Abstract
Simulations of administration of razupenem at 1 g every 12 h by 1-h intravenous (i.v.) infusion were performed in an in vitro pharmacokinetic model of infection. The antibacterial effect of this razupenem dosing regimen against six strains of Staphylococcus aureus (one methicillin-sensitive S. aureus [MSSA] strain [MIC, 0.015 μg/ml] and five methicillin-resistant S. aureus [MRSA] strains [MIC range, 0.09 to 3 μg/ml]) and five strains of Enterobacteriaceae (three Escherichia coli strains [two containing extended-spectrum β-lactamases {ESBLs}] and two Enterobacter sp. strains [one with an AmpC enzyme and the other with a raised razupenem MIC; MIC range, 0.09 to 6 μg/ml]) was assessed. Against the MSSA and MRSA strains, razupenem produced a >3.5-log-unit reduction in viable count after 24 h. There were no changes in population profiles. In a second series of experiments, over 5 days there was rapid initial clearance of MRSA from the model followed by regrowth after 48 h. MRSA colonies appeared on 2× MIC recovery medium after 72 h with strain 33820 (MIC, 3.0 μg/ml) and at 120 h with strain 27706 (MIC, 1.5 μg/ml). Against E. coli and Enterobacter spp., razupenem produced a >3.5-log-unit reduction in bacterial counts for all strains except that with an MIC of 6 μg/ml, where razupenem had a notably poorer antibacterial effect. Population profiles were unchanged after 48 h of exposure to razupenem except for Enterobacter strain 34425 (MIC, 6.0 μg/ml), where colonies were recovered from media containing 2×, 4×, and 8× MIC. In dose-ranging studies with MRSA strains, the percentage of the dosing interval that the free drug concentration remained higher than the pathogen MIC (fT>MIC) for a 24-h bacteriostatic effect was 5.0% ± 1.4%, and that for a 1-log-unit reduction in count was 12.5% ± 5.8%. Population profiles indicated growth on 2× MIC recovery medium at fT>MIC values of 1 to 35% but not at a value of >35%. In a similar set of experiments with Enterobacteriaceae, the fT>MIC for a 24-h bacteriostatic effect was 34.2% ± 7.6% and that for a 1-log-unit reduction in count was 42.5% ± 7.8%. Population analysis profiles indicated growth on recovery media with 2×, 4×, and 8× MIC at fT>MICs in the range of 1 to 69% but rarely at values of ≥70%. In conclusion, razupenem at simulated human doses of 1 g i.v. every 12 h has a marked antibacterial effect on MSSA and MRSA strains with MICs of ≤3.0 μg/ml and Enterobacteriaceae with MICs of ≤0.4 μg/ml. fT>MIC targets of ≥35% for MRSA and ≥70% for Enterobacteriaceae should provide significant antibacterial effects combined with low risks of changing pathogen antibiotic population profiles.
Razupenem (also called SM-216601, SMP601, PZ601, and PTZ601) is a developmental 1β-methyl carbapenem with in vitro potency against a broad range of Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. (16, 10). The razupenem MIC90s (μg/ml) for methicillin-sensitive S. aureus (MSSA), MRSA, and penicillin-resistant S. pneumoniae are ≤0.06, 2, and 0.25 μg/ml, respectively, and the MIC90s for E. coli, Klebsiella pneumoniae, and Enterobacter cloacae are 0.5, 0.2, and 8 μg/ml, respectively. Serratia marcescens, Pseudomonas aeruginosa, and Bacteroides fragilis all have MIC90 of ≥16 μg/ml (18). The model MICs for razupenem against ESBL-producing E. coli and Klebsiella spp. are 0.5 and 2 μg/ml, respectively, but ESBL-producing Enterobacter spp. have model MICs of 8 μg/ml, and Enterobacter spp which produce AmpC enzymes and have impermeability characteristics have model MICs of 16 μg/ml (6, 10). Early pharmacodynamic studies performed on only two strains of MRSA in a neutropenic thigh infection model indicated that the percentage of the dosing interval that the free drug concentration remained higher than the pathogen MIC (fT>MIC) was best related to antibacterial effect (ABE) for S. aureus and Enterococcus spp. and that an fT>MIC of 23% was associated with a 24-h bacteriostatic effect and an fT>MIC of 32 to 37% with a 2-log-unit reduction in counts (9).
The aims of this study were to describe the antibacterial effects of razupenem at simulated human doses of 1 g every 12 h intravenously (i.v.) against MSSA, MRSA, and Enterobacteriaceae and to establish the relationship of fT>MIC to antibacterial effect and the risk of emergence of resistance as measured by changes in bacterial population profiles. A dilutional single-compartment in vitro pharmacokinetic model was used.
MATERIALS AND METHODS
In vitro pharmacokinetic model.
A New Brunswick (Hatfield, Herefordshire, England) Bioflo 1000 in vitro pharmacokinetic model was used to simulate serum drug concentrations of razupenem associated with administration of 1 g i.v. every 12 h in humans. The apparatus, which has been described before, consists of a single central chamber connected to a reservoir containing broth. The central chamber is connected to a collecting vessel for overflow (11). The contents of the central chamber were diluted with broth using a peristaltic pump (Ismatec; Bennett & Co, Weston-super-Mare, England) at a flow rate of 313 ml/h for razupenem. The temperature was maintained at 37°C, and the broth in the central chamber was agitated with a magnetic stirrer.
Media.
Fifty percent cation-supplemented Mueller-Hinton broth (MHB) was used in the experiments with S. aureus strains and Enterobacteriaceae. Nutrient agar plates (Oxoid, Basingstoke, England) were to recover S. aureus and Enterobacteriaceae strains from the in vitro model. Five microliters β-lactamase/ml was used to neutralize razupenem. The β-lactamase neutralized razupenem up to a concentration of 75 μg/ml. Razupenem was added to nutrient agar plates in the studies on the emergence of resistance.
Strains.
A single MSSA strain (strain 5956; razupenem MIC, 0.015 μg/ml), five MRSA strains (strain 33922 [MIC, 0.09 μg/ml], strain 36895 [MIC, 0.38 μg/ml], strain 27706 [MIC, 1.5 μg/ml], strain 19898 [VISA] [MIC, 1.5 μg/ml], and strain 33820 [MIC, 3.0 μg/ml]) and five Enterobacteriaceae strains were employed. The Enterobacteriaceae were strain 31053, an E. coli strain containing SHV-12 (razupenem MIC, 0.09 μg/ml); strain 39136, an ampicillin-sensitive E. coli strain (MIC, 0.12 μg/ml); strain 35776, an E. coli strain containing a CTX-M enzyme (MIC, 0.1 μg/ml); strain 35054, an Enterobacter sp. strain with an AmpC enzyme (MIC, 0.4 μg/ml); and strain 34425, an Enterobacter sp. strain (MIC, 6 μg/ml).
Antibiotics.
Razupenem was supplied by Protez Pharmaceuticals, Malvern, PA. Stock solutions were prepared according to British Society for Antimicrobial Chemotherapy guidelines (5) and stored at −70°C.
MICs.
MICs were determined by a standard broth dilution method according to CLSI guidelines (7). MIC determinations were performed in 50% MHB and at nondoubling dilutions to more accurately determine MICs.
Pharmacokinetics.
The target maximum drug concentration in serum (Cmax) was 57.8 mg/liter after a 1-h infusion with a half-life (t1/2) of 1.5 h, and dosing was every 12 h for 48 h, that is, four dose exposures. The target area under the concentration-time curve from 0 to 12 h (AUC0-12) was 100.2 mg·h/liter. This dose simulation was used in simulations against MSSA, MRSA, and Enterobacteriaceae. In addition, between 7 and 16 doses were simulated per strain in dose-ranging experiments designed to achieve an fT>MIC range of 0 to 100% for each strain in experiments to define the relationship between fT>MIC and antibacterial effect. Concentrations of razupenem were determined by bioassay methodology using E. coli NCTC 1041895 as the indicator organism on DST agar (2). The limit of detection was 1.0 mg/liter.
ABE.
Experiments were performed at an initial inoculum of 106/ml, prepared as described previously (12). Samples were taken throughout the simulation period for detection of viable counts. Bacteria were quantified by using a spiral plater (Don Whitley Spiral Systems, West Yorkshire, United Kingdom). The minimum level of detection is 102 CFU/ml. Dosing simulations of 1 g every 12 h were performed over 48 h for S. aureus and Enterobacteriaceae, and a separate 5-day simulation was performed with S. aureus alone. Aliquots, taken each hour, were stored at −70°C for measurement of razupenem concentration.
Emergence of resistance.
Resistance to razupenem was assessed using population analysis profiles (11) at time zero (preexposure) and every 24 h postexposure. Samples were plated onto agar containing no antibiotic and antibiotic at 1×, 2×, 4×, and 8× MIC to quantify any resistant subpopulation. The limit of detection was 102 CFU/ml.
All pharmacokinetic simulations of human doses to determine ABE and emergence of resistance were performed at least in triplicate.
Pharmacodynamics and measurement of ABE.
The ABE of razupenem was calculated by determining the log change in viable counts between time zero and 12 h (d12), 24 h (d24), 36 h (d36) and 48 h (d48). The times for the inoculum to fall to 99% and 99.9% of its initial value were recorded as T99 and T99.9, respectively. The area under the bacterial kill curve (AUBKC) (log CFU/ml·h) was calculated using the log-linear trapezoidal rule for the times from 0 to 24 h (AUBKC24) and 0 to 48 h (AUBKC48) with a −4-log-unit baseline. The relationship between T>MIC and ABE was delineated using Boltzmann sigmoidal Emax models (Graph Pad Prism; Graph Pad Software, San Diego, CA).
RESULTS
Pharmacokinetic curves and pharmacodynamic parameters.
There was good agreement between the target and simulated pharmacokinetic parameters; the achieved Cmax was 61.4 ± 3.1 mg/liter, the Cmin at 12 h was <0.1 mg/liter, and the AUC0-12 was 98.1 ± 5.1. The free drug T>MIC for razupenem at 1 g every 12 h by 1-h infusion were as follows: for MSSA strain 5956, razupenem MIC of 0.015 μg/ml and fT>MIC of 100%; for MRSA strain 33922, MIC of 0.09 μg/ml and fT>MIC of 98%; for MRSA strain 36995, MIC of 0.38 μg/ml and fT>MIC of 75%; for MRSA strain 27706 MIC of 1.5 μg/ml and fT>MIC of 64%; for MRSA strain 19898, MIC of 1.5 μg/ml and fT>MIC of 64%; for MRSA strain 33820, MIC of 3.0 μg/ml and fT>MIC of 44%; for E. coli strain 31053, MIC of 0.09 μg/ml and fT>MIC of 100%; for E. coli strain 35776, MIC of 0.12 μg/ml and fT>MIC of 90%; for E. coli strain 35776, MIC of 0.1 μg/ml and fT>MIC of 96%; for Enterobacter sp. strain 35054, MIC of 0.4 μg/ml and fT>MIC of 75%; and for Enterobacter sp. strain 34425, MIC of 6.0 μg/ml and fT>MIC of 33%.
Effect against S. aureus. (i) Forty-eight-hour simulations against MSSA and MRSA.
The effects of simulations of razupenem given at 1 g by 1-h infusion every 12 h against a single strain of MSSA (MIC, 0.015 μg/ml) and five MRSA strains (MIC range, 0.09 to 3.0 μg/ml) are shown in Table 1. Against MSSA, razupenem produced a >3.5-log10-unit reduction in viable count by 24 h and a >4-log10-unit reduction in viable count at 48 h. Similarly, for the five MRSA strains, a >4-log-unit reduction in count at 24 h occurred with strains 33922 and 36895, a >3-log-unit reduction in count occurred with strain 27706, and a >2-log-unit reduction in count occurred with strains 19898 and 33820. At 48 h, the reduction in count was >3 log10 units for all strains except strain 33820. Comparison of the five MRSA strains using d24, d48, AUBKC24, and AUBKC48 (data not shown) indicated that razupenem had a poorer antibacterial effect against strain 33820 (MIC, 3.0 μg/ml) at 48 h than against strains 33922 and 36895 (AUBKC48) and strain 36895 (d48) (P < 0.05 by analysis of variance [ANOVA]).
TABLE 1.
Antibacterial effect of simulations of razupenem at 1 g given as a 1-h infusion against S. aureus strains
| Time (h) | Log change in viable count (mean ± SD) for strain (MIC, mg/liter): |
|||||
|---|---|---|---|---|---|---|
| MSSA 5956 (0.015) | MRSA 33922 (0.09) | MRSA 36895 (0.19) | MRSA 27706 (0.38) | MRSA 19898 (0.75) | MRSA 33820 (3.0) | |
| 6 | −3.8 ± 0.1 | −3.3 ± 1.3 | −3.7 ± 0.3 | −4.0 ± 0.4 | −4.3 ± 0.1 | −4.0 ± 0.4 |
| 12 | −3.4 ± 0.8 | −4.1 ± 0.1 | −4.5 ± 0.1 | −3.8 ± 0.8 | −4.2 ± 0.1 | −3.5 ± 1.0 |
| 24 | −3.8 ± 0.5 | −4.1 ± 0.1 | −4.1 ± 0.4 | −3.1 ± 0.3 | −2.6 ± 1.5 | −2.3 ± 0.7 |
| 36 | −4.3 ± 0.1 | −3.6 ± 0.1 | −4.4 ± 0.2 | −4.2 ± 0.2 | −3.8 ± 0.2 | −2.4 ± 1.0 |
| 48 | −4.3 ± 0.1 | −4.1 ± 0.1 | −4.3 ± 0.2 | −3.2 ± 1.0 | −3.8 ± 0.2 | −2.8 ± 0.9 |
| Maximum | −4.3 ± 0.1 | −4.1 ± 0.1 | −4.5 ± 0.1 | −4.3 ± 0.1 | −4.3 ± 0.1 | −4.2 ± 0.1 |
| T99 (h) | 1 | 2 | 2 | 2 | 4 | 3 |
| T99.9 (h) | 3 | 3 | 5 | 4 | 7 | 4 |
(ii) Five-day simulations against MRSA.
Three MRSA strains, with razupenem MICs of 0.38 μg/ml (strain 36895), 1.5 μg/ml (strain 22706), and 3.0 μg/ml (strain 33820), were selected for 5-day simulations. In no case were MRSA strains eradicated from the model, and for all three strains there was regrowth, with viable counts of >3 log CFU/ml occurring after 48 h. This was less for strain 27706 than for strains 36895 and 33820 (Fig. 1). Comparisons of the population analysis profiles of the three strains indicated that subpopulations able to grow on 2× MIC plates emerged at 120 h with strain 27706 (MIC, 1.5 μg/ml; viable count, 3.0 log CFU/ml) and at 72 h with strain 33820 (MIC, 3.0 μg/ml). The viable counts on 2× MIC plates at 72 h were 4.1 ± 0.7 log CFU/ml, and those at 120 h were 5.2 ± 0.1 log CFU/ml.
FIG. 1.
Antibacterial effect of razupenem against MRSA strains 36895 (MIC, 0.38 mg/liter), 27706 (MIC, 1.5 mg/liter), and 33820 (MIC, 3.0 mg/liter) over 5 days.
Dose-ranging studies with S. aureus.
A range of doses (n = 7 to 13) was used to provide an fT>MIC of 0 to 100% for each of the six S. aureus strains (one MSSA strain and five MRSA strains). Antibacterial effect was measured by d24, d48, AUBKC24, and AUBKC48. Using d24 as the primary outcome measure, the fT>MIC for a 24-h static effect was 5.0% ± 1.4% and that for a 1.0-log-unit reduction in count was 12.5% ± 5.8%; this increased to 36.6% ± 20.1% for a 3.0-log-unit reduction in count (Table 2). The fT>MIC could also be related to d48; the value associated with a static effect over 48 h was 9.0fT>MIC ± 2.8%, and that associated with a 3-log-unit reduction in count was 32.6% ± 16.3% (Table 2). An fT>MIC of 30 to 40% was associated with an almost-maximal antibacterial effect as measured by AUBKC24 or AUBKC48 (data not shown). Changes in population profiles of the S. aureus strains were related to razupenem exposure (Table 3). The proportion of experiments where a subpopulation was able to grow on 2× MIC recovery plates was >80% with an fT>MIC of 0.5 to 5.0% but zero with an fT>MIC of ≥35%. The number of bacteria (log CFU/ml) recovered on 2× MIC media was also related to the fT>MIC. After 48 h of razupenem exposure, colonies were recovered from media containing 4×MIC and 8×MIC. Again, the proportion of experiments and the absolute numbers of resistant colonies were related to fT>MIC.
TABLE 2.
Razupenem T>MIC targets required for a bacteriostatic effect and for 1-, 2-, and 3-log-unit reductions in S. aureus viable counts at 24 h and 48 h
| Time (h) | Effect |
T>MIC (%) for strain (MIC, mg/liter): |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| MSSA 5956 (0.015) | MRSA 33922 (0.09) | MRSA 36895 (0.38) | VISA 19898 (1.5) | MRSA 27706 (1.5) | MRSA 33820 (3.0) | MRSA, avg ± SD | MRSA, pooled analysis | ||
| 24 | Static | 23.6 | 6.7 | 4.5 | 3.9 | 6.4 | 3.6 | 5.0 ± 1.4 | 4.0 |
| 1.0-log-unit reduction | 31.5 | 13.4 | 18.4 | 7.3 | 17.7 | 5.7 | 12.5 ± 5.8 | 9.0 | |
| 2.0-log-unit reduction | 40.9 | 23.5 | 31.9 | 11.8 | 33.3 | 9.2 | 21.9 ± 11.1 | 16.1 | |
| 3.0-log-unit reduction | 53.0 | 40.9 | 18.9 | 59.1 | 36.6 ± 20.1 | 31.4 | |||
| r2 | 0.685 | 0.881 | 0.910 | 0.958 | 0.807 | 0.882 | 0.800 | ||
| 48 | Static | 31.5 | 12.8 | 9.7 | 10.3 | 6.4 | 6.0 | 9.0 ± 2.8 | 7.8 |
| 1.0-log-unit reduction | 35.9 | 23.5 | 16.8 | 13.7 | 11.8 | 7.6 | 14.7 ± 5.9 | 14.1 | |
| 2.0-log-unit reduction | 39.6 | 36.2 | 25.2 | 17.2 | 19.5 | 9.2 | 21.5 ± 10.4 | 22.1 | |
| 3.0-log-unit reduction | 45.0 | 55.0 | 38.0 | 22.3 | 35.5 | 12.2 | 32.6 ± 16.3 | 34.9 | |
| r2 | 0.972 | 0.984 | 0.963 | 0.966 | 0.8639 | 0.895 | 0.881 | ||
TABLE 3.
Changes in S. aureus razupenem population profiles at 24 h and 48 h for MRSA strains
| Time (h) | fT>MIC range (%) | Growth on: |
|||||
|---|---|---|---|---|---|---|---|
| 2× MIC recovery plates |
4× MIC recovery plates |
8× MIC recovery plates |
|||||
| No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | ||
| 24 | 0.5-2.5 | 5/6 (83) | 4.3 ± 0.8 | 0/6 | <2 | 0/6 | <2 |
| >2.5-5.0 | 5/6 (83) | 4.8 ± 0.8 | 0/6 | <2 | 0/6 | <2 | |
| >5-10 | 3/5 (60) | 4.1 ± 0.6 | 0/5 | <2 | 0/5 | <2 | |
| >10-15 | 2/6 (33) | 3.4 | 0/6 | <2 | 0/6 | <2 | |
| >15-35 | 3/7 (43) | 3.2 ± 0.2 | 0/7 | <2 | 0/7 | <2 | |
| >35-70 | 0/9 | <2 | 0/9 | <2 | 0/9 | <2 | |
| >70 | 0/6 | <2 | 0/6 | <2 | 0/6 | <2 | |
| 48 | 0.5-2.5 | 6/6 (100) | 6.2 ± 1.3 | 3/6 (50) | 5.2 ± 1.4 | 1/6 (17) | 2.4 |
| >2.5-5.0 | 5/6 (83) | 7.1 ± 0.4 | 3/6 (50) | 5.3 ± 2.0 | 1/6 (17) | 6.5 | |
| >5-10 | 5/5 (100) | 5.0 ± 2.0 | 2/5 (40) | 3.8 | 0/6 | <2 | |
| >10-15 | 3/6 (50) | 4.4 ± 1.7 | 3/6 (50) | 4.3 ± 1.7 | 1/6 (17) | 2.4 | |
| >15-35 | 3/7 (43) | 3.7 ± 1.0 | 3/7 (43) | 3.7 ± 1.2 | 0/6 | <2 | |
| >35-70 | 1/9 (11) | 2.1 | 0/9 | <2 | 0/9 | <2 | |
| >70 | 0/6 | <2 | 0/6 | <2 | 0/9 | <2 | |
Effect against Enterobacteriaceae in 48-h simulations.
Administration of 1 g razupenem every 12 h by 1-h infusion was simulated and its effect on five strains of E. coli and Enterobacter spp. assessed (Table 4). Against the three E. coli strains, two of which (strains 31053 and 35776) were ESBL producers, razupenem was rapidly bactericidal, producing a >4-log-unit reduction in viable counts by 24 h. With strain 31053, there was some regrowth after 36 h, but not with the other two strains. Against Enterobacter sp. strain 34425 (MIC, 6.0 μg/ml), razupenem had a markedly poorer effect than against Enterobacter sp. strain 35054 (MIC 0.4 μg/ml), with a 0.1 ± 0.2-log10-unit reduction in viable count at 24 h and regrowth at 36 h and 48 h (Table 4). There was no change in population profiles with these simulations except for Enterobacter sp. strain 34425 (MIC, 6.0 μg/ml), where the bacterial population on 2× MIC, 4× MIC, and 8×MIC plates increased from undetectable at 0 h to 4.6 ± 0.1, 4.0 ± 0.1, and 3.7 ± 0.2 log CFU/ml at 24 h and 5.0 ± 0.1, 3.3 ± 0.2, and <2 log CFU/ml at 48 h, respectively.
TABLE 4.
Antibacterial effect of simulations of razupenem at 1 g given as a 1-h infusion against Enterobacteriaceae
| Time (h) | Log change in viable count (mean ± SD) for strain (MIC, mg/liter): |
||||
|---|---|---|---|---|---|
| E. coli 31053 (SHV-12 ESBL) (0.09) | E. coli 39136 (ampicillin sensitive) (0.1) | E. coli 35776 (CTX-M ESBL) (0.12) | Enterobacter sp. strain 35054 (AmpC) (0.4) | Enterobacter sp. strain 34425 (6) | |
| 6 | −4.2 ± 0.1 | −4.4 ± 0.1 | −4.3 ± 0.1 | −4.2 ± 0.1 | −4.0 ± 0.3 |
| 12 | −4.2 ± 0.1 | −3.4 ± 0.5 | −4.3 ± 0.1 | −3.8 ± 0.4 | −1.5 ± 0.4 |
| 24 | −4.2 ± 0.1 | −4.4 ± 0.1 | −4.3 ± 0.1 | −3.7 ± 0.7 | −0.1 ± 0.2 |
| 36 | −4.2 ± 0.3 | −4.4 ± 0.1 | −4.3 ± 0.1 | −3.4 ± 0.2 | +0.2 ± 0.2 |
| 48 | −1.4 ± 1.7 | −4.4 ± 0.1 | −4.3 ± 0.1 | −3.0 ± 0.3 | +0.2 ± 0.1 |
| Maximum | −4.2 ± 0.1 | −4.4 ± 0.1 | −4.3 ± 0.1 | −4.2 ± 0.1 | −4.3 ± 0.2 |
| T99 (h) | 1 | 1 | 1 | 1 | 1 |
| T99.9 (h) | 2 | 1 | 1 | 2 | 2 |
Dose-ranging studies with Enterobacteriaceae.
A range of doses were simulated (n = 12 to 16 per strain) to provide fT>MIC ranges from 0 to 100% for each of the five Enterobacteriaceae tested. The antibacterial effect was measured by d24, d48, AUBKC24, and AUBKC48. Using d24 as the primary antibacterial effect measure, the fT>MIC for a 24-h static effect was 34.3% ± 7.6%, that for a 1-log-unit reduction in count was 42.5% ± 7.8%, and that for a 3-log-unit reduction in count was 61.3% ± 9.2% (Table 5). The fT>MIC could also be related to d48; the value associated with a static effect over 48 h was 42.2% ± 5.3%, and that associated with a 3-log-unit reduction in count was 61.2% ± 13.7% (Table 5). An fT>MIC of >70 to 80% was associated with an almost-maximal antibacterial effect as measured by AUBKC24 or AUBKC48 (data not shown). Changes in the population profiles of E. coli and Enterobacter sp. strains were related to razupenem exposure over 24 h or 48 h (Table 6). At 24 h the proportion of simulations in which bacteria were able to grow on 2×, 4×, or 8× MIC recovery plates was related to fT>MIC, with simulations with an fT>MIC of 1 to 69% being more likely to produce changes in population profiles than those with an fT>MIC of ≥70%.
TABLE 5.
Razupenem T>MIC targets required for a bacteriostatic effect and for 1-, 2-, and 3-log-unit reductions in Enterobacteriaceae viable counts at 24 h and 48 h
| Time (h) | Effect |
T>MIC (%) for strain (MIC, mg/liter): |
||||||
|---|---|---|---|---|---|---|---|---|
| E. coli 31053 (SHV-12+) (0.09) | E. coli 35776 (CTXM+) (0.1) | E. coli 39136 (Amps) (1.2) | Enterobacter sp. strain 35054 (0.4) | Enterobacter sp. strain 34425 (6) | Avg ± SD | Pooled analysis | ||
| 24 | Static | 31.7 | 47.0 | 35.0 | 27.5 | 30.2 | 34.3 ± 7.6 | 32.4 |
| 1.0-log-unit reduction | 40.9 | 55.5 | 40.9 | 34.2 | 40.9 | 42.5 ± 7.8 | 41.5 | |
| 2.0-log-unit reduction | 49.7 | 64.1 | 46.7 | 42.3 | 51.3 | 50.6 ± 8.2 | 50.5 | |
| 3.0-log-unit reduction | 61.7 | 74.0 | 52.6 | 52.3 | 65.8 | 61.3 ± 9.2 | 61.5 | |
| 4.0-log-unit reduction | 92.6 | 89.4 | 63.6 | 87.9 | 83.4 ± 13.3 | 83.9 | ||
| r2 | 0.981 | 0.975 | 0.963 | 0.957 | 0.943 | 0.929 | ||
| 48 | Static | 39.8 | 50.6 | 44.8 | 37.6 | 39.3 | 42.2 ± 5.3 | 38.5 |
| 1.0-log-unit reduction | 40.3 | 61.2 | 47.4 | 39.6 | 43.5 | 46.4 ± 8.8 | 45.2 | |
| 2.0-log-unit reduction | 40.9 | 71.0 | 50.6 | 41.6 | 47.7 | 50.4 ± 12.2 | 52.5 | |
| 3.0-log-unit reduction | 81.6 | 52.6 | 57.0 | 53.6 | 61.2 ± 13.7 | 63.9 | ||
| 4.0-log-unit reduction | 95.4 | 58.4 | 76.9 | |||||
| r2 | 0.829 | 0.949 | 0.960 | 0.944 | 0.803 | 0.853 | ||
TABLE 6.
Changes in Enterobacteriaceae razupenem population profiles at 24 h and 48 h
| Time (h) | T>MIC range (%) | Growth on: |
|||||
|---|---|---|---|---|---|---|---|
| 2× MIC recovery plates |
4× MIC recovery plates |
8× MIC recovery plates |
|||||
| No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | No. (%) of expts with >2-log-unit growth/total | Viable count (log CFU/ml), mean ± SD | ||
| 24 | 1-9 | 4/4 (100) | 8.1 ± 0.1 | 4/4 (100) | 7.3 ± 1.1 | 4/4 (100) | 5.6 ± 2.4 |
| 10-19 | 7/8 (87) | 7.3 ± 1.8 | 6/8 (75) | 7.0 ± 1.4 | 6/8 (75) | 6.0 ± 2.2 | |
| 20-29 | 7/7 (100) | 5.4 ± 2.6 | 3/7 (43) | 7.9 ± 0.1 | 3/7 (43) | 7.9 ± 0.1 | |
| 30-39 | 3/4 (75) | 6.4 ± 1.6 | 2/4 (50) | 5.9 | 2/4 (50) | 5.8 | |
| 40-49 | 5/9 (55) | 4.2 ± 1.1 | 3/9 (33) | 3.8 ± 1.3 | 3/9 (33) | 3.5 ± 0.9 | |
| 50-69 | 6/11 (54) | 3.8 ± 1.0 | 4/11 (36) | 4.2 ± 0.8 | 4/11 (36) | 3.2 ± 1.0 | |
| ≥70 | 3/14 (21) | 2.7 | 0/14 | <2 | 0/14 | <2 | |
| 48 | 1-9 | 4/4 (100) | 8.2 ± 0.1 | 4/4 (100) | 7.5 ± 1.3 | 4/4 (100) | 6.5 ± 1.6 |
| 10-19 | 8/8 (100) | 7.6 ± 1.5 | 7/8 (87) | 7.7 ± 0.9 | 6/8 (75) | 7.1 ± 1.4 | |
| 20-29 | 7/7 (100) | 7.0 ± 2.2 | 6/7 (86) | 6.5 ± 2.6 | 5/7 (71) | 7.2 ± 2.1 | |
| 30-39 | 4/4 (100) | 7.2 ± 1.9 | 4/4 (100) | 5.7 ± 2.9 | 3/4 (75) | 6.8 ± 2.3 | |
| 40-49 | 8/9 (89) | 4.0 ± 1.3 | 4/9 (44) | 3.7 ± 1.0 | 3/9 (33) | 3.6 ± 0.9 | |
| 50-69 | 8/11 (73) | 3.9 ± 1.5 | 4/11 (36) | 5.2 ± 0.2 | 4/11 (36) | 4.8 ± 0.1 | |
| ≥70 | 5/14 (36) | 2.9 ± 0.4 | 2/14 (14) | 2.9 ± 0.1 | 2/14 (14) | 2.3 ± 0.2 | |
A similar relationship between fT>MIC and population profiles was observed at 48 h, with the main differences being that a greater proportion of simulations produced colonies on antibiotic-containing media and that the absolute number of CFU/ml was greater.
DISCUSSION
The pharmacodynamics of carbapenems have been extensively studied in preclinical models over the last 15 years. The dominant pharmacodynamic index is the percentage of the dosing interval that antibiotic concentrations remain higher than the pathogen MIC (T>MIC) (8). The size of the T>MIC for a 24-h bacteriostatic effect varies between bacterial species, being 20 to 40% for Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. (1, 15, 17, 19), but also depends on the bacterial strain being tested (13). The T>MIC for bactericidal effects are larger; a T>MIC of 30 to 55% is required for a 2-log-unit reduction in viable bacterial counts (15, 17), and the maximum effect for some strains may require a T>MIC of >60% (14, 15). The relationships between carbapenem T>MICs and antibacterial effect are less clear for S. aureus, including MRSA strains. The T>MICs for four strains of S. aureus (MSSA and MRSA) were 25 to 35% for a static effect and 31 to 50% for a 2-log-unit kill (17). In contrast, lower T>MICs have been reported when the anti-MRSA carbapenem tomopenem (formerly RO4908463/CS-023) was tested against six S. aureus strains, five of which were MRSA. T>MIC values of 2 to 13% were associated with a static effect, 6 to 25% with a 2-log-unit drop in bacterial count, and 9 to 55% with a maximum 4-log-unit reduction in count (12). For razupenem the T>MIC associated with a bacteriostatic effect has been reported as 23% and that for a 2-log-unit kill as 32 to 37%, based on two MRSA strains tested in a neutropenic murine thigh infection model (9). Using the same murine model, Andes and Craig (1) reported a 24-h bacteriostatic effect with a T>MIC of 2 to 13% and 2-log-unit reductions in count with a T>MIC in the range of 16 to 25%. Our data are more in keeping with the findings of Andes and Craig (1), with the MRSA strains tested having a 24-h bacteriostatic effect T>MIC target of 3.6 to 6.7% and with a T>MIC of 9.2 to 33.3% being required for a 2-log-unit reduction in count at 24 h. The single MSSA strain that we tested had larger T>MIC values for these antibacterial effects; it is not clear if this represents a true difference between MSSA and MRSA or, more likely, strain-to-strain variation in the pharmacodynamic index as has been described with other drug classes (13).
The T>MIC values for the Enterobacteriaceae strains we tested were 27.5 to 47% for a bacteriostatic effect at 24 h, 42.3 to 64.1% for a 2-log-unit reduction in count, and 63.6 to 92.6% for a maximum, 4-log-unit reduction in count; these are in keeping with the published literature on carbapenems.
Given these relationships between T>MIC and antibacterial effect, it is unsurprising that razupenem at dose simulations of 1 g every 12 h i.v. is highly effective against S. aureus strains, as the T>MICs are ≥44%. S. aureus strains with MICs of 3.0 μg/ml (T>MIC of 44% with simulations of 1 g every 12 h) are rare, with the upper limit of the MIC range for MRSA being reported at 2 or 4 μg/ml (16, 18). Similarly, razupenem at dosing simulations of 1 g every 12 h was also highly effective against the Enterobacteriaceae strains with MICs of ≤0.4 μg/ml (T>MIC, ≥75%) but was notably less active against the strain with an MIC of 6 μg/ml (T>MIC, 33%). The MIC90 of Enterobacter cloacae is 8 μg/ml and that of Serratia marcescens is 16 μg/ml, indicating that not all the stains of these species may be treated by razupenem at 1 g every 12 h.
Changes in population profiles of both Gram-negative organisms and S. aureus have been reported following exposure to a variety of antibiotics in in vitro models (4, 11, 12). In the 48-h exposure experiments that we performed with the simulations of 1 g every 12 h and S. aureus, no changes in population profiles were noted. When the same dose simulations were performed over 5 days, the MRSA strains with the higher MICs (1.5 and 3.0 μg/ml) were recovered on 2× MIC-containing media after 120 and 72 h, respectively. With the Enterobacteriaceae exposed to 1 g razupenem every 12 h over 48 h, no changes in the population profiles were noted, except with the Enterobacter sp. strain with a MIC of 6 μg/ml, where new growth on 2× MIC, 4× MIC, and 8× MIC recovery media was noted at 24 h and 48 h.
We have previously reported changes in the population profiles of S. aureus exposed to tomopenem, daptomycin, and telavancin (4, 12). With each of these agents the size of the pharmacodynamic index associated with the greatest change in the bacterial population profile was below the 24-h bacteriostatic effect target. However, with all three antibiotics the size of the pharmacodynamic index associated with a 24-h static effect was also associated with a significant risk of changes in the bacterial population analysis profile. The same is also true of razupenem and S. aureus, with growth on 2× MIC recovery plates occurring at T>MIC values of ≤35%, that is, values below the 3-log-unit reduction in count T>MIC of 36%. A similar situation was noted with Enterobacteriaceae, with growth on 2× MIC, 4× MIC, and 8× MIC recovery media after 24 h in more than 30% of experimental simulations when the T>MIC was ≤69%. If the T>MIC was >70%, the risk of change in the population profile was much less, and such T>MIC values are associated with 3- to 4-log-unit reductions in viable count at 24 h. As we have previously observed, the length of time that the bacterial population is exposed to antibiotic as well as the size of the pharmacodynamic index determined the amount of change that occurs in population profiles (11). Again, with razupenem, changes in MRSA and Enterobacteriaceae population profiles occurred to a greater extent at 48 h than at 24 h of drug exposure. In addition, the T>MIC sizes required to produce a bacteriostatic effect at 48 h were generally greater than those required at 24 h (4).
Preliminary Monte Carlo simulations of razupenem based on pharmacokinetics in healthy volunteers have indicated >90% target attainment rates for bacterial strains with MICs of ≤ 2μg/ml using a T>MIC target of 30%, ≤4 μg/ml with a T>MIC target of 25%, and ≤8 μg/ml with a T>MIC target of 18% (3). It is usual practice to use bacteriostatic to 1-log-unit reduction in bacterial count pharmacodynamic index sizes to set antibacterial clinical breakpoints. Accordingly, a suitable T>MIC target to treat MRSA would be 5 to 20%, implying that all MRSA strains should be treated effectively by 1 g razupenem every 12 h, as MRSA strains have MICs of up to 2 to 4 μg/ml. As T>MIC targets for Enterobacteriaceae are larger, that is, 25 to 55%, and some species of Enterobacteriaceae have higher MICs than S. aureus, different dosing regimens will be required. However, as E. coli, Klebsiella spp., and Proteus mirabilis have razupenem MIC50 values 4- to 8-fold lower than those for MRSA, this compensates for the higher T>MIC targets and suggests that those species could be treated with 1 g razupenem by 1-h infusion every 12 h (18); our simulation data of this dose support this conclusion.
Finally, these preclinical pharmacodynamic data show that razupenem at a dose of 1 g every 12 h can be used to treat MRSA strains. A T>MIC target of 35% would render most S. aureus strains susceptible to razupenem and reduce the risk of emergence of resistance. It is likely that the same razupenem dose could be used to treat E. coli, Klebsiella sp., and P. mirabilis infections. In all situations, short-course therapy is less likely to lead to changes in the pathogen population profile and the emergence of resistance than longer courses.
Acknowledgments
We thank Luigi Xerri, Chief Scientific Officer of Protez Pharmaceuticals, for helpful advice and discussions during these studies.
This project was funded by Protez Pharmaceuticals, Malvern, PA.
Footnotes
Published ahead of print on 24 January 2011.
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