Frequency of diminazene-resistant trypanosomes in populations of Trypanosoma congolense arising in infected animals following treatment with diminazene aceturate. M Mamman, G Gettinby, N B Murphy, S Kemei and A S Peregrine Antimicrob. Agents Chemother. 1995, 39(5):1107. DOI: 10.1128/AAC.39.5.1107. These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV Updated information and services can be found at: http://aac.asm.org/content/39/5/1107 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1995, p. 1107–1113 0066-4804/95/$04.0010 Copyright q 1995, American Society for Microbiology Vol. 39, No. 5 Frequency of Diminazene-Resistant Trypanosomes in Populations of Trypanosoma congolense Arising in Infected Animals Following Treatment with Diminazene Aceturate† MOHAMMED MAMMAN,1 GEORGE GETTINBY,2 NOEL B. MURPHY,1 SAMMY KEMEI,1 AND ANDREW S. PEREGRINE1* International Laboratory for Research on Animal Diseases, Nairobi, Kenya,1 and Department of Statistics and Modelling Science, University of Strathclyde, Glasgow, Scotland2 The frequency of trypanosomes resistant to diminazene aceturate at a dose of 25 mg/kg of body weight was investigated for populations of Trypanosoma congolense IL 3274 which reappeared in infected mice after intraperitoneal treatment with diminazene aceturate at the same dosage. At inoculum sizes of 102, 103, 104, 105, and 106 trypanosomes per mouse, the relapse populations were used to initiate infections in five groups of 100 mice each by the intravenous route. Immediately after infection, 50 mice in each group were treated intraperitoneally with diminazene aceturate at the aforementioned dosage; the other 50 mice functioned as untreated controls. Thereafter, all animals were monitored for 100 days for the presence of trypanosomes. In each group, trypanosomes were detected in 50 of 50 control mice, indicating 100% infectivity for all five inoculum sizes. In contrast, in the groups of 50 mice infected with 102, 103, 104, 105, and 106 trypanosomes and treated with diminazene aceturate, trypanosomes were detected in 4, 11, 13, 28, and 39 of 50 mice, respectively. By logistic regression, a good fit was found between the number of mice identified as parasitemic and the inoculum sizes. Maximum likelihood estimates for the proportions of trypanosomes resistant to diminazene aceturate at 25 mg/kg of body weight for the inoculum of 102, 103, 104, 105, and 106 organisms were 8.335 3 1024, 2.485 3 1024, 3.02 3 1025, 8.3 3 1026, and 1.6 3 1026, respectively. These findings indicate that the majority of the relapse trypanosomes were susceptible to the drug dosage used for selecting the population and that, surprisingly, the calculated proportion of organisms which survived drug exposure varied inversely with the inoculum size. Further experiments with mice indicated that the inverse relationship did not result from alterations in the pharmacokinetics of the drug with different inoculum sizes. The data therefore suggest that parasite inoculum size and drug dosage are important factors in estimating the apparent frequency of diminazeneresistant trypanosomes in populations of T. congolense occurring in vivo. (17) have shown that at levels of parasitemia exceeding 108 trypanosomes per ml of blood, a strain of Trypanosoma brucei gambiense exhibited a significantly prolonged generation time compared with that of the same strain at lower levels of parasitemia. Growth and differentiation of trypanosomes appear to be regulated by both host (7, 8, 38) and parasite (4, 9, 37, 39) factors. There is also evidence that cyclic AMP (cAMP) is directly involved in such regulation, since high levels of parasitemia have been shown to be associated with an elevation in intracellular levels of cAMP and prolongation of the doubling time in Trypanosoma brucei brucei (29). The dynamics of growth of mixed populations of trypanosomes within the same host have received considerable interest, and it appears that rapidly growing strains competitively outgrow, and dominate, slowly growing subpopulations (46). There is also evidence to suggest that some drug-susceptible trypanosomes grow at relatively faster rates than drug-resistant strains, which may explain why, in the absence of chemotherapy, drug-susceptible populations appear to outgrow drug-resistant populations in vivo (12, 21, 36, 49). While the frequencies with which drugsusceptible and drug-resistant trypanosomes occur in vivo are unknown, we have shown in goats that the majority of trypanosomes which repopulate the blood following treatment with diminazene aceturate appear to remain susceptible to the drug dosage that was used (28). However, in that study, the proportions of the populations which survived drug treatment were not determined. In this report, we present estimates of these African trypanosomiasis is a disease complex caused by unicellular protozoan parasites belonging to the genus Trypanosoma. Pathogenic trypanosomes cause clinical disease in humans and are an important factor limiting development of domestic livestock in Africa (33). Diminazene aceturate (Berenil [Hoechst AG, Frankfurt, Germany] or Veriben) is an aromatic diamidine that was introduced in 1955 as a therapeutic agent for treatment of animal trypanosomiasis. In trypanosomatids, the drug appears to selectively block replication of kinetoplast DNA (27, 35). However, the primary target(s) for diminazene’s trypanocidal activity remain(s) unknown. Experimental studies of rodents indicate that the number of trypanosomes used to initiate an infection has a significant influence on the subsequent growth of trypanosomes in vivo (9, 16–18, 31); an increase of the inoculum size shortened the parasitemia prepatent period and the time to reach the first peak of parasitemia. However, it also resulted in an increased density of trypanosomes at the first peak of parasitemia (10, 18). Such differences in density appear to inhibit trypanosome proliferation in vivo in a density-dependent fashion, characteristic of the parasite strain (9, 10). For example, Diffley et al. * Corresponding author. Mailing address: ILRAD, P.O. Box 30709, Nairobi, Kenya. Phone: 254-2-630743. Fax: 254-2-631499. Electronic mail address: a.peregrine@cgnet.com. † International Laboratory for Research on Animal Diseases publication 1208. 1107 Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV Received 25 July 1994/Returned for modification 13 December 1994/Accepted 4 March 1995 1108 MAMMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Proportions of mice with diminazene-resistant trypanosomes after infection with relapsea populations of T. congolense IL 3274 and estimated proportions of diminazeneresistant trypanosomes of each inoculum size Inoculum size (no. of trypanosomes/ mouse) Observed parasitemic/treated 102 103 104 105 106 4/50 11/50 13/50 28/50 39/50 TABLE 2. Diminazene susceptibility of T. congolense IL 3274 that reappeared in infected mice after i.p. treatment with 25 mg of diminazene aceturate per kg of b.w. Predicted parasitemicb/treated Estimated proportion of resistant trypanosomes Dose of diminazene aceturate (mg/kg of b.w.) 3.9/50 8.6/50 16.9/50 27.8/50 37.8/50 8.335 3 1024 2.485 3 1024 3.020 3 1025 8.300 3 1026 1.600 3 1026 0.0 10.0 20.0 30.0 40.0 50.0 No. of mice proportions based on studies carried out with mice that simulate the dose-response relationship previously observed for goats. MATERIALS AND METHODS Experimental animals. Swiss white mice (25 to 38 g each) obtained from the International Laboratory for Research on Animal Diseases breeding colony were used throughout the study. They were allowed free access to both water and a pelleted ration (Unga Feeds Ltd., Nairobi, Kenya). Trypanosomes. Trypanosoma congolense IL 3274 (40) is a cloned population which was derived from a stock, Banankeledaga/83/CRTA/67, that was isolated from a bull in Burkina Faso (41). In goats, infections with this parasite are refractory to treatment with diminazene aceturate at an intramuscular dose of 7.0 mg/kg of body weight (b.w.) when treatment is administered on day 19 of infection (47). Treatment. Diminazene aceturate (Berenil) freshly prepared in sterile distilled water was used for intraperitoneal (i.p.) treatment of mice. Calculations for the different doses of diminazene aceturate used in the study were corrected such that the required amounts of drug were contained in a volume of 0.2 ml of sterile distilled water. The same volume of sterile distilled water was administered i.p. to infected, but untreated, control mice. Study design. T. congolense IL 3274 was initially expanded in sublethally (650-rad) irradiated mice. On day 9 of infection, the resultant population was used to infect nonirradiated mice i.p. at a dose of 107 trypanosomes per mouse. After detection of trypanosomes, the mice were treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. This dosage was used since it resulted in a parasitemia profile that was similar to that seen for goats treated intramuscularly with diminazene aceturate at a dose of 7.0 mg/kg of b.w.; the treatment caused temporary remission of parasitemia, which was subsequently followed by the reappearance of trypanosomes. At this stage, the mice were anaesthetized with diethyl ether, and exsanguinated by cardiac puncture and the blood was collected into sodium citrate (final concentration, 1.0% [wt/vol]). The number of trypanosomes per milliliter in the pooled blood was quantified with a Neubauer hemocytometer. Thereafter, the blood was diluted in phosphate saline glucose to prepare trypanosome inocula of five different sizes. With an inoculum volume of 0.2 ml, mice were infected intravenously (i.v.), via the tail vein, with 102, 103, 104, 105, or 106 trypanosomes. Inocula of each size were used to infect a group of 100 nonirradiated mice. Within a minute of infection, mice were either treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. or administered 0.2 ml of distilled water i.p. Administration of diminazene aceturate and distilled water was carried out in an alternating manner: i.e., mice 1, 3, 5, and 7, etc., were administered diminazene aceturate; mice 2, 4, 6, and 8, etc., were administered only distilled water. Immediate treatment following infection was carried out in order to minimize the chances of parasites invading drug-inaccessible sites. In this way, 50 mice in each group were administered diminazene aceturate; the other 50 mice were administered only sterile distilled water and functioned as infectivity controls. Following infection, development of parasitemia in all mice was monitored for 100 days by examining wet films of tail blood at a magnification of 3250; any mouse detected as parasitemic was removed from the experiment. Estimation of the proportion of diminazene-resistant trypanosomes in inocula. The relationship between the fraction of the mice detected as parasitemic following treatment with diminazene aceturate and the trypanosome inoculum sizes was investigated by using logistic regression. Parameter estimation using maximum likelihood methods was undertaken with GLIM version 3.77 (44). Characterization of the diminazene susceptibility of T. congolense IL 3274 that reappeared in mice after treatment. In a similar manner to that described above 103 106 25/25 25/25 15/25 2/25 4/25 2/25 25/25 25/25 25/25 13/25 4/25 2/25 a Number of mice detected as parasitemic/number of mice treated (25 mg of diminazene aceturate per kg of b.w. administered i.p. immediately after infection by the i.v. route). in the paragraph on study design, the subpopulation of T. congolense IL 3274 which reappeared in mice after treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. was diluted in phosphate saline glucose to prepare inocula of 103 and 106 trypanosomes in 0.2 ml. Inocula of each size were used to i.v. infect six groups of 25 mice each. Thereafter, the mice in each group were treated immediately with diminazene aceturate i.p. at doses of 0, 10, 20, 30, 40, or 50 mg/kg of b.w. For 100 days following treatment, all mice were monitored for development of parasitemia by examining wet films of tail blood at a magnification of 3250; mice detected as parasitemic were removed from the experiment. Values of the doses of diminazene aceturate required to cure 50% of the infected mice (CD50) were determined by logit analysis and the minimum chisquare method (22). Characterization of the diminazene susceptibility of clones derived from T. congolense IL 3274. Experiments to determine the diminazene sensitivity of clones of T. congolense IL 3274 that arose in mice prior to, or following, treatment with diminazene aceturate were carried out. Bloodstream forms of the parasite were used to infect a group of nonirradiated mice i.p. (107 trypanosomes per mouse). Following detection of trypanosomes, on day 3 of infection, each mouse was treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. Subsequently, all mice exhibited temporary remission of parasitemia followed by reappearance of trypanosomes. Trypanosome populations occurring in these animals were cloned on days 2 and 3 of infection (prior to treatment) by the method of Barry and Gathuo (6). They were also cloned on days 18 to 20 of infection, when trypanosomes had reappeared following treatment. Each clone was thereafter expanded in sublethally (650-rad) irradiated mice. The resultant populations were then used to infect groups of nonirradiated mice at an inoculum size of 107 trypanosomes per mouse. On day 3 of infection, the mice were treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. All mice were then monitored for 100 days for the development of parasitemia, as described above. Pharmacokinetics of diminazene in mice. In the first mouse study, all five groups of mice were treated with diminazene aceturate at a dose of 25 mg/kg of b.w. In order to determine whether differences in the disposition of diminazene occurred as the trypanosome inoculum size was increased, the pharmacokinetics of the drug for three groups of mice were determined. Group A consisted of noninfected mice which were treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. Mice in group B were infected i.v. (106 trypanosomes per mouse) with a population of T. congolense IL 3274 that had reappeared in infected mice after treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. Immediately after infection, the mice were treated i.p. with diminazene aceturate at a dose of 25 mg/kg of b.w. Mice in group C were not infected but were treated i.v. with diminazene aceturate at a dose of 15 mg/kg of b.w. This last group was included to provide data required to determine values of a number of pharmacokinetic parameters. Following treatment, blood samples were obtained from mice in each of groups A, B, and C after 5, 10, 15, 30, 40, and 45 min and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, and 48 h. Since ion pair extraction of diminazene from blood (2) requires a volume of 1 ml, samples from two treated mice were pooled in a 3-ml potassium-EDTA-containing Vacutainer (Becton Dickinson) tube for each time. Furthermore, at each sampling time, 10 mice were used to obtain five replicate samples. The methods used for plasma separation, storage, and analysis of diminazene by high-performance liquid chromatography were as previously described (1, 2). Plasma concentration-time data sets were modelled by using NONLIN version 3.0 (Statistical Consultants Inc.) and STATIS version 3.0 (ClydeSoft), with JANA (Statistical Consultants Inc.) to determine initial parameter estimates. The Akaike and Schwartz information criteria (45, 53) were used for selection of the compartmental model that best fitted each concentration-time data set. Symbols of compartmental parameters are used in this paper as commonly defined (3, 20, 25, 43). Noncompartmental parameters, such as the Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV a The population had relapsed in infected mice following i.p. treatment with 25 mg of diminazene aceturate/kg of b.w. b Estimated by using a logistic model (see the text). Proportion of parasitemic micea by inoculum size (no. of trypanosomes/mouse) VOL. 39, 1995 T. CONGOLENSE RESISTANCE TO DIMINAZENE ACETURATE 1109 TABLE 3. Coefficients and exponents describing disposition of diminazene following its administration at single doses of 15.0 mg/kg of b.w. i.v. or 25.0 mg/kg of b.w. i.p. to noninfected and T. congolense-infected mice Route of drug administration (group) i.p. (noninfected) i.p. (infected) i.v. (noninfected) a C1 (mg/ml) C2 (mg/ml) Cz (mg/ml) l1 (h21) l2 (h21) lz (h21) 14.24 6 10.37*a 13.21 6 8.23‡ 2.01 6 1.44*‡ 2.79 6 0.43* 1.42 6 1.77 0.93 6 0.40* 0.46 6 0.45 0.28 6 0.17 0.24 6 0.08 23.30 6 17.10* 15.17 6 16.45 5.36 6 1.67* 1.31 6 0.64* 0.83 6 0.96 0.45 6 0.10* 0.07 6 0.05 0.03 6 0.03 0.04 6 0.01 Arithmetic mean values (6 standard deviations) with the same symbols (* and ‡) in the same column are significantly different (P , 0.05). RESULTS Estimation of the proportions of resistant trypanosomes in inocula. Five groups of 100 mice each were infected i.v. with a population of T. congolense IL 3274 that relapsed in infected mice following i.p. treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. The results obtained for each of the five groups are presented in Table 1. In each group, 50 of 50 control mice were detected as parasitemic, thus indicating 100% infectivity for all five inoculum sizes. In contrast, in the groups of mice inoculated with 102, 103, 104, 105, or 106 trypanosomes and treated with diminazene aceturate at a dose of 25 mg/kg of b.w., trypanosomes were detected in 4, 11, 13, 28, or 39 of 50 mice, respectively. Blood from the mice that remained aparasitemic was not infective to irradiated mice (data not shown). In addition, sera from the same parasite-negative mice were negative for T. congolense antigen when examined by enzyme-linked immunosorbent assay (34) (data not shown). The data presented in Table 1 show that not all mice infected with T. congolense IL 3274 and treated with diminazene aceturate became parasitemic. Furthermore, mice receiving smaller inocula appeared less likely to become parasitemic than mice receiving larger inocula. Logistic regression was used to investigate the relationship between the probability of a mouse being parasitemic and the size of the inoculum. A relationship of the following form was found to be a good fit: ln (p/[1 2 p]) 5 24.27 1 0.90 log10(IS), where p is the proportion of mice detected as parasitemic following treatment with diminazene aceturate and IS is the trypanosome inoculum size. The 95% confidence intervals for the intercept and slope were (25.38, 23.16) and (0.66, 1.14), respectively. Table 1 shows the closeness of fit between the observed number of mice that were detected as parasitemic and the number that were predicted to be parasitemic by the logistic equation. The possibility that each inoculum contained a mixture of susceptible and resistant trypanosomes, and that sampling vari- ation resulted in some inocula not containing any resistant trypanosomes, was investigated as a possible explanation for the relationship between the inoculum size and the proportion of mice that became parasitemic. For an inoculum of size n trypanosomes containing a proportion Q of resistant trypanosomes, the probability of a mouse receiving at least one resistant trypanosome is calculated as follows: 1 2 (1 2 Q)n. By using the observed proportion of animals detected as parasitemic (Table 1) as an estimate of this proportion, it was possible to obtain an estimate of Q for each inoculum size. For inocula of 102, 103, 104, 105, and 106 trypanosomes, the maximum likelihood estimates for Q were 8.335 3 1024, 2.485 3 1024, 3.02 3 1025, 8.3 3 1026, and 1.6 3 1026, respectively. Likelihood ratio testing confirmed that the estimates were significantly different. Diminazene susceptibility of relapse populations of T. congolense IL 3274. Table 2 summarizes the diminazene aceturate susceptibility data for T. congolense IL 3274 that appeared in mice after treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. Regardless of the inoculum size, trypanosomes were detected in all mice that were treated with diminazene aceturate at doses of either 0 or 10 mg/kg of b.w. Similarly, trypanosomes were detected in 25 of 25 mice which were treated with 20 mg/kg of b.w. after infection with 106 trypanosomes. In the groups of mice that were infected with 103 trypanosomes and treated with diminazene aceturate at doses of 20, 30, 40, or 50 mg/kg of b.w., 15, 2, 4, or 2 of 25 mice, respectively, were detected as parasitemic. In the groups of mice that were infected with 106 trypanosomes and treated with diminazene aceturate at doses of 30, 40, or 50 mg/kg of b.w., 13, 4, or 2 of 25 mice, respectively, were detected as parasitemic. Based on these data, the diminazene aceturate CD50 value determined for mice infected with 106 trypanosomes was 29.88 mg/kg of b.w. and was significantly (P , 0.001) greater than that determined for mice infected with 103 trypanosomes (CD50, 22.28 mg/kg of b.w.). Characterization of the diminazene susceptibility of clones derived from T. congolense IL 3274. A total of 15 clones were examined: 5 were prepared on day 2 of infection, 6 were prepared on day 3, 1 was prepared on day 18, 1 was prepared on day 19, and 2 were prepared on day 20. All clones were refractory to treatment with a single dose of 25 mg of diminazene aceturate per kg of b.w. Pharmacokinetics of diminazene in mice. Values of the pharmacokinetic parameters determined from the noninfected and T. congolense-infected groups of mice after treatment with diminazene are shown in Tables 3 to 5. The Cmax of diminazene for both the noninfected (17.49 6 10.25 mg/ml) and T. congolense-infected (14.89 6 10.05 mg/ml) groups were observed at 5 min following treatment. For the noninfected group treated i.p., F, AUC0–`, and t1/2lz were 66.16% 6 20.97%, 9.70 6 1.83 mg z h/ml, and 10.71 h, respectively. Values of these parameters for the infected group were 87.00% 6 15.71%, 13.33 6 3.83 mg z h/ml, and 25.70 h, respectively. Although the Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV area under the concentration-time curve (AUC0–`) and the area under the (first) moment curve (AUMC0–`) determined by the trapezoidal rule from time zero to infinity, mean residence time (MRT), apparent total body clearance (CL) and apparent volume of distribution at steady state [Vd(ss)], were calculated in accordance with standard equations (20). Bioavailability (F) represents the percentage ratio of AUC0–` (i.p.) to AUC0–` (i.v.). Other parameters used in this paper are as follows: maximum concentration in plasma (Cmax), volume of the central compartment (Vc), zero time intercepts of the rapid (C1) and slow (C2) distribution phases, zero time intercept of the terminal elimination phase (Cz), rapid (l1) and slow (l2) exponents of the distribution phases, hybrid rate constant for the terminal elimination phase (lz), and rapid (t1/2l1) and slow (t1/2l2) distribution half-life (t1/2), terminal elimination t1/2 (t1/2lz), elimination rate constants from the central compartment (kel), t1/2 for elimination from central compartment (t1/2kel), transfer rate constant from central-to-shallow (k12) and central-to-deep (k13) peripheral compartments, and transfer rate constants from deep (k31) and shallow (k21) peripheral compartments to the central compartment. All statistical analyses were carried out with Minitab version 8.2 (Minitab Inc.). The geometric means (range) are given for t1/2 parameters. The values of other parameters are expressed as arithmetic means 6 standard deviations. A single classification analysis of variance was used to test differences between values of pharmacokinetic parameters determined in the three groups at the 5% significance level. a Values with the same symbols (* and ‡) in the same column are significantly different (P , 0.05). Geometric mean (range) values are given for t1/2l1, t1/2l2, t1/2lz and t1/2kel; all other parameters are arithmetic means 6 standard deviations. 2.00 6 1.18* 14.59 6 14.78 2.88 6 1.77* 0.25 6 0.29 4.96 6 0.46* 1.92 6 1.25* 1.88 6 1.46‡ 8.64 6 9.86 3.60 6 2.36‡ 0.14 6 0.13 2.64 6 3.40 2.17 6 1.04‡ 0.37 6 0.12*‡ 2.45 6 1.73 0.53 6 0.26*‡ 0.12 6 0.04 2.36 6 1.27* 5.30 6 2.12*‡ 0.38 (0.17–0.68)* 0.45 (0.15–0.79)‡ 1.94 (1.26–3.20)*‡ 0.036 (0.013–0.086)* 0.57 (0.29–0.84)* 10.71 (4.11–24.54) 0.07 (0.02–0.028) 1.86 (0.28–19.23) 25.70 (8.07–105.92) 0.13 (0.10–0.25)* 1.51 (1.23–2.27)* 17.37 (12.59–31.80) i.p. (noninfected) i.p. (infected) i.v. (noninfected) k13 (h21) k12 (h21) kel (h21) t1/2kel (h) t1/2 lz (h) t1/2 l2 (h) t1/2 l1 (h) Route of drug administration (group) ANTIMICROB. AGENTS CHEMOTHER. values of these parameters were higher for the infected group than for the noninfected group treated i.p., they did not differ significantly. For the noninfected group treated i.p., CL (28.25 6 5.73 ml/min/kg) was very similar to that for the infected group (28.62 6 5.73 ml/min/kg). Similarly, the MRT for the infected group (50.04 6 46.24 h) was not significantly longer than that for the same noninfected group (17.89 6 5.39 h). A similar trend was observed with Vd(ss), which was large for the infected group (84.29 6 54.68 liters/kg) but not significantly different from that for the noninfected group (47.32 6 15.24 liters/kg). There was also no significant difference between the Vc of the noninfected (1.92 6 1.25 liters/kg) and infected (2.17 6 1.04 liters/kg) groups. However, the Vc of the noninfected group treated i.v. (5.30 6 2.12 liters/kg) was significantly greater than those of both the noninfected and infected i.p. treatment groups. Similarly, the geometric mean t1/2kel from the central compartment of the i.v. group was significantly higher than those of the i.p. treatment groups. In contrast, the Cmax for the i.v. group (3.19 6 1.17 mg/ml) was significantly lower than those for both i.p. treatment groups. There were no significant differences between the i.v. and i.p. treatment groups with respect to AUC0–`, AUMC0–`, MRT, CL, and Vd(ss). DISCUSSION In the work presented here, experiments were carried out to investigate the dynamics of expression of diminazene resistance amongst trypanosomes which reappear in infected mice after treatment with diminazene aceturate. T. congolense IL 3274 was used for these studies. In initial work, experiments were carried out to examine the trypanosome populations which reappeared in infected mice after treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. Inocula of 102, 103, 104, 105, and 106 trypanosomes were injected i.v. into large numbers of mice. Immediately thereafter, their susceptibility to i.p. treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. was determined. In the absence of treatment, trypanosomes were detected in the blood of all mice that were infected. In additional work, infectivity was demonstrated at the level of individual trypanosomes (data not shown). In mice which were infected and treated, however, different proportions of animals were cured, depending on the inoculum size. The number of mice that became parasitemic, despite treatment, progressively increased from 4 in 50 to 39 in 50 mice from the smallest (102-trypanosome) to the largest (106-trypanosome) inoculum sizes used. A close fit to these data was obtained when the data were modelled by logistic regression. The observed data were also analyzed by using a mathematical model to determine the proportions of diminazene-resistant and -susceptible trypanosomes that were present in the inoculum populations. The model assumed that these proportions were fixed constants, regardless of the inoculum size. The resultant data indicated that with all five inoculum sizes, less than 0.1% of the trypanosomes were resistant to the drug dosage used. Thus, the majority of the populations appeared to be susceptible to diminazene aceturate at this dosage. Surprisingly, however, the proportion of the population that contained resistant trypanosomes did not remain constant but varied inversely with the inoculum size. The above data indicate that the subpopulation of T. congolense IL 3274 which reappeared in infected animals after treatment with diminazene aceturate at a dose of 25 mg/kg of b.w. contained trypanosomes that were both resistant and susceptible to the drug dosage used. There could be several ex- Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV k31 (h21) k21 (h21) Vc (liters/kg) MAMMAN ET AL. TABLE 4. Pharmacokinetic parametersa of diminazene in noninfected and T. congolense-infected mice after administration of the drug at doses of 15.0 mg/kg of b.w. i.v. and 25.0 mg/kg of b.w. i.p. 1110 VOL. 39, 1995 T. CONGOLENSE RESISTANCE TO DIMINAZENE ACETURATE 1111 TABLE 5. Values of noncompartmental pharmacokinetic parametersa of diminazene for noninfected and T. congolense-infected mice after administration of the drug at single doses of 15.0 mg/kg of b.w. i.v. and 25 mg/kg of b.w. i.p. Route of drug administration (group) i.p. (noninfected) i.p. (infected) i.v. (noninfected) a b Cmax (mg/ml) AUC0–` (mg z h/ml) AUMC0–` (mg z h2/ml) MRT (h) CL (ml/min/kg) Vd(ss) (liters/kg) F (%) 17.49 6 10.25* 14.89 6 10.05‡ 3.19 6 1.17*‡ 9.70 6 1.83 13.33 6 3.83 9.14 6 1.92 174.76 6 67.23 785.41 6 914.91 218.40 6 111.29 17.89 6 5.39 50.04 6 46.24 22.97 6 8.65 28.25 6 5.73 28.62 6 5.73 28.28 6 5.73 47.32 6 15.24 84.29 6 54.68 36.99 6 10.52 66.16 6 20.97 87.00 6 15.71 NAb Values with the same symbols (* and ‡) in the same column are significantly different (P , 0.05). The values are arithmetic means 6 standard deviations. NA, not applicable. FIG. 1. Relationship between T. congolense IL 3274 inoculum size and the proportion of the population resistant to diminazene aceturate at a dose of 25 mg/kg of b.w. (using data on observed parasitemia from Table 1). mice. The results indicated that the diminazene aceturate CD50 value for mice infected with 106 trypanosomes was 29.88 mg/kg of b.w. and was significantly greater than that for mice infected with 103 trypanosomes, 22.28 mg/kg of b.w. These observations therefore indicate that there is a direct relationship between the inoculum size and the level of resistance of a population, as described by Sones and Holmes (48). The object of the second experiment was to determine whether the inverse relationship between trypanosome inoculum size and the proportion of surviving trypanosomes occurred because the parasites were exposed to decreasing concentrations of the drug as the inoculum size was increased as a result of the effects of parasite numbers on drug pharmacokinetics. The resultant data indicated that the disposition of diminazene did not vary significantly with the trypanosome inoculum size. Lastly, it is possible that the observed apparent decrease in the proportion of drug-resistant trypanosomes with increasing inoculum sizes was associated with improvements in the anti-variant-specific surface glycoprotein antibody responses as the inoculum size was increased. However, in light of the inverse linear relationship between log10(trypanosome inoculum) and log10(proportion of trypanosome population resistant to diminazene), we do not believe that this is a logical explanation for a number of reasons. Firstly, the relationship between these two parameters, as shown in Fig. 1, indicates that for every 10-fold increase in the size of the trypanosome inoculum there was a decrease in log10 (proportion of diminazene-resistant trypanosomes) by 0.692 6 0.031. Thus, if antibody responses were responsible for these observations the functional activity of the antibody response would have had to increase by the same proportion with each 10-fold increase in inoculum size. Such a linear relationship between the antibody response and the quantity of trypanosome antigen is inconsistent with experimental findings (8). Furthermore, by extrapolation from experiments carried out with cattle, it would appear that the smallest inoculum sizes used in our experiment would be insufficient to elicit an anti-variant-specific surface glycoprotein response (32). The maximum concentration that a trypanosome population attains in rodents is regulated by both host factors (7, 8) and host-independent mechanisms associated with the parasite population (9). The results described here have demonstrated that diminazene susceptibility is dependent upon the concentration of a trypanosome population in vivo. Other studies have shown that the concentration of a population may influence the rate of proliferation of individual trypanosomes (9, 10), thus potentially altering the proportions of the population in the different stages of the cell division cycle. While little is known about the mechanism of action of diminazene on trypanosomes, evidence from other systems indicates that diminazene acts on organisms at specific stages of the cell division cycle (11, 26, 42, 50). A full explanation of the results observed Downloaded from http://aac.asm.org/ on April 25, 2014 by PENN STATE UNIV planations for this observation. First, it has been shown that populations of T. brucei in mice (23) and Trypanosoma vivax in goats (52) can evade treatment and establish relapse parasitemias by invading the central nervous system. It is therefore possible that a similar phenomenon accounted for the presence of the drug-susceptible trypanosomes that were observed in the relapse populations described herein, although immediate treatment should have circumvented this possibility. Secondly, sites other than the central nervous system may serve as cryptic foci (51). Thus, since T. congolense can localize in the microvasculature (5, 24, 30), such sites may serve as cryptic foci for the parasite if they are poorly perfused with blood. A third possibility is that the diminazene resistance of T. congolense IL 3274 is unstable, as has previously been reported for isometamidium in T. congolense (40). It is therefore possible that during expansion of the trypanosome population, a drug-susceptible variant with a growth rate more rapid than that of a drug-resistant population dominates the relapse population (49). However, since there appeared to be little variation in diminazene susceptibility among clones derived from T. congolense IL 3274, this appears to be unlikely. The present study also demonstrated that the level of diminazene resistance of T. congolense IL 3274 varies directly with the inoculum size. However, the calculated drug-resistant proportion of each population varied in an inverse linear manner with the inoculum size (Fig. 1). The reasons underlying this last observation were investigated in two experiments. In the first, we examined whether the resistance of the trypanosome population decreased as the inoculum size was increased. This question was investigated by characterizing the susceptibilities of relapse populations of two inoculum sizes (103 and 106 trypanosomes) to different doses of diminazene aceturate in 1112 MAMMAN ET AL. ACKNOWLEDGMENTS We thank C. Robertson of the University of Strathclyde, Glasgow, United Kingdom, and J. Rowlands, International Livestock Centre for Africa, for discussion concerning statistical analyses. B. Williams of the London School of Tropical Medicine and Hygiene, London, United Kingdom, is also thanked for helpful discussions. M. Mulindi is thanked for secretarial assistance. REFERENCES 1. Aliu, Y. O., M. Mamman, and A. S. Peregrine. 1993. Pharmacokinetics of diminazene in female Boran (Bos indicus) cattle. J. Vet. Pharmacol. Ther. 16:291–300. 2. Aliu, Y. O., and S. 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