N. McHardy
Coopers Animal Health Limited
Berkhamsted, Hertfordshire, United Kingdom
Buparvaquone is a second-generation hydroxynaphthoquinone related to parvaquone, with novel features that make it a promising compound for the therapy and prophylaxis of all forms of theileriosis. It has been tested extensively against Theileria annulata, T. parva and T. sergenti, both in laboratory studies and in field trials, and it has undergone a rigorous programme of toxicology and safety studies. Formulated as a solution for intramuscular injection (Butalex, Coopers Animal Health), it offers a safe and convenient alternative to existing antitheilerial products.
FEATURES OF BUPARVAQUONE
Buparvaquone was selected for development using the following three principal criteria:
· In vitro activity: EC50 of 0.0003 mg/L (10-9M) against Theileria parva
· Long plasma half-life (at least 7 days)
· Low toxicity: oral LD50 in rats > 8000 mg/kg
The high in vitro activity against T. parva (Muguga) E174 and the persistence in plasma following intramuscular injection may both be related to the tertiary-butyl moiety in the buparvaquone molecule, which results in far slower metatolism to the hydroxy derivative at the 4-position on the cyclohexyl ring (Figure 1). Parvaquone, which is much more readily metabolized, has an EC50 of 0.006 mg/L (1.3 × 10-8M) and persists in plasma for only about two days. A high-pressure liquid chromatography study (Kinabo and Bogan, 1988) confirms that buparvaquone persists longer in plasma than parvaquone. Acute oral toxicity in the rodent is similarly very low (> 8000 mg/kg) for both compounds.
EFFICACY AGAINST STABILATE-INDUCED THEILERIA INFECTIONS
When formulated as a 5% solution for injection (Butalex) and injected by the intramuscular route, a dose of 2.5 mg/kg is highly effective against T. parva (McHardy et al., 1985), T. annulata (McHardy et al., 1985; Dhar et al., 1986) and T. sergenti (Minami et al., 1985). However, Linyoni et al. (in preparation) obtained less satisfactory results, especially with T. parva lawrencei.
Figure 1. The structure of the antitheilerial compounds parvaquone and buparvaquone.
Dolan et al. (1988) showed that parvaquone (formulated as Clexon) is liable to eliminate stabilate infection of T. parva if infection and drug are injected simultaneously. Clexon therefore is unsuitable for use in a single-treatment infection-and-treatment system of immunization. McHardy and Wekesa (1985) reported that buparvaquone (2.5 mg/kg) injected at the same time as infection with T. parva suppressed but did not eliminate the infection at the schizont stage, and no piroplasms were detected in any of the 10 treated calves. All 10 untreated controls died of theileriosis (Table 1). Although treated animals were not challenge-infected, serology on five of them indicated homologous immunity.
Similarly encouraging results have been obtained since by various workers using T. parva. Mutugi et al. (1988) have reported good protection with T. p. lawrencei, and other such reports occur elsewhere in these proceedings. Similar results were obtained with T. annulata by Dhar et al. (1987).
In view of the prolonged plasma persistence of buparvaquone following intramuscular injection, McHardy and Wekesa (1985) also investigated the prophylactic effect of the compound by injecting livestock with 2.5 mg/kg buparvaquone seven days before infecting them with T. parva (Muguga) stabilate. Clinical disease was delayed significantly, and 7 of 10 calves survived to day 28, whereas all 10 untreated controls had died by day 23. U.V. Shastri (unpublished results) has demonstrated that in the face of heavy and consistent challenge with T. annulata, calves can be protected against clinical disease by administering buparvaquone. In a herd of calves in which theileriosis was endemic, he treated 18 clinical cases with 2.5 mg/kg buparvaquone. Ten were cured and of the seven that died, three also contracted enteritis. Ten in-contact but clinically normal calves were also treated and none developed clinical theileriosis. Of six similar in-contact calves, which were left untreated, four died of theileriosis (Table 2).
Table 1. Effect of simultaneous infection with Theileria parva (Muguga) and treatment with buparvaquone 2.5 mg/kg, injected intramuscularly
|
Parameter |
Untreated controls |
Buparvaquone 2.5 mg/kg at time of infection |
|
Survival |
0/10 |
10/10 |
|
Day of first schizont |
8.2±0.92 |
9.7±1.6 |
|
Days of healthy schizonts |
12.8±1.3 |
0 |
|
Days of damaged schizonts |
0 |
6.5±1.9 |
|
Max. % piroplasms |
57.7±10.9 |
0 |
Table 2. Prophylactic trial of buparvaquone, 2.5 mg/kg in calves infected with Theileria annulata (U.V. Shastri, personal communication)
|
Clinical state |
No. |
No. treated |
No. survived |
|
Sick |
17 |
17 |
10* |
|
Normal, in contact |
10 |
10 |
10 |
|
Normal, in contact |
6 |
0 |
2 |
* 3 deaths due to theileriosis and enteritis.
FIELD TRIALS WITH BUTALEX
Three major field trials have been conducted to observe the efficacy of Butalex in the therapy of theileriosis. Musisi et al. (unpublished results) tested a single dose of 2.5 mg/kg injected intramuscularly in 68 cattle diagnosed as infected with East Coast fever in Zambia. A cure rate of 91% was achieved (Table 3).
Butalex has also been tested against T. annulata infection in several countries. The largest trials have been in India and the Soviet Union. The Indian results are summarized in Table 4.
Table 3. Efficacy of buparvaquone at 2.5 mg/kg against field cases of Theileria parva, Zambia (F.L. Musisi, personal communication)
|
Severity of infection |
No. treated |
No. cured |
|
Mild |
20 |
20 |
|
Moderate |
18 |
16 * |
|
Severe |
30 |
26 |
|
Total |
68 |
62 (91 %) |
* 1 died of babesiosis.
Table 4. Efficacy of buparvaquone at 2.5 mg/kg against field cases of Theileria annulata, India 1987
|
Triallist |
No. treated |
No. cured |
% cured |
|
S. Dhar et al. |
19 |
19 |
100.0 |
|
B.B. Verma |
16 |
15 * |
93.7 |
|
R.D. Sharma et al. |
48 |
44 |
91.7 |
|
Total |
83 |
78 |
94 |
* 1 animal died within 12 hours of treatment.
In all of these cases a single dose of Butalex was given. Dhar et al. (1986) have found that if haemoglobin is 6 g/100 ml or greater at the time of treatment, no supportive treatment is necessary to combat anaemia. If haemoglobin is 3-6 g/100 ml, treatment with iron and vitamins is advisable, but recovery can then be expected. When haemoglobin is below 3 g/100 ml, the prognosis is not good, even if supportive therapy is given.
In the trials in the Soviet Union; conducted under the aegis of the All-Union Institute of Experimental Veterinary Medicine, in Moscow, a total of 402 cattle infected with T. annulata were treated, most with a single dose of Butalex (2.5 mg buparvaquone/kg), though some received up to three doses. An overall cure rate of 86% was achieved (Table 5).
Table 5. Efficacy of buparvaquone 2.5 mg/kg against field cases of Theileria annulata in the Soviet Union (V.T. Zablotsky, personal communication)
|
Clinical state |
No. treated |
No. cured |
% cured |
|
Mild (< 1% piroplasms) |
97 |
96 |
98.9 |
|
Moderate (1-40% piroplasms) |
293 |
244 |
83.3 |
|
Severe (> 40% piroplasms) |
12 |
6 |
50.0 |
|
Total |
402 |
346 |
86.1 |
In a further field trial in Egypt, Michael, el Refaii and McHardy (in press) treated dairy cattle known to be carrying low-grade infections of T. annulata and whose milk yields were very low. In a herd of 44, 22 were selected at random and injected with a single 2.5 mg/kg dose of buparvaquone. Over the next seven weeks the treated animals were clinically normal and their milk yield (though relatively low) was twice that of the untreated group of 22 cattle.
Thus, in addition to showing real promise as a therapeutic for theileriosis, particularly T. annulata infection and in the infection-and-treatment method of immunization, buparvaquone treatment may also improve the productivity of cattle carrying sub-clinical theilerial infections. Further research in all of these areas therefore seems justified.
MODE OF ACTION OF BUPARVAQUONE
The mode of action of buparvaquone on Theileria is not yet established, although studies by M. Fry et al. (unpublished) indicate an effect on energy generation, as demonstrated in coccidia (Fry et al., 1984). Electron microscopical studies on the effect of buparvaquone on T. parva schizonts in cultured lymphoid cells (N. McHardy and J. Beesley, unpublished results) show progressive vacuolation of the cytoplasm as the principal lesion. The surface membrane and the nuclear membrane remain apparently unaffected until the cytoplasmic disruption is very advanced. There is no apparent adverse effect on the structure of host lymphocytes, including mitochondria, which emphasizes the parasite-specificity of the mode of action of buparvaquone. It may also help to explain the narrow spectrum of action of the compound, which is confined to certain sporozoan parasites (Table 6).
SAFETY STUDIES
An extensive range of safety studies has been conducted on buparvaquone and on the formulated injection, Butalex. Safety is established both for treated cattle and for humans consuming milk and meat from treated animals. The product is very safe. A milk-withholding period of two days is recommended, and cattle should not be slaughtered for human consumption within 42 days of treatment.
Thus, buparvaquone has a range of features that would justify further studies in the following three major areas for the control of theileriosis:
a) therapy of clinical disease
b) productivity improvement in carrier infections
c) infection-and-treatment immunization
Table 6. Spectrum of activity of buparvaquone against selected protozoa and rickettsia
|
Efficacy |
Source |
|
Effective |
|
|
Babesia equi |
G. Zaugg (in press) |
|
B. gibsoni |
Lu & Wong (personal communication) |
|
B. canis |
N. McHardy & P.K.I. Mackenzie (unpublished results) |
|
B. ovis |
N. McHardy & P.K.I. Mackenzie (unpublished results) |
|
Theileria sergenti |
T. Minami et al. (1985) |
|
T. buffeli |
A.J. de Vos (personal communication) |
|
T. hirci |
Z. Abbas and O.M. Osman (personal communication) |
|
Plasmodium spp. |
A.T. Hudson et al. (1986) |
|
Eimeria spp. |
A.T. Hudson et al. (1986) |
|
Ineffective |
|
|
Anaplasma marginale |
N. McHardy and P.K.I. Mackenzie (unpublished results) |
|
Cowdria ruminantium |
N. McHardy and P.K.I. Mackenzie (Unpublished results) |
|
B. bigemina |
N. McHardy and P.K.I. Mackenzie (unpublished results) |
|
Cytauxoon felis |
E. Mitema (personal communication) |
ACKNOWLEDGEMENT
The involvement and enthusiasm of research workers around the world in studies on the efficacy and value of buparvaquone is gratefully acknowledged.
REFERENCES
Dhar, S., Malhotra, D.V., Bhushan, C. and Gautam, O.P. (1986). Chemotherapy of Theileria annulata infection with buparvaquone. Veterinary Record 119:635-636.
Dhar, S., Malhotra, D.V., Bhushan, C. and Gautam, O.P. (1987). Chemoprophylaxis with buparvaquone against theileriosis in calves. Veterinary Record 120:375.
Dolan, T.T., Linyoni, A., McHardy, N., Bond, A.L. and Clampitt, R.B. (1988). Chemotherapy of East Coast fever: parvaquone treatment of Theileria parva parva at intervals after infection. Research in Veterinary Science 44:15-20.
Fry, M., Hudson, A.T., Randall, A.W. and Williams, R.B. (1984). Potent and selective hydroxynaphthoquinone inhibitors to mitochondrial electron transport in Eimeria tenella (Apicomplexa: Coccidia). Biochemical Pharmacology 33:2115-2120.
Hudson, A.T., Pether, M.J., Randall, A.W., Fry, M., Latter, S. and McHardy, N. (1986). European Journal of Medicinal Chemistry-Chimie Therapeutique 21:271-275.
Kinabo, L.D.B. and Bogan, J.A. (1988). Parvaquone and buparvaquone: high performance liquid chromatographic analysis and comparative pharmacokinetics in cattle. Acta Tropica 45:87-94.
McHardy, N., Wekesa, L.S., Hudson, A.T. and Randall, A.W., (1985). Antitheilerial activity of BW 720C (buparvaquone): a comparison with parvaquone. Research in Veterinary Science 39:29-33.
McHardy, N. and Wekesa, L.S. (1985). Buparvaquone BW 720C: a new antitheilerial napthoquinone - its role in the therapy and prophylaxis of theileriosis. In: Irvin, A.D., ed. Immunization against Theileriosis in Africa: Proceedings of a Workshop Held in Nairobi, Kenya, 1-5 October 1984. Nairobi: International Laboratory for Research on Animal Diseases, p. 88.
Michael, S.A., el Refaii, A.H., McHardy, N. and Rae, D.G. (in press). The effect of treatment of chronic theileriosis with buparvaquone on milk yields. Tropical Animal Health and Production.
Minami, T.M., Nakano, T., Shimizu, S., Shimura, K., Fujinaga, T. and Ito, S. 1985. Efficacy of naphthoquinones and imidocarb dipropionate on Theileria sergenti infections in splenectomized calves. Japanese Journal of Veterinary Science 47:297-300.
Mutugi, J.J., Young, A.S., Maritim, A.C., Linyoni, A., Mbogo, S.K. and Leitch, B.W. (1988). Immunization of cattle using varying doses of Theileria parva lawrencei sporozoites derived from the African buffalo (Syncerus caffer) and treatment with buparvaquone. Parasitology 96:391-402.
