In Vitro Anaerobic Pharmacokinetic/Pharmacodynamic Model to Simulate the Bactericidal Activity of Levornidazole Against Bacteroides fragilis.

PURPOSE: This study was designed to correlate the pharmacokinetic/pharmacodynamic (PK/PD) parameters with PD indices of levornidazole against Bacteroides fragilis and to calculate the PK/PD target value for levornidazole to attain its expected maximal bactericidal effect using an in vitro anaerobic dynamic PK/PD model. METHODS: An anaerobic dynamic PK/PD model was developed in vitro. The scheme for PK modeling was designed according to the PK parameters of levornidazole in the human body. The device of 2-compartment PK/PD model was constructed by using digital control of flow rate to simulate 4 regimens of single-dose intravenous infusion of levornidazole to determine the bactericidal activity of levornidazole against the 3 strains of B fragilis within 72 hours. PD parameters such as reduction of colony count within 24 hours (∆Log24h), area under bactericidal curve (AUBC), and 2-hour initial killing rate (IKR) were calculated and correlated with PK/PD parameters. Sigmoid Emax model of levornidazole was established to calculate PK/PD target values to attain corresponding PD effect. FINDINGS: PK and PD validation proved the stability of the model in simulating levornidazole against B fragilis and the precision and accuracy in the results of PK modeling. Cmax and AUC0-24h found only -1.46% and -6.72% differences from the values in vivo. Our study found that ∆Log24h, AUBC, and IKR were more correlated with AUC0-24h/MIC and Cmax/MIC than with %T>MIC. According to ∆Log24h, the PK/PD target values of AUC0-24h/MIC, Cmax/MIC, and %T>MIC of levornidazole against B fragilis were 157.6%, 14.1%, and 56.4%, respectively. IMPLICATIONS: Our findings are useful for optimizing the clinical dosing regimen of levornidazole sodium chloride injection to attain maximal bactericidal effect.

A new pharmacological effect of levornidazole: Inhibition of NLRP3 inflammasome activation.

Levornidazole, which was originally used to inhibit anaerobic and protozoal infections, is currently known to possess a novel pharmacological effect. In this study, we investigated the possible modulation by levornidazole of NOD-like receptor protein 3 (NLRP3) inflammasome-mediated IL-1ß and IL-18 release from macrophages. The NLRP3 inflammasome could be activated by lipopolysaccharide (LPS) plus ATP or monosodium urate (MSU) in PMA-pretreated THP-1 macrophages. Surprisingly, an in vitro study showed that levornidazole suppressed IL-1ß and IL-18 secretion by blocking the activation of the NLRP3 inflammasome. However, dextrornidazole barely suppressed the NLRP3 inflammasome. Levornidazole displays activity similar to that of dextrornidazole against clinical anaerobic bacteria, and they possess the same pharmacokinetic properties. Moreover, both of these compounds were unable to ameliorate T cell-mediated inflammation. Therefore, we used the widely applied NLRP3 inflammasome-related models of dextran sodium sulfate (DSS)-induced colitis and LPS-induced endotoxin shock to confirm the novel pharmacological effect of levornidazole in vivo. The in vivo studies verified the novel activity of levornidazole because the inhibition of NLRP3 inflammasome by levornidazole contributed to a better ameliorating effect than that of dextrornidazole in the in vivo models tested. Furthermore, this inhibitory effect of levornidazole was found to be at least partially achieved by decreasing the mitochondrial ROS generation without inhibiting NF-κB activation. In summary, these data describe a new pharmacological effect of levornidazole as an inhibitor of NLRP3 inflammasome activation.

Quantification of levornidazole and its metabolites in human plasma and urine by ultra-performance liquid chromatography-mass spectrometry.

We developed and validated an ultra-performance liquid chromatographic (UPLC) method coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry for simultaneous determination of levornidazole and its first-pass metabolites, l-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol (Ml), 2-methyl-5-nitroimidazole (M2) and 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4), in human plasma and urine. The biological samples were pretreated by protein precipitation and liquid-liquid extraction and analyzed using an ACQUITY UPLC CSH C18 column (2.1×50 mm, 1.7 µm) and a QTRAP mass spectrometer in multiple reaction monitoring mode via APCI. Acetonitrile and 0.1% formic acid in water was used as the mobile phase in gradient elution at a flow rate of 0.6 mL/min. The lower limit of quantification of this method was 0.0100, 0.00500, 0.0200 and 0.00250 µg/mL for levornidazole, M1, M2 and M4, respectively. The linear calibration curves were obtained for levornidazole, M1, M2, and M4 over the range of 0.0100-5.00, 0.00500-2.50, 0.0200-10.0 and 0.00250-1.25 µg/mL, respectively. The intra- and inter-batch precision was less than 12.2% in plasma and less than 10.8% in urine. The intra- and inter-batch accuracy was 87.8-105.7% in plasma and 92.8-109.2% in urine. The mean recovery of levornidazole, M1, M2 and M4 was 91.1-105.1%, 95.8-103.8%, 87.8-96.8%, 96.8-100.6% from plasma and 96.0-100.9%, 96.9-107.9%, 95.1-102.7%, 103.7-105.9% from urine respectively. This method was validated under various conditions, including room temperature, freeze-thaw cycles, long-term storage at -40 ± 5°C, after pretreatment in the autosampler (at 10°C), and 10- and 100-fold dilution. This newly established analytical method was successfully applied in a pharmacokinetic study following single intravenous infusion of levornidazole in 24 healthy Chinese subjects.

Development and validation of a sensitive enantiomeric separation method for new single enantiomer drug levornidazole by CD-capillary electrophoresis.

A fast and sensitive chiral capillary electrophoresis method has been developed to determine levornidazole and its enantiomeric impurity at a 0.05% level in levornidazole injection solution. Several chemical and instrumental parameters which have an effect on chiral separation were investigated, including chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature and rinsing procedure. After optimizing all the effective parameters, the ideal separation conditions were 20 mM Tris phosphate buffer at pH 2.1, containing 2.0% (w/v) sulfated-α-cyclodextrin with short end injection at 0.5 psi for 5.0 s. Online UV detection was performed at 277 nm. A voltage of 30 kV was applied and the capillary temperature was kept at 25 °C. 2,4,6-triaminopyrimidine was chosen as internal standard to improve the injection precision. The total analysis time is less than 7 min, which is faster than the existing chiral HPLC method (65 min). The validation of the method was performed in terms of factorial analysis, stability of the solution, different cyclodextrin batches study, selectivity, linearity (from 2.5 µg/mL to 6000 µg/mL, y=0.0015 x+0.0304; R(2)=0.9999 and the residuals were randomly scattered around 0), LOD and LOQ (0.3 and 1.0 µg/mL, respectively), precision and accuracy. The proposed method was then applied to the enantiomeric purity control of the starting material and injection solution of levornidazole (0.5 mg/100 mL).

Solid-state studies of the triclinic (Z' = 2) antiprotozoal drug ternidazole.

The solid-state properties of antiprotozoal ternidazole (3-(2-methyl-5-nitroimidazol-1-yl)-propan-1-ol) have been studied. Crystals are triclinic in the temperature interval between 100 and 333 K (melting point) with two different molecular conformations present in the asymmetric unit (Z' = 2) and two of each conformer make up a tetramer held together by hydrogen bonding. Its melting enthalpy at 333 K is 25.65 (± 1.29) kJ · mol(-1). Linear plots were obtained for the melting temperature versus pressure (dP/dT = 5.67 (± 0.08) MPa · K(-1)] and the glass transition versus pressure [dP/dT = 7.73 (±1.76) MPa · K(-1)]. No crystalline polymorphism could be detected; thus, the single-crystal structure that has been found is most likely the stable one.

The effect of (R,S)-ornidazole on the fertility of male mice and the excretion and metabolism of 36Cl-(R,S)-ornidazole and 36Cl-(R,S)-alpha-chlorohydrin in male mice and rats.

(R,S)-Ornidazole, an effective antifertility agent for male rats at 400 mg/kg/day, was ineffective at this dose in male mice and at 1000 mg/kg/day caused neural effects. The compound was not excreted unchanged and more polar metabolites and Cl- were detected in 0-8 h urine following a single injection (400 mg/kg). In 8-24 h urine even these metabolites and most Cl ion were absent, indicating rapid metabolism of ornidazole. There was no organ specific accumulation of 36Cl-(R,S)-ornidazole in murine tissues. After injection of 36Cl-(R,S)-alpha-chlorohydrin, another antifertility agent in the rat but not the mouse, there was also no tissue-specific accumulation of radioactivity in the reproductive tract of either species. Urinary excretion rates of alpha-chlorohydrin were twice as rapid in mice as in rats. In mice, alpha-chlorohydrin was the major urinary metabolite, but in the rat metabolites included Cl-, 3-chlorolactate (BCLA) at 5 and 10 h and BCLA only at 24 h. BCLA was the major metabolite detected in most tissues at 10 and 24 h. In the rat cauda (but not caput) epididymidis the glycolytic inhibitor 3-chlorolactaldehyde was present at 5 h (but not 10 h), indicative of early metabolism. These results demonstrate a greater metabolism and excretion of putative antifertility agents in the mouse than the rat, lowering the amount of effective inhibitor circulating in the animal, which may explain why (R,S)-alpha-chlorohydrin and (R,S)-ornidazole are ineffective in this species at the dosages and injection times used, despite their spermatozoa being sensitive to inhibition by (R,S)-alpha-chlorohydrin in vitro.

Antifertility potential of ornidazole analogues in rats.

In order to determine which part of the ornidazole molecule [1-(3-chloro-2-hydroxy)propyl-2-methyl-5-nitroimidazole] is responsible for its antifertility action, structural analogues were fed to male rats of proven fertility at doses equivalent to the antifertility dose of ornidazole (1.82 mmol/kg/day). The fertility of the males was tested, before oral gavage (control mating) and after 10 and 14 days of feeding, by counting the number of fetuses and corpora lutea present in females 12 days after mating. The day after the last mating, the kinematic parameters of sperm from the cauda epididymidis were assessed objectively with a Hamilton-Thorne motility analyzer. Analogues bearing the 2-nitro and 5-methyl groups on the imidazole ring were inactive if the (chlorohydroxy)propyl group were substituted by proton or methyl, hydroxyethyl, chloroethyl, or (sulfonylethyl)ethyl groups, indicating that the three-carbon side chain of ornidazole was necessary for the antifertility action. Only ornidazole and its acetate were effective antifertility agents, but a compound bearing the (chlorohydroxy)propyl side chain attached to a nitrogen atom of a heterocyclic phthalimide produced a partial but temporary reduction in fertility. Similarities of the action of ornidazole with the male antifertility agent, alpha-chlorohydrin [3-chloro-1,2-propanediol], are discussed.

Toxicity of ornidazole and its analogues to rat spermatozoa as reflected in motility parameters.

Ornidazole, a 5-nitro-imidazole derivative, has contraceptive properties in rats. As some ornidazole passes through the body unmetabolized after administration, the aim of this study was to investigate if ornidazole itself has a direct effect on sperm motility and whether these effects are limited or potentiated by the epididymal epithelium or structural changes to the molecule. Cauda epididymal spermatozoa or cauda epididymal tubules were incubated with ornidazole or ornidazole analogues, and motility parameters were subsequently measured by means of a computer-assisted sperm analysis (CASA) system. Incubation of spermatozoa in 2.5 mmol/L ornidazole for 4 h reduced their motility significantly, whereas incubation of epididymal tubules for 8 h in 10 mmol/L ornidazole was required to alter the velocity parameters of the enclosed spermatozoa upon release, suggesting that extratubular non-metabolized ornidazole can participate in inhibiting the motility in vivo. The in vitro toxicity of ornidazole derivatives depends on the halogen present and on the position of the nitro-group. The putatively inactive (R)- and the active (S)-ornidazole exhibited equivalent depression of sperm motility by direct incubation. This observation, and the differences between the in vitro and the in vivo efficacies of various ornidazole analogues, indicates distinct mechanisms of motility inhibition in the two experimental systems.

Disposition of ornidazole and its metabolites during pregnancy.

The disposition of ornidazole and its two major hydroxylated metabolites was studied in five pregnant women (gestational ages 25 5/7 to 38 4/7 weeks) with either chorioamnionitis or pyelonephritis treated with ceftriaxone 2 g, tobramycin 3 mg/kg body weight and ornidazole 1 g all administered once-daily. Two series of blood samples were obtained, the first on the first day of treatment and the second at steady-state on day 5. Local and systemic tolerability of ornidazole was excellent and patients showed complete remission without premature delivery. There was no evidence of ornidazole accumulation, and the pharmacokinetic parameters were very similar to those seen in healthy subjects. The dosage regimen of ornidazole therefore requires no adjustment during pregnancy. Trough concentrations of ornidazole measured at 24 h post dose were above the MIC of sensitive organisms. Children born to the trial patients showed normal initial development and their growth was normal.