WO2010077903A1

WO2010077903A1 - Antioxidant-containing food composition for use in enhancing antiviral immunity in companion animals

Info

Publication number: WO2010077903A1
Application number: PCT/US2009/068166
Authority: WO
Inventors: Steven C. Zicker; Jeffrey A. Brockman; Nolan Zebulon Frantz
Original assignee: Hill's Pet Nutrition, Inc.
Priority date: 2008-12-16
Filing date: 2009-12-16
Publication date: 2010-07-08
Also published as: AU2009333295B2; BRPI0922701A8; AU2009333295A1; RU2485799C2; CN102245033B; JP5743903B2; BRPI0922701A2; RU2011129617A; US8592479B2; JP2012511932A; ZA201103884B; CA2744056A1; EP2358216A1; EP2358216B1; US20090149529A1; CA2744056C; CN102245033A

Abstract

The invention encompasses compositions for enhancing the ability of a companion animal to resist and/or overcome viral infections. The compositions of the invention include an amount of lipoic acid that is effective in enhancing the antiviral immunity of a companion animal.

Description

ΛNTIOXIDA\Ύ-CONTΛINING FOOD COMPOSITION FOR USE IN ENHANCING ANTIVIRAL IMMUNITY IN COMPANION ANIMALS

CROSS REFERENCE TO RELATED APPLICiVTIONS

[OQOIj This application is a continuation-in-part of pending application Ser. No. 1 1/154.210. filed June 16. 2005, which is a continuation of application Ser. No. 09, 978.132 filed October 16, 2001, which issued as U.S. patent number 6,914,071, which is a continuation-in-part of application Ser. No. 09/922.660 filed August 6, 2001. which claims benefit of Provisional Application Ser. No. 60/253,448 filed November 28, 2000 and Provisional Application Ser. No. 60/244.504. filed October 31, 2000, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002 J The invention encompasses compositions for enhancing the ability of a companion animal to resist and/or overcome viral infections. The compositions of the invention include an amount of lipoic acid that is effective in enhancing the antiviral immunity of a companion animal.

BACKGROUND OF THE INVENTION

[0003] Companion animals such as dogs and cats seem to suffer from aging problems. Some of these are manifested in commonplace savings. One of these is "You can't teach an old dog new tricks." This saying arises from the observation that as dogs age, their mental capacity seems to diminish as well as physical abilities. Mental activities associated with thinking, learning and memory seem to be lessened (Cummings. B. J.. Head. E., Ruehl. W., Milgram, N. W. & Cotman, C W . (1996): The canine as an animal model of aging and dementia. Neurobiology of Aging 17:259-268). Additional!) , behavioral change can be manifested in the aging animals in association with the changing mental capacity.

causes ha\ e been assigned to this lessening of capacity . 10004J These losses in capacity are generally observed in aged canines and felines. Dogs of sc\en years or older and felines of seven years or older are considered aged and can experience this problem,

10005J The presence of significant levels of at least one antioxidant in the diet of an adult companion pet or fed to a pet outside Ms diet can inhibit the onset of deterioration of the mental capacity of the aged companion pet and/ or maintain the mental capacit} of the adult companion pet further into the aged years.

SUMMARY OF THE INVENTION

[0006] In accordance with the invention, there is a companion pet diet meeting ordinary nutritional requirements of an adult pet and further comprising a sufficient amount of an antioxidant or mixture thereof to inhibit the onset of deterioration of the mental capacity of said companion pet in its aged years.

[0007] Another embodiment encompasses a method for inhibiting the deterioration of the mental capacity of an aged companion pet, which comprises feeding said pet in his adult years an antioxidant or mixture thereof at sufficient levels to accomplish this inhibition.

[0008] Another embodiment encompasses a companion adult pet diet meeting ordinary nutritional requirements of an adult companion pet and further comprising an antioxidant selected from the group consisting of Vitamin E. vitamin C. alpha-lipoic acid, L-carnitine and any mixtures thereof in quantities sufficient to inhibit the deterioration of the mental capacity of said pet in its aged years.

[0009] Another embodiment of the invention encompasses methods for increasing the mental capacity of an aged companion pet. which comprises feeding the pet in its adult years an amount of an antioxidant or mixture thereof sufficient to increase the mental capacity.

[OOlOj Another embodiment encompasses methods for increasing the mental capacity of an adult companion pet which comprises feeding the pet an amount of an antioxidant or mixture thereof sufficient to increase the mental capacity of said pet. |001 IJ Another embodiment encompasses pet food compositions including an amount of one or more antioxidants, for example, lipoie acid, effective to enhance antiviral activity in a companion animal.

DETAILED DHSCRIPl ION OF THE INVENTION General Description

[0012] The invention encompasses pet food compositions including an amount of lipoie acid effective amount to enhance antiviral activity in a companion animal. In certain embodiments, the effective amount of lipoie acid to enhance antiviral activity in a companion animal is at least about 25 ppm.

[0013] In certain embodiments, the effective amount is at least about 50 ppm.

[0014] In certain embodiments, the effective amount is at least about 100 ppm.

[0015] In certain embodiments, the effective amount is about 100 ppm to about 600 ppm.

[0016] In certain embodiments, the effective amount is about 100 ppm to about 200 ppm.

[0017] In certain embodiments, the companion animal is a dog.

[0018] In certain embodiments, the companion animal is a cat.

[0019] In certain embodiments, the effective amount is effective to enhance innate antiviral activity in a companion animal.

[0020] In certain embodiments, the pet food composition comprising lipoie acid is administered at least 15 da>s.

|002l| In certain embodiments, the pet food composition comprising lipoie acid is administered at least 30 days.

[0022] In certain embodiments, the pet food composition comprising lipoie acid is administered at least 45 days.

[0023] In certain embodiments, the pet food composition comprising lipoie acid is administered daih [0024] The diet fed to the adult companion pet. for example canine and feline, is the standard normal diet fed to an animal of that age. Below is a typical diet for a canine of 1 to 6 years of age.

Table 1

Component Target

i Protein (% of dry matter) ! 23

! Fat (% of dry matter) 15

I Phosphorus (% of dry matter) 0.6

Sodium (% of dry matter) 0.3

[0025] Adding significant quantities of an antioxidant or mixture thereof to the companion adult pet diet can bring about delay of the onset of demonstrative changes in the behavior, particularly the deterioration of mental capacity, as specifically shown by problem-solving capacity, in an aged pet. The addition of one or more antioxidants to the companion adult pet diet can also bring about enhancement of antiviral activity in companion animals. The term, adult, is intended to mean, in general, a canine of at least 1 to 6 years and a feline of at least 1 to 6 years. An aged dog or cat is 7 years and abo\e.

[0026] The loss of mental capacity for canines and felines has been observed for a number of years. This loss of mental capacity is manifested in numerous wa\ s. For a canine, for example, it can be manifested as disorientation, house soiling, altered sleep-wake patterns, decreased or altered interaction with humans and other pets, and inability to learn and concentrate. These conditions can be manifested in felines as well. Alzheimer's, as exhibited IE man, is not found in canines and felines.

[0027] Many theories have been advanced for this loss in mental capacity. To date, the inventors are unaware of any dietarj course of action, which inhibits this loss of mental capacity or can actual!}' bring about a positive change in mental capacity as measured by an objective parameter in dogs and cats,

[0028] The in\entors

succeeded in accomplishing delating the onset of this deterioration. By using the diet of their invention in adult companion pets it can be shown that aged pets' mental capacity can be maintained for a longer period of time. Essentially the deterioration of mental capacity can be stopped or delayed. Memory and learning ability can be

Overall mental alertness can be enhanced. Age related cognitive decline could be slowed. With respect to Cognitive Disfunction Syndrome its progress can be slowed in aged dogs and clinical signs associated with this syndrome can be controlled. Prophylaxis where appropriate and pets in need of these components are the target group.

[0029] The inventors have also surprisingly found that the addition of one or more antioxidants, for example lipoic acid, is useful in enhancing the innate antiviral immune function in companion animals, for example, dogs and cats. Λs used herein, the term '"enhance" or "enhancing" when referring to antiviral immune function refers to the ability of a companion animals to have an increased immune response to an antigen and thereby be more resistant to infection or clear viral infections from the system of the companion animal faster. Accordingly, a companion animal, for example, a dog, eating a pet food containing an antioxidant, for example, lipoic acid will be more resistant to and will clear viral infections faster than an animal not consuming antioxidants.

[0030] The component in the diet which accomplishes this is an antioxidant or mixture thereof.

An antioxidant is a material that quenches a free radical. Examples of such materials include foods such as ginkgo biloba, citrus pulp, grape pomace, tomato pomace, carrot and spinach, all preferably dried, as well as various other materials such as beta-carotene, selenium, coenzyme QlO (ubiquinone), lutein, tocotrienols. soy isoflavones, S-adenosylmethionine. gluthathione, taurine. N-acetylcysteine. vitamin E. vitamin C. alpha-lipoic acid. L-camitine and the like.

Vitamin E can be administered as a tocopherol or a mixture of tocopherols and various derhathes thereof such as esters like

Ii acetate, succinate, palmitate. and the like. I he alpha form is preferable but beta, gamma and delta forms can be included. 1 he D form is preferable but racemic mixtures are acceptable. The forms and derivatives will function in a Vitamin E like acti\ ity after ingestion by the pet. Vitamin C can be administered in this diet as

. ^ _ ascorbic acid and its various derivathes thereof such as calcium phosphate salts, cholcsteryl salt, 2-monophosphate, and the like, which will function in a vitamin C like activity after ingesting by the pet. They can be in any form such as liquid, semisolid, solid and heat stable form. Alpha- Iipoic acid can be administered into the diet as alpha-lipoic acid or as a lipoate derivative as in U.S. Pat. No. 5.621,117, racemic mixtures, salts, esters or amides thereof. L-earnitine can be administered in the diet and various derivatives of carnitine such as the salts such as the hydrochloride, fumarate and succinates, as well as acetylated carnitine and the like, can be used.

|0031J The quantities administered in the diet, all as wt % (dry matter basis) of the diet, are calculated as the active material, per se, that is measured as free material. The maximum amounts employed should not bring about toxicity.

[0032] At least about 100 ppm or at least about 150 ppm of vitamin E can be used. In certain embodiments, the range of about 500 to about 1,000 ppm can be employed. Although not necessary a maximum of about 2,000 ppm or about 1,500 ppm is generally not exceeded.

[0033] With respect to vitamin C at least about 50 ppm is used, desirably at least about 75 ppm and more desirably at least about 100 ppm. A nontoxic maximum can be employed.

[0034] The quantity of lipoic acid can vary from at least about 25 ppm, desirably at least about 50 ppm, more desirably about 100 ppm. In various embodiments, the range of lipoic acid that can be administered dogs is about 150 ppm to about 4500 ppm. In various embodiments, the range of lipoic acid that can be administered cats is about 65 ppm to about 2600 ppm. Maximum quantities can vary from about 100 ppm to 600 ppm or to an amount which remains nontoxic to the pet. In certain embodiments, a range is from about 100 ppm to about 200 ppm.

[0035] For L-carnitinc about 50 ppm, desirably about 200 ppm, more desirably about 300 ppm for canines are a useful minimum. For felines, slightl} higher minimums of L -carnitine can be employed such as about 100 ppm, 200 ppm, and 500 ppm. A nontoxic maximum quantity can be employed, for example, less than about 5,000 ppm. For canines, icwer quantities can be employed, for example, less than about 5,000 ppm. tor canines a preferred range is about 200 ppm to about 400 ppm. For felines a preferred range is about 400 ppm to about 600 ppm.

[0036 j Beta-carotene at about 1 - 15 ppm can be employed. {0037 j Selenium at about 0.1 up to about 5 ppm can be employed [0038] Lutein: at least about 5 pm can be employed.

[0039] locotrienols: at least about 25 ppm can be emploved.

|0040| Coenzyme QlO: at least about 25 pprn can be emploved.

[0041] S-adenos\imelhionine: at least about 50 ppm can be emploved.

[0042J Taurine: at least about 1000 ppm can be emploved.

[0043] So> isoflavones: at least about 25 ppm can be used.

[0044] N-acetylcysteine: at least about 50 ppm can be used.

[0045] Glutathione: at least about 50 ppm can be used.

[0046] Gingko biloba: at least 50 ppm of extract can be used.

[0047] The following are raw ingredients that are high in ORΛC (Oxygen radical absorbing capacity) content: When added to the diet as 1% inclusions (for a total of 5% substitution for a low ORAC ingredient such as corn) they increased the ORAC content of the overall diet and increased the ORAC content of the plasma of the animals which ate the diet containing these components. Preferably, any ingredient with an ORΛC content >25 .mu.mole of Trolox equivalents per gram of dry matter could be used if added at 1% in combination with four other 1 % ingredients for a total of 5% addition to the diet.

[0048] Spinach pomace, Tomato pomace. Citrus pulp. Grape pomace, Carrot granules, Broccoli, Green tea, Ginkgo biloba. and Com gluten meal.

Example 1

[0049] Seventeen adult beagle dogs 2-4 \ears of age (control n=8, antioxidant-enriched n— 9) were randomly placed into a control or enriched diet group. The control diet contained 59 ppm vitamin E and <32 ppm vitamin C. Fhe test diet had 900 ppm vitamin E and 121 ppm vitamin C,

260 ppm L-carnitinc and 135 ppm alpha-Upoie acid. Approximately 1 month after starting the diet, the first problem-solving task given to dogs was a landmark discrimination learning task, which is a test of spatial attention (Milgram, V W., Adams, B.» Callahan, I!.. Head. E., Maekej. B.. Thjflweli, C. & Cotman. C. W. ( 1999): Landmark discrimination learning in the dog. Learning Sc Mernorv. 6:54-61). (0050J Landmark discrimination learning requires subjects to select a particular object based on proximity to an object. The initial learning, however, is based on the dogs' ability to learn an object discrimination task. We have previous!} found that the effects of age on discrimination learning depend on task difficulty.

[0051] The adult dogs on the enriched diet made fewer errors than the adult dogs on the control food when learning the landmark 0 test (control mean-31.1, enriched mean^~l5.1 ). The adult dogs proceeded on to landmark 1 and 2 testing, where the landmark is moved further away from the positive well. Adult dogs on enriched diet learned landmark 0-2 with less errors than those on the control (number of mean errors landmark 0^J-l+2 (control)- 132.9; number of mean errors landmark O-t-1+2 (dogs on enriched diet)=87.1 ).

Example 2

[0052] 30 adult, random source, dogs were utilized for this study. Dogs were at least 10 months of age, not pregnant, not lactating and of reasonable body weight prior to start of test. Animals were randomized into 5 groups for dietary treatment with 3 males and 3 females per each group.

[0053] All dogs were fed a control food (0 ppm DL-alpha-lipoic acid added) that met or exceeded all recommendations for nutrients as proposed by the American Association of Feed Control Officials (AAFCO 2000) during a 2 week prefeeding period (Table 1). Following the prefeeding period dogs were randomized into 5 treatment groups with one of the following DL- alpha-lipoic acid target inclusions (dry matter basis): 0 ppm, 150 ppm. 1,500 ppm, 3,000 ppm, 4,500 ppm. In all diets, control and alpha-lipoic acid, vitamin E was added and was present at a level of 600-1000 International Units, and vitamin C was added at levels of 100-200 ppm.

[0054] Test foods were the sole source of nutrients except for water. Fresh water was provided ad libitum. After dogs were selected and initial bod} weights taken, a food dose was calculated for each dog based on the expected VIE of the food. Initial food dose calculations were based on the maintenance energ} requirement (MtR) for the dog modified b} a factor to account for norma! activ ity as calculated by the following formula:

MER(kcai'da> }=^rl .ό.tϊmes.RERs Resting Energy Requirement) where: RER

weight (kg).sup.0.75. [0055] Dogs were weighed weekly and had food doses adjusted as needed in order to feed enough food to maintain their optimal body weight. Optimal body weight was determined to be 3 on a 5 point scale. If a dog did not maintain bod} weight within -10% of initial body weight, after adjustment of food dose, it was removed from the study. All measures of body weight and food intake were recorded.

[0056] Samples were ground and 0.100.+-.0.001 g of sample was extracted twice into 5.0 ml phosphate buffer ( 10 niM Na₂HPO₄, 2 niM ethylcnediaminetetraacetic acid (ED^'l^'A), 0.9% NaCL pH 7.4). 250 μL of extract was placed into a 5 ml glass centrifuge tube with a Teflon lined cap. 15 μL EDTA solution (100 mM EDTA. adjusted to pH 7.8 with .about. LM NaOH) and 50 .mu.l freshly prepared 5 mM dithioerythritol (DTE) were added. The solutions were vortexed and incubated at room temperature for 5 minutes. Then 10 .mu.l of IM H₃PO₄ and 2.0 ml diethyl ether were added. The tubes were capped, vortexed, and eentrifuged at 1500.times.g for 3 minutes at room temperature. The ether layer was transferred to a separate 5 ml glass centrifuge tube, while the aqueous layer was extracted twice more with 1.5 ml ether. All extractions from the same sample were combined. The extracts are then dried in a nitrogen evaporator in a water bath at room temperature. At this point, the samples were capped and frozen overnight.

[0057] The dried extracts were then thawed and reconstituted with 70 .mu.l SDS/EDTΛ solution (0.1 1% sodium dodecyl sulfate (SDS), 15 mM EDTA, 0.9% NaCl) and 5 μL freshly prepared 1 mM DTE. 50 μL of freshly prepared NaBH₄ was then added to each tube. The tubes were vortexed and incubated at room temperature for 10 minutes. After 10 minutes, the samples were frozen at -70 ⁰C. Before the solutions were thawed, 20 μL 2 M HCl was added. After the solutions were thawed, 800 μL 100 mM NH4HCO₃ was added. The solutions are vortexed and 5 .mu.l of 100 mM monobromobimane in acetonitrile solution (mBBr) was added. The solutions were then incubated in the dark for 90 minutes at room temperature.

{§0581 Excess mBBr and the DTt derhatrve were removed from the samples after incubation by extraction with 1.5 ml dichioromethane. The aqueous la} er mas placed on the HPLC. The lipoic acid was separated using a mobile phase that consisted of 30% acetonitrile, 1% acetic acid, adjusted to pH 3.95 with about 2 M NII₄OH and was pumped at a flow rate of 1.0 mL'min with an isocratic elution for 15 minutes per injection, i^'his preparation assumes that the density of the extruded food is equal to i g,^'ml. J0059] Blood was collected aseplically for complete blood count and blood biochemisir>^r analysis 2 weeks prior to start, and again at 0. 28. 56, 84, 112, 140 and 168 days of the study. In addition, 15 ml of whole blood was collected for isolation of lymphocytes at days 0. 28 and 84 of the dietary intervention.

[0060J Heparinized whole blood was layered onto a 50 ml Aecuspin conical centrifuge tube (Sigma Chemical) and an equal volume of phosphate buffered saline (PBS) was added. Samples were centrifuged at 700.times.g for 30 minutes without brake. The monocyte layer was harvested, transferred to a 15 ml conical centrifuge tube, resuspended in 1-3 ml of PBS. and centrifuged as before (first wash). A second wash was performed as the first wash. Finally , cells were harvested and suspended in perchloric acid (10% w/v) and frozen at 70. degree. C. until analysis.

[0061] Samples were transferred from -70 ⁰C freezer into a cooler with dry ice in it. Vials were centrifuged at 12,000 rpm for 5 minutes in a refrigerated centrifuge. Λn aliquot of supernatant for glutathione (GSH) analysis was transferred to a conical test tube.

[0062] Derivatization of the acid soluble extracts was by the method of Reed and coworkers (Fariss et al) as modified by Jones (Jones et al).

[0063] Briefly. 150 μL extract or external standards were added into a 1.5 ml eppendorf tube followed by addition of 20 μL .gamma.-glu-glu internal standard and 50 μL IAΛ added followed by mixing. The solution was adjusted to pH about 10 (purple color) by using KOH — KHCO₃ working solution. Solutions were incubated 1 hr under room temperature in the dark. Sanger's reagent was added at the same volume as of the total volume and the solution was incubated overnight (20 hrs) in the dark at room temperature.

[0064] After incubation, the solution was eentrifuged at 12.000 rpm for 5 minutes with the supernatant transferred into another 1.5 ml eppendorf tube. 200 μL supernatant was added into an amber autøviai which had a 300 μL inlet, fix the top with a crimper for HPLC analysis

[0065] Solvents and separation conditions were as described {Fariss, Jones;. Le\els of GSlI and GSSG were quantified relatήe Io authentic standards. Gamma-glutamyl-glutamate was used as an internal standard to assess derivatization efficiencv . [0O66J Comparison of values for clinical chemistry, hematology and body weights vs baseline were analyzed by wav of paired t-test on SAS for Windows with significance set at P<0.05. Means of \alues at each measured time point were separated by a one-way ANO VΛ with signiilcancc set at P<0.05. The difference in GSϊI:GSSG between day 84 and baseline were analyzed between groups by way of SAS for Windows in a one-way ANOYA with significance set at PO.05.

Results

[0067] Concentrations of lipoic acid (ppm) in food as determined over 7 successive assays (0, 28, 56. 84. 112, 140, 168 days) were within the range of expected assay sensitivity and production parameters typically encountered at our facility (Table 2).

[0068J The food intake data were unremarkable. Most animals in all groups ingested more food at 6 months, on average, than at the beginning of the study. Body weight data were unremarkable except that some weight loss occurred initially in the 4,500 ppm inclusion group but that change appeared to reverse by 6 months time. Body condition scores did not appear to be affected by this minor loss of weight.

[0069] The routine physical examinations did not reveal any evidence of nutrition related abnormalities or DL-alpha-lipoic acid toxicity. All animals in the study population remained normal during the entire course of the study. Occasional vomiting was observed in several animals during the course of the study; however, a trend was not observed that would lead one to the conclusion that the vomiting may be attributable to lipoic acid. One animal, in the highest inclusion group, was dropped from the study at day 21 for weight loss and leukocytosis. The leukocytosis in this animal had not resolved by the end of the study and is suspected to be attributable to some other disease process.

[0070] When serum biochemistry values for days 28. 56, 84. 112. 140. and 168 were compared with the initial for the same group of dogs, several statistical differences were noted, however, none of these were considered biologically significant because these values were within or very near the laboratory reference range and consistent trends over months were noted. Comparisons between the controls and the other treatment groups at each time period also revealed several statistical differences, however, none of these were considered biologically

_ i significant because these values were within or very near the clinical laboratory reference ranges and no trends were present,

[0071] When the hematolog) \alues for da> s 28. 56. 84, 1 12. 140 and 168 were compared with the initial for the same group of dogs, several statistical differences were noted; howe\er, none of these were considered biologicall) significant because these values were within or very near the laboratory reference range and no trends were present. Comparison between the controls and the other treatment groups at each time period repealed several statistical differences; however, none of these were considered biologically significant because these values were within or very near the clinical laboratory reference ranges and no trends were present.

GSH:GSSG Ratio

[0072] The change in GSH;ϋSSG ratio over 84 days of feeding displayed a significant overall effect of diet (P^~0.024) with all supplemented groups having an increase in the ratio (Table 3). ANOVA revealed a significant difference, compared to the basal food, for the lowest and highest inclusions, however, the largest numerical increase was in the lowest inclusion level. That is to say, the changes in the GSH:GSSG ratio for the highest and lowest inclusion were significantly different from the change observed over this same time period in the basal food. Ratios for 4 points could not be determined at day 84 as no GSSG was detectable in any of these samples (1 control. 3 treatment groups). As such, the values for supplemented groups may have displayed even higher ratios of GSH:GSSG if the assa> had been sensitive enough to detect the low levels of GSSG at day 84.

TABLE 2

3.000 1869 250 96

- 1.

TABLE 3

Change In Mean Ratio Of GSHiGSSG From Day 0 To Day 84 In Dogs Consuming DL-Alpha Lipoic Acid In An Extruded Food

Inclusion Difference in N P value GSH:GSSG ratio - day 0 to day 84 Inclusion compared to baseline food

0 ppm -9.2 +/- 26 5* NA

150 ppm 70 +/- 20 0.003

1,500 ppm 24 +/- 7 0.16

3,000 ppm 10 +/- 4 4* 0.46

4,500 ppm 50 +/- 36 4* 0.03

* 1 dog in the control and 4,500 ppm group had no detectable GSSG at da> 84 while 2 dogs in the 3.000 ppm group had no detectable GSSG at dav 84.

[0073] Further observations with respect to alpha-lipoic acid are applicable. Chronic feeding of alpha-lipoic acid in diet is safe and effective. It improves the reduced glutathione (GSH) to oxidi/ed glutathione (GSSG) ratio. The chronic administration of alpha-lipoic acid in the diet can be for periods of one. two. three, four.

or six months minimum up through a period of one, two. three, four, fhe jears or e\en more including the lifetime of the animal. I he alplia-lipoic acid functions without an} special protection in the diet such as encapsulation and need not be present in the diet in a unit dosage form buch as those used in pharmaceuticals, for example, tablet, pill, capsule and the like The lipoic acid is provided in the diet in a minimum of about 25. 50, 75. or 100 ppm of did. The uppermost range is just below its toxic level, all the wa> down to about 400. 300, or 200 ppm of diet. General!} , one does not go beyond about 6 or 7 mg/kg body weight of animal per da} , more generally not abo\c about 5. The alpha-lipoie acid improves antioxidant defense capabilities as well as improves the animal's ability to resist oxidative damage. All this is done with the proper quantities of other antioxidants present such as vitamin E and \itamin C. This demonstrates that the action of alpha-lipoic acid is beyond that of vitamin C and/ or vitamin E.

Example 3 Experimental Conditions

[0074] Twenty dogs were fed for 30 days. Ten were fed an AAFCO level control food and 10 other dogs were fed the ΛΛFCO level control food containing 150 ppm alpha-lipoic acid. At the end of the end of the 30 days whole blood samples were collected from each dog in Paxgene tubes.

[0075] Total RNAs were isolated from whole blood samples using the PΛXgene RNA isolation kit. All measurements were done with the canine 2 Λffymetrix genechips. For statistical analysis, all measurements were normalized with RMA. All analysis was preformed using Partek. An ANOVA t-test was performed for genes that are differentially expressed between the control and test foods, (at least a 20% change in expression with a pvalue < 0.05)

[0076] Differentially expressed genes were analyzed with the GeneGo pathway analysis software. Dogs fed lipoic acid for 30 days exhibited an interferon mediated antiviral response. Genes up-regulated by feeding dogs lipoic acid for 30 days that are involved in interferon mediated antiviral response are listed in Table 4.

Table 4

30-days lipoic acid canine adult

Gene Symbol Protein Protein Name Fold 1 p- p-yalue regulated

CREBBP i CBP Human CRLB-binding

i protein

EIF2AIC E2AK2 Human , Interferon- 1 4 0 04

! induced double j stranded RNA- i activated protein

1FNAR2 INAR2 Human | interferon- 1.3 alpha/beta receptor beta chain precursor

IFNGR2 1NGR2 Human interferon- 1.2 gamma receptor beta chain precursor

1RF9 IRF9 Human interferon ' 1.3 regulatory factor , 9

JΛK2 JAK2 Human T} rosine protein 1 4 kinase JAK2

RXASFL KSt 54 Human 2-5Λ-dcpcndent 1.5 πbonuc lease [0077J Based on the studies of dogs fed lipoic acid for 30 days, the in\entors have surprisingly found that cell surface receptors for interferon alpha/ beta and interferon gamma are increased leading to the potential for increasing the entire interferon mediated antiviral defense mechanism. The inventors

found that JAK2, a ke> activator of STAT 1 and STΛT2, is up regulated. Interferon regulatory factor 9 (IFR9) is up-regulated, 1FR9, STATl and STAT2 form a complex (ISFG3) that translocates to the nucleus and up regulates the antiviral genes, interferon-induced, double stranded RNA-activated protein kinase (PKR) and 2-5A-dependent ribonuclease (RnaseL). PKR inhibits elF2Sl via phosphorylation leading to an inhibition of viral protein synthesis. RnaseL cleaves viral RNA inhibiting viral replication and function.

[0078J The invention is not to be limited in scope by the specific embodiments disclosed in the examples, which are intended as illustrations of a few aspects of the invention, and any embodiments, which are functionally equivalent, are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the appended claims.

[0079] For any references that have been cited, the entire disclosures of which are incorporated herein by reference.

Claims

CLAIMS What is claimed is:

1. A pet food composition comprising an effecth e amount of lipoic acid to enhance antiviral activity in a companion animal, wherein said effective amount of lipoic acid to enhance antiviral activity in a companion animal is at least about 25 ppm.

2. The composition of claim 1, wherein the effective amount is at least about 50 ppm.

3. The composition of claim 1 , wherein the effective amount is at least about 100 ppm.

4. The composition of claim 1. wherein the effective amount is about 100 ppm to about 600 ppm.

5. The composition of claim 1, wherein the effective amount is about 100 ppm to about 200 ppm.

6. The composition of claim 1, v\ herein the companion animal is a dog.

7. The composition of claim 1, wherein the companion animal is a cat.

8. The composition of claim 1, wherein the effective amount is effective to enhance innate antiviral activity in a companion animal.