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The effects of drug-sensitive and drug-resistant Trypanosoma congolense infections on the pharmacokinetics of homidium in Boran cattle
Acta Tropica 81 (2002) 185– 195 www.parasitology-online.com
The effects of drug-sensitive and drug-resistant Trypanosoma congolense infections on the pharmacokinetics of homidium in Boran cattle Grace A. Murilla a,*, Andrew S. Peregrine b,1, Joseph M. Ndung’u a, Peter H. Holmes c, Mark C. Eisler c a
Kenya Trypanosomiasis Research Institute (KETRI), P.O. Box 362, Kikuyu, Kenya International Li6estock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya c Uni6ersity of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK
b
Received 5 July 2001; received in revised form 10 September 2001; accepted 23 October 2001
Abstract Two groups of five Boran (Bos indicus) cattle were infected with one of two populations of Trypanosoma congolense; one drug-sensitive (IL1180), and one drug-resistant (IL3330). The animals were then treated intramuscularly with homidium bromide at a dose rate of 1.0 mg kg − 1 bodyweight 7 days after trypanosomes were detected in the peripheral blood of all the five animals in each group. Following treatment of cattle infected with drug-sensitive trypanosomes, parasites could no longer be detected in the bloodstream of four out of five cattle after 24 h, and after 48 h for the fifth animal. The animals remained aparasitaemic up to the end of the observation period of 90 days and serum drug concentrations determined by enzyme-linked immunosorbent assay (ELISA) remained above the detection limit of 0.1 ng ml − 1 for the entire period. Following treatment of cattle infected with drug-resistant trypanosomes, parasites did not disappear from the bloodstream in any of the five animals. The rate of drug elimination was greater in cattle infected with drug-resistant trypanosomes and the drug was no longer detectable approximately 3 weeks after treatment. Non-compartmental pharmacokinetic analysis showed that the values for t1i 2 of 75.5 916.9 h, the area under the curve (AUC0 − ) of 1.3390.156 mg h ml − 1 and the MRT0 − of 32.8 9 4.45 h obtained in cattle infected with the drug-resistant trypanosome population were significantly lower than the values of 424 9146 h for t1i, 1.67 90.233 mg h ml − 1 for AUC0 − and 297 9 159 h for MRT0 − obtained in cattle infected 2 with the drug-sensitive population. The persistence of drug-resistant infections in cattle following homidium treatment was associated with more rapid drug elimination than in those in which infections with drug-sensitive parasites were cleared by the drug. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Homidium; ELISA; Drug resistance; Pharmacokinetics; Trypanosoma congolense
* Corresponding author. Tel.: + 254-154-32960/4; fax: + 254-154-32397. E-mail address: [email protected] (G.A. Murilla). 1 Present address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont., Canada N1G 2W1. 0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 1 ) 0 0 2 0 9 - 1
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1. Introduction 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 which is generally classified mainly as a chemotherapeutic drug but has been reported to offer some prophylactic activity against trypanosome infections in the field (Mwambu, 1971; Dolan et al., 1990; Stevenson et al., 1995). Following its introduction, several studies were carried out to determine the minimum curative and maximum tolerated doses (Wilde and Robson, 1953; Wilson and Fairclough, 1953; Unsworth, 1954a,b). Homidium is usually used in the treatment of infections due to either Trypanosoma congolense or Trypanosoma 6i6ax in cattle, sheep and goats at the recommended dose rate of 1.0 mg kg − 1 body weight (b.w.) and several million doses of trypanocidal drugs are administered to livestock in sub-Saharan Africa each year (Holmes and Torr, 1988). Results of a study by Gilbert and Newton (1982) carried out in non-infected and trypanosome-infected calves using 14C-homidium showed that the drug concentrations in blood and tissue fluids in both groups of animals were not significantly different. They also demonstrated that approximately 80% of the total radioactivity detected in the blood of the calves at 1, 6 and 12 h after treatment with 1.0 mg kg − 1 b.w. was bound to trypanosomes. However, the level of sensitivity to homidium of the trypanosome populations used was not indicated. Detailed pharmacokinetic evaluations of the drug have also been reported in both non-infected and T. congolense-infected cattle using 14C-homidium (Murilla et al., 1996) and an enzyme-linked immunosorbent assay (ELISA) for non-labelled drug (Murilla et al., 1999a,b). Both studies showed that the concentrations of homidium declined rapidly during the first 24 h after intramuscular (i.m.) treatment. Furthermore, an increased rate of drug elimination was observed in cattle infected with trypanosomes. Despite these observations on homidium as a chemotherapeutic drug, information regarding the effects of trypanosome infections of varying drug susceptibility on the pharmacokinetics of the drug
is lacking. The main objective of the present study was, therefore, to characterise the effects of two populations of T. congolense, one drug-sensitive and the other drug-resistant, on the pharmacokinetics of homidium in cattle. The availability of a highly sensitive ELISA method (Murilla et al., 1999a) for the detection of homidium levels in cattle made the present study possible.
2. Materials and methods
2.1. Experimental cattle Ten castrated male Boran (Bos indicus) calves were obtained from Ol Maisor Farm, Laikipia District, Central Province, Kenya, an area free from endemic trypanosomosis. The calves were 6 months old and weighed between 120 and 140 kg. The procedures followed in the management of the animals, before and during the experimental period, were as described by Murilla et al. (1999b). Briefly, cattle were de-wormed using a suspension containing 3.0% oxyclozanide and 1.5% levamisole hydrochloride at a dose rate of 0.5 ml per kg b.w. Blood smears were examined microscopically after Giemsa staining for Theileria, Babesia and Anaplasma species with negative results. The animals were offered on hay ad libitum and concentrate (0.5 kg daily; Unga feeds Ltd., Nairobi, Kenya) and had free access to water.
2.2. Pre-infection sera Collection and storage of large pools of pre-infection, pre-treatment sera were carried out as described previously (Murilla et al., 1999a). Briefly, a 50 ml blood sample was collected from each of the ten animals 2 days before trypanosome inoculation. Serum was separated, a large pool prepared, aliquoted and stored at − 20 °C until required. The pooled serum samples were used as negative controls, for preparation of homidium-spiked standard solutions, and for quality control standards.
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2.3. Trypanosomes Two populations of T. congolense were used that differed in their sensitivity to the recommended therapeutic dose of 1.0 mg homidium kg − 1 b.w. in cattle; one homidium-sensitive (HS) and the other homidium-resistant (HR). The HS population IL1180 is a derivative of an isolate from a lion in Serengeti, Tanzania (Nantulya et al., 1984) and the HR population IL3330 was isolated from a bovine in the Ghibe Valley, Ethiopia (Codjia et al., 1993). To prepare the inoculum for infection of cattle, trypanosome stabilates were inoculated intraperitoneally into sub-lethally (600 rad) irradiated male Swiss White mice. Each mouse received 1× 106 trypanosomes. Wet blood smears were prepared from tail blood and examined for trypanosomes from day two of infection. At the first peak of parasitaemia, the mice were bled out by cardiac puncture under chloroform anaesthesia, using EDTA as an anticoagulant and the blood pooled. The trypanosome density in the pooled blood from the infected mice was determined and the blood subsequently diluted in EDTA saline glucose (ESG) to give a parasite density of 1×106 trypanosomes per millilitre.
2.4. Experimental design On the day of infection the ten cattle were randomly assigned to two groups. One group of five calves was inoculated with 1×105 T. congolense IL1180 parasites (Group HS) whereas the other group of five calves was inoculated with 1× 105 T. congolense IL3330 parasites (Group HR). The inoculum was given by intravenous injection into the jugular vein of all animals. In each group, cattle were treated with homidium bromide 7 days after trypanosomes were first demonstrated in the peripheral blood of all the animals. Cattle were removed from the experiment if their PCV values fell to 15% or below, or on clinical grounds.
2.5. Assessment of parasitaemia and packed cell 6olume Ear vein blood samples were collected into heparinised capillary tubes. The tubes were then cen-
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trifuged in a micro-haematocrit centrifuge at 12 000× g for 10 min and the PCV determined using a haematocrit reader. The buffy-coat was then examined for the presence of trypanosomes using the method of Murray et al. (1977).
2.6. Drug treatment Homidium bromide (Ethidium®, Camco, UK; Lot No. B4B3) was used to treat the animals and a 2.5% (w/v) solution of the drug was prepared in sterile water immediately prior to treatment. A dose of 1.0 mg kg − 1 b.w. was then given as a single bolus by deep i.m. injection into the muscles of the neck.
2.7. Sample collection Sampling for parasitaemia and PCV was carried out daily following infection until trypanosomes were detected in the peripheral blood. This sampling frequency was then continued for the first week following treatment. Thereafter samples were collected weekly for 8 weeks. In addition, blood samples were collected for drug assays at the following intervals after treatment, 5, 10, 15, 30 min, 1, 2, 4, 6, 8 and 12 h, twice a day during the first week, daily during the second week, thrice during the third week, twice during the fourth week, and thereafter weekly to the end of the observation period of 90 days following treatment.
2.8. Determination of total serum proteins Total serum protein was determined on all serum samples using the Coomassie® Plus Protein Assay Reagent (Pierce, Rockford, IL, USA).
2.9. Total serum albumin For the determination of total serum albumin, a quantitative, colorimetric method (Procedure No. 631, Sigma Diagnostics, Poole, England) was used. The absorbances of the solutions were read at 628 nm exactly 30 s after the addition of bromocresol green since the coloured complex was extremely unstable. In order to ensure reproducibility and consistency in the readings, all absorbances of the blanks, standards and test samples were deter-
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mined at exactly 30 s after the addition of the albumin reagent.
2.10. Serum globulin le6els The total serum globulin levels were determined as the difference between the total serum protein and the total serum albumin levels.
2.11. Determination of serum homidium concentrations Serum homidium concentrations were analysed using the direct homidium competition ELISA described by Murilla et al. (1999a). Briefly, microtitre plates (Immulon 4, Dynatech, UK) were coated with hyper-immune serum produced against homidium in sheep (100 ml per well, optimally diluted in carbonate/bicarbonate buffer, pH 9.2). The plates were incubated at + 4 °C overnight after which they were washed. This was followed by addition of test sera, diluted ten-fold in homidium-horseradish peroxidase conjugate in phosphate-buffered saline. The plates were then shaken (Varishaker, Dynatech, UK) for 10 min and incubated overnight at +4 °C. After washing, tetramethylbenzidine (TMB)/hydrogen peroxide (Cambridge Veterinary Services, England) were added, incubated at 37 °C for 15 min, the reaction stopped using 2 M sulphuric acid and the optical density (OD) determined in an ELISA reader (Multiskan Plus Mk II, Labsystems, Oy, Helsinki, Finland). In each plate homidiumspiked calibration standards and quality controls were included, as described previously (Murilla et al., 1999a). All samples were analysed in duplicate and mean ODs, standard deviations (S.D.) and coefficients of variation (CVs) were calculated for all sample replicas. Serum homidium concentrations of unknown samples were obtained with the aid of the calibration curve fitted using the fourparameter logistic method. The detection limit of the assay was 0.1 ng ml − 1 (Murilla et al., 1999a).
2.12. Pharmacokinetic and statistical e6aluation Non-compartmental analysis was carried out using the PCNONLIN, SCI Software. Pharmacoki-
netic evaluation of the serum homidium concentration-versus-time data was as described by Carceles et al. (1995), Murilla et al. (1996). The Student’s t-test was used to compare results of cattle infected with drug-sensitive and drug-resistant T. congolense. The pharmacokinetic parameters shown below were determined using formulae described by Gibaldi and Perrier (1982): the elimination halflife (t1i= 0.693/i) where i is the elimination rate 2 constant; the area under the curve (AUC, ng h ml − 1) and the area under the moments curve (AUMC, ng h2 ml − 1), which were estimated by the trapezoidal rule from the serum drug concentration-versus-time plots and the mean residence time (MRT, hours =AUMC/AUC).
3. Results
3.1. Parasitaemia and clinical findings Fig. 1 shows the level of parasitaemia in cattle following inoculation with either drug-sensitive or drug-resistant trypanosome populations, and the subsequent response to treatment with homidium. Trypanosomes were first detected in the peripheral blood of four of the five cattle in Group HS 7 days following infection; the remaining animal was first detected parasitaemic on the eighth day. All five cattle were, therefore, treated with homidium bromide at 1.0 mg kg − 1 b.w. on day 15. Individual animals exhibited different levels of parasitaemia at the time of treatment (Table 1). However, following treatment trypanosomes were cleared from the blood within 24 h in four of the five cattle. The remaining animal became aparasitaemic within 48 h. This latter animal had the highest parasite concentration at the time of treatment. All the animals remained aparasitaemic until the end of the 90-day observation period. Following inoculation of Group HR calves with T. congolense IL3330, parasites were first demonstrated in the peripheral blood of all five animals after 7 days. Treatment was, therefore, carried out 7 days later, on day 14. In a similar manner to Group HS, individual animals exhibited different levels of parasitaemia on the day of
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a drop in parasite concentration that lasted approximately 10 days (Fig. 1). Animals in both groups showed loss of appetite following infection, which was reversed in Group HS following treatment, but persisted in Group HR cattle and was accompanied by loss in body weight.
3.2. Serum drug concentrations and pharmacokinetics
Fig. 1. Mean level of parasitaemia in groups of five cattle infected with T. congolense, before and after treatment with homidium bromide at 1.0 mg kg − 1 b.w. Closed symbols, cattle infected with IL1180, a drug-sensitive T. congolense population. Open symbols cattle infected with IL3330, a drugresistant T. congolense population. Error bars, S.D., note: animals in Group HR were removed from experiment on day 77.
treatment (Table 1). Following treatment, trypanosomes did not disappear from the blood of any of the Group HR calves, although there was
Fig. 2 is a semi-log plot of the mean serum homidium concentration-versus-time data for Group HS. An exponential decline in the serum drug concentrations over time was observed. The mean (9 S.D.) drug concentration 5 min following treatment was 76.49 51.1 ng ml − 1. The individual concentrations are shown in Table 1. The mean (9S.D.) peak serum concentration (Cmax) was 1809 34.8 ng ml − 1, and occurred at 10 min in one animal and 15 min in the remaining four. By day 36 post-treatment, the concentration of homidium in serum ranged from 0.15 to 0.30 ng ml − 1. Pharmacokinetic parameters obtained from the concentration-versus-time data by non-compartmental analysis are given in Table 2. The mean
Table 1 Level of parasitaemia and homidium concentration in blood collected from cattle that were infected with either (a) a drug-sensitive or (b) a drug-resistant trypanosome population Animal number Parasitaemia on day of treatment (Parasites/ml×104)a
Homidium conc. 5 min. after treatment (ng ml−1)
Time to clearance of parasitaemia following treatment (h)
(a) Homidium-sensiti6e trypanosome population 410 16 411 12 412 20 413 8 414 8 Mean9 S.D. 12.8 9 4.7
34.1 57.9 74.1 40.9 174.7 76.4 9 51.1
24 24 48 24 24
(b) Homidium-resistant trypanosome population 415 10 416 20 417 8 418 14 419 3 Mean9 S.D. 11.0 9 5.7
78.7 104.3 121.2 173.1 113.4 118.1 931.0
not not not not not
a
cleared cleared cleared cleared cleared
Parasitaemia estimated by microscopic examination (×400) of a thick blood smear (Uilenberg, 1998).
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Fig. 2. Mean serum homidium concentrations of five cattle infected with IL1180, a drug-sensitive T. congolense population (open symbols) and of five non-infected cattle (Murilla et al., 1999b; closed symbols), following treatment with homidium bromide at 1 mg kg − 1 b.w. Error bars, S.D.
biological half-life (t1i) was 424 9 146 h and the 2 mean AUC0 − (observed) value was 1.679 0.233 −1 mg h ml . The mean ( 9S.D.) MRT0 − ob-
served value was 2979159 h. There was wide variation in the MRT values between individual infected animals (data not shown). The mean serum homidium concentration-versus-time plot for the five animals in Group HR is shown in Fig. 3. Five minutes following treatment, the mean drug level in serum was 1189 31.0 ng ml − 1. The mean peak serum drug concentration (Cmax) was 1809 29.3 ng ml − 1 and occurred at 10–15 min. These values did not differ significantly from those observed in Group HS (P\ 0.05). Over the first 10 days, the decline in serum homidium concentration was similar to that in Group HS cattle. However, 20 days following treatment, no drug was detectable in serum of any of the five animals in Group HR. This was in contrast to Group HS in which the drug was detectable for over 60 days post-treatment. The results of non-compartmental pharmacokinetic analyses for Group HR are given in Table 2. The mean biological half-life (t1i) was 2 75.5916.9 h. From the AUC0− and AUMC0− values of 1.3390.156 mg h ml − 1 and 43.59 7.55 mg h2 ml − 1, respectively, the MRT0 − value was calculated as 32.89 4.46 h. The values for t1i and 2 MRT0 − were both significantly smaller (PB 0.05) than those observed in Group HS cattle.
Table 2 Pharmacokinetic parameters for homidium bromide in Boran cattle infected with either a drug-sensitive (IL1180) or drug-resistant (IL3330) population of T. congolense Parameter
Boran cattle infected with IL1180 (n=5) (Group HS)
Boran cattle infected with IL3330 (n =5) (Group HR)
tmax (h) Cmax (ng ml−1) i T1i (h) 2 AUC0−last (mg h ml−1) AUC0− observed (mg h ml−1) AUC0− predicted (mg h ml−1) AUMC0−last (mg h2 ml−1) AUMC0− observed (mg h2 ml−1) AUMC0− predicted (mg h2 ml−1) MRT0−last (h) MRT0− observed (h) MRT0− predicted (h)
0.2269 0.0288 180934.8 0.00189 0.0006 4249146 1.6290.261 1.6790.233 1.65 90.247 3409 96.9 4819 239 4309 173 216.29 72.0 296.79 158.8 269.59 120.2
0.197 90.0353 180 929.3 0.0097 9 0.0022 75.5 9 16.9** 1.32 9 0.157* 1.33 9 0.156* 1.33 9 0.157 38.0 95.88 43.5 97.55 41.0 97.09 28.82 9 2.78* 32.78 9 4.46* 30.97 9 3.78
nd, Not determined. Statistical significance of differences between of means for Group HS and Group HR cattle: **, PB0.01; *, PB0.05.
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Fig. 3. Mean serum homidium concentrations of five cattle infected with IL3330, a drug-resistant T. congolense population (open symbols) and of five non-infected cattle (Murilla et al., 1999b; closed symbols), following treatment with homidium bromide at 1.0 mg kg-1 b.w. Error bars, S.D.
3.3. Haematological indices From the onset of parasitaemia in Group HS cattle, there was a rapid drop in PCV from a mean ( 9 S.D.) pre-infection value of 38.49 4.4– 24.8 9 1.6% within 7 days of demonstration of parasites in the peripheral blood (Fig. 4). However, the PCV values returned to pre-infection levels in all animals within 1 week of drug administration. In contrast, the drop in PCV in the animals infected with the HR trypanosomes was more gradual, decreasing from a mean pre-infection value of 38.49 2.5– 31.0 93.1% at the time of homidium treatment. There was then a slight elevation in PCV, to approximately 35%, after which the levels fell rapidly, reaching values of 19.09 1.2% at 70 days (Fig. 4). The value of 31.093.1% obtained at 7 days following detection of parasites in Group HR was markedly higher than 24.891.6% observed in Group HS cattle.
3.4. Total serum protein le6els The mean (9S.D.) total serum protein of the five cattle in Group HS, obtained with pre-infection
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sera, was 46.192.3 mg ml − 1. Within 15 days of trypanosome inoculation (i.e. at the time of homidium treatment) this value was 39.59 0.4 mg ml − 1, which was significantly lower than the pre-infection value (PB 0.05). After treatment of cattle, the levels increased to the pre-infection values within 1 week and continued to rise, reaching a value of approximately 50 mg ml − 1 within 14 days of treatment, which was maintained to the end of the experimental period. The mean ( 9 S.D.) pre-infection total serum protein concentration in Group HR cattle was 47.129 1.34 mg ml − 1. Fourteen days following infection, at the time of treatment, the mean total serum protein concentration was 42.059 4.63 mg ml − 1. Thus, the pre-infection value was slightly higher but not significantly different (i.e. P\ 0.05). Following homidium treatment, protein concentrations rose to pre-infection values within 1 week, despite the fact that the animals remained parasitaemic. Thereafter, there was a gradual drop to 39.6892.18 mg ml − 1 on day 36 following treatment. At day 65 the value was approximately 18 mg ml − 1, which was significantly lower than the pre-infection value of approximately 47 mg ml − 1.
Fig. 4. Mean packed cell volumes in groups of five cattle infected with T. congolense, before and after treatment with homidium bromide at 1.0 mg kg − 1 b.w. Closed symbols, cattle infected with IL1180, a drug-sensitive T. congolense population. Open symbols cattle infected with IL3330, a drugresistant T. congolense. Error bars, S.D.
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3.5. Serum albumin le6els The mean (9S.D.) pre-infection serum albumin concentration for the five cattle in Group HS was 26.09 2.5 mg ml − 1. Fifteen days following trypanosome inoculation, this value fell to 22.29 2.4 mg ml − 1, which was lower but not significantly different from that obtained before trypanosome inoculation. Pre-infection serum albumin levels were generally maintained to the end of the observation period. Pre-infection serum albumin concentrations in Group HR were 25.791.4 mg ml − 1. Fourteen days after trypanosome inoculation, levels of 24.5 92.5 mg ml − 1 were attained, which were lower but not significantly different from the preinfection values. Within 36 days of trypanosome inoculation, the serum albumin levels had dropped to 21.59 0.37 mg ml − 1. These levels were significantly lower than the pre-infection values (P B0.05).
3.6. Total serum globulin le6els The results obtained in Group HS cattle showed a significant drop in globulin levels during the first 14 days post-infection, from a pre-infection value of 20.491.5 mg ml − 1 to approximately 169 1.9 mg ml − 1. Approximately 13 days following treatment, concentrations of 24.192.0 mg ml − 1 were attained, peaking at approximately 15 days after treatment). Values of 24.991.8 mg ml − 1 were attained approximately 25 days following treatment. These levels, which were markedly higher than the pre-infection values, were generally maintained to the end of the observation period. Fourteen days following infection of Group HR cattle, there was a drop in the total serum globulin concentrations, from a pre-infection value of 22.690.98 mg ml − 1 to approximately 20.69 0.5 mg ml − 1. Concentrations did not appear to change markedly over a 2-week period following treatment, after which two peaks corresponding to approximately 25 mg ml − 1 were observed at about 40 and 65 days post-infection, which appeared to coincide with parasitaemia peaks. A mean value of 21.59 0.4 mg ml − 1,
recorded 36 days following homidium treatment, was not significantly different from the pre-infection values.
4. Discussion The present study has demonstrated that T. congolense infections can have profound effects on homidium serum concentrations and pharmacokinetics in cattle that are treated with the recommended therapeutic dose of 1.0 mg kg − 1 b.w. In particular, the drug elimination rate was markedly accelerated in the presence of HR infections, resulting in low values for the mean residence time when compared with these parameters in the presence of drug-sensitive infections (Table 2). Within 48 h of treatment, trypanosomes were cleared from the peripheral blood of all five cattle infected with a HS T. congolense population. Thereafter, no trypanosomes were detected in the blood of any of the animals for the remainder of the 90-day observation period. By contrast, in cattle infected with a drug-resistant T. congolense population, parasites did not clear from the circulation after treatment with homidium although there was a drop in the level of parasitaemia for approximately 10 days (Fig. 1). The drug-resistant population was, therefore, confirmed to be highly resistant to the recommended therapeutic dose of 1.0 mg kg−1 b.w. However, the fact that there was a temporary drop in parasitaemia suggested that this population consisted of individual trypanosomes of varying sensitivities to the drug. Five minutes following homidium treatment, the serum homidium concentrations in cattle infected with the HS trypanosome population (mean9S.D. 76.49 51.1 ng ml − 1) were significantly lower than those (mean9 S.D. 2619 136 ng ml − 1) reported in non-infected Boran cattle at the same time after treatment with the same dose of homidium (PB 0.01; Murilla et al., 1999b). Similarly, the mean serum drug concentrations (1189 131 ng ml − 1) 5 min following treatment of cattle infected with the HR trypanosome population were also significantly lower than those obtained in non-infected Boran cattle (PB 0.05; Murilla et al., 1999b) but not significantly differ-
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ent to those in Group HS cattle. These observations could be associated with drug uptake by both HS and HR trypanosome populations, or with the pathophysiological effects of trypanosome infection on the host. Following treatment of both groups of infected cattle in the present study, significant differences were observed in rates of homidium elimination (Table 2). An accelerated rate of drug elimination was observed in cattle infected with the HR trypanosome population that continued until the drug was no longer detectable, at approximately 3 weeks following treatment (Fig. 3). Similar observations of increased rates of drug elimination in cattle infected with drug-resistant T. congolense were made by Murilla et al. (1996) using 14C-homidium, and by Eisler et al. (1994, 1997) using isometamidium. The accelerated rate of homidium elimination in Group HR cattle was reflected in the mean residence time of the drug (32.89 4.46 h) in these cattle, which was nearly ten-fold lower than that obtained in cattle infected with HS trypanosomes (2969158.8 h). The most probable explanations for these differences are drug uptake by trypanosomes and trypanosome-induced metabolic changes in the bovine host. Differences in the metabolism of homidium by trypanosomes might also have played a role, although the study did not attempt to address this little known area. Packed cell volume (as a measure of anaemia) has been used in trypanosomosis to determine severity of the disease (Katunguka-Rwakishaya et al., 1999). The results of the present study showed that the drop in PCV during the first 7 days of infection was more gradual in cattle infected with HR trypanosomes than cattle infected with HS trypanosomes, suggesting that the HS trypanosome population was more virulent. Whilst the PCV levels returned to pre-infection values within 1 week of treatment in cattle infected with the HS population, levels in the group infected with HR trypanosomes continued to decline until 7 weeks after infection, and thereafter remained at a low level (Fig. 4). Katunguka-Rwakishaya et al. (1993) showed that the observed anaemia in T. congolense-infected sheep was, in part, due to haemodilution. Van Miert et al. (1976) observed that haemodynamic changes may influence the
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kinetics of amoxycillin in goats. Therefore, the haemodilution associated with trypanosome infections could have contributed to the changes in drug pharmacokinetics observed in the present study. The changes in pharmacokinetics may also be linked with low albumin levels and possibly tissue damage in trypanosome-infected cattle. Finally, trypanosome infections in animals are commonly associated with intermittent periods of fever, which usually coincide with peaks of parasitaemia. Studies with other drugs (Young, 1973; Van Miert et al., 1976; Groothuis et al., 1978) have shown that fever can alter their absorption, distribution, bio-transformation and/or excretion. Trypanocidal drug failure has been reported for all current drugs in use. Holmes and Torr (1988) suggested several possible explanations for drug failure, which included underdosing, preparation, administration, fraud, not treated, cryptic foci, re-infection and drug resistance. Underdosing may be as a result of economic difficulties faced by farmers and is the major contributor to the development of drug resistance. In the field, these situations may not occur singly, but in combination or sequentially. Of these, drug resistance may be the cause of greatest concern, because of the very limited number of compounds available for the treatment of bovine trypanosomosis. The demonstration of enhanced drug elimination in cattle infected with drug-resistant trypanosomes is particularly noteworthy. It is possible that strain-related factors other than drug resistance, such as effects on host metabolism, tissue damage or metabolic differences between the two trypanosome populations were responsible for the differences in elimination rate observed between the two populations. For example differences in the PCVs of cattle in the two groups were noticeable prior to homidium treatment (Fig. 3). However, differences between the two populations in terms of effect on drug concentrations were not observed immediately after drug administration, but only later when the drug sensitive population had been eliminated. Hence, the findings suggest that the persistence of trypanosomes can markedly alter the pharmacokinetics of trypanocidal drugs. This has important implications for the efficacy of homidium, not only as a curative drug for drug-resistant infections but also in terms of
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its prophylactic activity, since the period of protection could be markedly reduced even against subsequent drug-sensitive infections. The effects of serum homidium concentrations and the level of drug sensitivity (or resistance) of subsequent trypanosome challenges on the period of prophylaxis conferred by this drug are the subject a subsequent paper.
Acknowledgements The authors wish to thank the International Atomic Energy Agency and the UK Department for International Development (DFID; formerly the Overseas Development Administration) for funding this work. We are grateful to Raymond Mdachi, Benard Wanyonyi, Benza Mavuti and Phortunatus Sifuna of the KETRI Residue Analysis Laboratory, and to the staff of the Divisions of Veterinary Physiology and Pharmacology at the University of Glasgow. We also thank ILRI for kindly donating the protein and albumin assay kits. This paper has been published with permission from the Director, KETRI.
References Carceles, C.M., Escudero, E., Baggot, J.D., 1995. Comparative pharmacokinetics of amoxicillin/clavulanic acid combination after intravenous administration to sheep and goats. J. Vet. Pharmacol. Ther. 18, 132 – 136. Codjia, V., Mulatu, W., Majiwa, P.A.O., Leak, S.G.A., Rowlands, G.J., Authie, E., d’Ieteren, G.D.M., Peregrine, A.S., 1993. Epidemiology of bovine trypanosomiasis in the Ghibe Valley, South West Ethiopia. 3. Occurrence of populations of T. congolense resistant to diminazene, isometamidium and homidium. Acta Trop. 53, 151 – 163. Dolan, R.B., Oketch, G., Alushula, H., Mutugi, M., Stevenson, P., Sayer, P.D., Njogu, A.R., 1990. Homidium bromide as a chemoprophylactic for cattle trypanosomiasis in Kenya. Acta Trop. 47, 137 –144. Eisler, M.C., Arowolo, R.O.A., Gault, E.A., Moloo, S.K., Holmes, P.H., Peregrine, A.S., 1994. Isometamidium concentrations in the serum of treated cattle: correlation with prophylaxis against tsetse-transmitted Trypanosoma congolense. Acta Trop. 56, 39 –50. Eisler, M.C., Gault, E.A., Moloo, S.K., Holmes, P.H., Peregrine, A.S., 1997. Concentrations of isometamidium concentrations in the sera of cattle challenged with
drug-resistant Trypanosoma congolense. Acta Trop. 63, 89 – 100. Gibaldi, M., Perrier, D., 1982. Non-compartmental analysis based on the statistical moment theory. In: Pharmacokinetics, second ed. Marcel Dekker, New York, pp. 409 – 417. Gilbert, R.J., Newton, B.A., 1982. Ethidium bromide; pharmacokinetics and efficacy against trypanosome infections in rabbits and calves. Parasitology 85, 127 – 148. Groothuis, D.G., van Miert, A.S.J.P.A.M., Ziv, G., Nouws, J.F.M., 1978. Effects of experimental Escherichia coli endotoxaemia on ampicillin:amoxycillin blood levels after oral and parental administration in calves. J. Vet. Pharmacol. Ther. 1, 81 – 84. Holmes, P.H., Torr, S.J., 1988. The control of animal trypanosomiasis in Africa. Current methods and future trends. Outlook Agric. 17, 54 – 60. Katunguka-Rwakishaya, E., Parkins, J.J., Fishwick, G., Murray, M., Holmes, P.H., 1993. The pathophysiology of Trypanosoma congolense infection in Scottish Blackface sheep-Influence of dietary protein. Vet. Parasitol. 47, 189 – 204. Katunguka-Rwakishaya, E., Murray, M., Holmes, P.H., 1999. The influence of dietary intake on some blood biochemical parameters in Scottish Blackface sheep infected with Trypanosoma congolense. Vet. Parasitol. 84, 1 – 11. Murilla, G.A., Mdachi, R.E., Ismail, A.A., Karanja, W.M., 1996. Bioavailability, pharmacokinetics and tissue distribution of 14C homidium after parenteral administration to Boran cattle. J. Vet. Pharmacol. Ther. 19, 142 – 148. Murilla, G.A., Eisler, M.C., Peregrine, A.S., Ndung’u, J.M., Holmes, P.H., 1999a. Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle. J. Vet. Pharmacol. Ther. 22, 301 – 307. Murilla, G.A., Holmes, P.H., Peregrine, A.S., Eisler, M.C., Ndung’u, J.M., 1999b. Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA). J. Vet. Pharmacol. Ther. 22, 295 – 300. Murray, M., Murray, P.K., McIntyre, W.I.M., 1977. An improved parasitological technique for the diagnosis of African trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 71, 325 – 326. Mwambu, P.M., 1971. The effect of a block-treatment regimen, using ethidium, on cattle trypanosomiasis in an endemic area. E. Afr. Agric. and For. J. April, 414 – 417. Nantulya, V.M., Musoke, A.J., Rurangirwa, F.R., Moloo, S.K., 1984. Resistance of cattle to tsetse-transmitted challenge with Trypanosoma brucei and Trypanosoma congolense after spontaneous recovery from syringe passaged infections. Inf. Immun. 43, 735 – 738. Stevenson, P., Sones, K.R., Gicheru, M.M., Mwangi, E.K., 1995. Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya. Acta Trop. 59, 77 – 84.
G.A. Murilla et al. / Acta Tropica 81 (2002) 185–195 Uilenberg, G., 1998. A Field Guide for the Diagnosis, Treatment and Prevention of African Animal Trypanosomosis. FAO, Rome. Unsworth, K., 1954a. The curative effect of ethidium bromide against Trypanosoma 6i6ax infections of Zebu cattle in West Africa, with observations on the toxicity of the drug. Ann. Trop. Med. Parasitol. 48, 229 –236. Unsworth, K., 1954b. Further observations on the curative effect of Ethidium bromide against Trypanosoma 6i6ax infections in Zebu cattle in West Africa. Ann. Trop. Med. Parasitol. 48, 237 – 241.
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Van Miert, A.S.J.P.A.M., van Gogh, H., Wit, J.G., 1976. The influence of pyrogen induced fever on absorption of sulpha drugs. Vet. Rec. 99, 480 – 481. Wilde, J.K.H., Robson, J., 1953. The effect against Trypanosoma congolense in Zebu of 3 new phenanthridine compounds. Vet. Rec. 65, 49 – 51. Wilson, S.G., Fairclough, R., 1953. A preliminary note on the treatment of Trypanosoma congolense infections with ethidium bromide in Kenya colony. Vet. Rec. 65, 201 – 202. Young, J.K., 1973. Blood sulfonamide levels in feedlot cattle with respiratory disease. Mod. Vet. Pract. 54, 21.
The effects of drug-sensitive and drug-resistant Trypanosoma congolense infections on the pharmacokinetics of homidium in Boran cattle Grace A. Murilla a,*, Andrew S. Peregrine b,1, Joseph M. Ndung’u a, Peter H. Holmes c, Mark C. Eisler c a
Kenya Trypanosomiasis Research Institute (KETRI), P.O. Box 362, Kikuyu, Kenya International Li6estock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya c Uni6ersity of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK
b
Received 5 July 2001; received in revised form 10 September 2001; accepted 23 October 2001
Abstract Two groups of five Boran (Bos indicus) cattle were infected with one of two populations of Trypanosoma congolense; one drug-sensitive (IL1180), and one drug-resistant (IL3330). The animals were then treated intramuscularly with homidium bromide at a dose rate of 1.0 mg kg − 1 bodyweight 7 days after trypanosomes were detected in the peripheral blood of all the five animals in each group. Following treatment of cattle infected with drug-sensitive trypanosomes, parasites could no longer be detected in the bloodstream of four out of five cattle after 24 h, and after 48 h for the fifth animal. The animals remained aparasitaemic up to the end of the observation period of 90 days and serum drug concentrations determined by enzyme-linked immunosorbent assay (ELISA) remained above the detection limit of 0.1 ng ml − 1 for the entire period. Following treatment of cattle infected with drug-resistant trypanosomes, parasites did not disappear from the bloodstream in any of the five animals. The rate of drug elimination was greater in cattle infected with drug-resistant trypanosomes and the drug was no longer detectable approximately 3 weeks after treatment. Non-compartmental pharmacokinetic analysis showed that the values for t1i 2 of 75.5 916.9 h, the area under the curve (AUC0 − ) of 1.3390.156 mg h ml − 1 and the MRT0 − of 32.8 9 4.45 h obtained in cattle infected with the drug-resistant trypanosome population were significantly lower than the values of 424 9146 h for t1i, 1.67 90.233 mg h ml − 1 for AUC0 − and 297 9 159 h for MRT0 − obtained in cattle infected 2 with the drug-sensitive population. The persistence of drug-resistant infections in cattle following homidium treatment was associated with more rapid drug elimination than in those in which infections with drug-sensitive parasites were cleared by the drug. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Homidium; ELISA; Drug resistance; Pharmacokinetics; Trypanosoma congolense
* Corresponding author. Tel.: + 254-154-32960/4; fax: + 254-154-32397. E-mail address: [email protected] (G.A. Murilla). 1 Present address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ont., Canada N1G 2W1. 0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 1 ) 0 0 2 0 9 - 1
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1. Introduction 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 which is generally classified mainly as a chemotherapeutic drug but has been reported to offer some prophylactic activity against trypanosome infections in the field (Mwambu, 1971; Dolan et al., 1990; Stevenson et al., 1995). Following its introduction, several studies were carried out to determine the minimum curative and maximum tolerated doses (Wilde and Robson, 1953; Wilson and Fairclough, 1953; Unsworth, 1954a,b). Homidium is usually used in the treatment of infections due to either Trypanosoma congolense or Trypanosoma 6i6ax in cattle, sheep and goats at the recommended dose rate of 1.0 mg kg − 1 body weight (b.w.) and several million doses of trypanocidal drugs are administered to livestock in sub-Saharan Africa each year (Holmes and Torr, 1988). Results of a study by Gilbert and Newton (1982) carried out in non-infected and trypanosome-infected calves using 14C-homidium showed that the drug concentrations in blood and tissue fluids in both groups of animals were not significantly different. They also demonstrated that approximately 80% of the total radioactivity detected in the blood of the calves at 1, 6 and 12 h after treatment with 1.0 mg kg − 1 b.w. was bound to trypanosomes. However, the level of sensitivity to homidium of the trypanosome populations used was not indicated. Detailed pharmacokinetic evaluations of the drug have also been reported in both non-infected and T. congolense-infected cattle using 14C-homidium (Murilla et al., 1996) and an enzyme-linked immunosorbent assay (ELISA) for non-labelled drug (Murilla et al., 1999a,b). Both studies showed that the concentrations of homidium declined rapidly during the first 24 h after intramuscular (i.m.) treatment. Furthermore, an increased rate of drug elimination was observed in cattle infected with trypanosomes. Despite these observations on homidium as a chemotherapeutic drug, information regarding the effects of trypanosome infections of varying drug susceptibility on the pharmacokinetics of the drug
is lacking. The main objective of the present study was, therefore, to characterise the effects of two populations of T. congolense, one drug-sensitive and the other drug-resistant, on the pharmacokinetics of homidium in cattle. The availability of a highly sensitive ELISA method (Murilla et al., 1999a) for the detection of homidium levels in cattle made the present study possible.
2. Materials and methods
2.1. Experimental cattle Ten castrated male Boran (Bos indicus) calves were obtained from Ol Maisor Farm, Laikipia District, Central Province, Kenya, an area free from endemic trypanosomosis. The calves were 6 months old and weighed between 120 and 140 kg. The procedures followed in the management of the animals, before and during the experimental period, were as described by Murilla et al. (1999b). Briefly, cattle were de-wormed using a suspension containing 3.0% oxyclozanide and 1.5% levamisole hydrochloride at a dose rate of 0.5 ml per kg b.w. Blood smears were examined microscopically after Giemsa staining for Theileria, Babesia and Anaplasma species with negative results. The animals were offered on hay ad libitum and concentrate (0.5 kg daily; Unga feeds Ltd., Nairobi, Kenya) and had free access to water.
2.2. Pre-infection sera Collection and storage of large pools of pre-infection, pre-treatment sera were carried out as described previously (Murilla et al., 1999a). Briefly, a 50 ml blood sample was collected from each of the ten animals 2 days before trypanosome inoculation. Serum was separated, a large pool prepared, aliquoted and stored at − 20 °C until required. The pooled serum samples were used as negative controls, for preparation of homidium-spiked standard solutions, and for quality control standards.
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2.3. Trypanosomes Two populations of T. congolense were used that differed in their sensitivity to the recommended therapeutic dose of 1.0 mg homidium kg − 1 b.w. in cattle; one homidium-sensitive (HS) and the other homidium-resistant (HR). The HS population IL1180 is a derivative of an isolate from a lion in Serengeti, Tanzania (Nantulya et al., 1984) and the HR population IL3330 was isolated from a bovine in the Ghibe Valley, Ethiopia (Codjia et al., 1993). To prepare the inoculum for infection of cattle, trypanosome stabilates were inoculated intraperitoneally into sub-lethally (600 rad) irradiated male Swiss White mice. Each mouse received 1× 106 trypanosomes. Wet blood smears were prepared from tail blood and examined for trypanosomes from day two of infection. At the first peak of parasitaemia, the mice were bled out by cardiac puncture under chloroform anaesthesia, using EDTA as an anticoagulant and the blood pooled. The trypanosome density in the pooled blood from the infected mice was determined and the blood subsequently diluted in EDTA saline glucose (ESG) to give a parasite density of 1×106 trypanosomes per millilitre.
2.4. Experimental design On the day of infection the ten cattle were randomly assigned to two groups. One group of five calves was inoculated with 1×105 T. congolense IL1180 parasites (Group HS) whereas the other group of five calves was inoculated with 1× 105 T. congolense IL3330 parasites (Group HR). The inoculum was given by intravenous injection into the jugular vein of all animals. In each group, cattle were treated with homidium bromide 7 days after trypanosomes were first demonstrated in the peripheral blood of all the animals. Cattle were removed from the experiment if their PCV values fell to 15% or below, or on clinical grounds.
2.5. Assessment of parasitaemia and packed cell 6olume Ear vein blood samples were collected into heparinised capillary tubes. The tubes were then cen-
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trifuged in a micro-haematocrit centrifuge at 12 000× g for 10 min and the PCV determined using a haematocrit reader. The buffy-coat was then examined for the presence of trypanosomes using the method of Murray et al. (1977).
2.6. Drug treatment Homidium bromide (Ethidium®, Camco, UK; Lot No. B4B3) was used to treat the animals and a 2.5% (w/v) solution of the drug was prepared in sterile water immediately prior to treatment. A dose of 1.0 mg kg − 1 b.w. was then given as a single bolus by deep i.m. injection into the muscles of the neck.
2.7. Sample collection Sampling for parasitaemia and PCV was carried out daily following infection until trypanosomes were detected in the peripheral blood. This sampling frequency was then continued for the first week following treatment. Thereafter samples were collected weekly for 8 weeks. In addition, blood samples were collected for drug assays at the following intervals after treatment, 5, 10, 15, 30 min, 1, 2, 4, 6, 8 and 12 h, twice a day during the first week, daily during the second week, thrice during the third week, twice during the fourth week, and thereafter weekly to the end of the observation period of 90 days following treatment.
2.8. Determination of total serum proteins Total serum protein was determined on all serum samples using the Coomassie® Plus Protein Assay Reagent (Pierce, Rockford, IL, USA).
2.9. Total serum albumin For the determination of total serum albumin, a quantitative, colorimetric method (Procedure No. 631, Sigma Diagnostics, Poole, England) was used. The absorbances of the solutions were read at 628 nm exactly 30 s after the addition of bromocresol green since the coloured complex was extremely unstable. In order to ensure reproducibility and consistency in the readings, all absorbances of the blanks, standards and test samples were deter-
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mined at exactly 30 s after the addition of the albumin reagent.
2.10. Serum globulin le6els The total serum globulin levels were determined as the difference between the total serum protein and the total serum albumin levels.
2.11. Determination of serum homidium concentrations Serum homidium concentrations were analysed using the direct homidium competition ELISA described by Murilla et al. (1999a). Briefly, microtitre plates (Immulon 4, Dynatech, UK) were coated with hyper-immune serum produced against homidium in sheep (100 ml per well, optimally diluted in carbonate/bicarbonate buffer, pH 9.2). The plates were incubated at + 4 °C overnight after which they were washed. This was followed by addition of test sera, diluted ten-fold in homidium-horseradish peroxidase conjugate in phosphate-buffered saline. The plates were then shaken (Varishaker, Dynatech, UK) for 10 min and incubated overnight at +4 °C. After washing, tetramethylbenzidine (TMB)/hydrogen peroxide (Cambridge Veterinary Services, England) were added, incubated at 37 °C for 15 min, the reaction stopped using 2 M sulphuric acid and the optical density (OD) determined in an ELISA reader (Multiskan Plus Mk II, Labsystems, Oy, Helsinki, Finland). In each plate homidiumspiked calibration standards and quality controls were included, as described previously (Murilla et al., 1999a). All samples were analysed in duplicate and mean ODs, standard deviations (S.D.) and coefficients of variation (CVs) were calculated for all sample replicas. Serum homidium concentrations of unknown samples were obtained with the aid of the calibration curve fitted using the fourparameter logistic method. The detection limit of the assay was 0.1 ng ml − 1 (Murilla et al., 1999a).
2.12. Pharmacokinetic and statistical e6aluation Non-compartmental analysis was carried out using the PCNONLIN, SCI Software. Pharmacoki-
netic evaluation of the serum homidium concentration-versus-time data was as described by Carceles et al. (1995), Murilla et al. (1996). The Student’s t-test was used to compare results of cattle infected with drug-sensitive and drug-resistant T. congolense. The pharmacokinetic parameters shown below were determined using formulae described by Gibaldi and Perrier (1982): the elimination halflife (t1i= 0.693/i) where i is the elimination rate 2 constant; the area under the curve (AUC, ng h ml − 1) and the area under the moments curve (AUMC, ng h2 ml − 1), which were estimated by the trapezoidal rule from the serum drug concentration-versus-time plots and the mean residence time (MRT, hours =AUMC/AUC).
3. Results
3.1. Parasitaemia and clinical findings Fig. 1 shows the level of parasitaemia in cattle following inoculation with either drug-sensitive or drug-resistant trypanosome populations, and the subsequent response to treatment with homidium. Trypanosomes were first detected in the peripheral blood of four of the five cattle in Group HS 7 days following infection; the remaining animal was first detected parasitaemic on the eighth day. All five cattle were, therefore, treated with homidium bromide at 1.0 mg kg − 1 b.w. on day 15. Individual animals exhibited different levels of parasitaemia at the time of treatment (Table 1). However, following treatment trypanosomes were cleared from the blood within 24 h in four of the five cattle. The remaining animal became aparasitaemic within 48 h. This latter animal had the highest parasite concentration at the time of treatment. All the animals remained aparasitaemic until the end of the 90-day observation period. Following inoculation of Group HR calves with T. congolense IL3330, parasites were first demonstrated in the peripheral blood of all five animals after 7 days. Treatment was, therefore, carried out 7 days later, on day 14. In a similar manner to Group HS, individual animals exhibited different levels of parasitaemia on the day of
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a drop in parasite concentration that lasted approximately 10 days (Fig. 1). Animals in both groups showed loss of appetite following infection, which was reversed in Group HS following treatment, but persisted in Group HR cattle and was accompanied by loss in body weight.
3.2. Serum drug concentrations and pharmacokinetics
Fig. 1. Mean level of parasitaemia in groups of five cattle infected with T. congolense, before and after treatment with homidium bromide at 1.0 mg kg − 1 b.w. Closed symbols, cattle infected with IL1180, a drug-sensitive T. congolense population. Open symbols cattle infected with IL3330, a drugresistant T. congolense population. Error bars, S.D., note: animals in Group HR were removed from experiment on day 77.
treatment (Table 1). Following treatment, trypanosomes did not disappear from the blood of any of the Group HR calves, although there was
Fig. 2 is a semi-log plot of the mean serum homidium concentration-versus-time data for Group HS. An exponential decline in the serum drug concentrations over time was observed. The mean (9 S.D.) drug concentration 5 min following treatment was 76.49 51.1 ng ml − 1. The individual concentrations are shown in Table 1. The mean (9S.D.) peak serum concentration (Cmax) was 1809 34.8 ng ml − 1, and occurred at 10 min in one animal and 15 min in the remaining four. By day 36 post-treatment, the concentration of homidium in serum ranged from 0.15 to 0.30 ng ml − 1. Pharmacokinetic parameters obtained from the concentration-versus-time data by non-compartmental analysis are given in Table 2. The mean
Table 1 Level of parasitaemia and homidium concentration in blood collected from cattle that were infected with either (a) a drug-sensitive or (b) a drug-resistant trypanosome population Animal number Parasitaemia on day of treatment (Parasites/ml×104)a
Homidium conc. 5 min. after treatment (ng ml−1)
Time to clearance of parasitaemia following treatment (h)
(a) Homidium-sensiti6e trypanosome population 410 16 411 12 412 20 413 8 414 8 Mean9 S.D. 12.8 9 4.7
34.1 57.9 74.1 40.9 174.7 76.4 9 51.1
24 24 48 24 24
(b) Homidium-resistant trypanosome population 415 10 416 20 417 8 418 14 419 3 Mean9 S.D. 11.0 9 5.7
78.7 104.3 121.2 173.1 113.4 118.1 931.0
not not not not not
a
cleared cleared cleared cleared cleared
Parasitaemia estimated by microscopic examination (×400) of a thick blood smear (Uilenberg, 1998).
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Fig. 2. Mean serum homidium concentrations of five cattle infected with IL1180, a drug-sensitive T. congolense population (open symbols) and of five non-infected cattle (Murilla et al., 1999b; closed symbols), following treatment with homidium bromide at 1 mg kg − 1 b.w. Error bars, S.D.
biological half-life (t1i) was 424 9 146 h and the 2 mean AUC0 − (observed) value was 1.679 0.233 −1 mg h ml . The mean ( 9S.D.) MRT0 − ob-
served value was 2979159 h. There was wide variation in the MRT values between individual infected animals (data not shown). The mean serum homidium concentration-versus-time plot for the five animals in Group HR is shown in Fig. 3. Five minutes following treatment, the mean drug level in serum was 1189 31.0 ng ml − 1. The mean peak serum drug concentration (Cmax) was 1809 29.3 ng ml − 1 and occurred at 10–15 min. These values did not differ significantly from those observed in Group HS (P\ 0.05). Over the first 10 days, the decline in serum homidium concentration was similar to that in Group HS cattle. However, 20 days following treatment, no drug was detectable in serum of any of the five animals in Group HR. This was in contrast to Group HS in which the drug was detectable for over 60 days post-treatment. The results of non-compartmental pharmacokinetic analyses for Group HR are given in Table 2. The mean biological half-life (t1i) was 2 75.5916.9 h. From the AUC0− and AUMC0− values of 1.3390.156 mg h ml − 1 and 43.59 7.55 mg h2 ml − 1, respectively, the MRT0 − value was calculated as 32.89 4.46 h. The values for t1i and 2 MRT0 − were both significantly smaller (PB 0.05) than those observed in Group HS cattle.
Table 2 Pharmacokinetic parameters for homidium bromide in Boran cattle infected with either a drug-sensitive (IL1180) or drug-resistant (IL3330) population of T. congolense Parameter
Boran cattle infected with IL1180 (n=5) (Group HS)
Boran cattle infected with IL3330 (n =5) (Group HR)
tmax (h) Cmax (ng ml−1) i T1i (h) 2 AUC0−last (mg h ml−1) AUC0− observed (mg h ml−1) AUC0− predicted (mg h ml−1) AUMC0−last (mg h2 ml−1) AUMC0− observed (mg h2 ml−1) AUMC0− predicted (mg h2 ml−1) MRT0−last (h) MRT0− observed (h) MRT0− predicted (h)
0.2269 0.0288 180934.8 0.00189 0.0006 4249146 1.6290.261 1.6790.233 1.65 90.247 3409 96.9 4819 239 4309 173 216.29 72.0 296.79 158.8 269.59 120.2
0.197 90.0353 180 929.3 0.0097 9 0.0022 75.5 9 16.9** 1.32 9 0.157* 1.33 9 0.156* 1.33 9 0.157 38.0 95.88 43.5 97.55 41.0 97.09 28.82 9 2.78* 32.78 9 4.46* 30.97 9 3.78
nd, Not determined. Statistical significance of differences between of means for Group HS and Group HR cattle: **, PB0.01; *, PB0.05.
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Fig. 3. Mean serum homidium concentrations of five cattle infected with IL3330, a drug-resistant T. congolense population (open symbols) and of five non-infected cattle (Murilla et al., 1999b; closed symbols), following treatment with homidium bromide at 1.0 mg kg-1 b.w. Error bars, S.D.
3.3. Haematological indices From the onset of parasitaemia in Group HS cattle, there was a rapid drop in PCV from a mean ( 9 S.D.) pre-infection value of 38.49 4.4– 24.8 9 1.6% within 7 days of demonstration of parasites in the peripheral blood (Fig. 4). However, the PCV values returned to pre-infection levels in all animals within 1 week of drug administration. In contrast, the drop in PCV in the animals infected with the HR trypanosomes was more gradual, decreasing from a mean pre-infection value of 38.49 2.5– 31.0 93.1% at the time of homidium treatment. There was then a slight elevation in PCV, to approximately 35%, after which the levels fell rapidly, reaching values of 19.09 1.2% at 70 days (Fig. 4). The value of 31.093.1% obtained at 7 days following detection of parasites in Group HR was markedly higher than 24.891.6% observed in Group HS cattle.
3.4. Total serum protein le6els The mean (9S.D.) total serum protein of the five cattle in Group HS, obtained with pre-infection
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sera, was 46.192.3 mg ml − 1. Within 15 days of trypanosome inoculation (i.e. at the time of homidium treatment) this value was 39.59 0.4 mg ml − 1, which was significantly lower than the pre-infection value (PB 0.05). After treatment of cattle, the levels increased to the pre-infection values within 1 week and continued to rise, reaching a value of approximately 50 mg ml − 1 within 14 days of treatment, which was maintained to the end of the experimental period. The mean ( 9 S.D.) pre-infection total serum protein concentration in Group HR cattle was 47.129 1.34 mg ml − 1. Fourteen days following infection, at the time of treatment, the mean total serum protein concentration was 42.059 4.63 mg ml − 1. Thus, the pre-infection value was slightly higher but not significantly different (i.e. P\ 0.05). Following homidium treatment, protein concentrations rose to pre-infection values within 1 week, despite the fact that the animals remained parasitaemic. Thereafter, there was a gradual drop to 39.6892.18 mg ml − 1 on day 36 following treatment. At day 65 the value was approximately 18 mg ml − 1, which was significantly lower than the pre-infection value of approximately 47 mg ml − 1.
Fig. 4. Mean packed cell volumes in groups of five cattle infected with T. congolense, before and after treatment with homidium bromide at 1.0 mg kg − 1 b.w. Closed symbols, cattle infected with IL1180, a drug-sensitive T. congolense population. Open symbols cattle infected with IL3330, a drugresistant T. congolense. Error bars, S.D.
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3.5. Serum albumin le6els The mean (9S.D.) pre-infection serum albumin concentration for the five cattle in Group HS was 26.09 2.5 mg ml − 1. Fifteen days following trypanosome inoculation, this value fell to 22.29 2.4 mg ml − 1, which was lower but not significantly different from that obtained before trypanosome inoculation. Pre-infection serum albumin levels were generally maintained to the end of the observation period. Pre-infection serum albumin concentrations in Group HR were 25.791.4 mg ml − 1. Fourteen days after trypanosome inoculation, levels of 24.5 92.5 mg ml − 1 were attained, which were lower but not significantly different from the preinfection values. Within 36 days of trypanosome inoculation, the serum albumin levels had dropped to 21.59 0.37 mg ml − 1. These levels were significantly lower than the pre-infection values (P B0.05).
3.6. Total serum globulin le6els The results obtained in Group HS cattle showed a significant drop in globulin levels during the first 14 days post-infection, from a pre-infection value of 20.491.5 mg ml − 1 to approximately 169 1.9 mg ml − 1. Approximately 13 days following treatment, concentrations of 24.192.0 mg ml − 1 were attained, peaking at approximately 15 days after treatment). Values of 24.991.8 mg ml − 1 were attained approximately 25 days following treatment. These levels, which were markedly higher than the pre-infection values, were generally maintained to the end of the observation period. Fourteen days following infection of Group HR cattle, there was a drop in the total serum globulin concentrations, from a pre-infection value of 22.690.98 mg ml − 1 to approximately 20.69 0.5 mg ml − 1. Concentrations did not appear to change markedly over a 2-week period following treatment, after which two peaks corresponding to approximately 25 mg ml − 1 were observed at about 40 and 65 days post-infection, which appeared to coincide with parasitaemia peaks. A mean value of 21.59 0.4 mg ml − 1,
recorded 36 days following homidium treatment, was not significantly different from the pre-infection values.
4. Discussion The present study has demonstrated that T. congolense infections can have profound effects on homidium serum concentrations and pharmacokinetics in cattle that are treated with the recommended therapeutic dose of 1.0 mg kg − 1 b.w. In particular, the drug elimination rate was markedly accelerated in the presence of HR infections, resulting in low values for the mean residence time when compared with these parameters in the presence of drug-sensitive infections (Table 2). Within 48 h of treatment, trypanosomes were cleared from the peripheral blood of all five cattle infected with a HS T. congolense population. Thereafter, no trypanosomes were detected in the blood of any of the animals for the remainder of the 90-day observation period. By contrast, in cattle infected with a drug-resistant T. congolense population, parasites did not clear from the circulation after treatment with homidium although there was a drop in the level of parasitaemia for approximately 10 days (Fig. 1). The drug-resistant population was, therefore, confirmed to be highly resistant to the recommended therapeutic dose of 1.0 mg kg−1 b.w. However, the fact that there was a temporary drop in parasitaemia suggested that this population consisted of individual trypanosomes of varying sensitivities to the drug. Five minutes following homidium treatment, the serum homidium concentrations in cattle infected with the HS trypanosome population (mean9S.D. 76.49 51.1 ng ml − 1) were significantly lower than those (mean9 S.D. 2619 136 ng ml − 1) reported in non-infected Boran cattle at the same time after treatment with the same dose of homidium (PB 0.01; Murilla et al., 1999b). Similarly, the mean serum drug concentrations (1189 131 ng ml − 1) 5 min following treatment of cattle infected with the HR trypanosome population were also significantly lower than those obtained in non-infected Boran cattle (PB 0.05; Murilla et al., 1999b) but not significantly differ-
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ent to those in Group HS cattle. These observations could be associated with drug uptake by both HS and HR trypanosome populations, or with the pathophysiological effects of trypanosome infection on the host. Following treatment of both groups of infected cattle in the present study, significant differences were observed in rates of homidium elimination (Table 2). An accelerated rate of drug elimination was observed in cattle infected with the HR trypanosome population that continued until the drug was no longer detectable, at approximately 3 weeks following treatment (Fig. 3). Similar observations of increased rates of drug elimination in cattle infected with drug-resistant T. congolense were made by Murilla et al. (1996) using 14C-homidium, and by Eisler et al. (1994, 1997) using isometamidium. The accelerated rate of homidium elimination in Group HR cattle was reflected in the mean residence time of the drug (32.89 4.46 h) in these cattle, which was nearly ten-fold lower than that obtained in cattle infected with HS trypanosomes (2969158.8 h). The most probable explanations for these differences are drug uptake by trypanosomes and trypanosome-induced metabolic changes in the bovine host. Differences in the metabolism of homidium by trypanosomes might also have played a role, although the study did not attempt to address this little known area. Packed cell volume (as a measure of anaemia) has been used in trypanosomosis to determine severity of the disease (Katunguka-Rwakishaya et al., 1999). The results of the present study showed that the drop in PCV during the first 7 days of infection was more gradual in cattle infected with HR trypanosomes than cattle infected with HS trypanosomes, suggesting that the HS trypanosome population was more virulent. Whilst the PCV levels returned to pre-infection values within 1 week of treatment in cattle infected with the HS population, levels in the group infected with HR trypanosomes continued to decline until 7 weeks after infection, and thereafter remained at a low level (Fig. 4). Katunguka-Rwakishaya et al. (1993) showed that the observed anaemia in T. congolense-infected sheep was, in part, due to haemodilution. Van Miert et al. (1976) observed that haemodynamic changes may influence the
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kinetics of amoxycillin in goats. Therefore, the haemodilution associated with trypanosome infections could have contributed to the changes in drug pharmacokinetics observed in the present study. The changes in pharmacokinetics may also be linked with low albumin levels and possibly tissue damage in trypanosome-infected cattle. Finally, trypanosome infections in animals are commonly associated with intermittent periods of fever, which usually coincide with peaks of parasitaemia. Studies with other drugs (Young, 1973; Van Miert et al., 1976; Groothuis et al., 1978) have shown that fever can alter their absorption, distribution, bio-transformation and/or excretion. Trypanocidal drug failure has been reported for all current drugs in use. Holmes and Torr (1988) suggested several possible explanations for drug failure, which included underdosing, preparation, administration, fraud, not treated, cryptic foci, re-infection and drug resistance. Underdosing may be as a result of economic difficulties faced by farmers and is the major contributor to the development of drug resistance. In the field, these situations may not occur singly, but in combination or sequentially. Of these, drug resistance may be the cause of greatest concern, because of the very limited number of compounds available for the treatment of bovine trypanosomosis. The demonstration of enhanced drug elimination in cattle infected with drug-resistant trypanosomes is particularly noteworthy. It is possible that strain-related factors other than drug resistance, such as effects on host metabolism, tissue damage or metabolic differences between the two trypanosome populations were responsible for the differences in elimination rate observed between the two populations. For example differences in the PCVs of cattle in the two groups were noticeable prior to homidium treatment (Fig. 3). However, differences between the two populations in terms of effect on drug concentrations were not observed immediately after drug administration, but only later when the drug sensitive population had been eliminated. Hence, the findings suggest that the persistence of trypanosomes can markedly alter the pharmacokinetics of trypanocidal drugs. This has important implications for the efficacy of homidium, not only as a curative drug for drug-resistant infections but also in terms of
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its prophylactic activity, since the period of protection could be markedly reduced even against subsequent drug-sensitive infections. The effects of serum homidium concentrations and the level of drug sensitivity (or resistance) of subsequent trypanosome challenges on the period of prophylaxis conferred by this drug are the subject a subsequent paper.
Acknowledgements The authors wish to thank the International Atomic Energy Agency and the UK Department for International Development (DFID; formerly the Overseas Development Administration) for funding this work. We are grateful to Raymond Mdachi, Benard Wanyonyi, Benza Mavuti and Phortunatus Sifuna of the KETRI Residue Analysis Laboratory, and to the staff of the Divisions of Veterinary Physiology and Pharmacology at the University of Glasgow. We also thank ILRI for kindly donating the protein and albumin assay kits. This paper has been published with permission from the Director, KETRI.
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