| Nemonoxacin, a novel non-fluorinated quinolone, is different from the traditional fluoroquinolones. It acts by inhibitig bacterial DNA gyrase. In vitro activity results showed it retained potent broad-spectrum activities against a variety of gram-positive organisms, gram-negative organisms, anaerobic bacterium and non-classical pathogen, including antibiotic-resistant organisms like multiple-drug resistant (MDR) Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). Currently it is approved to conduct the phase I clinical trial of nemonoxacin by State Food and Drug Administration (SFDA) in China. The study of safety and tolerability for nemonoxacin has been finished, which indicated that all doses between 125mg and 1000mg were well tolerated by the Chineses healthy subjects. Thus, we can carry out the pharmacokinetics study following single oral dose of nemonoxacin (250mg, 500mg and 750mg) under fasting state, as well as the food effect. Moreover, we conduct the the pharmacokinetics study following multiple daily doses of nemonoxacin (500mg and 750mg) for 10 days. Based on the results from phamacokinetics studies following the single dose and multiple doses of nemonoxacin, which combined with the results from the previous pharmacodynamics study, the indication and the dosing regimen may be recommended for the phase II clinical trial of nemonoxacin.This study included four parts:Part 1 Development and validation of the Method for the Determination of Nemonoxacin in Human Plasma and Urine Using LC-MS/MSA high performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) method was developed to determine the nemonoxacin concentration in the plasma and urine samples collected from the pharmacokinetics studies. Waters Alliance 2690 series HPLC instrument and Finnigan TSQ Quantum Discovery Max tandem mass spectrometer apparatus was applicated in the determination. Nemonoxacin was separated using a Waters Symmetry Shield RP18 column (2.1 by 50mm,5μm) with mobile phase of 0.1% methanoic acid and acetonitrile (82:18) and 0.1% methanoic acid and acetonitrile (85:15) to determine the plasma and urine samples respectively. A rapid sensitive convenient LC-MS/MS method was developed, in which the biological specimens of plasma and urine were pretreated by directed protein precipitation and liquid-liquid extraction, respectively. The internal standard was gatifloxacin. The lower limit of quantification for nemonoxacin in plasma and urine were both 0.005μg/mL. For plasma and urine samples, the recoveries of nemonoxacin were (98.49±4.69)% and (84.92±6.81)%,the intra-day precision (RSD)≤5.87% and≤8.30%, the inter-day precision (RSD)≤6.94% and≤5.43%,and the intra-day accuracy ranged from 99.27% to 103.75% and 103.52% to 115.94%, respectively. In room temperature, nemonoxacin kept stable over 24 h in plasma and urine samples before pretreatment, and also stable over 24 h in the post-preparative plasma and urine samples. Nemonoxacin was stable in plasma over three cycles of freeze (-40℃)and thawing (room temperature).The recovery of nemonoxacin in plasma and urine samples were (92.14～110.87)% and (83.55～101.95)%, respectively after three months storage in refrigerator (-40℃).The method was accorded with the demands of the relevant documents released by SFDA drug evaluation.Part 2 Pharmacokinetic Study of Nemonoxacin following single dose in Healthy VolunteersThe pharmacokinetic study of nemonoxain following single dose included two studies. The first study was a randomized, open, placebo-controlled, three periods, three doses of cross-over study in which to evaluate the safety and pharmacokinetics of nemonoxacin.12 subjects (male:female=6:6) were randomized to three groups and administered orally 250mg,500mg and 750mg nemonoxacin under fasting. After washout of seven days, these 12 subjects received 500mg nemonoxacin with high-fat meal to study the food effect. The plasma and urine samples were collected on schedule. The LC-MS/MS method was used to determine the concentration of nemonoxacin in these samples. Pharmacokinetic parameters were calculated by the data of plasma and urine concentration using the non-compartment model of WinNolin 5.2.1 software (Pharsight Corp.CA USA).The pharmacokinetic findigns of this study show that following oral administration of 250mg,500mg and 750 mg, nemonoxacin rapidly reached its Cmax (within 1 to 2h), which were 3.237μg/mL,5.909μg/mL and 8.525μg/mL, respectively. AUCo-72h were 21.40μg·h/mL,42.17μg·h/mL and 66.26μg·h/mL, respectively, while AUCo-∞were 21.52μg·h/mL,42.41μg·h/mL and 66.54μg·h/mL, respectively. T1/2 were 10.73h,12.83h and 11.01h, respectively. CLt/F were 11.88L/h,11.98L/h and 4.41L/h, respectively. Ae0-72h were 70.28%, 67.05% and 66.92%, respectively. The statistic analysis show that AUC0-∞,AUC0-72h and Cmax were linear and dose proportional over the dose range from 250mg to 750mg.The extents of nemonoxacin absorption were different under the fasting and fed conditions. The Cmax decreased to 3.895μg/mL and Tmax increased to 3.64h. The AUC0-∞decreased to 34.53 μg·h/mL by approximately 20%. Dosing in the fed state also led to a decrease in the Ae72h which was 54.25%,and the T1/2 was similar to that in the fasting state, which were 14.99h and 12.83h, respetively. The statistic analysis show that the differences of AUC0-72h, AUC0-∞, Cmax, Tmax, CLt/F, UA and Ae between two groups were significantly.The study show that AUC0-∞AUC0-72h and Cmax were linear and dose proportional over the dose range from 250mg to 750mg. In the fed state, the absorption of nemonoxacin was slowed and decreasedPart 3 Pharmacokinetic Study of Nemonoxacin following multiple doses in Healthy VolunteersThis study was designed to evaluate the safety, tolerability and pharmacokinetic parameters of multiple daily doses of 500mg and 750mg nemonoxacin for 10 days in healthy adult subjects. 24 subjects (male:female=12:12) were enrolled in the study. Each subjects received a once-daily oral dose of nemonoxacin at two levels (500mg or 750mg) under fasting condition during a 10-day dosing period. The plasma and urine samples were collected on schedule to carry out the safey and pharmacokinetic analysis. The full course PK samples were collected on day 1 and day 10 after doseing, while trough and peak plasma samples were also collected on day 3,day5,day 8 and day 9 prior to dosing and lh after dosing, respectively. The LC-MS/MS method was used to determine the concentration of nemonoxacin in these samples. Pharmacokinetic parameters were calculated and safety results were assessed.For group of 500mg nemonoxacin, the Cmax, C24h, AUC0-24h and Tmax of median on day 1 were 6.462μg/mL,0.320μg/mL,43.94μg·h/mL and 1h, respectively. The Cmax, C24h and AUC0-24h in the steady state on day 10 were similar to those on day 1.The Cavg, CLss/F and Vd/f were calculated as 1.955μg/mL,11.32 L/h and 107.64L, respectively. The DF was 345.85%, and the accumulation factor was 1.09.The Ae0-24h on day 10 compared to that on day 1 were 54.81% and 50.81%,respectively.For group of 750mg nemonoxacin, the Cmax, C24h, AUC0-24h and Tmax of median on day 1 were 9.378μ,g/mL,0.541μg/mL,63.25μg-h/mL and 1.75h, respectively. The Cmax, C24h and AUC0-24h in the steady state on day 10 were similar to those on day 1.The Cavg, CLss/F and Vd/f were calculated as 2.739μg/mL,11.64 L/h and 113.27L, respectively. The DF was 319.15%,and the accumulation factor was 1.10.The Ae0-24h on day 10 compared to that on day 1 were 56.24% and 65.38%, respectively.The statistic analysis show that the differences of Cmax, C24h, AUC0-24, UA0-24h and AE0-24h between day 1 and day 10 were not significantly (P>0.05). Part 4 The PK/PD Profiles of nemonoxacin and the Evaluation of the Therapeutic RegimensThe fluoroquinolones are concentration-dependent killing antimicrobials according to the concept of PK/PD.The principal determinant of efficacy is AUC/MIC (ratio by the area under the concentration-time curve and minimal inhibitory concentration) and Cmax/MIC (ratio by the maximum plasma concentration and minimal inhibitory concentration).Nemonoxacin is NFQ, however, none reference was presented to determine its PK/PD parameter. We presumed that nemonoxacin is also the concentration-dependent killing antimicrobial and with the same PK/PD parameter as other quinolones. Based on the results from phamacokinetics studies following the multiple doses of nemonoxacin, which combined with the results from the previous pharmacodynamics study, we conducted 10,000-subject Monte Carlo simulation (@Risk software) to estimate the probability of critical pharmadynamic target attainment for 2 different regimens of 500mg and 750mg nemonoxacin once daily for 10 days.The Cmax over the MIC90 ratio (Cmax/MIC90) of 5 and the AUC24h over the MIC90 ratio (AUC24h/MIC9o) of 25 were used to assess bacterial eradication, respectively. The Cmax over the MIC90 ratio of 8 and the AUC24h over the MIC90 ratio of 60 were used to assess the most baterial eradication and resistance development, respectively. When the Cmax/MIC9o of 5 and AUC241/MIC90 were target attainment values, the two regimens could achieve high probabilities of bacteriostatic/bactericidal target attainment against the tested species except for MRSA. When the Cmax/MIC90 of 8 was target attainment value, the two regimens could achieve high probabilities of bacteriostatic/bactericidal target attainment against the tested species except for MRSA. When the AUC24h/MIC9o was target attainment value, the probabilities of bacteriostatic target attainment against PSSP, PISP and Hemophilus spp for the 500mg qd regimen were lower than 90%, while the probability of bacteriostatic target attainment against Hemophilus spp for the 750mg qd regimen were lower than 90%.But the probabilities of bactericidal target attainment against MRSA for two regimen wrer both 0.
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