In Vivo Comparison of CXA-101 (FR264205) with and without Tazobactam versus Piperacillin-Tazobactam Using Human Simulated Exposures against Phenotypically Diverse Gram-Negative Organisms

Abstract

CXA-101 is a novel antipseudomonal cephalosporin with enhanced activity against Gram-negative organisms displaying various resistance mechanisms. This study evaluates the efficacy of exposures approximating human percent free time above the MIC (%fT > MIC) of CXA-101 with or without tazobactam and piperacillin-tazobactam (TZP) against target Gram-negative organisms, including those expressing extended-spectrum β-lactamases (ESBLs). Sixteen clinical Gram-negative isolates (6 Pseudomonas aeruginosa isolates [piperacillin-tazobactam MIC range, 8 to 64 μg/ml], 4 Escherichia coli isolates (2 ESBL and 2 non-ESBL expressing), and 4 Klebsiella pneumoniae isolates (3 ESBL and 1 non-ESBL expressing) were used in an immunocompetent murine thigh infection model. After infection, groups of mice were administered doses of CXA-101 with or without tazobactam (2:1) designed to approximate the %fT > MIC observed in humans given 1 g of CXA-101 with or without tazobactam every 8 h as a 1-h infusion. As a comparison, groups of mice were administered piperacillin-tazobactam doses designed to approximate the %fT > MIC observed in humans given 4.5 g piperacillin-tazobactam every 6 h as a 30-min infusion. Predicted piperacillin-tazobactam %fT > MIC exposures of greater than 40% resulted in static to >1 log decreases in CFU in non-ESBL-expressing organisms with MICs of ≤32 μg/ml after 24 h of therapy. Predicted CXA-101 with or without tazobactam %fT > MIC exposures of ≥37.5% resulted in 1- to 3-log-unit decreases in CFU in non-ESBL-expressing organisms, with MICs of ≤16 μg/ml after 24 h of therapy. With regard to the ESBL-expressing organisms, the inhibitor combinations showed enhanced CFU decreases versus CXA-101 alone. Due to enhanced in vitro potency and resultant increased in vivo exposure, CXA-101 produced statistically significant reductions in CFU in 9 isolates compared with piperacillin-tazobactam. The addition of tazobactam to CXA-101 produced significant reductions in CFU for 7 isolates compared with piperacillin-tazobactam. Overall, human simulated exposures of CXA-101 with or without tazobactam demonstrated improved efficacy versus piperacillin-tazobactam.

INTRODUCTION

With the steadily increasing worldwide rise of multidrug-resistant Gram-negative organisms, the growing need for new treatment options has become ever more apparent (7, 10, 17). Multidrug resistance in Pseudomonas aeruginosa has negated the effectiveness of the majority of agents and may be mediated by the development of resistance either through the selection of a mutant or through the transfer of resistance determinants from other pathogens (16, 21). For other Gram-negative organisms, such as Klebsiella pneumoniae and Escherichia coli, drug resistance can often be attributed to the expression of extended-spectrum β-lactamases (ESBLs) (19).

Piperacillin-tazobactam is a frequently used β-lactam/β-lactamase inhibitor combination due to its broad spectrum of activity, and while P. aeruginosa retains high rates of susceptibility to piperacillin-tazobactam, reductions in its in vitro potency against many Gram-negative organisms have been noted recently. Unfortunately, in recent surveillance, piperacillin-tazobactam has experienced reduced susceptibility rates for P. aeruginosa, with 16% MICs of 64 μg/ml in almost all of the hospitals examined (8). Moreover, piperacillin-tazobactam, along with many other antibiotics, has demonstrated increasing rates of resistance in Enterobacteriaceae from 2002 to 2007 (10). In an effort to maximize outcomes when considering these Gram-negative pathogens, piperacillin-tazobactam is generally utilized at a dosage of 4.5 g every 6 h (q6h) (2).

CXA-101, formally known as FR264205, is a novel parenteral antipseudomonal cephalosporin that shares a similar spectrum of activity with ceftazidime. It has recently been shown to have in vitro activity against multidrug-resistant Gram-negative organisms, especially when β-lactamase inhibitors were added to this agent (11, 5). It demonstrates increased stability against AmpC β-lactamase-producing organisms and, through increased binding to penicillin-binding protein 3 (PBP3), has been shown to have activity against organisms with porin deficiencies and efflux mechanisms (23). Here, we compared the in vivo bactericidal efficacy of CXA-101 with or without tazobactam to that of piperacillin-tazobactam through the use of human simulated exposures in an immunocompetent murine thigh infection model with multidrug-resistant P. aeruginosa and ESBL-positive and -negative E. coli and K. pneumoniae.

MATERIALS AND METHODS

Antimicrobial test agent.

Analytical-grade CXA-101 and tazobactam (Cubist Pharmaceuticals, Lexington, MA) were utilized for all in vitro and in vivo studies. Based on the sponsor's supplied potency, CXA-101–tazobactam powder was weighed in a quantity sufficient to achieve the required concentration and reconstituted immediately prior to use. All CXA-101–tazobactam solutions were made in a ratio of 2:1. Analytical-grade piperacillin and tazobactam were used for all in vitro studies (Wyeth Pharmaceuticals, Inc., Philadelphia, PA), while commercially available piperacillin-tazobactam (8:1; Wyeth Pharmaceuticals, Inc., Philadelphia, PA; lot E01394; expiration date, August 2012) was used for all in vivo studies. Each vial was reconstituted immediately prior to use with 10 ml of normal saline. The resultant piperacillin-tazobactam solution was further diluted with normal saline to achieve the required concentration. All CXA-101, CXA-101–tazobactam, and piperacillin-tazobactam solutions were stored refrigerated, protected from light, and discarded after 24 h.

Bacterial isolates.

A total of 16 clinical Gram-negative isolates were used in this analysis; 6 P. aeruginosa isolates (piperacillin-tazobactam MIC range, 8 to 64 μg/ml); 4 E. coli isolates, 2 ESBL positive and 2 ESBL negative; and 4 K. pneumoniae isolates, 3 ESBL positive and 1 ESBL negative. The MIC of each isolate for CXA-101, CXA-101–tazobactam, and piperacillin-tazobactam was determined in triplicate by broth microdilution, using methods outlined by the Clinical and Laboratory Standards Institute (6), and the modal MIC was reported.

Animal infection model.

Pathogen-free female ICR mice weighing approximately 18 to 20 g were acquired from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and utilized throughout these experiments. The study was reviewed and approved by the Hartford Hospital Institutional Animal Care and Use Committee. The animals were maintained and used in accordance with National Research Council recommendations and provided food and water ad libitum. Two hours prior to the initiation of antimicrobial therapy, each thigh was inoculated intramuscularly with a 0.1-ml solution containing approximately 10⁸ CFU/ml of the test isolate. In this murine model, there is an apparent interaction of decreased elimination between the β-lactam and β-lactamase inhibitor components. As a result, we decreased the dose of CXA-101 when it was in combination with tazobactam relative to CXA-101 alone in order to adjust for this interaction.

Pharmacokinetic studies and dosing regimen determination.

Preliminary pharmacokinetic studies were undertaken in immunocompetent infected mice given single doses of CXA-101, CXA-101–tazobactam, and piperacillin-tazobactam subcutaneously 2 h after inoculation. Blood samples were collected by intracardiac puncture from groups of 6 mice per time point for a total of 8 time points per dose over 8 h. Serum or plasma samples were separated after centrifugation and stored at −80°C until analysis.

Concentrations of CXA-101 with or without tazobactam in murine plasma were determined by MicroConstants Inc., San Diego, CA, using liquid chromatography-tandem mass spectrometry (LC–MS-MS). The assay was linear over a range of 0.1 to 50 μg/ml for CXA-101 and from 0.02 to 10 μg/ml for tazobactam. Interday coefficients of variation for the low (0.3-μg/ml) and high (40-μg/ml) quality control samples for CXA-101 were 0.07% and 0.026%, respectively. Interday coefficients of variation for the low (0.06-μg/ml) and high (8-μg/ml) quality control samples for tazobactam were 0.08% and 0.1%, respectively.

Concentrations of piperacillin-tazobactam in murine serum were determined using a previously validated high-performance liquid chromatography assay (12). The assay was linear over a range of 2 to 100 μg/ml (R² = 0.996) for piperacillin and 1 to 50 μg/ml (R² = 0.995) for tazobactam. The intraday coefficients of variation for the low (6-μg/ml) and high (80-μg/ml) quality control samples for piperacillin were 4.76% and 1.95%, respectively. The intraday coefficients of variation for the low (3-μg/ml) and high (40-μg/ml) quality control samples for tazobactam were 3.74% and 2.68%, respectively. The interday coefficients of variation for the quality control samples for piperacillin were 5.64% and 4.75%, respectively. The interday coefficients of variation for the quality control samples for tazobactam were 6.92% and 5.40%, respectively.

Pharmacokinetic parameters for single doses of CXA-101 were calculated, using first-order elimination, by nonlinear least-squares techniques (WinNonlin version 5.2; Pharsight, Mountain View, CA). Compartment model selection was based on visual inspection of the fit and correlation between the observed and calculated concentrations by Akaike's information criterion. Mean pharmacokinetic parameters were calculated from the individual parameter estimates and applied to WinNonlin in order to simulate various CXA-101 exposures expressed as free time above the MIC (fT > MIC) over a wide range of MICs. Dosing regimens in mice were designed to approximate %fT > MIC observed in humans following CXA-101 at 1,000 mg every 8 h given as a 1-h infusion. Human concentration-time profiles were derived from healthy human volunteers, and protein binding was estimated to be 20% in humans and 9% in mice (data on file at Calixa Therapeutics Inc., San Diego, CA). The resulting regimen for 1-h infusions of CXA-101 involved 6 doses of 80, 40, 50, 30, 12.5, and 7.5 mg/kg of body weight administered at 0, 0.25, 2, 4, 6, and 7.25 h every 8 h over 24 h.

This procedure was repeated for CXA-101–tazobactam, simulating the % fT > MIC observed in humans administered CXA-101–tazobactam (2:1) at 1,000/500 mg every 8 h given as a 1-h infusion. Due to the fact that this combination entailed a fixed ratio of tazobactam, only the non-tazobactam β-lactam component was modeled to simulate human exposures because the fixed ratio allowed the tazobactam to follow the CXA-101 pharmacokinetic profile, as one would observe in humans. Additionally, due to the observation of a significant drug interaction between the β-lactam and the β-lactam inhibitor, the resulting dosing regimen of the combination was adjusted to account for this interaction. As the concentration of β-lactam inhibitor can affect the activity of the β-lactam, we made sure to match the tazobactam exposures (see Table 3). The resulting regimen for the 1-h infusions of CXA-101–tazobactam involved 5 doses of 30/15, 15/7.5, 15/7.5, 6.25/3.125, and 3.125/1.5625 mg/kg of body weight administered at 0, 2, 4, 5.5, and 7 h every 8 h over 24 h. The pharmacokinetic studies were repeated multiple times to confirm exposures, including a final pharmacokinetic study in which CXA-101–tazobactam was dosed to steady state before samples were taken.

Table 3.

Comparison of the tazobactam component % fT > MIC values achieved with TZP and CXAT at each potential tazobactam MIC observed in humans and in mice receiving humanized exposures

Druga	Speciesb	% fT > MIC for a MIC (μg/ml) of:
		0.5	1	2	4
TZP	H	98.3	83.3	66.7	51.7
	M	86.7	68.3	56.7	45.0
CXAT	H	52.5	42.5	32.5	23.8
	M	63.8	51.3	36.3	22.5

^aTZP, 4.5 g q6h, 30-min infusion; CXAT, 2:1, 1,000/500 mg q8h, 1-h infusion.

^bH, human; M, mouse.

Finally, the piperacillin-tazobactam % fT > MIC values observed in humans administered 4.5 g every 6 h as a 30-min infusion were simulated using concentration-time profiles derived from 12 healthy human volunteers (15). A protein binding value of 20% was used for humans and mice (3, 13, 20). Once again, because this combination also entailed a fixed ratio of tazobactam (8:1), only the non-tazobactam β-lactam component was modeled to simulate human exposures because the fixed ratio allowed the tazobactam to follow the piperacillin concentration, as one would observe in human dosing regimens. Again, due to the observation of a significant drug interaction between the β-lactam and the β-lactam inhibitor, the resulting dosing regimen of the combination was adjusted to account for this interaction. The resulting dosing regimen for the 30-min infusion involved 4 doses of 500/62.5, 100/12.5, 200/25, and 75/9.375 mg/kg administered at 0, 0.25, 2.5, and 5 h every 6 h over 24 h. Prior to the efficacy studies, confirmation of the exposures attained by these 3 simulated dosing regimens was performed in separate pharmacokinetic experiments in infected mice.

Efficacy as assessed by bacterial density.

Two hours after infection, CXA-101 1-h infusion, CXA-101-tazobactam (2:1) 1-h infusion, and piperacillin-tazobactam 30-min infusion regimens were administered as subcutaneous 0.2-ml injections and studied for each isolate in groups of three mice over a 24-h period. All 16 isolates were studied with all three regimens. Control animals received sterile normal saline with the same volume, route, and schedule as the active-drug regimens. Untreated control mice (three per group) were sacrificed just prior to antibiotic initiation (0 h) and after 24 h. After the 24-h treatment period, all animals were euthanized by CO₂ exposure, followed by cervical dislocation. After sacrifice, the thighs were removed and individually homogenized in normal saline. Serial dilutions of the thigh homogenate were plated on Trypticase soy agar with 5% sheep blood for CFU determination. The efficacy, designated as the change in bacterial density, was calculated as the log₁₀ change in bacterial CFU/ml obtained for antibiotic-treated mice after 24 h from the preantibiotic CFU/ml measured for 0-h control animals. In an effort to confirm the intrarun observed efficacy for several “drug-bug” combinations, KP 51 and KP 393 were studied during two independent periods, while isolates EC 45 and EC 114 were assessed independently three times. As the quantitative efficacies were similar during each of these assessments, the data were averaged for the purposes of the analysis. Differences in efficacy between the antimicrobial regimens were assessed with a one-way analysis of variance (ANOVA) and Tukey test when appropriate using SigmaStat version 2.03 (SPSS Inc., Chicago, IL). A P value of less than 0.05 was considered significant for the statistical analysis.

RESULTS

In vitro susceptibility.

The CXA-101, CXA-101–tazobactam, and piperacillin-tazobactam MICs for all P. aeruginosa, K. pneumoniae, and E. coli isolates are shown in Table 1. The MIC ranges for the 16 organisms were 1 to 64 μg/ml for piperacillin-tazobactam, 0.25 to >64 μg/ml for CXA-101, and 0.125 to >64 μg/ml for CXA-101–tazobactam. On average, the CXA-101 and CXA-101–tazobactam MICs were between 2- and 6-fold lower than that for piperacillin-tazobactam.

Table 1.

MICs and pharmacodynamic exposures of human simulated TZP, CXA-101, and CXA-101–tazobactam against selected P. aeruginosa, K. pneumoniae, and E. coli isolates

Isolate (ESBL phenotype)a	Predicted % fT > MIC (MIC [μg/ml])b
	TZPc	CXA-101d	CXA-101–tazobactame
P. aeruginosa
PSA 7-17	81.7 (8)	100 (0.5)	100 (0.5)
PSA 14-2	81.7 (8)	97.5 (1)	100 (1)
PSA 20-20	63.3 (16)	92.5 (2)	96.3 (2)
PSA 8-18	63.3 (16)	97.5 (1)	100 (1)
PSA 10-34	45.0 (32)	100 (0.5)	100 (1)
PSA 11-15	45.0 (32)	100 (0.5)	100 (0.5)
PSA 24-2	28.3 (64)	56.3 (8)	96.3 (2)
PSA 11-18	28.3 (64)	37.5 (16)	32.5 (16)
K. pneumoniae
KP 393 (+)	45 (32)	0 (>64)	0 (>64)
KP 394 (+)	91.7 (4)	56.3 (8)	100 (0.5)
KP 51 (−)	63.3 (16)	97.5 (1)	100 (1)
KP 320 (+)	63.3 (16)	21.3 (32)	0 (>64)
E. coli
EC 315 (+)	96.7 (2)	56.3 (8)	100 (0.25)
EC 348 (+)	81.7 (8)	8.3 (64)	100 (0.25)
EC 114 (−)	100 (1)	100 (0.25)	100 (0.125)
EC 45 (−)	96.7 (2)	100 (0.25)	100 (0.25)

^a+, positive; −, negative.

^bDue to the fact that inhibitor combinations were utilized and therefore only piperacillin or CXA-101 was simulated, the resultant % fT > MIC values are based solely on the piperacillin or CXA-101component.

^cAt 4.5 g q6h; 30-min infusion.

^dAt 1,000 mg q8h; 1-h infusion.

^eAt 2:1; 1,000/500 mg q8h; 1-h infusion.

Pharmacokinetic determinations.

A one-compartment model was used to characterize the free-concentration–time profile of piperacillin-tazobactam in murine serum. The free-concentration–time profile of the resulting murine regimen (equivalent to a 30-min infusion of piperacillin-tazobactam at 4.5 g q6h), and the corresponding piperacillin exposures in humans given this combination are displayed in Fig. 1a. The actual pharmacodynamic exposures for piperacillin for both humans and mice with corresponding % fT > MIC values are depicted in Table 2. While the optimal pharmacodynamic exposure profile for tazobactam has not been described, the TZP regimen simulated and utilized here produce measured tazobactam concentrations that we found to be greater than any potential target tazobactam MICs observed in humans (Table 3).

Fig 1.

(a) Free-concentration–time profiles of piperacillin-tazobactam at 4.5 g q6h in healthy human volunteers versus mice. (b) Free-concentration–time profiles of CXA-101 at 1,000 mg q8h in healthy human volunteers versus mice. (c) Free-concentration–time profiles of CXA-101–tazobactam (2:1) at 1,000/500 mg q8h in healthy human volunteers versus mice. Each value represents the mean ± standard deviation of 6 infected mice.

Table 2.

Comparison of piperacillin and CXA-101 component % fT > MIC values achieved with piperacillin-tazobactam (TZP), CXA-101, and CXA-101–tazobactam at each tazobactam MIC in humans and in mice receiving humanized exposures

Druga	Speciesb	% fT > MIC for a MIC (μg/ml) of:
		1	2	4	8	16	32	64	>64
TZP	H	100	100	95	75	58	45	32	18
	M	100	97	92	82	63	45	28	13
CXA-101	H	100	100	83	58	33	9	0	0
	M	98	93	80	56	38	21	9	0
CXAT	H	100	100	83	58	33	9	0	0
	M	100	96	80	55	33	9	0	0

^aTZP, 4.5 g q6h, 30-min infusion; CXA-101, 1,000 mg q8h, 1-h infusion; CXAT, CXA-101–tazobactam (2:1; 1,000/500 mg q8h, 1-h infusion).

^bH, human; M, mouse.

A one-compartment model was used to characterize the free-concentration–time profile of CXA-101 in murine plasma. The free-concentration–time profiles of the resulting murine regimen (1-h infusion of CXA-101 at 1,000 mg q8h) and the corresponding exposures in humans are displayed in Fig. 1b. The actual pharmacodynamic exposures for both humans and mice with matched % fT > MIC values are depicted in Table 2.

Finally, a two-compartment model was used to characterize the free-concentration–time profile of CXA-101–tazobactam in murine plasma. The free-concentration–time profile of the resulting murine regimen (equivalent to a 1-h infusion of CXA-101–tazobactam [2:1] at 1,000/500 mg q8h) and the corresponding CXA-101 exposures in humans given this combination are displayed in Fig. 1c. Table 2 displays the similarities in the pharmacodynamic exposures of the nontazobactam β-lactam in mice and humans. In addition, as the tazobactam component of the combination therapies is critical to the activity of the nontazobactam β-lactam component, Table 3 shows that these data support a human simulated profile of tazobactam.

Efficacy as assessed by bacterial density.

At the start of dosing, 0-h control mice displayed mean P. aeruginosa bacterial burdens of 6.81 ± 0.24 log₁₀ CFU per thigh, E. coli bacterial burdens of 7.16 ± 0.16 log₁₀ CFU per thigh, and K. pneumoniae bacterial burdens of 7.28 ± 0.08 log₁₀ CFU per thigh. The bacterial load of P. aeruginosa in untreated mice increased by an average of 1.74 ± 0.50 log₁₀ CFU after 24 h, that of K. pneumoniae increased by 0.96 ± 1.10 log₁₀ CFU after 24 h, and that of E. coli increased by an average of 1.98 ± 0.44 log₁₀ CFU after 24 h. The results of the efficacy studies for all three dosing regimens are shown in Fig. 2, 3, and 4. Predicted piperacillin-tazobactam fT > MIC exposures of greater than 40% resulted in CFU numbers that were static to >1 log unit decreased in non-ESBL-expressing organisms with MICs of ≤32 μg/ml. Predicted CXA-101 and CXA-101–tazobactam fT > MIC exposures of ≥37.5% resulted in 1- to 3-log-unit decreases in CFU in non-ESBL-expressing organisms with MICs of ≤16 μg/ml. For the ESBL-expressing organisms, combinations that included tazobactam showed enhanced CFU decreases versus CXA-101 alone. Due to enhanced in vitro potency and resultant increased in vivo exposure, CXA-101 produced statistically significant reductions in CFU in 9 isolates compared with piperacillin-tazobactam. The addition of tazobactam to CXA-101 produced significant reductions in CFU for 7 isolates compared with piperacillin-tazobactam; however, all ESBL-positive isolates except KP 320 had increased efficacy with CXA-101–tazobactam.

Fig 2.

Comparative efficacies of human simulated piperacillin-tazobactam (black bars), CXA-101 (light-gray bars), and CXA-tazobactam (dark-gray bars) against a distribution of P. aeruginosa, presented as mean ± standard deviation. Statistical significance (SS) between antimicrobial agents is denoted as follows: * indicates SS between piperacillin-tazobactam and CXA-101–tazobactam, δ indicates SS between piperacillin-tazobactam and CXA-101, and ψ indicates SS between CXA-101 and CXA-101–tazobactam.

Fig 3.

Comparative efficacies of human simulated piperacillin-tazobactam (black bars), CXA-101 (light-gray bars), and CXA-tazobactam (dark-gray bars) against a distribution of ESBL-positive and ESBL-negative K. pneumoniae isolates, presented as mean ± standard deviation. SS between antimicrobial agents is denoted as follows: * indicates SS between piperacillin-tazobactam and CXA-101–tazobactam, δ indicates SS between piperacillin-tazobactam and CXA-101, and ψ indicates SS between CXA-101 and CXA-101–tazobactam. +, positive; −, negative.

Fig 4.

Comparative efficacies of human simulated piperacillin-tazobactam (black bars), CXA-101 (light-gray bars), and CXA-tazobactam (dark-gray bars) against a distribution of ESBL-positive and ESBL-negative E. coli isolates, presented as mean ± standard deviation. SS between antimicrobial agents is denoted as follows: * indicates SS between piperacillin-tazobactam and CXA-101–tazobactam, δ indicates SS between piperacillin-tazobactam and CXA-101, and ψ indicates SS between CXA-101 and CXA-101–tazobactam. +, positive; −, negative.

DISCUSSION

Gram-negative pathogens remain a common etiology of hospital-based infections, and resistance in these organisms is associated with poor treatment outcomes (17). One of the agents considered first-line therapy against these pathogens is piperacillin-tazobactam. In this study, we sought to compare the efficacy of CXA-101, a novel antipseudomonal cephalosporin, with and without the addition of tazobactam, with piperacillin-tazobactam against a host of phenotypically diverse Gram-negative pathogens.

Within our data, there are clear distinctions between the results of the piperacillin-tazobactam, CXA-101, and CXA-101–tazobactam regimens when focusing on multidrug-resistant P. aeruginosa. The dosing regimens with CXA-101 demonstrated statistically significant ≥1-log-unit decreases in bacterial density against all P. aeruginosa isolates except PSA 14-2 and 11-18. The piperacillin-tazobactam regimens demonstrated anywhere between a 0.5-log-unit increase and a <1-log-unit decrease in bacterial density. While the resistance mechanism(s) in the above-mentioned pseudomonads have not been characterized, it is postulated that the enhanced activity of the CXA-101 regimens may in part be due to the compound's improved stability against AmpC β-lactamases and/or increased affinity for PBP3 (23). This increased affinity for PBP3 allows CXA-101 to be less affected and, in most cases, unaffected by porin deficiencies or efflux mechanisms (23). The addition of tazobactam to this agent further serves to increase CXA-101's stability against AmpC β-lactamases, rendering the combination very efficacious against multidrug-resistant P. aeruginosa.

When focusing on the ESBL-positive and -negative K. pneumoniae isolates, the presence of the ESBL enzyme appeared to influence the efficacy of CXA-101 so that only with the ESBL-negative isolate KP 51 did CXA-101 demonstrate statistically significant efficacy compared with piperacillin-tazobactam (P < 0.001) or compared with CXA-101–tazobactam (P = 0.05). For the K. pneumoniae isolates tested, CXA-101–tazobactam human simulated exposures resulted in ≥1-log-unit killing with MICs of ≤1 μg/ml. Additionally, as KP 320 (ESBL positive) did not respond to CXA-101–tazobactam treatment, as was expected based upon the MIC of >64 μg/ml, and KP 393 (ESBL positive), which also had an CXA-101–tazobactam MIC of >64 μg/ml, did show marked efficacy with CXA-101–tazobactam exposure, it is apparent that these isolates, being clinical isolates, may have additional mechanisms of resistance to CXA-101 that are not indicated by the MIC results. Among the K. pneumoniae isolates studied, the piperacillin-tazobactam regimen produced the most profound reduction in CFU (0.75 and >1-log-unit decreases) over the 24-h treatment period. The efficacy of piperacillin-tazobactam against ESBL-positive organisms has been demonstrated in several studies, with some studies showing that piperacillin-tazobactam was the most active agent studied (9, 14, 18). In these, efficacy was dependent not only on the actual MIC of the organism, but also on the % fT > MIC achieved by the piperacillin-tazobactam regimen, striving for a % fT > MIC of >30 to 35% (1, 9). In our study, the simulated piperacillin-tazobactam regimen obtained 45%, 63.3%, and 91.7% fT > MIC against the ESBL-positive isolates, well above the indicated target (1). In contrast to the results for K. pneumoniae, when the focus was shifted to the E. coli isolates, the simulated regimens of CXA-101–tazobactam had increased efficacy against the ESBL-positive isolates. In fact, this regimen demonstrated statistically significant decreases in bacterial density of 1.2 to 1.5 log units over 24 h compared with piperacillin-tazobactam (P < 0.001). The superior efficacy of CXA-101–tazobactam against ESBL-positive isolates has also been well documented in other studies (4, 22). In these studies, there is a recurrent trend in which CXA-101 combined with tazobactam showed increased potency over CXA-101 alone, other cephalosporins, or piperacillin-tazobactam against ESBL-positive isolates (4, 22). This trend is seen again in our data, where the most efficacious agent against the ESBL-negative isolates is CXA-101 alone but the addition of tazobactam increases this efficacy to include ESBL producers. Overall, when combining the results for both the E. coli and K. pneumoniae isolates, it is clear that the regimens containing tazobactam demonstrated the most efficacy against ESBL-positive isolates.

Overall, CXA-101 with or without tazobactam exhibited MICs that were 2- to 6-fold lower than those of piperacillin-tazobactam. This in turn equated to increased in vivo efficacy when using human simulated regimens against a diverse Gram-negative population, including multidrug-resistant P. aeruginosa and ESBL-positive and -negative E. coli and K. pneumoniae. When focusing on isolates that were ESBL positive, human simulated combinations that contained tazobactam showed increased efficacy compared with CXA-101 alone. These results support previous data that indicate that CXA-101 with or without tazobactam is a formidable agent against multidrug-resistant Gram-negative pathogens, including ESBL-producing E. coli and K. pneumoniae.