Pharmacokinetics and Disposition of Contezolid in Humans: Resolution of a Disproportionate Human Metabolite for Clinical Development

ABSTRACT

Contezolid (MRX-I), a novel oxazolidinone antibiotic, was recently approved for the treatment of serious Gram-positive infections. The pharmacokinetics and disposition of [¹⁴C]contezolid were investigated in a single-dose human mass balance study. Cross-species comparison of plasma exposure for contezolid and metabolites was performed, and the safety of the disproportionate metabolite in human was evaluated with additional nonclinical studies. After an oral administration of 99.1 μCi/602-mg dose of [¹⁴C]contezolid, approximately 91.5% of the radioactivity was recovered in 0 to 168 h postdose, mainly in urine followed by that in feces. The principal metabolic pathway of contezolid in human comprised an oxidative ring opening of the 2,3-dihydropyridin-4-one fragment into polar metabolites MRX445-1 and MRX459, with recovery of approximately 48% and 15% of the dose, respectively, in urine and feces. Contezolid, MRX445-1, and MRX459 accounted for 68.0%, 19.5%, and 4.84% of the plasma exposure of the total radioactivity, respectively. Metabolites MRX445-1 and MRX459 were observed in disproportionately larger amounts in human plasma than in samples from rat or dog, the rodent and nonrodent species, respectively, used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies, wherein the primary metabolite, MRX445-1, was further characterized. The no-observed-adverse-effect level (NOAEL) of MRX445-1 was determined as 360 mg/kg body weight/day in a 14-day repeat-dose test in pregnant and nonpregnant Sprague Dawley rats. Furthermore, MRX445-1 exhibited no antibacterial activity in vitro. Thus, MRX445-1 is not expected to exert clinically relevant pharmacology and toxicity.

TEXT

Contezolid (MRX-I) is a novel oxazolidinone antibiotic against Gram-positive pathogens; it inhibits bacterial protein synthesis by binding to the 50S subunit of bacterial ribosomes (1). In nonclinical studies, contezolid demonstrated promising efficacy results across a range of animal infection models (2, 3). There was a very low propensity for Staphylococcus aureus to develop resistance to contezolid compared to that with linezolid (4), the first oxazolidinone antibiotic approved by the U.S. Food and Drug Administration to treat serious Gram-positive infections (5). Furthermore, contezolid showed the potential to minimize the limiting adverse effects encountered in linezolid therapy, primarily associated with bone marrow toxicity and serotonergic drug interactions (1).

In a phase I study, single-dose oral administrations of 800, 1,200, and 1,600 mg of contezolid were well tolerated in healthy subjects in the fed state (6). In a multiple-dose study, oral contezolid was well tolerated at doses of 800 mg every 12 h for up to 28 days; all adverse events were mild to moderate in severity, and there was no drug discontinuation due to adverse events occurred (7). In humans, contezolid was rapidly absorbed, and it reached peak plasma concentration at approximately 2 h postdose. The maximum concentration of drug in serum (C_max) values were 8.07, 12.24, and 15.25 mg/liter, and the corresponding area under the concentration-time curve from 0 to infinity (AUC_0−∞) values were 29.21, 48.27, and 59.60 h·mg/liter in 300-, 600-, and 900-mg dosing groups, respectively. High-fat diet increased the exposure of contezolid. No discernible drug accumulation was observed after 15 days of continuous drug administration. Approximately 2% contezolid was excreted via kidneys in an unchanged form (8). A preliminary metabolism study in humans demonstrated that contezolid was mainly metabolized into MRX445-1 and MRX459 (Fig. 1) by the oxidative 2,3-dihydropyridin-4-one (DHPO) ring opening mainly catalyzed by flavin-containing monooxygenase 5 (9). However, the fate of contezolid in humans has not been fully explored, and whether any metabolite contributes substantially to the safety profile and pharmacological activity of the drug substance must be identified (10–12).

FIG 1.

Metabolic pathways of contezolid in humans reported in a previous study (9).

This report presents the characterization of the pharmacokinetics (PK) and disposition of [¹⁴C]contezolid in human and the cross-species comparison of plasma exposure for contezolid and metabolites. Contezolid forms disproportionately larger amounts of circulating metabolites (MRX445-1 and MRX459) in humans than in nonclinical species. On the basis of experimental results, nonclinical repeat-dose studies were performed with MRX445-1 to satisfy the regulatory requirements on safety testing of drug metabolites. In addition, the antibacterial activity of contezolid metabolites was examined.

RESULTS

Human mass balance/absorption, distribution, metabolism, and excretion study and safety.

Six healthy male subjects between the ages of 24 and 30 years were enrolled in the study, with a mean weight of 69.5 ± 2.4 kg (68.9 to 71.4 kg), a mean height of 1.77 ± 0.08 m (1.7 to 1.9 m), and a mean body mass index (BMI) of 22.6 ± 1.4 (20.0 to 23.8). All subjects are Han Chinese.

No serious or severe adverse event was observed during the clinical trial. All subjects well tolerated the drug and finished the study. Six adverse drug reactions (ADRs) occurred in three subjects (3/6 [50%]), including rash, increased alanine transaminase (ALT), and increased aspartate transaminase (AST). All ADRs were mild and transient, and all subjects recovered without treatment.

Mass balance/excretion in urine and feces.

The cumulative recovery of radioactivity from the six subjects is shown graphically in Fig. 2. An average of 91.5% ± 0.6% of the administered dose was recovered over the 168-h study. The majority of the administered radioactivity was recovered in urine, with a mean cumulative percentage of 76.7% ± 2.8% of the administered radioactivity. The radioactivity recovered in feces cumulatively accounted for 14.8% ± 2.9% of the administered radioactivity. The majority of the radioactivity (∼80% of the dose) was recovered within the first 24 h postdose.

FIG 2.

Cumulative percentage of dose recovered as total radioactivity in urine and feces after a single oral dose of [¹⁴C]contezolid (602 mg, 99.1 μCi) given to six healthy subjects (mean + standard deviation [SD]).

Metabolite profiles in excreta and plasma.

Contezolid metabolism involved oxidation and scission of the DHPO ring, reduction of the DHPO ring, reduction and scission of the isoxazole ring, oxidative deamination, hydration, glucuronidation, sulfation, etc. (Fig. 3). A summary of contezolid and its metabolites observed in excreta and plasma is provided in Table 1. The high-resolution mass spectrometry (HRMS) data and structure assignments for contezolid and its metabolites are provided in Fig. S1 to S18 in the supplemental material. The primary metabolite excreted in urine was MRX445-1 (approximately 48% of the dose), followed by MRX459 (approximately 15% of the dose), MRX401 (4.24% of the dose), and coeluted MRX423 and MRX445-2 (2.59% of the dose). Unchanged contezolid accounted for 2.45% and 0.365% of the dose in urine and feces, respectively. The other metabolites in urine or feces each accounted for less than 1% of the dose.

FIG 3.

Proposed [¹⁴C]contezolid biotransformation pathways in human (* denotes ¹⁴C label position). Structures of contezolid, MRX343, MRX357, MRX407, MRX413, MRX445-1, MRX445-2, and MRX459 were confirmed with synthetic or isolated standards. For all other metabolites, the proposed structures are consistent with available high-resolution mass spectrometry data and the biotransformation pathways leading to their formation.

TABLE 1.

Mean percentages of the ¹⁴C radioactivity of the most abundant metabolites in AUC pooled human plasma (0 to 8 h) and their corresponding percentages of the dose in pooled urine (0 to 168 h) and feces (0 to 168 h)

Parent drug and metabolite	Metabolic pathways	Matrixa
		Plasma	Urine	Feces
		% 14C AUC0–8	% dose	% dose
Contezolid	Parent drug	71.0	2.45	0.365
MRX343	Oxidative deamination	√	0.330	0.111
MRX357	Oxidative deamination	√	0.492	0.186
MRX359	Hydroxylation of MRX343	√	0.265	ND
MRX401	N-Dealkylation of MRX445-1 or MRX459	0.857	4.24	0.097
MRX405	Reductive isoxazole ring opening of MRX401	ND	ND	0.176
MRX407	Oxidative dehydrogenation of DHPO	√	0.302	ND
MRX413	Reductive isoxazole ring opening	ND	N. D.	0.141
MRX423	Sulfation of MRX343	ND	2.59b	ND
MRX427	Hydration	ND	ND	√
MRX429	Reduction plus hydration	√	0.412	ND
MRX445-1	Oxidative ring opening of DHPO by BVO	19.4	47.7	0.089
MRX445-2	Oxidative ring opening of DHPO	0.267	2.59b	0.082
MRX449	Reductive isoxazole ring opening of MRX445-1	ND	ND	0.331
MRX459	Oxidative ring opening of DHPO by BVO	7.45	15.2	0.148
MRX463	Reductive isoxazole ring opening of MRX459	ND	ND.	0.154
MRX489	Sulfation	√	0.286	ND
MRX601	Hydroxylation plus glucuronidation	0.568	0.549	ND

^aND, not detected; √, not detected by radiochromatography but detected by MS.

^bMRX423 coeluted with MRX445-2.

Plasma radioactivity mainly consisted of contezolid and six metabolites, as shown in Fig. 4 and Table 1. Contezolid accounted for the largest percentage of radioactivity in plasma (71.0%), followed by MRX445-1 (19.4%) and MRX459 (7.45%). Each of the other metabolites accounted for less than 1% of the radioactivity in plasma.

FIG 4.

Representative radiochromatogram of pooled human plasma from human subjects administered 602 mg [¹⁴C]contezolid (99.1 μCi). Unchanged contezolid, MRX445-1, and MRX459 are the main circulating drug-related materials.

Pharmacokinetics.

The pharmacokinetics of total radioactivity, contezolid, MRX445-1, and MRX459 were evaluated, and the mean concentration-time profiles are presented in Fig. 5, with the PK parameter estimates summarized in Table 2. The total radioactivity, contezolid, MRX445-1, and MRX459 median times to reach C_max (T_max) were observed at 3, 3, 3.5, and 4 h, respectively. The mean C_max for total radioactivity in plasma was 21.3 μg eq/g, while the mean C_maxs for contezolid, MRX445-1, and M459 in plasma were 14.2, 3.95, and 1.18 μg eq/ml, respectively. The total radioactivity, contezolid, MRX445-1, and MRX459 concentrations in plasma declined after reaching C_max, with mean terminal half-life (t_1/2) values of 2.84, 1.58, 1.72, and 1.86 h, respectively. The mean AUC_0–∞ values for contezolid (68.0 h · μg eq/ml), MRX445-1 (20.5 h · μg eq/ml), and MRX459 (1.57 h · μg eq/ml) in plasma were approximately 68.0%, 19.5%, and 4.84% of the mean total radioactivity AUC_0–∞ in plasma (110 h · μg eq/g). MRX445-1 was the only major circulating metabolite present in more than 10% of the circulating radioactivity, the threshold above which safety testing for metabolites may be required (13).

FIG 5.

Plasma concentration-time curves for total radioactivity, contezolid, MRX445-1, and MRX459 after a single oral dose of [¹⁴C]contezolid (602 mg, 99.1 μCi) given to six healthy subjects (mean + SD). Inset graph indicates the time profiles for 3 h following administration.

TABLE 2.

Pharmacokinetic parameters for total radioactivity, contezolid, MRX445-1, and MRX459 in plasma following a single oral dose of [¹⁴C]contezolid (602 mg, 99.1 μCi) given to six healthy male subjects^a

Parameter	Cmax (μg eq/ml)	Tmax (h)	AUC (h · μg eq/ml)		t1/2 (h)	AUC (%)c
			0 to last	0 to ∞
Radioactivityb	21.3 (3.6)	3.0 (2, 3)	107 (28)	110 (29)	2.84 (0.23)	NA
Contezolid	14.2 (3.6)	3.0 (2, 4)	67.6 (22.3)	68.0 (22.4)	1.58 (0.23)	60.8 (6.4)
MRX445-1	3.95 (0.60)	3.5 (3, 4)	20.3 (1.3)	20.5 (1.3)	1.72 (0.26)	19.5 (4.3)
MRX459	1.18 (0.81)	4.0 (2, 4)	5.06 (1.68)	5.17 (1.67)	1.86 (0.36)	4.84 (1.80)

^aData for all parameters are means (SDs) except for the time to reach C_max (T_max), which are the median (minimum, maximum) values.

^bUnits of μg eq/g for C_max or h · μg eq/g for AUC.

^cRatio of AUC_0-∞ contezolid, MRX445-1, and MRX459 to total radioactivity. NA, not applicable.

Cross-species comparison of contezolid and metabolite exposure in plasma.

The relative plasma AUC exposures of contezolid and its metabolites (MRX445-1 and MRX459) at the first and steady-state doses in humans, rats, and dogs are presented in Table 3. The first dose and steady-state plasma AUCs of intact contezolid were similar between dogs and humans. The plasma AUC of contezolid in rats was 3.25-fold and 2.06-fold more abundant than in humans at the first dose and at steady state. However, the MRX445-1 and MRX459 plasma AUCs were much less abundant in rats and dogs than in humans, whether at the first dose or at steady state. Furthermore, the plasma exposures of contezolid, MRX445-1, and MRX459 were similar at the first dose and at steady state whether in rat, dog, and human subjects, indicating no accumulation and no metabolic induction at repeat dosing.

TABLE 3.

Relative AUC exposure levels of contezolid and metabolites in rat and dog at the corresponding NOAEL dose and human at the dose of 600 mg^a

Drug	AUC relative to that in humans
	At the first dose		At steady state
	Rat	Dog	Rat	Dog	Humanb
Contezolid	3.25	1.27	2.06	1.08	1.29
MRX445-1	0.052	0.012	0.036	0.012	1.11
MRX459	0.189	0.047	0.218	0.030	1.27

^aNOAEL, no-observed-adverse-effect level.

^bAUC exposure levels in humans at stead state relative to that at the first dose.

PK of MRX445-1 following intravenous administration of MRX445-1 to rats.

The PK of MRX445-1 was evaluated, and the mean concentration-time profiles are presented in Fig. 6, with the PK parameter estimates summarized in Table 4. The MRX445-1 AUC_0–last values were 59.6, 98.5, and 166 h · μg/ml at 160, 240, and 360 mg/kg body weight, respectively, for the first dose and 47.8, 75.3, 134 h · μg/ml, respectively, for the second dose on day 1. On day 14, the MRX445-1 AUC_0–last values were 51.3, 85.5, and 141 h · μg/ml at 160, 240, and 360 mg/kg body weight, respectively, for the first dose and 48.8, 83.0, and 123 h · μg/ml, respectively, for the second dose. The clearance (CL), volume of distribution at steady state (V_ss), and t_1/2 of MRX445-1 in rats, which were ∼3 liters/kg body weight/h, 0.34 liters/kg body weight, and ∼0.2 h, respectively, were similar at different doses (Table 4).

FIG 6.

Plasma concentration-time curves for MRX445-1 after intravenous dose of MRX445-1 given to rats. The data in the plot are mean values.

TABLE 4.

Pharmacokinetic parameters for MRX445-1 in plasma following intravenous doses of [¹⁴C]MRX445-1 given to rats

Dose (mg/kg/day)	Day	1st dose							2nd dose
		Tmax (h)	Cmax (μg/ml)	AUC (h · μg/ml)		t1/2 (h)	Vss (liters/kg)	CL (liters/h/kg)	Tmax (h)	Cmax (μg/ml)	AUC (h · μg/ml)		t1/2 (h)	Vss (liters/kg)	CL (liters/h/kg)
				0 to last	0 to ∞						0 to last	0 to ∞
160	1	0.033	303	59.6	59.7	0.222	0.299	2.70	0.033	253	47.2	47.2	0.121	0.309	3.39
	14	0.033	257	51.3	51.4	0.364	0.382	3.12	0.033	256	48.8	49.0	0.135	0.335	3.27
240	1	0.033	492	98.5	98.6	0.338	0.298	2.44	0.033	386	75.3	75.3	0.110	0.323	3.23
	14	0.033	398	85.5	85.6	0.477	0.417	2.81	0.033	410	83.0	83.2	0.116	0.326	2.92
360	1	0.033	785	166	166	0.426	0.294	2.24	0.033	669	134	134	0.097	0.280	2.69
	14	0.033	652	141	141	0.520	0.354	2.58	0.033	487	122	123	0.120	0.465	2.95
Mean						0.391	0.340	2.64					0.116	0.340	3.07
SD						0.107	0.052	0.31					0.012	0.064	0.27

Antibacterial activity of contezolid metabolites.

The MICs of the contezolid metabolites, MRX445-1, and MRX459, were >16 μg/ml in all strains tested (Table 5). In contrast, the high potency of contezolid was consistent with the results of previous studies conducted in Gram-positive pathogens (1). In line with the structure-activity relationships for the oxazolidinone class, the loss of antibacterial activity of MRX445-1 and MRX459 (formed by DHPO ring opening), demonstrated the importance of a relatively planar DHPO ring for contezolid activity and the poor tolerance of acyclic and highly polar structures at this position of the fluorinated aromatic ring (1).

TABLE 5.

Antimicrobial activity of contezolid, MRX445-1, and MRX459 in Gram-positive pathogens

Species	ATCC no.	MIC (μg/ml)
		MRX-I	MRX445-1	MRX459
Staphylococcus aureus	ATCC 29213	2	>16	>16
Enterococcus faecium	ATCC 35667	2	>16	>16
Enterococcus faecalis	ATCC 29212	2	>16	>16
Streptococcus pneumoniae	ATCC 6305	0.25	>16	>16
	ATCC 49619	0.5	>16	>16

DISCUSSION

Absorption, distribution, metabolism, and excretion (ADME) studies with [¹⁴C]contezolid enabled a holistic understanding of drug disposition. Overall, almost complete recovery (91.5% of dose) was obtained, of which 76.7% of the dose was recovered in urine and 14.8% in feces over 168 h after the oral administration of [¹⁴C]contezolid. The elimination was rapid, with ∼80% of the dose recovered within the first 24 h postdose. The unchanged contezolid recovered in urine and feces accounted for <3% of the dose over 168 h. The major elimination pathway was metabolism and elimination by the urinary route. The principal metabolic pathway of contezolid in humans was the DHPO ring opening via Baeyer-Villiger oxidation generating MRX445-1 and MRX459, which was first disclosed in the preliminary metabolism study (9). The present study showed that MRX445-1 and MRX459 recoveries were approximately 48% and 15% of the dose, respectively, in urine and feces. The in vitro phenotyping study demonstrated that multiple noncytochrome P450 enzymes are involved in the formation of MRX445-1 and MRX459, including flavin-containing monooxygenase 5, short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (9). Furthermore, MRX401, which was also generated via Baeyer-Villiger oxidation of DHPO ring, was >4% of the dose in urine and feces. In addition to the oxidation of DHPO ring opening, the human ADME study suggested additional biotransformation, including oxidative deamination (MRX343, MRX357, and MRX359), reductive isoxazole ring opening (MRX405, MRX413, MRX449, and MRX463), oxidation of the DHPO ring (MRX407), sulfation (MRX423 and MRX489), and glucuronidation (MRX601). The recovery of each of these metabolites accounted for <3% of the dose.

Following single-dose [¹⁴C]contezolid administration, the most abundant circulating radioactivity in humans was composed of the parent drug, representing 60.8% of the AUC of total radioactivity in plasma. The PK profile of contezolid at 602 mg was consistent with the phase I study, with similar exposure (AUC and C_max) and absorption characteristics (8). The most abundant circulating metabolites in humans were MRX445-1 and MRX459, representing 19.5% and 4.84%, respectively, of the AUC of total radioactivity in plasma. The exposures of MRX445-1 and MRX459 were similar after single-dose and multiple-dose administrations. The ratios of the relative AUC exposure levels for MRX445-1 and MRX459 were 1.11 and 1.27, respectively (Table 3). These findings indicated that no major metabolite accumulation was observed after 600 mg twice a day (b.i.d.) for 15 days.

The systemic exposure of MRX445-1 and MRX459 was disproportionately higher in humans than in rats and dogs. This was consistently observed in comparisons of the relative plasma AUC exposure for contezolid and its metabolites (MRX445-1 and MRX459) at the first dose and at steady state in rats, dogs, and humans. Given that MRX445-1 was the major metabolite (>10% of the circulating drug-related material [DRM]) in humans and disproportionately higher MRX445-1 human systemic AUC exposure was observed, MRX445-1 was synthetized and directly administered to rats for further safety evaluation. The preliminary PK studies showed that MRX445-1 was hardly absorbed after intragastrical administration, and the absolute bioavailability of MRX445-1 was less than 50% after subcutaneous injection or intraperitoneal injection (data not published). Therefore, MRX445-1 was intravenously injected to increase its systemic AUC exposure. After intravenous injection to rats, the CL, V_ss, and t_1/2 of MRX445-1 were similar at the first dose and at steady state within the dose range of 160 to 360 mg/kg body weight. The V_ss of MRX445-1 in rats was 0.34 liters/kg body weight, which was similar to the volume of extracellular fluid in rats (0.3 liters/kg body weight) (14), indicative of localization within the extracellular fluid compartment. The high CL (∼3 liters/kg/h) and short t_1/2 (∼0.2 h) of MRX445-1 indicated the rapid elimination of MRX445-1 in rats.

The systemic AUC exposure of MRX445-1 increased in the range of 160 to 360 mg/kg. At the lowest level of 160 mg/kg, the AUC_0–last values of MRX445-1 were 59.6 and 47.2 h · μg/ml for the first and second doses on day 1, respectively, and 51.3 and 48.8 h · μg/ml on day 14, respectively. Those values were higher than the AUC_0–∞ values of MRX445-1 in humans after oral administration of 602 mg contezolid. Therefore, the intravenous administration of MRX445-1 was considered sufficient to allow for MRX445-1 safety assessment. The dosing approach was adopted in a subsequent subchronic toxicity study and embryo-fetal development study with MRX445-1 to meet regulatory guidelines (13), and the no-observed-adverse-effect level (NOAEL) was 360 mg/kg body weight once a day (QD). In addition, in vitro genotoxicity testing was conducted for MRX445-1, with a negative outcome (data not published). Considering contezolid could not be used long term in the clinical study, the rats with MRX445-1 were exempted from the subsequent carcinogenicity study.

MRX445-1 and MRX459, the major metabolites of contezolid, essentially lack the antibacterial activity. Thus, contezolid exposure determined its efficacy against serious infections caused by Gram-positive bacteria. In addition, given that unchanged contezolid was not eliminated in significant amounts in urine or feces and the major metabolites exhibited lower antibacterial activity, significant antibacterial effects in the bladder or large intestine after oral administration of contezolid were not expected.

In summary, contezolid was metabolized via Baeyer-Villiger oxidation generating the DHPO ring metabolites, MRX445-1 and MRX459, which were recovered as >47% and >15% of the dose, respectively, in urine and feces in humans. MRX445-1 accounted for 19.5% of the plasma exposure of the total radioactivity, and it was identified as the disproportionate metabolite. The repeat-dose NOAEL of MRX445-1 was determined as 360 mg/kg/day in nonpregnant and pregnant Sprague Dawley rats for intravenous administration, and MRX445-1 showed no antibacterial activities in vitro. Therefore, MRX445-1 was not expected to exhibit clinically relevant pharmacological activities and toxicity.

MATERIALS AND METHODS

Human mass balance study.

The primary objectives of this study were to determine the rates and routes of excretion of [¹⁴C]contezolid-related radioactivity, identify and (semi)quantify the metabolites of contezolid in plasma, urine, and feces to elucidate the key biotransformation pathways and clearance mechanisms of contezolid in humans, and characterize the pharmacokinetics of metabolites. The secondary objective was to assess the safety and tolerability of a single 602-mg oral dose of [¹⁴C]contezolid (Shanghai Institute of Materia Medica, Shanghai, China; MicuRx Pharmaceuticals, Inc., Shanghai, China) administered to healthy male volunteers. The protocol of the study was approved by an independent ethics committee, and the study was conducted in accordance with the ethical principles that originated in the Declaration of Helsinki and with good clinical practices and applicable regulatory requirements. Written informed consent was obtained from all participants. An overview of the purpose and design of this study is presented in Table 6.

TABLE 6.

Summary of nonclinical and clinical PK study design following oral administration of contezolid or intravenous administration of MRX445-1

Speciesa	No. of subjects or animals and sexb	Dose	Dose regimenc	Purpose and time points
Mass balance study in healthy  subjects
Human	6 M	602 mg total	Single dose	PK in plasma, mass balance, and metabolic profiling; Plasma: predose, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8., 12, 16, 24, 36, 48, 72, 96, 120, 144, and 168 h; Urine: predose (−12 to 0), 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h; Feces: predose (−24 to 0), 0–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h
Cross-species comparison of  plasma exposure for  contezolid and metabolite
Rat	9 M and 9 F	100 mg/kg/day	Repeat dose: QD for the first day, TD for the following 90 days	Plasma PK on days 1 and 91: predose, 1, 2, 4, 8, 10, 12, and 24 h
Dog	5 M and 5 F	60 mg/kg/day	Repeat dose: QD for the first day, TD for the following 91 days	Plasma PK on days 1 and 92: predose, 0.5, 2, 4, 9, and 24 h
Human	6 M and 6F	600 mg	Repeat dose: QD for the first day, TD for the following 14 days	Plasma PK on days 1 and 15: predose, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h
PK of MRX445-1 following  intravenous administration
Rat	5 M and 5 F	160 mg/kg/day	Repeat dose: TD for 14 days	Plasma PK on days 1 and 14: predose, 0.033, 0.25, 0.5, 1, 2, 4, 10, 10.033, 10.25, 10.5, 11, and 24 h
Rat	5 M and 5 F	240 mg/kg/day	Repeat dose: TD for 14 days	Plasma PK on days 1 and 14: predose, 0.033, 0.25, 0.5, 1, 2, 4, 10, 10.033, 10.25, 10.5, 11, and 24 h
Rat	5 M and 5 F	360 mg/kg/day	Repeat dose: TD for 14 days	Plasma PK on days 1 and 14: predose, 0.033, 0.25, 0.5, 1, 2, 4, 10, 10.033, 10.25, 10.5, 11, and 24 h

^aRat, Sprague Dawley; dog, beagle.

^bF, female; M, male.

^cQD, once daily; TD, twice daily.

(i) Study design.

Six healthy male volunteers were enrolled in this open-label, single-center ADME study of a single oral dose of 602 mg [¹⁴C]contezolid conducted at the Clinical Trial Center of Shanghai Huashan Hospital. Volunteers who met the inclusion criteria and were confirmed to be eligible according to predose safety evaluations received a single oral dose of [¹⁴C]contezolid. The key inclusion criteria included male volunteers 18 to 45 years of age and in good health, as determined by past medical history, physical examination, electrocardiography (ECG), and laboratory tests at screening, and weight of more 50 kg, with a body mass index (BMI) of 19 to 24 kg/m². The key exclusion criteria included a history of hypersensitivity to the study drug or to drugs of similar chemical classes or excipients, relevant radiation exposure (>0.2 mSv) within 12 months prior to scheduled dosing with [¹⁴C]contezolid, and use of prescribed or nonprescribed concomitant medications within 14 days and medications with CYP450 enzyme-inducing or inhibiting properties within 30 days before the study. Volunteers were also excluded if they had any intake of any product containing grapefruit 7 days before and during the study.

(ii) Medication and sampling.

On the day of dosing, the subjects received a single oral dose of 50 ml [¹⁴C]contezolid suspension (99.1 μCi/602 mg) after breakfast. The cup was then rinsed three times with 50 ml of warm water, and the water was swallowed by the subjects. Blood samples for PK assessment were collected from −1 to 0 h before drug administration and at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, 96, 120, 144, and 168 h after administration. At each time point, 4 ml of blood sample was collected into a lithium heparin tube. Plasma was then obtained by centrifugation at 1,500 × g at 4°C for 10 min within 30 min after collection and stored at −20°C until concentrations were determined. Plasma samples for metabolite tracking and identification were collected at −1, 0, 0.5, 1, 3, 8, and 24 h after drug administration. Urine samples were collected and pooled at the following intervals: 0 to 24 h before drug administration and 0 to 4, 4 to 8, 8 to 12, and 12 to 24 h after administration, and then every 24 h during 24 to 168 h after administration. Fecal samples were collected and pooled at the following intervals: 0 to 24 h before administration and once every 24 h within 168 h after administration. Except for special reasons, the subjects were discharged from the clinical unit if two consecutive collections of urine and feces demonstrated recoveries less than three times the background amount or if the accumulative radioactivity in the excreta within 1 day was less than 1% of the total radioactivity of the administered drug. If neither discharge criteria were achieved on day 7 of administration, the subjects were required to remain in the clinical unit, and the collection of samples was continued until the amount of radioactivity met one of the criteria.

(iii) Determination of radioactivity by LSC.

The radioactivity at each collection interval of urine was directly assessed using a Packard Tri-Carb 3100 TR liquid scintillation counter (PerkinElmer, Waltham, MA, USA). Fecal residues were combusted using a Harvey OX-501 biological oxidizer (R. J. Harvey Instrument Corp., Tappan, New York, USA) prior to liquid scintillation counting (LSC). Radioactive counts were assessed in duplicate determinations and normalized to total radioactivity collected per interval, accounting for the total weight. The total recovery of radioactivity (Fe_tot; percentage or fraction of administered radioactive dose recovered in urine and feces overall) was computed as the sum of the cumulative excretion in urine and feces across all collection periods. The radioactivity at each collection time point was also assessed by direct LSC of the plasma samples. Radioactive counts were converted into the concentration of drug-related materials in accordance with the specific activity of the dose (0.164 μCi/mg).

(iv) Metabolite profiling and identification in plasma, urine, and feces.

Plasma samples obtained at 0.5, 1, 3, 8, and 24 h postdose were pooled across subjects evenly (0.5 ml) at each individual time point and then used to quantify metabolites at these individual time points. These data were used to calculate the AUCs for [¹⁴C]contezolid and individual metabolites. In brief, 3.0 ml of pooled plasma was loaded onto Oasis HLB solid-phase extraction (SPE) columns (3-ml barrel; Waters Corporation, Milford, MA, USA). The retained radioactivity on the SPE columns was eluted with 6 ml methanol and evaporated to dryness. The residues were reconstituted in 300 μl of 5 mM ammonium formate and acetonitrile (5:95) and profiled by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with eluent fraction collections made at 12-s intervals on a 96-well plate containing solid scintillant and counted via TopCount analysis (PerkinElmer, Waltham, MA, USA). The pooled urine samples (2.0 g) were centrifuged at 11,000 × g prior to analysis. For fecal homogenates, 1 ml sample was extracted with 2 ml methanol and centrifuged at 11,000 × g, and the pellet was resuspended in another 2 ml methanol and centrifuged again. The supernatants were combined, evaporated to dryness, and reconstituted in 500 μl of 5 mM ammonium formate and acetonitrile (5:95) prior to TopCount analysis and LC-MS/MS profiling. The metabolites in urine, feces, and plasma extracts were separated on a Synergi Hydro-RP C₁₈ (150 mm by 4.6-mm inside diameter [i.d.], 4 μm) column in line with a Phenomenex C₁₈ guard column (4 mm by 3.0-mm i.d., 5 μm). The gradient schedule for plasma and urine samples was programmed at 95:5 (A:B) for 0 min, 75:25 for 40 min, 55:45 for 50 min, and 0:100 for 55 min with 5 mM ammonium formate (solvent A) and acetonitrile (solvent B) as the mobile phase. The gradient schedule for the plasma and urine samples was programmed at 95:5 (A:B) for 0 min, 50:50 for 40 min, and 0:100 for 55 min, with 0.05% formic acid (solvent A) and methanol (solvent B) as the mobile phase. The LC flow rate was 0.6 ml/min. Metabolite identification or characterization was performed via LC-MS/MS using a triple time of flight (TOF) 5600+ MS/MS system (AB Sciex, Concord, ON, Canada) as described previously (9).

(v) Quantitation of contezolid and metabolites by LC-MS/MS.

The LC-MS/MS analysis of contezolid and its major metabolites, MRX4451-1 and MRX459 (MicuRx Pharmaceuticals, Inc., Shanghai, China), was performed on a Shimadzu ultraperformance liquid chromatography (UPLC) system (Shimadzu Corporation, Kyoto, Japan) coupled to a triple quadrupole mass spectrometer TSQ Quantum Ultra with a turbo-electrospray ionization source (Thermo Finnigan, San Jose, CA, USA). Chromatographic separations were performed on an Eclipse XDB-C₈ column (150 mm by 4.6-mm i.d., 5 μm) from Agilent Technologies, Inc. (Santa Clara, CA, USA) using isocratic elution. The mobile phase consisted of acetonitrile and 5 mM ammonium acetate containing 0.05% formic acid (45:55), with a flowrate of 0.65 ml/min. Quantitative data were acquired in multiple-reaction monitoring–positive ionization mode. Contezolid, MRX445-1, and MRX459 were detected at the selected reaction monitoring transitions of m/z 409.1 to m/z 269.1, m/z 445.1 to m/z 385.2, and m/z 459.0 to m/z 399.0, respectively. Linezolid was used as the internal standard and detected at the selected reaction monitoring transitions of m/z 338.0 to m/z 296.0. Calibration curves were prepared in blank plasma at concentrations ranging from 0.0400 μg/ml to 20.0 μg/ml for contezolid and from 0.0200 μg/ml to 5.00 μg/ml for MRX445-1 and MRX459. The plasma samples were extracted using protein precipitation. In particular, 100-μl plasma samples were mixed with 20 μl linezolid solution (0.0500 μg/ml, internal standard) and 300 μl of acetonitrile, followed by vortex mixing on a mixer for 1 min. The mixed solutions were then centrifuged at 14,000 × g for 5 min by a CT15RE centrifuge (Hitachi, Minato, Tokyo, Japan), and 20 μl supernatant was mixed with 80 μl mobile phase. Five microliters of the sample was injected for analysis.

(vi) Pharmacokinetic analysis.

The pharmacokinetic parameters for plasma contezolid, MRX445-1, MRX459, and ¹⁴C radioactivity were assessed using standard noncompartmental methods with Phoenix WinNonLin Professional version 6.3 (Pharsight Corporation, Mountain View, CA). The following pharmacokinetic parameters were assessed: maximum concentration (C_max), time to reach C_max (T_max), elimination half-life time (t_1/2), AUC from time zero to time of the last quantifiable concentration (AUC_0–last), and AUC from time zero to infinite time (AUC_0–∞), calculated as the sum of AUC_0–last and C_last/λz, in which C_last is the last observed quantifiable concentration and λz is the elimination half-life time to last quantifiable concentration.

(vii) Safety evaluation.

The physical examination and laboratory safety test parameters were assessed on screening day, day before dosing (D-1), dosing day (D1), follow-up day (D8), and ending day for all subjects. ECG examination, including corrected QT interval, was conducted before dosing and on D1, D8, and ending day. Vital sign examination was conducted on screening day, within 1 h before dose, 2, 4, 12, 24, 48, and 96 h postdose, D8, and ending day. The adverse events (AEs) that occurred during the whole study were recorded.

Nonclinical in vivo PK studies.

An overview of the purpose and design of nonclinical studies is provided in Table 6. All nonclinical samples were provided by a contract research organization (Covance, Shanghai, China). All protocols were approved by the Institutional Animal Care and Use Committee.

(i) Cross-species comparison of contezolid and metabolite exposure in plasma.

The relative plasma exposures of contezolid and key metabolites (MRX445-1 and MRX459) were compared in AUC pooled samples (15) collected from rats, dogs, and humans (Table 6).

The rat AUC pool (0 to 24 h) was prepared from day-1 or day-91 samples from animals administered the 100-mg/kg/day dose of contezolid. The dog AUC pool (0 to 24 h) was derived from day-1 or day-92 samples from animals provided with a 60-mg/kg/day dose of contezolid. Dose levels of 100 and 60 mg/kg per day were selected for rat and dog samples, respectively, because they represented the no-observed-adverse-effect level (NOAEL) doses for these species. The human AUC pool (0 to 24 h) was prepared from day-14 samples from subjects administered a 600-mg/day dose of contezolid in tablets. All AUC pools contained plasma from equal numbers of males and females. The rat control pool was derived from plasma samples taken 3 h after dosing of the vehicle alone and the predose samples of selected animals on day 1. The rat, dog, and human control pools were derived from predose samples. For each pairwise comparison (rat to human, dog to human, etc.), three samples were prepared by mixing equal volumes of the AUC pool of each species with a control pool of the comparator species to ensure that the three samples would have exactly the same matrix (16). In particular, for the rat-to-human and dog-to-human comparisons, a rat AUC pool was mixed with a human control pool and dog control pool, a dog AUC pool was mixed with a rat control pool and human control poll, and a human AUC pool was mixed with a rat control pool and dog control pool. The plasma samples were prepared and analyzed by LC-MS/MS using a triple TOF 5600+ MS/MS system (AB Sciex, Concord, ON, Canada) as described previously (9).

(ii) PK of MRX445-1 following intravenous administration to rats.

Given the limited MRX445-1 formation in rats (Table 3), whether the administration of MRX445-1 in rats could produce sufficient systemic AUC exposure must be determined to allow for MRX445-1 safety assessment, as per FDA guidelines (13). An overview of the purpose and design of this study is provided in Table 6. Synthetized MRX445-1 was formulated with sterile normal saline and intravenously administered at 0 h for the first dose and 10 h for the second dose (twice daily [TD]) for 14 days. Plasma PK samples were collected at specific time points and analyzed by LC-MS/MS.

Antibacterial activity of contezolid, MRX445-1, and MRX459.

MICs were determined by broth microdilution in accordance with Clinical and Laboratory Standards Institute-approved methods (17). Staphylococci (S. aureus strain ATCC 29213) and enterococci (Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 35667) were incubated in Mueller-Hinton agar or cation-adjusted Mueller-Hinton broth. Streptococci (Streptococcus pneumoniae ATCC 49619 and ATCC 6305) were cultured in tryptic soy agar with 5% sheep blood. Stock solutions of 5 mg/ml contezolid, MRX445-1, and MRX459 were prepared separately in 100% dimethyl sulfoxide and serially diluted in 2-fold steps for the final concentration range of 0.016 to 16.0 μg/ml. The plates were incubated 20 h at 35°C. The MIC values were determined as the concentration with no visible color change.