Patent Application
20100137404
The present invention relates to compositions and methods regulating gene expression or transcription in a growing animal.
Commercially available canine and feline foods include compositions specially formulated to address many different nutritional needs. These include, for example, formulations designed for different breed types, sizes and body conditions. They also include formulations designed to address the nutritional needs of animals in the different stages of their life cycle. Typically, these stages include the growth, adult and senior stages of life. For example, U.S. Pat. No. 5,851,573 discloses a pet food composition for large breed puppies; U.S. Pat. No. 6,426,100 discloses compositions to provide improved bone modeling and chondrocyte functioning in growing animals; U.S. Pat. No. 6,582,752 discloses gender specific puppy food. Despite the availability of such pet food formulations, however, the need remains for the development of additional formulations comprising innovative ingredients and nutrients designed to enhance the development of growing animals.
It is known in the art that certain nutrients have an effect on gene expression. Nutrigenomics is the study of such a relationship. Despite what is already known, there is a need to develop compositions and methods which may positively influence gene expression of an animal.
In certain aspects, the present invention relates to compositions that are useful to enhance the development of a growing animal. Particularly, the compositions of the present invention comprise one or more nutrients or bioactive substances that can enhance neurologic development, bone and joint health, immune function, and promote healthy body composition in a growing animal. In certain embodiments, the nutrients and bioactive substances include, but are not limited to, fatty acids, antioxidants, essential nutrients, amino acids, minerals and trace elements, vitamins and vitamin-like substances. Other aspects of the invention relate to methods to enhance the development of a growing animal comprising administration of effective amounts of the compositions of the present invention directly to a growing animal or to the dam of said animal while the animal is in utero or is a nursling.
Thus, in one aspect, the present invention includes Composition 1.0, a pet food composition comprising:
about 5 to about 70% protein,
about 0.5 to about 1.6% methionine,
about 50 to about 200 ppm manganese,
about 0.1 to about 0.5% DHA,
about 0.1 to about 0.7% EPA,
about 1200 to about 7500 ppm choline,
about 1000 to about 2000 ppm taurine,
about 2.5 to about 6% linoleic acid,
about 1 to about 3% total n-3 fatty acids,
about 50 to about 1200 IU/kg vitamin E,
about 50 to about 500 ppm vitamin C,
about 50 to about 500 ppm carnitine, and
about 2.5 to about 7 g lysine/1000 kcal.
The preset invention also includes the following compositions:
1.1 Composition 1.0 comprising:
In another aspect, the invention relates to methods to cause a beneficial modification in gene expression in an animal, specifically, down regulation in expression of a gene or genes associated with an undesirable biological condition or pathway or disease state and/or up regulation in expression of a gene or genes associated with a desired biological condition or pathway or which may have a positive or preventive effect on a disease state for any one or more biological conditions, pathways or disease states and genes described in Tables 2-15, comprising administering an effective amount of a composition of according to any one of compositions 1.0-1.14 to an animal, either directly to the animal or to the dam while the animal is in utero.
In another aspect, the invention is directed to Method 2.0, a method to regulate gene expression in a canine comprising administering to the canine any one of compositions the compositions of the present invention, e.g., compositions 1.0-1.14.
The present invention also includes the following methods:
In a further aspects, the invention relates to the use of any of the formulae of the present invention in the manufacture of a composition to modify gene expression in an animal as described herein.
Other features and advantages of the present invention will be understood by reference to the detailed description of the examples that follow.
It is contemplated that the invention described herein is not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, all references cited herein are hereby incorporated by reference in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.
The present invention relates to any animal, preferably a mammal, more preferably a companion animal. The term “companion animal” refers to any animal that lives in close association with humans and includes, but is not limited to, canines and felines of any breed. It is contemplated herein, however, that any animal whose diet may be controlled by humans may benefit from feeding the formulations disclosed herein. These animals may include, for example, domesticated farm animals (e.g., cattle, horses, swine, etc.) as well as undomesticated animals held in captivity, e.g., in zoological parks and the like. Preferred animals include canines, e.g., dogs, including growing dogs, e.g., puppies.
“Beneficial modification in the expression of genes” as used herein includes, e.g., down regulation of genes expressing proteins associated with disease states and/or up regulation of genes expressing proteins which have a beneficial or healthful effect as compared to appropriate controls, as may be determined using conventional methods, e.g., by microarray (e.g., Affymetrix gene chip) techniques familiar to one of skill in the art. Further, one of skill in the art is familiar with the known associations between diseases and specific genes as those listed in the tables provided hereinbelow such that it is understood whether an increase or decrease in expression of a particular gene is desirable.
The “growth” life stage of an animal refers to the period from birth or weaning (approximately 8 weeks of age) to about 1 year of age or beyond, depending on the species and breed of the animal.
As used herein, the term “puppy” refers to an immature canine, typically between the ages of birth and 12 months.
“Essential amino acids” as used herein refers to those amino acids that cannot be synthesized de novo by an organism and thus must be supplied in the diet. It is understood by one of skill in the art that the essential amino acids varies from species to species, depending upon the organism's metabolism. For example, it is generally understood that the essential amino acids for dogs and cats (and humans), are phenylalanine, leucine, methionine, lysine, isoleucine, valine, threonine, tryptophan, histidine and arginine.
As understood by one of skill in the art, a “limiting amino acid” refers to an amino acid which if present in insufficient quantities in a diet, results in the limitation in usefulness of other essential amino acids, even if the other essential amino acids are present in otherwise large enough quantities. Lysine is the limiting essential amino acid in the compositions disclosed herein. Thus, the remaining essential amino acids are quantitatively formulated or “balanced” in relationship to the amount of lysine determined critical to affect the desired biological result. As used herein, “balanced amino acids” refers to the relationship of the essential amino acid lysine to energy to assure optimal animal growth and development.
“Essential nutrients” as used herein refers to nutrients required for normal body functioning that cannot be synthesized by the body. Categories of essential nutrient include vitamin dietary minerals, fatty acid, and amino acid. It is understood by one of skill in the art that the nutrients deemed essential varies from species to species, depending upon the organism's metabolism. For example, essential nutrients for dogs and cats include Vitamins A, D, E, K, B1, B6 B12, riboflavin, niacin, pantothenic acid, folic acid, calcium, phosphorous, magnesium, sodium, potassium, chlorine, iron, copper, zinc, manganese, selenium and iodine. Choline, generally regarded as a B complex vitamin, may be included among the semi-essential nutrients. In addition, taurine, while technically not an amino acid but a derivative of cysteine, is an essential nutrient for cats.
Carnitine, also known as L-carnitine, (levocarnitine) is a quaternary ammonium compound synthesized from the amino acids lysine and methionine and is responsible for the transport of fatty acids from the cytosol into the mitochondria.
Without being limited to any theories or particular modes of action, the present invention is based on the surprising discovery that the addition of certain ingredients to pet food compositions and administration of these compositions to animals can enhance the development of a growing animal. For example, data indicate that animals fed the compositions of the present invention (or those whose dams were fed the compositions during gestation and prior to weaning but continued throughout growth of their litters), demonstrate enhanced neurologic development, bone and joint health, immune function, and have overall healthier body composition. Interestingly, microarray data also indicate a differential change in gene expression in these animals compared to controls which is generally reflective of a beneficial modification (i.e., up or down regulation) in the expression of many genes associated with biological processes including those involved in growth and development. Thus, in one aspect, the invention relates to compositions and methods to enhance the development of a growing animal as described in detail herein.
As contemplated herein, the compositions of the present invention comprise nutritionally complete and balanced animal feed compositions. Such compositions include, among other nutrients and ingredients, recommended healthful amounts of protein, carbohydrate and fat. “Nutritionally complete and balanced animal feed compositions”, as well as nutrients and ingredients suitable for animal feed compositions, and recommended amounts thereof, are familiar to one of skill in the art (see, for example, National Research Council, 2006 Nutritional Requirements for Dogs and Cats, National Academy Press, Washington D.C. or the Official Publication of the Association of American Feed Control Officials, Inc. Nutrient Requirements for Dogs and Cats 2006).
It is contemplated herein that the compositions disclosed herein may also comprise antioxidants, additives, stabilizers, thickeners, flavorants, palatability enhancers and colorants in amounts and combinations familiar to one of skill in the art. “Antioxidants” refers to a substance that is capable of reacting with or decreasing the production of free radicals and neutralizing them. Examples include, but are not limited to, beta-carotene, selenium, coenzyme Q10 (ubiquinone), lutein, tocotrienols, soy isoflavones, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, vitamin E, vitamin D, vitamin C, flavanoids, anthocyanindins, and lipoic acid.
While foods of any consistency or moisture content are contemplated, preferably the compositions of the present invention may be, for example, a wet, semi-dry, or dry animal food composition. “Wet” food refers to food which is sold in cans or foil bags and has a moisture content of about 70 to about a 90%. “Dry” food refers to compositions with about 5 to about 15% moisture content and is often manufactured in the form of small bits or kibbles. Semi-dry compositions refers to food which has a moisture content greater than dry foods, but less than wet foods. Also contemplated herein are compositions of intermediate moisture consistency and those that may comprise components of various consistency as well as components that may include more than one consistency, for example, soft, chewy meat-like particles as well as kibble having an outer cereal component and an inner cream component as described in, e.g., U.S. Pat. No. 6,517,877.
Various processes for manufacturing and packaging the compositions of the present invention may be employed and are familiar to one of skill in the art.
It is also contemplated herein that the methods of the present invention include methods to cause a beneficial modification in gene expression in an animal, specifically, down regulation in expression of a gene or genes associated with an undesirable biological condition or pathway or disease state and/or up regulation in expression of a gene or genes associated with a desired biological condition or pathway or which may have a positive or preventive effect on a disease state, as the case may be, for any one or more biological conditions, pathways or disease states and genes described in Tables 14-27, comprising administering an effective amount of a composition of the present invention to the animal, either directly to the animal or to the dam while the animal is in utero. Indeed, as discussed in Examples 7 and 8 here-in-below, by effecting the animal at the genomic level, the administration of the compositions of the present invention, either directly to the animal or to the dam while the animal is in utero, may have beneficial, even prophylatic, health effects on the animal by effecting the expression of any one or more genes listed in Tables 14-27. For example, the administration of an effective amount of a composition of the present invention to an animal may enhance bone and joint health in the animal by causing the down regulation in genes associated with cartilage and joint damage associated with arthritis, e.g. interleukin 1-beta, fibronectin, lactoransferrin, etc. (see Table 14).
It is contemplated herein that the compositions of the present invention may be administered to an animal alone as a complete nutritionally balanced diet, or in conjunction with dietary supplements, vitamins and/or other nutritionally beneficial agents familiar to one of skill in the art, as part of an overall wellness program for the animal. Compositions of the invention may also be useful as a veterinary therapeutic product. As such, the compositions may optionally contain a carrier, diluent, or an excipient, the suitability of which for the intended use being familiar to one of skill in the art.
It is also contemplated that, in addition to administering the compositions disclosed herein directly to a growing animal, e.g., to a growing puppy or kitten, the compositions may be administered to the dam of the animal while the animal is still in utero or while the animal is a nursling.
The following examples further illustrate the present invention and are not intended to limit the invention. As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. It is understood that when formulations are described, they may be described in terms of their ingredients, as is common in the art, notwithstanding that these ingredients may react with one another in the actual formulation as it is made, stored and used, and such products are intended to be covered by the formulations described.
The following examples further describe and demonstrate illustrative embodiments within the scope of the present invention. The examples are given solely for illustration and are not to be construed as limitations of this invention as many variations are possible without departing from the spirit and scope thereof. Various modifications of the invention in addition to those shown and described herein should be apparent to those skilled in the art and are intended to fall within the appended claims.
Except to the extent stated otherwise, all percentages used in this specification are weight percentages on a dry matter basis. The phrase “dry matter basis” means the component concentration in the composition after any moisture in the composition is removed.
In order to characterize the genotypic effects of feeding a composition formulated to enhance the development of a growing animal, an experiment is performed to identify changes in gene expression in puppies at one year of age from dams fed the compositions disclosed in Table 1.
Composition P is a commercially available dog food. Composition H is an experimental dog food composition.
Dams are fed either Composition H or Composition P for at least 10 days prior to conception. Dams are maintained in group lodging until confirmed pregnant via palpation, and are then moved to maternity lodging. Puppies from dams are kept on the same foods fed to the dams until one year of age (including pre-weaning and post-weaning period). Blood samples are then taken from the puppies and mRNA isolated according to conventional methods. Microarray assays are performed using the Affymetrix Canine-2 gene chip according to conventional methods.
The tables below show the genes grouped by function and the direction of expression, wherein up regulation in Composition H (“H”) vs Composition P (“P”) demonstrates increased gene expression in Composition H fed puppies compared to Composition P fed puppies. Similarly, down regulation of a gene in the Composition H fed puppies vs Composition P fed puppies represents decreased gene expression in puppies fed Composition H relative to those fed Composition P.
Gene expression profiles from puppies fed Composition H and P are obtained and compared. Results indicate that 143 genes are differentially expressed between the study groups and, in general, their functions may be associated with cell assembly, cell cycle regulation, DNA replication and repair, cell compromise, arthritis and cancer. At a minimum of 1.5 fold change, 143 genes are differentially expressed in the two groups.
Genes associated with arthritis/inflammation are down-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests reduced cartilage/joint damage in Prototype pups. A separate study performed also indicates that levels of bone alkaline phosphatase are lower in puppies fed composition H (data not shown). See Table 2.
Genes associated with DNA fragmentation are down-regulated and genes associated with DNA processing control are up-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests reduced DNA damage and improved DNA protection in Prototype pups (also supported by elevated Vitamin E levels in blood, data not shown). See Table 3.
Genes associated with cancer incidence are down-regulated and genes associated with tumor suppression are up-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests the possibility of reduced cancer susceptibility in these Prototype animals; see Table 4.
Genes associated with cellular compromise are down-regulated and genes associated with cellular integrity are up-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests the possibility of reduced cell damage and enhanced cellular protection in these Prototype animals (also supported by elevated Vitamin E levels in blood, data not shown). See Table 5.
Genes associated with disruption of cellular assembly are down-regulated and genes associated with proper cellular organization are up-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests improved cellular organization and function in Prototype pups. See Table 6.
Genes associated with proper cell cycle regulation are up-regulated and genes associated with disruption of cell cycle control are down-regulated in puppies fed Composition H compared to puppies fed Composition P, which suggests proper control of cell cycle progression and cell survival. See Table 7.
Thus, it is contemplated herein that the nutritional benefits of the compositions of the present invention as described herein may involve modification in gene expression which results in the enhancement of the development of a growing animal. In addition, feeding proper nutrients during early development may have a prophylatic effect and influence disease processes later in life, i.e., lessen the chance of disease in the animal, as the expression of genes associated with common diseases and disorders may be influenced due to the given nutritional compositions of the foods provided maternally and during early development.
An experiment is performed to determine the effects on gene expression in puppies fed Composition H vs. Composition P from weaning until one year of age.
Dams utilized in this study are fed Composition P prior to, and during pregnancy. Following weaning, puppies are divided into two groups and provided with either Composition H or maintained on Composition P until one year old. Blood samples are then taken from the puppies and mRNA isolated according to conventional methods. Microarray assays are performed using the Affymetrix Canine-2 gene chip according to conventional methods.
Results indicate that 99 genes are differentially expressed between the two study groups, and are presented in Tables 9-15. Of the genes identified, many are related to biological functions or pathways such as, e.g., immune activation, lipid metabolism, cardiovascular development, skeletal and muscular disorders, contraction and function and cell compromise and cancer.
Gene expression profiles from puppies fed Composition H or P from weaning to one year of age are obtained and compared. Microarray data indicate that, at a minimum of 1.5 fold change, 99 genes are differentially expressed in the two study groups.
Genes associated with immune activation are down-regulated in puppies fed Composition H when compared to puppies fed Composition P, which suggests improved immune system function (Table 9).
Genes associated with lipid metabolism are down-regulated in puppies fed Composition H when compared to puppies fed Composition P, which suggests reduced lipid processing (Table 11).
Genes associated with cardiovascular development are also down-regulated in puppies fed Composition H when compared to puppies fed Composition P, which suggests enhanced cardiovascular health (Table 12).
Genes associated with skeletal muscular disorders are down-regulated and genes associated with muscle contraction/function are up-regulated in puppies fed Composition H when compared to puppies fed Composition P, which suggests reduced skeletal disorder risk and improved muscle contraction (Table 13).
Genes associated with cellular compromise are down-regulated in puppies fed Composition H when compared to puppies fed Composition P, which suggests reduced cellular damage (Table 14).
Genes known to have an association with cancer are down-regulated in puppies fed Composition H when compared to puppies fed Composition P, which may suggest reduced cancer susceptibility. (Table 10).
As discussed above, genetic data such as these indicate that the nutritional benefits of the compositions of the present invention include the beneficial modification of gene expression in the animal such that there is an overall enhancement in the development of the animal. In addition, the beneficial modification of gene expression may also result in a decrease in the incidence of disease in the animal due to an inhibition in expression of disease related genes and/or an increase in the expression of genes which play a role in disease prevention.
This application claims priority to U.S. Provisional Application No. 60/891,171, filed on Feb. 22, 2007, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2008/054800 | 2/22/2008 | WO | 00 | 1/19/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60891171 | Feb 2007 | US |
