Method of treating heartworm with emodepside

Abstract

The disclosure provides methods of preventing and / or treating parasitic infections and / or infestations in animals by administering such compounds or compositions to the animals.

Description

METHOD OF TREATING HEARTWORM WITH EMODEPSIDE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 643,644, filed May 7, 2025, and which is herein incorporated by reference in its entirety. FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods of preventing and / or treating parasitic infections in humans or animals. BACKGROUND

[0003] Various compounds have been developed as antiparasitic agents against one or more parasites. However, certain such compounds suffer from one or more drawbacks, e.g., insufficient antiparasitic efficacy, slow speed of action, limited duration of action, or high cost of goods. Some such compounds have a low therapeutic index and / or may require significant care in storing and administering to animals. Some antiparasitic agents are becoming ineffective due to parasite resistance. For at least these reasons, there is continuing interest in the art to develop additional compounds capable of preventing and / or controlling various parasites, which can be used to prevent or treat certain infections, infestations, and / or diseases transmitted by such parasites. BRIEF SUMMARY

[0004] The present disclosure relates to a method of treating and / or preventing heartworm infection. Heartworm disease, caused by the adult form of Dirofilaria immitis, is a major veterinary concern and represents an economically important concern of global proportion. From both a pathologic and animal welfare standpoint, heartworm is one of the most significant endoparasites in companion animal health. From an animal health perspective, dogs are the most important species amongst the numerous mammalian hosts that can be infected by D. immitis. In dogs, heartworm disease is caused by the adult parasite eliciting pathology in the pulmonary arteries. Canines act as the definitive host, so sexual reproduction occurs in the pulmonary arteries and microfilariae are released into the canine’s circulatory system (McCall et al., Advances in Parasitology 66(2008), 193-285; Bowman and Atkins, Vet Clin North Am Small Anim Pract. 2009 Nov; 39(6):1127-58,2009; Selzer et al., Trends Parasitol. 2021 -1- WBD (US) 4892-1795-8322v3Jan;37(1):77-89.2021). Treatment of mature heartworm infection with an adulticide (e.g., melarsomine dihydrochloride) is expensive and can cause serious adverse side effects. Particularly, treatment of an established D. immitis infection in dogs requires a prolonged regimen of drug treatment, exercise restriction, and sometimes even surgical intervention (Bowman and Atkins, 2009). The occurrence of dogs infected with drug-resistant heartworms has been spreading across the U.S. with high prevalence particularly in the Mississippi delta region. D. immitis infection also occurs in cats. In cats, severe cases of heartworm disease, and even death, can be caused by infection with just a few developing immature or adult filariae.

[0005] Current practice is to control heartworm disease in dogs and cats through prevention. The goal of marketed heartworm preventive therapies in dogs is to prevent the development of the parasite to adult heartworms by interrupting the Dirofilaria life cycle post-infection. The macrocyclic lactones (MLs; e.g., ivermectin, eprinomectin, milbemycin oxime, moxidectin, and selamectin) are the most commonly used chemoprophylaxis agents and have been effective against Dirofilaria immitis infective third-stage larvae (L3) as well as maturing fourth-stage larvae (L4). Recently, however, it has been reported that some populations of Dirofilaria immitis have developed selectional resistance to heartworm preventives (see, e.g., "Heartworm Preventive Resistance. Is it Possible?", Bulletin of the American Heartworm Society vol. 37, 2010, p. 5). Cyclic depsipeptides with antiparasitic activity are also known. PF-1022a, a 24- membered cyclooctadepsipeptide isolated from the fungus Mycelia sterilia by Sasaki et al. (J. Antibiotics 45, 1992, 692-697), has been found to exhibit broad spectrum anthelmintic activity against a variety of endoparasites in vivo with low toxicity. These compounds are described in, for example, U.S. Patent Nos. 5,514,773; 5,747,448; 5,646,244; and 5,874,530, which are incorporated herein by reference. Emodepside is a semi-synthetic analog of PF-1022a containing a morpholine group at the para position of the aryl ring in the phenyl lactate groups and is a potent anthelmintic. Emodepside has the following structure: -2- WBD (US) 4892-1795-8322v3O N .

[0006] Emodepside ishookworms), where it is believed to act at the neuromuscular junction by stimulating presynaptic receptors belonging to the secretin receptor family, resulting in paralysis and death of the parasites. A combination of emodepside and praziquantel for the treatment of parasitic worms is marketed as the prescription topical product PROFENDER® and is approved in the US for the treatment of multiple parasitic infections in cats. Praziquantel is a pyrazinoisoquinoline effective against tapeworms. Praziquantel is rapidly adsorbed via the surface of the parasites and acts primarily by changing the Ca2+permeability of the parasite membranes, resulting in severe damage to the parasite integument, contraction and paralysis, disruption of metabolism, and death of the parasite.

[0007] However, it has been reported that emodepside lacks sufficient efficacy to treat or prevent infection with D. immitis. See, for example, U.S. Patent No. 11,382,949 to de Fallois et al. Further, no commercial products containing emodepside as the sole active agent have been developed or marketed. According to the present disclosure (and contrary to prior reports), it has surprisingly been found that emodepside possesses potent activity against D. immitis microfilaria and L3 stage larvae, and further possesses in vitro and in vivo pharmacokinetic properties suggesting utility as a solo agent for the prevention and treatment of heartworm. -3- WBD (US) 4892-1795-8322v3

[0008] Accordingly, provided herein is a method of treating or preventing heartworm in an animal, the method comprising administering to the animal an effective amount of emodepside, or a composition comprising emodepside.

[0009] In some embodiments, the animal is a companion animal. In some embodiments, the companion animal is a dog. In some embodiments, the companion animal is a cat.

[0010] In some embodiments, the method treats or prevents canine heartworm.

[0011] In some embodiments, the method treats or prevents feline heartworm.

[0012] In some embodiments, the method prevents canine heartworm.

[0013] In some embodiments, the method prevents canine heartworm.

[0014] In some embodiments, the method prevents parasitic infection of a companion animal by Dirofilaria immitis.

[0015] In some embodiments, the emodepside is administered once a month.

[0016] In some embodiments, the emodepside is administered orally.

[0017] In some embodiments, the emodepside is administered orally once a month in an amount from about 0.5 mg / kg to about 5 mg / kg, based on the body weight of the animal.

[0018] In some embodiments, the minimum effective dose (MED), defined as the concentration required to inhibit motility by 80%, is below 1 ^M for L1 stage Dirofilaria immitis larvae. In some embodiments, the MED is about 0.1 ^M for L1 stage Dirofilaria immitis larvae.

[0019] In some embodiments, the method prevents maturation of L1 to L2 larvae.

[0020] In some embodiments, the MED is below 1 ^M for L3 stage Dirofilaria immitis larvae. In some embodiments, the MED is below 0.1 ^M for L3 stage Dirofilaria immitis larvae. In some embodiments, the MED is about 0.03 ^M for L3 stage Dirofilaria immitis larvae.

[0021] In some embodiments, the method prevents maturation of L3 to L4 larvae.

[0022] In some embodiments, the emodepside is administered as a pharmaceutical composition comprising one or more excipients. In some embodiments, the pharmaceutical composition is a chewable form.

[0023] These and other features, aspects, and advantages of the present disclosure will be apparent from a reading of the following detailed description. The present disclosure includes any combination of two, three, four or more features or elements set forth in this disclosure, regardless of whether such features or elements are expressly combined or otherwise recited in a specific example implementation described herein. This disclosure is intended to be read holistically such that any separable features or elements of the disclosure, in any of its aspects -4- WBD (US) 4892-1795-8322v3and example implementations, should be viewed as combinable, unless the context of the disclosure clearly dictates otherwise. It will therefore be appreciated that this Summary is provided merely for purposes of summarizing some example implementations so as to provide a basic understanding of some aspects of the disclosure. Accordingly, it will be appreciated that the above-described example implementations are merely examples and should not be construed to narrow the scope or spirit of the disclosure in any way. Other example implementations, aspects, and advantages will become apparent from the following detailed description. DETAILED DESCRIPTION

[0024] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof. Before describing several example embodiments of the technology, it is to be understood that the technology is not limited to the details of construction or process steps set forth in the following description. The technology is capable of other embodiments and of being practiced or being carried out in various ways.

[0025] The following description sets forth numerous exemplary configurations, methods, parameters, and the like in order to provide a thorough understanding of various embodiments of the disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments. Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. With respect to the terms used in this disclosure, the following definitions are provided. This application will use the following terms as defined below unless the context of the text in which the term appears requires a different meaning.

[0027] The articles "a" and "an" as used in this disclosure may refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0028] As used herein, the term "and / or" as used in this disclosure may mean either "and" or "or" unless indicated otherwise. -5- WBD (US) 4892-1795-8322v3

[0029] Unless the context requires otherwise, throughout the present specification and claims, as used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0030] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0031] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0032] The phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0033] The term "carrier," as used in this disclosure, may encompass carriers, excipients, and diluents and may mean a material, composition or vehicle, such as a liquid or solid filler, -6- WBD (US) 4892-1795-8322v3diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent (i.e. emodepside), from one organ, or portion of the body, to another organ, or portion of the body of a subject. Carriers should be selected on the basis of compatibility and the release profile properties of the desired dosage form. Exemplary carrier materials may include, e.g., adjuvants, binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, spray-dried dispersions, and the like. See, e.g., Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975. Exemplary carrier materials may also include without limitation any adjuvant, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0034] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" may refer to a material which is not biologically, or otherwise, undesirable—the material may be administered to an individual without causing any substantially undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0035] A "pharmaceutical composition" may refer to a formulation of emodepside and a medium generally accepted in the art for the delivery of the emodepside to a subject, e.g., mammals. Such a medium may include all pharmaceutically acceptable carriers therefor.

[0036] The terms "subject," "individual," and "patient" may be used interchangeably and refer to non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like).

[0037] As used herein, the phrase "a subject in need thereof" refers to a subject, as described infra, that suffers from, or is at risk for, heartworm to be prophylactically or therapeutically treated with emodepside.

[0038] The terms "administer", "administered", "administers" and "administering" are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In certain embodiments, oral routes of administering a composition can be used. -7- WBD (US) 4892-1795-8322v3

[0039] The term "effective amount" or "therapeutically effective amount" refers to that amount of emodepside that is sufficient to affect the intended application including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject, e.g., the weight and age of the subject, the severity of the infection, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose can vary depending on the dosing regimen to be followed, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. Method of Treating and / or Preventing Heartworm Disease

[0040] The disclosure provides a method of preventing, treating, and / or controlling a parasitic disease, e.g., in an animal. In some embodiments, the parasitic disease is dirofilariasis (i.e., heartworm, such as canine heartworm), which can be caused, e.g., by Dirofilaria immitis, Dirofilaria repens, or Dirofilaria honkongensis. The most common hosts are dogs and cats but other mammals such as ferrets and raccoons may also be infected.

[0041] Heartworm infection is a severe and life-threatening disease. Heartworms go through several life stages before they become adults infecting the pulmonary artery of the host mammal, particularly dogs. The worms require the mosquito as an intermediate host to complete their life cycle. The period between the initial infection when a host mammal (e.g., dog) is bitten by a mosquito and the maturation of the transferred worms into adults living in the heart and pulmonary arteries is six to seven months in dogs and is known as the "prepatent period".

[0042] In the life cycle of Dirofilaria immitis, a mosquito ingests Dirofilaria immitis larvae microfilariae (first stage juvenile, L1), after a blood meal from an infected host, e.g., a dog. The L1 larvae develop into second stage larvae (L2) in the malpighian tubules of the mosquito, followed by development of third stage larvae (L3) which enter the mosquito body cavity. Development from the L1 to the L3 stage takes 15-16 days. L3 larvae migrate during blood feeding of the mosquito to the tip of the mosquito’s mouth parts (labium), leave the mosquito, and are deposited on the skin of the host mammal (e.g., dog), where they then migrate through the bite wound into the host. Most L3 larvae molt to fourth-stage larvae (L4) in the definitive host 0-14 days after infection. The L4 larvae migrate to submuscular membranes and subcutaneous tissue of the host and remain dormant. The L4 larvae then migrate to the muscles of the host’s chest and abdomen, and 45 to 60 days after infection, molt to the fifth stage (L5, -8- WBD (US) 4892-1795-8322v3immature adult). Between 75 and 120 days after infection, these immature heartworms then enter the host’s bloodstream and are carried through the heart to reside in the pulmonary artery. The L5 immature adults mature into adults that further migrate to the host’s right ventricle or pulmonary artery 85-120 days after infection. The adults reach maturity in a further 2-month period, sexually reproduce in the pulmonary arteries and right ventricle of the host, and shed microfilariae into the host’s blood, repeating the cycle. Adult male heartworms are around 15 cm in length, and females are around 25 cm in length and their normal life span as adults is calculated to be about 5 years.

[0043] As described herein above, prevention or treatment of early stages of infection (i.e., at the L1 to L3 stage) is desirable. According to the present disclosure, it has been surprisingly found that emodepside is efficacious in suppressing activity of L1 and L3 stage Dirofilaria immitis larvae both in vitro and in vivo.

[0044] With reference to Examples 1-3, emodepside has been demonstrated to have a minimum effective dose of 0.1 ^M against L1 larvae and 0.03 ^M against L3 larvae and to have high activity against the SLO1 ion channel. With reference to Examples 4 and 5, emodepside has in vitro and in vivo pharmacokinetic properties that demonstrate exposure levels above 0.1 ^M in mice. Further, with reference to Example 6, the disclosed combination of efficacy and desirable pharmacokinetic properties support a surprising pharmacodynamic effect when administered to mice at a dose of about 0.5 mg / kg to about 5 mg / kg.

[0045] Accordingly, provided herein is a method of treating and / or preventing heartworm disease in an animal. The method generally comprises administering an effective amount of emodepside or a veterinary / pharmaceutical composition comprising emodepside to the animal. It is noted that pharmaceutical and veterinary compositions are typically provided in dosage units; according to the disclosed method, one or more such dosage units can be administered to an animal. As described herein above, emodepside is a semi-synthetic analog of PF-1022a.

[0046] The route of administration may depend, e.g., on the extent of the infection and / or the type of animal. One of skill in the art will be able to select a suitable composition for the desired route of administration. As such, emodepside or a composition thereof can be administered to the subject enterally (e.g., orally or rectally), topically, or parenterally (e.g., intravenously, intramuscularly, or subcutaneously).

[0047] In some embodiments, emodepside is administered topically. In some embodiments, emodepside is administered orally. In some embodiments, emodepside is administered orally -9- WBD (US) 4892-1795-8322v3as a composition, such as a chewable composition. The formulation of chewable compositions for veterinary administration is known to one of skill in the art. In some embodiments, a chewable emodepside composition comprises fillers such as calcium hydrogen phosphate and cellulose, bindres such as povidone, disintegrants such as croscarmellose sodium, flow aids such as colloidal silica and magnesium stearate, and optionally one or more flavorants.

[0048] In some embodiments, emodepside is the sole active ingredient administered to the animal for treating and / or preventing heartworm disease. For example, in some embodiments, an administered composition does not comprise any other compound known to exhibit heartworm disease treatment and / or prevention.

[0049] The amount of emodepside administered can vary widely, depending on, e.g., the type of animal being treated, the animal’s body weight, the animal’s age, the desired effect, and the extent of infestation / infection. It is to be understood that the dosages may vary depending upon the requirements of each subject and the severity of the disorders or diseases being treated (e.g., the stage(s) of heartworm present within the animal). One of skill in the art will be able to discern a specific efficacious dose. Generally, a therapeutically effective amount is the amount of emodepside sufficient to provide a desired beneficial effect or to otherwise reduce a detrimental non-beneficial event to the subject to whom the emodepside is administered. A therapeutically effective dose can be a dose that produces one or more desired or desirable (e.g., beneficial) effects, such administration occurring one or more times over a given period of time. Also, it is to be understood that an initial, higher dosage (i.e., one or more loading doses) may be administered in order to rapidly achieve the desired plasma concentration. On the other hand, the initial dosage may be smaller than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation (i.e., dose titration).

[0050] In some embodiments, emodepside is administered at a dose of about 0.5 mg / kg to about 5 mg / kg, based on the body weight of the animal. In some embodiments, emodepside is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg based on the body weight of the animal.

[0051] Administration of emodepside may vary in treatment timing. Administration may, for example, be intermittent in time and can be administered daily, weekly, biweekly, monthly, bimonthly, quarterly, or even for longer durations of time. In some embodiments, administration of the desired dose may be presented as a single dose or as divided doses -10- WBD (US) 4892-1795-8322v3administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete, loosely spaced administrations, such as multiple oral dose forms. In some embodiments, the administration is daily for a period of time of at least about one week, two weeks, three weeks, a month, or two months, and up to six months, a year, or multiple years, including for the lifetime of the subject. The time period between treatments can depend upon factors such as the type of animal being treated, and the environment in which the animal resides. One of skill in the art will be able to develop a specific administration protocol for a particular situation. In some embodiments, emodepside is administered as a single dose every day, every week, every month, every three months, every six months, or every year. In some embodiments, emodepside is administered as a single dose once a month.

[0052] In some embodiments, emodepside is orally administered as a single dose once a month in an amount from about 0.5 mg / kg to about 5 mg / kg, based on the body weight of the animal.

[0053] In some embodiments, emodepside is administered orally once a month at a dose of about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg, based on the body weight of the animal.

[0054] It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of emodepside will be determined by the nature and extent of the infection being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a veterinarian will ultimately determine appropriate dosages, frequency and treatment duration to be used. The selected dosage may be repeated as often as appropriate. If side effects develop the amount and / or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice. One of skill in the art will be able to develop a specific administration protocol for a particular situation. Animal

[0055] Animals to which emodepside is administered according to the disclosed method is generally a mammal. The animal can be of any age. In some embodiments, the method relates to the treatment of adult animals. In some embodiments, the method relates to the treatment of juvenile animals. In some embodiments, the method relates to treatment of companion animals (e.g., dogs, cats, llamas, and horses). In some embodiments, the method relates to treatment of livestock (e.g., swine, camel, rabbits, goat, sheep, deer, elk, cattle, and bison). In particular embodiments, the animal is a companion animal, for example, a dog or cat. In specific -11- WBD (US) 4892-1795-8322v3embodiments, the animal is a dog. However, it is to be noted that the disclosed methods and compositions may be relevant to any animal suspected of or known to be infected with heartworm disease. EXEMPLIFICATION

[0056] Aspects of the present disclosure are more fully illustrated by the following examples, which are set forth to illustrate certain aspects of the present invention and are not to be construed as limiting thereof. Example 1. Screening of emodepside against L1 microfilaria of Dirofilaria immitis by immersion assay

[0057] To evaluate the efficacy of emodepside against Dirofilaria immitis, L1 stage larvae were exposed to various concentrations of emodepside by immersion to determine survival or paralysis of the larvae. To perform the assay, L1 microfilaria of Dirofilaria immitis were added to wells of a microtiter plate containing buffer and emodepside (in varying amounts) in DMSO. Microfilaria exposed to DMSO alone served as control. The plate was incubated for up to 144 hours. At 24 hours, the plates were observed and motility assessed to determine survival of the microfilaria. The minimum effective dose (MED), defined as the concentration required to inhibit motility by 80%, was about 0.1 ^M. Example 2. Screening of emodepside against L3 larvae of Dirofilaria immitis by immersion assay

[0058] To evaluate the efficacy of emodepside against Dirofilaria immitis, L3 stage larvae were exposed to various concentrations of emodepside by immersion to determine survival or paralysis of the larvae. The Berkeley isolate of D. immitis was used, and the isolate was obtained as follows: a dog from Berkeley County, South Carolina, USA tested positive for D. immitis in April 2014. The age of the heartworm infection is unknown. Blood was collected from the dog on April 7th, 2014, and blood from the dog was used to infect mosquitoes on April 8th, 2014. The Berkeley heartworm isolate was validated in December 2014 via microfilarial testing, antigen testing, and worm recovery.

[0059] To perform the assay, approximately five L3 stage larvae of D. immitis were added to each of three individual wells of a 24-well plate and incubated with growth media and emodepside. Development to the L4 stage as well as viability was assessed after seven days. A negative control (DMSO, three wells of 5 L3s) was also included in each larval development assay cycle. Each well was filled with 890 μL of in vitro media, 100 μL in vitro media -12- WBD (US) 4892-1795-8322v3containing approximately 5 infective L3 D. immitis, and 10 μL of emodepside stock solution to give the appropriate final concentration in a total volume of 1 mL and 1% of DMSO. As a negative control, 10 μL DMSO was used. The plate was incubated at 37ºC and 5% CO2 with saturated humidity. On Day 7 (±2 hrs), the number of fourth stage larvae (L4) were counted and the viability and / or motility described. The motility of larvae was qualitatively scored as dead, immobile, sluggish, or active. Data for L3 (failure to molt / survive) and L4 stages were reported separately. The minimum effective dose (MED), defined as the concentration required to inhibit motility by 80%, was about 0.03 ^M. Example 3. Screening for activity at the SLO-1 calcium-gated potassium channel

[0060] Emodepside was evaluated for activity at the SLO-1 calcium-gated potassium ion channel. To perform the assay, the SLO-1 channel derived from the nematode C. elegans was expressed in Xenopus oocytes. The membrane potential was clamped at a fixed value and voltage steps applied at regular intervals. At a concentration of 1 ^M, emodepside provided a current potentiation of 276%, demonstrating that emodepside has potent activity at the SLO-1 channel. Example 4. Liver microsome stability

[0061] Emodepside was diluted with DMSO and acetonitrile to provide a 20% DMSO final concentration. Liver microsomes were freshly thawed in a 37 °C water bath and diluted to 0.629 mg / mL (mouse) or 1.258 mg / mL (human) with phosphate buffered saline. NADPH working solution (5 mM) is phosphate buffer was prepared. Compound controls for use as internal standards for LC / MS analysis, terfenadine and tolbutamide, were prepared at 1 mg / mL stock solutions in DMSO. The concentration of terfenadine was 5 ng / mL and the concentration of tolbutamide was 10 ng / mL. Control / emodepside working solution (1.5 µL) was added to 238.5 µL liver microsome working solution in a 1.1 mL mini tube and gently mixed. Samples were pre-incubated in a 37°C shaking water bath for 5 min. The reaction was started by adding 60 µL NADPH working solution. The reaction was mixed by pipetting up and down. After 0, 5, 15, 30 and 60 minutes of incubation, 30 µL of the reaction mixture was transferred to 300 µL quenching solution and mixed well by pipetting. All samples were vortexed vigorously for 1 minute and centrifuged at 4,000 rpm at 4 °C for 15 minutes. Supernatant (100 µL) was mixed with 100 µL distilled water for LC-MS / MS analysis. Emodepside has a half-life of 65.7 minutes, which demonstrates slow conversion of emodepside to its oxidative metabolites. Example 5. Mouse pharmacokinetic assay -13- WBD (US) 4892-1795-8322v3

[0062] Male BALB / c mice weighing 20−30 grams were socially housed and allowed free access to food and water. Studies were conducted in groups of mice with each group containing 3 animals. All groups were dosed with emodepside dissolved in 100% DMSO to the desired concentration and dosing volume. Mice received an oral dose administered by gavage. Sequential blood samples were obtained from a saphenous vein of each animal for 24 hours after dosing. Plasma was separated by centrifugation and stored frozen until analysis.

[0063] For bioanalysis of the plasma samples, standards and quality control samples (QCs) were first prepared in blank male Balb / c mice plasma (K2EDTA anticoagulant). The resulting standard curve in plasma ranged from 1 to 1000 ng / mL. Concentration levels of the QCs in mouse plasma were 2, 200 and 800 ng / mL. To prepare samples for LC-MS / MS bioanalysis, 10 μL of plasma (standard, QC, control blank or sample) was added to 200 µL of internal standard (5 ng / mL terfenadine) in methanol / acetonitrile (1:1, v / v). For double blank samples, 200 µL of blank methanol / acetonitrile (1:1, v / v) was added to the blank matrix. The samples were mixed for 1 minute and centrifuged at 4000 rpm at 4°C for 15 min. The supernatant was transferred and 5x diluted with methanol / water (1:1, v / v, with 0.1% formic acid) for injection into the LC / MS-MS instrument. Pharmacokinetic parameters after oral dosing were calculated by noncompartmental methods using WinNonlin (Certara USA Inc., St. Louis, MO). Data are provided in Table 1, which demonstrate plasma concentrations above 0.1 ^M in the evaluated dose range. This data supports the potential for achieving efficacious plasma concentrations following oral dosing. Table 1. Mouse PK data Avg t1 / 2(hr) Avg Cmax (ng / mL) Avg AUClast(hr*ng / mL) [1 / 3 / 10 mg / kg] 1 / 3 / 10 mg / kg] [1 / 3 / 10 mg / kg] 8.1 / 11.4 / 38.6 67.6 / 255.7 / 813.7 786 / 2200 / 6660 Example 6. Mouse in vivo efficacy

[0064] Mice were inoculated subcutaneously with fifty infective, third-stage larvae (L3) of D. immitis (SC-20 Isolate) harvested from infected mosquitoes. The SC-20 Isolate originated in Greenwood, South Carolina USA and has been maintained under laboratory conditions since October 2017. The isolate was validated June 2019 and has had four passages. Infective L3 -14- WBD (US) 4892-1795-8322v3were harvested from Aedes aegypti (Liverpool strain) into Hanks’ balance salt solution (HBSS) 15 days after membrane feeding on blood (collected from TRS dog 401), which was heparinized, containing 50 to 100 microfilariae / 20 µl. A dissecting stereomicroscope and a small Pasteur pipette were used to remove the infective L3 from the petri dishes in which they had been collected. One hundred D. immitis L3 were counted into separate petri dishes for each individual animal. While counting, the petri dishes containing the L3 were tilted slightly to allow the larvae to settle to one end of the dish. Excess fluid was then removed from the dish using a Pasteur pipette, such that less than 1 mL of fluid (containing L3) remained in the dish. The fluid containing the L3 was drawn into a 1 mL tuberculin syringe.

[0065] Dimethyl sulfoxide with 0.5% TWEEN® 80 was added to the required amount of emodepside based on the desired dosage and sonicated to dissolve. The mice received the formulations orally or as a subcutaneous injection. The individual doses were calculated (rounding up to the next 10 µL) using body weights as determined prior to administration. For subcutaneous administration, a 22-gauge needle was attached to a syringe and the contents of the syringe were injected into each mouse subcutaneously over the caudal dorsum. For oral administration of emodepside, a suitable commercially available feeding tube of sufficient length was used. Mice were restrained by holding near the thoracic region and supporting the lower body. The gavage tube was placed into the diastema of the mouth and gently passed along the upper palate until the esophagus was reached. The tube was passed down the esophagus in one smooth motion. Once proper placement was verified, emodepside was administered via a syringe attached to the gavage tube.

[0066] After humane euthanasia via gradual carbon dioxide asphyxiation followed by cervical dislocation, mice were skinned and the skins were placed epidermis down and pinned to a styrofoam board. The subcutaneous layer was scarified with a dulled #10 surgical blade. The skin and carcass were then allowed to soak in warmed Hanks’ balance salt solution (HBSS) at37° ֯C for a minimum of 1 hour. The larvae from the soaks were then examined and countedusing a stereo dissecting microscope. The minimum effective does (MED 85%) was determined as 50 mg / kg. Example 7. Cell cytotoxicity assay for HEK293 cell line protocol

[0067] HEK-293 is a cell line exhibiting epithelial morphology that was isolated from the kidney of a human embryo. This cell line is routinely used in industrial biotechnology and toxicology research. The viability of HEK-293 cells exposed to emodepside was measured -15- WBD (US) 4892-1795-8322v3using CellTiter-Glo® (Promega cat. # G7570; CTG) based on quantitation of ATP present, which is an indicator of metabolically active cells. In preparation for the assay, CTG buffer and substrate were brought to room temperature. The appropriate volume of CTG buffer, based on manufacturer’s instructions, was combined with the CTG substrate to reconstitute the enzyme / substrate mixture forming the CTG “Reagent”. The CTG Reagent was mixed gently by vortexing, swirling, or inverting the contents to obtain a homogeneous solution. Emosdepside was dissolved in DMSO to make a 10 mM stock solution. The positive control reference compound, staurosporine, was dissolved in DMSO to make a 50 mM stock solution.

[0068] On Day 1, 30 uL of HEK-293 cell suspension (~ 6000 cells) was added to each well of a 384-well plate according to a pre-defined plate layout. The plate was incubated at 37 °C with 5% CO2for 24 hours. On Day 2, the reference compound and emodepside were prepared in a 96-well plate according to the desired testing concentrations. The plate was centrifuged at 1000 rpm for 1 minute. Cell media (164.66 uL per well / Dulbecco’s Modified Eagle’s Medium; DMEM) was added to another 96-well plate. Two microliters of solutions from the intermediate 96-well plate were then added to the plate containing the cell media and shaken at room temperature for 5 minutes. An aliquot (10 μL) was then transferred to the plate containing the cells to achieve the final desired concentrations of cells and assay compounds. The assay plate was centrifuged at 1000 rpm for 1 minute and then incubated at 37 °C with 5% CO2for 48 hours.

[0069] On Day 4, 30 microliters of CTG Reagent was added to each well of the assay plate. The plate was mixed using an orbital shaker at room temperature for 10 minutes. Luminescence from each well was then read using a plate reader (SPARK 10M instrument; Settle time 0 ms; integration time 1000 ms). Raw readings were normalized and converted to a “percent toxicity” using several readings from control wells using only cell media as the “low signal” and control wells using cells with no compound added as the “high signal”. Percent toxicity for emodepside was computed as: = [1 – (sample signal – average low signal) / (average high signal – average low signal)]*100

[0070] Dose-response data was fit using Model 205 in XLfit statistical software with concentration on the x-axis and the computed percent toxicity on the y-axis to yield IC50 values. For emodepside, no statistically significant percent toxicity could be computed up to 250 μM, resulting in an IC50 limit of >250 μM. -16- WBD (US) 4892-1795-8322v3

Claims

CLAIMS What is claimed is:

1. A method of preventing heartworm infection in an animal, the method comprising administering to the animal a parasiticidally effective amount of emodepside.

2. The method of claim 1, wherein the emodepside is administered once a month.

3. The method of claim 1 or 2, wherein the emodepside is administered orally.

4. The method of any one of claims 1-3, wherein the emodepside is administered orally once a month in an amount from about 0.5 mg / kg to about 5 mg / kg, based on the body weight of the animal.

5. The method of any one of claims 1-4, wherein the animal is a companion animal.

6. The method of claim 5, wherein the companion animal is a dog.

7. The method of claim 5, wherein the companion animal is a cat.

8. The method of any one of claims 1-7, wherein the method prevents parasitic infection by Dirofilaria immitis.

9. The method of claim 8, where the minimum effective dose (MED), defined as the concentration required to inhibit motility by 80%, is below about 0.1 ^M for L3 stage Dirofilaria immitis larvae.

10. The method of claim 8, where the MED is about 0.03 ^M for L3 stage Dirofilaria immitis larvae.

11. The method of claim 8, where the MED is below 1 ^M for L1 stage Dirofilaria immitis larvae.

12. The method of claim 8, where the MED is about 0.1 ^M for L1 stage Dirofilaria immitis larvae.

13. The method of any one of claims 1-12, wherein the method prevents maturation of L1 to L2 larvae. -17- WBD (US) 4892-1795-8322v314. The method of any one of claims 1-13, wherein the method prevents maturation of L3 to L4 larvae.

15. The method of any one of claims 1-14, wherein the emodepside is administered as a pharmaceutical composition comprising one or more excipients.

16. The method of claim 15, wherein the pharmaceutical composition is a chewable form. -18- WBD (US) 4892-1795-8322v3

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