| Laevo-ornidazole is a new5-nitroimidazoles antimicrobial drug and is the laevo isomer of ornidazole. In vivo, the cytotoxic active compound and intermediates of laevo-ornidazole can make the DNA spiral structure breakage or block its transcription to replication, and finally cause the death of bacteria to reach the antibacterial purpose. In vitro, laevo-ornidazole has similar antianaerobic activity with ornidazole, although the activity is slightly stronger on some test strARns, it isn’t statistically significant. Laevo-ornidazole has significantly lower central neurotoxicity than ornidazole.Laevo-ornidazole and sodium chloride injection was manufactured by Sanhome Pharmaceutical Co., Ltd (Nanjing, China). Laevo-ornidazole is a1.3class of new chemical drug and was approved by the State Food and Drug Administration on August13,2009. Currently the phase IV clinical study of laevo-ornidazole and sodium chloride injection is being carried out. The completed phase I clinical study shows that the500mg, q12h continuous administration of laevo-ornidazole can cause drug accumulation in human body. The preliminary pharmacokinetic studies indicate that the accumulation factor can be reduced from1.93to1.3when the dose regime changed to750mg, q24h. The above results combined with the in vitro pharmacodynamic results of anaerobic bacteria suggested the dose regimen of currently500mg twice daily should be changed to750mg, once a day. But this is only the preliminary findings and needs further clinical validation.Ornidazole had5metabolites in human urine:M1,1-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol; M2,2-methyl-5-nitroimidazole; M3, N-(3-chloro-2-hydroxypropy1)acetamide; M4,3-(2-methyl-5-nitro-l-imidazolyl)-1,2-propanedio; M5, acetamide. Oxidation leads to the formation of the oxymethyl analogue M1, or to the rupture of the side-chARn with formation of M2. Hydrolysis of chlorine in the side-chARn leads to the formation of M4, whereas hydrolytic cleavage of the imidazole ring, presumably after reduction of the nitro group, gives rise to M3and M5.The metabolites of laevo-ornidazole has not been researched yet, this study will complement the metabolites study of laevo-ornidazole. Based on the above findings, we designed a single-center, open, parallel, multi-dose study to further evaluate the safety and PK of laevo-ornidazole and sodium chloride injection in healthy Chinese volunteers. The dose regime were500mg,12h and750mg, q24h respectively. The PK parameters of the two dose regimes were calculated by the qualification of laevo-ornidazole and its metabolites in the blood and urine samples of the24healthy subjects (male and female).①To research the PK characteristics of laevo-ornidazole and its metabolites in human body.②By the combination of the PK information and the previous PD results, we conducted PK/PD investigation to optimize the dose regimen, and to provide evidence for the reasonable dose regimen determination of the phase IV clinical study of laevo-ornidazole.The study included three parts:Part I Development and Validation of the LC-MS/MS Method for the Simultaneous Determination of Laevo-ornidazole and its Metabolites in Human Plasma and UrineThe liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed to simultaneously determine the concentration of laevo-ornidazole and its metabolites in human plasma and urine. Waters ultra performance liquid chromatography (UPLC) instrument and API4000QTRAP mass spectrometer were applied. The analyses were carried out on a ACQUITY UPLC CSH Cig column (2.1×50mm,1.7μm) using a mobile phase composed of0.1%formic acid and acetonitrile. The biological specimens of plasma and urine were all pretreated by protein precipitation and liquid-liquid extraction. The internal standard (IS) was metronidazole. Both analytes and IS were determined by atmospheric pressure ionization via the multiple reaction monitoring in positive ion mode. The detection channels of laevo-ornidazole, M1, M2, M4and IS were m/z220.0to127.5, m/z236.2to170.9, m/z127.9to110.7, m/z202.5to127.7and m/z172.1to127.8, respectively.The lower limit of quantification (LLOQ) of laevo-ornidazole was0.0100μg/mL and the calibration curves were linear in the concentration range of0.0100-5.00μg/mL. The LLOQ of Ml was0.00500μg/mL and the calibration curves were linear in the concentration range of0.00500-2.50μg/mL. The LLOQ of M2was0.0200μg/mL and the calibration curves were linear in the concentration range of0.0200-10.0μg/mL. The LLOQ of M4was0.00250μg/mL and the calibration curves were linear in the concentration range of0.00250-1.25μg/mL. The matrix effect factors of laevo-ornidazole, M1, M2and M4for plasma and urine samples were96.3±8.9%and106.1±2.2%,114.1±2.1%and98.1±2.0%,92.7±3.0%and101.9±3.0%,98.8±5.2%and102.1±1.9%, respectively. The extraction recoveries of laevo-ornidazole、M1、M2and M4for plasma and urine samples were104.5±3.5%and108.3±8.2%,97.7±4.4%and104.7±4.5%,105.4±3.9%and112.0±6.9%,91.3±3.5%and87.7±5.9%, respectively. The intra-day precision (RSD) of laevo-ornidazole for plasma and urine samples were≤5.1%and≤10.8%, and the intra-day accuracy were ranged from91.1%to105.1%and98.3%to100.9%. The inter-day RSD were≤9.3%and≤6.4%, and the inter-day accuracy were ranged from96.2%to101.2%and92.8%to105.6%, respectively. The intra-day RSD of M1for plasma and urine samples were≤8.7%and <8.2%, and the intra-day accuracy were ranged from95.8%to103.8%and92.8%to105.6%. The inter-day RSD were≤8.6%and≤7.1%, and the inter-day accuracy were ranged from104.2%to105.7%and99.1%to109.2%, respectively. The intra-day RSD of M2for plasma and urine samples were≤12.2%and≤5.8%, and the intra-day accuracy were ranged from87.8%to96.8%and95.1%to102.7%. The inter-day RSD were≤9.7%and≤9.4%, and the inter-day accuracy were ranged from91.5%to103.2%and95.9%to104.0%, respectively. The intra-day RSD of M4for plasma and urine samples were≤8.9%and≤8.1%, and the intra-day accuracy were ranged from96.8%to100.6%and103.7%to105.9%. The inter-day RSD were≤7.2%and≤7.0%, and the inter-day accuracy were ranged from96.5%to103.2%and97.9%to105.5%, respectively. The stock sloutions of laevo-ornidazole, M1, M2and M4were kept stable over5months in-40±5℃. In room temperature, laevo-ornidazole, M1, M2and M4were kept stable over24h in plasma and urine samples before treatment, over48h in the post-preparative plasma and urine samples, and over three cycles of freeze (-40±5℃)and thawing (room temperature). The plasma and urine samples were kept stable for3and4months storage in refrigerator (-40±5℃), respectively. The recoveries of laevo-ornidazole, M1, M2and M4after10fold dilution in plasma samples were110.5±4.1%,103.7±6.8%,104.9±4.1%and106.8±1.3%respectively. The recoveries of laevo-ornidazole, M1, M2and M4after10fold dilution in urine samples were103.5±1.8%,118.9±5.1%,93.9±3.4%and111.0±2.1%respectively. The recoveries of laevo-ornidazole, M1and M4after100fold dilution in urine samples were98.2±3.6%,101.2±3.2%and97.6±1.8%respectively. The method was convenient, fast and high sensitive, and can be used to the concentration determination of laevo-orinazole and its metabolites in the human plasma and urine samples.Part II PK Study of Laevo-ornidazole following Single and Multi-Dose Intravenous Infusion in Healthy Chinese VolunteersThis study was a single-center, open, parallel PK study following single and multiple continuous intravenous infusion of laevo-orinazole and sodium chloride injection in healthy Chinese subjects to evaluate the safety and PK profile of laevo-orinazole.Twenty-four subjects (male:female=1:1) were enrolled in the study. The plasma and urine samples were collected by schedule and were analysised by LC-MS/MS method to determine the concentration of laevo-orinazole and the metabolites. PK parameters were calculated using the non-compartment model and compartment model with Phoenix WinNonlin6.0software.The study shows that:①After a single dose of500mg laevo-ornidazole was intravenous infused, the average Cmax and AUCo-72h of laevo-ornidazole were12.5±3.4μg/mL and174±40μg-h/mL L, the t1/2was11.6±0.8h. The Cmax of M1and M4were0.118±0.043μg/mL and0.131±0.027μg/mL. The MR of M1and M4were2.12±0.53%and2.57±0.13%; the urine cumulative excretion rate Aeo-24h of laevo-ornidazole,Ml, M2and M4were77.62±1.91%,0.702±0.172%,0.117±0.072%and1.72+0.18%, respectively. After a single dose of750mg laevo-ornidazole was intravenous infused, the average Cmax and AUCo-72h of laevo-ornidazole were17.3±2.2μg/mL and262±48μg-h/mL, the t1/2was11.9±1.3h. The Cmax of M1and M4were0.150±0.060μg/mL and0.184±0.030μg/mL. The MR of M1and M4were2.57±0.13%and2.45±0.38%. The urine cumulative excretion rate Aeo-24h of laevo-ornidazole, Ml, M2and M4were7.54±3.08%、0.717±0.352%、0.080±0.030%and1.32±0.75%, respectively. The plasma concentrations of M2in both of the two groups were all lower than the detection limit (0.0200μg/mL).②After multipile dose of laevo-ornidazole was intravenous infused, the difference of Cmax_ss and AUC0-∞between the750mg group and500mg group was not statistically significant. The Cmin_ss of750mg group was44.8%lower than the500mg group, the CLss and Vss were38.6%and42.5%higher than the500mg group, the AR of750mg group was64.0%of the500mg group, the differences were all statistically significant (P<0.05). The results showed that compared with the500mg, q12h dose regimen, the750mg,qd administration can reduce the accumulation of laevo-ornidazole in the body. After multipile dose of laevo-ornidazole was intravenous infused, the Ae0-24h were19.7±4.6%and14.3±20.4%respectively in the500mg and750mg group.③The Cmax and AUC0-τ of female subjects were higher than male subjects in both of the500mg group and750mg group. The AR of women was higher than men in the500mg group, but the gender difference of AR was not statistically significant in the750mg group, which showed that500mg, q12h continuous administration can result in higher drug accumulation in the female subjects. The gender differences of urine excretion parameters in the two groups were not statistically significant.④Both groups have no serious adverse events.500mg group reported a total of7adverse events (AEs), including4cases of abnormal laboratory AE reported by3subjects. There was no clinical releated ADR.750mg group reported a total of4AEs, including5cases of abnormal laboratory AE reported by3subjects. The blood routine examination neutrophils of subject10increased and alveolar abscess occurred the second day after the leavo-ornidazole was intravenous infused. The subject terminated the study.This maybe irrelevant with the tested drug.There were no clinical ADR in this group. Vital signs such as blood pressure and heart rate in both of the two groups and12-lead ECG before and after administration showed no significant differences.In short, the pharmacokinetic characteristics of leavo-ornidazole were similar between the500mg and750mg single dose administration. Compared with500mg q12h,750mg qd multi-dose administration can significantly reduce the accumulation of leavo-ornidazole in the body. The urinary excretion rates of leavo-ornidazole and its metabilites M1, M2and M4were low, showed that leavo-ornidazole did not meet the material balance in the body. Both of500mg ql2h and750mg qd dose regimen can obtain good safety profile.Part Ⅲ PK/PD Study of Laevo-ornidazole and Evaluation of the two Dose RegimensLaevo-ornidazole is a5-nitroimidazole drug, the PK/PD parameters are 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) as other nitroimidazole drugs (refered to metronidazole). Based on the results from our pharmacokinetics studies in Part II and the previous pharmacodynamic study, we estimated the cumulative fraction of response (CFR) of laevo-ornidazole against anaerobes and the probability of target attainment (PTA) under various MIC levels using Monte Carlo simulation.For both of the two dose regimes of750mg,q24h and500mg,q12h to B. fragilis, other Bacteroides, Clostridium difficile, Clostridium perfringens Fusobacterium, Digestive Streptococcus, the CFR for AUC0-24h/MIC=70were all higher than99.9%. In addition, the results of the PTA calculation showed that when the MIC<2μg/mL, the pharmacodynamic target value probability were all100%for both of the2dose regimens, all can obtain good clinical and microbiological effect. |
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