Source: UNIVERSITY OF KENTUCKY submitted to
NEW THERAPEUTIC APPROACHES FOR EQUINE PROTOZOAL MYELITIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0183222
Grant No.
(N/A)
Project No.
KY014009
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 1999
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Project Director
Tobin, T.
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
VETERINARY SCIENCE
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30538101180100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1180 - Pharmacology;
Goals / Objectives
Preliminary data on benzeneacetonirile anti-coccidials suggests high specificity for S. neurona, low mammalian toxicity and orally bioavailability; as such they are prime candidates for prophylaxis and treatment of Equine Protozoal Myelitis. Dicalzuril and Toltrazuril appear to be the most promising members of this chemical group.
Project Methods
To establish in vitro anti-S neurona efficacy, develop dosing schedule that maintains minimum inhibitory concentrations (MICs) of selected agents in the Central Nervous System, establish clinical efficacy and define the adverse reaction rates and relapse rate on withdrawal of therapy.

Progress 10/01/99 to 09/30/04

Outputs
Equine Protozoal Myelitis (EPM) is an important infectious neurological disease of horses in the Americas. It is caused by Sarcocystis neurona; 95% of US horses may be exposed to S. neurona; up to 30% of exposed horses have antibodies to S.neurona in their cerebrospinal fluid; 0.5% of horses in endemic areas are believed to develop clinical evidence of the disease and without effective treatment many of these horses die. Additionally, young horses purchased and exported from the US may carry the organism and may develop the disease during training. In the past, the widespread prevalence of the infection in the US has adversely affected the marketability of American-bred horses. When this project commenced, the only available treatment for EPM was a combination of pyrimethamine and sulfonamide; more effective treatments for EPM were urgently required. Reviewing the scientific literature, we proposed the clinical efficacy of anti-apicomplexan triazine derivatives such as diclazuril, toltrazuril and toltrazuril sulfone and various analogs and formulations thereof as treatments and preventatives for EPM. We soon established proof of principle and shortly thereafter demonstrated the clinical efficacy of the clazuril drugs. A use patent for the drug was filed for and awarded (University of Kentucky Patent #5,883,095). This patent was licensed to Bayer and in August, 2001 Marquis, a paste formulation of toltrazuril sulfone, formulated for use in the treatment of EPM, was approved for marketing by the Center of Veterinary Medicine of the US Food and Drug Administration (FDA). It was the first FDA-approved treatment for EPM. Research since then has focused on identifying improved oral formulations for diclazuril, one of the more clinically effective of the anti-apicomplexan clazurils. The ultimate goal of this research has been to develop safe, effective, and economically viable approaches to the prophylaxis of EPM and other apicomplexan-based animal diseases, including that caused by Neospora caninum diseases. The preferred prophylactic approach is the inclusion of low part per million concentrations, of diclazuril or other triazine agents in the food of susceptible animals in areas in which there is high risk of exposure to apicomplexan parasites. Recent research has shown that sodium salt formulations of diclazuril result in substantially better oral bioavailability for diclazuril, including substantially higher serum and cerebrospinal fluid concentrations of diclazuril than the marketed formulation of diclazuril (Clinacox). These results bear directly on the important second goal of this research, namely widespread use of the triazines in the prophylaxis/metaphylaxis of EPM. An approximately 10 fold improvement in bioavailability for these sodium salt formulations compared with Clinacox has been demonstrated, which should improve the prophylactic efficacy, safety and cost effectiveness of these approaches for EPM and related apicomplexan diseases of domestic animals.

Impacts
Equine Protozoal Myeloencephalitis (EPM) is an important infectious neurological disease of Kentucky and American horses. At the start of this project, a new and highly specific and non-toxic prophylactic and therapeutic approach to EPM was proposed based on the clazuril group of drugs. Proof of principle was demonstrated and a use patent obtained. Based on this patent, Bayer formulated Marquis, which was rapidly approved for marketing in the US in August, 2001, and was the first FDA approved treatment for EPM. Horse breeding, and especially Thoroughbred horse breeding, is Kentucky's signature industry and leading agricultural commodity. This research project has provided a highly effective therapeutic approach to a disease that has been economically damaging to that industry. This project clearly demonstrated the therapeutic efficacy of the clazurils as treatments for systemic, as opposed to intestinal, apicomplexan related disease in large animals, and as such may lead to wider prophylactic and therapeutic use of these highly specific and virtually nontoxic anti-apicomplexan drugs.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
Equine Protozoal Myelitis (EPM) is an infectious neurological disease of horses in the Americas caused by Sarcocystis neurona. Between 50 and 95% of horses in endemic parts of the US show serological evidence of exposure to S. neurona, and up to 30% of horses may have antibodies to S.neurona in their cerebrospinal fluid. Approaching one-half percent of North American horses in endemic areas can be clinically affected, and in the absence of treatment, a significant proportion of these horses die. A further concern is that young racehorses purchased and exported from the United States may be incubating the disease, which then becomes clinically apparent during training, adversely affecting the international reputation of American-bred horses. When this project started the available treatments for EPM were based on combinations of pyrimethamine and sulfonamide. New and more effective treatments for EPM were urgently required, and the goal of this project was to establish the clinical efficacy of anti-apicomplexan triazine derivatives such as diclazuril, toltrazuril and toltrazuril sulfone and various analogs and formulations thereof, as treatments and/or preventatives for EPM. Our research clearly established the efficacy of this approach, and during the calendar year 2001 the paste formulation of toltrazuril sulfone, formulated for use in the treatment of EPM and known as Ponazuril (as set forth under the University of Kentucky Patent 5,883,095), was approved by the Center of Veterinary Medicine at the Food and Drug Administration (FDA) for marketing as the first FDA-approved treatment for EPM. This formulation was first marketed on August 7, 2001, as Marquis. Our research during 2003 was concerned with identifying improved oral formulations for diclazuril, the most potent and therapeutically effective of all of the anti-apicomplexan triazines. The goal of this research is to develop and provide a safe, effective, and economically viable approach to the prophylaxis of EPM and other coccidial-based animal diseases, including the closely related Neospora caninum organism, by including low part per million concentrations of diclazuril or other triazine agents in the food of horses in areas in which S. neurona is endemic, as is routinely done in the poultry business, as set forth under the University of Kentucky Patent 5,883,095. Research has shown that sodium salt formulations of diclazuril and related formulations result in substantially better oral bioavailability for diclazuril, along with substantially higher serum concentrations and cerebrospinal fluid concentrations of diclazuril when compared with the marketed oral Clinacox formulations of diclazuril with which this project initiated. These results bear directly on the second goal of this research, namely to use anti-apicomplexan triazines in the prophylaxis/metaphylaxis of EPM. The approximately 10 fold improvement in bioavailability for these sodium salt formulations compared with Clinacox may be expected to result in an equivalent increase in the prophylactic efficacy of these agents along with an equivalent improvement in the safety and cost effectiveness of this prophylactic approach.

Impacts
Horse breeding, especially the breeding of Thoroughbred horses, is Kentucky's signature and major agricultural product. EPM is endemic in Kentucky and adjacent states. EPM results in substantial economic loss and loss of reputation for Kentucky's most important agricultural product. Attainment of the first goal of this project, namely development and marketing of the first FDA-approved treatment for EPM, represents a major step forward in combating this disease. Achievement of the second goal of this project, namely development of a safe, effective andeconomically viable prophylactic approach to EPM, would further serve the industry by effectively removing a significant local disease and its associated stigma from the business of breeding and raising Thoroughbred and other bloodstock in Kentucky and the United States. Together, attainment of the primary and secondary goals of this project will mean that high cost Thoroughbreds and other horses can be raised and marketed in Kentucky and elsewhere in the United States without fear that EPM represents a significant health threat to the industry.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
Equine Protozoal Myelitis (EPM) is an infectious neurological disease of horses in the Americas principally caused by Sarcocystis neurona. Approximately 50 percent or more of horses in endemic parts of the United States show serological evidence of exposure to S. neurona, and up to 30 percent of horses reportedly have antibodies to S. neurona in their cerebrospinal fluid. Up to one-half percent of North American horses in some endemic areas can be clinically affected, and in the absence of effective treatment, a significant proportion of these horses die. New and more effective treatments for EPM were urgently required at the commencement of this project. The goal of this project has been to establish the clinical efficacy of the anti-apicomplexan triazine derivative, toltrazuril sulfone, and its various analogs, as treatments and/or preventatives for EPM. During the calendar year 2001 the paste formulation of toltrazuril sulfone, formulated for use in the treatment of EPM and known as Ponazurilr (as set forth under the University of Kentucky Patent #5,883,095), was approved by the Center of Veterinary Medicine at the Food and Drug Administration (FDA) for marketing as the first FDA-approved treatment for EPM. This formulation was first marketed on August 7, 2001, as Marquisr. Moving on from this major advance in the treatment of EPM, our laboratory research during the calendar year of 2002 focused on identifying improved oral formulations for diclazuril, the most potent and therapeutically effective of all of the anti-apicomplexan triazines. The goal of this research is to develop and provide a safe, effective and economically viable approach to the prophylaxis of EPM by including low part per million concentrations of diclazuril or other triazine agents in the food of horses in areas in which S. neurona is endemic, as is routinely done in the poultry business, as set forth under the University of Kentucky Patent #5,883,095. Research performed in 2002 showed that sodium salt formulations of diclazuril and related formulations resulted in substantially better oral bioavailability for diclazuril, along with substantially higher serum concentrations and cerebrospinal fluid concentrations of diclazuril when compared with the marketed oral Clinacoxr formulations of diclazuril with which this project initiated. These results bear directly on the second goal of this research, namely to use anti-apicomplexan triazines in the prophylaxix/metaphylaxis of EPM. The approximately 10 fold improvement in bioavailability for these sodium salt formulations compared with Clinacox may be expected to result in an equivalent increase in the prophylactic efficacy of these agents along with an equivalent improvement in the safety and cost effectiveness of this prophylactic approach.

Impacts
Horse breeding, especially the breeding of Thoroughbred horses, is Kentucky's signature and major agricultural product. EPM is endemic in Kentucky and adjacent states. EPM results in substantial economic loss and loss of reputation for Kentucky's most important agricultural product. Attainment of the first goal of this project, namely development and marketing of the first FDA-approved treatment for EPM represents a major step forward in combating this disease. Achievement of the second goal of this project, namely development of a safe, effective and economically viable prophylactic approach to EPM would further serve the industry by effectively removing a significant local disease and its associated stigma from the business of breeding and raising Thoroughbred and other bloodstock in Kentucky and the United States. Together, attainment of the primary and secondary goals of this project will mean that high cost Thoroughbreds and other horses can be raised and marketed in Kentucky and elsewhere in the United States without fear that EPM represents a significant health threat to the industry.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
Equine Protozoal Myelitis is an infectious neurological disease of horses in the Americas principally caused by Sarcocystis neurona. Approximately 50 percent of horses in endemic parts of the United States have had exposure to S. neurona, and up to 30 percent of horses reportedly have antibodies to S. neurona in their cerebrospinal fluid. Up to one-half percent of North American horses in some endemic areas can be clinically affected, and in the absence of effective treatment, some will die. New and more effective treatments for Equine Protozoal Myelitis were urgently required during the calendar year 2001. The goal of this project has been to establish the clinical efficacy of the triazine derivative, toltrazuril sulfone, and its various analogs, as a preventatives and or treatments for Equine Protozoal Myelitis. During the calendar year 2001 laboratory research focused and establishing improved oral formulations for toltrazuril sulfone. This research showed that formulations of toltrazuril sulfone in dimethyl sulfoxide and related formulations yielded substantially better oral bioavailability, substantially higher serum concentrations of toltrazuril sulfone and also substantially higher cerebrospinal fluid concentrations of totrazuril and diclazuril than previously developed paste formulations of toltrazuril sulfone. Beyond this, during the calendar year 2001 the paste formulation of toltrazuril sulfone, formulated for use in the treatment of equine protozoal myelitis known as Ponazuril(r) as set forth under the University of Kentucky Patent #5,883095 was approved by the Bureau of Veterinary Medicine of the Food and Drug Administration for marketing as the first FDA approved treatment for Equine Protozoal Myelitis. This formulation was first marketed on August 7th, 2001 as Marquis, the first FDA approved treatment for Equine Protozoal Myelitis in the United States. More recent research has focused on developing oral formulations of toltrazuril sulfone and related substances suitable for use in the prophylaxis of Equine Protozoal Myelitis in North American horse husbandry operations.

Impacts
Horsebreeding, and especially the breeding of Thoroughbred horses is Kentucky's major agricultural product. Equine Protozoal Myelitis is endemic in Kentucky and adjacent states. EPM can result in economic loss for Kentucky's most important agricultural product. The identification, development and marketing of an FDA approved treatment for Equine Protozoal Myelitis represents a major step forward in combating this disease. High cost Thoroughbred horses and other horses are raised and marketed in Kentucky without fear that Equine Protozoal Myelitis represents a significant health threat for the industry.

Publications

  • Publications during 2001 with reference to this project were limited to the promotional materials/package inserts associated with FDA approval and marketing of Marquis for the treatment of EPM.


Progress 01/01/00 to 12/31/00

Outputs
Equine Protozoal Myeloencephalitis [EPM] is a disease of the nervous system caused by the protozoan Sarcocystis neurona. During the year 2000, work continued on evaluating the clinical efficacy of diclazuril and toltrazuril sulfone in the prophylaxis and treatment of EPM. Diclazuril was available to us as Clinacox r, a chicken feed premix containing 0.5 percent diclazuril and 99.5 inert proteins carrier. Diclazuril is a triazine derivative used in the prophylaxis of coccidiosis, an apicomplexan disease in poultry. No information, however, was available about its efficacy in the treatment of central nervous system infections. The purpose of the study was to 1/evaluate the clinical efficacy of diclazuril as Clinacoxr in the treatment of severe clinical cases of EPM, 2/to establish an effective daily dosage schedule and treatment duration, 3/to estimate the rate of relapse at six months after cessation of therapy and 4/to develop preliminary information on the rate of occurrence of adverse effects associated with this treatment. We solicited clinical cases of EPM from practitioners nationwide for this trial. Our ideal clinical candidate was Western blot positive in its cerebrospinal fluid [CSF] was multifocally and asymmetrically neurologic, and was at least grade 3, ideally grade 4 neurologic. Neurologic and diagnostic evaluations were obtained from board-certified large animal internists and were graded on a scale from 0 [normal] to 5 [recumbency due to neurological disease] Additionally, our ideal candidate had been treated with pyrimethamine-sulfonamide therapy, had responded to treatment but relapsed when therapy was withdrawn. In the absence of effective treatment, these horses were expected to die. Horses were treated with Clinacoxr for 21 days, then therapy stopped and the response to therapy was evaluated over the following six months. Our starting clinical dose of diclazuril was 5 mg/kg of diclazuril, as Clinacoxr, and our initial duration of treatment was 21 days. Six-month follow-up examinations were completed for all 40 horses that completed treatment. Of these 40 horses, 28 [70%] improved. Four horses improved 3 or more neurologic grades, 11 improved 2 to 3 grades, and 13 improved 1 to 2 grades. 12 horses [30 %] did not improve upon treatment. Using a student's t-test, the null hypothesis was rejected at an alpha value of 0.025, suggesting that there was a statistically significant decrease in the neurologic score between the pre and post-treatment animals. Few adverse responses were reported, and none that could be unequivocally associated with treatment. At six months after completion of the study the relapse rate was reported a 5 percent [two out of 40] of the horses. Based on early review of the data, we extended the duration of treatment from 21 to 28 days. These preliminary results provided good evidence for a positive outlook in the response to diclazuril in a group of animals which otherwise would have died or required euthanasia.

Impacts
EPM is the most common infectious neurological equine disease in North America. In endemic areas, which includes much of the non-desert United States, 90 percent of horses have been exposed to the causative organism, Sarcocystis neurona, and 30 percent of clinically normal horses shed antibodies to this organism in its spinal fluid. About 1/2 to 1 percent of horses in endemic areas are clinically affected. Losses in performance and productivity to sub-clinical infection are not known, but are presumably substantial. Currently available treatments for EPM are generally prolonged, expensive and relapse rate is high when treatment is withdrawn. Better treatment modalities are urgently required, and an effective and inexpensive prophylactic approach is highly desirable. Horses severely clinically affected with equine protozoal myelitis were considerably improved by the 3 to 4 week course of therapy, and the relapse rate was substantially less than that found with a current therapy. These results suggest that we have identified a new and highly effective clinical approach to the treatment of EPM. Because Thoroughbred racehorses constitutes Kentucky's major agricultural export, a cost-effective mechanism of effectively eliminating this disease will substantially increase its market value.

Publications

  • Bentz BG, Dirikolu L, Carter WG, Saville W, Williams NM, Bernard WV, Wulff-Strobel C, Baker CB, McCrillis S, Reed S, Harkins JD, Granstrom DE, Tobin T: Diclazruil and equine protozoal myeloencephalitis (EPM): A clinical report. Equine Vet Education, 2:258-263, 2000.


Progress 01/01/99 to 12/31/99

Outputs
Equine Protozoal Myelitis (EPM) is a common infectious neurological disease of horses caused by Sarcocystis neurona (S. neurona), an apicomplexan parasite. S. neurona has the opossum as its primary host and another unidentified species as its secondary host. The opossum is a New World species, and cases of EPM almost always trace back to New World exposure. Currently available treatments for EPM are derived from anti-malarial therapy and are generally based on combination therapies using pyrimethamine and sulfonamides. Clinical experience suggests that these treatments must be continued for at least three to nine months, and a significant proportion of horses relapse after treatment ceases. There is, therefore, a pressing need for new and better treatments for this disease. S. neurona is an apicomplexan parasite, and apicomplexan parasites are uniquely sensitive to a family of benzeneacetonitrile based anti-coccidial drugs. Diclazuril (4-chlorophenyl [2,6-dichloro-4-(4,5-dihydro-3H-3,5-dioxo-1,2,4-triazin-2-yl)phenyl] acetonitrile), is a benzeneacetonitrile anti-protozoal agent (Janssen Research Compound R 64433) marketed as Clinacox. Review of the literature and clinical research suggested that diclazuril may have clinical application in the treatment of Equine Protozoal Myeloencephalitis (EPM). To evaluate its bioavailability and preliminary pharmacokinetics in the horse we developed a sensitive quantitative high-pressure liquid chromatography (HPLC) method for diclazuril in equine biological fluids. In other experiments MS/MS analysis of diclazuril in our HPLC solvent system yielded mass spectral data consistent with the presence of diclazuril. Our pharmacokinetic work showed that after a single oral dose of diclazuril at 2.5 g/450kg (as 500 g Clinacox), plasma samples from 4 horses yielded good plasma concentrations of diclazuril, which peaked at 1.077 microgram/ml plus or minus 0.174 (SEM) at 24 hours after administration and then declined with an apparent plasma half-life of about 43 hours. When this dose of Clinacox was administered daily for 21 days to two horses, mean steady state plasma concentrations of 7-9 microgram/ml were attained and maintained. Steady-state levels in the cerebrospinal fluid ranged between 100-250 ng/ml. There was no detectable parent diclazuril in the urine samples of dosed horses by HPLC analysis or by routine post-race thin layer chromatographic (TLC) analysis. These results show that diclazuril is absorbed well after oral administration and attains therapeutically useful steady-state concentrations in plasma and CSF. This is because the steady state concentrations attained in CSF are, based on studies reported in the literature, more than sufficient to interfere with S. neurona, whose proliferation is reportedly 95 % inhibited by concentrations of diclazuril as low as 1 ng/ml. These results are therefore entirely consistent with and support the concept of there being a very high probability of therapeutic efficacy associated with the treatment of clinical cases of EPM with diclazuril.

Impacts
Current therapy for EPM is prolonged and expensive and the relapse rate after therapy is withdrawn can be substantial. As such, there is an acute need for better treatments for this disease. These preliminary pharmacokinetic data and the published in vitro minimal inhibitory concentration (MIC) data for dicalzuril suggest that this benzene acetonitrile anti-coccidial has the potential to be a highly effective treatment for Equine Protozoal Myelitis.

Publications

  • Dirikolu, L.,Lehner, F., Nattrass, C., Bentz, B.G., Woods, W.E., Carter, W.G., Karpiesiuk, W., Jacobs,J., Boyles, J., Harkins, J.D., Granstrom, D.E., Tobin, T. 1999. Diclazuril in the horse: its identification and detection and preliminary pharmacokinetics. J. vet Pharmacol. Therap. 22, 374-379.