Isometamidium in pigs: disposition kinetics, tissue residues and adverse reactions

Researchin VeterinaryScience1991,50, 6-13

Isometamidium in pigs: disposition kinetics, tissue residues and adverse reactions L. D. B. KINABO, Q. A. McKELLAR, Department of Veterinary Pharmacology, P. D. ECKERSALL, Department of Veterinary Clinical Biochemistry, University of Glasgow

Veterinary School, Bearsden Road, G61 1QH

The disposition and adverse effects of the antitrypanosomal drug isometamidinm in pigs were evaluated. Following intramuscular administration of the drug at doses of 0"5, 15 and 35 mg kg -1, the drug was rapidly absorbed within 15 to 30 minutes to reach maximum plasma concentrations of 12 to 477 ( n = 6 ) , 302 to 655 ( n = 4 ) and 1620 ( n = l ) ng m1-1, respectively. No drug was detectable in plasma (less than 5 ng m l - 1) 24 hours after drug administration at the three doses used. The half-lives of disappearance of the drug from plasma during the terminal phase were 7,12 h for the pigs given a dose of 15 mg kg -1, and 7.20 h for the pig which received a dose of 35 mg kg -1. At all the intramuscular injection sites, high drug concentrations were found six weeks after administration. The most dramatic adverse reactions observed were: one death after intramuscular administration at a dose of 35 mg kg -~ to two animals, and two deaths after intravenous administration at a dose of 2 mg kg- 1 to two animals. For all these cases, the immediate cause of death was acute cardiovascular collapse. Biochemical analyses and gross and histological examinations showed that the animals that tolerated the high doses of 15 and 35 mg kg-1 given intramuscularly had extensive and severe tissue damage at the injection sites. Significant increases in plasma 3,-glutamyltransferase and alanine aminotransferase following drug administration suggested a degree of hepatobiliary damage.

INFECTION in pigs due to Trypanosoma simiae is an acute syndrome characterised by pyrexia, respiratory distress and prostration, and frequently it progresses to death within 48 hours of the onset of symptoms. T simiae is widely distributed in tsetse-infested areas of tropical Africa, particularly West, Central and East Africa. The primary vertebrate host is the warthog (Phacochoerus aethiopicus) and transmission is cyclical mainly by various species of Glossina, including Glossina morsitans, G palpalis and G fuscipes. Mechanical transmission by other biting

flies is also common. Although precise data on the incidence of pig trypanosomiasis in affected areas is limited, it is well known that both morbidity and mortality are usually high (Stephen 1966, Hoare 1972, Leach and Roberts 1981). To date, no effective drugs have been available for either curative or prophylactic purposes. Quinapyramine and combinations of quinapyramine and suramin have, however, been employed with limited success (Williamson 1980). The use of other antitrypanosomal drugs that are effective in other animal species have focused primarily on isometamidium, and this drug has been claimed to be effective in controlling clinical outbreaks and protecting pigs against Tsimiae for up to five months (Steel 1966, Finelle 1973, Otaru and Nsengwa 1987) although the manufacturers do not have a registered claim in the pig (Samorin; RM~ Animal Health, data sheet). The doses that have been considered effective in the pig range from 12.5 to 35.1 mg k g - 1given intramuscularly. These doses are considerably higher than those used in other animal species. They are about 20- to 70-fold higher than the recommended standard dose of 0"5 mg kg -1 for other animal species and such doses are usually lethal in other animals (Robson 1962, Philips et al 1967). Whether pigs have an inherent capacity different from other animals to eliminate isometamidium, or to tolerate the toxicodynamic actions of the drug is not known. The pharmacokinetics and the adverse effects of isometamidium in pigs following intramuscular and intravenous administration at doses that are effective in other animal species and at those considered effective against swine trypanosomiasis were therefore examined. Materials and methods

Animals Fourteen healthy pigs (Camborough hybrids, Landrace cross Large White) aged two to three

lsometamidium in pigs months and weighing 20 ± 4 kg were used. They were fed on a standard cereal diet and had free access to water.

Drug admin&tration Isometamidium at four different doses was administered to pigs randomly allocated to different groups as follows.

O. 5 mg kg-1 intramuscularly: six pigs received the drug as a I per cent w v - 1 aqueous solution administered into the gluteal muscles; 15 mg kg -1 intramuscularly: four pigs received the drug as a 4 per cent w v - 1 aqueous solution administered into the gluteal muscles; 35 mg kg-1 intramuscularly: two pigs received the drug as a 4 per cent aqueous solution administered into the gluteal muscles. Because one of the first two animals that received this dose died 15 minutes after drug administration, the number of animals at this dose was deliberately limited to two on ethical considerations. 2 mg kg 1 intravenously: this dose was used in two pigs as a 1 per cent w v - ~ aqueous solution administered into the anterior vena cava. The number of animhls was limited to two because this dose also proved fatal to the first two pigs that were administered the drug. Sampling Blood samples were collected from the anterior vena cavae from conscious unsedated pigs into heparinised tubes (Monovettes; Sarstedt) before and at predetermined times after drug administration for up to 14 days. Samples were immediately centrifuged and plasma separated and kept at - 2 0 ° C until analysed. Various tissues for drug analysis were collected when the animals were killed six weeks after drug administration. These were kept at - 20°C until analysed. The time between sample collection and drug analysis was about three weeks.

Drug analysis Plasma and tissue samples were analysed for isometamidium as described previously (Kinabo and Bogan 1988a).

Pharmacokinetic calculations The isometamidium plasma concentration versus time data for the pigs that received the high doses (15 and 35 mg kg-1) were analysed for each animal using

non-compartmental methods. The maximum plasma concentration (Crnax) and the time of occurrence (tmax) were obtained from the measured Values. The terminal phase rate constant (13) was determined by linear regression of the terminal phase log concentration versus time data, and the terminal phase half-life (tv2) was calculated as 0.693//3. The area under the plasma concentration versus time curve (AUC) was obtained by the trapezoidal rule and extrapolated to infinity using the equation C*//3, where C* is the last measured concentration. The area under the moment curve (AUMC)was determined from the area under the curve of the product of time and plasma concentration versus time data by the trapezoidal rule, and extrapolated to infinity using the equation t*. C*//3 + C*//32 where t* is the time for C*. The mean residence time (MRTim)was calculated as AUMC/AUC. The apparent volume of distribution (Vd(area)/F) was determined as dose//3-AUC, and body clearance (eL/F) was calculated as dose/Auc. The fraction absorbed (F) or bioavailabilitycould not be estimated because the animals that received the drug intravenously died.

Biochemical analyses Plasma samples from the pigs given the high doses were analysed for creatine kinase (¢K), aspartate a m i n o t r a n s f e r a s e (AST), alkaline phosphatase (ALKV), alanine aminotransferase (ALT), urea and creatinine on a Mira biochemical analyser (Roche Diagnostica) using commercial kits from the same source. Plasma haptoglobin (Hp) was assayed after modification of the method of Makimura and Suzuki (1982), described in detail by Eckersall et al (1988). For the pigs dosed at 15 mg kg- 1, the mean values for day - 1 samples were considered as baseline values and compared by the Student's t test for paired data with those for the samples collected on the other days. A probability level of P = 0" 05 was considered significant. The results are given as mean ± SD.

Pathological examination Post mortem examination was carried out on the three animals that died shortly after drug administration and various tissues were collected for histological examination. When the pigs were killed six weeks after drug administration, they were also examined for any gross lesions in the parenchymatous organs and at the injection site. Tissue samples from the injection sites for examination of microscopic lesions were collected in 10 per cent buffered formalin solution and processed routinely for haematoxylin and eosin staining.

8

L. D. B. Kinabo, Q. A. McKellar, P. D. Eckersall 2000 -

Results

Disposition kinetics The concentrations of isometamidium in plasma after administration in pigs are shown in Table 1 (0.5 mg kg -1) and Fig 1 (15 and 35 mg k g - l ) , and the disposition kinetics are shown in Table 2. The concentrations attained in plasma after intramuscular administration of 0.5 mg kg-1 were very low and could not be measured for more than two hours to permit calculation of pharmacokinetic parameter estimates. Following isometamidium administration at doses of 15 and 35 mg kg - I , the Cmax values were high (470 and 1620 ng ml-1, respectively) and rapidly attained (0.31 and 0.25 hours, respectively) but in both cases, the plasma concentrations were less than 5 ng ml-1 24 hours after drug administration. The extreme interanimal variation seen at the 0.5 mg kg-1 dose level was not apparent in the pigs given a dose of 15 mg kg -1. The rapid decay in plasma concentration had a terminal phase half-life of 7.12 hours for the pigs dosed at 15 mg kg -1, and w a s similar to that found for the pig dosed at 35 mg k g (7.20 hours). The AUC value for the pig dosed at 35 mg kg-~ was about twice that of the mean value for the group dosed at 15 mg kg-1, indicating that in this dose range, disposition of the drug obeyed firstorder kinetics.

Tissue residues

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Time (h) FIG 1: Plasma concentration versus time profile of isometamidium after intramuscular administration of the drug to four pigs at a dose of 15 mg kg - 1 (open circles; mean 4- SD values) and to one pig at a dose of 35 mg k g - 1 (closed circles)

tissues are shown in Table 3. Highest drug concentrations in all the pigs were found at the injection site, with very much lower concentrations in the liver and kidney. In the muscle, the concentrations were not detectable (less than 0.4/tg g - 1 wet tissue) in the pigs dosed at 0- 5 mg k g - 1, and only traces were present in those dosed at 15 mg kg -1.

Clinical signs

The concentrations of isometamidium measured in

The pigs administered isometamidium at doses of

TABLE 1: Plasma concentrations (ng m l - 1 ) of isometamidium in pigs after intramuscular administration at a dose of 0 - . 6 mg kg - 1

Time (h)

76

77

Pig number 78 79

0 0-25 0.50 0.75 1.0 2"0 4-0

0 29 16 8 ND ND ND

0 12 8 6 ND ND ND

0 78 20 12 8 ND ND

0 38 12 ND ND ND ND

80

81

0 477 132 16 8 6 ND

0 24 12 8 6 ND ND

Range (minimum-maximum) 12 8 ND ND ND

0 -----ND

477 132 16 8 6

ND Not detectable (less than 5 ng m l - 1) TABLE 2: Disposition kinetics of isometamidium in pigs after intramuscular administration at doses of 15 and 3 5 mg kg - 1

Parameter Cma x (ng m1-1) tma x (h) /~(h - 1 ) t,/= (h) AUC ( n g h m 1 - 1 ) MRTim (h) , Vd(area)/F(I k g - 1 ) CL/F(Ikg 1 h - l ) * Harmonic mean

21 302 O- 50 0"085 8.14 1020 8"16 172 14"68

Dose = 1 5 mg kg - 1 Pig number 23 24 25 505 O" 25 0"104 6-66 1255 6'74 115 11 " 9 5

655 O" 25 0"091 7"62 944 5-49 166 15'09

424 O" 25 0"109 6'34 1430 6"39 96 10"48

Mean ± SD 470 O" 31 0-097 7.12" 1175 6"69 137 13.05

4- 147 4- O" 13 4- 0 . 0 1 1 4444-

210 1.11 38 2"21

Dose = 35 mg kg -1 Pig number 27 1620 O' 25 0"096 7"20 2310 4.96 157 15.14

Isometamidium in pigs TABLE 3: Concentrations of isometamidium in tissues from pigs killed six weeks after intramuscular administration at three different doses

Tissue Injection site Liver Kidney Muscle

C o n c e n t r a t i o n (mean ± SD, #g g - 1 w e t tissue) D o s e = 0 - 5 mg k g - 1 D o s e = 15 mg k g - 1 D o s e = 3 5 mg k g - 1 (n = 6) (n = 4) (n = 1 ) 31 • 6 ~- 3 5 . 9 (1 • 7-91 • 9)

540 ± 290* (368-875)

Trace 0.62 ± 0.49 (0.3-1.3) ND

1690

0.63 ± 0.16 (0.5-0.9)

2 - 10

1 .0 ± 0-82 (0.4-2.2)

1 -80

Trace

0' 40

ND N o t d e t e c t a b l e (less t h a n 0 " 4/zg g - 1 w e t tissue) * n = 3 (no samp!e w a s collected f r o m one of t h e f o u r pigs) ( ) Range; m i n i m u m - m a x i m u m

15 and 35 mg kg -l intramuscularly showed muscle tremors, restlessness and excitement within the first 30 minutes. Additionally, the first pig to receive a dose of 35 mg kg-1 fell in lateral recumbency within the first 10 minutes. Cessation of respiration and loss of eye and pedal reflexes followed, and death occurred after 15 rain. The pigs that survived the high doses had marked swellings at the injection sites eight hours after drug administration, and they became lame for up to five days. The two pigs given the drug at a dose of 2 mg kg- 1 intravenously also showed sudden excitement, shivering and fell in lateral recumbency. This was followed by cessation of respiration, loss of reflexes and death after 15 minutes.

Biochemical changes"

± 15 iu litre-1 24 hours after drug administration. C o n c e n t r a t i o n s of ALT changed significantly (t=3"93, P=0"029) from 28 ± 3 iu litre -1 on day 1 to 39 ± 8 iu litre- 1eight hours after drug administration. This significant increase persisted for up to 14 days, with concentrations of ALT ranging from 40 ± 5 to 46 ± 9 iu litre- 1(t ranged from 3.57 to 8- 49 with corresponding P values of 0.038 and 0.003). There was a significant decrease in ALKP from 263 ± 53 to 158 ± 26 iu litre-1 on day 2 (t = 3.66, P = 0-035) and to 167 4- 24 iu litre -1 on day 4 (t=3.20, P = 0 . 0 5 ) . The responses with 'y-GT, ALT and ALKP for the pig dosed at 35 mg kg-l paralleled those shown by the group dosed at 15 mg kg -l. Alterations in plasma urea and creatinine (Fig 4) were measured to assess kidney function. For the pigs dosed at 15 mg kg-1, urea concentrations rose from 4.8 ± I-0 mmol litre- 1 on day - I to 5"7 ± 0"8 mmol litre-i eight hours after drug administration. This increase was significant with t = 6.65 and P = 0-007. No significant changes were found with creatinine concentrations. -

Alterations in plasma concentrations of CK and AST which reflect muscle damage in the pigs dosed at 15 and 35 mg kg- i are shown in Fig 2. Also included are the plasma concentrations of an acute phase protein Hp which reflect general tissue damage (Eckersall and Conner 1988). For the pigs dosed at 15 mg kg -1, cK increased significantly eight hours (t=6"23, Pathological changes P=0-008) and 24 hours ( t = 3 . 8 0 , P=0"032) after drug administration. There was also a significant Post mortem examination of the pig which died increase in AST (t = 3" 36, P = 0-044) eight hours after shortly after administration of the drug at a dose of 35 drug administration. In the pig dosed at 35 mg kg- 1, mg kg-1 revealed marked congestion of the heart, increase in Hp was about 60 per cent on days 1, 2 and liver, stomach and intestines. There was extensive dis4 after drug administration. In this animal, changes in tribution of the red drug solution between the muscles ¢K and AST were consistent with those found in the of the hindquarters at the injection site. The muscle group dosed at 15 mg kg -~. bundles appeared normal, indicating that the drug Changes in ALT as an indicator of hepatocellular was deposited intermuscularly and not in the muscle damage and ALKP and 3,-glutamyl transferase ('y-GT) mass. Histological examination confirmed the as indication of damage to the biliary tract are shown presence of general congestion including marked conin Fig 3. For the group dosed at 15 mg kg- 1, the con- gestion of vessels in the brain and meninges. There centration of 3,-GT on day - 1 was 30 ± 8 iu litre- 1 was also acute degeneration of muscle fibres at the and this rose significantly (t = 4.17, P = 0-025) to 52 periphery of muscle bundles at the injection site. The

L. D. B. Kinabo, Q. A. McKellar, P. D. Eckersall

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FIG 2: Concentrations of creatine kinase (CK), aspartate a m i n o transferase (AST) and haptoglobin (Hp) in plasma samples from pigs before and after administration of isometamidium i n t r a muscularly at doses of 15 (open circles; mean ± SD values, n = 4 ) and 35 (closed circles; n = 1 ) mg k g - 1 Drugadministra tion was at day 0 HbBC Haemoglobin binding capacity

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Time (days) FIG 3: Concentrations of 3, glutamyltransferase ('y-GT), alanine aminotransferase (ALT) and alkaline phosphatase (ALKP) in plasma samples from pigs before and after administration of i s o metamidium intramuscularly at doses of 15 (open circles; mean 4 SDvalues, n = 4 ) and 35 (closed circles; n = 1 ) mg k g - 1 Drug administration was at day 0

Isometamidium in pigs 7-

11

ing about 250 to 500 ml of reddish inflammatory fluid. Large parts of the cysts were lined by fibrous tissue which also encapsulated long tracts of necrotic muscle tissue. No discernible lesions were found in the parenchymatous organs. Histological examination of the lesions at the injection sites six weeks after administration revealed wide areas of coagulative necrosis, infiltration of leucocytes, macrophages and multinucleated giant cells. There was also evidence of capillary ingrowth.

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Time (days) FIG 4: Concentrations of urea and creatinine in plasma samples from pigs before and after administration of isometamidium intramuscularly at doses of 15 (open circles; mean ± SD values, n = 4 ) and 35 (closed circles; n = l ) mg kg - 1 . Drug administration was at day 0

cause of death appeared to be acute cardiovascular collapse. The carcases of the pigs that died after intravenous administration showed similar gross changes, with generalised organ and tissue congestion. Microscopic examination confirmed marked congestion especially in the centrilobular areas of the liver, and meninges. The cause of death for both pigs also appeared to be acute cardiovascular collapse. Six weeks after drug administration gross examination of the intramuscular injection sites for all the pigs given the drug intramuscularly showed extensive inflammatory reactions. In the pigs dosed at 0- 5 mg kg -1, the lesions were well defined by fibrous tissue encapsulating the necrotic muscle. In the pigs given the high doses, the lesions measured about 16 × 8 × 6 cm. These were characterised by large cysts contain-

In terms of the parameters examined in this study, pigs do not appear to exhibit considerable pharmacokinetic differences compared to other animal species such as cattle and goats. The plasma concentration versus time profile of the pigs dosed at 0.5 mg kg- z was similar to that found in cattle (Kinabo and Bogan 1988b) suggesting similar absorption and plasma clearance mechanisms. Furthermore, the mechanisms of clearing the drug from plasma in pigs did not appear to differ much from those in goats, judged by the similarity between their terminal phase half-lives (pigs: 7- 12 hours, Table 2; goats: 6.86 hours, Kinabo and McKellar 1990). However, goats had a much later tma× and higher plasma concentration after 24 hours than pigs following administration of similar doses. These differences are more likely to be related to variations in the kinetics of drug absorption than to differences in the elimination process. In the pigs, although the rate of absorption was initially rapid as reflected by the early tma× values, the extent of absorption was markedly limited since high concentrations of the drug were still found at the injection sites six weeks after drug administration. Since the Cmax for the pigs occurred at a time when the animals exhibited muscle tremors and excitement, it appears that higher plasma concentrations may produce greater adverse effects. This seems to explain the sudden death of the pig dosed at 35 mg k g - I In this pig, post mortem examination revealed that most of the drug was deposited in the intermuscular fascial sheaths, and it seems that the drug was exposed to a relatively large absorptive surface area (MacDiarmid 1983), resulting in high absorption and hence toxic concentrations in circulation. Problems of this kind are not uncommon with other drugs. Although it is not generally appreciated, it is known that, in practice, intramuscular injections are frequently made into fascial plane s and connective tissue, and not into the muscle mass (Baxter and Evans 1973, Marshall and Palmer 1980, Boyd 1987). These points might partly explain the wide variation in the duration of prophylaxis afforded by isometamidium in different trials and in the field (Kinabo and Bogan

12

L. D. B. Kinabo, Q. A. McKellar, P. D. Eckersall

1988c), the toxic reactions reported in cattle (Robson 1962) and goats (Kanyari et al 1983), and the much higher Cmax value for one of the pigs dosed at 0.5 mg kg -~ in this study (pig 80, Table 1). The concentrations of isometamidium found at the injection site in pigs given 0.5 mg k g - l in this study are lower than those reported for cattle given an equivalent dose and killed after a similar period (Kinabo and Bogan 1988c). This is also true with regard to the concentrations found in the kidney and liver. The reasons for these differences are unclear, but it could be speculated that the absorption mechanisms for the drug in pigs are more efficient than in cattle. Differences in absorption characteristics may also account for the lower concentrations of isometamidium found in tissue from goats (Kinabo and McKellar 1990) than in pigs in this study. The deaths of the pigs given the drug intravenously could also be attributed to the high concentrations that were attained in plasma. There is evidence from studies on isometamidium to suggest that the molecular mechanisms that could have resulted in acute cardiovascular collapse, the immediate cause of death of the three pigs, might be several, comprising: blockage of neuromuscular transmission, stimulation of cholinoceptors, potentiation of histamine (Philips et al 1967, Arowolo and Eyre 1984) and antagonism of Ca 2+ (Williamson 1979). Tissue damage at intramuscular injection sites is probably the major adverse effect of isometamidium in the various animal species in which the drug is routinely used. Previous studies that had investigated this aspect were based on gross and histological examination only (Braide and Isoun 1980, Kinabo and Bogan 1988b). Here, in addition to such methods, muscle damage was assessed using changes in plasma cK, AST and Hp as biochemical probes. Significant increases were rapidly seen in CK and AST which are released by damaged muscle tissue (Kaneko 1980). That these changes were related to muscle damage at the injection site was confirmed by the gross and histological findings. Because the liver and the kidney have been found to be the major parenchymatous organs in which isometamidium accumulates, at least in goats and cattle (Braide and Eghianruwa 1980, Kinabo and Bogan 1988b), biochemical functional tests were also carried out to evaluate whether, in pigs, these organs are adversely affected by the drug. The significant increases in "y-GTand ALT (Fig 3) suggest a degree of hepatobiliary damage has occurred. Kidney function did not appear to be greatly affected since only slight increases occurred in urea concentration eight hours after drug administration. The concentrations of the enzymes measured in the present study differ from those reported by Schillinger et al (1985) after intravenous administration of iso-

metamidium in cattle. These authors did not find any significant changes in serum AST, ALKP, and ",/-GT. Factors that may account for these differences include: the different routes of drug administration and the doses used, and variations between animal species (Rasmussen 1980). Animal drugs are usually marketed after being approved by drug regulatory bodies, which require that a given drug product is safe and effective for its labelled indications and for its use in the target species, and that its residues in edible products of food-producing animals do not constitute a health hazard to humans. Use of isometamidium in pigs is not indicated by the manufacturer (Samorin data sheet), but some workers have used it (Steel 1966, Otaru and Nsengwa 1987 ) and recommendations for use have been given (Finelle 1973). The high doses that are efficacious against T simiae infections in pigs, unlike ~rypanosomal infections of other animal species (Kinabo and Bogan 1988c) may reflect the sensitivity of this trypanosome species to isometamidium (Finelle 1973) rather than to pharmacokinetic differences between animal species since the concentrations found in plasma of pigs are similar or higher than those found in cattle (Kinabo and Bogan 1988a). No studies have been reported on isometamidium with regard to its in vitro or in vivo minimum effective concentrations against T simiae. The results obtained in this study show that pigs, like most other animal species, are also very susceptible to the toxic effects of isometamidium. It is concluded that the high doses that have been recommended for use in pigs against Tsimiae are too toxic to be acceptable for routine use. Additionally, the ecor~omic losses due to damage of meat would also be great, since in the pigs that received the high doses, all the hindquarters injected with the drug would have been condemned.

Acknowledgements This work was financially supported by the International Laboratory for Research on Animal Diseases (ILRAD), Nairobi, Kenya. The authors would like to thank Dr I. McCandlish for carrying out the post mortem examinations.

References A R O W O L O , R. O. A. & EYRE, P. (1984) Pharmacological interactions of three trypanocidal drugs with selected autonomic and autocoid mediator substances. Tropical Veterinarian 2, 6 8 - 7 5 BAXTER, J. S. & EVANS, J. M. (1973) Intramuscular injection in the cat. Journal o f Small Animal Practice 14, 297-302 BOYD, J. S. (1987) Selection of sites for intramuscular injection in the neck of the horse. Veterinary Record 121, 197-200 BRAIDE, V. & E G H I A N R U W A , K. I. (1980) Isometamidium residues in goat tissues after parenteral administration. Research in Veterinary Science 29, 111-113

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Received December 20, 1989 Accepted June 8, 1990