Dmd055335 932..942

Avibactam, a novel non-b-lactam b-lactamase inhibitor with activity against Ambler class A, class C, and some class D enzymes is being evaluated in combination with various b-lactam antibiotics to treat serious bacterial infections. The in vivo mass balance recovery and metabolite profile of [C] avibactam (500 mg/1-h infusion) was assessed in six healthy male subjects, and a series of in vitro experiments evaluated the metabolism and drug-drug interaction potential of avibactam. In the mass balance study, measurement of plasma avibactam (using a validated liquid chromatography-tandem mass spectrometry method) and total radioactivity in plasma, whole blood, urine, and feces (using liquid scintillation counting) indicated that most of the avibactam was excreted unchanged in urine within 12 hours, with recovery complete (>97% of the administered dose) within 96 hours. Geometric mean avibactam renal clearance (158 ml/min) was greater than the product of unbound fraction of drug and glomerular filtration rate (109.5 ml/min), suggesting that active tubular secretion accounted for some renal elimination. There was no evidence of metabolism in plasma and urine, with unchanged avibactam the major component in both matrices. Avibactam demonstrated in vitro substrate potential for organic anion transporters 1 and 3 (OAT1 and OAT3) proteins expressed in human embryonic kidney 293 cells (Km > 1000 mM; >10-fold the Cmax of a therapeutic dose), which could account for the active tubular secretion observed in vivo. Avibactam uptake by OAT1 and OAT3 was inhibited by probenecid, a potent OAT1/OAT3 inhibitor. Avibactam did not interact with various other membrane transport proteins or cytochrome P450 enzymes in vitro, suggesting it has limited propensity for drug–drug interactions involving cytochrome P450 enzymes. Introduction Infections caused by Gram-negative pathogens are becoming increasingly challenging to treat as a result of the global spread of multidrug resistance among many clinically important species (Peleg and Hooper, 2010) and a limited armamentarium of new agents with intrinsic Gram-negative activity (Boucher et al., 2009). A key facet of bacterial drug resistance, particularly among the Enterobacteriaceae, is the expression of b-lactamases, a large family of hydrolytic enzymes that confer reduced susceptibility to b-lactam antibiotics (Kanj and Kanafani, 2011). Extended spectrum b2lactamases inactivate extended-spectrum cephalosporins, as well as penicillins, and typically require treatment with a carbapenem or b2lactam/b2lactamase inhibitor combination. Recently, the emergence of molecular class A b-lactamases with carbapenemase activity (e.g., Klebsiella pneumoniae carbapenemase [KPC]) has been reported; treatment options for such infections are severely limited (Hirsch and Tam, 2010; Nordmann et al., 2011). Avibactam, formerly known as NXL104, is a novel non b-lactam inhibitor of Ambler class A and C (and some class D) b-lactamases, including KPC (Stachyra et al., 2009; Shlaes 2013; Zhanel et al., 2013) (Fig. 1). Avibactam has a unique mechanism of action involving covalent, slow, reversible b-lactamase inhibition (Ehmann et al., 2012), which restores the in vitro activity of b-lactam antibiotics, including ceftazidime, ceftaroline, and aztreonam against extended-spectrum b2lactamases–producing pathogens (Livermore The mass balance recovery and in vitro membrane transporter studies were supported by AstraZeneca. The in vitro cytochrome inhibition and induction studies and microsomal metabolism study were supported by Novexel. Ceftazidime-avibactam is now being developed by AstraZeneca and ForestCerexa. dx.doi.org/10.1124/dmd.113.055335. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: Ae, amount of total radioactivity or parent drug eliminated; AE, adverse event; AUC, area under the concentration-time curve; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; CYP450, cytochrome P450; ECG, electrocardiogram; HEK293, human embryonic kidney 293 cells; HPLC, high-performance liquid chromatography; KPC, Klebsiella pneumoniae carbapenemase; LC, liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry detection; LSC; liquid scintillation counting; MDCKII, Madin-Darby canine kidney II cells; MDR1, multidrug resistance 1 protein; MRP4, multidrug resistance-associated protein; OAT1/OAT3, organic anion transporters 1/3; OATP1B1/OATP1B3, organic anion transporting polypeptide proteins 1 and 3; OCT1/OCT2, organic cation transporters 1 and 2; PK, pharmacokinetic. 932 http://dmd.aspetjournals.org/content/suppl/2014/03/10/dmd.113.055335.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from et al., 2011). Previous investigations have demonstrated that the pharmacokinetic (PK) profile of i.v. avibactam is dose-proportional up to 2000 mg (the highest dose tested), that elimination is largely by the renal route (Felices et al., 2010; Merdjan et al., 2007, 2010), and that there are no drug-drug interactions between avibactam and ceftazidime (Edeki et al., 2013). In these studies, the amount of unchanged avibactam eliminated in urine varied between 80% and 100%. To fully understand and characterize the elimination pathways of avibactam and to understand the impact of covalent, reversible tissue binding, a [C] avibactam mass balance study was undertaken in healthy human subjects. This report describes the results of the mass balance study as well as a series of additional in vitro studies undertaken to further characterize the disposition of avibactam, including assessments of the potential of avibactam as a substrate or inhibitor of human hepatic and renal transporter proteins and cytochrome P450 enzymes. Materials and Methods Overall Study Design and Objectives The mass balance recovery study (ClinicalTrials.gov identifier NCT01448395) was an open-label, single-center phase I clinical trial conducted at Quotient Clinical, Nottingham, UK (with metabolite profiling of study samples conducted at AstraZeneca R&D, Waltham, MA). The study was performed in accordance with the Declaration of Helsinki and International Conference on Harmonization (ICH)/Good Clinical Practice (GCP) and applicable regulatory requirements, with protocol approval by an independent ethics committee; all participants provided written informed consent. The UK Department of Health Administration of Radioactive Substances Advisory Committee provided prior approval of the chosen dose of radioactivity. The primary objectives were to determine the mass balance and routes of [C] avibactam metabolism and excretion after a single 500-mg i.v. dose of [C] avibactam (this dose of avibactam is currently being investigated in combination with ceftazidime in phase III trials), to estimate the whole blood and plasma partitioning of total radioactivity, and to determine the urine and fecal recovery of radioactivity. Secondary objectives were to assess the PK of avibactam; identify and characterize avibactam metabolites in plasma, whole blood, urine, and feces; and obtain additional safety and tolerability information for avibactam. The in vitro studies comprised a human membrane transporter study (conducted at Netherlands Organisation for Applied Scientific Research, Zeist, Netherlands), two human cytochrome inhibition studies (respectively conducted at Biopredic, Saint Grégoire, France, and GenPharmTox Biotech AG, Planegg, Germany); a human cytochrome induction study (conducted at GenPharmTox Biotech AG), and a hepatic metabolism study using microsomes from various species (conducted at Biopredic). The studies were designed with reference to industry guidelines and carried out in accordance with relevant standards of the U.S. Food and Drug Administration (www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf), the Organisation for Economic Co-Operation and Development (http://search.oecd.org/ officialdocuments/publicdisplaydocumentpdf/?cote=ENV/MC/CHEM(98)16&docLanguage=En), and the International Transporter Consortium (Giacomini et al., 2010). The objective of the membrane transporter study was to evaluate the substrate and inhibitor potential of avibactam for a range of human transporter proteins expressed in cultured human and canine cells or human vesicles. The objectives of the cytochrome inhibition and induction and microsomal metabolism studies were to evaluate the possible hepatic metabolism and drug interaction potential of avibactam on hepatic drug-metabolizing enzymes. Mass Balance Study Subjects and Treatments. Healthy male subjects aged 30–65 years, with body mass index between 18 and 32 kg/m and body weight between 50 and 100 kg, were eligible to participate; volunteers were required to have a clinically normal physical examination (including negative test results for drugs of abuse, alcohol, carbon monoxide breath test, hepatitis B surface antigen, antibodies to hepatitis C virus, and antibodies to HIV at the screening visit) and had to be willing to use an adequate method of contraception for 3 months from the day of dosing with the study drug. Exclusion criteria included clinically significant disease or clinically relevant abnormal findings in physical examination, vital signs, clinical chemistry, hematology, or urinalysis that, in the investigator’s opinion, could put the volunteer at risk by participation in the study; QT interval corrected for heart rate .450 ms or QT . 500 ms or other electrocardiogram (ECG) abnormality; and history of drug or alcohol abuse or smoking. After an overnight fast of $10 hours, subjects received a single clinically relevant dose of [C] avibactam (Quotient Bioresearch Ltd, Nottingham, UK) of approximately 500 mg diluted in 100 ml of 0.9% saline by 60 minutes of i.v. infusion. The target dose of C was to be no more than 300 mCi (11.1 MBq). Assessments. Venous blood samples for analysis of whole blood and plasma radioactivity and plasma avibactam concentrations were collected via venipuncture/ indwelling catheter at predose and at 0.25, 0.5, 1, 1.25, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 16, 24, 36, 48, 60, 72, and 96 hours postdose. Urine and feces samples for analysis of total radioactivity and avibactam were collected at predefined intervals (urine:212–0 hours predose and 0–2, 2–4, 4–8, 8–12, 12–24, 24–48, 48–72, and 72–96 hours postdose; feces:224–0 hours predose and 0–24, 24–48, 48–72, and 72–96 hours postdose). Subjects were discharged from the study unit at 96 hours, with scheduled assessments planned to continue to up to 168 hours (240 hours for feces) if required. Concentrations of avibactam in plasma and urine were measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for which the limits of quantification were 10.0 ng/ml (plasma) and 500 ng/ml (urine). Whole blood, plasma, and urine total radioactivity concentrations were measured using a liquid scintillation counting (LSC) method (feces samples were first combusted and the resultant CO2 collected into scintillation fluid before counting). The limits of detection for total radioactivity were 0.028 mg equiv/ml (plasma), 0.089 mg equiv/g (whole blood), 0.012 mg equiv/g (urine), and 0.07 mg equiv/g (feces). Safety assessments included analysis of adverse events (AEs; coded using MedDRA version 14.1), laboratory assessments of blood and urine, vital signs, ECG, and physical examinations. Metabolite Profiling. Plasma samples obtained during the first 4 hours postdose were pooled across subjects in a time-interval proportional fashion (Hamilton et al., 1981) such that the final pool represented the area under the curve (AUC) over the 4-hour postdose period. Pooled plasma was denatured and precipitated with a 2-fold excess volume of acetonitrile to extract drugrelated material. The supernatant obtained after centrifugation was evaporated and reconstituted in water before analysis. Similarly, urine samples obtained during the 0to 24-hour collection interval were pooled across subjects. The volume of a given sample represented in the pool was weighted based on the net weight of urine collected for the time interval. The pooled urine was analyzed without further processing. LC-MS using LTQ-Orbitrap was used to analyze avibactam and its related components in pooled plasma and urine samples (see Supplemental Methods). Fig. 1. Chemical structure of avibactam showing the location of the C radiolabel used in the mass balance recovery study. Avibactam Mass Balance and Drug-Drug Interaction Studies 933 at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from Inline radiochromatographic data were collected using a b-RAM detector where the LC flow was split 1:9 between the MS and b-RAM detector. The LC flow to the b-RAM was complemented with IN-FLOW scintillant at a rate of 1.6 ml/min throughout the first 40 minutes of the chromatographic analysis (see Supplemental Methods on radiochromatographic monitoring). Total radioactivity of samples before extraction or analysis, and of column effluent in recovery experiments, was measured using LSC. Recovery of radioactivity from the LC column during a gradient run was determined using the same LSC method. An aliquot of sample before injection was analyzed by LSC. Another aliquot of the same sample was then injected onto the LC system, and the column effluent was collected quantitatively into a volumetric cylinder. Aliquots of the column eluate were likewise analyzed by LSC and compared with preinjection samples. Control human plasma, which was fortified with [C] avibactam after extraction, and [C] avibactam dose solution were also analyzed to assess possible degradation during sample processing. Statistical Analysis. No formal sample size calculation was performed. A planned sample size of six subjects was chosen based on previous experience with mass balance studies. PK parameter estimates and analyses of whole blood and plasma radioactivity were performed for all subjects who received the complete dose of [C] avibactam and who had sufficient concentration data (plasma, whole blood, urinary, and fecal) for mass balance determination and PK parameter estimation (PK population). Safety data were summarized for all enrolled subjects who received any fraction of the dose of [C] avibactam (safety population). Summary statistics were prepared using Statistical Analysis System software (v. 8.2; SAS Institute Inc., Cary, NC); however, no formal statistical analysis was planned. PK parameters were estimated usingWinNonlin (v. 6.1, Pharsight Corporation, St. Louis, MO) except for the amount of total radioactivity or parent drug eliminated (Ae), which was calculated using Statistical Analysis System software. In Vitro Studies Membrane Transporter Study. Assay systems to evaluate the inhibition and substrate (uptake) potential of avibactam for human membrane transporters were developed using transporter protein cDNA stably transfected into Madin-Darby canine kidney II (MDCKII) cells, human embryonic kidney (HEK293) cells, or human membrane vesicles expressing native transporter protein. Avibactam substrate and inhibition potential for breast cancer resistance protein (BCRP) and multidrug resistance 1 protein (MDR1) were assessed with transporters expressed in MDCKII cells. Human multidrug resistance-associated protein (MRP4), organic anion transporting polypeptide proteins 1 and 3 (OATP1B1 and OATP1B3), organic anion transporters 1 and 3 (OAT1 and OAT3), and organic cation transporters 1 and 2 (OCT1 and OCT2) expressed in HEK293 cells were assessed for avibactam inhibition potential. In addition, MRP4, OAT1, OAT3, and OCT2 expressed in HEK293 cells were assessed for avibactam substrate potential. Inhibition potential of avibactam for human bile salt export pump (BSEP) was assessed in membrane vesicles isolated from cells overexpressing BSEP and control membrane vesicles (see Supplemental Methods). Inhibition potential of avibactam was assayed by measurement of radiolabeled model substrate uptake in the presence of avibactam (AstraZeneca, Macclesfield, UK). Substrate potential of avibactam was assessed by measuring the bidirectional transport (MDCKII cells) or uptake (HEK293 cells) of [C] avibactam in the absence or presence of the following model inhibitors: MDR1, ketoconazole (Sigma-Aldrich, Zwijndrecht, Netherlands); BCRP, Ko143 (Allen et al., 2002; synthesized by Prof. G. J. Koomen, Van’t Hoff Institute for Molecular Sciences, Netherlands/Tocris Bioscience, Bristol, UK); MRP4, dipyridamole; OAT1 and OAT3, probenecid; OCT2, quinidine (all from Sigma-Aldrich). Empty vectors treated with test substances were used as negative controls. Effects of avibactam at concentrations of 0.5–300 mM on cytotoxicity or monolayer cell viability were assessed for the MDCKII and HEK293 control cells using a neutral red uptake assay (Borenfreund and Puerner, 1985) with Triton-X100 (1%) as positive control. Cytochrome P450 Inhibition Studies. The in vitro potential of avibactam to inhibit the activity of human cytochrome P450 isoforms 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4/5 was investigated in human liver microsomes (see Supplemental Table 1 and Supplemental Methods). In one study, microsomes (individual and pooled samples from 15–30 donors) were preincubated with avibactam with and without NADPH (2 mM) for 20 minutes to evaluate timedependent inhibition of cytochrome enzymes. Parallel incubations were done with reference inhibitors as positive controls. Since a minor inhibition of CYP2C9 was observed in the first study, a further study was conducted using a similar method to evaluate the potential of avibactam to inhibit CYP2C9 activity at concentrations up to 5000 mM in pooled microsomes from 50 donors with preincubation with NADPH (2 mM) for 10 minutes. Enzymatic activities were determined by measuring the concentration of formed metabolite using high-performance liquid chromatography (HPLC) with ultraviolet detection method for all enzymes except CYP2A6, which was determined by fluorimetry. Cytochrome P450 Induction Study. The in vitro induction potential of avibactam on the catalytic activity of the human hepatic cytochrome isoforms 1A2, 2B6, 2C9, 2E1, and 3A4 was investigated in freshly isolated human hepatocytes from three donors (see Supplemental Table 2 and Supplemental Methods). The induction potential of avibactam was tested at 200–5000 mM, TABLE 1 Key pharmacokinetic (PK) parameters for total radioactivity and avibactam after a single intravenous infusion of [C] avibactam (PK population; n = 6) Terminal slopes (lz) for radioactivity in whole blood could not be reliably defined for any subject and could be defined for only one subject for plasma radioactivity but were reliably defined for plasma avibactam; hence, all lz-dependent parameters (lz, t1/2, AUC0–‘, MRT, Vss, and CLT) were estimated only for plasma avibactam. Parameter Total Radioactivity in Plasma Total Radioactivity in Whole Blood Avibactam in Plasma Cmax (mg/ml) c 22.4 (17.4) 14.6 (15.2) 23.1 (11.1) tmax (h) 1.0 (1.0–1.0) 1.0 (1.0–1.0) 1.0 (1.0–1.0) AUC0–t (mg.h/ml) d 47.6 (22.7) 36.2 (24.0) 47.10 (20.0) AUC0–‘ (mg.h/ml) d NC NC 47.20 (20.0) t1/2 (h) NC NC 2.778 (0.6) MRT (h) NC NC 1.887 (16.0) Vss (ml) NC NC 21 200 (10.7) CLT (ml/min) NC NC 11 200 (18.6) CLr (ml/min) NC NC 158.0 (21.2) Total radioactivity in urine Total radioactivity in feces Avibactam in urine Fe 0–96 h (%) 97.0 (1.0) 0.2 (0.03) 84.9 (11.8) AUC0-‘, area under the concentration-time curve from dosing extrapolated to infinity; AUC0-t, area under the concentration-time curve from dosing to the last measurable time point; CLr, renal clearance; CLT, total clearance; Cmax maximum (peak) concentration; Fe, amount of total radioactivity or parent drug eliminated expressed as a percentage of the dose administered; MRT, mean residence time; NC, not calculated; t1/2, terminal half-life; tmax, time to reach Cmax; Vss, volume of distribution at steady state. Values shown are geometric mean (%CV) except for tmax, which are median (range), and t1/2 and Fe%, which are arithmetic mean (S.D.) Determined by liquid chromatography tandem mass spectrometry Units are mg equiv/ml for total radioactivity in plasma and mg equiv/g for total radioactivity in whole blood. Units are mg equiv.h/ml for total radioactivity in plasma and mg equiv.h/g for total radioactivity in whole blood. 934 Vishwanathan et al. at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from expected to cover therapeutic concentrations and span a safety margin .50-fold the Cmax of a 500-mg dose. Hepatocytes were incubated in 0.5 ml of hepatocyte incubation medium at 37 6 2°C for 72 6 4 hours (induction phase) with avibactam, a fixed concentration of reference inducer (positive controls), or hepatocyte incubation medium only (negative controls). After completion of the induction phase, supernatants were removed and hepatocytes were incubated in Krebs’ Henseleit buffer containing 3 mM salicylamide and marker substrate for 3 hours 6 15 minutes (reaction phase). Proteins were then precipitated in acetonitrile, and substrate metabolites (markers of enzyme activity) were analyzed by assay-specific HPLC and fluorimetry methods. Fig. 2. Geometric mean concentrations of avibactam in plasma (determined using LC-MS/MS detection) and total C radioactivity in plasma and whole blood after a single i.v. infusion of 500 mg of [C] avibactam in healthy male subjects (PK population; n = 6). Error bars represent the interval of geometric mean/ geometric S.D., geometric mean geometric mean S.D. Fig. 3. Mean cumulative recovery of avibactam and total radioactivity in urine as a percentage of the dose administered (Fe%) after a single i.v. infusion of 500 mg of [C] avibactam in healthy male subjects (PK population; n = 6). Error bars represent S.D. Fig. 4. Representative LC radiochromatograms (A) 0to 4-hour pooled human plasma after a single i.v. infusion of 500 mg of [C] avibactam in healthy male subjects (PK population; n = 6) and (B) control human plasma fortified with [C] avibactam. Avibactam Mass Balance and Drug-Drug Interaction Studies 935 at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from