Exploring the Pharmacokinetic/Pharmacodynamic Relationship of Relebactam (MK-7655) in Combination with Imipenem in a Hollow-Fiber Infection Model

Antibiotics and bacterial strains.Imipenem and REL were dissolved in 3-(N-morpholino)propanesulfonic acid (MOPS) and dimethyl sulfoxide (DMSO) at 10 mM, respectively, made to the appropriate concentrations needed, frozen at −70°C, and thawed immediately prior to use. Imipenem is extensively metabolized by a renal dipeptidase (dehydropeptidase I) present in the brush border of renal tubular epithelium in animal species and humans. To prevent its renal metabolism, imipenem is generally coadministered with cilastatin (a dehydropeptidase inhibitor) in animals and humans. However, there was no dehydropeptidase I present in the medium in the in vitro hollow-fiber studies described in the present report; therefore, cilastatin was not required. Imipenem was utilized alone or in combination with REL.

Sixteen isolates were evaluated in the hollow-fiber infection model. Five P. aeruginosa strains produced inducible or constitutive AmpC of various PDC alleles. P. aeruginosa and 11 Enterobacteriaceae isolates produced various alleles of KPC, CTX-M, TEM, CMY, and DHA, alone or in combination, as detailed in Table 1. All isolates were obtained from the Merck Clinical Culture Collection or the IHMA freezer collection of recent clinical isolates on the basis of previous molecular characterization (Table 1).

These strains were chosen to include strains with a range of imipenem MICs, either alone or in the presence of REL at 4 μg/ml. The in vitro susceptibility concentration of REL of 4 μg/ml was chosen on the basis of a phase 2 average concentration in patients of 4.94 μg/ml (10). Strains were grown from frozen glycerol stocks on Trypticase soy agar plates with 5% sheep blood (BD Laboratories, Franklin Lakes, NJ, USA), and susceptibility testing was performed in cation-adjusted Mueller-Hinton Broth (CAMHB). The MICs of imipenem were determined by the broth microdilution method, using the CLSI guidelines (28, 29), and all isolates were resistant to imipenem on the basis of CLSI breakpoints.

In vitro hollow-fiber model.The two-compartment hollow-fiber model was adapted from models previously published by Grasso et al. (17) and Blaser et al. (15) and was developed as described earlier by Wang et al. (18). A schematic of the system is presented in Fig. 1.

The volume of distribution of the central compartment (V_c) was 150 ml, and imipenem and REL were added under computer control using a peristaltic pump. Fresh CAMHB was continuously supplied and removed from the central compartment via a peristaltic pump set to simulate the half-lives (t_1/2) of the antibiotics and the free fraction of drug in plasma (set at 1.5 h to replicate the clinical t_1/2 of imipenem and REL and the fraction of unbound drug [f_u] of 0.8 for both imipenem and REL). The flow rate was calculated to be 1.16 ml/min on the basis of the V_c and the t_1/2 per the equation ln 2 · (V_c/t_1/2).

The central compartment was incubated at 37°C with shaking at 80 rpm to ensure proper mixing of the drug. Samples of 0.2 ml were collected by the automated sample collector from the extracapillary space of the hollow-fiber cartridge (FiberCell Systems, Frederick, MD, USA) at 0, 3.5, 6.5, 12.5, 18.5, 24.5, 30.5, 36.5, 42.5, 48.5, 54.5, 60.5, 66.5, and 70 h for PK analysis.

For the PD evaluations, the initial challenge inoculum was prepared from overnight cultures grown on Trypticase soy agar plates with 5% sheep blood at 35°C. Colonies were picked from each overnight culture and suspended in 5 ml of Trypticase soy broth, and the turbidity was adjusted to match that of a 0.5 McFarland standard using a Siemens turbidity meter (preferred optical density, 0.10 to 0.12). This resulted in a concentration of approximately 1 × 10⁸ CFU/ml. This standardized suspension was then diluted 1:1,000 into CAMHB and inoculated into the extracapillary space of the preconditioned hollow-fiber cartridges (FiberCell Systems, Frederick, MD, USA). Inoculated hollow-fiber cartridges were incubated at 37°C for 4 h prior to the start of the experiment; the resulting exponentially growing cultures reached ∼10⁶ CFU/ml. Samples of 0.25 ml were collected by the automated sample collector from the extracapillary space of the hollow-fiber cartridge at 0, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and 70 h for PD analysis.

Measurement of drug concentrations.Upon collection, samples were diluted 1:1 with a preservative composed of equal parts of ethylene glycol and a buffer [1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0], which was prepared by adding MES (10.88 g; Sigma-Aldrich, St. Louis, MO, USA) and MES sodium salt (9.62 g) to 100 ml of water, mixing, and subsequently adding 100 ml of ethylene glycol. The stabilized samples were frozen at −80°C. Stable isotopically labeled internal standards of imipenem and REL were used for the high-performance liquid chromatography–mass spectrometry analysis. Sample preparation was performed over ice. CAMHB, preservative, acetonitrile, and a 10 mM MES buffer (pH 6.7) in acetonitrile/water (50/50, prepared by dilution of 1 M MES buffer, pH 6.7, 0.384 g MES, and 1.74 g MES sodium salt in 10 ml water) were used in appropriate ratios during sample preparation to ensure matrix matching of the prepared unknown samples, standards, quality controls, as well as single and double blanks. A standard curve for concentrations ranging from 100 to 0.195 μg/ml was prepared. Protein precipitation was performed by the addition of a 5:1 ratio of acetonitrile to the matrix-matched samples. Following centrifugation and transfer of supernatant, samples were diluted 3-fold by the addition of 80% aqueous acetonitrile, chromatographed using hydrophilic interaction chromatography (HILIC), and detected via tandem mass spectrometry. Liquid chromatography was performed using a Waters HILIC (particle size, 3 μm; 2.1- by 50-mm column) held at 35°C, eluting isocratically with ammonium acetate 5 mM (pH 4.5) in acetonitrile/water (80/20) at a flow rate of 0.3 ml/min on a Thermo Fisher Scientific (Waltham, MA, USA) Transcend II multiplexed ultra-high-performance liquid chromatography system. An Applied Biosystems (Foster City, CA, USA) Sciex API 4000 triple-quadrupole tandem mass spectrometer equipped with an electrospray source was operated in positive-ion mode using multiple-reaction monitoring. The ion spray voltage was set to 4.5 kV, and the auxiliary gas temperature was maintained at 500°C. Nitrogen was used for gas 1, gas 2, curtain, and collision gas. The mass resolution was set to a peak width of 0.7 mass unit at half height for both quadrupole (Q)1 and Q3. The electron multiplier was set at 2,000 V.

Quantification of antibacterial effect.Bacterial viable counts were determined by serially diluting samples 10-fold in cold sterile saline. Aliquots of 100 μl of nondiluted and diluted samples (10⁻¹ to 10⁻⁷) were plated onto Trypticase soy agar plates with 5% sheep blood using an Eddy Jet spiral plater (Neutec Group Inc., Farmingdale, NY, USA) with subsequent incubation at 37°C for 18 to 24 h. Drug carryover was negligible after serial dilutions. After incubation, the resulting colonies were counted and the numbers of CFU per milliliter were determined using a Flash & Go colony counter (Neutec Group Inc., Farmingdale, NY, USA).

Data analysis.The simulated PK were captured with a one-compartment model fitted to the drug concentrations in the in vitro model central compartment (WinNonlin software; Pharsight Corp). The PK/PD relationship was analyzed by fitting a sigmoidal E_max relationship between the REL exposure (C_max, AUC, and T_>CT) and the associated response (antimicrobial effect, as measured by the change in bacterial counts). These relationships were assessed both by using the absolute value of the PK parameters C_max and AUC and also by normalizing the PK parameters to the potentiated MIC of the strain in the presence of 4 μg/ml REL. The time above the threshold concentration was explored using a range of concentration threshold values.

Analysis of REL PK/PD indices.REL PK/PD were assessed using the traditional indices frequently used in antibiotic PK/PD, namely, T_>CT, C_max, the C_max/MIC ratio, AUC_0–24, and the AUC_0–24/MIC ratio. The PK/PD index that best correlates with efficacy would be considered the driver for REL. In such a traditional analysis, different thresholds could be considered for the T_>CT index. The relationship between efficacy and various indices is analyzed by a sigmoid E_max model. The model-predicted efficacy and observed efficacy at a specified time point are compared, in order to assess the best r². The index that provides the best r² is considered the driver for efficacy.

Influence of REL on the MIC of imipenem.The relationship between the MIC of imipenem and the REL concentration was captured by a nonlinear maximum-effect (E_max) model using a population approach, wherein each strain is considered an individual. The MIC₀ is the baseline, or unpotentiated, MIC for each strain. E_max is the maximum response to the drug (in this case REL), and EC₅₀ is the drug concentration at which half of the maximum effect was observed. Populated parameters calculated for P. aeruginosa were E_max (0.96) and EC₅₀ (0.6 μg/ml). Interstrain variability was accounted for as a random effect in the modeling. Checkerboard static exposure data from 93 strains of P. aeruginosa and the following equation were used to calculate the dynamic MIC of imipenem as a function of the time-varying concentration of REL: MIC = MIC₀{1 − [(E_max · C_REL)/(EC₅₀ + C_REL)]} = MIC₀{1 − [(0.96 · C_REL)/(0.6 + C_REL)]}.

Percentage of the dosing interval that the imipenem-REL concentration remains above the dynamic MIC.The duration of the dosing interval that the imipenem concentration remains above the MIC is important for assessing the effectiveness of a dosing regimen. Therefore, the changing MIC or dynamic MIC against the P. aeruginosa and K. pneumoniae strains was studied in the hollow-fiber system using the same procedures and sample collection described earlier. To assess the REL PK driver, a subtherapeutic dose of imipenem of 200 mg q6h was applied in combination with a range of REL doses (13, 27, 50, 100, and 200 mg q6h). In addition, the phase 2B doses of imipenem at 500 mg plus REL at 125 mg or 250 mg q6h were also simulated.

Checkerboard methodology.All strains were streaked from frozen stocks onto Trypticase soy agar plates with 5% sheep blood on the day before use, and the plates were incubated at 37°C overnight. On the next day, 4 to 6 colonies from each plate were selected with a BD Prompt wand and resuspended in 1 ml sterile saline. The stock was estimated to be ∼1.5 × 10⁸ CFU/ml. Bacterial stock solutions were diluted to 5.25 × 10⁵ CFU per ml (15 ml CAMHB plus 52.5 μl bacterium-inoculated saline from the Prompt wand) to achieve a final starting concentration of bacteria of ∼5 × 10⁵ CFU/ml when 95 μl of bacterial stock was added to the assay plate. Imipenem stock solutions were prepared in 2.5 mM MOPS, pH 7.4, and REL stocks were prepared in 100% DMSO. Twenty-times-titration plates were prepared, and 5 μl of the titration was transferred to replicate 96-well, round-bottom (clear) plates to achieve complete overlapping dilutions series of imipenem at concentrations ranging from 32 to 0.5 mg/ml and REL at concentrations ranging from 64 to 0.0625 mg/ml. Imipenem and REL were run in titration alone. Plates were shaken for 5 min on a rotary shaker and then incubated overnight (∼19 h) at 37°C. On the next morning, the plates were read by eye for bacterial growth in the wells using a mirrored plate reader.