Microdialysis Study of Aztreonam-Avibactam Distribution in Peritoneal Fluid and Muscle of Rats with or without Experimental Peritonitis

Chemicals.Aztreonam (Azactam; Sanofi-Aventis Laboratories, Paris, France) and avibactam (provided as a dry powder by Astrazeneca, Macclesfield, United Kingdom) were used to prepare ATM-AVI solutions in 0.9% NaCl or Ringer solution for intravenous (i.v.) administration and probe perfusion, respectively. All chemicals used were of analytical grade.

Animals.Experiments were carried out in accordance with EC Directive 2010/63/EU. They were approved by the local ethics committee (COMETHEA) and registered by the French Ministry of Higher Education and Research (authorization number 2016011111381822). Fourteen male Sprague-Dawley rats from Charles River Laboratories (Saint-Germain-Nuelles, France) weighing 291 ± 16 g (mean ± the standard deviation [SD]) were used and divided into two groups: a peritonitis group (n = 9) and a control group (n = 5). All animals were acclimatized in ventilated rack in temperature regulated environment with a 12-h light-dark cycle, with free access to food and water for a minimum of 5 days before the beginning of the experiment.

Surgery. (i) Catheter, vein and muscle probes insertion.The day before the experiment, rats received subcutaneous (s.c.) administration of buprenorphine at a dose of 0.05 mg · kg⁻¹ before being anesthetized by isoflurane (Forene; Abbot, Rungis, France) inhalation (3% in inhalation chamber, followed by 1% under mask). A polyethylene cannula was then inserted into the left femoral vein for drug administration, as previously described (25). Two CMA/20 probes (polycarbonate; cutoff, 20,000 Da; membrane length, 10 mm; CMA microdialysis; Phymep, Paris, France) were inserted into the right jugular vein and the right hind leg muscle, as previously described too (25). In brief, the microdialysis CMA/20 probe, perfused with 1% low-molecular-weight heparin at a flow rate of 2 μl · min⁻¹ (CMA 100 microdialysis pump; Phymep), was inserted through the pectoral muscle into the right jugular vein with the help of an introducer (needle inserted into a tube) and then secured by suturing it to the pectoral muscle. The probe, perfused with Ringer solution (perfusion fluid T1 for peripheral tissue; CMA microdialysis; Phymep) was inserted into the right hind leg muscle as previously described and finally secured by suturing it to the muscle. After insertion, the microdialysis probes were flushed at 10 μl · min⁻¹ for approximately 15 min to remove bubbles. The flow rate was then decreased to 2 μl · min⁻¹ until the end of the surgery. The inlet and the outlet of probes as the femoral catheters were then passed subcutaneously to exit at the nape.

(ii) Induction of peritonitis.Directly after installation of vein catheter and muscle microdialysis probe, peritonitis was induced in rats of the infected group, as previously described (31, 32). Briefly, after a laparotomy, the cecum was ligated just below the ileocecal valve, punctured three times below the ligature, and kneaded to get the contents out before being placed back into the peritoneal cavity. The abdomen was closed and, at the end of surgery, the rats were allowed to recover consciousness. Food was withdrawn approximately 12 h before the experiment, but the animals had free access to water and, at this time, they received a second s.c. injection of buprenorphine at the same dose as previously.

(iii) Intraperitoneal probe implantation.On the day of the experiment, the rats were reanesthetized by isoflurane (Forene; Abbot) inhalation. Another CMA/20 probe, perfused with Ringer solution (perfusion fluid T1 for peripheral tissue; CMA microdialysis; Phymep), was inserted into the rat peritoneal cavity between intestinal loops through laparotomy, as previously described (16). After insertion, the intraperitoneal microdialysis probe was flushed as previously done for the muscle probe. The probe was then sutured to the abdominal muscle, and the abdominal cavity was closed.

Pharmacokinetic study. (i) Recovery calculations.The pharmacokinetic experiment started with a retrodialysis by drug period, during which probes were perfused for 15 min at 2 μl · min⁻¹ and then for 45 min at 1 μl · min⁻¹ with Ringer containing ATM-AVI (10 and 10 μg · ml⁻¹) to equilibrate the system. After this equilibration period, microdialysate samples were collected for 1 h by fractions corresponding to 20-min intervals. To determine the in vivo recovery by loss (RL_in vivo), the ATM and AVI concentrations in the perfusate (C_in) and in dialysates (C_out) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The RL_in vivo was expressed as a percentage and was calculated for each interval of time as follows: RL_in vivo = [(C_in − C_out)/C_in] × 100. The in vivo recovery used to correct the dialysate concentrations was the mean value obtained from the three individual determinations. A washout period of 1 h (15 min at 2 μl · min⁻¹ and 45 min at 1 μl · min⁻¹) with blank Ringer solution perfusion was allowed before i.v. ATM-AVI administration to remove the ATM and AVI from the probes. The flow rate was maintained at 1 μl · min⁻¹ for the rest of the study.

(ii) ATM-AVI administration.The aztreonam-avibactam combination was administered as an i.v. bolus at a dose of 100-25 mg · kg⁻¹.

(iii) Microdialysis experiment.In each group, 10 min after the beginning of the i.v. ATM-AVI administration (the time needed to flush the dead volume), the dialysates in muscle, peritoneal fluid (PF), and blood were collected automatically by a Univentor/820 microfraction collector (Phymep) over 120 min at 10-min intervals. At the end of the experiment, two blood samples were collected via an intracardiac puncture, one in a tube containing fluoride and one in a heparinized tube, and then centrifuged at 1,000 × g for 10 min at 4°C. The supernatants were used for lactate and glucose determinations.

Microdialysis sample analysis.Analysis of ATM and AVI in the dialysates was performed by an adaptation of two separate LC-MS/MS methods, previously described and developed for the quantification of ceftazidime and avibactam in human plasma samples (36). Calibration curves were established in Ringer solution over 0.05 to 10 μg · ml⁻¹ (AVI) and 0.1 to 200 μg · ml⁻¹ (ATM). Directly after collection, microdialysates were diluted (1:3 [vol/vol]) with Ringer solution and were directly subjected to onto LC-MS/MS. The system included a Shimadzu high-performance liquid chromatography system module (Nexera XR; Shimadzu, Marne la Vallée, France) coupled with an API 3000 mass spectrometer (Sciex, Les Ulis, France). ATM was analyzed on an XBridge C₁₈ column (3.5 μm, 50 by 2.1 mm [inside diameter]; Waters, Saint-Quentin en Yvelines, France). The mobile phase A consisted of water with 0.05% trifluoroacetic acid, and mobile phase B was acetonitrile with 0.05% trifluoroacetic acid. The gradient for mobile phase B was set at 5, 90, and 5% at 0.01, 1, and 3 min, respectively, with a flow rate of 0.3 ml · min⁻¹. AVI was analyzed on an Acquity BEH amide column (1.7 μm, 50 by 2.1 mm [inside diameter]; Waters), and a mobile phase composed of 100 mM ammonium formate and acetonitrile (5:95 [vol/vol]) was delivered isocratically at 0.2 ml · min⁻¹. Electrospray ionization in negative mode was used for the detection of ATM and AVI. Ions were analyzed in the multiple reaction monitoring, and the following transitions were inspected: m/z 434.1→122 for ATM, m/z 440.1→122 for its deuterated internal standard, and m/z 264.1→96 for AVI and m/z 270.1→96 for the AVI labeled internal standard. The intraday variability was characterized at four concentration levels (200, 5, 0.2, and 0.1 μg · ml⁻¹ for ATM; 10, 2, 0.1, and 0.05 μg · ml⁻¹ for AVI) with a precision and bias of <10% for both compounds (n = 5 per molecule). Corresponding between-day variability was determined with a precision and a bias of <10% (n = 15).

Glucose and lactate determinations.Glucose and lactate concentrations were measured, respectively, by enzymatic and colorimetric methods in Cobas 8000 device (Roche Diagnostics, Meylan, France). Glucose levels could be determined in only 11 of 14 rats (2 control rats and 9 infected rats) due to technical issues.

Noncompartmental pharmacokinetic analysis.Pharmacokinetic parameters were determined in each individual rat by a noncompartmental approach according to standard procedures and with the software Phoenix WinNonLin 6.2 (Certara, Princeton, NJ). The total unbound body clearance (CL_u) was calculated as CL_u = dose/AUC_u,blood, where AUC_u,blood is the total area under the unbound blood concentration-versus-time curve from zero to infinity, calculated by the trapezoidal rule. The area remaining under the curve after the last measured concentration, C(last)_u,blood, was determined from C(last)_u,blood/k_el,blood. The elimination rate constant (k_el,blood) and its corresponding half-life (t_1/2,blood) were estimated by a least-squares fit of the data points (log concentration-time) in the terminal phase of the decline. The volume of distribution (V_u) was obtained from CL_u/k_el,blood. The AUC and t_1/2 in tissues were also estimated by the same procedure and are referred as AUC_muscle, AUC_PF, t_1/2,muscle, and t_1/2,PF.

Statistical analysis.Concentrations were expressed as means ± the standard deviations (SD). However, when probe recoveries were lower than 10% in some tissues, concentrations were excluded from the analysis since small recovery values result in large errors in the estimation of the concentrations. Comparisons of AUC and t_1/2 estimated in blood, muscle and PF were performed for each group of rats by a nonparametric Kruskal-Wallis test. The AUC, t_1/2, CL_u, V_u, AUC_muscle/AUC_u,blood ratio, AUC_PF/AUC_u,blood ratio, and lactate and glucose concentrations between the two groups were compared by a Mann-Whitney test. Significance was set at a P level of <0.05.