WO2001087290A1

WO2001087290A1 - Decoquinate, 4-hydroxyquinolones and napthoquinones for the prevention and treatment of equine protozoal myeloencephalitis caused by sarcocystis neurona hughesi and other apicomplexan protozoans

Info

Publication number: WO2001087290A1
Application number: PCT/US2001/015931
Authority: WO
Inventors: David S. Lindsay
Original assignee: Virginia Tech Intellectual Properties, Inc.
Priority date: 2000-05-17
Filing date: 2001-05-17
Publication date: 2001-11-22
Also published as: AU2001261704A1

Abstract

Apicomplexan parasites such as Sarcocystis neurona and Neospora hughesi may be killed with decoquinate, a 4-hydroxyquinolone and/or a naphthoquinone. Based on such parasite-killing activity, these compounds are used in methods of preventing and treating neurological disease or dysfunction such as protozoal myeloencephalitis, especially EPM.

Description

DECOQUINATE, 4-HYDROXYQUINOLONES AND NAPTHOQUINONES FOR THE PREVENTION AND

TREATMENT OF EQUINE PROTOZOAL MYELOENCEPHALITIS CAUSED BY SARCOCYSTIS NEURONA HUGHESI AND OTHER APICOMPLEXAN

PROTOZOANS

DESCRIPTION

Field of the Invention The invention generally relates to neurologic disease and dysfunction, and particularly relates to apicomplexan parasites causing equine neurologic syndromes.

BACKGROUND OF THE INVENTION

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasites, Sarcocystis neurona and Neospora hughesi. EPM is considered the most important protozoal disease of horses in the United States, and usually is considered in any horse with neurological signs. Serological surveys using the Western blot test demonstrate that about 45 to 53% or more of horses have antibodies to S. neurona indicating high exposure to that parasite. There are about 6.9 million horses in the United States, corresponding to a $112 billion dollar annual industry. Clinical EPM occurs in 0.5 to 1.0% of horses.

In a recent survey of 2599 respondents, 76% of which were horse owners with the rest being veterinarians and other representatives of the horse industry, of the infectious diseases listed, EPM was listed by 24% and ranked first (USD A, APHIS Report May, 1997). The number of cases of EPM diagnosed in horses with neurological signs at the Ohio State University veterinary school has increased from 24.9% in 1992 to 50% in 1996 indicating an increasing prevalence.

The Virginia opossum, Didelphis virginiana is the only known definitive host in North America . Dubey, J. P., Lindsay, D. S., 1998, "Isolation of Sarcocystis neurona from opossum (Didelphis virginiana) faeces in immunodeficient mice and its differentiation from Sarcocystis falcatula, Int. J. Parasitol. 29,1823-1828. The riine-banded armadillo, Dasypus novemcinctus, is a natural intermediate host (Cheadle et al., 2001) and domestic cats (Felis domesticus) are experimental intermediate hosts (Dubey et al., 2000). Horses become infected by ingesting S. neurona sporocysts excreted in opossum feces. Particularly, horses become infected with EPM-causing agents by ingesting sporocysts or oocysts while grazing or from contaminated feed or water. It is virtually impossible to prevent horses from encountering EPM-causing agents.

Conventionally, pyrimethamine and sulfonamides are used to treat EPM, with a prolonged course of treatment (twelve weeks being about the average length of treatment time). Usual treatment involves the use of sulfadiazine at a dose of 20 mg/kg, once or twice a day. In addition, affected horses are placed on pyrimethamine, at a dosage of 1.0 mg/kg daily for 120 days or longer. Duration of treatment maybe longer if the CSF remains positive and/or the horse continues to demonstrate clinical signs of neurological disease. Complications of anemia and/or leukopenia have been observed, especially for doubling the pyrimethamine dose, and in some horses diarrhea occurs.

For these conventional therapies, a determination to discontinue treatment is based on either significant improvement of the clinical signs or the horse returning to normal and Western blot testing of CSF returning to negative. The combination of sulfadiazine and pyrimethamine results in a sequential blockade of folic acid metabolism. The specific concentration of pyrimethamine required to achieve an anti-protozoal level for S. neurona is not known.

Diclazuril (Clinicox, Pharmacia Upjohn, Canada), a coccidiostat, is an alternative treatment for horses not responding to the above- mentioned traditional therapies or in horses having developed complications. The drag is absorbed quickly and has been found in serum one hour after feeding to horses. It is a triazine and has been used as a prophylactic agent against coccidiosis in poultry and has been used experimentally in the treatment of similar problems in rabbits. It has anti-S. neurona activity in cell cultures infected with S. neurona .

Toltrazuril (Baycox 5% suspension; Bayer, Canada) is an anti- coccidial drug used in several species. The mechanism of action is to disrupt intracellular pathways important in energy metabolism as well as cell division. This drug and its major metabolite, ponazuril have potential efficacy for the treatment of EPM. Toltrazuril appears to have good oral absorption and fairly long elimination time (48-72 hours). The drug has good lipid solubility and is well absorbed into CSF. hi horses given toltrazuril at 5 mg/kg daily for 10 days, plasma levels of toltrazuril were 20 mcg/ml with a mean CSF concentration of 160 mcg/ml. The use of this drug has not been shown to result in any complications nor have elevations of serum chemistry values or changes in complete blood counts been observed. A metabolite, ponazuril (toltrazuril sulfone), was recently used in a multi-center treatment study (7 sites) involving 100 horses. The drug appeared to show favorable clinical results and is currently under review by the FDA for marketing in the United States.

For horses treated with the above-mentioned conventional therapies, relapse may occur. For example, relapse may occur if horses are not treated long enough. Reactivation of the infection may occur during periods of unusual stress. The conventional chemotherapy regimens do not completely remove all disease-causing parasites from the central nervous system. Also, if too-low a concentration of the conventional drugs is used, intracellular stages of the parasites are not killed.

However, even if in most horses that contract EPM relief ultimately can be provided, the costs of treating EPM are substantial. Diagnostic neurological evaluation may cost $456 per horse. Treatment of horses for EPM can be expensive, especially since most affected horses are treated for a period of 120 to 150 days, and sometimes longer. The monthly cost of treatment is approximately $200.00 for a 450 kg horse. Reevaluation of the horse at 30 to 60 day intervals and a subsequent spinal tap at 90 to 120 days after initiation of treatment adds to the cost. If clinical signs persist, therapy is continued and reevaluated every 30 days. Treating horses with toltrazuril is estimated at $1,200 (5 mg/kg) to 2400 (10 mg/kg) for 30 days and diclazuril at $770 for 30 days. In addition to direct treatment costs, indirect costs also are associated with EPM, such as decreased performance time, loss of stake payments, transport costs, death or euthanasia.

Thus, improved treatments for equine EPM are wanted, as are methods for avoiding EPM in the first instance. In treating EPM, for example, there remains the hope that horses can be restored to health more rapidly than with current treatments and with more permanent results.

SUMMARY OF THE INVENTION

The present invention exploits the discovery that decoquinate is highly active against S. neurona and N. hughesi, and the further discovery that decoquinate even at low concentrations accomplishes intracellular stage killing. According to the invention, decoquinate, a 4- hydroxyquinolone, and or a naphthoquinone or pharmaceutically acceptable salts (e.g., anionic or cationic, hydrochlorides, ammonium, sodium, etc.), esters (C^ alkyl) or other derivatives (e.g., amine) thereof may be fed prophylatically to horses, to prevent EPM, and may be administered to horses to treat EPM.

In order to accomplish these and other objects of the invention, the present invention in a preferred embodiment provides a method of killing Sarcocystis neurona and Neospora hughesi, comprising contacting a population comprising Sarcocystis neurona and/or Neospora hughesi with an agent selected from the group consisting of decoquinate, a 4-hydroxyquinolone and a naphthoquinone, and salts, esters or derivatives thereof. In one embodiment of the inventive killing method, the population may be in an equine host. In a further embodiment of the killing method, contacting includes the step of orally providing said equine host with said agent.

In another preferred embodiment, the invention provides a method of preventing or treating equine neurological disease or dysfunction, comprising administration to an equine of a pharmaceutically effective dose of an agent selected from the group consisting of decoquinate, a 4-hydroxyquinolone and a naphthoquinone, and salts, esters, or derivatives thereof. In a further embodiment of such an inventive method, the neurological disease or dysfunction may be associated with infection with an apicomplexan parasite. In another embodiment of the invention, the neurological disease or dysfunction may be protozoal myeloencephalitis. The apicomplexan parasite may be Sarcocystis neurona or Neospora hughesi. In a further preferred embodiment, the invention provides a method used when the equine has not yet demonstrated symptoms associated with protozoal myeloencephalitis.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be better understood from the following detailed description of the preferred embodiments of the invention with reference to the drawings, in which:

Figures 1A-G depict chemical structures of Buquinolate, Decoquinate, Nequinate, Buparvaquone, Parvaquone, and Atovaquone used in the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In a first preferred embodiment, a parasite population comprising Sarcocystis neurona and/or Neospora hughesi is killed by being contacted with decoquinate, a hydroxyquinolone (such as 4- hydroxyquinolone) and/or a naphthoquinone or pharmaceutically acceptable salts (e.g., anionic or cationic, hydrochlorides, ammom^'um, sodium, etc.), esters (C,.₁₂ alkyl) or other derivatives (e.g., amine). Such compounds are known, such as the compounds with structures set forth in Figures 1 A-G. See also Figure 47.7 and pages 966-967 of David S. Lindsay and Byron L. Blagburn, Antiprotozoan Drugs, Section 11 ("Chemotherapy of Parasitic Diseases"), Chapter 47. Compounds for use in the present invention (hereinafter anti-EPM compounds or agents) include those shown in Figures 1 A-G, but are not particularly limited thereto:

Buquinolate (4-hydroxy-6,7-diisobutoxy-3-quinoline- carboxylic acid ethyl ester, C₂₀H₂₇NO₅),

Decoquinate (6-declyoxy-7-ethoxy-4-hydroxy-3-quinoline- carboxylic acid ethyl ester, C₂₄H₃₅NO₅),

Nequinate (7-(benzyloxy)-6-n-butyl-l,4-dihydro-4-oxo-3- quinoline-carboxylic acid methyl ester, C₂₂H₂₃NO₄),

Buparvaquone (2-tr «5-(4-t-butylcyclo-hexyl)methyl-3- hydroxy-l,4-naphthoquinone, C₂₁H₂₆O₃),

Parvaquone (2-cyclohexy-3-hydroxy- 1 ,4-naphthoquinone, C₁₆H₁₆O₃) and Ato vaquone (2- [tra«5-4-(4-chlorophenyl)cyclohexyl] -3 - hydroxy-1 ,4-naphthoquinone, C₂₂H₁₉ClO₃).

By way of non-limiting example, a commercially available example of decoquinate is Deccox^R which is an anticoccidial feed additive containing 6% decoquinate, marketed by Alpharma Inc. of Fort Lee, New Jersey. Alphapharma's FDA clearances are for Deccox ^R use for prevention of coccidiosis in cattle, goats, sheep and broiler chickens. Alpharma's product sheet data indicate that Deccox ^R is non-toxic to horses.

The invention may be used for prevention and treatment of neurologic disease such as equine EPM. In the case of prevention, horses which may be exposed to agents that cause EPM are provided with a sufficient quantity of the anti-EPM agent of this invention to kill or immobilize the EPM disease causing agents. In the case of treatment of horses suspected to have EPM, administration of an anti-EPM agent according to the present invention should begin as quickly as possible after clinical signs of the disease are recognized. At least as good recovery is expected using the invention as for conventional treatments, which are said to result in successful recovery in 70 to 75% of the EPM- affected horses. In either case, treatment or prevention, the anti-EPM agent may be administered orally as part of feed, or by other means such as injection.

Regular (such as daily) feeding of the above-mentioned anti- EPM compounds to horses may have further beneficial effects, such as (1) prevention of abortions due to Neospora caninum, Neospora hughesi or Toxoplasma gondii; (2) prevention of equine babesiosis (because the piroplasmas have mitochondria that are sensitive to mitochondrial inhibitors); and (3) prevention of intestinal coccidiosis caused by Eimeria leuckarti in foals and other equids.

The following experimentation was conducted for Sarcocystis neurona isolates and cell culture. Sarcocystis neurona merozoites (SN2, SN3, or SN6 strains, isolated from a horse with EPM (Dubey et al., 2001) were grown and maintained in bovine turbinate (BT cells, ATTC CRL 1390, American Type Culture Collection) or African green monkey (Cercopithecus aethiops) kidney cells (CV-1 cells, ATTC CCL-70, American Type Culture Collection, Rockville, Maryland, USA) according to the method in Lindsay, D.S., and Dubey, J.P., "Determination of the activity of diclazuril against Sarcocystis neurona and Sarcocystis falcatula in cell cultures, J. Parasitol. 86, 164-166 (2000). The host cells were grown to confluence in 25 cm² plastic cell culture flasks in growth media that consisted of 10% (v/v) fetal bovine serum (FBS) in RPMI 1640 medium supplemented with 100 U penicillin G/ml, and 100 mg streptomycin/ml. Cell cultures were maintained in growth medium in which the FBS content was lowered from 10% to 2%. Cell cultures were incubated at 37 C in a humidified atmosphere containing 5% CO₂and 95% air.

For quantitative studies, merozoites were harvested from infected cell cultures by removing the medium and replacing it with Hanks' balanced salt solution without calcium and magnesium. The host cells were then removed from the plastic growth surface by use of a cell scraper. This cell mixture was passed through a 27-gauge needle attached to a 10-ml syringe to rupture host cells. The suspension was then filtered through a sterile 3 μm filter to remove cellular debris. The number of merozoites in the filtrate was determined using a hemacytometer. The final volume of suspension was adjusted so 2.5 x 10⁵ merozoites were present for inoculation.

For general maintenance of merozoites, monolayers were examined with an inverted microscope for the development of lesions (areas devoid of host cells caused by parasite replication) in the monolayer or the presence of many extracellular merozoites. Once lesions were observed or many extracellular parasites were present, the monolayer was scraped with the tip of a 5 ml pipette and 1 to 3 drops of the merozoite containing fluid was transferred to 2 flasks of BT cells. Merozoites of S. neurona were passaged in this manner every 3 to 7 days.

Decoquinate (lot 7916Z4) was dissolved in DMSO to make a stock solution of 1 mg/ml. Dilutions were made from this stock solution and used in the following studies.

Experiment 1. Merozoites (200,000/flask) of the SN6 strain were inoculated on to cell cultures and allowed to penetrate host cells for 2 hours. The host cells were then treated with 0.1 microgram/ml decoquinate for 5 minutes or 15 minutes. Control flasks contained merozoites but no decoquinate. The decoquinate containing medium was washed off the infected host cells at 5 or 15 minutes and they were rinsed with Hanks balanced salt solution 5 times to remove any residual decoquinate. Cell cultures were maintained for 6 weeks. No parasites or parasite induced lesions were seen in the decoquinate treated infected flasks at 6 weeks. The control flask was destroyed by this time due to parasite multiplication. These results demonstrated that decoquinate can effectively kill Sarcocystis neurona after a 5 or 15 minute exposure period.

Experiment 2. Merozoites (1 million /flask) of the SN2 and SN3 strains were inoculated separately on to cell cultures and allowed to penetrate host cells for 2 hours. The host cells were then treated with 0.1 microgram/ml decoquinate for 10 or 20 minutes. Control flasks contained merozoites but no decoquinate. The decoquinate containing medium was washed off the infected host cells at 10 or 20 minutes and they were rinsed with Hanks balanced salt solution 5 times to remove any residual decoquinate. Cell cultures were maintained for 16 days. No parasites or parasite induced lesions were seen in the decoquinate treated SN3 infected flasks. Flasks containing the SN2 strain had 5% cytopathic effect (CPE). The control flasks had 25% (SN3 strain controls) to 40% (SN2 strain controls) CPE at this time. These results demonstrated that decoquinate can effectively inhibit several strains of Sarcocystis neurona.

Experiment 3. Merozoites of the SN3 strain were inoculated on to cell cultures and allowed to penetrate host cells for 2 hours. The host cells were then treated with 0.01 (2 flasks), 0.001 (2 flasks), or 0.0001 (2 flasks) microgram ml decoquinate continuously for 10 days. Control flasks contained merozoites but no decoquinate. The numbers of merozoites produced were determined at 10 days and a percent reduction in merozoite production determined. Treatment with 0.01 microgram/ml caused a 98% reduction in merozoite production. Treatment with 0.001 microgram/ml caused a 87% reduction in merozoite production. Treatment with 0.0001 microgram/ml caused a 40% reduction in merozoite production. These findings indicate that there is a dose response to decoquinate and suggests setting a target dose of 0.01 microgram or greater.

The results of Experiments 1, 2 and 3 above establish killing activity of decoquinate. Moreover, decoquinate is superior to diclazuril and other conventional agents used to treat EPM because decoquinate kills the EPM-causing parasite more rapidly and at lower concentrations. The above data are particularly important considered in view of EPM being a neurologic syndrome in horses caused primarily by infection with Sarcocystis neurona and rarely with Neospora hughesi, and further considering that EPM is the most important protozoal disease of horses in the United States and is present in the Americas wherever the definitive host the opossum is found. The present inventor has considered that treatment of EPM conventionally has often been with pyrimethamine combined with trimethoprim and sulfonamides, which are agents that are potentially toxic to the horse. Thus, prevention is a rational alternative to treatment of clinically ill animals and new effective agents are needed to treat or better prevent EPM. The present inventor has identified such new anti-EPM agents, such as decoquinate.

Decoquinate is a quinolone antiparasitic agent. Decoquinate inhibits the parasites' mitochondria. The experimental data set forth above indicate that decoquinate can quickly kill stages of Sarcocystis neurona in cell cultures. Decoquinate also exerts its anti-Sarcocystis neurona activity at low doses in cell cultures, and is safe in the horse and not readily toxic to other vertebrate species. This indicates it will be safe when used by lay people (i.e. horse owners).

Without limiting the invention to such an example, an example of preventing EPM in a horse not exhibiting any current EPM symptoms would be to add decoquinate in powdered form to the dry feed daily, a daily preventative for EPM in horses. It can be fed alone or in combination with other agents (antibiotics, vitamin supplements, herbal supplements, mineral supplements, etc.) which are fed daily. Another delivery mechanism would be for feed suppliers to mix decoqumate in the ration. Decoqumate also can be placed in free choice vehicles such as mineral blocks for preventative dosing. Weekly or monthly administered sustained release formulations of decoquinate also may be used for the prevention of EPM. Horses on preventative decoquinate treatment are expected to be protected against EPM caused by Neospora hughesi, as delivery systems that are preventative against Sarcocystis neurona will be preventative against Neospora hughesi.

Again without limiting the invention to such an example, an example of treating EPM would be as follows. Upon a horse exhibiting an EPM symptom, decoquinate is administered to the horse.

Common structural features and other common properties between decoquinate and the other compounds in Figure 1 suggest that the other compounds of Figure 1 are highly likely to have such killing activity. Thus, the compounds of Figure 1 are concluded to have anti- EPM activity, as a prophylactic for preventing EPM and for treating EPM (including treatment of acute EPM), based on the activity of such compounds on EPM-causing parasites.

While horses in the United States, Canada and South America may particularly benefit from the present invention, the invention may be used with regard to horses anywhere.

While the invention above has been discussed with particular reference to horses, it will be appreciated that the invention is not particularly limited and may be used for treating other animals, such as, by way of non-limiting examples, Australian marsupials, arboreal monkey species (including endangered monkey species), sea otters, sea lions, skunks, raccoons, mink, and other animals in aquaria, zoos or farms. Administration of the compounds of Figure 1 to such animals may aid in preventing fatal toxoplasmosis in highly susceptible animals.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims.

Claims

CLAIMSWe claim:

1. A method of killing Sarcocystis neurona and Neospora hughesi, comprising contacting a population comprising Sarcocystis neurona and/or Neospora hughesi with an agent selected from the group consisting of decoquinate, a 4-hydroxyquinolone and a naphthoquinone, and salts, esters or derivatives thereof.

2. The killing method of claim 1, wherein said population is in an equine host.

3. The killing method of claim 2, wherein contacting includes the step of orally providing said equine host with said agent.

4. A method of preventing or treating equine neurological disease or dysfunction, comprising administration to an equine of a pharmaceutically effective dose of an agent selected from the group consisting of decoquinate, a 4-hydroxyquinolone and a naphthoquinone, and salts, esters, or derivatives thereof.

5. The method of claim 4, wherein said agent is decoquinate.

6. The method of claim 4, wherein said agent is a naphthoquinone.

7. The method of claim 4, wherein said agent is a 4- hydroxyquinolone.

8. The method of claim 4, wherein the neurological disease or dysfunction is associated with infection with an apicomplexan parasite.

9. The method of claim 4, wherein the neurological disease or dysfunction is protozoal myeloencephalitis.

10. The method of claim 5, wherein the apicomplexan parasite is Sarcocystis neurona or Neospora hughesi.

11. The method of claim 4, wherein the equine has not yet demonstrated symptoms associated with protozoal myeloencephalitis.