change) of comparing the changes in cfu/mL from 0 h (cfu0) versus 24 h (cfu24) was calculated as shown in Equation (2).



Formula 133M1

1



Formula 133M2

2

Using non-linear regression, a four-parameter concentration–effect Hill-type model was fitted to the effect parameter Systat (Version 11, Richmond, VA, USA) using:


Formula 133M3

3
Where the dependent variable (E) is either log ratio area or log ratio change, E0 the measured effect at zero drug concentration, Emax the maximal effect, C:MIC the concentration of drug divided by the MIC, EC50 the C:MIC for which there is 50% maximal effect and H the Hill or sigmoidicity constant.


   Results
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ABI-0043 displayed MICs and MBCs against all MRSA and MSSA isolates of 0.008 mg/L. The activity of ABI-0043 is depicted in Figure 1. At concentrations of 2x and 4x MIC, ABI-0043 displayed bacteriostatic activity, with a reduction in bacterial counts at 24 h of <3 log10 cfu/mL when compared with the starting inoculum against both MRSA and MSSA. Bactericidal activity was achieved within 4 h at concentrations ≥16x MIC against all isolates and was generally maintained through 8 h. However, against S293 and S288 isolates, a rebound in bacterial growth was observed at 24 h. With increasing ABI-0043 concentrations, a concentration-dependent trend towards greater bacterial killing was observed. Bacterial reductions were greatest at higher concentrations of 64x and 128x MIC against MRSA and MSSA isolates: a rapid reduction in bacterial counts >4 log10 cfu/mL was observed as early as 2 h, reaching undetectable limits, which was sustained throughout 24 h. Growth on resistance plates at 4x MIC was detected at concentrations 0.5x MIC against S285, S288, S290 and S293 providing a potential explanation for the regrowth that was demonstrated in time–kill experiments. Concentrations ≥2x MIC suppressed the development of resistance as no growth was detected on medium containing 4x or 8x MIC of drug.


Figure 1
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  		Figure 1. Time–kill experiments evaluating the bactericidal activity of ABI-0043 versus MRSA S285 (a), MRSA S293 (b), MSSA 288 (c) and MSSA 290 (d), and the pharmacodynamic relationship between concentration and log ratio change or log ratio area for MRSA S285 (e and i), MRSA S293 (f and j), MSSA 288 (g and k) and MSSA 290 (h and l).

 
Analysis of pharmacodynamics revealed excellent model fits of the data to the Hill model (Table 1 and Figure 1). R2 values were slightly higher using the log ratio area approach, as all model fits were >0.99 versus >0.96 for the log ratio change approach. Bactericidal activity for ABI-0043 occurred in a concentration-dependent manner, as seen with the steep sigmoidicity constant (H) and low EC50 against all isolates. Rapid concentration-dependent activity was most evident against MRSA S285, displaying the steepest H among all isolates and a low EC50. ABI-0043 against MSSA 288 displayed the shallowest H and highest EC50, partially explained by the different concentration-killing profiles, with slight regrowth and less activity at 16x MIC, versus other isolates where this exposure resulted in bactericidal activity. The maximal effect for ABI-0043 was a >7 log10 reduction in cfu/mL and a reduction of >4 log10 of area for ABI-0043 against all isolates.


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  		Table 1. Model-fitted parameter estimatesa for ABI-0043 versus S. aureus clinical isolates

 

   Discussion
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ABI-0043 is a representative of the most recent series of benzoxazinorifamycins, which displays potent activity against S. aureus, including rifampicin-resistant isolates. ABI-0043 is one of a number of rifamycins, such as ABI-0369, ABI-0699 and rifalazil, which also displays extremely low MICs for staphylococci.2,3,7,8 Although rifampicin, the oldest representative rifamycin, possesses rapid concentration-dependent activity against staphylococci, its therapeutic use has been limited due to the rapid emergence of resistant organisms, poor tolerance and interactions with CYP3A4.9 Rifampicin has typically been utilized in combination with other agents in persistent, difficult-to-treat infections to overcome a high bacterial burden. However, numerous reports on the emergence of resistance, questionable efficacy and clinical failure raise uncertainty regarding its utility, especially in the treatment S. aureus endocarditis where it has been used in combination with other agents.10,11 ABI-0043 possesses more potent activity than rifampicin in vitro and retains some activity against rifampicin-resistant strains.2,3 Comparable efficacy has also been established for ABI-0043 at lower doses than rifampicin in murine models of infection, and ABI-0043 has also displayed efficacy equivalent to rifampicin in combination, in an experimental model of foreign body infection.3–5 In addition, ABI-0043 does not induce the P450 system, whereas rifampicin is a strong inducer of CYP3A4 and other P450 isoenzymes.7 In the present study, ABI-0043 displayed rapid bactericidal activity against clinical isolates of S. aureus, at concentrations as low as 16x MIC. Concentrations above the MIC prevented the amplification of resistant mutants, although exposure to 0.5x MIC resulted in the development of resistance at 24 h. The pharmacodynamics of ABI-0043 were well described by a Hill-type sigmoidal, maximal effect model, displaying concentration-dependent activity against both MRSA and MSSA, where maximal effect (>7 log10 cfu/mL) was achieved at higher thresholds of ≥64x MIC. Our findings are also similar to other investigations in which ABI-0043 demonstrated a dose-dependent response with a high correlation to bacterial kill in a murine pneumococcal pneumonia model.4 Additionally, the pharmacokinetic profile of ABI-0043 is reported to have a prolonged terminal-phase half-life in animal studies, which may be consistent with once daily dosing in humans.7,12 In conclusion, ABI-0043 is a novel rifamycin that may provide an alternative option for the treatment of MRSA infections. Further pharmacokinetic and pharmacodynamic, resistance and clinical studies are warranted to determine its ultimate place in antibacterial therapy.


   Funding
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This study was funded by the University at Buffalo, State University of New York and the Gustavus and Louise Pfeiffer Research Foundation.


   Transparency declarations
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B. T. T. and A. F. have received grant support through Pfizer Pharmaceuticals, New York, NY, USA. P. F. S. is an employee of F. Hoffmann-La Roche Ltd. J. C. Y. and P. A. K.: none to declare.


   Footnotes
 
{dagger} Present address. Roche Pharmaceuticals, Nutley, NJ, USA. Back


   Acknowledgements
 
We thank Dr Alan Lesse for the gift of strains S285, S293, S288 and S290.

A portion of these results were presented at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2006. We thank David Rothstein and Christopher Murphy for critical review of this manuscript.


   References
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1 Fujii K, Saito H, Tomioka H, et al. Mechanism of action of antimycobacterial activity of the new benzoxazinorifamycin KRM-1648. Antimicrob Agents Chemother (1995) 39:1489–92.[Abstract]

2 Murphy CK, Mullin S, Osburne MS, et al. In vitro activity of novel rifamycins against rifamycin-resistant Staphylococcus aureus. Antimicrob Agents Chemother (2006) 50:827–34.[Abstract/Free Full Text]

3 Rothstein DM, Farquhar RS, Sirokman K, et al. Efficacy of novel rifamycin derivatives against rifamycin-sensitive and -resistant Staphylococcus aureus isolates in murine models of infection. Antimicrob Agents Chemother (2006) 50:3658–64.[Abstract/Free Full Text]

4 Lee SY, Tessier PR, Murphy CK, et al. Bactericidal efficacy of ABI-0043, a novel rifamycin, in a murine pneumococcal pneumonia model. J Antibiot (Tokyo) (2006) 59:804–7.[Medline]

5 Trampuz A, Murphy CK, Rothstein DM, et al. Efficacy of a novel rifamycin derivative, ABI-0043, against Staphylococcus aureus in an experimental model of foreign-body infection. Antimicrob Agents Chemother (2007) 51:2540–5.[Abstract/Free Full Text]

6 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition: Approved Standard M7-A7 (2006) Wayne, PA, USA: CLSI.

7 Rothstein DM, Shalish C, Murphy CK, et al. Development potential of rifalazil and other benzoxazinorifamycins. Expert Opin Investig Drugs (2006) 15:603–23.[CrossRef][Web of Science][Medline]

8 Flamm RK, Karginova E, Rothstein DMDM, et al. In vitro potency and spectrum of novel rifamycins ABI-0043, ABI-0094 and ABI-0299. Abstracts of the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, 2005: Washington, DC, USA. Washington, DC, USA: American Society for Microbiology. Abstract F-2043, p. 222.

9 Zavasky DM, Sande MA. Reconsideration of rifampin: a unique drug for a unique infection. JAMA (1998) 279:1575–7.[Free Full Text]

10 Levine DP, Fromm BS, Reddy BR. Slow response to vancomycin or vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis. Ann Intern Med (1991) 115:674–80.[CrossRef][Web of Science][Medline]

11 Eng RH, Smith SM, Tillem M, et al. Rifampin resistance. Development during the therapy of methicillin-resistant Staphylococcus aureus infection. Arch Intern Med (1985) 145:146–8.[Abstract/Free Full Text]

12 Chen Y, Larsson M, Pieniaszek, et al. The pharmacokinetics of ABI-0043, a novel rifamycin antibacterial, in healthy beagle dogs and male Sprague–Dawley rats following oral and intravenous administration. Abstracts of the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, 2005: Washington, DC, USA. Washington, DC, USA: American Society for Microbiology. Abstract F-2047, pp. 223–4.


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