Application of urine antigen assay to evaluate outcomes of praziquantel treatment and reinfection in opisthorchiasis in northeast Thailand.

BACKGROUND: A urine antigen assay was applied to evaluate chemotherapeutic outcomes and reinfection patterns of opisthorchiasis in Thailand. METHODS: We used a prospective study design by following opisthorchiasis subjects at baseline and post-treatment using a urine antigen assay and faecal examination by the formalin-ethyl acetate concentration technique (FECT). RESULTS: The antigen of Opisthorchis viverrini in urine diminished within 4 weeks after praziquantel treatment. Concurrent faecal examinations by FECT showed that faecal eggs were negative at 4 weeks after treatment. In a subsequent study, reinfection rates and intensity patterns of O. viverrini were evaluated at 48 weeks after praziquantel treatment. Within a group of subjects with curative treatment (n=137), 16.8% became reinfected according to FECT and 27.7% according to the urine antigen assay (p<0.05). There were significant correlations in intensity of infection between pretreatment and at 48 weeks post-treatment in both faecal egg counts and antigen levels in urine. CONCLUSIONS: The results suggested that in addition to screening, the urine antigen assay is an efficient tool for monitoring outcomes of drug treatment and reinfection in opisthorchiasis. Due to the ease of urine sample collection and handling, the urine assay becomes an alternative method to faecal examination for diagnosis and monitoring of opisthorchiasis.

Characterizing the Biochemical Response to Schistosoma mansoni Infection and Treatment with Praziquantel in Preschool and School Aged Children.

Schistosomiasis is a widespread chronic neglected tropical disease prevalent mostly in children in under-resourced rural areas. Its pathological effects have been clinically characterized, yet the molecular-level effects are understudied. In this study, the biochemical effects of Schistosoma mansoni infection and praziquantel treatment were studied in 130 preschool aged and 159 school aged infected children and 11 noninfected children in Azaguié, Côte d'Ivoire. Urine samples were collected prior to receiving 20, 40, or 60 mg/kg of praziquantel or a placebo, as well as 24 h post-treatment, and at the 3-week follow up. Urinary metabolic phenotypes were measured using 1H NMR spectroscopy, and metabolic variation associated with S. mansoni infection and praziquantel administration was identified using multivariate statistical techniques. Discriminatory metabolic signatures were detected between heavily infected and noninfected children at baseline as well as according to the dose of praziquantel administered 24 h post treatment. These signatures were primarily associated with the metabolic activity of the gut microbiota, gut health and growth biomarkers and energy and liver metabolism. These analyses provide insights into the metabolic phenotype of schistosomiasis and treatment with praziquantel in two important demographics.

Metabolic profiling of praziquantel enantiomers.

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing, indicating that the major metabolic reactions encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity toward the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in substrate location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Isolation and identification of 8-hydroxypraziquantel as a metabolite of the antischistosomal drug praziquantel.

Praziquantel, a broad spectrum anthelmintic drug, is extensively metabolized in the liver, yielding mainly monohydroxylated and dihydroxylated phase-I metabolites. However, the exact chemical structure of most metabolites is still unknown. One of these unidentified phase-I metabolites was isolated from human urine by high performance liquid chromatography using an isocratic separation method. This metabolite was identified as 8-hydroxypraziquantel. For the structure elucidation, electrospray ionization-mass spectrometry, 1H and 13C NMR spectroscopy have been used.

Voltammetric determination of praziquantel in tablets and biological fluids.

A simple method is described for the determination of praziquantel (CAS 55268-74-1) in its pure form, tablet formulations and biological fluids. The proposed method depends upon the polarographic activity of praziquantel at the dropping mercury electrode (DME) in Britton Robinson buffers, whereby a well-defined catholic wave is produced over the pH range 7-12. The wave was characterized as being irreversible diffusion-controlled with limited adsorption properties. The diffusion current constant (Id) was 0.56 +/- 0.004 (n = 11). The current-concentration relationship was found to be rectilinear over the range 8-48, 3.2-38.4 and 0.48-20 micrograms.ml-1 using direct current (DCt), differential pulse polarographic (DPP) and alternating current (ACt) odes, respectively, with minimum detection limit (S/N = 2) of 0.32 microgram.ml-1 (1.02 x 10(-6) mol/l and 0.02 microgram.ml-1 (6.4 x 10(-8) mol/l) for DPP and ACt modes respectively. The average percent recovery was favourably compared to a reference method with a satisfactory standard deviation. The proposed method was applied to spiked human urine and plasma. The percentage recoveries were 99.33 +/- 0.79 and 98.23 +/- 0.53, respectively.

Capillary electrophoresis-mass spectrometry, liquid chromatography-mass spectrometry and nanoelectrospray-mass spectrometry of praziquantel metabolites.

Capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS) coupling was used for the investigation of metabolites of the anthelmintic drug praziquantel. Human urine samples and microsomal incubation mixtures were investigated after preparation by solid-phase extraction. CE- and LC-MS coupling was performed using an electrospray ionization interface. An ion trap mass spectrometer equipped with a laboratory-made nanoelectrospray ion source was used for the investigation of the glucuronide conjugates by consecutive fragmentations. The nanoelectrospray interface offers the opportunity to perform complicated MSn spectrometric investigations. Different phase I metabolites of praziquantel and their glucuronidated and sulfated conjugates were detected.

Sensitive high-performance liquid chromatographic assay for praziquantel in plasma, urine and liver homogenates.

A high-performance liquid chromatographic method for the determination of praziquantel in plasma, urine and rat liver homogenates has been developed. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, a 0.05 M phosphate buffer solution (pH 5.0) for equilibrating and washing and ethyl acetate-diisopropyl ether for drug elution. The analysis was performed on an Ultrasphere ODS C18 column with a mobile phase of acetonitrile-water with ultraviolet detection at 217 nm. The results showed that the assay is sensitive (31.2 ng/ml), linear between 0.125 and 4.0 micrograms/ml, precise (coefficient of variation = 10%) and selective with other drugs currently administered with praziquantel.

Metabolism studies of the antischistosomal drug praziquantel using tandem mass spectrometry: qualitative identification of 17 hydroxylated metabolites from mouse urine.

Schistosomiasis is a parasitic liver infection which is known to affect many aspects of drug metabolism. Praziquantel (PZQ) is the drug of choice for treating this disease. PZQ is known to be highly metabolized, but the effect of the disease on its metabolism has not been investigated. Control mice and mice infected with Schistosoma mansoni were dosed with PZQ and their urines were examined for the presence of metabolites using a triple-quadrupole mass spectrometer (tandem mass spectrometer). The collisionally induced dissociation of PZQ was remarkable in its structurally significant fragments. From this we were able to identify 17 hydroxylated metabolites of PZQ from purified urine samples without further chemical separation, including three monohydroxylated, six dihydroxylated, and eight trihydroxylated metabolites. There were no qualitative differences in metabolite production between control and infected animals.

Metabolism studies of the antischistosomal drug praziquantel using tandem mass spectrometry: distribution of parent drug and ten metabolites obtained from control and schistosome-infected mouse urine.

The collisionally activated dissociation spectrum of the antischistosomal drug praziquantel (PZQ) has many structure-specific fragmentations which permit identification of PZQ and seventeen of its hydroxylated metabolites in mouse urine. These fragmentations may also be used to quantify the metabolic pattern of PZQ. In the present study, a triple-quadrupole mass spectrometer system has been used to generate [M + H]+ ions for PZQ and its monohydroxy, dihydroxy and trihydroxy metabolites which yield daughter ions capable of quantifying PZQ and ten of these metabolites. The goal of these experiments was to evaluate the effect of this hepatic infection on drug metabolism. This was accomplished in two steps. First, the amount of unmetabolized, mono- and dihydroxylated PZQ was established from the [M + H]+ ions. Then the specific metabolites at each level of hydroxylation were determined from daughter ion spectra. The product of these two values produces the metabolite pattern. The reproducibility of these assays ranged from good, with a coefficient of variation of 3% for the most abundant metabolite, to poor (43%) for PZQ, which is only 1% of the total elimination pattern. The excretion of unchanged PZQ and two dihydroxylated metabolites was enhanced in animals bearing schistosomiasis compared to control mice, while a third dihydroxylated metabolite was depressed.

Quantitative determination of praziquantel in body fluids by gas liquid chromatography.

Praziquantel was determined in body fluids by gas liquid chromatography as follows: A known amount of an internal standard and 0.1 N sodium hydroxide solution was added to the sample to be analyzed. After extraction with methyl acetate/diisopropyl ether = 30/70, the organic extract was evaporated, the residue taken up in methylacetate and an aliquot injected for glc analysis. Separation was accomplished on a OV3 silicon oil phase, and for detection and quantitation, a thermoionic FID sensitized for nitrogen-containing compounds was used. The determination limit in serum is about 0.01 micrograms/ml. The relative standard deviation for serum concentrations of 0.1 micrograms/ml was found to be 4.5%.

A fluorometric method for the determination of praziquantel in blood-plasma and urine.

Some physicochemical data of praziquantel which may have analytical relevance are reported. For the quantitative determination praziquantel is extracted from plasma or urine by means of organic solvents and then hydrolyzed in an aqueous alkaline solution. The hydrolyzed product is reacted with dansyl-chloride (5-dimethylaminonaphthalene-sulfonylchloride). The dansylated compound is separated and quantified fluorometrically. The limit of determination is 3 micrograms/l in blood plasma and 30 micrograms/d in urine. For both fluids, the imprecision is approximately 7.5%. The method is suitable for the determination of praziquantel in patients or healthy volunteers treated with therapeutic doses.