HK1059894B - Praziquantel compounds for treating diseases due to sarcocystis, neospora, toxoplasma and isospora - Google Patents

Description

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

Technical Field

The present invention relates to a praziquantel composition for treating abortion (aboriginic) or a neurological disease in an animal caused by a parasitic infection, and more particularly, to a praziquantel composition for treating abortion or a neurological disease in a mammal caused by a parasite such as coccidia.

Brief introduction to the prior art

Several compounds have been used in the prior art for the prevention and treatment of various mammalian, insect and fish diseases caused by protozoal infections which may cause neurological and/or abortive diseases. Among them, triazinone (triazoneone) compounds have recently been used. Generally, protozoa that are sensitive to these compounds include parasites that infect the digestive tract of birds, mammals, and insects, causing symptoms of diarrhea, wasting disease, nausea, vomiting, and the like. More recently it has been found that triazinones can be used to treat parasites that cross the blood-brain barrier and cross the placental barrier. Parasites which are known to cross the blood brain barrier or placenta barrier are those which can cause coccidiosis, such as Toxoplasma gondii, Sarcocystis fascicularis, neospora caninum, neospora fradiae and swine isospora. Representative triazinones are triazindiones, such as diclazurils, and triazinetriones, such as toltrazuril (see U.S. Pat. Nos. 4,933,341, 4,935,423, 5,114,938, 5,141,938, 5,188,832, 5,196,562, 5,256,631 and 5,464,837).

Although other technically relevant compounds, including praziquantel (2-acyl-4-oxo-hexahydro-4H-pyrazine [2, 1-a [ isoquinoline derivatives ], have been disclosed for use in the treatment of parasitic diseases, the disclosure does not teach or suggest any use of such compounds for the treatment of parasitic diseases that can cross the blood brain barrier or placenta barrier.

U.S. Pat. No. 5,663,155 discloses praziquantel for use in the control of diseases caused by parasitic infections such as malaria, trypanosomiasis, Leishmania, schistosomiasis and elephantiasis. The patent also suggests praziquantel that may be used to treat parasitic infections confined to blood, lymph and tissues. Toxoplasma and Sarcocystis infections in muscle tissue and the intestinal tract are specifically mentioned. There is no mention of the control of toxoplasma, sarcocystis, neospora or isospora into the brain of adult mammals or infection of fetal central nervous tissue through the placental barrier, resulting in abortion or hypoplasia of the newborn.

Generally, the mode of action of triazinones is to attack parasites found in the gut and gut wall cell vector stages, causing swelling of the endoplasmic reticulum, the perinuclear cavity and the mitochondria of the parasite. This alleged disruption of nuclear fission capacity causes schizonts and microgametes to remain small, forming only a few merozoites and microgametes, respectively. The end result reported is a loss of the parasite's ability to penetrate new mammalian cells at a later stage, effectively preventing the parasite from replicating in the host.

As disclosed in U.S. Pat. No. 5,663,155 (supra), the mode of action of praziquantel is associated with interruption of the enzymatic process by the parasite, which involves the metabolism of purine derivatives and purine-like chemical structural compounds. Parasites are very sensitive to interference with these processes and therefore compositions specific for these processes can selectively eliminate parasitic infections. Praziquantel is administered to attack parasites infected in muscle tissue, intestinal tract or blood. There is no mention of treating parasites of the central nervous system (brain and spinal cord) or parasites that are able to cross the placental barrier.

Although the technical field of protozoa suspected to cause diseases of the nervous system and/or abortion in mammals has received attention since 1970, it has proven difficult to successfully isolate and culture these protozoa in vitro. For example, successful isolation of parasites from the brain or cerebrospinal fluid was not completed until the end of the 80 s. However, once it has been determined that neurological diseases are caused by brain-infected protozoa and that abortion is caused by protozoa infected with a fetus, there is a need for antiprotozoal drugs that can effectively cross the blood-brain barrier or placental barrier without deleterious side effects. Very few drugs can cross the blood brain barrier or placental barrier in mammals. Unfortunately, many drugs known in the art that can cross the blood brain barrier or placental barrier of mammals, and that are effective in treating parasitic infections in the brain, all produce deleterious side effects. There is a great risk associated with these drugs. Thus, to date, no effective drug has proven to be effective in treating such neurological and/or abortive diseases.

It is noted that the mode of action of praziquantel disclosed in U.S. Pat. No. 5,663,155 involves a wide range of action on enzymes affecting purine metabolism, and it is expected that the compound may also produce deleterious side effects if used to treat parasitic infections in the brain of mammals.

The following outlines parasitic diseases that can be treated with praziquantel. Equine protozoal Encephalomyelitis (EPM) is a neurological disease of horses, and young horses undergoing heavy burden are particularly susceptible to disease (e.g., elite racehorses and purebred performance horses), and thus, the disease has a great economic impact on the equine industry. It was initially recognized that EPM disease was not cultured in the seventies until 1991 from a horse with EPM and was named Sarcocystis neurona. In 1997, a neospora, now designated neospora textilis, was isolated from a horse brain with EPM. It is therefore now proposed that EPM may be caused only by this newly identified organism, or only by Sarcocystis indica, or by a combination of both organisms.

Another species of parasitic coccidia, toxoplasma gondii, has been known for some time for the first time to be isolated from the cat's intestinal and muscle tissues. The defined host for this parasite is a cat, which can carry the organism for long periods of time, distributing oocysts to other animals, such as cattle, sheep, pigs and humans. Infections in sheep, cattle and humans are associated with abortions and congenital diseases affecting the central nervous system. It has also been recently discovered that seronegative pregnant queens before infection are associated with post-infection abortion and malformation in newborn kittens. Non-feline hosts, such as cattle, sheep, pigs and humans do not produce oocysts, but can also develop disease through invasion of the muscle and brain by tachyzoites and bradyzoites, developing clinical symptoms of neurological disease and abortion with fetal defects. It has been reported that 60% of cats are seropositive for Toxoplasma gondii. Furthermore, there is currently no effective method for treating or preventing toxoplasmosis.

However, neospora caninum, another parasitic coccidia first isolated from dogs in 1988, can cause neurologic and abortive disease in animals. It was previously confused with Toxoplasma gondii. The disease caused by this parasite occurs mainly and severely in the placenta-infected puppies and is characterized by progressive, ascending paralysis, especially of the hind limbs. Polymyositis and hepatitis may also occur. It has recently been recognized that the disease is the major causative agent of abortion and is associated with neurological deficits that lead to newborn calf limb disability. Non-suppurative encephalitis and myocarditis in the brain, spinal cord and heart of the aborted fetus were observed under a microscope. The definitive host for neospora caninum was recently identified as dog. There is currently no satisfactory treatment or prevention of neospora caninum or neospora caninum in dogs or cattle or neospora caninum in horses.

In view of the above prior art, including the references cited above, no suggestion or guidance is given for the treatment of animals infected with coccidia, more specifically with sarcocystidae protozoa causing abortion or neurological diseases, with praziquantel without producing intolerable side effects. The present invention provides an improved safe treatment for animals suffering from parasitic infections, exhibiting neurological and abortive diseases.

Summary of The Invention

As noted above, the present invention includes a composition and a method for treating a parasitic neurologic or abortigenic disease in an animal that is susceptible to treatment with a pharmaceutically effective amount of praziquantel. The method comprises administering a pharmaceutically effective amount of praziquantel to the animal.

As used herein, the term "pharmaceutically effective amount" means an amount of praziquantel or a derivative thereof administered sufficient to inhibit parasite growth in vitro and in vivo without causing adverse effects in the administered animal. Representative of said parasitic protozoa are protozoa of the coccidia class causing neurological and/or abortive diseases. The effective amount of the drug controls parasites in the infected tissue, thereby improving the health of the animal. Furthermore, a pharmaceutically effective amount of praziquantel or a derivative thereof is capable of crossing the blood-brain barrier or placental barrier to treat a parasite infected with the central nervous system without causing adverse effects in the treated mammal.

In addition, the invention includes a method of remedial prophylactic (methacylicallyy) treatment of animals infected with parasites that cause abortion or neurological disease and are susceptible to praziquantel treatment. The therapy comprises administering praziquantel to the animal in an effective remedial prophylactic treatment regimen. The term "effective remedial prophylactic treatment regimen" means that the praziquantel compound or derivative thereof is administered at regular intervals over an extended period of time until the animal defeats the invading parasite, e.g., by generating a protective immune response or by eliminating the parasite. For example, effective control of parasites and prevention of clinical symptoms are representative means. The animal may also be administered the effective remedial prophylactic dose of the drug for a period of up to 5 years or for the life of the animal, particularly for those parasites that are difficult to control.

The invention also includes a method of treating an animal with a single bolus. The method comprises administering to the animal suffering from the parasitic neurologic or abortigenic disease a large dose of a pharmaceutically effective amount of a praziquantel compound or derivative thereof in a single dose. The term "single bolus" refers to an amount that controls the disease-causing parasite taken only once. This amount is much higher than the amount used for therapy or prophylaxis and is effective in controlling the parasites responsible for the disease, and also does not produce harmful effects, such as toxicity. Therefore, the one-time large dose of the praziquantel is more than 10 mg/Kg. This and other aspects of the invention will be described in more detail.

Detailed Description

As described above, the present invention relates to a composition or method for treating infections or suffering from parasitic neurological or abortive diseases in mammals that can be treated with praziquantel compounds or derivatives thereof. The method comprises administering to the mammal a composition comprising a pharmaceutically effective amount of praziquantel. By way of illustration, but not limitation, animals may be horses, cattle, cats, dogs, pigs, sheep, birds, insects, and humans. The infectious or pathogenic parasite is a coccidia of the family Sarcocystidae, which may cause a neurological or abortive disease. Thus, by way of illustration, but not limitation, examples are selected from the genera Sarcocystis, neospora and Toxoplasma. The coccidia of the family Sarcocystidae is typically selected from Sarcocystis fascicularis, neospora immitis, neospora caninum and Toxoplasma gondii. The protozoal infection or disease includes, but is not limited to, EPM, neosporosis, and toxoplasmosis.

In the practice of the present invention, treatment of parasitic infections or diseases caused by protozoa as described herein may alleviate the symptoms of neurological or abortive diseases. Typically, these symptoms include limb disability, ataxia, paralysis, abortion, newborn weakness and other related diseases. As a treatment, animals with symptoms of the above-mentioned diseases are administered a praziquantel compound. The treatment period is usually 28 to 90 days, preferably 28 to 60 days. Depending on the severity of the disease and the type of pathogenic parasite, the treatment regimen may be set to once daily, twice or more daily, or every other day, or even once weekly. In some cases, however, the duration of the treatment regimen is uncertain and sometimes requires the animal to be taken for its lifetime. The latter treatment regimen is required in the case of an animal infected with a strongly resistant strain of parasite. However, treatment may be extended as long as necessary until the condition is eliminated. The preferred treatment regimen is once daily for 28 days. The preferred dosage range for therapeutic administration is 1mg/Kg to 100mg/Kg, the preferred range is 2.5mg/Kg to 25 mg/Kg.

For remedial prophylactic treatment, infected animals are treated to protect them against the clinical symptoms of the disease. This treatment ultimately results in these animals gaining the ability to control parasites, such as establishing an effective immune response to combat further infection, without the need for further administration of praziquantel. This remedial prophylactic treatment, as well as the present invention, is the use of praziquantel compounds at scheduled intervals (an effective remedial prophylactic treatment regimen) to control the protozoa infecting an animal. Thus, the effective remedial prophylactic treatment regimen is by taking the drug to reduce the protozoan pathogenic ability, e.g., killing them, or reducing their numbers. In essence, the effective remedial preventive regimen can be administered about once a month, throughout the life of the animal or until an intrinsic clearance mechanism, such as an effective immune response, is developed to protect the animal from further infection. The latter case may occur within 5 years or less. As will be appreciated, remedial prophylactic treatment is based on the recognition that after a significant period of time (e.g., 2-6 months) has elapsed since the animal was infected with a protozoan as described herein, clinical symptoms, such as neurological symptoms or abortion, are not manifest. In contrast, intestinal protozoal infections show symptoms shortly after infection. According to the present invention, remedial prophylactic treatment can prevent the parasite from sticking in the body and causing clinical disease. The regimen is an intermittent schedule, i.e., about once a month, once every two months, or once every two weeks, at a dosage of about 1.0 to about 100mg/Kg, preferably at a dosage of about 1.0 to about 25mg/Kg, and more preferably at a dosage of about 2.5 to about 10 mg/Kg. High doses are used in cases where the protozoa are extremely resistant (e.g., animals are infected with resistant strains of protozoa). The required dosage and treatment time are determined by one of ordinary skill in the art. For horses or cattle and dogs with EPM or neosporosis, the preferred treatment regimen is to administer about 1.0-25mg/Kg praziquantel or derivative thereof every 28 days, more preferably at a dose of about 2.5-10 mg/Kg.

For one-time bolus therapy, praziquantel is administered in a pharmaceutically effective amount of greater than 10mg/Kg to about 100 mg/Kg. A unique feature of the present invention is that the compounds used in the present invention are non-toxic and therefore can be used in very large doses. The advantage of a large dose is that repeated administration is not required.

Without being bound by any particular theory, it is believed that the unexpected success of the described treatment is due to the ability of praziquantel to cross the blood brain barrier or placental barrier. It is believed that the compounds of the present invention readily cross the blood brain barrier and also penetrate the placenta, killing protozoa in the brain and in the cerebrospinal fluid/spinal cord. It has also been found that such compounds are non-toxic and mutagenic even when administered in the required high doses in a single high dose regimen described herein.

To date, no such low cost, easily administered drug has been available for the effective treatment and prevention of these diseases in mammals without producing unacceptable side effects such as toxicity or mutagenicity.

In the practice of the present invention, praziquantel can be formulated into any convenient dosage form for administration to an animal. Preferred formulations herein are those suitable for oral administration, and may be in the form of suspensions, tablets, capsules, gels, pastes, pellets, or in the form of powders, granules, or pellets. The most preferred oral formulations are pastes or food additives. Other types of formulations are injections, topical, intramuscular and intramucosal, or other routes of administration known to those skilled in the art. Topical administration in the form of infusion is also preferred.

Classical pharmaceutically acceptable carriers and adjuvants may be used in the formulations of the present invention. Examples thereof may be thickeners selected from the following: polycarbonylethylene, inorganic thickeners such as silicates, bentonite or silica gel; an organic thickener such as a fatty alcohol or fatty acid ester, and a wetting agent selected from polyethylene glycol and sodium lauryl sulfate bound to a polycarbonyl ethylene. The thickeners used herein to prepare the pastes are carbopol 974P and carbopol 934P. Preservatives selected from the group consisting of parabens, alcohols and aldehydes may also be employed. Can be a liquid, solid or gaseous substance, which is otherwise inert or medically acceptable, and is compatible with the active ingredient.

The pastes of the invention are surprisingly effective in the treatment of parasitic diseases. It is particularly surprising that the pastes of the present invention can be administered in neuroactive doses. Praziquantel can be effectively made to cross the blood brain barrier or placenta barrier, attacking parasites that have invaded the brain or infected the fetus of a pregnant animal. For convenience, a preferred paste formulation and an example of how to prepare it is provided herein. Preferred pastes according to the invention comprise micronized praziquantel suspension, polyethylene glycol, a thickener, such as polycarbonyl ethylene, preservatives, such as methyl and propyl parabens, and water. Preparation of the paste water (typically purified water) and propylene glycol may be mixed, the mixture heated to 70 ℃, and preservatives added at this temperature. After the addition of the polycarbonylethylene, preferably in the form of polycarbonylethylene 974P or 934P, the resulting mixture is cooled to room temperature and the praziquantel is finally added. After complete mixing, the pH was adjusted to about 6.0 with sodium hydroxide. The most preferred paste comprises 15% w/w praziquantel, 20% w/w propylene glycol, 0.5% w/w carbopol 974P, 0.14% w/w methylparaben, 0.02% w/w propylparaben, 0.1% w/w sodium hydroxide, and the balance pure water. Sweeteners including glucose, sucrose, lactose, fructose, sorbitol, xylitol, artificial sweeteners and molasses may be added to improve taste. In addition, yeast and liver flavoring agents may be added for the same purpose.

The invention is further illustrated by, but is not limited to, the following examples.

Example 1

To determine the scope of protection afforded by praziquantel, in vitro experiments were performed. The sensitivity of Sarcocystis fasciatus to this compound was evaluated. Praziquantel was tested at two concentrations, 10. mu.g/mL and 20. mu.g/mL, using SN3 Sarcocystis.

All in vitro experiments were performed with bovine nasal turbinate (BT) cells. Cells at 25cm²Growing to confluent state in flasks, using RMP 11640 medium and supplemented with 10% v/v Fetal Bovine Serum (FBS), 100 units penicillin (g/mL), streptomycin 100mg/mL and 5X 10^-22 mM mercaptoethanol. After cell fusion, the cells were stored in the same medium with a reduced amount of FBS (2% v/v). The cell culture was carried out at 37 ℃ in a humidified gas containing 5% carbon dioxide and 95% air.

For parasite growth, a monolayer of BT cells was infected with the parasite and examined with an inverted microscope for the extent of cell damage (cytopathic effect, "CPE") or the presence of many merozoites outside the cell. Once lesions or the presence of many extracellular parasites were observed, the monolayer was scraped off with the top of a 5mL pipette and 1-3 drops of merozoite-containing liquid were transferred to two flasks containing fresh BT cells. The merozoites of Sarcocystis fasciatus are passaged in this manner once every 5-10 days.

The test to determine the effectiveness of praziquantel is the micro monolayer disruption test (MMDA). This assay is used to determine whether a parasite or compound is toxic to BT cells. BT cells were seeded in 96-well flat-bottomed microtiter plates and tested for CPE (cytopathology effect plaque formation)The resulting monolayer cells were used to determine the effect of praziquantel on merozoite production. Parasites (50000 Sarcocystis fasciatus per well) were plated on a monolayer of cells, and appropriate concentrations of praziquantel were plated 2 hours after infection, with untreated and uninfected monolayer wells as parasite controls and untreated BT wells as toxicity controls. Each group treatment was repeated 6 times. Each well was observed daily. The experiment was stopped when 90-100% of the cells not dosed but infected with merozoites alone were lysed (90-100% CPE). All wells on the plate were washed with Phosphate Buffered Saline (PBS) and fixed in 100% methanol for 5 minutes, and then stained with crystal violet solution, and the crystal violet was not absorbed by damaged areas caused by merozoites or by BT cells dead due to toxicity. The amount of crystal violet bound was measured using an ELISA plate reader and the data used to determine the concentration of praziquantel (half maximal inhibitory concentration, or IC) that inhibited cell damage by 50%₅₀). Data confirming inhibition are shown in table 1. Note that a small dose of praziquantel at 10. mu.g/mL inhibited 51% cell rupture and a concentration of praziquantel at 20. mu.g/mL inhibited 69% cell rupture, indicating that praziquantel is effective in treating those diseases caused by coccidia associated with the symptoms of neurological and abortive diseases, including those caused by Sarcocystis ciliate, neospora caninum, neospora fradiae, Toxoplasma gondii, and swine isospora. In addition, praziquantel is not toxic to BT cells.

TABLE 1 in vitro Effect data of Praziquantel on Sarcocystis fasciatus

Biological body	Percent inhibition of cell disruption
				0 microgram/mL	10 microgram/mL	20 microgram/mL
	Sarcocystis ciliate (Royle) Rehd	0％	51％				69％

These data indicate that a dose of 2.5-10mg/Kg is effective in the nervous system when treating mammals suffering from diseases caused by Sarcocystis fasciatus.

Claims (14)

1. Use of a pharmaceutically effective amount of a composition of praziquantel and a pharmaceutically acceptable carrier for the manufacture of a medicament for treating a parasitic neurologic or abortigenic disease in a mammal suffering from a neurologic or abortigenic disease that is treatable with a praziquantel compound, wherein the disease is caused by a coccidia.

2. The use of claim 1, wherein the coccidia is one of the genera Sarcocystis, neospora, Toxoplasma and Isospora.

3. The use as claimed in claim 2, wherein the coccidia of the genus Sarcocystis is Sarcocystis indica, the coccidia of the genus neospora is neospora caninum or neospora immitis, the coccidia of the genus Toxoplasma is Toxoplasma gondii, and the coccidia of the genus Isospora is porcine isospora.

4. The use as claimed in claim 3, wherein said Sarcocystis fascicularis is the causative agent of equine protozoal encephalomyelitis.

5. The use of claim 2 wherein the neospora caninum is the causative agent of bovine or canine neosporosis.

6. The use of claim 2, wherein said neospora fradiae is the causative agent of equine protozoal encephalomyelitis.

7. The use of claim 2, wherein the Toxoplasma gondii is the causative agent of Toxoplasma-associated abortion in mammals.

8. Use of a composition of a pharmaceutically effective amount of a praziquantel compound and a pharmaceutically acceptable carrier for the preparation of a medicament for the remedial prophylactic treatment of a mammal having a neurologic or abortigenic disease caused by coccidia, and said disease is treatable with a praziquantel compound.

9. The use of claim 8, wherein the coccidia is one of the genera Sarcocystis, neospora, Toxoplasma and Isospora.

10. The use as claimed in claim 9, wherein the coccidia of the genus Sarcocystis is Sarcocystis indica, the coccidia of the genus neospora is neospora caninum or neospora immitis, the coccidia of the genus Toxoplasma is Toxoplasma gondii, and the coccidia of the genus Isospora is porcine isospora.

11. The use of claim 10, wherein said Sarcocystis fascicularis is the causative agent of equine protozoal encephalomyelitis.

12. The use of claim 9 wherein the neospora caninum is the causative agent of bovine or canine neosporosis.

13. The use of claim 9, wherein said neospora fradiae is the causative agent of equine protozoal encephalomyelitis.

14. The use of claim 9, wherein the Toxoplasma gondii is the causative agent of Toxoplasma-associated abortion in mammals.