HK1043315B - Triazineone compounds for treating diseases due to sarcosystis, neospora and toxoplasma - Google Patents

Description

Triazinone compounds for the treatment of diseases caused by Sarcocystis, neospora and Toxoplasma

Technical Field

The present invention relates to triazineone compounds useful in treating animals infected with parasites that cause abortigenic (abortigenic) or neurological diseases. In particular, the invention relates to compounds useful in the treatment of parasitic protozoa such as triazineone of the coccidian family, which cause abortigenic or neurological diseases.

Brief description of the Prior Art

Triazineone compounds such as triazinediones, e.g., diclazuril (diclazuril) compounds, and triazinetriones, e.g., Toltrazuril (Toltrazuril) compounds, are used to control a wide variety of animal, insect and fish diseases caused by a wide range of protozoa. See U.S. patent nos.: 4933341, respectively; 4935423, respectively; 5114938, respectively; 5141938, respectively; 5188832, respectively; 5196562, 5256631 and 5464837. Protozoa sensitive to these compounds infect birds, mammals and insects and exhibit diarrhea, wasting, nausea and vomiting. Generally, the mode of action of triazineones is to attack the metaphase parasitic stage found in the gut and gut wall cells, causing the endoplasmic reticulum, perinuclear space and mitochondria of the parasite to enlarge. This purposely interferes with nuclear division, so that shizonts and microgamete parents remain small and only a few merozoites and microgametes are formed, respectively. The end result reported is that these later parasites lose the ability to penetrate new mammalian cells, effectively stopping their replication in the host.

In particular, this relates to a certain protozoan suspected of causing neurological and/or abortigenic diseases of animals since the 70's of the 20 th century. Successful isolation and in vitro culture of these protozoa has proven difficult. For example, separation from the brain or cerebrospinal fluid was not successfully accomplished until the 80's of the 20 th century. Once it has been determined that neurological diseases are caused by certain brain-infecting parasites and abortigenic genetic diseases are caused by certain embryo-infecting parasites, the need for anti-parasitic drugs that are able to cross the blood-brain and placental barriers without causing harmful side effects has become imperative. Many of the drugs known in the art to cross the blood-brain barrier and/or the placental barrier to effectively treat parasitic infections of the brain have deleterious side effects such that their use entails a significant risk. Thus, to date, there has been no approved effective drug for providing effective treatment of such neurological or abortigenic genetic diseases. The following is a major description of parasitic diseases.

Equine protozoal Encephalomyelitis (EPM) is a neurological disease of horses that has a significant monetary effect on the Equine industry due to a preference for small horses that are in short supply (e.g. elite racehorses and purebred show horses). EPM, first identified as a disease in the 70's 20 th century, was cultured from horses with EPM and was not named as neuronal Sarcocystis neurona until 1991. In 1997, Neospora was isolated from equine brains with EPM (Neospora spp.) and is now named giant Neospora (Neospora hugesi). Thus, it is now proposed that EPM may be caused by this newly identified organism alone, by the neuronal Sarcocystis alone or in combination. EPM often causes imbalance (dyskinesia), weakness and rigidity. The disease can mimic almost any neurological condition. It can occur in either acute or chronic form. The chronic form is often insidious at the outset, difficult to diagnose until the disease reaches a late stage, and can lead to death. In the lightest state, the only clinical symptom may be undefined limping of the lower extremities or light breathing noise. In the most severe state, horses are unable to swallow or stand. It is now known that in the most severe state, parasites, such as the neuronal Sarcocystis, infect and cause severe damage to the brain. Like brain injury, edema, and neuronal death associated with merozoites and submucous variants in the nervous system (CNS), resulting from infiltration of inflammatory cells, the clinical symptoms of EPM are caused by direct neuronal (brain and spinal) injury by parasites. Currently, there is no effective treatment or prevention approved for the control of EPM. The human drug trimethoprim-sulfamoyl conjugate has been used. However, the treatment is expensive and requires multiple administrations.

Another coccidial parasite, the murine toxoplasma, has been known for some time and was first isolated from the intestine and muscle tissues of cats. The final host of this parasite is a cat that can colonize the organism for a long period of time and transmit oocysts to other animals including cattle, sheep, pigs and humans. Infections in sheep, cattle and humans are known to be associated with abortions and acquired sexual dysfunction, which initially infects the central nervous system. It has recently been shown to be associated with abortion and malformations in kittens born to infected queens who were seronegative prior to infection during pregnancy. Non-feline hosts such as cattle, sheep, pigs and humans do not produce oocysts but during development muscles and brain may be attacked by tachyzoites and bradyzoites, the clinical symptoms of the disease being neurological symptoms and fetal defective abortion. It was reported that 60% of cats were seropositive for murine toxoplasma. Again, there is no approved treatment or prevention of toxoplasmosis.

Yet another coccidioid parasite, Neospora caninum (Neospora caninum), causes both neurological and abortigenic diseases in animals. It was first isolated from dogs in 1988 and previously fused with murine Toxoplasma gondii. The disease caused by this parasite is most severe in the case of placenta-infected puppies and is characterized by an increasing paralysis, especially of the hind limbs; polymyositis and hepatitis may also be caused. This disease has more recently been recognized as a major cause of neonatal calf limb deficits associated with abortion and neurology. Microscopic lesions of non-suppurative encephalitis and myocarditis of aborted fetuses can be seen in the brain, spinal cord, and heart. The ultimate host for neospora caninum was recently identified as dog. Neither treatment or prevention of Neospora caninum in dogs or cattle nor treatment or prevention of megaspora (Neospora hugesi) in horses was approved during this period.

None of the references in the art, including the references mentioned above, suggest or teach the use of triazineone compounds such as toltrazuril (Toltrazuil) or toltrazuril sulfone (recently renamed "Ponazuril"), for treating animals infected with coccidia causing abortive genetic or neurological diseases or, in particular, with Sarcocystidae (Sacocystidae), without causing tolerogenic side effects. There is therefore a need for an improved and safe method of treating animals suffering from parasitic diseases manifested as neurological or abortigenic genetic diseases.

Summary of The Invention

In light of the foregoing, the invention includes a method of treating a diseased animal suffering from a parasitic neurologic or abortigenic disease that is treatable with a triazineone compound, with the proviso that if the disease is caused by Sarcocystis neurona, the compound diclazuril or Toltrazuril (Toltrazuril) cannot be used. The method comprises administering to the animal a pharmaceutically effective dose of the compound. The term "pharmaceutically effective dose" as used herein means a dose of triazineone administered that is high enough to inhibit the growth of parasitic protozoa, typically coccidia causing neurological diseases and/or abortions, in vivo or in vitro. The pharmaceutically effective dose controls parasites in the infected tissue and thus improves the health of the animal.

Furthermore, the invention includes a prophylactic (metaphylactically) treatment of animals infected with parasites that cause neurological or abortigenic diseases that can be treated with triazineone compounds. Prophylactic treatment includes administering the triazineone compound to an animal by a prophylactically-effective treatment regimen. The term "prophylactically-effective treatment regimen" refers to an extended period of time for a predetermined intermittent administration of the triazineone compound until the animal defeats the infested parasite, that is, an immune protective response is established or the parasite is eliminated. Typically, the treatment regimen is such that it is effective to control parasites and prevent clinical symptoms of the disease. A prophylactically effective dose can also be administered for a prolonged period of five years or the animal's life, especially in cases where parasite control is difficult. For prophylactic treatment, preferred triazineone compounds are triazinetriones, including but not limited to toltrazuril and ponazuril (Ponazruil).

The invention also includes a method of treating a single high dose animal. The method comprises administering a single high dose of a pharmaceutically effective dose of a triazineone compound to an animal suffering from a neurologic or abortigenic parasitic disease that is treatable with triazineone. The term "single high dose" refers to a dose that is administered only once. The dosage is significantly higher than the dosage used during therapeutic or prophylactic treatment; is effective in controlling parasites that cause disease and does not produce deleterious effects such as toxicity. Single high doses of triazineone are typically greater than 10 mg/Kg. This and other aspects of the present invention are described more fully below.

Detailed description of the invention

As indicated above, the present invention is directed to a method of treating an infected or diseased animal suffering from a neurologic or abortigenic parasitic disease treatable with triazineone, comprising administering to the animal a pharmaceutically effective amount of the compound. Illustrative, but not limiting, examples of animals are horses, cattle, cats, dogs, pigs, birds, insects, and humans. The disease causing parasite is a coccidia of the genus Sarcocystidae (Sarcocystidae) which manifests as a neurological or abortigenic disease. Examples of parasites, which are given as an illustration and not as a limitation, may be selected from the group of parasites consisting of: the genera Sarcocystis (Sarcocystis), Neospora (Neosporia) and Toxoplasma. Sarcocystidae (Sarcocystidae) is typically selected from the group of parasites consisting of: neuronal sarcocystis (s. neurona), neospora megaterium (n. hugesi), neospora caninum (n. caninum), and toxoplasma gondii. Protozoan infectious diseases or diseases include, but are not limited to, EPM, neospora (neospora), and toxoplasma.

In the practice of the present invention, treatment of parasitic infectious diseases or diseases caused by protozoa as described herein results in a reduction of clinical symptoms of neurological and abortigenic genetic diseases. Often, clinical symptoms include lameness, movement disorders, paralysis, miscarriage, weak pups, and other related disorders. In terms of therapeutic treatment, the treatment regimen may be once a day, twice or more a day, once every other day or even once a week, depending on factors such as the severity of the disease and the parasite species responsible for the disease. In some cases, however, the treatment regimen may continue indefinitely, sometimes for the entire lifetime of the animal. For example, in the case of animals infected with a more resistant strain of parasite, treatment may be extended for a longer period of time until the condition is eliminated. A typical treatment period is from about 28 to 90 days and preferably from about 28 to 60 days. The most preferred treatment is once a day for about 28 days.

With respect to prophylactic treatment, infected animals are treated to the point where they do not manifest clinical symptoms themselves. Such treatment ultimately allows the animal to gain the ability to control parasites, that is, to be given the ability to resist infection in the future by establishing an effective immune response, without the need for further administration of the triazineone compound. According to the present invention, the prophylactic activity involves the use of triazineone compounds to control protozoa according to a predetermined intermittent treatment regimen (prophylactically-effective treatment regimen). Thus, a prophylactically-effective treatment regimen is to reduce the pathogenic potential of the parasites by administration, that is, to kill them or reduce their numbers. In essence, a prophylactically-effective treatment regimen can be administered two or more times, typically from about once a month to the entire life of the animal or until a genetic clearance mechanism, such as an effective immune response, is established in the animal to combat future infections. The latter may be present for 5 years or less than 5 years. As recognized, prophylactic treatment is based on the recognition that when animals become infected with the protozoa described herein, they do not manifest themselves until a significant period of time has elapsed (e.g., 2-6 months after infection). Clinical symptoms such as neurological symptoms or abortion. In contrast, intestinal protozoan infections exhibit their own symptoms immediately after infection. According to the invention, the prevention and treatment of the growth of parasites and the resulting clinical diseases is carried out. The treatment regimen is an intermittent regimen that is about once a month, once every two months or once every two weeks.

In the therapeutic and prophylactic treatment, a dose corresponding to about 1.0 to 100mg/Kg, preferably about 1.0 to 25mg/Kg and more preferably about 2.5 to 10mg/Kg, can be used. For a particular case of resistance (e.g., when the animal is infected with a resistant strain), a high range is required. The desired dosage level and treatment course will be within the skill of the art. A preferred treatment regimen for horses with EPM or cattle with neospora (Neosporosis) is to take triazinetrione in the range of about 1.0 to 25mg/Kg and more preferably in the range of about 2.5 to 10mg/Kg every 28 days.

In single high dose therapy, triazineone is administered at a pharmaceutically effective dose of greater than 10mg/Kg and up to about 100 mg/Kg. This is a significant feature of the present invention, and the compounds of the present invention may be non-toxic so that they may be administered at high dosage levels. The advantage of high dose administration is that repeated administration is virtually unnecessary. Upon single high dose treatment, ponazuril was found to be both safe and effective at doses up to 100mg/kg body weight. Unlike prior related compounds, the compounds of the triazinones equivalent to ponazuril are preferred, wherein they do not cause toxic side effects if administered at very high dosage levels.

Without being bound by any particular theory of the invention, it is believed that the unexpected success of the treatment of the present invention is due to the ability of the triazineone compounds to cross the blood brain barrier or placental barrier. The compounds of the invention must be able to readily penetrate the blood brain barrier and also be able to penetrate the placenta and kill protozoa in situ in the brain and cerebrospinal fluid/spinal cord. It has further been found that such compounds are non-toxic and non-mutagenic even at the high doses necessary for the single high dose treatment regimen described herein.

Thus, inexpensive, easily administered drugs are suitable for the effective treatment and protection of the above-mentioned diseases in animals without producing unacceptable side effects such as toxicity and mutagenicity. Triazinones are specifically described below but are not limited to toltrazuril. The disclosure and invention also includes other triazineone compounds that are applied in the manner of toltrazuril. Toltrazuril as used herein has the following formula (1):

wherein

R¹The expression haloalkylthio, haloalkyl-sulfinyl or haloalkylsulfonyl,

R²represents hydrogen, alkyl, alkoxy, alkoxyalkyl, alkylmercapto, halogen, haloalkyl or optionally substituted sulfamoyl, such as dialkylsulfamoyl,

R³and R⁴May be identical or different and represents hydrogen, alkyl, alkenyl or alkynyl and X is O or S, and their physiologically acceptable salts.

Furthermore, it has been found, inter alia, that the present invention may employ compounds of the following formula Ia:

wherein

R^IRepresents halo (C)₁-C₄) Alkylthio, halo (C)₁-C₄) -alkylsulfinyl or halo (C)₁-C₄) -an alkylsulfonyl group,

R^IIrepresents hydrogen, alkyl (C)₁-C₄) Alkoxy (C)₁-C₄) Halogen, alkoxy (C)₁-C₄) Alkyl radical (C)₁-C₄) Alkyl (C)₁-C₄) Mercapto, dialkyl (C)₁-C₄) Aminosulfonyl or haloalkyl (C)₁-C₄) And

R^IIIand R^IVMay be the same or different and represent hydrogen, alkyl (C)₁-C₄) Or alkenyl (C)₂-C₄) And X is O or S. Finally, it has now been found that:

(a)1- (4-phenoxy-phenyl) -1, 3, 5-triazines are obtained by the following process when compounds of formula II

Wherein

R¹、R²、R³And X has the above-mentioned meaning; with a substituted carbonyl isocyanate of formula III:

wherein R is⁵Represents a halogen atom, an alkoxy group or an aryloxy group, and optionally separates the substituted 1, 3, 5-triazine derivative of formula IV formed in the process:

wherein R is¹、R²、R³And X has the above meaning and is optionally reacted with a compound of formula V:

A---Z (V)

wherein:

a represents alkyl, alkenyl or alkynyl and

z represents halogen; or

(b) 1- (4-phenoxy-phenyl) -1, 3, 5-triazine derivatives of the general formula I, wherein R is obtained by¹、R²、R³And X has the meaning given above, with a bis (chlorocarbonyl) -amine of the formula VI, optionally in the presence of an acid acceptor:

wherein

R⁶Represents alkyl, or

(c) To obtain a compound in which the substituent R²、R³And R⁴And X has the abovementioned meaning and R¹A compound of formula I, representing a haloalkylsulfinyl or haloalkylsulfonyl group, a compound of formula (VII) is reacted with a suitable amount of a suitable oxidizing agent:

wherein

R²、R³And R⁴Have the above meanings, and

R^1’represents haloalkylthio.

If N- [ 3-chloro-4- (4 '-trifluoromethylsulfanyl-phenoxy) -phenyl ] -N' -methyl-urea and chlorocarbonyl isocyanate are used in process (a), the course of the reaction can be represented by the following reaction scheme:

if N- [ 3-ethoxy-4- (4' -trifluoromethylsulfanyl-phenoxy) -phenyl ] -thiourea and N-methyl-bis (chlorocarbonyl) amine are used as starting materials in process (b), the course of the reaction can be represented by the following reaction scheme:

wherein R is obtained according to the process (a) or (b)₁Compounds of the general formula I ═ haloalkylthio and X ═ O can be oxidized according to process (c) to the corresponding haloalkylsulfinyl or haloalkylsulfonyl derivatives. If hydrogen peroxide is used as the oxidizing agent, the course of the reaction can be represented by the following equation:

in the formulae I, II, IV, V, VI and VII, R²、R³、R⁴、R⁶Or alkyl as defined under A is a straight or branched chain alkyl group having 1 to 6, preferably 1 to 4, carbon atoms. Examples which may be mentioned are optionally substituted methyl, ethyl, n-and i-propyl and n-, i-and t-butyl.

In formulae I, II, IV, V and VII, R³、R⁴Alkenyl as defined under A is straight-chain or branched alkenyl having preferably 2 to 6, in particular 2 to 4, carbon atoms. Examples which may be mentioned are optionally substituted vinyl, propen-1-yl, propen-2-yl and buten-3-yl.

In formulae I, II, IV, V and VII, R³、R⁴Or alkynyl as defined under A is straight-chain or branched alkynyl having preferably 2 to 6, in particular 2 to 4, carbon atoms. Examples which may be mentioned are optionally substituted ethynyl, propyn-1-yl, propyn-2-yl and butyn-3-yl.

In formulae I, II, III, IV and VII, R²Or R⁵The alkoxy group as defined in (1) is a straight or branched chain alkoxy group having preferably 1 to 6, especially 1 to 4 carbon atoms. Examples which may be mentioned are optionally substituted methoxy, ethoxy, n-and i-propoxy andn-and i-butoxy.

In the formulae I, II, III, IV, V and VII, R²、R⁵Halogen as defined in Z is preferably fluorine, chlorine, bromine and iodine, especially chlorine and bromine.

In the formulae I, II, IV and VII, R¹Haloalkylthio is defined as a haloalkylthio having preferably 1 to 4, especially 1 or 2, carbon atoms and preferably 1 to 5, especially 1 to 3 identical or different halogen atoms, preferably fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylthio, chloro-difluoromethylthio, bromomethylthio, 2, 2, 2-trifluoroethylthio and perfluoroethylthio.

In formulae I, II and IV, R¹Haloalkylsulfinyl as defined in (a) is haloalkylsulfinyl having 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, preferably fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulfinyl, chloro-difluoromethylsulfinyl, bromomethylsulfinyl, 2, 2, 2-trifluoroethylsulfinyl and perfluoroethylsulfinyl.

In formulae I, II and IV, R¹The haloalkylsulfonyl group as defined in (1) is a haloalkylsulfonyl group having 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, preferably fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulfinyl, chloro-difluoromethylsulfinyl, bromomethyl-sulfonyl, 2, 2, 2-trifluoroethylsulfonyl and perfluoroethylsulfonyl.

In formulae I, II and IV, R²The optionally substituted sulfamoyl group as defined in (1) is preferably one of the following groups:

SO₂NH₂，SO₂NH-CH₃，SO₂N(CH₃)₂，

SO₂NH-C₂H₅，SO₂-N(C₂H₅)₂，

and

in formula III, R⁵The aryloxy group as defined in (1) is preferably a monocyclic carbocyclic aryloxy group or a bicyclic carbocyclic aryloxy group, in particular a phenoxy group.

In formula III, aryloxy R⁵Phenoxy is preferred.

The majority of substituted ureas or thioureas of the formula II used as starting materials have not been known to date, but they can be readily prepared by known methods, or by (a) reacting a substituted 4-aminodiphenyl ether with the corresponding substituted isocyanate or isothiocyanate in an inert solvent at a temperature of from 0 ℃ to 100 ℃, or reversing the order, (b) reacting ammonia or a substituted amine with the corresponding substituted isocyanate or 4-isothiocyanatodiphenyl ether with each other under the same conditions, or by (c) condensation reaction of a 4-hydroxyphenyl-urea or-thiourea compound with an active haloaromatic compound in an aprotic solvent (e.g., dimethyl sulfoxide, dimethyl polyamide or hexamethylphosphoric triamide) in the presence of a base (e.g., sodium hydride, potassium hydroxide, potassium carbonate, etc.) at a temperature of 20 ℃ to 150 ℃.

When the amount of solvent is suitably selected, the reaction product generally crystallizes upon cooling of the solution. Other documents relating to the preparation of urea compounds from amines and isocyanates are: method of organic chemistry (Methoden der org. Chemie) (Houben-Weyl), IV edition, volume VIII, page 157 & 158.

Some bis (chlorocarbonyl) amine compounds of the formula VI which can be employed in process (b) according to the invention are already known (cf. synthesis 1970, 542-543), and if they are unknown compounds, can be prepared analogously from cyclic diacyldisulfides and chlorinated in an inert organic solvent, preferably carbon tetrachloride.

Possible diluents for the reaction of the ureas or thioureas of the formula II with carbonyl isocyanates of the formula III (process a) and with bis (chlorocarbonyl) amines of the formula VI (process b) and the reaction of the 1, 3, 5-triazine derivatives of the formula IV with compounds of the formulae A to Z are all organic solvents which are inert in these reactions.

These include, in addition to pyridine, aromatic hydrocarbons such as benzene, toluene and xylene; halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene; and ethers such as tetrahydrofuran and dioxane.

Hydrochloric acid which may be formed in the reaction is either liberated as a gas or may be bound by an organic or inorganic acid acceptor. Preferred acid acceptors include: tertiary organic bases, such as trialkylamine compounds, e.g. triethylamine; azamono-or bi-cyclic aromatic amines, such as pyridine; mono-or bicyclic azacyclic alkylamines, such as diazabicyclononene, diazabicycloundecene and various other compounds; or an inorganic base such as an alkali metal carbonate, oxide or hydroxide, or an alkaline earth metal carbonate, oxide or hydroxide.

The reaction temperature during the above reaction can vary within wide limits. Typically, the reaction is carried out at a temperature of between about 0 ℃ and about 150 ℃, preferably between about 20 ℃ and about 100 ℃.

In the above reaction process, the reaction may be carried out under normal pressure or under high pressure. Generally, the reaction is carried out at atmospheric pressure.

Possible oxidizing agents which are suitable for converting the trifluoromethylthio compound of the formula 1 wherein Y represents oxygen into the corresponding sulfinyl or sulfonyl compound in process (c) are: h₂O₂Glacial acetic acid; h₂O₂Acetic anhydride; h₂O₂Methanol; peracids such as m-chloroperbenzoic acid, and chromic acid; potassium permanganate; the content of the sodium periodate is as follows,cherry-colored ammonium nitrate; and nitric acid.

The resulting compounds may be converted into the corresponding addition salts, for example by reaction with an inorganic or organic base.

In the practice of the present invention, the triazineone compounds can be formulated in any conventional manner into compositions or formulations for administration to an animal. Preferred formulations herein for oral administration may be in the form of suspensions, tablets, capsules, gels, pastes, boluses, or formulations in the form of powders, granules or pellets. Preferred formulations for oral administration are in the form of pastes or food additives. Other modes of administration that may be employed include parenteral, topical, intramuscular, and intramucosal or by other routes known to those skilled in the art. Topical administration in a pour-on form is also preferred.

Pharmaceutically acceptable carriers and adjuvants are commonly used in formulations. Examples thereof may be: thickeners selected from Carbopol (Carbopol), inorganic thickeners such as silicates, bentonite or colloidal silica, and organic thickeners such as fatty alcohols or fatty acid esters; a wetting agent selected from the group consisting of polyethylene glycol and sodium lauryl sulfate; for carboxyvinyl materials, carboxyvinyl 974P is the most preferred thickener for the preferred pastes of the present invention. Preservatives selected from the group consisting of parabens, alcohols and aldehydes may also be employed in the present invention. These may be liquid, solid or gaseous materials which are either inert or medically acceptable and compatible with the active ingredient.

Surprisingly, the pastes of the present invention are effective in delivering triazineone compounds, particularly toltrazuril, and ponazuril, across the blood brain or placenta barrier and attack parasites that have invaded the brain or infected the placenta of pregnant animals. For convenience, the present invention provides a description of specific embodiments of preferred pastes and how to make them. A preferred paste according to the invention comprises a micronized suspension of triazinetrione (e.g. ponazuril), propylene glycol, a thickener (e.g. carbopol), a preservative (e.g. methyl and propyl parabens) and water. It can be prepared at such temperatures by mixing water, usually pure water, and propylene glycol, and adding a preservative. The resulting mixture is cooled to room temperature, after which carbopol is added, preferably in the form of carbopol 974P. The triazinetrione is finally added. After complete mixing, the pH was adjusted to about 6.0 with sodium hydroxide the preferred paste comprised 15% w/w toltrazuril, 20% w/w propylene glycol, 0.5% carbopol 974P, 0.14% w/w methylparaben, 0.02% w/w propylparaben, 0.1% w/w sodium hydroxide, and the balance purified water. Sweeteners including glucose, sucrose, lactose, fructose, sorbitol, xylitol, artificial sweeteners and molasses may be added to improve mouthfeel. In addition, yeast or liver-type flavors may be added for the same purpose.

The present invention will be further described with reference to the following examples, but not limited thereto.

Example 1

Pharmacokinetic studies were performed comparing the concentration of toltrazuril, ponazuril and toltrazuril sulfoxide in horse blood at different times after single dose administration of toltrazuril. All horses received a single dose of 10mg/Kg, which was administered orally in suspension. Blood samples were drawn at treatment time (0) and at 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hours post-treatment. The results for the samples are listed in Table 1. it is surprising that horses receiving toltrazuril showed a higher concentration of ponazuril in their sera. Thus, toltrazuril sulfoxide is found in very high concentrations in blood. This indicates that ponazuril alone is capable of producing acceptable blood concentrations that are expected to pass through the blood-brain barrier, a desirable characteristic for the treatment of neurological diseases such as those caused by Sarcocystis neurona, Toxoplasma gondii, Neospora caninum (Neosporium caninum), and Neosporozoea giganticum (Neosporihugesi).

TABLE 1 pharmacokinetics of toltrazuril in a single dose

D	Test Compounds	Concentration in blood of mg/l00.250.5124
			A	Toltrazuril sulfoxide ponazuril	0.027 0.773 2.863 4.511 3.119＜0.01 0.077 0.070 0.159 0.1420.010 0.089 0.08B 0.171 0.110
B	Toltrazuril sulfoxide ponazuril	0.061 0.393 2.61 7 4.296 6.820＜0.01 0.025 0.047 0.083 0.157＜0.01 0.029 0.036 0.040 0.050
			C	Toltrazuril sulfoxide ponazuril	0.061 0.560 3.286 5.788 9.079＜0.01 0.024 0.041 0.097 0.218＜0.01 0.013 0.019 0.026 0.032
D	Toltrazuril sulfoxide ponazuril	0.01 7 0.295 3.286 2.165 3.328＜0.01 0.027 0.039 0.058 0.100＜0.01 0.011 0.021 0.024 0.029
			E	Toltrazuril sulfoxide ponazuril	＜0.01 0.039 1.146 3.175 8.410＜0.01 ＜0.01 0.021 0.064 0.194＜0.01 ＜0.01 0.017 0.015 0.044
F	Toltrazuril sulfoxide ponazuril	0.110 0.428 1.741 - 8.144＜0.01 0.026 0.044 - 0.183＜0.01 0.012 ＜0.01 - 0.041

D	Test Compounds	Concentration in blood mg/l 612244872
			A	Toltrazuril sulfoxide ponazuril	5.1 49 5.066 6.434 7.607 6.6530.1 67 0.230 0.407 0.732 0.5920.108 0.170 0.324 1.622 1.933
B	Toltrazuril sulfoxide ponazuril	11.474 11.670 11.690 6.677 5.0580.320 0.451 0.566 0.454 0.3460.131 0.254 0.255 0.831 0.880
			C	Toltrazuril sulfoxide ponazuril	14.202 13.751 - 9.758 7.6330.280 0.436 - 0.477 0.3770.061 0.135 - 0.540 0.642
D	Toltrazuril sulfoxide ponazuril	3.816 10.544 7.236 8.234 -0.133 0.668 0.461 0.749 -0.030 1.651 0.315 0.986 -
			E	Toltrazuril sulfoxide ponazuril	11.335 12.032 8.694 6.869 -0.259 0.430 0.481 0.741 -0.074 0.268 0.231 0.501 -
F	Toltrazuril sulfoxide ponazuril	10.966 6.660 10.224 7.096 -0.245 0.453 0.633 0.642 -0.061 0.725 0.192 0.532 -

Example 2

Ponazuril, 1-methyl-3- [ 4-p- [ trifluoromethyl) sulfonylphenoxy ] -m-tolyl ] -s-triazine-2, 4, 6(1H, 3H, 5H) -trione, a representative triazinetrione, is formulated into a paste suitable for administration to horses. The components listed in table 2 were used to prepare the following formulations.

TABLE 2 composition of Ponaculam paste

Composition (I)	Theoretical quantity	Actual amount% w/w
			Ponazuril-micronized propylene glycol polyhydroxy ethylene 974P methylparaben, NF sodium hydroxide and NF pure water	22.5Kg30.0Kg0.750Kg0.210Kg0.030Kg0.150Kg96.365Kg	15.020.00.50.140.020.1064.24

The following methods (A) and (B) were employed to prepare a medicament. The first method (a) comprises: 1) mixing a portion of the water with propylene glycol; 2) adding preservatives (methyl paraben and propyl paraben); 3) slowly adding 974P until a homogeneous suspension is formed; 4) adding ponazuril in a particulate form; 5) sodium hydroxide was added to bring the PH of the suspension to about 6.0; and 6) adding the remaining water to the QS volume. The final suspension is a paste that can be administered orally to horses.

The second method (B) comprises: 1) mixing a portion of the water with propylene glycol; 2) heating to 70 deg.C; 3) adding preservatives (methyl paraben and propyl paraben) while maintaining the temperature of the solution at 70 ℃; 4) cooling the solution to room temperature; 5) slowly adding 974P until a homogeneous suspension is formed; 6) adding ponazuril in a particulate form; 7) sodium hydroxide was added to bring the PH of the suspension to about 6.0; and 8) adding the remaining water to the QS volume. The final suspension is also pasty and can be administered to horses in oral form.

The resulting paste was administered to horses and found to be palatable and acceptable.

Example 3

Ponazuril, 1-methyl-3- [ 4-p- [ trifluoromethyl) sulfonylphenoxy ] -m-tolyl ] -s-triazine-2, 4, 6(1H, 3H, 5H) -trione, a representative triazinetrione, was tested for its ability to treat horses that had exhibited symptoms of Equine protozoal Encephalomyelitis (EPM). The compounds were formulated as pastes with ponazuril as described in example 1 as 15% of the active ingredient (a.i.). It is administered to horses already diagnosed with EPM once a day for 28 days, in a dosage ratio of 2.5mg/Kg to 10 mg/Kg.

The clinical symptoms of naturally occurring EPM are well characterized by special markers and laboratory diagnostic analysis. The diagnostic method for performing this test on EPM-positive horses is as follows: confirmation of the presence of off-site neurological deficits determined by one standardized neurological test method includes radiography, EPM positive; positive western blot for neuronal sarcocystis IgG; red blood cell count below 500 cells/M1; CSF index-total protein < 90, IgG index > 0.3, AQ coefficient < 2.2.

With the proviso that the horse does not suffer from a disease other than EPM. Therefore, they need to satisfy the following conditions: negative CSF (< 1: 4) versus RHV-1; standard serum values relative to vitamin E (2.0 ug/mL); absence of an epileptic condition; no behavioral disorder. The horses to be diagnosed were arbitrarily divided into several groups. The horses of group 1 were dosed daily with 5mg/Kg dose and the horses of group 2 were dosed daily with 10mg/Kg dose. Therapeutic doses are based on body weight. To confirm that the treatment was indeed effective, the horses were evaluated for 90 days (approximately 60 days after termination of the treatment). The following systems were used to evaluate the effect of the treatment:

1) 0-total success-clinical criteria with negative CSF; 2)1 ═ defects detected with normal gait; 3)2 ═ defects that are easily detected and exaggerated by retreat, rotation, rocking jaw waist compression and neck elongation; 4) 3-a very prominent defect when walking, facial rotation, waist compression or neck elongation; 5) stumbling, stumbling and falling naturally; 6)5 lying down, can't get up. The improvement of one (1) unit was considered to be a significant improvement in the evaluation process.

Table 3 shows the results of this study. All horses (100%) in the 28-day-treated 10mg/Kg group showed a significant improvement from clinical evaluation at day 90 after the start of treatment with ponazuril (day 0). Eight out of nine (88.9%) horses treated with a 5mg/Kg dose showed a recognized improvement. If all the evaluation scores of each group for each day of treatment are added, a total score is obtained. The improvement in the overall score shown by group 1 and group 2 was almost the same. It was therefore concluded that: a dose of 5mg/Kg or 10mg/Kg of ponazuril is effective for treating EPM in horses.

TABLE 3 response to treatment of EPM infected horses with toltrazuril sulfone

Horse ID	5mg/Kg dose			10mg/Kg dose
							Day 0	28 days	90 days	Day 0	28 days	90 days
A							2	1	2
	B	2	1	1
C										4	2	1
	D				3	2							0
E										2	2	1
	F	3	2	0
G										2	1	1
	H				2	2							1
1							2	1	0
	J	2	0	0
K										3	0	0
	L	2	3	3
M							2	2	0
	N	2	2	0
O							3	3	2
	Total of	17	13	6	19	15							4

Example 4

To determine the protection afforded by ponazuril, in vitro experiments were performed. The sensitivity of the following parasite strains to this compound was evaluated: SN3 strain of neuronal sarcocystis; strain SF1 of fusarium sporotrichum (Sarcocystis falcatala); RH strain of mouse common toxoplasma; and NC-1 strain of Neospora caninum (Neosporium caninum). Ponacuril was determined at two concentrations (1. mu.g/ml and 10. mu.g/ml).

Bovine gyroscope (BT) cells were used for in vitro studies. Cells were grown in RMPI1640 medium supplemented with 10% V/V bovine serum (FBS), 100 units of penicillin (G/mL), 100mg streptomycin/mL and 5X 10 in 25cm2 flasks for polymerization in RMPI1640 medium^-2mM 2-mercaptoethanol. After the cells were polymerized, the cells were maintained in the same medium with a reduction in bovine serum FBS (2% V/V). The cell culture was cultured at 37 ℃ in a humid atmosphere containing 5% carbon dioxide and 95% air.

As a result of parasite growth, the BT cell monolayer was infected with the parasite and the occurrence of damage (cytopathic effect, "CPE") or the appearance of many extracellular merozoites was detected using inverse microscopy. Once damage was observed, or the appearance of many extracellular parasites, the monolayer was broken up with the tip of a 5mL pipette and 1 to 3 drops of merozoite containing liquid were transferred to two flasks containing fresh BT cells. Merozoites of neuronal sarcocystis and fusarium were passaged in this manner every 5 to 10 days while tachyzoites of toxoplasma murine and neospora caninum (n.caninum) were passaged every 3 to 4 days.

The analytical test used to determine the effectiveness of ponazuril is the Microtiter Monolayer lysis test (MMDA). This assay is intended to determine whether a parasite or compound is toxic to BT cells. Flat-bottomed microtiter plates of 96 wells were seeded with BT cells and the resulting monolayers were used to determine the effect of toltrazuril and ponazuril on merozoite production as measured by CPE (plaque formation). A monolayer inoculated with parasites (either Sarcocystis neurona (S.neurona) or Fusarium (S.falcatala) at 50000/well counts, Toxoplasma gondii at 10000/well levels, and Neosporosis caninum (N.caninum) at 20000/well levels 2 hours after infection all wells were loaded with test compound, untreated and uninfected monolayer wells served as parasite controls, and uninfected reagent-treated BT cells served as toxicity controls, each treatment was assayed in 6 parallel assays, each well was visually monitored daily and the assay was terminated when 90-100% of the untreated merozoite-infected cells lysed (90-100% CPE). all wells on the plate were washed with Phosphate Buffer (PBS) and fixed with 100% ethanol for 5 minutes, then stained in crystal violet solution Was used to quantify the incorporation of crystal violet and these data were used to determine the concentration of ponazuril that inhibited 50% of damage (inhibitory concentration)₅₀Or IC₅₀). Data showing inhibition are presented in table 4. It is noteworthy that as little as 1 μ g/mL of ponazuril provides 100% inhibition of cell destruction by neospora caninum, toxoplasma murine and sarcocystis sickle (s.falcatala), while the need for 10 μ g/mL of ponazuril results in 100% inhibition of cell destruction by the neuronal sarcocystis (s.neurona). This suggests that triazinones such as toltrazuril and ponazuril are effective in treating known and neurological and abortive remains caused by coccidiaDiseases associated with the hereditary disease syndromes, including those caused by neuronal Sarcocystis (S.neurona), Neosporosis canis, Neosporosis megaterium, and Toxoplasma gondii. In addition, ponazuril is not toxic to BT cells.

Table 4 in vitro data for ponazuril

Biological body	Percent inhibition of cell destruction 0.1. mu.g/mL 1. mu.g/mL 5.0. mu.g/mL 10. mu.g/mL
		Neuron sarcocystis	0 40 90 100
Fusarium shaped sarcocystis sp	61 100 100 100
Biological body	Percent inhibition of cell destruction 0.001. mu.g/mL 0.01. mu.g/mL 0.1. mu.g/mL 1.0. mu.g/mL
		Neospora caninum	3 13 100 100
Mouse toxoplasma gondii	11 16 100 100

Example 5

This test was performed in order to determine whether triazinones such as toltrazuril could pass the blood-brain barrier. Normal horses were divided into three groups of three horses. Horses of group 1 were orally administered toltrazuril in the form of a 5% suspension at a dosage level of 2.5 mg/Kg. Horses of group 2 were orally administered toltrazuril in the form of a 5% suspension at a dosage level of 5.0 mg/Kg. Horses of group 3 were orally administered toltrazuril in the form of a 5% suspension at a dosage level of 7.5 mg/Kg. Such administration is repeated daily for 10 days. Blood samples were drawn at 48, 96 and 240 hours and serum was assayed for toltrazuril, toltrazuril sulfoxide and ponazuril. On day 10 after treatment was initiated (day 10), one cerebrospinal fluid sample was taken from each horse and these samples were again assayed for toltrazuril, toltrazuril sulfoxide and ponazuril. The concentrations of toltrazuril, toltrazuril sulfone and ponazuril in serum and cerebrospinal fluid are listed in tables 5a and 5 b. The concentration of ponazuril in the serum and cerebrospinal fluid of horses treated with toltrazuril is so high that the concentration of ponazuril in cerebrospinal fluid of horses treated with toltrazuril is comparable to the concentration of toltrazuril itself. This demonstrates that both toltrazuril and ponazuril can effectively pass through the blood-brain barrier and that ponazuril can pass this barrier more effectively than toltrazuril. For those skilled in the art, this data demonstrates that toltrazuril also effectively passes the placental barrier.

TABLE 5a drug concentration in horses following repeated toltrazuril administration

Horse ID	10 days dose (mg/Kg)	Concentration of toltrazuril			Cerebrospinal fluid for-10 days
						48 hours	96 hours	240 hours
		123456789	2.52.52.55.05.05.07.57.57.5	4.494.011.67.289.189.26N/A9.9010.46					9.859.0913.114.1714.0318.1927.7419.5518.47	15.299.6015.2124.9216.5417.5930.0824.1523.53	0.230.060.150.190.120.260.500.210.45
AVGAVGAVG	2.5mg/Kg dose 5.0mg/Kg dose 7.5mg/Kg dose				6.708.5710.18	10.6815.4621.92	13.3719.6825.95	0.150.190.39

TABLE 5b drug concentrations in horses following repeated toltrazuril administration

Horse ID	10 days dose (mg/Kg)	Ponacuril concentration			Cerebrospinal fluid for-10 days
						48 hours	96 hours	240 hours
		123456789	2.52.52.55.05.05.07.57.57.5	0.290.243.700.460.630.486.350.780.52					0.991.153.132.092.032.662.692.893.09	2.612.364.045.442.035.616.316.377.06	0.090.070.110.120.140.210.230.170.27
AVGAVGAVG	2.5mg/Kg dose 5.0mg/Kg dose 7.5mg/Kg dose				1.410.532.55	1.762.262.89	3.005.026.58	0.090.160.22

Although the invention has been described in detail in the foregoing for the purpose of illustrating the invention, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.

Claims (22)

1. Use of a triazineone compound in the manufacture of a medicament for the prevention or treatment of a parasitic-induced neuropathy or abortion disease in an animal, with the proviso that when the disease is sarcocystis neurona, the compound is not diclazuril or toltrazuril.

2. The use as claimed in claim 1, wherein the parasite-caused disease is caused by the order Coccidia.

3. The use as claimed in claim 2, wherein the coccidioideae is a member of the family Sarcocystidae.

4. The use as claimed in claim 3, wherein the member of the family Sarcocystidae is selected from the group of parasites consisting of Sarcocystis, neospora and Toxoplasma.

5. The use as claimed in claim 4, wherein the genus Sarcocystis is selected from the group of parasites consisting of the species Sarcocystis, the genus neospora is selected from the group of parasites consisting of the species neospora, and the genus Toxoplasma is selected from the group of parasites consisting of the species Toxoplasma.

6. The use as claimed in claim 5, wherein the species of the genus Sarcocystis is Sarcocystis neurona, the species of the genus neospora is neospora caninum or neospora macrocephala and the species of the genus Toxoplasma is Toxoplasma gondii.

7. The use as claimed in claim 4, wherein the Sarcocystis is Sarcocystis neurona causing equine protozoal encephalomyelitis.

8. The use as claimed in claim 4, wherein the neospora is neospora caninum causing neosporosis in cattle or dogs.

9. The use of claim 4, wherein the Toxoplasma is Toxoplasma gondii.

10. Use according to claim 1, wherein the triazineone compound is selected from toltrazuril, ponazuril and diclazuril.

11. The use as claimed in claim 1, wherein the triazineone compound is ponazuril.

12. The use according to claim 1, wherein the triazineone compound is administered in two or more repeated doses.

13. The use as claimed in claim 12, wherein the repeated administration is in an amount of 1.0 to 100 mg/Kg.

14. The use of claim 1, wherein the triazineone compound is administered until protective immunity is established in the animal.

15. Use according to claim 1, wherein triazineone is administered in an amount of 2.5 to 10 mg/Kg.

16. The use of claim 15, wherein the triazineone is toltrazuril or ponazuril.

17. The use according to claim 1, wherein the triazineone compound is administered in a single high dose of more than 10 mg/Kg.

18. The use of claim 8, wherein the triazineone compound is administered according to a repeated periodic dosing regimen until immune protection is established.

19. The use according to claim 1, wherein the triazineone compound is administered according to a dosing regimen of 2.5 to 10mg/Kg per day for 28 days.

20. Use according to claim 1, wherein the parasite caused disease is equine protozoal encephalomyelitis.

21. Use according to claim 20, wherein the triazineone used is diclazuril.

22. The use as claimed in claim 20 wherein the equine animal is a horse.