Investigations On Pharmacokinetics And Toxicology Of Nitazoxanide In Goats

Nitazoxanide is a nitrothiazole benzamide compound that has a wide range of antimicrobial activity against parasites, bacterial, viral pathogens. The broad spectrum of in vivo activity is related to its desacetyl derivative, tizoxanide, and includes intracellular and extracellular protozoa, helminthes, aerobic and anaerobic bacteria, and virus. Therefore, the investigations on pharmacology and toxicology of nitazoxanide play an important role in its further development and application.A sensitive and specific method for the identification of nitazoxanide metabolites in goat feces by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) with negative ion mode was firstly developed. After extraction procedure the pretreated samples were injected on an XTerra MS C8 column with mobile phase (0.2 ml/min) of acetonitrile and 10 mM ammonium acetate (adjusted to pH 2.5 with formic acid) followed a linear gradient elution, and detected by MS–MS. Identification and structural elucidation of the metabolites were performed by comparing their retention times (Rt), full scan, product ion scan, precursor ion scan and neutral loss scan MS–MS spectra with those of the parent drug or other available standard. The parent drug (nitazoxanide) and its deacetyl metabolite (tizoxanide) were found in goat feces after the administration of a single oral dose of 200 mg/kg of nitazoxanide. Tizoxanide was detected in goat feces for up 96 h after ingestion of nitazoxanide.According to the method described above, a rapid, sensitive and specific liquid chromatography–electrospray ionization (ESI) tandem mass spectrometry (LC–MS–MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitirle and 10 mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS–MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS–MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200 mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.A simple and specific high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance detection has been developed for the determination of tizoxanide, the active metabolite of nitazoxanide, in goat plasma. The plasma samples were deproteinized with acetonitrile. The analysis was performed on a Diamonsil C18 reversed-phase column (250 mm×4.6 mm, 5μm) with acetonitrile– 0.02 mol/l KH2PO4 (65:35, v/v) as mobile phase at a flow rate of 1.0 ml/min over 10 min. Detection wavelength was set at 360 nm. The linear range was 0.2–10μg/ml, the limit of detection (LOD) and the limit of quantification was 0.02 and 0.04μg/ml, respectively. The intra- and inter-day coefficient of variation (CV%) were less than 10%, respectively, and accuracy as relative error (RE%) between 1.42 and 9.05%. Mean extraction recovery was above 94.5%. There was no interference of sources on the determination of tizoxanide. The validated method was successfully applied to the pharmacokinetic study of tizoxanide in goat plasma after oral administration of nitazoxanide capsules at a dose of 100 mg/kg.The pharmacokinetics of nitazoxanide, in form of its active metabolite, tizoxanide, and its to its protein binding ability in goat plasma and in the solutions of albumin andα-1-acid-glycoprotein were investigated. The plasma and protein binding samples were analyzed using a high-performance liquid chromatography (HPLC) assay with UV detection at 360 nm. The plasma concentration of tizoxanide were detectable in goats up to 24 h. Plasma concentrations versus time data of tizoxanide after 200 mg/kg oral administration of nitazoxanide in goats were adequately described by one-compartment open model with first order absorption. The values of t1/2Ka, t1/2Ke, Tmax, Cmax, AUC, V/F(c) and CL(s) were 2.51±0.41 h, 3.47±0.32 h, 4.90±0.13 h, 2.56±0.25μg/ml, 27.40±1.54 (μg/ml)×h, 30.17±2.17 l/kg and 7.34±1.21 l/(kg×h), respectively. Afterβ-glucuronidase hydrolysis, t1/2ke, Cmax, Tmax, AUC increased, while the V/F(c) and CL(s) decreased. Study of the protein binding ability showed that tizoxanide with 4μg/ml concentration in goat plasma and in the albumin solution achieved a protein binding rate of more than 95%, while in the solution ofα-1-acid-glycoprotein the rate was only about 49%. This result suggested that tizoxanide might have much more potent binding ability with albumin than withα-1-acid-glycoprotein, since its acidic property.Finally, the toxicological effects of nitazoxanide in vivo and in vitro, as well as its potential mechanism were were investigated. Single oral gavage doses of 200 mg per kg body weight were administered to goats. Systemic toxicity was evaluated with urine routine analysis including 11 indexes and Histology examination including heart, liver, spleen, lung, kidney, urinary bladder and gall bladder evaluation. Nitazoxanide has been shown to be nontoxic, with no abnormalities in urine routine analysis as well as gross and histopathological examination of animals. The cytotoxicity results from MTT assay demonstrated nitazoxanide at concentrations of under 10μg/ml with no obvious effects on the growth of cells, show different sensitivity to Chang liver cells and Vero cells. However, nitazoxanide at concentrations of 10~100μg/ml can inhibit the cell growth in a dose-dependent manner, and IC50 for Chang liver cells and Vero cells were 37μg/ml and 41μg/ml, respectively. Compared with control group, the 293T cells treated with nitazoxanide especially more than 10μg/ml show quantitatively reduced, abnormal or wizened morphology, and the number of apoptosis cells was increased. Futher analysis with Western blot was carried out, and results showed that nitazoxanide could enhance the expression of p53, although not significantly, and did not affect cell cycle (10μg/ml). This study demonstrated that nitazoxanide appears to be well tolerated, and could enhance p53 expression to a certain extent, and thus induce apoptosis.