In Vitro Anti-anaerobe Pharmacodynamic And Pharmacokinetic/Pharmacodynamic Study Of Laevo-ornidazole

Laevo-ornidazole, the laevo isomer of ornidazole, is the third generation nitroimidazole derivative following metronidazole, tinidazole and 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.Laevo-ornidazole has favourable antianaerobic and antiprotozoal activity. Adverse reactions of laevo-ornidazole are significantly lower than Ornidazole, especially its central nervous system toxicity.Laevo-ornidazole and sodium chloride injection was approved by China Food and Drug Administration on August 13,2009. Currently, its phase IV clinical study is being carried out mainly to investigate the drug efficacy and safety when treating anti-anaerobic infections in a wide range of conditions and to assess the relationship between benefits and risks of its use in the general population or specific population.Howerver, due to a lack of in vitro pharmacodynamic study data and corresponding PK/PD research of laevo-ornidazole in the previous study, the problems displayed as follows:①Laevo-ornidazole was short of a comparison of antibacterial activity with metronidazole and its tested strains were less. ② The metabolic pathway of laevo-ornidazole was found mainly in liver metabolic and we had obtained the phase I metabolites but still didn’t know the antimicrobial activity of metabolites; ③There were inadequate data of bactericidal kinetics study and PK/PD target values research of laevo-ornidazole. In this study, we aim to provide a scientific basis for the development and optimization of dosing regimens of laevo-ornidazole in clinical practice in order to fully clarify its in vitro pharmacodynamic and PK/PD characteristics.Laevo-ornidazole has 5 metabolites of phase I in human liver:Ml, 1-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol;M2,2-methyl-5-nitroimida zole;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 theoxymethyl analogue M1, or to the rupture of the side-chARn with formation of M2. Hydrolytic cleavage of the imidazole ring, presumably after reduction of the nitro group, gives rise to M3 and M5, whereas hydrolysis of chlorine in the side-chARn leads to the formation of M4. This five phase I metabolites of laevo-ornidazole were developed.Because the metabolites activity of laevo-ornidazole has not been researched yet, this study will complement the metabolites study of laevo-ornidazole.In summary, ①this study was designed to compare the antimicrobial activity of laevo-ornidazole with metronidazole, ornidazole and dextrornidazole and add the antibacterial activity study of 5 metabolites (Ml, M2, M3, M4, M5) to elucidate antibacterial spectrum antimicrobial activity of laevo-ornidazole and its metabolites;② The bactericidal kinetics of laevo-ornidazole was confimed by measuring the static kill curve of laevo-ornidazole against Bacteroides fragilis and using pharmacodynamic model to analyze, providing a reference for future research; ③In vitro PK/PD dynamic model of laevo-ornidazole against Bacteroides fragilis was established and verified to lay a foundation for exploring PK/PD target value;④In vitro PK/PD target of laevo-ornidazole was calculated and achieved to optimize two kinds of dosing regimens (500mg, q12h and 750mg, q24h) and provide basis for phase IV clinical trials.The study included four parts:Part I In Vitro Antibacterial Activity of Laevo-ornidazole and its Five MetabolitesObjectives:We determine the in vitro antibacterial activity and spectrum of laevo-ornidazole and its metabolites against 375 clinical isolates of anaerobic bacteria. Methods:Agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of laevo-ornidazole,3 comparators and 5 metabolites of laevo-ornidazole against 375 anaerobic isolates by using RapID ERIC method identifying. The minimum bactericidal concentrations (MBCs) of laevo-ornidazole and metronidazole were measured against 22 strains of Bacteroides fragilis. Results:375 clinical isolates consisted of 181strains of anaerobic gram-negative bacilli,11 strains of Gram-negative cocci,139 strains of gram-positive bacteria and 44 strains of Gram-positive cocci,covering 34 species.Laevo-ornidazole showed a good activity against gram-negative bacilli (B. fragilis, other Bacteroides), gram-positive bacteria (Clostridium difficile, Clostridium perfringens), and Gram-positive cocci (Peptostreptococcus magnus), since the value of MIC90 for laevo-ornidazole against above-mentioned anaerobic isolates is 0.5 mg·L-1,! mg·L-1,0.25 mg·L-1,2 mg·L-1 and 1 mg·L-1, respectively. However, laevo-ornidazole and its comparators had poor antibacterial activity against Veillonella spp among anaerobic gram-negative cocci but needed to be supported by a large number of datas. For anaerobic gram-negative bacilli, anaerobic gram-positive bacilli, and anaerobic gram-positive cocci, laevo-ornidazole displayed activity similar to or slightly higher than that of metronidazole, ornidazole and dexornidazole,especially the better activity against Bacteroides fragilis. Favorable anti-anaerobic activity was also seen with laevo-ornidazole metabolites M1 and M4 except M2、M3 and M5. MBC50 of laevo-ornidazole was 2 mg·L-1 and MBC904 mg·L-1 against 22 clinical B. fragilis strains. MBC90/MIC90 ratio of laevo-ornidazole was 4, similar to that of metronidazole. Conclusions:The in vitro anti-anaerobic activity of laevo-omidazole is similar to or slightly higher than that of metronidazole, ornidazole and dextrornidazole. Laevo-ornidazole has good activity against most anaerobic species. Favorable anti-anaerobic activity is also seen with laevo-ornidazole metabolites M1 and M4. Laevo-ornidazole is a bactericidal agent, similar to metronidazole.Part II In Vitro Bactericidal Activity and Bactericidal Kinetics Study of Laevo-ornidazole against Bacteroides fragilisObjectives:Setting metronidazole against Bacteroides fragilis ATCC 25285 as the control, we explored the bactericidal kinetics of laevo-ornidazole by determining the static time-kill curve of clinical strains and the control strain of Bacteroides fragilis in different MIC values. Methods:The control strain ATCC 25285 and 3 clinical isolates of Bacteroides fragilis in different MIC values, respective namely 06-W6-57 (0.5 mg·L-1), 13-W45-64 (1 mg·L-1) and 13-W45-69 (2 mg·L-1), were selected from the first part.The static time-kill curve was constructed at different concentrations (0.5MIC-64MIC) of laevo-ornidazole and metronidazole. A pharmacodynamic model was built to analyze the relationship between ALogCFU24h and drug concentration, compare the efficacy of the two drugs and define the bactericidal kinetics of laevo-ornidazole using Matlab 7.0 software. Results:Static killing curve and sigmoid Emax model analysis suggested that laevo-ornidazole was a concentration-dependent antibiotic.Model analysis showed that the EC90 value of laevo-ornidazole against Bacteroides fragilis ATCC 25285 was only 1/5 value of metronidazole, respectively 0.648 and 3.42 mg·L-1; When △LogCFU24h was -3 against ATCC 25285, the concentration of laevo-ornidazole was 1/2 concentration of metronidazole, respectively 0.499 and 1.09 mg·L-1, indicating that bactericidal activity of laevo-ornidazole was more powerful than metronidazole. Meanwhile, with the MIC values of Bacteroides fragilis from 0.5 mg·L-1 increased to 2 mg·L-1, the bacterial killing rate and bactericidal activity of laevo-ornidazole gradually decreased, requiring higher drug concentration to achieve a good efficiency. Conclusions:Laevo-omidazole is a concentration-dependent antibiotic and has a faster bacterial killing rate than metronidazole.Part III Construction and Verification of the in vitro PK/PD Model on Laevo-ornidazole against Bacteroides fragilisObjectives:The present study we initially carried out was to build the in vitro PK/PD anaerobic bacteria model and validate the reliability of this model. Methods:We establish anaerobic model system to simulate the concentration time curve of 750mg single dose of laevo-ornidazole in healthy volunteers in the case of an analog dynamic velocity condition. We also observed dynamic anti-bacteria process against Bacteroides fragilis ATCC 25285 and normal growth of ATCC 25285 in the absence of laevo-ornidazole. Results:By PK and PD verification of the model, anaerobic conditions in the model were stable and controllable in 72 hour, controlling the variation of the flow velocity. We successfully simulated two-compartment model and ensured the accuracy and controllability of pharmacokinetics with only-10.6% relative deviation, without adding the configuration of the model; the model also provided an anaerobic environment in line with required growth conditions, in which Bacteroides fragilis ATCC 25285 can achieve rapid growth phase in 6h. Conclusions:The PK/PD anaerobic bacteria model can be applied to the vitro PK/PD target value research of laevo-ornidazole.Part IV In Vitro PK/PD Study of Laevo-ornidazole against Bacteroides fragilisObjectives:The correlation investigation of PK/PD parameters with pharmacodynamics index and PK/PD target of laevo-ornidazole to reach the maximum drug effect were conducted in our study. Methods:In this study, we set up PK/PD model in accordance with the PK parameters of healthy volunteers in clinical trial of the phase IV and observed antibacterial effect of laevo-ornidazole in four single dose of 250mg, 500mg,750mg and 1000mg against Bacteroides fragilis (Bacteroides fragilis ATCC 25285 and 2 clinical isolates of 13-W45-64 and 13-W45-69 choosing from the second part) in PK/PD models. △Log24h (△LogCFU24h=LogCFU24h-LogCFUo), AUBC (the area under bacteria-kill curve within 24 hours) and IKR (the initial killing rate) were calculated as three pharmacodynamic indexes. Correlation analysis was quantized between three pharmacodynamic indexes (△Log24h, AUBC and IKR) of laevo-ornidazole with its PK/PD parameter (AUC/MIC, Cmax/MIC and %T>MIC).We also established pharmacodynamic model (Emax model) to calculate the PK/PD parameters of achieving the desired pharmacodynamic effect. Results:The results indicated that antibacterial effect of laevo-ornidazole on Bacteroides fragilis represented by ALog24h and AUBC had a better correlation with above-mentioned PK/PD parameters than IKR. The relativity of three pharmacodynamic indexes with AUC/MIC and Cmax/MIC showed higher than %T>MIC. When the pharmacodynamic index of Log24h declined 3-log 10 compared to 0 h, the PK/PD parameters (AUC/MIC, Cmax/MIC and % T>MIC) values were 157.6,14.1 and 56.4%, respectively. Conclusions:The laevo-ornidazole target values against main pathogenic bacteria(Bacteroides fragilis) were 157.6 and 14.1, respectively. We hope the study would provide a guidance to optimize dosing regimen of laevo-ornidazole sodium chloride injection to achieve maximum bactericidal effect.