New antitrypanosomal tetranotriterpenoids from Azadirachta Indica

Organic extracts of the leaves of Azadirachta indica A. Juss. yielded ten antitrypanosomal terpenoids. Three of these (1 – 3), are novel and are derivatives of nimbolide and nimbin. They were extracted from chloroformfraction of methanol extract. These compounds were found to exhibit strong antitrypanosomal activities against Trypanosoma brucei rhodesiense with MIC values ranging of 6.9, 15.6 and 7.8 μg/ml respectively and were more active than Cymerlarsan ( a standard drug), which had an MIC value of 187.5 μg/ml when tested against T. b. rhodesiense The structures were elucidated by spectroscopic methods including; NMR, MS, UV and IR.Key words: Meliaceae, limonoids, Trypanosoma brucei rhodesiense, Azadirachta indica, antitrypanosomal activity

Pharmacology of DB844, an orally active aza analogue of pafuramidine, in a monkey model of second stage human African trypanosomiasis

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-yl]-nicotinamide (DB820), exhibiting plasma C(max) values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible

KENYAN PURPLE TEA ANTHOCYANINS ABILITY TO CROSS THE BLOOD BRAIN BARRIER REINFORCING BRAIN ANTIOXIDANT CAPACITY IN MICE

Studies on antioxidants as neuroprotective agents have been hampered by the impermeability of the blood brain barrier (BBB) to many compounds. However, previous studies have shown that a group of tea ( Camellia sinensis ) flavonoids, the catechins, are brain permeable and neuroprotective. Despite this remarkable observation, there exists no data on the bioavailability and pharmacological benefits of tea anthocyanins (ACNs) in the brain tissue. This study investigated the ability of Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity. Mice were orally administered with purified and characterised Kenyan purple tea ACNs or a combination of Kenyan purple tea ACN\u2019s and coenzyme-Q10, at a dose of 200 mg kg-1 body weight in an experiment that lasted for 15 days. Twenty four hours post the last dosage of antioxidants, CO2 was used to euthenise the mice. Then the brain was excised and used for various biochemical analyses. Kenyan purple tea ACNs significantly (P<0.05) raised brain Glutathione (GSH) levels, implying a boost in brain antioxidant capacity. Notably, ACN metabolites were detected in brain tissue of ACN fed mice. This is the first demonstration that Kenyan purple tea ACNs can cross the BBB, reinforcing the brain\u2019s antioxidant capacity. Hence, there is need to study ACNs as suitable candidates for dietary supplements that could support antioxidant capacity in the brain and have potential to provide neuroprotection in neurodegenerative conditions.Les \ue9tudes sur les antioxydants comme agents neuroprotecteurs ont \ue9t\ue9 handicap\ue9es par l\u2019imperm\ue9abilit\ue9 de la barri\ue8re de sang du cerveau (BBB) \ue0 plusieurs produits. Par ailleurs, les \ue9tudes ant\ue9rieurs ont montr\ue9 qu\u2019un groupe de flavono\uefdes du th\ue9 ( Camellia sinensis ) , les cat\ue9chines, permettent la perm\ue9abilit\ue9 du cerveau et prot\ue8gent les nerfs. Malgr\ue9 cette observation importante, il n\u2019ya pas de donn\ue9es sur la biodisponibilit\ue9 et les vertus pharmacologiques des anthocyanines du th\ue9 (ACNs) dans le tissue du cerveau. Cette \ue9tude a \ue9valu\ue9 l\u2019aptitude des anthocyanines du th\ue9 pourpre du Kenya de traverser le BBB et am\ue9liorer la capacit\ue9 antioxydante du cerveau. Des rats \ue9taient oralement administr\ue9s des anthocianines du th\ue9 pourpre purifi\ue9s et caract\ue9ris\ue9s ou une combinaison des ACNs avec le coenzyme-Q10, \ue0 une dose de 200 mg kg-1 de poids vif dans un essai d\u2019une dur\ue9e de 15 jours. Vingt quatre heures apr\ue8s le dernier dosage d\u2019antioxydants, le CO2 \ue9tait utilis\ue9 pour euthanasier les rats. Ensuite, le cerveau \ue9tait excis\ue9 et soumis \ue0 diverses analyses biochimiques. Les ACNs ont augment\ue9 significativement (P<0.05) les niveaux du Glutathione (GSH) du cerveau,,ce qui implique une am\ue9lioration de la capacit\ue9 antioxydante du cerveau. Remarquablement, les m\ue9tabolites ACN \ue9taient d\ue9tect\ue9s dans le tissu des rats nourris aux ACNs. Ceci est la premi\ue8re d\ue9monstration que les anthocyanines (ACNs) du th\ue9 pourpre Kenyan peuvent franchir la barri\ue8re h\ue9mato-enc\ue9phalique (BHE), renfor\ue7ant ainsi la capacit\ue9 antioxydante du cerveau. Ainsi, le besoin s\u2019impose de les \ue9tudier comme des candidats appropri\ue9s pour les aliments de suppl\ue9ment qui pourraient renforcer la capacit\ue9 antioxydante dans le cerveau et avoir le potentiel d\u2019offrir la neuroprotection dans les conditions neurod\ue9g\ue9n\ue9ratrices

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense

Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.IS

Reduced Mitochondrial Membrane Potential is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo- ATPase

Background: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. Methodology/Principal Findings: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. Conclusions/Significance: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle

Two enzyme-linked immunosorbent assays (ELISA) for the determination of homidium in serum of treated cattle have been developed and evaluated. One is a direct competition (Assay 1) and the other an indirect competition assay (Assay 2). Both assays are highly sensitive with a limit of detection of 0.1 ng homidium per mL serum. Homidium levels were measurable in serum of cattle for over 2 months following administration of a single intramuscular (i.m.) dose at 1 mg/kg bodyweight. The level of sensitivity afforded by these assays makes them potentially useful tools in the pharmacokinetic evaluation of homidium and for invetigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies

Investigation into the effects of Trypanosoma congolense infections on the pharmacokinetics of Homidium in Boran cattle

Control of trypanosomosis in cattle, sheep and goats in endemic areas has depended largely on the use of chemotherapeutic or chemoprophylactic agents. One such agent is homidium. The normal use of homidium has been in the treatment of infections due to both Trypanosoma congolense and T. vivax in cattle, sheep and goats at the recommended dose rate of 1.0 mg kg -1 b.w. This paper describes the results of an investigation into the chemotherapeutic activity of homidium against t. congolense infections in cattle. Two groups of five Boran cattle were infected with two populations of T. congolense; one drug-sensitive IL 1180) and one drug-resistant (IL 3330). Parasitaemia was estimated using the methods of Murray et al. (1977); the serum drug levels by the method described by Murilla (1996) and the pharmacokinetic parameters using the formulae described by Baggot (1977). After infection, there was a rapid drop in packed cell volume (PCV) values from a mean pre-infection value of approximately 40 percent to approximately 25 percent within 14 days of infection in cattle infected with IL 1180. However, in cattle infected with IL 3330, the drop in PCV was more gradual from approximately 40 percent to approximately 30 percent within the same period of time. The animals were treated with homidium bromide at a dose rate of 1.0 mg kg -1 body weight (b.w.) seven days after the last animal in each group was detected positive. Following intramuscular (i.m.) treatment of cattle infected with drug-sensitive trypanosomes, no parasites were detected in the bloodstream of four out of five cattle within 24 hours; the fifth within 48 hours. During this period and for the next 10 days after treatment, an acceleration in the rate of drug elimination was observed. Thereafter, the rate of elimination reverted back to that observed in non-infected cattle (Murilla, 1996). This was accompanied by an elevation in PCV to pre-infection values. The animals remained aparasitaemic up to the end of the 90 days observation period with low serum drug concentrations of between 0.1 and 0.3 ng ml-1 in circulation

Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA)

Pharmacokinetic studies on the trypanocidal drug homidium bromide using a competitive enzyme immunoassay (detection limit 0.1 ng/mL) are reported for non-infected Friesian and Boran steers following treatment with homidium bromide at a dose of 1.0 mg/kg b.w. Following intravenous (i.v.) treatment of Friesian steers (n = 5), the mean serum drug concentrations were 31.9 ± 2.1 and 3.9 ± 0.4 ng/ml at 1 and 24 h, respectively. The decline in serum drug concentration was tri-exponential with half-lives of 0.064 ± 0.037 h for t( 1/2 ?), 7.17 ± 1.87 h for T( 1/2 ?) and 106.3 ± 6.6 h for t( 1/2 ?) for distribution and elimination phases 1 and 2, respectively. Drug was detectable in serum for 17 days following treatment. The mean residence time (MRT) was 63.4 ± 7.5 h. Following intramuscular (i.m.) treatment of Friesian steers (n = 5), the drug concentration at 1 h after treatment was 72.5 ± 2.2 ng/mL. This declined to 9.8 ± 1.8 ng/mL at 24 h. Low concentrations of between 0.1 and 0.3 ng/mL remained in circulation for up to 90 days post-treatment. Following intramuscular treatment of Boran steers (n = 5), the mean serum drug concentration at 1 h after treatment was 112.1 ± 40.3 ng/mL. By 24 h after treatment, the concentration had fallen to 13.0 ± 3.3 ng/mL. Thereafter, the serum drug concentration-versus-time profile and the pharmacokinetic parameters obtained following non-compartmental analysis were similar to those obtained following intramuscular treatment of Friesian steers