Pharmacokinetics, Safety, and Tolerability of Cefiderocol, a Novel Siderophore Cephalosporin for Gram-Negative Bacteria, in Healthy Subjects

ABSTRACT Cefiderocol is a novel parenteral siderophore cephalosporin that shows potent efficacy against various Gram-negative bacteria, including carbapenem-resistant strains, in vitro and in preclinical models of infection. The aim of the present study was to evaluate the pharmacokinetics (PK), safety, and tolerability of cefiderocol after both single and multiple dosing by intravenous infusion over 60 min in healthy adult subjects. A single-ascending-dose study at doses of 100, 250, 500, 1,000, and 2,000 mg was conducted in 40 healthy Japanese males and females (6 individuals receiving the active drug and 2 individuals receiving a placebo per cohort). A multiple-ascending-dose study at doses of 1,000 (two groups) and 2,000 mg every 8 h (q8h) was conducted in 30 healthy Japanese and Caucasian males (8 individuals receiving the active drug and 2 individuals receiving a placebo per cohort). There were no serious or clinically significant adverse events (AEs) observed in either study. A single subject receiving 1,000 mg cefiderocol q8h was withdrawn due to AEs. Dose-proportional increases in the maximum plasma concentration (Cmax), the area under the concentration-time curve (AUC) from time zero to the time of the last quantifiable concentration after dosing, and the area under the concentration-time curve extrapolated from time zero to infinity were observed across the dose range of 100 to 2,000 mg. The mean plasma half-life of cefiderocol was 1.98 to 2.74 h. Cefiderocol was primarily excreted unchanged in the urine (61.5% to 68.4% of the dose). There was little accumulation of Cmax and AUC by dosing q8h, and the PK of cefiderocol did not change with multiple dosing. This study indicates that single and multiple intravenous doses of cefiderocol at up to 2,000 mg are well tolerated in healthy subjects and exhibit linear PK at doses up to 2,000 mg.

T here is an urgent need to develop new antibiotics to combat the recent worldwide increase in the incidence in multidrug-resistant Gram-negative bacterial strains (1). In particular, it is becoming more challenging to treat serious nosocomial infections caused by Gram-negative pathogens, such as Pseudomonas aeruginosa and Acinetobacter baumannii, as well as carbapenem-resistant Enterobacteriaceae (2). The 2017 global priority pathogens list from the World Health Organization has carbapenemresistant A. baumannii and P. aeruginosa and carbapenem-resistant, third-generation cephalosporin-resistant Enterobacteriaceae as the three pathogens with the highest priority for the research and development of new antibiotics (3). The mechanisms responsible for carbapenem resistance in Gram-negative bacteria include the spread of exogenous carbapenemases (4,5), overexpression of efflux pumps, overexpression of chromosomal ␤-lactamases, and a deficiency in outer membrane porins (4,(6)(7)(8).
Cefiderocol (also known as S-649266) is a novel catechol-substituted siderophore cephalosporin with potent in vitro and in vivo activity against a variety of Gramnegative bacteria, including carbapenem-resistant Enterobacteriaceae, P. aeruginosa, and A. baumannii (9)(10)(11)20). The catechol moiety, which is found at the three-position side chain, forms a chelating complex with ferric iron (12). This allows cefiderocol to function as a siderophore and enter bacterial cells through active transport via a Trojan horse mechanism that utilizes the bacterial iron transport system (12). In an in vitro study in P. aeruginosa, the iron-mediated uptake of cefiderocol was shown to contribute to its antibacterial activity (12).
No siderophore antibiotics have been approved for clinical use to date, although naturally occurring and synthetic siderophore-conjugated antibiotics have been under investigation for several decades (13,14). Cefiderocol is the first siderophore antibiotic to advance into late-stage development (15,16). The primary objective of the present study was to evaluate the pharmacokinetics (PK), safety, and tolerability of single-and multiple-dose administration of cefiderocol by intravenous infusion in healthy Japanese and Caucasian adult subjects. A secondary objective was to evaluate the production of any cefiderocol metabolites.

Subjects.
A total of 70 subjects were enrolled in the study (40 in the single-dose study, 30 in the multiple-dose study). Of these, 54 received cefiderocol and 16 received placebo. Table 1 shows the demographics and baseline characteristics of all dosed subjects. The data obtained from all subjects receiving cefiderocol were included in the PK analysis. Safety and tolerability. Cefiderocol was generally well tolerated in the single-dose and multiple-dose studies (Tables 2 and 3).
In the single-dose study, nine adverse events (AEs) reported by six subjects in the cefiderocol groups were considered possibly or probably related to study treatment: diarrhea (two events in two subjects), rash (two events in two subjects), and one event each of abdominal pain, blood present in urine, urine positive for red blood cells, white blood cell count increased, and urine positive for white blood cells. In the placebo group, four AEs reported by three subjects were considered possibly related to study treatment (one event each of diarrhea, dizziness, nausea, and urine positive for white blood cells).
In the multiple-dose study, 16 AEs reported by seven subjects in the cefiderocol 1,000-mg 1st group were considered possibly or probably related to the study treatment: rash (5 events in five subjects), a blood thyroid-stimulating hormone (TSH) level increase (3 events in three subjects), pyrexia (2 events in two subjects), and 1 event each of an alanine aminotransferase level increase, an aspartate aminotransferase level increase, a blood TSH level decrease, a blood urea level increase, urine present in blood, and headache. In the cefiderocol 1,000-mg 2nd and 2,000-mg groups, 22 AEs reported by 12 subjects were considered possibly or probably related to the study treatment: an alanine aminotransferase level increase (4 events in four subjects), an aspartate aminotransferase level increase (3 events in three subjects), diarrhea (3 events in two subjects), rash (3 events in two subjects), pyrexia (2 events in two subjects), a white blood cell count increase (2 events in two subjects), and 1 event each of abdominal pain, a blood creatine phosphokinase level increase, headache, oropharyngeal pain, and urine positive for white blood cells. TSH abnormalities were reported only in the 1,000-mg 1st group.
In the multiple-dose study, there was a higher frequency of rash (five events in five of eight subjects [62.5%]) in the 1,000-mg 1st group than in the 1,000-mg 2nd (no events) and 2,000-mg groups (three events in two of eight subjects [25%]). Allergy tests conducted for the two subjects with rash in the 2,000-mg group showed levels almost within normal ranges, and measurement of cefiderocol-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) yielded nondetectable levels.
All of the AEs were mild in intensity, except for one AE that was moderate (pyrexia in the 1,000-mg 1st group). There were no deaths, serious AEs, abnormal 12-lead or  continuous electrocardiogram (ECG) findings, or abnormal changes in vital signs, except in the subjects in the multiple-dose study with pyrexia. One subject in the cefiderocol 1,000-mg 2nd group withdrew due to pyrexia on the last day of study drug administration. There was no dose-response trend in the incidence of AEs, which were relatively evenly spread among the dose groups, including the placebo groups. Blood iron and total iron-binding capacity levels. In the single-dose study, the mean value of blood iron was slightly below the lower limit of normal (LLN) (reference range, 80 to 199 g/dl for males, 70 to 179 g/dl for females) in the 500-mg group on day 5 (71.2 g/dl) and day 8 (68.3 g/dl) and in the 1,000-mg group on day 8 (76.8 g/dl); no mean value below the LLN was observed in the 2,000-mg group or the placebo group. In the multiple-dose study, the mean value of blood iron was slightly below the LLN in the 1,000-mg 1st group on days 5, 11, and 17 and in the 1,000-mg 2nd group on days 5, 11, 13, 14, and 17; no mean value below the LLN was observed in the 2,000-mg group or the placebo group (Table 4). Pharmacokinetics in plasma. The mean plasma concentration profiles of unchanged cefiderocol after the infusion of single doses ranging from 100 to 2,000 mg are shown in Fig. 1. A summary of the values of the PK parameters following the singledose administration of cefiderocol is shown in Table 5. Geometric mean values ranged from 7.76 to 156 g/ml (coefficient of variation for geometric mean [CV], 4.6% to 10.7%) .08), respectively, indicating dose-proportional increases in these parameters across the dose range of 100 to 2,000 mg. Statistical analyses showed no dose dependency for t 1/2,z , total clearance (CL), the mean residence time (MRT), the urinary excretion ratio relative to the dose over 48 h (Feu 0 -48 ), or renal clearance (CL R ). Figure 2 shows the mean plasma concentration profiles of cefiderocol following multiple infusions in the 1,000-mg 1st, 1,000-mg 2nd, and 2,000-mg groups. The mean trough concentrations from 48 to 192 h after the start of the initial infusion were 5.18 to 5.60, 5.11 to 5.39, and 9.66 to 13.0 g/ml for the 1,000-mg 1st, 1,000-mg 2nd, and 2,000-mg groups, respectively. A summary of the values of the PK parameters for cefiderocol after multiple-dose administration is shown in Table 6. Plasma concentra-    tion profiles and linear regression for plasma trough concentrations indicated that steady state was achieved within 1 day after the initiation of multiple dosing (day 3). Plasma concentrations were similar in the 1,000-mg 1st and 1,000-mg 2nd groups. The ratios of C max and the area under the concentration-time curve over dosing interval (AUC 0 -) between dose groups on day 10 were close to the dose ratio (i.e., 2), suggesting dose-proportional increases in C max and AUC 0 -following multiple dosing. Accumulation ratios of C max and AUC with dosing every 8 h (q8h) were 1.069 and 1.053, respectively, at 1,000 mg and 1.084 and 1.164, respectively, at 2,000 mg. The comparisons of AUC (AUC 0 -inf on day 1, AUC 0 -on day 10), CL, and CL R between days 1 and 10 indicated no change in PK with multiple dosing.
In the single-dose groups, the plasma concentrations of cefiderocol were below the lower limit of quantitation (BLQ) in most samples after 12 h at 100 mg, 16 h at 250 mg, 24 h at 500 and 1,000 mg, and 36 h at 2,000 mg. In the multiple-dose groups, the plasma cefiderocol concentration was BLQ in most samples after 24 h in both 1,000-mg groups and 36 h in the 2,000-mg group.
Urinary excretion. The mean urine concentration profiles of unchanged cefiderocol after the infusion of single doses ranging from 100 to 2,000 mg are shown in Fig. 3. Dose-dependent increases in the urine concentrations appeared across the dose range of 100 to 2,000 mg. The geometric mean urinary excretion ratio relative to the dose of cefiderocol ranged from 61.5% to 68.4% unchanged drug product (Fig. 4). The amount of cefiderocol metabolites was estimated to be less than 10%, as described below. The urine concentration of cefiderocol was BLQ in most urine samples collected from 24 to Exploratory study of human metabolites in plasma and urine. In plasma and urine, the most prominent peak identified by mass spectrometry (MS) was unchanged cefiderocol. Two types of methylated cefiderocol with a low MS response were detected, along with an additional nine metabolites. These metabolites had previously been identified in animal studies; there were no human-specific metabolites identified in plasma or urine. All urine metabolites were present at trace levels, and the total amount was 10% of the administered dose or less. Plasma metabolites showed no notable differences between single and multiple dosing, suggesting that the accumulation of metabolites is unlikely with multiple dosing.

DISCUSSION
Cefiderocol is a novel parenteral siderophore cephalosporin with in vitro and in vivo efficacy against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, P. aeruginosa, and A. baumannii (9)(10)(11)20). In the present study, cefiderocol was well tolerated at doses of up to 2,000 mg q8h in healthy volunteers. There were no serious or clinically significant AEs, and only one subject withdrew due to an AE (fever). The PK analysis showed dose-proportional increases in exposure at doses ranging from 100 to 2,000 mg, with little accumulation with multiple dosing. The majority of cefiderocol was excreted unchanged in the urine. The results from this study and a phase 1 single-dose study in subjects with renal impairment (17) were used to develop a population PK model for adjusting the dose of cefiderocol on the basis of renal function (18).
Because the proposed mechanism of action of cefiderocol involves iron chelation, the present study included laboratory tests to evaluate the effect of cefiderocol on iron homeostasis. Several multiple-dose cefiderocol groups had blood iron levels slightly below the lower limit of normal, which the investigator attributed to bone marrow iron uptake due to hematopoiesis from the frequent blood samplings. In our opinion, the fluctuation of the iron concentration in the blood was not clinically significant or related to the study drug. Further supporting our hypothesis, the total iron-binding capacity in the cefiderocol groups did not show a significant change from that in the placebo group (see Table S1 in the supplemental material).
With 60% to 70% of the administered dose of cefiderocol being excreted in the urine as unchanged parent drug and Ͻ10% being excreted as metabolites, the excretory fate of the remaining ϳ20% is not known. Further study, such as a mass-balance trial, is needed to characterize the metabolism and excretion of cefiderocol.
Due to iodide contamination in the initial drug supply, additional safety measures were added to the multiple-dose study. The high frequency of rash and the TSH abnormalities in the 1,000-mg 1st group in the multiple-dose study were likely attributed to iodide exposure (19). As the formulation no longer contains iodide, these safety concerns are not relevant to the ongoing clinical development of cefiderocol.
In conclusion, this study indicates that single and multiple intravenous doses of cefiderocol are well tolerated in healthy subjects, and dose proportionality for the PK parameters was observed.

MATERIALS AND METHODS
Subjects. Eligible subjects were healthy Japanese and Caucasian males or Japanese females not of childbearing potential (i.e., permanently sterilized or postmenopausal women) aged 20 to 60 years with body weights of 50.0 to 80.0 kg (40.0 to 80.0 kg for Japanese females, Ն50.0 kg for Caucasian males) and body mass indexes of Ն18.5 and Ͻ25.0 kg/m 2 (Ն18.5 and Ͻ30.0 kg/m 2 for Caucasian males). Subjects The dose proportionality of the PK parameters was examined by using a power model. Dose proportionality, dose independency, the effect of multiple doses, and the accumulation ratio of PK parameters were examined by analysis of variance. Achievement of steady state was assessed by visual inspection of the plots and by linear regression for plasma trough concentrations. SAS software (version 9.1; SAS Institute Inc., Cary, NC) was used for statistical analyses.
Exploratory study of human metabolites in plasma and urine. Analysis and identification of cefiderocol and its metabolites were determined by use of an LC (Agilent 1100 series; Agilent Technologies)-MS/MS n (LTQ Orbitrap Velos; Thermo Fisher Scientific) system. Exploratory investigations of major or human-specific minor metabolites were performed using pooled plasma samples (for the 2,000-mgdose group in the single-dose study, 1, 2, 4, 12, and 24 h after dosing; for the 1,000-mg 1st and 2nd groups in the multiple-dose study, 1, 2, 4, 8, and 12 h after the morning dose on day 1 and 0, 1, 2, 4, and 8 h after the morning dose on day 10) and pooled urine (for the 2,000-mg-dose group in the single-dose study, 0 to 8, 8 to 12, 12 to 24, and 24 to 48 h after dosing).

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC .02163-17.