Efficacy and Pharmacokinetics of the Combination of OP0595 and Cefepime in a Mouse Model of Pneumonia Caused by Extended-Spectrum-Beta-Lactamase-Producing Klebsiella pneumoniae

ABSTRACT

OP0595 (RG6080) is a novel diazabicyclooctane that inhibits class A and C serine beta-lactamases. Although the combination of OP0595 and cefepime (FEP) showed good in vitro activity against extended-spectrum-beta-lactamase (ESBL)-producing pathogens, the effect of the combination therapy against severe infections, such as pneumonia or bacteremia, remains unknown in vivo. In this study, we investigated the efficacy and pharmacokinetics of the combination therapy of OP0595 and FEP in a mouse model of pneumonia caused by Klebsiella pneumoniae harboring SHV- and CTX-M-9-type ESBLs. The infected BALB/c mice were intraperitoneally administered saline (control), 100 mg/kg of body weight of FEP, 20 mg/kg of OP0595, or both FEP and OP0595, twice a day. The MIC of FEP against the bacteria was 8 mg/liter and markedly improved to 0.06 mg/liter with the addition of 0.5 mg/ml of OP0595. In the survival study, the combination of FEP and OP0595 significantly improved the survival rate compared with that reported with either OP0595 or FEP alone (P < 0.001). The number of bacteria in the lungs and blood significantly decreased in the combination therapy group compared to that reported for the monotherapy groups (P < 0.001). In addition, the in vivo effect depended on the dose of FEP. However, pharmacokinetic analysis revealed that the percentage of time above MIC remained constant when increasing the dose of FEP in combination with 20 mg/kg of OP0595. The results of our study demonstrated the in vivo effectiveness of the combination of OP0595 and FEP.

INTRODUCTION

Extended-spectrum beta-lactamases (ESBLs) are enzymes classified as class A in the Ambler classification of beta-lactams (1) and are responsible for multiresistance to most beta-lactam antibiotics, including penicillins, cephalosporins, and monobactams. The global increase in ESBL-producing pathogens, particularly Escherichia coli and Klebsiella pneumoniae, is a major clinical concern. The current effective therapeutic option for severe infections caused by ESBL-producing pathogens is carbapenems (2, 3). However, it is necessary to develop other therapeutic options because of the emergence and global spread of carbapenemase-producing Enterobacteriaceae.

Recently, combination agents consisting of a cephalosporin and beta-lactamase inhibitor, such as ceftazidime-avibactam and ceftolozane-tazobactam, have been developed. Ceftolozane-tazobactam showed good activity against most ESBL-producing E. coli isolates but lower activity against ESBL-producing K. pneumoniae isolates (4, 5). In contrast, the addition of avibactam significantly increased the activity of ceftazidime against ESBL-producing pathogens, including K. pneumoniae (6). OP0595 (RG6080) is a novel diazabicyclooctane that, similarly to avibactam, inhibits class A and C serine beta-lactamases. In addition to the inhibition of beta-lactamases, OP0595 showed antimicrobial activity by inhibiting penicillin binding protein 2 (PBP2) and enhanced the antimicrobial activity of the beta-lactams that bind to PBP3 (7, 8). The combination of OP0595 and beta-lactams showed good in vitro activity against ESBL-, AmpC-, and carbapenemase-producing pathogens, including those producing OXA-48-class beta-lactamases (7–9). Additionally, the combination of OP0595 and cefepime (FEP) showed a dose-dependent effect in a neutropenic murine thigh infection model (9). However, the effect of the combination therapy against severe infections, such as pneumonia or bacteremia, remains unknown.

In this study, we investigated the efficacy and pharmacokinetics of the combination therapy of OP0595 and FEP in a mouse model of pneumonia caused by ESBL-producing K. pneumoniae.

RESULTS

MICs of antimicrobial agents against KEN-11 strain.

The MICs of FEP and OP0595 against the KEN-11 strain of K. pneumoniae were 8 and 4 mg/liter, respectively. The MIC of FEP against the bacterial strain markedly improved in combination with OP0595 (Table 1).

TABLE 1.

MICs of FEP in combination with OP0595 against KEN-11 strain

Concn (mg/liter) of OP0595	MIC (mg/liter) of FEP
0	8
0.5	0.06
1.0	0.03
2.0	0.015

Pharmacokinetics of antimicrobial agents.

The plasma concentration profiles and calculated pharmacokinetic parameters of FEP and OP0595 are presented in Fig. 1 and Table 2. The concentration of OP0595 in plasma with 20 mg/kg of body weight of OP0595 was significantly lower than that with 100 mg/kg of OP0595 at 5, 15, 30, 45, 60, and 180 min posttreatment (P < 0.05) (Fig. 1). The areas under the plasma concentration-time curve from 0 to infinity (AUC_0–inf) of FEP in combination with 20 and 100 mg/kg of OP0595 were 93.89 and 82.18 μg · h/ml, respectively. The half-lives (t_1/2s) of FEP in combination with 20 and 100 mg/kg of OP0595 were 0.31 and 0.44 h, respectively. Based on the MICs and pharmacokinetic parameters of FEP and OP0595, the percentage of time above MIC (%TAM) was calculated. The %TAM of FEP was increased by dose escalation of OP0595 (Fig. 2). The %TAMs of 4, 20, and 100 mg/kg of FEP in combination with 20 mg/kg of OP0595 administered twice a day were 27.1, 27.7, and 27.7%, respectively.

FIG 1.

Plasma concentrations of cefepime (FEP) and OP0595. The mice were treated with 100 mg/kg of FEP and 20 (gray squares) or 100 (black circles) mg/kg of OP0595 (n = 4 in each group). The pharmacokinetics of FEP (A) and OP0595 (B) were measured at 5, 15, 30, 45, 60, 90, 120, and 180 min. *, the concentration of FEP in the plasma of mice treated with 100 mg/kg OP0595 was significantly lower than that in the plasma of mice treated with 20 mg/kg OP0595 at 45 min (P < 0.05). †, the concentration of OP0595 in the plasma of mice treated with 100-mg/kg OP0595 was significantly higher than that in the plasma of mice treated with 20-mg/kg OP0595 at 4, 15, 30, 45, 60, and 180 min (P < 0.05).

TABLE 2.

Selected pharmacokinetic parameters estimated for FEP and OP0595 in plasma^a

Compound	Dose (mg/kg)	Combined drug (concn in mg/kg)	CL (liters/h/kg)	V/F (liters/kg)	t1/2 (h)	AUC0–inf (μg · h/ml)
FEP	100	OP0595 (20)	1.07	0.47	0.31	93.89
	100	OP0595 (100)	1.22	0.78	0.44	82.18
OP0595	20	FEP (100)	0.68	0.36	0.37	29.55
	100	FEP (100)	0.75	0.49	0.46	133.42

^aAbbreviations: FEP, cefepime; CL, clearance; V/F, apparent volume of distribution; t_1/2, half-life; AUC_0–inf, area under the concentration-time curve from 0 to infinity.

FIG 2.

%TAM of cefepime (FEP) in combination with OP0595. Based on the pharmacokinetic analysis, we calculated the percentage of time above MIC (%TAM) of FEP in combination with OP0595 against the bacteria. The %TAM of FEP twice a day was increased by dose escalation of OP0595 twice a day.

Therapeutic effects of antimicrobial agents on survival rate.

Based on the results of pharmacokinetic studies, we decided the doses of OP0595 and FEP. Since we mainly investigated the effects of OP0595, such as inhibition of beta-lactamases and enhanced effects of the antimicrobial activity of the beta-lactams that bind to PBP3, the mice were administered saline (control), 100 mg/kg of FEP, 20 mg/kg of OP0595, or FEP combined with OP0595, twice a day.

In the survival study, the mice were treated using the prescribed methods until 108 h postinoculation and the survival rates were observed until 120 h postinoculation (n = 7 in each group). As shown in Fig. 3A, the survival rates were significantly higher in the combination treatment group than in the other groups (P < 0.001).

FIG 3.

Therapeutic effects of FEP in combination with OP0595. Twelve hours postinoculation, the mice were treated twice a day with saline (control), 20 mg/kg of OP0595, 100 mg/kg of FEP, or 100 mg/kg of FEP in combination with 20 mg/kg of OP0595 (combination) until 108 h postinoculation. (A) The survival rates were observed until 120 h after inoculation (n = 7 in each group). *, the survival rate in the combination treatment group was significantly higher than those in the other treatment groups (P < 0.001). (B and C) Thirty-six hours postinoculation, the mice were sacrificed, and the numbers of bacteria in the lungs (B) and the blood (C) were analyzed (n = 6 in each group). Box-and-whisker plots show the range and median of the number of bacteria. The number of bacteria in the lungs significantly decreased in the combination therapy group compared with the other groups (P < 0.001). The number of bacteria in the blood significantly decreased in the combination therapy group compared with the other groups (P < 0.001 versus the control, P < 0.05 versus the OP0595 treatment group, and P < 0.01 versus the FEP treatment group).

In the bacteriological examinations, the mice were sacrificed 36 h postinoculation (n = 6 in each group). The numbers of bacteria in the lungs in the control, OP0595, FEP, and combination treatment groups were 8.65 ± 0.58, 8.35 ± 0.40, 8.94 ± 0.48, and 4.47 ± 0.99 log₁₀ CFU/ml, respectively (Fig. 3B). The number of bacteria in the lungs was significantly lower in the combination treatment group than in the other groups (P < 0.001). The numbers of bacteria in the blood in the control, OP0595, FEP, and combination treatment groups were 7.41 ± 0.93, 6.44 ± 0.71, 7.07 ± 1.20, and 4.49 ± 1.29 log₁₀ CFU/ml, respectively (Fig. 3C). The number of bacteria in the blood was significantly lower in the combination treatment group than in the other groups (P < 0.001 versus the control, P < 0.05 versus the OP0595 treatment group, and P < 0.01 versus the FEP treatment group).

Effects of combination therapy that depend on doses of FEP.

Twelve hours postinoculation, the mice were treated twice a day with saline (control) or 4, 20, or 100 mg/kg of FEP in combination with 20 mg/kg of OP0595 until 108 h postinoculation. In the survival study, the survival rates were observed until 120 h postinoculation (n = 9 in each group). As shown in Fig. 4A, the survival rates were significantly higher in the 100-mg/kg FEP treatment group than in the other groups (P < 0.001). The survival rates in the 4- and 20-mg/kg FEP treatment groups were significantly higher than that in the control group (P < 0.05 for 4 mg/kg of FEP and P < 0.01 for 20 mg/kg).

FIG 4.

Effects of combination therapy that depended on the doses of FEP. Twelve hours postinoculation, the mice were treated twice a day with saline (control) or 4, 20, or 100 mg/kg of FEP in combination with 20 mg/kg of OP0595 until 108 h postinoculation. (A) The survival rates were observed until 120 h after inoculation (n = 9 in each group). The survival rate in the 100-mg/kg FEP treatment group was significantly higher than those in the other treatment groups (*, P < 0.001). The survival rates in the 4- and 20-mg/kg FEP treatment groups were significantly higher than that in the control (‡, P < 0.05, 4 mg/kg of FEP, and †, P < 0.01, 20 mg/kg of FEP, respectively). (B and C) Thirty-six hours postinoculation, the mice were sacrificed, and the numbers of bacteria in the lungs (B) and the blood (C) were analyzed (n = 4 in each group). The numbers of bacteria in the lungs and blood were significantly lower in the 100-mg/kg FEP group than in the other groups (P < 0.001). The numbers of bacteria in the lungs were significantly decreased in the 4- and 20-mg/kg treatment groups compared with the control (§, P < 0.001). The number of bacteria in the blood was significantly lower in the 20-mg/kg-FEP treatment group than in the control (¶, P < 0.001).

In the bacteriological examinations, the mice were sacrificed 36 h postinoculation (n = 4 in each group). The numbers of bacteria in the lungs in the control group and the groups with 4-, 20-, and 100-mg/kg FEP with OP0595 treatment were 9.42 ± 0.35, 7.46 ± 0.24, 6.53 ± 0.80, and 4.38 ± 0.69 log₁₀ CFU/ml, respectively (Fig. 4B). The number of bacteria in the lungs was significantly lower in the group with 100-mg/kg FEP with OP0595 treatment than in the other groups (P < 0.001). The number of bacteria in the lungs was significantly lower in the groups with 4- and 20-mg/kg FEP with OP0595 treatment than in the control group (P < 0.001). The numbers of bacteria in the blood in the control group and the groups with 4-, 20-, and 100-mg/kg FEP with OP0595 were 8.14 ± 0.42, 7.17 ± 0.65, 5.46 ± 1.05, and 2.96 ± 0.68 log₁₀ CFU/ml, respectively (Fig. 4C). The number of bacteria in the blood was significantly lower in the group with 100-mg/kg FEP with OP0595 treatment than in the other groups (P < 0.001). The number of bacteria in the blood was significantly lower in the group with 20-mg/kg FEP with OP0595 treatment than in the control group (P < 0.001).

DISCUSSION

The combination therapy of OP0595 and FEP showed good in vivo activity against ESBL-producing K. pneumoniae in the mouse model. K. pneumoniae often colonizes the human digestive tract and nasopharynx (10) and has been one of the major causes of both community-acquired and nosocomial pneumonia (11). When bacteremia is complicated with pneumonia, the mortality rate of patients with K. pneumoniae infection is 2-fold higher than that of patients with Streptococcus pneumoniae infection (12). In addition, the proportion of drug-resistant K. pneumoniae ESBL producers is relatively high, and the use of appropriate therapy is independently associated with lower mortality (2). Therefore, there is a need to develop a novel agent, including OP0595, against ESBL-producing K. pneumoniae.

In the antimicrobial susceptibility test, the MIC of OP0595 against the KEN-11 strain was 4 mg/liter, which was lower than that of FEP. A previous study reported that OP0595 directly inhibits the growth of many Enterobacteriaceae strains at concentrations of 1 to 8 mg/liter by inhibiting PBP2, and in some strains, the MIC of OP0595 was lower than that of beta-lactams, such as piperacillin, FEP, and ceftazidime (8). However, in the mouse model of pneumonia caused by K. pneumonia, the mice were treated with 20 mg/kg of OP0595, and monotherapy with OP0595 did not improve survival or decrease the number of bacteria in the lungs and blood (Fig. 3). Hence, we consider that the effect of OP0595 in combination therapy did not depend on the direct antimicrobial activity of OP0595 against the KEN-11 strain.

OP0595 is a novel diazabicyclooctane that, similarly to avibactam, inhibits class A and C serine beta-lactamases. In the previous study, the 50% inhibitory concentrations (IC₅₀s) of OP0595 for the class A and C beta-lactamases were similar to or slightly higher than those of avibactam (8). OP0595 also improved the MICs of beta-lactams in a dose-dependent manner as avibactam does (7–9). In this study, the MICs of FEP against KEN-11 markedly improved in combination with 0.5, 1.0, and 2.0 mg/liter of OP0595 (Table 1). Additionally, in the mouse model, the effects of combination therapy with OP0595 and FEP depended on the dose of FEP (Fig. 4). From these results, the function of OP0595 in the combination therapy seems to be as a beta-lactamase inhibitor.

However, this does not explain the dose-dependent in vivo effects since the %TAMs of 4, 20, and 100 mg/kg of FEP in combination with 20 mg/kg of OP0595 were almost the same (Fig. 2). If the in vivo effect depended on the inhibitory activity of OP0595 against beta-lactamase, then the %TAM of FEP would increase according to the FEP dose. Moreover, the %TAM of FEP in combination with OP0595 was much lower than the effective %TAM in the previous study (13, 14). Previous studies on OP0595 revealed that OP0595 enhances the activity of beta-lactams that bind to other PBPs besides PBP2 (7–9). In a neutropenic mouse model of thigh infection, the number of bacteria decreased in an FEP dose-dependent manner in combination with OP0595, and the action of OP0595 in the combination therapy was considered to be as a beta-lactamase inhibitor and beta-lactam enhancer (9). In our mouse model of pneumonia as well, OP0595 in the combination therapy might act as both a beta-lactamase inhibitor and a beta-lactam enhancer.

There are concerns that the low dose of OP0595 will lead to the development of OP0595-resistant pathogens. A previous study revealed that subinhibitory concentrations of OP0595 caused resistance to OP0595 via activation of RpoS (15). In addition, natural OP0595-resistant strains were reported (8, 9). However, OP0595 showed a synergistic effect with beta-lactams, including FEP, against OP0595-resistant strains via inhibition of beta-lactamases and enhancement of the activity of beta-lactams (8, 9). Since the OP0595-resistant mutants caused small increases of MICs of beta-lactams (8, 9), further investigation of the OP0595 resistance is needed.

This study has some limitations. First, only one clinical strain of K. pneumoniae harboring SHV- and CTX-M-9-type ESBLs was used in this study. The activities of beta-lactams against ESBL producers vary according to ESBL subgroups, such as TEM, SHV, and CTX-M types (16). In previous studies, OP0595 showed good in vitro activity against various kinds of ESBLs (7–9), but the in vivo efficacy against ESBLs other than SHV and CTX-M types remains unknown. Second, there was no comparison of the combination therapy of OP0595 and FEP with the other novel combination therapies, such as ceftazidime-avibactam and ceftolozane-tazobactam. Third, it is unverifiable whether the combination therapy shows the same effect in humans, because there are no pharmacokinetic data in humans. Finally, the toxicity of OP0595 was not investigated in this study.

In conclusion, the results of our study demonstrated the in vivo effects of the combination of OP0595 and FEP. Further investigations, including clinical trials, are needed to study the effect of the combination therapy in patients with pneumonia caused by ESBL-producing K. pneumoniae.

MATERIALS AND METHODS

Bacterial strains.

The K. pneumoniae strain used in this study was the KEN-11 strain, a clinical isolate obtained at the Nagasaki University Hospital (17). The KEN-11 strain is positive for SHV- and CTX-M-9-type ESBLs and showed positive results in the string test (17). The bacteria were stored at −80°C in a Microbank bead preservation system (Pro-Lab Diagnostics, Ontario, Canada) until use.

Antimicrobial agents.

OP0595 was supplied by Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). Cefepime dihydrochloride hydrate was purchased from Bristol-Myers Squibb Company (Tokyo, Japan).

Animals.

We purchased specific-pathogen-free BALB/c male mice (6 to 7 weeks old) from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed in a pathogen-free environment and received sterile food and water at the Biomedical Research Centre of Nagasaki University.

Ethics.

All the experimental protocols used in this study were approved by the Ethics Review Committee for Animal Experimentation of Nagasaki University (approval number 1503101199).

Antimicrobial susceptibility test.

We tested the MICs of the agents against the KEN-11 strain by a microdilution method with cation-adjusted Mueller-Hinton broth (Becton, Dickinson & Co., Franklin Lakes, NJ) in accordance with the guidelines of the Clinical and Laboratory Standards Institute (18, 19). The final inoculum was approximately 5 × 10⁵ CFU/well. The MICs were defined as the lowest concentration that inhibits visible growth after incubation at 35°C for 16 to 18 h.

Pharmacokinetic studies.

The mice were infected with the bacterial suspension intratracheally (0.05 ml; 1 × 10⁶ CFU/mouse). At 12 h postinoculation, the mice were treated with 100 mg/kg of FEP and 20 or 100 mg/kg of OP0595 and were then sacrificed by cervical dislocation at 5, 15, 30, 45, 60, 90, 120, and 180 min postadministration. Blood was collected via a right ventricular puncture using heparin-coated syringes. Four mice were used for each group. The blood samples were centrifuged, and the isolated plasma samples were stored at −80°C until use.

The concentration of FEP and OP0595 in plasma was measured using a liquid chromatograph (Acquity ultraperformance liquid chromatography [UPLC] system; Waters, Milford, MA, USA) coupled with a tandem mass spectrometer (Qtrap 5500; AB Sciex, Tokyo, Japan). For pharmacokinetic analysis, WinNonlin Professional ver. 6.3 (Certara) was used.

Plasma concentration was analyzed using a noncompartmental model to calculate the elimination half-life (t_1/2), apparent volume of distribution (V), area under the plasma concentration-time curve from 0 to infinity (AUC_0–inf), and total body clearance (CL).

Preparation of bacteria and mouse model of pneumonia.

The method of preparation and inoculation of bacteria has been previously reported (17). The KEN-11 strain was cultured overnight on Muller-Hinton II agar (Becton Dickinson, Le Pont de Claix, France). To prepare the inoculum, a single colony of the bacteria was preincubated in Luria-Bertani (LB) broth containing 100 μg/ml penicillin at 37°C for 18 h with shaking at 250 rpm. After 8 h of additional incubation in LB broth, the bacterial suspension was adjusted to appropriate concentrations using turbidimetry. The nonneutropenic mice were infected with the bacterial suspension intratracheally (0.05 ml; 1 × 10⁶ CFU/mouse).

Treatment protocol.

OP0595 and FEP were diluted with saline. Treatment commenced 12 h postinoculation. Mice were intraperitoneally administered saline (control), 100 mg/kg of FEP, 20 mg/kg of OP0595, or FEP combined with OP0595, twice a day.

Bacteriological examinations.

Mice were sacrificed from each group at 36 h postinoculation. Subsequently, they were dissected under aseptic conditions. Blood was collected via a right ventricular puncture using heparin-coated syringes. The lungs were removed, suspended in 1 ml of normal saline, and homogenized with a homogenizer (AS One Co., Osaka, Japan). The lungs were then cultured, and the serially diluted blood samples were spread onto the Mueller-Hinton II agar plates. After overnight incubation at 37°C, the number of visible colonies in the plates was evaluated. The lowest level of detectable bacterial count was 1 × 10² CFU/ml.

Statistical analysis.

A statistical software package (StatMate V; ATMS Co., Ltd., Tokyo, Japan) was used for all the statistical comparisons, and the survival rates were calculated using the Kaplan-Meier method. The survival analysis was performed using the log rank test, and the data were expressed as mean and standard deviation (SD). In the graph of the bacterial count in the lungs and the blood, the data were depicted by a box-and-whisker plot and the differences between the groups were analyzed using a one-way analysis of variance with Tukey's post hoc test. All the tests of significance were two-tailed, and the alpha level for denoting statistical significance was set at <0.05.