Study design and participants

Written informed consent was obtained from all participants, and the protocols were approved by the institutional review board of the study site (West Coast Clinical Trials, LLC, Cypress, CA, USA). These randomized, double-blind, placebo-controlled, phase 1 studies consisted of a single ascending dose (SAD) study (protocol number LCD-SAD-08-04, NCT02835105) and a multiple ascending dose (MAD) study (protocol number LCD-MAD-08-08, NCT02835118). Both studies were conducted in accordance with the ethical principles originating from the Declaration of Helsinki and its amendments, consistent with Good Clinical Practices and local regulatory requirements.

Male and female subjects aged 18–75 years were eligible for these studies if considered by the investigator to be in good health with unremarkable current and past medical history before the first day of study. Subjects were required to have no clinically significant abnormalities in prestudy physical examination, electrocardiogram (ECG), and laboratory evaluations. Subjects with findings outside of the normal range were included in the study only if these findings were deemed not clinically significant by the investigator or medical monitor. Subjects had no evidence of prior chronic gastrointestinal inflammatory disease.

Exclusion criteria included incidence of C. difficile disease within 1 year before study entry (SAD); prior exposure to surotomycin (MAD); known hypersensitivity to lipopeptide antibacterials; any comorbid disease judged by the investigator to be clinically significant; any concomitant medication, except low-dose aspirin, paracetamol, and multivitamins, in the 2 weeks before dosing (investigator- and medical monitor-approved concomitant medications were permitted in patients aged 49 years and above); or any antibiotic within 30 days before the first dose of the study drug. Women who were unwilling or unable to use an acceptable method to avoid pregnancy or who were pregnant or lactating during the conduct of the study and until 1 month after last surotomycin dose were excluded.

For the SAD study, eligible subjects were sequentially enrolled into 1 of 4 dose cohorts: 500 mg, 1000 mg, 2000 mg, or 4000 mg surotomycin (Fig. 1a). In total, 10 subjects were intended to be randomized into each dose cohort in a 4:1 ratio to receive surotomycin (n = 8) or placebo (n = 2). At least 24 h before dosing the first full cohort, 2 subjects received surotomycin or placebo (randomized 1:1). The remaining 8 subjects were randomly assigned to surotomycin (n = 7) or placebo (n = 1) only if no significant safety findings or clinically significant abnormal laboratory values were reported for the first 2 subjects. Randomization was assigned by blinded study personnel and stratified by gender to achieve an equal number of male and female subjects in each cohort. Subjects aged 18–49 years and 50–75 years were equally distributed in each dosing cohort. Subjects received a single dose of surotomycin or placebo during the morning of day 1 (1 h after breakfast) and were followed as an inpatient through day 4 when they were discharged to return for a follow-up visit on day 8.

Eligible subjects were recruited and sequentially enrolled into 1 of 3 MAD dose cohorts: 250 mg, 500 mg, or 1000 mg surotomycin twice daily (BID) (Fig. 1b). A total of 10 subjects were randomized (4:1) to receive surotomycin (n = 8) or placebo (n = 2) in each cohort. Randomization was also stratified by gender and distributed by age to ensure that dosing cohorts were balanced. Subjects were dosed BID, once in the morning and once in the evening, for 14 consecutive days, with at least 8 ounces of water and approximately 1 h after breakfast and 1 h after dinner. Subjects were observed as inpatients through day 15 and were then discharged to return for follow-up on day 21.

In both studies, dose escalation to the next cohort occurred sequentially, and only after review of key safety data obtained from the previous cohort indicated that it was safe to proceed. The investigator and all personnel involved in the clinical or analytical evaluations of the study remained blinded to treatment until all cohorts had completed and the database was locked. Treatment doses were administered according to a randomization code by a pharmacist who was not an investigator or involved in study evaluations.

Pharmacokinetic analysis

Any subject receiving at least one full dose of the study drug was included in the PK analysis population. PK analysis during the SAD study was conducted on serial plasma samples collected predose and at 30 min, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after dosing, and analyzed using a validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method, with a lower limit of quantification (LLOQ) of 1 ng/mL. Urine and stool samples were also collected during this period and analyzed using LC/MS/MS. Urine and feces were collected for 7 days after dose administration. The samples were analyzed using an API 5000 triple quadrupole mass spectrometer (Applied Biosystems/ScieEx, Concord, ON, Canada) using electrospray ionization in positive ion mode. Analyst^TM software (version 1.4.2., Applied Biosystems, Foster City, CA, USA) was used for data acquisition. In total, 8 calibration solutions with a range of 1.00 ng/mL to 1000 ng/mL were used as internal standards in addition to a blank. Inter-assay bias was determined to be –2.6 to 2.5% with inter-assay precision of 3.9 to 9.4%.

During the MAD study, serial plasma samples for PK analysis were collected predose and at 30 min, 3, 6, and 9 h after the morning dose on days 1 and 14, and before the morning dose on days 4, 7, 10, and 12 (trough levels). Stool was also collected in its entirety from all bowel movements for PK analysis following the morning dose on day 5 through predose day 6 in the 1000-mg BID dose cohort. Samples were analyzed using the same bioanalytical LC/MS/MS method as in the SAD study (LLOQ of 1 ng/mL).

Plasma PK parameters were calculated using standard noncompartmental methods in a validated version of WinNonlin (version 5.2, Pharsight, Mountain View, CA, USA). All concentrations that were below the LLOQ prior to the first detectable concentration were assigned a value of 0. All concentrations that were below the LLOQ after the first quantifiable concentration were designated as missing and replaced with a period. Actual sample collection times were used in the analysis of the concentration versus time profiles for individual subjects. Integration of plasma concentrations versus time was conducted using the linear-up, log-down function in WinNonlin. The following parameters were determined: maximum plasma concentration (C_max), time of C_max (T_max), area under the concentration-time curve (AUC) from 0 to last measurable plasma concentration (AUC_0-t), AUC from 0 to infinity (AUC_0-∞), percent of dose excreted in the urine, and terminal exponential half-life (t_½).

Sample sizes were chosen based primarily on clinical considerations and were considered sufficient for the exploratory evaluation of single- and multiple-dose safety and PK.

Safety analysis

Safety was monitored throughout the studies and on return for follow-up assessment on day 8 (SAD group) or day 21 (MAD group), by observation or reports of adverse events (AEs), and by changes in physical examination findings, vital signs, ECG, and laboratory tests. Concomitant medications and procedures were recorded. Any subject who received any dose of the study drug was included in the safety analysis population.

Statistics

Statistical methods were primarily descriptive and no formal hypothesis tests were planned or completed. Data were summarized and analyzed using Statistical Analysis System SAS^® (version 9.1.3; SAS Institute, Cary, NC, USA).

Subject demographics and characteristics

Both study groups were enrolled to completion with four 10-subject cohorts randomized in the SAD study and three cohorts of the same size randomized in the MAD study. All 40 subjects in the SAD group received all scheduled study medication. One subject (2000 mg) withdrew from the study early and missed the follow-up visit due to a family emergency. In all, 28 of 30 subjects completed the MAD study as planned. One subject (500 mg BID) discontinued treatment due to AEs of anxiety and dyspnea after 5 doses, and one subject (250 mg BID) did not complete the day-21 follow-up visit (considered lost to follow-up).

Pharmacokinetic results

The median plasma concentration-time profiles of surotomycin following administration of single oral doses (SAD) and following administration of a single dose and repeated doses (MAD) are presented in Fig. 2. However, although quantifiable levels of surotomycin were observed in 4 subjects in the SAD 2 g cohort and 6 subjects in the 4 g cohort, a full plasma concentration-time curve was obtained in only 2 subjects, 1 in each dose cohort. Following a brief lag after the administration of a single dose, median plasma concentrations of surotomycin were quantifiable and seemed to increase with an increase in dose (Fig. 2a and b). Peak median plasma surotomycin concentrations were observed from 6 to 12 h after the first single dose in both the SAD and MAD groups. For patients receiving repeated surotomycin dosing, the median plasma concentration versus time profile on day 14 was flat (Fig. 2c), indicating that steady state had been reached and that the concentrations were essentially steady or constant during the dosing interval.

In the SAD group, median C_max ranged from 10.5 ng/mL in the 500-mg dose cohort to 86.7 ng/mL in the 4000-mg dose cohort, and the AUC_0-∞ ranged from 317 ng*h/mL in the 500-mg dose cohort to 2572 ng*h/mL in the 4000-mg dose cohort. While the overall exposure of surotomycin as measured by C_max and AUC_0–∞ increased with increases in dose, the median elimination half-life was independent of the dose administered and ranged between 14.8 and 21.1 h. In the MAD study, C_max on day 1 (following a single dose) was consistent with the findings of the SAD group and ranged from 6.8 ng/mL to 21.0 ng/mL. On day 14, the median C_max ranged from 25.5 ng/mL to 93.5 ng/mL, possibly suggesting an accumulation of surotomycin in the body following repeated dosing. No additional PK parameters for surotomycin could be computed due to the nature of the plasma concentration-time profile on days 1 and 14, and limited sampling. Figure 3 shows the dose-normalized C_max and AUC_0–∞ for surotomycin after a single dose (SAD), indicating that surotomycin exposure increased in a dose-dependent manner.

In the SAD study, concentrations of surotomycin in the urine were detected in 12 of 32 subjects treated, and the median cumulative amount of the administered dose recovered in the urine was <0.01% over a 7-day period. The median cumulative fraction of the administered surotomycin dose excreted in the feces over a 4-day period ranged from 20.8 to 60.2% after a single dose (SAD). Stool levels of surotomycin increased proportionally with the dose administered. In the MAD study, the mean concentration of surotomycin from the 1000-mg BID dose cohort on day 5 was 6394 μg/g.

Safety analysis

Although a total of 18 (60.0%) subjects experienced at least one AE during the MAD study, all reported AEs were considered unrelated to the study drug. The most common AE in the MAD group was headache, reported in 2 (8.3%) of the 24 subjects who received surotomycin (1 each in the 250-mg BID and 500-mg BID dose cohorts) and 2 (33.3%) of the 6 placebo subjects (Table 3). Constipation, back pain, oropharyngeal pain, and pruritus were each reported in 2 (8.3%) of the subjects who received surotomycin.

All reported AEs in both studies were mild to moderate in severity. No serious AEs were reported and none of the subjects discontinued the study due to AEs in the SAD group. One subject in the MAD group (500 mg BID) discontinued due to transient moderate anxiety and associated mild dyspnea. The events were reported 2 h after the 5^th dose of study treatment. The anxiety resolved within 4 h and the dyspnea within 3 min, both without the need for additional treatment.