The pharmacology of halogenated salicylanilides and their anthelmintic use in animals

INTRODUCTION Salicylanilides are a very large group of compounds, originally developed as fungicides for topical use and as antimicrobial agents in soaps. Later these compounds were shown to possess potent anthelmintic activity of which the niclosamides tribromsalans and clioxanide were some of the 1st agents to be used. Since then, a wide range of halogenated salicylanilide congeners active against helminth parasites have been synthesised. The halogenated salicylanilides available in South Africa are summarised in Table 1. Halogenated salicylanilides, in particular closantel and rafoxanide, are important anthelmintics that are used extensively in the control of Haemonchus spp. and Fasciola spp. infestation in sheep and cattle, and Oestrus ovis in sheep in many parts of the world. Niclosamide is widely used for the treatment and control of cestode infections in several animal species. Despite being available over many years and the extensive use of these products, no definitive review of these products has been published.


INTRODUCTION
Salicylanilides are a very large group of compounds, originally developed as fungicides for topical use and as antimicrobial agents in soaps 64 .Later these compounds were shown to possess potent anthelmintic activity of which the niclosamides 54 tribromsalans 8 and clioxanide 9 were some of the 1st agents to be used.Since then, a wide range of halogenated salicylanilide congeners active against helminth parasites have been synthesised 11,12,32,37,66,76,102,110,126 .The halogenated salicylanilides available in South Africa are summarised in Table 1 113 .
Halogenated salicylanilides, in particular closantel and rafoxanide, are important anthelmintics that are used extensively in the control of Haemonchus spp.and Fasciola spp.infestation in sheep and cattle, and Oestrus ovis in sheep in many parts of the world.Niclosamide is widely used for the treatment and control of cestode infections in several animal species.Despite being available over many years and the extensive use of these products, no definitive review of these products has been published.

Chemical structure
Salicylanilides, also referred to as benzamides or salicylamides 54 , are weak, acidic phenolic compounds.The basic chemical structure consists of a salicylic acid ring and an anilide ring (Fig. 1).
The structural criteria for fasciolicidal action has been described as the need for electron-withdrawing substitutes on both the salicylic and anilide rings, together with a lipophilic group, such as tert-butyl in the 3-position 66 .Improved fasciolicidal activity of salicylanilides has been achieved in compounds such as closantel and rafoxanide by the incorporation of an aryl chain in the aniline moiety of the anilide 1 2 .In both drugs the active pharmacophore is 3,5-diiodosalicyloyol 110 .
Depending on the type of structural substitution, salicylanilides have been subdivided into a number of different groups, including halogenated and non-halogenated derivatives, acetylsalicylanilides, dichlorosalicylanilides and benzoylsalicylanilides.Acetylsalicylanilide derivatives, e.g.clioxanide, are activated in vivo.This is presumably achieved by hydrolysis of the acetyl group to free the hydroxyl derivative 38 .

Crystal structure and polymorphic forms
The 2 aromatic rings of the salicylanilide moeity are approximately co-planar with a dihedral angle between them 101 .There are significant conformational differences between the various halogenated salicylanilides and analogues regarding their crystalline forms.Interatomic dimensions in the salicylanilide moiety are consistent within the halogenated salicylanilides.The crystalline forms of closantel have not been reported.
Polymorphic forms of salicylanilides have been characterised by X-ray powder diffraction for oxyclozanide 82 .The polymorphic forms exhibit variable behaviour, both towards aqueous solubility and stability in suspension.Absorbed or occluded impurities are thought to play an important role in the polymorphic behaviour of the drug.
Anthelmintic activity of rafoxanide is inversely related to the size of crystalline particles present in a 2.5 % oral suspension 4 .

Physicochemical characteristics
The salicylanilides are generally weakly acidic, highly lipid-soluble compounds.Very little information on the physicochemical characteristics of the individual salicylanilides has been reported.Of the salicylanilide group, rafoxanide and closantel are the most widely used and are therefore more extensively described in literature.Rafoxanide has a molecular weight of 626.01 and a melting point of 173-7 °C2 .It is a greyish-white crystalline powder, moderately soluble in acetone, chloroform, ethyl acetate, acetonitrile and methanol but insoluble in water 76 .
Ultraviolet light absorption of a 0.004 % w/v solution in 0.1 M methanolic hydrochloric acid is in the range 230-350 nm and exhibits maximal excitation at 280 nm and 335 nm.Absorbance at 280 nm is about 0.97 and at 335 nm, about 0.59 2 .

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 phosphorylation 127 .
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 closantel 24,25,26,62,121,123 .Several workers have subsequently confirmed the proposed mechanism in vivo 25,87,123 .
In vitro inhibition of succinate dehydrogenase activity with a wide range of salicylanilides 38 and inhibition of the fumarate reductase system by oxyclozanide and rafoxanide in F. hepatica (unpublished information as cited in Coles 23 ) have also been reported.These effects may be due to the interrelationship of the succinate dehydrogenase activity to the fumarate reductase system and oxidative phosphorylation.It has been suggested that rafoxanide diminishes ATP-synthesis, resulting in increased internal intermediate pools in the pathway.Later, as an independent effect, the further metabolism of succinate is inhibited.
According to the chemiosmotic theory of Mitchell 73 , hydrogen-ionophores act by uncoupling of phosphorylation by the translocation of protons through the inner mitochondrial membrane.The maintenance of the proton gradient across the inner mitochondrial membrane is essential for the continued production of ATP.The inner mitochondrial membrane is normally impermeable to protons, but can be rendered permeable by the addition of uncouplers of oxidative phosphorylation that act by destroying the proton gradient.This activity selectively prevents the utilisation of the chemical energy derived from electron transport for the net phosphorylation of ADP to ATP, thus depriving the cell of its normal source of energy 103 .
Closantel was shown to have in vitro and in vivo effects on both the motility and ultrastructure of F. hepatica 103 .It induces rapid spastic paralysis of the fluke, severe sloughing of the integument, swelling of the basal infoldings, mitochondrial deformation and a reduced output of secretory products, especially in the tegumental and gastrodermal cells.The spastic paralysis seen with closantel is similar to that observed with rafoxanide and oxyclozanide 41 .Although these changes have been ascribed to uncoupling of oxidative phosphorylation, the increased muscle tone may be due to an increase in calcium ions within the muscle cells of the fluke 103 .Closantel has also been shown to rapidly decrease intrategumental pH in Schistosoma mansoni and F. hepatica at concentrations that were lower than those that affect ATP concentration 80 .The sensitivity of flukes to uncoupling agents may be due to the fact that Kreb's cycle activity is restricted to the 'outer layer ' of the fluke 119 .

PHARMACOKINETICS
Very few pharmacokinetics studies, other than metabolic studies, have been reported for salicylanilides in ruminants 30,31,55,72,75,114,115 .These pharmacokinetic studies involve only closantel, oxyclozanide and rafoxanide.No intravascular pharmacokinetic studies have been reported.
Halogenated salicylanilides generally share common phar macokinetic features 70 .The anthelmintic activity of these compounds has been directly associated with their pharmacokinetic profile 6,47,66,72,75,86 .A summary of the main pharmacokinetic parameters reported for closantel, rafoxanide and oxyclozanide in sheep and cattle are given in Table 2.
These parameters were derived from both model-independent procedures and non-linear compartmental analysis.The elimination rate constants and half-lives of closantel were determined by a single exponential compartmental model in 2 of the studies 55,72 , whereas in 1 study a tri-exponential equation determined the best-fitted cur ves for closantel, oxyclozanide and rafoxanide 75 .
A lag time for oral absorption was included in the model in one of the rafoxanide studies evaluated by a single exponential compartment 114 .

Absorption
Following oral administration of closantel and rafoxanide in ruminants, maximum plasma concentrations (Cmax) are reached 24-48 h later 7,114,115 .Hennessy et al. 55 reported a longer time of 65 h to maximum plasma concentrations (Tmax), indicating a slower rate of absorption of closantel administered intraruminally in sheep.The rate of gastrointestinal absorption of closantel is slower in goats than in sheep, most likely due to a reduced digesta flow rate 55 .
Reduced digesta flow rate resulted in significantly lower peak plasma concentrations and extent of absorption, as measured by area under the drug concentration versus time curve (AUC) of rafoxanide administered orally in grazing lambs compared to housed lambs fed hay and concentrates 118 .Peak rafoxanide plasma concentrations were significantly higher in sheep infected with 6-week-old F. hepatica as compared to uninfected sheep 75 .A 2-3-fold increase in the bioavailability of rafoxanide was reported in suckling lambs, aged 5-8 weeks of age compared to weaned lambs aged 4-5 months 115 .
In ruminants the oral bioavailability of closantel relative to intramuscular administration is approximately 50 % 72 .A similar relative oral bioavailability, based on the difference in efficacy against 6-week-old F. hepatica, after oral (36.1 %) and intramuscular (60.4 %) administration at 3 mg/kg, has been proposed for rafoxanide in cattle 75 .
The extent and rate of absorption of closantel is dose independent 72 .A linear increase in closantel plasma concentrations was reported in cattle and sheep at doses between 2.5 mg/kg and 10 mg/kg.Niclosamide is restricted to the gastrointestinal tract due to very poor absorption following oral administration 20 .
The activity of clioxanide in sheep is reduced if it is passed directly into the abomasum 9,117,123 .It is suggested that deacetylation of clioxanide takes place in the rumen to form a hydroxyl derivative that is more readily absorbed 86,87  persistence of the active constituents in blood.Acetylsalicylanilide derivatives such as clioxanide show poor anthelmintic activity in vitro, but in vivo are potent anthelmintics 38 .

Distribution
Halogenated salicylanilides are extensively plasma-bound and poorly distributed to tissues.Michiels et al. 72 reported the ratio of closantel in the plasma relative to that in the lung and kidney tissue is 6-7; 9-12 for heart and liver; 30 for muscle; and about 100 for fat.A plasma to liver concentration ratio of 17.5 for rafoxanide; and a plasma to bile ratio for closantel and rafoxanide of >50 % and 35 %, respectively, have been reported 75 .
Extensive binding to plasma albumin (>97 %) has been reported for both closantel and rafoxanide 72,75 .The binding to plasma albumin is characterised by very high affinity and high capacity.Closantel-binding to erythrocytes (c. 4 %) and in plasma water (1 %) is limited 72 .
The extensive plasma binding of salicylanilides is responsible for the long elimination half-life (T½el) associated with these products 75,86,88 .A T½el of 2-3 weeks has been reported for closantel and rafoxanide in sheep and cattle, and 6 days for oxyclozanide in sheep 30,31,55,72,75 .The T½el for closantel in different animal species varies from 6 days in goats, 1 week in the rat and 10 days in the dog 55,72 .Faster elimination resulted in an almost 3-fold lowering of the AUC in goats 55 .
Comparable data in rats and dogs have been reported for rafoxanide 6 .Elimination half-life of closantel and rafoxanide was unaffected by route and dose administered 31,55,72 .
It has been suggested 75 that the T½el of salicylanilides may be correlated with the turnover of plasma albumin to which they are bound, which is about 16.6 days 56 .

Metabolism and excretion
Salicylanilides are poorly metabolised and are excreted mainly unchanged.Only 1.0-2.5 % of rafoxanide is metabolised in the liver in cattle 31 and about 90 % of closantel is excreted unchanged in the faeces and urine in sheep and cattle 72 .
A reductive monodeiodination reaction appears as the main metabolic pathway for closantel in sheep, resulting in the formation of 3 and 5-monoiodoclosantel isomers 72 .Similar results have not been confirmed in cattle and goats 99 .Incubation of clioxanide with sheep liver microsomal enzymes revealed that monodeiodination is a non-enzymatic reaction 32 .Monodeiodination was not considered in the metabolic degradation of rafoxanide 29 .
Rafoxanide is metabolised to 3,5-diiodosalicylic acid by amide hydrolysis and is found in blood, milk and muscle of cattle 29 .Amide hydrolysis is unlikely in closantel, niclosamide and resorantel owing to steric hindrances by substituents ortho to the amide bond 72 .
Metabolic dehalogenation of both iodine atoms has not been observed for closantel or other deiodosalicylanilide derivatives 29,34,72 .Rapid and complete photolytic dehalogenation of rafoxanide by exposure to daylight has been reported 44 .
Glucuronide formation occurs with salicylanilides and accounts for the presence of an enterohepatic circulation proposed for these compounds 74 .In rats 10-15 % of a salicylanilide dose is reabsorbed from the gastrointestinal tract.A glucuronide metabolite of oxyclozanide has also been identified 11 .Halogenated salicylanilides are not metabolised by glutathione conjugation 33 .
Closantel is excreted mainly via the faeces.More than 80 % of 14 C-closantel was recovered from total 8-week faecal output and less than 0.5 % from the urine in sheep 72 .Within 48 h of dosing c. 43 % of the oral and 10 % of the i.m. dose was excreted with the faeces.Thereafter, faecal excretion proceeded more slowly with an average of 1-2 % of the dose per day.A faecal elimination half-life of 15.9-23 days was reported.Two to three percent of closantel is excreted at a maximum concentration of 1 µg/m in milk in cattle.Comparable excretion of rafoxanide occurs in the urine of sheep and milk in cattle 29,30,31,54 .No data on biliary and faecal excretion of rafoxanide have been reported in ruminants.Oxyclozanide is concentrated and excreted in the bile in the environment of adult flukes 11 .
In the rat the urine is the most important (>70 %) route for excretion of the salicylanilide derivative of benzanilide, an intermediate compound used in the dye and perfume industry 74 .Only 20 % is excreted in the faeces.Thirty-five percent of the [ 14 C]salicylanilide was excreted in the bile in 24 h.Incubation with rat caecal contents produced no hydrolysis 104 .In the case of rafoxanide there is extensive metabolism in the bile of rats 46 .

ANTIPARASITIC ACTIVITY
Salicylanilides have variable activity against a wide range of helminths and a number of ectoparasites.The antiparasitic spectrum of halogenated salicylanilides used in sheep and cattle is summarised in Tables 3 and 4. Except for niclosamide, these products are mainly restricted for use in ruminants, although antiparasitic activity has been shown in a number of other animal species.Niclosamide is recommended for use in ruminants, dogs and cats.Closantel and rafoxanide have the broadest antiparasitic spectrum and are the most widely used of the salicylanilides.Oxyclozanide, niclosamide and resorantel have a very narrow anthelmintic spectrum.

Nematodes
The anthelmintic activity of salicylanilides, except niclosamide and oxyclozanide, is specifically directed towards haematophagous nematodes 86 .Niclosamide and oxyclozanide are not effective against nematodes.Closantel and rafoxanide are highly effective against both immature and mature stages of Haemonchus contortus, Geigeria pachyscelis and Chabertia ovina in sheep 47,60 and H. placei, Bunostomum phlebotomum and Oesophagostomum radiatum in cattle 47,98,106 .Activity against the immature parasitic stages are partly due to persistent activity related to their long biological half-lives 70 .Reduced effectivity occurs against non-blood sucking immature H. contortus (before 8 days of age) 100 and hypobiotic larval stages 108 .Oxyclozanide is poorly active against Haemonchus spp.and is ineffective against Bunostomum sp., Oesophagostomum sp. and Chabertia sp. in either cattle or sheep 126 .
Persistent antiparasitic activity of closantel has been reported against a number of different parasite species and in different animals 47,48,50,52 .Activity is maintained against infective 3rd larval stages (L3) of H. contortus and G. pachyscelis when administered orally to sheep at 10 mg/kg for up to 7 weeks and 8 weeks before infection, respectively 47 .Prolonged activity has also been reported against L 3 H. placei, B. phlebotomum and O. radiatum in cattle when administered subcutaneously (s.c.) at 5 mg/kg.Anthelmintic persistence of closantel protects sheep against reinfection for up to 28 days 52 .
A single s.c.injection of closantel at 5 mg/kg completely cleared adult Capillaria bovis infections in cattle 47 .
Marked effectivity against natural and experimentally-induced infestations of Strongylus vulgaris was reported in foals given repeated doses of an oral closantel formulation 48,49 .Fourth larval and immature adult stages of S. vulgaris present in the mesenteric arteries had been completely cleared in foals treated 5 times with closantel at 20 mg/kg every 2 months starting at 1 month of age and was 86 % effective when foals were given 3 treatments of 8 mg/kg at the same interval.At the higher dose, closantel was also highly effective against adult S. vulgaris, S. edentatus and Triodontophorus spp.Closantel at 7.5 and 10 mg/kg has marked anthelmintic effect on adult stages of Ancylostoma caninum in dogs 50 .The authors propose that the maturation of the larval stage may be affected at 20 mg/kg, although closantel does not affect the abundance of arrested hookworm larvae nor prevents their subsequent development.
Early studies showed that rafoxanide was highly effective against a thiabendazole tolerant H. contortus K-strain 40 .Closantel was also shown to have activity against benzimidazole resistant strains of H. contortus in sheep 52,53 and against fenbendazole-and levamisole-resistant H. contortus strains 120 .Anthelmintic resistance to rafoxanide and closantel has been identified in H. contortus in sheep in South Africa 124 .
Closantel, in combination with broad spectrum anthelmintics, has been used successfully in a preventative anthelmintic programme to control haemonchosis and trichostrongylosis in sheep and was reported to retard the selection for anthelmintic resistance in Trichostrongylus spp. 27.
Eradication of H. contortus using closantel in sheep has been proposed 5 .

Trematodes
Salicylanilides are effective against a wide range of hepatic and intestinal trematodes in a variety of animals 20,122 .Closantel, rafoxanide and oxyclozanide are of the most important drugs used for the treatment and control of fascioliasis, whereas niclosamide and resorantel form the mainstay of Paramphistomum control in ruminants.
Closantel administered at 5 and 10 mg/kg orally in sheep and at 2.5 mg/kg s.c. in cattle is highly effective against adult F. hepatica 47,69 .In sheep similar high efficacy occurs at the same dosage against adult and immature F. gigantica (8-week-old) and immature F. hepatica (4-and 6-week-old) 47 .Only moderate efficacy against immature F. hepatica (6-week-old) at 7.5 mg/kg s.c. was reported 47 .Given intramuscularly at 2.5 mg/kg in cattle, closantel is highly effective against adult F. gigantica, but only slightly effective (55.8 %) against the 6-week-old immature stages 47 .
Closantel is ineffective against immature stages of 2 commonly occurring paramphistome species in Australia 92 .
The efficacy of rafoxanide oral suspension at doses of 2.5-20 mg/kg against immature and adult F. hepatica and F. gigantica has been confirmed in both cattle 63,85,96,98,106,107 and sheep 3,10,19,26,39,60 .An improved efficacy against F. gigantica has been ascribed to the more pathogenic nature, voracious feeding habits and higher metabolic rate of this parasite, in relation to F. hepatica 10,26,96 .Rafoxanide also appears to be more efficacious in sheep than in cattle against fluke of comparable age 60,106,107 .Administered s.c., rafoxanide injectable solution is approximately 2.5 times more effective than the oral suspension formulation when administered i.r. in cattle against Fasciola infestation 96,122 .Rafoxanide at 15 mg/kg orally is moderately effective against immature Paramphistomum microbothrium 58 in sheep, but poorly effective up to 9 mg/kg s.c. in cattle 96,98 .
Putative efficacy of closantel and rafoxanide against immature F. hepatica has been attributed to the persistent plasma concentrations affecting the flukes as they mature 75 .The presence of stunted or arrested fluke forms following closantel and rafoxanide treatment in sheep, is in part also ascribed to the persistent effect of the drug 69,75 .Nevertheless there is other evidence that indicate that these drugs act prior to F. hepatica reaching maturity.Campbell et al. 19 found that rafoxanide was virtually 100 % effective against 6-week-old fluke when sheep were necropsied 6-10 days after treatment, whereas Maes et al. 69 showed that closantel was equally effective against 6-week-old and 8-week-old fluke when necropsied either 1 week or 12 weeks after treatment.
According to Maes et al. 69 , the flukicidal effect of closantel is related more to peak plasma concentrations and less to residual persistent effect.
Closantel given orally at 15 and 20 mg/kg is highly effective in reducing 8-week-old Fascioloides magna infestation in sheep 111,112 .A 7.5 mg/kg dose administered i.m. was equivalent to a 15 mg/kg oral dose 1 1 2 .Rafoxanide at 10 and 15 mg/kg administered orally was shown to be 100 % effective against both immature and mature F. magna 42 .Oxyclozanide only had a slight effect.
At doses of 10 and 15 mg/kg oxyclozanide is highly effective against mature stages of F. hepatica and against the adult and immature (6 weeks) stages of F. gigantica in sheep and cattle 11,93,126 .
Higher doses are required against 6-week-old F. hepatica.It was suggested that the poor activity against immature F. hepatica is due to the high plasma binding of oxyclozanide in blood that bathes the immature fluke in the liver parenchyma 11 .According to Froyd et al. 43  cor relation between oxyclozanide concentrations in plasma and effectivity against liver fluke as judged by the number of parasites found at slaughter.A fixed dose of 3.4 g of oxyclozanide was shown to have equivalent efficacy in cattle with mass greater than 350 kg in comparison with a dose of 10 mg/kg.Oxyclozanide has been extensively used for the treatment of paramphistomiasis 122 .Oxyclozanide alone or in combination with levamisole is highly effective against immature and mature paramphistomes at oral doses of 15 and 18.7 mg/kg 91,122 .Rolfe and Boray 91 found that 2 doses of oxyclozanide given 3 days apart was more effective than a single dose against Calicophoron calicophorum in cattle.Single doses gave varying activity.According to Van den Bossche et al. 122 oxyclozanide does not remove all parasites present in the host.Where complete success is claimed, it is usually on the basis of negative faecal egg counts.In horses, oxyclozanide is successful against Gastrodiscus aegypticus 90 .
The efficacy of clioxanide against imma-ture and mature F. hepatica has been reported by a number of workers 8,9,18,19,81,94 .Large differences in fasciolicidal efficacy of clioxanide, particularly against immature stages occurs between oral, i.r. and i-a routes of administration 8,9,17 .A marked reduction in efficacy occurs following i-a administration.Reduction in efficacy reported after oral treatment is most likely due to a proportion of the drug that bypasses the rumen and which is deposited into the abomasum 9,19 .As described previously, the activity of clioxanide depends on activation by rumen microbes.The efficacy of rafoxanide against F. hepatica is not affected when administered either orally, intraruminally or intraabomasally 17 .Niclosamide at 50-100 mg/kg and resorantel at 65 mg/kg given orally to cattle and sheep are highly effective against immature P. microbothrium 45,57 .The effect of niclosamide in calves against the immature stages is erratic and it is not effective against adult stages in both sheep and cattle 20 .Erratic efficacy is also reported for resorantel against both immature and mature paramphistomes in sheep, goats and cattle.However, according to Van den Bossche et al. 122 resorantel is the most consistently successful drug used in the treatment of paramphistomiasis in cattle and sheep.Resorantel has also been used for the treatment of G. aegypticus 90 .

Cestodes
Anticestodal activity of salicylanilides is predominantly restricted to niclosamide and resorantel, although some activity has also been reported for closantel and oxyclozanide.The effect of oxyclozanide against Moniezia sp. in sheep has been restricted to reduction in faecal egg counts and voiding of gravid segments 126 .At necropsy it was noted that the scolices had remained in situ and were capable of continued growth.Closantel at 40 mg/kg orally and 20 mg/kg administered i.m. has been shown to have high anthelmintic activity against the larval stages of Taenia pisiformis in experimentally-infected rabbits 22,47 .No effect against the cysticercus stages in the peritoneal cavity has been found.Activity against Anoplocephala perfoliata was demonstrated in horses that had received 5 monthly doses of closantel at 20 and 40 mg/kg 48 .

Ectoparasites
Although salicylanilides are primarily effective against helminths, their effect against a number of insects and arthropods has been demonstrated.As far as could be determined, except for Oestrus ovis, these activities have not been recognised as official claims by regulatory authorities for these products.
Closantel and rafoxanide are 2 of the major products recommended for the control of O. ovis in sheep 47,58,95,108 .According to Snijders et al. 108 , rafoxanide at 7.5 mg/kg given orally was highly effective against overwintering 1st instar larvae and against 2nd and 3rd larval instars within 96 h of treatment.A residual effect against the re-establishment of O. ovis in recently-treated sheep was proposed 58,59 .Closantel is highly effective against O. ovis at 5 mg/kg administered orally to sheep 47 .
Other than their effect against O. ovis in sheep, closantel and rafoxanide have been shown to be effective against a range of other Oestridae in different animals.Studies conducted in foals treated with closantel 3 or 5 times every 2 months at doses between 2-40 mg/kg demonstrated a high efficacy against Gastrophilus intestinalis larvae 48,49 .In yearlings, 3 doses of at least 8 mg/kg were required.Closantel has also been shown be effective against Der matobia hominis Anti-arthropod activity of closantel has been reported against Boophilus microplus in cattle that were naturally infected 67,125 and against Amblyomma americanum in experimentally-infected cattle 36,47,67 , and against Linognathus ovillus 15 , Cochlyomia hominivorax 47 and Psoroptes communis var.ovis 83 in sheep.Closantel is active against Demodex canis in dogs 68 and Ornithonyssus sylvarium when given as a feed additive to chickens 28 .

SAFETY AND TOXICITY
Salicylanilides are moderately safe compounds and have safety factors of approximately 3-6 times the recommended dose levels 1,116 .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 been reported in lambs that had allegedly been treated at the recommended dose 84 .A LD50 of rafoxanide for either sheep or cattle has not been determined, but for the rat an oral LD50 of approximately 2300 mg/kg has been calculated.According to Adams 1 , no adverse effects are seen with closantel in sheep after repeated doses of 10 or 40 mg/kg orally or 5 or 20 mg/kg i.m. every 4 weeks over a 40-week period.Closantel is safe for use in breeding rams, ewes and bulls.
Salicylanilides are potent uncouplers of oxidative phosphorylation 127 .The pharmacokinetic behaviour of salicylanilides most likely contribute mostly to the selective toxicity for parasites.

Clinical signs of toxicity
The classical signs of salicylanilide toxicity in animals include blindness, paresis and ultimately death (Fig. 3).Blindness is an inconsistent toxic effect in cattle.General signs related to uncoupling of phosphorylation, i.e. hyperventilation, hyperthermia, convulsions and tachycardia may also be present.
The clinical signs of rafoxanide and closantel toxicity in sheep include inappetence, blindness, mydriasis and ophthalmoscopic papilloedema 13,51,76,78,79,84,89 .Prozesky and Pienaar 89 reported slight ataxia of the hindquarters as accompanying signs, while others 51 reported intense dyspnoea, diarrhoea and recumbency in sheep receiving doses of 450 mg/kg or more of rafoxanide.Susceptibility to rafoxanide toxicity appears to be more severe in sheep infected with liver fluke than in uninfected animals 76 .
Signs of toxicity occur within 24-72 h of treatment 16,51,89 .However, sheep inadvertently overdosed with rafoxanide, at an estimated dose of 450 mg/kg, exhibited signs of toxicity on the same day 79 .Recovery of some affected animals over a period of 3-4 weeks was reported in goats overdosed with closantel 16 .Cattle overdosed with rafoxanide s.c. at 45-60 mg/kg presented signs of tachypnoea, muscle tremors, clonic spasms, opisthotonus, paddling movements of the forelimbs, prolapse of the nictitating membrane and blindness with mydriasis 97 .At an oral dose of 80 mg/kg in cattle, inappetance and diarrhoea are observed 1 .
Oxyclozanide signs of toxicity in sheep and cattle include depression, anorexia and diarrhoea at oral doses of 25 mg/kg per day 126 .The toxic manifestations of clioxanide included neuropathy and cerebral oedema in rats 65 and blindness and vacuolation of the white matter of the central nervous system in sheep 77 .Experimental papilloedema induced by rafoxanide in the dog has been reported 13 .
Other toxic manifestations included increased glutamic oxaloacetic transaminase and serum alkaline phosphatase, neutrophilia, lymphopaenia, focal hepatic necrosis and lymphoid necrosis in lymphnodes and intestine lymphoid tissue.
Photodermatitis and skin irritation in man have been reported for halogenated salicylanilides which have been incorporated in soaps as antimicrobial agents or when used topically as fungicides 61,64 .
Complete absence of nerve cells in the ganglionic cell layer of the retina has also been reported 89 .
Odiawo et al. 79 suggests that optic nerve lesions, but not retinal lesions, appear to contribute to blindness in acute poisoning.Dogs given 3-11 doses of rafoxanide orally at 100 mg/kg developed bilateral equatorial cataracts, papilloedema, vacuolation of the optic nerve, optic chiasma, white matter of the brain and spinal cord and focal vacuolation of the sciatic nerve 13 .The pathogenesis of the ocular and neural lesions was ascribed to increased cerebrospinal fluid pressure, probably due to an increased amount of fluid in the cranial cavity, brain swelling and meningeal inflammation around the optic nerve and optic chiasma.Similar findings, i.e. vacuolation of the white  matter of the brain and lens opacities, have also been observed in experiments using multiple doses of 250 mg/kg rafoxanide per day in rats 65 .

CONCLUSIONS
The halogenated salicylanilides, in particular closantel and rafoxanide, are important anthelmintics that are used extensively in the control of Haemonchus spp.and Fasciola spp.infestation in sheep and cattle, and O. ovis in sheep.Niclosamide and resorantel are used for the control of paramphistomes and cestodes.Halogenated salicylanilides share a number of common pharmacokinetic features, including extensive plasma binding, prolonged elimination half-life and limited metabolism.The persistent anthelmintic effect and predilection against haematophagous parasites substantiate the importance of the pharmacokinetics of these compounds on their efficacy.In addition, the margin of safety of halogenated salicylanilides, as toxicity is dosedependent, may be affected by changes in bioavailability and in extent of plasma binding.Despite the importance of pharmacokinetics on the efficacy and safety of halogenated salicylanilides, very few studies have been reported that examine factors that potentially may influence their absorption and disposition in ruminants.

Fig. 4 :
Fig. 4: Brain and optic nerve histopathological lesions in sheep following rafoxanide and closantel poisoning (left: optic nerve vacuolation; right: vacuolation of white matter of brain).

Table 4 : Summary of the antiparasitic spectrum of salicylanilides commonly used in cattle. Type of parasite Antiparasitic spectrum Closantel Rafoxanide Oxyclozanide Niclosamide Resorantel
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