Evaluation of the Effect of Contezolid (MRX-I) on the Corrected QT Interval in a Randomized, Double-Blind, Placebo- and Positive-Controlled Crossover Study in Healthy Chinese Volunteers

Contezolid (MRX-I), a new oxazolidinone, is an antibiotic in development for treating complicated skin and soft tissue infections caused by resistant Gram-positive bacteria. This was a thorough QT study conducted in 52 healthy subjects who were administered oral contezolid at a therapeutic (800 mg) dose, a supratherapeutic (1,600 mg) dose, placebo, and oral moxifloxacin at 400 mg in four separate treatment periods. The pharmacokinetic profile of contezolid was also evaluated.

ABSTRACT

Contezolid (MRX-I), a new oxazolidinone, is an antibiotic in development for treating complicated skin and soft tissue infections caused by resistant Gram-positive bacteria. This was a thorough QT study conducted in 52 healthy subjects who were administered oral contezolid at a therapeutic (800 mg) dose, a supratherapeutic (1,600 mg) dose, placebo, and oral moxifloxacin at 400 mg in four separate treatment periods. The pharmacokinetic profile of contezolid was also evaluated. Time point analysis indicated that the upper bounds of the two-sided 90% confidence interval (CI) for placebo-corrected change-from-baseline QTc (ΔΔQTc) were <10 ms for the contezolid therapeutic dose at each time point. The upper bound of the 90% CI for ΔΔQTc was slightly more than 10 ms with the contezolid supratherapeutic dose at 3 and 4 h postdose, and the prolongation effect on the QT/QTc interval was less than that of the positive control, moxifloxacin, at 400 mg. At 3 and 4 h after the moxifloxacin dose, the moxifloxacin group met the assay sensitivity criteria outlined in ICH Guidance E14 by having a lower confidence bound of ≥5 ms. The results of a linear exposure-response model which were similar to that of a time point analysis demonstrated a slightly positive relationship between contezolid plasma levels and ΔQTcF interval with a slope of 0.227 ms per mg/liter (90% CI, 0.188 to 0.266). In summary, contezolid did not prolong the QT interval at a therapeutic dose and may have a slight effect on QT interval prolongation at a supratherapeutic dose.

INTRODUCTION

Skin and soft tissue infections are mainly caused by Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), one of the leading microorganisms threatening human health (1). Enterococci are becoming one of the important major nosocomial pathogens due to their intrinsic resistance to many antimicrobials, including last-resort antimicrobials (e.g., vancomycin, linezolid, daptomycin, and tigecycline) (2).

Linezolid was the first oxazolidinone approved by the U.S. Food and Drug Administration (FDA) in 2000 and has excellent activity against many important Gram-positive bacteria (3). However, the use of linezolid is somewhat limited because of myelotoxicity when used long term (>21 days) (4). Contezolid (MRX-I) is a new oxazolidinone, which is being developed by MicuRx Pharmaceuticals, Inc. Contezolid exhibits potent activity against Gram-positive bacteria, including MRSA, methicillin-resistant Streptococcus epidermidis, penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant enterococci (5). Compared to linezolid, a major potential advantage of contezolid is its significantly improved safety profile with minimal myelosuppression and monoamine oxidase inhibition (5, 6). The safety and efficacy of contezolid in treating complicated skin and soft tissue infections have been evaluated in a recently completed phase III trial in China (CTR20150855) which used 800 mg every 12 h (q12h) as the therapeutic dose regimen and linezolid as the positive control. According to the phase III study, the most common adverse events related to contezolid were gastrointestinal disorders, and the incidence of myelosuppression related hematological disorders was significantly less compared to linezolid (MicuRx Pharmaceuticals, Inc. [unpublished data]).

Drugs other than antiarrhythmia drugs may have the potential to prolong the QT interval and lead to the occurrence of torsade de points (TdP), so the degree of QT prolongation has been regarded as a biomarker for TdP which may lead to sudden death (7). In 2005, International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) released the ICH E14 guidance to request that new drugs with systemic bioavailability be evaluated for the potential to delay repolarization with thorough QT (TQT) studies (8). The typical QT prolongation evaluation of E14 TQT study mainly is a time point analysis or intersection-union test (IUT). With the development of modeling and simulation, ICH E14 was updated (E14 Q&A [R3]) to highlight the assessment of concentration-response analysis in TQT studies in 2015 (9). Compared to time point analysis, concentration-QT model was more based on pharmacology of drug-induced QT prolongation (10), and it could predict the effect of drugs on QT interval at different doses (10, 11).

Nonclinical studies have demonstrated that contezolid inhibited the human Ether-à-go-go-Related Gene (hERG) ion tail current with a 50% inhibitory concentration of 160 μM, which was similar to the effect of linezolid (MicuRx Pharmaceuticals, unpublished). A retrospective evaluation of contezolid on the effect of QT interval conducted in a phase I study and did not find a positive influence (12), but a well-designed thorough QT study was needed due to the lack of a positive control. A dosing regimen of contezolid at 800 mg q12h was evaluated as the therapeutic dose regimen in a phase III study, and a tolerability assessment showed that a single oral dose of 1,600 mg in the fed state could be used as a supratherapeutic dose (13). Since the mechanism of drug-induced QT prolongation mainly focused on inhibiting hERG passage, a number of studies proposed that a direct linear mixed-model with time as a factor variable provides accuracy of the concentration-QT slope estimates (14, 15). This study was designed to meet the requirements for a TQT study and to assess QT interval prolongation at therapeutic and supratherapeutic doses of contezolid using both time point analysis and concentration-response methods.

RESULTS

Demographics.

Fifty-two participants (26 females, 26 males) completed the study, and none dropped out because of adverse events. The mean age (standard deviation [SD]) of the subjects was 26.1 (4.74) years old, and the mean height was 165 (7.73) cm. The mean weight and body mass index were 59.6 (7.64) kg and 21.9 (1.80) kg/m², respectively.

Pharmacokinetics.

The pharmacokinetic (PK) parameters of contezolid and its major metabolite in human M2 in 800- and 1,600-mg contezolid groups are presented in Table 1. Following a single oral administration of 800 or 1,600 mg, mean contezolid C_max values for all subjects were 26.5 and 44.0 mg/liter, respectively. The mean plasma exposures of M2 following single oral administrations of 800 and 1,600 mg of contezolid were about 25% of that for contezolid.

TABLE 1.

Pharmacokinetic parameters of mean contezolid and metabolite M2 in healthy Chinese subjects

Compound	Parametera	Mean (SD)
		800 mg (n = 52)	1,600 mg (n = 52)
Contezolid	Cmax (mg/liter)	26.5 (6.06)	44.0 (9.04)
	AUC0–23.5 (h·mg/liter)	96.6 (26.2)	188 (49.7)
	AUC0–∞ (h·mg/liter)	96.8 (26.2)	190 (50.0)
	t1/2 (h)	1.99 (0.89)	4.09 (1.70)
	Tmax (h)	4.0 (1.5, 6.0)	4.0 (2.02, 6.0)
	Vz/F (liters)	25.62 (15.1)	55.12 (32.79)
	CL/F (liters/h)	8.84 (2.29)	9.03 (2.41)
	MRT0–∞ (h)	4.76 (0.94)	5.25 (0.97)
M2	Cmax (mg/liter)	5.13 (1.42)	8.27 (2.26)
	AUC0–23.5 (h·mg/liter)	22.8 (4.77)	42.4 (9.26)
	AUC0–∞ (h·mg/ml)	22.8 (4.77)	42.9 (9.25)
	t1/2 (h)	2.23 (0.68)	4.02 (1.61)
	Tmax (h)	4.0 (2.02, 6.0)	4.0 (3.0, 6.0)
	MRT0–∞ (h)	5.46 (0.92)	6.12 (1.00)
	MR (%)	25.09 (8.02)	23.94 (7.33)

^aT_max values are presented as medians with minimum and maximum values in parentheses.

Heart rate and QT data.

Similar trends in changes of heart rate from baseline (ΔHR) were seen in the contezolid therapeutic, contezolid supratherapeutic, moxifloxacin, and placebo periods (Fig. 1). Contezolid had no effect on the heart rate. Due to the inverse relationship between QT interval and heart rate, QT intervals were needed to be corrected to compare the QT intervals changes at different heart rates. Figure 2 presents scatterplots of QT/QTc versus RR; these show that there were no relationships between QTcF, QTcI, and heart rate. Since a slight negative correlation between QTcB and RR interval was found, QTcB was mildly undercorrected or overcorrected to heart rate when the heart rate was too fast or slow.

FIG 1.

Change from baseline heart rate (ΔHR, bpm) with time. Bars show means ± the 95% CI.

FIG 2.

Evaluation of the relationship between the QT/QTc interval and RR interval. (a to d) Scatterplots of QT, QTcF, QTcB, and QTcI and RR interval in the placebo group, moxifloxacin treatment, and contezolid treatment groups, respectively. The black dots represent the QT interval, the green dots represent the QTcF intervals, the blue dots represent the QTcB intervals, and the red dots represent the QTcI intervals.

Categorical analysis.

Table 2 summarizes the categorical analysis of QT/QTc interval prolongation. No subjects with QTcF and QTcI intervals exceeding 480 ms were observed in any of the cohorts. Similarly, no subjects had QTcF, QTcB, and QTcI prolongation (compared to baseline) exceeding 60 ms in the four cohorts. Two subjects (4%) with QTcI prolongation exceeding 30 ms were observed in the contezolid 1,600-mg group and another two subjects (4%) in the moxifloxacin cohorts, while one subject (2%) with QTcF prolongation exceeding 30 ms was reported in the moxifloxacin cohort. No heart rate, PR, and QRS outliers were reported in the study.

TABLE 2.

Number of observations (ratios of observations) with categorical analysis of QT, QTcF, QTcB, and QTcI prolongation

Outlier	Parameter(s)	Treatment group, no. (%)
		Contezolid, 800 mg	Contezolid, 1,600 mg	Moxifloxacin, 400 mg	Placebo
>480 ms	QT	0 (0)	3 (0.15)	1 (0.04)	0 (0)
	QTcB	0 (0)	0 (0)	1 (0.04)	0 (0)
	QTcF/QTcI	0 (0)	0 (0)	0 (0)	0 (0)
>500 ms	QT/QTcF/QTcB/QTcI	0 (0)	0 (0)	0 (0)	0 (0)
>30-ms increase from baseline	QT	136 (5.05)	153 (5.68)	155 (5.76)	160 (5.94)
	QTcF	1 (0.037)	2 (0.074)	3 (0.11)	8 (0.30)
	QTcB	0 (0)	6 (0.022)	10 (0.37)	2 (0.074)
	QTcI	1 (0.037)	2 (0.074)	2 (0.074)	10 (0.37)
>60-ms increase from baseline	QT	1 (0.037)	11 (0.41)	10 (0.37)	6 (0.22)
	QTcF/QTcB/QTcI	0 (0)	0 (0)	0 (0)	0 (0)

Time point analysis.

The time-matched analysis for QTcF was conducted as the primary endpoint as recommended by ICH E14. The time-matched analysis for the QTcF endpoint revealed that the moxifloxacin group met the assay sensitivity criteria outlined in E14, with a lower confidence bound ≥5 ms at h 3 and 4 (Table 3), a typical moxifloxacin profile, as seen in Fig. 3.

TABLE 3.

Estimates and 90% confidence intervals for placebo-corrected changes from baseline in the QTcF interval from the mixed-effect model

Time (h) postdose	Estimate and rangea
	Contezolid, 800 mg			Contezolid, 1,600 mg			Moxifloxacin, 400 mg
	Estimate	Lower	Upper	Estimate	Lower	Upper	Estimate	Lower	Upper
0.25	−1.0	–3.1	1.2	–0.9	–3.0	1.3	–0.5	–3.4	2.4
0.5	–0.8	–2.9	1.3	0.2	–2.0	2.3	0.3	–2.6	3.2
1	–0.6	–2.7	1.5	0.4	−1.7	2.6	0.6	–2.3	3.5
1.5	0.8	−1.4	2.9	2.4	0.3	4.5	0.5	–2.4	3.4
2	0.9	−1.3	3.0	2.9	0.8	5.0	3.4	0.5	6.3
3	4.9	2.8	7.0	9.7	7.6	11.9	10.5	7.6	13.4
4	5.6	3.5	7.7	11.5	9.4	13.6	13.2	10.3	16
6	1.6	–0.5	3.7	7.7	5.5	9.8	9.3	6.4	12.2
8	0.4	–1.7	2.6	4.7	2.5	6.8	8.7	5.8	11.6
12	–0.6	−2.8	1.5	2.3	0.1	4.4	8.7	5.8	11.6
16	0.7	–1.4	2.8	2.2	0.1	4.3	9.5	6.6	12.3
23.5	−2.9	–5.0	−0.7	0.5	−1.6	2.7	5.4	2.4	8.3

^aThe mean estimates and upper and lower confidence intervals are mixed-effect model-based estimates. The lower and upper bounds are the lower/upper two-sided 90% data-based confidence limits.

FIG 3.

Placebo-corrected change from baseline in QTc intervals (ΔΔQTc intervals) over time. Bars show means ± the 95% CI.

Table 3 details the two-sided 90% or the equivalent one-sided 95% upper confidence boundary in milliseconds for each treatment at each time point with the placebo and baseline adjusted ΔΔQTcF analysis for each of the contezolid dose groups and moxifloxacin group. At 3 and 4 h after contezolid 1,600-mg administration, the upper bound of the two-sided 90% CI exceeded 10 ms (12.2 and 13.1 ms, respectively). However, at each time point for the contezolid 800-mg group, the upper bounds of the two-sided 90% CI were <10 ms. The ΔΔQTcF profiles are presented in Fig. 3. The results of ΔΔQTcB and ΔΔQTcI were similar to that of the ΔΔQTcF.

Concentration-QT relationships.

Table 4 illustrates the pharmacokinetic-pharmacodynamic (PK/PD) model results for the relationship between plasma concentration of contezolid and the predicted ΔΔQTc change at C_max for QTcF, QTcI, and QTcB. In the contezolid 800-mg group, the predicted ΔΔQTcF at mean C_max was 7.42 ms, and the two-sided upper bound of 90% CI was 8.27 ms, which was less than 10 ms. Similar results were reported based on QTcI and QTcB corrected methods. The two-sided upper bound of the 90% CI predicted ΔΔQTcF at mean C_max in the contezolid supratherapeutic dose group, slightly exceeded 10 ms, as well as that of ΔΔQTcB and ΔΔQTcI. Figure 4 displays the relationship between observed contezolid concentration and ΔQTcF according to the linear exposure-response model. The C-QTc quantile plot of observed data overlaid with the model predictions provides an acceptable fit (Fig. 4). The estimated slope of the contezolid concentration-ΔQTcF relationship was 0.227 ms per mg/liter (90% CI, 0.188 to 0.266), with a treatment effect-specific intercept of 1.4 ms (90% CI, −0.299 to 3.099). The relationships between concentration of M2 and QTc intervals were consistent with contezolid.

TABLE 4.

Predictions of ΔΔQTc changes at C_max of contezolid based on PK/PD models in contezolid 800- and 1,600-mg treatment groups

Treatment dose	Mean Cmax (mg/liter)	Corrected method	Placebo-adjusted change (ms) from baseline
			Predicteda	Lower CI	Upper CI
Contezolid, 800 mg	26.5	QTcF	7.42	6.56	8.27
		QTcI	7.35	6.50	8.20
		QTcB	4.85	3.81	5.88
Contezolid, 1,600 mg	44	QTcF	11.39	9.96	12.81
		QTcI	10.97	9.56	12.38
		QTcB	9.36	7.64	11.08

^aThe predicted mean effect at mean C_max.

FIG 4.

(Top panel) Scatterplot with observed ΔΔQTcF versus contezolid concentration and exposure-response model predicted effect (solid red line) with the 90% CI (dotted green lines). The blue squares and red triangles represent the 800- and 1,600-mg contezolid groups, respectively. The prediction line is from the mixed-effect concentration-ΔQTcF model with the mean and pooled treatment-specific intercept. Band (green) above/below the prediction line represents the 90% CI. (Bottom panel) Predicted and observed ΔΔQTcF versus contezolid concentration. Red circles with vertical bars show the observed ΔΔQTcF with the 90% CI displayed at the mean drug concentration in plasma with each decile for contezolid. The solid black line with the gray-shaded area represents the model-predicted ΔΔQTcF with the 90% CI. The red line at the bottom represents each contezolid plasma concentration decile.

Safety and tolerability.

All 52 subjects completed the four-crossover trial and no severe adverse events (AEs) were observed. In the four groups, 21 subjects (40.4%) reported experiencing drug-related adverse events; these included 15 gastrointestinal disorders and 6 laboratory tests abnormalities, and the others were dizziness and a headache (Table 5). More AEs occurred in the contezolid 1,600-mg group (14, 26.9%) than in the other three groups. The most common adverse events were gastrointestinal disorders, with nausea (10, 19.2%) and vomiting (5, 9.6%) occurring more often in the contezolid 1,600-mg group. Vomiting in the five subjects (one male and four females) was observed 8 to 13 h postdose, but no PK profiles were observed to be altered for these subjects. In the moxifloxacin group, uric acid elevation and white blood count and neutrophil decreases were observed. No hematological disorders were observed in the contezolid treatment groups except for a hemoglobin decrease in the contezolid 1,600-mg group. No significantly abnormal liver function test results were observed in the four groups. All the drug-related adverse events were classified as mild in severity and resolved quickly without any intervention.

TABLE 5.

Drug-related adverse events after single oral dose of contezolid, moxifloxacin, or placebo in healthy Chinese subjects

Variable	No. (%) of subjects with TEAEs
	Placebo (n = 52)	Moxifloxacin (n = 52)	Contezolid, 800 mg (n = 52)	Contezolid, 1,600 mg (n = 52)	Sum (n = 52)
Drug-related adverse event	3 (5.8)	7 (13.5)	5 (9.6)	14 (26.9)	21 (40.4)
Gastrointestinal disorders	2 (3.8)	3 (5.8)	3 (5.8)	10 (19.2)	15 (28.8)
Nausea	1 (1.9)	1 (1.9)	2 (3.8)	10 (19.2)	11 (21.2)
Vomiting	0	0	1 (1.9)	5 (9.6)	5 (9.6)
Diarrhea	1 (1.9)	0	1 (1.9)	0	2 (3.8)
Abdominal bloating	0	2 (3.8)	0	0	2 (3.8)
Upper abdominal discomfort	0	0	1 (1.9)	0	1 (1.9)
Laboratory test	1 (1.9)	4 (7.7)	2 (3.8)	3 (5.8)	6 (11.5)
Serum uric acid elevation	0	2 (3.8)	2 (3.8)	1 (1.9)	3 (5.8)
White blood cell count decrease	0	1 (1.9)	0	0	1 (1.9)
Alanine aminotransferase elevation	1 (1.9)	0	0	0	1 (1.9)
Direct bilirubin elevation	0	0	0	1 (1.9)	1 (1.9)
Aspartate aminotransferase elevation	1 (1.9)	0	0	0	1 (1.9)
Bilirubin elevation	1 (1.9)	0	0	0	1 (1.9)
Hemoglobin decrease	0	1 (1.9)	0	1 (1.9)	1 (1.9)
Neutrophil percent decrease	0	1 (1.9)	0	0	1 (1.9)
Neutrophil count decrease	0	1 (1.9)	0	0	1 (1.9)
Neurological disorders	0	0	0	2 (3.8)	2 (3.8)
Headache	0	0	0	1 (1.9)	1 (1.9)
Dizziness	0	0	0	1 (1.9)	1 (1.9)

DISCUSSION

This thorough QT/QTc study was conducted to evaluate the effects on cardiac repolarization of therapeutic and supratherapeutic doses of contezolid in healthy subjects. The primary finding was that the therapeutic dose of contezolid did not prolong the placebo-corrected change from baseline in the QTcF interval, and the largest two-sided upper bound of the 90% CI was 7.7 ms according to time point analysis. The largest two-sided upper bound of the 90% CI was >10 ms (13.6 ms) at the supratherapeutic dose of contezolid but less than that of the moxifloxacin group (16 ms). The results of the PK/PD model showed that the slope for QTcF interval for contezolid was slightly positive (0.227 ms per mg/liter, 90% CI = 0.188 to 0.266). According to the results of categorical analysis, it can be concluded that contezolid has no effect on heart rate, PR, and QRS interval, and there were no new clinically relevant morphological changes. There was only a small signal with the supratherapeutic contezolid on cardiac repolarization, as evidenced by the results of the time-matched and PK-PD analyses at 3 and 4 h postdose. No significant safety concerns were found with the therapeutic and supratherapeutic doses of contezolid in this study, and the results were consistent with previous studies (6, 13).

A variety of intrinsic and extrinsic factors have potential impacts on the drug exposure. On one hand, no drug accumulation was found when contezolid was given twice daily in multiple dose evaluations according to the previous studies (6, 16). On the other hand, contezolid is not metabolized by P450 enzymes in humans, and the principal metabolic pathway proposed for the oxidative ring opening of 2,3-dihydropyridin-4-one (DHPO) is via flavin-containing monooxygenase 5 (FMO5), short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (ALDH) (17). Further study should be conducted to determine whether exposure of contezolid will be increased in patients with hepatic or renal dysfunction.

In some circumstances, a drug has no risk of QT prolongation but a metabolite may (18). M2 is the principal metabolite of contezolid in human with an elemental composition of C₁₈H₁₉N₄0₆F₃ (17), which was regarded as metabolite through the dehydration (2H₂O) of contezolid and the mean plasma exposure of M2 to that of contezolid exceeded 20% in both the therapeutic and the supratherapeutic dose groups. Nevertheless, the effect of M2 on the QT interval was consistent with that of contezolid, indicating that the production of M2 did not prolong the QT interval in healthy subjects in addition to the effects of contezolid.

Since this TQT study was well designed (six ECG samples with intensive sampling around C_max) and adequately powered (52 subjects), a time point analysis could be used as a reference. The linear mixed-effect model in this study was able to predict accurately the drug-induced QTc prolongation in which the maximum ΔΔQTc effect and two-sided upper bound of the 90% CI predicted by slope estimates and C_max were consistent with the results of time point analysis. Since QT interval changes exhibit circadian rhythm during the day, a great number of studies have employed oscillatory time functions to characterize the biological rhythms in QTc profiles (15, 19). However, a fitting biological QTc model is not feasible because it requires sufficient ECG sampling. Several authors have shown that linear mixed-effect (LME) model provides similar accuracy, precision, and even less bias compared to a complex biological model since the LME model could be used prespecified without any model building (15, 19). Garnett et al. proposed that C-QTc models incorporating time- and treatment-specific terms give the least-biased ΔΔQT C_max predictions (14). In this current concentration-QT model, time was regarded as a factor in the model that could represent the circadian rhythm of the QTc interval. Moreover, the treatment-specific intercept incorporated in the model provides the model with more flexibility, especially when the true relationships between concentration and QT interval are not known (14). In addition, more and more studies have proposed that the use of concentration-QT model analysis of early-phase studies could be a substitute for a TQT study (20–22). This study may provide several points that should be considered in establishing an LME model.

However, there were several limitations of this type of study noted. First, risk factors, including cardiac failure, hypokalemia, genes mutation on encoding IKr, or drug-metabolizing enzymes, were well recognized to be related to drug-induced torsade de points (TdP) (18). The study was conducted in healthy Chinese volunteers 18 to 45 years of age. The intended patient population may vary substantially in real-world cases. Thus, even though no QT prolongations were observed in the safety analysis in phase II and III studies, further evaluations needed to continue through postmarketing surveillance. Second, only single doses of contezolid were evaluated here, while the therapeutic dose will be 800 mg q12h. However, no drug accumulation was observed in multiple-dose studies, so this may not be a concern.

Conclusion.

We demonstrated here that a therapeutic dose of contezolid at 800 mg did not prolong the placebo-corrected QTc interval in healthy subjects (the largest 90% CI upper bound was 7.7 ms). Contezolid at a supratherapeutic dose of 1,600 mg had slightly positive effects on QT interval (the largest 90% CI upper bound was 13.6 ms) but was less than that of moxifloxacin group. Furthermore, no major tolerability concerns for contezolid were encountered in the study.

MATERIALS AND METHODS

Study design and subjects.

A total 52 healthy subjects (26 females, 26 males) were enrolled in a blinded (except for moxifloxacin), randomized, single-site, four-period, four-arm crossover design TQT study (Chinese Clinical Trial registration number CTR20161074). Subjects were screened with detailed medical histories, physical examinations, laboratory tests, and standard 12-lead electrocardiograms (ECGs). Subjects randomly received a single oral therapeutic dose of contezolid at 800 mg, a supratherapeutic dose of contezolid at 1,600 mg, a 400-mg dose of moxifloxacin, and an administration of placebo. The washout period between each period was 7 days, and all subjects received the assigned study drug after a normal breakfast on day 1 of each treatment period. Subjects stayed in the study site during all four periods until the end of the fourth period.

The study was approved by the Ethics Committee of Huashan Hospital, Fudan University, and was conducted in compliance with the International Conference on Harmonization (ICH) good clinical practice guidelines. All subjects provided informed consent before participating the study.

Pharmacokinetic sampling.

Blood samples were obtained from all subjects in the study at following time points: predose (within –3 h) and postdose at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 23.5 h. Only samples from subjects that received therapeutic or supratherapeutic doses of contezolid were analyzed. Plasma concentrations of contezolid and metabolite M2 were determined by a validated UPLC-MS/MS assay (13). Noncompartmental analysis of PK parameters was conducted using Phoneix WinNonlin software (version 6.3.0; Pharsight Corp., Sunnyvale, CA).

12-Lead ECG acquisition.

Intensive ECG sampling was obtained digitally using a Mortara Instrument (Milwaukee, WI) H-12+ ECG continuous 12-lead digital recorder to evaluate the effect of contezolid on the QT/QTc interval. Triplicate 12-lead ECGs (approximately 1 min apart) were collected at three time points at −60, −45, and −30 min (a total of nine ECGs as a baseline) before each dose and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 23.5 h postdose to match PK sampling time points in each treatment period of the crossover trial. To obtain good-quality data, subjects rested on beds 15 min before each of these time points.

ECG analysis methods.

ECGs used in the analysis were read using a high-resolution manual on-screen caliber semiautomatic method. The average of RR, PR, QRS, heart rate (HR), and QT/QTc interval durations were calculated based on the triplicate ECGs collected at each time point. The correction formulae QTcB, QTcF, and QTcI were calculated as follows: Bazett correction, QTcB = QT/RR^1/2; Fridericia correction, QTcF = QT/RR^1/3; and individual correction, QTcI = QT/RR^β. The heart rate (HR) was calculated as follows:

$$\text{HR = 60/RR}$$ (1)

Statistical methods.

After the TQT study, the flash cards from the ECG machines were sent to ERT, along with data transmittal forms (with flash card serial number, subject identification number, and demographic information), and ECG data were analyzed by eResearch Technology (ERT). Time point analysis for each group was performed as the primary analysis for QT/QTc data, which was based upon the endpoint “change from baseline in the QTcF interval, placebo-corrected – the ΔΔ approach.” The baseline for each period was regarded as the average of the measured QTc intervals from the three predose ECG time points (−60, −45, and −30 min) in that period on day 1.

These changes from baseline ECG data, for each treatment period, were then subjected to a mixed-effect model (using the SAS Procedure PROC MIXED) with the following covariates: time (categorical), treatment, time by treatment interaction, gender, period and sequence, and the baseline value of the parameter. Subjects were included as a random effect. The estimates of the ΔΔQTcF and its 90% two-sided confidence intervals (CI) were determined using a DIFF option in LSMEANS statement within PROC MIXED (SAS V.9.1; SAS Institute, Cary, NC). Analyses for QTcB and QTcI were also implemented according to this method. A single oral administration of 400 mg of moxifloxacin was used as a positive control to determine the assay sensitivity of the study with at least one time point where the lower confidence bound of the placebo-corrected moxifloxacin’s QTc interval was >5 ms.

Pharmacokinetic-pharmacodynamic analysis.

A linear mixed-effect model was also performed to explore the relationship between the change from baseline in QTc intervals (QTcF, QTcB, and QTcI) and plasma concentrations (contezolid and its main metabolite M2) using the model proposed in the 2017 white paper by Garnett et al. (23). PK/PD analyses were implemented using NONMEM (version 7.3; ICON Development Solutions, Ellicott City, MD) and independent variables for treatment (active or placebo), time (categorical), baseline adjustment, and plasma concentration. The calculation is presented below in equation 2:

$$\Delta {\text{QTc}}_{\mathit{ijk}}=\left({\theta }_0+{\eta }_{0,i}\right)+{\theta }_1\text{TRT}+({\theta }_2+{\eta }_{2,i})C_{\mathit{ijk}}+{\theta }_3\hspace{0.17em}\left(\text{BASELINE-Mean BASELINE}\right)+{\theta }_4{\text{TIME}}_k+{\epsilon }_{\mathit{ijk}}$$ (2)

where the dependent variable ΔQTc for the jth treatment, ith subject, and kth time point and parameters are as follows: is the treatment specific intercept, θ₁ is the treatment-specific intercept (contezolid, placebo = 0), θ₂ is the slope, C is the concentration (C_ijk is the concentration at the jth treatment for the ith subject at the kth time point, θ₄ is the time effect on the intercept, θ₃ is the baseline adjustment, η_0,i is the random subject effect on the intercept, η_2,i is the random subject effect on the slope, and ε_ijk is the residual error.

The placebo-adjusted change from baseline at the mean C_max and its upper two-sided 90% CI is calculated using the following equation(s):

$$\Delta \Delta {\text{QTc}}_{\text{max}}={\theta }_{1,\mathit{Est}}+C_{\mathit{max}}{\theta }_{2,\mathit{Est}}$$

$$\text{Estimated SE}=\sqrt{\mathit{var}\left({\theta }_{1,\mathit{Est}}\right)+C^{2}v\mathit{ar}\left({\theta }_{2,\mathit{Est}}\right)+2C\left(\mathit{COV}\left({\theta }_{1,\mathit{Est}}{\theta }_{2,\mathit{Est}}\right)\right)}$$

$$\text{90\% CI}=\Delta \Delta {\text{QTc}}_{\text{max}}\pm t(0.90,\text{DF)}\times \text{Estimated SE}$$ (3)

Model parameters were estimated by the first-order conditional estimation method with the INTERACTION option (FOCEI). Interindividual variability (IIV) was estimated by exponential functions, and residual variability was expressed with an additive variation model. The objective function value (OFV) was calculated based on the NONMEM analysis.