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Clinical efficacy and plasma concentrations of two formulations of buparvaquone in cattle infected with East Coast fever (Theileria parva infection)
Research in Veterinary Science 81 (2006) 119–126 www.elsevier.com/locate/rvsc
Clinical efficacy and plasma concentrations of two formulations of buparvaquone in cattle infected with East Coast fever (Theileria parva infection) G.R. Muraguri a, P.N. Ngumi a, D. Wesonga a, S.G. Ndungu a, J.M. Wanjohi a, K. Bang b, A. Fox b, J. Dunne b, N. McHardy c,* b
a National Veterinary Research Centre, Muguga, P.O. Box 32, Kikuyu, Kenya Cross Vetpharm Group Ltd., Broomhill Road, Tallaght, Dublin 24, Republic of Ireland c TBD Consulting Ltd., Baltyboys, Blessington, Co. Wicklow, Republic of Ireland
Accepted 29 September 2005
Abstract East Coast fever, caused by the protozoan parasite Theileria parva, kills about 600,000 cattle annually in Africa. The hydroxynaphthoquinone compound buparvaquone (BPQ) is curative. Sixteen calves were infected with T. parva. On manifestation of disease symptoms, eight were injected with the original (pioneer) BPQ product and eight with a test product containing BPQ. All 16 calves were cured by one injection of 2.5 mg BPQ/kg bodyweight. The concentration of BPQ in blood plasma was monitored by HPLC. The mean observed Cmax of BPQ was 0.229 and 0.253 lg/mL of plasma, the mean observed time to reach this concentration (Tmax) was 2.62 and 2.12 h and the AUC (area under curve) was 4.785 and 4.156 lg h/mL, respectively, for the pioneer and test product. Considerable variations occurred in the plasma concentration of BPQ within each group. They showed no relationship with either clinical or parasitological parameters following treatment. 2005 Elsevier Ltd. All rights reserved. Keywords: Buparvaquone; Plasma concentrations; Theileria; Chemotherapy
1. Introduction This study on the relationship between clinical response to treatment and plasma concentrations of the curative drug buparvaquone (BPQ) was part of a programme to develop a generic BPQ product for the treatment of East Coast fever (ECF) of cattle. Kinetics studies in support of a drugÕs development are usually conducted in uninfected animals. However, an additional objective of this study was to obtain information on the product that might be helpful to veterinary practitioners who may use it, so the study was done in cattle infected with ECF. ECF is a tick-transmitted disease caused by the protozoan parasite Theileria parva. It kills at least 600,000 cattle *
Corresponding author. Tel.: +353 1 4515522; fax: +353 14515803. E-mail address: [email protected] (N. McHardy).
0034-5288/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.09.012
annually in Eastern and Southern Africa (Mukhebi et al., 1992). The mortality rate is more than 90% in European breeds of cattle and around 50% in indigenous breeds. Extrapolation from MukhebiÕs findings to the present indicates that the total cost of East Coast fever is around US$250 million per year in production losses and the cost of attempts at controlling the disease. BPQ and parvaquone (PQ) are both hydroxynaphthoquinone (HNQ) drugs. Their efficacy in the treatment of ECF was first reported by McHardy et al. (1983) and McHardy et al. (1985), respectively. Separate field trials in clinical cases of ECF (Chema et al., 1986; Dolan et al., 1992; Wanjohi et al., 1995) and a comparative field trial (Muraguri et al., 1999) all obtained cure rates of around 90% with each drug. Kinabo and Bogan (1988) investigated the plasma pharmacokinetics of commercially available injectable products
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containing PQ or BPQ in uninfected cattle, using a highperformance liquid chromatography (HPLC) method. PQ was injected at a dose of 20 mg/kg bodyweight (twice the recommended dose), BPQ at 2.5 mg/kg, its recommended dose rate. PQ reached a maximum concentration in plasma of about 6.4 lg/mL 0.8 h after injection; BPQ reached 0.10 lg/mL after 3.2 h. The area under the curve (AUC) was 31.27 and 3.43 lg h/mL, respectively, for PQ and BPQ. McHardy and Mercer (1984), using a bioassay, found no relationship between plasma concentrations of parvaquone (PQ) in individual cattle infected with ECF and their clinical and parasitological response to treatment. 2. Materials and methods
2.3. Infection with ECF Sixteen of the calves were selected at random from the 20 candidates and infected by the subcutaneous injection of 1 mL of a 1-in-10 dilution of T. parva KARI sporozoite stabilate 316, above and in front of the left prescapular lymph node. Stabilate 316 is the NVRC ‘‘standard’’ stabilate of the Marikebuni isolate of T. parva. The day of infection was designated as day 0 of the study. In numerous previous studies using this infective dose of this stabilate, it consistently produced mortality rates of about 90% in untreated cattle, usually between 15 and 20 days after infection (data on file at NVRC). For this and for ethical and animal welfare reasons, no infected untreated control cattle were included in the study.
2.1. Buparvaquone 2.4. Treatments BPQ, 2-trans(4-t-buytylcyclohexyl)methyl-3-hydroxy1,4-naphthoquinone was used as the pioneer product (PP) Butalexe (Schering-Plough Animal Health, Harefield, Great Britain) or the test product (TP) Buparvexe (Bimeda, Dublin, Ireland). Each product is a solution for intramuscular injection containing 50 mg BPQ/mL dissolved in N-methylpyrrolidone and fractionated coconut oil, with a surfactant and an emulsifier as necessary to give a stable, non-irritant, pharmaceutical preparation. The recommended dosage for each product is 1 mL/20 kg bodyweight (2.5 mg BPQ/kg) delivered into the neck muscles. 2.2. Cattle Twenty male calves, approximately 75% Friesian (Bos taurus) phenotype and 25% Zebu (B. indicus) were used. They were aged 6–9 months and in good health. Their mean bodyweight was 145.2 kg (range 93–228). They were obtained from a farm that practiced strict tick control and had no recent history of ECF. Their susceptibility to ECF was confirmed by the indirect fluorescent antibody test (IFAT, Burridge and Kimber, 1972) before they were transported by road to the test site, the National Veterinary Research Centre (NVRC), Kikuyu, Kenya. On arrival they were ear-tagged with unique numbers and subjected to a thorough clinical examination. They were weighed and a jugular venous blood sample was taken for a confirmatory IFAT, and from which a blood smear was prepared for staining and microscopical examination for the presence of haemoparasites. The cattle were treated for endoparasites and ectoparasites, respectively, with levamisole/oxyclozanide (Nilzan Plus) and deltamethrin (CooperÕs Spot-on) (both products Schering-Plough Animal Health, Harefield, Great Britain). They were then quarantined at NVRC for five days before the start of the study. The cattle were housed in a purpose-built tick-free barn and provided with good quality tick-free hay and clean drinking water at all times. Concentrated rations were provided twice daily in amounts necessary to maintain a good plane of nutrition.
Alternate infected calves were treated with a single injection of either the PP or TP presentation of BPQ in the order in which they developed clear clinical and parasitological signs of ECF. Seven calves were injected with each product on the second consecutive day on which their rectal temperature was 39.5 C or higher and macroschizonts of T. parva were present in stained lymph node smears, designated as day ‘‘TS2’’. This treatment time was chosen to maximise the chances of curing with a single injection so as not to complicate the drug kinetics aspects of the study. Treatment was administered intramuscularly on the right side of the neck. Both products were used at the rate of 2.5 mg BPQ/kg bodyweight. Two calves failed to attain TS2 by day 14 although one had shown fever and both had shown parasitosis on at least one day. One of these calves was treated with PP and one with TP on day 14-post infection. Two of the four uninfected calves were similarly injected with each of the products between day 9 and day 13, to fit conveniently into the complex treatment and sampling schedule. 2.5. Observations and collection of samples All calves were examined clinically twice daily throughout the quarantine period and the study. Any signs of abnormality, particularly those symptomatic of ECF, were noted. These include enlarged superficial lymph nodes, respiratory distress, nasal discharge and other signs of pulmonary oedema, inappetance, weight-loss and dehydration. From the time of injection of PP or TP, the calves were examined for signs of reaction to, or intolerance of, the treatment, including injection site reactions. Rectal temperature was taken daily before 10.00 hrs using a clinical thermometer and the calves were weighed weekly. A sample of jugular blood was taken by heparinised vacutainer on the first day of the 5-day quarantine period (day –5), the day of infection with T. parva (day 0), then three times per week until clinical recovery and thereafter weekly until the end of the study. All blood samples were
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taken from the left jugular vein, i.e., contra-lateral to the site of drug injection. The blood was used to assay packed cell volume (PCV) and to prepare blood smears. The smears were stained with GiemsaÕs stain and examined for parasitaemia of T. parva piroplasms and for intercurrent haemoparasites, using an oil immersion objective. Needle biopsy smears, using a disposable 18 G · 1.500 hypodermic needle, were taken from the left prescapular lymph node (LPN), i.e., the node draining the site of infection but contra-lateral to the site of BPQ injection, daily from day 5 until clinical and parasitological recovery, then twice weekly. They were stained with GiemsaÕs stain and examined for macroschizonts and microschizonts of T. parva and for signs of lymphoblastosis, using an oil immersion objective. Daily sampling was resumed in the event of clinical or parasitological signs of ECF. Right prescapular lymph node (RPN) smears were prepared daily from the day after schizonts were first observed in the LPN and subsequently at the same times that LPN smears were prepared. Lymphoblastosis and macroschizont parasitosis were each ‘‘scored’’ on a scale of 0–3, representing a range from ‘‘absent’’ to ‘‘advanced’’ blastosis or parasitosis (Muraguri et al., 1999). The presence of microschizonts, and any signs of damage to the macroschizonts following drug treatment, were also noted.
each calf, then at 0.5, 1, 2, 4, 8 and 24 h and 2, 3, 4, 7 and 10 days after treatment. When more than one calf was treated on a single day, care was taken to allow sufficient time between treating them to ensure that all blood samples could be taken at the intended times. The samples were centrifuged at 3000g for 20 min to sediment blood cells then at least 4.5 mL of plasma was withdrawn using a Pasteur pipette and placed in plastic, screw-cap, 5 mL vials. The vials were labelled with the calf number, date and time of sampling, but not the identity of the product with which the calf was treated, so as to maintain a degree of ‘‘blinding’’ of samples during the analytical phase. The samples were then frozen to 20 C. When all the samples had been collected and frozen, they were thawed and heated for 30 min in a water bath at 56 C then re-frozen at 20 C. All manipulations were performed out of direct sunlight. The samples were then transported by air to Cross VetpharmÕs analytical laboratory in Ireland in an insulated container packed with ice at 20 C. Heating the samples to 56 C for 30 min was a condition of the licence to import the samples into Ireland. Repeated heating and freezing of plasma samples in this way was shown not to affect either the accuracy or sensitivity of the assay of their buparvaquone content using the methods employed in this study (data on file).
2.6. Serology
2.9. Assay of buparvaquone in plasma samples
Blood samples were taken on four occasions from the left jugular vein into untreated vacutainers while on the supplying farm, immediately following arrival at NVRC, and then on days 0 and 35 of the study. The blood was allowed to clot at ambient temperature and serum was withdrawn approximately 2 h later. The samples were assayed for antibody to T. parva by the IFAT (Burridge and Kimber, 1972) using a macroschizont antigen.
The plasma samples were stored frozen at 20 C, then thawed by immersion in a water bath at 37 C immediately before assay. The remainder of the samples were then refrozen. The samples were analysed for their buparvaquone content according to Kinabo and Bogan (1988) by highperformance liquid chromatography (HPLC, Walters Millennium) using a Waters Spherisorb S50DS2, 25 cm · 4.6 mm analytical column and ultra-violet (UV) detection at 252 nm. The methodology was validated for accuracy by assaying control plasma samples spiked with a range of five concentrations of buparvaquone from 0.025 to 0.150 lg/mL of plasma, in six replicates (Table 3). Precision and between-day variability of the analytical method was monitored by assaying plasma samples spiked with 0.04 or 0.14 lg BPQ/mL on each of the six days on which test samples were analysed (Table 4). All plasma samples were spiked with parvaquone as the internal standard. Both compounds were extracted from the plasma samples with diethyl ether. The ether extract was dried under nitrogen at 50 C and the residue was dissolved in methanol. Analyses were performed using a mobile phase of 850:150 methanol:0.05 M sodium acetate buffer, pH 5.0. Analytical results were collated using Waters Millennium 32 Chromatography data handling software. These data were used to demonstrate Cmax (maximum observed concentration), Tmax (observed time after treatment to reach Cmax) and AUC (area under the curve) calculated using the trapezoidal method, of BPQ. Sample identity was not ‘‘unblinded’’ until the time of data analysis. For clarity in the presentation
2.7. Analysis of clinical and parasitological data Data were entered into appropriate data sets in Excel (Microsoft Corporation, USA). Descriptive analyses were carried out in Excel. Descriptive statistics involved calculation of means and standard deviations to assess variability. A multiple regression was done with the CSS Statistical package (Statsoft Inc., Tulsa, USA) using AUC or Cmax as the independent variable and time to clinical cure and time to clear parasitosis as dependent variables from the 14 infected and treated animals. The treatment groups were combined as there was no difference between them with regard to any of the parameters used. 2.8. Blood sampling for the assay of BPQ concentrations in plasma A 10 mL sample of blood was taken from the left jugular vein, i.e., contra-lateral to the site of drug injection, by heparinised vacutainer immediately before treatment of
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of results, BPQ concentrations are expressed either as lg/mL or ng/mL of plasma in some instances. 2.10. Sensitivity and reproducibility of analytical methodology Buparvaquone recovery from plasma samples spiked with five concentrations of BPQ from 0.0250 to 0.1500 lg/ mL was between 123.97% and 80.87%, mean 86.87%, SD 2.87–6.52 (n = 6). The calibration curve was rectilinear (r = 0.99) over the concentration range studied. The limit of detection (LOD) for BPQ was 0.013 lg/mL (13.0 ng/ mL), the limit of quantitation (LOQ) was 0.025 lg/mL (25.0 ng/mL). The analytical methodology was shown to be both accurate and reproducible (Tables 3 and 4). LOD (0.013 lg/mL) was almost identical to that found by Kinabo and Bogan (1988) i.e., 0.015 lg/mL, and the mean % recovery of BPQ from the samples (86.87%) was also comparable to that of Kinabo and Bogan (91.88%). Kinabo and Bogan make no mention of LOQ, with which the value reported here (0.025 lg/mL) can be compared. 3. Results 3.1. Clinical observations during quarantine All 20 cattle were in good health on arrival at NVRC and during the quarantine period at NVRC, apart from two that had mild conjunctivitis with lachrymation. Treatment with eye ointment containing cloxacillin benzathine (Orbenin Opthalmic Ointment, Pfizer Animal Health, New York, USA) quickly controlled the condition in one calf but in the other, 678, the condition was incompletely resolved and treatment continued throughout the study. PCV ranged from 24% to 41%, which is within normal ranges. No intercurrent haemoparasite infections were detected in blood smears. The T. parva IFAT titres of all 20 calves were negative (at 1 in 40 dilution) while on the farm and on day 0 of the study. The four uninfected calves remained clinically normal throughout the study, apart from the eye condition of 678, and their IFAT titres were still negative on day 35.
smears. All 16 had IFAT titres of at least 1 in 160 on day 35, confirming that they had been infected with T. parva. Fourteen calves attained the infection status of ‘‘TS2’’, the criterion for treatment, between days 9 and 14 (Table 1). Maximum temperatures were 39.9–40.7 C. There were no significant differences between the two treatment groups. Two calves did not attain TS2 status. Calf 674 developed fever (39.9 C) on day 12 only and calf 664 developed its highest temperature, 39.2 C, on day 13. These two calves were treated on day 14 none-the-less. They are excluded from the clinical and parasitological analyses (Tables 1 and 2) but they are included in the kinetics results shown in Table 5. At the time of treatment all 14 calves that attained TS2 status had macroschizonts in both prescapular lymph node smears. Schizont scores were 1–3 in the LPN and 1–2 in RPN smears. Small numbers of microschizonts were observed in seven calves. Only five calves had developed piroplasm parasitaemia by day TS2. It was less than 1% in all cases. PCV did not vary significantly throughout the study (ECF causes anaemia only rarely) and no intercurrent haemoparasitic infections were observed. All 14 calves showed mild or moderate clinical signs of ECF, including greatly enlarged superficial lymph nodes in all cases and dullness and mild signs of pulmonary oedema in some. None was regarded as suffering from severe ECF. The two calves that did not attain TS2 status showed no clear signs of ECF except enlarged lymph nodes. 3.3. Response to treatment Rectal temperature fell below 39.5 C within one day in all seven calves treated with TP and in five treated with PP (Table 2). In the other two it was below 39.5 C on the second day after treatment. Subsequently, temperatures
Table 2 Comparison of rectal temperature (C) in groups of calves infected with T. parva and treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight, group mean and (standard deviation) Days after treatment
3.2. Response to infection with T. parva stabilate All 16 calves developed parasitological signs of T. parva infection, with macroschizonts observed in lymph node
Treated PP (n = 7) Treated TP (n = 7) p Value of difference
0
1
2
3
39.9 (0.29) 40.0 (0.39) 0.82
38.9 (0.53) 38.6 (0.29) 0.66
38.4 (0.25) 38.5 (0.27) 0.58
38.4 (0.21) 38.3 (0.20) 0.56
Table 1 Time to first parasitosis, first fever and to treatment in groups of calves treated with BPQ 2.5 mg/kg bodyweight Days after infection to:
Treated PP (n = 7) Treated TP (n = 7) p Value of difference
Macroschizonts in LPN
Fever 39.5 C
Mean
SD
Mean
SD
Mean
SD
8.6 9.7
0.53 1.60
9.9 11.0
2.10 0.82
11.4 12.1
1.10 1.10
0.07
PP, pioneer product (Butalexe); TP, test product (Buparvex).
0.06
Treatment (day TS2)
0.07
G.R. Muraguri et al. / Research in Veterinary Science 81 (2006) 119–126 Table 3 Accuracy of analytical methodology BPQ concentration in spiked sample (lg/mL)
BPQ recovery % mean
SD
%CV
0.0250 0.0500 0.1000 0.1250 0.1500
123.967 90.534 80.867 84.2130 84.0530
6.399 6.519 3.252 2.871 5.319
5.162 7.200 4.022 3.409 6.328
(n = 6) (n = 6) (n = 6) (n = 6) (n = 6)
Mean recovery of BPQ from plasma samples (six replicates) spiked with five concentrations of BPQ. %CV = % coefficient of variation.
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With the exception of enlargement of superficial lymph nodes, all clinical signs of ECF had resolved within two days of treatment with either product. None of the animals required additional treatments for ECF or for any other condition. No adverse signs of any sort due to the treatments were observed in any animal. Mean bodyweight in the group treated with PP was 141.4 kg on day 0 and 147.8 kg on day 35. For those treated with TP it was 149.7 and 154.9 kg, respectively. The differences between groups are not significant. 3.4. BPQ concentrations in blood plasma
Table 4 Day-to-day variability of analytical methodology BPQ concentration in spiked sample (lg/mL)
BPQ concentration detected (mean) lg/mL
SD
%CV
0.040 0.140
0.0386 (n = 5) 0.1459 (n = 5)
0.0025 0.0131
6.4008 8.9786
Mean concentrations of BPQ found in plasma samples spiked with two concentrations of BPQ on five occasions.
remained within normal ranges in all calves. Mean temperatures, throughout, were not significantly different between the treated groups. Following treatment, the mean number of days taken for both lymph node smears to become negative for macroschizonts was 4.14 days (SD 0.69) for treatment PP and 3.86 days (SD 1.21) for TP; the difference is not significant. On the day following treatment most macroschizonts appeared to be undamaged by either product but on day 2 damage was obvious in 43% of the smears of calves treated with PP (two smears, LPN and RPN, from each calf) and 71% of those treated with TP. By day 3, schizonts were clearly damaged in all smears in which they were still observable. The damage appeared first as a blurring of the outline, and reduction in the intensity of staining, of the schizont nuclei followed by apparent ‘‘dissolution’’ of the schizont remains into the cytoplasm of the host cell, leaving pyknotic nuclear debris in some instances. All blood smears were negative for piroplasms from three days after treatment until the end of the study.
Sensitivity and reproducibility of the analytical methodology is shown in Tables 3 and 4. The results of analyses of BPQ concentration in plasma (Table 5) include the two calves that did not attain TS2, but which were treated, none-the-less, on day 14. The mean highest observed plasma concentration (Cmax) of BPQ following treatment with PP was 229.0 ng/mL (0.229 lg/mL). It ranged from 32 to 589 ng/mL in individual animals. Observed Cmax after TP treatment was 252.7 ng/mL but the range was smaller, 183 to 449 ng/mL. The mean time after treatment at which maximum BPQ concentration occurred (Tmax) was 2.62 h for PP and 2.12 h for TP. Mean area under the curve (AUC) was 4.7847 lg h/mL (range 1.0822–7.2524) for PP and 4.1588 (range 3.1299–5.8775) for TP. None of these differences is statistically significant. BPQ concentration data from the pairs of uninfected calves treated with PP or TP were within the ranges of the equivalent groups of calves infected with T. parva (Table 6), but one, 678, had lost 5.2% bodyweight by day 35, possibly due to its poorly responsive eye infection. Table 6 Plasma concentration of BPQ parameters in pairs of uninfected calves treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight Parameter
Treatment
Range
Mean
Cmax BPQ (ng/mL)
PP TP PP TP
131 and 319 279 and 402 2 and 2 2 and 2
225.0 340.5 2.0 2.0
Tmax BPQ (h)
Table 5 Comparison of kinetic, clinical and parasitological parameters in groups of calves infected with T. parva and treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight Parameter
Treated with
Range
Mean (SD)
AUC (lg h/mL) (n = 8)
PP TP TP TP PP TP PP TP PP TP PP TP
1.082–7.252 3.130–5.877 32–589 183–449 1–4 1–4 0–1.8 0–1.9 2–5 2–5 1.0 to +4.0 1.6 to +3.5
4.785 (2.097) 4.156 (0.924) 229.0 (187.6) 252.7 (81.8) 2.62 (1.19) 2.12 (0.83) 0.81 (0.70) 1.29 (0.60) 4.15 (0.69) 3.86 (1.21) +1.39 (1.51) +0.85 (1.77)
Cmax BPQ (ng/mL) (n = 8) Tmax BPQ (h) (n = 8) Fall in temperature 24 h after treatment (C) (n = 7) Days from treatment to parasitic cure (n = 7) % weight change day 14–35 (n = 7)
p Value of difference 0.75 0.34 0.18 0.06 0.32 0.70
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Table 7 Comparisons between two kinetic parameters Cmax and AUC, and two clinical observations CURE and CLEAR, in 14 calves infected with ECF and treated with buparvaquone, 2.5 mg/kg bodyweight Comparison
R2
Adjusted R2
F
‘‘p’’ Value
Significance
Cmax vs. CURE Cmax vs. CLEAR AUC vs. CURE AUC vs. CLEAR
0.01403 0.08562 0.11609 0.00007
0.06814 0.00942 0.04244 0.08326
0.17072 1.12361 1.57611 0.00086
0.68676 0.31001 0.23322 0.97713
ns ns ns ns
Following multiple regression, the multiple R2 for AUC and Cmax for ÔTime to CUREÕ and ÔTime to CLEARÕ are given in Table 7. These show that there was little correlation between kinetic and clinical parameters. 4. Discussion The response of the calves to infection with this dose of T. parva (Marikebuni) sporozoite stabilate KARI 316 was as expected from previous findings (NVRC files, unpublished data). Fourteen of the 16 infected calves attained TS2 between nine and 14 days after infection. Based on extensive past experience, it is likely that if they had not been treated with BPQ to control the infection at least 12 and possibly all 14 would have died of ECF within about one week. Some isolates of T. parva produce variable patterns of ECF, including temporary remission of symptoms followed by severe, often fatal, disease (Dolan et al., 1984). Stabilate 316 typically induces a progressive disease syndrome without remission so it seems unlikely that the two calves that did not reach TS2 would have developed more severe ECF if they had not been treated. Their exclusion from the clinical assessment, but their inclusion in the kinetics section of this study, for which the stipulation was only that they should be infected with T. parva, therefore seems justified. Their infection status was confirmed by the observation of macroschizonts in lymph node smears between days 9 and 13 and by their positive IFAT titres on day 35. At the time of treatment, day TS2, the 14 calves in the clinical study were mostly suffering from mild clinical signs of ECF. None was severely affected and all 14 were quickly cured with a single injection of either product, PP or TP. There was no statistically significant difference in the rate of cure or clearance of parasites (Table 5) with the two products. None of the calves developed a recrudescence of the disease that required additional treatment following initial cure, which may be necessary when more severe cases are treated (Dolan et al., 1992). The objective of avoiding the need for additional treatments that would have complicated the plasma concentration study, by treating the calves relatively early in the disease syndrome, therefore was achieved. The study produced results on the mean concentration of BPQ in plasma broadly similar to those of Kinabo and Bogan (1988) for product PP. The present study gave higher Cmax of BPQ than Kinabo and Bogan found
(0.229 compared with 0.102 lg/mL), slightly earlier Tmax (2.62 compared with 3.17 h) and higher AUC (4.785 compared with 3.43 lg h/mL). These differences are probably within the expected range of variation between studies by different researchers in different laboratories. Kinabo and Bogan used healthy, uninfected calves while in the present study the calves were undergoing a clinical episode of ECF. The fever and widespread oedema associated with ECF might have been expected to affect the rate of distribution and metabolism of BPQ, resulting in greater differences between results of the two studies than were actually found. However, the observed concentrations of BPQ in the pairs of uninfected control calves in this study were similar to those of the infected principals, albeit that the control group size, two calves, is too small for statistical comparison. This said, the results suggest that the moderate disease state did not greatly influence the kinetics of BPQ. Our results also demonstrate no obvious relationships between any of the plasma concentration parameters and any of the clinical or parasitological parameters in individual calves – apart from the obvious one, that the disease was cured quickly and completely by treatment with BPQ. The observed Cmax of BPQ in the plasma of individual calves treated with PP ranged from 32 to 589 ng/mL, Tmax was reached in 1–4 h and AUC was 1.082 to 7.252 lg h/mL. The range of Cmax following treatment with TP was less broad (183–449 ng/mL), Tmax was similar (1–4 h) and the range of AUC was less (3.130– 5.877 lg h/mL). These differences are not significant. McHardy and Mercer (1984), using a bioassay, not HPLC, also found no relationship between the concentration of another HNQ compound, parvaquone, in the blood of individual cattle suffering from naturally acquired ECF of varying severity and their clinical response to the treatment. It is possible that if more severe cases had been treated, particularly if some animals died despite treatment, correlation with plasma kinetics might be found. Similarly, a relationship might have been found if additional dose rates of BPQ had been studied, particularly lower ones. However, the present study was conducted in accordance with guidelines for the marketing approval of a generic product which stipulate only that the comparison should investigate the recommended dose rate of the pioneer and test products. The in vitro EC50 (the concentration that eliminates the schizonts from 50% of cultured lymphoblasts infected with
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T. parva in an exposure period of 48 h) for BPQ is 3 ng/mL (0.003 lg/mL, 6.1 · 1010 M, Hudson et al., 1986). This concentration was exceeded in the plasma of all 16 infected cattle and it was maintained for at least three days in most of them. The mean Cmax of BPQ in the animals treated with PP was 76-times, and in those treated with TP 84-times, its in vitro EC50. Even the animal with the lowest AUC of BPQ concentration (no. 672, treated with PP) may have met EC50 criteria. Its plasma concentration was 0.032 lg/ mL in the 4-h sample (i.e., 10-times EC50) and 0.028 lg/ mL at 24 h. A concentration of 0.008 lg/mL (2.7-times EC50) was indicated in the 48 and 72-h samples. However, these latter values are below the validated LOD level of 0.013 lg/mL, so no reliance should be placed on them. Clear signs of damage to macroschizonts, characterised by disorganisation of their nuclear particles, were not seen until two days following treatment in most animals. Under the electron microscope, signs of vaccuolation of the schizont cytoplasm and disruption of membranous organelles in the cytoplasm were seen within 3 h of treatment with PP at the dosage used in this study, in cattle suffering from ECF (N. McHardy and T.T. Dolan, unpublished data). Similar changes were seen in T. parva piroplasms in the blood of these animals, starting only 1 h after treatment. Damage to schizont and piroplasm nuclei and to the limiting membranes of the parasites, however, was not obvious until 48 h after treatment. This tallies well with light-microscopical observations in the present study and with the proposed mode of action of the hydroxynaphthoquinones against sporozoan parasites, on their mitochondrial electron transport mechanisms (Hudson et al., 1985). Under the electron microscope, host cell mitochondria appeared to be unaffected which indicates the specificity of the anti-theilerial action of the hydroxynaphthoquinones, and it helps to explain their wide therapeutic index, indicated by the complete absence of adverse signs of treatment in the present study. By far the greatest biomass of T. parva schizonts is located within lymphoblastoid cells in lymphoid tissues, not in the blood. Medlicott et al. (2004) state that low molecular weight (mw) molecules (which would include BPQ, mw 326.44) predominantly enter blood capillaries, not lymphatics, after intramuscular injection. Subsequently, the concentration in lymph should be related directly to that in the plasma, but BPQ may show a propensity to concentrate in lymphoid tissue. The kinetics of BPQ in lymph and lymphoid tissue have not been studied, but it seems possible that they could relate more closely with clinical and parasitological responses than the plasma-based findings reported here. In earlier studies (McHardy et al., 1985; Muraguri et al., 1999), damage to T. parva piroplasms in Giemsa-stained blood smears was obvious within one day of treatment with BPQ. Two days after treatment almost all the piroplasms were pyknotic. This tallies well with the findings in the present study and with the electron microscopic observations of McHardy and Dolan (unpublished).
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Also of interest is the lack of statistical difference between the mean values for plasma concentrations and parasitological and clinical parameters for the two BPQ products studied here, PP (Butalex) and TP (Buparvex). This formulation of BPQ as a solution for intramuscular injection was ‘‘optimised’’ in a series of studies with three HNQs, menoctone (McHardy and Rae, 1981), parvaquone (McHardy et al., 1983) and buparvaquone (McHardy et al., 1985). It was found that apparently minor differences in formulation could result in major changes in plasma concentrations and clinical performance. Products PP and TP are formulated essentially similarly but it is likely that their constituents – BPQ and excipients, are sourced from different manufacturers. Small differences between constituents from different manufacturers may account for the greater variability in plasma concentrations, but apparently not of the clinical performance, of PP compared with TP in this study. Clinical field trials of products to treat ECF have consistently shown that cure rates approach 100%, even in B. taurus breeds, when mild cases are treated with a single injection of BPQ or two of PQ. Additional injections may be necessary to cure more advanced cases. Cure rates fall to around 40% in severe cases, even if multiple treatments are given (Azuba Musoke et al., 2004). It is possible that small differences between the performance of PP and TP may be more apparent under field conditions, and particularly in cases more severe than in the present study. However, an alert cattle owner, and certainly a veterinary practitioner, should be able to recognise the clinical signs of ECF that were present at the time of treatment in this study – fever and clearly enlarged superficial lymph nodes in all 14 calves and laboured respiration in some. In all 14 cases, examination of a lymph node smear would have confirmed the provisional diagnosis. Cattle owners, therefore, should be encouraged to recognise the early signs of ECF and to summon a veterinary practitioner immediately if they are seen. If this were done, it is likely that cure rates would increase substantially. The number of treatments necessary to produce cure and the number of veterinary attendances would be less, greatly reducing the cost of management of ECF. Acknowledgements We thank the laboratory and farm staff, at both NVRC and Cross Vetpharm, for their expert assistance with this study, and M.J. Hope-Cawdery for statistical consultancy. We are grateful to the Director, Kenya Agricultural Research Institute (KARI) and the Director, NVRC, for permission to conduct this study at NVRC. References Azuba Musoke, R., Tweyongyere, R., Bizimenyera, E., Waiswa, C., Mugisha, A., Biryomumaisho, S., McHardy, N., 2004. Treatment of East Coast fever of cattle with a combination of parvaquone and frusemide. Tropical Animal Health and Production 36, 233–245.
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Burridge, M.J., Kimber, C.D., 1972. The indirect fluorescent antibody test for experimental East Coast fever (Theileria parva infection of cattle); evaluation of a cell culture schizont antigen. Research in Veterinary Science 13, 451–455. Chema, S., Waghela, S., James, A.D., Dolan, T.T., Young, A.S., Masiga, W.N., Irvin, A.D., Mulela, G.H.M., Wekesa, L.S., 1986. Clinical trial of parvaquone for the treatment of East Coast fever in Kenya. The Veterinary Record 118, 588–589. Dolan, T.T., Young, A.S., Leitch, B.L., Stagg, D.A., 1984. Chemotherapy of East Coast fever: parvaquone treatment of clinical disease induced by isolates of Theileria parva. Veterinary Parasitology 15, 103–116. Dolan, T.T., Injairu, R., Gisemba, F., Maina, J.N., Mbadi, G., Mbwiria, S.K., Mulela, G.H.M., Othieno, D.A.O., 1992. A clinical trial of buparvaquone in the treatment of East Coast fever. The Veterinary Record 130, 536–538. Hudson, A.T., Randall, A.W., Fry, M., Ginger, C.D., Hill, B., Latter, V.S., McHardy, N., Williams, R.B., 1985. Novel anti-malarial hydroxynaphthoquinones with potent broad-spectrum anti-protozoal activity. Parasitology 90, 45–55. Hudson, A.T., Pether, M.J., Randall, A.W., Fry, M., Latter, V.S., McHardy, N., 1986. In vitro activity of 2-cycloalkyl-3-hydroxy-1,4naphthoquinones against Theileria, Eimeria and Plasmodia species. European Journal of Medicinal Chemistry – Chemistry and Therapeutics 21, 271–275. Kinabo, L.D.B., Bogan, J.A., 1988. Parvaquone and buparvaquone: HPLC analysis and comparative pharmacokinetics in cattle. Acta Tropica 45, 87–94.
McHardy, N., Mercer, J.P., 1984. Bioassay of parvaquone in the serum of cattle infected with Theileria parva and treated with Clexon. The Kenya Veterinarian 9, 9–11. McHardy, N., Rae, D.G., 1981. Treatment of stabilate-induced East Coast fever with menoctone. Tropical Animal Health and Production 13, 227–239. McHardy, N., Hudson, A.T., Morgan, D.W.T., Rae, D.G., Dolan, T.T., 1983. Activity of 10 naphthoquinones, including parvaquone (993C) and menoctone, in cattle artificially infected with Theileria parva. Research in Veterinary Science 35, 347–352. McHardy, N., Wekesa, L.S., Hudson, A.T., Randall, A.W., 1985. Antitheilerial activity of BW720C (buparvaquone): a comparison with parvaquone. Research in Veterinary Science 39, 29–33. Medlicott, N.J., Waldron, N.A., Foster, T.P., 2004. Sustained release veterinary parenteral products. Advanced Drug Delivery Reviews 56, 1345–1365. Mukhebi, A.W., Perry, B.D., Kruska, R., 1992. Estimated economics of theileriosis control in Africa. Preventive Veterinary Medicine 12, 73– 85. Muraguri, G.R., Kiara, H.K., McHardy, N., 1999. Treatment of East Coast fever: a comparison of parvaquone and buparvaquone. Veterinary Parasitology 87, 25–37. Wanjohi, J.M., Muraguri, G.R., Rumberia, R.M., Kariuki, D.P., McHardy, N., 1995. Chemotherapy of East Coast fever: buparvaquone treatment of field cases occurring in Kikuyu and Limuru Divisions of Kiambu District, Kenya. East African Agricultural and Forestry Journal 60, 131–135.
Clinical efficacy and plasma concentrations of two formulations of buparvaquone in cattle infected with East Coast fever (Theileria parva infection) G.R. Muraguri a, P.N. Ngumi a, D. Wesonga a, S.G. Ndungu a, J.M. Wanjohi a, K. Bang b, A. Fox b, J. Dunne b, N. McHardy c,* b
a National Veterinary Research Centre, Muguga, P.O. Box 32, Kikuyu, Kenya Cross Vetpharm Group Ltd., Broomhill Road, Tallaght, Dublin 24, Republic of Ireland c TBD Consulting Ltd., Baltyboys, Blessington, Co. Wicklow, Republic of Ireland
Accepted 29 September 2005
Abstract East Coast fever, caused by the protozoan parasite Theileria parva, kills about 600,000 cattle annually in Africa. The hydroxynaphthoquinone compound buparvaquone (BPQ) is curative. Sixteen calves were infected with T. parva. On manifestation of disease symptoms, eight were injected with the original (pioneer) BPQ product and eight with a test product containing BPQ. All 16 calves were cured by one injection of 2.5 mg BPQ/kg bodyweight. The concentration of BPQ in blood plasma was monitored by HPLC. The mean observed Cmax of BPQ was 0.229 and 0.253 lg/mL of plasma, the mean observed time to reach this concentration (Tmax) was 2.62 and 2.12 h and the AUC (area under curve) was 4.785 and 4.156 lg h/mL, respectively, for the pioneer and test product. Considerable variations occurred in the plasma concentration of BPQ within each group. They showed no relationship with either clinical or parasitological parameters following treatment. 2005 Elsevier Ltd. All rights reserved. Keywords: Buparvaquone; Plasma concentrations; Theileria; Chemotherapy
1. Introduction This study on the relationship between clinical response to treatment and plasma concentrations of the curative drug buparvaquone (BPQ) was part of a programme to develop a generic BPQ product for the treatment of East Coast fever (ECF) of cattle. Kinetics studies in support of a drugÕs development are usually conducted in uninfected animals. However, an additional objective of this study was to obtain information on the product that might be helpful to veterinary practitioners who may use it, so the study was done in cattle infected with ECF. ECF is a tick-transmitted disease caused by the protozoan parasite Theileria parva. It kills at least 600,000 cattle *
Corresponding author. Tel.: +353 1 4515522; fax: +353 14515803. E-mail address: [email protected] (N. McHardy).
0034-5288/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.09.012
annually in Eastern and Southern Africa (Mukhebi et al., 1992). The mortality rate is more than 90% in European breeds of cattle and around 50% in indigenous breeds. Extrapolation from MukhebiÕs findings to the present indicates that the total cost of East Coast fever is around US$250 million per year in production losses and the cost of attempts at controlling the disease. BPQ and parvaquone (PQ) are both hydroxynaphthoquinone (HNQ) drugs. Their efficacy in the treatment of ECF was first reported by McHardy et al. (1983) and McHardy et al. (1985), respectively. Separate field trials in clinical cases of ECF (Chema et al., 1986; Dolan et al., 1992; Wanjohi et al., 1995) and a comparative field trial (Muraguri et al., 1999) all obtained cure rates of around 90% with each drug. Kinabo and Bogan (1988) investigated the plasma pharmacokinetics of commercially available injectable products
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containing PQ or BPQ in uninfected cattle, using a highperformance liquid chromatography (HPLC) method. PQ was injected at a dose of 20 mg/kg bodyweight (twice the recommended dose), BPQ at 2.5 mg/kg, its recommended dose rate. PQ reached a maximum concentration in plasma of about 6.4 lg/mL 0.8 h after injection; BPQ reached 0.10 lg/mL after 3.2 h. The area under the curve (AUC) was 31.27 and 3.43 lg h/mL, respectively, for PQ and BPQ. McHardy and Mercer (1984), using a bioassay, found no relationship between plasma concentrations of parvaquone (PQ) in individual cattle infected with ECF and their clinical and parasitological response to treatment. 2. Materials and methods
2.3. Infection with ECF Sixteen of the calves were selected at random from the 20 candidates and infected by the subcutaneous injection of 1 mL of a 1-in-10 dilution of T. parva KARI sporozoite stabilate 316, above and in front of the left prescapular lymph node. Stabilate 316 is the NVRC ‘‘standard’’ stabilate of the Marikebuni isolate of T. parva. The day of infection was designated as day 0 of the study. In numerous previous studies using this infective dose of this stabilate, it consistently produced mortality rates of about 90% in untreated cattle, usually between 15 and 20 days after infection (data on file at NVRC). For this and for ethical and animal welfare reasons, no infected untreated control cattle were included in the study.
2.1. Buparvaquone 2.4. Treatments BPQ, 2-trans(4-t-buytylcyclohexyl)methyl-3-hydroxy1,4-naphthoquinone was used as the pioneer product (PP) Butalexe (Schering-Plough Animal Health, Harefield, Great Britain) or the test product (TP) Buparvexe (Bimeda, Dublin, Ireland). Each product is a solution for intramuscular injection containing 50 mg BPQ/mL dissolved in N-methylpyrrolidone and fractionated coconut oil, with a surfactant and an emulsifier as necessary to give a stable, non-irritant, pharmaceutical preparation. The recommended dosage for each product is 1 mL/20 kg bodyweight (2.5 mg BPQ/kg) delivered into the neck muscles. 2.2. Cattle Twenty male calves, approximately 75% Friesian (Bos taurus) phenotype and 25% Zebu (B. indicus) were used. They were aged 6–9 months and in good health. Their mean bodyweight was 145.2 kg (range 93–228). They were obtained from a farm that practiced strict tick control and had no recent history of ECF. Their susceptibility to ECF was confirmed by the indirect fluorescent antibody test (IFAT, Burridge and Kimber, 1972) before they were transported by road to the test site, the National Veterinary Research Centre (NVRC), Kikuyu, Kenya. On arrival they were ear-tagged with unique numbers and subjected to a thorough clinical examination. They were weighed and a jugular venous blood sample was taken for a confirmatory IFAT, and from which a blood smear was prepared for staining and microscopical examination for the presence of haemoparasites. The cattle were treated for endoparasites and ectoparasites, respectively, with levamisole/oxyclozanide (Nilzan Plus) and deltamethrin (CooperÕs Spot-on) (both products Schering-Plough Animal Health, Harefield, Great Britain). They were then quarantined at NVRC for five days before the start of the study. The cattle were housed in a purpose-built tick-free barn and provided with good quality tick-free hay and clean drinking water at all times. Concentrated rations were provided twice daily in amounts necessary to maintain a good plane of nutrition.
Alternate infected calves were treated with a single injection of either the PP or TP presentation of BPQ in the order in which they developed clear clinical and parasitological signs of ECF. Seven calves were injected with each product on the second consecutive day on which their rectal temperature was 39.5 C or higher and macroschizonts of T. parva were present in stained lymph node smears, designated as day ‘‘TS2’’. This treatment time was chosen to maximise the chances of curing with a single injection so as not to complicate the drug kinetics aspects of the study. Treatment was administered intramuscularly on the right side of the neck. Both products were used at the rate of 2.5 mg BPQ/kg bodyweight. Two calves failed to attain TS2 by day 14 although one had shown fever and both had shown parasitosis on at least one day. One of these calves was treated with PP and one with TP on day 14-post infection. Two of the four uninfected calves were similarly injected with each of the products between day 9 and day 13, to fit conveniently into the complex treatment and sampling schedule. 2.5. Observations and collection of samples All calves were examined clinically twice daily throughout the quarantine period and the study. Any signs of abnormality, particularly those symptomatic of ECF, were noted. These include enlarged superficial lymph nodes, respiratory distress, nasal discharge and other signs of pulmonary oedema, inappetance, weight-loss and dehydration. From the time of injection of PP or TP, the calves were examined for signs of reaction to, or intolerance of, the treatment, including injection site reactions. Rectal temperature was taken daily before 10.00 hrs using a clinical thermometer and the calves were weighed weekly. A sample of jugular blood was taken by heparinised vacutainer on the first day of the 5-day quarantine period (day –5), the day of infection with T. parva (day 0), then three times per week until clinical recovery and thereafter weekly until the end of the study. All blood samples were
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taken from the left jugular vein, i.e., contra-lateral to the site of drug injection. The blood was used to assay packed cell volume (PCV) and to prepare blood smears. The smears were stained with GiemsaÕs stain and examined for parasitaemia of T. parva piroplasms and for intercurrent haemoparasites, using an oil immersion objective. Needle biopsy smears, using a disposable 18 G · 1.500 hypodermic needle, were taken from the left prescapular lymph node (LPN), i.e., the node draining the site of infection but contra-lateral to the site of BPQ injection, daily from day 5 until clinical and parasitological recovery, then twice weekly. They were stained with GiemsaÕs stain and examined for macroschizonts and microschizonts of T. parva and for signs of lymphoblastosis, using an oil immersion objective. Daily sampling was resumed in the event of clinical or parasitological signs of ECF. Right prescapular lymph node (RPN) smears were prepared daily from the day after schizonts were first observed in the LPN and subsequently at the same times that LPN smears were prepared. Lymphoblastosis and macroschizont parasitosis were each ‘‘scored’’ on a scale of 0–3, representing a range from ‘‘absent’’ to ‘‘advanced’’ blastosis or parasitosis (Muraguri et al., 1999). The presence of microschizonts, and any signs of damage to the macroschizonts following drug treatment, were also noted.
each calf, then at 0.5, 1, 2, 4, 8 and 24 h and 2, 3, 4, 7 and 10 days after treatment. When more than one calf was treated on a single day, care was taken to allow sufficient time between treating them to ensure that all blood samples could be taken at the intended times. The samples were centrifuged at 3000g for 20 min to sediment blood cells then at least 4.5 mL of plasma was withdrawn using a Pasteur pipette and placed in plastic, screw-cap, 5 mL vials. The vials were labelled with the calf number, date and time of sampling, but not the identity of the product with which the calf was treated, so as to maintain a degree of ‘‘blinding’’ of samples during the analytical phase. The samples were then frozen to 20 C. When all the samples had been collected and frozen, they were thawed and heated for 30 min in a water bath at 56 C then re-frozen at 20 C. All manipulations were performed out of direct sunlight. The samples were then transported by air to Cross VetpharmÕs analytical laboratory in Ireland in an insulated container packed with ice at 20 C. Heating the samples to 56 C for 30 min was a condition of the licence to import the samples into Ireland. Repeated heating and freezing of plasma samples in this way was shown not to affect either the accuracy or sensitivity of the assay of their buparvaquone content using the methods employed in this study (data on file).
2.6. Serology
2.9. Assay of buparvaquone in plasma samples
Blood samples were taken on four occasions from the left jugular vein into untreated vacutainers while on the supplying farm, immediately following arrival at NVRC, and then on days 0 and 35 of the study. The blood was allowed to clot at ambient temperature and serum was withdrawn approximately 2 h later. The samples were assayed for antibody to T. parva by the IFAT (Burridge and Kimber, 1972) using a macroschizont antigen.
The plasma samples were stored frozen at 20 C, then thawed by immersion in a water bath at 37 C immediately before assay. The remainder of the samples were then refrozen. The samples were analysed for their buparvaquone content according to Kinabo and Bogan (1988) by highperformance liquid chromatography (HPLC, Walters Millennium) using a Waters Spherisorb S50DS2, 25 cm · 4.6 mm analytical column and ultra-violet (UV) detection at 252 nm. The methodology was validated for accuracy by assaying control plasma samples spiked with a range of five concentrations of buparvaquone from 0.025 to 0.150 lg/mL of plasma, in six replicates (Table 3). Precision and between-day variability of the analytical method was monitored by assaying plasma samples spiked with 0.04 or 0.14 lg BPQ/mL on each of the six days on which test samples were analysed (Table 4). All plasma samples were spiked with parvaquone as the internal standard. Both compounds were extracted from the plasma samples with diethyl ether. The ether extract was dried under nitrogen at 50 C and the residue was dissolved in methanol. Analyses were performed using a mobile phase of 850:150 methanol:0.05 M sodium acetate buffer, pH 5.0. Analytical results were collated using Waters Millennium 32 Chromatography data handling software. These data were used to demonstrate Cmax (maximum observed concentration), Tmax (observed time after treatment to reach Cmax) and AUC (area under the curve) calculated using the trapezoidal method, of BPQ. Sample identity was not ‘‘unblinded’’ until the time of data analysis. For clarity in the presentation
2.7. Analysis of clinical and parasitological data Data were entered into appropriate data sets in Excel (Microsoft Corporation, USA). Descriptive analyses were carried out in Excel. Descriptive statistics involved calculation of means and standard deviations to assess variability. A multiple regression was done with the CSS Statistical package (Statsoft Inc., Tulsa, USA) using AUC or Cmax as the independent variable and time to clinical cure and time to clear parasitosis as dependent variables from the 14 infected and treated animals. The treatment groups were combined as there was no difference between them with regard to any of the parameters used. 2.8. Blood sampling for the assay of BPQ concentrations in plasma A 10 mL sample of blood was taken from the left jugular vein, i.e., contra-lateral to the site of drug injection, by heparinised vacutainer immediately before treatment of
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of results, BPQ concentrations are expressed either as lg/mL or ng/mL of plasma in some instances. 2.10. Sensitivity and reproducibility of analytical methodology Buparvaquone recovery from plasma samples spiked with five concentrations of BPQ from 0.0250 to 0.1500 lg/ mL was between 123.97% and 80.87%, mean 86.87%, SD 2.87–6.52 (n = 6). The calibration curve was rectilinear (r = 0.99) over the concentration range studied. The limit of detection (LOD) for BPQ was 0.013 lg/mL (13.0 ng/ mL), the limit of quantitation (LOQ) was 0.025 lg/mL (25.0 ng/mL). The analytical methodology was shown to be both accurate and reproducible (Tables 3 and 4). LOD (0.013 lg/mL) was almost identical to that found by Kinabo and Bogan (1988) i.e., 0.015 lg/mL, and the mean % recovery of BPQ from the samples (86.87%) was also comparable to that of Kinabo and Bogan (91.88%). Kinabo and Bogan make no mention of LOQ, with which the value reported here (0.025 lg/mL) can be compared. 3. Results 3.1. Clinical observations during quarantine All 20 cattle were in good health on arrival at NVRC and during the quarantine period at NVRC, apart from two that had mild conjunctivitis with lachrymation. Treatment with eye ointment containing cloxacillin benzathine (Orbenin Opthalmic Ointment, Pfizer Animal Health, New York, USA) quickly controlled the condition in one calf but in the other, 678, the condition was incompletely resolved and treatment continued throughout the study. PCV ranged from 24% to 41%, which is within normal ranges. No intercurrent haemoparasite infections were detected in blood smears. The T. parva IFAT titres of all 20 calves were negative (at 1 in 40 dilution) while on the farm and on day 0 of the study. The four uninfected calves remained clinically normal throughout the study, apart from the eye condition of 678, and their IFAT titres were still negative on day 35.
smears. All 16 had IFAT titres of at least 1 in 160 on day 35, confirming that they had been infected with T. parva. Fourteen calves attained the infection status of ‘‘TS2’’, the criterion for treatment, between days 9 and 14 (Table 1). Maximum temperatures were 39.9–40.7 C. There were no significant differences between the two treatment groups. Two calves did not attain TS2 status. Calf 674 developed fever (39.9 C) on day 12 only and calf 664 developed its highest temperature, 39.2 C, on day 13. These two calves were treated on day 14 none-the-less. They are excluded from the clinical and parasitological analyses (Tables 1 and 2) but they are included in the kinetics results shown in Table 5. At the time of treatment all 14 calves that attained TS2 status had macroschizonts in both prescapular lymph node smears. Schizont scores were 1–3 in the LPN and 1–2 in RPN smears. Small numbers of microschizonts were observed in seven calves. Only five calves had developed piroplasm parasitaemia by day TS2. It was less than 1% in all cases. PCV did not vary significantly throughout the study (ECF causes anaemia only rarely) and no intercurrent haemoparasitic infections were observed. All 14 calves showed mild or moderate clinical signs of ECF, including greatly enlarged superficial lymph nodes in all cases and dullness and mild signs of pulmonary oedema in some. None was regarded as suffering from severe ECF. The two calves that did not attain TS2 status showed no clear signs of ECF except enlarged lymph nodes. 3.3. Response to treatment Rectal temperature fell below 39.5 C within one day in all seven calves treated with TP and in five treated with PP (Table 2). In the other two it was below 39.5 C on the second day after treatment. Subsequently, temperatures
Table 2 Comparison of rectal temperature (C) in groups of calves infected with T. parva and treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight, group mean and (standard deviation) Days after treatment
3.2. Response to infection with T. parva stabilate All 16 calves developed parasitological signs of T. parva infection, with macroschizonts observed in lymph node
Treated PP (n = 7) Treated TP (n = 7) p Value of difference
0
1
2
3
39.9 (0.29) 40.0 (0.39) 0.82
38.9 (0.53) 38.6 (0.29) 0.66
38.4 (0.25) 38.5 (0.27) 0.58
38.4 (0.21) 38.3 (0.20) 0.56
Table 1 Time to first parasitosis, first fever and to treatment in groups of calves treated with BPQ 2.5 mg/kg bodyweight Days after infection to:
Treated PP (n = 7) Treated TP (n = 7) p Value of difference
Macroschizonts in LPN
Fever 39.5 C
Mean
SD
Mean
SD
Mean
SD
8.6 9.7
0.53 1.60
9.9 11.0
2.10 0.82
11.4 12.1
1.10 1.10
0.07
PP, pioneer product (Butalexe); TP, test product (Buparvex).
0.06
Treatment (day TS2)
0.07
G.R. Muraguri et al. / Research in Veterinary Science 81 (2006) 119–126 Table 3 Accuracy of analytical methodology BPQ concentration in spiked sample (lg/mL)
BPQ recovery % mean
SD
%CV
0.0250 0.0500 0.1000 0.1250 0.1500
123.967 90.534 80.867 84.2130 84.0530
6.399 6.519 3.252 2.871 5.319
5.162 7.200 4.022 3.409 6.328
(n = 6) (n = 6) (n = 6) (n = 6) (n = 6)
Mean recovery of BPQ from plasma samples (six replicates) spiked with five concentrations of BPQ. %CV = % coefficient of variation.
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With the exception of enlargement of superficial lymph nodes, all clinical signs of ECF had resolved within two days of treatment with either product. None of the animals required additional treatments for ECF or for any other condition. No adverse signs of any sort due to the treatments were observed in any animal. Mean bodyweight in the group treated with PP was 141.4 kg on day 0 and 147.8 kg on day 35. For those treated with TP it was 149.7 and 154.9 kg, respectively. The differences between groups are not significant. 3.4. BPQ concentrations in blood plasma
Table 4 Day-to-day variability of analytical methodology BPQ concentration in spiked sample (lg/mL)
BPQ concentration detected (mean) lg/mL
SD
%CV
0.040 0.140
0.0386 (n = 5) 0.1459 (n = 5)
0.0025 0.0131
6.4008 8.9786
Mean concentrations of BPQ found in plasma samples spiked with two concentrations of BPQ on five occasions.
remained within normal ranges in all calves. Mean temperatures, throughout, were not significantly different between the treated groups. Following treatment, the mean number of days taken for both lymph node smears to become negative for macroschizonts was 4.14 days (SD 0.69) for treatment PP and 3.86 days (SD 1.21) for TP; the difference is not significant. On the day following treatment most macroschizonts appeared to be undamaged by either product but on day 2 damage was obvious in 43% of the smears of calves treated with PP (two smears, LPN and RPN, from each calf) and 71% of those treated with TP. By day 3, schizonts were clearly damaged in all smears in which they were still observable. The damage appeared first as a blurring of the outline, and reduction in the intensity of staining, of the schizont nuclei followed by apparent ‘‘dissolution’’ of the schizont remains into the cytoplasm of the host cell, leaving pyknotic nuclear debris in some instances. All blood smears were negative for piroplasms from three days after treatment until the end of the study.
Sensitivity and reproducibility of the analytical methodology is shown in Tables 3 and 4. The results of analyses of BPQ concentration in plasma (Table 5) include the two calves that did not attain TS2, but which were treated, none-the-less, on day 14. The mean highest observed plasma concentration (Cmax) of BPQ following treatment with PP was 229.0 ng/mL (0.229 lg/mL). It ranged from 32 to 589 ng/mL in individual animals. Observed Cmax after TP treatment was 252.7 ng/mL but the range was smaller, 183 to 449 ng/mL. The mean time after treatment at which maximum BPQ concentration occurred (Tmax) was 2.62 h for PP and 2.12 h for TP. Mean area under the curve (AUC) was 4.7847 lg h/mL (range 1.0822–7.2524) for PP and 4.1588 (range 3.1299–5.8775) for TP. None of these differences is statistically significant. BPQ concentration data from the pairs of uninfected calves treated with PP or TP were within the ranges of the equivalent groups of calves infected with T. parva (Table 6), but one, 678, had lost 5.2% bodyweight by day 35, possibly due to its poorly responsive eye infection. Table 6 Plasma concentration of BPQ parameters in pairs of uninfected calves treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight Parameter
Treatment
Range
Mean
Cmax BPQ (ng/mL)
PP TP PP TP
131 and 319 279 and 402 2 and 2 2 and 2
225.0 340.5 2.0 2.0
Tmax BPQ (h)
Table 5 Comparison of kinetic, clinical and parasitological parameters in groups of calves infected with T. parva and treated with either PP or TP, equivalent to 2.5 mg BPQ/kg bodyweight Parameter
Treated with
Range
Mean (SD)
AUC (lg h/mL) (n = 8)
PP TP TP TP PP TP PP TP PP TP PP TP
1.082–7.252 3.130–5.877 32–589 183–449 1–4 1–4 0–1.8 0–1.9 2–5 2–5 1.0 to +4.0 1.6 to +3.5
4.785 (2.097) 4.156 (0.924) 229.0 (187.6) 252.7 (81.8) 2.62 (1.19) 2.12 (0.83) 0.81 (0.70) 1.29 (0.60) 4.15 (0.69) 3.86 (1.21) +1.39 (1.51) +0.85 (1.77)
Cmax BPQ (ng/mL) (n = 8) Tmax BPQ (h) (n = 8) Fall in temperature 24 h after treatment (C) (n = 7) Days from treatment to parasitic cure (n = 7) % weight change day 14–35 (n = 7)
p Value of difference 0.75 0.34 0.18 0.06 0.32 0.70
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Table 7 Comparisons between two kinetic parameters Cmax and AUC, and two clinical observations CURE and CLEAR, in 14 calves infected with ECF and treated with buparvaquone, 2.5 mg/kg bodyweight Comparison
R2
Adjusted R2
F
‘‘p’’ Value
Significance
Cmax vs. CURE Cmax vs. CLEAR AUC vs. CURE AUC vs. CLEAR
0.01403 0.08562 0.11609 0.00007
0.06814 0.00942 0.04244 0.08326
0.17072 1.12361 1.57611 0.00086
0.68676 0.31001 0.23322 0.97713
ns ns ns ns
Following multiple regression, the multiple R2 for AUC and Cmax for ÔTime to CUREÕ and ÔTime to CLEARÕ are given in Table 7. These show that there was little correlation between kinetic and clinical parameters. 4. Discussion The response of the calves to infection with this dose of T. parva (Marikebuni) sporozoite stabilate KARI 316 was as expected from previous findings (NVRC files, unpublished data). Fourteen of the 16 infected calves attained TS2 between nine and 14 days after infection. Based on extensive past experience, it is likely that if they had not been treated with BPQ to control the infection at least 12 and possibly all 14 would have died of ECF within about one week. Some isolates of T. parva produce variable patterns of ECF, including temporary remission of symptoms followed by severe, often fatal, disease (Dolan et al., 1984). Stabilate 316 typically induces a progressive disease syndrome without remission so it seems unlikely that the two calves that did not reach TS2 would have developed more severe ECF if they had not been treated. Their exclusion from the clinical assessment, but their inclusion in the kinetics section of this study, for which the stipulation was only that they should be infected with T. parva, therefore seems justified. Their infection status was confirmed by the observation of macroschizonts in lymph node smears between days 9 and 13 and by their positive IFAT titres on day 35. At the time of treatment, day TS2, the 14 calves in the clinical study were mostly suffering from mild clinical signs of ECF. None was severely affected and all 14 were quickly cured with a single injection of either product, PP or TP. There was no statistically significant difference in the rate of cure or clearance of parasites (Table 5) with the two products. None of the calves developed a recrudescence of the disease that required additional treatment following initial cure, which may be necessary when more severe cases are treated (Dolan et al., 1992). The objective of avoiding the need for additional treatments that would have complicated the plasma concentration study, by treating the calves relatively early in the disease syndrome, therefore was achieved. The study produced results on the mean concentration of BPQ in plasma broadly similar to those of Kinabo and Bogan (1988) for product PP. The present study gave higher Cmax of BPQ than Kinabo and Bogan found
(0.229 compared with 0.102 lg/mL), slightly earlier Tmax (2.62 compared with 3.17 h) and higher AUC (4.785 compared with 3.43 lg h/mL). These differences are probably within the expected range of variation between studies by different researchers in different laboratories. Kinabo and Bogan used healthy, uninfected calves while in the present study the calves were undergoing a clinical episode of ECF. The fever and widespread oedema associated with ECF might have been expected to affect the rate of distribution and metabolism of BPQ, resulting in greater differences between results of the two studies than were actually found. However, the observed concentrations of BPQ in the pairs of uninfected control calves in this study were similar to those of the infected principals, albeit that the control group size, two calves, is too small for statistical comparison. This said, the results suggest that the moderate disease state did not greatly influence the kinetics of BPQ. Our results also demonstrate no obvious relationships between any of the plasma concentration parameters and any of the clinical or parasitological parameters in individual calves – apart from the obvious one, that the disease was cured quickly and completely by treatment with BPQ. The observed Cmax of BPQ in the plasma of individual calves treated with PP ranged from 32 to 589 ng/mL, Tmax was reached in 1–4 h and AUC was 1.082 to 7.252 lg h/mL. The range of Cmax following treatment with TP was less broad (183–449 ng/mL), Tmax was similar (1–4 h) and the range of AUC was less (3.130– 5.877 lg h/mL). These differences are not significant. McHardy and Mercer (1984), using a bioassay, not HPLC, also found no relationship between the concentration of another HNQ compound, parvaquone, in the blood of individual cattle suffering from naturally acquired ECF of varying severity and their clinical response to the treatment. It is possible that if more severe cases had been treated, particularly if some animals died despite treatment, correlation with plasma kinetics might be found. Similarly, a relationship might have been found if additional dose rates of BPQ had been studied, particularly lower ones. However, the present study was conducted in accordance with guidelines for the marketing approval of a generic product which stipulate only that the comparison should investigate the recommended dose rate of the pioneer and test products. The in vitro EC50 (the concentration that eliminates the schizonts from 50% of cultured lymphoblasts infected with
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T. parva in an exposure period of 48 h) for BPQ is 3 ng/mL (0.003 lg/mL, 6.1 · 1010 M, Hudson et al., 1986). This concentration was exceeded in the plasma of all 16 infected cattle and it was maintained for at least three days in most of them. The mean Cmax of BPQ in the animals treated with PP was 76-times, and in those treated with TP 84-times, its in vitro EC50. Even the animal with the lowest AUC of BPQ concentration (no. 672, treated with PP) may have met EC50 criteria. Its plasma concentration was 0.032 lg/ mL in the 4-h sample (i.e., 10-times EC50) and 0.028 lg/ mL at 24 h. A concentration of 0.008 lg/mL (2.7-times EC50) was indicated in the 48 and 72-h samples. However, these latter values are below the validated LOD level of 0.013 lg/mL, so no reliance should be placed on them. Clear signs of damage to macroschizonts, characterised by disorganisation of their nuclear particles, were not seen until two days following treatment in most animals. Under the electron microscope, signs of vaccuolation of the schizont cytoplasm and disruption of membranous organelles in the cytoplasm were seen within 3 h of treatment with PP at the dosage used in this study, in cattle suffering from ECF (N. McHardy and T.T. Dolan, unpublished data). Similar changes were seen in T. parva piroplasms in the blood of these animals, starting only 1 h after treatment. Damage to schizont and piroplasm nuclei and to the limiting membranes of the parasites, however, was not obvious until 48 h after treatment. This tallies well with light-microscopical observations in the present study and with the proposed mode of action of the hydroxynaphthoquinones against sporozoan parasites, on their mitochondrial electron transport mechanisms (Hudson et al., 1985). Under the electron microscope, host cell mitochondria appeared to be unaffected which indicates the specificity of the anti-theilerial action of the hydroxynaphthoquinones, and it helps to explain their wide therapeutic index, indicated by the complete absence of adverse signs of treatment in the present study. By far the greatest biomass of T. parva schizonts is located within lymphoblastoid cells in lymphoid tissues, not in the blood. Medlicott et al. (2004) state that low molecular weight (mw) molecules (which would include BPQ, mw 326.44) predominantly enter blood capillaries, not lymphatics, after intramuscular injection. Subsequently, the concentration in lymph should be related directly to that in the plasma, but BPQ may show a propensity to concentrate in lymphoid tissue. The kinetics of BPQ in lymph and lymphoid tissue have not been studied, but it seems possible that they could relate more closely with clinical and parasitological responses than the plasma-based findings reported here. In earlier studies (McHardy et al., 1985; Muraguri et al., 1999), damage to T. parva piroplasms in Giemsa-stained blood smears was obvious within one day of treatment with BPQ. Two days after treatment almost all the piroplasms were pyknotic. This tallies well with the findings in the present study and with the electron microscopic observations of McHardy and Dolan (unpublished).
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Also of interest is the lack of statistical difference between the mean values for plasma concentrations and parasitological and clinical parameters for the two BPQ products studied here, PP (Butalex) and TP (Buparvex). This formulation of BPQ as a solution for intramuscular injection was ‘‘optimised’’ in a series of studies with three HNQs, menoctone (McHardy and Rae, 1981), parvaquone (McHardy et al., 1983) and buparvaquone (McHardy et al., 1985). It was found that apparently minor differences in formulation could result in major changes in plasma concentrations and clinical performance. Products PP and TP are formulated essentially similarly but it is likely that their constituents – BPQ and excipients, are sourced from different manufacturers. Small differences between constituents from different manufacturers may account for the greater variability in plasma concentrations, but apparently not of the clinical performance, of PP compared with TP in this study. Clinical field trials of products to treat ECF have consistently shown that cure rates approach 100%, even in B. taurus breeds, when mild cases are treated with a single injection of BPQ or two of PQ. Additional injections may be necessary to cure more advanced cases. Cure rates fall to around 40% in severe cases, even if multiple treatments are given (Azuba Musoke et al., 2004). It is possible that small differences between the performance of PP and TP may be more apparent under field conditions, and particularly in cases more severe than in the present study. However, an alert cattle owner, and certainly a veterinary practitioner, should be able to recognise the clinical signs of ECF that were present at the time of treatment in this study – fever and clearly enlarged superficial lymph nodes in all 14 calves and laboured respiration in some. In all 14 cases, examination of a lymph node smear would have confirmed the provisional diagnosis. Cattle owners, therefore, should be encouraged to recognise the early signs of ECF and to summon a veterinary practitioner immediately if they are seen. If this were done, it is likely that cure rates would increase substantially. The number of treatments necessary to produce cure and the number of veterinary attendances would be less, greatly reducing the cost of management of ECF. Acknowledgements We thank the laboratory and farm staff, at both NVRC and Cross Vetpharm, for their expert assistance with this study, and M.J. Hope-Cawdery for statistical consultancy. We are grateful to the Director, Kenya Agricultural Research Institute (KARI) and the Director, NVRC, for permission to conduct this study at NVRC. References Azuba Musoke, R., Tweyongyere, R., Bizimenyera, E., Waiswa, C., Mugisha, A., Biryomumaisho, S., McHardy, N., 2004. Treatment of East Coast fever of cattle with a combination of parvaquone and frusemide. Tropical Animal Health and Production 36, 233–245.
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Burridge, M.J., Kimber, C.D., 1972. The indirect fluorescent antibody test for experimental East Coast fever (Theileria parva infection of cattle); evaluation of a cell culture schizont antigen. Research in Veterinary Science 13, 451–455. Chema, S., Waghela, S., James, A.D., Dolan, T.T., Young, A.S., Masiga, W.N., Irvin, A.D., Mulela, G.H.M., Wekesa, L.S., 1986. Clinical trial of parvaquone for the treatment of East Coast fever in Kenya. The Veterinary Record 118, 588–589. Dolan, T.T., Young, A.S., Leitch, B.L., Stagg, D.A., 1984. Chemotherapy of East Coast fever: parvaquone treatment of clinical disease induced by isolates of Theileria parva. Veterinary Parasitology 15, 103–116. Dolan, T.T., Injairu, R., Gisemba, F., Maina, J.N., Mbadi, G., Mbwiria, S.K., Mulela, G.H.M., Othieno, D.A.O., 1992. A clinical trial of buparvaquone in the treatment of East Coast fever. The Veterinary Record 130, 536–538. Hudson, A.T., Randall, A.W., Fry, M., Ginger, C.D., Hill, B., Latter, V.S., McHardy, N., Williams, R.B., 1985. Novel anti-malarial hydroxynaphthoquinones with potent broad-spectrum anti-protozoal activity. Parasitology 90, 45–55. Hudson, A.T., Pether, M.J., Randall, A.W., Fry, M., Latter, V.S., McHardy, N., 1986. In vitro activity of 2-cycloalkyl-3-hydroxy-1,4naphthoquinones against Theileria, Eimeria and Plasmodia species. European Journal of Medicinal Chemistry – Chemistry and Therapeutics 21, 271–275. Kinabo, L.D.B., Bogan, J.A., 1988. Parvaquone and buparvaquone: HPLC analysis and comparative pharmacokinetics in cattle. Acta Tropica 45, 87–94.
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