Ureaplasma

The purpose of these studies was to establish human-simulated regimens of cefepime and taniborbactam in the murine pneumonia model equivalent to clinical doses of 2 and 0.5 g, respectively, administered every 8 h as 4 h infusions based on the unbound (free) plasma exposures. Following the selection and the confirmation of the cefepime and taniborbactam human-simulated regimens, additional studies were conducted to quantify the plasma and bronchopulmonary exposures achieved following the administration of the taniborbactam dosages utilized in the dose-ranging studies in combination with the cefepime human-simulated regimen.
Cefepime pharmacokinetic data in healthy adult volunteers collected in Phase I studies upon taniborbactam co-administration^{8 (link)} and cefepime and taniborbactam pharmacokinetic parameters in the murine model were utilized for simulation. Cefepime protein-binding percentages utilized in the simulations were 20% and 0% in humans and mice, respectively,^{9 (link)} while taniborbactam protein-binding percentages were 0% and 19.4% in humans¹⁰ and mice,^{11 (link)} respectively.
Infected mice (6–10 groups of six mice each) received the estimated human-simulated regimen of cefepime as monotherapy or in combination with that of taniborbactam or fractions of the established taniborbactam human-simulated regimen: 1.56% or 12.5% of the taniborbactam human-simulated regimen doses (equivalent to 7.8 or 62.5 mg every 8 h as 4 h infusion, respectively).
At 6–10 different timepoints, groups of six mice were euthanized by CO₂ asphyxiation followed by blood collection via intracardiac puncture and cervical dislocation. Following blood collection, but prior to cervical dislocation, bronchoalveolar lavage (BAL) fluid was collected from the mice at the same timepoints using methods previously described.¹² Formic acid in water (2% v/v) was added to BAL samples (equal parts) prior to freezing. All sample tubes were stored at −80°C until drug and urea concentration determination. Cefepime, taniborbactam and urea concentrations in plasma and BAL fluid were assayed by either Keystone Bioanalytical, Inc. (North Wales, PA, USA) or by Venatorx Pharmaceuticals, Inc. using qualified LC-MS/MS methods.
Cefepime and taniborbactam concentrations in the epithelial lining fluid (ELF) were estimated by correcting the drug concentration in BAL fluid for the dilution with NS during lavage using the following formula^{13 (link)}:
Compound_ELF = Compound_BAL × (Urea_plasma/Urea_BAL), where Compound_BAL is the measured concentration of either cefepime or taniborbactam in the BAL fluid sample and Urea_plasma and Urea_BAL are the concentrations of urea in paired plasma and BAL fluid samples from each mouse, respectively.
Statistical outliers for each respective analyte were removed by the IQR method. A pharmacokinetic model was fitted to the plasma and ELF concentrations of each of cefepime and taniborbactam and the best-fit estimate parameters were determined by the non-linear least-squares techniques (WinNonlin, Version 8.3, Pharsight Corp., Mountain View, CA, USA). Compartment model selection was based on visual inspection of the fit and comparison of model diagnostics.
These parameters were utilized to estimate the plasma and ELF exposures and the ELF penetration ratios as the ratio of the ELF AUC_0–24 to unbound (free) plasma AUC_0–24.

Taniborbactam and cefepime concentrations were assayed using validated LC-MS/MS methods. The lower limit of quantitation (LLOQ) was 100 ng/mL for both taniborbactam and cefepime in plasma, and 5.00 ng/mL and 3.00 ng/mL for taniborbactam and cefepime, respectively, in ELF. Assay accuracy and precision were demonstrated for all assays in the validations.^{19 (link)} Urea concentrations in plasma and BAL fluid were determined by Keystone Bioanalytical, Inc. (North Wales, PA, USA) using validated LC-MS/MS methods.
The ELF volume and drug concentration were calculated by the urea dilution method and described below:^{20 (link)} $$V_{\mathrm{ELF}}=V_{\mathrm{BAL}}\times (\mathrm{Ure}{\mathrm{a}}_{\mathrm{BAL}}//\mathrm{Ure}{\mathrm{a}}_{\mathrm{PLASMA}}),$$ where V_ELF is the volume of ELF sampled by BAL, V_BAL is the volume of BAL fluid recovered from aspiration, Urea_BAL is the concentration of urea in the BAL fluid and Urea_PLASMA is the concentration of urea in the plasma. The concentration of the antibiotic in ELF (ABX_ELF) was determined by the following relationship: $$\mathrm{AB}{\mathrm{X}}_{\mathrm{ELF}}=\mathrm{AB}{\mathrm{X}}_{\mathrm{BAL}}\times (V_{\mathrm{BAL}}//V_{\mathrm{ELF}}),$$ where ABX_BAL is the concentration of the antibiotic determined in the BAL fluid sample.
The number of AM within the BAL fluid was determined from the mean proportion of histiocytes and monocytes present in two manual cell counts, and the total volume of these cells in the cell pellet was calculated by using a mean AM cell volume of 2.42 µL/10⁶ cells.^{21 (link)} To determine the total amount (Amt) of drug in the cell pellet, the following equation was used: $$\mathrm{Am}{\mathrm{t}}_{\mathrm{PELLET}}=\mathrm{AB}{\mathrm{X}}_{\mathrm{PELLET}}\times {\mathrm{V}}_{\mathrm{PELLET}},$$ where ABX_PELLET is the concentration of drug within the reconstituted cell pellet and V_PELLET is the volume used to reconstitute the cell pellet. The amount of drug in the AM could then be calculated using the following equation: $$\mathrm{Dru}{\mathrm{g}}_{\mathrm{AM}}=\mathrm{Am}{\mathrm{t}}_{\mathrm{PELLET}}//(\mathrm{mean}\mathrm{AM}\mathrm{in}\mathrm{BAL}\times V_{\mathrm{AM}}),$$ where V_AM is the mean cell volume of an AM (2.42 µL/10⁶ cells). AM drug concentrations were determined for taniborbactam only.
Taniborbactam and cefepime plasma pharmacokinetics were described by non-compartmental methods using Phoenix WinNonlin (Certara, Princeton, NJ, USA). Estimated pharmacokinetic parameters included the maximum concentration (C_max), AUC over the dosing interval (AUC_0–8) calculated using the linear/log trapezoidal rule, the free drug exposure (fAUC_0–8) calculated by correcting concentrations for protein binding, the volume of distribution at steady state (V_ss), and CL. PK parameters in ELF (C_max, T_max and AUC_0–8 by linear/log trapezoidal rule) were calculated manually based on mean ELF concentrations. Drug penetration was estimated by the ratio of the AUC_0–8 for ELF or AM to the mean fAUC_0–8 in plasma.