WO2010126857A1

WO2010126857A1 - Parasiticidal combinations of macrocyclic lactones and polyether antibiotics

Info

Publication number: WO2010126857A1
Application number: PCT/US2010/032490
Authority: WO
Inventors: Cecil Mark Eppler; Thomas Gerard Cullen; Daniel Howard Cohen; Zhenpeng Xi; Robert Alan Pollet
Original assignee: Wyeth LLC
Current assignee: Wyeth LLC
Priority date: 2009-04-28
Filing date: 2010-04-27
Publication date: 2010-11-04
Anticipated expiration: 2011-10-28

Abstract

Veterinary compositions including a combination of a macrocyclic lactone; and a polyether antibiotic are provided. Also provided is a method of treating or controlling a parasitic infection in a mammal with these veterinary compositions.

Description

PARASITICIDAL COMBINATIONS OF MACROCYCLIC LACTONES AND POLYETHER ANTIBIOTICS

FIELD OF THE INVENTION

The present invention relates to parasiticidal compositions including at least two active agents, and in particular, to parasiticidal compositions in which the two active agents are a macrocyclic lactone and a polyether antibiotic.

BACKGROUND QF THE INVENTION

Parasiticidal formulations are widely used in veterinary medicine to control parasite infestations in and on animals, particularly farm animals. There are numerous types of parasites known to infest animals. These parasites are typically characterized as ectoparasites (which live on the outside of an animal's body, such as fleas, ticks and lice) and endoparasites or helminthes (which live inside an animal's body, such as tape worms, flat worms and round worms).

Parasite infestations, particularly in farm animals, are responsible for significant economic losses throughout the world. Major anthelmintic drug classes used in the control of parasitic roundworms (nematodes) of small ruminants and horses include benzimidazoles, imidothiazoles-tetrahydropyrimidines, and avermectin-milbemycins. However, many parasitic nematodes have developed drug resistance against these major anthelmintic classes. Two new classes of anthelmintics have emerged recently: the cyclooctadepsipeptides and the oxindole alkaloid, paraherquamide. While various analogs of these new drugs have demonstrated promising efficacy against nematodes in a variety of animal hosts, at the present time, no public information is available on the plans for the development of these drugs, and it is unknown whether a new product will be marketed in the foreseeable future.

It is unlikely that development of new anthelmintics will rescue livestock producers from the inevitable losses in productivity and problems of animal welfare that result from a failure to adequately control drug resistant worms. Therefore, there is a need in the art to preserve and/or enhance the efficacy of existing anthelmintics.

Individual animals can often be infected with numerous types of parasites, and other types of organisms, such as bacteria, fungi, viruses, protozoans, insects, etc. requiring multiple treatments with different drugs. Polyether antibiotics exhibit a broad range of biological, antibacterial, antifungal, antiviral, anticoccidal, antiparasitic, and insecticidal activities. Polyether antibiotics improve feed efficiency and growth performance in ruminant and monogastric animals. However, only the anticoccidial activity in poultry and cattle, and the effect on feed efficiency in ruminants such as cattle and sheep, are currently of commercial interest. K-41 is one example of a polyether antibiotic. The manufacturing method and certain properties of K-41 are described in The Journal of Antibiotics 29, 10-14 (1976). JP2000109495 discloses the use of K-41 as an anti-coccidial, anti-nematodal and antibacterial agent and its agrochemical uses are disclosed in JP2000063395. EP0326417 discloses anti-coccidial derivatives of K-41. K-41 is disclosed as an antimalarial agent in JP 2003335667 and in The Journal of Antibiotic (2002) 55(9) 832-834.

The need to treat large numbers of farm animals with multiple parasiticidal or other drug formulations is time-consuming, labor intensive and costly. As a result, formulations containing more than one active agent have been developed. A need exists for alternative formulations effective at treating multiple parasite or other infestations in animals.

SUMMARY QF THE INVENTION

The present invention provides a veterinarily effective combination of at least two active agents. The present invention further provides a synergistic combination of at least two active agents, wherein the interaction of the two active agents has a combined effect that is greater than the sum of their individual effects. An advantage of synergism between two active agents is that lower doses of each active agent can be administered to achieve the same therapeutic effect, while reducing any side effects associated with either active agent. Another advantage is that of enhancement of clinical utility. That is to say, since the combination of active agents according to the present invention shows a better parasiticidal effect than the individual active agents, it is more effective for the treatment of parasites.

The present invention also provides veterinary formulations that are useful for treating parasitic infections in animals, as well as other types of infections/infestations in animals, such as bacterial, viral, fungal, protozoan or insect infections/infestations.

The present invention provides a veterinarily effective combination comprising at least two active agents, one of which is a macrocyclic lactone and one of which is a polyether antibiotic or veterinarily acceptable salts or derivatives thereof. Further, the present invention provides a veterinary composition comprising: a macrocyclic lactone; a polyether antibiotic; and a veterinarily acceptable carrier.

The present invention further provides a method of treating or controlling a parasitic infection, the method comprising administering to an animal in need thereof an effective amount of a combination of a macrocyclic lactone and a polyether antibiotic.

DETAILED DESCRIPTION QF THE INVENTION

The present invention relates to veterinarily effective combinations of at least two active agents, one of which is a macrocyclic lactone and one of which is a polyether antibiotic. As used throughout the specification and claims, whenever the terms "macrocyclic lactone" and "polyether antibiotic" are used it is to be understood that these terms are intended to include veterinarily acceptable salts or veterinarily acceptable derivatives of these agents.

The invention relates to veterinary compositions including a combination of a macrocyclic lactone and a polyether antibiotic. Applicants have surprisingly discovered that these combinations of a polyether antibiotic and a macrocyclic lactone have a synergistic effect.

As used herein, the term "synergy", "synergism" and the like is intended to mean that the interaction of two or more agents produces a combined parasiticidal effect that is greater than predicted from the separate parasiticidal effects of the individual agents. Predictions may be based on the sum of the separate effects or use of a specialized mathematical formula (e.g., Colby equation [Colby, S. R. in Calculating Synergistic and Antagonistic Responses of Herbicide Combinations, Weeds, (1967), 15 (1 ), 20-22] ). Synergy may alternatively, or additionally, be defined as the process in which two or more agents work together to enhance the functions and effects of one another. It is to be understood that synergism may be obtained irrespective of whether the at least two active agents are administered simultaneously, separately or sequentially.

In particular, in tests employing a model non-parasitic nematode (C. elegans) cultured in vitro, Applicants have found that concentrations of polyether antibiotics not giving apparent anti-nematodal effects enhanced the anti-nematodal activities of macrocyclic lactones. Furthermore, through in vivo tests employing rodents infected with a parasitic nematode (T. colubriformis),, Applicants have found that an in vivo administered combination of a polyether antibiotic and a macrocylic lactone provides increased anti-nematodal activity as compared to in vivo administration of either the polyether antibiotic or the macrocyclic lactone alone.

The term "polyether antibiotic" as used herein refers to a class of antibiotics characterized by multiple tetrahydrofuran and tetrahydropyran rings connected by aliphatic bridges, direct carbon-carbon linkages, or spiro linkages. Other features include a free carboxyl group, many lower alkyl groups, and a variety of functional oxygen groups. These structural features enable transport of cations across lipid membranes. Examples of polyether antibiotics include K-41 , monensin, maduramicin, lasalocid, salinomycin and narasin, among others. Brimble, M.A. in Polyether Antibiotics, Kirk-Othmer Encyclopedia of Chemical Technology, 5th Edition [2006], A. Seidel, Ed., John Wiley & Sons, Inc., Volume 20, pp. 119-148.

The term "macrocyclic lactone" as used herein designates a pharmaceutical compound in the avermectin or milbemycin family of compounds including avermectins such as ivermectin, abamectin or doramectin, and milbemycins such as milbemycin D or moxidectin, or combinations thereof.

The term "veterinarily acceptable salts", "salts" and the like as used herein refers to salts prepared from veterinarily and/or pharmaceutically acceptable non-toxic acids, including inorganic salts, and organic salts. Suitable non-organic salts include inorganic and organic acids such as acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, malic, maleic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic and the like.

The term "veterinarily acceptable derivatives", "derivatives" and the like as used herein is intended to include salt derivatives and analogs of the compound(s). The term "veterinarily acceptable carriers", "carrier" and the like as used herein are those that are compatible with the other ingredients in the formulation and biologically acceptable. The compounds of the present invention may be administered neat or in combination with conventional veterinary or pharmaceutical carriers. Applicable solid carriers can include one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents or an encapsulating material. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Liquid carriers may be used in preparing solutions, suspensions, emulsions, syrups, and elixirs. The active ingredients of this invention can be dissolved or suspended in a veterinarily or pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or veterinarily or pharmaceutically acceptable oils or fat. The liquid carrier can contain other suitable veterinary additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo- regulators. Suitable examples of liquid carriers for oral and parenteral administration include water, alcohols and their derivatives, and oils. As used herein, "benzimidazole compound" designates a veterinary compound of the benzimidazole chemical family such as thiabendazole, cambendazole, parbendazole, mebendazole, fenbendazole, oxfendazole, oxibendazole, albendazole, albendazole sulfoxide, thiophanate, febantel, netobimin, or thiabendazole.

As used herein, the term "w/v" designates weight/volume, and the term "mg/kg" designates milligrams per kilogram of body weight.

As described herein, the compositions of the present invention include a combination of a macrocyclic lactone and a polyether antibiotic. It is desirable to administer the polyether compound in combination with a macrocyclic lactone to enhance the parasiticidal activity of the composition. Furthermore, it is desirable to combine a macrocyclic lactone with a polyether antibiotic in order to enhance the spectrum of activity. For example, polyether antibiotics are active against a broad range of organisms, including bacteria, fungi, viruses, coccidia (small protozoans), parasites and insects. Macrocyclic lactones, such as moxidectin, are highly active against round worms and arthropods, such as insects, ticks, and lice. Therefore, by combining a macrocyclic lactone with a polyether antibiotic, the spectrum of organisms to be controlled is broadened.

It has now been found that a veterinary composition wherein the active agents are macrocyclic lactone and a polyether antibiotic may be formulated as a parasiticidal composition wherein the active agents synergistically interact to provide enhanced anti- nematodal activity relative to either active agent alone. The compositions are suitable for administration to a homeothermic animal. The composition is effective against nematodes. Furthermore, in view of the broad range of activity of polyether antibiotics, the composition is expected to provide efficacy against other organisms as well, such as, but not limited to, protozoans, bacteria, fungi and viruses. Macrocyclic lactones suitable for use in the present invention include avermectins, milbemycins or combinations thereof. Suitable avermectins for use in the present invention include ivermectin, abamectin and combinations thereof. Suitable milbemycins for use in the present invention include, but are not limited to, moxidectin, doramectin, nemadectin, salts thereof and combinations thereof.

Polyether antibiotics suitable for use in the present invention include, but are not limited to, the following: K-41 , maduramicin, monensin, lasalocid, salinomycin, narasin and combinations thereof. Derivatives, including salts and analogs, of these polyether antibiotics may also be employed, alone or in combination. The effective amounts of the macrocyclic lactone compound and the polyether antibiotics may vary according to the potency of the compounds, the method of application, the host animal, the target parasite, the degree of infestation, or the like. In general, amounts of about 0.01-15.0% w/v, preferably 0.05-10.0% w/v and more preferably, 0.5% w/v of a macrocyclic lactone, such as moxidectin, are suitable. Furthermore, suitable amounts of the polyether antibiotic are, in general, amounts of about 1-10% w/v preferably 3-7% w/v.

In one embodiment, the weight ratio of macrocyclic lactone to polyether antibiotic is about 0.001 - 0.05:1. In a preferred embodiment, the weight ratio of macrocyclic lactone to polyether antibiotic is about 0.01 - 0.03:1. In one preferred embodiment, the macrocyclic lactone is moxidectin and the polyether antibiotic is K-41.

In another preferred embodiment, the macrocyclic lactone is moxidectin and the polyether antibiotic is maduramicin.

In a further preferred embodiment, the macrocyclic lactone is moxidectin and the polyether antibiotic is monensin.

In a still further embodiment, the macrocyclic lactone is ivermectin and the polyether antibiotic is K-41.

In yet another embodiment, the macrocyclic lactone is nemadectin and the polyether antibiotic is K-41. In yet another embodiment, the macrocyclic lactone is ivermectin and the polyether antibiotic is maduramicin.

The combination/composition of the present invention may further include other anthelmintics in addition to the macrocyclic lactone and the polyether antibiotic. In one embodiment, the compositions may include a benzimidazole compound or a salt thereof. Benzimidazoles suitable for use in the composition include thiabendazole, cambendazole, parbendazole, mebendazole, fenbendazole, oxfendazole, oxibendazole, albendazole, albendazole sulfoxide, thiophanate, febantel, netobimin, thiabendazole, salts thereof, and combinations thereof. In one preferred embodiment, the benzimidazole compound is oxibendazole, albendazole, fenbendazole, mebendazole, thiabendazole or a salt thereof or combination thereof. In another embodiment, the benzimidazole anthelmintic is thiabendazole or a salt thereof. Triclabendazole is highly effective against liver fluke at all stages of their life cycle.

In one embodiment, a benzimidazole compound or a salt thereof may be present in the composition in an amount of about 10% to about 40% w/v. In general, amounts of about 15-25% w/v, of a benzimidazole compound, such as triclabendazole, are preferred.

In one embodiment, closantel or an organic amine salt of closantel may be present in the composition. Organic amine salts suitable for use in the present invention include, but are not limited to, alkanol amine salts such as ethanolamine, diethanolamine, methylpropanol amine, or the like; N-methyl glucamine, piperidine, piperazine, triethylamine, methyl amine, α-methylbenzyl amine, or the like. Typically, such salts are prepared by contacting closantel with a solution of the organic amine. Organic amine salts are generally described, for instance, in U.S. Patent No. 4,005,218. In general, amounts of about 5% w/v to 60% w/v of closantel or an organic amine salt thereof are suitable for use in the present invention.

In one embodiment, compositions of the present invention may include one or more anthelmintics belonging to the class of imidothiazoles-tetrahydropyrimidines, which are used in the control of parasitic nematodes. Drugs belonging to this class include, for example, levamisole and pyrantel.

In one embodiment, the composition of the present invention includes pyrantel, which is a tetrahydropyrimidine. Pyrantel may be present in the composition in an amount of about 0-15% %w/v.

In another embodiment, the composition includes an imidothiazole, such as levamisole or tetramisole or a salt thereof. In general, suitable amounts of levamisole or tetramisole or a salt thereof are in the range of about 0-30% %w/v.

In a further embodiment, the composition includes praziquantel. Praziquantel has high efficacy against cestode parasites. Praziquantel may be present in the composition in an amount of about 2-20% w/v. The compositions may include other classes of anthelmintics, such as cyclooctadepsipeptides or oxindole alkaloids. In one embodiment, the composition includes emodepside or paraherquamide.

Compositions of the present invention may also contain other typical formulation excipients, such as surfactants, solvents, stabilizing agents or combinations thereof. Buffering agents or preservatives may also be included.

Surfactants may be included in the composition in amounts of about 0-30% w/v, preferably 0.01-20% w/v, and more preferably 0.1-15% w/v. A surfactant may be selected from non-ionic, cationic, anionic or amphoteric surfactants. Nonionic surfactants may be preferable.

Surfactants suitable for use in the composition include, but are not limited to, one or more of the following: Cs-Cio alkylphenol ethoxylates (e.g., Teric N9, Teric N20, Teric N100); C₉-Ci₇ alcohol ethoxylates (e.g., Teric 9A2, Teric 16A16); C₈-C₂O alkyl amine ethoxylates (e.g., Teric 13M15, Teric 18M20); castor oil ethoxylates (e.g., Cremophor EL, Acconon CA-5, Teric 380); lanolin alcohol ethoxylates (e.g., Polycol 5, Polycol 40); sorbitan fatty acid ester ethoxylates (e.g., Polysorbate 20, Polysorbate 60, Polysorbate 80); sorbitan fatty acid esters (e.g., sorbitan monoisostearate, sorbitan monostearate, Hodag SML and Span 25); polyoxyethylenated alkyl ethers; polyethylene glycol fatty acid esters (e.g., polyethylene glycol mono- or distearate); polyglycerol esters; polyoxyethylenated fatty alcohols; polyoxyethylenated fatty acids; copolymers of ethylene oxide; copolymers of propylene oxide; polyethylene glycol glycerol fatty acid esters; alcohol-oil transesterification products; polyglycerized fatty acids; propylene glycol fatty acid esters; mono-, di- and triglycerides and combinations thereof. In one embodiment, the surfactant is a mixture of mono-, di-, and triglycerides and mono- and di-fatty acid esters of polyethylene glycol. For example, a suitable surfactant is

Labrasol® (Gattefosse, Saint-Priest, France), which is composed mainly of PEG esters and glycerides with medium acyl chains. Labrasol® is also known as PEG-8 caprylic/capric glycerides. Another suitable surfactant is PEG-6 caprylic/capric glycerides. Other surfactants suitable for use in the compositions are those such as polyethylene glycol monolaurate, polyethylene glycol dilaurate, polyethylene glycol monooleate, polyethylene glycol dioleate or glycerin polyethylene glycol coconut oil.

A stabilizing agent may be added to the composition. Stabilizing agents may be present in the composition in amounts of about 0-5% w/v, and preferably 0-2% w/v. Suitable stabilizing agents include, but are not limited to, C₁-₁₂ alkyl gallate; d-₆ alkyl hydroxybenzoate or a salt thereof; benzyl hydroxytoluene; a quinone or a salt thereof; nordihydroguaiaretic acid; a tocopherol; dilauryl thiodipropionate; monothioglycerol; potassium metabisulfite; sodium formaldehyde sufoxylate; sodium thiosulfate; thioglycolic acid; thiourea; ascorbyl palmitate; cysteine or a salt thereof; ethoxyquin, isoascorbic acid; ethylene diamine tetra-acetic acid or a salt thereof; potassium bisulfate; sodium metabisulfite; sodium bisulfite; thiosorbitol; fumaric acid; malic acid; and combinations thereof. In one embodiment, the stabilizing agent is butylated hydroxytoluene (BHT). Organic solvents may be present in the composition. Suitable organic solvents include, but are not limited to, glycols, glycol ethers, glycol ether acetate, Ci-Cs alkyl pyrrolidones, lactone solvents (e.g., butyrolactone, γ-hexalactone), aliphatic hydrocarbons, aromatic hydrocarbons, and combinations thereof. The preferred alilphatic hydrocarbons are those with a boiling point between 30 and 320⁰C, such as hexane, heptane and pentane. The preferred aromatic hydrocarbons are those with a boiling point between 100 and 240⁰C, such as toluene and xylene.

The composition may further include excipients, which are commonly used in parasiticidal compositions including, but not limited to, dyes, perfumes, preservatives, viscosity modifiers, suspending agents, antifoam agents or combinations thereof. Advantageously, the veterinary composition provides easy administration and effective biovailability of the anthelmintic ingredients. Accordingly, the present invention provides a method of treating or controlling a parasitic infection, the method comprising administering to an animal in need thereof an effective amount of a combination of a macrocyclic lactone and a polyether antibiotic. The macrocyclic lactone and the polyether antibiotic may be administered in a therapeutically effective amount simultaneously (such as individually at the same time, or together in a veterinary composition), and/or successively with one or more compounds of the present invention. In either case, the treatment regimen will provide beneficial effects of the synergistic combination of active agents in treating parasite infections/infestations. In one embodiment, the macrocyclic lactone and the polyether antibiotic are administered simultaneously. In another embodiment, the macrocyclic lactone and the polyether antibiotic are administered separately, such as sequentially.

The parasiticidal composition may be formulated as an oral composition, such as an oral drench, a gavage, a feed additive, such as a granule or grain capsule, a tablet, powder, pill, liquid suspension, emulsion or the like; a topical composition, such as a pour-on, spray, dip, spot-on, ointment, salve, patch or the like; an injectable composition, a suppository or implant, and the like.

Animals suitable for treatment in the method include: swine, cattle, sheep, horses, goats, camels, water buffalos, donkeys, fallow deer, reindeer, or the like, preferably swine, cattle, horses or sheep.

The method of this invention is useful for the treatment and control of endo- and/or ectoparasitic infections or infestations in an animal. Infections and infestations that may be treated or controlled by the method of the invention include, but are not limited to, Helminthiases caused by helminthes, such as Ostertagia circumcincta,

Haemonchus contortus, Thchostrongylus colubriformis, or the like; nematode infections caused by, for example, Haemonchus, Ostertagia, Cooperia, Oesphagostomum, Nematodirus, or the like; acarid infestations, such as biting lice, i.e., Damalinia ovis, Damalinia bovis, or the like; chorioptic, sarcoptic or demodectic mange caused by Chorioptes bovis, Sarcoptes scabiei and Demodex, respectively; Psoroptic mange caused by Psoroptes ovis; arthropod endo-parasites, such as cattle grubs; and the like. In actual practice, the composition of the invention may be administered in dose rates of mg of active agent per kg of body weight of the host animal. Dose rates suitable for use in the method of invention will vary depending upon the mode of administration, the species and health of the host animal, the target parasite, the degree of infection or infestation, the breeding habitat, the potency of the additional parasiticidal compound, and the like. In general, the macrocyclic lactone and the polyether antibiotic are each administered in an amount of about 1 to about 100 mg/Kg.

The present invention also provides a process of preparing a veterinary composition. The process includes combining a macrocyclic lactone and a polyether antibiotic with a carrier solvent. In one embodiment, said combining comprises dissolving the macrocyclic lactone in the solvent to form a macrocyclic lactone/solvent solution; and mixing the macrocyclic lactone/solvent solution with the polyether antibiotic to form the composition. In another embodiment, said combining comprises dissolving the polyether antibiotic in the solvent to form a polyether antibiotic/solvent solution; and mixing the polyether antibiotic/solvent solution with the macrocyclic lactone to form the composition. Macrocyclic lactones suitable for use in the process include milbemycin D, avermectin, ivermectin, abamectin, doramectin, moxidectin, or combinations thereof, preferably moxidectin.

Polyether antibiotics suitable for use in the process include K-41 , maduramicin, monensin, lasalocid, salinomycin, narasin and combinations thereof.

In one embodiment, the process further includes adding additional active agents to the composition, examples of which are the same as those described above.

As will be clear to persons skilled in the art, where compositions according to the present invention are to be used, or are to be prepared for use, in veterinary medicine, they may also contain additional carriers, stabilizing agents, buffering agents, preservatives or other excipients well know in the art.

For a more clear understanding of the invention, the following examples are set forth herein below. These examples are merely illustrative and are not understood to limit the scope or underlying principles of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the examples set forth hereinbelow and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. It is noted that the abbreviation n.a. [or] NA, which appears in some of the tables in the Example section below stands for Not applicable; Not available.

EXAMPLES

Example 1 -Method for In vitro testing of the efficacy of various macrolide:polvether combinations against nematodes C. elegans is a non-parasitic soil-dwelling nematode. It has morphology and drug sensitivities similar to parasitic nematodes and is useful in early-stage evaluation of nematicidal compounds (see Simpkin, K. G and Coles, G. C. in The Use of Caenorhabditis Elegans for Anthelmintic Screening [1981] J. Chem. Tech. Biotechnol. Vol. 31 : pp. 66-69) Test compounds were dissolved in solvent and added (1 μl) to each well of a 96-well plate at the final concentrations indicated in Tables 2-7 below. Concentrations of the macrolide (when present) were varied by serially diluting macrolide stock solutions such that the doses of the macrolide decreased to 1/3 of the previous dose in each dilution step. A mixture of C. elegans in buffer is added to each well, such that worms of mixed stages (L1 , l_2, L3, l_4 and adults) are present in each well. In each experiment, the macrolide and polyether were each tested individually and in combination, with each condition being assayed in duplicate. Observations were made under a dissecting microscope. A rating was based on the motility of the larvae and adults, using the rating system shown in Table 1 below. The results obtained using the method of this example are presented in Tables 2-7 below for the specific macrolide:polyether combinations. A higher score indicates greater efficacy.

Table 1. Activity Score

Rating Description

0 no effect

7 reduced movement in 24 h

8 dead in 24 h

9 dead in 4 h

Example 2-ln vitro testing of the efficacy of Ivermectin :K-41 combination

Employing the method described in Example 1 , the efficacy of the lvermectin:K- 41 combination was evaluated. The results are presented in Table 2 below, where scores for each duplicate are shown separately.

Table 2. Ivermectin and K-41

The results presented in Table 2 surprisingly indicate that, when testing a combination of ivermectin and K-41 , concentrations of K-41 not giving apparent effects on C. elegans enhanced the activity of ivermectin. These results support the synergistic effect of the ivermectin: K-41 combination.

Example 3- In vitro testing of the efficacy of nemadectin:K-41 combination

Using the method described in Example 1 , the efficacy of the nemadectin:K-41 combination was evaluated. The results are presented in Table 3 below, where scores for each duplicate are shown separately. Table 3. nemadectin and K-41

The results presented in Table 3 surprisingly indicate that, when testing a combination of nemadectin and K-41 , concentrations of K-41 not giving apparent effects on C. elegans enhanced the activity of nemadectin. These results support the synergistic effect of the nemadectin: K-41 combination.

Example A-In vitro testing of the efficacy of moxidectin:K-41 combination The method described in Example 1 was used to evaluate the efficacy of the moxidectin:K-41 combination. The results are presented in Table 4 below, where scores for each duplicate are shown separately.

Table 4. Moxidectin and K-41

The results presented in Table 4 surprisingly indicate that, when testing a combination of moxidectin and K-41 , concentrations of K-41 not giving apparent effects on C. elegans enhanced the activity of moxidectin. These results support the synergistic effect of the moxidectin: K-41 combination.

Example 5-ln vitro testing of the efficacy of moxidectin:maduramicin combination

Employing the method described in Example 1 , the efficacy of the moxidectin:maduramicin combination was evaluated. The results are presented in Tables 5 and 6 below, where scores for each duplicate are shown separately. Table 5. Moxidectin and Maduramicin (6.3 ppm - 100 ppm)

The results presented in Table 5 surprisingly indicate that, when testing a combination of moxidectin and maduramicin, concentrations of maduramicin (6.3 ppm - 100 ppm) not giving apparent effects on C. elegans enhanced the activity of moxidectin. These results together with the results shown in Table 6 below support the synergistic effect of the moxidectin: maduramicin combination.

Table 6. Moxidectin and Maduramicin (3.2 ppm - 1.6 ppm)

The results presented in Table 6 surprisingly indicate that, when testing a combination of moxidectin and maduramicin, concentrations of maduramicin (1.6 ppm and 3.2 ppm) not giving apparent effects on C. elegans enhanced the activity of moxidectin. These results together with the results shown in Table 5 above support the synergistic effect of the moxidectin: maduramicin combination. Example 6- In vitro testing of the efficacy of moxidectin:monensin combination

The method described in Example 1 was used to evaluate the efficacy of the moxidectin:monensin combination. The results are presented in Table 7 below, where scores for each duplicate are shown separately.

Table 7. Moxidectin and Monensin

The results presented in Table 7 surprisingly indicate that, when testing a combination of moxidectin and monensin, concentrations of monensin not giving apparent effects on C. elegans enhanced the activity of moxidectin. These results support the synergistic effect of the moxidectin: monensin combination.

Example 7- Method for In vivo testing of the efficacy of various macrolide:polvether combinations against nematodes

Larvae of the parasite nematode strain T. colubriformis were obtained from fecal cultures of infected sheep. Animals were de-wormed prior to experimental infection and propagation of nematodes. Fecal material collection began on day 21 post-infection for T. colubriformis. Larvae were harvested, rinsed with deionized water and held in a refrigerator until use.

Gerbils of were used in all studies. On the day of infection (day-7), gerbils were weighed and infected via oral gavage with T. colubriformis. Test compounds were formulated as a suspension in dosing solution and administered orally. Nemadectin was used as the positive control in the studies. Dosing solution alone was administered to a group of gerbils and served as the negative control (untreated) in all studies.

Experimental compounds were tested for activity at the various point doses indicated in Tables 8-15 below. K-41 , maduramicin, nemadectin, moxidecin were used in all experiments. A minimum of three gerbils per treatment were used for each condition.

Approximately 4 days post-treatment, gerbils were euthanized and the small intestine of the gastrointestinal tract was removed and processed for enumeration of T. colubhformis. After mixing, the gut contents were transferred to a Petri dish and worms were enumerated under a microscope.

Example 8-ln vivo analysis of nemadectin:K-41 combination

The method described in Example 7 above was used to evaluate the efficacy of the nemadectin:K-41 combination against the nematode strain T. colubriformis.

Representative results from two independent studies are presented in Tables 8 and 9 below.

Table 8. Analysis of Nemadectin:K-41 Combination

The results presented in Table 8 above demonstrate that both nemadectin:K-41 combinations had substantially lower worm counts than 0.03 mg/kg nemadectin alone. Moreover, both nemadectin:K-41 combinations had substantially lower worm counts than that of 2 mg/kg K-41 alone. These results support the synergistic interaction between nemadectin and K-41.

Table 9. Analysis of Nemadectin:K-41 Combination

The results presented in Table 9 above demonstrate that the 0.02 mg/kg nemadectin + 2mg/kg K-41 combination was more effective against T. colubriformis than either active agent alone. These results support the synergistic interaction between nemadectin and K-41.

Example 9- In vivo analysis of moxidectin:K-41 combination The method described in Example 7 above was used to evaluate the efficacy of the moxidectin:K-41 combination against the nematode strain T. colubriformis. Representative results from an individual study are presented in Table 10 below.

Table 10. Analysis of Moxidectin:K-41 Combination

The experimental results presented in Table 10 show that moxidectin doses over a range from 0.135 to 0.005 mg/kg gave dose related control of T. colubriformis from 100% to 0%. Furthermore, the results show that the combination of 0.015 mg/kg moxidectin + 2 mg/kg K-41 gave a parasiticidal effect substantially greater than that of either active agent alone and at least equivalent to the added effects of the individual active agents (% decrease for treatment group G > the combined % decreases for treatments D and F). The combination of 0.005 mg/kg moxidectin + 2 mg/kg K-41 gave an effect substantially greater than that of 0.005 mg/kg moxidectin, but not significantly greater than that of 2 mg/kg K-41 (H vs. E and H vs. F, respectively). These trends support the synergistic interaction between moxidectin and K-41.

Summary of moxidectin: K-41 results from four separate experiments Results obtained from four separate experiments where 0.015 mg/kg moxidectin and 2 mg/kg K-41 were tested individually and in combination against T. colubriformis in gerbils are summarized in Table 11 below. It is noted that Experiment 1 in Table 11 corresponds to the individual study presented in Table 10.

Table 11. Summary of moxidectin:K-41 results

As shown in Table 11 above, in three of the four experiments, the % decrease in worm counts in gerbils treated with the combination of moxidectin + K-41 was substantially greater than predicted by the sum of the effects of each active agent alone. This was also seen in the combined results, representing pooled worm counts from all four experiments, where the ratio of observed/expected effect was 1.4 indicating a synergistic interaction between moxidectin and K-41. Synergistic effect of moxidectin:K-41 combination further evidenced by the Colby equation

Synergism between moxidectin and K-41 was further evidenced with the aid of the Colby eguation (see Colby, S. R. In Calculating Synergistic and Antagonistic Responses of Herbicide Combinations, Weeds, (1967), 15 (1 ), 20-22):

Expected % Effect = X + Y - ([X x Y] /100 X=Observed % Effect of component A Y=Observed % Effect of component B

The Colby equation compensates for the fact that two compounds, each with

50% efficacy against the same target, should not give 100% efficacy when added together because each compound depletes the other compound's target. The Expected % Effect is the % effect expected from the additive contribution of the individual components. If the Expected % Effect of the combination is lower than the experimentally observed effect of the combination, synergism has occurred.

The results of the moxidectin:K-41 combination are presented in Table 12 below for four separate experiments where the concentrations of the components employed in the treatments are shown as mg/kg amounts. The % decrease in worm counts of the components individually and in combination is based on differences of the geometric means from the negative control.

Table 12. Effects of Moxidectin, K-41 , and Moxidectin:K-41 Combinations on Worm Counts in Gerbils

The results presented in Table 12 above support a synergistic interaction between moxidectin and K-41 , as evidenced by actual/expected ratios of > 1 in at least three of the four experiments.

Example 10- In vivo analysis of moxidectin:maduramicin combination

The method described in Example 7 above was used to evaluate the efficacy of the moxidectin:maduramicin combination against the nematode strain T. colubriformis. Representative results from an individual study are presented in Table 13 below. Table 13. Analysis of Moxidectin:Maduramicin Combination

With reference to Table 13 above, worm counts with the combination containing

0.50 mg/kg maduramicin and 0.015 mg/kg moxidectin (group G) were significantly lower than with either component alone (groups C and E). These results support a synergistic interaction between moxidectin and maduramicin.

Summary of moxidectin:maduramicin results from three separate experiments

Results obtained from three separate experiments where 0.015 mg/kg moxidectin and 0.5 mg/kg maduramicin were tested individually and in combination against T. colubriformis in gerbils are summarized in Table 14 below. It is noted that

Experiment 5 in Table 14 corresponds to the individual study presented in Table 13.

Table 14. Summary of Moxidectin:Maduramicin Results.

As shown in Table 14 above, in all three experiments, the % decrease in worm counts in gerbils treated with the combination of moxidectin + maduramicin was substantially greater than predicted by the sum of the effects of each active agent alone. This was also seen in the combined results, representing pooled worm counts from all three experiments, where the ratio of observed/expected effect was 1.2, indicating a synergistic interaction between moxidectin and maduramicin.

Example 11- In vivo analysis of ivermectin :K-41 combination

The method described in Example 7 above was used to evaluate the efficacy of the moxidectin:maduramicin combination against the nematode strain T. colubriformis. Representative results from an individual study are presented in Table 15 below.

Table 15. Analysis of lvermectin:K-41 Combination

With reference to Table 15 above, worm counts with the combination containing 2.0 mg/kg K-41 and 0.030 mg/kg ivermectin (group F) were lower than with either component alone (groups C and E). These results support a synergistic interaction between ivermectin and K-41. In addition, worm counts with the combination containing 2.0 mg/kg K-41 and 0.015 mg/kg ivermectin (group G) were lower than with either component alone (groups D and E). These results also support a synergistic interaction between ivermectin and K-41.

Example 12- Comparative Evaluation of the Efficacy of moxidectin - K41 Against Blowfly Larvae In this evaluation, filter paper discs were treated with an acetone solution of test compound and allowed to dry. Bovine serum and newly emerged larvae of blowfly, Lucilia sericata, were added to the treated filter paper. Mortality was assessed at 24h and 48h. When more than one evaluation was performed, the data were averaged.

The results of the effects of moxidectin:K-41 combinations on insect (blowfly) larvae are shown in Table 16 below. Table 16. Effects of Moxidectin:K-41 Combinations on Insect (Blowfly) Larvae

Referring to Table 16, a series of moxidectin doses ranging from 3 to 0.0013 ppm was tested in blowfly larvae, either alone or in the presence of K-41 at doses ranging from 4 to 0.44 ppm. As summarized in Table 16, K-41 at the doses tested was inactive. The 0.44 ppm dose of K-41 produced strong synergy with moxidectin, with lethality to blowfly larvae at doses of moxidectin 30- to 100-fold lower than when tested alone. These results support a synergistic interaction between moxidectin and K-41.

Claims

What is Claimed is:

1. A veterinarily effective combination comprising at least two active agents, one of which is a macrocyclic lactone and one of which is a polyether antibiotic or veterinarily acceptable salts or derivatives thereof.

2. The combination of claim 1 , wherein the macrocyclic lactone is an avermectin a milbemycin, or a combination thereof.

3. The combination of claim 2, wherein the avermectin is selected from the group consisting of ivermectin, abamectin and combinations thereof

4. The combination of claim 2, wherein the milbemycin is selected from the group consisting of moxidectin, nemadectin and combinations thereof

5. The combination of any of claims 1 to 4, wherein the polyether antibiotic is selected from the group consisting of K-41 , maduramicin, monensin, lasalocid, salinomycin, narasin and combinations thereof.

6. The combination of claim 1 , wherein the macrocyclic lactone is moxidectin and the polyether antibiotic is K-41.

7. The combination of claim 1 , wherein the macrocyclic lactone is moxidectin and the polyether antibiotic is maduramicin.

8. The combination of claim 1 , wherein the macrocyclic lactone is moxidectin and the polyether antibiotic is monensin.

9. The combination of claim 1 , wherein the macrocyclic lactone is ivermectin and the polyether antibiotic is K-41.

10. The combination of claim 1 , wherein the macrocyclic lactone is nemadectin and the polyether antibiotic is K-41.

11. The combination of claim 1 , wherein the macrocyclic lactone is ivermectin and the polyether antibiotic is maduramicin.

12. The combination of any of claims 1 to 11 , further comprising an active selected from the group consisting of pyrantel, levamisole or a salt thereof, tetramisole or a salt thereof, praziquantel, a benzimidazole compound or a salt thereof, closantel or an organic amine salt thereof, emodepside, paraherquamide, and combinations thereof.

13. The combination of any of claims 1 to 12, wherein the weight ratio of the macrocyclic lactone to the polyether antibiotic is about 0.001 - 0.05:1.

14. A veterinary composition comprising: a macrocyclic lactone; a polyether antibiotic; and a veterinarily acceptable carrier.

15. The composition of claim 14, wherein the macrocyclic lactone is an avermectin a milbemycin, or a combination thereof.

16. The composition of claim 14 or claim 15, wherein the polyether antibiotic is selected from the group consisting of K-41 , maduramicin, monensin, lasalocid, salinomycin, narasin and combinations thereof.

17. The composition of any of claims 14 to 16, wherein the weight ratio of the macrocyclic lactone to the polyether antibiotic is about 0.001 - 0.05:1.

18. The composition of any of claims 14 to 17, wherein the macrocyclic lactone is present in the composition at a concentration of about 0.01-15.0% w/v.

19. The composition of any of claims 14 to 18, wherein the polyether antibiotic is present in the composition at a concentration of about 1-10% w/v.

20. The composition of any of claims 14 to 19, further comprising an active selected from the group consisting of pyrantel, levamisole or a salt thereof, tetramisole or a salt thereof, praziquantel, a benzimidazole compound or a salt thereof, closantel or an organic amine salt thereof, emodepside, paraherquamide, and combinations thereof.

21. The composition of any of claims 14 to 20, further comprising a component selected from the group consisting of a surfactant, a solvent, a stabilizing agent and combinations therof.

22. A method of treating or controlling a parasitic infection, the method comprising administering to an animal in need thereof an effective amount of a combination of a macrocyclic lactone and a polyether antibiotic.

23. The method of claim 22, wherein the macrocyclic lactone and the polyether antibiotic are each administered in an amount of about 1 to about 100 mg/Kg.

24. The method of claim 22 or claim 23, wherein the macrocyclic lactone and the polyether antibiotic are administered simultaneously or separately.

25. The method of claim 22 or claim 23, wherein the macrocyclic lactone and the polyether antibiotic are administered sequentially.