Sulfonamides and Sulfonamide Combinations

Sulfonamides and Sulfonamide Combinations

            Sulfonamides are the oldest and remain among the most widely used antibacterial agents in veterinary medicine, chiefly because of low cost and their relative efficacy in some common bacterial diseases. The synergistic action of sulfonamides with specific diaminopyrimidines renders these drugs much more effective compared to sulfonamides alone.

History

            In 1935, Domagk, a German scientist demonstrated the antibacterial therapeutic potency of Prontosil – an azo dye possessing p-amino-benzene sulfonamide group. He was awarded Nobel prize of medicine in 1939 for his outstanding contribution. The antibacterial activity of the drug was due to its sulfanilamide component.

General Properties

            The sulfonamides are derivatives of sulfanilamide. All have the same nucleus to which various functional groups have been added to the amido group or in which various substitutions on the amino group are made. These changes produce compounds with varying physical, chemical, pharmacologic, and antibacterial properties. Although amphoteric, sulfonamides generally behave as weak organic acids and are much more soluble in an alkaline than in an acidic environment. Those of therapeutic interest have pK_a values of 4.8–8.6. Water-soluble sodium or disodium salts are used for parenteral administration. Such solutions are highly alkaline, somewhat unstable, and readily precipitate with the addition of polyionic electrolytes. In a mixture of sulfonamides (e.g., the sulfa-pyrimidine group), each component drug has its own solubility; therefore, a combination of sulfonamides is more water-soluble than a single drug at the same total concentration. This is the basis of triple sulfonamide mixtures used clinically. The N-4 acetylated sulfonamides, except for the sulfapyrimidine group (sulfamethazine, sulfamerazine, sulfadiazine), are less water-soluble than their non-acetylated forms. This has bearing in the development of sulfonamide crystalluria. The highly insoluble sulfonamides (phthalylsulfathiazole and succinylsulfathiazole) are retained in the lumen of the GI tract for prolonged periods and are known as “gut-active” sulfonamides. The sulfacetamide is neutral in pH and is used to combat eye infections. 

             The nitrogen of amino group at para position is designated as N⁴ while nitrogen of SO₂NH₂ is designated as N¹. Systemic sulfa drugs are evolved by substitution at N¹ position whereas gut active sulfa drugs are produced by substituting N⁴ position. By substitution at N¹ and N⁴ positions about 5000 compounds are synthesized. Among them 30 are of clinical significance.

Structure activity relationship

1. Free para amino group is essential for antibacterial activity.
2. Substitution of heterocyclic aromatic components at N⁴ position produces more potent sulfa drugs.
3. Any substitution in benzene ring causes loss of activity.
4. SO₂NH₂ group is not essential as such however, sulfur atom is directly linked with benzene ring.
5. The more negative SO₂ group at N¹ exhibits greater antibacterial activity.
6. Substitutions made in the amide NH₂ (N¹) have variable antibacterial activity.
7. The para NH₂ group (N⁴) can be replaced or substituted by such chemical groups that can be converted into free NH₂ group in the body.

Classes

            Sulfonamides are classified based on their chemical structure as:

1. Both N¹ and N⁴ substituted sulfonamides: Pthalyl sulfathiazole and Succinyl sulfathiazole.

2. N¹ substituted sulfonamides: Sulfadimidine (Sulfamethazine), Sulfamerazine, Sulfaphenazole, Sulfamethoxazole, Sulfadimethoxine, Sulfacetamide, Sulfaquinoxaline, Sulfaethoxypyridazine, Sulfamethoxypyridazine, Sulfasomidine, Sulfisoxazole (Sulfafurazole), Silversulfadiazine, Sulfamylon (Mafenide), Sulfasimazole, Sulfaguanidine, etc.

            The many available sulfonamides and sulfonamide derivatives can be categorized into several types, based mainly on their indications and duration of action in the body.

Standard Use Sulfonamides

            In most species, members of this large group are administered 1–4 times/day, depending on the drug, to control systemic infections caused by susceptible bacteria. In some instances, administration of the sulfonamide can be less frequent if the drug is eliminated slowly in the species being treated. Sulfonamides included in this class, depending on the species, are sulfathiazole, sulfamethazine (sulfadimidine), sulfamerazine, sulfadiazine, sulfapyridine, sulfabromomethazine,  sulfaethoxy-pyridazine, sulfamethoxypyridazine, sulfadimethoxine, and sulfachlorpyridazine.

Highly Soluble Sulfonamides Used for Urinary Tract Infections

            A few very water-soluble sulfonamides, e.g., sulfisoxazole (sulfafurazole) and sulfasomidine, are rapidly excreted via the urinary tract (>90% in 24 hr) mostly in an unchanged form; because of this, they are primarily used for the treatment of urinary tract infections.

Poorly Soluble Sulfonamides Used for Intestinal Infections

            Some sulfonamide derivatives, such as sulfaguanidine, are so insoluble that they are not absorbed from the GI tract (<5%). Phthalylsulfathiazole and succinylsulfathiazole undergo bacterial hydrolysis in the lower GI tract with the consequent release of active sulfathiazole. Salicylazosulfapyridine (sulfasalazine) is also hydrolyzed in the large intestine to sulfapyridine and 5-aminosalicylic acid, an anti-inflammatory agent that might be used for the management of ulcerative colitis in dogs.

Potentiated Sulfonamides

            Certain diaminopyrimidines when used in combination with sulfonamides cause a sequential blockade of microbial tetrahydrofolate synthesis, which ultimately kills the organism. Sulfonamides are used in combination with pyrimethamine to treat protozoal diseases such as leishmaniasis and toxoplasmosis.

Topical Sulfonamides

            Several sulfonamides are used topically for specific purposes. Sulfacetamide is not highly efficacious but is occasionally used to treat ophthalmic infections. Mafenide and silver sulfadiazine are used on burn wounds to prevent invasion by many gram-negative and gram-positive organisms. Sulfathiazole is commonly included in wound powders for the same purpose.

            Sulfonamides are also classified based on their duration of action:

1. Short acting: Sulfathiazole, sulfasomidine
2. Intermediate acting: Sulfasimazole, sulfamethoxazole
3. Long acting: sulfamethazine, sulfamerazine, sulfamethoxypyridazine, sulfaethoxypyridazine, sulfadoxine.

Antimicrobial Activity

Mode of Action

            The sulfonamides are structural analogs of para-aminobenzoic acid (PABA) and competitively inhibit dihydropterate synthetase, an enzyme that facilitates PABA as a substrate for the synthesis of dihydrofolic acid (folic acid). Dihydrofolate is a precursor for formation of tetrahydrofolate (folinic acid), an essential component of the coenzymes responsible for single carbon metabolism in cells. Sulfonamides are antimetabolites that substitute for PABA, resulting in blockade of several enzymes needed for the biogenesis of purine bases; for the transfer of desoxy-uridine to thymidine; and for the biosynthesis of methionine, glycine, and formylmethionyl-transfer-RNA. Protein synthesis, metabolic processes, and inhibition of growth and replication occur in organisms that cannot use preformed (eg, dietary) folate. The effect is bacteriostatic, although a bactericidal action is evident at the high concentrations that may be found in urine.

            Sulfonamides are most effective in the early stages of acute infections when organisms are rapidly multiplying. They are not active against quiescent bacteria. Typically, there is a latent period before the effects of sulfonamide therapy become evident. This lag period occurs because the bacteria use existing stores of folic acid, folinic acid, purines, thymidine, and amino acids. Once these stores are depleted, bacteriostasis occurs. Bacterial growth can resume when the concentration of PABA increases or when the level of sulfonamide falls below an enzyme-inhibitory concentration. Because of the bacteriostatic nature of sulfonamides, adequate cellular and humoral defense mechanisms are critical for successful sulfonamide therapy.

            Although all of the sulfonamides have the same mechanism of action, differences are evident with respect to activity, pharmacokinetic fate, and even antimicrobial spectrum at usual concentrations. The differences are due to the variety of physiochemical characteristics seen among the sulfonamides.

            The efficacy of sulfonamides can be reduced radically by excess PABA, folic acid, thymine, purine, methionine, plasma, blood, albumin, tissue autolysates, and endogenous protein-degradation products.

Antimicrobial Spectrum

            The spectrum of all sulfonamides is generally the same. Sulfonamides inhibit both gram-positive and gram-negative bacteria, Nocardia, Actinomyces spp, and some protozoa such as coccidia and Toxoplasma spp. More active sulfonamides may include several species of Streptococcus, Staphylococcus, Salmonella, Pasteurella, and even Escherichia coli in their spectra. Strains of Pseudomonas, Klebsiella, Proteus, Clostridium, and Leptospira spp are most often highly resistant, as are rickettsiae, mycoplasmas, and most Chlamydia.

Bacterial Resistance

            Both chromosomal and R-factor-mediated resistance to sulfon-amides have been attributed to altered forms of dihydropterate synthetase (for which sulfonamides have a lowered affinity). Because sulfonamides act in a competitive fashion, overproduction of PABA can preclude inhibition of dihydropterate synthetase. Cross-resistance between sulfonamides is common. Resistance emerges gradually and is widespread in many animal populations. Plasmid-mediated sulfonamide resistance in intestinal gram-negative bacteria is often linked with ampicillin and tetracycline resistance.

Pharmacokinetic Features

            There are notable differences among the many sulfonamides with respect to their pharmacokinetic fate in the various species. The standard classification of short-, medium-, and long-acting sulfonamides that is used in human therapeutics is usually inappropriate in veterinary medicine because of species differences in disposition and elimination.

Absorption

            Sulfonamides may be administered PO, IV, IP, IM, intrauterine, or topically, depending on the specific preparation. Except for the poorly absorbed sulfonamides intended for intestinal use, most are rather rapidly and completely absorbed from the GI tract of monogastric animals. Absorption from the ruminoreticulum is delayed, especially if ruminal stasis is present. Therapeutic doses of sulfonamides are usually administered PO except in acute life-threatening infections when IV infusions are used to establish adequate blood concentrations as rapidly as possible. Sulfonamides are frequently added to drinking water or feed either for therapeutic purposes or to improve feed efficiency. A few highly water-soluble preparations may be injected IM (e.g., sodium sulfadimethoxine) or IP (some irritation of the peritoneum can be seen). Absorption is rapid from these parenteral sites. Generally, sulfonamide solutions are too alkaline for routine parenteral use.

Distribution

            Sulfonamides are distributed throughout all body tissues. The distribution pattern depends on the ionization state of the sulfonamide, the vascularity of specific tissues, the presence of specific barriers to sulfonamide diffusion, and the fraction of the administered dose bound to plasma proteins. The unbound drug fraction is freely diffusible. Sulfonamides are bound to plasma proteins to a greater or lesser extent, and concentrations in pleural, peritoneal, synovial, and ocular fluids may be 50–90% of that in blood. Sulfadiazine is 90% or more bound to plasma proteins. Concentrations in the kidneys exceed plasma concentrations, and those in the skin, liver, and lungs are only slightly less than the corresponding plasma concentrations. Concentrations in muscle and bone are ∼50% of those in the plasma, and those in the CSF may be 20–80% of blood concentrations, depending on the particular sulfonamide. Low concentrations are found in adipose tissue. After parenteral administration, sulfamethazine is found in jejunal and colonic contents at about the same concentration as in blood. Passive diffusion into milk also occurs; although the concentrations achieved are usually inadequate to control infections, sulfonamide residues may be detected in milk.

Biotransformation

            Sulfonamides are usually extensively metabolized, mainly by several oxidative pathways, acetylation, and conjugation with sulfate or glucuronic acid. Species differences are marked in this regard. The acetylated, hydroxylated, and conjugated forms have little antibacterial activity. Acetylation (poorly developed in dogs) reduces the solubility of most sulfonamides except for the sulfapyrimidine group. The hydroxylated and conjugated forms are less likely to precipitate in urine.

Excretion

            Most sulfonamides are excreted primarily in the urine. Bile, feces, milk, and sweat are excretory routes of lesser significance. Glomerular filtration, active tubular secretion, and tubular reabsorption are the main processes involved. The proportion reabsorbed is influenced by the inherent lipid solubility of individual sulfonamides and their metabolites and by urinary pH. Urinary pH, renal clearance, and the concentration and solubility of the respective sulfonamides and their metabolites determine whether solubilities are exceeded and crystals precipitate. This can be prevented by alkalinizing the urine, increasing fluid intake, reducing dose rates in renal insufficiency, and using triple-sulfonamide or sulfonamide-diaminopyrimidine combinations.

Pharmacokinetic Considerations

            There are great differences between the pharmacokinetic values of various sulfonamides in animals, and extrapolation of these values is rarely appropriate; for example, the plasma half-life of sulfadiazine is 10.1 hr in cattle and 2.9 hr in pigs. The recommended dose rates and frequencies reflect this disparity in elimination kinetics.

Therapeutic Indications and Dose Rates

            The sulfonamides are commonly used to treat or prevent acute systemic or local infections. Disease syndromes treated with sulfonamides include actinobacillosis, coccidiosis, mastitis, metritis, colibacillosis, pododermatitis, polyarthritis, respiratory infections, and toxoplasmosis.

            Sulfonamides are more effective when administered early in the course of a disease. Chronic infections, particularly with large amounts of exudate or tissue debris present, often are not responsive. In severe infections, the initial dose should be administered IV to reduce the lag time between dose and effect. For drugs with a long elimination half-life, the initial dose should be double the maintenance dose. Adequate drinking water should be available at all times, and urine output monitored. A course of treatment should not exceed 7 days under usual circumstances. If a favorable response is seen within 72 hr, treatment should be continued for 48 hr after remission to prevent relapse and the emergence of resistance. The ability to mount an immune response must be intact for successful sulfonamide therapy.

            A selection of general dosages for some sulfonamides is listed. The dose rate and frequency should be adjusted as needed for the individual animal.

Sulfonamide                           Species                        Dosage, Route, and Frequency

Sulfathiazole                           Horses                         66 mg/kg, PO, tid

                                                Cattle, sheep, pigs       66 mg/kg, PO, every 4 hr

Sulfamethazine                       Cattle                          220 mg/kg, PO or IV, sid (initial dose; half for                                                                                  subsequent doses)

Sulfadiazine                            All                               50 mg/kg, PO, bid

Sulfadimethoxine                    All                               55 mg/kg, PO, sid (initial dose; half for                                                                                             subsequent doses)

Sulfaethoxypyridazine            Cattle                          55 mg/kg, PO, sid

                                                Pigs                             110 mg/kg, PO, sid (initial dose, half for                                                                                           subsequent doses)

Sulfapyridine                          Cattle                          132 mg/kg, PO, bid (initial dose, half for                                                                                           subsequent doses)

Succinylsulfathiazole              All                               160 mg/kg, PO, bid (initial dose, half for                                                                                           subsequent doses)

Sulfaguanidine                        All                               264 mg/kg, PO, sid (initial dose, half for                                                                                           subsequent doses)

Special Clinical Concerns

Adverse Effects and Toxicity

            Adverse reactions to sulfonamides may be due to hypersensitivity or direct toxic effects. Possible hypersensitivity reactions include urticaria, angioedema, anaphylaxis, skin rashes, drug fever, polyarthritis, hemolytic anemia, and agranulocytosis. Crystalluria with hematuria, and even tubular obstruction, is not common in veterinary medicine. Acute toxic manifestations may be seen after too rapid IV administration or if an excessive dose is injected. Clinical signs include muscle weakness, ataxia, blindness, and collapse. GI disturbances, in addition to nausea and vomiting, may occur when sulfonamide concentrations are sufficiently high in the tract to disturb normal micro-floral balance and vitamin B synthesis. Sulfonamides depress the cellulolytic function of ruminal microflora, but the effect is usually transient (unless excessively high concentrations are reached). Several adverse effects have been reported after prolonged treatment, including bone marrow depression (aplastic anemia, granulocytopenia, thrombocytopenia), hepatitis and icterus, peripheral neuritis and myelin degeneration in the spinal cord and peripheral nerves, photosensitization, stomatitis, conjunctivitis, and keratitis sicca. Mild follicular thyroid hyperplasia may be associated with prolonged administration of sulfonamides in sensitive species such as dogs, and reversible hypothyroidism can be induced after treatment with high doses in dogs. Several sulfonamides can lead to decreased egg production and growth. Topically, the sulfonamides retard healing of uncontaminated wounds.

Interactions

            Sulfonamide solutions are incompatible with calcium- or other polyionic-containing fluids as well as many other preparations. Sulfonamides may be displaced from their plasma-protein-binding sites by other acidic drugs with higher binding affinities. Antacids tend to inhibit the GI absorption of sulfonamides. Alkalinization of the urine promotes sulfonamide excretion, and urinary acidification increases the risk of crystalluria. Some sulfonamides act as microsomal enzyme inhibitors, which may lead to toxic manifestations of concurrently administered drugs such as phenytoin.

Effects on Laboratory Tests

            Bilirubin, BUN, sulfobromophthalein (BSP^®), eosinophils, methemoglobin, AST, and ALT may be increased. Platelet, RBC, and WBC counts are often decreased. Urinalysis may show a change in color, glucose, porphyrins, and urobilinogen. Sulfonamide crystals may also be found.

Drug Withdrawal and Milk Discard Times

            Regulatory requirements for withdrawal times for food animals and milk discard times vary among countries. These must be followed carefully to prevent food residues and consequent public health implications.

Sulfonamide                           Species            Withdrawal Time (days)       Milk Discard Time (hr)

Sulfamethazine                       Cattle                          10^a                                           96

                                                Pigs                             14

Sulfabromethazine                  Cattle                          10                                            96

Triple sulfonamide solution^b   Cattle                          10                                            96

Sulfadimethoxidine                Cattle                          7                                              60

                ^a 28 days for slow-release bolus

                ^b 8% sodium sulfamethazine, 8% sodium sulfapyridine, 8% sodium sulfathiazole

Potentiated Sulfonamides

            A group of diaminopyrimidines (trimethoprim, methoprim, ormetoprim, aditoprim, pyrimethamine) inhibit dihydrofolate reductase in bacteria and protozoa far more efficiently than in mammalian cells. Used alone, these agents are not particularly effective against bacteria, and resistance develops rapidly. However, when combined with sulfonamides, a sequential blockade of microbial enzyme systems occurs with bactericidal consequences. Examples of such potentiated sulfonamide preparations include trimethoprim/sulfadiazine (co-trimazine), trimethoprim/sulfamethoxazole (co-trimoxazole), trimethoprim/sulfadoxine (co-trimoxine), and ormetoprim/sulfadimethoxine.

General Properties

            Trimethoprim and ormetoprim are basic drugs that tend to accumulate in more acidic environments such as acidic urine, milk, and ruminal fluid.

Antimicrobial Features

            In susceptible bacteria, the sulfonamide component blocks the synthesis of dihydrofolic acid, and the particular diaminopyrimidine used in combination inhibits the next enzyme in the sequence (dihydrofolate reductase) to prevent the formation of tetrahydrofolic acid (folinic acid). Folinic acid is required for the synthesis of DNA. This sequential blockade produces a bactericidal rather than bacteriostatic effect under usual conditions, but in the presence of thymidine, only bacteriostasis is evident because the block is circumvented.

            The optimal ratio in vitro for the combination of trimethoprim or ormetoprim and a sulfonamide depends on the type of microorganism but is usually ∼1:20. However, the commercially available preparations use a ratio of 1:5 because of pharmacokinetic considerations.

            Bacterial resistance to trimethoprim readily develops, but resistance to the combination occurs much more slowly. Resistance may take 2 forms: mutant resistance, with bacteria becoming dependent on exogenous folinic acid or thymidine; and plasmid-mediated resistance, based on enzyme modification.

Antibacterial Spectrum

            Sulfonamide-diaminopyrimidine combinations are active against gram-negative and gram-positive organisms, including Actinomyces, Bordetella, Clostridium, Arcanobacterium, Fusobacterium, Haemophilus, Klebsiella, Pasteurella, Proteus, Salmonella, Shigella, and Campylobacter spp, as well as Escherichia coli, streptococci, and staphylococci. Some streptococcal strains are only moderately sensitive, as are Brucella, Erysipelothrix, Nocardia, and Moraxella spp. The antibacterial spectrum does not include Pseudomonas or Mycobacterium spp. Cell wall-deficient microbes generally are not susceptible.

Pharmacokinetic Features

            Trimethoprim is rapidly absorbed after administration PO (plasma concentrations peak in ∼2–4 hr) except in ruminants, in which it tends to be trapped in the ruminoreticulum and appears to undergo a degree of microbial degradation.

            Absorption occurs readily from parenteral injection sites; effective antibacterial concentrations are reached in <1 hr, and peak concentrations in ∼4 hr. Trimethoprim diffuses extensively into tissues and body fluids. Tissue concentrations are often higher than the corresponding plasma concentrations, especially in lungs, liver, and kidneys. About 30–60% of trimethoprim is bound to plasma proteins. The extent of metabolic transformation of trimethoprim has not yet been established, although there is a suggestion that hepatic biotransformation can be extensive, at least in ruminants. This may not be the case in all species; >50% of a dose is excreted unchanged in many instances. Trimethoprim is largely excreted in the urine by glomerular filtration and tubular secretion. A substantial amount may also be found in the feces. The concentrations in milk are often 1–3.5 times higher than those in plasma. The plasma half-life of trimethoprim is quite prolonged in most species; effective concentrations may be maintained for >12 hr, with the result that the frequency of administration is usually 12–24 hr. The elimination rates of trimethoprim in sheep seem to be much shorter than for monogastric species.

Elimination of ormetoprim appears to be prolonged.

Dose Rates

            A selection of general dosages is listed in dosages of potentiated sulfonamides. The dose rate and frequency should be adjusted as needed for the individual animal.

Combination                                      Dosage, Route, and Frequency

Trimethoprim/sulfadiazine                  15–60 mg/kg, PO, IV, or IM, sid

Ormetoprim/sulfadimethoxine            55 mg/kg, PO, sid (initial dose; half for subsequent doses)

Adverse Effects and Toxicity

            Adverse effects due to the potentiated sulfonamides are quite rare, although adverse reactions to the sulfonamide components still occur. Up to 10 times the recommended dose of trimethoprim has been given with no adverse effects. Prolonged administration of trimethoprim at reasonably high concentrations leads to maturation defects in hematopoiesis due to impaired folinic acid synthesis. This effect is readily reversible by supplementation with folinic acid.

Drug Withdrawal and Milk Discard Times

            Regulatory requirements for withdrawal times for food animals and milk discard times vary among countries. These must be followed carefully to prevent food residues and consequent public health implications. The withdrawal times in drug withdrawal and milk discard times of potentiated sulfonamides serve only as general guidelines.

Combination                          Withdrawal Time (days)                   Milk Discard Time (days)

Trimethoprim/sulfadiazine                              3                                                          7

Trimethoprim/sulfadoxine       5 (PO); 28 (parenteral)

Sulphonamides as anticoccidials

            Sulphonamides have longest history of use as anticoccidial drugs. The common drugs of this group which are used as anticoccidials are sulphadimidine, sulphaquinoxaline, sulphadimethoxine, sulphanitran and sulphaguanidine. Sulphonamides have broad spectrum of activity against eimerian species and have coccidiostatic action. They are used for prevention and treatment of coccidia and in outbreaks. They are more effective against intestinal than caecal forms of coccidia. They stop the onset of the disease by acting against the second generation schizonts of E. tenella and E. necatrix. They can act upon first generation schizonts and possibly against sexual stages but much higher doses are required. Use of these drugs does not impair immunity development.

            Mechanism of action: Wood and Fildes proposed mechanism of action of sulphonamides, coccidian is synthesizing their own folic acid utilizing PABA (p-amino-benzoic acid) from growing medium because folic acid is required for growth/replication of DNA. Sulfonamides are structural analogues (PABA and Sulfonamide is similar in nature) of PABA inhibit bacterial folate synthetase resulting into folic acid is not formed and a number of essential metabolic reactions suffer. Animal cells also require folic acid but they utilize performed folic acid supplied in diet and are unaffected by sulfonamides. Therefore they prevent proper development of schizonts. Diaminopyrimidines inhibits the conversion of folic acid to tetrahydrofolic acid and are used in combination with Sulphonamides to potentiate their anticoccidial action.

            Sulphadimidine: This compound is still used as a curative drug in certain parts of the world, but its use has largely been discontinued in Western Europe and North America where it has been replaced by other compounds. It is given @ 0.4% in feed or in drinking water as 0.2% solution of the sodium salt. It is active against E. tenella, E. necatrix and other species of coccidia. It has been used in the control of clinical outbreaks of coccidiosis. The problem of this drug is that it interferes with vitamin K synthesis in the intestine and resulting into prolongation of blood coagulation time. At higher doses it causes loss of egg production in laying hens and hyperplasia of the somniferous tubules of testicles of male birds.

            Sulphaquinoxaline: It is an important, effective and commonly used coccidiostat throughout the world. For therapeutic purposes a dose of 0.5% in the feed is given. In drinking water, a dose of 0.043% is given for two durations each for 2 days with 3-5 days intervals, is satisfactory. Doses ranging from 0.025 to 0.033% over fairly long periods may be used as preventive medication. It is also active against E. acervulina in addition to E. necatrix and E. tenella. It exerts marked inhibitory effects on schizogony. Drug at a level of 0.1% in the ration inhibited invasion by the sporozoites. When used at higher dose for long duration it produces toxic effects which include multiple hemorrhages in many organs accompanied by necrotic lesions in the spleen, hypoplasia of bone marrow and agranulocytosis. This toxicity is associated with an interference with vitamin K metabolism. This compound has 6 days withdrawal premarketing requirement and eggs from treated birds should not be used for human consumption.