Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/06—Antiabortive agents; Labour repressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis

Definitions

• the present invention relates to triazineone compounds for treating animals infected with parasites that cause abortigenic or neurologic diseases. More specifically, the present invention relates the triazineone compounds that are useful in treating parasitic protozoa such as coccidia that cause abortigenic or neurologic diseases.
• Triazineone compounds such as triazinediones, e.g., diclazuril compounds, and triazinetriones, e.g., toltrazuril compounds have been used in treating and protecting various mammals, insects and fish from diseases caused by a broad range of protozoa. See U.S. Patents; 4,933,341 ; 4,935,423; 5,114,938; 5,141 ,938; 5,188,832, 5,196,562, 5,256,631 and 5,464,837. Protozoa sensitive to these compounds include parasites, which infect the intestines of birds, mammals and insects and manifest as diarrhea, wasting, nausea and vomiting.
• the mode of action of the triazineones is to attack the intermediate parasite stages found in the gut and intestinal wall cells, causing the endoplasmic reticulum, the perinuclear space and the mitochondria of the parasite to swell.
• This purportedly disturbs the ability for nuclear divisions causing the shizonts and microgamonts to remain small forming only a few merozoites and microgametes respectively.
• the end result is reported to be the loss of the ability of these latter stages of the parasites to penetrate new mammalian cells, effectively halting the replication of the parasite in the host.
• Equine Protozal Myeloencephalitis is a neurologic disease of horses, with a predilection for young horses undergoing stress (e.g., thoroughbred race horses and purebred performance horses), and is thus a disease with significant monetary impact for the horse industry.
• EPM first recognized as a disease in the 1970's, was not cultured from a horse with EPM and given the name Sarcocystis neurona until 1991.
• a Neospora spp. now named Neospora hugesi, was isolated from the brain of a horse with EPM. Accordingly, it is now proposed that EPM may be caused by this newly recognized organism alone, by Sarcocystis neurona alone or the combination of the two.
• EPM most often results in asymmetric incoordination (ataxia), weakness, and spasticity.
• the disease can mimic almost any neurologic condition. It can occur as a peracute or chronic condition. The chronic form is often insidious at onset, difficult to diagnose until late in the course of the disease, and can result in death. In the mildest cases, the only clinical sign may be ill-defined pelvic limb lameness or a minor respiratory noise. In the most severe cases, horses are unable to swallow or stand. It is now known that in the most severe cases, the parasite, e.g., S. neurona infects the brain and produces significant damage therein.
• EPM central nervous system
• Toxoplasma gondii Another coccidian parasite, Toxoplasma gondii, has been known for some time and was first isolated from the intestines and muscle tissue of cats.
• the definitive host for this parasite is the cat that can harbor the organism for long periods of time spreading oocysts to other animals including bovines, ovines swine and humans.
• Infection of sheep, cattle and humans has been associated with abortion and congenitally acquired disorders, which primarily affect the central nervous system. It has also recently been associated with abortion and malformation in kittens born to infected queens that had been seronegative prior to infection during pregnancy.
• Non-feline hosts such as bovines, ovines swine and humans do not produce oocysts but develop and may suffer from invasion of muscle and brain by tachyzoites and bradyzoites which produce the clinical signs of disease - neurological symptoms and abortion with fetal defects. It has been reported that 60% of cats are serologically positive to T. gondii. Once again, there is no approved treatment or prophylactic for toxoplasmosis.
• Neospora caninum produces both a neurologic and abortigenic disease in animals. It was first isolated from dogs in 1988. It was previously confused with Toxoplasma gondii. The disease caused by this parasite occurs most severely in transplacentally infected puppies and is characterized by progressive ascending paralysis in the puppies, particularly of the hind limbs; polymyositis and hepatitis may also occur. This disease has more recently been recognized as a major cause of abortion and neurologically-associated limb defects in newborn calves. Microscopic lesions of non-suppurative encephalitis and myocarditis in aborted fetuses may be seen in the brain, spinal cord and heart. A definitive host for Neospora caninum has recently been identified to be the dog. At this time there is no approved treatment or prophylaxis for either Neospora caninum of dogs or bovines or Neospora hugesi of horses.
• the present invention encompasses a method of therapeutically treating a diseased animal suffering from a parasitic neurologic or abortigenic disease that is susceptible to being treated with a triazineone compound, comprising administering to the animal a pharmaceutically effective amount of the compound, with the proviso that when the disease is Sarcocystis neurona the compound is not diclazuril or toltrazuril.
• pharmaceutically-effective amount means that the amount of triazineone being administered is high enough to inhibit the in vivo or in vitro growth of the parasitic protozoa, typically coccidia which, produce neurological disease and/or abortions.
• the pharmaceuticallly-effective amount controls the parasites in the infected tissues; consequently, the animal's health improves.
• the present invention encompasses a method of metaphylactically treating an animal infected with a parasite that can cause a neurologic or abortigenic disease, that is susceptible to being treated with a triazineone compound.
• the metapylactic treatment comprises administering to the animals the triazineone compound using a metaphylactically-effective regimen.
• a metaphylactically-effective regimen is meant administering scheduled intermittent doses of triazineone compounds for a prolonged period until said animal overcomes the invading parasites by, say, developing a protective immune response or otherwise clearing the parasite.
• the regimen is such as would effectively control the parasites and prevent clinical signs of disease.
• the metaphylactically-effective dose can also be administered for a prolonged period up to five years or the lifetime of the animal, especially in instance when the parasite is difficult to control.
• the preferred triazineone compounds are triazinetriones, which include but are not limited to Toltrazuril, and Ponazruil.
• the present invention encompasses a single high dose treatment of the animals.
• This method comprises administering to the animals a single high dose of a pharmaceutically effective amount of the triazineone compound to a diseased animal suffering from a parasitic neurologic or abortigenic disease that is susceptible to being treated with a triazineone.
• single high dose is meant an amount that is administered only once. This amount is significantly higher than the dose amount employed in the therapeutic or metapylactic treatment; is effective in controlling the disease-causing parasites, and as such would not result in detrimental effects such as toxicity.
• the single high dose of triazineone is accordingly greater than 10 mg/Kg.
• the present invention relates to a method of treating a infected or diseased animal suffering from a parasitic disease that manifests as neurologic or abortigenic disease that is susceptible to being treated with a triazineone compound, comprising, administering thereto a pharmaceutically effective amount of said.
• the animals can be equines, bovines, felines, canines, swine, ovines, birds, insects and humans.
• the parasites infecting or causing disease are coccidia of the Family Sarcocystidae that can that can manifest as neurologic or abortigenic diseases.
• Illustrative but non- limiting examples thereof can be selected from the group consisting of Sarcocystis spp., Neospora spp. and Toxoplasma spp.
• the Sarcocystidae are typically selected from the group consisting of S. neurona, N. hugesi, N. caninum and T. gondii.
• the protozoan infections or diseases include but are not limited to EPM, Neosporosis, and Toxoplasmosis.
• treatment of the parasitic infections or diseases caused by the protozoa described herein results in the alleviation of the symptoms of the neurologic and abortigenic diseases.
• the symptoms include lameness, ataxia, paralysis, abortion, weak newboms and other related disorders.
• animals already suffering the above signs of disease are treated with the triazineone compounds.
• the duration of treatment is from about 28 days to 90 days and preferably from about 28 to 60 days. It is understood that for therapeutic treatment, the treatment regimen can be once a day, two or more times a day, once every other day or even once per week, depending on factors such as the severity of the disease and the type of disease-producing parasite.
• the treatment regimen can last indefinitely, sometimes for the remaining life of the animal.
• the latter treatment would be required in the case of infection of an animal with a more resistant strain of parasite.
• the treatment can be extended for longer periods of time as necessary until the signs of disease are eliminated.
• the preferred treatment is once, daily for about 28 days.
• metaphylactic activity refers to the use of the triazineone compounds on a scheduled intermittent treatment regimen (metaphylactically-effective regimen) to control the protozoa, which may have infected the animal, since the previous treatment. Accordingly, the metaphylactically-effective regimen is administered to reduce their ability to cause disease by, say, killing them or reducing them in number.
• the metaphylactically effective regimen can be administered about once per month, over the lifetime of the animal or until an inherent clearance mechanism, e.g., an effective immune response, develops within the animal to protect it from future infections.
• an inherent clearance mechanism e.g., an effective immune response
• the latter can occur within 5 years or less.
• the metaphylactic treatment is based on the recognition that when animals are infected with the protozoa described herein, they do not demonstrate clinical signs such as neurological signs or abortion until a significant time has passed (e.g., 2-6 months post infection). In contrast, the enteric protozoan infections manifest themselves shortly after infection.
• the metaphylactic treatment prevents the parasite from establishing itself and causing a clinical disease.
• the treatment regimen is on an intermittent schedule of about once per month, once per two months or once per two weeks at a dose equivalent to about between 1.0 and 100 mg/Kg, preferably about 1.0 to 25mg/Kg and more preferably about 2.5 to 10mg/Kg.
• the high range would be required in particularly resistant cases (e.g. when an animal is infected with a resistant strain).
• the required dose level and duration of treatment are within the purview of one of ordinary skill in the art.
• a preferred treatment regimen for horses with EPM or bovines with Neosporosis is about 1.0 to 25 mg/Kg, and a more preferred range is about 2.5 to 10 mg/Kg of triazinetrione every 28 days.
• the triazineone is administered in pharmaceutically effective amounts that are greater than 10 mg/Kg and up to about 100 mg/Kg. It is a distinct feature of the invention the compounds of this invention can be non-toxic, thus they can be administrated at high dose levels.
• the advantage of the high dose administration resides in the fact repeated doses are not required and that some triazineone compounds can cause detrimental side effects if administered at very high dose levels.
• Particularly preferred is Ponazuril, which has been found to be both safe and effective at doses as high as 100 mg/Kg body weight.
• the unexpected success of the treatments described herein results from the ability of the triazineone compounds to cross the blood- brain barrier or placental barrier. It is believed that the compounds of this invention easily cross the blood-brain barrier and, also, are able to penetrate the placenta and kill the protozoa in situ in the brain and cerebral spinal fluid/spinal cord. It has been a further been found that the compounds of this class are non-toxic and non-mutagenic even at the high doses necessary for the single high dose treatment regimen described herein.
• Toltrazuril compounds useful herein are of formula (1 ):
• R 1 represents halogenoalkylthio, halogenoalkyl-sulphinyl or halogenoalkylsulphonyl
• R 2 represents hydrogen, alky!, alkoxy, alkoxyalkyl, alkylmercapto, halogen, halogenoalkyl or an optionally substituted sulphamoyl, such as dialkyl sulphamoyl, radical, R 3 and R 4 can be identical or different and represent hydrogen, alkyl, alkenyl or alkinyl and X is 0 or S, and their physiologically acceptable salts.
• R' represents halogenoalkyl(C C 4 )-thio, halogenoalkyl(C C 4 )-sulphinyl or halogenoalkyl(C C 4 )-sulphonyl
• R" represents hydrogen, alkyl (0 ⁇ 4 ), alkoxy (C, ⁇ ), halogen, alkoxy(C 1 -C 4 )alkyl(C 1 -C 4 ), alkyl (C ⁇ mercapto, dialkyl
• (C 1 -C 4 )aminosulphonyl or halogenoalkyl (C C 4 ) and R 1 " and R ⁇ v can be identical or different and represent hydrogen, alkyl
• R ⁇ R 2 , R 3 and X have the meaning indicated above, are reacted with a substituted carbonyl isocyanate of the formula III (in)
• R 5 represents a halogen atom, an alkoxy group or an aryloxy group, and the substituted 1 ,3,5-triazine derivatives, formed during this procedure, of the formula IV
• R 1 , R 2 , R 3 and X have the meaning indicated above, are optionally isolated and optionally reacted with a compound of the formula V
• A represents alkyl, alkenyl or alkinyl and Z represents halogen; or that (b) 1 -(4-phenoxy-phenyl)-1 ,3,5-triazine derivatives of the general formula I are obtained when compounds of the formula II, in which R ⁇ R 2 , R 3 and X have the meaning indicated above, are reacted with bis- (chlorocarbonyl)-amines of the formula VI
• R 6 represents alkyl, optionally in the presence of acid acceptors or that
• R ' represents halogenalkylthio
• R ' represents halogenalkylthio
• R 6 or A is straight-chain or branched alkyl with preferably 1 to 6, in particular 1 to 4, carbon atoms. Examples that may be mentioned are optionally substituted methyl, ethyl, n- and i-propyl and n-, i- and t-butyl.
• alkenyl as defined in R 3 , R 4 or A is straight-chain or branched alkenyl with preferably 2 to 6, in particular 2 to 4, carbon atoms. Examples, which may be mentioned, are optionally substituted ethenyl, propen-1-yl, propen-2-yl and buten-3-yl.
• alkinyl as defined in R 3 , R 4 or A is straight-chain or branched alkinyl with preferably 2 to 6, in particular 2 to 4, carbon atoms. Examples which may be mentioned are optionally substituted ethinyl, propen-1-yi, propin-2-yl and butin-3-yl.
• alkoxy as defined in R 2 or R 5 is straight-chain or branched alkoxy with preferably 1 to 6, in particular 1 to 4, carbon atoms. Examples which may be mentioned are optionally substituted methoxy, ethoxy, n- and i-propoxy and n- and i-butoxy.
• halogen as defined in R 2 , R 5 or Z is preferably fluorine, chlorine, bromine and iodine, especially chlorine and bromine.
• halogenoalkylthio as defined in R 1 is halogenoalkylthio preferably 1 to 4, in particular 1 or 2, carbon atoms preferably 1 to 5, in particular 1 to 3, identical or different halogen atoms, halogen atoms preferably being fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylthio, chloro-di-fluoromethylthio, bromomethylthio, 2,2,2-tri- fluoroethylthio and pentafluoroethylthio.
• halogenoalkylsulphinyl as defined in R 1 is halogenoalkylsulphinyl with preferably 1 to 4, in particular 1 or 2, carbon atoms and preferably 1 to 5, in particular 1 to 3, identical or different halogen atoms, halogen atoms preferably being fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulphuryl, chloro-di-fluoromethylsulphuryl, bromomethylsulphinyl, 2,2,2-trifluoroethylsulphinyl and pentafluoroethyl- sulphinyl.
• halogenoalkylsulphonyl as defined in R 1 is halogenoalkylsulphonyl with preferably 1 to 4, in particular 1 or 2, carbon atoms and preferably 1 to 5, in particular 1 to 3, identical or different halogen atoms, halogen atoms preferably being fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulphonyl, chloro-di-fluoromethylsulphonyl, bromomethyl-sulphonyl, 2,2,2-trifluoroethylsulphonyl and pentafluoroethyl- sulphonyl.
• optionally substituted sulphamoyl as defined in R2 is preferably one of the following radicals:
• aryloxy as defined in R 5 is preferably monocyclic carbocyclic aryloxy or bicyclic carbocyclic aryloxy, particularly phenoxy.
• aryloxy R 5 is preferably phenoxy.
• Most of the substituted ureas or thioureas of the formula II which are used as starting materials have not been known hitherto, but they can be easily prepared by methods which are in themselves known by (a) either reacting substituted 4-aminodiphenyl ethers with the corresponding substituted isocyanates or isothiocyanates in an inert solvent at temperatures between 0°C.
• Possible diluents for the reaction of the ureas or thioureas of the formula II both with carbonyl isocyanates of the formula III (process variant a) and with bis(chlorocarbonyl)-amines of the formula VI (process variant b) as well as for the reaction of the 1 ,3,5-triazine derivatives of the formula IV with compounds of the formula A-Z are all the organic solvents which are inert in these reactions.
• aromatic hydrocarbons such as benzene, toluene and xylene
• halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene
• ethers such as tetrahydrofurane and dioxane.
• the hydrochloric acid which may form during the reaction escapes as a gas or can be bonded by organic or inorganic acid acceptors.
• the acid acceptors include, preferably, tertiary organic bases, such as trialkyla- mines, for example, triethylamine, N-hetero mono- or bi-cyclic aromatic amines, such as pyridine aza-cyclo alkyl amines which are mono- or bi- cyclic, such as diazabicyclononene, diazabicycloundecene and many others, or inorganic bases, such as alkali metal carbonates, oxides or hydroxides or alkaline earth metal carbonates, oxides or hydroxides.
• tertiary organic bases such as trialkyla- mines, for example, triethylamine, N-hetero mono- or bi-cyclic aromatic amines, such as pyridine aza-cyclo alkyl amines which are mono- or bi- cyclic, such as diazabicyclononene, diazabicycloundecene and many others
• inorganic bases such as alkali metal carbon
• reaction temperatures for the above-mentioned reaction stages can be varied within a wide range. In general, the reaction is carried out between about 0°C. and about 150°C, preferably between about 20°C. and about 100°C.
• the reaction can be carried out under normal pressure or under elevated pressure. In general, the reaction is carried out under normal pressure.
• Possible oxidizing agents for the conversion, according to process variant (c) of the trifluoromethylthio compounds of the general formula 1 , in which Y represents oxygen, into the corresponding sulphinyl or sul- phonyl compounds are, appropriately: ⁇ O ⁇ glacial acetic acid; H ⁇ acetic anhydride; H ⁇ methanol; peracids, such as, for example, m-chloroper- benzoic acid, and chromic acid; potassium permanganate; sodium periodate, cerise ammonium nitrate; and nitric acid.
• a resulting compound can be converted into a corresponding addition salt, for example by reacting it with an inorganic or organic base.
• the triazineone compound can be formulated in any convenient manner for administration to animals.
• Formulations suitable for oral administration can be suspensions, tablets, capsules, gels, pastes, boluses, or preparations in the form of powders, granules, or pellets.
• the preferred orally administered formulation is in the form of a paste or a feed additive.
• Other modes of administration that can be employed include parenteral, topical, intramuscular, and intramucosal or by other routes known to those skilled in the art. Topical administration in the form of a pour-on is also preferred.
• pharmaceutically acceptable carriers and auxiliaries are employed in the formulations.
• examples thereof can be a thickening agents selected from the group consisting of: Carbopol, inorganic thickeners such as silicates, bentonites or colloidal silica and organic thickeners such as fatty alcohols or fatty acid esters and the wetting agent is selected from the group consisting of polyethylene glycol and sodium lauryl sulfate with Carbopols, more specifically, Carbopol 974P being the most preferred thickening agent for the paste formulation preferred herein.
• preservatives selected from the group consisting of parabens, alcohols and aldehydes. These may be liquid, solid, or gaseous materials, which are otherwise inert or medically acceptable and are compatible with the active ingredients.
• the pastes, according to the invention are effective when used in treating the parasites. More specifically, it is surprising that the pastes of the present invention are effective in delivering the triazineones, particularly Toltrazuril, and Ponazuril to cross the blood- brain or placenta barrier and attack the parasites which have already invaded the brain or infected the fetus of a pregnant animals. As a matter convenience, there is provided herein a description of a specific embodiment of the pastes preferred herein and how it is prepared.
• a preferred paste, according to the present invention contains a micronized suspension of the triazinetrione (e.g.
• Ponazuril propylene glycol, a thickening agent such as Carbopol, preservatives such as Methylparaben and Propylparaben, and water. It can be made by combining water, typically, purified water and Propylene Glycol, heating the combination to about 70°C, and adding the preservatives, at this temperature. The resulting mixture is cooled to room temperature after which Carbopol, preferably in the form of Carbopol 974P, is added. Finally the triazinetrione is added. After complete mixing, the pH is adjusted to approximately 6.0 with sodium hydroxide.
• the most preferable paste includes 15% w/w Ponazuril, 20% w/w Propylene Glycol, 0.5% w/w Carbopol 974P, 0.14% w/w Methylparaben, 0,02%w/w Propylparaben, 0.1 %w/w sodium hydroxide with the remainder being purified water.
• Sweeteners including dextrose, sucrose, lactose, fructose, sorbitol, xylitol, artificial sweeteners and molasses may be added to improve palatability. Additionally, yeast or liver flavoring may be added for the same purpose.
• the invention is further described by following illustrative but non- limiting examples.
• Ponazuril 1-methyi-3-[4-p-[trifluoromethyl)sulfonylphenoxy]-m-tolyl]- s-triazine-2,4,6 (1 H,3H,5H)-trione, a representative Triazinetrione, was formulated into a paste for administration to horses.
• the components listed in Table 2 were used in preparing formulations as follows.
• Table 2 Components of Ponazuril Horse Paste
• the formulations were prepared using process (A) and (B) as follows.
• the first process (A) comprised: 1 ) Mixing a portion of the water with the Propylene Glycol; 2) adding the preservatives (Methylparaben and Propylparaben; 3) slowly adding the Carbopol 974P until an even suspension was prepared; 4) adding the Ponazuril in a micronized form;
• the final suspension was in the form of a paste, which can be delivered orally to a horse.
• the second process (B) comprised: 1 ) Mixing a portion of the water with the Propylene Glycol; 2) heating to 70°C; 3) adding the preservatives (Methylparaben and Propylparaben while holding the solution at 70°C; 4) cooling the solution to -room temperature; 5) slowly adding the Carbopol 974P until an even suspension was prepared; 6) adding the Ponazuril in a micronized form; 7) adding the Sodium Hydroxide to bring the suspension to a pH of approximately 6.0; and 8) adding the remainder of the water to QS to volume.
• the final suspension was also in the form of a paste, which can be delivered orally to a horse.
• Ponazuril 1-methyl-3-[4-p-[trifluoromethyl)sulfonylphenoxy]-m-toIyl]- s-triazine-2,4,6 (1 H,3H,5H)-trione, a representative Triazinetrione, was tested for its ability to treat horses already demonstrating signs of Equine Protozoal Myoencephalitis (EPM).
• the compound was formulated into a paste using Ponazuril as a 15% active ingredient (a.i.) as described in EXAMPLE 1. It was administered to horses already diagnosed with EPM once a day for 28 days at a dose rate between 2.5 mg/Kg and 10mg/Kg. Naturally occurring clinical cases of EPM were well characterized by signalment and laboratory diagnosis.
• the diagnosis used for incorporation of EPM-positive horses into this trial was as follows: Confirmed asymmetrical neurological deficit as determined by a standardized neurological examination, to include radiography, indicative of EPM; Positive Western Blot for Sarcocystis neurona IgG; Red Blood Cell count below 500 cells/mL; CSF indices - Total Protein ⁇ 90, IgG index >0.3. AQ quotient ⁇ 2.2.
• Ponazuril In order to determine the scope of protection provided by Ponazuril, in vitro testing was conducted. The following strains of parasites were evaluated for their sensitivity to this compound: Strain SN3 of Sarcocystis neurona; strain SF1 of Sarcocystis falcatula; strain RH of Toxoplasma gondii; and the NC-1 strain of Neospora caninum. Ponazuril was tested at 2 concentrations (1 ⁇ g/mL and 10 ⁇ g/mL).
• Bovine turbinate (BT) cells were used for all in vitro studies. Cells were grown to confluency in 25 cm 2 flasks in RMPI 1640 media supplemented with 10% v/v fetal bovine serum (FBS), 100 Units penicillin (G/mL), 100 mg streptomycin/mL and 5 X 10 "2 mM 2-mercpatoethanol. After cell confluence was obtained, cells were maintained in the same media with reduced FBS (2% v/v). Cell cultures were incubated at 37 C in a humidified atmosphere containing 5% carbon dioxide and 95% air.
• FBS v/v fetal bovine serum
• G/mL penicillin
• streptomycin/mL 100 mg streptomycin/mL
• 5 X 10 "2 mM 2-mercpatoethanol 5 X 10 "2 mM 2-mercpatoethanol. After cell confluence was obtained, cells were maintained in the same media with reduced FBS (2% v/v). Cell cultures were incuba
• BT cell monolayers were infected with parasites and examined with an inverted microscope for the development of lesions (cytopathic effect, "CPE") or the presence of many extracellular merozoites. Once lesions were observed, or many extracellular parasites were present, the monolayer was scraped with the tip of a 5 mL pipette and 1 to 3 drops of the merozoite-containing fluid was transferred to two flasks of fresh BT cells.
• Merozoites of S. neurona and S. falcatula were passaged in this manner every 5 to 10 days while the tachyzoites of T. gondii and N. caninum were passaged every 3 to 4 days.
• the assay used to determine the effectiveness of Ponazuril was the Microtiter Monolayer Disruption Assay (MMDA). This assay was used to determine if the parasites or compound were toxic for BT cells. Flat bottomed 96-well microtiter plates were inoculated with BT cells and the resulting monolayers were used to determine the effects of Toltrazuril and Ponazuril on merozoite production as measured by CPE (plaque formation). Monolayers were inoculated with parasites (S. neurona or S. falcatula at a count of 50,000/well, T. gondii at a level of 10,000/well, and N. caninum at 20,000/well.
• MMDA Microtiter Monolayer Disruption Assay
• Sarcycystis 61 100 100 100 falcatula
• Organism Percent Inhibition of Cell Destruction 0.001 ⁇ g/mL 0.01 ⁇ g/mL 0.1 ⁇ g/mL 1.O ⁇ g/rnL
• Neospora caninum 3 13 100 100 NC-1
• This experiment was conducted in order to determine whether triazineones such as Toltrazuril can pass the blood-brain barrier.
• Normal horses were divided into three groups of three horses per group.
• Group 1 horses received Toltrazuril administered orally as a 5% suspension at a dose level of 2.5 mg/Kg.
• Group 2 horses received Toltrazuril administered orally as a 5% suspension at a dose level of 5.0 mg/Kg.
• Group 3 horses received Toltrazuril administered orally as a 5% suspension at a dose level of 7.5 mg/Kg.
• the dosing was repeated daily for 10 days.

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• 1999
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• 2001
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