Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin increasingly found in drinking water sources worldwide. Toxicity studies have shown CYN can induce effects in a range of different cell types with primary hepatocytes consistently shown to be the most sensitive cellular model. How CYN enters the intracellular environment is not clear, although the size and hydrophilic nature of the toxin suggest it would not readily cross a lipid bilayer. In this study, a Vero cell line expressing green fluorescent protein (GFP) was used to monitor for CYN uptake based on the toxin's potent effects on protein synthesis. Effects on the GFP signal were compared with inhibitors cycloheximide (CHEX) and emetine. While CYN potency was demonstrated in a cell-free system (CYN>CHEX>emetine) it was considerably reduced in the Vero-GFP cell model (CHEX, emetine>>CYN). In contrast to other inhibitors, CYN effects on GFP signal increased 6 fold over 4-24 h incubation indicating slow, progressive uptake of the toxin. Confirming that the uptake process is not energy dependent CYN entry also occurred at 4 degrees C, while competition experiments excluded the uracil nucleobase transporter system as potential mechanism for CYN uptake. Dilution of media enhanced CYN uptake by Vero-GFP cells although mechanism by which this occurred is unknown.

Urinary excretion of ipecac alkaloids in human volunteers.

Limited data are available regarding urinary excretion of ipecac alkaloids in humans. In this study, ipecac syrup was administered po to 12 healthy human volunteers at a dose of either 20 mL or 30 mL, and urinary excretions of cephaeline and emetine as well as blood and vomit concentrations were detected by HPLC. All participants showed vomiting after the 30 mL dose within 1 h, whereas 2/6 did not show vomiting within 4 h after the 20 mL dose. Percentage recovery of alkaloids in vomit were 39 +/- 38 or 76 +/- 14% after the 20 mL or 30 mL doses, respectively. In most participants, plasma alkaloids reached their maximum levels within I h and became undetectable after 6 h. Total excretions of ipecac alkaloids into the urine within the first 48 h were less than 2%, but both alkaloids were detectable in the urine at 2w in all participants and could be detected up to 12w in 1/2 participants who did not vomit. These results show that ipecac alkaloids may be detectable in urine several weeks after ingestion and suggest that their detection in urine may be helpful to identify the Munchausen syndrome by proxy using ipecac syrup.

Absorption, distribution and excretion of 3H-labeled cephaeline- and emetine-spiked ipecac syrup in rats.

The maximum plasma radioactivity levels of tritium (3H)-labeled cephaeline, (24.3, 28.7 and 40.6 ng eq./mL) were reached at 2.00-3.33 hours following oral dosing of ipecac syrup. The maximum plasma radioactivity levels of 3H-emetine (2.71, 6.47 and 9.62 ng eq./mL) were reached at 1.08-2.33 hours following ipecac syrup administration. The Cmax values of 3H-cephaeline were followed by a biexponential decrease with half-lives t 1/2(lambda z) of 3.45-9.40 hours. On the other hand, the t 1/2 (lambda z)of 3H-emetine were 65.4-163 hours, which revealed a biexponential decrease. The radioactivity of both tritium-labeled compounds was distrbuted maximally in most tissues at 24 hours. For 3H-cephaeline, the maximum radioactivity levels in tissues were approximately 100-150 times greater than in plasma. For 3H-emetine, the radioactivity levels in tissues were approximately 1000-3000 times greater than in plasma. Tissue radioactivity levels decreased at a substantially slower rate than that observed in plasma. Tissue radioactivity of 3H-emetine decreased more slowly than that of 3H-cephaeline. For 3H-cephaeline, the cumulative biliary excretion of radioactivity was 57.5% at 48 hours. The cumulative urinary and fecal excretion of radioactivity in these rats was 16.5% and 29.1%, respectively, of the dose at 48 hours following dosing. For 3H-emetine, the cumulative biliary excretion of radioactivity was 12.5% at 48 hours. The cumulative urinary and fecal excretion of radioactivity was 9.4% and 34.1%, respectively, of the administered dose at 48 hours. The radioactivity level of 3H-emetine remaining in the carcasses at 48 hours was equivalent to approximately 50% of the dose. A portion of each tritium-labeled compound was subjected to entero-hepatic circulation. Thus, the absorption rate of 3H-cephaeline and 3H-emetine was estimated to be approximately 70% on the basis of the data obtained from excretion studies. There was no difference in the absorption process between these two compounds. However, the difference was admitted in the biliary clearance, which is the main excretion route of both compounds. Delayed excretion of 3H-emetine may be primarily due to its resorption as related to entero-hepatic circulation and tissue retention. This study has determined the absorption, distribution and excretion of 3H-cephaeline and 3H-emetine in rats.

Biotransformation of the ipecac alkaloids cephaeline and emetine from ipecac syrup in rats.

The metabolism of cephaeline and emetine, which are the primary active components of ipecac syrup, were investigated in rats. Cephaeline-6'-O-glucuronide was found to be a biliary metabolite of cephaeline. Cephaeline (6'-O-demethylemetine) and 9-O-demethylemetine were observed to be enzyme-hydrolyzed biliary metabolites of emetine. Cephaeline was conjugated to glucuronide, while emetine was demethylated to cephaeline and 9-0-demethylemetine, and may be conjugated to glucuronides afterwards. Urine, feces and bile were collected from rats within 48 hours following the administration of ipecac syrup containing tritium (3H)--labeled cephaeline or emetine. Metabolites were separated and quantified by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Biliary and urinary excretion rates of 3H-cephaeline were 57.5% and 16.5% of the dose, respectively. Cephaeline-6'-O-glucuronide was comprised 79.5% of biliary radioactivity and 84.3% of urinary radioactivity. Unchanged cephaeline was detected in 42.4% of the dose in feces. Biliary excretion rate of 3H-emetine was 6.9% of the dose. Emetine, cephaeline and 9-0-demethylemetine comprised 5.8%, 43.2% and 13.6% in hydrolyzed bile, respectively. There were no emetine-derived metabolites in urine or feces. The occurrence of unchanged emetine was 6.8% and 19.7% of the dose in urine and feces, respectively.

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine.

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C18 columns, plasma analysis; 15 cmx4.6 mm I.D., 5 microm particle size, urine analysis; 7.5 cmx4.6 mm I.D., 5 microm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)-methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.

Single dose pharmacokinetics of syrup of ipecac.

Syrup of ipecac (SI) has been used medicinally since the 1500s; however, little is known about the pharmacokinetics in humans of SI's active ingredients, emetine and cephaeline. The objective of this study was to evaluate the rate of absorption and the rate of elimination of emetine and cephaeline. Ten healthy, adult, human volunteers between 18 and 45 years of age who were of ideal body weight (body mass index 20-25) completed this study. After an overnight fast, 30 mL SI were ingested. Blood samples were collected 30, 45, 60, 90, 120, 150, and 180 minutes post-ingestion and urine was collected throughout the study period. Plasma and urine concentrations of emetine and cephaeline were measured by reverse-phase HPLC with fluorescence detection. In virtually all subjects, emetine and cephaeline were detected within 5-10 minutes of dosing with the time to maximum concentration being approximately 20 minutes. The mean areas under the concentration-time curve (AUC) for both emetine and cephaeline were similar; however, the ratio of mean cephaeline maximum concentration (Cmax) to emetine Cmax was approximately 1.5. Four of the ten subjects exhibited a type of concentration-time profile in which the levels of cephaeline were substantially higher than those of emetine and the levels of cephaeline were substantially higher than noted for the other six subjects. In these remaining six subjects, the levels of emetine and cephaeline were lower than 10 ng/mL at all time-points. An initial elimination phase was noted in some subjects but not in others. Individuals in whom an initial elimination phase was not observed also exhibited low levels of both alkaloids as compared with the other subjects suggestive of a slower distribution phase. Less than 0. 15% of the administered emetine and cephaeline was recovered in the urine at 3 hours. No relationship between vomiting episodes and peak concentrations of emetine or cephaeline was found. Administration of SI results in rapid appearance and disappearance of emetine and cephaeline in plasma becoming almost undetectable at 3 hours. Very little of either alkaloid is eliminated in the urine within this time period, suggesting extensive distribution. The length of time that an administered dose of SI can result in the detection of emetine and/or cephaeline in the urine has not been determined; future studies in humans are required.

Susceptibility of an emetine-resistant mutant of Entamoeba histolytica to multiple drugs and to channel blockers.

Previously a cloned emetine-resistant mutant of the protozoal parasite Entamoeba histolytica was shown to overexpress a gene for an ameba homolog of the mammalian P-glycoprotein, a plasma membrane pump that removes hydrophobic drugs from multidrug-resistant tumor cells. Three sets of experiments were performed to better characterize the multidrug-resistant phenotype of the emetine-resistant amebae. First, the emetine resistance of the mutant amebae was reversed by concentrations of calcium and sodium channel blockers effective in reversing drug resistance by multidrug-resistant tumor cells, but it was reversed only in the presence of very high concentrations of the tricyclic antidepressants. Second, the mutant amebae showed cross-resistance to antiamebic drugs used to treat luminal infection (iodoquinol and diloxanide) but were not cross-resistant to drugs used to treat invasive disease (chloroquine and metronidazole). Third, when amebae were loaded with radiolabeled emetine, the mutant parasites released the drug at approximately 1.6 times the rate of the wild-type organisms. We conclude that the emetine-resistant E. histolytica parasites have some but not all the features of the multidrug-resistant phenotype.

Entamoeba histolytica: physiology of multidrug resistance.

Cross-resistance to unrelated drugs has been previously observed in multidrug-resistant carcinoma cells and the goal of this work was to determine whether a similar mechanism existed in Entamoeba histolytica. An emetine and a colchicine-resistant clone, C2(90) (IC50 = 62 microM, and 1.5 mM, respectively), and the parental clone, A (IC50 = 5 microM and 1 mM, respectively), were analyzed for resistance to other drugs and for the effect of verapamil. Both clones, C2(90) and A, exhibited similar resistance to both daunomycin (IC50 = 50 microM) and actinomycin D (IC50 = 13 nM). In the presence of verapamil, the IC50 for emetine was reduced to 0.5 microM, while the IC50 for colchicine was reduced to 0.3 mM. These results demonstrate that verapamil reverses both emetine and colchicine resistance in the mutant C2(90). In uptake experiments with [3H]emetine, drug accumulation was lower in resistant trophozoites. However, in the presence of verapamil, drug accumulation was increased in clone C2(90) to a level close to that of the parental strain, clone A. These results are consistent with observations made using malaria and multidrug-resistant tumor cells and suggest that a P-glycoprotein-like molecule may play a role in drug resistance in E. histolytica.

Development of dehydroemetine nanoparticles for the treatment of visceral leishmaniasis.

The preparation and physico-chemical characterization of lyophilized polyisohexylcyanoacrylate nanoparticles loaded with dehydroemetine (DHE) for the treatment of visceral leishmaniasis disease is described. The resulting formulation was found to efficiently absorb DHE and gave very reproducible preparations with regard to the size and drug adsorption rate. Stability has been confirmed for at least 24 months. The acute toxicity of DHE was reduced in intravenous administration by its association with nanoparticles. Data concerning tissue distribution in mice showed that DHE nanoparticles were rapidly cleared from the blood stream and that they mainly concentrated in the reticuloendothelial system. Furthermore, DHE linkage to the carrier reduced the cardiac concentrations of the drug.