The Pharmacology of Halogenated Salicylanilides and Their Anthelmintic Use in Animals

Review article — Oorsigartikel

The pharmacology of halogenated salicylanilides and their anthelmintic use in animals

G E Swana

The structural criteria for fasciolicidal ABSTRACT action has been described as the need for The halogenated salicylanilides are a large group of compounds developed mainly for their electron-withdrawing substitutes on both antiparasitic activity in animals. Several halogenated salicylanilides with potent the salicylic and anilide rings, together antiparasitic activity have been synthesised of which only closantel, niclosamide, with a lipophilic group, such as tert-butyl oxyclozanide, rafoxanide and resorantel are commercially available. Closantel and in the 3-position66. Improved fasciolicidal rafoxanide, which represent the most important drugs in the group, are used extensively activity of salicylanilides has been for the control of Haemonchus spp. and Fasciola spp. infestations in sheep and cattle and achieved in compounds such as closantel Oestrus ovis in sheep in many parts of the world. Niclosamide is used extensively for its and rafoxanide by the incorporation of an anticestodal activity in a wide range of animals. Antiparasitic activity of the halogenated aryl chain in the aniline moiety of the salicylanilides has also been demonstrated against a large number of other internal anilide12. In both drugs the active parasites, in particular haematophagous helminths, and external parasites including ticks and mites, in a variety of animal species. Several cases of toxicity and mortality have been pharmacophore is 3,5-diiodosalicyl- 110 reported for closantel and rafoxanide in sheep and goats. Their unique pharmacokinetic oyol . behaviour appears to play an important role in the efficacy and safety of these compounds. Depending on the type of structural The chemical and physical characteristics, mode of action, pharmacokinetics, antiparasitic substitution, salicylanilides have been activity and toxicity of the halogenated salicylanilides in animals are reviewed. subdivided into a number of different groups, including halogenated and Key words: animals, antiparasitic activity, halogenated salicylanilides, pharmacology, review, safety. non-halogenated derivatives, acetyl- salicylanilides, dichlorosalicylanilides Swan G E The pharmacology of halogenated salicylanilides and their anthelmintic use in and benzoylsalicylanilides. Acetylsalicyl- animals. Journal of the South African Veterinary Association (1999) 70(2): 61–70 (En.). Depart- ment of Pharmacology and Toxicology, Faculty of Veterinary Science, University of anilide derivatives, e.g. clioxanide, are Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa. activated in vivo. This is presumably achieved by hydrolysis of the acetyl group to free the hydroxyl derivative38. INTRODUCTION many years and the extensive use of these All salicylanilides developed for use as Salicylanilides are a very large group of products, no definitive review of these anthelmintics in animals are halogenated compounds, originally developed as products has been published. and include: clioxanide (N-(4’-chloro- fungicides for topical use and as antimi- phenol)-3,5-diiodo-acetylsalicylamide); crobial agents in soaps64. Later these com- CHEMICAL AND PHYSICAL closantel (N-(5-chloro-4[(4-chlorophenyl) pounds were shown to possess potent CHARACTERISTICS cyano-methyl]-2-methylphenyl)-2-hydro anthelmintic activity of which the niclo- xy-3,5-diiodo-benzamide), dibromsalan samides54 tribromsalans8 and clioxanide9 Chemical structure (4’,5’-dibromosalicylanilide); oxycloza- were some of the 1st agents to be used. Salicylanilides, also referred to as nide (3,3’,5,5’,6-pentachloro-2-hydroxy- Since then, a wide range of halogenated benzamides or salicylamides54, are weak, salicylanilide), niclosamide (5-chloro-N- salicylanilide congeners active against acidic phenolic compounds. The basic (2-chloro-4-nitrophenyl-2-hydroxy- helminth parasites have been synthe- chemical structure consists of a salicylic salicylanilide); rafoxanide (3’-chloro-4’- sised11,12,32,37,66,76,102,110,126. The halogenated acid ring and an anilide ring (Fig. 1). (p-chlorophenoxy)-3,5-diiodosalicyl- salicylanilides available in South Africa are summarised in Table 1113. Halogenated salicylanilides, in particu- Table 1: Halogenated salicylanilides registered for use in animals in South Africa. lar closantel and rafoxanide, are impor- Drug Trade names Manufacturer/Supplier tant anthelmintics that are used extensively in the control of Haemonchus Closantel Flukiver, Seponver Janssen AH spp. and Fasciola spp. infestation in sheep Pro-inject Yellow, Prodose Yellow, and cattle, and Oestrus ovis in sheep in Ranide Super, Vetdose 4, Vetdose 4 injectable Logos Agvet many parts of the world. Niclosamide is Oxyclozanide Milborrow Liverfluke Remedy Bayer AH widely used for the treatment and control Niclosamide Ex-A-Lint Hoechst Roussel Vet of cestode infections in several animal Lintex Bayer AH species. Despite being available over Prodose Lint Logos Agvet Rafoxanide Nasalcur Hoechst Roussel Vet Ovinox Bayer AH aDepartment of Pharmacology and Toxicology, Faculty of Ranide Logos Agvet Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa. Ranox Pfizer AH Resorantel Terenol-S Hoechst Roussel Vet Received: December 1998. Accepted: May 1999.

0038-2809 Jl S.Afr.vet.Ass. (1999) 70(2): 61–70 61 Closantel has a molecular weight of 663.0714. It is weakly acidic (pKa = 4.28) and is highly lipid-soluble (log P = 7.15, n-octanol/buffer pH 6.98)72.

MODE OF PHARMACOLOGICAL ACTION The primary action of salicylanilides has generally been associated with the uncoupling of oxidative phosphorylation. Early in vitro studies, using houseflies as well as rat liver mitochondria, showed several salicylanilides as potent inhibitors of this electron transport associated phosphorylation127. Salicylanilides were approximately 1000–10000 times more potent than dinitrophenol, an earlier compound known to inhibit oxidative phosphorylation. In vitro activity of inhibition of electron transport associated phosphorylation in Fasciola hepatica and Ascaris lumbricoides were later reported for oxyclozanide, rafoxanide and closan- tel24,25,26,62,121,123. Several workers have subsequently confirmed the proposed mechanism in vivo25,87,123. In vitro inhibition of succinate dehydrogenase activity with a wide range of salicylanilides38 and inhibition of the fumarate reductase system by oxyclozanide and rafoxanide in F.hepatica (unpub- lished information as cited in Coles23) Fig. 1: Chemical structures of the halogenated salicylanilides developed for use as anthelmintics in animals1,14. have also been reported. These effects may be due to the interrelationship of the both towards aqueous solubility and succinate dehydrogenase activity to the stability in suspension. Absorbed or fumarate reductase system and oxidative occluded impurities are thought to play phosphorylation. It has been suggested an important role in the polymorphic that rafoxanide diminishes ATP-syn- behaviour of the drug. thesis, resulting in increased internal Fig. 2: Basic structure of salicylanilides: salicylic ring (A) and anilide ring (B). Anthelmintic activity of rafoxanide is intermediate pools in the pathway. Later, inversely related to the size of crystalline as an independent effect, the further particles present in a 2.5 % oral suspen- metabolism of succinate is inhibited. anilide); resorantel (N-(4’-bromo- sion4. According to the chemiosmotic theory phenyl)-6-hydroxysalicylamide) and of Mitchell73, hydrogen-ionophores act by tribromsalan (3,4’,5-tri-bromosalicyl- Physicochemical characteristics uncoupling of phosphorylation by the anilide). The chemical structures of these The salicylanilides are generally weakly translocation of protons through the compounds are given in Fig.2.1,14. acidic, highly lipid-soluble compounds. inner mitochondrial membrane. The Very little information on the physico- maintenance of the proton gradient Crystal structure and polymorphic chemical characteristics of the individual across the inner mitochondrial mem- forms salicylanilides has been reported. Of the brane is essential for the continued pro- The 2 aromatic rings of the salicylanilide salicylanilide group, rafoxanide and duction of ATP. The inner mitochondrial moeity are approximately co-planar with closantel are the most widely used and membrane is normally impermeable to a dihedral angle between them101. There are therefore more extensively described protons, but can be rendered permeable are significant conformational differences in literature. Rafoxanide has a molecular by the addition of uncouplers of oxidative between the various halogenated salicyl- weight of 626.01 and a melting point of phosphorylation that act by destroying anilides and analogues regarding their 173–7 °C2. It is a greyish-white crystalline the proton gradient. This activity selec- crystalline forms. Interatomic dimensions powder, moderately soluble in acetone, tively prevents the utilisation of the in the salicylanilide moiety are consistent chloroform, ethyl acetate, acetonitrile and chemical energy derived from electron within the halogenated salicylanilides. methanol but insoluble in water76. transport for the net phosphorylation of The crystalline forms of closantel have Ultraviolet light absorption of a 0.004 % ADP to ATP,thus depriving the cell of its not been reported. w/v solution in 0.1 M methanolic hydro- normal source of energy103. Polymorphic forms of salicylanilides chloric acid is in the range 230–350 nm Closantel was shown to have in vitro have been characterised by X-ray powder and exhibits maximal excitation at 280 nm and in vivo effects on both the motility and diffraction for oxyclozanide82. The poly- and 335 nm. Absorbance at 280 nm is ultrastructure of F. hepatica103. It induces morphic forms exhibit variable behaviour, about 0.97 and at 335 nm, about 0.59 2. rapid spastic paralysis of the fluke, severe

62 0038-2809 Tydskr.S.Afr.vet.Ver. (1999) 70(2): 61–70 Table 2: Summary of pharmacokinetic parameters reported for closantel, rafoxanide and oxyclozanide in sheep and cattle.

Drug and species Dosage Pharmacokinetic parameter Reference n Dose Routea AUC Cmax Tmax T½el (mg/kg) (µg.d/m ) (µg/m ) (h) (d)

Closantel — sheep 4 7.5b i.r. 786 ± 335 32 ± 9 64.8 ± 21.6 14.3 ± 1.9 55 4 3.75b i.m. 575 ± 278 31 ± 4 12 ± 12 12.2 ± 4.6 5 10 p.o. 1303 ± 447 47 ± 11 24 23 72 5 5 i.m. 1027 ± 101 48 ± 4 8–24 16 3 5 p.o. 852 ± 215 22 ± 5 – – 3 5 i.m. 1497 ± 428 55 ± 10 – – 310c p.o. 1541 ± 272 48 ± 6 – – 310d p.o. 1578 ± 223 51 ± 3 – – 5 7.5 p.o. 1035 ± 212 48 ± 5 – 14.5 ± 2.3 75 — cattle 3 10 p.o. 1108 ± 112 51 ± 3 48 – 72 3 5 i.m. 1205 ± 323 54 ± 5 48 – 6 2.5 i.m. 756 ± 333 28 ± 7 – – 3 5 i.m. 1205 ± 323 54 ± 5 – – 310c p.o. 1118 ± 327 48 ± 6 – – 310d p.o. 1108 ± 112 51 ± 3 – – Rafoxanide — sheep 5 7.5 p.o. 605 ± 79 23 ± 2 – 16.6 ± 1.2 75 8 15 p.o. 203 ± 74 26±7 39 ± 14 6.5 ± 1.5 114 10 7.5 p.o. 414 ± 106 35±5 26 ± 2.4 7.5 ± 2.0 115 Oxyclozanide — sheep 5 15 p.o. 51±8 19.0 ± 2.3 – 6.4 ± 0.8 75 ap.o. = orally; i.r. = intraruminal; i.m. = intramuscular. b14C-closantel. cSolution; dsuspension. sloughing of the integument, swelling of pharmacokinetic parameters reported for lambs compared to housed lambs fed hay the basal infoldings, mitochondrial defor- closantel, rafoxanide and oxyclozanide in and concentrates118. Peak rafoxanide mation and a reduced output of secretory sheep and cattle are given in Table 2. plasma concentrations were significantly products, especially in the tegumental These parameters were derived from higher in sheep infected with 6-week-old and gastrodermal cells. The spastic paral- both model-independent procedures and F. hepatica as compared to uninfected ysis seen with closantel is similar to that non-linear compartmental analysis. The sheep75. A 2–3-fold increase in the observed with rafoxanide and oxycloza- elimination rate constants and half-lives bioavailability of rafoxanide was reported nide41. Although these changes have been of closantel were determined by a single in suckling lambs, aged 5–8 weeks of age ascribed to uncoupling of oxidative exponential compartmental model in 2 of compared to weaned lambs aged 4–5 phosphorylation, the increased muscle the studies55,72, whereas in 1 study a months115. tone may be due to an increase in calcium tri-exponential equation determined the In ruminants the oral bioavailability of ions within the muscle cells of the fluke103. best-fitted curves for closantel, closantel relative to intramuscular admin- Closantel has also been shown to oxyclozanide and rafoxanide75. istration is approximately 50 %72. A similar rapidly decrease intrategumental pH in A lag time for oral absorption was in- relative oral bioavailability, based on the Schistosoma mansoni and F. hepatica at con- cluded in the model in one of the difference in efficacy against 6-week-old centrations that were lower than those rafoxanide studies evaluated by a single F. hepatica, after oral (36.1 %) and intra- that affect ATP concentration80. The sensi- exponential compartment114. muscular (60.4 %) administration at tivity of flukes to uncoupling agents may 3 mg/kg, has been proposed for rafoxa- be due to the fact that Kreb’s cycle activity Absorption nide in cattle75. is restricted to the ‘outer layer’ of the Following oral administration of The extent and rate of absorption of fluke119. closantel and rafoxanide in ruminants, closantel is dose independent72. A linear maximum plasma concentrations (Cmax) increase in closantel plasma concentra- PHARMACOKINETICS are reached 24–48 h later7,114,115. Hennessy tions was reported in cattle and sheep at Very few pharmacokinetics studies, et al.55 reported a longer time of 65 h to doses between 2.5 mg/kg and 10 mg/kg. other than metabolic studies, have been maximum plasma concentrations (Tmax), Niclosamide is restricted to the gastroin- reported for salicylanilides in rumi- indicating a slower rate of absorption of testinal tract due to very poor absorption nants30,31,55,72,75,114,115. These pharmaco- closantel administered intraruminally in following oral administration20. kinetic studies involve only closantel, sheep. The rate of gastrointestinal absorp- The activity of clioxanide in sheep is oxyclozanide and rafoxanide. No intra- tion of closantel is slower in goats than in reduced if it is passed directly into the vascular pharmacokinetic studies have sheep, most likely due to a reduced abomasum9,117,123. It is suggested that been reported. digesta flow rate55. deacetylation of clioxanide takes place in Halogenated salicylanilides generally Reduced digesta flow rate resulted in the rumen to form a hydroxyl derivative share common pharmacokinetic significantly lower peak plasma concen- that is more readily absorbed86,87. Efficacy features70. The anthelmintic activity of trations and extent of absorption, as of clioxanide is reduced if administered these compounds has been directly asso- measured by area under the drug concen- into the abomasum, most likely due to ciated with their pharmacokinetic tration versus time curve (AUC) of rafoxa- slow deacetylation affecting the extent profile6,47,66,72,75,86. A summary of the main nide administered orally in grazing and rate of absorption, with reduced

0038-2809 Jl S.Afr.vet.Ass. (1999) 70(2): 61–70 63 persistence of the active constituents in Rafoxanide is metabolised to 3,5-diio- other animal species. Niclosamide is blood. Acetylsalicylanilide derivatives dosalicylic acid by amide hydrolysis and recommended for use in ruminants, dogs such as clioxanide show poor anthel- is found in blood, milk and muscle of and cats. Closantel and rafoxanide have mintic activity in vitro, but in vivo are cattle29. Amide hydrolysis is unlikely in the broadest antiparasitic spectrum and potent anthelmintics38. closantel, niclosamide and resorantel are the most widely used of the owing to steric hindrances by substitu- salicylanilides. Oxyclozanide, niclosa- Distribution ents ortho to the amide bond72. mide and resorantel have a very narrow Halogenated salicylanilides are exten- Metabolic dehalogenation of both anthelmintic spectrum. sively plasma-bound and poorly distrib- iodine atoms has not been observed for uted to tissues. Michiels et al.72 reported closantel or other deiodosalicylanilide Nematodes the ratio of closantel in the plasma relative derivatives29,34,72. Rapid and complete The anthelmintic activity of salicyl- to that in the lung and kidney tissue is 6–7; photolytic dehalogenation of rafoxanide anilides, except niclosamide and oxy- 9–12 for heart and liver; 30 for muscle; and by exposure to daylight has been clozanide, is specifically directed towards about 100 for fat. A plasma to liver concen- reported44. haematophagous nematodes86. Niclosa- tration ratio of 17.5 for rafoxanide; and a Glucuronide formation occurs with mide and oxyclozanide are not effective plasma to bile ratio for closantel and salicylanilides and accounts for the against nematodes. Closantel and rafoxanide of >50 % and 35 %, respec- presence of an enterohepatic circulation rafoxanide are highly effective against tively, have been reported75. proposed for these compounds74. In rats both immature and mature stages of Extensive binding to plasma albumin 10–15 % of a salicylanilide dose is Haemonchus contortus, Geigeria pachyscelis (>97 %) has been reported for both reabsorbed from the gastrointestinal and Chabertia ovina in sheep47,60 and H. closantel and rafoxanide72,75. The binding tract. A glucuronide metabolite of oxy- placei, Bunostomum phlebotomum and to plasma albumin is characterised by clozanide has also been identified11. Oesophagostomum radiatum in cattle47,98,106. very high affinity and high capacity. Halogenated salicylanilides are not me- Activity against the immature parasitic Closantel-binding to erythrocytes (c. 4%) tabolised by glutathione conjugation33. stages are partly due to persistent activity and in plasma water (1 %) is limited72. Closantel is excreted mainly via the related to their long biological half-lives70. The extensive plasma binding of faeces. More than 80 % of 14C-closantel Reduced effectivity occurs against salicylanilides is responsible for the long was recovered from total 8-week faecal non-blood sucking immature H. contortus elimination half-life (T½el) associated output and less than 0.5 % from the urine (before 8 days of age)100 and hypobiotic with these products75,86,88.AT½el of 2–3 in sheep72. Within 48 h of dosing c. 43%of larval stages108. Oxyclozanide is poorly weeks has been reported for closantel and the oral and 10 % of the i.m. dose was active against Haemonchus spp. and is rafoxanide in sheep and cattle, and 6 days excreted with the faeces. Thereafter, ineffective against Bunostomum sp., for oxyclozanide in sheep30,31,55,72,75. The faecal excretion proceeded more slowly Oesophagostomum sp. and Chabertia sp. in T½el for closantel in different animal with an average of 1–2 % of the dose per either cattle or sheep126. species varies from 6 days in goats, 1 week day. A faecal elimination half-life of Persistent antiparasitic activity of in the rat and 10 days in the dog55,72. Faster 15.9–23 days was reported. Two to three closantel has been reported against a elimination resulted in an almost 3-fold percent of closantel is excreted at a maxi- number of different parasite species and lowering of the AUC in goats55. mum concentration of 1 µg/m in milk in in different animals47,48,50,52. Activity is Comparable data in rats and dogs have cattle. Comparable excretion of rafoxa- maintained against infective 3rd larval 6 been reported for rafoxanide . Elimina- nide occurs in the urine of sheep and milk stages (L3)ofH. contortus and G. pachyscelis tion half-life of closantel and rafoxanide in cattle29,30,31,54. No data on biliary and when administered orally to sheep at was unaffected by route and dose admin- faecal excretion of rafoxanide have been 10 mg/kg for up to 7 weeks and 8 weeks istered31,55,72. reported in ruminants. Oxyclozanide is before infection, respectively47.Pro- It has been suggested75 that the T½el of concentrated and excreted in the bile in longed activity has also been reported 11 salicylanilides may be correlated with the the environment of adult flukes . against L3 H. placei, B. phlebotomum and O. turnover of plasma albumin to which they In the rat the urine is the most important radiatum in cattle when administered are bound, which is about 16.6 days56. (>70 %) route for excretion of the subcutaneously (s.c.) at 5 mg/kg. Anthel- salicylanilide derivative of benzanilide, mintic persistence of closantel protects Metabolism and excretion an intermediate compound used in the sheep against reinfection for up to 28 Salicylanilides are poorly metabolised dye and perfume industry74. Only 20 % is days52. and are excreted mainly unchanged. excreted in the faeces. Thirty-five percent A single s.c. injection of closantel at Only 1.0–2.5 % of rafoxanide is metabo- of the [14C]salicylanilide was excreted in 5 mg/kg completely cleared adult Capil- lised in the liver in cattle31 and about 90 % the bile in 24 h. Incubation with rat caecal laria bovis infections in cattle47. of closantel is excreted unchanged in the contents produced no hydrolysis104.In Marked effectivity against natural and faeces and urine in sheep and cattle72. the case of rafoxanide there is extensive experimentally-induced infestations of A reductive monodeiodination reaction metabolism in the bile of rats46. Strongylus vulgaris was reported in foals appears as the main metabolic pathway given repeated doses of an oral closantel for closantel in sheep, resulting in the ANTIPARASITIC ACTIVITY formulation48,49. Fourth larval and imma- formation of 3 and 5-monoiodoclosantel Salicylanilides have variable activity ture adult stages of S. vulgaris present in isomers72. Similar results have not been against a wide range of helminths and a the mesenteric arteries had been com- confirmed in cattle and goats99. Incuba- number of ectoparasites. The antiparasitic pletely cleared in foals treated 5 times tion of clioxanide with sheep liver spectrum of halogenated salicylanilides with closantel at 20 mg/kg every 2 months microsomal enzymes revealed that used in sheep and cattle is summarised in starting at 1 month of age and was 86 % monodeiodination is a non-enzymatic Tables 3 and 4. Except for niclosamide, effective when foals were given 3 treat- reaction32. Monodeiodination was not these products are mainly restricted for ments of 8 mg/kg at the same interval. At considered in the metabolic degradation use in ruminants, although antiparasitic the higher dose, closantel was also highly of rafoxanide29. activity has been shown in a number of effective against adult S. vulgaris, S.

64 0038-2809 Tydskr.S.Afr.vet.Ver. (1999) 70(2): 61–70 Table 3: Summary of the antiparasitic spectrum of all halogenated salicylanilides immature and adult F. hepatica and F. commonly used in sheep. gigantica has been confirmed in both cattle63,85,96,98,106,107 and sheep3,10,19,26,39,60.An Type of parasite Antiparasitic spectrum improved efficacy against F. gigantica has Closantel Rafoxanide Oxyclozanide Niclosamide Resorantel been ascribed to the more pathogenic nature, voracious feeding habits and NEMATODES higher metabolic rate of this parasite, in H. contortus relation to F.hepatica10,26,96. Rafoxanide also Adult +++ +++ + appears to be more efficacious in sheep Immatures +++ ++ than in cattle against fluke of comparable G. pachyscelis age60,106,107. Administered s.c., rafoxanide Adult +++ +++ injectable solution is approximately 2.5 Immature +++ +++ times more effective than the oral suspen- C. ovina sion formulation when administered i.r. Adult +++ +++ in cattle against Fasciola infestation96,122. Immatures +++ +++ Rafoxanide at 15 mg/kg orally is moder- TREMATODES ately effective against immature Param- 58 F. hepatica phistomum microbothrium in sheep, but Adult +++ +++ +++ poorly effective up to 9 mg/kg s.c. in Immatures +++ +++ ++ cattle96,98. F. gigantica Putative efficacy of closantel and Adult +++ +++ +++ rafoxanide against immature F. hepatica Immatures +++ +++ +++ has been attributed to the persistent Paramphistomum sp. plasma concentrations affecting the Adult +++ ++ flukes as they mature75. The presence of Immatures ++ ++ ++ stunted or arrested fluke forms following CESTODES closantel and rafoxanide treatment in Moniezia expansa +++ ++ sheep, is in part also ascribed to the persistent effect of the drug69,75. Nevertheless ECTOPARASITES there is other evidence that indicate that Oestrus ovis +++ +++ these drugs act prior to F.hepatica reaching Linognathus ovillus +++ maturity. Campbell et al.19 found that Psoroptes sp. ++ rafoxanide was virtually 100 % effective

+Effectivity only demonstrated; ++moderately effective; +++highly effective. against 6-week-old fluke when sheep were necropsied 6–10 days after treatment, whereas Maes et al.69 showed that edentatus and Triodontophorus spp. Trematodes closantel was equally effective against Closantel at 7.5 and 10 mg/kg has Salicylanilides are effective against a 6-week-old and 8-week-old fluke when marked anthelmintic effect on adult wide range of hepatic and intestinal necropsied either 1 week or 12 weeks after stages of Ancylostoma caninum in dogs50. trematodes in a variety of animals20,122. treatment. The authors propose that the maturation Closantel, rafoxanide and oxyclozanide According to Maes et al.69, the flukicidal of the larval stage may be affected at are of the most important drugs used for effect of closantel is related more to peak 20 mg/kg, although closantel does not the treatment and control of fascioliasis, plasma concentrations and less to resid- affect the abundance of arrested hook- whereas niclosamide and resorantel form ual persistent effect. worm larvae nor prevents their subse- the mainstay of Paramphistomum control Closantel given orally at 15 and quent development. in ruminants. 20 mg/kg is highly effective in reducing Early studies showed that rafoxanide Closantel administered at 5 and 8-week-old Fascioloides magna infestation was highly effective against a thiaben- 10 mg/kg orally in sheep and at 2.5 mg/kg in sheep111,112. A 7.5 mg/kg dose adminis- dazole tolerant H. contortus K-strain40. s.c. in cattle is highly effective against tered i.m. was equivalent to a 15 mg/kg Closantel was also shown to have activity adult F. hepatica47,69. In sheep similar high oral dose112. Rafoxanide at 10 and against benzimidazole resistant strains of efficacy occurs at the same dosage against 15 mg/kg administered orally was shown H. contortus in sheep52,53 and against adult and immature F. gigantica to be 100 % effective against both imma- fenbendazole- and levamisole-resistant (8-week-old) and immature F. hepatica ture and mature F. magna42. Oxyclozanide H. contortus strains120. Anthelmintic resis- (4- and 6-week-old)47. Only moderate effi- only had a slight effect. tance to rafoxanide and closantel has been cacy against immature F. hepatica At doses of 10 and 15 mg/kg oxy- identified in H. contortus in sheep in South (6-week-old) at 7.5 mg/kg s.c. was clozanide is highly effective against Africa124. reported47. Given intramuscularly at mature stages of F.hepatica and against the Closantel, in combination with broad 2.5 mg/kg in cattle, closantel is highly adult and immature (6 weeks) stages of spectrum anthelmintics, has been used effective against adult F. gigantica, but F. gigantica in sheep and cattle11,93,126. successfully in a preventative anthelmin- only slightly effective (55.8 %) against the Higher doses are required against tic programme to control haemonchosis 6-week-old immature stages47. 6-week-old F. hepatica. It was suggested and trichostrongylosis in sheep and was Closantel is ineffective against imma- that the poor activity against immature F. reported to retard the selection for anthel- ture stages of 2 commonly occurring hepatica is due to the high plasma binding mintic resistance in Trichostrongylus spp.27. paramphistome species in Australia92. of oxyclozanide in blood that bathes the Eradication of H. contortus using closan- The efficacy of rafoxanide oral suspen- immature fluke in the liver parenchyma11. tel in sheep has been proposed5. sion at doses of 2.5–20 mg/kg against According to Froyd et al.43, there is no

0038-2809 Jl S.Afr.vet.Ass. (1999) 70(2): 61–70 65 Table4: Summary of the antiparasitic spectrum of salicylanilides commonly used in cattle. immature and mature paramphistomes in sheep, goats and cattle. However, Type of parasite Antiparasitic spectrum according to Van den Bossche et al.122 Closantel Rafoxanide Oxyclozanide Niclosamide Resorantel resorantel is the most consistently successful drug used in the treatment of NEMATODES paramphistomiasis in cattle and sheep. H. placei Resorantel has also been used for the Adult +++ + + treatment of G. aegypticus90. Immatures +++ B. phlebotomum Cestodes Adult +++ ++ Anticestodal activity of salicylanilides is Immatures ++ predominantly restricted to niclosamide O. radiatum and resorantel, although some activity Adult +++ ++ has also been reported for closantel and Immatures +++ oxyclozanide. The effect of oxyclozanide Capillaria sp. against Moniezia sp. in sheep has been TREMATODES restricted to reduction in faecal egg counts and voiding of gravid segments126. F. hepatica At necropsy it was noted that the scolices Adult +++ +++ +++ Immatures +++ ++ had remained in situ and were capable of continued growth. Closantel at 40 mg/kg F. gigantica orally and 20 mg/kg administered i.m. has Adult +++ +++ Immatures +++ +++ been shown to have high anthelmintic activity against the larval stages of Taenia Paramphistomum sp. pisiformis in experimentally-infected Adult + +++ +++ +++ 22,47 Immature + +++ +++ rabbits . No effect against the cysti- cercus stages in the peritoneal cavity has CESTODES been found. Activity against Anoplocephala ECTOPARASITES perfoliata was demonstrated in horses that Dermatobia sp. +++ had received 5 monthly doses of closantel Hypoderma sp. +++ at 20 and 40 mg/kg48. Gedoelstia sp. ++ Chrysomya bezziana ++ Ectoparasites Although salicylanilides are primarily Boophilus microplus ++ effective against helminths, their effect Amblyomma sp. ++ against a number of insects and arthro-

+Effectivity only demonstrated; ++moderate efficacy; +++highly effective. pods has been demonstrated. As far as could be determined, except for Oestrus ovis, these activities have not been recog- correlation between oxyclozanide ture and mature F. hepatica has been nised as official claims by regulatory concentrations in plasma and effectivity reported by a number of workers8,9,18,19,81,94. authorities for these products. against liver fluke as judged by the num- Large differences in fasciolicidal efficacy Closantel and rafoxanide are 2 of the ber of parasites found at slaughter. A fixed of clioxanide, particularly against imma- major products recommended for the dose of 3.4 g of oxyclozanide was shown ture stages occurs between oral, i.r.and i-a control of O. ovis in sheep47,58,95,108. Accord- to have equivalent efficacy in cattle with routes of administration8,9,17. A marked ing to Snijders et al.108, rafoxanide at mass greater than 350 kg in comparison reduction in efficacy occurs following i-a 7.5 mg/kg given orally was highly effec- with a dose of 10 mg/kg. administration. Reduction in efficacy re- tive against overwintering 1st instar Oxyclozanide has been extensively ported after oral treatment is most likely larvae and against 2nd and 3rd larval used for the treatment of paramphisto- due to a proportion of the drug that by- instars within 96 h of treatment. A resid- miasis122. Oxyclozanide alone or in combi- passes the rumen and which is deposited ual effect against the re-establishment of nation with levamisole is highly effective into the abomasum9,19. As described previ- O. ovis in recently-treated sheep was against immature and mature param- ously, the activity of clioxanide depends proposed58,59. Closantel is highly effective phistomes at oral doses of 15 and on activation by rumen microbes. The against O. ovis at 5 mg/kg administered 18.7 mg/kg91,122. Rolfe and Boray91 found efficacy of rafoxanide against F. hepatica orally to sheep47. that 2 doses of oxyclozanide given 3 days is not affected when administered Other than their effect against O. ovis in apart was more effective than a single either orally, intraruminally or intra- sheep, closantel and rafoxanide have dose against Calicophoron calicophorum in abomasally17. been shown to be effective against a range cattle. Single doses gave varying activity. Niclosamide at 50–100 mg/kg and of other Oestridae in different animals. According to Van den Bossche et al.122 resorantel at 65 mg/kg given orally to Studies conducted in foals treated with oxyclozanide does not remove all para- cattle and sheep are highly effective closantel 3 or 5 times every 2 months at sites present in the host. Where complete against immature P.microbothrium45,57. The doses between 2–40 mg/kg demonstrated success is claimed, it is usually on the basis effect of niclosamide in calves against the a high efficacy against Gastrophilus of negative faecal egg counts. In horses, immature stages is erratic and it is not intestinalis larvae48,49. In yearlings, 3 doses oxyclozanide is successful against effective against adult stages in both of at least 8 mg/kg were required. Gastrodiscus aegypticus90. sheep and cattle20. Erratic efficacy is also Closantel has also been shown be effec- The efficacy of clioxanide against imma- reported for resorantel against both tive against Dermatobia hominis21,

66 0038-2809 Tydskr.S.Afr.vet.Ver. (1999) 70(2): 61–70 Hypoderma bovis47 and H. lineatum35 in cattle. Effectivity of rafoxanide against Gedoelstia in blesbuck105 and D. hominis in cattle21 has also been reported.Efficacy of closantel against Chrysomya bezziana has also been shown109. Closantel had no effect on the larvae of Stomoxys calcitrans and Haematobia irritans36. Anti-arthropod activity of closantel has been reported against Boophilus microplus in cattle that were naturally infected67,125 and against Amblyomma americanum in 36,47,67 experimentally-infected cattle ,and Fig. 3: Clinical signs of rafoxanide and closantel toxicity in sheep at more than double the against Linognathus ovillus15, Cochlyomia recommended dose (left: weakness and recumbency; right: mydriasis). hominivorax47 and Psoroptes communis var. ovis83 in sheep. Closantel is active against Demodex canis in dogs68 and Ornithonyssus sylvarium when given as a feed additive to chickens28.

SAFETY AND TOXICITY Salicylanilides are moderately safe compounds and have safety factors of approximately 3–6 times the recommended dose levels1,116. No untoward effects are generally seen with rafoxanide when using single doses of 58 mg/kg in cattle or 45 mg/kg in sheep. However, toxicity has Fig. 4: Brain and optic nerve histopathological lesions in sheep following rafoxanide and been reported in lambs that had allegedly closantel poisoning (left: optic nerve vacuolation; right: vacuolation of white matter of been treated at the recommended dose84. brain).

ALD50 of rafoxanide for either sheep or cattle has not been determined, but for severe in sheep infected with liver fluke Photodermatitis and skin irritation in 76 the rat an oral LD50 of approximately than in uninfected animals . man have been reported for halogenated 2300 mg/kg has been calculated. Accord- Signs of toxicity occur within 24–72 h of salicylanilides which have been incorpo- ing to Adams1, no adverse effects are seen treatment16,51,89. However, sheep inadver- rated in soaps as antimicrobial agents or with closantel in sheep after repeated tently overdosed with rafoxanide, at an when used topically as fungicides61,64. doses of 10 or 40 mg/kg orally or 5 or estimated dose of 450 mg/kg, exhibited 20 mg/kg i.m. every 4 weeks over a signs of toxicity on the same day79. Recov- Pathological lesions 40-week period. Closantel is safe for use ery of some affected animals over a period No gross post mortem lesions have been in breeding rams, ewes and bulls. of 3–4 weeks was reported in goats over- reported in either closantel or rafoxanide Salicylanilides are potent uncouplers of dosed with closantel16. Cattle overdosed toxicity. Histopathologically, symmetrical oxidative phosphorylation127. The phar- with rafoxanide s.c. at 45–60 mg/kg status spongiosis (Fig. 4), oedema, macokinetic behaviour of salicylanilides presented signs of tachypnoea, muscle haemorrhage, demyelination and lytic most likely contribute mostly to the selec- tremors, clonic spasms, opisthotonus, necrosis of the optic fasciculi, accompa- tive toxicity for parasites. paddling movements of the forelimbs, nied by neurophagocytosis and chroma- prolapse of the nictitating membrane and tolysis, have been reported79,89,116. Clinical signs of toxicity blindness with mydriasis97. At an oral Complete absence of nerve cells in the The classical signs of salicylanilide toxic- dose of 80 mg/kg in cattle, inappetance ganglionic cell layer of the retina has also ity in animals include blindness, paresis and diarrhoea are observed1. been reported89. and ultimately death (Fig. 3). Blindness is Oxyclozanide signs of toxicity in sheep Odiawo et al.79 suggests that optic nerve an inconsistent toxic effect in cattle. and cattle include depression, anorexia lesions, but not retinal lesions, appear to General signs related to uncoupling of and diarrhoea at oral doses of 25 mg/kg contribute to blindness in acute poison- phosphorylation, i.e. hyperventilation, per day126. The toxic manifestations of ing. Dogs given 3–11 doses of rafoxanide hyperthermia, convulsions and tachycar- clioxanide included neuropathy and orally at 100 mg/kg developed bilateral dia may also be present. cerebral oedema in rats65 and blindness equatorial cataracts, papilloedema, The clinical signs of rafoxanide and clos- and vacuolation of the white matter of the vacuolation of the optic nerve, optic antel toxicity in sheep include inap- central nervous system in sheep77. Experi- chiasma, white matter of the brain and petence, blindness, mydriasis and mental papilloedema induced by spinal cord and focal vacuolation of the ophthalmoscopic papilloedema13,51,76,78,79,84,89. rafoxanide in the dog has been reported13. sciatic nerve13. The pathogenesis of the Prozesky and Pienaar89 reported slight Other toxic manifestations included in- ocular and neural lesions was ascribed to ataxia of the hindquarters as accompany- creased glutamic oxaloacetic transami- increased cerebrospinal fluid pressure, ing signs, while others51 reported intense nase and serum alkaline phosphatase, probably due to an increased amount of dyspnoea, diarrhoea and recumbency in neutrophilia, lymphopaenia, focal fluid in the cranial cavity, brain swelling sheep receiving doses of 450 mg/kg or hepatic necrosis and lymphoid necrosis in and meningeal inflammation around the more of rafoxanide. Susceptibility to lymphnodes and intestine lymphoid optic nerve and optic chiasma. Similar rafoxanide toxicity appears to be more tissue. findings, i.e. vacuolation of the white

0038-2809 Jl S.Afr.vet.Ass. (1999) 70(2): 61–70 67 matter of the brain and lens opacities, ica in sheep. Australian Veterinary Journal 45: northern fowl mites 1983. Insecticidal have also been observed in experiments 363–365 Acaricidal Tests 9: 435–435 10. Boray J C, Wolff K, Trepp H C 1973 Testing 29. Dedek W, Grahl R, Schwarz H, Ludwig P using multiple doses of 250 mg/kg of new fasciolicides: I. Efficacy and toxicity 1978 Metabolismus Rückstände und 65 rafoxanide per day in rats . of rafoxanide in sheep experimentally in- Ausscheidung des Anthelminthikums fected with Fasciola hepatica and F. gigantica. 131J-Rafoxanid in Blut Milch, Fleisch und CONCLUSIONS Schweizer Archiv für Tierheilkunde 115: Urin am laktierenden Rind. Archiv für The halogenated salicylanilides, in 367–371 Experimentelle Veterinarmedizin 32: 951–955 11. Broome A W,Jones W G M 1966 A new drug 30. Dedek W, Schwarz H, Liebaug E 1976 particular closantel and rafoxanide, are for the treatment of fascioliasis in sheep and Untersuchungen zum Abbau und zur Aus- important anthelmintics that are used cattle. Nature (London) 210: 744–745 scheidung des Anthelminthikums 131J- extensively in the control of Haemonchus 12. Brown G R, Chesterson G J, Coles G C 1985 Rafoxanid an Rind und Schaf. Archiv für spp. and Fasciola spp. infestation in sheep Potentiation of fasciolicidal agents by Experimentelle Veterinarmedizin 30: 423–426 and cattle, and O. ovis in sheep. Niclosa- benzoyl side chains. Synthesis of Benzoyl- 31. Dedek W, Schwarz H, Liebaug E 1977 salicylanilides. Journal of Medical Chemistry Abbau un Rückstandsdynamik des mide and resorantel are used for the 28: 143–146 Anthelminthikums 131J-Rafoxanid in Blut control of paramphistomes and cestodes. 13. 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