Sleeping sickness and the brain.

Recent progress in understanding the neuropathological mechanisms of sleeping sickness reveals a complex relationship between the trypanosome parasite that causes this disease and the host nervous system. The pathology of late-stage sleeping sickness, in which the central nervous system is involved, is complicated and is associated with disturbances in the circadian rhythm of sleep. The blood-brain barrier, which separates circulating blood from the central nervous system, regulates the flow of materials to and from the brain. During the course of disease, the integrity of the blood-brain barrier is compromised. Dysfunction of the nervous system may be exacerbated by factors of trypanosomal origin or by host responses to parasites. Microscopic examination of cerebrospinal fluid remains the best way to confirm late-stage sleeping sickness, but this necessitates a risky lumbar puncture. Most drugs, including many trypanocides, do not cross the blood-brain barrier efficiently. Improved diagnostic and therapeutic approaches are thus urgently required. The latter might benefit from approaches which manipulate the blood-brain barrier to enhance permeability or to limit drug efflux. This review summarizes our current understanding of the neurological aspects of sleeping sickness, and envisages new research into blood-brain barrier models that are necessary to understand the interactions between trypanosomes and drugs active against them within the host nervous system.

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.

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.

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.

[Human African trypanosomiasis, contributions of experimental models]. / La trypanosomose humaine africaine, apport des modèles expérimentaux.

Melarsoprol has remained the chosen drug for the late-stage treatment of human African trypanosomiasis (HAT) due both to Trypanosoma brucei (T.b.) gambiense and T.b. rhodesiense; however, arsenical encephalopathies, which are often fatal, occur in 5-10% of the treated cases. To date, two major problems have not been solved. The first one is the precise diagnosis of early involvement of the central nervous system (CNS) which determines the therapeutics to be administered. The second one is linked to the lack of data on in vivo efficacy of products which are effective in vitro against trypanosomes. Answers have to be provided by experimental animal models of HAT. Such models would allow for better studies of the pathology and pathogenesis of the disease, as well as therapeutic trials of potentially effective new drugs or combinations. We have developed acute and chronic murine and sheep experimental animal models of HAT infected by T. b. brucei. Meningoencephalitis and neurological signs are relatively difficult to obtain in murine models and require artificial means, such as suramin treatment on day 21 after-infection. The chronic murine model has demonstrated CNS involvement with meningitis, followed by meningoencephalitis with progressive astrocytosis. The sheep model develops a disease with CNS complications and cerebrospinal fluid can be collected. In the sheep model, we have described anti-galactocerebrosides antibodies, which represent major components of myelin, which may indicate an autoimmune process in the CNS. We then described these antibodies in the cerebrospinal fluids and sera from patients at a late-stage of the disease. From a therapeutic point of view, we have cured mice or sheep with low doses of melarsoprol, or with the nitroimidazole derivatives Ro 15-0216 and megazol, alone or combined with suramin. Further studies of these nitroimidazole compounds, which could be proposed for human use, have to be carried out on a-primate model infected by T.b. gambiense. To our knowledge, this primate model is not available. This is why we have recently developed a T. b. gambiense primate model of HAT on Cercopithecus aethiops.

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.

Trypanosoma brucei brucei: a long-term model of human African trypanosomiasis in mice, meningo-encephalitis, astrocytosis, and neurological disorders.

The search for a chronic experimental model for human African trypanosomiasis (HAT) in animals with cerebral lesions and neurological disorders has been difficult. Models with meningo-encephalitis have been proposed using Trypanosoma brucei gambiense or T. b. rhodesiense. Meningo-encephalitis is rare in infection with T. b. brucei. It has been shown that the treatment of mice infected with T. b. brucei with diminazene aceturate (Berenyl) led to development of a rapid meningo-encephalitis. In this study, we report the development of a chronic experimental model of HAT in mice infected with T. b. brucei AnTat 1.1E. To obtain a chronic evolution of the infection, on Day 21 postinfection, mice were treated with a dose of suramin (Moranyl) at 20 mg x kg(-1) body weight, a dose which failed to eliminate trypanosomes in the central nervous system (CNS). This treatment, repeated after each parasitemic relapse in the blood, allowed animals to survive more than 300 days postinfection. After a few weeks of infection, mice displayed neurological signs. Histological studies showed the appearance of increasing inflammatory lesions, from meningitis to meningo-encephalitis, with progression of lesions throughout the perivascular spaces in cerebral and cerebellum parenchyma. No demyelination or neuronal alteration were observed except in the necrotic spaces. Trypanosomes were observed in different structures in CNS. An immunohistochemical study of glial fibrillary acidic protein (GFAP) showed an increasing astrocytosis according to the duration of the infection. This model reproduces neurological and histological pathology observed in the human disease and can be useful for further immunopathological, neurohistological and therapeutic studies on this condition.

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.

Evidence for adenylyl cyclase-dependent receptors for parathyroid hormone (PTH)-related protein in rabbit kidney glomeruli.

Studies were conducted to test whether parathyroid hormone-related protein (PTHrP) is able to stimulate adenylate cyclase activity in isolated rabbit glomeruli. Maximal stimulations were reached at 10(-7) M of human PTHrP-(1-34) or rat PTH-(1-34) and showed a 3-3.3 fold increase over basal activity. The potency (EC50) values were close to 10(-9) M. The guanyl nucleotide GTP, at 10(-5) M, potentiated the effect of PTH and PTHrP but reduced their potency. The combined effect of maximal concentrations of PTHrP and PTH was not additive, and the PTH antagonist [Nle8.18, Tyr34]-bPTH-(3-34)amide inhibited both PTHrP- and PTH-stimulated adenylate cyclase activities. These findings suggest that PTHrP could affect glomerular function through changes in glomerular cAMP content by interaction with PTH receptors.