Method for screening drugs and animal model for same

Abstract

Acetylcholine and its receptors appeared in evolution before development of a nervous system. Cholinergic agonists functions include proliferation, differentiation, and cell-to-cell contact in protozoa] as well as vertebrate cells. Animal models for infection by Apicomplexan parasites require cell-to-cell contact followed by differentiation of parasite and host cells to produce clinical disease. Experimental infections are produced by introducing parasite infected leukocytes into a host. Binding cholinergic receptors on the parasites and leukocytes with levamisole HCl induces non-progressive infections and absence of signs of disease.

Claims

1. A method for screening a candidate drug for treating a disease in an equine host, the disease being caused by Sarcocystis neurona, comprising: obtaining an equine host model that has been infected with Sarcocystis neurona and treated with a sufficient amount of a cholinergic agonist agent to prevent clinical signs of the disease caused by Sarcocystis neurona without killing the Sarcocystis neurona, thereby exhibiting a parasitemia in the equine model; administering the candidate drug to the equine model; and determining whether the candidate drug reduces the parasitemia in the equine model.

2. The method of claim 1, wherein the cholinergic agonist agent is levamisole HCL, imidazothiazole, salts thereof, esters thereof or derivatives thereof.

3. The method of claim 1, wherein the cholinergic agonist is administered by a route selected from the group consisting of: oral, intranasal, topical, inhalation, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intratracheal injection, intrathecal injection and combinations thereof.

4. The method of claim 1 wherein the dose of cholinergic agonist agent used to obtain a parasitemia in the mammalian host is between 0.001 and 100 mg/kg.

5. The method of claim 1, wherein the dose of cholinergic agonist agent used to obtain a parasitemia in the equine host is between 0.1 and 10 mg/kg.

6. The method of claim 1, wherein the dose of cholinergic agonist agent used to to obtain a parasitemia in the equine host is 1 mg/kg.

7. The method of claim 1, wherein the dosage form of the cholinergic agonist agent is a member selected from the group consisting of a powder, a solid, a liquid, a crystal, a pill, a tablet, a capsule, a thin film, a suspension, a paste, a cream, a gel, a liniment, a balm, a lotion, an ointment, and a skin patch.

8. The animal model as described in claim 1.

Description

DETAILED DESCRIPTION

(1) Production of Persistent Parasitemia in a Horse:

(2) In a first preferred embodiment a parasite population was isolated from horses as described in an experimental model (Experimental infection of horses with culture derived Sarcocystis neurnoa merozoites as a model for equine protozoal myeloencephalitis, 2004) and used to infect a yearling thoroughbred filly. The challenge (6000 organisms/day) was used daily for 10 days. Concurrent with the challenge infection the filly was administered 1 mg/kg levamisole HCl orally for 21 days. The serum antibody titer to SAG 1, 5, 6 was negative at 2 for each antigen at the onset of challenge. Daily blood samples were taken and 5 microliters of blood were used to prepare thin layer smears followed by staining with Geimsa. Ten fields were counted to determine the presence of S. neurona. A hemocytometer was used to determine the number of parasites per ml of peripheral blood. Day 10, the filly had no clinical signs and a parasitemia of one million organisms in the serum. At day 14, the filly had no clinical signs of disease and a parasitemia of one million organisms. At the end of fourteen days the filly was treated with decoquinate orally at 0.5 mg/kg for 10 days. At day 30, the horse had no visible parasites in the blood and no clinical signs of disease. It was previously established that infection for 10 days without the additional of levamisole HCl reliably results in clinical signs of disease in 93% of horses by day 30.

(3) Modulating Cytokine Responses in Horses Using Levamisole HCl:

(4) Field cases of idiopathic encephalitis associated with EPM due to S. neurona were identified by veterinary clinical examination. In a one-year period (2013), 54 horses received levamisole HCl at 1 mg/kg for signs of EPM that had not resolved with conventional treatment as indicated on submission forms for serum antibody analysis. Signs were assessed by the Field Veterinarian by a neurological examination of gait and scored on a scale of 1-5. Attributable data was obtained from 27 Field Veterinarians. Thirty-three animals were included into the study, missing data from 21 horses prevented inclusion of these 21 animals into the study analysis. In the included data set, one Field Veterinarian submitted data for 6 animals, one Field Veterinarian submitted data from two animals, and 25 veterinarians submitted data for one animal.

(5) Horses were evaluated for a change in gait score by subtracting the gait score after treatment from the gait score before treatment. Twenty-seven (82%) of the horses had a gait score that improved by at least one grade after levamisole HCl treatment determined by veterinary neurological examination. The change in gait score ranged from 1 to 3 in this group. Six animals (18%) had a gait score that was worse or did not change after treatment. The change in gait score ranged from 2 to 0 in this group.

(6) The results of this study indicate that 82% of horses showed a potential clinical benefit by alleviation of neuro inflammation when treated with levamisole HCl. Horses had not improved before treatment and had shown clinical decline that prompted veterinary attention. The positive treatment response in this study suggests that EPM treatment failures are due to a lack of recognition of an associated inflammatory condition that is levamisole responsive.

(7) The following experimentation was conducted for the pharmacokinetics of levamisole HCl in horses:

(8) Levamisole HCl is rapidly metabolized in horses. Horses given 5 mg/kg of levamisole HCl (2800 mg) orally reportedly have 485 ng/ml in plasma at one hour. In order to determine the plasma concentrations of low dose levamisole HCl three horses were given a single treatment. In this experiment adult male horses were given 550 mg, 1650 mg, or 2750 mg of levamisole HCl orally (1 mg/kg, 3 mg/kg, and 5 mg/kg). The plasma levels measured at 15 minutes were 606, 591, and 1082 ng/ml in plasma respectively. At one hour the plasma levels were 139, 690, and 750 ng/ml. The rapid elimination of levamisole HCl from plasma in horses may indicate that secondary messengers are responsible for the immune modulating effects apparent with treatment. Binding of levamisole to levamisole sensitive receptors are anticipated to increase cGMP, attenuation of CAMP, and attenuation of proinflammatory molecules that are immune regulating effectors.

(9) 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 other animals such as, by way of non-limiting examples, people, sea otters, sea lions, skunks, raccoons, mink, and other animals in aquaria, zoos or farms.

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

(11) The present invention provides animal models for Apicomplexan infections including but not limited to Toxoplasma, Plasmodium, Neospora, and/or Sarcocystis.

(12) The present invention provides the models for development of preventive and/or therapeutic agents (drugs and vaccines) by demonstrating their effectiveness against Apicomplexan infections such as Toxoplasma, Plasmodium, Neospora, and/or Sarcocystis by specific acetylcholine receptor binding.