Pharmacodynamics of Tebipenem: New Options for Oral Treatment of Multidrug-Resistant Gram-Negative Infections

Drug.Tebipenem (pure active compound) was supplied by Spero Therapeutics as SPR859 (pure active compound) for liquid chromatography-tandem mass spectrometry (LC-MS/MS), hollow-fiber studies, MIC determinations, and incorporation into agar for evaluation of the emergence of resistance. The orally bioavailable prodrug was used for all murine studies. Dose formulations were stored on the bench at ambient temperature for the length of the study, and stability under these conditions was established (no longer than 24 h).

All test articles were prepared based on a dosing volume of 10 ml/kg of body weight. Prodrug was stored at room temperature. Solutions of prodrug at the appropriate concentration were prepared in 2.5% ethanol–2.5% Tween 80–95% citric acid-sodium citrate buffer (pH 4.0) prior to administration. Prodrug powder was initially suspended by vortex mixing for 30 s in 200 μl 50% ethanol–50% Tween 80, after which the mixture was allowed to stand for 2 min. Subsequently, the mixture was subjected to sonication for 10 min in a water bath at ambient temperature before being diluted with 3.8 ml filter-sterilized (pore size, 0.22 μm) citric acid-sodium citrate buffer (pH 4.0), and the pH was adjusted to between 4.5 and 5.0 with 6 N HCl. Further sonication followed by alternating cycles of vortex mixing and sonication (if required) were used to completely solubilize the prodrug. The formulation solutions were stable for up to 24 h at ambient temperature.

Isolates.Challenge organisms with a range of resistance mechanisms and tebipenem MICs were chosen (Table 1). A variety of wild-type and non-wild-type isolates of the Enterobacteriacae were investigated. The isolates were stored long term at −80°C. Colonies were confirmed to possess the correct characteristics and purity at the time of experimentation.

Molecular characterization of β-lactam resistance mechanisms.Molecular characterization of β-lactam resistance mechanisms was performed as JMI study 18-SPT-06.

(i) DNA extraction. Total genomic DNA of selected isolates was extracted using fully automated Thermo Scientific KingFisher Flex magnetic particle processors (Cleveland, OH, USA).

(ii) Whole-genome sequencing. Total genomic DNA was used as input material for library construction. DNA libraries were prepared using the Nextera XT library construction protocol and index kit (Illumina, San Diego, CA, USA) and sequenced on a MiSeq sequencer (Illumina) using a MiSeq reagent kit (v2, 500 cycles; v3, 600 cycles) with a minimum of 20× coverage.

(iii) DNA assembly and data analysis. FASTQ format sequencing files for each sample set were quality ensured, error corrected, and assembled independently using the de novo assembler SPAdes (v3.9.0). In-house-designed software was applied to the assembled sequences to align against the sequences of known β-lactam resistance genes. E. coli sequences of OmpC/OmpF and the respective homologues from K. pneumoniae, OmpK36/OmpK35 and OmpK37, were retrieved from FASTQ format sequencing files for each sample set and compared to those of reference, wild-type, and surveillance control isolates.

Measurement of tebipenem.Total drug concentrations of tebipenem in murine plasma were measured using an Agilent 6420 LC-MS/MS. A working solution of the internal standard, cefotaxime (Sigma-Aldrich, Dorset, UK), was prepared in methanol and acetonitrile (0.05 mg/liter in 50:50 methanol-acetonitrile) before being added to a 96-well Waters Sirocco protein precipitation plate (300 μl). Mouse plasma samples along with blanks, calibrators, and quality control samples were then aliquoted (30 μl) into the plate and mixed with the cefotaxime working solution on an orbital shaker for 2 min. Using a positive-pressure manifold, the liquid was drawn through the Sirocco plate into a collection plate. Supernatant (200 μl) from each well in the collection plate was transferred into a 96-well plate, which was then sealed and was ready for analysis by LC-MS/MS. The lower limit of quantification (LLOQ) was 0.05 mg/liter. The coefficient of variation (CV) was 0.29 to 11.55% over the concentration range of 0.05 to 10 mg/liter. The intra- and interday variation was −12.21 to 10.25%.

Murine thigh model of infection.All murine experiments were conducted under UK Home Office project license PAC022930 and approved by the Animal Welfare Ethics Review Board at the University of Liverpool. Male CD1 mice were provided by Charles River and weighed approximately 25 to 30 g at the time of experimentation. Food and water were provided ad libitum. Mice were housed in individually ventilated cages.

The challenge organism was recovered from beads that had been stored at −80°C, cultured onto Mueller-Hinton (MH) agar, and grown overnight at 37°C. A sweep of colonies was then placed in 2 × 30 ml of Mueller-Hinton broth (Sigma-Aldrich, Dorset, UK) and placed on an orbital shaker at 37°C for 4 or 24 h, depending on the species and strain. The final inoculum was prepared by centrifuging 30 ml at 1,438 × g for 5 min. The supernatant was removed and discarded. The final inoculum was dependent on the species, strain, and route of infection and was adjusted to the desired optical density with progressive dilution in sterile phosphate-buffered saline (PBS) using a spectrophotometer. The inoculum was checked with quantitative cultures.

A well-characterized neutropenic murine thigh infection model was used throughout (20, 32). Neutropenia was induced with 150- and 100-mg/kg cyclophosphamide (Baxter Healthcare Ltd., UK) administered intraperitoneally on study days −4 and −1, respectively, relative to the time of infection. Mice were inoculated while they were under temporary general anesthesia, induced with 2% isoflurane. A total of 0.05 ml per thigh of the diluted inoculum suspension was injected into each lateral thigh muscle of each mouse. Mice were anesthetized for approximately 5 min.

At 2 h postinfection, three animals were sacrificed using pentobarbitone overdose to provide a pretreatment control group. Thigh muscles (from immediately above the knee joint to the hip joint) were removed, weighed, and homogenized using a stainless steel sawtooth dispersing element (VWR, UK) in sterile PBS (2 ml). Thigh homogenates were serially diluted 10-fold in sterile PBS and plated onto Mueller-Hinton (MH) agar. Total bacterial counts were determined following overnight incubation at 37°C. The remaining mice were treated orally every 8 h (q8h) with a range of tebipenem doses. Ertapenem was administered on the same schedule. Groups of 3 mice were used for each regimen. At 26 h postinfection, the remaining mice were euthanized using a pentobarbitone overdose, and the thighs were processed in the same manner described above and used as the 2-h controls. The endpoint of the studies was the average bacterial density (number of CFU per gram of tissue) of both thighs.

Murine dose fractionation study.Male CD-1 mice were housed and rendered neutropenic as described above in the thigh model of infection. Mice were infected intramuscularly with E. coli ATCC 25922. Treatment was initiated at 2 h postinfection with total daily doses of prodrug at 5, 9, 14, and 25 mg/kg. These dosages of prodrug were determined from the dose-ranging studies (Fig. 1) and produced 20% of the maximal effect (EC₂₀), EC₄₀, EC₆₀, and EC₈₀ calculated from dose-ranging studies (Fig. 1). The doses were fractionated to be given 4 times, twice, or once a day over a period of 24 h (i.e., prodrug at 1.25 mg/kg q6h, 2.5 mg/kg q12h, and 5 mg/kg q24h, respectively, for the EC₂₀; 2.25 mg/kg q6h, 4.5 mg/kg q12h, and 9 mg/kg q24h, respectively, for the EC₄₀; 3.5 mg/kg q6h, 7 mg/kg q12h, and 14 mg/kg q24h, respectively, for the EC₆₀; and 6.25 mg/kg q6h, 12.5 mg/kg q12h, and 25 mg/kg q24h, respectively, for the EC₈₀). Groups of 4 mice were used per regimen. All mice were sacrificed at a single time point at 26 h postinfection. The study endpoint was the average bacterial density (CFU per gram of tissue) of both thighs, processed as described above.

Persistent antibacterial effect.The neutropenic murine thigh model of infection described above was used with E. coli ATCC 25922 to determine the persistent effect of tebipenem over 26 h. Treatment was intitiated at 2 h postinfection with doses of prodrug at 6.67, 16.67, and 33.33 mg/kg q24h, and ertapenem at 200 mg/kg q24h. An untreated cohort was also included. Groups of 3 mice per dosage-time point were euthanized via pentobarbitone injection at 2, 6, 10, 14, 18, 22, and 26 h postinfection. At each time point postinfection, the thighs were dissected and processed as previously described. The study endpoint was the average bacterial density (number of CFU per gram of tissue) of both thighs for each mouse.

Pharmacokinetic study.The murine pharmacokinetics were performed in immunosuppressed infected mice, using the murine thigh infection model described above with E. coli ATCC 25922. The regimens chosen for study were selected after preliminary pharmacodynamic experiments had been completed. Mice received prodrug at 3.33, 8.33, 16.67, or 33.33 mg/kg q8h over 24 h. Plasma samples for PK bioanalysis of tebipenem were taken in the 1st and 3rd dosing intervals. Mice from each cohort were euthanized in a serial sacrifice design at 0.083, 0.25, 0.5, 1, 2, 4, and 8 h postdose. Groups of 3 mice were used for each dose-time point combination. A combination of 5% isofluorane and 95% oxygen was used to anesthetize the mice for at least 5 min, until a deep plane of anesthesia was achieved. Whole blood was taken from the mice via terminal cardiac puncture using heparinized syringes at the required time postdose. Whole blood was centrifuged, and the plasma supernatant was stored at −80°C for bioanalysis.

Hollow-fiber infection model.E. coli SPT 719 (supplied by Spero Therapeutics) was used as the challenge strain against tebipenem in a hollow-fiber infection model (HFIM). The MIC was 0.03 mg/liter (Table 1). Cation-adjusted MH broth (Ca-MHB) was pumped from the central compartment through a hollow-fiber cartridge (FiberCell Systems, Frederick, MD, USA) before being returned to the central compartment. A peristaltic pump (model 205 U; Watson-Marlow, United Kingdom) was used. SPR859 (tebipenem) was administered to the central compartment via a programmable syringe driver (CME Medical, Blackpool, UK) over a 1-h period to achieve the required C_maxs. Fresh Ca-MHB was pumped into the central compartment from a peripheral reservoir. Concomitantly, drug-containing broth in the central compartment was removed (at the same rate) into a waste reservoir. The simulated half-life was 35 min.

The extracapillary space of each HFIM was inoculated with ∼40 ml of bacterial suspension, and the desired inoculum was confirmed by quantitative cultures. The HFIM system was incubated at 37°C in ambient air. The PK were estimated by sampling from the central reservoir using the same bioanalytical assay described above. Samples were taken throughout the dosing interval (the specific times depended on the schedule). Bacterial densities were determined by removing 1 ml from the extracapillary space via a sampling port. Serial dilutions were then plated onto both drug-free and drug-containing (4× tebipenem MIC at 0.125 mg/liter) Ca-MH agar to enumerate the total and resistant subpopulations.

Pharmacokinetic-pharmacodynamic modeling.The drug exposure-response relationships were modeled using an inhibitory sigmoid E_max model that took the following form:effect=Econ−Emax⋅drugexposureHEC50H+drugexposureH where effect is the bacterial burden (number of log₁₀ CFU per gram of thigh tissue), E_con is the bacterial density in the vehicle-treated controls, E_max is the maximal antibacterial effect, EC₅₀ is the drug exposure that causes a half-maximal antibacterial effect, drug exposure is the dose or the relevant pharmacodynamic index (e.g., AUC/MIC), and H is the slope function. The model was fitted to the data using both the ADAPT (version 5) and Pmetrics programs.

The PK of tebipenem were estimated using a population methodology and the Pmetrics program. The following structural model was used:XP(1)=−Ka⋅X(1) (1)XP(2)=Ka⋅X(1)−SCL/V⋅X(2)−Kcp⋅X(2)+Kpc⋅X(3) (2)XP(3)=Kcp⋅X(2)−Kpc⋅X(3) (3)with output equation Y(1)=X(2)/V

Equations 1 to 3 represent the rate of change of the mass of drug in the gut, bloodstream, and peripheral compartment, respectively. K_a is the first-order rate constant describing the absorption of drug from the gut into the central compartment, K_cp and K_pc are the respective first-order intercompartmental rate constants, SCL is the first-order clearance of drug from the central compartment, and V is the volume of the central compartment. XP(1), XP(2), and XP(3) are the rates of change of mass in compartments 1, 2, and 3, respectively. Y(1) is the output of the model.

The PD data from the experiments to describe the persistent effect were modeled using the following differential equation, which was combined with PK equations 1 to 3 above. The value of the PK parameters estimated from fitting of the PK model were fixed.dN/dt=Kgmax⋅(1−[X(2)/V]⋅Hg/{C50g⋅Hg+[X(2)/V]⋅Hg})⋅{1⋅[X(4)/popmax]}⋅N− Kkmax⋅[X(2)/V]⋅Hk/{C50k⋅Hk+[X(2)/V]⋅Hk}⋅N (4)with output equationY(1)=Dlog10[X(4)] (5)

Equation 4 describes that rate of change (dN/dt) of the number of organisms (N) in the thigh at time t, which is a balance between bacterial growth and drug-induced bacterial killing. The maximum rate of growth is given by Kgmax (number of log₁₀ CFU per gram of thigh tissue per hour), which is modulated by the drug concentrations, given by X(2)/V from equation 2 above. popmax is the maximum theoretical bacterial density. C_50g is the concentration of drug at which there is half-maximal suppression of growth, and H_g is the associated slope function. The maximum rate of drug-induced killing is given by Kkmax (number of log₁₀ CFU per gram of thigh tissue per hour), which is similarly affected by the drug concentrations. C_50k and H_k are the concentration at which the rate of killing is half maximal and the slope function, respectively.

The hollow-fiber data were modeled using the following set of inhomogeneous differential equations that enabled the PK in each cartridge to affect bacterial killing of a susceptible and resistant bacterial subpopulation.XP(1)=R(1)−SCL/[V⋅X(1)] (6)XP(2)=Kgmax_s⋅{1.D0−[X(2)/popmax]}⋅X(2)−Kkmax_s⋅[X(1)/V]⋅Hks/{C50k_s⋅Hks+[X(1)/V]⋅Hks}⋅X(2) (7)XP(3)=Kgmax_r⋅{1.D0−[X(3)/popmax]}⋅X(3)−Kkmax_r⋅[X(1)/V]⋅Hkr/{C50k_r⋅Hkr+[X(1)/V]⋅Hkr}⋅X(3) (8)with output equations: Y(1)=X(1)/V (9)Y(2)=Dlog10[X(2)+X(3)] (10)Y(3)=Dlog10[X(3)] (11)

Equation 6 represents a 1-compartment model that describes the PK of tebipenem in the hollow-fiber circuit with input of drug represented by R(1). Equations 7 and 8 represent the pharmacodynamics of susceptible and resistant subpopulations, respectively. In each case, the rate of change of the organism is modeled as growth minus drug-induced killing. The pharmacodynamic equations take the same structural form with parameters that describe the maximum rate of growth of susceptible and resistant subpopulations (Kgmax_s and Kgmax_r, respectively), the maximum rate of drug-induced killing of susceptible and resistant subpopulations (Kkmax_s and Kkmax_r, respectively), the concentrations of tebipenem where the rate of killing is half maximal (C_{50k_s} and C_{50k_r}, respectively), and the respective slope functions for the susceptible and resistant subpopulations (H_ks and H_kr, respectively). The total bacterial population consists of the sum of the susceptible and resistant subpopulations and is given in equation 10, while the resistant subpopulation is given by equation 11.