HK1043065A1 - Triazineone compounds for treating diseases due to sarcosystis, neospora and toxoplasma - Google Patents

Abstract

Disclosed herein is a method of treating therapeutically, or metaphylactically infected animals susceptible to, or infected animal suffering from parasitic neurologic or abortigenic diseases such as Sarcocystidiae or Toxoplasmosis that are treatable with triazineone compounds by administering thereto a pharmaceutically effective amount of the compound, including a single high dose therapeutic treatment.

Description

Triazinone compounds for the treatment of diseases caused by Sarcocystis, Neospora (Neospora) and Toxoplasma

Background

Technical Field

The present invention relates to triazineone compounds for the treatment of animals infected with parasites that cause abortive or neurological diseases. More particularly, the present invention relates to triazineone compounds useful in the treatment of parasitic protozoa of the order coccidia that cause abortigenic or neurological diseases.

Brief description of the Prior Art

Triazinone compounds, for example triazinediones such as diclazuril compounds, and triazinetriones such as toltrazuril compounds, have been used to treat and protect a wide variety of mammals, insects, and fish from a wide range of protozoan-caused diseases. See U.S. patent 4,933,341; 4,935,423, respectively; 5,114,938, respectively; 5,141,938, respectively; 5,188,832, respectively; 5,196,562, respectively; 5,256,631, and 5,464,837. Protozoa susceptible to these compounds include those parasites that infect the gut of birds, mammals and insects and manifest as diarrhea, wasting, nausea and vomiting. Generally, the mode of action of triazineones is to attack the intermediate parasitic stages found in the gut and gut wall cells, causing swelling of the endoplasmic reticulum, perinuclear space and mitochondria of the parasite. This intentionally interferes with nuclear resolving power, leaving the shinzots and microgamete precursors still small, forming only a few merozoites and microgametes, respectively. The net result is reported to be that these later stages of the parasite lose their ability to penetrate new mammalian cells, effectively preventing the parasite from replicating within the host.

Of particular concern here are certain protozoa that have been suspected of causing neurological and/or abortigenic diseases in animals since the 1970 s. Successful isolation and ex vivo culture of some of these protozoa has proven difficult. For example, successful isolation from brain or brain marrow fluid was not achieved until late 1980 s. It has been established that parasites infecting the brain may produce neurological diseases, whereas infected fetuses may produce abortigenic diseases, and there is a need for effective anti-protozoal drugs that penetrate the blood-brain barrier and the placental barrier without producing harmful side effects. Only a few drugs are able to pass through the blood-brain barrier or the placental barrier of animals. However, many of the art-recognized drugs that cross the blood-brain barrier and/or the placental barrier and thus are effective in treating parasitic infections of the brain have deleterious side effects that render them at great risk for use. Thus, to date, no effective drug has been approved that provides an effective treatment for such neurological diseases or abortive diseases. The following is a brief description of parasitic diseases.

Equine protozoan Encephalomyelitis (EPM) is a neurological disease of horses, and those young horses that are in tension (e.g. purebred racehorses and purebred performance horses) are particularly susceptible to disease and are thus a disease that has a significant monetary impact on the equine industry. EPM, which was first recognized as a disease in the 1970 s, was not cultured from horses suffering from EPM until 1991 and has been named as Sarcocystis neurona before. In 1997, a Neospora species (Neospora spp.) was isolated from the brain of an EPM-bearing horse and is now designated Neospora hugesi. Thus, it is now proposed that EPM may be caused by this newly recognized organism alone, by the neuronal Sarcocystis alone or by a combination of both. EPM most commonly causes asymmetric ataxia (ataxia), weakness, and spasticity. The disease may mimic almost any neurological condition. It may manifest as either an extremely acute or chronic symptom. Chronic disease forms are often insidious until late in the disease process and can lead to death. In the slightest case, the only clinical sign may be an insignificant limp lower limb or negligible respiratory noise. In the most severe cases, horses are unable to swallow or stand. It is now known that in the most severe cases parasites such as the neuronal Sarcocystis infect the brain and cause significant damage there. Clinical signs of EPM are caused by direct neuronal (brain and spinal cord) damage by the parasite as well as brain damage caused by inflammatory cell infiltration, edema, and neuronal death associated with merozoites and submucous variants in the Central Nervous System (CNS). Currently, there is no approved effective treatment or prevention available for EPM control. Human drugs trimethoprim-sulfonamide cocktails have been used. However, the treatment is expensive and requires numerous repeated doses.

Another coccidioidomycosis Toxoplasma gondii (Toxoplasma gondii) has been known for some time and was first isolated from the intestinal and muscle tissues of cats. The ultimate host for this parasite is the cat, which harbors the organism for extended periods of time, spreading oocysts to other animals, including cattle, sheep, pigs and humans. Infections in sheep, cattle and humans are associated with abortions and congenital acquired diseases, mainly affecting the central nervous system. Recently, it has also been associated with abortion and malformation of kittens born to infected female cats who are seronegative during pregnancy before infection. Non-feline hosts such as cattle, sheep, pigs and humans do not produce oocysts, but develop and may harbor muscle and brain invasion by tachyzoites and bradyzoites, producing clinical signs of disease-neurological symptoms and abortion and fetal defects. It has been reported that 60% of cats are seropositive for toxoplasma gondii (t.gondii). There is also no approved therapeutic or prophylactic agent for toxoplasmosis.

Yet another coccidioidomyomata caninum, a parasite of the order coccidioidomyomata, produces abortive disease in animals with neurological disease. It was first isolated from dogs in 1988. It has previously been mixed with the Toxoplasma gondii (T.gondii). This parasite-induced disease occurs most severely in placenta-infected puppies, characterized by progressive ascending paralysis of the puppies, particularly their hind limbs; polymyositis and hepatitis may also occur. Recently, it has been recognized that this disease is a major cause of neurologically-related limb deficits in miscarriages and newborn babies. Microscopic lesions of non-suppurative encephalitis and myocarditis in aborted fetuses can be seen in the brain, spinal cord, and heart. The ultimate host of neospora caninum (n. caninum) has recently been identified as a dog. Currently, there are no approved therapeutic or prophylactic agents for Neospora caninum, whether dog or cow, or Neospora hugesi, horse.

None of the references known in the art, including the references cited above, suggest or teach the use of triazineone compounds such as toltrazuril or toltrazuril sulfone (recently re-named Ponazuril ") in the treatment of animals infected with the Coccidia, particularly of the Coccidia, which cause abortigenic or neurological diseases, without causing intolerable side effects. Thus, there is a need for an improved and complete treatment for animals afflicted with parasitic diseases manifested as neurological diseases or abortigenic diseases.

Summary of The Invention

1. In accordance with the foregoing, the invention encompasses a method of therapeutically treating an animal suffering from a parasitic neurological or abortigenic disease that is susceptible to treatment 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 (s.neurona) the compound is not diclazuril or toltrazuril. The term "pharmaceutically effective amount" as used herein refers to an amount of triazineone administered in an amount sufficient to inhibit the growth in or ex vivo of a parasitic protozoan, typically an organism of the order coccidia, that is associated with neurological disease and/or abortion. The pharmaceutically effective amount is effective to control parasites in the infected tissue; as a result, the health of the animal is improved.

Furthermore, the invention also encompasses a method for the quasi-prophylactic treatment of animals infected with parasites that are susceptible to treatment with triazineone compounds and that cause neurological or abortigenic diseases. The quasi-prophylactic treatment comprises administering the triazineone compound to the animal using a quasi-prophylactically effective treatment regimen. The term "quasi-prophylactically effective treatment regimen" refers to the administration of a predetermined intermittent dose of a triazineone compound for an extended period of time until the animal is able to overcome the invasive parasite by, for example, developing a protective immune response or otherwise eliminating the parasite. Typically, the treatment regimen is one that is effective in controlling the parasite and preventing clinical signs of disease. The quasi-prophylactically effective dose may also be administered over a long period of up to 5 years or the life of the animal, especially if the parasite is difficult to control. For quasi-prophylactic treatments, preferred triazineone compounds are triazinetriones, including but not limited to toltrazuril, and ponazuril.

In addition, single high dose treatment of these animals is also contemplated by the present invention. This method comprises administering to a patient suffering from a parasitic neurological disease or abortigenic disease that is susceptible to treatment with triazineone a single high dose of a pharmaceutically effective amount of a triazineone compound. The term "single high dose" refers to a quantity that is administered only once. This amount is significantly higher than the dose amount employed for therapeutic or quasi-prophylactic treatment; the pathogenic parasite can be effectively controlled and thus does not cause deleterious effects such as toxicity. Thus, a single high dose of triazineone is greater than 10 mg/kg. This and other aspects of the present invention are described more fully below.

Detailed description of the invention

As set forth above, the present invention relates to a method of treating an infected or diseased animal suffering from a parasitic disease manifested as a neurological disease or abortigenic disease susceptible to treatment with a triazineone compound, comprising administering thereto a pharmaceutically effective amount of the compound. Illustrative, but non-limiting, examples of such animals can be horses, cattle, cats, dogs, pigs, sheep, birds, insects, and humans. The parasite infecting or causing the disease is of the order Sarcocystidae Coccidia which can manifest as a neurological disease or an abortigenic disease. Illustrative, but non-limiting, examples thereof may be selected from the group consisting of a Sarcocystis species (Sarcocystis spp.), a Neospora species (Neosporia spp.) and a Toxoplasma species (Toxoplasma spp.). The family sarcodictyidae is typically selected from the group consisting of the neuronal sarcocystis (s. neurona), n.hugesi, neospora caninum (n.caninum) and toxoplasma gondii (t.gondii). Protozoan infections or diseases include, but are not limited to, EPM, neosporosis, and toxoplasmosis.

In the practice of the present invention, treatment of parasitic infections or diseases caused by protozoans as described herein results in the relief of the symptoms of the neurological disease and abortigenic disease. Generally, symptoms include lameness, movement disorders, paralysis, abortion, neonatal weakness, and other related disorders. For therapeutic treatment, animals that have developed signs of the above disease are treated with the triazinone compound. Typically, the duration of treatment is from about 28 days to 90 days, preferably from about 28 days to 60 days. It is understood that for therapeutic treatment, the treatment regimen may be once a day, twice or more a day, every other day, or even weekly, depending on factors such as the severity of the disease and the type of parasite producing the disease. However, in some cases, the treatment regimen may continue indefinitely, sometimes for the remainder of the life of the animal. In the case of an animal infected with a more resistant parasite strain, the latter treatment would be required. However, the treatment can be extended for a longer period of time, if necessary, until the signs of the disease are eliminated. Preferably, the treatment is once a day for about 28 days.

For quasi-prophylactic treatment, infected animals are treated to protect them from the clinical manifestations of the disease. This treatment ultimately results in the animal gaining the ability to control the parasite, for example, by establishing an effective immune response that protects against future infections without further administration of the triazineone compound. According to the invention, the quasi-preventive activity refers to the use of the triazineone compound in a predetermined intermittent treatment regimen (quasi-prophylactically effective treatment regimen) to control protozoa that may have infected the animal since the previous treatment. Therefore, quasi-prophylactically effective treatment regimens are implemented in order to reduce their ability to cause disease, such as by killing them or reducing their number. In essence, the quasi-prophylactically effective treatment regimen may be carried out about once a month during the life of the animal, or until an intrinsic clearance mechanism, such as an effective immune response, has developed in the animal to protect it from future infections. The latter case may occur in 5 years or less. To be able to do this, the quasi-preventive treatment is based on the following recognition: when animals are infected with the protozoa described herein, they do not develop clinical signs such as neurological signs or miscarriage until a significant period of time has elapsed (e.g., 2-6 months after infection). In sharp contrast, intestinal protozoan infections manifest themselves soon after infection. According to the invention, the quasi-prophylactic treatment prevents the parasite from self-colonizing and causing clinical disease. The treatment regimen is carried out on an intermittent schedule of about once a month, once every two months, or once every two weeks at a dosage equivalent to about 1.0 to about 100mg/kg, preferably about 1.0 to about 25mg/kg, more preferably about 2.5 to about 10 mg/kg. High dosage ranges may be required in particularly resistant situations (e.g., when an animal is infected with a resistant strain). The required dose level and duration of treatment are within the knowledge of one of ordinary skill in the art. A preferred treatment regimen for horses with EPM or cattle with neosporosis is triazinetrione at about 1.0-25 mg/kg, more preferably about 2.5-10 mg/kg every 28 days.

For single high dose treatments, triazineone is administered in a pharmaceutically effective amount of above 10mg/kg and up to about 100 mg/kg. It is a unique feature of the present invention that the compounds of the present invention may be non-toxic so that they can be administered at high dosage levels. The advantages of high dose administration are attributed to the fact that: repeated dosing is not required and there are some triazineone compounds which cause harmful side effects when administered at very high dosage levels. Particularly preferred is Ponazuril (Ponazuril), which has been found to be both safe and effective when administered at doses up to 100mg/kg body weight.

Without being bound by any one particular theory of the invention, it is believed that the unexpected success of the treatments described herein is a result of the ability of the triazineone compound to cross the blood-brain barrier or the placental barrier. It is believed that the compounds of the present invention readily cross the blood-brain barrier and also penetrate the placenta and kill protozoa in the brain and cerebral spinal fluid/cord in situ. It has further been found that this class of compounds is non-toxic and non-mutagenic even at the high doses required for the single high dose treatment regimen described herein.

Heretofore, there has been no cost-effective, easily administered drug that can be used to effectively manage and protect against these diseases without producing unacceptable side effects such as toxicity or mutagenicity in animals. The following is a description of specific triazineone compounds, but is not limited to Toltrazuril (Toltrazuril). This disclosure and the claimed patent also covers other triazineone compounds that can be used in the manner of toltrazuril compounds. The toltrazuril compounds which may be used herein have the formula (I):in the formula R¹Represents an alkyl halideA thio group, a haloalkylsulfinyl group or a haloalkylsulfonyl group,

R²represents hydrogen, alkyl, alkoxy, alkoxyalkyl, alkylmercapto, halogen, haloalkyl or sulfamoyl which may also be substituted, such as dialkylsulfamoyl,

R³and R⁴Which may be identical or different, represent hydrogen, alkyl, alkenyl or alkynyl, X is O or S, and the physiologically acceptable salts thereof.

Furthermore, it has been found that, in particular, the following compounds of the formula Ia:in the formula R^IRepresents an alkyl halide (C)₁-C₄) Thio group, alkyl halide (C)₁-C₄) Sulfinyl or alkyl halide (C)₁-C₄) A sulfonyl group, a carboxyl group,

R^IIrepresents hydrogen, alkyl (C)₁-C₄) Alkyl, alkyl (C)₁-C₄) Oxy, halogen, alkyl (C)₁-C₄) Siloxane (C)₁-C₄) Alkyl, alkyl (C)₁-C₄) Mercapto, dioxane (C)₁-C₄) Aminosulfonyl or haloalkane (C)₁-C₄) A base, and

R^IIIand R^IVMay be the same or different and represents hydrogen, alkyl (C)₁-C₄) Alkyl or alkenyl (C)₁-C₄) And X is O or S.

Finally, it has been found that

(a) 1- (4-phenoxyphenyl) -1, 3, 5-triazines of formula (I) are compounds of formula (II)In the formula R¹、R²、R³And X is as defined above, with a substituted carbonyl isocyanate of the formula (III)In the formula R⁵Represents a halogen atom, an alkaneOxy or aryloxy, and substituted 1, 3, 5-triazine derivatives of the formula (IV) formed during this procedureIn the formula R¹、R²、R³And X is as defined above, optionally isolated and optionally reacted with a compound of formula (V)

A-Z (V) wherein A represents alkyl, alkenyl or alkynyl, and

z represents halogen, obtained in the reaction; or

(b) 1- (4-phenoxyphenyl) -1, 3, 5-triazine derivatives of formula (I) are compounds of formula (II) wherein R is¹、R²、R³And X have the meaning given above, with a di (chlorocarbonyl) amine of the formula (VI)In the formula R⁶Represents alkyl, optionally obtained when reacted in the presence of an acid acceptor; or

(c) To obtain compounds of the formula (I), in which the substituents R²、R³And R⁴And X has the meaning given above, and R¹Represents haloalkylsulfinyl or haloalkylsulfonyl, or a salt thereof, a compound of the formula (VII)In the formula R²、R³And R⁴Has the same meaning as above, and

R^1′represents a haloalkylthio group, with an appropriate amount of a suitable oxidizing agent.

If N- [ 3-chloro-4- (4 '-trifluoromethylthiophenoxy) phenyl ] -N' -methylurea and chlorocarbonyl isocyanate are used in variant (a), the course of the reaction can be represented by the following equation

If N- [ 3-ethoxy-4- (4' -trifluoromethylthiophenoxy) phenyl ] thiourea and N-methyldi (chlorocarbonyl) are used in variant (b)) Amine as starting material, the reaction progress can be represented by the following equation

A compound of the general formula (I) [ wherein R is₁X ═ O ] can be oxidized to haloalkylsulfinyl or haloalkylsulfonyl derivatives according to process variant (c). If hydrogen peroxide is used as the oxidizing agent, the progress of the reaction can be expressed by the following equation

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

In formulae I, II, IV, V and VI, R³、R⁴The alkenyl groups defined under A are preferably straight-chain or branched alkenyl groups having 2 to 6, in particular 2 to 4, carbon atoms. Examples which may be mentioned are vinyl, propen-1-yl and buten-3-yl which may also be substituted.

In formulae I, II, IV, V and VII, R³、R⁴The alkynyl group as defined in A is preferably a straight-chain or branched alkynyl group having 2 to 6, particularly 2 to 4, carbon atoms. Examples which may be mentioned are ethynyl, propyn-1-yl and butyn-3-yl which may also be substituted.

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

In the formulae I, II, III, IV, V and VII, R²、R⁵Or in ZThe halogen of (A) is preferably fluorine, chlorine, bromine and iodine, especially chlorine and bromine.

In the formulae I, II, IV and VII, R¹The haloalkylthio group as defined in (1) is preferably a haloalkylthio group 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, chlorodifluoromethylthio, bromomethylthio, 2, 2, 2-trifluoroethylthio and pentafluoroethylthio.

In formulae I, II and IV, R¹The haloalkanesulfinyl group as defined in (1) is preferably a haloalkanesulfinyl group having 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 trifluoromethylsulfinyl, chlorodifluoromethylsulfinyl, bromomethylsulfinyl, 2, 2, 2-trifluoroethylsulfinyl and pentafluoroethylsulfinyl.

In formulae I, II and IV, R¹The haloalkanesulfonyl group as defined in (1) is preferably a haloalkanesulfonyl group having 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 trifluoromethylsulfonyl, chlorodifluoromethylsulfonyl, bromomethylsulfonyl, 2, 2, 2-trifluoroethylsulfonyl and pentafluoroethylsulfonyl.

In formulae I, II and IV, the aminosulfonyl group, which may also be substituted, as defined in R2 is preferably one of the following groups:

SO₂NH₂，SO₂NH—CH₃，SO₂N(CH₃)₂，

SO₂NH—C₂H₅，SO₂—N(C₂H₅)₂，

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

In formula III, aryloxy R⁵Phenoxy group is preferred.

The substituted ureas or thioureas of formula II used as starting materials are mostly not known before, but they can be prepared easily by methods known per se, i.e. (a) either by reacting a substituted 4-aminophenyl ether with the corresponding substituted isocyanate or isothiocyanate in an inert solvent at a temperature between 0 ℃ and 100 ℃ or by reversing the order; (b) reacting ammonia or substituted amine with corresponding substituted isocyanato or 4-isothiocyanatodiphenyl ether under the same conditions; or (c) condensation of a substituted 4-hydroxyphenyl-urea or thiourea with an activated haloaromatic compound in an aprotic solvent such as dimethyl sulfoxide, dimethylformamide or hexamethyl phosphoric triamide in the presence of a base such as sodium hydride, potassium hydroxide, potassium carbonate z.a.m. at a temperature between 20 ℃ and 150 ℃.

When the amount of solvent is suitably selected, the reaction product generally crystallizes out as the solution cools. Alternative preparation documents of ureas from amines and isocyanates are: chemie (method of organic chemistry) (Houben-Weyl), fourth edition, volume 8, page 157-158.

The bis (chlorocarbonyl) amines of the formula (VI) which can be used according to the invention in the process (b) are known in some cases (compare synthesis 1970, pages 542-543). And if they are not known, they can be prepared in a similar manner from cyclic diacyl disulfides and chlorination 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 variant a) and with di (chlorocarbonyl) amines of the formula VI (process variant b) and also for the reaction of the 1, 3, 5-triazine derivatives of the formula IV with compounds of the formulae A to Z are organic solvents which are inert in these reactions.

These preferably 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 during the reaction may escape as a gas or may be bound by an organic or inorganic acid acceptor. The acid acceptor preferably comprises a tertiary organic base such as a trialkylamine, for example triethylamine, an N-heteromonocyclic or bicyclic aromatic amine, for example pyridine, a mono-or bicyclic azacycloalkylamine, for example diazabicyclononene, diazabicycloundecene and many other organic bases, 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 in the above-mentioned reaction stages can be varied within a wide range. Generally, the reaction is carried out at a temperature of from about 0 ℃ to about 150 ℃, preferably from about 20 ℃ to about 100 ℃.

In each of the above reaction stages, the reaction may be carried out under normal pressure or under high pressure. Generally, the reaction is carried out under normal pressure.

Possible oxidizing agents for the conversion of trifluoromethylthio compounds of general formula (I) (in which Y represents oxygen) according to process variant (c) into the corresponding sulfinyl or sulfonyl compounds are suitably: h₂O₂Glacial acetic acid; h₂O₂Acetic anhydride; h₂O₂Methanol; peracids such as m-chloroperbenzoic acid, and chromic acid; potassium permanganate; sodium periodate, ammonium cerium nitrate; and nitric acid.

The resulting compound may be converted into a corresponding addition salt, such as by reacting it with an inorganic or organic base.

In the practice of the present invention, the triazineone compounds can be formulated in any manner that facilitates administration to an animal. Preferred formulations for oral administration herein are in the form of suspensions, tablets, capsules, gels, pastes, boluses, or preparations in powder, granulated, or pelletized form. Preferred formulations for oral administration are in the form of a paste or feed supplement. Other modes of administration that may be employed include parenteral, topical, intramuscular, intramucosal or other routes known to those skilled in the art. Topical administration in the form of perfusion (pour-on) is also preferred.

Typically, these formulations employ pharmaceutically acceptable carriers and adjuvants. An example thereof may be a thickener selected from the group consisting of: carbopol, inorganic thickeners such as silicates, bentonites or colloidal silicas, 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 Carbopol, more specifically Carbopol974P is the best thickener for the preferred paste formulations herein. Also employed herein are preservatives selected from the group consisting of parabens, alcohols and aldehydes. These may be liquid, solid or gaseous materials that are otherwise inert or pharmaceutically acceptable and compatible with the active ingredient.

Surprisingly, the paste according to the invention is effective when used for treating parasites. More specifically, it is surprising that the ointments of the invention are effective in delivering the triazineones, especially toltrazuril and ponazuril, across the blood-brain barrier or the placental barrier and attack parasites that have invaded the brain or infect the fetus of a pregnant animal. For ease of understanding, a description is provided herein of one specific embodiment of the preferred pastes herein and how they are prepared. According to the present invention, a preferred ointment comprises an ultra-fine suspension of triazinetrione (e.g., ponazuril), propylene glycol, a thickening agent (e.g., Carbopol), a preservative (e.g., methylparaben and propylparaben), and water. It can be made by combining water (typically purified water) and propylene glycol, heating the composition to about 70 c and adding the preservative at this temperature. The resulting mixture is cooled to room temperature and then Carbopol, preferably in the form of Carbopol974P, is added. Finally the triazinetrione is added. After thorough mixing, the pH was adjusted to about 6.0 with sodium hydroxide. The most preferred ointment comprises 15% by weight of ponazuril, 20% by weight of propylene glycol, 0.5% by weight of Carbopol974P, 0.14% by weight of methylparaben, 0.02% by weight of propylparaben, 0.1% by weight of sodium hydroxide, and the balance purified water. Sweeteners including dextrose, sucrose, lactose, fructose, sorbitol, xylitol, artificial sweeteners, and molasses may be added to improve palatability. In addition, yeast or liver flavoring agents may also be added for the same purpose.

The following illustrative but non-limiting examples further illustrate the invention.

Examples

Example 1

Pharmacokinetic studies were performed in horses comparing blood levels of toltrazuril, ponazuril and toltrazuril sulfoxide at different times after single doses of toltrazuril. All horses received a single dose of 10mg/kg, administered orally as a suspension. Blood samples were drawn at treatment time (0) and 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hours post-treatment. The sampling results are shown in table 1. It was surprisingly noted that horses receiving toltrazuril showed relatively high levels of ponazuril in their sera. In addition, significant levels of toltrazuril vanadium are found in its bloodstream. This suggests that ponazuril alone produces acceptable blood levels that are expected to pass through the blood-brain barrier, a feature required to treat neurological diseases such as those caused by Sarcocystis neurona (S.neurona), Toxoplasma gondii (T.gondii), neospora caninum (N.caninum) and N.heugesi. TABLE 1 Matt Totrazuril Single dose pharmacokinetics

D	The compounds tested	Blood concentration, mg/l
								0	0.25	0.5	1	2	4
		A	The mixture of trazuril toltrazuril sulfoxide ponazuril		0.027＜0.010.010	0.7730.0770.089	2.8630.0700.088							4.5110.1590.171	3.1190.1420.110
B	Totrazuril trazuril sulfone Ponacazuril								0.061＜0.01＜0.01	0.3930.0250.029	2.6170.0470.036	4.2960.0830.040	6.8200.1570.050
		C	Toltrazuril sulfoxide ponazuril		0.061＜0.01＜0.01	0.5600.0240.013	3.2860.0410.019							5.7880.0970.026	9.0790.2180.032
D	The mixture of trazuril toltrazuril sulfoxide ponazuril								0.017＜0.01＜0.01	0.2950.0270.011	3.2860.0390.021	2.1650.0580.024	3.3280.1000.029
		E	Toltrazuril sulfoxide ponazuril		＜0.01＜0.01＜0.01	0.039＜0.01＜0.01	1.1460.0210.017							3.1750.0640.015	8.4100.1940.044
F	Toltrazuril sulfoxide ponazuril								0.110＜0.01＜0.01	0.4280.0260.012	1.7410.044＜0.01	---	8.1440.1830.041

D	The compounds tested	Blood concentration, mg/l
							6	12	24	48	72
		A	ToltrazurilToltrazuril sulfoxide ponazuril	5.1490.1670.108	5.0660.2300.170	6.4340.4070.324						7.6070.7321.622	6.6530.5921.933
B	Toltrazuril sulfoxide ponazuril						11.4740.3200.131	11.6700.4510.254	11.6900.5660.255	6.6770.4540.831	5.0580.3460.880
		C	Toltrazuril sulfoxide ponazuril	14.2020.2800.061	13.7510.4360.135	---						9.7680.4770.540	7.6330.3770.642
D	Toltrazuril sulfoxide ponazuril						3.8160.1330.030	10.5440.6681.851	7.2360.4610.315	8.2340.7490.986	---
		E	Toltrazuril sulfoxide ponazuril	11.3350.2590.074	12.0320.4300.268	8.6940.4810.231						6.8690.7410.501	---
F	Toltrazuril sulfoxide ponazuril						10.9660.2450.061	6.6600.4530.725	10.2240.6330.192	7.0960.6420.532	---

Example 2

Ponacularine, 1-methyl-3- [ 4-p- (trifluoromethylsulfonyl) phenoxy ] m-tolyl-s-triazine-2, 4, 6- (1H, 3H, 5H) trione, a representative triazinetrione, is formulated into a paste formulation for administration to horses. The ingredients listed in table 2 were used to prepare the formulations as follows.

TABLE 2 Ponazurima cream compositions

Composition (I)	Theoretical quantity	Actual amount% (by weight)
			Ponacularia micro-fine powder propylene glycol Carbopol974P methylparaben, NF propylparaben, NF sodium hydroxide, NF purified water	22.5kg30.0kg0.750kg0.210kg0.030kg0.150kg96.365kg	15.020.00.50.140.020.1064.24

NF ═ united states pharmacopeia

These formulations were formulated as follows using procedures (A) and (B). The first process (a) comprises: 1) mixing part of water with propylene glycol; 2) adding antiseptic (methyl p-hydroxybenzoate and propyl p-hydroxybenzoate); 3) slowly adding Carbopol974P until a uniform suspension is prepared; 4) adding ponazuril in the form of a fine powder; 5) adding sodium hydroxide to bring the suspension to a pH of about 6.0; and 6) adding the remaining water to a sufficient volume. The final suspension is in the form of a paste that can be delivered to the horse's mouth.

The second process (B) comprises: 1) mixing part of water with propylene glycol; 2) heating to 70 deg.C; 3) adding preservatives (methyl and propyl parabens) while the solution is maintained at 70 ℃; 4) cooling the solution to room temperature; 5) slowly adding Carbopol974P until a uniform suspension is prepared; 6) adding ponazuril in the form of a fine powder; 7) adding sodium hydroxide to bring the suspension to a pH of about 6.0; and 8) adding the remaining water to a sufficient volume. The final suspension is also in the form of a paste which can be delivered to the horse's mouth.

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

Example 3

Ponacularine, 1-methyl-3- [ 4-p- (trifluoromethylsulfonyl) phenoxy ] m-tolyl-s-triazine-2, 4, 6- (1H, 3H, 5H) trione, a representative triazinetrione, was tested for its ability to treat horses that had shown signs of equine protozoal Encephalomyelitis (EPM). The compound was formulated as an ointment with ponazuril as 15% active ingredient (a.i.) as in example 1. It is administered once a day to horses already diagnosed with EPM at a dose rate of between 2.5 and 10mg/kg during 28 days.

Naturally occurring clinical cases of EPM are well characterized by signs and laboratory diagnoses. The diagnosis used to incorporate EPM-positive horses into this assay is as follows: evidence of asymmetric neurological deficits determined using standardized neurological investigations, including radiography, EPM cues; positive Western blotches of neuronal sarcocystis IgG; red blood cell count below 500 cells/mL; CSF index-Total protein < 90, IgG index > 0.3, AQ quotient < 2.2.

It is further required that these horses do not suffer from diseases other than EPM. Therefore, they must comply with the following criteria: negative CSF (< 1: 4) to EHV-1; normal serum values of vitamin E (2.0. mu.g/mL); absence of seizure disorders; there was no behavioral disorder.

The diagnosed horses were randomly grouped. Group 1 horses received the paste formulation daily at a dose rate of 5mg/kg, while group 2 horses received the paste formulation daily at a dose rate of 10 mg/kg. Treatment doses were based on body weight. To determine that treatment was indeed effective, the horses were evaluated over a period of 90 days (about 60 days after treatment interruption). Responses to treatment were scored using the following system:

1) 0-complete success, clinically normal, CSF negative; 2)1, detecting the defect just enough, and ensuring that the gait is normal; 3) 2-defects are easily detected and aggravated by backing, turning, rocking, jaw waist pressure and neck stretching; 4)3, the defects are very obvious when people walk, face twist, waist press or neck stretch; 5) 4-spontaneous toddlers, atrophi36396and falls; 6)5, the user cannot get up when lying on a slant. An improvement of- (1) units in the fraction is considered a significant improvement.

The results of this study are listed in table 3. All horses (100%) in the 10mg/kg group treated for 28 days showed significant improvement in clinical score by day 90 after the start of the ponazuril treatment (day 0). 8 of 9 horses treated at 5mg/kg dose (88.9%) showed acceptable improvement. When the scores of each group are all summed up per treatment day, a total score is obtained. The overall score improvement exhibited by both group 1 and group 2 horses was approximately comparable. Thus, it was concluded that ponazuril was effective for the active treatment of EPM in horses, both at a dose rate of 5mg/kg and 10 mg/kg.

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

Horse mark	5mg/kg dose			1mg/kg dose
							Day 0	Day 28	Day 90	Day 0	Day 28	Day 90
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
I							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 score	17	13	6	19	15							4

Example 4

To determine the scope of protection afforded by ponazuril, ex vivo experiments were performed. The following parasite strains were evaluated for their sensitivity to this compound: SN3 strain of neuronal sarcocystis; strain SF1 of sarcocystis falcate; RH strain of murine toxoplasma; and NC-1 strain of neospora caninum. Ponacril was tested at 2 concentrations (1. mu.g/mL and 10. mu.g/mL).

Bovine nasal turbinate (BT) cells were used to perform all ex vivo studies. Cells at 25cm²Flasks were grown to confluence with RMPI1640 medium supplemented with 10% by volume Fetal Bovine Serum (FBS), 100 units penicillin (G/mL), 100mg streptomycin/mL and 5X 10^-2mM 2-mercaptoethanol. After the resulting cells were synthesized, the cells were maintained in the same medium with less FBS (2% by volume). Cell cultures were incubated at 37 ℃ in a humidified atmosphere containing 5% carbon dioxide and 95% air.

For parasite growth, a BT cell monolayer was infected with the parasite and examined using a reverse microscope for the development of foci (cytopathic effect "CPE") and the presence of numerous intracellular merozoites. Once the foci, or the presence of many intracellular parasites, was observed, the monolayer was rubbed with the tip of a 5mL pipette and 1-3 drops of merozoite-containing fluid were transferred to 2 fresh BT cell flasks. Merozoites of neuronal Sarcocystis and Sarcocystis sickle were passed in this manner every 5-10 days, while tachyzoites of Toxoplasma gondii and neospora caninum were passed in this manner every 3-4 days.

The test used to determine the effectiveness of ponazuril is the droplet monolayer disruption test (MMDA). This assay was used to determine whether the parasite or compound was toxic to BT cells. Flat bottom 96-well microtiter plates were incubated with BT cells,the resulting monolayers were used to determine the effect of toltrazuril and ponazuril on merozoite production (spot formation) as measured by CPE. Each monolayer was incubated with the parasite (either Sarcocystis neurona or Sarcocystis sickle at a count of 50,000/well, Toxoplasma gondii at a level of 10,000/well, neospora caninum at a level of 20,000/well). All wells were incubated with test compound 2 hours after infection. Untreated and uninfected single wells were used as parasite controls, while uninfected agent treated BT cells were used as toxicity controls. Each treatment was examined in 6 replicates. Each well was monitored daily by visual inspection and the experiment was stopped when 90-100% of the non-treated merozoite-infected cells were lysed (90-100% CPE). All wells of the test plate were rinsed with Phosphate Buffered Saline (PBS), fixed with 100% methanol for 5 minutes, and then stained with crystal violet solution. Areas where merozoites induce destruction or BT cells die due to toxicity do not take up crystal violet. The amount of crystal violet bound was quantified using an ELISA plate reader and the data used to determine the concentration of Ponazuril that inhibited 50% of the destruction (inhibitory concentration)₅₀Or IC₅₀). Data showing inhibition are listed in table 4. It is noted that ponazuril as low as 1. mu.g/mL provides 100% inhibition of cell destruction by neospora caninum, Toxoplasma gondii and Sarcocystis sickle. Whereas 10. mu.g/mL of ponazuril was required to produce 100% inhibition of cell destruction by Sarcocystis neurona. This suggests that triazinones such as toltrazuril and ponazuril are effective in treating diseases caused by the coccidia family, known to be associated with neurological and abortigenic disease symptoms, including those caused by Sarcocystis neurona, neospora caninum, N.hugesi and Toxoplasma gondii. Furthermore, ponazuril is non-toxic to BT cells.

Table 4 ex vivo test data on ponazuril

Biological organisms	Percent inhibition of cell destruction
					0.1μg/mL	1μg/mL	5.0μg/mL	10μ/mL
	Neuron sarcocystis	0	40	90					100
Sarcocystis falciparum					61	100	100	100
Biological organisms						Percent inhibition of cell destruction
	0.001μg/mL	0.01μg/mL	0.1μg/mL	1.0μg/mL
					Neosporozoon caninum NC-1	3	13	100	100
Bow-shaped body of mouse	11	16	100	100

Example 5

This experiment was performed to determine whether triazinones such as toltrazuril can pass the blood-brain barrier. Normal horses were divided into three groups of three horses. Group 1 horses received toltrazuril orally administered as a 5% suspension at a dose level of 2.5 mg/kg. Group 2 horses received toltrazuril orally administered as a 5% suspension at a dosage level of 5.0 mg/kg. Group 3 horses received toltrazuril orally administered as a 5% suspension at a dose level of 7.5 mg/kg. The administration is repeated once a day for 10 days. Blood samples were drawn at 48 hours, 96 hours and 240 hours and serum concentrations of toltrazuril, toltrazuril sulfoxide and ponazuril were determined. 10 days after the start of the treatment (day 10), a sample of the cerebral spinal fluid of each horse was taken, and the concentration of toltrazuril, toltrazuril sulfoxide and ponazuril in these samples was determined again. The concentrations of toltrazuril, toltrazuril sulfoxide and ponazuril in serum and in brain marrow are reported in tables 5a and 5 b. The concentration of ponazuril in blood and brain marrow following treatment of horses with toltrazuril is significant in that the concentration of ponazuril in brain marrow following treatment of horses with toltrazuril is substantially equivalent to the concentration of toltrazuril itself. This is evidence that both toltrazuril and ponazuril can effectively cross the blood-brain barrier and that ponazuril crosses this barrier more effectively than toltrazuril. For those skilled in the art, these data indicate that triazineones are also effective in crossing the placental barrier.

TABLE 5a drug levels in horses following repeated dosing of toltrazuril

Product of horse mark	10 days dose (mg/kg)	Toltrazuril level			Medullary fluid on day 10
						48 hours	96 hours	240 hours
		123456789	2.52.52.55.05.05.07.57.57.5	4.494.011.67.289.189.26 not tested 9.9010.46					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
Mean average	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 levels in horses following repeated toltrazuril administration

Product of horse mark	10 days dose (mg/kg)	Ponacuril levels			Medullary fluid on day 10
						48 hours	96 hours	240 hours
		123456789	2.52.52.55.05.05.07.57.57.5	0.290.243.700.480.630.486.350.780.52					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
Mean average	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 illustration, 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 (25)

1. A method of therapeutically treating a diseased animal suffering from a parasitic neurological disease or abortigenic disease that is susceptible to treatment 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 (Sarcocystis neurona) the compound is not diclazuril or toltrazuril.

2. The method of claim 1, wherein the parasitic disease is caused by coccidia.

3. The method of claim 2, wherein the coccidia is a member of the family Sarcocystidae.

4. The method of claim 3 wherein the member of the family Sarcocystidae is selected from the group consisting of Sarcocystis, neospora and Toxoplasma.

5. The method of claim 4 wherein the Sarcocystis is selected from the group consisting of Sarcocystis species, neospora is selected from the group consisting of neospora species, and Toxoplasma is selected from the group consisting of Toxoplasma species.

6. The method of claim 5 wherein the Sarcocystis species is Sarcocystis neurona, the Neospora species is Neospora caninum or Neosporia hugesi, and the Toxoplasma species is Toxoplasma gondii.

7. The method of claim 4 wherein the Sarcocystis is Sarcocystis neurona causing equine protozoan Encephalomyelitis (EPM).

8. The method of claim 4 wherein the neospora is neospora caninum causing neosporosis in cattle or dogs.

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

10. A method for the quasi-prophylactic treatment of animals infected with a parasite that is causative of a neurological disease or an abortigenic disease susceptible to treatment with a triazineone compound, comprising administering thereto a quasi-prophylactically effective amount of said triazineone.

11. The method of claim 1 or claim 10, wherein the triazineone compound is selected from the group consisting of toltrazuril, ponazuril and diclazuril.

12. The method of claim 1 or claim 10, wherein the triazineone compound is ponazuril.

13. The method of claim 1 or claim 10, wherein the triazineone compound is administered in two or more repeated doses.

14. The method of claim 13, wherein the repeat dose is administered in an amount of 1.0 to 100 mg/kg.

15. The method of claim 10, wherein the triazineone compound is administered until the animal develops protective immunity.

16. The method of claim 1 or claim 10, wherein the triazineone is administered in an amount of between 2.5mg/kg and 10 mg/kg.

17. The method of claim 16, wherein the triazineone is toltrazuril or ponazuril.

18. The method of claim 1, wherein the triazineone compound is administered in a single high dose of greater than 10 mg/kg.

19. The method of claim 8, wherein the triazineone compound is administered in a repeated periodic dosing regimen until immunological protection is established.

20. The method of claim 1, wherein the triazineone compound is administered on a treatment regimen of 2.5mg/kg to 10mg/kg per day for 28 days.

21. A therapeutic composition comprising (a) a pharmaceutically effective amount of a triazineone compound intended for treating a diseased animal to which it is susceptible, (b) a carrier, and (c) optionally, adjuvants.

22. The composition of claim 21 in the form of a paste.

23. A method of treating equine protozoan Encephalomyelitis (EPM) comprising administering to a horse suspected of having EPM a therapeutically effective amount of one or more triazinediones.

24. The method of claim 23, wherein the triazinedione is diclazuril.

25. The method of claim 23, wherein the horse is a horse.