Pharmacokinetics, metabolism and excretion of megazol in a Trypanosoma brucei gambiense primate model of human African trypanosomiasis. Preliminary study.

The pharmacokinetics of megazol (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazol, CAS 19622-55-0) was investigated after a 100 mg/kg oral administration to six primates infected with Trypanosoma brucei gambiense. The plasma levels of megazol were between 0.2 microgram/ml and 46 micrograms/ml 24 h after dosing in all animals. In animals with prolonged infection, megazol absorption was accelerated (Tmax was 4 h compared with 8 h, for day 53 and day 39 post inoculation) but the amount absorbed was not modified. The megazol concentrations in the cerebrospinal fluid represented between 5.5% and 10.6% of the plasma levels at the same times. Unchanged megazol was eliminated predominantly via the kidneys: 46-96% of the ingested dose was recovered in the urine, compared with 0-5% in the faeces. Furthermore, this urinary elimination of megazol was altered in animals with prolonged infections. In the urine, 4 unknown metabolites were observed, unchanged megazol was characterized by LC-MS/MS. This study indicates that megazol crosses the blood-brain barrier after oral administration. Prolonged infections affect the absorption of megazol and its urinary elimination.

Uptake of the nitroimidazole drug megazol by African trypanosomes.

Megazol, CL 64,855 (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]-1,3, 4-thiazole) has been shown to be extremely effective in clearing experimental infections of African trypanosomes. An unusual amino-purine transporter termed P2, implicated in the transport of both the diamidine and melaminophenyl arsenical classes of drug in Trypanosoma brucei, recognised chemical groups on compounds which are also present on megazol. Megazol interacted with this carrier protein, as judged by its ability to inhibit P2 adenosine transport and to abrogate in vitro arsenical-induced lysis in a dose-dependent manner. However, parasites resistant to melaminophenyl arsenical and diamidine drugs due to lack of the P2 transporter showed no resistance to megazol. This is because passive diffusion represented the major route of entry. Initial rates of uptake were not saturable within the limit of megazol's solubility and did not conform to thermodynamic precepts compatible with carrier-mediated uptake. Adenosine and other P2 transporter substrates, even at high concentration, had little impact on megazol uptake. Uptake was biphasic, with a very rapid equilibration across the membrane followed by a slower accumulation over time. The equilibration phase represented a simple passive diffusion, with the subsequent uptake probably being due to metabolism of the drug.

Pharmacokinetics, metabolism and excretion of megazol, a new potent trypanocidal drug in animals.

The pharmacokinetics of megazol (CAS 19622-55-0) was investigated after intraperitoneal and oral administration of the drug (80 mg/kg) to mice. The plasma levels were significantly higher after oral administration of drug than after intraperitoneal route (33.8 micrograms/ml compared with 19.0 micrograms/ml for Cmax, 158714 micrograms.h/l compared with 96057 micrograms.h/l for AUC). When suramin (CAS 145-63-1) was administered 24 h before oral administration of megazol, megazol absorption was accelerated (2 h compared with 4 h for Tmax) but the amount absorbed was lower (19.9 micrograms/ml compared with 33.8 micrograms/ml for Cmax and 95547 micrograms.h/l vs 158714 micrograms.h/l for AUC). In the infected mice previously treated with suramin, all estimated pharmacokinetic parameters of plasma megazol were significantly modified, in particularly an increase in the apparent volume of distribution (5.6 l/kg compared with 0.9 l/kg) with a prolongation of the elimination half-life (3 h compared with 0.7 h) of megazol. Excretion of the total radioactivity of megazol was also evaluated after oral administration of 3H-megazol to rats. Total radioactivity was eliminated predominantly via the urinary route (80%) vs. 10.5% in the faeces, 9.5% remaining in the body 8 days after dosing. When unlabelled megazol was orally administered to rats with absence or presence of suramin, megazol recovered in urine and faeces 72 h dosing was: 55.7%/2% vs 20.6%/1.6%, respectively. In the urine, unchanged megazol was present as characterized by LC-MS/MS as well as 4 unknown metabolites. This study indicates that suramin significantly affects the pharmacokinetics of megazol and its elimination.

Enzymatic reduction studies of nitroheterocycles.

The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.

Megazol combined with suramin: a chemotherapy regimen which reversed the CNS pathology in a model of human African trypanosomiasis in mice.

Chemotherapy for human African trypanosomiasis (HAT), or sleeping sickness, is unreliable because of resistance, refraction and toxic and adverse side-effects. Using a long-term experimental model of HAT with involvement of the central nervous system (CNS), we tested the ability of a megazol and suramin combination treatment to eliminate CNS trypanosomes. This consisted of 20 mg suramin per kg body weight administered intraperitoneally (i.p.), followed 24 h later by 4 daily doses (80 mg/kg) of megazol given either i.p. or per os. One week post-treatment, neurological disorders had disappeared. One of 15 mice relapsed in each application group at 81 and 98 days after treatment, respectively. At six months, no signs of relapse were seen in remaining mice, indicating that this chemotherapy regimen was curative. Immunohistochemical (astrocytosis) and histological (inflammatory lesions) examinations of brain tissues showed that animals returned to normal from 2 months post-treatment. These results suggest that the megazol-suramin combination reversed the CNS pathology in this model.

Simple high-performance liquid chromatographic method to analyse megazol in human and rat plasma.

A simple and sensitive high-performance liquid chromatographic method has been developed to measure megazol in human plasma. The method was optimized and validated according to the Washington Concensus Conference on the Validation of Analytical Methods (V.P. Shah et al., Eur. J. Drug Metab. Pharmacokinet., 15 (1991) 249). The criteria of complete validation were specificity, linearity, precision, analytical recovery, dilution and stability. It involved extraction of the plasma with dichloromethane, followed by reversed-phase high-performance liquid chromatography using a Kromasil C8 column and UV detection at 360 nm. The retention times of the internal standard (tinidazol) and megazol were 6.10 and 9.60 min, respectively. The standard curve was linear from 2 ng ml-1 (limit of quantification) to 2000 ng ml-1. The coefficients of variation for all the criteria of validation were less than 6%; 85 to 92% extraction efficiencies were obtained. Megazol was stable during the storage period (one month at -20 degrees C) in plasma and for two months at 25 degrees C in standard solution. The method was tested by measuring the plasma concentration following oral administration to rat and was shown to be suitable for pharmacokinetic studies.

Topical chemotherapy for experimental African trypanosomiasis with cerebral involvement: the use of melarsoprol combined with the 5-nitroimidazole, megazol.

Megazol, one of a number of related 5-nitroimidazoles, can be dissolved in dimethylsulphoxide and the solution can be converted into a gel by the addition of hydroxypropylcellulose which facilitates the ease and accuracy of administration. This megazol gel, when used in combination with melarsoprol (3.6%) in propylene glycol gel, will cure experimental CNS-trypanosomiasis in mice. A single application of 0.1 ml of melarsoprol (3.6%) gel plus 0.1 ml of either 8 or 16 mg/ml megazol gel successfully treated experimental CNS-trypanosomiasis while two consecutive days' treatment with 0.05 ml melarsoprol and 0.1 ml of 16 or 32 mg/ml megazol gels also produced satisfactory cures.

Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice.

Human African trypanosomiasis (HAT) or sleeping sickness is a major public health problem in 36 sub-Saharan African countries and is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. About 25,000 new cases of the disease are reported annually, and around 50 million people are classed as at risk of contracting the disease. Until now; the only effective drug available for treatment of advanced HAT was the trypanocide melarsoprol. The mortality rate of melarsoprol treated patients is 1-5%. Megazol is a nitroimidazole derivative shown to be effective in vitro against T. b. brucei with an EC50 of 0.01 micrograms.ml-1. When this compound was tested for its in vivo activity in T. b. brucei infected Swiss mice, it was shown to cure the acute disease. However, megazol alone did not cause cure of mice carrying a subacute infection with involvement of the central nervous system (CNS). Combined suramin and megazol treatment did prove effective and the mice were shown to have remission without further relapse from the CNS. The study of three megazol derivatives is also described here. Substitution of a bromine, methyl or trifluoromethyl moiety at the 4 position of the imidazole ring abolished trypanocidal activity both in vivo and in vitro. Intermediates of megazol synthesis (imidazole sulfoxide and imidazole sulfone) were also tested, but were shown not to be active. It is thought that megazol trypanocidal effect may be due to the triggering of radical production by the compound, which have toxic effects on the trypanosomes metabolism. In depth study of megazol is needed to fully elucidate its pharmacokinetics and to precisely pin down its mode of action.

[Rational concepts and the study of active molecules against various trypanosomiases]. / Conception rationnelle et étude de molécules actives contre les différentes trypanosomiases.

Several sets of compounds, active against different trypanosomes, Trypanosoma brucei and Trypanosoma cruzi are presented, the lethal doses for some of them being less than the micro-molar concentration. These compounds are designed by taking advantage of two metabolic features of these parasites, glucose metabolism and oxidative stress.

[An efficacy trial on Trypanosoma brucei brucei of molecules permeating the blood-brain barrier and of megazol]. / Essais d'efficacité sur Trypanosoma brucei brucei de molécules franchissant la barrière hématoméningée et du mégazol.

Human African trypanosomiasis (HAT) is a major public health problem in 36 sub-Saharan African countries and around 50 million people are classed as "at risk". About 25,000 new cases of the disease are reported annually by the World Health Organisation (WHO). This disease is fatal if untreated. As for now, chemotherapy is unsatisfactory and relies on a few drugs which show two major problems. The first is pharmacokinetics involving the passage through the blood-brain barrier. The second concerns toxicity and adverse side-effects of drugs used to treat this disease. New trypanocides should be safe, effective without toxicity. This study reports the action of 45 drugs, known to pass through the blood-brain barrier and belonging to different therapeutic classes, and also the megazol, a nitrothiadiazole derivative, on Trypanosoma brucei brucei AnTat 1-9 in vitro in acellular semi-defined medium. Results showed that some drugs did not modify the parasitic growth, and others were either trypanostatic or trypanocide. These last drugs were tested in vivo on T. b. brucei An-Tat 1-9 infected Swiss mice. Only megazol was shown to be effective and trypanocide. This compound might trigger the production of oxygen derivatives and free radicals-which have toxic effects on the trypanosome metabolism.

Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells.

Heat-killed L. acidophilus, strain LB, was tested for its ability to adhere in vitro onto human enterocyte-like Caco-2 and muco-secreting HT29-MTX cells in culture. The heat-killed LB bacteria exhibited a high adhesive property. A diffuse pattern of adhesion was observed to the undifferentiated cells, the apical brush border of the enterocytic cells, and to the mucus layer that covered the surface of the mucus-secreting cells. The inhibitory effect of heat-killed LB organisms against the human intestinal Caco-2 cell-adhesion and cell-invasion by a large variety of diarrhoeagenic bacteria was investigated. The following dose-dependent inhibitions were obtained: (i) against the cell-association of enterotoxigenic, diffusely-adhering and enteropathogenic Escherichia coli, Listeria monocytogenes, Yersinia pseudotuberculosis, and Salmonella typhimurium; (ii) against the cell-invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, Listeria monocytogenes and Salmonella typhimurium.

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion.

Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.

Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells.

Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.

Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture.

To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used. Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation. We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells. The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA. No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells. However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity. This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present. ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed. Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture. This indicated that the expression of the ETEC CFA receptors was a growth-related event. Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.

Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus.

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.

Use of purified F1845 fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture.

Whole diffusely adhering Escherichia coli (DAEC) C1845 cells bearing the F1845 adhesive factor bind diffusely to differentiated human colon carcinoma cell lines HT-29 and Caco-2. By using antibodies directed against the purified fimbrial adhesin F1845 factor, the expression of the DAEC F1845-specific brush border receptors in the polarized human intestinal HT-29 and Caco-2 epithelial cells was studied by indirect immunofluorescence. A low level of DAEC F1845 receptors in undifferentiated intestinal cells was detected; they were localized in a cluster of cells. DAEC F1845 receptors were expressed at a high level in differentiated HT-29 and Caco-2 cells. DAEC F1845 receptors were expressed at a strikingly high level in the apical domains of the cells and developed during enterocytic differentiation in culture, in parallel with the apical expression of the intestinal brush border hydrolase, sucrase-isomaltase.

Adhesion of enterotoxigenic Escherichia coli to the human colon carcinoma cell line Caco-2 in culture.

Enterotoxigenic Escherichia coli (ETEC) strains possessing colonization factor antigen I (CFA/I), CFA/II, CFA/III, and antigen 2230 were tested for their ability to adhere to the following cell lines: HeLa, HEp-2, HRT 18, Hutu 80, MDBK, MDCK, Vero, and Caco-2. ETEC strains adhered only to the Caco-2 cell line. Irrespective of the known adhesive factors, the ETEC strains that adhered to the brush border of human enterocytes also adhered to the Caco-2 cell line. The negative variants, which were cured of the plasmid encoding the adhesive factor, did not adhere. Adhesion of ETEC strains no longer occurred when the Caco-2 cells were pretreated with the homologous colonization factor antigen or when the bacterial cells were pretreated with homologous antibodies raised against the adhesive factors. This indicates that this adhesion is specific and that a different receptor exists for each type of adhesion factor. Electron micrographs of cross sections of the monolayer showed that the adhesion of ETEC strains to the brush border microvilli does not induce any lesion. Therefore, the Caco-2 cell line behaves in the same way as human enterocytes do.

Iridoids from Scaevola racemigera1.

Five iridoids have been isolated from the aerial parts of SCAEVOLA RACEMIGERA Däniker, namely, loganin, loganic acid, sylvestroside III, cantleyoside, and scaevoloside. This latter is a novel compound whose structure 1 has been elucidated on the basis of its spectral data, mainly (1)H- and (13)C-NMR.