Efficacy of the novel diamidine compound 2,5-Bis(4-amidinophenyl)- furan-bis-O-Methlylamidoxime (Pafuramidine, DB289) against Trypanosoma brucei rhodesiense infection in vervet monkeys after oral administration.

Owing to the lack of oral drugs for human African trypanosomiasis, patients have to be hospitalized for 10 to 30 days to facilitate treatment with parenterally administered medicines. The efficacy of a novel orally administered prodrug, 2,5-bis(4-amidinophenyl)-furan-bis-O-methlylamidoxime (pafuramidine, DB289), was tested in the vervet monkey (Chlorocebus [Cercopithecus] aethiops) model of sleeping sickness. Five groups of three animals each were infected intravenously with 10(4) Trypanosoma brucei rhodesiense KETRI 2537 cells. On the seventh day postinfection (p.i.) in an early-stage infection, animals in groups 1, 2, and 3 were treated orally with pafuramidine at dose rates of 1, 3, or 10 mg/kg of body weight, respectively, for five consecutive days. The animals in groups 4 and 5 were treated with 10 mg/kg for 10 consecutive days starting on the 14th day p.i. (group 4) or on the 28th day p.i. (group 5), when these animals were in the late stage of the disease. In the groups treated in the early stage, 10 mg/kg of pafuramidine completely cured all three monkeys, whereas lower doses of 3 mg/kg and 1 mg/kg cured only one of three and zero of three monkeys, respectively. Treatment of late-stage infections resulted in cure rates of one of three (group 4) and zero of three (group 5) monkeys. These studies demonstrated that pafuramidine was orally active in monkeys with early-stage T. brucei rhodesiense infections at dose rates above 3 mg/kg for 5 days. It was also evident that the drug attained only minimal efficacy against late-stage infections, indicating the limited ability of the molecule to cross the blood-brain barrier. This study has shown that oral diamidines have potential for the treatment of early-stage sleeping sickness.

Estimation of tsetse challenge and its relationship with trypanosomosis incidence in cattle kept under pastoral production systems in Kenya.

In an on-farm trial conducted amongst the Maasai pastoralists in Nkuruman and Nkineji areas of Kenya between April 2004 and August 2005 designed to evaluate the effectiveness of a synthetic tsetse repellent technology, we assessed the relationship between tsetse challenge and trypanosomosis incidence in cattle. Six villages were used in each area. Each of these villages had a sentinel cattle herd that was screened for trypanosomosis on monthly basis using buffy coat technique. Animals found infected at each sampling were treated with diminazene aceturate at 7 mg kg(-1) body weight. Treatments administered by the owners over the sampling intervals were recorded as well. Tsetse flies were trapped at the time of sampling using baited stationary traps and apparent tsetse density estimated as flies per trap per day (FTD). A fixed proportion (10%) of the flies was dissected and their infection status determined through microscopy. Blood meals were also collected from some of the flies and their sources identified using enzyme-linked immunosorbent assay (ELISA). Tsetse challenge was obtained as a product of tsetse density, trypanosome prevalence and the proportion of blood meals obtained from cattle. This variable was transformed using logarithmic function and fitted as an independent factor in a Poisson model that had trypanosomosis incidence in the sentinel cattle as the outcome of interest. The mean trypanosomosis incidence in the sentinel group of cattle was 7.2 and 10.2% in Nkuruman and Nkineji, respectively. Glossina pallidipes was the most prevalent tsetse species in Nkuruman while G. swynnertoni was prevalent in Nkineji. The proportions of tsetse that had mature infections in the respective areas were 0.6 and 4.2%. Most tsetse (28%) sampled in Nkuruman had blood meals from warthogs while most of those sampled in Nkineji (30%) had blood meals from cattle. A statistically significant association between tsetse challenge and trypanosomosis incidence was obtained only in Nkuruman when data was pooled and analyzed at the area but not at the village-level. In the later scenario, lagging tsetse challenge by 1 month improved the strength but not the significance of the association. These findings show that when the spatial unit of analysis in observational studies or on-farm trials is small, for instance a village, it may not be possible to demonstrate a statistically significant association between tsetse challenge and trypanosomosis incidence in livestock so as to effectively control for tsetse challenge.

A competitive enzyme-linked immunosorbent assay for determination of chloramphenicol.

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.

Evaluation of isometamidium levels in the serum of sheep and goats after prophylactic treatment against trypanosomosis.

Isometamidium chloride has been used for the control of trypanosomosis in animals for over 36 years, but recently there have been reports of prophylaxis failure under natural conditions. In this study, use of the drug for prophylactic purpose against trypanosomosis in small ruminants was investigated. Forty-two sheep and 44 goats were divided into four treatment groups. Groups 1 and 2 were treated with isometamidium chloride (Samorin, Rhone Merieux, Lyon, France) at 3-month intervals while groups 3 and 4 were used as controls. All the animals were exposed to natural tsetse challenge and monitored for serum isometamidium levels and anti-trypanosome antibodies. Seven days after drug administration, isometamidium levels were significantly higher in goats 13.7+/-0.07 ng/ml than in sheep 6.2+/-0.06 ng/ml. However, the elimination half-life in the sheep was 14.2+/-0.92 days and was significantly higher (P> 0.05) than that of the goats 12+/-0.5 days. This study established that isometamidium metabolism differs between sheep and goats and this difference may have important implications in high tsetse challenge areas.

Resistance to trypanocidal drugs--suggestions from field survey on drug use in Kwale district, Kenya.

A household survey was conducted in 21 villages of Kwale district, Kenya, to assess farmers' trypanocidal drug use characteristics for treatment of bovine trypanosomosis and their relationship to drug effectiveness. Descriptive statistical tools were used to summarize the farmers' drug use patterns. The chi-square test was the done to establish the relationship between proper drug use and recovery. The results indicate that the farmers had considerable knowledge about trypanocidal drugs with 82% (n = 65) having used these drugs within 6 months preceding the survey. Cases of incorrect drug use were reported. This study established that there was no significant relationship between correct drug use and recovery of the treated animals, suggesting the presence of drug resistance in Kwale district.

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle.

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

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 t1/2 alpha, 7.17 +/- 1.87 h for t1/2 beta and 106.3 +/- 6.6 h for t1/2 gamma 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.

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 investigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies.

Anti-trypanosomal effects of some compounds isolated from the extracts of Warburgia ugandensis.

The plant kingdom has been used as a source of compounds employed in the treatment of many disease conditions for many years. Even with the new technology in synthetic chemistry, plants are still being used as a source of lead compounds in drug development. In the treatment of trypanosomiasis, the drugs that are currently in the market were developed between 1950-1960's. These drugs are expensive and associated with a number of toxic effects, therefore there is still need to develop newer drugs in the management of trypanosomiasis. The plant Warburgia ugandansis is a common plant that has been used traditionally to treat many disease conditions. The crude and pure compounds from this plant were tested against trypanosomes: T. congolense, T. evansi and T. bruceL In vitro tests using tissue culture method and in vivo tests using mice were carried out The results of the in vitro method indicated that the pure compound was more active than the crude extract The in vivo method indicated that the total extract was not effective, while one of the pure compounds was too toxic, and the other one showed activity. The two compounds investigated were basically of the same structure type with a slight difference on the functional groups. These preliminary results indicate that there is a possibility of finding active compounds against Trypanosomes in plants.

Pharmacokinetics, bioavailability and tissue residues of [14C]isometamidium in non-infected and Trypanosoma congolense-infected Boran cattle.

In this paper, pharmacokinetics, bioavailability and tissue residues are reported in non-infected and Trypanosoma congolense-infected Boran steers following either intravenous or intramuscular injection of [14C]isometamidium at a dose rate of 1 mg kg-1 body weight. Two differently labelled compounds of isometamidium were used; 6-14C (ISMM-1) and ring-U-14C (ISMM-2). The cattle were divided into 5 groups: group 1 consisted of 3 non-infected cattle treated with ISMM-1 by intravenous injection; group 2 consisted of 2 non-infected cattle treated with ISMM-1 by intramuscular injection; group 3 consisted of 2 Trypanosoma congolense-infected cattle given similar treatment as group 2 cattle; group 4 consisted of 3 non-infected and group 5 of 2 infected cattle treated with ISMM-2 by intramuscular injection. Radioactivity was measured in plasma, urine, faeces and tissues, and drug concentrations were calculated. Data obtained following i.v. treatment were best described by tri-exponential equations with half-lives of 0.13, 1.22 and 120.7 h. Bioavailability of the intramuscular dose was 58% in group 2 cattle. The major route of excretion was in faeces. Approximately 80% of the intravenous dose given was excreted within 21 days out of which only 18% was through urine. Total residues accounted for approximately 15% the total dose given. Drug residues remained high in organs with excretory functions including the liver and kidneys.

Bioavailability, pharmacokinetics, and tissue distribution of 14C homidium after parenteral administration to Boran cattle.

The absorption, distribution and elimination characteristics of 14C homidium have been described in non-infected and Trypanosoma congolense-infected cattle treated with 14C homidium chloride by either intramuscular (i.m.) or intravenous (i.v.) injection at a dose level of 1 mg/kg body weight. Results show that the mean (+/-SD) elimination of the drug from plasma followed a biexponential process, with half-lives of 0.084 +/- 0.006 h and 97.66 +/- 16.28 h for the distribution and elimination phases after intravenous injection, respectively. Bioavailability of the intramuscular dose was 62.5% and 57.8% in non-infected and trypanosome-infected cattle, respectively. Absorption was rapid, with a tmax of 15 min and a mean Cmax (+/-SD) of 268.4 +/- 4.09 ng/mL following the intramuscular dose in non-infected cattle. The major route of excretion was via faeces. Approximately 90% of the total dose given to non-infected i.m.-treated cattle was excreted within 14 days. Following intramuscular administration of the drug, residues remained high in the major excretory organs, with the liver having concentrations of 1411 and 1199 ng/g after 14 and 28 days, respectively. Over the same period, the values in the kidneys were 649 and 448 ng/g. Concentrations in the liver 14 and 21 days following i.v. treatment were 2195 and 2454 ng/g, respectively. These results show that there was no significant difference in liver drug residues between 14 and 21 days, or 28 days depending on the treatment given, suggesting that once the drug is in this organ, it is released back into the circulation at an extremely slow rate.

Disposition of diminazene aceturate (Berenil) in trypanosome-infected pregnant and lactating cows.

Three cows were repeatedly infected with different strains of Trypanosoma congolense and treated intramuscularly each time with a different dose of diminazene aceturate (Berenil). Biphasic decline was observed of the maximal plasma drug levels, which were attained at 15 min after the first treatment and at 30 min after the second and third treatments. The rate constants for the distribution and terminal phases depended on the period of exposure to parasitaemia of the animal at the time of treatment. Maximal diminazene aceturate residue levels were found in milk 8 h post treatment and declined biexponentially to 4.56 ng ml-1 and 8.76 ng ml-1 at 21 days post treatment after 3.5 mg kg-1 and 7.0 mg kg-1 doses, respectively. In the three cows, higher drug residues were found in the kidney (7.04, 3.92 and 7.99 micrograms g-1) than in liver (3.26, 2.87 and 1.24 micrograms g-1) and heart (1.79, 1.25 and 1.03 micrograms g-1). The results of this study indicate that the level of parasitaemia (degree of anaemia) in the animal at the time of treatment affects the distribution, disposition and elimination of diminazene aceturate in the animal. Furthermore, the residue level in milk after treatment depends on the treatment dose and could easily be bioavailable to the consumer.

Retention reproducibility of thiazide diuretics and related drugs in reversed-phase high-performance liquid chromatography.

A method has been developed for the separation of thiazide diuretics and a number of related drugs by high-performance liquid chromatography on an ODS-Hypersil column with acetonitrile-1% aqueous acetic acid as the eluent. The effects caused by changes in the separation conditions on the reproducibility and robustness of alternative methods for recording retentions (including capacity factors, retention indices based on the alkyl aryl ketone scale, and relative capacity factors compared to a thiazide standard) have been examined. The results confirm that good interlaboratory reproducibility will only be achieved when operators control the temperature of the column and use the same brand of column packing material. The retentions should be recorded using a relative method, as these were found to be virtually independent of minor variations in the eluent composition.