WO2004009053A2 - Oral delivery system containing a gel matrix and liposomes - Google Patents

Abstract

A transmucosal delivery system for oral delivery of one or more biologically active agent associated with an artificial membrane vesicle (AMV), such as a liposome, is provided. The system is especially suited for the delivery of biologically active agents with poor solubility, that are easily degraded in the gastrointestinal tract or that tend to have an unpleasant taste and mouth feel. The delivery system comprises a two-phase system whereby one or more AMV-associated biologically active agents are substantially uniformly dispersed in a semi-solid matrix that allows for maintenance of bioactivity. The dual phase system may further comprise one or more non-AMV associated biologically active agents incorporated within the matrix. Methods of preparing the delivery system and methods of use for delivering biologically active agents to animals, including humans, are also provided.

Description

TRANSMUCOSAL DELIVERY SYSTEM

FIELD OF INVENTION

The present invention relates to the field of delivery systems for biologically active agents and in particular to a dual phase transmucosal delivery system for biologically active agents.

BACKGROUND OF THE INVENTION

The therapeutic effect of an administered substance is usually directly related to the quantity and rate at which the substance reaches the bloodstream. There are many factors that affect the ability of the substance to reach the systemic circulation including; the site of entry into the body, the physical form of the substance, the design of the formulation of the product, various physicochemical properties of the compound and the excipients, and control and maintenance of the location of the substance at the proper absorption site.

Oral delivery of a therapeutic substance is the most common form of delivery today because of convenience and ease of administration, however, it is not the most reliable route of administration and can often be inefficient and erratic. Factors that influence the absorption, and thus the ability of the substance to reach the bloodstream, of an orally administered substance are related to the physicochemical properties of the substance, the physiologic factors in the gastrointestinal tract and the variables in the dosage form. Conventional oral dosage forms consist of solutions, suspensions, powders, two-piece gelatin capsules, soft gelatin capsules, compressed tablets, and coated tablets. It is generally the case that gastrointestinal absorption is most rapid with solutions and progressively slower as you move toward coated tablets in the above continuum. Liquid dosage forms are generally much faster absorbed than solid forms because dissolution is not a rate-determining step in the absorption process. Much work has been conducted to both identify agents that are capable of increasing the permeability of the gastrointestinal tract and to protect the biologically active agent from the digestive degradative process. The discovery of liposomes as a carrier system also resulted in great improvements in the oral administration of biologically active agents.

Liposomes are microscopic, three-dimensional vesicles comprising phospholipid which spontaneously form into concentric bi-layers when they come into contact with aqueous solutions. Particles contained in solution become trapped between the bilayers which, being biodegradable, slowly release their contents as they are broken down witliin biological systems. Today, the medical applications of liposomes range from systemic anticancer therapy to enhancing gene topical anesthesia and gene delivery.

Liposomes have been used primarily in the human health market to deliver various compounds to target organs and to reduce the toxicity of certain chemotherapeutic agents. They are also used as a method to administer lipophilic substances (U.S. Patent No. 5,989,583). In mammalian studies, liposomes are considered to be suitable carriers since they can: a) serve as a depot system for the sustained release of a compound; b) alter the biodistribution of biologically active substances (Profitt et al., 1983; Lui and Huang, 1992); c) protect the encapsulated materials from inactivation by the host defence mechanisms (Ahmad et al. 1993; Naage et al. 1993); and d) reduce side effects (Graner et al. 1985; Philips et al. 1991). Moreover, liposomes have been used as drug delivery vehicles since they can be safely administered without serious side effects due to their biodegradable and non-toxic nature.

There are, however, a number of pharmaceutical related problems associated with administering liposomes orally including the pH of the stomach and the presence of bile salts and digestive enzymes, primarily lipases. The unbuffered pH of the stomach can range from 1.5 to 2.5 and causes chemical instability of the liposome membrane surface. Bile salts act as detergents causing instability of the liposome bilayer by emulsification and exposure of lipases and other enzymes can lead to cleavage of the polar head groups or the acyl chains of the phospholipids thus rupturing the liposome vesicle.

In the past, administration of oral liposomes has been as a liquid, by intubation directly into the small intestine, to the back of the throat by a lavage syringe or by dropper directly into the mouth. It will be appreciated that these modes of administration are impractical, messy, may result in inaccurate dosing and can be difficult for patients to handle. In addition many biologically active agents have a bitter, astringent taste that can be unpalatable and difficult to mask.

Chemical and stearic modifications have been made to liposomes to help stability (International Patent Application WO 01/56548), and encapsulation of the liposome in capsules (International Patent Application WO 99/11242) or in powder form (U.S. Patent No. 5,989,583) have also been reported. While these improvements have resulted in making the formulations easier to administer, more convenient, less aggressive and better tolerated by patients, they are not always capable of overcoming shortcomings such as unpleasant taste and mouth-feel.

"Pro-liposomes" have also been reported that improve the stability of liposome formulations. Typically pro-liposomes are dry formulations of lipid-coated carrier which form liposomal solutions when mixed with water (see, for example, Payne et al, (1986) J Pharm. Sci, 75:325-329). Pro-liposomes that spontaneously form liposomes on contact with water have also been described, for example pro-liposomes comprising phospholipids dispersed in a water-miscible organic solvent (see, European Patent Nos. EP 0 158 441 and EP 0 309 464) and gel-like compositions comprising liposomes and hydrating agents (see, European Patent No. EP 0 211 647).

Drug delivery systems known as niosomes that are similar to liposomes but comprise surfactants rather than phospholipids have been described. Proniosomes are also known (see, for example, Hu & Rhodes, (1999) Int. J. Pharm., 185:23-25; Blazek- Welsh, et al., (2001) AAPS Pharmsci., 3: 1-8) in which the surfactants are coated onto sorbitol or maltodextrin powder. A number of attempts have been made to encapsulate or retain functional agents into various glassy, sintered or chewy matrixes. In general, the confectionery serves as a solid continuous matrix for the functional agent. The functional agent is delivered according to the dissolution rate of the confectionery matrix that confers a solid taste in the mouth. Crushing the confectionery is a solution for the consumer to speed up the release of the functional agent but this solution maybe undesirable as dental problems may arise and/or the release rate of the functional agent may be altered. Depending upon the method of manufacturing the confectionery matrix, the functional agent may suffer from deterioration or damages due to heat and/or mechanical stresses in the manufacturing process. Often an "overdose" of the functional agents is included in the confectionery matrix in order to overcome a high deterioration rate due to strong processing conditions, however, this can result in high production costs. The "solid" taste a pressed tablet or glassy matrix may provide in the mouth may also be considered as not very attractive in the context of delivering active agents, especially if the product is supposed to be primarily a confectionery.

A number of publications have reported the encapsulation of functional agents. For example, U.S. Patent No. 5,897,897 describes the encapsulation of medications, pesticides, vitamins, preservatives and flavouring agents within a glassy matrix consisting of modified starch and polyhydric alcohol, and U.S. Patent No. 5,648,092 relates to pharmaceutical compositions in the form of pleasant-tasting chewable tablets or chewable coated tablets which besides the pharmaceutically active agent sulfacrate, essentially contain at least one rapidly swellable physiologically acceptable gel former plus sugar or sugar substitutes.

Encapsulation of probiotic bacteria in a variety of formats has been described. European Patent No. EP 0904 784 describes a probiotic preparation with health promoting action comprising bacteria cells, novelose, arabic gum included in a 3- gram proteinic capsule. U.S. Patent No. 4,396,631 describes a bifidobacterium- containing confectionery tablet including one or more of substances selected from the group consisting of starch, starch hydrolyzate and protein. Japanese Patent No. JP 2893021 relates to a boiled sweet enclosing bifidobacteria encapsulated with a protective coating film and diluted with a mixture of powdered sugar or sugar alcohol as a filling. Japanese Patent No. JP 60083535 describes a preparation of candies containing lactobacilli activated with spores made by mixing sugar and millet honey, chilling, pulverizing and adding activated lactobacilli powder. Japanese Patent No. JP 57032221 discloses candy tablets containing bifidus microorganism made by mixing microorganism powder with fat, adding further raw materials and tabletting. European Patent No. EP 704164 discloses a confectionery composition containing a long-life lactic bacteria, fats and/or oil, fermented milk powder and saccharide. DE 19830528 discloses a multi-layer tablet comprising nutritious substances and microorganisms that can be stored without cooling.

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 dual phase transmucosal delivery system that allows for the uniform and complete dispersion within a matrix of one or more biologically active agents associated with artificial membrane vesicles. In accordance with one aspect of the present invention, there is provided an oral delivery system for biologically active agents comprising a matrix having one or more biologically active agent associated with artificial membrane vesicles (AMNs) substantially uniformly dispersed therein, said matrix comprising: (i) a sugar component comprising one or more sugar, sugar syrup, sugar alcohol, or a combination thereof; (ii) one or more carbohydrate; (iii) a hydrocolloid component comprising one or more hydrocolloid; (iv) a solvent component comprising one or more polyhydric alcohol, and (v) one or more source of water, wherein said delivery system is a semi-solid at room temperature and has a final moisture content of between about 10% and about 30% by weight, a pH between about 4.0 and about 9.0 and a water activity of less than about 0.7. In accordance with another aspect of the invention, there is provided a process for preparing an oral delivery system for biologically active agents associated with artificial membrane vesicles (AMNs), said process comprising (a) 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; (b) optionally adjusting the moisture content of the blend; (c) reducing the temperature of the blend to about 60°C or below; (d) adding to said blend one or more AMN-associated biologically active agent and a solvent component comprising one or more polyhydric alcohols to form a matrix whereby the AMN-associated biologically active agent is substantially uniformly dispersed throughout said matrix, and (e) forming said matrix to provide said oral delivery system, wherein said oral delivery system has a moisture content between about 10% and about 30% by weight.

In accordance with another aspect of the invention, there is provided an oral delivery system for bioactive agents produced by the above process.

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

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 demonstrates the enhanced uptake of creatine into the blood following administration to humans of a delivery system prepared according to Example 7.

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. The present invention provides a dual phase transmucosal delivery system comprising a semi-solid matrix incorporating one or more biologically active agents associated with artificial membrane vesicles (AMNs), such as liposomes. The delivery system may further comprise one or more unassociated biologically active agents incorporated directly into the matrix.

The characteristics of the matrix provide for substantially uniform dispersion of both the AMN-associated and non-AMN associated biologically active agent(s) with no phase separation should the matrix liquefy at a high ambient temperature. The delivery system is especially suited for oral administration due to its palatability.

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, birds, and reptiles.

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

The terms "antigen" and "antigenic material," as used interchangeably 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 (AMN)," 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 "AMN-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 AMN. The term also encompasses biologically active agents to which one or more AMN is adhered.

The term "unassociated," as used herein with reference to a biologically active agent means that the agent is not associated with an AMV.

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 AMN.

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 AMNs, 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. 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.

The Delivery System The dual phase transmucosal delivery system according to the present invention comprises one or more AMV-associated biologically active agent substantially uniformly dispersed within a semi-solid matrix. The delivery system may further comprise one or more unassociated bioactive agents incorporated directly into the matrix. The AMVs may be incorporated into the matrix in the form of pre-hydrated solutions, or they may be incorporated as pro-AMV preparations. Association of a bioactive agent with an AMV can be used, for example, to facilitate the incorporation of hydrophobic and amphiphilic agents into the delivery system and/or for efficient transmucosal delivery of the agent in the gastro-intestinal tract.

h one embodiment, the dual-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 separate phases (i.e. associated with an AMN or incorporated into the matrix).

In one embodiment of the present invention, the transmucosal 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, transmucosal 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. The delivery systems of the present invention comprise one or more AMV-associated bioactive agent substantially uniformly dispersed within a matrix which comprises 1) one or more carbohydrate that exhibits good moisture binding and low gelatinisation temperature; 2) a sugar component comprising one or more sugar, sugar syrup and/or sugar alcohol; 3) a hydrocolloid component; 4) a solvent component comprising one or more polyhydric alcohol and 5) one or more source of water. The use of one or more carbohydrate 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 artifiςial flavourings, colourings, acidulants, buffers and sweeteners can also be included in conventional amounts in the matrix.

Due to the substantially uniform and complete dispersion of the AMV-associated bioactive agents within the matrix, the transmucosal delivery systems are suitable for division into sub-units. For example, if a single unit of a delivery system of the invention is divided into three subunits, each subunit will contain a third of the dose of the original unit. Such division would not be possible with other delivery systems in which the bioactive agents are not evenly dispersed.

The matrix of the delivery systems provides for minimised degradation of the bioactive agents 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 delivery systems are prepared at a temperature of 100°C or less. In one embodiment of the present invention, the delivery systems are prepared at or below a temperature of 75°C. In another embodiment, the delivery systems are prepared at or below a temperature of 70°C. In a further embodiment, the delivery systems are prepared at or below a temperature of 65°C. Low temperatures can be employed in the preparation of the delivery system because the matrix is formulated to remain flowable at temperatures at or above 35°C. hi one embodiment of the invention, the matrix remains flowable at or above 45°C.

In addition, the transmucosal delivery systems have a low moisture content and low water activity, which can contribute to the stability of the AMVs or pro-AMVs contained therein. The low moisture content and low water activity also contribute to the stability of any sensitive bioactive agents incorporated into the delivery system in a non-AMV associated form.

In accordance with the present invention, the final moisture content of the delivery systems is between about 10% and about 30%. In one embodiment, the final moisture content of the delivery systems is between about 11% and about 25%. In another embodiment, the moisture content is between about 13% and about 20%). In other embodiments, the moisture content is between about 15% and about 18%, and between about 15% and about 16%.

Typically, the delivery systems of the present invention have a water activity (a_w) below about 0.7. In one embodiment of the invention, the water activity of the final delivery systems is below about 0.6. In another embodiment, the water activity is below about 0.55. In further embodiments, the water activity is 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.

The matrix also provides for minimized degradation of the bioactive agents incorporated therein during storage of the final delivery systems under normal storage conditions (i.e. at temperatures of 30°C or below), hi accordance with the present invention, therefore, degradation of the bioactive agents during storage of the delivery systems under normal conditions is less than about 20%. In one embodiment, degradation of the bioactive agents 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 delivery systems of the present invention can be formulated such that the matrix has a final pH in the range of about 2.5 to about 9.0. In order to maintain the stability of the AMVs within the matrix, however, the matrix is typically formulated with a pH between about 4.0 and about 9.0. One skilled in the art will appreciate that the final pH of the delivery system must be adjusted in accordance with the type of AMV and bioactive agent(s) incorporated therein. Selection of an appropriate final pH for the delivery system is within the ordinary skills of a worker in the art. In one embodiment of the present invention, the matrix is formulated with a final pH of between about 4.5 and about 8.5. In another embodiment, the matrix is formulated with a final pH of between about 5.0 and about 8.0. In a further embodiment, the matrix is formulated with a final pH of between about 5.5 and about 7.8.

The final form of the transmucosal delivery systems of the present invention 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 of confectioneries. The delivery systems, therefore, are formulated to be semi-solid at normal room temperature, hi the event, however, that the delivery system liquefies due to exposure to elevated temperatures, the formulation of the delivery system matrix is such that no phase separation of the matrix components occurs and the delivery system can be readily re-solidified by cooling (for example, by cooling to room temperature, or more rapidly by cooling to temperatures of around 4°C). The reformed product maintains the substantially uniform dispersion of the AMV- associated bioactive agents contained therein, hi one embodiment of the present invention, the delivery systems are formulated to be semi-solid at temperatures at or below about 40°C. In another embodiment, the delivery systems are semi-solid at or below about 35°C. hi other embodiments, the delivery systems are semi-solid at or below about 32°C, 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 the final properties of the delivery systems are maintained, i.e. substantially uniform and complete dispersion of the bioactive agents, minimisation of the degradation of the bioactive agents and a final moisture content for the delivery systems 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.

The delivery systems according to the present invention are suitable for oral administration to both human and non-human 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. Due to their highly portable format, the delivery systems are simple and convenient to administer and to consume for both humans and other animals.

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.

One skilled in the art will 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 provided below that are significantly toxic or cause other types of significant harm to animal health are explicitly excluded from the description of the invention.

1. The Matrix

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 AMVs without significant degradation of these components or the bioactive agents associated with them. 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. h 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 mould (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. In 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 corn, 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, corn, waxy corn, 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.

In accordance with the present invention, the carbohydrate component of the matrix ranges from about 0.6% to about 15%o 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. In another embodiment, the amount of modified starch included in the matrix is between about 2% and 10%, In 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 sugar component of the matrix comprises one or more sugars, sugar syrups, sugar alcohols and/or sugar alcohol solids. Examples include, but are not limited to, sugars such as sucrose, glucose, xylose, ribose, maltose, galactose, dextrose, and fructose; syrups such as corn 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 corn syrup. Corn 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 corn syrup. In another embodiment, the matrix comprises a corn syrup that exhibits a D.E. of between 20 D.E. and 99 D.E. In other embodiments, the matrix comprises a "high" DE corn syrup with a D.E. of between 40 and 70, or with a D.E. of between 62 and 65. In another embodiment, the corn syrup is a high fructose corn syrup.

Various corn syrups are commercially available. For example, 62 D.E. 1600 Corn Syrup (Casco Inc./ Canada Starch Operating Co. Inc.), SWEETOSE 4300 corn syrup (a 63 D. E. corn syrup; A. E. Staley Manufacturing Company; Decatur, IL) and Clearsweet^® 63/43 IX corn syrup (a 63 D. E. corn 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 syrup and high fructose corn syrup, a high DE corn syrup and high fructose corn syrup and maltitol syrup and high fructose corn syrup.

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, structural and functional properties. In 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 hydrocolloids that may be 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, carageenan, gellan, alginate, or various combinations thereof. The use of hydrocolloids is well-known in the art and many hydrocolloids 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^® hydrocolloids from 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 carageenan 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 bioactive agent(s) to be incorporated into the delivery system. For AMVs and bioactive agents that are unstable at higher temperatures a hydrocolloid or mixture of hydrocolloids that have a low set temperature will be required, whereas for AMVs and bioactive agents that are more stable hydrocolloids 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. In one embodiment, the Bloom value is about 250 BL. h 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%o by weight. In one embodiment, the total amount of hydrocolloid in the matrix is between about 0.5% and about 6.8% by weight, i another embodiment, the total amount is between about 1.0%> and about 6.6%. In 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 AMVs and unassociated bioactive agents (if used) to allow for substantially uniform and complete incorporation of these ingredients into the matrix. 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 hydrocolloids, such as gellan and carageenan, require cations for proper gelation to occur, whereas other hydrocolloids 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. In 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. In one embodiment, it is added in an amount between about 1% and about 3%. In another embodiment, it is added in an amount between about 1.2% and about 2.5%.

1.7 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 syrup, 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 AMNs or bioactive agents 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%ι 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. AMNs

AMVs are vesicles comprising hydrated uni- or multi-lamellar systems of bilayers of amphipathic 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 AMNs in this manner. The AMN 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 AMNs include, but 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-Ν-ρolyethylene 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 l,2-diacyl-3-trimethylammoniumpropane (TAP), l,2-diacyl-3- dimethylammom^'umpropane (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 l,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 dipalmitoylphosphatidylgycerol.

As is also known in the art, AMVs can be formulated to have an overall positive, neutral, or negative charge. Amphoteric AMNs are also known in the art. AMNs 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 AMNs 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) maybe 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 AMNs 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 AMN. AMNs may be made from extracts containing lipids from natural sources.

Biologically active agents can be combined with AMN 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 AMN of known charge, size and lamellarity and will be dependent on the type of biologically active agent to be associated with the AMN. Selection of the appropriate AMN 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 AMNs, AMN 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.

Νiosomes 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 may be 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 AMNs is 20nm to lOOOnm. hi one embodiment of the invention, AMNs 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, AMNs having an average size between about 80nm and about 200nm are used. In a further embodiment, AMNs having an average size between about 80nm and about 120nm are used.

2.1 Preparation of the AMVs The AMNs 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 AMN 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, AMNs can be formed by the method disclosed in GB-A-2,134,869. h this method, microspheres (lOμm or less) of a hydrosoluble carrier solid (ΝaCl, 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.

AMNs 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. Set, 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 Νos. 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 AMNs passively (i.e. during AMN formation) or actively (i.e. after AMN formation) using standard techniques.

Other methods of preparing AMNs 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 AMNs or pro-AMNs 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 AMNs or pro-AMNs may be prepared according to specific requirements.

3. Biologically Active Agents

One skilled in the art will appreciate that the delivery system according to the present invention can be used to deliver a wide variety of biologically active agents to an animal or human. A person skilled in the art will understand that certain bioactive agents are best incorporated directly in the matrix while others may be more suited for association with AMVs. 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 and that certain agents will fall into more than one category.

Biologically active agents alone or associated with AMNs 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, unassociated biologically active agents incorporated into the matrix constitute less than about 20%> by weight of the final product. In one embodiment, unassociated biologically active agents constitute between 1% and about 10% by weight of the final product. In another embodiment, unassociated biologically active agents constitute between 5% and about 10% by weight of the final product. It is understood that the total daily intake 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 bioactive agents 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 bioactive agents suitable for administration to animals may differ from those suitable for humans. Selection of appropriate bioactive agents for incorporation into the delivery system for administration to a given animal is considered to be within the ordinary skills of a worker in the art. In addition, it will be apparent that inappropriate combinations of bioactive agents, for example those that may interact, should be included in different phases of the delivery system. Thus, the present invention contemplates various combinations of bioactive agents for use with the delivery system.

3.1 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, doxyrubicin, taxol, cisplatin; anti- viral compounds such as ddl and ddA, anti-inflammatory compounds such as NSAfl s and steroids; antibiotic compounds such as antifungal and antibacterial compounds; cholesterol lowering drugs and contrast agents for medical diagnostic imaging. In general, drugs suitable for use with AMNs can be classified as water-soluble, AMN permeable; water-soluble, AMN-impermeable and lipophilic.

Water-soluble, AMN-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 AMV 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 drugs tend to partition into the 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 drugs include prostaglandins, amphotericin B, progesterone, isosorbide dinitrate, testosterone, nitroglycerin, estradiol, doxorubicin, epirubicin, beclomethasone and esters, vitamin E, cortisone, dexamethasone and esters, and betamethasone valerete.

3.2 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 spp. 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), histitut 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 1-2168. Bifidobacterium lactis (Bbl2) maybe 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, hi 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. In a related embodiment, this mixture comprises PREBI01^® or a mixture of commercially available RAFTILOSE^® and RAFTILL E^® 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. In 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 (niacinamide 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 B 1 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 and other tocopherols, lycopenes, beta-carotene or other carotenoids, quertin, rutin, flavonoids, catechins anthocyanes, eleutherosides and ginsenosides. Some of these antioxidants may be found in significant amounts in plant extracts. Examples include Ginko Biloba leaves that contain Gingko flavanoids, Blueberry fruits which 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 may be used in amounts of up to 160 mg, typically between 60 mg and 90 mg in a single unit.

For example, 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 No. EP 283675. 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 U.S. 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 lipases), shark cartilage extracts, Brewer's yeast, blue green algae and the like.

In 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 Pariax ginseng, lemon balm, Kava Kava, matte, bilberry, soy, grapefruit, seaweed, hawthorn, lime blossom, sage, clove, basil, curcurnin, 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.

3.3 Antigens

Antigenic material includes but is not limited to, proteins, polypeptides, polysaccharides, lipopolysaccharides, nucleic acids such as DNA and mRNA, 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.

Antigenic material may be isolated, purified or synthesized using a variety of techniques familiar to a worker skilled in the art.

Polypeptide antigens used as antigenic material for the purposes of the present invention can be synthesized by methods familiar to a worker skilled in the art, for example, by the well-known solid phase method. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963), Houghten et al., Int. J. Pept. Proc. Res. 16: 311- 320 (1980) and Parker and Hodges, J. Prot. Chem. 3: 465-478 (1985), for a complete discussion of these techniques. The solid phase method of polypeptide synthesis can be practiced utilizing a Beckman Model 990B Peptide Synthesizer, available commercially from Beckman Instruments Co., Berkeley, Calif, U.S.A.

4. Process for Preparing the Delivery System

In accordance with the present invention, the delivery system is prepared at temperatures below 100°C. Various standard methods known in the confectionery manufacturing industry can be used to prepare the delivery systems 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 delivery system.

In order to allow for full dispersion and incorporation of the AMV-associated bioactive agents into the matrix while minimising or preventing degradation of the AMNs and/or the bioactive agents, the matrix of the delivery system remains flowable at or below about 60°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 AMN-associated bioactive agents into the matrix at temperatures at or below about 60°C. The AMN-associated bioactive agents may be in the form of an AMN solution or a pro-AMN preparation. In one embodiment of the present invention, the step of incorporating the AMV-associated bioactive agents into the matrix is conducted at temperatures at or below about 55°C. In another embodiment, the step of incorporating the AMV-associated bioactive agents into the matrix is conducted at temperatures at or below about 50°C. h a further embodiment, this step is conducted at temperatures at or below about 45°C.

The present invention also contemplates the addition of certain compounds to the AMV or pro-AMN preparation that may stabilise the preparation to heat and allow for incorporation of the AMN-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-liposomal preparations.

The AMN solution may be incorporated into the matrix delivery system up to a level of approximately 50% by weight. In one embodiment, the AMN preparation constitutes between about 1% and about 50% by weight of the delivery system. In another embodiment, the AMV preparation constitutes between about 5% and about 40% by weight. In a further embodiment, the AMV preparation constitutes between about 10%) and about 25% by weight.

One skilled in the art will appreciate that, when an AMV solution is used, the maximum amount of solution that may be incorporated into the delivery system can vary and will depend upon the total liquid content and AMV concentration of the solution. Additionally, 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 AMN preparation is considered to be within the skills of a worker in the art.

The following description represents a general method of preparing a delivery system of the present invention.

Briefly, the AMN preparation is blended with the solvent and is kept at or below about 60°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 about 60°C or below, at which point the AMN/solvent mixture is added. 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.

In 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. In 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 AMN/solvent 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 particular AMN preparation and 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 can be formed into the 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 delivery systems 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 AMN- associated 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. In contrast, the use of other hydrocolloids or combinations of other hydrocolloids 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 the AMN or pro-AMN preparation 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.

In one embodiment of the present invention, a transmucosal delivery system is prepared using (a) modified starch; (b) gelatine: gellan as the hydrocolloid; (c) a mixture of corn syrup and high fructose corn syrup 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. The process comprises blending the glycerol and propylene glycol, adding the liposome solution and holding the resulting blend at about 25 - 45°C. The fructose syrup, glucose syrup, potassium citrate and sweetener are blended with water. The gelatine is blended with the gellan and then blended into the fructose syrup mixture together with the starch and the temperature is raised to about 80 - 85°C in order to hydrate the starch. The temperature is maintained until the moisture content has been reduced 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 solution is then cooled to about 40 — 45°C and the liposome solution is added, together with any desired colouring and flavouring. The resulting matrix is then formed into the desired shape using standard procedures.

hi another embodiment of the present invention, a transmucosal delivery system containing the same matrix components as indicated above together with an AMN- associated bioactive agent in the form of a pre-liposomal solution is prepared by the following process. Glycerol and propylene glycol are blended together, 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 is then formed into the desired shape, for example, by injection filled into preformed packaging.

In a further embodiment of the present invention, a transmucosal delivery system is prepared using (a) modified starch; (b) gelatine :pectin as the hydrocolloid; (c) a mixture of corn syrup and high fructose corn syrup as the sugar component, (d) a mixture glycerol and propylene glycol as the solvent component, (e) water and (f) an AMN-associated bioactive agent in the form of a pre-liposomal solution. The process comprises blending the pre-liposomal solution with the glycerol and propylene glycol and warming the resulting solution to about 40°C - 60°C. The sugar syrups are blended with water and heated to about 70°C. The starch, pectin and gelatine are mixed and combined with the warmed syrups under high shear. The mixture is maintained at a temperature of between 45°C and 60°C. The pre-liposome/solvent blend is incorporated into the mixture, which is maintained at a temperature of between 45°C and 55°C. Colouring or flavouring is then added, if desired, and the resulting delivery system is formed using standard techniques.

If one or more unassociated bioactive agent is to be incorporated directly into the matrix in addition to the AMN-associated bioactive agent, this may be accomplished by blending the bioactive agent into the solvent mixture at an appropriate temperature and adding this to the blended matrix components. This temperature will be dependent on the stability of the agent and can be readily determined by a skilled technician. When the temperatures for addition of the unassociated and AMN- associated bioactive agents are compatible, they may be added to the solvent component together, if desired. Alternatively, if heat stable bioactive agent(s) are used, they can be combined with the dry ingredients or the sugar component during the preparation of the matrix.

5. Testing the Delivery System

5.1. Physical Properties

As is standard in the art, a sample of the delivery system incorporating the desired AMN-associated bioactive agent(s) can be prepared prior to large-scale production and tested in order to determine whether the matrix retains the desired physical properties, i.e. that the AMN-associated bioactive agent(s) are substantially uniformly dispersed, that degradation of the bioactive agents during the preparation of the matrix is below 20% and that the water activity of the delivery system is below 0.7.

For example, dispersion of the AMN-associated bioactive agent(s) in the delivery system can be determined by dividing a single unit of the final delivery system 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 LR spectrometry, or chromatographic techniques, such as gas chromatography or high-performance liquid chromatography (HPLC). If the % by weight of AMV-associated bioactive agent in each subunit is similar, then the bioactive agent is said to be substantially uniformly dispersed throughout the product. One skilled in the art will appreciate that the % by weight need not be identical for each subunit to indicate substantially uniform dispersion. In one embodiment the present invention, the %> by weight of AMN- associated bioactive agent in each subunit of the final delivery system varies by less than 5%. In another embodiment, the % by weight of AMN-associated bioactive agent in each subunit of the final delivery system varies by less than 2%>. hi further embodiments, the %> by weight of bioactive agent in each subunit varies by less than 1.5% and less than 1.0%.

Similarly, the degradation of the AMN-associated bioactive agents can be determined by standard analytical techniques taking into account the total amount of each AMN- associated bioactive agent included in the preparation of the delivery system. 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 techniques, such as HPLC. As indicated above, the degradation of the bioactive agents is minimised during the preparation of the delivery system and is less than about 20%> in the final product.

The water activity (a_w) of the final delivery system 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/p₀- In accordance with the present invention, the water activity of the final delivery system is less than about 0.7. Other parameters, such as the release rate of the AMN-associated bioactive agents from a 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 containing a known amount of AMN-associated 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 stirred. 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 agents contained therein is analysed under conditions that are known to accelerate the degradation of food products and can be correlated 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.

5.2 Efficacy

The delivery systems of the present invention may also 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 pharmacokinetic 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 bioactive agent(s) 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 a bioactive agent in a delivery system brings 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 bioactive agents, 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 phosphorus compounds can also be utilized. The total concentration of creatinine can also be measured after 24 hours in order to examine clearance of creatine.

6. Format Of The Delivery System

The present invention contemplates various formats for the delivery systems. For example, the delivery systems may be in the form of a confectionery, such as a jujube, in which case it may be formulated alone or it may further comprise a coating, such as a chocolate or yoghurt coating. Preparation of jujube or jelly type confectionery products are known in the art and include, for example, the use of moulds, injection- filling of pre-formed packages and extrusion processes. It will be readily apparent to one skilled in the art that such standard techniques can be applied to prepare a wide variety of different shaped confectioneries.

For example, a variety of differently shaped moulds or pre-formed packages may be used. Jelly candies such as imitation fruit pieces, fruit bars, and sugared jellies are typical. These confections have a firm, but soft, texture that contributes to their desirable mouth feel. Jelly candies can be manufactured by, for example, the open kettle or batch process, or various kinds of continuous processes and formed by, for example, the traditional Mogul system using starch moulds or by injection-filling packages pre-formed into an appropriate size and shape with the liquid mixture and allowing the mixture to set up. Alternatively, the delivery system can be fonned as a confectionery product by an extrusion process in which the matrix mass is forced at relatively low pressure through a die which confers on the matrix the desired shape and then the resultant extrudate is cut off at an appropriate position to yield products of the desired weight. For example, the matrix can be forced through a die of relatively small cross-section to form a ribbon, which is carried on a belt under a guillotine-type cutter which cuts the moving ribbon into pieces of equivalent weight and dimensions. Alternatively, the mass may also be extruded as a sheet, which is then cut with a stamp or cookie type cutter into appropriate shapes. After moulding or shaping, the delivery system confectionery product is moved by a conveyor to an area where it may be further processed or simply packaged.

Methods of making and applying coatings to confectionery products are also well- known in the art. Coatings are in general compound coatings the major ingredients of which are sugar and fat. Flavours and colours are often added. Chocolate coatings are usually based on cocoa butter whereas yoghurt coatings typically comprise powdered yoghurt, hi 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. Typically, application of the coating to the confection takes place while the coating is molten, for example, by passing the formed confection 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 confection. Excess coating is blown off by means of air jets and the coated confection passes through a cooling tunnel where refrigerated air currents solidify the applied coating, hi accordance with the present invention, the properties and method of application of the coating must not interfere with, or compromise, the properties of the delivery system. For example, the application of the coating must not require elevated temperatures that would affect the stability of the AMN-associated bioactive agent(s) incorporated into the delivery system.

The present invention further contemplates the delivery system as a filling or a coating, for example, for baked goods such as wafers or cookies. For example, the matrix can be used as a layer between two wafers, or a jelly layer on the top of a cookie or sponge, in which case the product may be further coated with a chocolate or other flavoured coating, if desired, as described above for confectionery products. Alternatively, the matrix may be used to fill doughnut type baked goods. Methods of filling and coating baked goods are also well known in the art.

7. Administration

The organoleptic properties of the delivery systems of the present invention ensure that they are easy to take and/or to administer, h one embodiment, the delivery systems are formulated for administration 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 another embodiment, the delivery systems provide a means of easily administrating certain bioactive agents, such as multi- vitamins and minerals, to children.

h another embodiment, the delivery systems are formulated for administration to a non-human animal. In a related embodiment the non-human animal is 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.

Kits The present invention additionally provides for kits containing a delivery system for administration to a human or non-human 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 agent(s) contained in the delivery system. The instructions typically include information as to the appropriate dosage and dosing schedule for the bioactive agent(s) in terms of units of 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. All percentages throughout the specification and claims are by weight of the final delivery system unless otherwise indicated.

EXAMPLES

EXAMPLE 1: Preparation of a Delivery System containing Creatine and Dimethylglycine One example of a matrix comprising unassociated creatine and dimethylglycine is as follows:

Ingredient % by weight Ingredient % by weight

Glycerol 14.57%

Propylene Glycol 5.30%

Creatine monohydrate 11.71%

Corn Syrup 62DE 31.79%

Sucralose 0.04%

Modified Starch ( Staley Softset^®) 2.65%

Potassium citrate 2.15%

Dimethylglycine 1.67%

High fructose corn syrup 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. hi a separate container, the two types of gelatine and the gellan were blended together. The fructose syrup 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. In a third container, the com syrup was warmed to 30-35°C and the sucralose, potassium citrate, dimethylglycine and starch were then blended in. The corn syrup 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 delivery system was then moulded using standard techniques. EXAMPLE 2: Preparation of a Delivery System containing a Liposome-Associated Bioactive Agent

The following formulation yields a 12gram product containing approximately 5 grams of CoQIO.

Ingredient % by weight

Glycerol 17.19%

Propylene Glycol 5.93%

CoQIO liposome solution 10.37%

Corn Syrup 62DE 22.81%

High fructose corn syrup 26.67%

Modified Starch ( Staley Miraquick^®) 2.96%

Potassium citrate 1.51%

Water 5.33%

Gelatine type A 5.^'93%

Gellan (Kelcogel^® LT100) CP Kelco 0.36%

Colour 0.30%

Flavour 0.59%

Total: 100.00%

Note: the concentration of the CoQIO liposome solution is approximately 4.2gm CoQIO/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. In a separate container, the gelatine and the gellan were blended together. The fructose syrup, glucose syrup, 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 hydrocolloids. 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 3: Preparation of a Delivery System containing a Liposome-Associated Bioactive Agent

Ingredient % by weight

High fructose corn syrup 24.77%

62 DE Corn syrup 21.19%

1 -Testosterone pre- -liposomal solution¹ 16.70%

Glycerol 15.96%

Propylene Glycol 5.50%

Gelatine 5.50%

Water 4.95%

Modified Starch (Staley Miraquick®) 2.75%

Potassium citrate 1.40%

Flavour 0.55%

Gellan 0.33%

Colour² 0.28%

Sucralose³ 0.11%

Total: 100.00%

* 1.25 mg/ml solution 10%) w/w solution ³ 25%o 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 hydrocolloids. 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 4: Preparation of a Delivery System containing a Liposome-Associated Bioactive Agent

Another example of a delivery system containing a liposome-associated bioactive agent is provided below:

Ingredient % by weight

Tocopherols 0.03%

63 DE Corn syrup 8.54%

High fructose corn syrup 27.33%

Pre-liposomal/bioactive solution 41.00%

Glycerol 15.96%

Propylene Glycol 4.78%

Gelatine 7.52%

Water 7.17%

Modified Starch (Staley Miraquick®) 2.05%

Flavour 0.68%

Pectin 0.41%

Colour¹ 0.34%

Sucralose² 0.14%

Total: 100.00%

¹ 10%) w/w solution ² 25%o w/w solution

Glycerol and propylene glycol were first blended with the tocopherol and the pre- liposome solution. The blend was warmed to between 40°C and 60°C. In a separate container, the starch, gelatine and the pectin were blended together. The syrups were blended together and heated to about 70 - 80°C. The blended solids were then added to the heated syrups under high shear and the mixture was maintained at a temperature of about 45 - 60°C. The liposome/solvent mixture was blended in and the mixture was maintained at about 45 - 55°C. Colour and flavour additives were then added and the delivery system was then moulded in silicone rubber sample moulds.

EXAMPLE 5:

Ingredient % by weight

Corn 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 UN 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, transfened 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 6: Preparation of a Delivery System containing Creatine

Another example of a delivery system containing unassociated creatine is as follows:

Ingredient % by weight

Glycerol 15.97%

Propylene Glycol 5.51% Ingredient % by weight

Creatine Monohydrate 16.71%

63 DE Corn syrup 21.20%

High Fructose Corn Syrup 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 syrup 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 hydrocolloids. The solid content can then adjusted by addition of water. The temperature of the syrup 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 UN detection to determine 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 transfened 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 and the percentage by weight of creatine contained within each sample are 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 7: Preparation of a Delivery System containing Creatine

Another example of a delivery system containing unassociated creatine is as follows:

Ingredient % by Weight

Glycerol 27.9990%

Propylene Glycol 3.4145%

Potassium Hydroxide 0.1208%

Creatine Monohydrate 24.0154%

High Fructose Corn Syrup 15.7068%

Corn 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%

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. h 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.

In vivo Testing 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 (prepared as described in Example 1) 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 8: Accelerated Shelf-Life Determination

An accelerated shelf life test was conducted on the creatine delivery system prepared as described in Example 6 and demonstrates the stability of the matrix 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 (Nolume 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 delivery system 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 jujubes after 35 days exposure to the above-described conditions. Prior to the start of the experiment, three jujubes had an average of 13.4% by weight of creatine monohydrate. After 35 days, four jujubes 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 9: Analysis of Water Activity of the Delivery System

Water activity was measured in samples of jujubes that had been prepared according to the method described in Example 6 and demonstrates the low water activity of the matrix 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 milliamperes (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 of NaCl.

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.

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 (1039 u_g mL) x (500 mL)/(1000)

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. An oral delivery system for biologically active agents comprising a matrix having one or more biologically active agent associated with artificial membrane vesicles (AMNs) substantially uniformly dispersed therein, said matrix comprising:

i) a sugar component comprising one or more sugar, sugar syrup, sugar alcohol, or a combination thereof; ii) one or more carbohydrate; iii) a hydrocolloid component comprising one or more hydrocolloid; iv) a solvent component comprising one or more polyhydric alcohol, and v) one or more source of water, wherein said delivery system is a semi-solid at room temperature and has a final moisture content of between about 10%o and about 30% by weight, a pH between about 4.0 and about 9.0 and a water activity of less than about 0.7.

2. The oral delivery system according to claim 1, wherein said matrix 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.

3. The oral delivery system according to claim 1 or 2, wherein said one or more carbohydrate comprises a starch or a modified version thereof.

4. The oral delivery system according to claim 3, wherein said starch or modified version thereof, is present in an amount between about 1% and about 15% by weight.

5. The oral delivery system according to any one of claims 1 - 4, further comprising one or more modified cellulose.

6. The oral delivery system according to any one of claims 1 — 5, wherein said hydrocolloid component comprises a mixture of gelatine and gellan in a ratio between about 15:1 to about 40:1.

7. The oral delivery system according to any one of claims 1 - 6, wherein said hydrocolloid component comprises a mixture of gelatine and pectin in a ratio between about 15:1 to about 35:1.

8. The oral delivery system according to any one of claims 1 — 7, wherein said sugar component comprises one or more sugar syrup.

9. The oral delivery system according to any one of claims 1 - 8, wherein said solvent component comprises glycerol and propylene glycol.

10. The oral delivery system according to any one of claims 1 - 9, wherein said one or more bioactive agents are selected from the group of drugs, botanicals, nutritional supplements, vitamins, minerals, enzymes, hormones, proteins, polypeptides and antigens.

11. The oral delivery system according to any one of claims 1 — 10, further comprising a sweetener, a buffer, a natural or artificial flavouring, a colouring agent or a combination thereof.

12. The oral delivery system according to any one of claims 1 - 11, wherein said AMN is a liposome.

13. A process for preparing an oral delivery system for biologically active agents associated with artificial membrane vesicles (AMNs), said process comprising:

(a) 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;

(b) optionally adjusting the moisture content of the blend; (c) reducing the temperature of the blend to about 60°C or below;

(d) adding to said blend one or more AMN-associated biologically active agent and a solvent component comprising one or more polyhydric alcohols to form a matrix whereby the AMV-associated biologically active agent is substantially uniformly dispersed throughout said matrix, and

(e) forming said matrix to provide said oral delivery system, wherein said oral delivery system has a moisture content between about 10%o and about 30%> by weight.

14. The method according to claim 13, 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 one or more sugar, sugar alcohol or sugar syrup, or a combination thereof, preheated to a temperature of less than 100°C whereby said hydrocolloid and said carbohydrate are hydrated.

15. The method according to claim 13, 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 one or more sugar, sugar alcohol or sugar syrup, or a combination thereof, and simultaneously increasing the temperature to between about 60°C and about 100°C to hydrate said hydrocolloid and said carbohydrate.

16. The method according to claim 13, wherein said blend is prepared by (i) blending one or more hydrocolloid with a solution comprising water and one or more sugar, sugar alcohol or sugar syrup, or a 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.

17. The method according to any one of claims 13 — 16, wherein the AMN- associated biologically active agent is dispersed in the solvent component prior to addition to the blend in step (d).

18. The method according to any one of claims 13 - 17, wherein the AMN- associated biologically active agent is in the form of a pro-AMN preparation.

19. The method according to any one of claims 13 - 18, further comprising adding one or more modified cellulose to the solvent component in step (d).

20. The method according to any one of claims 13 - 19, further comprising adding a natural or artificial flavouring, a colouring agent or a combination thereof to the matrix in step (d).

21. The method according to any one of claims 13 - 20, further comprising adding a sweetener, a buffer or a combination thereof to said blend in step (a) or (d).

22. An oral delivery system for AMN-associated biologically active ingredients prepared by the process according to any one of claims 13 - 21.

23. Use of a delivery system according to any one of claims 1 - 12 or 22 for oral administration of one or more biologically active agent to an animal in need thereof.