Pharmacokinetics and Pharmacodynamics of Multiple-Dose Intravenous Nemonoxacin in Healthy Chinese Volunteers

Abstract

This study evaluated the safety and pharmacokinetic/pharmacodynamic profiles of nemonoxacin in healthy Chinese volunteers following multiple-dose intravenous infusion once daily for 10 consecutive days. The study was composed of two stages. In the open-label stage, 500 mg or 750 mg of nemonoxacin (n = 12 each) was administered at an infusion rate of 5.56 mg/min. In the second stage, with a randomized double-blind placebo-controlled design, 500, 650, or 750 mg of nemonoxacin (n = 16 in each cohort; 12 subjects received the drug and the other 4 subjects received the placebo) was given at an infusion rate of 4.17 mg/min. The results showed that, in the first stage, the maximal nemonoxacin concentrations (mean ± SD) at steady state (C_{max_ss}) were 9.60 ± 1.84 and 11.04 ± 2.18 μg/ml in the 500-mg and 750-mg cohorts, respectively; the areas under the concentration-time curve at steady state (AUC_{0–24_ss}) were 44.03 ± 8.62 and 65.82 ± 10.78 μg · h/ml in the 500-mg and 750-mg cohorts, respectively. In the second stage, the nemonoxacin C_{max_ss} values were 7.13 ± 1.47, 8.17 ± 1.76, and 9.96 ± 2.23 μg/ml in the 500-mg, 650-mg, and 750-mg cohorts, respectively; the AUC_{0–24_ss} values were 40.46 ± 9.52, 54.17 ± 12.10, and 71.34 ± 17.79 μg · h/ml in the 500-mg, 650-mg, and 750-mg cohorts, respectively. No accumulation was found after the 10-day infusion with any regimen. The drug was well tolerated. A Monte Carlo simulation indicated that the cumulative fraction of response of any dosing regimen was nearly 100% against Streptococcus pneumoniae. The probability of target attainment of nemonoxacin therapy was >98% when the MIC of nemonoxacin against S. pneumoniae was ≤1 mg/liter. It is suggested that all of the studied intravenous nemonoxacin dosing regimens should have favorable clinical and microbiological efficacies in future clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01944774.)

INTRODUCTION

Nemonoxacin is a novel nonfluorinated quinolone with a wide antimicrobial spectrum covering Gram-positive cocci and Gram-negative bacilli, including the common pathogens of community-acquired pneumonia (CAP) (1, 2). Nemonoxacin is generally more active than other fluoroquinolones against Streptococcus pneumoniae strains that are resistant to penicillin (PRSP) or quinolone. The MIC₉₀s of nemonoxacin and levofloxacin against PRSP are 0.03 mg/liter and 1 mg/liter, respectively (3–10). Nemonoxacin is also active against Staphylococcus aureus strains that are resistant to methicillin (MRSA) or vancomycin. The MIC₉₀s of nemonoxacin and levofloxacin against community-acquired MRSA are 0.5 mg/liter and 8 mg/liter, respectively (3–10).

There are two formulations available for nemonoxacin, an oral capsule formulation of nemonoxacin malate and an intravenous (i.v.) formulation of nemonoxacin malate sodium chloride. The premarketing clinical studies for the nemonoxacin malate capsule have been completed, and it was first approved by the Taiwan Food and Drug Administration in November 2014. Series of pharmacokinetic (PK) and pharmacodynamic (PD) studies in the United States and China revealed that single oral doses of 25 to 1,500 mg nemonoxacin malate in a capsule were safe in healthy subjects (11), and nemonoxacin was also well tolerated and safe in healthy subjects after oral administration of nemonoxacin capsule doses ranging from 75 to 1,000 mg once daily for 10 consecutive days (12). The PK study in healthy Chinese subjects also reported that nemonoxacin in the capsule formulation was rapidly absorbed and reached the peak blood concentration in about 1 to 2 h under fasting conditions. The half-life (t_1/2) of nemonoxacin in the capsule formulation was about 11 h, and about 70% of the administered dose was excreted by the kidneys in an unchanged form. The two oral regimens, 500 mg or 750 mg once daily for 10 consecutive days under fasting conditions, were also well tolerated in Chinese subjects (13).

TaiGen Biotechnology Co., Ltd., has completed PK/PD studies for the i.v. formulation of nemonoxacin malate sodium chloride. The results of the single-dose tolerability and PK study of the i.v. nemonoxacin formulation indicated that the range of tolerated doses was 25 to 1,250 mg, with acceptable i.v. infusion rates ranging from 0.42 to 5.56 mg/min. The area under the concentration-time curve from time zero to infinity (AUC_0–∞) showed a good linear relationship with nemonoxacin doses in the range of 250 mg to 750 mg, with a half-life of about 11 h. About 65% to 77% of the administered nemonoxacin was excreted by the kidneys in unchanged form (14).

In this study (registered at ClinicalTrials.gov under registration no. NCT01944774), the PK properties and safety of nemonoxacin were further examined in healthy Chinese subjects after multiple i.v. infusions of nemonoxacin at different doses and infusion rates. Five dosing regimens were studied, including 500 or 750 mg at an infusion rate of 5.56 mg/min and 500 mg, 650 mg, or 750 mg at an infusion rate of 4.17 mg/min; all doses were administered once daily for 10 consecutive days. The PK results from this study were also analyzed in combination with PD findings to explore the appropriate dosing regimens for late-phase clinical trials in the target patient population.

MATERIALS AND METHODS

This study was conducted in a university teaching hospital according to the Declaration of Helsinki and all its amendments. The study protocol and informed consent form were reviewed and approved by the Ethics Committee of Huashan Hospital, Fudan University. Each subject provided written informed consent before the initiation of screening procedures.

Overall study design.

This study was a multiple-parameter PK study designed with five arms. The first two arms used an open-label design in which the healthy subjects received 500 or 750 mg (12 subjects per dose group) nemonoxacin by i.v. infusion, with infusion times of 1.5 h and 2.25 h at an infusion rate of 5.56 mg/min, once daily for 10 consecutive days. The last three arms used a randomized double-blind placebo-controlled design in which the healthy subjects received 500, 650, or 750 mg (12 subjects per dose group) nemonoxacin or placebo by i.v. infusion (4 subjects per dose group), with infusion times of 2 h, 2.6 h, and 3 h at an infusion rate of 4.17 mg/min, once daily for 10 consecutive days.

Study subjects.

Healthy Chinese male or female volunteers aged 18 to 45 years were screened for eligibility as study subjects within 22 days before enrollment. The baseline evaluation included a medical history, a physical examination, vital signs, laboratory tests, and a 12-lead electrocardiogram (ECG). The individuals were excluded if they had any of the following conditions: history of acute or chronic disease of any major organ or system, infection with viral hepatitis or HIV, QTc prolongation on ECG, clinically significant abnormality of laboratory tests, body mass index (BMI) beyond the range of 19 to 24 kg/m², or history of allergy to any drug, substance abuse, or participation in another clinical study in the past 3 months.

Investigational drug.

Nemonoxacin 0.5 g (volume, 100 ml; lot 08011904; containing sodium chloride 0.9 g; pH 3.9) was used in the first stage. Nemonoxacin 0.5 g (volume, 100 ml; lot 10101606; containing sodium chloride 0.9 g; pH 4.5) and placebo (volume, 100 ml; lot 10101506; containing sodium chloride 0.9 g) were used in the second stage. The two nemonoxacin formulations and the placebo were manufactured by Huayu (Wuxi) Pharmaceutical Co., Ltd.

PK study. (i) Blood and urine sample collection.

Blood samples were obtained via a peripheral venous catheter at the following time points: before the infusion (0 h), in the middle of the infusion, at the end of the infusion, and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 16 h after the intravenous infusion of the first dose (day 1) and the last dose (day 10). The last sampling point was 24 h after the start of the infusion for the first dose (day 1) and the last dose (day 10). In stage 1, the infusion times were 1.5 h and 2.25 h for the 500- and 750-mg cohorts, respectively. In stage 2, the infusion times were 2 h, 2.6 h, and 3 h for the 500-, 600-, and 750-mg cohorts, respectively. Additional blood samples were collected 36, 48, 60, and 72 h after the intravenous infusion of the last dose. In addition, blood samples were also obtained before the infusion and at the end of the infusion on days 3, 5, 8, and 9 to determine the peak (C_max) and trough (C_min) concentrations of nemonoxacin. The blood samples were centrifuged at 1200 × g (for 10 min at 4°C) and stored at −40°C for future experiments.

Urine samples were collected at the following time periods: −1 to 0 h (predose) and 0 to 2, 2 to 4, 4 to 8, 8 to 12, and 12 to 24 h after intravenous infusion of the first dose and −1 to 0 h (predose) and 0 to 2, 2 to 4, 4 to 8, 8 to 12, 12 to 24, 24 to 36, 36 to 48, and 48 to 72 h after intravenous infusion of the last dose. The samples were stored at −40°C for future experiments.

(ii) Concentrations of nemonoxacin in blood and urine samples.

The concentrations of nemonoxacin in human blood and urine samples were determined using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods as reported by Guo et al. (15). The LC-MS/MS system included a Waters Alliance 2690 high-pressure liquid chromatograph and Finnigan TSQ Quantum mass spectrometer (Waters Co., Milford, MA, USA). Samples were analyzed by a mass spectrometer equipped with an electrospray ionization source operating in positive-ion selected reaction monitoring mode. Chromatography was performed with a Waters Symmetry Shield RP18 column (50 mm by 2.1 mm, 5-μm pore size; Waters Co.), and the mobile phase was 0.1% formic acid–acetonitrile (82:18) for plasma samples and 0.1% formic acid–acetonitrile (85:15) for urine samples. The flow rate of the mobile phase was set at 0.2 ml/min, and the injection volume used was 5 μl. The method was validated over a nemonoxacin concentration range of 0.005 to 1.0 μg/ml in both plasma and urine matrices. Quality control (QC) samples at four concentration levels (lower limit of quantitation [LLOQ], low QC, medium QC, and high QC) were prepared and tested in each run of this study. The coefficients of variation (CV) were less than 12.4% and 13.2% in plasma and urine matrices, respectively.

(iii) Calculation of PK parameters and statistical analysis.

The PK parameters of nemonoxacin were obtained using noncompartmental analysis. The parameters reflecting the exposure, distribution, and elimination of nemonoxacin in plasma included the C_max, AUC_0–24, AUC_0–∞, t_1/2, V, CL_z, MRT_0–∞, C_min, and C_avg. The C_max is the peak plasma concentration; the AUC_0–24 is the area under the concentration-time curve from 0 to 24 h since the drug infusion, which was calculated using a linear trapezoidal method. The AUC_0–∞ is the area under the concentration-time curve from time zero to infinity. t_1/2 is the half-life calculated according to 0.693/λ_z, where λ_z is the terminal elimination rate obtained using concentration data during 0 to 24 h from day 1 or day 10. The CL_z is the total clearance obtained from dose/AUC_0–∞. V is the apparent distribution volume, calculated as CL_z/λ_z. MRT_0–∞ is the mean residence time calculated as AUMC/AUC_0–∞, where AUMC is the integration of C × t versus time from 0 to infinity. C_min is the minimal concentration of nemonoxacin at steady state, and C_avg is the plateau concentration at steady state calculated as AUC_{0–24_ss}/24. Nemonoxacin accumulation was evaluated by comparing the C_max and AUC after the first dose and the last dose. The parameters describing the urinary excretion of nemonoxacin are U_0–24 and CL_r, where U_0–24 is the accumulative urinary excretion amount of nemonoxacin during the period from 0 to 24 h, and CL_r is the renal clearance calculated as the ratio of U_0–24 to AUC_0–24. These PK parameters of nemonoxacin were calculated after the first dose as well as at steady state after the last dose for the subjects receiving an i.v. infusion of nemonoxacin at 500 mg (4.17 mg/min and 5.56 mg/min), 650 mg (5.56 mg/min), or 750 mg (4.17 mg/min and 5.56 mg/min) once daily for 10 consecutive days. All the calculations of PK parameters were performed using WinNonlin software version 5.2.1 (Pharsight Corp., USA).

A two-compartment model was employed to analyze the PK profile of nemonoxacin in healthy volunteers using WinNonlin software. The nemonoxacin concentration data from the first to last dosing were combined to estimate individual compartmental parameters. The clearance of nemonoxacin from a central compartment is represented by CL, while the clearance between central and peripheral compartments is represented by Q. The distribution volumes of nemonoxacin in central and peripheral compartments are indicated by V₁ and V₂, respectively. The rate of the distribution phase and the elimination phase are expressed as α and β, and the corresponding half-lives are t_1/2,α and t_1/2,β, respectively. The weight of 1/C² was used in the calculation.

The statistical analysis of the PK parameters of nemonoxacin was conducted as follows. First, the PK parameters in each dose group were analyzed after stratification by gender to evaluate any gender effect on the PK profile. Then, the PK data of all the subjects in the five dose groups (excluding the subjects receiving placebo in the second stage) were combined to perform the multiple linear regression analysis. Stepwise regression was conducted to identify the possible independent factors affecting drug exposure, including gender, body weight, and BMI, on the C_max, C_min, or AUC_0–24 after the first dose, the C_max, C_min, or AUC_0–24 at steady state after the last dose, and the C_avg after continuous dosing. All analyses were conducted using SAS software version 9.1.3 (SAS Institute, Inc., USA).

In vitro susceptibility testing.

A total of 302 clinical bacterial isolates of common pathogens for community-acquired pneumonia were collected from the hospitals in Shanghai and Beijing during the period 2004 to 2005. Isolates included penicillin-susceptible S. pneumoniae (PSSP; n = 44), penicillin-intermediate S. pneumoniae (PISP; n = 44), penicillin-resistant S. pneumoniae (PRSP; n = 14), Haemophilus influenzae (n = 50), Moraxella catarrhalis (n = 50), methicillin-susceptible S. aureus (MSSA; n = 52), and methicillin-resistant S. aureus (MRSA; n = 48). The MICs of nemonoxacin against these bacterial strains were determined using the agar dilution method.

PK/PD analysis.

Nemonoxacin is a concentration-dependent antimicrobial agent (16). The main PK/PD parameters are the AUC/MIC and the C_max/MIC. PK/PD analysis of nemonoxacin was performed using a Monte Carlo simulation. Specifically, the simulated data of the AUC_0–24 and the C_max were generated based on logarithmic normal distribution. The simulated MIC data were generated based on discrete distribution according to a specified probability at each MIC level. The PK/PD targets of nemonoxacin (fC_max/MIC, ≥5.07; fAUC_0–24/MIC, ≥47.05) were used for predicting the favorable bacteriological efficacy of the drug against S. pneumoniae (16), where f indicates the free fraction of nemonoxacin (0.84) (12). The cumulative fraction of response (CFR) of five different dosing regimens of nemonoxacin (500 or 750 mg at an infusion rate of 5.56 mg/min or 500, 650, or 750 mg at an infusion rate of 4.17 mg/min, once daily for 10 days) was calculated as the percentage of the PK/PD parameters attaining above-target values. The probability of target attainment (PTA) of nemonoxacin was calculated as the percentage of the PK/PD parameters reaching the target value when the MIC was at the specified level. The simulation was performed on data from 5,000 patients with Matlab software version 7.0.1 (MathWorks, Inc., USA) (17).

Safety evaluation.

All 72 subjects, including those receiving placebo, were included in the safety analysis. The safety of the subjects was evaluated by physical examination, vital signs, and safety laboratory tests, such as hematological tests, urinalysis, serum biochemical tests, and a 12-lead ECG.

RESULTS

Subject demographics and baseline characteristics.

A total of 72 subjects completed this study. The subjects in the two dose groups of the first stage and in the three dose groups and the placebo group of the second stage were similar in terms of age and BMI (Table 1).

TABLE 1.

Demographic data of the healthy Chinese subjects^a

Demographic	Data for subjects in stageb:
	1		2
	500 mg	750 mg	500 mg	650 mg	750 mg	Placebo cohort data
Age (mean ± SD) (yr)	25 ± 3	24 ± 3	23 ± 3	26 ± 4	27 ± 3	26 ± 4
No. of males/females	6/6	6/6	5/7	7/5	5/7	7/5
Ethnicity (no. of Han/other)	10/2	12/0	10/2	12/0	12/0	12/0
Wt (mean ± SD) (kg)	59.7 ± 6.1	60.6 ± 7.5	57.8 ± 7.3	61.0 ± 7.8	59.4 ± 8.3	61.2 ± 9.5
BMIc (mean ± SD) (kg/m2)	21.5 ± 1.1	21.2 ± 1.1	21.2 ± 1.7	21.8 ± 1.6	22.2 ± 1.4	21.7 ± 1.7
CrClc (mean ± SD) (ml/min · 1.73 m2)	112.3 ± 17.2	119.6 ± 13.4	114.2 ± 16.5	128.7 ± 20.7	120.3 ± 19.3	122.2 ± 12.9

^an = 12 subjects in each group.

^bThe infusion rates of nemonoxacin were 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2.

^cBMI, body mass index; CrCl, creatine clearance.

PK parameters of multiple-dose i.v. nemonoxacin.

The maximal (mean ± SD) concentrations of nemonoxacin at steady state (C_{max_ss}) after the last dose of nemonoxacin in the first stage were 9.60 ± 1.84 μg/ml and 11.0 ± 2.2 μg/ml in the 500-mg and 750-mg cohorts (once daily for 10 days, at an infusion rate of 5.56 mg/min), respectively. The areas under the concentration-time curve at steady state from 0 to 24 h (AUC_{0–24_ss}) were 44.03 ± 8.62 and 65.82 ± 10.78 μg · h/ml in the 500-mg and 750-mg cohorts, respectively (Table 2). The drug concentration-time curve is presented in Fig. 1. Based on the statistical analysis, the accumulation factors were 1.08 for both dose groups, which indicates that the two dose levels showed no or little accumulation of nemonoxacin after a 10-day i.v. infusion. Generally, the PK parameters did not show statistical difference (P > 0.05) between males and females in either dose group, except for the C_max value on day 10 in the 500-mg group (P < 0.05).

TABLE 2.

Pharmacokinetic parameters of nemonoxacin in healthy Chinese subjects after multiple-dose intravenous infusion of 500, 650, or 750 mg once daily for 10 consecutive days^a

Dosing regimen and timing	Cmax(μg/ml)	Cmin(μg/ml)	Cavg(μg/ml)	AUC0–24(μg · h/ml)	AUC0–∞(μg · h/ml)	t1/2c(h)	V(liter)	CLz(liter/h)	CLr(liter/h)	MRT0–∞(h)
500 mg at 5.56 mg/min
Day 1	10.0 ± 1.6	0.302 ± 0.098		40.25 ± 8.59	43.30 ± 9.33	6.96 ± 1.42	86.67 ± 14.23	11.97 ± 2.15	7.76 ± 1.75	7.34 ± 0.89
Day 10b	9.60 ± 1.84	0.324 ± 0.103	1.84 ± 0.36	44.03 ± 8.62	46.97 ± 9.49	6.20 ± 1.39	81.77 ± 14.75	11.72 ± 2.08	6.95 ± 1.36	7.05 ± 0.91
750 mg at 5.56 mg/min
Day 1	11.8 ± 2.7	0.407 ± 0.094◻		61.93 ± 9.06◻◻	65.41 ± 9.65◻◻	5.90 ± 0.59◻	78.93 ± 11.16	11.71 ± 1.79	6.56 ± 1.25	6.77 ± 0.61
Day 10	11.0 ± 2.2	0.492 ± 0.136◼◼	2.74 ± 0.45◼◼	65.82 ± 10.78◼◼	70.45 ± 12.22◼◼	6.34 ± 1.21	84.01 ± 13.34	11.69 ± 2.02	6.89 ± 0.98	7.24 ± 0.88
500 mg at 4.17 mg/min
Day 1	7.54 ± 0.86◻◻	0.220 ± 0.064◻		33.97 ± 4.66◻	35.91 ± 4.84◻	6.01 ± 1.18	95.23 ± 17.86	14.16 ± 1.94◻	8.34 ± 2.12	6.75 ± 1.05
Day 10	7.13 ± 1.47◼◼	0.290 ± 0.101	1.69 ± 0.40	40.46 ± 9.52*	44.74 ± 10.77*	6.30 ± 1.15	101.13 ± 23.64◼	11.77 ± 2.84*	7.22 ± 1.99	8.68 ± 1.34**,◼◼
650 mg at 4.17 mg/min
Day 1	7.21 ± 1.54	0.340 ± 0.082**		46.45 ± 7.98*	49.47 ± 8.05**	6.05 ± 1.15	96.97 ± 22.00	13.46 ± 2.16	8.06 ± 2.34	7.18 ± 1.04
Day 10	8.17 ± 1.76	0.407 ± 0.137&	2.26 ± 0.50&	54.17 ± 12.10&	60.52 ± 13.89&,▵	5.91 ± 1.21	99.72 ± 18.89	11.20 ± 2.20▵	7.42 ± 2.04	9.03 ± 1.48▵▵
750 mg at 4.17 mg/min
Day 1	9.03 ± 3.23▵☆	0.462 ± 0.133**,▵▵		61.19 ± 17.30**,▵▵	65.03 ± 17.45**,▵▵	5.67 ± 0.93	91.57 ± 29.19	12.26 ± 2.58*	7.92 ± 1.42▵	7.43 ± 1.22
Day 10	9.96 ± 2.23&&,▴	0.534 ± 0.161&&,▴▴	2.97 ± 0.75&&,▴▴	71.34 ± 17.79&&,▴▴	79.86 ± 19.52&&,▴▴	5.72 ± 0.99	90.25 ± 23.38	9.84 ± 2.04+,⋆	7.94 ± 1.62	9.19 ± 1.52++,⋆⋆

^an = 12 in each group. All data are means ± SD. Compared to 500-mg group (at 5.56 mg/min): day 1: ^◻, P < 0.05; ^◻◻, P < 0.01; day 10: ^◼, P < 0.05; ◼◼, P < 0.01. Compared to 750-mg group (at 5.56 mg/min): day 1: ^☆, P < 0.05; ^☆☆, P < 0.01; day 10: *, P < 0.05, **, P < 0.01. Compared to 500-mg group (at 4.17 mg/min): day 1: *, P < 0.05; **, P < 0.01; day 10: ^&, P < 0.05; ^&&, P < 0.01. Compared to 650-mg group (at 4.17 mg/min): day 1: ^▵, P < 0.05; ^▵▵, P < 0.01; day 10: ^▴, P < 0.05, ^▴▴, P < 0.01. Compared to 750-mg group (at 4.17 mg/min): day 1: ⁺, P < 0.05; ⁺⁺, P < 0.01.

^bAll of the PK parameters on day 10 were values at steady state.

^ct_1/2 was calculated using concentration data during 0 to 24 h from day 1 or day 10.

FIG 1.

The plasma concentration (mean ± SD) versus time curves of nemonoxacin following multiple-dose intravenous infusions of 500 mg (at 5.56 mg/min over 1.5 h) or 750 mg (at 5.56 mg/min over 2.25 h) nemonoxacin malate once daily for 10 consecutive days in healthy subjects (n = 12 each). The symbols and lines indicate the observed values and predictions, respectively, by a two-compartment model.

In the second stage, the C_{max_ss} values were 7.13 ± 1.47, 8.17 ± 1.76, and 9.96 ± 2.23 μg/ml after the last dose of nemonoxacin in the 500-mg, 650-mg, and 750-mg cohorts (once daily for 10 days, at an infusion rate of 4.17 mg/min), respectively. The AUC_{0–24_ss} values in the 500-mg, 650-mg, and 750-mg cohorts were 40.46 ± 9.52, 54.17 ± 12.10, and 71.34 ± 17.79 μg · h/ml, respectively (Table 2). The concentration-time curve is presented in Fig. 2. The results of the statistical analysis show no or little accumulation after a 10-day infusion in 3 cohorts. The accumulation factors ranged from 1.06 to 1.08. PK parameters that showed significant differences (P < 0.05) between males and females were the C_min after the first dose and the last dose in the 500-mg group, the C_max, C_min, and AUC_0–t after the first dose in the 650-mg group, and the C_min after the first dose in the 750-mg group.

FIG 2.

The plasma concentration (mean ± SD) versus time curves of nemonoxacin following multiple-dose intravenous infusions of 500 mg (at 4.17 mg/min over 2 h), 650 mg (at 4.17 mg/min over 2.6 h), or 750 mg (at 4.17 mg/min over 3 h) nemonoxacin malate once daily for 10 consecutive days in healthy subjects (n = 12 in each cohort). The symbols and lines indicate the observed values and predictions, respectively, by a two-compartment model.

In the first stage (infusion rate, 5.56 mg/min), the cumulative amounts of nemonoxacin in urine over 24 h were approximately the same after the first and last doses (60.14% ± 5.85% versus 59.79% ± 8.38%, respectively) in the 500-mg group. For the 750-mg group, the cumulative amounts of nemonoxacin were 53.59% ± 9.12% and 59.48% ± 6.63% for the first dose and last dose, respectively. In the second stage (infusion rate, 4.17 mg/min), the cumulative amounts of nemonoxacin over 24 h after the first dose were 55.62% ± 11.45%, 55.51% ± 8.75%, and 62.00% ± 7.42% in the 500, 650, and 750-mg groups, respectively. After multiple-dose treatments, the cumulative amounts of nemonoxacin following the last dose were 55.94% ± 10.6%, 58.86% ± 8.0%, and 72.77% ± 11.0% in the 500, 650 and 750-mg groups, respectively.

The PK profiles of nemonoxacin in healthy volunteers were well described by the two-compartment model (Fig. 1 and 2). The distribution volumes of nemonoxacin in the central compartment were in the range of 64.5 to 83.2 liters, while the distribution volumes of the peripheral compartment were in the range of 24.7 to 40.9 liters (Table 3). When the nemonoxacin dose increased from 500 mg to 750 mg (infusion rate, 5.56 mg/min), the distribution half-life elevated from 2.42 to 3.37 h (P < 0.01), while the elimination half-life increased from 10.8 to 12.7 h (P < 0.01). The similar tendency of increased t_1/2,α and t_1/2,β of nemonoxacin was also observed in stage 2 (Table 3). In contrast, the intercompartmental clearance showed a weak tendency to decrease when the nemonoxacin dose increased. The clearance rates of nemonoxacin from the central compartment were in the range of 12.0 to 14.6 liter/h.

TABLE 3.

Compartmental parameters of nemonoxacin in healthy Chinese subjects after multiple-dose intravenous infusions of 500, 650, or 750 mg once daily for 10 consecutive days^a

Parameter	Data for subjects in stageb:
	1		2
	500 mg	750 mg	500 mg	650 mg	750 mg
V1 (liter)	64.5 ± 6.77	73.5 ± 13.8	81.2 ± 12.8◻◻	83.2 ± 13.8	81.7 ± 20.5
V2 (liter)	40.9 ± 10.8	28.5 ± 14.9	37.0 ± 13.4	35.5 ± 14.3	24.7 ± 13.0*
t1/2,α (h)	2.42 ± 0.478	3.37 ± 0.778◻◻	3.00 ± 0.456◻◻	3.29 ± 0.528	4.03 ± 0.504☆,**,▵▵
t1/2,β (h)	10.8 ± 1.30	12.7 ± 1.70◻◻	12.1 ± 1.73◻	13.7 ± 5.26	16.6 ± 5.88☆,*
CL (liter/h)	13.0 ± 2.37	12.3 ± 2.02	14.6 ± 2.48	13.7 ± 2.23	12.0 ± 2.48*
Q (liter/h)c	4.16 ± 2.14	2.26 ± 2.01	2.93 ± 1.55	2.76 ± 1.74	1.48 ± 1.15*,▵

^an = 12 in each group. Data reported are means ± SD. The weight was 1/C² in the PK calculation.

^bThe infusion rates of nemonoxacin were 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2. Compared to 500-mg group (at 5.56 mg/min): ^◻, P < 0.05; ^◻◻, P < 0.01. Compared to 750-mg group (at 5.56 mg/min): ^☆, P < 0.05; ^☆☆, P < 0.01. Compared to 500-mg group (at 4.17 mg/min): *, P < 0.05; **, P < 0.01. Compared to 650-mg group (at 4.17 mg/min): ^▵, P < 0.05; ^▵▵, P < 0.01.

^cQ = k₁₂ × V₁, where k₁₂ is the rate constant from the central compartment to the peripheral compartment.

The results of multiple linear regression analysis indicated that sex, body weight, and BMI were not independent factors affecting the C_min, C_{min_ss}, or AUC_0–t. Gender was an independent factor for C_max. The C_{max_ss} of female subjects was about 1.11- to 1.28-fold higher than that of male subjects. However, this did not have a significant effect on drug exposure.

Results of susceptibility testing.

The MIC₉₀s of nemonoxacin were 0.09 mg/liter, 0.09 mg/liter, and 0.04 mg/liter against PSSP, PISP, and PRSP strains, respectively. For H. influenzae, M. catarrhalis, MSSA, and MRSA, nemonoxacin had MIC₉₀s of 0.18 mg/liter, 0.04 mg/liter, 0.04 mg/liter, and 0.72 mg/liter, respectively. The distribution of nemonoxacin MIC values against these strains is presented in Table 4.

TABLE 4.

Frequency distribution of nemonoxacin MICs against the primary pathogens of community-acquired pneumonia

Bacterial straina (no. of isolates)	Frequency distribution (%) of MIC (mg/liter)b:
	0.011	0.022	0.043	0.090	0.181	0.361	0.722	1.444	2.888	5.776	11.55	23.10	46.21	92.42
PSSP (44)	6.8	18.2	45.5	29.5	0	0	0	0	0	0	0	0	0	0
PISP (44)	0	0	81.8	18.2	0	0	0	0	0	0	0	0	0	0
PRSP (14)	0	21.4	78.6	0	0	0	0	0	0	0	0	0	0	0
Haemophilus spp. (50)	56.0	10.0	12.0	6.0	6.0	0	4.0	6.0	0	0	0	0	0	0
Moraxella catarrhalis (50)	2.0	62.0	28.0	4.0	2.0	0	0	0	0	2.0	0	0	0	0
MSSA (52)	0	0	94.3	0	3.8	1.9	0	0	0	0	0	0	0	0
MRSA (48)	0	0	0	0	8.3	66.7	22.9	0	2.1	0	0	0	0	0

^aPSSP, penicillin-susceptible S. pneumoniae; PISP, penicillin-intermediate S. pneumoniae; PRSP, penicillin-resistant S. pneumoniae; MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus.

^bThe bold and underlined data indicate the corresponding concentrations of the MIC₅₀ and MIC₉₀, respectively.

Monte Carlo simulation and PK/PD analysis.

The Monte Carlo simulation indicated that the CFR of nemonoxacin in all dosing regimens achieved 99.99% or higher for S. pneumoniae (including PSSP, PISP, and PRSP) in terms of the fAUC_0–24/MIC, which was ≥47.05 (Table 5). The PTA reached 99% or higher when the MIC of nemonoxacin against S. pneumoniae was ≤0.361 mg/liter. When the MIC was ≤0.722 mg/liter, all the dosing regimens except the two 500-mg regimens achieved a PTA of 90% or higher (Fig. 3). When the target of fC_max/MIC was set as 5.07, all 5 dosing regimens achieved the CFR of 100% for S. pneumoniae (including PSSP, PISP, and PRSP) (Table 5); when the MIC of nemonoxacin against S. pneumoniae was ≤0.722 mg/liter, the PTA of all 5 dosing regimens attained 98% or higher (Fig. 4). These results indicate that all 5 studied dosing regimens of nemonoxacin have a high probability of reaching clinical and microbiological efficacy in treating patients with community-acquired pneumonia, mainly caused by S. pneumoniae.

TABLE 5.

Cumulative fraction of response of nemonoxacin against various pathogens in terms of different multiple-dose intravenous infusion regimens in healthy Chinese subjects

Parametera	Bacterial strainb	Response by nemonoxacin stage and dose cohortc
		1		2
		500 mg	750 mg	500 mg	650 mg	750 mg
fAUC0–24h/MIC	PSSP	100	100	99.99	100	100
	PISP	100	100	99.99	100	100
	PRSP	100	100	100	100	100
	Haemophilus spp.	91.88	95.16	91.43	94.15	95.65
	Moraxella catarrhalis	98.16	98.46	98.32	97.80	97.88
	MSSA	99.99	99.99	99.98	99.98	99.99
	MRSA	89.39	97.98	85.18	96.20	97.46
fCmax/MIC	PSSP	100	100	100	100	100
	PISP	100	100	100	100	100
	PRSP	100	100	100	100	100
	Haemophilus spp.	97.73	98.93	95.36	96.55	98.02
	Moraxella catarrhalis	98.14	98.08	97.98	98.14	98.10
	MSSA	99.99	99.99	99.99	99.99	99.99
	MRSA	97.82	98.17	97.68	97.52	97.88

^af indicates the free fraction of nemonoxacin (0.84).

^bPSSP, penicillin-susceptible S. pneumoniae; PISP, penicillin-intermediate S. pneumoniae; PRSP, penicillin-resistant S. pneumoniae; MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus.

^cThe infusion rates of nemonoxacin were 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2.

FIG 3.

Probability of target attainment (PTA) of nemonoxacin in terms of fAUC_0–24/MIC (target = 47.05) following intravenous infusion of nemonoxacin malate sodium chloride in healthy subjects. The horizontal dotted line indicates the PTA value of 90%. The infusion rate of nemonoxacin was 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2.

FIG 4.

Probability of target attainment (PTA) of nemonoxacin in terms of fC_max/MIC (target = 5.07) following intravenous infusion of nemonoxacin malate sodium chloride in healthy subjects. The horizontal dotted line indicates the PTA value of 90%. The infusion rate of nemonoxacin was 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2.

Safety and tolerability.

The adverse events (AEs) of nemonoxacin were mild and transient, and no serious or severe AEs were observed (Table 6). The most common AE in the clinical disorder category was an injection site reaction, and rash. For the laboratory assays, the most common AEs were increased aspartate aminotransferase and alanine aminotransferase levels and elevation of the total bilirubin level. The AEs described above spontaneously resolved during infusion or within 24 h after administration. One subject receiving placebo was discontinued from the study because of important adverse event of rash. No clinically significant abnormalities were observed in the vital signs or on the physical examination of the subjects. The healthy Chinese subjects showed good tolerability to an i.v. infusion of 500 mg to 750 mg nemonoxacin malate sodium chloride once daily for 10 consecutive days at infusion rates of 4.17 mg/min to 5.56 mg/min.

TABLE 6.

Common drug-related adverse events in the multiple-dose pharmacokinetic study of intravenous nemonoxacin^a

Adverse event	No. (%) of subjects with indicated adverse event
	1b		2
	500 mg	750 mg	500 mg	650 mg	750 mg	Placebo
Clinical disorder
Injection site reaction	7 (58.3)	8 (66.7)	7 (58.3)	12 (100.0)	6 (50.0)	0 (0.0)
Rash	1 (8.3)	1 (8.3)	0 (0.0)	3 (25)	3 (25)	1 (8.3)
Abnormal laboratory assay
White blood cell count decreased	1 (8.3)	0 (0.0)	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)
Aspartate aminotransferase increased	1 (8.3)	4 (33.3)	2 (16.7)	1 (8.3)	1 (8.3)	1 (8.3)
Alanine aminotransferase increased	1 (8.3)	4 (33.3)	2 (16.7)	1 (8.3)	1 (8.3)	1 (8.3)
Total bilirubin increased	2 (16.7)	0 (0.0)	0 (0.0)	2 (16.7)	1 (8.3)	1 (8.3)
Conjugated bilirubin increased	0 (0.0)	0 (0.0)	0 (0.0)	2 (16.7)	1 (8.3)	1 (8.3)
γ-Glutamyltransferase increased	0 (0.0)	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)	1 (8.3)
Percentage of eosinophils increased	0 (0.0)	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Percentage of neutrophils decreased	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Lymphocyte percentage increased	1 (8.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Creatine phosphokinase increased	0 (0.0)	0 (0.0)	1 (8.3)	1 (8.3)	0 (0.0)	0 (0.0)
Abnormal electrocardiogram
QTc difference > 60 msc,d	0 (0.0)	1 (8.3)	1 (8.3)	0 (0.0)	1 (8.3)	0 (0.0)
30 ms ≤ QTc difference ≤ 60 msc,d	5 (41.7)	2 (16.7)	1 (8.3)	2 (16.7)	2 (16.7)	0 (0.0)
QTc ≥ 450 msc,d	1 (8.3)	2 (16.7)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
QTc prolongationd	0 (0.0)	0 (0.0)	1 (8.3)	0 (0.0)	1 (8.3)	0 (0.0)
Supraventricular extrasystoles	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (8.3)

^an = 12 in each group.

^bThe infusion rates of nemonoxacin were 5.56 mg/min in stage 1 and 4.17 mg/min in stage 2.

^cThese events were verbatim adverse events, which are not coded by MedDRA.

^dQTc, corrected QT interval. QTc in this study was calculated using Bazet's formula (QTcB = QT/RR^0.5). Electrocardiogram data were measured only once at each scheduled time point.

DISCUSSION

Based on the PK results, nemonoxacin showed no or little accumulation after i.v. infusions of 500 or 750 mg once daily for 10 days at an infusion rate of 5.56 mg/min or after i.v. infusions of 500, 650, or 750 mg once daily for 10 days at an infusion rate of 4.17 mg/min. There were no significant differences between the male and female subjects in terms of C_max, C_min, or AUC_0–24 after the first dose (day 1) or the last dose (day 10) with all treatments. Therefore, we combined the PK data of all 60 subjects in the 5 dose groups (excluding the subjects receiving placebo in the second stage) to perform multiple linear regression analysis. The evaluation revealed that only gender was an independent factor of C_max, but it did not have a significant effect on drug exposure, possibly because all volunteers for the study were healthy and had similar demographic data except for gender. Based on the results of drug exposure, it is not necessary to adjust the dose of nemonoxacin in exploratory clinical trials in healthy subjects.

In this study, we used a two-stage method to conduct a PK/PD analysis rather than a population analysis, which is common in many reports. We did this because of the following considerations: (i) All subjects were healthy volunteers, and the population was homogeneous, with similar ages and body mass index (BMI). As shown in Table 1, the average coefficients of variation (CV) for age and BMI were 13.2% and 6.64%, suggesting that the variations of these two indices are small. We believe that the results from multiple linear regression analysis, such as a post hoc test, would not change greatly from results of a population analysis. (ii) We developed a population pharmacokinetic (PPK) model for an oral preparation of nemonoxacin in CAP patients. The results showed that the PK parameters of nemonoxacin were significantly affected by seven covariates (X. J. Wu, Y. C. Chen, J. Zhang, J. C. Yu, G. Y. Cao, B. N. Guo, J. F. Wu, D. M. Zhu, Y. G. Shi, and Y. Y. Zhang, unpublished data). The relative difference of five covariates between healthy subjects and CAP patients were in the range of ±20%. At steady state, the nemonoxacin AUC_0–24 in healthy subjects obtained from a Bayesian analysis of the PPK model (44.5 µg · h/ml) was close to that from noncompartmental analysis using actual data (46.8 µg · h/ml). Meanwhile, the CV of the C_max obtained from Bayesian analysis of the PPK model (25.0%) was almost the same as that from noncompartmental analysis in healthy subjects (25.2%). In the future, we will develop a PPK model for an intravenous preparation of nemonoxacin using data from healthy volunteers and CAP patients. A phase II clinical trial of intravenous nemonoxacin in CAP patients was completed, and a phase III clinical trial of intravenous nemonoxacin is still under way. (iii) We found several reports using a two-stage method (18–21) rather than a population approach in the PK/PD analysis of antimicrobial agents. During the Monte Carlo simulation, the authors of these reports used the results of a noncompartmental or compartmental analysis to generate simulated data of PK parameters based on a specified assumption of distribution. This is similar to our work.

The profile of intravenous nemonoxacin had characteristics similar to those of the nemonoxacin capsule in healthy volunteers (13). For example, the C_max of i.v. nemonoxacin (at an infusion rate of 4.17 mg/min) at steady state was close to that of the oral capsule form of nemonoxacin in the 500-mg group (7.13 mg/liter versus 7.02 mg/liter, respectively) and the 750-mg group (9.96 mg/liter versus 9.13 mg/liter, respectively; P < 0.05). The AUC_0–24 of i.v. nemonoxacin (at an infusion rate of 4.17 mg/min) was also near that of the nemonoxacin capsule in the 500-mg group (40.46 μg · h/ml versus 46.8 μg · h/ml, respectively) and the 750-mg group (71.34 μg · h/ml versus 65.6 μg · h/ml, respectively; P < 0.01). In contrast, the MRT_0–∞ of i.v. nemonoxacin (at an infusion rate of 4.17 mg/min) at steady state was shorter than that of the oral capsule form of nemonoxacin in the 500-mg group (8.68 h versus 9.9 h, respectively; P < 0.05) and the 750-mg group (9.19 h versus 10.25 h, respectively). The main reason is that the nemonoxacin capsule will undergo an absorption process following oral administration. These results are similar to those of an absolute bioavailability study of nemonoxacin in healthy Chinese volunteers (B. N. Guo, G. L. He, J. Zhang, X. J. Wu, J. C. Yu, G. Y. Cao, Y. C. Chen, Y. G. Shi, and Y. Y. Zhang, unpublished data).

In addition to the Monte Carlo simulation, we used a single-point method to calculate the PK/PD parameters of nemonoxacin. The results show that 500 mg nemonoxacin at an infusion rate of 4.17 mg/min or 5.56 mg/min can achieve a target fC_max/MIC₉₀ value of ≥67 and an fAUC_0–24/MIC₉₀ value of ≥378 for penicillin-susceptible or -resistant S. pneumoniae. Zhang et al. reported in 2005 (22) that levofloxacin has a higher probability of reaching the PD target if the C_max/MIC₉₀ of levofloxacin is 7.56 and the AUC_0–24/MIC₉₀ is 38.28 against S. pneumoniae after a single-dose i.v. infusion of 500 mg levofloxacin in healthy Chinese subjects. The PK/PD parameters of nemonoxacin are much higher than those of levofloxacin at the same clinical dose. This study also demonstrates that all five dosing regimens of nemonoxacin can achieve a target fC_max/MIC₉₀ of ≥8 and a target fAUC_0–24/MIC₉₀ of ≥51 against MRSA. Based on the promising microbiological efficacy data, nemonoxacin can be expected to be useful for treating CAP caused by MRSA, which should be confirmed in further clinical studies.

In conclusion, the dosing regimens of nemonoxacin ranging from 500 mg to 750 mg showed a good safety profile without accumulation in healthy Chinese subjects via i.v. infusion at a rate of 4.17 or 5.56 mg/min administered once daily for 10 consecutive days. The PK/PD analysis predicted that all 5 studied dosing regimens can achieve favorable clinical and microbiological efficacies for infections caused by common community-acquired pneumonia pathogens.