ANTHELMINTIC LABORATORY ANIMAL MODEL FOR HEARTWORM

Abstract

The current invention describes a laboratory animal model for dirofilarial nematodes wherein said laboratory animal is fed a dietary admixture of laboratory feed containing an immunosuppressing agent, for example, a glucocorticoid. The invention also describes the use of the laboratory animal model for screening endoparasiticides for the treatment and/or prevention of filarial nematode infections in animals.

Claims

1. An immunosuppressed laboratory animal model for dirofilarial heartworm nematodes wherein said animal is fed a dietary admixture of an immunosuppressing agent before and after inoculation of dirofilarial L3 larvae.

2. The animal model according to claim 1, wherein the immunosuppressing agent is a glucocorticoid.

3. The animal model according to claim 2, wherein the laboratory animal is a rodent.

4. The animal model according to claim 1, wherein the dirofilarial heartworm nematode is Dirofilaria immitis or Dirofilaria repens.

5. The animal model according to claim 2, wherein the glucocorticoid is hydrocortisone or a salt thereof.

6. The animal model according to claim 5, wherein the glucocorticoid is hydrocortisone acetate and the rodent is a rat.

7. The animal model according to claim 6, wherein the dietary admixture comprises about 20 ppm to about 250 ppm hydrocortisone.

8. The animal model according to claim 7, wherein the dietary admixture comprises about 50 ppm or about 200 ppm hydrocortisone.

9. The animal model according to claim 8, wherein the rat is fed the dietary admixture comprising about 200 ppm hydrocortisone for at least three days before inoculation with Dirofilaria immitis L3 larvae.

10. The animal model according to claim 9, wherein the rat is fed the dietary admixture comprising about 200 ppm hydrocortisone for about 8 days before inoculation and then for about 8 to about 14 days after inoculation.

11. The animal model according to claim 10, wherein the rat is fed the dietary admixture comprising about 200 ppm hydrocortisone on the day of L3 inoculation and for about 12 days after L3 inoculation.

12. The animal model according to claim 11, wherein the rat is fed the dietary admixture comprising about 200 ppm hydrocortisone for about 8 days prior to L3 inoculation, fed the same amount of hydrocortisone on the day of L3 inoculation and fed the same amount of hydrocortisone for about 12 days after L3 inoculation totaling about 21 days and then fed a dietary admixture comprising about 50 ppm hydrocortisone through necropsy.

13. The animal model according to claim 12, wherein the rat is fed the dietary admixture comprising about 50 ppm hydrocortisone for at least 70 days through necropsy.

14. A method for preparing an immunosuppressed rodent for a dirofilarial heartworm nematode animal model comprising: a. administering about 200 ppm of a glucocorticoid in a dietary feed admixture for at least 3 days; b. inoculating the rodent with dirofilarial L3 larvae; c. administering about 200 ppm of a glucocorticoid in a dietary feed admixture for at least 3 days post-inoculation; and subsequently d. administering about 50 ppm of a glucocorticoid in a dietary feed admixture for at least 70 days through necropsy.

15. The method according to claim 14, wherein the rodent is a rat and the glucocorticoid is hydrocortisone, or salt thereof.

16. The method of claim 15, wherein the hydrocortisone is hydrocortisone acetate and the dirofilarial larvae is a Dirofilaria immitis or Dirofilaria repens larvae.

17. The method of claim 14 for preparing an immunosuppressed rodent for a dirofilarial heartworm nematode animal model comprising: a. administering about 200 ppm of a glucocorticoid in a dietary feed admixture for about 5 to about 10 days; b. inoculating the rodent with dirofilarial L3 larvae wherein the L3 larvae are Dirofilaria immitis or Dirofilaria repens larvae; c. administering about 200 ppm of a glucocorticoid in a dietary feed admixture for about 5 to about 20 days post-inoculation; and subsequently d. administering about 50 ppm of a glucocorticoid in a dietary feed admixture for at least 70 days through necropsy; and e. wherein the glucocorticoid is hydrocortisone, or salt thereof, and the rodent is a rat.

18. The method of claim 17 wherein the rat is administered about 200 ppm hydrocortisone for about 7 to about 9 days, inoculated with dirofilarial L3 larvae, administered about 200 ppm hydrocortisone for about 8 to about 14 days after L3 inoculation and then administered about 50 ppm hydrocortisone for at least 70 days through necropsy.

19. The method of claim 18 wherein dirofilarial L3 larvae is a Dirofilaria immitis larvae.

20. The method of claim 18 wherein the dirofilarial larvae is a Dirofilaria repens larvae.

Description

DETAILED DESCRIPTION

[0043] The current immunosuppressed laboratory animal model, particularly rodent model, was developed by evaluating different doses of hydrocortisone using a dietary admixture of animal feed and hydrocortisone acetate fed to rodents ad libitum prior to and after inoculation with D. immitis L3 larvae. Additional in vivo studies have investigated other parameters, such as non-immunosuppression, rat age and gender, single and multiple heartworm inoculation levels and post-inoculation durations. Infected rats were maintained for nearly 7 months when up to 8 adult worms 6″ inches in length were recovered and microfilariae observed, confirming successful life cycle completion to fecundity. For D. immitis rat model efficacy validation, oral efficacy of compounds representing multiple antiparasitic classes, including ivermectin, moxidectin, emodepside, novel cyclooctadepsipeptides, an isoxazoline, and a bisimide were evaluated.

[0044] Although the heartworm life cycle is similar in the rat, ferret and dog, we have determined that preventative efficacy can be assessed 1 month earlier in the rat (˜106 days) than in the ferret and dog (˜140 days). The macrocyclic lactones, ivermectin and moxidectin, which are currently used in heartworm preventive therapies, achieved 96.5% and 100% efficacy at their use-dose. Emodepside and some cycloocta-depsipeptide analogs also demonstrated 100% prevention of heartworm.

[0045] The model has also been adapted to support evaluation of safety in adult heartworm infected dogs, both with an in vivo model and by providing adult heartworms for in vitro testing. Approximately 4000 adult heartworms have been generated for in vitro testing of over 200 compounds. This has substantially reduced the number of heavily infected dogs required to obtain a similar number of worms. Ongoing model optimization has resulted in further reduction of the immunosuppression regimen to the minimum level needed to ensure optimal infection and overall rodent health. In addition, heartworms from rat can be excised and transferred (transplanted) into a dog for in vivo safety assessments with heartworm endoparasiticides.

[0046] Laboratory animal models are an essential component in the discovery of new drugs reducing the need for testing in higher order target species, which often cannot sustain the number of compounds and evaluations necessary for early triage in lead seeking and lead optimization. The novel rodent heartworm model has demonstrated good correlation with the dog prevention efficacy model and the capability to reduce the number of dogs required to evaluate compounds both in vitro and in vivo, while shortening timelines for key decisions. There are different laboratory strains of rat and include the non-limiting examples: Wistar rat, Sprague-Dawley rat, including the CD (Sprague Dawley) IGS rat, Hairless rat, Long-Evans rat, Wistar Han IGS rat, Brown Norway rat, Copenhagen rat, Fischer rat, F344 rat, and the Lewis rat. The CD (Sprague Dawley) IGS rat is a preferred rat due to its weight and size characteristics.

[0047] Immunomodulating agents include corticosteroids. Corticosteroids are involved in a wide range of physiological processes, including stress response, immune response, regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids down regulate the immune system and affect white blood cell functionality. Corticosteroids are synthetic drugs that closely resemble cortisol, a naturally occurring hormone produced by mammalian adrenal glands. By chemical structure, the corticosteroids are classed into different groups. Group A is defined as hydrocortisone type corticosteroids that include hydrocortisone and its respective salts (e.g., acetate, butyrate, hem i-succinate, sodium phosphate, sodium succinate, and valerate), loteprednol etabonate, betamethasone, dexamethasone, fluorometholone, methylprednisolone, prednisolone, prednisone, rimexolone, cortisol, and triamcinolone. Group B are defined as acetonides, and include, for example, amcinonide, budesonide, desonide, fluocinolone, fluocinonide, halcinonide, and triamcinolone acetonide. Group C are defined as betamethasone types, and include, for example, beclomethasone, betamethasone, dexamethasone, fluocortolone, halometasone, and mometasone. Corticosteroid refers to both the glucocorticoids and mineralocorticoids. The preferred corticosteroids are glucocorticoids that modulate inflammation and the immune system. The term glucocorticoid is a portmanteau (glucose+cortex (adrenal)+steroid) composed from its role in the regulation of glucose metabolism, synthesis in the adrenal cortex (cortisol) and its steroid structure. The preferred glucocorticoid is hydrocortisone and the hydrocortisone salts thereof, prednisolone, and dexamethasone. The more preferred glucocorticoid is hydrocortisone and hydrocortisone salts thereof. The even more preferred glucocorticoid is hydrocortisone acetate. In particular, the more preferred glucocorticoid is hydrocortisone-21-acetate. The manipulation of the immune system in the rodent in this way does not appear to have affected the function of known heartworm preventives in delivering similar efficacies as is found in similar studies using the dog model, offering confidence that this rat model will be predictive of novel compounds that will provide similar preventive heartworm efficacy in the dog.

[0048] In addition to glucocorticoids, other immunomodulating agents can be used in the laboratory animal model. These additional immunomodulating agents include hormones that include, but are not limited to estrogen, progesterone, androgen, progestin, testosterone, epinephrine, and dehydroepiandrosterone (DHEA). In addition, immunosuppressive agents, including, but not limited to alkylating agents (e.g., cyclophosphamide, nitrosoureas, platinum compounds); antimetabolites (e.g., folic acid analogues (e.g., methotrexate), purine analogues (e.g., azathioprine and mercaptopurine), pyrimidine analogues (e.g., fluorouracil), and protein synthesis inhibitors; cytotoxic antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, and the like); and immunomodulating drugs like ciclosporin, tacrolimus, sirolimus, and everolimus.

[0049] The immunosuppressing agent can be administered to the laboratory animal, particularly rodents, orally, topically, and by injection (intramuscular, subcutaneously, and intravenously). Oral routes of administration are preferred routes and include gavage and dietary admixture. The preferred route is by dietary admixture.

[0050] The dietary feed admixture was prepared in a two-step process. First, a Type A concentrated mixture (20,000 ppm; 2% w/w) of hydrocortisone was prepared. To about 977.22 g (˜98% w/w) LabDiet 5002 rodent (meal), 22.78 grams of hydrocortisone acetate was added (˜20 g hydrocortisone). The rodent meal and hydrocortisone acetate were mixed uniformly. Second, a portion (or all) of the concentrated hydrocortisone mixture was mixed with more LabDiet 5002 rodent feed in a mixer for about 20 minutes. Once uniform, water (10% w/w) was added to the mixture and mixed for an additional 10 minutes. The wet mixture was pelletized and dried. The pelleted dietary admixture contains about 200 ppm hydrocortisone. Lesser (e.g., 50 ppm) and greater (e.g. 250 ppm) amounts of ready to feed dietary admixture pellets were made by adjusting the amount of the concentrated hydrocortisone mixture that was added to the feed. Immunosuppression dosing is based on the amount of hydrocortisone in the feed admixture. The molecular weight of hydrocortisone is 362.46 g/mol and the molecular weight of hydrocortisone acetate is 404.5 g/mol. Therefore, about 1.116 mg of hydrocortisone acetate is added to about 998.884 g of feed to prepare a 1 mg/kg (˜1 ppm) dietary admixture of hydrocortisone.

[0051] Upon receipt, newly acquired rats are fed a normal laboratory rat chow diet for a few days for acclimation. Once the immunosuppression diet begins, rats can be fed a glucocorticoid (e.g., hydrocortisone) dietary admixture for about 130 days using at least two dosing concentrations at about 50 ppm, 75 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, and 250 ppm. Intermittent dose amounts of a glucocorticoid (e.g., hydrocortisone) also include, for example, 65 ppm, 80 ppm, 115 ppm, 135 ppm, 160 ppm, 180 ppm, 190 ppm, 210 ppm, 230 ppm, and 245 ppm, and the like.

[0052] Earlier studies showed that rats fed a 200 ppm hydrocortisone diet for about 120 days had an increased incidence of mortality. Therefore, to maintain normal rat growth, health, and behavior, the 200 ppm hydrocortisone diet should be fed for a duration of about 85 days; preferably about 60 days, and more preferably about 30 days, and even more preferably about 21 days. This 200 ppm dietary duration includes the pre-L3 inoculation period, L3 inoculation day, and the post-L3 inoculation period. Thereafter, rats can be fed the immunosuppressant diet with a reduced amount of hydrocortisone at about 20 ppm, 30 ppm, 40 ppm, 50 ppm, or 60 ppm through necropsy. The preferred reduced amount of hydrocortisone is about 50 ppm. Rats should be maintained on the immunosuppressant diet containing about 50 ppm hydrocortisone for at least 60 days through necropsy, and preferably for at least 70 days through necropsy. A preferred immunosuppression diet plan includes feeding the rodent a 200 ppm hydrocortisone dietary admixture for about 8 days prior to D. immitis L3 inoculation; then continue feeding the rat the same 200 ppm hydrocortisone dietary admixture for about 13 days post-inoculation (including inoculation day); then continue feeding the rat a 50 ppm hydrocortisone dietary admixture for at least 60 days through necropsy, and preferably for at least 70 days through necropsy. Preferably, the rodent is fed the 50 ppm hydrocortisone dietary admixture for about 90 days to about 100 days, and preferably about 94 days, before necropsy, which allows the immature adult worms to migrate to the heart and lungs, where they can be more easily harvested. A longer duration (about 180 to about 260 days post L3 inoculation) of the 50 ppm hydrocortisone diet can be fed to the rat to allow the immature adult worms to mature and begin producing and releasing microfilariae (L1) into the blood stream. In addition, the reduced amount of hydrocortisone can be administered within the dietary admixture for greater than 260 days post-inoculation to maintain the mature adult worms in vivo for future studies.

[0053] Heparinized blood from infected dogs with patent adult D. immitis worms and circulating D. immitis microfilaria were obtained and membrane fed to Aedes aegypti mosquitoes. The infected blood contained about 50-100 microfilariae/20 μL. After about 15-17 days following feeding, mosquitoes were collected and crushed to obtain the infective L3 larvae. Larvae were stored in a Roswell Park Memorial Institute (RPMI) medium which is a cell and tissue culture medium used for growing mammalian cell lines. The RPMI is a nutrient blend of amino acids, vitamins, organic and inorganic supplements, and salts. The L3 larvae were injected into the rat in about a 0.2 mL volume of the RPMI culture media.

[0054] D. immitis antigen can be monitored using an enzyme-linked immunosorbent assay (ELISA) for the detection of antigen to adult D. immitis antigen in canine and feline plasma or serum. For example, the DiroCHECK™ test kit can be used to detect the D. immitis antigen, particularly from adult female worms. Other IgG antibody and serological tests can be used to monitor for the antigen(s). In addition, polymerase chain reaction (PCR) tests can be performed to further substantiate Dirofilaria spp.

[0055] Dirofilaria is a genus of nematodes, or roundworms, in the family Onchocercidae. Some species cause dirofilariasis, a state of parasitic infection, in humans and animals. There are about 27 species in the genus. These are generally divided into two subgenera, Dirofilaria and Nochtiella. Some species are well-known parasites, including D. immitis, the dog heartworm, Dirofilaria repens, which affects many types of nonhuman mammals, and Dirofilaria tenuis, which usually parasitizes raccoons, but can infect humans, as well. Human dirofilariasis is generally caused by D. immitis and D. repens. The former can cause pulmonary dirofilariasis, which may have no symptoms. Another form of the infection can be characterized by a painful lump under the skin or infection of the eye. The nematode infection is spread by mosquitoes. Species in the genus include: D. acutiuscula, D. aethiops, D. ailure, D. asymmetrica, D. cancrivori, D. conjunctivae, D. corynodes, D. desportesi, D. fausti, D. freitasi, D. genettae, D. hystrix, D. immitis (dog heartworm), D. indica, D. louisianensis, D. macacae, D. macrodemos, D. magalhaesi, D. magnilarvata, D. pongoi, D. reconditum, D. repens, D. timidi, D. uniformis, D. ursi, D. tawila, and D. tenuis. The preferred dirofilarial nematodea are D. immitis and D. repens. The more preferred dirofilarial nematode is D. immitis.

Examples

[0056] In an early study, thirty-two male CD (Sprague Dawley) IGS rats weighing 175-250 grams were randomized into 4 treatment groups (n=8/group). Each group received a medicated diet containing hydrocortisone acetate. T01, T02, and T03 received a 200 ppm, 100 ppm, and 50 ppm hydrocortisone dose, respectively, for 120 days. The fourth group, T04, received a 200 ppm dose through Day 17; subsequent dosing through necropsy was with 50 ppm hydrocortisone. Feed was administered ad-libitum. On Day 6, animals were inoculated with approximately 15 L3 D. immitis larvae (Michigan strain) by subcutaneous injection into the inguinal area. On Day 114 post-inoculation, rats were necropsied and adult worm counts assessed. In total, about 3, 11, 5, and 19 adult worms were collected from the T01, T02, T03, and T04 groups, respectively.

[0057] In a second study, duration of immunosuppression and timing of inoculation was assessed to establish criteria for consistent adult dog heartworm infections in the rat. Male and female CD (Sprague Dawley) IGS rats weighing 40-50 grams (Group A) and 151-175 grams (Group B) were used in the study. The study design is described below in Table 1.

TABLE-US-00001 TABLE 1 Study Design for the duration of immunosuppression and timing of D. immitis inoculation in rats Day L3 Days of Days of post- # of inocu- immuno- immuno- Nec- inocu- animals lation suppression suppression ropsy lation Group (gender) day 200 ppm 50 ppm day (±1) T01 (A) 6M 0 None None 98 98 T02 (A) 6M 0 None None 112 112 T03 (A) 6M 0 None None 126 126 T04 (A) 6M 0 None None 140 140 T05 (A) 6M 0 −8 to 7  7-98 98 98 T06 (A) 6M 0 −8 to 7   7-112 112 112 T07 (A) 6F 0 −8 to 7   7-112 112 112 T08 (A) 6M 0 −8 to 7   7-126 126 126 T09 (A) 6M 0 −8 to 7   7-140 140 140 T10 (A) 6M 7 −8 to 21 21-98  105 98 T11 (A) 6M 7 −8 to 21 21-112 119 112 T12 (A) 6M 7 −8 to 21 21-126 133 126 T13 (A) 6M 7 −8 to 21 21-140 147 140 T14 (B) 6M 7 −8 to 21 21-98  105 98 T15 (B) 6M 7 −8 to 21 21-112 119 112 T16 (B) 6F 7 −8 to 21 21-112 119 112 T17 (B) 6M 7 −8 to 21 21-126 133 126 T18 (B) 6M 7 −8 to 21 21-140 147 140
On Day −8, medicated hydrocortisone diet at 200 ppm was provided to treatment groups T05-T18. On Day 0, treatment groups T01-T09 received an inoculation of approximately 50 L3 D. immitis larvae (Michigan strain) in 0.2 mL RPMI, subcutaneously injected into the inguinal area. On Day 7, T10-T18 received an inoculation of approximately 50 (2×25) L3 D. immitis by subcutaneous injection into the inguinal area. On Day 7, Groups T05-T09 were converted from the 200 ppm hydrocortisone diet to the 50 ppm hydrocortisone diet. On Day 21, groups T10-T18 were converted from the 200 ppm hydrocortisone diet to the 50 ppm hydrocortisone diet. Diet for all groups was administered ad-libitum. Necropsy occurred on Days 98, 112, 126 and 140 (±1) post-inoculation. Blood was obtained at necropsy to test for D. immitis antigen using the DiroCHECK ELISA based serological test. Data from this study is presented below in Tables 2 and 3.

TABLE-US-00002 TABLE 2 Study results for the duration of immunosuppression and timing of D. immitis inoculation in rats. Mean worm Percent of Worm counts count rats with from body Group (range) infection (n) cavity digestion T01 0 (0)   0 (6) 0 T02 0 (0)   0 (6) 0 T03 0.3 (0-2)  17 (6) 0 T04 0 (0)   0 (6) 0 T05 4.2 (2-8) 100 (5) 0 T06 6.0 (5-7) 100 (5) 2 T07 0 (0)   0 (6) 0 T08  5.3 (4-11) 100 (4) 7 T09 2.4 (0-6)  60 (5) 5 T10 2.2 (2-7)  60 (5) 0 T11  5.5 (2-11) 100 (4) 2 T12 5.3 (0-9)  83 (6) 4 T13 4.0 (3-6) 100 (4) 4 T14 3.6 (0-6)  83 (6) 0 T15 5.7 (2-8) 100 (6) 5 T16 0.2 (0-1)  17 (6) 0 T17  7.3 (2-16) 100 (6) 8 T18  7.7 (4-18) 100 (6) 14

[0058] As can be seen in Table 2, only 1 non-immunosuppressed rat had worms (T06) showing immunosuppression is necessary to establish meaningful worm burdens for studies. Female rats were almost wormless clearly demonstrating a gender component in rats causing a non-permissive state, or perhaps, a semi-permissive state. For the male immunosuppressed groups, 96% of mature rats with an average of 5.9 worms and 87% of newly weaned rats with an average of 4.3 worms was achieved. Extending the 200 ppm diet duration to or after inoculation did not have a significant positive or negative effect. An increased dual inoculation challenge produced an increase in worm burden and a greater percentage of infected rats. Newly weaned rats are susceptible to adverse effects including mortality on an immunosuppressed 200 ppm diet. The number of worms recovered from the body cavity increased as the study necropsy day was extended. Lung digestion assisted in recovery of further worms.

[0059] A third study was conducted to evaluate drug efficacy of selected compounds, with compounds and dosages selected based on efficacy from prior ferret and dog studies in the D. immitis rat model; and to determine the effect of withdrawal of immunosuppression at Day 70. Male CD (Sprague Dawley) IGS rats weighing 150-175 grams were randomized into specific treatment groups (n=6/group). On Day −8, medicated hydrocortisone diet at 200 ppm was provided ad-libitum to all treatment groups. On Day 0, infected larvae were harvested from Aedes aegypti approximately 15-17 days following membrane feeding on heparinized blood containing 50-100 microfilariae/20 μl. Rats were inoculated with approximately 50 (2×25) L3 D. immitis larvae (Michigan strain) by subcutaneous injection into the inguinal area. On Day 21, all animals had the 200 ppm hydrocortisone diet removed and replaced with 50 ppm hydrocortisone diet. On Day 30, all groups received a treatment medication (moxidectin (MOX), ivermectin (IVM), emodepside (EMO), a bisimide, a cyclooctoadepsipeptide, or an isoxazoline) by subcutaneous injection. On Days 34 and 38, the T09, T10 and T11 groups received further subcutaneous injections of the treatment medication. On Day 70, group T13 received un-medicated diet until necropsy. On Day 112 (±1) post-inoculation, all animals were necropsied for the presence of adult D. immitis worms. A blood sample was taken at necropsy for D. immitis antigen ((Ag); Ag+ (positive)). A lung digestion procedure was conducted to collect additional worms. Treatment medications were dosed by subcutaneous injection. The study design is shown below in Table 3 and the study results are described in Tables 4 and 5.

TABLE-US-00003 TABLE 3 Study design for the efficacy of various compounds in the D. immitis immunosuppressed rat model Necropsy day post- Days of Days of inocu- Treat- Dose Dosing 200 ppm 50 ppm lation Group ment (mg/kg) day(s) diet diet (±1) T01 Control 0 30 −8 to 21 21-112 112 T02 IVN 0.001 30 −8 to 21 21-112 112 T03 IVN 0.003 30 −8 to 21 21-112 112 T04 MOX 0.001 30 −8 to 21 21-112 112 T05 MOX 0.003 30 −8 to 21 21-112 112 T06 EMO 5 30 −8 to 21 21-112 112 T07 EMO 1 30 −8 to 21 21-112 112 T08 Bisamide 10 30 −8 to 21 21-112 112 T09 Depsi-1 10 30, 34 −8 to 21 21-112 112 and 38 T10 Depsi-2 10 30, 34 −8 to 21 21-112 112 and 38 T11 Depsi-3 10 30, 34 −8 to 21 21-112 112 and 38 T12 Iso- 30 30 −8 to 21 21-112 112 xazoline T13 Control W 0 N/A −8 to 21 21-70  112 T14 Control L 0 N/A −8 to 21 21-112 112 T15 Control 0 N/A −8 to 21 21-112 120 T16 Control 0 N/A −8 to 21 21-112 128 Depsi (1-3) = Novel cyclooctadepsipeptides Control L (T14) = Lung digestion only

[0060] On Day −8, medicated hydrocortisone diet at 200 ppm was provided to all treatment groups. On Day 0, infected larvae were harvested from Aedes aegypti approximately 15-17 days following membrane feeding on heparinized blood containing 50-100 microfilariae/20 μl. Rats were inoculated with approximately 50 (2×25) L3 D. immitis larvae (Michigan strain) by subcutaneous injection into the inguinal area. On Day 21, all animals had the 200 ppm hydrocortisone diet removed and replaced with 50 ppm hydrocortisone diet. On Day 30, all groups received a treatment medication (moxidectin, ivermectin, emodepside, a bisimide, a cyclooctoadepsipeptide, or an isoxazoline) by subcutaneous injection. On Days 34 and 38, the T09, T10 and T11 groups received further subcutaneous injections of the treatment medication. On Day 70, group T13 received un-medicated diet until necropsy. On Day 112 (±1) post inoculation, all animals were necropsied for the presence of D. immitis. A blood sample was also taken at necropsy for D. immitis antigen. A lung digestion procedure was also conducted to collect additional worms.

TABLE-US-00004 TABLE 4 Arithmetic mean heartworm counts and % efficacy in rats Dosing Necropsy Mean # rats day(s) day worm with Group Dose post- post- count infection # of % (n = 6) Treatment (mg/kg) inoculation inoculation (range) (n = 6) Ag+ Efficacy T01 Control 0 30 112-114 7.5 6 2 N/A  (5-13) T02 IVM 0.001 30 112-114 0.8 5 2 89.3 (0-2) T03 IVM 0.003 30 112-114 0.3 1 2 96.5 (0-2) T04 MOX 0.001 30 112-114  0.67 4 1 91.7 (0-1) T05 MOX 0.003 30 112-114 0   0 0 100 T06 EMO 5.00 30 112-114 0   0 1 100 T07 EMO 1.00 30 112-114 1.8 3 1 75.6 (2-5) T08 Bisamide 10 30 112-114 5.8 6 3 24.6  (2-10) T09 Depsi-1 10 30, 34 112-114 0   0 0 100 and 38 T10 Depsi-2 10 30, 34 112-114 0   0 2 100 and 38 T11 Depsi-3 10 30, 34 112-114 0   0 2 100 and 38 T12 Isoxazoline 30 30 112-114 5.3 6 4 28.9  (2-11)

[0061] Table 4 demonstrates that rats from the control (T01) group had an infection with an average of 7.5 worms per rat. The ivermectin (IVM) and moxidectin (MOX) groups achieved a similar dose efficacy response of 89.3% and 96.5% for IVM at 0.001 and 0.003 mg/kg and 91.7% and 100% for moxidectin at the same doses, respectively. Emodepside also achieved a dose response percent efficacy of 75.6 and 100 at 1.0 and 5.0 mg/kg, respectively. The three cyclooctadepsipeptides dosed at 10 mg/kg on days 30, 34 and 38 all achieved 100% efficacy. The bisimide and isoxazoline administered at 10 and 30 mg/kg were ineffective, achieving 24.6% and 28.9% efficacy, respectively.

[0062] Overall, the study achieved comparable efficacies to compounds that have been dosed in dogs and/or ferrets. Antigen (DiroCHECK) tests were not fully predictive of worm infection; possibly because the test only detects tiny pieces of female (not male) heartworm skin proteins circulating in the blood.

TABLE-US-00005 TABLE 5 Arithmetic mean heartworm counts in rats Necropsy Mean # # day worm rats with recovered Group post- count infection by (n = 6) Treatment inoculation (range) (n = 6) digestion T13 Control (medicated 112-114 4.5 6 Not diet withdrawal (1-8)  Recorded day 70) T14 Control (lung 112-114 4.5 6 *12 digest) (2-7)  T15 Control (extend 120 8.2 6 3 +8 days) (3-13) T16 Control (extend 128 3.8 6 +16 days) (2-10) *No dissection of lung employed

[0063] Table 5 shows that substitution of the medicated diet at Day 70 with un-medicated (T13) moderately decreased worm burden as did worm recovery by lung digestion (T14) when compared to T13. Extending the duration of infection to 120 (T15) and 128 (T16) days after inoculation achieved slightly increased and decreased worm burden means, respectively. All control rats had worms and no adverse effects were observed throughout the study. Lung digestion was shown to recover low numbers of additional worms and was best employed as a contributory procedure to dissection.

[0064] A fourth study was conducted to determine the effect of immunosuppressed diet withdrawal at strategic life stages of D. immitis infection in rats. Male CD (Sprague Dawley) IGS rats weighing 150-175 grams were used for this study (n=6/group). On Day −8, medicated hydrocortisone diet at 200 ppm was provided to all treatment groups, ad libitum. On Day 0, L3 were harvested from A. aegypti approximately 15-17 days following membrane feeding on heparinized blood containing 50-100 microfilariae/20 μl. Rats were inoculated with approximately 60 (2×30) L3 D. immitis larvae by subcutaneous injection into the inguinal area. On Day 21, all 200 ppm hydrocortisone diets were replaced with a 50 ppm hydrocortisone diet. On Day 31, cages 1, 2 and 3 had the 50 ppm hydrocortisone diet removed and replaced with standard rat non-medicated diet. On Day 45, cages 4, 5 and 6 had the 50 ppm hydrocortisone diet removed and replaced with standard rat non-medicated diet. On Day 62, cages 7, 8, 9 and 10 had the 50 ppm hydrocortisone diet removed and replaced with standard rat non-medicated diet. All rats were necropsied on Day 119 (±1) post-inoculation. The study design is presented in Table 6 and the study data is presented in Table 7.

TABLE-US-00006 TABLE 6 Study design for diet withdrawal Necropsy Days of Days of day (±1) Day of 200 ppm 50 ppm post- # of Group Treatment inoculation diet diet inoculation Cage # rats T01 Control 0 −8 to 21 21-31 119 1, 2, 3 6 T02 Control 0 −8 to 21 21-45 119 4, 5, 6 6 T03 Control 0 −8 to 21 21-62 119 7, 8, 9, 10 8

TABLE-US-00007 TABLE 7 Mean worm counts with varied diet withdrawal Day of diet Necropsy withdrawal day Live Arith- Geo- (post- (post- worm metic metric Group inoculation) inoculation) Animal count mean mean T01 31 119 1 0 0 0 2 0 3 0 4 0 5 0 6 0 T02 45 119 7 1 3.17 1.91 8 2 9 5 10 0 11 0 12 11 T03 62 119 13 2 4.25 2.85 14 1 15 3 16 6 17 0 18 15 19 5 20 2

[0065] On 3 separate previous occasions (all data not shown), using 12 rats in total, withdrawal of immunosuppressed diet on Day 70 post-inoculation demonstrated little or no effect on worm burdens. Overall, reducing immunosuppression to the minimum level required is beneficial to the general health status of the animals on study and potentially reduces any effect on treatments administered following medicated diet withdrawal.

[0066] Data from numerous studies were compiled to correlate dose data from rat and dog. As can be seen in Table 8, the efficacy achieved between rat and dog at recommended (approved) macrocyclic lactone (ML) doses for dog were generally comparable in rat. Dogs were inoculated with approximately 50 D. immitis L3 larvae (UGA (University of Georgia) susceptible strain) and were orally administered the ML or emodepside at 1, 1.5, and/or 2 months post-inoculation. Dogs were necropsied at about 5 months post-inoculation. Rats were inoculated with approximately 50 D. immitis L3 larvae (Michigan susceptible strain) and were administered the ML or emodepside by subcutaneous injection at 1 month post-inoculation. Rats were necropsied at about 4 months post-inoculation. As can be observed in Table 8, the efficacy between the subcutaneous dosing in the rat and oral dosing in the dog models correlated well. Thus, the efficacy of antifilarial drugs in the rat model correlates well with efficacy in the dog model. Studies are ongoing to directly compare oral dosing in the rat versus subcutaneous dosing using susceptible heartworm strains. It was also found that the D. immitis life cycle in the immuno-suppressed rat was about 1 month shorter than in dogs. This time savings provides an additional reduction in housing cost of the animal colony.

TABLE-US-00008 TABLE 8 Correlation of drug efficacy in rat (SQ) and dog (oral) heartworm (D. immitis) models Approved oral Oral dose SQ dose Month(s) post- dose in tested in dog tested in rat inoculation % dog (μg/kg) (μg/kg) (μg/kg) dosed Efficacy Moxidectin 1.25 — 1.2 100 (3) 0.625 — 2 100 0.5 — 2 100 — 1 1 92 — 3 1 100 Ivermectin 1 — 1 53 (6) 2 — 1 83-97  3.3 — 1 98 6 — 1 100 2 — 1.5 64 6 — 1.5 100 — 1 1 89 — 3 1 97-100 — 6 1 100 Emodepside 1 — 1 58 (N/A) 3 — 1 88 5 — 1 97 10 — 1 97 — 1 1 76 — 5 1 100

[0067] In yet another study, moxidectin was used to treat immunosuppressed CD (Sprague Dawley) IGS male rats (250-350 g) infected with 2 characterized macrocyclic lactone resistant D. immitis strains to assess efficacy and plasma exposure. Rats were inoculated with L3 larvae of ZoeMI-01 (ZM1) and JYD-34 D. immitis strains. T01 and T06 were control (C) groups. Animals received either a 3, 12, or 24 μg/kg dose of moxidectin 28 days post L3 inoculation. In addition, two groups, T05 and T10, received 3 μg/kg doses 28, 56, and 84 days post L3 inoculation. Moxidectin was administered by oral gavage. Animals were necropsied 120 days post L3 inoculation and worms were harvested. Data for this efficacy study is provided in Table 9.

TABLE-US-00009 TABLE 9 Geometric Mean Worm Counts and Efficacy in Rats Post- Oral inoculation Necropsy Mean Number Group dose dosing day (post- worm of rats % (n = 4) Strain Treatment (μg/kg) (day(s)) inoculation) counts infected efficacy T01 ZM1 C 0 28 120 5.6 4 N/A T02 ZM1 MOX 3 28 120 1.1 3 81 T03 ZM1 MOX 12 28 120 0 0 100 T04 ZM1 MOX 24 28 120 0 0 100 T05 ZM1 MOX 3 28, 56, 84 120 0 0 100 T06 JYD-34 C 0 28 120 3.9 4 N/A T07 JYD-34 MOX 3 28 120 6.0 4 0 T08 JYD-34 MOX 12 28 120 2.8 *3  28 T09 JYD-34 MOX 24 28 120 0.5 1 87 T10 JYD-34 MOX 3 28, 56, 84 120 2.4 4 38 *One rat died pre-necropsy

[0068] Moxidectin administered by oral gavage at 28 days post infection against a macrocyclic lactone (ML) susceptible D. immitis strain (ZM1; T-02) at 3 μg/kg achieved 81% efficacy. When administered similarly at 12 and 24 μg/kg at 28 days post infection and at 28, 56, and 84 days post infection at 3 μg/kg 100% efficacy was achieved. Moxidectin administered by oral gavage at 28 days post infection against a JYD ML-resistant D. immitis strain at 24 μg/kg was 87% efficacious. Overall, the efficacy and plasma exposure of oral moxidectin using varying dosing regimens, against two strains of D. immitis, demonstrated that the rat model can accurately evaluate in vivo drug susceptibility of ML-resistant strains that correlate with similar results in the dog. Additional studies are being planned to evaluate more compounds and strains.

D. immitis Adult (Immature and Mature) Worm Assays (In Vitro and In Vivo)

[0069] Adult heartworms were obtained from rat heart and lungs aseptically following euthanasia. The adult worms were maintained in a general purpose cell culture media for about 7-10 days at 37° C. For the in vitro assay, test compounds were dissolved and serially diluted in dimethylsulfoxide (DMSO). Aliquots were added to the empty wells of assay plates. The cell culture media and a single adult (immature/mature) D. immitis worm was added to each well to dilute the test compounds to the desired concentrations. Assay plates were incubated for approximately 24 hours, and the worms in the assay wells were observed visually for drug effect. Worms were assessed subjectively for survival or paralysis, and results were reported as Minimum Effective Dose (MED). Data for these assays are not reported.

[0070] For future in vivo studies, immature adult heartworms extracted from immunosuppressed rats will be surgically or directly transferred into the dogs' circulatory system. These studies, if successful, will reduce or eliminate the need for dog donors of adult heartworms for experimental studies. These recipient dogs will be monitored for microfilaria levels and adult heartworm antigen to determine the success of the transplant procedures from rat to dog.