WO2004014152A1

WO2004014152A1 - Multi-phase delivery system comprising a semi-solid matrix phase

Info

Publication number: WO2004014152A1
Application number: PCT/CA2003/001206
Authority: WO
Inventors: Jonathan Farber; Michael Farber
Original assignee: Vitalstate Canada Ltd.
Priority date: 2002-08-13
Filing date: 2003-08-13
Publication date: 2004-02-19
Also published as: AU2003257313A1

Abstract

A multi-phase delivery system that provides for the oral delivery of one or more biologically active agents, methods for the production of the delivery system and uses thereof for the delivery of biologically active agents to animals, as food supplements and as meal replacements are provided. The delivery system comprises at least two phases, one of which is a semi-solid matrix phase having at least one bioactive agents substantially uniformly dispersed therein.

Description

MULTI-PHASE ORAL DELIVERY SYSTEM COMPRISING A SEMI-SOLID MATRIX PHASE

FIELD OF THE INVENTION

The present invention pertains to the field of delivery systems for biologically active agents and in particular to a multi-phase delivery system comprising two or more discrete phases.

BACKGROUND

Nutritional food bars and snack products are convenient nutritional supplements, particularly for those persons too busy or unable to eat regular meals, for dieters as meal replacements, and for hikers, cyclists, runners or other athletes who need high- energy nutritional snacks while they are exercising. Such bars are also convenient nutritional supplements for the elderly or those who need prepackaged, ready-to-eat snacks. Additionally, such food supplements can supply consumers with the necessary vitamins and minerals specified in the recommended daily allowances provided by various governmental and health authorities.

A number of nutritional products have been described. For example, U. S. Patent No. 4,018,427 describes a ready-to-eat breakfast cereal in which various toasted cereal flakes or puffed cereals are preserved and agglomerated within a fat-syrup double or single coating and U.S. Patent No. 4,543,262 describes a high protein, low or no lactose, vitamin and mineral fortified, nutritionally-balanced snack bar. Additionally, U.S. Patent No. 3,814,819 describes a protein-fortified food bar composed of several layered baked crisp wafers with a creamy filling between the layers. The creamy filling contains added vitamins, providing twenty-five percent (25%) of the recommended daily allowance of vitamins and minerals. U.S. Patent Nos. 4,152,462 and 4,152,463 describe protein and vitamin enriched food bars, having a marshmallow base, and their method of manufacture. U.S. Patent No. 3,901,799 describes a high protein chocolate bar, in which caseinate and peanut butter are added to a mixture of chocolate and cocoa butter and to which vitamins compatible with the ingredients can be added. U.S. Patent No. 4,039,688 describes a food bar having a high protein, flavored outer shell and an inner filling. The food bar is described as comprising an outer shell of protein-supplemented marshmallow, and an inner filling comprising an intermediate moisture food product such as cheese, fruit jam or jelly. U.S. Patent No. 6,432,457 describes a confectionery product intended for use as a meal replacement. The product contains protein and carbohydrate materials present in a relative weight ratio higher than 1. U.S. Patent Application No. 2002/0054944 describes a snack product mainly comprising an amylaceous material and milk solids which is rich in protein and calcium and which may optionally contain added vitamins, oligoelements, sodium chloride and/or a source of dietary fibre.

hi addition to nutritional products incorporating basic dietary supplements, some food- or confectionery-based products incorporating other supplements have been described. For example, U.S. Patent No. 5,906,833 describes a nutritional supplement comprising separately identifiable parts each containing a chronologically appropriate dosage of drugs, vitamins, herbs, hormones, minerals, enzymes or other nutrients.

The nutritional supplement is described as being contained in a palatable base, such as a food bar. U.S Patent No. 6,365,209 describes an encapsulated product that may be incorporated into a food item. The encapsulated product can be a vitamin, mineral, herbal or drug. International Patent Application WO02/00033 describes a confectionery products, such as chocolate, containing one or more active ingredients incorporated into carrier bodies. The carrier bodies are described as pellets, capsules, strands or layers that are visually or texturally distinct and contain active ingredient(s) such as vitamins, enzymes, minerals, trace elements and botanical extracts.

While a number of nutritional products and food- or confectionery-based supplements are known in the art a need still exists for a system into which a variety of biologically active agents may be incorporated and which can optionally contain nutritional quantities of protein, carbohydrates, fats, vitamins and minerals, or other nutritional supplements. Such a system would be convenient for individuals who desire a more balanced nutritional product or one with specific types of bioactive agents. Furthermore, while consumers are concerned about health many are unwilling to sacrifice taste to achieve good health. There exists, therefore, a need in the art for a delivery system for bioactives that provides similar taste, texture, and/or convenience to a food bar.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a multi-phase delivery system. In accordance with an aspect of the present invention, there is provided a multi-phase oral delivery system for biologically active agents comprising:

(a) one or more biologically active agent(s);

(b) a matrix phase comprising: i) one or more sugar; ii) one or more carbohydrate; iii) one or more hydrocolloid; iv) one or more polyhydric alcohol, and v) one or more source of water, and having at least one of said biologically active agent(s) substantially uniformly dispersed therein, wherein said matrix phase is a semi-solid at room temperature, has a final moisture content between about 15% and about 30% by weight and a water activity of less than about 0.7, and

(c) one or more other phase associated with the matrix phase, said one or more other phase comprising at least one of said biologically active agent(s), one or more nutritional component(s), or a combination thereof, i accordance with another aspect of the invention, there is provided a multiphase oral delivery system for biologically active agents comprising: (a) one or more biologically active agent(s); (b) a matrix phase comprising; i) one or more sugar; ii) one or more carbohydrate; iii) one or more hydrocolloid; iv) one or more polyhydric alcohol, and v) one or more source of water, and having at least one biologically active agent(s) substantially uniformly dispersed therein, wherein said matrix phase is a semi-solid at room temperature, has a final moisture content between about 15% and about 30% by weight and a water activity of less than about 0.7; (c) one or more other phase associated with the matrix phase, said one or more other phase comprising at least one of said biologically active agent(s), one or more nutritional components), or a combination thereof, and (d) an artificial membrane vesicle (AMV) phase dispersed in the matrix phase, the one or more other phase, or in both the matrix phase and the one or more other phase, said AMV phase comprising at least one of said biologically active agent(s) associated with an AMV.

In accordance with another aspect of the present invention, one or more biologically active agent included in the multi-phase delivery system is a drug, a botanical, a nutritional supplement, a vitamin, a mineral, an enzyme, a hormone, a protein, a polypeptide, an antigen, or a combination thereof.

In accordance with another aspect of the present invention, there is provided a process for preparing a multi-phase delivery system for biologically active agents, said process comprising:

(a) preparing a matrix phase by

(i) preparing a blend comprising one or more hydrated carbohydrate, one or more hydrated hydrocolloid, one or more sugar, sugar alcohol or sugar syrup, or a combination thereof, and water at a temperature of less than 100°C;

(ii) optionally adjusting the moisture content of the blend; (iii) reducing the temperature of the blend to between about 50°C and about 80°C; (iv) adding to said blend one or more biologically active agent and a solvent component comprising one or more polyhydric alcohols thereby forming said matrix phase;

(b) preparing one or more other phase comprising one or more biologically active agent, one or more nutritional component, or a combination thereof, and

(c) combining said matrix phase and said one or more other phase to form said multi-phase delivery system.

In accordance with another aspect of the present invention, there is provided a multiphase delivery system prepared by a process of the present invention.

In accordance with another aspect of the present invention, there is provided a use of a multi-phase delivery system for oral administration of one or more biologically active agent to an animal in need thereof.

In accordance with another aspect of the present invention, there is provided a kit comprising a multi-phase delivery system and optionally instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 demonstrates the enhanced uptake of creatine into the blood following administration to humans of jujubes prepared according to Example 6.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to the particular process steps and materials disclosed herein, but is extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, weight percentages (% weight) are based on the total weight of the composition for the present delivery system.

The term "animal" as used herein includes, but is not limited to mammals, including humans, birds, and reptiles.

The terms "biologically active agent" and "bioactive agent" as used interchangeably herein include physiologically or pharmacologically active substances that produce a localized or systemic effect or effects in animals and includes, for example, drugs, nutritional supplements, botanicals, botanical extracts, vitamins, minerals, enzymes, hormones, proteins, polypeptides, antigens and other pharmaceutically or therapeutically useful compounds.

The term "binder", when used herein refers to a composition that essentially acts as a "glue" for combining relatively dry ingredients. The composition can be a mixture of various humectants, such as water, fat, syrup, sugars. Certain solvents (for example, glycerol and other polyols), lecithin, vegetable and other oils (such as sunflower oil) can also act as binders. A binder may also be combined with other ingredients such as protein, vitamins, minerals, macronutrients, flavouring and colourings.

The terms "antigen" and "antigenic material" as used herein refer to a molecule or molecules, a portion of a molecule or a combination of molecules, up to and including whole cells, capable of provoking an immune response in an animal.

The term "artificial membrane vesicle (AMV)," as used herein, refers to a preparation of single-lamellar vesicles, multi-lamellar vesicles, or a combination thereof. The term encompasses both traditional phospholipid-based liposomes as well as newer artificial membrane vesicles including, but not limited to, niosomes, lederosomes and archeosomes that comprise compounds such as ether lipids, fluorinated lipids, synthetic double chain amphiphiles, single chain amphiphiles, polyhydroxyl lipids, polyhedral non-ionic surfactants, polymerized liposomes, cationic amphiphiles, plasmalogens and the like.

The term "AMV-associated," as used herein with reference to a biologically active agent means that the agent is encapsulated by, adhered to, embedded in, trapped between, mixed with or otherwise combined with one or more AMV. The term also encompasses biologically active agents to which one or more AMV is adhered.

The term "encapsulated by," as used herein with reference to an AMV-associated biologically active agent, refers to an agent that is completely encapsulated or enclosed in the interior portion of an AMV.

The term "adhered to," as used herein with reference to an AMV-associated biologically active agent, refers to an agent that is attached to or trapped between AMVs, or clusters of AMVs, but is not necessarily enclosed within the interior of a portion of an AMV.

The terms "pro-AMV" and "pre- AMV" are used interchangeably herein to refer to a preparation of AMVs that has been treated such that the shelf-life of the AMVs has been extended when compared to a normal AMV solution. Typically, extension of shelf life is achieved through removal of the AMVs from contact with water, for example, by dehydration (including lyophilisation) of a pre-formed AMV solution, or suspension of pre-formed AMVs or materials that will form AMVs in an organic solvent, or dispersion of materials that will form AMVs in a minimal amount of water to form a gel or paste. Thus, the terms can refer to solutions, powders, suspensions or gels.

The term "carbohydrate" as used herein refers to both simple (mono- and disaccharides) and complex (polysaccharide) carbohydrates.

The term "nutritional component,' as used herein, refers to a substance that replenishes, or promotes growth of, tissue or which generally promotes the health of an organism. In the context of the present invention, a nutritional component is typically a substance that falls into one of the basic food groups, i.e. a protein, carbohydrate or fat (including oils and lipids). Dietary fibre is also considered to be a nutritional component in that it promotes digestive tract health.

As used herein, the term "about" refers to a +/-10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

I. MULTI-PHASE DELIVERY SYSTEM

The multi-phase delivery system according to the present invention comprises at least two discrete phases, of which one is a semi-solid matrix phase that comprises one or more bioactive agents dispersed therein. Bioactive agents may further be incorporated into one or more of the other phases. The composition of the other phases may vary considerably provided that they are capable of remaining distinct from one another and from the matrix phase. Examples of non-matrix phases suitable for incorporation into the delivery system include, but are not limited to, solid, typically low-moisture content, phases (such as biscuit, cookie, or wafer-type formats); soft, typically intermediate-moisture, phases (such as caramel, soft nougat or marshmallow type formats); coatings (for example, compound coatings, chocolate or yoghurt coatings) and various combinations thereof. Another example of a non-matrix phase is an artifical membrane vesicle (AMV) phase. One or more bioactive agents may be associated with the AMVs in this phase. When an AMV phase is included in the delivery system it is dispersed in one or more of the other phases.

In one embodiment, the multi-phase delivery system of the present invention provides for the physical separation of different bioactive agents, which may be incompatible or reactive when found in a homogenous system, into the separate phases.

hi another embodiment of the present invention, the delivery system comprises at least two discrete phases, these phases being one or more solid, soft or coating non-matrix phase having one or more bioactive agent dispersed therein and/or comprising one or more nutritional components and a matrix phase comprising one or more bioactive agent substantially uniformly dispersed therein. In a fiirther embodiment, the delivery system comprises three or more discrete phases. In accordance with this embodiment, these phases are at least one solid, soft or coating non-matrix phase having one or more bioactive agent dispersed therein and/or comprising one or more nutritional components, a matrix phase comprising one or more bioactive agent and an AMV phase having one or more bioactive agent associated therewith. The AMV phase may be dispersed in one or more non-matrix phase, in the matrix phase, or in both the matrix and non-matrix phases.

h accordance with one embodiment of the present invention, the multi-phase delivery system comprises between about 10% and about 50% by weight of the matrix phase. another embodiment, the multi-phase delivery system comprises between about 10% and about 30% by weight of the matrix phase, hi a further embodiment, the multi-phase delivery system comprises between about 15% and about 25% by weight of the matrix phase.

The delivery system is especially suited for oral administration due to its palatability. Additionally, due to its highly portable format, the delivery system is simple and convenient to administer and to consume for both humans and animals. One skilled in the art will appreciate that each delivery system can be formulated differently according to the type of animal to which it is to be administered. For example, for administration to an animal such as a cat or a dog, meat or fish-based flavours may be added. For administration to a human, the delivery system may be formulated, for example, as a confectionery using fruit-based or other flavours or as a snack bar or cookie. Due to their highly portable format, the delivery systems are simple and convenient to administer and to consume for both humans and other animals.

In one embodiment of the present invention, the multi-phase delivery systems are tailored for specific purposes, i.e. the delivery systems are formulated with specific combinations of bioactive agents in order to produce specific physiological effects. For example, multi-phase delivery systems can be formulated for therapeutic or diagnostic applications and accordingly would comprise drugs or diagnostic agents as the bioactive agent(s). Other examples include delivery systems to promote sexual potency, promote endurance, promote cardiovascular health, control fat and/or cholesterol, promote healthy joints, maintain or improve bone density, enhance cellular anti-oxidant capacity, control appetite, promote energy, increase endurance, promote weight loss, promote muscle enhancement, improve digestion, prevent colds, fight infection, and the like. In one embodiment of the invention, the multi-phase delivery system is formulated for use as a protein supplement.

The present invention further contemplates delivery systems that comprise one or more nutritional components in sufficient quantities to constitute a food supplement or meal replacement. Such delivery systems may comprise additional bioactives to the nutritional components and may be useful, for example, for athletes, for weight management, for diabetics and the like. Thus, one embodiment of the present invention provides for food supplement or meal replacement delivery systems. Typically in this embodiment of the invention, the nutritional components are dispersed in one or more non-matrix phases, although the dispersion of one or more nutritional component in the matrix phase is also contemplated, hi the context of the present invention, a "food supplement" refers to a delivery system that provides at least part of an individual' s daily caloric, protein, fat and/or fibre requirement, and a "meal replacement" refers to a delivery system that comprises a combination of carbohydrate, protein and fat or lipid in amounts that provide at least part of an individual's daily requirement for these components. A meal replacement may additionally comprise dietary fibre and both food supplement and meal replacement formulations may optionally comprise other nutritional compounds such as vitamins, minerals, and the like.

One skilled in the art would appreciate that specific selections of the possible components provided below, must be safe for animal consumption. Components for inclusion in the delivery systems are, therefore, substances that are generally regarded as safe (GRAS) and/or meet regulatory standards, such as those of the Codex

Alimentarius. Examples falling within the general descriptions that are significantly toxic or cause other types of significant harm to animal health are explicitly excluded from the description of the invention.

1. Matrix Phase The texture, physical attributes, form and shape of the matrix as described below, can be varied by altering the ratio of ingredients within the given ranges using the methods described herein or by methods familiar to a worker skilled in the art.

The matrix phase of the delivery system provides for substantially uniform and complete dispersion of the bioactive agents contained therein while minimizing their degradation. The matrix phases which comprises 1) one or more carbohydrates that exhibit good moisture binding and low gelatinisation temperature; 2) a sugar component comprising one or more sugars, sugar syrups and/or sugar alcohols; 3) a hydrocolloid component; 4) a solvent component comprising one or more polyhydric alcohols; and 5) one or more sources of water. The use of one or more carbohydrates and a hydrocolloid component in amounts within the ranges indicated below results in a matrix that readily retains the solvent component and thereby prevents separation of the solvent from other components of the matrix. The delivery system may optionally further comprise one or more sources of mono- or divalent cations. Additives such as natural or artificial flavourings, colourings, acidulants, buffers and sweeteners can be included in conventional amounts in the matrix.

The matrix of the delivery systems provides for minimised degradation of the bioactive agents dispersed therein during the preparation of the matrix and the storage of the final delivery systems. The use of relatively low temperatures in the preparation of the matrix, when compared to typical manufacturing procedures for confectioneries, ensures that the bioactive agents are not degraded by excessive heat. In accordance with the present invention, the matrix is prepared at a temperature of 100°C or less, hi one embodiment of the present invention, the matrix is prepared at or below a temperature of about 75°C. In other embodiments, the matrix is prepared at or below a temperature of about 70°C, and at or below a temperature of about 65°C. Low temperatures can be employed in the preparation of the matrix because it is formulated to remain flowable at temperatures at or above about 35°C. In one embodiment of the invention, the matrix remains flowable at or above about 45°C.

In addition, the matrix has a low water content, which also contributes to the stability of the bioactive agents dispersed therein. In accordance with the present invention, the final moisture content of the matrix is between about 10% and about 30%. In one embodiment, the final moisture content of the matrix is between about 11% and about 25%. In other embodiments, the moisture content is between about 13% and about 20%, between about 15% and about 18%, and between about 15% and about 16%.

Furthermore, the matrix of the present invention has a low water activity (a_w), typically below about 0.7. hi one embodiment of the invention, the water activity of the matrix is below about 0.6. In other embodiments, the water activity is below about 0.55, between about 0.45 and about 0.55, between about 0.5 and about 0.55, and between about 0.47 and about 0.52.

In accordance with the present invention, degradation of the bioactive agents during the process of preparing the matrix is less than about 20%. In one embodiment, degradation of the bioactive agents during preparation of the matrix is less than about 15%. hi other embodiments, degradation during preparation is less than about 10%, less than about 5%, less than about 2% and less than about 1%.

The matrix also provides for minimised degradation of the bioactive agents dispersed therein during storage of the final delivery systems under normal storage conditions (i.e. at temperatures of 30°C or below). In accordance with the present invention, therefore, degradation of the bioactive agents in the matrix during storage of the delivery systems under normal conditions is less than about 20%. In one embodiment, degradation of the bioactive agents in the matrix during storage is less than about 15%. In other embodiments, degradation during storage is less than about 10%, less than about 5%, less than about 2% and less than about 1%.

The matrix can be formulated to have a final pH in the range of about 2.5 to about 8.5. In one embodiment, the matrix has a final pH of between about 3.0 and about 8.5. Acidic pH is known in the art to promote degradation of certain bioactive agents. Accordingly, when bioactive agents which are sensitive to, or reactive at, acidic pH are to be incorporated into the matrix, it is formulated to have a final pH that is neutral to mildly basic. By neutral to mildly basic pH it is meant that the final pH is between about 6.0 and about 8.5. hi one embodiment of the present invention, the matrix is formulated to have a final pH between about 6.2 and about 8.5 and thus is suitable for delivery of bioactive agents that are sensitive to, or reactive at, acidic pH. In other embodiments, the final pH of the matrix of the delivery systems is between about 7.0 and about 8.5, and between about 7.1 and about 8.0.

For those bioactive agents that are more stable in acidic form, such as trimethylglycine, or bioactive agents which may react with other components at neutral pH such as glucosamine hydrochloride, the pH of the matrix of the delivery systems may be below neutral. By below neutral, it is meant that the final pH is between about 2.5 and about 6.0. hi another embodiment of the present invention, therefore, the matrix is formulated to have a final pH between about 3.0 and about 6.0 and thus is suitable for delivery of bioactive agents that are stable at acidic pH and/or interact with other components at neutral pH.

In its final form, the matrix is a semi-solid, intermediate moisture system, having some properties clearly identified with those of jellies and some properties that are similar to the jujube variety ofconfectioneri.es. The matrix of the delivery systems, therefore, is formulated to be semi-solid at normal room temperature, hi the event, however, that the matrix liquefies due to exposure to elevated temperatures, the formulation of the matrix is such that no phase separation of the components occurs and the matrix can be readily re-solidified by cooling (for example, by cooling to temperatures of around 4°C). The reformed product maintains the substantially uniform dispersion of the bioactive agents contained therein, hi one embodiment of the present invention, the matrix is formulated to be a semi-solid at temperatures at or below about 40°C. hi another embodiment, the matrix is semi-solid at or below about 35°C. In other embodiments, the matrix is semi-solid at or below about 30°C and at or below about 25°C.

It will be readily apparent to one skilled in the art that new formulations of carbohydrate and hydrocolloid or modifications or substitutes thereof are being developed within the food industry. The present invention therefore contemplates the use of such new formulations to prepare the matrix of the present invention provided that its final properties are maintained, i.e. substantially uniform and complete dispersion of the bioactive agents, minimisation of the degradation of the bioactive agents, a final moisture content of between about 10% and about 30% and a water activity below about 0.7. For example, a whey-based polymer has recently been developed that acts as a gelling agent (Dairy Management Inc™). The polymer mimics gelatine functionality and forms strong gels at room temperature that exhibit large deformation without fracture and may be suitable for use in the matrix in accordance with the present invention.

1.1 Carbohydrate

The carbohydrate component of the matrix typically performs the functions of water binding and gelation and contributes to the overall texture and body of the final delivery system. The carbohydrate contributes to the structural integrity of the matrix and its low set temperature. The carbohydrate can also provide heat stability to the finished product as well as the ability to bind a limited quantity of fats/oils if required.

The carbohydrate to be included in the matrix is selected for its ability to hydrate and develop its viscosity in the presence of the other matrix-forming components at a temperature below 100°C. The selected carbohydrate should thus be capable of dispersing without clumping in a sugar syrup or in water, and of becoming hydrated with or without heating either in the presence of a sugar syrup or another source of water. While the majority of carbohydrates hydrate upon heating, certain starches, which are commercially available and are known in the art as "cold set" or "pre- gelatinised" starches are capable of hydrating at room temperature and are also suitable for use in the matrix according to the present invention.

In accordance with the present invention, therefore, the selected carbohydrate is capable of hydrating and developing its viscosity at a temperature below 100°C. In one embodiment, the carbohydrate is capable of hydrating at or below 70°C. In another embodiment, the carbohydrate is capable of hydrating at or below 50°C. In other embodiments, the carbohydrate is capable of hydrating at or below 40°C, 35°C or 25°C.

Furthermore, the selected carbohydrate should allow the final matrix to remain in a free-flowing state at a sufficiently low temperature for addition of the bioactive agents or AMVs without significant degradation of the bioactive agents or AMV. In accordance with the present invention, therefore, the hydrated carbohydrate remains free-flowing at or below 100°C. In one embodiment of the present invention, the hydrated carbohydrate remains free-flowing between about 35°C and about 85°C. In another embodiment, the hydrated carbohydrate remains free-flowing between about 35°C and about 70°C. In a further embodiment, the hydrated carbohydrate remains free-flowing between about 45°C and about 70°C.

The viscosity development of the selected carbohydrate should allow for sufficient ease of mechanical handling and pumping during production as well as allowing sufficient time to incorporate all the ingredients and to mold (form) the final product before it sets. As is known in the art, some carbohydrates develop their viscosity upon heating, whereas others develop viscosity upon cooling. Both types of carbohydrates are considered to be suitable for use in the matrix of the present invention. In one embodiment, the selected carbohydrate will develop its viscosity upon cooling, hi another embodiment, the viscosity of the carbohydrate will develop completely after being formed into the shape of the final delivery system.

Carbohydrates that meet the above criteria are known in the art. Examples include cellulose (or vegetable) gums, starches and other amylaceous ingredients that have been modified such that they have a low set temperature. An amylaceous ingredient as used herein refers to a food-stuff that contains a preponderance of starch and/or starch-like material. Examples of amylaceous ingredients include cereal grains and meals or flours obtained upon grinding cereal grains such as com, oats, wheat, milo, barley, rice, as well as the various milling by-products of these cereal grains such as wheat feed flour, wheat middlings, mixed feed, wheat shorts, wheat red dog, oat groats, hominy feed, and other such material. Other sources of amylaceous ingredients include tuberous foodstuffs, such as potatoes, tapioca, and the like.

Suitable starches are typically modified starches and include those derived from a natural source, such as those obtained from various plant species. Examples of plant sources of starch include, but are not limited to, com, waxy com, wheat, rice, tapioca, potato, pea and other sources known in the art. Modified starches are known in the art and the term generally refers to starch that has been physically or chemically altered to improve its functional characteristics. Suitable modified starches include, but are not limited to, pre-gelatinised starches, low viscosity starches (such as dextrins, acid- modified starches, oxidized starches and enzyme modified starches), derivatised starches, stabilised starches (such as starch esters and starch ethers), cross-linked starches, starch sugars (such as glucose syrup, dextrose and isoglucose) and starches that have been submitted to a combination of treatments (such as cross-linking and gelatinisation) and mixtures thereof. The carbohydrate may also be a synthetic starch substitute provided that it meets the criteria outlined herein.

In one embodiment of the present invention, the carbohydrate is a modified starch. In another embodiment, the modified starch is a modified cornstarch. Examples of commercially available modified cornstarches include Soft-Set® and MiraQuick® (A.E. Staley Manufacturing Co.).

Suitable cellulose gums for use in the preparation of the matrix are typically modified cellulose gums. Examples of modified cellulose gums include, for example, methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), hydroxyethyl cellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose acetate, hydroxyethyl methylcellulose, hydroxyethylcellulose acetate, hydroxyethyl ethylcellulose and combinations thereof. Such modified celluloses are well known in the food industry, for example, a range of modified celluloses known as Methogel Food Gums are manufactured by Dow Chemical Company. In one embodiment of the present invention, the carbohydrate used in the preparation of the matrix is methylcellulose, hydroxypropyl methylcellulose or a combination thereof.

The use of combinations of modified starches and modified celluloses as the carbohydrate component of the matrix is also contemplated by the present invention as discussed below in Section 1.7.

hi accordance with the present invention, the carbohydrate component of the matrix ranges from about 0.6% to about 15% by weight. The selection of the actual amount of carbohydrate from within this range to be included in the matrix will be dependent upon the type of carbohydrate being used and on desired texture of the final product. Determination of this amount is considered to be within the ordinary skills of a worker in the art. Thus, for example, when modified starch is used as the carbohydrate and a delivery system with a final texture similar to gum drop confectionery is desired, the amount of carbohydrate will be between about 9% and about 14%.

In one embodiment of the present invention, the carbohydrate used in the preparation of the matrix is one or more modified starch, which is included in the matrix in a total amount between about 2% and about 15% by weight, h another embodiment, the amount of modified starch included in the matrix is between about 2% and 10%, h other embodiments, the amount of modified starch included in the matrix is between about 2% and about 8%, between about 2% and about 5%, or between about 2% and about 4%.

In still another embodiment of the present invention, the carbohydrate used in the preparation of the matrix is one or more modified cellulose, which is included in the matrix in a total amount between about 0.6% and about 3% by weight. In another embodiment, the amount of modified cellulose included in the matrix is between about 0.6% and 1.5%.

1.2 Sugar Component

Sugar is generally used in a confection primarily for sweetness; however, it is known in the art that sugar can also play an important role in the physical properties of a matrix, such as crystallinity, gel strength, bodying/texture, humectancy, and water activity.

The matrix comprises one or more sugar. The term "sugar" as used herein includes sugars per se, sugar syrups, sugar alcohols, sugar alcohol solids and the like and various combinations thereof. Examples include, but are not limited to, sugars such as sucrose, glucose, xylose, ribose, maltose, galactose, dextrose, and fructose; syrups such as com syrups, hydrogenated glucose syrups, high fructose corn syrups; polydextrose; and sugar alcohols such as isomalt, maltitol, sorbitol, lactitol and mannitol. The latter are also often in the form of syrups. One skilled in the art will appreciate that if a sugar or sugar alcohol solid is used in the matrix, it should be first dissolved, for example, by heating in water or in another syrup, prior to being added to the mixture. When the sugar used to prepare the matrix is dextrose, it is generally provided in the form of a com syrup. Com syrups are prepared by hydrolysis of starch and are characterised by dextrose equivalent (D.E.) values such that they are classified as low, medium or high D.E. syrups, with high D.E. syrups having a high concentration of dextrose and low D.E. syrups having a low concentration of dextrose. In one embodiment of the present invention, the sugar component used in the preparation of the matrix comprises a com syrup, hi another embodiment, the matrix comprises a com syrup that exhibits a D.E. of between 20 D.E. and 99 D.E. In other embodiments, the matrix comprises a "high" DE com syrup with a D.E. of between 40 and 70, or with a D.E. of between 62 and 65. In another embodiment, the com symp is a high fructose corn syrup.

Various corn syrups are commercially available. For example, 62 D.E. 1600 Com Syrup (Casco Inc./ Canada Starch Operating Co. h e), SWEETOSE 4300 com symp (a 63 D. E. co syrup; A. E. Staley Manufacturing Company; Decatur, IL) and Clearsweet^® 63/43 IX com sy p (a 63 D. E. com syrup; Cargill / North America Sweeteners).

Combinations of sugars or sugar syrups are also suitable for use in the preparation of the matrix. Examples of suitable combinations of syrups include, but are not limited to, isomalt symp and high fructose com syrup, a high DE corn syrup and high fructose com syrup and maltitol symp and high fructose com symp.

One skilled in the art will appreciate that the total amount of sugar in the matrix will vary depending upon the combination of sugar sources used. For example, when sugar syrups are used, lower viscosity sugar syrups will produce a matrix with less body and lower rigidity. The total amount of sugar present in the matrix is about 20% to about 60% by weight.

In one embodiment of the present invention, a mixture of sugar syrups is used as the sugar component in a total amount between about 35% and about 55% by weight. In another embodiment, a mixture of sugar syrups is used as the sugar component in a total amount between about 40% and about 50% by weight. 1.3 Hydrocolloid Component

The matrix according to the present invention further comprises one or more hydrocolloid. HydrocoUoids are hydrophilic polymers of vegetable, animal, microbial or synthetic origin, naturally present or added to aqueous foodstuffs for a variety of reasons due to their unique textural, stractural and functional properties, hi general, they are used for their thickening and/or gelling properties as well as their water binding and organoleptic properties. HydrocoUoids can also be used to improve and/or stabilise the texture of a food product while inhibiting crystallisation.

Examples of hydrocoUoids that maybe used in the delivery systems of the invention include, but are not limited to, tragacanth, guar gum, acacia gum, karaya gum, locust bean gum, xanthan gum, agar, pectin, gelatine, carrageenan, gellan, alginate, or various combinations thereof. The use of hydrocoUoids is well-known in the art and many hydrocoUoids for use in products for human or animal consumption are available commercially, for example, Type B gelatine from Leiner Davis, Kelcogel^® Gellan Gum manufactured by CP Kelco and a range of Ticagel^® hydrocoUoids firom TIC Gums.

One skilled in the art will appreciate that the selection of the hydrocolloid to be used in the matrix will depend on the pH of the matrix, the interaction of the hydrocolloid with the carbohydrate component of the matrix and the particular texture and consistency required for the final product. The type of hydrocolloid used will also affect the set temperature of the matrix. For example, the use of a gelatine/gellan mixture or a gelatine/pectin mixture provides a set temperature around 35°C, whereas the use of carrageenan or locust bean gum will result in a set temperature closer to 60°C. Thus, the choice of hydrocolloid for use in the matrix is also dependent upon the properties of the AMVs and/or bioactive agent(s) to be incorporated into the delivery system. Foπ AMVs and bioactive agents that are unstable at higher temperatures a hydrocolloid or mixture of hydrocoUoids that have a low set temperature will be required, whereas for AMVs and bioactive agents that are more stable hydrocoUoids having a higher set temperature can be used. Selection of an appropriate hydrocolloid is considered to be within the ordinary skills of a worker in the art. In one embodiment of the present invention, the matrix comprises gelatine. The term "gelatine" refers to a heterogeneous mixture of water-soluble proteins of high average molecular weight derived from the collagen-containing parts of animals, such as skin, bone and ossein by hydrolytic action, usually either acid hydrolysis or alkaline hydrolysis. Different types of gelatine can be prepared by altering the process parameters. Gelatine is defined generally using a "Bloom value" which indicates the strength of the gel formed under certain circumstances using the gelatine, hi the preparation of confectionery, when a harder gel is desired, gelatine having a higher Bloom value is used. Conversely, when the final product is required to be more flowing, gelatine having a lower Bloom value is used. One skilled in the art will appreciate that the water holding capacity of gelatine alone is lower than that of a combination of gelatine with another hydrocolloid, such as gellan or pectin, and may necessitate the use of a higher amount of gelatine to achieve the desired gelation/texture of the matrix than when a combination is used. When the hydrocolloid in the matrix of the present invention comprises gelatine, the Bloom value (BL) is generally about 100 to 260 BL. hi one embodiment, the Bloom value is about 250 BL. In another embodiment, a mixture of gelatines with different Bloom values is used.

As indicated above, gelatine can be combined with one or more other hydrocolloid to impart slightly different characteristics to the matrix. For example, combinations of gelatine with gellan or with pectin provide a good texture to the matrix. When combinations of gelatine and gellan are used in the preparation of the matrix, the ratio of gelatine:gellan is typically in the range between about 15 : 1 to about 40: 1. These relative amounts provide a cohesive structure to the delivery system. When a combination of gelatine and pectin are used in the preparation of the matrix, the ratio of gelatine:pectin is typically in the range between about 15 : 1 to about 40: 1.

In one embodiment of the present invention, a combination of gelatine and gellan is used in the preparation of the matrix in a gelatine: gellan ratio of about 15:1 to about 35:1. hi another embodiment, a combination of gelatine and pectin is used in the preparation of the matrix in a gelatine :pectin ratio of about 15:1 to about 25:1. The total amount of hydrocolloid incorporated into the matrix is generally between about 0.1% and about 7.0% by weight. In one embodiment, the total amount of hydrocolloid in the matrix is between about 0.5% and about 6.8% by weight. In another embodiment, the total amount is between about 1.0% and about 6.6%. hi other embodiments, it is between about 2.0% and about 6.0%, between about 4.0% and about 6.0%, between about 5.0% and about 6.0% and between about 6.0% and about 7.0%.

1.4 Solvent Component

The primary role of the solvent component of the matrix is to dissolve or disperse the bioactive agents to allow for substantially uniform and complete incorporation of these ingredients into the matrix. Similarly, when an AMV phase is incorporated into the matrix the solvent aids in the dispersion of the AMVs. The solvent also provides for improved flow characteristics of the mixture and functions somewhat as a humectant.

The solvent used in the preparation of the matrix is typically colourless, non- volatile with no strong odour or flavour and is substantially miscible with water and/or alcohols, hi accordance with the present invention, the solvent component can be one or more polyhydric alcohol. The term "polyhydric" as used herein means that the compound contains two or more hydroxyl groups. Examples of polyhydric alcohols include, but are not limited to, glycerol and/or its lower alkyl ester derivatives, sorbitol, propylene glycol, and short chain polyalkylene glycols, such as polyethylene glycol, and mixtures thereof. In one embodiment of the present invention, the solvent component comprises glycerol. In another embodiment, the solvent component comprises glycerol and a short chain polyalkylene glycol. In another embodiment, the solvent component comprises glycerol and polyethylene glycol.

The solvent component may act as a carrier to effectively solubilize the bioactive agent(s) being incorporated into the delivery system. Typically, the delivery system according to the present invention contains about 5% to about 35% by weight of the solvent component. In one embodiment, the delivery system contains about 20% to about 30% of the solvent component. 1.5 Mono- or Divalent Cations

The matrix may optionally comprise one or more sources of mono- and/or divalent cations when required for proper gelation of the matrix. One skilled in the art will appreciate that the requirement for cations will be dependent on the particular hydrocolloid being used in the matrix. Some hydrocoUoids, such as gellan and carageenan, require cations for proper gelation to occur, whereas other hydrocoUoids do not.

Suitable sources of mono- and divalent cations for incorporation into food products are known in the art and are commercially available. Examples include mono- or divalent salts such as sodium chloride, potassium chloride, calcium chloride or potassium citrate. Mono- and/or divalent cations may also be provided by one or more ingredients in the matrix, for example, by a buffering agent or the salt form of a bioactive agent, hi one embodiment of the present invention, potassium citrate is added to the matrix as a source of monovalent cations.

When a source of mono- or divalent cations is required and is added to the matrix in the form of a mono- or divalent salt, then it is typically added in an amount between about 0.1% and about 5% by weight, h one embodiment, it is added in an amount between about 1% and about 3%. h another embodiment, it is added in an amount between about 1.2% and about 2.5%.

1.6 Water

As indicated above, the delivery system according to the present invention has a final moisture content between about 10% and about 30% and a water activity below about 0.7. It will be readily apparent to one skilled in the art that the appropriate amount of water may be provided by one or more of the various components of the system, for example, a sugar symp, a hydrated starch or a hydrated hydrocolloid, or it may be added in the course of the manufacturing process (for example, if steam injection is used) or additional water may need to be added separately. Additional water can be provided alone or as a solution containing other additives, for example, as a buffer solution or as a solution containing a sweetener, flavouring or colouring. The total amount of water from the one or more sources will be sufficient to provide the final delivery system with a moisture content and water activity within the ranges indicated above.

1.7 Other Additives

The matrix can optionally contain other additives such as sweeteners, flavourings, colourings, modified vegetable gums or celluloses, or combinations thereof. It will be readily apparent that additives for inclusion in the matrix should be selected such that they do not affect the properties of the matrix, do not exhibit substantial reactivity with the bioactive agents or AMVs dispersed in the matrix, and are stable during preparation of the matrix.

The sweetener can be selected from a wide variety of suitable materials known in the art. Representative non-limiting examples of sweeteners include xylose, ribose, sucrose, mannose, galactose, fructose, dextrose, maltose, partially hydrolysed starch, lactose, maltodextrins, hydrogenated starch hydrolysate and mixtures thereof. In addition to these sweeteners, polyhydric alcohols such as sorbitol, mannitol, xylitol, and the like may also be incorporated. Alternatively, one or more artificial sweeteners can be used, for example, sucrose derivatives (such as Sucralose), amino acid based sweeteners, dipeptide sweeteners, saccharin and salts thereof, acesulfame salts (such as acesulfame potassium), cyclamates, steviosides, dihydrochalcone compounds, thaumatin (talin), glycyrrhizin, aspartame, neotame, alitame, and mixtures thereof.

When an additional sweetener is used, it can be used in amounts as low as 0.01% by weight. The actual amount of sweetener required will be dependent on the type of sweetener selected and on the desired sweetness of the final product. Amounts of various sweeteners to be added to food products are well known in the art. The total amount of the sugar component, which forms a structural part of the matrix, and additional sweetener(s) in the matrix, however, remains less than 60% by weight.

Suitable flavourings that can be added to the delivery system are known in the art and include, both synthetic flavour oils and oils derived from various sources, such as plants, leaves, flowers, fruits, nuts, and the like. Representative flavour oils include spearmint oil, peppermint oil, cinnamon oil, and oil of wintergreen (methylsalicylate). Other useful oils include, for example, artificial, natural or synthetic fruit flavors such as citrus oils including lemon, orange, grape, lime and grapefruit, and fruit essences including apple, strawberry, cherry, pineapple, banana, raspberry and others that are familiar to a worker skilled in the art.

The amount of flavouring agent employed is normally a matter of preference subject to such factors as concentration dilution of the flavour stock, flavour type, base type and strength desired, hi general, amounts of about 0.01% to about 5.0% by weight of a final product are useful. In one embodiment of the present invention, a flavouring agent is included in the matrix in amounts of about 0.02% to about 3%. In another embodiment, the flavouring agent is added in amounts of about 0.03% to about 1.5%.

Colourings suitable for use in foodstuffs are well known in the art and can be optionally included in the matrix to add aesthetic appeal. A wide variety of suitable food colourings are available commercially, for example, from Warner Jenkins, St. Louis, MO. Where a synthetic colouring agent is used in the matrix, the amount ranges from about 0.01% to about 2% by weight. In one embodiment of the present invention, a synthetic colouring agent is added to the matrix in an amount between about 0.03% to about 1% by weight. A worker skilled in the art will appreciate that when a colouring agent derived from a natural source is used in the matrix, an increased amount of the colouring agent is generally required to achieve the same effect as a synthetic colouring agent.

The present invention also contemplates that, when the carbohydrate component of the matrix is a modified starch, that a modified vegetable gum or cellulose may be included in the matrix in order to improve the texture, lubricity and/or elasticity of the matrix. These compounds can be used, for example, to increase the viscosity of the delivery system if it is warmed, thus reducing potential melting and lessening water activity which will help to improve the stability of the system in the event it is left in an excessively hot environment. Examples of modified vegetable gums or celluloses are provided above. The modified vegetable gum or cellulose can be included in the matrix in amounts between about 0.01 % and 2.0% by weight. In one embodiment, modified vegetable gum or cellulose is included in the matrix in an amount between about 0.05% and about 1.5%. In another embodiment, modified vegetable gum or cellulose is included in the matrix in an amount between about 0.1% and about 1.0%. In a further embodiment, modified vegetable gum or cellulose is included in the matrix in an amount between about 0.1% and about 0.7%.

2. Non-Matrix Phases

Various other phases known in the art are suitable for use in the multi-phase delivery system of the present invention. Examples of non-matrix phases that may be incorporated into the delivery system include, for example, cookies (baked and unbaked, including granola-type cookies or bars), cakes and cake-like products (including products based on brownie and muffin mixes), wafers, caramel, marshmallow, nougat, fruit-based phases, such as jams and jellies, praline, fondant cream, paste, chocolate or yoghurt pieces (such as chips or chunks), and chocolate, yoghurt or other flavoured coatings. These non-matrix phases may be formulated such that they comprise one or more nutritional components, for example, proteins, carbohydrates, lipids or fats and fibre as major ingredients by techniques known in the art. When nutritional components are included in the non-matrix phases, they may be included in sufficient amounts for the delivery system to act as a food supplement or meal replacement. In one embodiment of the present invention, one or more of the non-matrix phases comprise one or more nutritional components, hi another embodiment, one or more nutritional components are included in a solid phase, such as in a cookie or wafer format. In a further embodiment, one or more non-matrix phases comprise one or more nutritional component as a major ingredient.

The delivery system may comprise one or more texture additives incorporated into a non-matrix phase. Alternatively, a non-matrix phase may itself serve as a texture additive. Texture additives are ingredients that have a particular associated mouthfeel and include, but are not limited to, cereals in piece form such as rolled cereals, gun puffed grains, cereal flakes, rice crisps and/or cooked-extruded cereals; granola pieces; marshmallow pieces; candy pieces; cookie pieces; white and milk chocolates and chocolate products (e.g. chocolate chips, candy bars, etc.); other types of edible particulates (e.g. peanut butter chips, butterscotch chips, carob chips, etc.); dried fruit pieces; nuts; caramel pieces; nougat; wafers; fruit preparations and the like. The texture additives can be manufactured in a variety of suitable sizes and shapes depending on the manufacturing conditions, machine die plates, formulation, and so forth as is known in the art.

The non-matrix phase of the delivery system may also be a coating. Coatings are in general compound coatings, the major ingredients of which are sugar and fat. Various confectioner's coating materials are commercially available and are suitable for use in the present invention. The fat or partially hydrogenated vegetable oil may be, for example, an oil derived from cottonseed, coconut, soybean, palm kernel, palm, peanut and the like. Compound coatings may be unflavoured, or flavoured, for example, with chocolate, vanilla, peanut, coconut, yoghurt, fruit flavours and the like. Chocolate coatings are typically based on cocoa butter, whereas yoghurt coatings typically comprise yoghurt powder. Milk and milk products, including milk powders and whey, are also commonly used in coatings. In general, the coating material comprises a fat that is solid at room temperature, but liquid at temperatures in excess of, for example, 35°C, together with other materials that confer appropriate organoleptic attributes on the final coating, hi accordance with one embodiment of the present invention, a commercially available coating material is used as one phase of the delivery system which comprises sugar, modified palm kernel oil, non-fat milk, maltodextrin and non- fat yoghurt powder.

Fat substitutes may also be used in coatings to reduce the fat content of the phase. Examples include, but are not limited to caprocaprylobehenic triacylglyceride (Caprenin™), and short and long acyl triglyceride molecule (e.g. SALATRIM or Benefat™).

In accordance with one embodiment of the present invention, the coating comprises between about 5% and about 25% of the total weight of the delivery system.

2.1 Artificial Membrane Vesicle (AMV)-Phase

The present invention contemplates delivery systems in which one of the non-matrix phases is an AMV-phase. AMVs are vesicles comprising hydrated uni- or multi- lamellar systems of bilayers of ampbipathic compounds that are dispersed in an aqueous medium. True liposomes are derived from phospholipids, but AMVs can comprise other non-toxic, physiologically acceptable, and metabolizable lipid and non-lipid substances, as is known in the art. Compounds contained in aqueous medium become trapped within the AMV or between the bilayers as the AMV forms. Both water-soluble and water insoluble bioactive agents may be associated with

AMVs in this manner. The AMV will slowly release the associated compounds as the biolayers are broken down within biological systems.

Examples of lipids that can be used to make the AMVs include, hut are not limited to, lipids and phospholipids such as soy lecithin, egg lecithin, partially refined lecithin, hydrogenated phospholipids, lysophosphate, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine, phosphatidylinositol (PI), cardiolipin, sphingolipids, gangliosides, cerebrosides, ceramides, other ester analogues of phosphatidylcholine (such as PAF, lysoPAF); synthetic phospholipids such as L-α-lecithin, dilauroylphosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC), dilinoloylphosphatidylcholine, distearoylphosphatidyl choline, diarachidoylphosphatidylcholine; PE derivatives, such as l,2-diacyl-sn-glycero-3- phosphoethanolamine, 1 -acyl-2-acyl-sn-glycero-3-phosphoethanolamine, dinitro- phenyl- and dinitrophenylamino- caproylphosphatidylethanolamine, 1,2-diacyl-sn- glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG-PE), N-biotinyl-PE, N- caproylamine PE, N-dodecylamine-PE, N-MPB-PE, N-PDD-PE, N-succinyl-PE, N- glutaryl-PE; phosphatidyl glycerols such as dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol; phosphatidic acids, l,2-diacyl-sn-glycero-3-phosphate salt, l-acyl-2-acyl-sn-glycero-3-phosphate sodium salt; phosphatidylserine such as l,2-diacyl-sn-glycero-3-[phospho-L-serine] sodium salt, l-acyl-2-acyl-sn-glycero-3- [phospho-L-serine] sodium salt, lysophosphatidic acid; cationic lipids such as 1,2- diacyl-3-trimethylammoniumpropane (TAP), l,2-diacyl-3- dimethylammoniumpropane (DAP), N-[l-(2,3-dioleoyloxy)propyl]-N,N',N"- trimethylammonium chloride (DOTMA); phospholipids with multivarious headgroups such as phosphatidylethanol, phosphatidylpropanol and phosphatidylbutanol, phosphatidylethanolamine-N-mono-methyl, 1 ,2-distearoyl(dibromo)-sn-glycero-3- phosphocholine; polymerizable lipids such as diyne PC, diynePE for example 1,2- bis(10,12-tricosadiynoyl-sn-glycero-3-phosphocholine; phospholipids with partially or fully fluorinated fatty acid chains, and various combinations thereof.

An emulsifying or surfactant agent may also be incorporated in the AMVs or used for AMV preparation. Many such agents are commercially available and examples include various Pluronic™ compounds, Poloxamer™ compounds, Span™ compounds, Brij™ compounds, Tween™ compounds, Triton-X™ compounds, and fluorinated surfactants such as Zonyl™.

Other compounds that may be incorporated into the AMVs include sterols, such as cholesterol, cholesterol hemisuccinate and histidinyl cholesterol hemisuccinate, and amphiphiles, such as stearylamine (SA), phosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, phosphatidylglycerol, diphytanylphosphatidylcholine and dipahnitoylphosphatidylgycerol.

As is also known in the art, AMVs can be formulated to have an overall positive, neutral, or negative charge. Amphoteric AMVs are also known in the art. AMVs can also be formulated to be homogeneous (i.e. composed of only one type of lipid) or heterogeneous (i.e. composed of two or more types of lipid). For example, the selected lipids may be mixed together to form AMVs of the formula X:Y:Z, where X represents a phospholipid or sphingolipid, Y represents a neutral lipid and Z is a charged amphipathic molecule or amphiphile. The phospholipid or sphingolipid (X) may be one or more than one, of a group of natural and/or synthetic compounds including, but not limited to, phospatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and sphingomyelin; the neutral lipid (Y) may include, but is not limited to, cholesterol and its precursors and derivatives, diglycerides or triglycerides, and the charged amphiphile (Z) may include, but is not limited to, amphiphiles such as stearylamine, fatty acids and other phospholipids that have either overall positive, neutral or negative charges. Ratios of the lipid components used to form AMVs may range from about 4:3:3 to about 1:0:0, including ratios in between these ranges, and will depend upon the desired formulation of the AMV. AMVs may be made from extracts containing lipids from natural sources. Biologically active agents can be combined with AMV formulations that are positively charged, neutral, or negatively charged. As is known in the art the type of lipid, natural or synthetic, the overall charge of the lipid or other components, positive, negative, or neutral, the size of the lipids or other components, large or small, and the number of lipids or other components need to be selected in order to make an AMV of known charge, size and lamellarity and will be dependent on the type of biologically active agent to be associated with the AMV. Selection of the appropriate AMV for association with a particular biologically active agent is considered to be within the normal skills of a worker in the art.

By choosing a desired combination of lipids or other components to form AMVs,

AMV formulations of the invention can be designed, for example, to target organs of mammals with the biologically active agents, increase the loading capacity and/or the carrying ability of a particular biologically active agent, increase or decrease the uptake of biologically active agents at the target site or increase the association of biologically active agents with certain lipids of an AMV formulation or make the formulations more benign to the animal in terms of decreasing side effects or increasing potency or adjuvanticity.

Niosomes are nonionic surfactant vesicles that form multi-lamellar structures similar to those of phospholipid-based liposomes. The use of niosomes as vesicles for association with a bioactive agent is also contemplated in the present invention.

Examples of surfactants that maybe employed to make noisomes include, but are not limited to, various Pluronic™ compounds, Poloxamer™ compounds, Span™ compounds, Brij™ compounds, Tween™ compounds, Triton-X™ compounds, Triton-™ compounds, fluorinated surfactants such as Zonyl™, cholesterol, dicetylphosphate, polyoxyethylene (4) lauryl ether, sorbitan esters, octyl and decyl polyglucosides, polyglyceryl-3-diisostearate, polysorbates, myristyl alcohol, D-alpha tocopheryl, polyethylene glycols (PEGs) and PEG derivatives, hexadecyl diglycerol ether (C16G2), poly(24)oxyethylene cholesteryl ether (Solulan C24) and various combinations thereof. In addition, lipids used to make AMVs may be "ether" lipids. Such lipids may be derivatized, functionalized or modified using techniques known to a worker skilled in the art.

Cochleates, which are stable phospholipid-calcium precipitates composed of phosphatidylserine, cholesterol and calcium are non-toxic reagents that can be used in AMV formulations of the instant invention.

The typical average size range for the AMVs is 20nm to lOOOnm. In one embodiment of the invention, AMVs having an average size between about 50nm and about 700nm are used in the delivery system. In another embodiment, AMVs having an average size between about 75nm and about 500nm are used. In another embodiment, AMVs having an average size between about 80nm and about 200nm are used, hi a further embodiment, AMVs having an average size between about 80nm and about 120nm are used.

2.1.1 Preparation of the AMVs The AMVs for use with the delivery system according to the present invention can be prepared according to established techniques [for example, see "Liposomes as Drug Carriers" G. Gregoriadis, Wiley & Sons, New- York (1988); Gregoriadis, G., "Liposome preparation and related techniques," in: G. Gregoriadis (Ed.) "Liposome Technology" Vol. 1, 2^nd Edition, CRC Press, Baton Rouge, FL, (1993), pp.1-63]. For example, the lipid composition can be dissolved in an organic solvent, such as an alcohol, ether, halohydrocarbon or mixtures thereof, and the solvent removed from the resulting solution, for example by rotary evaporation or freeze-drying. The resulting lipid film can then be hydrated by dispersing in an aqueous medium, such as phosphate-buffered saline or an aqueous solution of a sugar such as lactose, which medium also contains the biologically active agent, to give an aqueous suspension of AMVs in the form of multi-lamellar vesicles. If required, the aqueous AMV suspension may be treated to reduce the AMV size or to give small uni-lamellar vesicles using established techniques such as sonication, reversed phase evaporation, membrane extrusion e.g. using polycarbonate membranes of selected size, or detergent-based procedures. Alternatively, AMVs can be formed by the method disclosed in GB-A-2, 134,869. In this method, microspheres (lOμm or less) of a hydrosoluble carrier solid (NaCl, sucrose, lactose and other carbohydrates) are coated with a phospholipid mixture and then, this coated carrier is dissolved in an aqueous phase to yield liposomic vesicles. Similarly, in GB-A-2,135,647 insoluble particles, e.g. glass or resin microbeads are coated by moistening in a solution of lipids in an organic solvent followed by removal of the solvent by evaporation. The lipid-coated microbeads are thereafter contacted with an aqueous carrier phase, whereby liposomic vesicles will form in that carrier phase.

AMVs comprising non-phospholipid components, such as niosomes, can also be prepared using standard techniques known in the art, see for example, Synthetic Surfactant Vesicles: Niosomes and Other Non-Phospholipid Vesicular Systems, Drug Targeting and Delivery, Volume 11 (2000) Uchegbu, ed., Amsterdam: Harwood Academic Publishers.

The present invention also contemplates the use of pro-AMV preparations. Methods of preparing pro-AMVs are known in the art. For example, one method of preparing pro-AMV powders is by spraying lipid solution onto a suitable carrier followed by evaporation of the solvent (see, for example, Payne et al, (1986) J. Pharm. Sci., 75:325-329). The process can be repeated several times if necessary in order to achieve the desired lipid loading on the carrier. Examples of suitable carriers include, but are not limited to, sorbitol powder, sodium chloride and maltodextrin. Alternatively, pro-AMVs can be formed in an appropriate organic solvent as described in European Patent Nos. EP 1 158 441 and EP 0 309 464 or as aqueous gels as described in European Patent No. EP 0 211 647.

Bioactive agents can be loaded into AMVs passively (i.e. during AMV formation) or actively (i.e. after AMV formation) using standard techniques.

Other methods of preparing AMVs associated with bioactive agents are known in the art and are considered to be within the scope of the present invention. In addition, many preparations comprising bioactive agents associated with AMVs or pro-AMVs are available commercially for use in products for human or animal consumption and are suitable for use with the delivery systems of the invention. Commercial laboratory services are also available whereby bioactive associated AMVs or pro-AMVs may be prepared according to specific requirements.

The AMV preparation may be incorporated into the matrix at between about 1% and about 40% by weight of the matrix. In one embodiment, the AMV preparation constitutes between about 5% and about 30% by weight of the matrix. In a further embodiment, the AMV preparation constitutes between about 10% and about 25% by weight of the matrix.

3. Nutritional Components

The non-matrix phases of the delivery system may comprise one or more nutritional component, such as protein, carbohydrate, fat (including oils and lipids) and fibre. One skilled in the art will appreciate that these categories overlap and that a single ingredient may provide a combination of nutritional components. For example, nuts and nut products can provide protein, carbohydrate and fat in varying proportions to the delivery system.

3.1 Proteins

The protein content of the delivery system may be derived from either animal or non- animal sources. Non-limiting examples of sources of protein include soy (for example, commercially available soy protein isolates and soy lecithin), carob, wheat (for example, wheat germ and wheat gluten), com, legumes, eggs (for example, egg albumin), milk (for example, various milk powders, whey, whey powders, whey protein concentrate, whey protein hydrolysate, casein, caseinates, including calcium, potassium and sodium caseinates), hydrolysed collagens, and nuts (such as peanuts, almonds and soy nuts and products derived therefrom, such as nut butters or defatted nut flour). The protein may be added as an ingredient per se, such as, for example, a whey protein isolate, or may be sourced from other ingredients such as, for example, peanut pieces, or it may be a mixture of both. According to the type and purpose of delivery system desired, the protein content may vary in the delivery system, h accordance with the present invention, when protein is included as a nutritional component, the delivery system comprises between about 10% and about 75% by weight of protein, hi one embodiment of the invention, the delivery system comprises between about 20% and about 70% by weight of protein as a nutritional component, hi another embodiment, the delivery system comprises between about 30% and about 60% by weight of protein as a nutritional component. In a further embodiment, the delivery system comprises at least 50% by weight of protein as a nutritional component. It is considered to be within the skills of a worker in the art to determine the appropriate protein level based on the purpose of the delivery system. For example, delivery systems intended for use as a protein supplement or high protein delivery system will contain protein levels at the higher end of these ranges, whereas delivery systems intended for use as a meal replacement will contain protein at the low to mid range.

3.2 Carbohydrates

Carbohydrates are a well-known source of energy, which are readily absorbed by the body and can be used to provide the major proportion of the caloric content of food supplement and meal replacement delivery systems. Carbohydrate material that can be used in the delivery system includes, for example, digestible carbohydrate, poorly digestible carbohydrate, indigestible carbohydrate or mixtures thereof. Typically, carbohydrates range between complex carbohydrates and simple sugars. Structurally, these carbohydrates differ in the number of sugars in the molecule and in the degree of branching. Functionally, they differ by how readily the body can absorb and process them to derive energy. Thus, the correct ratio of the different types of carbohydrates can supply short-term, mid-term, and long-term supplies of energy to the body.

Simple carbohydrates are selected from, but not limited to, high fructose com syrup; high maltose com syrup; rice syrup; malted cereal syrup from com, barley or brown rice; date paste; brown sugar; sucrose; fructose; maltodextrin; lactose; glucose; dextrose and maltose. Complex carbohydrates are provided by, but not limited to, sources such as cereal grains, for example, wheat, oat, com, barley, rice, rye, sorghum; legumes both mature and dry, such as soybeans; and nuts such as peanuts, and the like. The carbohydrates can be in the form of, for example, grains, flakes, flours and meals.

The carbohydrate may be in the form of an indigestible carbohydrate, which is used to achieve sweetness without a consequential increase in calorific value. Such carbohydrates can, therefore, be used in delivery systems formulated for calorie- conscious consumers. The carbohydrate material may be material that has been processed, prepared, extracted or otherwise produced in such a manner as to render a proportion or all of it indigestible by the consumer, but is not considered to be fibre as such. A number of carbohydrate materials comprising indigestible carbohydrate are known in the art and are available commercially. Non-limiting examples include sugar alcohols or a mixture of sugar alcohols, or a polymer of dextrose. Specific examples include lactitol as described in British Patent Application No. 1,253,300, and mixtures of various polyglucoses as described in British Patent Application Nos. 1,262,842 and 1,317,746. Similar non-nutritive carbohydrate substitutes are disclosed in U.S. Patent Nos. 3,766,165 and 3,876,794. An example of a commercially available indigestible carbohydrate is Malbit®, which is based on maltitol.

One skilled in the art will appreciate that the amount of carbohydrate will vary according to the purpose of the delivery system. For example, products formulated to promote weight gain will contain a higher level of carbohydrates compared to other products not formulated for this purpose. Additionally, products that are formulated to be taken more frequently each day, or to supplement a meal will not have the same caloric intake as products designed to be meal replacements. In general, the caloric content of the delivery system will be based on the daily-recommended dietary allowances issued by countries and adjusted according to the desired purpose of the final product. Typically, the caloric content of a delivery system formulated as a food supplement or meal replacement will be between about 100 and about 400 calories.

In accordance with the present invention, when carbohydrate is included as a nutritional component, the delivery system comprises between about 40% and about 85% by weight of carbohydrate. In one embodiment of the invention, the delivery system comprises between about 50% and about 80% by weight of carbohydrate as a nutritional component. In another embodiment, the delivery system comprises between about 60% and about 75% by weight of carbohydrate as a nutritional component.

3.3 Fats/Lipids

Vegetable or animal fats can be used as sources to provide lipids and include both fats and oils. Hydrogenated fats can act as a moisture barrier and lubricant for the final product. Alternatively, if the fat is not hydrogenated, it can first be rendered to a liquid at room temperature. The choice of fat used depends on the desired nutritional value and the desired viscosity of the phase into which it is to be incorporated.

Examples of fat that may be used in the delivery system of the present invention, include, but are not limited to, dairy sources such as butter, butter oil, dried milk/cream powder and vegetable sources such as coconut, palm kernel, palm, cottonseed, canola, rapeseed, com, soybean, sesame seed, sunflower, safflower, peanut and olive oils, which can be used per se or may be partially hydrogenated prior to use. Fats can be short, medium or long chain triglycerides. According to the desired fat content of the final product, a worker skilled in the art may determine the fat content of each phase and select the most appropriate fat source.

In accordance with the present invention, when fat is included as a nutritional component, the delivery system comprises between about 2% and about 15% by weight of fat. In one embodiment of the invention, the delivery system comprises between about 3% and about 12% by weight of fat as a nutritional component. In another embodiment, the delivery system comprises between about 4% and about 10% by weight of fat as a nutritional component.

3.4 Dietary fibre

Dietary fibre refers to the indigenous components of plant materials in the diet that are resistant to digestion by enzymes produced by animals. Dietary fibre consists of polysaccharides and lignin that are not digested by secretions of the digestive tract. Although "fibre" generally refers to filamentous, stringy materials, "dietary fibre" is often gelatinous or mucilaginous. In recent years, the apparent physiological benefits of adequate levels of dietary fibre in the diet have been widely reported, including normalization of bowel function and reduction of occurrence of certain colonic diseases.

Dietary fibre can be divided into two broad categories: insoluble dietary fibre and water-soluble dietary fibre. One example of a water-insoluble fibre source is cereal bran. Non-limiting examples of cereal brans include rice, wheat, com, barley, rye, oats, pea and mixtures thereof. The components of the insoluble dietary fibre derived from these cereal brans are cellulose, hemicellulose and lignin.

The soluble dietary fibres may be film-forming hydrocolloid materials such as alginates, gums, pectin, mucilage and similar plant exudates. Non-limiting examples of useful soluble fibres include arabic; tragacanth; karaya; ghatti; seaweed extracts including agar, alginates, canageenans, and furcellan; pectin; and mucilage such as psyllium.

Dietary fibre may constitute about 2-15% of the carbohydrates. Ratios of insoluble to soluble fibre may range from 50:50 to about 99:1, for example, from about 80:20 to about 99:1, which have been shown to add to improve taste characteristics of the food product. Other ranges of ratios of insoluble to soluble fibre are known in the art and may be used according to the desired texture, taste and purpose of the delivery system.

4. Biologically Active Agents

A wide variety of biologically active agents are suitable for delivery to animals using the delivery system according to the present invention. A person skilled in the art will appreciate that certain bioactive agents are best incorporated directly in the matrix while others may be more suited to be associated with AMVs or incorporated directly into one of the other non-matrix phases.

In general, biologically active agents that are suitable for use with the present delivery system fall under one of the following broad categories: drugs, diagnostic agents, nutritional supplements, botanicals, botanical extracts, vitamins, minerals, enzymes, hormones, proteins, polypeptides, and antigens. One skilled in the art will appreciate that these categories overlap to some extent and that certain agents will, therefore, fall into more than one category. One skilled in the art will further appreciate that compounds that fall within the group of "nutritional supplements" (see section 4.1 below) may also be classified as "nutritional components" as described above. In the context of the present invention, the term "nutritional component" is used to refer to a compound that is present in the delivery system in an amount up to about 75% by weight, whereas a "nutritional supplement" is generally present in amounts of about 25% by weight or less.

Biologically active agents either alone or associated with AMVs may be incorporated into the delivery system at levels sufficient to affect the structure or function of the body when taken regularly. Such levels are known in the art or can readily be determined by a skilled technician. Typically, biologically active agents constitute less than about 25% by weight of the phase into which they are incorporated, in one embodiment, the biologically active agents constitute between 5% and about 20% by weight of the phase. In another embodiment, the biologically active agents constitute between 10% and 25% by weight of the phase. It is understood that the total daily intake for the bioactive agents may be based on administration of one unit of the delivery system, or it may be based on administration of more than one unit. The amount of each bioactive agent in the final product will thus vary depending on the format of the units and the number to be administered daily.

The following sections provide non-limiting examples of biologically active agents that may be used with the delivery system according to the present invention. It is understood that bioactives suitable for administration to different animals may differ from those suitable for humans. The selection of appropriate bioactives to incorporate into the delivery system for administration to a given animal is considered to be within the ordinary skills of a worker in the art. hi addition, it will be apparent that inappropriate combinations of bioactives, for example those that may interact, should be included in different phases of the delivery system. Thus, the present invention contemplates various combinations of bioactives for use with the delivery system.

4.1 Nutritional Supplements

Examples of nutritional supplements suitable for use with the delivery system according to the present invention include, probiotic bacteria, prebiotics, vitamins, enzymes, co-enzymes, antioxidants, mineral salts, amino-acids, peptides, proteins, gums, carbohydrates, phytochemicals, dextroses, phospholipids, other trace nutrients, oxygenators, brain-stimulating substances, energy providers, minerals, mineral salts, botanical extracts, fatty acids, oat beta-glucan or other functional fibres, creatine, carnitine, bicarbonate, citrate, caffeine or a combination thereof.

Examples of nutritional supplement formulations that maybe administered through the use of the above-described delivery system include the following: L-arginine, co- enzyme Q10, human growth hormone, glutathione precursors, N,N dimethylglycine, chromium-niacin complex with hydroxycitric acid and devil's club, glucosamine, multi vitamins and minerals, methoxyisoflavones, chitosan, methylsulfonylmethane, and conjugated linoleic acids.

Probiotic microorganisms in the form of live microbial nutritional supplements and which are recognized as conferring a beneficial effect on an animal can be delivered using the delivery system according to the present invention. Probiotic microorganisms are microorganisms which beneficially affect a host by improving its intestinal microbial balance (Fuller, R; 1089; J. Applied Bacteriology, 66: 365-378). There are a variety of probiotic microorganisms that are suitable, in particular, having regard to activation of the immune system, prevention of the bacterial overgrowth by pathogens, prevention of diarrhoea and/or restoration of intestinal flora. Examples of probiotic microorganisms include Bifidobacterium, Lactobacillus, Streptococcus,

Saccharomyces. Typically, the microorganism is in a spray dried or freeze-dried form.

In one embodiment of the present invention, the biologically active agent is a probiotic bacterium selected from the group of Lactobacillus johnsonii, Lactobacillus paracasei, Bifidobacterium longum B129, Bifidobacterium longum B128, Bifidobacterium adolescentis Bad4, and Bifidobacterium lactis Bbl2.

The above bacterial strains were deposited under the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Lactobacillus johnsonii (NCC 533) has been deposited on the 30.06.1992 under reference CNCM 1-1225, Lactobacillus paracasei (NCC 2461) has been deposited on the 12.01.1999 under reference CNMC 1-2116, Bifidobacterium longum (B129) (NCC490) has been deposited on 15.03.1999 under reference CNCM 1-2170, Bifidobacterium longum (B128) (NCC481) has been deposited on 15.03.1999 under reference CNCM 1-2169, and Bifidobacterium adolescentis (Bad4) (NCC251) has been deposited on 15.03.1999 under CNCM I- 2168. Bifidobacterium lactis (Bbl2) may be obtained at Hanzen A/S, 10-12 Boege Alle, P.O. Box 407, DK-2970.

The amount of probiotic incorporated into the delivery system will vary according to the specific needs, h one embodiment, the amount of lactic acid bacteria in one unit of the delivery system is between 10² and 10¹² count/gram, typically between 10⁷ and 10¹¹ count/gram, or 10⁸ and 10¹⁰ count/gram.

Prebiotics can be delivered alone or in combination with probiotic bacteria in the delivery vehicle. Prebiotics comprise carbohydrates, generally oligosaccharides. This type of prebiotic has the ability to resist hydrolysis by enzymes in the animal digestive tract and thus can reach the colon undegraded to provide a carbohydrate substance particularly suited to growth of probiotic bacteria. Oligosaccharides may be produced from glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, or a mixture thereof. Purified commercially available products such as fructooligosacchari.de contain greater than about 95% solids in the form of oligosaccharides. In one embodiment of the present invention, the prebiotic comprises a mixture of fructooligosaccharide and inulin. hi a related embodiment, this mixture comprises PREBI01^® or a mixture of commercially available RAFTILOSE^® and RAFTJLINE^® commercialized by Orafti. A prebiotic of this kind has been demonstrated to improve the response of the immune system.

Other suitable nutritional supplements include vitamins and minerals that the body is usually not capable of synthesizing and which are necessary for ensuring normal growth and/or daily body maintenance, hi the context of the present invention, the vitamins can be hydrosoluble or liposoluble vitamins. Examples includes, but are not limited to, Vitamin A (axerophtol or retinol), Vitamin D, Vitamin E (alpha- tocopherol), Vitamin K, Vitamin B and/or PP (niacinarnide or nicotinic acid amide) and Vitamin C (L-ascorbic acid). The dosage of vitamins in the delivery system can be adapted to specific needs. In general, one unit of the delivery system may contain a fraction of the recommended daily amount (RDA) of the desired vitamin. For example, assuming a daily consumption of five units of the delivery system, and following European RDA recommendations, Vitamin A can be used up to 160 μg typically between 70 μg and 90 μg a single unit; Vitamin C up to 12 mg typically between 5 mg and 7 mg a single unit; Vitamin E up to 2 mg typically between 0.8 mg and 1.2 mg a single unit; Vitamin D up to 1 μg typically between 0.4 μg and 0.6 μg a single unit; Vitamin Bl up to 0.28 mg typically between 0.12 mg and 0.15 mg a single unit.

Antioxidants can be delivered using the delivery system of the present invention, alone or in combination with other biologically active agents, such as glutathione, peroxidase, superoxide dismutase, catalase, co-enzyme Q10, honey tocopherols, lycopenes, beta-carotene or other carotenoids, quertin, rutin, flavonoids, catechins anthocyanes, eleutherosides and ginsenosides. Some of these antioxidants maybe found in significant amounts in plant extracts. Examples include Ginko Biloba leaves that contain Gingko flavanoids, Blueberry fruits that contains anthocyanids, Ginseng roots which contains ginsenosides, Eleutherococcus roots which contains eleutherosides. The biologically active agent may also be a phytochemical such as polyphenol, procyanidin, phenolic acid, catechin or epicatechin, isoflavone, terpene or other phytonutritive plant material.

Suitable minerals include macro-nutrients such as sodium, potassium, calcium, magnesium, phosphorus or oligo-elements such as iron, zinc, copper, selenium, chromium, iodine. Macro-nutrients are known to play an essential role in complex metabolisms of the body such as in cellular cation exchange, for example, calcium is an essential constituent of the skeleton. Following EU RDA recommendations and assuming, for instance, an average daily consumption of 5 units of the delivery system. Calcium maybe used in amounts of up to 160 mg, typically between 60 mg and 90 mg in a single unit.

Trace elements are minerals present in the human body in quantity of usually less than 5 g. An example of a trace element is zinc that has antioxidant properties, helps in the synthesis of metallothionein, is an essential factor for protein synthesis and helps improve the function of the immune system. Following EU RDA recommendations and assuming a daily consumption of 5 units of the delivery system, zinc may be used in amounts of up to 3 mg per unit, typically between 1.3 mg and 1.7 mg.

Selenium is also an antioxidant and is a co-factor for glutathione peroxidase. Selenium is known to contribute to the integrity of muscles and sperm and also plays a role in hepatic metabolism. Selenium deficiencies may lead to sever cardiac, bone or neuromuscular damage. For example, following the European RDA recommendations and assuming a daily consumption of 5 units of the delivery system, Selenium may be used in amounts of up to 11 μg per unit, typically between 4 μg and 6 μg in humans.

Other nutritional supplements include amino-acids, di-peptides or polypeptides or proteins or essential fatty acids. A suitable example of an amino-acid is glutamine which provides fuel to gastro-intestinal and immune cells, reduces bacterial translocation and helps prevent muscle loss and improves nitrogen balance. Examples of peptides are the glycopeptides of lactic origin active in inhibiting the adhesion of the bacteria responsible for dental plaque and caries. More particularly, dental and anti-plaque caries agents of this type comprise active principle(s) selected from kappa- caseino-glycopeptides and deacylated derivatives thereof (also known as "CGMP"). Such active principles have an effectiveness on the dental plaque only after a few seconds in the mouth. A detailed description of these active glycopeptides is given in the European Patent 283675; the content of which is incorporated herewith by reference. Other peptides may also be a phosphopeptide or a salt thereof having anticaries properties such as those having from 5 to 30 amino acids including the sequence A-B-C-D-E where, A, B, C, D and E being independently phosphoserine, phosphothreonine, phosphotyrosine, phosphohistidine, glutamate and aspartate and compositions particularly compositions to teeth including same. A detailed description of those phosphopeptides is provided in US Patent No. 5,015,628.

Other examples of polypeptides are cysteine, acetylcysteine, cysteine methionine or a combination thereof. Cysteine and its derivatives are known to aid in defense against oxidative stress and in protein synthesis. Other nutritional supplements include functional fibres, phospholipids and caffeine, which is known as CNS stimulant, enzymes known to aid digestion (such as papain, bromelain and Upases), shark cartilage extracts, Brewer's yeast, blue green algae and the like.

hi one embodiment of the present invention, the nutritional supplement can be a botanical extract, such as guarana, gingko biloba, kola nut, goldenseal, goto kola, schizandra, elderberry, St. John's Wort, valerian and ephedra, evening primrose oil, beta-sitosterol, caffeine, cafestol, D-limonene, kabweol, nomilin, oltipraz, sulphoraphane, tangeretin, black tea, white tea, Java tea, folic acid, garlic oil, fiber, green tea extract, lemon oil, mace, licorice, menthol, onion oil, orange oil, rosemary extract, milk thistle extract, Echinacea, Siberian ginseng or Panax ginseng, lemon balm, Kava Kava, matte, bilberry, soy, grapefruit, seaweed, hawthorn, lime blossom, sage, clove, basil, curcumin, taurine, wild oat herb, dandelion, gentian, aloe vera, hops, cinnamon, peppermint, grape chamomile, fennel, marshmallow, ginger, slippery elm, cardamon, coriander, anise, thyme, rehmannia, eucalyptus, menthol, kava kava, and schisandra.

4.2 Drugs

A variety of drugs or therapeutic and/or diagnostic compounds are suitable for use with the present delivery system for administration to an animal. Representative examples include, anti-tumour compounds such as tamoxiphen, doxymbicin, taxol, cisplatin; anti-viral compounds such as ddl and ddA, anti-inflammatory compounds such as NSAEDs and steroids; antibiotic compounds such as antifungal and antibacterial compounds; cholesterol lowering drugs and contrast agents for medical diagnostic imaging. A worker skilled in the art will appreciate that certain drugs are best incorporated directly into the matrix while others may be more suited to be associated with the AMV phase or other non-matrix phases. In general, drugs suitable for use with AMVs can be classified as water-soluble, AMV permeable; water- soluble, AMV-impermeable and lipophilic.

Water-soluble, AMV-permeable drugs are characterized by a tendency to partition preferentially into the aqueous compartments of the AMV suspension, and to equilibrate, over time, between the inner liposomal spaces and outer bulk phase of the suspension. Representative drugs in this class include terbutaline, albuterol, atropine methyl nitrate, cromolyn sodium, propranalol, flunoisolide, ibuprofin, gentamycin, tobermycin, pentamidine, penicillin, theophylline, bleomycin, etoposide, captoprel, n- acetyl cysteine, verapamil, vitamins, and radio-opaque and particle-emitter agents, such as chelated metals.

Water-soluble, AMV-impermeable drugs tend to be peptide or protein molecules, such as peptide hormones, enzymes, enzyme inhibitors, apolipoproteins, and higher molecular weight carbohydrates characterized by long-term stability of encapsulation. Representative compounds in this class include calcitonin, atriopeptin, α-1 antitrypsin (protease inhibitor), interferon, oxytocin, vasopressin, insulin, interleukin-2, superoxide dismutase, tissue plasminogen activator (TPA), plasma factor 8, epidermal growth factor, tumor necrosis factor, lung surfactant protein, interferon, lipocortin, α- interferon, macrophage colony stimulating factor, and erythropoietin.

Lipophilic d gs tend to partition into the lipid bilayer phase of the AMVs, and are therefore associated with the AMVs predominantly in a membrane-entrapped form. The drugs in this class are defined by an oil/water partition coefficient, as measured in a standard oil/water mixture such as octanol/water, of greater than 1 and typically greater than about 5. Representative lipophilic drags include prostaglandins, amphotericin B, progesterone, isosorbide dinitrate, testosterone, nitroglycerin, estradiol, doxorubicin, epirubicin, beclomethasone and esters, vitamin E, cortisone, dexamefhasone and esters, and betamethasone valerete.

4.3 Antigens

Antigenic material includes but is not limited to, proteins, polypeptides, polysaccharides, lipopolysaccharides, nucleic acids such as DNA and m- NA, lipids, complete, large parts or fragments of bacterial, viral, parasitic or fungal material including whole-cell live or attenuated mammalian pathogens or combinations thereof, cell extracts, cell secretions, toxins, glycolipids, viruses, cell organelles, cell membranes or fragments thereof, and extracts of tissues from multicellular systems. AMVs themselves may act as vaccine-adjuvants because of the slow release of antigen. Interestingly, phosphatidylcholine is itself a poor antigen (Alving, In: Sela, M. (ed.) The Antigens, Vol 4, Academic Press. New York, 1-15, 1977), but it has been used successfully as an immuno-adjuvant (van Rooijen & van Nieuwmegan, Immunol. Commun. 9:747-757, 1980). Thus, not only do AMVs function in the capacity as a carrier for vaccines, for example as a replacement to mineral oil, but they may also be used as an adjuvant.

5. Additional Ingredients

The various phases of the delivery system may contain other food product ingredients that improve certain characteristics of the final product such as taste, appearance, stability, smell or texture. Non-limiting examples of additional ingredients include emulsifiers, flavourants, aromas, colourants, preservatives (such as anti-oxidants), humectants, and high intensity sweeteners. The amount of additional ingredients included in the delivery system will vary depending on the formulation, end use and consumer preferences and can be readily determined by a worker skilled in the art.

Emulsifers maybe added to improve the stability of the product. Non-limiting examples of emusifiers include lecithin, polyglycerol esters, sorbitan, fatty acid esters, and mono-and di-glycerides.

Humectants may be added to control the moisture level in the product. Examples of humectants include, but are not limited to, fruit juice and fruit juice concentrate, glycerol, glycerine, sorbitol, xylitol, fructose, dextrose, propylene glycol and other polyols.

Flavourants can be used in both the coating and the delivery system. The amount of flavouring employed is normally a matter of preference subject to such factors as flavour type, base type and strength desired. In general, amounts of above 0.01% by weight of a final product are useful. Flavours that may optionally be added to the delivery system are well known in the art. For example, synthetic flavour oils, and/or oils derived from plants, leaves, flowers, fruits, nuts, and so forth, and combinations thereof may be useful. Representative flavour oils include spearmint oil, peppermint oil, cinnamon oil, and oil of wintergreen (methylsalicylate), clove, bay, anise, eucalyptus, thyme, cedar leaf, oil of nutmeg, allspice, oil of sage, mace, oil of bitter almonds, and cassia oil. Other useful oils that may be used include, but are not limited to, artificial, natural or synthetic fruit flavors such as citrus oils including lemon, orange, grape, lime and grapefruit, and fruit essences including apple, strawberry, cherry, pineapple, banana, raspberry and others that are familiar to a worker skilled in the art; chocolate flavourings, peanut butter flavouring, rum, butterscotch, toffee, cocoa, coconut, carob, honey, pecan, pistashio, almond, butter, yogurt, and the like. Generally flavourings or food additives such as those described in Chemicals Used in Food Processing, publication 1274, pages 63258, by the National Academy of Sciences, maybe used.

Perservatives or antioxidants have been reported to prevent the formation of free radicals and oxidant wastes and to act as preservatives. Non-limiting examples suitable for inclusion in one or more of the phases of the delivery system include vitamin E, vitamin C, propyl gallate, octyl gallate, deodecyl gallate, tert- butylhyroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) and mixtures thereof.

The sweetener can be selected from a wide variety of suitable materials known in the art. When used, a sweetener is typically present in amounts above 0.01% by weight. Representative, but non-limiting, examples of sweeteners include fruit juice concentrate, honey, xylose, ribose, sucrose, mannose, galactose, fructose, dextrose, maltose, partially hydrolyzed starch, lactose, maltodextrins, hydrogenated starch hydrolysate and mixtures thereof. In addition to these sweeteners, polyhydric alcohols such as sorbitol, mannitol, xylitol, and the like may also be incorporated. Alternatively, one or more artificial sweeteners can be used, for example, sucrose derivatives (such as sucralose), amino acid based sweeteners, dipeptide sweeteners, saccharin and salts thereof, acesulfame salts, cyclamates, steviosides, dihydrochalcone compounds, talin, glycynhizin and mixtures thereof.

Colourings suitable for use in foodstuffs are well known in the art and can be optionally included in the delivery system to add aesthetic appeal. A wide variety of suitable food colourings are available commercially, for example, from Warner Jenkins, St. Louis, MO. Non-limiting examples include natural colourings such as anthocyanins, carotenoids, and po hyrins. Synthetic colourings include armoisine, tatrazine, and canthaxanthin and the like.

II. PROCESS OF MAKING THE DELIVERY SYSTEM

In accordance with the present invention, the multi-phase delivery system comprises a matrix phase and one or more non-matrix phases.

1. Preparation of the Matrix Phase

In accordance with the present invention, the matrix phase is prepared at temperatures below 100°C. Various standard methods known in the confectionery manufacturing industry can be used to prepare the matrix phase and selection of the appropriate method is considered to be within the ordinary skills of a worker in the art. Batch processes, such as kettle cooking, as well as continuous processes, such as direct steam injection jet cookers and indirect steam tubular heat exchangers, are suitable for preparing the matrix phase.

In order to allow for full dispersion and incorporation of the bioactive agent(s) into the matrix while minimising or preventing degradation of the bioactive agent(s), the matrix of the delivery system remains flowable at or below about 10O°C. Thus, although the actual methodology used to prepare the delivery systems may vary depending on the individual components selected to make up the matrix, the process of preparing the matrix comprises the step of incorporating the bioactive agent(s) into the matrix at temperatures at or below about 100°C. hi one embodiment of the present invention, the step of incorporating the bioactive agent(s) into the matrix is conducted at temperatures at or below about 80°C. In another embodiment, the step of incorporating the bioactive agent(s) into the matrix is conducted at temperatures at or below about 70°C. In a further embodiment, this step is conducted at temperatures at or below about 60°C. One skilled in the art will appreciate that the temperature at which a particular bioactive agent is incorporated into the matrix will be dependent on the properties of the bioactive agent and can be readily determined by a worker skilled in the art

Typically, when an AMV phase is included in the matrix, the AMVs with the associated bioactive agent(s) are added to the matrix at or below a temperature of about 60 °C. The AMV-associated bioactive agent(s) may be in the form of an AMV solution or a pro-AMV preparation. The present invention also contemplates the addition of certain compounds to the AMV or pro-AMV preparation that may stabilise the preparation to heat and allow for incorporation of the AMV-associated bioactive agent into the matrix at slightly higher temperatures than would otherwise be possible. For example, certain anti-oxidants (such as vitamin E) may have a heat stabilising effect on pro-AMV preparations. The temperature at which the AMV preparation is added to the solvent mixture will vary according to the type and characteristics of the AMV being used. Amending the process due to the type and characteristics of the AMV preparation is considered to be within the skills of a worker in the art.

The following description represents a general method of preparing a matrix phase of the present invention.

Briefly, the bioactive agent(s) is combined with the solvent at or below about 70°C. A blend comprising the sugar, hydrocolloid and carbohydrate components and water is prepared at a temperature below 100°C, for example between about 60°C and about 85°C, and allows for hydration of the carbohydrate and hydrocolloid. Optionally, the temperature is maintained until the desired moisture content is achieved. Once the starch and hydrocolloid are hydrated, the blend is cooled down to between about 50°C to about 80 °C, at which point the bioactive agent(s)/solvent mixture is added. Alternatively, bioactive agent(s) may be directly added to the blend without prior mixing with a solvent. The resultant delivery system can then be moulded (formed) into the desired shape. As indicated above, the final product has a moisture level between 10% and 30% and a water activity of less than 0.7.

hi one embodiment of the present invention, the blend is prepared by combining under high shear a mixture of the carbohydrate and hydrocolloid components with a solution comprising water and the sugar component, which has been preheated to a temperature of less than 100°C. Hydration of the hydrocolloid and carbohydrate subsequently takes place in the warmed sugar solution. In another embodiment, the blend is prepared by combining under high shear a mixture of the carbohydrate and hydrocolloid components with a solution comprising water and the sugar component, and simultaneously increasing the temperature to between about 60°C and about 100°C to hydrate the hydrocolloid and carbohydrate, hi a further embodiment, the blend is prepared by (i) blending the hydrocolloid component with a solution comprising water and the sugar component, (ii) adding the carbohydrate component, and (iii) subsequently heating the blend to a temperature of less than 100°C to hydrate the hydrocolloid and the carbohydrate.

Flavourings and colourings may optionally be added during preparation of the matrix. Typically these are added in the final step and can be added prior to, together with, or after, addition of the bioactive agent(s) mixture.

The hydrocolloid may require the presence of mono or divalent cations for optimum development of its gel strength and if no intrinsic source is present in the ingredients. When a separate source of mono or divalent cations is required it can be added to the hydrated hydrocolloid.

The pH of the matrix can be adjusted, if necessary, to a desired final value. Adjustment of the pH can be made at a number of points during the preparation of the matrix as will be apparent to one skilled in the art. The pH of the delivery systems can range from an upper end of alkalinity to a lower end of acidity that is selected based on taste perception and physiological acceptability and on the ability to support the stability of the bioactive agent(s) being incorporated into the delivery system. Suitable methods of adjusting the pH of food products are known in the art and include, for example, the addition of buffers, acids or bases, such as citric acid, sodium citrate, phosphates, sodium hydroxide or potassium hydroxide.

Once the delivery system has been prepared as described above, it may be formed, if required, into a desired shape using a number of techniques known in the art, for example, the standard Mogul process or by injection-filling of pre-formed moulds. One skilled in the art will appreciate that the matrix can also be readily adapted to extrusion methods.

In final form, the matrix phase of the present invention are semi-solid, intermediate moisture systems, having some properties clearly identified with those of jellies and some properties that are similar to the jujube variety of confectioneries. The matrix of the delivery systems is thus formulated to be semi-solid at normal room temperature (i.e. at temperatures between about 20°C and about 30°C). It will be readily apparent that depending on the particular components selected for use in the preparation of the matrix, the amount of each to be included in the matrix may need to be manipulated within the ranges indicated in order to achieve a semi-solid, intermediate moisture product. One skilled in the art of confectionery design can readily determine which component(s) will need to be adjusted in order to achieve an end-product with these physical properties.

Similarly, it will be readily apparent to one skilled in the art that variations can be made to the described process dependent on the type and the actual amount of each component used (within the given ranges) in order to obtain an end product with the described properties. For example, if the carbohydrate component is a starch, it is known in the art that the gelatinisation temperature of the starch maybe affected when certain sugars and sugar alcohols are used. If required, therefore, starch, hydrated hydrocolloid and the sugar component can be heated above 100°C to allow gelatinisation of the starch to occur and the desired moisture content to be reached. The temperature of the mixture can then be reduced prior to addition of the bioactive agent(s) and optionally flavourings and colourings.

As is known in the art, modified celluloses, such as methylcellulose and hydroxypropyl methylcellulose, have unique properties resulting in the ability to delay hydration of these carbohydrates during preparation processes. Thus, when these compounds are used a "delayed hydration technique" may be employed in which the cellulose is first dispersed in the solvent component of the matrix and then mixed with the other components in aqueous solution. The hydration of the cellulose then takes place gradually as the processing is complete and the formed matrix cools. Delayed hydration and non-aqueous fluid carrier techniques using modified celluloses are standard in the art.

Similarly, the choice of hydrocolloid can affect the set up temperature of the matrix. The use of a combination of gelatine and gellan, such as a gelatine: gellan ratio of between about 20: 1 and about 40: 1 , as the hydrocolloid, for example, results in a matrix set-up temperature of about 35°C, as does a combination of gelatine and pectin at a ratio between about 15:1 and about 25:1. hi contrast, the use of other hydrocoUoids or combinations of other hydrocoUoids with or without gelatine or gellan, alters the set up temperature of the matrix. For example, the use of locust bean gum or carageenan results in set up temperatures of around 60°C. The choice of an appropriate hydrocolloid or hydrocolloid combination is within the ordinary skills of a worker in the art.

The manner in which the individual components are combined may also be varied although typically at least one bioactive agent is dispersed in solvent prior to addition to the remainder of the components. For example, the hydrocolloid and part of the sugar component can be mixed and heated prior to being blended with the carbohydrate and remainder of the sugar component. Alternatively, the carbohydrate and the sugar component can be mixed and heated prior to addition of the hydrated hydrocolloid, or the carbohydrate maybe added to the solvent component and then blended with the hydrocolloid and sugar component. These and other variations are considered to be within the scope of the present invention.

hi one embodiment of the present invention, the matrix is prepared using (a) modified starch; (b) gelatine:gellan as the hydrocolloid; (c) a mixture of com symp and high fructose co symp as the sugar component, (d) a mixture of glycerol and propylene glycol as the solvent component, (e) potassium citrate as a source of monovalent cations, and (f) water. The process comprises blending the glycerol and propylene glycol, adding the bioactive agent(s) and warming the resulting blend to 65 - 70°C. The fructose symp is blended with water and warmed to 60°C. The gelatine is blended with the gellan, added to the fructose symp with under high shear and the temperature is raised to 75°C in order to dissolve all the components. The com symp is warmed to 30 - 35°C and the starch and potassium citrate, and optionally other sweeteners, are blended in. The gelatine:gellan blend and the starch blend are then combined and the solution is maintained at 75 - 80°C in order to reduce the moisture content to the desired solids content level. The solids content can be measured using standard techniques, such as measurement of the refractive index to estimate production moisture level. Once the desired level has been achieved, the bioactive agent/solvent blend is added, together with any desired colouring and flavouring. The resulting matrix is then formed using standard procedures.

In another embodiment of the present invention, a matrix containing the same components as indicated above is prepared by the following process. Glycerol and propylene glycol are blended together, the bioactive agent(s) is added and the resulting solution is blended and warmed to 40°C - 60°C. The com and fructose syrups are blended with water and heated. The dry ingredients are blended and combined with the warmed syrups under high shear. The mixture is then heated to at least 80°C. In an alternative embodiment, the blended dry ingredients are added under high shear with simultaneous heating to at least 80°C. The solid content is then adjusted by addition of water to provide a final moisture content of 10% to 30%. The temperature of the symp mixture is lowered to between 50°C and 80°C and the bioactive agent/solvent blend is incorporated. Finally, colouring and flavouring is added, if desired. The matrix is formed into the desired shape, for example, by then injection filling into preformed packaging or extrusion.

In a further embodiment of the present invention, the matrix is prepared using (a) modified starch; (b) gelatine:pectin as the hydrocolloid; (c) a mixture of maltitol symp and high fructose com syrup as the sugar component, (d) a mixture glycerol and propylene glycol as the solvent component, (e) potassium citrate as a source of monovalent cations, and (f) water. The process comprises blending the solvents, adding the bioactive agent(s) and warming the mixture to 60°C - 70°C. The starch, gelatine and pectin are blended together with any additional sweeteners required. Tins blend is added to the syrups under high shear and the temperature is maintained at 60°C - 70°C until the moisture content reaches the desired level. Colouring or flavouring is then added, if desired, and the resulting matrix is formed using standard techniques.

In still another embodiment of the present invention, the matrix phase comprises an AMV-associated bioactive agent and is prepared using (a) modified starch; (b) gelatine: gellan as the hydrocolloid; (c) a mixture of corn symp and high fructose com symp as the sugar component, (d) a mixture of glycerol and propylene glycol as the solvent component, (e) potassium citrate as a source of monovalent cations, (f) water, and (g) a liposome-associated bioactive agent as pre-liposomal solution. The process comprises blending the glycerol and propylene glycol, the pre-liposomal solution is added and the resulting solution is held at a temperature between about 25°C - 45°C. The sugar syrups are blended with water and heated. The dry ingredients are blended and combined with the warmed syrups under high shear. The mixture is then heated to a temperature of between about 70°C and about 85°C. hi an alternative embodiment, the blended dry ingredients are added under high shear to the syrup mixture with simultaneous heating to about 70°C and about 85°C. The solid content is then adjusted by addition of water to provide a moisture content of 10% to 30% in the final product. The temperature of the syrup mixture is lowered to between 40°C and 45°C and the pre-liposome/solvent blend is incorporated. Finally, colouring and flavouring is added, if desired. The matrix may then formed into the desired shape, for example, by injection filled into preformed packaging.

2. Preparation of the Non-Matrix Phases

Non-matrix phases suitable for incorporation into the delivery system include, but are not limited to, solid, typically low-moisture content, phases (such as biscuit, cookie, cake, or wafer-type formats, which may be baked or non-baked); soft, typically intermediate-moisture, phases (such as caramel, soft nougat or marshmallow type formats), coatings (for example, compound coatings, chocolate or yoghurt coatings), and combinations thereof. Thus, the delivery system can have a number of different suitable moisture contents. It is known in the art that water activity plays an important role in relation to product stability, and that the water activity will vary according to the choice of ingredients and the desired texture of the various phases of the delivery system. In general, a low moisture content is necessary for phases that have a crisp or crunchy texture while a higher moisture content is required for phases that are soft and chewy. The ratio of binder to dry ingredients and the inherent moisture in the raw ingredients will affect the moisture content and the stability of each phase and the final product. It is considered within the ordinary skills of a worker in the art to appreciate that moisture content will have to be adjusted depending upon the desired consistency of the different phases.

Processes for making suitable non-matrix phases and for the production of food products in which various non-matrix phases are combined are known in the art. Specific examples are described in U.S. Patent Application No. 2002/0051835 and U.S. Patent No. 6,346,284, PCT Application Nos. WO 01/22835 and WO 99/62351 and European Patent Application No. 1,151,676. Processes for incorporation of various bioactive agents into a variety of non-matrix phases have also been described in the art, and may be employed in the manufacture of various bioactive-containing phases for incorporation into the delivery system of the present invention. Examples include U.S. Patent Nos. 6,143,335 and 6,391,864.

It is understood that when a temperature-sensitive bioactive agent is used that it is incorporated into the present delivery system at a temperature that ensures the activity of the bioactive is maintained. In addition, when an AMV phase is to be incorporated into one of the non-matrix phases, the temperature at which the AMV solution is added should be such that the AMV remains stable. This temperature will vary according to the type or characteristic of the AMV being used and can be readily determined by one skilled in the art.

The non-matrix phases may be pre-formed and then combined with the matrix phase. Alternatively, the various phases can be combined and then formed into the desired shape. The non-matrix phases may be prepared and shaped by conventional confectionery technique. For example, the final mixture may be extruded as known in the art and the extruded material or extrudate is then cut to a desired size. In the manufacturing process binders such as glycerine, lecithin, vegetable and other oils may be added to help bind the ingredients of the non-matrix phase together thereby facilitating the formation a uniform shape in the extrusion machinery. The delivery system may also be formed by sheeting-cutting, sheeting-moulding, moulding, rolling, pressing and the like. The non-matrix phases may also be baked, rather than extruded or moulded.

The present invention contemplates that, in its a final form, the delivery system may be coated. The coating may form one phase of the delivery system and may or may not incorporate a bioactive or an AMV-associated bioactive. Methods of coating food products with a variety of coatings, such as compound coatings, chocolate or yoghurt based coatings, are known in the art.

Typically, application of the coating to the confection takes place while the coating is molten, for example, by passing the formed delivery system simultaneously through a falling curtain of liquid coating and over a plate or rollers which permit coating to be applied to the under surface of the product. Excess coating is blown off by means of air jets and the coated product passes through a cooling tunnel where refrigerated air cunents solidify the applied coating. The present invention also contemplates the use of edible protein-based coatings such as those based on casein (see, for example, U.S. Patent No. 6,379,726).

3. Forming of the Delivery System

The delivery system according to the present invention can be readily prepared and formed into various final formats by conventional techniques known in the food and confectionery industries.

As indicated above, the matrix and non-matrix phases may be pre-formed into a desired shape and then combined, and optionally coated, to form the final delivery system. For example, the matrix phase may be formed into the desired shape using a number of techniques known in the art, such as, the standard Mogul process, by injection-filling pre-formed moulds or by extrusion. The non-matrix phase may be formed by, for example, extrusion, moulding, cutting, pressing or baking in a predetermined shape. The matrix phase may then be sandwiched between, layered or coated with the non-matrix phase(s). Alternatively, the matrix and non-matrix phases can be combined and then formed into the final desired format. The following provides an exemplary method of preparing the multi-phase delivery system using a co-extruder. In this embodiment, the delivery system comprises an extruded non-matrix hollow shell sunounding an inner matrix-filing layer.

After preparation of the matrix phase as described above, the matrix blend may be kept either in a liquid state, or in a solid state that is subsequently liquefied prior to extrusion. A worker skilled in the art will appreciate that the temperature sensitivity of the bioactive agent(s) incorporated into the various phases will determine the temperature at which the delivery system is prepared. In one embodiment of the present invention, the matrix phase is liquefied below 40-45°C.

The non-matrix phase can be prepared according to methods known in the art for use with conventional co-extruders. In one embodiment of the present invention, the non- matrix phase is a protein-rich, solid, low-moisture content phase.

The final product is prepared and shaped through the use of a co-extruder that allows the matrix phase to be extruded within the hollow core of the non-matrix phase. Methods of co-extruding food products are well known in the art. See, for example, U.S. Patent No. 5,686,128 which describes an apparatus and method for triple co- extruding a snack product. If desired, the final product can be coated after extrusion using one of the various coatings known in the art. In one embodiment of the present invention, the final product is coated, hi another embodiment, the coating comprises a bioactive agent.

The present invention contemplates various formats for the delivery system. For example, the delivery system may be in the form of a bar, such as a snack, food or nutritional bar; a confection, such as a candy or candy bar; a pudding; a snack food, or a baked good, such as a cookie or biscuit that can be filled and/or coated.

The delivery system can be provided in a variety of shapes and sizes, for example, the final form of the delivery system maybe substantially rectangular, square, round, oval or O-shaped containing a hole of suitable diameter in the centre. Alternatively, the delivery system may be provided in bite size pieces of suitable shapes, such as regular geometric shapes (e.g. squares, rounds, triangles, hexagonals, rectangles, etc.), inegular shapes, which can be patterned (e.g. animals, stars, twists, figurines, trees, etc.) or unpatterned, such as a nugget shape. The present invention also contemplates the delivery system being provided in the form of a large bar or block which is scored such that the bar or block may be divided into equal sized pieces.

The different phases of the delivery system can be provided in various anangements for example, in discrete layers of variable thickness that are arranged horizontally, vertically or a combination thereof. Alternatively, the phases may be ananged such that a non-matrix phase forms a hollow tube that is filled with matrix phase, or the phases may be intertwined to create a swirled pattern. Texture additives can be randomly interspersed throughout the phases, if desired.

4. Method of Testing Inclusion of One or More Biologically Active Agents

One skilled in the art will appreciate that the molecular interaction between the biologically active agent and the ingredients of the different phases may alter the physical attributes of the phase itself. For example, acidic bioactive agents may prevent the proper gelation of the hydrocoUoids in the matrix phase and will require the addition of suitable buffer salts to conect the pH. Hence, as is standard in the art a sample of the delivery system can be prepared prior to large scale production in order to determine whether the chemistry of the agents can be combined to bring about the desired physical properties, as described above. Selection of appropriate tests is considered to be within the ordinary skills of a worker in the art.

4.1. Physical Properties

For example, dispersion of the bioactive agent(s) within a phase of the delivery system can be determined by dividing a sample of the phase into several subunits and analysing the content of bioactive agent(s) in each subunit, for example as a % by weight. The levels of bioactive agents can readily be measured by standard analytical techniques such as mass spectrometry, UV or IR spectrometry, or chromatographic techniques, such as gas chromatography or high-performance liquid chromatography (HPLC). If the % by weight of bioactive agent in each subunit is similar, then the bioactive agent is said to be substantially uniformly dispersed throughout the phase. One skilled in the art will appreciate that the % by weight need not be identical for each subunit to indicate substantially uniform dispersion. In accordance with the present invention, "substantially uniform dispersion" indicates that the % by weight of bioactive agent for each subunit varies by less than 2.5%.

In accordance with the present invention, the dispersion of the one or more bioactive agent in the matrix phase is substantially uniform. In one embodiment, the % by weight of bioactive agent for each subunit of a sample of the matrix phase varies by less than 2%. In other embodiments, the % by weight of bioactive agent for each subunit varies by less than 1.5%, by less than 1% and by less than 0.5%.

Similarly, the degradation of the bioactive agent within a phase or within the final delivery system can be determined by standard analytical techniques taking into account the total amount of each compound included in the preparation of the phase. Many bioactive agents degrade to yield specific breakdown products, the presence or absence of which can be determined in the final product. As an example, the bioactive agent creatine is hydrolysed to creatinine, which can be distinguished from creatine using chromatographic techmques, such as HPLC. In accordance with the present invention, degradation of the bioactive agent incorporated into the matrix phase is minimised during the preparation of the matrix and is less than about 20%.

The water activity (a_w) of the various phases of the final product can also be analysed by standard techniques. The a_w of a food product is a physical property that has direct implications on the microbial safety of the product and influences storage stability. Lower a_w values generally indicate a food product that is more stable and more resistant to microbial contamination than one with a high a_w value due to the requirement for water of most microbes and the fact that most deteriorative processes in food products are mediated by water. As is known in the art, the a_w value of a food product is the ratio of the water vapour pressure of the product (p) to that of pure water (p₀) at the same temperature, i.e. a_w= p/ρ₀. In accordance with the present invention, the water activity of the matrix phase is less than about 0.7, while that of the non-matrix phases varies according to the particular phase being employed.

Other parameters, such as the release rate of the bioactive agent from a phase of the delivery system can also be tested by standard methods (for example, the USP Basket Method or Paddle Method; see U.S. Pharmacopoeia XXII (1990)). Typically, a sample of the delivery system or a phase thereof containing a known amount of bioactive agent(s) (for example, a unit dose) is placed in an aqueous solution of a predetermined pH, for example around pH 1.2 to simulate stomach conditions and/or around pH 7.4 to simulate colon conditions. The suspension may or may not be st red. Samples of the aqueous solution are removed at predetermined time intervals and are assayed for their content of the bioactive by standard analytical techniques, such as those indicated above.

In addition, the delivery system may undergo testing to evaluate such factors as the microbial content of the product and the shelf-life of the product. Such quality control testing is standard in the art and can be conducted using known methods.

For example, microbial analysis of the delivery system can be conducted using techniques approved by the appropriate regulatory board, such as those described in "The Compendium of Analytical Methods: HPB Methods for the Microbiological Analysis of Foods" issued by the Health Products and Food Branch of Health Canada. Shelf life is typically evaluated using accelerated shelf life tests in which the stability of the system and the degradation of the bioactive agent contained therein is analysed under conditions that are known to accelerate the degradation of food products and can be conelated to the stability of the product under normal storage conditions.

Palatability can also be tested using standard techniques. Methods of evaluating the organoleptic properties of foods are well-known in the art. For example, sensory evaluations can be performed using individuals who are spatially separated from each other, for example, in individual partitioned booths, as testers and a hedonic nine- point scale that ranges from 1 (most disliked) to 9 (most liked), with 5 indicating no preference [Larmond, Laboratory methods for Sensory Evaluation of Foods, Research branch of Agriculture Canada (1977)]. Odour and taste are generally evaluated under a red light, which masks any differences in the colour of the product. Another nine- point hedonic scale test can be carried out under normal light to evaluate the acceptability of the appearance of the product.

4.2 Efficacy If desired, the various delivery systems of the present invention can be tested for efficacy in vivo. Typically, the efficacy is tested by conducting bioavailability studies using standard techniques in the pharmaceutical art, such as peak plasma levels and pharmokinetic analyses (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, New York, NY).

Bioavailability studies are usually conducted by administering to groups of subjects various doses of the delivery system under study over a pre-determined period of time and comparing plasma levels of the functional ingredients in these groups at varying intervals with an appropriate control or controls. Appropriate controls include groups of subjects taking recommended doses of competitor's products. The subjects may or may not have fasted prior to administration of the doses of the delivery system. Single dose or multiple dose studies may be conducted. The studies can also be used to monitor any side-effects of the dosing regimens of the delivery system under investigation by compiling reports of any adverse effects encountered during the course of the study and comparing them to side-effects reported by the control group(s). Optimal dosing schedules can also be determined in this manner.

Studies to determine that the combination of functional ingredients in a delivery system bring about the desired effect in a subject can also be conducted in a similar manner to the bioavailability studies indicated above. Such studies are routine in the art and can be readily designed and conducted by a skilled technician. End effects are measured dependent on the type of effect the delivery system is intended to bring about. For example, for weight loss or thermogenic delivery systems, the body weight and/or body fat percentage of individual subjects to whom varying doses of the delivery system is being administered can be monitored over a period of time and compared to that of individuals in control groups, for example, placebo groups or groups taking competitor's products. For muscle enhancement delivery systems, criteria such as percentage increase in muscle mass can be monitored, for bone health formulations, criteria such as bone density can be monitored. Other factors and end effects that can be monitored for various formulations will be readily apparent to one skilled in the art. In addition, for certain specific functional ingredients, characteristic metabolic products can be analysed. For example, the effect of creatine on muscle phospho- creatine can be measured by performing muscle biopsy on individuals following a controlled dosing regimen. Extraction and measurement of phosphorus compounds from the biopsy using standard techniques is then conducted to determine changes in muscle phosphor-creatine. Non-invasive measurements, for example, using P-NMR to measure changes in phosphoms compounds can also be utilized. The total concentration of creatinine can also be measured after 24 hours in order to examine clearance of creatine.

III. USE OF THE MULTI-PHASE DELIVERY SYSTEM

The present invention provides for the use of the multi-phase delivery system to administer biologically active agents to an animal, hi one embodiment, the delivery systems may be used to administer biologically active agents to humans and thus contain flavours that would appeal to humans, such as fruit-based flavours. Delivery systems of the present invention that are formulated with confectionery-like qualities and flavours are also appealing to children who are often resistant to taking medications or supplements due to unpleasant tastes or mouthfeel. Thus, in one embodiment, the multi-phase delivery systems provide a means of easily administrating certain bioactive agents, such as multi- vitamins, minerals and/or food supplements, to children.

In accordance with the present invention, the delivery system can be formulated for a number of uses depending on the selection and proportions of the nutritional components and bioactives contained therein. For example, as indicated above, the delivery system can be formulated as a meal replacement, which provides a balanced amount of essential nutrients and additional bioactives in a ready-to-eat format. The nutrients can be provided by the non-matrix phase of the delivery system or they can be dispersed in both the non-matrix and matrix phases. Alternatively, the delivery system can be formulated as a high source of protein, carbohydrate, fat and/or dietary fibre, for example by comprising a protein, carbohydrate, fat and/or dietary fibre enriched non-matrix phase. Other examples of uses contemplated for the delivery system include, but are not limited to special nutritional applications, such as delivery systems formulated for high-protein, high-cafbohydrate, high-lipid, muscle-building, energy, mineral supplements, diabetic, menopausal, prenatal, postnatal, low-fat, high-calcium, weight- loss, lactation, woman's health applications.

The delivery system may also be used for the administration of pharmaceuticals or therapeutically or diagnostically useful compounds. The use of the delivery system of the present invention is particularly advantageous to ensure the consumption of biologically active agents due to its high palatability.

Generally, based on the requirements of the animal, optimal levels of the bioactive agent(s) may be calculated and an administration schedule determined in order to maximize the bioavailability of each of the bioactive agents. The delivery system may be used as single dose units or as multi-dose units.

The delivery systems may also be used to administer biologically active agents to a non-human animal, for example, a domestic animal, such as a dog or a cat.

Administration of bioactive agents to an animal in conventional solid dosage forms, such as tablets and capsules, can be problematic in that the animal often expels them, and multiple dosing is often difficult because the animal learns to resist the dosing procedure. It will be readily apparent that the delivery systems of the present invention, which is formulated as a foodstuff, is ideally suited for administration of bioactive agents to animals. When formulated for this purpose, the matrix may contain flavours that more typically appeal to non-human animals, for example, fish or meat flavours. Additional bioactive agents more suited to animal use, such as desiccated liver, may also be included.

IV. KITS

The present invention additionally provides for kits containing the multi-phase delivery system for administration to an animal. The kit would provide an appropriate dosing regimen for a prescribed period for the bioactive agents contained in the delivery system. The kits of the invention comprise one or more packages containing the delivery system in combination with a set of instructions, generally written instructions, relating to the use and dosage of the bioactive agents contained in the delivery system. The instructions generally include information as to the appropriate dosage and dosing schedule for the bioactive agents within the delivery system. The packages containing the delivery system may in the form of unit doses, bulk packages (for example, multi- dose packages) or sub-unit doses. The doses may be packaged in a format such that each dose is associated, for example, with a day of the week. There may also be associated with the kit a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of biological products, which notice reflects approval by the agency of manufacture, use or sale for human or animal administration.

To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

EXAMPLES

EXAMPLE 1: Matrix Phase containing a Bioactive Agents

One example of a matrix phase containing bioactive agents is as follows: Ingredient % by weight

Glycerol 14.57%

Propylene Glycol 5.30%

Creatine monohydrate 11.71%

Com Symp 62DE 31.79%

Sucralose 0.04%

Modified Starch ( Staley Softset^®) 2.65%

Potassium citrate 2.15%

Dimethylglycine 1.67%

High fructose com symp 9.27%

Water 14.57%

Gelatine 100 bloom type B 1.32%

Gelatine 250 bloom type A 3.97%

Gellan (Kelcogel^® LT100) CP Kelco 0.32%

Colour 0.21%

Flavour 0.45%

Total: 100.00%

Glycerol and propylene glycol were first blended and the creatine was added. The blend was heated to 45-70°C. In a separate container, the two types of gelatine and the gellan were blended together. The fructose symp and water were mixed and heated to 60°C, after which the gelatine:gellan mixture was added with constant agitation. The mixture was then heated to 75°C to allow the components to dissolve, hi a third container, the com symp was warmed to 30-35°C and the sucralose, potassium citrate, dimethylglycine and starch were then blended in. The com symp mixture was combined with the gelatine:gellan mixture and heated to 75-80°C until the moisture content was reduced and the desired solids level achieved. The solution was cooled to about 40 - 60°C. The creatine mixture was then added together with the colour and flavour additives. The matrix phase was then moulded using standard techniques.

EXAMPLE 2: Matrix Phase containing a Bioactive Agent

Another example of a matrix phase comprising a bioactive is as follows:

Ingredient % by weight

Com syrup 40.58%

Sucrose 7.25%

Creatine magnesium chelate 7.25%

Glycerol 14.49%

Gelatine Type B 4.20%

Water 18.84%

Modified Starch (Staley Softset®) 5.80%

Flavour 0.58%

Gellan 0.14% α-Lipoic acid 0.14%

Taurine 0.72%

Total: 100.00%

Moisture of the final preparation was adjusted to yield approximately 18% in the final product.

HPLC Analysis of Creatine Stability Samples of the above delivery system were analysed by high performance liquid chromatography (HPLC) using UV detection to determine the percentage of creatine. Prior to injection, each sample was subject to a dissolution procedure wherein the sample was cut into small pieces and heated in 400 ml of Type 1 water at 90°C for 10 minutes. The samples were then transfened to a water bath at 4°C and 50ml of 1% perchloric acid was added. The mixture was then heated to 28°C, transferred to a 500 ml volumetric flask and the volume made up to 500 ml with Type 1 water. A 60μL aliquot of this solution was then added to 140μL of methanol and vortexed. Three replicates were prepared for each sample. Samples of lOμL of the final solution were used to inject into the HPLC.

The percentage of creatine (by weight) was determined by comparing the mean response of creatine in each sample to the mean response of a stock solution at known concentrations. For each replicate prepared as described above, the solution was injected in triplicate.

Tables 1 and 2 outline the quantity and percentage creatine in the samples of the delivery system. Of particular note is the only slight variation between the percentage creatine by weight of each jujube despite the larger variation in the weight of the jujubes. The percentage by weight of creatine determined for each jujube varied between 7.71% and 9.04% (%CV-^: 14.1%), while the weight of the jujubes varied from 7082.40 mg to 11124.16 mg. The mean percentage creatine by weight for the samples was 8.0%. This is consistent with the expected amount of 9% of chelate in the final product.

EXAMPLE 3: Matrix Phase containing a Bioactive Agent associated with AMVs

This formulation yields a 12 gram matrix phase containing approximately 5milligrams ofCoQIO.

Ingredient % by weight

Glycerol 17.19%

Propylene Glycol 5.93%

CoQIO liposome solution 10.37% Com Symp 62DE 22.81% high fructose com symp 26.67%

Sucralose® 0.059%

Modified Starch ( Staley Miraquick ®) 2.96%

Potassium citrate 1.51%

Water 5.33%

Gelatin Type A 5.93%

Gellan (Kelcogel LT100) CP Kelco 0.36% colour 0.30%

Flavour 0.59%

Total: 100.00%

Note: Concentration of the CoQIO liposome solution is approximately 4.2 mg CoQ/ml solution.

Glycerol and propylene glycol were first blended and the liposome solution was added. The blend was held at a temperature between 25°C and 45°C. hi a separate container, the gelatine and the gellan were blended together. The fructose symp, glucose symp, water, citrate and Sucralose® were blended at room temperature and the starch was blended into the mixture followed by the gelatine: gellan mixture. The mixture was then heated to 80 - 85°C to fully hydrate the starch and hydrocoUoids. The temperature was maintained until the desired moisture content was achieved. The solution was cooled to about 40 - 45°C and the liposome/solvent mixture blended in followed by the colour and flavour additives. The delivery system was then moulded using standard techniques.

EXAMPLE 4: Matrix Phase containing a Bioactive

Another example of a matrix phase containing a bioactive is as follows: Ingredient % by weight Ingredient % by weight

Glycerol 15.97%

Propylene Glycol 5.51%

Creatine Monohydrate 16.71%

63 DE Com symp 21.20%

High Fmctose Com Symp 24.78%

Gelatine 250 Bloom Type A 5.51%

Gellan 0.33%

Sucralose 0.06% potassium citrate 1.40%

Modified Starch (Staley Miraquick®) 2.75%

Water 4.96%

Flavour 0.56%

Colour 0.28%

Total: 100.00%

Creatine was added to a mixture of glycerol and propylene glycol, and heated to 40- 60°C. The syrups were blended with water and heated to 60 - 80°C. The dry ingredients were pre-blended and then mixed into the symp mixture under high shear. Alternatively, the blended dry ingredients can be added under high shear with simultaneous heating to raise the temperature of the mixture to 70 - 85°C and effect full hydration of the starch and hydrocoUoids. The solid content can then adjusted by addition of water. The temperature of the symp mixture was then lowered to between 50°C and 80°C and the glycerol-glycol mixture was added. Colour and/or flavouring additives were then added and the delivery system was injection filled into the preformed packaging.

HPLC Analysis of Creatine Stability

Samples of the above delivery system were analysed by HPLC using UV detection to detennine the percentage of creatine monohydrate by weight of each sample. Prior to injection, each sample was subject to a dissolution procedure wherein the sample was cut into small pieces and heated in 200 ml of water at 90°C for 10 minutes, then transferred to a water bath at 4°C. The mixture was subsequently heated to 28°C, transfened to a 250 ml volumetric flask and the volume made up to 250 ml with water. After mixing, a 1 ml aliquot of the mixture was placed into an Eppendorf tube and centrifuged at 10 000 rpm. The supernatant was filtered through a 0.2μ filter and centrifuged again at 10 OOOrpm. A 5μl sample of the supernatant was then taken for HPLC analysis. Three injections were made for each sample preparation.

The results of the HPLC analysis are given in Tables 3 and 4. Both the weight of the jujubes produced by the above method and the percentage by weight of creatine that they contained can be seen to be notably uniform. The weight of the jujubes varied from 26 262.37mg to 26 954.56mg, with an average value of 26 774.37mg, and the percentage by weight of creatine varied from 11.75% to 11.85%, with an average value of 11.80%.

EXAMPLE 5: Multi-Phase Delivery System

One example of a multi-phase delivery system is:

Ingredient % by Weight

Protein blend (whey protein isolate, whey protein 52.04 concentrate, whey protein hydrolysate, soy protein isolate, calcium caseinate)

Matrix phase 16.43

Fruit j uice concentrate (apple, pear, grape) 6.85

Glycerol 5.48

Rice Crisps 2.74

Water 1.37

Dried Fruit Pieces (apple, cranberry, etc.) 1.37

Flavouring 0.03

Yoghurt coating (sugar, modified palm 13.69 Ingredient % by Weight kernel oil, non-fat milk, maltodextrin, non-fat yoghurt powder)

Total: 100.00%

The fruit juice concentrate, glycerol, water and flavouring were mixed together, hi a separate container, the protein blend was mixed with the rice crisps and dried fruit pieces. The fruit juice blend was mixed with the protein blend. The matrix phase was prepared. The protein containing blend and matrix phase were molded using a co- extruder. The multi-phase delivery system was then coated with a prepared yoghurt coating.

EXAMPLE 6: In Vivo Testing of a Matrix Phase comprising a Bioactive Agent

Ingredient % by Weight

Glycerol 27.9990%

Propylene Glycol 3.4145%

Potassium Hydroxide 0.1208%

Creatine Monohydrate 24.0154%

High Fructose Com Symp 15.7068%

Com syrup 14.7962%

Starch (Mira-quik MGL™ ) 2.5040%

Water 3.9836%

Potassium phosphate 0.4234%

Sucralose 0.0381%

Potassium citrate 0.9526%

Gelatine Type A 4.7803%

Pectin 0.2732%

Flavour 0.5464% Ingredient % by Weight

Colour 0.2982%

Total: 100.0000%

Glycerol and propylene glycol were first blended and the creatine was added. The blend was heated to 45-50°C. hi a separate container, the gelatine, pectin, starch and sucralose were blended together. The fructose and glucose syrups and water were mixed and heated to 60°C, after which the salts and pH modifying agents were added with constant agitation and heated to 60-70°C to dissolve the solids. The powder blend was then incorporated into the syrup mixture using high shear. Finally, the creatine mixture was added, together with the colour and flavour additives, and blended. The delivery system was then moulded using standard techniques.

Serum concentration levels of creatine of subjects who ingested either 3.5 gram of micronized creatine powder in capsule format or 3.5 gram of micronized creatine in jujubes (as described above) were analysed by mass spectroscopy. Seven individuals were enrolled in the test, with an age range between 18 and 50 years. Individuals fasted overnight prior to administration of the creatine. The test protocol was as follows. Individuals were administered jujube containing 3.5g creatine with 8 oz water. Blood samples were taken every 15 minutes for the first hour, every 30 minutes for the second hour and subsequently at hourly intervals for a total of 8 hours after administration. After sufficient period of time to allow blood creatine levels to return to normal, the subjects were administered 5 capsules containing a total of 3.5g creatine with 8 oz water. Blood samples were taken at the same time intervals as indicated above. Results are shown in Figure 1.

EXAMPLE 7: Accelerated Shelf-Life Determination

An accelerated shelf life test was conducted on the matrix phase prepared as described in Example 4 and demonstrates the stability of the matrix phase that is included in the delivery system of the present invention. Microbial analysis was conducted using approved methods as described in The Compendium of Analytical Methods: HPB Methods for the Microbiological Analysis of Foods (Volume 2 issued by the Health Products and Food Branch of Health Canada. After subjecting samples of the delivery system to a temperature of 35°C and a relative humidity of 45-55% for a period of 35 days, the samples were tested for the presence of various micro-organisms as listed in Table 5. The average water activity of the samples tested was approximately 0.51.

In addition to the above microbial analysis, the creatine level in each sample was determined by HPLC prior to the test and after 35 days. The average creatine content for four samples randomly selected for analysis after 35 days was compared to the average creatine content for three samples taken prior to the shelf life test. HPLC analysis of creatine monohydrate levels was conducted as described in Example 6.

The results, as shown in Table 5, indicate that after a period of 35 days at the above- described conditions, microbial contamination was minimal and well below accepted levels. Based on these results, the matrix phase is shown to have a stable shelf life of at least one year from the date of manufacture.

Results from the HPLC analysis also indicated that levels of creatine monohydrate remained stable in the matrix phase after 35 days exposure to the above-described conditions. Prior to the start of the experiment, three matrix phase samples had an average of 13.4% by weight of creatine monohydrate. After 35 days, matrix phase samples were shown to have an average of 14.2% by weight of creatine monohydrate, which is within the enor limits of the analysis performed.

EXAMPLE 8: Analysis of Water Activity of the Matrix Phase

Water activity was measured in samples of matrix phase that had been prepared according to the method described in Example 4 and demonstrates the low water activity of the matrix phase included in the delivery system of the present invention.

The procedure for measuring water activity is based on the fact that the water activity of a sample is equal to the relative humidity created by the sample in a closed environment when in equilibrium. The procedure uses a water activity meter constructed by David Brookman & Associates (DB&A). The DB&A Water Activity Meter uses an Omega Engineering HX92C Relative Humidity indicator to measure the relative humidity within a closed environment containing the sample. The Omega probe converts the relative humidity (R.H.) into milhamperes (ma), where 4 ma equals 0% R.H. and 20 ma equals 100% R.H. The water activity meter is calibrated to 11.3% R.H. using a saturated solution of LiCl and to 75.3% R.H. using a saturated solution ofNaCl.

The samples are manually macerated in a plastic bag and then transfened to a 30 ml sample bottle. The bottles are filled with sample to at least 1 cm from the shoulder. The bottles are capped until use and stored at room temperature. Measurements are taken by screwing the sample bottle onto the DB&A meter probe and the bottle probe assembly is maintained in a vertical position in a rack. Measurements are taken at hourly intervals at room temperature (20 - 22°C) until such time that successive readings do not vary more than 1 %.

Random sampling of the jujubes was conducted. The water activity (a_w) was determined to be 0.507, 0.515 and 0.544. These values are well below levels those that favour the growth of micro-organisms. It has been shown that micro-organisms generally grow best between a_w values of 0.995 - 0.980 and most microbes will cease to grow at a_w values less than 0.900.

EXAMPLE 9: Preparation of a Delivery System containing an AMV-Associated Bioactive Agent

Ingredient % by weight

High fructose com symp 24.77%

62 DE Com symp 21.19%

1 -Testosterone pre-liposomal solution¹ 16.70%

Glycerol 15.96%

Propylene Glycol 5.50% Ingredient % by weight

Gelatine 5.50%

Water 4.95%

Modified Starch (Staley Miraquick®) 2.75%

Potassium citrate 1.40%

Flavour 0.55%

Gellan 0.33%

Colour 0.28%

Sucralose³ 0.11%

Total: 100.00%

¹ 1.25 mg/ml solution

² 10% w/w solution

³ 25% w/w solution

Glycerol and propylene glycol were first blended and the liposome solution was added. The blend was held at a temperature between 25°C and 45°C. In a separate container, the starch, gelatine and the gellan were blended together. The syrups were blended together and heated to 70 - 80°C. The blended dry ingredients were then added to the heated syrups under high shear. Alternatively, the blended dry ingredients can be added under high shear with simultaneous heating to raise the temperature to 70 - 80°C and to effect full hydration of the starch and hydrocoUoids. The solid content may then be adjusted to the desired level by addition of water. The mixture was cooled to about 40 - 45°C and the liposome/solvent mixture blended in followed by the colour and flavour additives. The delivery system was then moulded using standard techniques.

EXAMPLE 10: Heart Health Delivery System

One example of a matrix phase for a delivery system formulated to promote heart health is as follows: Ingredient % by Weight

12.57%

Glycerol

Propylene Glycol 4.19%

Arginine 14.02%

Maltitol solution 33.52%

Modified Starch ( Staley Miraquick^®) 2.79%

Potassium citrate 1.17%

Sucralose 0.04%

High fructose corn symp 9.78%

Water 15.37%

Gelatine 250 bloom type A 5.59%

Gellan (Kelcogel^® LT100) CP Kelco 0.28%

Colour 0.168%

Flavour 0.503%

Total: 100.00%

Glycerol and propylene glycol were first blended and the arginine was added. The blend was heated to 65-70°C. hi a separate container, the gelatine and the gellan were blended together. The fructose symp and water were mixed and heated to 60°C, after which the gelatine: gellan mixture was added with constant agitation. The mixture was then heated to 75 °C to allow the components to dissolve, hi a third container, the maltitol solution was warmed to 30-35°C and the sucralose, potassium citrate and starch were then blended in. The maltitol mixture was combined with the gelatine:gellan mixture and heated to 75-80°C until the moisture content was reduced and the desired solids level achieved. The arginine mixture was then added together with the colour and flavour additives. The delivery system was then moulded using standard techniques. EXAMPLE 11: Energy Delivery System

An example of a matrix phase for a delivery system formulated to promote energy is as follows:

Ingredient % by Weight

13.82%

Glycerol

Propylene Glycol 5.53%

Creatine monohydrate(CM) 4.59%

Conjugated Linoleic Acid (CLA) 4.59%

Lecithin 1.05%

Isomalt syrup 33.17%

Sucralose 0.055%

Modified Starch (Staley Softset®) 2.76%

Potassium citrate 2.24%

N,N, dimethylglycine (dmg) 0.47%

Rhodiola / Seabuckthorn extract 0.21% solution

Chromium chelate 0.11%

High Fmctose Com syrup 9.68%

Water 15.20%

Gelatine 250 bloom type A 5.53%

Gellan (Kelcogel^® LT100) CP 0.33%

Kelco

Colour 0.08%

Flavour 0.08%

Total: 100.00%

The CLA, creatine and lecithin were first mixed together. The glycerol and propylene glycol were mixed and heated to 65-70°C. The CLA/creatine/lecithin blend was then added to the solvents and the resultant mixture was maintained at 65-70°C. In another container, the gelatine was mixed with the gellan. The fructose symp and water were combined and heated to 60°C and the gelatine:gellan mixture was then added, after which the temperature was raised to 75°C and maintained at this temperature until the solids dissolved. In another container, the isomalt symp was warmed to 30 -35°C and the sucralose, citrate, dmg, rhodiola/seabuckthorn extract, chromium chelate and starch were then blended in. This mixture was combined with the gelatine mixture and the temperature maintained at 75-80°C until the moisture content was reduced sufficiently to give the desired solids level. Once the proper moisture level was achieved, the glycerol- glycol mixture was blended in together with colour and flavouring additives. The mixture was then moulded using standard techniques.

EXAMPLE 11: Weight Loss or Maintenance Delivery System

An example of a matrix phase for a delivery system formulated to aid in weight loss or maintenance is as follows:

Ingredient % by Weight

Glycerol 16.67%

Propylene Glycol 7.86%

Conjugated linoleic acid - - Clarinol 80 7.86%

Citrus Aurantium 0.50%

Maltitol syrup 35.86%

High fructose com symp 15.73%

Sucralose 0.06%

Modified Starch ( Staley Miraquick^®) 3.15%

Potassium citrate 1.42%

Potassium hydroxide 0.92%

Inulin 0.63%

Caffeine 0.25%

Mixed tocopherols 0.04% Ascorbic acid 0.03%

Water 1.38%

Gelatine 6.29%

Pectin . 0.31%

Colour 0.3%

Flavour 0.74%

Total: 100.00%

The glycerol and propylene glycol were first blended together. The CLA, Citrus Aurantium and mixed tocopherols were then added and the resultant mixture was warmed to 60-70°C. hi another container, the syrups, water, potassium citrate and potassium hydroxide were combined and warmed to 60-70°C. The starch, gelatine, pectin, inulin and sucralose were pre-blended then added to the syrup mixture under high shear. This mixture was combined with the glycerol mixture and the temperature maintained at 60-70°C until the moisture content was reduced sufficiently to give the desired solids level. Colour and flavour were added and the mixture was then moulded using standard techniques.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

TABLE 1: Peak Height Responses and Determined Quantity (Mg) of Creatine Monohydrate Chelate in Jujubes

¹ Calculated as the (Mean Peak Height of Jujube Solutions) / (Mean Peak Height of Reference Stock Solutions) x (1Q39 -Ig mL) x (500 mL)/(lQ00)

TABLE 2: Percentage Creatine Monohydrate Chelate by Weight in Jujubes

TABLE 3: Percentage Creatine Monohydrate by weight in Jujubes

TABLE 4: Peak Height Responses of Creatine Monohydrate in Jujubes

TABLE 5: Microbial Analysis of Creatine Monohydrate Jujubes - Accelerated Shelf Life Determination

Water activity: approximately 0.51 Time: 35 days Temperature: 35°C Humidity: 45-55%

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A multi-phase oral delivery system for biologically active agents comprising:

(a) one or more biologically active agent(s);

(c) one or more other phase associated with the matrix phase, said one or more other phase comprising at least one of said biologically active agent(s), one or more nutritional component(s), or a combination thereof.

2. A multi-phase oral delivery system for biologically active agents comprising:

(a) one or more biologically active agent(s);

(b) a matrix phase comprising; i) one or more sugar; ii) one or more carbohydrate; iii) one or more hydrocolloid; iv) one or more polyhydric alcohol, and v) one or more source of water, and having at least one biologically active agent(s) substantially uniformly dispersed therein, wherein said matrix phase is a semi-solid at room temperature, has a final moisture content between about 15% and about 30% by weight and a water activity of less than about 0.7; (c) one or more other phase associated with the matrix phase, said one or more other phase comprising at least one of said biologically active agent(s), one or more nutritional component(s), or a combination thereof, and

(d) an artificial membrane vesicle (AMV) phase dispersed in the matrix phase, the one or more other phase, or in both the matrix phase and the one or more other phase, said AMV phase comprising at least one of said biologically active agent(s) associated with an AMV.

3. The multi-phase delivery system according to claim 1 or 2, wherein said one or more other phase comprises a solid, low-moisture content phase; a soft, intennediate-moisture phase; a confectioner's coating, or a combination thereof.

4. The multi-phase delivery system according to any one of claims 1 to 3, wherein said one or more nutritional component comprises protein, carbohydrate, fat, lipid, fibre, or a combination thereof.

5. The multi-phase delivery system according to any one of claims 1 to 4, wherein said matrix phase comprises between about 20% and about 60% by weight of said sugar component, between about 0.6% and about 15% by weight of said carbohydrate, between about 0.1% and about 7.0% by weight of said hydrocolloid component and between about 5% and about 35% by weight of said solvent component.

6. The delivery system according to any one of claims 1 to 5, wherein said one or more carbohydrate comprises a starch or a modified version thereof.

7. The delivery system according to claim 6, wherein said starch or modified version thereof, is present in an amount between 1% and about 15% by weight of said matrix phase.

8. The delivery system according to any one of claims 1 to 7, wherein said matrix phase further comprises one or more modified cellulose.

9. The delivery system according to any one of claims 1 to 8, wherein said one or more hydrocolloid comprises a mixture of gelatine and gellan in a ratio between about 15:1 to about 40:1.

10. The delivery system according to any one of claims 1 to 8, wherein said one or more hydrocolloid comprises a mixture of gelatine and pectin in a ratio between 15:1 to about 35:1.

11. The delivery system according to any one of claims 1 to 10, wherein said one or more sugar is sugar, sugar symp, sugar alcohol or combination thereof.

12. The delivery system according to claim 11, wherein said one or more sugar is a combination of sugar syrups.

13. The delivery system according to any one of claims 1 to 12, wherein said one or more polyhydric alcohol comprises glycerol and propylene glycol.

14. The delivery system according to any one of claims 1 to 13, wherein said AMV phase comprises liposomes.

15. The delivery system according to any one of claims 1 to 14, wherein said one or more biologically active agent is a drug, a botanical, a nutritional supplement, a vitamin, a mineral, an enzyme, a hormone, a protein, a polypeptide, an antigen, or a combination thereof.

16. A process for preparing a multi-phase delivery system for biologically active agents, said process comprising:

(a) preparing a matrix phase by (i) preparing a blend comprising one or more hydrated carbohydrate, one or more hydrated hydrocolloid, one or more sugar, sugar alcohol or sugar symp, or a combination thereof, and water at a temperature of less than 100°C;

(ii) optionally adjusting the moisture content of the blend;

(iii) reducing the temperature of the blend to between about 50°C and about 80°C;

(iv) adding to said blend one or more biologically active agent and a solvent component comprising one or more polyhydric alcohols thereby forming said matrix phase;

17. The process according to claim 16, wherein said blend is prepared by combining under high shear a solution comprising water and said one or more sugar, sugar alcohol or sugar symp, or combination thereof, preheated to a temperature of less than 100°C with a mixture of one or more carbohydrate and one or more hydrocolloid, whereby said hydrocolloid and said carbohydrate are hydrated.

18. The process according to claim 16, wherein said blend is prepared by combining under high shear a mixture of one or more carbohydrate and one or more hydrocolloid with a solution comprising water and said one or more sugar, sugar alcohol or sugar symp, or combination thereof, and simultaneously increasing the temperature to between about 60°C and about 100°C to hydrate said hydrocolloid and said carbohydrate.

19. The process according to claim 16, wherein said blend is prepared by (i) blending one or more hydrocolloid with a solution comprising water and said one or more sugar, sugar alcohol or sugar symp, or combination thereof, (ii) adding one or more carbohydrate, and (iii) heating the blend to a temperature of less than 100°C to hydrate said hydrocolloid and said carbohydrate.

20. A multi-phase delivery system prepared by the process according to any one of claims 16 to 19.

21. Use of the multi-phase delivery system according to any one of claims 1 to 15 or 20 for oral administration of one or more biologically active agent to an animal in need thereof.

22. A kit comprising the multi-phase delivery system according to any one of claims 1 to 15 or 20 and optionally instructions for use.