Maintaining diethylcarbamazine in an extruded daily ration animal feed product

Abstract

A method is disclosed for maintaining a therapeutically effective amount of diethylcarbamazine in an extruded daily ration feed product for animals. The diethylcarbamazine is introduced and mixed into a feed mixture upstream of an extruder die. Additionally, a preservative is introduced and mixed into the feed mixture upstream of the extruder die. The preservative comprises an antioxidant system having a water-soluble antioxidant. The feed mixture is then extruded to form the extruded daily ration feed product having a concentration of diethylcarbamazine that is between 200 milligrams per kilogram to 2,000 milligrams per kilogram. This method ensures stabilization of diethylcarbamazine in the extruded daily ration feed product stored at ambient conditions over a shelf-life period. An effective amount of the therapeutic compound can be delivered to animals as part of their daily ration.

Description

TITLEMaintaining Diethylcarbamazine in an Extruded Daily Ration Animal Feed ProductINVENTORSBradley S. Strahm, Sabetha, Kansas;Cole M. Ahivers, Topeka, Kansas; andJohn C. Kuenzi, Sabetha, Kansas.CROSS REFERENCESThis application is an international application which claims the benefit of U.S. Provisional Patent Application No. 63 / 516,987, entitled “Maintaining Diethylcarbamazine in an Extruded Daily Ration Animal Feed Product,” filed on August 1, 2023, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0001] This disclosure relates to a method of maintaining an effective amount of a therapeutic active in an extruded daily ration feed product by incorporation of an antioxidant.BACKGROUND

[0002] Significant research is directed to determining proper nutritional standards and effective drug treatments for animals. This is true not only in connection with domestic household pets such as dogs, cats, birds, and exotics, but also regarding economically significant animals such as farm animals (e.g., horses, sheep, and cattle) and zoo animals of all types.

[0003] Insofar as drug or active treatment of animals is concerned, these agents are generally administered from time to time by oral administration or injection, so that therapeutic amounts of the actives or drugs can be maintained in the bloodstreams of the animals either continuously or at least during a prescribed treatment period. Active treatments can treat acute or chronic conditions or provide pain management in animals.

[0004] Periodic dosing presents several difficulties. For example, the animal’s caretaker may simply forget to administer a given drug or active at the required time. This can have the effect of disrupting a treatment protocol and even causing harm to the animal. For instance,dogs are conventionally treated monthly with heartworm preventative drugs such as ivermectin. If the dog's owner forgets to timely administer the drug, the dog is susceptible to heart worm infection. Another problem with periodic dosing of animals stems from the fact that the animals may be very reluctant to cooperate, especially if the drug or active is to be orally administered in pill form or by syringe. Any pet owner can testify to the difficulty of persuading the pet to consume the drug product.

[0005] Attempts have been made in the past to provide daily ration feed products which include therapeutic drugs. For example, Hills Pet Food Products made and sold a Science Diet product referred to as “Maximum Stress Diet” which included small amounts of styrylpyridinium chloride and diethylcarbamazine in a canned dog food containing substantial quantities of animal fat which required refrigeration. However, the Maximum Stress Diet is no longer available, and was not optimum in that it required refrigeration and special handling. This is to be contrasted with conventional extruded feed products designed to be stored over extended periods at ambient temperatures without significant loss of nutrients.SUMMARY

[0006] This disclosure relates to a method of maintaining a therapeutically effective amount of diethylcarbamazine in an extruded daily ration feed product for animals including the steps of: (a) mixing into a feed mixture a diethylcarbamazine introduced upstream of an extruder die; (b) mixing into the feed mixture a preservative introduced upstream of the extruder die, wherein the preservative includes: (i) a water-soluble antioxidant; and (c) extruding the feed mixture to form the extruded daily ration feed product, wherein a concentration of the diethylcarbamazine is between 200 milligrams per kilogram to 2,000 milligrams per kilogram of the extruded daily ration feed product.

[0007] In some aspects, the techniques described herein relate to a method, where the preservative includes: (a) an amphiphilic antioxidant.

[0008] In some aspects, the techniques described herein relate to a method, where the preservative includes: (a) a metal chelator.

[0009] In some aspects, the techniques described herein relate to a method, where the preservative includes: (a) a fat-soluble antioxidant; and (b) a tocopherol.

[0010] In some aspects, the techniques described herein relate to a method, where a ratio of water- soluble antioxidant to the fat-soluble antioxidant is greater than 1:1.

[0011] In some aspects, the techniques described herein relate to a method, where a ratio of themetal chelator to the tocopherol is greater than 1:1.

[0012] In some aspects, the techniques described herein relate to a method, further including the step of: (a) distributing the diethylcarbamazine and water-soluble antioxidant throughout the feed.

[0013] In some aspects, the techniques described herein relate to a method, where a tocopherol is incorporated before a preconditioner mix.

[0014] In some aspects, the techniques described herein relate to a method, where the diethylcarbamazine is incorporated after a preconditioner mix.

[0015] In some aspects, the techniques described herein relate to a method, where a metal chelator is incorporated before a preconditioner mix.

[0016] In some aspects, the techniques described herein relate to a method, where the metal chelator includes: (a) a green tea extract.

[0017] In some aspects, the techniques described herein relate to a method, where the metal chelator includes: (a) a tetrasodium pyrophosphate.

[0018] In some aspects, the techniques described herein relate to a method, where the water- soluble antioxidant includes: a plant extract of (a) a rosemary extract; and (b) a spearmint extract.

[0019] In some aspects, the techniques described herein relate to a method, further including the step of: (a) maintaining the therapeutically effective amount of diethylcarbamazine for a period of 6 months up to 40°C and 75% relative humidity.

[0020] In some aspects, the techniques described herein relate to a method, further including the step of: (a) maintaining at least 95% of an amount of diethylcarbamazine for a period of 8 months up to 25 °C and 40% relative humidity.

[0021] In some aspects, the techniques described herein relate to a method, further including the steps of: (a) storing the extruded daily ration feed product in a package with an oxygen transmission rate of up to 0.05 cc / 100 in2-24hr for a period of up to 8 months; and (b) conserving the diethylcarbamazine in the extruded daily ration feed product from less than a five percent of start difference from extrusion.

[0022] In some aspects, the techniques described herein relate to a method, further including the step of: (a) blending the diethylcarbamazine with a carrier before an extruder mix.

[0023] In some aspects, the techniques described herein relate to a method, where the concentration of diethylcarbamazine is further between 500 milligrams per kilogram and 1,500 milligrams per kilogram and a concentration of the preservative is between 200 milligrams per kilogram and 1 ,000 milligrams per kilogram.

[0024] In some aspects, the techniques described herein relate to a method, where theconcentration of diethylcarbamazine is further between 800 milligrams per kilogram and 1,100 milligrams per kilogram and a concentration of the preservative is further between 500 milligrams per kilogram and 750 milligrams per kilogram.

[0025] In some aspects, the techniques described herein relate to a method, further including the step of: (a) stabilizing the diethylcarbamazine in the extruded daily ration feed product for a period of up to 8 months by the preservative being introduced at between 200 milligrams per kilogram and 1,000 milligrams per kilogram.

[0026] The above advantages and features are of representative embodiments only, and are presented only to assist in understanding the invention. It should be understood that they are not to be considered limitations on the invention as defined by the claims. Additional features and advantages of embodiments of the invention will become apparent in the following description, from the drawings, and from the claims.BRIEF DESCRIPTION OF DRAWINGS

[0027] Aspects are illustrated by way of example, and not by way of limitation, in the accompanying drawings, wherein:

[0028] FIG. 1 depicts equipment in an illustrative diagram of a production process that forms a shelf-stable extruded daily ration feed product to maintain a therapeutic amount of diethylcarbamazine.

[0029] FIG. 2 depicts an illustrative graph of stability results of diethylcarbamazine in the shelfstable extruded daily ration feed product stored at 25 °C and 40% relative humidity during storage.

[0030] FIG. 3 depicts an illustrative graph of stability results of diethylcarbamazine in the shelfstable extruded daily ration feed product stored at 40°C and 75% relative humidity during storage.

[0031] FIG. 4 depicts an illustrative flowchart of the production process that forms a shelf-stable extruded daily ration feed product that maintains a therapeutic amount of diethylcarbamazine.DETAILED DESCRIPTION

[0032] A production method is disclosed for maintenance of a therapeutically effective amount of diethylcarbamazine, an active pharmaceutical ingredient, within an improved extruded daily ration feed product that is capable to be stored for long shelf periods at ambient temperatures. The stability of diethylcarbamazine in feed products has been a concern, asthe compound can degrade or lose potency over time, rendering feed products less effective in providing the desired therapeutic benefits. Previous approaches have not adequately addressed the issue of maintaining a therapeutically effective amount of diethylcarbamazine over an extended period. This production method overcomes this problem and provides methods of preparing an extruded daily ration feed product that maintains the active content even when the extruded daily ration feed product is stored at ambient temperatures for long periods. A wide variety of feed product types may be improved in accordance with the disclosed production method, especially extrusion- processed feeds of either dry or semi-moist kind. When the feed products are produced by extrusion they usually contain respective quantities of protein, fat, and starch, together with a relatively minor amount of one or more actives. The potency of the active content of extruded daily ration feed products produced by the production method can be maintained for at least eight months up to past twelve months at ambient temperature storage conditions.

[0033] The production method may include mixing the active, such as diethylcarbamazine, throughout a feed mixture so that an extruded daily ration feed product contains the active at a concentration of between 200 parts per million (ppm) to 2,000 ppm. The diethylcarbamazine is distributed and incorporated into the feed mixture using an extruder. This method allows for a more controlled and uniform distribution of diethylcarbamazine throughout the extruded daily ration feed product. The diethylcarbamazine may be mixed into the feed mixture upstream of a die of an extruder. A water-soluble antioxidant may be mixed into the feed mixture upstream of the extruder die, which may be before a preconditioner. A metal chelator may be mixed into the feed mixture upstream of the extruder die, which may be before the preconditioner. A carrier may be incorporated with the diethylcarbamazine upstream of the extruder die, which may be after the preconditioner. An amphiphilic antioxidant may be incorporated into the feed mixture before mixing by the preconditioner. A fat-soluble antioxidant may be incorporated into the feed mixture before mixing by the preconditioner. A tocopherol may be incorporated into the feed mixture before mixing by the preconditioner.

[0034] The ratio amount of water-soluble antioxidant to fat-soluble antioxidant may be greater than 1:1. The ratio amount of the metal chelator to the tocopherol may be greater than 1:1. The diethylcarbamazine and the antioxidant may be incorporated, together, throughout the feed mixture during the step of extrusion. Distributing the diethylcarbamazine and a water- soluble antioxidant throughout the feed mixture may maintain at least 95% of an original amount of diethylcarbamazine from the end of extrusion to the end of a shelf-life storageperiod (approximately 8 months). The features of the preceding paragraphs may be used singly or in any combination.

[0035] Retention of specific active amounts will vary depending upon the preservative(s) utilized. The preservative(s) may comprise, but are not limited to, the parabens (methylparaben and / or propylparaben), benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butylparaben, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, ethylparaben, imidurea, methylparaben, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium benzoate, sodium propionate, sorbic acid, thimerosal, ascorbic acid, calcium, phydrox benzoic acid esters, and antioxidants.

[0036] “Antioxidants” are components which serve as preservatives to increase the stability of active ingredients which are not stable if exposed to oxygen. In the production method, the preservative is primarily an antioxidant system, singularly or in blended combinations, which may include substances such as a metal chelator, mixed tocopherols, propylene glycol, various acids like ascorbic acid with its water-soluble salts or liposoluble esters, and herbal extracts like rosemary composed of phenolic antioxidants such as rosmarinic acid, camosol, and camosidic acid. Such compounds can be added to bind reducing agents to decrease oxidation or degradation of components in the extruded daily ration feed product. The preservative may comprise antioxidants such as vitamins A, C, E, selenium, and the like to increase the stability of the active ingredients) which are not stable if exposed to oxygen.

[0037] Suitable antioxidants that resist oxidation and loss of bioactivity during processing may be found in Remington: The Science And Practice Of Pharmacy (Twenty Third Edition, Published by Elsevier Inc., 2021). Other suitable antioxidants that may be included in the preservative, but are not limited to, Tenox® 2; Tenox® PG; Tenox® s-i; BHA (2-t-butyl- 4-methoxyphenol); BHT (2,6-di-t-butyl-4-methylphenol); sodium metabisulfite reducing agents; antioxidant synergists such as tocopherols (alpha, beta, or delta-tocopherol, tocopherol esters, alpha-tocopherol acetate), alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, citric acid anhydrous and hydrous, edetic acid and its salts, lecithin, tartaric acid, ascorbic acid, ascrobyl palmitate, fumaric acid, malic acid, sodium ascorbate, sodium meta bisulfate, n-propyl gallate, monothioglycerol, resveratrol, quercetin, benzoic acid, dimethyl thiourea (DMTIJ), hesperetin, tetrahydrocurcumin, tetrahydrodemethoxycurcumin, Trolox (N-acetylcysteine, 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), butylhydroxyanisole, and sodium meta-bisulphit. rosemary extract, mixed tocopherols, cherry extract, vitamin C, acerola (Malpighiaemarginata) extract, green tea extract, sage extract, ascorbyl palmitate, 3,5,4'-trihydroxy- trans-stilbene, Mentha spicate extract, turmeric extract and combinations thereof.

[0038] For example, a chosen antioxidant system may contain a lipophilic antioxidant and a hydrophilic antioxidant. The two types of antioxidants may be incorporated into the feed mixture to preserve lipid components and maintain therapeutic effects of diethylcarbamazine, respectively, over prolonged shelf-life storage periods. Protection of the diethylcarbamazine and lipid components may serve as key parameters in evaluations of the antioxidant system efficiency. The antioxidant may be introduced into a dry mix of the feed mixture at a concentration of at least about 200 milligrams per kilogram (mg / kg) to 2,000 mg / kg of feed product, preferably from about 200 mg / kg to 1,500 mg / kg of the extruded daily ration feed product, more preferably from about 500 mg / kg to 1,500 mg / kg of feed product, and most preferably from about 750 mg / kg to 1 ,250 mg / kg of feed product.

[0039] For example, in extruded daily ration feed products made from this production method, the antioxidant(s) may be present at a concentration of about 0.2% by weight, preferably to about 0.15% by weight, and still more preferably about 0.1% by weight. Among a blend of hydrophobic and hydrophilic antioxidants or a hydrophilic antioxidant(s) that might be utilized are Kemin Nutrisurance, Inc. branded products such as a blend of NaturOx® brand Plus (fat-soluble) and NaturFort™ brand RSGT101 Dry (water-soluble) or Verdilox™ GT (fat- and water-soluble). Other antioxidant systems might be utilized which are made of water-soluble, a blend of fat- and water-soluble, or amphiphilic antioxidants.

[0040] Some possible amphiphilic antioxidants that may be a component of the preservative in the extruded daily ration feed product for animals are tannyl stearate, nitrone-trolox, or other food safe amphiphilic antioxidants identified in the future. Tannyl stearate is derived from reaction of tannic acid with stearic acid and may be synthesized to improve tannic acid solubility in lipid materials. This reaction may give many products having different degrees of esterification (including tannyl mono stearate, tannyl di stearate, tannyl tri stearate, tannyl tetra stearate, tannyl penta stearate, tannyl hexa stearate, and tannyl hepta stearate). Tannyl penta stearate is assumed to be the most suitable amphiphilic antioxidant derivative, where those derivatives with less degree of esterification would be less soluble in fat, and those of higher degree of esterification would exhaust more hydroxyl groups that can cause decreases of antioxidant activity. The addition of tannyl stearate to oil(s) or fat(s) may offer good protection against oxidation. Without being bound to theory, the effectiveness of tannyl stearate as an amphiphilic antioxidant may be attributed to its stability at high temperature. Therefore, tannyl stearate may be found to be a safe compound that can be used as feed additive that undergoes extrusion. Additionally, inhibitors of lipid oxidation,such as various agents that are protective against oxidation caused by free radicals, may be rendered amphiphilic by adding a perfluorinated chain as well as a sugar group to those molecules.In accordance with Table 1 below, a dry mix containing little to no added antioxidants was obtained from Lortscher Animal Nutrition Inc. (LANI) in Brownwood, Texas. The dry mix may comprise additional ingredients such as flaxseed (linseed) raw materials, partially defatted flaxseed flour, hempseed raw materials, partially defatted hempseed flour, rice flour, rice starch, tapioca starch, potato starch, pea protein concentrate, rice protein concentrate, pea protein isolate, oat flour, lentil flour, cereal flour honey, malt syrup, date syrup, maple syrup, salt, steviol glycosides, an antioxidant and / or other nutraceuticals such as turmeric, wheatgrass, chlorella spirulina, etc. The dry mix may be high in protein, high in fiber, and / or high in omega-3 fatty acids. An antioxidant treatment was added to the dry mix. Other preservative treatments may be introduced into the dry mix of the feed mixture. The preservative may be introduced into the feed mixture anywhere upstream of the extruder die.

[0041] Table 1, which follows, sets forth an illustrative dry mix:Table 1

[0042] Several types of antioxidant treatments may be considered for reducing or preventing degradation of diethylcarbamazine into diethylcarbamazine-N-oxide. In the case discussed further below, six different antioxidant treatments were utilized to identify an antioxidantsystem that effectively reduces the oxidation of diethylcarbamazine into diethylcarbamazine-N-oxide under both ambient and accelerated storage conditions. Reduction in antioxidant concentrations were monitored during the shelf-life storage period while sample extruded daily ration feed products continued to be stored at ambient conditions.

[0043] Table 2, which follows, sets forth an illustrative summary of the antioxidant treatments that were added to the dry mix:Table 2

[0044] For Treatments B, C, D, and E, 750 ppm of Naturox® brand Plus from Kemin Nutrisurance, Inc. in Des Moines, Iowa was added to the dry mix and well mixed in a ribbon blender. Kemin’s Naturox® brand Plus is a dry, free-flowing product containing natural dry fatsoluble antioxidants and other ingredients including silicon dioxide, citric acid, natural mixed tocopherols, vegetable oil, and rosemary extract. Application rates of Naturox® brand Plus may be approximately 0.3-0.6 pounds per ton (Ib. / ton) or about 0.15-0.3 kilograms per metric tonne (150-300 ppm) when incorporated into the dry mix. For Treatment F, 750ppm of Kemin’s Verdilox™ GT was added to the dry mix and well mixed in a ribbon blender. Verdilox™ GT is a dry, free-flowing product containing natural fat- and water-soluble antioxidants and a metal chelator such as tetrasodium pyrophosphate among other ingredients including silicon dioxide, vegetable oil, natural mixed tocopherols, rosemary extract, green tea extract, and spearmint extract. Application rates of Kemin’s Verdilox™ GT may be approximately 0.3-0.6 Ib. / ton or about 150-300 ppm when incorporated into the dry mix. For treatments G and H, 750 ppm of Kemin’s Naturox® brand Plus and 0.3% of Kemin’s NaturFORT™ brand RGST101 was added to the dry mix and well mixed in a ribbon blender. NaturFORT™ brand RGST101 is a dry, food-grade, water dispersible, blend of natural flavorings for use in feed products and contains natural water-soluble antioxidantsalong with other ingredients including maltodextrin, silicon dioxide, spearmint extract, rosemary extract, and a green tea extract. Application rates ofNaturFORT™ brand RGST101 may be up to 0.5% which may be based on total weight of the extruded daily ration feed product. The actual inclusion rate of the antioxidant treatments into the dry mix will depend upon the formulations, storage conditions, and desired storage time of the extruded daily ration feed product.

[0045] In accordance with FIG. 1, feed mixtures incorporating the dry mix and antioxidant treatment were introduced into a Wenger® Model 2 DDC preconditioner from Wenger Manufacturing in Sabetha, Kansas. A loss-in-weight feeding system assisted in feeding the dry mix at a dry feed rate of approximately 80 kilograms / hour (kg / hr.). PRO*TEMP Chicken, a stabilized meat product, was obtained from Simmons Feed Ingredients, Inc. in Siloam Spring, Arkansas. The stabilized meat product was added to the preconditioner at a ratio of approximately 26.3% of the dry feed rate (about 21.1 kg / hr.). Fish oil stabilized with mixed tocopherols was obtained from LANI and added to the preconditioner at a ratio of approximately 2.6% of the dry feed rate (about 2.1 kg / hr. or 35 g / min.). Steam was added to the preconditioner at a ratio of approximately 24% of the dry feed rate (about 19.2 kg / hr.) to preheat the material before it passed into the extruder.

[0046] This heated and mixed material, which is referred to herein as the feed mixture, passed from the preconditioner into the extrusion equipment. Extrusion equipment included a Wenger® Model 2 DDC preconditioner and a Wenger® TX-52 twin-screw extruder. The extruder barrel may be made up of a series of interconnected heads. The twin-screw configuration, dies, adaptor parts, preconditioner shafts and beater elements may be configured to subject the feed mixture to form the extruded daily ration feed product.

[0047] A therapeutic amount of one or more actives may be mixed into the feed mixture to form the drug-containing extruded daily ration feed product. One or more actives processed into the extruded daily ration feed product by the production method are stable throughout a shelf-life period. The extruded daily ration feed product is effective to maintain the therapeutic amount of the active within the bloodstream of an animal when consumed daily by the animal. The desired therapeutic amount of drug is achieved and beneficial effects of the active remain undiminished even though the one or more actives typically degrade when not processed by the disclosed production method and stored at ambient conditions. For example, diethylcarbamazine has been shown to oxidize into diethylcarbamazine-N-oxide during long-term storage at ambient conditions.

[0048] An animal consuming extruded daily ration feed products from this production method daily receives a maintenance quantity of the active, so that therapeutic effects are realized.The active may be introduced into the feed mixture so that a concentration of the active is at least about 200 milligrams per kilogram (mg / kg) to 2,000 mg / kg of the extruded daily ration feed product, more preferably from about 500 mg / kg to 1,500 mg / kg of the extruded daily ration feed product, and most preferably from about 800 mg / kg to 1,100 mg / kg of the extruded daily ration feed product. For example, in other in extruded daily ration feed products made by this feed production process, the active(s) may be present at a concentration of about 0.2% by weight, more preferably about 0.15% by weight, and still more preferably about 0.1% by weight.

[0049] Among actives that might be utilized, but are not limited to, are antibiotics, steroids, antiinflammatory agents, anti-parasitic agents, such as anti-protozoa agents, antihelminthics, (e.g. Albendazole, Mebendazole, Niclosamide, Suramin, Thiabendazole, Pyrantel pamoate Octadepsipeptides and the like), antinematodes including, but not limited to, benzimiadazoles (e.g., mebendazole, thiabendazole), avermectins (e.g., ivermectin), pyrantel pamoate, anticestodes, antitrematodes, antiamoebics, antiprotozoals, piperazine, piperazine salts and derivatives, for example diethylcarbamazine, dewormers such as heartworm preventative drugs, other endoparasiticides, ectoparasiticides which are drugs effective against fleas and ticks, and other active ingredients that include those actives against parasites or pests such as afoxolaner, fluralaner, lotilaner, sarolaner, albendazole, cambendazole, fenbendazole, flubendazole, mebendazole, oxfendazole, parabendazole, tiabendazole, triclabendazole, amitraz, demiditraz, clorsulon, closantel, oxyclonazide, rafoxanide, cyphenothrin, flumethrin, permethrin, cyromazine, derquantel, diamphenetide, dicyclanil, dinotefuran, imidacloprid, nitenpyram, thiamethoxam, abamectin, doramectin, emamectin, eprinomectin, moxidectin, selamectin, milbemycin oxime, emodepside, epsiprantel, fipronil, fluazuron, fluhexafon, indoxacarb, levamisol, lufenuron, metaflumizone, methoprene, monepantel, morantel, niclosamide, nitroscanate, nitroxynil, novaluron, oxantel, praziquantel, pyrantel, pyriprole, pyriproxyfen, sisapronil, spinosad, spinetoram, triflumezopyrim, or a salt of any of the foregoing, and combinations thereof.The active agent may also be an antimicrobial active ingredient, for example, various penicillins, tetracydines, sulfonamides, cephalosporins, cephamycins, aminoglucosids, trimethoprim, dimetridazoles, erythromycin, framycetin, fruazolidone, various pleuromutilins such as thiamulin, valnemulin, various macrolides, streptomycin and substances acting against protozoa, e.g. clopidol, salinomycin, monensin, halofuginone, narasin and robenidine.

[0050] The antioxidant system of the disclosed production method maintains a therapeutically effective amount of diethylcarbamazine from converting into diethylcarbamazine-N-oxidewithin the extruded daily ration feed product. In accordance with FIG. 1, diethylcarbamazine was mixed into feed mixtures at about a target dose concentration of 900 mg / kg to form the sample extruded daily ration feed products. Diethylcarbamazine was mixed into the feed mixtures by being first introduced into an active liquid mix. A carrier such as propylene glycol was utilized for incorporation of the diethylcarbamazine into the active liquid mix and / or water, and transferred into the barrel of the extruder. Antioxidants such as 2,4,5-trihydroxybutyrophenone (THBP) and butylated hydroxyanisole (BHA) may be soluble with the carrier and incorporated into the feed mixture alongside the diethylcarbamazine. The diethylcarbamazine was incorporated after a preconditioner. The diethylcarbamazine-containing active liquid mix was passed through a Wenger® TX-52 twin-screw extruder with the feed mixture. The active liquid mix containing the diethylcarbamazine was introduced upstream of an extruder die of the extruder at a ratio of approximately 2.4% of a dry feed rate (about 1.9 kg / hr. or 32 g / min.). Additional steam was added upstream of the extruder die at a ratio of approximately 10% of the dry feed rate (about 8 kg / hr.).

[0051] Materials for preparing active liquids were obtained from multiple sources. Diethylcarbamazine was obtained from ChemWerth, Inc. in Woodbridge, Connecticut. Propylene glycol was obtained from Bulk Apothecary in Aurora, Ohio. Phosphoric acid was obtained from Acres Chemical in Morris Plains, New Jersey. Erythorbic acid was obtained from Sigma- Aldrich in St. Louis, Missouri. Active liquids were prepared prior to sample feed production runs by blending the diethylcarbamazine, propylene glycol, water and sometimes phosphoric acid or erythorbic acid.

[0052] Active liquids of the extruded daily ration feed product can be added at different locations during the production process. One preferred technique is to prepare a dilute drug solution which can be pumped at a known rate into the feed mixture dining processing. For example, the active liquid may be added at the preconditioner, preferably adjacent the outlet thereof. Alternately, the drug may be injected directly into the extruder barrel during processing. Given the relatively small quantities of drug employed, it is appropriate to provide sufficient time in the process to adequately mix the active with other ingredient components so that they are substantially uniform throughout the feed mixture.

[0053] Table 3, which follows, sets forth an illustrative summary of the active liquid mixes that were added to the feed mixture before the extruder die:Table 3

[0054] Additional steam and active liquid mix was added to the feed mixtures in the extruder barrel at about 9-10 kg / hr. Extruder screw speed and cutter speed downstream of the extruder die were adjusted to obtain the sample extruded daily ration feed products, such as extruded kibble pieces having a wet bulk density of 400 - 430 grams / liter (g / 1) and a diameter and length of about 8 millimeters (mm) at the extruder discharge.

[0055] The kibble pieces were pneumatically conveyed to a Wenger® Model 4800 two pass, single section dryer. The drying temperature was set at approximately 99 degrees Celsius (°C) with a retention time of about 16 minutes. From the dryer, the kibble fell into a continuous cooler where it was cooled with ambient air for about 5 minutes. Kibble discharged from the dryer was placed in paper feed bags for storage prior to coating. Samples of dried kibble were labeled as indicated in Table 3 with the run number designation.

[0056] Dry kibble samples were transferred to the XIM Group, LLC in Sabetha, Kansas for coating and final packaging. Coating materials including Optimizer Bl 8060, a liquid palatability enhancer, obtained from AFB International in St. Charles, Missouri. Chicken fat with added antioxidant was obtained from Simmons Feed Ingredients in Siloam Springs, Arkansas. Dried kibble samples were coated in a small concrete mixer with a batch consisting of approximately 92% kibble, 6% chicken fat and 2% liquid palatability enhancer.

[0057] Bags for packaging of coated product were obtained from Pacific Bag, Inc in Woodinville, Washington. Bags had dimensions of approximately 7 and 7 / 8 inches (approx. 20 centimeters) by 10.5 inches (approx. 30 centimeters) by 3.5 inches (approx. 10 centimeters). Bags were constructed with a metallized barrier having multiple-layer construction. In a 3-ply construction, the bag may have an outer 40-gauge layer of polyethylene terephthalate (PET), a middle layer of 60-gauge, metallized, biaxially oriented nylon, and am inner layer of Linear low-density polyethylene (LLDPE) having a thicknessof approximately 4 mils (approx. 0.1 millimeters). The bag may have a moisture vapor transmission rate of 0.02 grams / 100 inches squared (in2), or less, in a 24-hour period (approx. 0.02 cc / 645 cm2-24hr). The bag may have an oxygen transmission rate of 0.05 cubic centimeter (cc) / 100 in2, or less, in a 24-hour period (approx. 0.05 cc / 645 cm2-24hr). Oxygen absorber packets of approximately 500 cc were obtained from IMPAK Corp in Los Angeles, California and added to bags for Treatments C and H. The antioxidant system with the product package bag selection may work together to protect fat components in the extruded daily ration feed product from undergoing oxidation at an accelerated rate.

[0058] Thirteen bags of each sample extruded daily ration feed product (with treatments) were shipped to PPD Inc. in Madison, Wisconsin for a shelf-life study conducted under PPD protocol P33768.01. One bag was analyzed for diethylcarbamazine concentration at the start of the study. Six of the thirteen bags were stored at 25 °C and 40% relative humidity (RH) which represent conditions under climate control and the remaining six bags were stored at 40°C and 75% RH which represent conditions without climate control in a hot and humid environment. Hot and humid environments are perceived to lead to accelerated degradation of feed products. Bagged samples of each extruded daily ration feed product were removed from each storage condition at 1, 2, 3, 6, 8, and 12 months for diethylcarbamazine analysis. All diethylcarbamazine analyses were performed using PPD Method M15607, which was modified to utilize 0.1N hydrochloric (HC1) acid as the diluent rather than water.

[0059] The drug-containing extruded daily ration feed products were produced at about the target dose concentration of 900 mg / kg of diethylcarbamazine, placed in the extruded daily ration feed product bags, stored at designated room temperatures, and sampled for a period of more than eight months. The sample extruded daily ration feed products with corresponding treatment stored at 25°C and 40% RH are shown in Table 4 and stored at 40°C and 75% RH are shown in Table 5. The average diethylcarbamazine concentrations were calculated from the two repetition analyses of each sample. The average percent of label claim (Ave % LC) is shown based on that calculation. Not all samples were close to the target dose concentration initially, so another calculation was made to compare the Ave % LC at each storage time to the starting average percent label claim (% of Start) to better assess the stability of the diethylcarbamazine in all samples. It should be noted that results for testing performed at the 12-month storage time were not included in the tables.

[0060] Table 4, which follows, sets forth an illustrative summary of the stability results of the diethylcarbamazine stored at about 25°C and 60% RH:Table 4Table 4 - continued

[0061] Table 5, which follows, sets forth an illustrative summary of the stability results of the diethylcarbamazine stored at about 40°C and 75% RH:Table 5Table 5 - continuedTable 5 - continued

[0062] The % of start data in Table 4 is shown graphically in FIG. 2 for the sample extruded daily ration feed products stored at 25°C and 60% RH. The % of start data in Table 5 is shown graphically in FIG. 3 for sample extruded daily ration feed products stored at 40°C and 75% RH. Diethylcarbamazine content is reported as a percent of the 0-month level for each treatment in FIG. 2 and FIG. 3.

[0063] At both storage conditions, the control product, which in this case was the sample extruded daily ration feed product with the Naturox® brand Plus antioxidant system, which contains only fat-soluble antioxidants, had the lowest diethylcarbamazine results at the end of twelve months. However, at the end of twelve-month shelf-life storage period storage under both storage conditions, the diethylcarbamazine level preserved by Kemin’s Verdilox™ GT antioxidant system, which contains both fat-soluble and water-soluble antioxidants as well as a chelator, had the highest diethylcarbamazine concentration at the end of the 8-month and 12-month storage periods.

[0064] Previous approaches have not explored the use or effectiveness of a water-soluble antioxidant or an antioxidant system containing a water-soluble antioxidant to preserve the therapeutic efficacy of diethylcarbamazine over a maintenance period. Water-soluble antioxidants refer to a group of antioxidant compounds that can dissolve in water. Watersoluble antioxidants that are generally recognized as safe may be utilized. The term "generally recognized as safe" (GRAS) is a designation used by regulatory agencies like the United States Food and Drug Administration (FDA) to classify that substances may besafe for consumption or considered safe for their intended use in food and other products. Based on available scientific evidence, examples of fat-soluble or water-soluble antioxidants that are generally recognized as safe and are not known to cause harm when used as intended are vitamin C (ascorbic acid), vitamin E (tocopherol), polyphenols, glutathione, conenzyme Q10 (CoQlO), among others now know or identified in the future.

[0065] Vitamin C is a co-substrate in metal catalyzed hydroxylations. Like beta-carotene, vitamin C has antioxidant properties. It interacts directly with superoxide hydroxyl radicals and singlet oxygen, and also provides antioxidant protection for folate and vitamin E, keeping vitamin E in its most potent form. Vitamin C may afford protective effects against preeclampsia by participating in the scavenging of free radicals. Vitamin C (ascorbic acid) is an antioxidant that may play a crucial role in boosting the immune system, collagen synthesis, and protecting cells from oxidative damage.

[0066] Vitamin E is a fat-soluble vitamin antioxidant found in biological membranes where it protects the phospholipid membrane from oxidative stress. Vitamin E inhibits the oxidation of unsaturated fatty acids by trapping peroxyl free radicals. In addition, vitamin E, like betacarotene and vitamin C, may participate in the scavenging of free radicals. Vitamin E may be included in the form of d-alpha-tocopheryl acetate or d-alpha tocopheryl succinate. Vitamin E (tocopherol), although primarily known as fat-soluble, has some water-soluble derivatives, such as tocopheryl polyethylene glycols (TPGS). These derivatives are often used as water-soluble antioxidants in various formulations.

[0067] Some polyphenolic compounds, such as flavonoids and catechins found in fruits, vegetables, and tea, are water-soluble and act as antioxidants. Glutathione, an endogenous antioxidant, may help protect cells from oxidative stress and detoxify harmful substances. While coenzyme Q10 (CoQlO) is mainly fat-soluble, water-soluble formulations of CoQlO (e.g., ubiquinol-10) are also available and used as antioxidants in certain products. These water-soluble antioxidants are generally considered safe within recommended doses. Other possible water-soluble antioxidants that may be utilized as a component of the preservative introduced into the dry mix are rosemary extract, spearmint extract, 2,4,5- Trihydroxybutyrophenone (THBP), dilauryl thiodipropionate, thiodipropionic acid, lipoic acid, uric acid, and thiourea. Such water-soluble antioxidants and their derivatives may react synergistically with tocopherols. Other food safe water-soluble antioxidants such as chitosan, high-protein concentrates, and amino acids may also be identified as providing stabilization of diethylcarbamazine over a shelf-life period.

[0068] Inclusion of a fat-soluble antioxidant may prevent oxidation of the fat components within the extruded daily ration feed product. Some possible fat-soluble antioxidants that may bea component of the preservative introduced into the dry mix are BHA (2-tert-buty-4- hydroxyanisole and 3-tert-butyl-4-hydroxyanisole), BHT (buty-hydroxy-toluol and 3,5-di- tert-butyl-4-hydroxytouluene), TBHQ (tertiary butylhydroquinone, terbutil hydroquinone) which can show a good synergy with citric acid, BHA or BHT, ethoxyquin, and propyl gallate or ethoxyquin. Other food safe fat-soluble antioxidants are available or may be identified such as carotenes, a-tocopherol (vitamin E), and ubiquinol (coenzyme Q). Betacarotene also has powerful antioxidant properties. Antioxidants are important for numerous reasons. For example, antioxidants are especially important in preventing lipid peroxidation due to oxidative stress.

[0069] Additionally, tocopherols, also in the class of primary antioxidants, are another natural antioxidant found in plants which may be included in extruded daily ration feed products formed by the production method. Tocopherols are a fat-soluble antioxidant formed by a mixture of four isomers. Tocopherols prevent the oxidation of fats by stopping chain reactions of free radicals through neutralization. Free radicals are considered harmful molecules which can be produced during normal metabolic processes or because of exposure to environmental factors such as pollution or radiation. An antioxidant system based on tocopherols may help stabilize fats and oils present in the feed mixture. Benefits to using tocopherols are their resistance to high temperatures of extrusion, high solubility within the feed mixture, and little to null impact on the color and odor of the final extruded daily ration feed product. Tocopherols may be used in combination with other antioxidants to bring about a synergistic effect between selected ingredients.

[0070] Chelators, a class of secondary antioxidants, may work as a component of the preservative system to bind with metal ions and prevent those metal ions from catalyzing and increasing the fat-oxidation process. Given the abundance of pro-oxidative metals that may naturally occur in many foods, sequestration of free metals through chelation can be critical for the mitigation of lipid oxidation and preservation of the extruded daily ration feed product. Free metals can reduce endogenous hydrogen and lipid hydroperoxides to generate free radicals which can propel further oxidation. To marginalize their reactivity, some metal chelators may be utilized as an add-on component of the preservative to effect the quality of the extruded daily ration feed product. Since many chelators bind metals through charged acid groups, they will be most active when pH is greater than their pKa (acid dissociation constant). Chelator concentrations may also be critical to efficacy. When chelator concentrations are similar to, or below the metal concentration in the extruded daily ration feed product, this may result in increased metal solubility. When chelator concentrations are greater than metal concentrations, the metal can be bound by multiple chelators thustying up all metal coordination sites and thereby preventing redox cycling.

[0071] Common chelators used in the food industry include acids like ethylenediaminetetraacetic acid (EDTA), organic acids like citric acid, tartaric acid, and phosphates like phosphoric acid, tetrasodium pyrophosphate, trisodium polyphosphate, sodium tripolyphosphate, hexametaphosphate, polyphosphates, as well as proteins and organic acids. The calcium and sodium salts of EDTA may be used in foods due to their high-water solubility. The catechins in green tea extract may act as an iron chelator, which can suppress the prooxidant iron that can be released for example from the hemoglobin released during the production of ground meat. Additionally, the green tea extract presents as a polyphenol antioxidant which can complement the activity of vitamins E and C in the extruded daily ration feed product.

[0072] Chelators like calcium phytate, ferritin, lactoferrin, phosvitin, whey, oxalic acid, aqueous phase anionic proteins, hydrolyzed proteins, highly phosphorylated proteins, and polysaccharides like xanthan gum will also bind metals within the continuous phase of oil- in-water emulsions and decrease their reactivity by decreasing metal-lipid interactions. Potential also exists for the use of another class of chelating agents, called metal protein attenuating compounds (MPACs), and novel applications of chelator technologies outside of their traditional use as food additives are emerging, particularly within active packaging. In addition, an inchoate class of iron chelators derived from microorganisms, siderophores, may be another chelator class to those commonly used today. Other off-label uses of specific chelating agents, such as calcium-EDTA (Versenate), disodium-EDTA, deferasirox, deferiprone (Ferriprox®), or deferoxamine which may be used in chelation therapies or under investigation may also be identified.

[0073] Particularly, the tocopherols may be synergistically affected when mixed with the metal chelator(s) incorporated into the feed mixture. In accordance with FIG. 1 , the tocopherol may be incorporated with the antioxidant treatment before the preconditioner or after the preconditioner to be mixed. Likewise, the metal chelator may be incorporated with the antioxidant treatment before the preconditioner or incorporated after the preconditioner. A ratio of the metal chelator to the tocopherols in the antioxidant system may be greater than 1:1. The ratio of metal chelator to tocopherols in the antioxidant system may be 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 40:1 or anywhere between 1:1 to 40:1, or further between 20:1 to 40:1, 10:1 to 40:1, 1:1 to 10:1, 1:1 to 5:1, or 2:1 to 4:l.

[0074] Kemin’s Verdilox™ GT preservative system, which includes similar components as described above, was found to perform well in maintaining the diethylcarbamazine at the target dose concentration over a long shelf-life storage period as well as decreasingoxidation of fat components based on peroxide values observed well below 1.0 milliequivalents per kilogram (meq. / kg). The combination of water- and fat-soluble antioxidants may contribute to the stronger protection of the entire food matrix. Specifically, the diethylcarbamazine, having greater than 75% solubility in water at or above 20°C, may be synergistically affected when mixed with the water-soluble antioxidants incorporated into the feed mixture. In particular, this antioxidant system may have a ratio of water-soluble antioxidants to the fat-soluble antioxidants that is greater than 1:1. The ratio of water-soluble antioxidants to fat-soluble antioxidants in the antioxidant system may be 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 40:1 or anywhere between 1:1 to 40:1, or further between 20:1 to 40:1, 10:1 to 40:1, 1:1 to 10:1, 1:1 to 5:1, or 2:1 to 4:1. Similar antioxidant systems incorporating a water-soluble antioxidant, metal chelator, or a blend of water- and fat-soluble antioxidants might be utilized to form a quality extruded pet food diet that protects and provides a therapeutic amount of pharmacological actives and / or nutraceuticals even though extended storage periods at temperatures and humidities exist without climate control.

[0075] FIG. 4 is a flowchart for an example method 400 for maintaining a therapeutically effective amount of an active in an extruded daily ration feed product. A preservative comprising a water-soluble antioxidant is added into a dry mix of the feed mixture at a point upstream of the extruder die, according to step 402. The feed mixture with the preservative added is mixed in a preconditioner, according to step 404. An active, such as diethylcarbamazine is blended with a carrier and added into the feed mixture at a point after the preconditioner and upstream of an extruder die, according to step 406. The active is distributed throughout the feed mixture with the water-soluble antioxidant, according to step 408. The activecontaining feed mixture is extruded to form the extruded daily ration feed product, according to step 410. At least 95% of the therapeutic amount of diethylcarbamazine in the extruded daily ration feed product is maintained for a period of up to 8 months, according to step 412. The extruded daily ration feed product may be stored in a package within an oxygen transmission rate of up to 0.05 cc / 100 in2-24hr (approx. 0.05 cc / 645 cm2-24hr) for a period of up to eight months to conserve the therapeutically effective amount of active. The therapeutically effective amount of active is conserved in the extruded daily ration feed product at a concentration of between 200 milligrams per kilogram to 2,000 milligrams per kilogram of the extruded daily ration feed product. For example, at least 95% of an original amount of diethylcarbamazine was maintained from extrusion in the extruded daily ration feed product after the storage period. In other words, there was less than a 5% difference from an original amount of diethylcarbamazine in the extruded daily ration feed product atthe end of the storage period.

[0076] It is understood that the invention is not confined to the particular construction and arrangement of parts herein described. That although the drawings and specification set forth a preferred embodiment, and although specific terms are employed, they are used in a description sense only and embody all such forms as come within the scope of the following claims.

[0077] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, are possible from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims.

[0078] For the convenience of the reader, the above description has focused on a representative sample of all possible embodiments, a sample that teaches the principles of the invention and conveys the best mode contemplated for carrying it out. Throughout this application and its associated file history, when the term “invention” is used, it refers to the entire collection of ideas and principles described; in contrast, the formal definition of the exclusive protected property right is set forth in the claims, which exclusively control. The description has not attempted to exhaustively enumerate all possible variations. Other undescribed variations or modifications may be possible. Where multiple alternative embodiments are described, in many cases it will be possible to combine elements of different embodiments, or to combine elements of the embodiments described here with other modifications or variations that are not expressly described. A list of items does not imply that any or all of the items are mutually exclusive, nor that any or all of the items are comprehensive of any category, unless expressly specified otherwise. In many cases, one feature or group of features may be used separately from the entire apparatus or methods described. Many of those undescribed variations, modifications and variations are within the literal scope of the following claims, and others are equivalent.

Claims

CLAIMSWe claim:

1. A method of maintaining a therapeutically effective amount of diethylcarbamazine in an extruded daily ration feed product for animals comprising the steps of: a. adding a preservative into a feed mixture upstream of an extruder die, wherein the preservative comprises: i. a water-soluble antioxidant; b. mixing the feed mixture in a preconditioner; c. adding a diethylcarbamazine into the feed mixture after the preconditioner and upstream of the extruder die; and d. extruding the feed mixture to form the extruded daily ration feed product with a concentration of diethylcarbamazine between 200 milligrams per kilogram to 2,000 milligrams per kilogram.

2. The method of claim 1 , wherein the preservative further comprises: a. a metal chelator; and b. a tocopherol, wherein a ratio of the metal chelator to the tocopherol is greater than 1:1.

3. The method of claim 1, wherein the preservative further comprises: a. a fat-soluble antioxidant, wherein a ratio of the water-soluble antioxidant to the fat-soluble antioxidant is greater than 1:1.

4. The method of claim 1, wherein the preservative is incorporated with a dry mix before the preconditioner.

5. The method of claim 4, wherein the preservative further comprises: a. a tocopherol.

6. The method of claim 4, wherein the preservative further comprises: a. a metal chelator.

7. The method of claim 6, wherein the metal chelator comprises: a. a tetrasodium pyrophosphate.

8. The method of claim 1 , wherein the diethylcarbamazine is added to the feed mixture within an extruder.

9. The method of claim 1 , further comprising: a. maintaining the therapeutically effective amount of diethylcarbamazine in the extruded daily ration feed product for a period of 6 months up to 40°C and 75% relative humidity.

10. The method of claim 1, further comprising:a. maintaining at least 95% of an original amount of diethylcarbamazine in the extruded daily ration feed product for a period of 8 months up to 25°C and 40% relative humidity.

11. The method of claim 1 , further comprising the step of: a. maintaining at least 95% of an original amount of diethylcarbamazine in the extruded daily ration feed product stored in a package with an oxygen transmission rate of up to 0.05 cc / 100 in2-24hr for a period of up to 8 months at ambient temperatures.

12. The method of claim 1, wherein the concentration of diethylcarbamazine in the extruded daily ration feed product is further between 500 milligrams per kilogram and 1,500 milligrams per kilogram and a concentration of the preservative added is between 200 milligrams per kilogram and 1 ,000 milligrams per kilogram.

13. The method of claim 1, wherein the concentration of diethylcarbamazine in the extruded daily ration feed product is further between 800 milligrams per kilogram and 1,100 milligrams per kilogram and a concentration of the preservative added is between 500 milligrams per kilogram and 750 milligrams per kilogram.

14. The method of claim 1, further comprising the step of: a. stabilizing the diethylcarbamazine in the extruded daily ration feed product for a period of up to 8 months by the preservative added being between 200 milligrams per kilogram and 1,000 milligrams per kilogram.

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