Received 30 March 2005; returned 16 June 2005; revised 8 September 2005; accepted 12 September 2005

Transcription

1 Journal of Antimicrobial Chemotherapy (2005) 56, doi: /jac/dki362 Advance Access publication 20 October 2005 Evaluation of PPI-0903M (T91825), a novel cephalosporin: bactericidal activity, effects of modifying in vitro testing parameters and optimization of disc diffusion tests Ronald N. Jones 1,2, Thomas R. Fritsche 1, Yigong Ge 3, Koné Kaniga 3 and Helio S. Sader 1,4 * 1 JMI Laboratories, Inc., 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317, USA; 2 Tufts University School of Medicine, Boston, MA, USA; 3 Peninsula Pharmaceuticals, Alameda, CA, USA; 4 Universidade Federal de São Paulo, São Paulo, Brazil Received 30 March 2005; returned 16 June 2005; revised 8 September 2005; accepted 12 September 2005 Objectives: To evaluate bactericidal activity of PPI-0903M and in vitro testing parameters for this compound. Methods: A total of 110 strains were tested to determine MIC and MBC values of PPI-0903M. MIC values were determined by reference broth microdilution whereas MBC was assessed by plating the broth from the MIC well and from those wells above the MIC for each organism onto appropriate drug-free growth media. Seventeen strains were tested by the kill-curve methodology to again evaluate the bactericidal activity of PPI-0903M. Broth microdilution results for 15 strains (in triplicate) were compared with the standard, reference method after modifying several testing parameters. Optimal disc content was assessed by testing seven reference strains with five PPI-0903M disc concentrations (5, 10, 30, 50 and 100 mg). Results: PPI-0903MMBC/MIC ratios were 4 in 90% of strains tested. Kill-curve studies showed >99.9% killing at concentrations 4 MIC within 8 to 24 h against staphylococci and Enterobacteriaceae tested. PPI- 0903M MIC results were rarely influenced by the test or medium changes evaluated [high inoculum, low calcium or reduced ph (5.0)]. Human serum had no effect on in vitro antimicrobial activity of PPI-0903M. The 10 mg disc content would be recommended for optimal correlation of MIC breakpoints of 1 4 mg/l. Conclusions: PPI-0903M was generally bactericidal against tested species, and the MIC results were stable across numerous susceptibility test condition changes. Keywords: PPI-0903M, anti-mrsa cephalosporins, MRSA Introduction PPI-0903 (formerly TAK-599) is a water-soluble prodrug of PPI- 0903M (T-91825), a new generation semi-synthetic N-phosphono cephalosporin. When PPI-0903 is administered to animals, it converts to PPI-0903M upon hydrolysis of the phosphonate group. Preliminary in vitro studies demonstrated that PPI-0903M was very active against many clinically important bacterial pathogens, including streptococci (b-haemolytic, viridans group and Streptococcus pneumoniae), staphylococci (Staphylococcus aureus and coagulase-negative species), Haemophilus influenzae and Moraxella catarrhalis among others. PPI-0903M activity against these pathogens was similar to that demonstrated by other new anti-methicillin-resistant (MRSA) cephalosporins. 1 6 Moreover, PPI-0903M showed potent activity against multidrug-resistant Gram-positive pathogens that may cause both community-acquired and hospital-acquired infections, especially MRSA and penicillin-resistant S. pneumoniae. In various animal models, PPI-0903 has demonstrated excellent activities against MRSA and vancomycin-intermediate (VISA). 6 Although a number of new agents have recently become available to treat infections caused by resistant Gram-positive organisms, all these agents (including daptomycin, linezolid and quinupristin/dalfopristin) lack activity against common Gramnegative pathogens, necessitating combination therapy for empirical treatment of many serious infections. There is a clinical need for a new broad-spectrum antimicrobial that covers both resistant Gram-positive and Gram-negative pathogens. PPI-0903 is currently being developed as a parenteral antimicrobial agent for the treatment of serious infections. 6,7 We evaluated bactericidal... *Corresponding author. Tel: ; Fax: ; helio-sader@jmilabs.com Ó The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Jones et al. action of PPI-0903M and optimized in vitro testing parameters for this new cephalosporin compound. Materials and methods A total of 110 strains were tested to determine MIC and MBC values of PPI-0903M. MIC values were determined by the Clinical and Laboratory Standards Institute [CLSI; formerly the National Committee for Clinical Laboratory Standards (NCCLS)] reference broth microdilution methods. 8 PPI-0903M reagent grade compound (lot no. M599-R10S01) was manufactured by Takeda Pharmaceuticals Co., Ltd (Osaka, Japan) and supplied by Peninsula Pharmaceuticals, Inc. (Alameda, CA, USA). Concurrent quality control (QC) procedures were performed by testing the following reference strains: S. pneumoniae ATCC 49619, Enterococcus faecalis ATCC 29212, ATCC 29213, Escherichia coli ATCC and Pseudomonas aeruginosa ATCC The MBC was assessed by plating the broth from the MIC well and from those wells above the MIC for each organism onto appropriate drug-free growth media. 8,9 Quantitative colony counts were performed on the starting inoculum. The MBC was defined as the lowest concentration of PPI-0903M that killed 99.9% of the starting test inoculum. 9 Seventeen strains (six, two Staphylococcus epidermidis, four S. pneumoniae, and one each of H. influenzae, E. coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens) were tested by the kill-curve methodology to again evaluate the bactericidal activity of PPI-0903M. Bacterial kill curves were performed in Mueller Hinton broth (MHB) for all isolates except S. pneumoniae (MHB supplemented with 2 5% lysed horse blood) and H. influenzae (Haemophilus Test Medium broth). PPI-0903M activity was tested at timed intervals of 0, 4, 8 and 24 h at 1, 2, 4 and 8 MIC. 9 Broth microdilution MIC results for 15 strains (in triplicate) were compared with the standard, reference method after modifying broth ph (5.0, 6.0 and 8.0), incubation environments (5% CO 2 and anaerobic), inoculum concentration ( and 10 7 cfu/ml), Ca 2+ content (trace and 50 mg/l), medium supplements (lysed horse blood and HTM) and human serum concentrations in the test broth (5, 10 and 50%). Optimal disc content was assessed by testing seven reference strains with five PPI-0903M disc concentrations (5, 10, 30, 50 and 100 mg). The strains tested in this experiment were: ATCC and 29213, E. faecalis ATCC 29212, S. pneumoniae ATCC 49619, E. coli ATCC 25922, P. aeruginosa ATCC and E. faecium D. The disc diffusion tests were performed according to CLSI/NCCLS methods. 10 Ceftriaxone and cefepime were tested concurrently as control agents of the same antimicrobial class. Results PPI-0903M exhibited bactericidal activity at or one log 2 dilution above the MIC for 86.4% of tested organisms and 90% of strains had an MBC/MIC ratio of 4 (preferred ratios). The majority of isolates with an MBC/MIC ratio >4 (6 of 11) were penicillinresistant S. pneumoniae (Table 1). Kill-curve kinetic studies confirmed the findings observed using MBC determinations for PPI-0903M (Table 2). Bactericidal ( 3 log 10 cfu/ml reductions) action was noted for PPI-0903M at all multiples of the reference MIC tested (2,4 and 8 ) against 12 of 17 strains (70.6%) tested. Three strains showed bactericidal activity only at 8 MIC (two oxacillin-resistant )or at 4 and 8 MIC ( ATCC Table 1. Listing of MBC results compared with the MIC for 110 organisms tested against PPI-0903M Occurrences at MBC/MIC ratio of: Organism group (no. tested) MIC (mg/l) range >4 oxacillin-resistant (10) oxacillin-susceptible (10) hvisa (10) Coagulase-negative staphylococci oxacillin-resistant (10) oxacillin-susceptible (10) S. pneumoniae penicillin-susceptible (10) penicillin-resistant (10) Viridans group streptococci penicillin-susceptible (5) penicillin-resistant (5) Enterobacteriaceae (20) a 2 b Quality control strains (10) c Total (110) Cumulative % a Includes E. coli (one strain) and S. marcescens (two strains). b Includes E. coli (one strain) and S. marcescens (one strain). c One strain, E. faecalis ATCC

3 Evaluation of PPI-0903M (T91825), a novel cephalosporin Table 2. Kill-curve results for PPI-0903M tested at three concentrations (2, 4 and 8 MIC) tested against (six strains; four oxacillin-resistant of which two were VISA), S. epidermidis (two strains; one oxacillin-resistant), S. pneumoniae (four strains; three penicillin-non-susceptible), Enterobacteriaceae (four wild-type strains) and H. influenzae (one quality control strain) Concentration (log 10 cfu/ml) at t (h) Organism (PPI-0903M MIC, mg/l) Test concentration Activity category a ATCC (0.12) 2 3.1E5 2.9E4 3.7E3 5.6E4 static b 4 2.2E4 3.8E3 1.3E2 cidal 8 2.7E4 9.5E2 3.0E2 cidal ATCC (0.25) 2 5.3E5 5.5E4 1.9E3 1.8E2 cidal 4 8.3E4 3.2E3 1.5E2 cidal 8 6.8E4 5.5E3 1.9E2 cidal 1-170A (0.5) 2 1.3E6 8.2E4 1.5E3 1.0E1 cidal 4 2.0E4 6.5E2 <1.0E1 cidal 8 1.3E4 4.0E2 3.0E1 cidal A (0.5) 2 5.6E5 4.2E4 1.5E3 1.5E7 c 4 1.8E4 8.2E2 3.1E3 static b 8 1.7E4 6.3E2 1.0E1 cidal (2) 2 3.8E5 3.0E5 1.1E5 7.3E2 static b 4 2.7E5 1.1E5 8.4E3 static b 8 3.5E5 8.2E4 2.8E2 cidal A (2) 2 3.0E5 3.5E4 3.4E3 1.9E2 cidal 4 2.2E4 1.9E3 1.1E2 cidal 8 3.0E4 1.1E3 6.0E1 cidal S. epidermidis A (0.5) 2 1.7E5 1.7E2 <1.0E1 6.4E7 c 4 4.8E2 <1.0E1 <1.0E1 cidal 8 1.3E3 1.0E1 1.0E1 cidal 51-70A (0.12) 2 1.6E5 7.0E1 <1.0E1 1.0E1 cidal 4 1.0E1 1.0E1 <1.0E1 cidal 8 1.0E2 1.0E1 6.0E1 cidal S. pneumoniae B (0.03) 2 2.8E5 2.5E4 4.8E3 7.1E2 static d 4 4.1E4 5.8E3 7.9E2 static d 8 3.4E4 8.1E3 7.1E2 static d B (0.06) 2 2.2E5 1.3E3 6.5E1 <1.0E1 cidal 4 1.6E3 1.7E2 <1.0E1 cidal 8 1.1E3 1.0E1 <1.0E1 cidal 90-71B (0.12) 2 3.3E5 3.8E4 5.5E3 2.0E1 cidal 4 3.1E4 4.3E3 3.0E1 cidal 8 2.8E4 5.6E3 <1.0E1 cidal B (0.25) 2 8.1E5 1.6E5 6.3E4 2.3E3 static d 4 1.4E5 5.7E4 2.4E3 static d 8 1.3E5 7.3E4 2.5E3 static d E. coli ATCC (0.06) 2 2.7E5 7.2E3 7.0E2 <1.0E1 cidal 4 1.2E3 5.0E2 1.0E1 cidal 8 1.3E4 1.1E3 <1.0E1 cidal K. pneumoniae 1-174A (0.12) 2 5.2E5 4.0E3 1.1E2 <1.0E1 cidal 4 2.1E3 9.0E1 <1.0E1 cidal 8 2.4E3 1.1E2 <1.0E1 cidal E. cloacae A (0.25) 2 4.3E5 1.2E4 1.4E3 5.0E1 cidal 4 6.7E4 1.4E4 2.1E2 cidal 8 5.9E4 9.6E3 4.0E1 cidal 1049

4 Jones et al. Table 2. (continued) Concentration (log 10 cfu/ml) at t (h) Organism (PPI-0903M MIC, mg/l) Test concentration Activity category a S. marcescens A (1.0) 2 4.6E5 9.6E4 6.0E3 9.5E1 cidal 4 5.3E4 8.8E3 1.8E2 cidal 8 2.0E4 6.5E3 3.7E2 cidal H. influenzae ATCC (0.06) 2 3.7E5 4.5E4 3.0E1 <1.0E1 cidal 4 6.1E3 3.0E1 <1.0E1 cidal 8 4.4E3 3.0E1 <1.0E1 cidal a Bactericidal (cidal) activity was defined as 3 log 10 reduction in the initial inoculum within 24 h of incubation. b All static activity results for staphylococci occurred at 2 or 4 MIC concentrations with cidal action detected at higher drug levels. c Indicates regrowth after initial killing (cidal for A) in the first 8 h of incubation. d Static activity of PPI-0903M versus the pneumococci represented between 2 and 3 log 10 cfu/ml killing. Table 3. Effects of varying reference NCCLS broth microdilution test conditions on the MIC of PPI-0903M using 15 selected organisms PPI-0903M MIC (mg/l) inoculum (cfu/ml) calcium conc. incubation supplements medium ph Organism Reference lysed MIC a trace 50 mg/l anaerobic 5% CO 2 HB b HTM c E. coli ATCC E. cloacae 32-43A >32 d P. aeruginosa ATCC > A. baumannii A ATCC ATCC VISA A CoNS 51-81A A E. faecalis ATCC A E. faecium A S. pneumoniae ATCC NT e a a NG f Viridans group streptococci A NT e a a NG f a MIC determined under CLSI/NCCLS method conditions, usually Mueller Hinton broth, ph , Ca 2+ at 25 mg/l, inoculum of cfu/ml incubated in ambient air. b Strains requiring 2 5% lysed horse blood (S. pneumoniae and viridans group streptococci) were incubated in that medium. c HTM, Haemophilus Test Medium. d Underlined results vary by >4-fold from reference MIC value. e NT, not tested. f NG, no growth ). In addition, a reduction of only 2 log 10 cfu/ml after 24 h of incubation was noted for two S. pneumoniae strains, one penicillin-susceptible (MIC 0.06 mg/l) and one penicillinresistant strain (MIC 4 mg/l; Table 2). PPI-0903M was bactericidal versus all Enterobacteriaceae (including one S. marcescens) and H. influenzae strains tested. Table 3 summarizes the results from 15 organisms tested against PPI-0903M using 11 variations of the NCCLS methods. 1050

5 Evaluation of PPI-0903M (T91825), a novel cephalosporin Table 4. Effects of increasing concentrations of human serum on the MIC values of PPI-0903M PPI-0903M MIC (mg/l) in: Organism broth +5% serum +10% serum +50% serum S. pneumoniae ATCC S. pneumoniae B ATCC C A A E. coli ATCC K. pneumoniae 1-174A P. aeruginosa ATCC MIC (mg/l) > <= µg disc P. aeruginosa 30 µg disc Ideal breakpoint region E. faecalis S. pneumoniae E. coli Zone diameter in mm Figure 1. Comparisons of zones of inhibition for two PPI-0903M disc concentrations (10 and 30 mg) tested against five quality control organisms. Only 16 (9.7%) MICs among 165 results varied by more than 4-fold when compared with the baseline, reference PPI-0903M MIC. One test condition (medium ph 5.0) was responsible for nine of 16 significant variations; results that were markedly lower than baseline MICs due to suboptimal growth concentrations or possible instability of PPI-0903M under acidic conditions. No significant increase in MICs (>1 log 2 dilution) was detected using MHB supplemented with three human serum concentrations (5, 10 and 50%) and nine organisms representing the streptococci, Enterobacteriaceae, non-fermentative Gram-negative bacilli and the staphylococci (Table 4). Susceptible Gram-positive QC strains all had PPI-0903M zone diameters of >20 mm for the 10 to 100 mg disc concentrations, and a corresponding MIC of 0.5 mg/l (Figure 1). Maximum zone diameter differences (10 mm) between possible PPI- 0903M-resistant strains (example: E. faecium) and susceptible species were achieved with PPI-0903M disc concentrations of 10 or 30 mg. Based on the results from this pilot experiment, the 10 mg disc content would be recommended for potential correlation of MIC breakpoints of 1 4 mg/l. Discussion PPI-0903M has demonstrated enhanced activity against resistant Gram-positive cocci and similar in vitro activity against common Gram-negative pathogens when compared with broad-spectrum cephalosporins. 5 PPI-0903M is very active against many clinically important bacterial pathogens, especially streptococci (bhaemolytic, viridans group and pneumococci), staphylococci [ and coagulase-negative staphylococci (CoNS)], H. influenzae and M. catarrhalis. PPI-0903M in vitro activity against these pathogens is similar or more potent when compared with other described anti-mrsa cephalosporins. 2 4 Moreover, PPI-0903M exhibited activity against several multidrug-resistant Grampositive pathogens that may cause both community-acquired and nosocomial infections, including MRSA and penicillin-resistant S. pneumoniae. 5 This spectrum of activity distinguishes PPI-0903 from currently available b-lactams and suggests that PPI-0903 has potential for use in the treatment of bacterial infections in the hospital environment. The prodrug, PPI-0903, is currently under Phase I clinical evaluation and has the potential to be a unique addition to the well-established, safe cephalosporin class. In this study, PPI-0903M showed bactericidal activity against a wide range of tested species with MBC values near the measured MIC. Most importantly, PPI-0903M has demonstrated bactericidal activity against MRSA. PPI-0903M MIC results were very stable across numerous susceptibility test technical variations and human serum proteins did not adversely influence activity. The standard CLSI method can be easily applied to in vitro susceptibility testing of PPI-0903M. Further evaluations should be performed to establish the clinical role of this promising new generation anti-mrsa cephalosporin. Transparency declarations No declarations were made by the authors of this paper. References 1. Chamberland S, Blais J, Hoang M et al. In vitro activities of RWJ (MC-02,479) against multiresistant Gram-positive bacteria. Antimicrob Agents Chemother 2001; 45: Fujimura T, Yamano Y, Yoshida I et al. In vitro activity of S-3578, a new broad-spectrum cephalosporin active against methicillin-resistant staphylococci. Antimicrob Agents Chemother 2003; 47:

6 Jones et al. 3. Jones RN, Deshpande LM, Mutnick AH et al. In vitro evaluation of BAL9141, a novel parenteral cephalosporin active against oxacillinresistant staphylococci. J Antimicrob Chemother 2002; 50: Sader HS, Johnson DM, Jones RN. In vitro activities of the novel cephalosporin LB against multidrug-resistant staphylococci and streptococci. Antimicrob Agents Chemother 2004; 48: Sader HS, Fritsche TR, Kaniga K et al. Antimicrobial activity and spectrum of PPI-0903M (T-91825), a novel cephalosporin, tested against a worldwide collection of clinical strains. Antimicrob Agents Chemother 2005; 49: Iizawa Y, Nagai J, Ishikawa T et al. In vitro antimicrobial activity of T-91825, a novel anti-mrsa cephalosporin, and in vivo anti- MRSA activity of its prodrug, TAK-599. J Infect Chemother 2004; 10: Ishikawa T, Matsunaga N, Tawada H et al. TAK-599, a novel N-phosphono type prodrug of anti-mrsa cephalosporin T-91825: synthesis, physicochemical and pharmacological properties. Bioorg Med Chem 2003; 11: National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, Moody J, Knapp C. Time kill assay. In: Isenberg HD, ed. Clinical Microbiology Procedures Handbook. Washington, DC, USA: ASM Press, 2004; National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests Eighth Edition: Approved Standard M2-A8. NCCLS, Wayne, PA, USA,