JP2002532552A - Triazine-on compounds for treating diseases caused by sarcocystis, neospora and toxoplasma - Google Patents

Abstract

  (57) [Summary] Disclosed herein are infected animals or those afflicted with a neurological or abortive parasite disease such as Sarcocystidiae or toxoplasmosis that can be treated with a triazine-on compound. A method of treating an infected animal therapeutically or post-prophylactically by administering to it a pharmaceutically effective amount of the compound, including a single high-dose therapeutic treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] BACKGROUND OF THE INVENTION Field of the Invention: The present invention relates to triazine ON (triazineone) compounds to treat animals infected with parasites that cause abortion or neurological disease. More particularly, the present invention relates to triazine-one compounds useful in treating protozoan parasites such as coccidium that cause abortion or neurological disease. BRIEF DESCRIPTION OF THE PRIOR ART: Triazine diones, such as dicrazuryl compounds, and triazine triones such as triazine triones, such as toltrazuril compounds, treat and protect a variety of mammals, insects and fish from diseases caused by a wide range of protozoa. It is used for U.S. Patents: 4,933,341; 4,935,423: 5,114,938: 5,141,938:
5,188,832: See 5,196,562: 5,256,631 and 5,464,837. Protozoa susceptible to these compounds include parasites that infect the intestines of birds, mammals and insects and manifest as diarrhea, wasting, nausea and vomiting. In general, the mode of action of triazineone is to attack the intermediate parasite stage present in gastric and intestinal parietal cells and to swell the endoplasmic reticulum, nuclear envelope and mitochondria of the parasite. This intentionally interferes with the ability of fission, leaving the schizont and microgerm cells small, forming only a few merozoites and microgametes, respectively. The end result is the loss of the ability of the parasite to invade new mammalian cells at these latter stages, and has been reported to effectively stop parasite replication in the host.

Of particular interest here are certain protozoa that have been suspected of causing neurological and / or abortion disorders in animals since the 1970s. Successful isolation and in vitro culture of some of these protozoa has proven difficult. For example, successful isolation from brain or cerebrospinal fluid was not achieved until the late 1980s. Once a neurological disorder can be caused by a parasite that infects the brain, and an abortion disorder can be caused by a parasite that infects the fetus, it produces harmful side effects. There is a need for effective antiprotozoal drugs that can cross the blood-brain and placental barriers without the need. Very few drugs can cross the blood-brain or placenta barrier of an animal. However, many drugs known in the art that can cross the blood-brain barrier and / or placenta barrier to effectively treat brain parasite infections cannot use them without great risk So have harmful side effects. Thus, to date, there are no approved and effective agents that provide effective treatment of such neurological or miscarriage diseases. The following is a brief description of the parasitic disease.

[0003] Equine protozoal myeloencephalitis (EPM) is a neurological disease in horses that prefers stressed young horses (eg, purebred racehorses and purebred runners), and is therefore It is a disease with significant financial consequences. EPM, first recognized as a disease in the 1970s, was not cultured from horses with EPM and was given the name Sarcocystis neurona until 1991. In 1997, a species of Neospora, now called Neospora hugesi,
Isolated from the brain of a horse with PM. Thus, at present, EPM is only due to this newly recognized organism, only to Sarcosis tis neuronas or to these two.
It is suggested that it may be caused by a combination of the two. EPM is
Usually results in asymmetric coordination impairment (ataxia), weakness and spasticity. The disease can appear to be almost any neurological condition. It can occur as a super-acute or chronic condition. Chronic forms are usually latent at onset, difficult to diagnose until the end of the course of the disease, and can result in death. At its worst, the only clinical signs may be unclear pelvic limb locomotion or slight breathing noise. At the heaviest, horses cannot swallow or stand. Current,
In the heaviest cases, parasites, such as Sarcocystis neurona, are known to infect the brain and cause significant damage thereto. Clinical signs of EPM include direct neuronal (brain and spinal cord) damage by parasites and infiltration of inflammatory cells, edema,
And brain damage caused by neuronal death associated with merozoites and the melonto phase in the central nervous system (CNS). Currently, there is no approved and effective treatment or prophylaxis to control EPM. The human drug trimethoprim-sulfonamide combination has been used. However, treatment is expensive and requires a very large number of repeated doses.

[0004] Another coccidian parasite, Toxoplasma gondii
Has been known for quite some time and was first isolated from cat intestine and muscle tissue. The indigenous host of the parasite is a cat, which can retain the organism for long periods of time while transmitting oocysts to other animals, including cattle, sheep, pigs and humans. Sheep, bovine and human infections are associated with miscarriage and congenital acquired diseases that primarily affect the central nervous system. It has also recently been linked to miscarriages and malformations in kittens born to infected kittens that were seronegative prior to infection during pregnancy. Non-feline hosts, such as cattle, sheep, pigs and humans, do not produce, but develop, oocysts, leading to clinical signs of disease-neurological symptoms and tachyzoites causing abortion with fetal defects. May suffer from muscle and brain invasion by bradyzoites. 60% of cats are reported to be serologically positive for Toxoplasma gondii. Again, there is no approved treatment or prevention for toxoplasmosis.

[0005] Yet another coccidian parasite, Neospora caninum
Causes both neurological and abortion disorders in animals. It was first isolated from dogs in 1988. Previously confused with Toxoplasma gondii.
The disease caused by this parasite is most severe in transplacentally infected puppies and is characterized by progressive ascending paralysis of the puppies, especially in the hind limbs; polymyositis and hepatitis can also occur. The disease has more recently been identified as a major cause of abortions and neurologically related limb defects in newborn calves. Microscopic damage of non-suppurative encephalitis and myocarditis in aborted fetus
It can be found in the spinal cord and heart. The endemic host of Neospora caninum has recently been identified as a dog. At this time, there is no approved treatment or prevention of either canine or bovine Neospora caninum or equine Neospora fugueshi.

[0006] References known in the art, including those cited above, are those of coccidium or, more specifically, those of the sarcosis family that cause abortion or neurological disease without unacceptable side effects. It does not suggest or teach the use of triazine-one compounds such as toltrazuril or toltrazurilsulfone (recently renamed "ponazuryl") in treating infected animals. Thus, there is a need for improved safe treatment of animals afflicted with parasitic diseases that manifest as neurological or abortive diseases.

[0007] In accordance summary above matters invention, the present invention provides a pharmaceutically effective amount of the triazine-one compound comprising administering to an animal, the compound in neurological or capable of treating A method of therapeutically treating an affected animal suffering from abortion parasitic disease,
Provided that the compound is not diclazuril or toltrazuril when the disease is sarcocystis neurona.

The term “pharmaceutically effective amount”, as used herein, refers to the amount of triazineone that is administered when the amount of triazineone administered is such that the protozoan parasite that causes neurological disease and / or miscarriage, typically Mean high enough to inhibit in vivo or in vitro growth. A pharmaceutically effective amount controls the parasites in the infected tissue; as a result, the animal's health is restored.

Further, the invention encompasses a method of post-prophylactically treating an animal that is infected with a parasite that can cause a neurological or miscarriage disease treatable with a triazine-on compound. . Post-prophylactic treatment comprises administering a triazine-on compound to an animal using a post-prophylactically effective regimen. The term "post-prophylactically effective regimen" refers to the use of triazine-on compounds for a long period of time until the animal overcomes the invading parasite, for example, by developing a protective immune response or otherwise removing the parasite. It means administering a scheduled intermittent dose. Typically, the regimen is such as to effectively control the parasite and prevent clinical signs of the disease. Post-prophylactically effective doses can also be administered for long periods of up to 5 years or throughout the life of the animal, especially when parasites are difficult to control. For post-prophylactic treatment, a preferred triazineone compound is triazinetrione, including but not limited to toltrazuril and ponazuryl.

[0010] The invention also encompasses a single high dose treatment of the animal. The method comprises administering to a diseased animal suffering from a neurological or abortive parasitic disease capable of being treated with a triazine on a single high dose of a pharmaceutically effective amount of the triazine on compound. Comprising. The term "single high dose" means a single dose. This amount is significantly higher than the dose used in therapeutic or post-prophylactic treatment; it is effective in controlling the disease-causing parasites, and as such does not have adverse effects such as toxicity. Thus, a single high dose of triazineone is 10 mg
More than / Kg. This and other aspects of the invention are described more fully below.

[0011] As detailed description above invention, the present invention provides pharmaceutically effective amount of the triazine-one compound comprising administering to an animal, neurology that can be treated with the compound A method for treating an infected or diseased animal suffering from a parasitic disease manifesting as a target or miscarriage disease. Illustrative but non-limiting examples of animals can be equines, bovines, felines, dogs, pigs, sheep, birds, insects and humans. The parasite that infects or causes disease is coccidiomycetes of the family Sarcocystis, which can manifest as a neurological or abortive disease. Illustrative but non-limiting examples can be selected from the group consisting of Sarcocystis species, Neospora species and Toxoplasma species. The Sarcocystis family is typically selected from the group consisting of Sarcocystis neurona, Neospora fugesi, Neospora caninum and Toxoplasma gondii. Protozoal infections or diseases include, but are not limited to, EPM, Neospora disease and toxoplasmosis.

In the practice of the present invention, treatment of a parasitic infection or disease caused by a protozoa described herein results in a reduction in the symptoms of neurological and abortion disease. Generally, these symptoms include locomotion, ataxia, paralysis, miscarriage, frail newborns and other related disorders. In therapeutic treatment, animals already suffering from the above symptoms of the disease are treated with the triazine-on compound. Typically, the duration of treatment is about 28-90 days, preferably about 28-60 days. For therapeutic treatment, the treatment regimen is
Depending on factors such as the severity of the disease and the type of parasite causing the disease,
It is understood that it can be twice daily, twice or more, once every other day, or even once a week. However, in some cases, treatment regimens can continue indefinitely, sometimes for the rest of the animal's life. The latter treatment is required in case of infection of the animal with a more resistant strain of the parasite. However, treatment can be extended for longer periods as necessary until the symptoms of the disease are eliminated. The preferred treatment is once daily for about 28 days.

In post-prophylactic treatment, infected animals are treated to protect them from developing clinical symptoms of the disease. This treatment ultimately gives the animal the ability to control parasites without the need for further administration of triazine-on compounds, for example, by establishing an effective immune response to provide protection against future infections. Bring that. The post-prophylactic activity according to the present invention is directed to triazine-on in a scheduled intermittent treatment regimen (post-prophylactically effective regimen) to control protozoa that may have infected an animal since previous treatments. Refers to the use of compounds. Thus, post-prophylactically effective regimens are administered to reduce their ability to cause disease, for example, by killing them or reducing their number. In essence, a post-prophylactically effective regimen is designed to provide a unique clearance mechanism, such as an effective immune response, in the animal about once a month, to protect the animal from its lifetime or from future infection. It can be administered until it occurs. The latter can occur within 5 years or less. As will be appreciated, post-prophylactic treatment is that when animals become infected with the protozoa described herein, they will take a significant amount of time (eg, 2-6 months after infection).
Based on the recognition that it does not show clinical signs such as neurological signs or miscarriage. In contrast, intestinal protozoal infections manifest immediately after infection. According to the present invention, post-prophylactic treatment prevents parasites from colonizing and causing clinical disease. The treatment regimen is
Between about 1.0-100 mg / Kg, preferably about 1.0-25 mg / Kg, more preferably about 2.5-10 mg
By an intermittent schedule about once a month, once every two months or once every two weeks at a dose equal to / Kg. Higher ranges are needed, especially if resistant (eg, if the animal is infected with a resistant strain). The required dose level and duration of treatment are
It is within the purview of those skilled in the art. A preferred treatment regimen for horses with EPM or cattle with Neospora disease is about 1.0-25 mg / Kg, with a more preferred range being about 2.5-10 mg / Kg triazinetrione every 28 days.

In a single high dose treatment, the triazineone is administered in a pharmaceutically effective amount that is greater than 10 mg / Kg and up to about 100 mg / Kg. It is another feature of the present invention that the compounds of the present invention can be non-toxic, and thus they can be administered at high dose levels. The advantages of high dose administration are that multiple doses are not required and that some triazineone compounds can cause deleterious side effects when administered at very high dose levels. Particularly preferred is ponazril, which has been found to be safe and effective at doses as high as 100 mg / kg body weight.

Without being bound by any particular theory of the present invention, the surprising success of the treatments described herein is due to triazine-on compounds being able to cross the blood-brain or placenta barrier. Conceivable. It is believed that the compounds of the invention can easily cross the blood-brain barrier and also enter the placenta, and kill protozoa in situ in the brain and cerebrospinal fluid / spinal cord. In addition, this class of compounds has been found to be non-toxic and non-mutagenic even at the high doses required for the single high-dose treatment regimes described herein. No cost-effective, easily-administered drugs were available to effectively treat and prevent these diseases without causing unacceptable side effects such as toxicity or mutagenicity. The following is a description of a triazine-on compound that is specific to, but not limited to, toltrazuril compounds. The present disclosure and claimed invention also encompass other useful triazine-on compounds, such as toltrazuril compounds. The toltrazuril compounds useful in the present invention are of formula (1):

[0016]

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Wherein R ¹ represents halogenoalkylthio, halogenoalkyl-sulfinyl or halogenoalkylsulfonyl; R ² is hydrogen, alkyl, alkoxy, alkoxyalkyl, alkylmercapto,
Halogen, represents halogenoalkyl or sulfamoyl group which is optionally substituted, such as dialkyl sulfamoyl, R ³ and R ⁴ can be the same or different and represent hydrogen, alkyl, alkenyl or alkynyl, and X is O or S, and their physiologically acceptable salts.

Furthermore, in particular, the following compounds of formula Ia and their physiologically acceptable salts:
It has been found that it may be useful in the present invention:

[0019]

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Where R^IIs a halogenoalkyl (C₁-C_Four) -Thio, halogenoalkyl (C₁-C_Four) -Sulfinyl
Or halogenoalkyl (C₁-C_Four) -Sulfonyl, R^IIIs hydrogen, alkyl (C₁-C_Four), Alkoxy (C₁-C_Four), Halogen, alkoxy (C₁-C _Four ) Alkyl (C₁-C_Four), Alkyl (C₁-C_Four) -Mercapto, dialkyl (C₁-C_FourAminos
Ruphonyl or halogenoalkyl (C₁-C_Four) And R^IIIAnd R^IVCan be the same or different and hydrogen, alkyl (C₁-C_Four)Also
Is alkenyl (C_Two-C_Four) And X is O or S. Finally, (a) 1- (4-phenoxy-phenyl) -1,3,5-triazine of formula I is converted to a compound of formula II

[0021]

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Wherein R ¹ , R ² , R ³ and X have the meanings indicated above, with a compound of formula III

[0023]

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Wherein R ⁵ represents a halogen atom, an alkoxy group or an aryloxy group, which is obtained by reacting with a substituted carbonyl isocyanate of formula IV

[0025]

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Wherein R ¹ , R ² , R ³ and X have the meanings indicated above, optionally a substituted 1,3,5-triazine derivative of the formula AZ (V) wherein A represents an alkyl, alkenyl or alkynyl, and Z represents a halogen; or (b) 1- (4-phenoxy-phenyl) of the general formula I The -1,3,5-triazine derivative is a compound of formula II wherein R ¹ , R ² , R ³ and X have the meanings indicated above, optionally in the presence of an acid acceptor.
Of formula VI

[0027]

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Wherein R ⁶ represents alkyl and is obtained by reacting with bis- (chlorocarbonyl) -amine, or (c) the substituents R ² , R ³ and R ⁴ and X are as defined above To obtain a compound of formula I having the meaning and R ¹ represents halogenoalkylsulfinyl or halogenoalkylsulfonyl,

[0029]

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Wherein R ² , R ³ and R ⁴ have the meanings indicated above, and R ¹ ′ has been found to react a compound of the formula (I) with a suitable amount of a suitable oxidizing agent. Was issued.

The course of the reaction when N- [3-chloro-4- (4′-trifluoromethylthio-phenoxy) -phenyl] -N′-methyl-urea and chlorocarbonyl isocyanate are used in process variant (a) Can be represented by the following equation:

[0032]

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N- [3-ethoxy-4- (4′-trifluoromethylthio-phenoxy) -phenyl] -thiourea and N-methyl-bis- (chlorocarbonyl) amine are the starting materials in process variant (b) When used, the course of the reaction can be described by the following equation:

[0034]

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The compounds of general formula I obtained according to process variant (a) or (b), wherein R ₁ = halogenoalkylthio and X = O, are the corresponding halogenoalkylsulfinyl or halogenoalkylsulfonyl derivatives according to process variant (c) Can be oxidized. When hydrogen peroxide is used as the oxidant, the course of the reaction can be described by the following equation:

[0036]

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In formulas I, II, IV, V, VI and VII, the alkyl as defined for R ² , R ³ , R ⁴ , R ⁶ or A is preferably from 1 to 6, especially from 1 to 4, Straight or branched alkyl having three carbon atoms. Examples which may be mentioned are optionally substituted methyl, ethyl, n- and i-propyl and n, i- and t-butyl.

In formulas I, II, IV, V and VII, alkenyl as defined for R ³ , R ⁴ or A is preferably straight-chain having 2 to 6, especially 2 to 4, carbon atoms Or a branched alkenyl. Examples which may be mentioned are optionally substituted ethenyl, propen-1-yl, propen-2-yl and buten-3-yl.

In formulas I, II, IV, V and VII, alkynyl as defined for R ³ , R ⁴ or A is preferably a straight-chain having 2 to 6, especially 2 to 4, carbon atoms Or a branched alkynyl. Examples which may be mentioned are optionally substituted ethynyl, propyn-1-yl, propyn-2-yl and butyn-3-yl.

In formulas I, II, III, IV and VII, the alkoxy as defined for R ² or R ⁵ is preferably straight-chain or branched having 1 to 6, especially 1 to 4, carbon atoms. It is a branched alkoxy. Examples which may be mentioned are optionally substituted methoxy, ethoxy, n- and i-propoxy and n- and i-butoxy.

In formulas I, II, III, IV, V and VII, the halogen as defined for R ² , R ⁵ or Z is preferably fluorine, chlorine, bromine and iodine, especially chlorine and bromine.

In formulas I, II, IV and VII, the halogenoalkylthio as defined for R ¹ is preferably 1-4, especially 1 or 2, carbon atoms, preferably 1-5, especially 1-5 ~ 3
Halogenoalkylthio having the same or different halogen atoms, preferably halogen, fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylthio, chloro-di-fluoromethylthio, bromomethylthio, 2,2,2-trifluoroethylthio and pentafluoroethylthio.

In the formulas I, II and IV, the halogenoalkylsulphinyl as defined for R ¹ is preferably 1 to 4, in particular 1 or 2 carbon atoms and preferably 1 to 5, especially 1 to 5, Halogenoalkylsulfinyl having three identical or different halogen atoms, the halogen atoms being preferably fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulfuryl, chloro-
Di-fluoromethylsulfuryl, bromomethylsulfinyl, 2,2,2-trifluoroethylsulfinyl and pentafluoroethylsulfinyl.

In the formulas I, II and IV, the halogenoalkylsulphonyl as defined for R ¹ is preferably 1 to 4, especially 1 or 2, carbon atoms and preferably 1 to 5, especially
Halogenoalkylsulfonyl having 1 to 3 same or different halogen atoms, the halogen atoms being preferably fluorine, chlorine and bromine, especially fluorine and chlorine. Examples which may be mentioned are trifluoromethylsulphonyl, chloro-di-fluoromethylsulphonyl, bromomethyl-sulphonyl, 2,2,2-trifluoroethylsulphonyl and pentafluoroethylsulphonyl.

In formulas I, II and IV, the optionally substituted sulfamoyl as defined for R ² is preferably any of the following groups:

[0046]

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In formula III, the aryloxy as defined for R ⁵ is preferably a monocyclic carbocyclic aryloxy or a bicyclic carbocyclic aryloxy, especially phenoxy.

In formula III, aryloxy R ⁵ is preferably phenoxy.

Most of the substituted ureas or thioureas of the formula II used as starting materials are not heretofore known, but (a) substituted 4-aminodiphenyl ethers with the corresponding substituted isocyanates or isothiocyanates (B) ammonia or a substituted amine and the corresponding substituted isocyanate or 4-isothiocyanate, either by reacting in an inert solvent at a temperature between Reacting the -diphenyl ethers with each other under the same conditions, or (c) reacting the substituted 4-hydroxyphenyl-urea or -thiourea with sodium hydride, potassium hydroxide, carbonate at a temperature between 20 ° C. and 150 ° C. Dimethyl sulfoxide, dimethyl formamide or hexam in the presence of a base such as potassium, zam It can be prepared them easily by originally known methods by subjecting to condensation reaction with activated halogeno aromatic compound in an aprotic solvent such as Chirurin triamide.

With an appropriate choice of the amount of solvent, the reaction products generally crystallize when the solution is cooled. Another reference for the preparation of urea from amines and isocyanates is: Meth
oden der Org.Chemie (Methods of Organic Chemistry) (Houben-Weyl), Chapter IV
Edition, Vol. VIII, pp. 157-158.

Some of the bis- (chlorocarbonyl) -amines of the general formula VI which can be used according to the invention in process (b) are already known (Synthesis 1970, 542-54)
Compare the literature on page 3), and if they are not already known, they can be prepared analogously from cyclic diacyl disulfides and chlorinations in inert organic solvents, preferably in carbon tetrachloride.

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

These include, in addition to pyridine, preferably aromatic hydrocarbons such as benzene, toluene and xylene, halogenated aromatic hydrocarbons such as chlorobenzene and dichlorobenzene, and such as tetrahydrofuran and dioxane. Ethers are included.

Hydrochloric acid, which may be formed during the reaction, can either escape as a gas or be bound by an organic or inorganic acid acceptor. Preferably, the acid acceptor
Tertiary organic bases such as trialkylamines, e.g. triethylamine, N-heteromono- or bicyclic aromatic amines such as pyridine, mono- or bicyclics such as diazabicyclononene, diazabicycloundecene Aza-cycloalkylamines of the formula and numerous others, or inorganic bases such as alkyl metal carbonates, oxides or hydroxides or alkyl earth metal carbonates, oxides or hydroxides are included .

The reaction temperature of the above reaction steps can be varied within a wide range. Generally, the reaction is carried out between about 0 ° C and about 150 ° C, preferably between about 20 ° C and about 100 ° C.

In the above reaction step, the reaction can be carried out under standard pressure or high pressure. Generally, the reactions are performed under standard pressure.

The possible oxidizing agents for the conversion of the trifluoromethylthio compound of the general formula 1 in which Y represents oxygen into the corresponding sulfinyl or sulfonyl compound by process variant (c) are suitably: H ₂ O ₂ / Glacial acetic acid; H ₂ O ₂ / acetic anhydride; H ₂ O ₂ / methanol; peracids and chromic acids such as m-chloroperbenzoic acid; potassium permanganate; sodium periodate, cerise ammonium nitrate; is there.

The resulting compound can be converted to the corresponding addition salt, for example, by reacting it with an inorganic or organic base.

In practicing the present invention, the triazine-on compounds can be formulated in any conventional manner for administration to animals. Formulations suitable for oral administration which are preferred according to the invention can be those in the form of suspensions, tablets, capsules, gels, pastas, boluses or powders, granules or pellets. Preferred orally administered formulations are in the form of pasta or feed additives. Other modes of administration that can be used 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.

[0060] Typically, pharmaceutically acceptable carriers and auxiliaries are used in the formulation. Examples can be thickeners selected from the group consisting of inorganic thickeners such as carbopol, silicates, bentonite or colloidal silica and organic thickeners such as fatty alcohols or fatty acid esters, and The wetting agent is selected from the group consisting of polyethylene glycol and sodium lauryl sulfate, Carbopol,
More specifically, Carbopol 974P is the most preferred thickener for the preferred pasta formulations in the present invention. In the present invention, a preservative selected from the group consisting of parabens, alcohols and aldehydes can also be used. These can be liquid, solid or gaseous substances, which are otherwise inert or medically acceptable and compatible with the active ingredient.

Unexpectedly, the pasta agents of the present invention are effective when used to treat parasites. More particularly, the pasta agents of the present invention may be used as triazines to cross the blood-brain or placenta barrier and attack parasites that have already entered the brain or infect fetuses of pregnant animals. It is surprising that it is effective in delivering toltrazril and ponazuril, especially. For convenience, certain embodiments of the pasta agents preferred in the present invention and a description of how to make them are provided herein. Preferred pasta agents of the present invention contain a micronized suspension of triazinetrione (eg, ponazuryl), a thickening agent such as propylene glycol, carbopol, preservatives such as methyl and propylparaben, and water. It can be made by combining water, typically purified water, and propylene glycol, heating the combination to about 70 ° C., and adding a preservative at this temperature. The resulting mixture is cooled to room temperature, after which time Carbopol, preferably in the form of Carbopol 974P, is added. Finally, the triazinetrione is added. After mixing thoroughly, adjust the pH to about 6.0 with sodium hydroxide. The most preferred pasta is 15% w / w ponazuryl, 20% w / w propylene glycol, 0.5% w / w carbopol 974P, 0.14% w / w methyl paraben, 0.02%
Contains w / w propylparaben, 0.1% w / w sodium hydroxide, the balance being purified water. Sweeteners, including dextrose, sucrose, lactose, fructose, sorbitol, xylitol, artificial sweeteners and molasses, can be added to enhance palatability. In addition, yeast or liver flavors can be added for the same purpose.

The present invention is further described by the following illustrative but non-limiting examples.

[0063] EXAMPLES Example 1: toltrazuril, a pharmacokinetic study comparing the blood level of Ponazuriru and Toruto lazuli sulfoxide was conducted in horses at various times after toltrazuril single dose. All horses received a single dose of 10 mg / kg, which was administered orally as a suspension. Blood samples were collected at the time of treatment (0) and 0.25, 0.5, 1, 2 after treatment.
, 4, 6, 12, 24, 48 and 72 hours. Table 1 shows the results of the sampling. It was surprising to see that horses fed toltrazuril exhibited relatively high levels of ponazuril in their serum. In addition, significant levels of toltrazuril sulfoxide were present in the bloodstream. It can cross the blood-brain barrier, a property required to treat neurological diseases such as those caused by Sarcocystis neurona, Toxoplasma gondii, Neospora caninum and Neospora fugesi. It was an indication that ponazuril alone produced predictable acceptable blood levels.

[0064]

[Table 1]

[0065]

[Table 2]

Example 2: Representative triazinetrione ponazuryl, 1-methyl-3- [4-p- (trifluoromethyl) sulfonylphenoxy] -m-tolyl] -s-triazine-2,4,6 ( (1H, 3H, 5H) -trione was formulated into a pasta for administration to horses. The components described in Table 2 were used in preparing a formulation as follows.

[0067]

[Table 3]

A formulation was prepared using methods (A) and (B) as follows. First method (A)
: 1) mixing part of the water with propylene glycol; 2) adding preservatives (methyl and propyl paraben); 3) slowly adding Carbopol 974P until a homogeneous suspension is prepared. 4) adding ponazuril in micronized form; 5) adding sodium hydroxide to bring the suspension to a pH of about 6.0;
And 6) adding an appropriate amount of the remaining water to the volume. The final suspension is in the form of a pasta, which can be delivered orally to the horse.

The second method (B) is: 1) mixing part of the water with propylene glycol; 2) 7
Heating to 0 ° C; 3) adding preservatives (methyl and propylparaben) while keeping the solution at 70 ° C; 4) cooling the solution to room temperature; 5) until a homogeneous suspension is prepared. Slowly adding Carbopol 974P; 6) adding ponazuril in micronized form; 7) adding sodium hydroxide to bring the suspension to a pH of about 6.0; and 8) removing remaining water. Adding an appropriate amount up to the volume.
The final suspension is likewise in the form of a pasta, which can be delivered orally to the horse.

The resulting pasta was administered to horses and found to be tasty and well-accepted. Example 3: Representative triazinetrione ponazuryl, 1-methyl-3- [4-p- (trifluoromethyl) sulfonylphenoxy] -m-tolyl] -s-triazine-2,4,6 (1H, 3H , 5H) -Trione was tested for its ability to treat horses already showing signs of equine parasite myeloencephalitis (EPM). This compound was formulated into a pasta formulation as described in Example 1 (Example 2?) Using ponazuryl as the 15% active ingredient (ai). It was administered to horses already diagnosed with EPM at a dose rate between 2.5 mg / Kg and 10 mg / Kg once a day for 28 days.

The naturally occurring clinical cases of EPM have been well characterized by signalling and laboratory diagnosis. The diagnoses used to include EPM-positive horses in this study were as follows: confirmed asymmetric nerves as determined by standardized neurological examination, including radiography, showing EPM Western blot positive for sarcocystis neurona IgG; red blood cell count less than 500 cells / mL; CSF index-total protein <90, IgG index> 0.3, AQ index <2.2.

An additional requirement was that these horses did not suffer from diseases other than EPM. Therefore, they had to meet the following criteria: CSF negative for EHV-1 (<1: 4); normal serum levels of vitamin E (2.0 μg / mL); absence of seizure disorders; lack.

The horses diagnosed were randomly assigned to groups. Group 1 horses receive the pasta formulation at a dose rate of 5 mg / Kg daily, while group 2 horses receive 1 pasta formulation.
They were given daily at a dose rate of 0 mg / Kg. Treatment doses were based on body weight. Horses were evaluated over a period of 90 days (about 60 days after discontinuation of treatment) to determine that the treatment was indeed effective. Response to treatment was scored using the following system: 1) 0 = Completely successful-clinically normal with negative CSF; 2) 1 = Barely detectable defect in normal gait; 3) 2 = Easily Defects detected and worsened by riding on the back, changing direction, moving back and forth, right and left, jaw-loin pressure and neck extension; 4) 3 = walking, face rotation, waist pressure Or a very noticeable defect when stretching the neck; 5) 4 = staggering, stumbling and spontaneous falling; 6
5) I can't get up and lie down. An improvement of one unit in the score was considered a significant improvement.

The results of this study are shown in Table 3. All of the 10 mg / Kg groups treated for 28 days (100
%) Of horses showed a significant improvement in clinical score by 90 days after the start of treatment with ponazril (day 0). Eight of the nine (88.9%) horses treated with the 5 mg / Kg dose showed acceptable improvement. Adding all of the scores for each group on each treatment day gives a total score. The improvement in total scores shown by both Group 1 and Group 2 horses is about equal. Therefore, it is concluded that either 5 mg / Kg or 10 mg / Kg ponazuril is effective for active treatment of EPM in horses.

[0075]

[Table 4]

Example 4 An in vitro test was performed to determine the extent of protection afforded by ponazuril. The following strains of parasites were evaluated for their susceptibility to this compound: strain SN3 of Sarcocystis neuronala; strain SF1 of Sarcocystis falcatula; strain RH of Toxoplasma gondii; and Neospora caninum. NC-1 strain. Ponazuril is prepared in two concentrations (1 μg / mL and 10 μg / m
L).

[0077] Bovine turbinate (BT) cells were used for all in vitro studies. 25c in RMPI 1640 medium supplemented with 10% v / v fetal bovine serum (FBS), 100 units of penicillin (G / mL), 100 mg of streptomycin / mL and 5 × 10 ⁻² mM 2-mercaptoethanol
were grown to confluency the cells in flasks m ^2. After cell fusion is obtained, FB
Cells were maintained in the same medium with reduced S (2% v / v). Cell cultures were incubated at 37 ° C. in a humidified atmosphere containing 5% carbon dioxide and 95% air.

For parasite growth, BT cell monolayers were infected with the parasite and examined with an inverted microscope for the development of damage (cytopathic effect, “CPE”) or the presence of numerous extracellular merozoites. Once damage is observed or a large number of extracellular parasites are present,
Strip the monolayer with the tip of a 5 mL pipette and pour the solution containing 1-3 drops of merozoite into fresh B
Transferred to two flasks of T cells. In this way, merozoites of Sarcocystis neurona and Sarcocystis farkatura were passaged every 5-10 days, while tachyzoites of Toxoplasma gondii and Neospora caninum were passaged every 3-4 days.

The assay used to determine the effect of ponazuril was a microtiter monolayer
Disruption assay (MMDA). This assay allows parasites or compounds to
It was used to determine if it was toxic. Flat bottom 96-hole microtiter
Plates are inoculated with BT cells, and the resulting monolayer is used for CPE (plaque formation).
Effects of toltrazuril and ponazuril on merozoite production as measured
It was determined. Parasites in the monolayer (50,000 cells / well with sarcosis
Rona or Sarcocystis farkatura, Toxo at a level of 10,000 / well
Plasma Gonzi, and Neospora caninum at 20,000 / well)
Was. All wells were inoculated with test compound 2 hours after infection. Unprocessed and uninfected
Monolayer wells are used as a parasite control and uninfected drug-treated BT cells.
Vesicles were used as a toxicity control. Each treatment was examined in six replicates. Each well daily
Monitor visually, if 90-100% of untreated merozoite-infected cells are lysed
The (90-100% CPE) assay was stopped. Fill all wells of the plate with a phosphate buffered diet
Rinse with saline (PBS), fix in 100% methanol for 5 minutes, then clean them
Stained in Listal Violet solution. Destruction caused by merozoites or
Areas of BT cell death due to toxicity do not incorporate crystal violet. Chris
Use an ELISA plate reader to quantify tall violet uptake.
Using these data, the concentration of ponazuryl that inhibits destruction by 50% (inhibition concentration ₅₀ Or IC₅₀)It was determined. The data showing the inhibition is shown in Table 4. Small amount of about 1μg / mL
Ponazurils from Neospora caninum, Toxoplasma gondii and Sarkosh
Provides 100% inhibition of cell destruction caused by Stis falcatula,
On the other hand, 10 μg / mL of ponazuril caused 10
It can be seen that it was required to produce 0% inhibition. This is the sarcosystem
Niss Neurona, Neospora caninum, Neospora hughesi and Toxopra
Neurological and miscarriage disorders, including those caused by Zuma Gondii
Treatment of diseases caused by coccidium known to be associated with a climate group
Triazineones such as toltrazuril and ponazuril are effective for
And In addition, ponazril was not toxic to BT cells.

[0080]

[Table 5]

Example 5 This experiment was performed to determine if a triazineone, such as toltrazuril, could cross the blood-brain barrier. 3 normal horses per group
The horses were divided into three groups. Group 1 horses at a dose level of 2.5 mg / Kg
Toltrazuril was given orally as a 5% suspension. Group 2 horses received toltrazuril administered orally as a 5% suspension at a dose level of 5.0 mg / Kg. Group 3 horses received toltrazuril administered orally as a 5% suspension at a dose level of 7.5 mg / Kg. Dosing was repeated daily for 10 days. Blood samples are drawn at 48, 96 and 240 hours, tortrazuril in serum,
The concentrations of toltrazuril sulfoxide and ponazuril were measured. 10 after the start of the procedure
On day (day 10), samples of cerebrospinal fluid were removed from each horse and the concentrations of toltrazuril, toltrazurilsulfoxide, and ponazuril in these samples were measured again. The concentrations of toltrazuril, toltrazuril sulfoxide and ponazuril in serum and cerebrospinal fluid are reported in Tables 5a and 5b. The concentration of ponazuril in blood and cerebrospinal fluid after treatment of horses with toltrazuril was significantly higher, since the concentration of ponazuril in cerebrospinal fluid after treatment of horses with toltrazuril was essentially equal to that of toltrazuril itself. Was. This is evidence that toltrazuril and ponazuril both cross the blood-brain barrier effectively, and that ponazuril crosses this barrier more effectively than toltrazuril. This data suggests to those skilled in the art that triazineone can also effectively cross the placental barrier.

[0082]

[Table 6]

[0083]

[Table 7]

The present invention has been described in detail in the foregoing for purposes of illustration, but such details are merely for that purpose and unless otherwise limited by the claims. It is to be understood that modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07D 251/34 C07D 251/34 B // C07D 251/38 251/38 A (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW

Claims (25)

[Claims] 1. A disease suffering from a neurological or miscarriage parasite disease treatable with a pharmaceutically effective amount of the compound comprising administering to the animal a triazine-on compound. A method of treating an animal therapeutically, provided that the compound is not diclazuril or toltrazuril if the disease is Sarcocystis neurona. 2. The method of claim 1, wherein the parasitic disease is caused by coccidiosis. 3. The method of claim 2, wherein the coccidium is a member of the sarcosis family. 4. A sarcocystis family member comprising Sarcocystis (Sarcoctis).
4. The method of claim 3, wherein the method is selected from the group consisting of ystis), Neospora, and Toxoplasma. 5. The method of claim 4, wherein sarcocystis is selected from the group consisting of sarcosis sp., Neospora is selected from the group consisting of Neospora sp., And toxoplasma is selected from the group consisting of toxoplasma species. 6. The sarcocystis species is Sarcocystis neurona, the Neospora species is Neospora caninum or Neospora hugesi, and the Toxoplasma species is Toxoplasma gondii. 6. The method of claim 5, wherein 7. The method of claim 4, wherein the sarcocystis is sarcocystis neurona, which causes equine parasite myeloencephalitis. 8. The method of claim 4, wherein the Neospora is Bovine or Canine Neospora disease causing Neospora caninum. 9. The method according to claim 4, wherein the toxoplasma is Toxoplasma gondii.
the method of. 10. A causative agent of a neurological or miscarriage disease treatable with a triazine-on which comprises administering to the animal a post-prophylactically effective regimen of the triazine-on compound. Post-prophylactic (me
taphylactically). 11. The method of claim 1 wherein the triazineone compound is toltrazuril or ponazuryl
11. The method of claim 1 or claim 10, wherein the method is selected from the group consisting of: nazuril) and diclazuril. 12. The method according to claim 1, wherein the triazineone compound is ponazuryl. 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 repeated dose is administered in an amount between 1.0 and 100 mg / Kg. 15. The method of claim 10, wherein the triazine-on 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 between 2.5 mg / Kg and 10 mg / Kg. 17. The method of claim 16, wherein the triazineone is toltrazuril or ponazuryl. 18. The method of claim 1, wherein the triazineone compound is administered in a single high dose of more than 10 mg / Kg. 19. The method of claim 8, wherein the triazine-on compound is administered in a repeated cycling dose regimen until immunological protection is established. 20. The method of claim 1, wherein the triazineone compound is administered daily on a regimen of 2.5 mg / Kg to 10 mg / Kg for a period of 28 days. 21. (a) a pharmaceutically effective amount of a triazine-on compound for treating a susceptible susceptible animal, (b) a carrier and (c)
A) therapeutic compositions optionally comprising auxiliaries. 22. The composition of claim 18, which is in the form of a pasta. 23. A method of treating an equine protozoal myeloencephalitis (EPM) comprising administering a therapeutically effective amount of one or more triazinedione to an equine suspected of having EPM. . 24. The method of claim 23, wherein the triazinedione is diclazuril. 25. The method of claim 23, wherein the equine is a horse.